hydrogels based on cucurbituril and methods for preparing said hydrogels

专利摘要:
CUCURBITURIL-BASED HYDROGELS. It is a hydrogel, in which the hydrogel has a crosslinked supramolecular network obtainable or obtained from the complexation of an aqueous composition comprising a host, such as cucurbituril, and one or more polymers having adequate guest functionality. One or more polymers in the aqueous composition can have a molecular weight of 50 kDa or greater, such as 200 kDa or greater. The hydrogel can hold a component, such as a therapeutic compound or a biological molecule. Hydrogels are suitable for use in medicine.
公开号:BR112014020450B1
申请号:R112014020450-0
申请日:2013-02-20
公开日:2020-12-08
发明作者:Oren Scherman；Eric Appel；Xian Jun Loh；Matthew Rowland；Frank Biedermann
申请人:Cambridge Enterprise Limited；
IPC主号:

专利说明:

RELATED REQUESTS
[001] The present case refers to and claims priority to document GB 1202834.6 deposited on February 20, 2012 and document GB 1301648.0 deposited on January 30, 2013, with the contents of both here incorporated in their totalities as a reference. FIELD OF THE INVENTION
[002] The present invention relates to hydrogels based on a supercellular lattice network of cucurbituril, and to methods for the preparation of these hydrogels, and their use in methods of distributing components retained within the hydrogel. FUNDAMENTALS
[003] Hydrogels are three-dimensional cross-linked polymeric networks that trap and store large amounts of water. Given their similarity to soft biological tissues and their mechanical properties, that is, from soft and delicate to hard and resistant, these hydrogels are progressively important in a variety of biomedical and industrial applications.
[004] Hydrogels can be prepared using a covalent or non-covalent approach. Most covalently crosslinked polymer hydrogels are fragile, have poor transparency and lack the ability to self-heal once the mesh is broken (Peppas et al. Annu. Rev. Biomed. Eng. 2 2000, 9-29) . These disadvantages were addressed by employing dynamic and reversible non-covalent interactions such as structural crosslinking in hydrogels (direct formation) or by exciting the formation of nanofibers, whose entanglement subsequently leads to the formation of hydrogels (indirect formation) (Estroff et al. Chem. Rev. 2004, 104, 1201-1217; Wojtecki et al. Nat. Mat. 2010, 10, 1427). There are a large number of small molecules that self-assemble into long fibers, leading to the formation of hydrogel. An excellent example of this is the series of amphiphilic peptides developed by Stupp and collaborators (Hartgerink et al. Science 2001, 294, 1684-1688). Relatively strong hydrogels (G '= 1 kPa) with low material loads in relation to water (0.5% by weight of peptide + 0.5% by weight of CaCl2) can be prepared in this way (Greenfield et al. Langmuir 2010, 26, 3641-3647). However, there are far fewer examples of direct hydrogel formation, especially those with a high water content (> 97%).
[005] From the wide precedence of supramolecular polymers, it is clear that there are only very few non-covalent systems that work in aqueous media (Sijbesma et al. Science 1997, 278, 1601-1604; Greef et al. Chem. Rev. 2009, 109, 5687-5754). These include host-guest interactions of cyclodextrins and cucurbit [n] urine, hydrophobic, ionic, and some metal-ligand interactions. Various attempts have been made to develop hydrogels from cyclodextrins, however, these have been intrinsically limited by the low affinity of binding CDs to their guests, which has resulted in poor mechanical properties (Kretschmann et al. Angew. Chem. Int. Edit 2006, 45, 4361-4365; Wu et al. Langmuir 2008, 24, 10306-10312; Koopmans et al. Macromolecules 2008, 41, 74187422). In addition, ionic interactions are extremely sensitive to the ionic resistance of aqueous media and, generally, to changes in pH although the use of transition metals in metal-ligand pairs should be avoided in many applications due to toxicity and environmental issues (Van Tomme et al. Biomaterials 2005, 26, 2129-2135; Hunt, J. et al. Adv. Mater. 2011, 23, 2327-2331; Wang et al. Nature 2010, 463, 339-343). Thermally activated hydrogels formed by the hydrophobic association of block copolymers, typically containing two or more blocks exhibiting a lower critical solution temperature (LCST), for example, N-isopropyl acrylamide or Pluronics, have been extensively studied (Loh et al. Macromol. Symp. 2010, 296, 161-169; Loh et al. Biomacromolecules 2007, 8, 585-593). Although these materials have demonstrated potential for bio-medical applications, such as engineering due to their spontaneous training in temperature and biocompatibility, their applicability in other industries has not been demonstrated.
[006] Biomaterials represent a rapidly developing field of designer materials that typically exhibit properties similar to bio-logical material or are biocompatible and useful for important biological applications, such as drug administration and tissue engineering. Hydrogels consist of a type of biomaterial that has been shown to be particularly important candidates for drug administration and tissue engineering applications given its similarity to soft biological tissues and highly variable mechanical properties (Lutolf et al. Nat. Mat. 2009, 8, 451 -453; Staats et al. Nat. Mat. 2010, 9, 537-538; Nochi et al. Nat. Mat. 2010, 9, 572-578). There are many systems that have been studied in detail in their sustained release of drugs for drug administration, wound coverage and chemosensitization for cancer therapy (Loh et al. J. Control. Release 2010, 143, 175-182; Loh et al. J Phys. Chem. B 2009, 113, 11822-11830; Loh et al. Biomaterials 2008, 29, 2164-2172; Loh et al. Biomaterials 2008, 29, 3185-194; Loh et al. Biomaterials 2007, 28, 4113 -4123; Li et al. Biomacromolecules 2005, 6, 2740-2747).
[007] Generally, a high concentration of polymeric constituents is required in these formulations, sometimes requiring more than 15% by weight, and often these formulations exhibit poor resilience and a strong and 'rapid' release of drugs . These disadvantages made the system unsuitable for many biomedical applications (Esposito et al. Int. J. Pharm. 1996, 142, 923; Katakam et al. Int. J. Pharm. 1997, 152, 53-58). Recently, biocompatible thermogelifying polymers based on poly (PEG / PPG / PHB urethane (s) have been described requiring relatively low polymer concentrations (5% by weight) to form hydrogels from aqueous solutions upon heating (Loh et al. Biomacromolecules 2007, 8, 585-593) In addition, the hydrolytic degradability of these triblock copolymers allowed an additional adjustability of their protein release, which has been shown to extend for approximately 75 days in vitro (Loh et al. Biomaterials 2007 , 28, 4113-4123).
[008] Some of the present inventors have previously described a hydrogel based on a network of polymers linked together by CB [8] (Appel et al. J. Am. Chem. Soc. 2010, 132, 14251-14260). This hydrogel aims to facilitate progress in the fields of intelligent self-healing materials, self-assembling hydrogels, and controlled solution viscosity. Polymers for use in the hydrogel have molecular weights in the range of 10.1 to 37.1 kDa and polydispersity values in the range of 1.11 to 2.42. At least one of the polymers has a molecular weight in the range of 10.1 to 21.8 kDa, and at least one of the polymers has a polydispersity value in the range of 1.53 to 2.42. The functionality of the polymers is in the range of 4.3 to 10.1%. The hydrogel is based on the non-covalent attachment of a polystyrene-based polymer to a polyacrylamide-based polymer using a cucurbituryl cuff.
[009] Although some of the present inventors have referred to the network described in the previous study as a hydrogel, this simply consists of a convenient term to refer to the material that has been obtained. It is recognized that the material can be referred to as a viscoelastic material. The rheological properties of this material are not ideal. For example, when the rheological properties of the materials are analyzed by dynamic oscillatory rheology at 10% tension, it is observed that the loss module (G '') dominates the storage module (G ') at higher frequencies. Therefore, the loss module is dominant at frequencies of 20 rad / s or greater. At lower frequencies, the storage module is dominant. G 'and G' 'are therefore non-linear and are not parallel in oscillatory rheology.
[010] The previous study also describes the Newtonian behavior of the material at a high shear (measured in stable shear measurements) with a subsequent catastrophic loss of viscosity. For example, materials have a viscosity in the range of 8 to 50 Pa s at shear rates in the range of 0.1 to 30 1 / s. Viscosity remains substantially constant over this range of shear rates. At shear rates greater than 30 l / s, viscosity decreases dramatically. SUMMARY OF THE INVENTION
[011] The present invention generally provides a hydrogel suitable for maintaining a distributable component. The present inventors have found that hydrogels based on a network of cucurbituril complexes are suitable for maintaining and distributing components. In particular, the present inventors have found that hydrogels obtainable from complexing cucurbituril with a high molecular weight polymer suitably functionalized with a cucurbituril guest functionality can be used to maintain and distribute components, such as proteins. Advantageously, the mesh, obtainable from cucurbituril and the polymeric component (s), consists only of a small percentage by weight of the total hydrogel. Therefore, the hydrogels of the present invention have a very high water content.
[012] The rheological properties of hydrogels are useful and desirable. These properties can be advantageously adjusted through appropriate changes to the hydrogel components and their reasons. A stable hydrogel can be prepared from a relatively small amount of material. As described in this document, hydrogels have a dominant storage module (G ') in relation to the loss module (G' ') for any frequency value in the range of 0.1 to 100 rad / s for a hydrogel analyzed by a frequency scan measurement at 37 ° C. The present hydrogels can be considered true hydrogels in view of the fact that G 'and G' 'are substantially linear and substantially parallel in oscillatory rheology, with G' dominant at all times. This is opposed to previously reported materials where G ’is not dominant throughout the oscillatory rheology.
[013] The shearing behavior of the hydrogels of the invention consists of an additional distinguishing feature. The hydrogels described in this document exhibit a continuous thinning behavior under shear when analyzed by stable shear methods. At low shear rates, for example, in the range of 0.1 to 0.3 1 / s, the viscosity value for the hydrogel material of the invention can be 100 Pa s or greater, for example 400 Pa s or greater, for example, 700 Pa s or greater. In contrast, at the same shear rates, prior art materials have significantly lower viscosity values (for example, in the range of 8 to 50 Pa s).
[014] The present inventors believe that the use of polymers with higher molecular weights, such as hydrophilic polymers with higher molecular weights, provides hydrogels having these advantageous rheological properties. The present inventors also recognize that hydrogels based on a cross-linked supramolecular network of cucurbituril are suitable for maintaining and releasing components as needed.
[015] In one aspect of the invention, a hydrogel is provided while maintaining a component, wherein the hydrogel has a crosslinked supramolecular network obtainable from the complexation of an aqueous composition comprising cucurbituryl and one or more polymers having a cucurbituryl guest functionality proper.
[016] In one embodiment, the aqueous composition comprises a polymer having a molecular weight of 50 kDa or greater, for example, 200 kDa or greater. In one embodiment, the component has a molecular weight of 200 kDa or greater.
[017] In a second aspect, the present invention provides a hydrogel, wherein the hydrogel has a crosslinked supramolecular network obtainable or obtained from the complexation of an aqueous composition comprising cucurbituril and one or more polymers having adequate cucurbituril functionality . One or more polymers in the aqueous composition have a molecular weight of 50 kDa or greater. The polymers in the composition can be hydrophilic polymers.
[018] In another aspect of the invention, a method is provided for preparing a hydrogel while maintaining a component, the method being in accordance with (a) or (b), in which
[019] (a) the method comprises the steps of (i) obtaining or forming a hydrogel, and (ii) introducing a component into the hydrogel to thereby form a hydrogel holding a component, wherein the hydrogel holding a component is formed by placing a mixture of cucurbituryl in contact with an aqueous solution and one or more polymers having a suitable cucurbituril guest functionality to thereby form a cross-linked supramolecular network; and
[020] (b) the method comprises the step of putting in contact in aqueous solution a mixture of cucurbituril, a component, and one or more polymers having a suitable cucurbituril guest functionality, to thereby form a hydrogel maintaining a component and the hydrogel is a cross-linked supramolecular network.
[021] In another aspect of the invention, a method is provided for the preparation of a hydrogel, the method comprising the step of contacting a mixture of cucurbituril in an aqueous solution, and one or more polymers having a suitable guest functionality of cucurbituril, to thereby form a cross-linked supramolecular network, in which the aqueous composition comprises a polymer having a molecular weight of 50 kDa or greater.
[022] Advantageously, the hydrogels of the present invention are obtained after cucurbituryl and one or more polymers having a suitable cucurbituril guest functionality, optionally together with a component, are co-located in contact. The methods of the invention can be carried out under mild conditions and environments.
[023] In a further aspect of the invention, a method of distributing a component to a location is provided, the method comprising the steps of:
[024] (i) providing a hydrogel while maintaining a component, according to the first aspect of the invention;
[025] (ii) making the hydrogel available at a target location;
[026] (iii) release the component from the hydrogel.
[027] In additional aspects of the invention, the use of hydrogels of the first or second aspects is provided, for example, in medicine.
[028] In any of the aspects described above, cucurbituryl can be replaced by an alternative host, for example, a host that is capable of forming a ternary host-guest complex.
[029] In one embodiment, the host is selected from cucurbituryl, cyclodextrin, calix [n] arene, and crown ether compounds.
[030] In one embodiment, the host is selected from cyclodextrin, calix [n] arene, and crown ether compounds.
[031] In one embodiment, the host is able to form a ternary host-guest complex.
[032] In a further aspect of the invention, a hydrogel is provided that is obtainable from a hydrogel of the first aspect of the invention by forming a covalent bond or cross-linking between the polymers in the hydrogel, for example, between the guest molecules of the polymers.
[033] In one embodiment of the invention, a hydrogel as described in this document is provided, such as a hydrogel of the first aspect of the invention, where the polymers are unbound or non-covalently cross-linked through a complex and are additionally bound or covalently crosslinked.
[034] In one aspect, a method of covalently bonding or crosslinking a polymer is provided, the method comprising the steps of:
[035] providing a non-covalently bonded polymer or polymers, wherein the non-covalent bonded polymer is formed from a ternary complex of a host maintaining first and second guest molecules provided in the polymer or polymers;
[036] allowing the polymer or polymers to react, thereby forming a covalent bond that binds the polymer or polymers.
[037] In a further aspect, a method of preparing a hydrogel having a crosslinked supramolecular network is provided, in which the hydrogel is formed from the covalent crosslinking of a polymer and / or the covalent bonding of one polymer to another polymer, the method comprising the steps of:
[038] providing a hydrogel having a crosslinked supramolecular network that is obtainable from the ternary complexation of an aqueous composition comprising a host, such as cucurbituril, and one or more polymers having adequate guest functionality, such as the guest functionality of cu - curbituril;
[039] allowing the polymer or polymers to react, thereby forming a covalent bond that binds the polymer or polymers. SUMMARY OF FIGURES
[040] Figure 1 is a schematic representation of the supramolecular hydrogel prepared by adding CB [8] to a mixture of first and second functional multivalent guest polymers in water.
[041] Figure 2 shows the recorded data referring to the rheological analysis of the hydrogels of the invention. The graphs in the top row show the effect of the HEC-Np load and the relative load of HEC-Np and PVA-MV @ CB [8] (ie, 1: 1 ratio between PVA-MV and CB [8]) in storage module, loss module. complex viscosity and tan delta; The graphs in the bottom row show the hydrogel thermal stabilizer determined by a dynamic sweep temperature sweep test (bottom left); and step rate time scan measurements showing the recovery of the hydrogel structure following a high magnitude deformation (bottom right). The symbolic indications are as follows: HEC-Np (1.5%, by weight) / PVA-MV (0.3%, by weight) (■), HEC-Np (1.0%, by weight) / PVA -MV (0.2% by weight) (•), HEC-Np (0.5% by weight) / PVA-MV (0.1% by weight) (▲), HEC-Np (0, 5% by weight) / PVA-MV (0.05% by weight) (▼), HEC-Np (0.25% by weight) / PVA-MV (0.05% by weight) (♦ ). The filled symbols refer to the parameter on the left geometric axis, and the unfilled versions of the symbols refer to the parameter on the right geometric axis.
[042] Figures 3 (a) and (b) are scanning electron microscopic images of freeze-dried and freeze-dried samples of (a) a hydrogel derived from an aqueous composition comprising HEC-Np (0.5 % by weight) / PVA-MV (0.1% by weight) / CB [8] (1 eq.), and (b) a hydrogel derived from an aqueous composition comprising HEC-Np (0, 5% by weight) / PVA-MV (0.05% by weight) / CB [8] (1 eq.). Figure 3 (c) shows the neutron scattering at low angle of the same two hydrogels in D2O.
[043] Figure 4 is a photograph of an inverted flask test that demonstrates the formation of the hydrogel from the aqueous mixture of PVA-MV (0.1% by weight), HEC-Np (0.5%, in weight) and CB [8] (0.1% by weight) exclusively. (a) HEC-Np; (b) HEC-Np and PVA-MV; (c) HEC-Np and PVA-MV and CB [7]; And (d) HEC-Np, PVA-MV and CB [8].
[044] Figure 5 shows the ITC data for (a) binding of HEC-Np to PVA-MV with and without CB [8] present; (b) binding of 2-naphthol (NpOH) to PVA-MV with and without CB [8] present; and (c) binding of HEC-Np to M2V with and without CB [8] present.
[045] Figure 6 shows the relationship between the rate, viscosity and shear stress for prepared supramolecular hydrogels, where HEC-Np (1.5%, by weight) / PVA-MV (0.3%, by weight) ) (■), HEC-Np (1.0% by weight) / PVA-MV (0.2% by weight) (•), HEC-Np (0.5% by weight) / PVA-MV (0.1%, by weight) (▲), HEC-Np (0.5% by weight) / PVA-MV (0.05%) (▼), and HEC-Np (0.25%, in weight) / PVA-MV (0.05% by weight) (♦). The relative amount of CB [8] in each case was 1 equiv. in relation to PVA-MV. The filled symbols represent viscosity values, and the unfilled versions of the symbols represent stress values.
[046] Figure 7 is a photograph of an inverted flask test that shows the stimulus responsiveness of a hydrogel formed from 0.5% HEC-Np / 0.1% PVA-MV / CB [8] 1 eq . This hydrogel is shown in (a) and the hydrogel has a variable responsiveness to the disturbance in the presence of (b) hexane; (c) toluene; (d) a second competitive 2,6-dihydroxy naphthalene guest; and (e) a sodium dithionite reducing agent. In the case of (c), the intact hydrogel can be seen in its original color and is indicated by the arrow, while the hexane layer flows to the bottom of the bottle.
[047] Figure 8 is a schematic representation of the preparation of a hydrogel modality of the invention from naphthyl and MV containing polymers in the presence of a therapeutic protein model.
[048] Figure 9 is a dynamic oscillatory rheological characterization performed at 37 ° C of the HEC-based hydrogels used in this study, where (a) is the storage and loss module and (b) is the complex and tan δ viscosity, from which these values are taken from voltage amplitude sweep measurements. In addition, (c) is the storage and loss module and (d) is the complex and tan δ viscosity, from which these values are taken from frequency sweep measurements. The black symbols refer to a hydrogel derived from an aqueous composition comprising HEC-Np 1.5% by weight, PVA-MV 0.3% by weight and CB [8] 1 eq. (black); while the blue symbols represent HEC-Np 0.5% by weight, PVA-MV 0.1% by weight and CB [8] 1 eq. Squares refer to the left geometric axis and triangles to the right geometric axis.
[049] Figure 10 shows scanning electron microscopy images of freeze-dried and cryophilized samples of a hydrogel derived from an aqueous composition comprising 1.5% HEC-Np, 0.3% PVA-MV , by weight and CB [8] 1 eq.
[050] Figure 11 is a graph showing the percentage viability of a population of 3TC cells exposed to different concentrations of a mixture comprising the polymers HEC-Np and PVA-MV. The concentration is given as the%, by weight, total of the polymers in an aqueous mixture.
[051] Figure 12 is a graph showing the cumulative release of BSA and Lysozyme from two different hydrogels over time. Polypeptides are present in 0.5%, by weight, of the hydrogel, and the total polymer content of the hydrogel is 0.5%, by weight, or 1.5%, by weight.
[052] Figure 13 is a graph showing the change in bioactivity of a BSA and Lysozyme released from a hydrogel over time. Polypeptides are present in 0.5%, by weight, of the hydrogel, and the total polymer content of the hydrogel is 0.5%, by weight, or 1.5%, by weight.
[053] Figure 14 is a schematic representation of hydrogel formation by mixing CB [8] with a polymer having tryptophan or phenylalanine guests. The amino acids (represented by the hatched cylinders) bind in 2: 1 with the host CB [8]. The R group of the amino acid is encapsulated within the hydrophobic cavity by non-covalent interactions. Additional interactions occur between the protonated N-terminus of the amino acid unit and the portal carbonyl groups CB [8].
[054] Figures 15 (a) and 15 (b) are graphs showing the frequency-dependent oscillatory rheology of StPhe-StAm hydrogels (Figure 15 (a)) and StTrp-StAm (Figure 15 (b)) with variable equivalents CB [8]. The open symbols are loss modules, G ”and the closed symbols are storage modules, G’. The amount of CB [8] varies from 0.00 equiv. (lower lines) 0.70 equiv. (upper lines).
[055] Figures 16 (a) and 16 (b) are graphs showing the rheological measurements of stable shear of hydrogels formed with StPhe-StAm (10% w / v) (Figure 16 (a)) and StTrp- StAm (10% w / v) (Figure 16 (b)) with increasing amounts of CB [8]. The amount of CB [8] varies from 0.00 equiv. (lower lines) 0.70 equiv. (top lines).
[056] Figure 17 is a graph showing the effect of CB concentration [8] on zero shear viscosity at an increasing shear rate for hydrogels formed with StPhe-StAm and StTrp-StAm. The three data points for StTrp-StAm are located approximately at a shear viscosity zeroed to 0.35, 0.5 and 0.7 equivalents of CB [8]. The remaining six data points refer to the behavior of StPhe-StAm.
[057] Figures 18 (a) and 18 (b) are graphs of voltage-dependent oscillatory shear measurements for the hydrogel derived from StPhe-StAm (Figure 18 (a)) and StTrp-StAm (Figure 18 (b )). The values of G ’, G’ ’and cv increase from bottom to top.
[058] Figure 19 shows (a) the chemical structures of compounds used in the dimerization study: CB [8], cationic anthracene species of small molecules 1a and their macromolecular analogues 1b (poly polymer) ethylene glycol) - terminal group level, PEG) and 1c (side-chained functionalized hydroxyethyl cellulose, HEC); (b) a schematic reaction of CB [8] “handcuffing” to two portions of anthracene in a π-π pile face-to-face to form a 1: 2 homologous complex in water; and (c) a schematic reaction of the 1: 2 ternary complex photoiradiation with a 350 nm light source leads to an almost quantitative photodimerization [4 + 4] in minutes.
[059] Figure 20 (a) consists of UV / vis spectra of a 1a (10 μM) in the presence of 0.5 equiv. CB [8] in H2O by photo-radiation with a 350 nm light source, spectra taken with 15 seconds of separation. The addendums show kinetic data compared to control experiments in the absence of the CB host [8], and in the presence of CB [7]. The continuous line shows the best mono-exponential fit of the kinetic data. Figure 20 (b) is a 1H NMR spectrum of CB [8] '1a2 (500 μM in D2O) before (bottom) and after (top) photo-radiation for 15 minutes. The addendums show the aromatic peak region.
[060] Figure 21 (a) is a photographic series of 1c in 1.0%, by weight, in H2O. From left to right: 1c before photo-radiation; after photo-radiation at 350 nm for 15 minutes; 1c in the presence of CB [8] (0.5 equiv. Per portion of anthracene); after photoiradiation at 350 nm for 15 minutes. Figure 21 (b) is a schematic representation of the formation of a non-covalent network (gelation) due to the addition of CB [8] to 1c followed by photo-crosslinking through anthracene dimerization.
[061] Figures 22 (a) and (b) are rheological analyzes at 20 ° C of a hydrogel formed by adding CB [8] to a solution of 1.0%, by weight, of 1c in water. Changes upon exposure to UV light (15 minutes) are indicated by an arrow. The squares refer to the left geometric axis and the circles to the right geometric axis. (a) Storage and loss modules obtained from a frequency scan performed at 5% voltage. (b) Stable shear rheological measurements. (c) and (d) Fluorescence spectra of aqueous solution di-liquid 1c (60 μg / mL) by (c) adding CB [8] and (d) subsequent photo-radiation with a 350 nm light source .
[062] Figure 23 is a series of graphs referring to the rheological properties of 1c in water. Changes upon exposure to UV light (15 minutes) are indicated by an arrow. The squares refer to the left geometric axis and the circles to the right geometric axis. (a) Storage module and complex viscosity obtained from a voltage amplitude scan performed at 10 rad s-1; (b) Storage and loss modules obtained from a frequency sweep performed at 5% voltage; and (c) Rheological measurements of stable shear.
[063] Figure 24 shows the oscillatory rheological analysis at 20 ° C for the hydrogel formed by adding CB [8] to a 1.0% solution by weight of 1c in water. Storage module and complex viscosity obtained from a voltage amplitude scan performed at 10 rad s-1. Changes upon exposure to UV light (15 minutes) are indicated by an arrow. The squares refer to the left geometric axis and the circles to the right geometric axis.
[064] Figure 25 shows the UV / vis spectra for 1c (60 μg / mL in water) by photo-radiation (350 nm) (a) in the presence of 0.5 equiv. CB [8] and (b) in the absence of the host CB [8]. The graph on the right shows the normalized absorbance at 254 nm as a function of the irradiation time. The solid red line shows the best mono-exponential fit of the kinetic data. DETAILED DESCRIPTION OF THE INVENTION
[065] The present inventors have established that structurally useful hydrogels can be readily prepared from relatively minimal amounts of cucurbituril and one or more suitably functionalized polymers with a cucurbituril guest functionality. Therefore, hydrogels can have a very high water content. The inventors also established that structurally useful hydrogels can be readily prepared from cucurbituril and one or more high molecular weight polymers suitably functionalized with a cucurbituril guest functionality.
[066] The supramolecular nature of these hydrogels provides scope to adjust mechanical properties and the hydrogel is usefully responsible for various external stimuli including temperature, electrical potential and competitive guests. Hydrogels are easily processed and the simplicity of their preparation and the high adjustability of their properties are distinctive for many important water-based applications. In addition, in many modalities, the components of the hydrogel network (polymer and cucurbituril) are available from cheap and renewable resources.
[067] Many of the hydrogels described in this document have an exceptionally low total polymer concentration. The high water content of the hydro-gel makes it highly attractive for biomedical applications, for example, due to the improved biocompatibility. The inventors established that certain polymers for use in the hydrogel, being that the polymers are suitably functionalized with a cucurbituril guest functionality, have low toxicity, for example, as measured in relation to a fibroblast cell line. The present inventors have also shown that the integrity of hydrogels is not substantially altered on heating to 75 ° C.
[068] In addition, rigid polymer chains with phenylalanine or tryptophan pendent amino acids have the ability to form hydrogels when in the presence of CB [8]. It has been determined that the phenylalanine unit provides hydrogel materials much stronger than its tryptophan counterpart. The study described in this document demonstrates the potential for improved biomedical systems for drug delivery since gel formation can now be achieved using biologically relevant guest moieties, such as biologically compatible guest moieties for CB-based crosslinking [8]. This avoids limitations of potentially toxic polymers and guest metabolites. When these guests are linked to biocompatible polymers, such as natural polymers, it is clear that the system may be suitable for use in 3D cell culture, drug administration and regenerative medicine. In these systems, the material properties of the hydrogel can be altered by appropriate changes in the host concentration (or equivalence) and / or in the nature of guests pending the polymer.
[069] Some of the present inventors have previously described a hydrogel based on a network of polymers linked together by CB [8] (see Appel et al. J. Am. Chem. Soc. 2010, 132, 14251-14260). Typically, the hydrogels described in this document are based on polymers having low functionality, high molecular weight and low polydispersity values. In contrast, at least one of the polymers described in previous studies by the inventors has high functionality, low molecular weight and / or a high polydispersity value. As discussed in the fundamentals section above, the materials described in previous studies do not exhibit dominant G 'values over the entire oscillatory range. In addition, the materials do not have high viscosity values at low shear rates.
[070] The comparative examples provided in this document also show that hydrogels prepared from a low molecular weight (Mn = approximately 10 kDa) and high polydispersity (PDI approximately 2.2) polymer have a loss modulus (G ' ') that dominates the storage module (G') at higher frequencies. Hydrogel
[071] The hydrogel of the invention is a three-dimensional cross-linked polymeric network that retains water, optionally next to a component. In the present case, the network is obtainable or obtained from the complexation of cucurbituryl hosts with a suitable guest molecule functionality provided in one or more polymers. Therefore, the hydrogel comprises this network and trapped water.
[072] In one aspect of the invention, a hydrogel is provided having a crosslinked supramolecular network obtainable or obtained from the complexation of an aqueous composition comprising cucurbituril and one or more polymers having adequate cucurbituryl guest functionality. The aqueous composition comprises a polymer having a molecular weight of 50 kDa or greater. The hydrogel of this aspect of the invention can be used in the first aspect of the invention, to maintain a component.
[073] In one embodiment, a network is obtainable from the complexation of (a) an aqueous composition comprising cucurbituril and (1) or (2); or (b) a composition comprising a plurality of covalently linked cucurbiturils and (1), (2) or (3).
[074] In one embodiment, the network is obtainable from the complexation of (a) an aqueous composition comprising cucurbituril and (1) or (2); or (b) an aqueous composition comprising a plurality of covalently linked cucurbiturils and (1), (2) or (3).
[075] In one embodiment, the network is obtainable from the complexation of an aqueous composition comprising cucurbituril and (1) or (2).
[076] In one embodiment, the network is obtainable from the complexation of an aqueous composition comprising cucurbituril and (2).
[077] In one embodiment, the network is obtainable from the complexation of an aqueous composition comprising cucurbituril and (1).
[078] In one embodiment, a composition as described above comprises a component, that component being kept in the resulting hydrogel.
[079] (1) comprises a first covalently attached to a plurality of first cucurbituryl guest molecules and a second polymer covalently attached to a plurality of second cucurbituryl guest molecules, wherein a first guest molecule and a second guest molecule together à cucurbiturila are suitable to form a ternary guest-host complex. The first and second guest molecules can be the same or different.
[080] (2) comprises a first polymer covalently linked to a plurality of first cucurbituryl guest molecules and a plurality of second cucurbituryl guest molecules, wherein a first and second guest molecules joined to the cucurbituryl are suitable to form one with - guesthouse-host complex. The first and second guest molecules can be the same or different. Optionally, the composition further comprises a second polymer covalently attached to one or more third cucurbituryl guest molecules, one or more fourth cucurbituryl guest molecules or both, wherein a third and fourth molecules joined to cucurbituryl are suitable to form a ternary guest-host complex, and / or the first and fourth molecules together with the cucurbituril are suitable to form a host-host complex, and / or the second and third molecules together with the cucurbituryl are suitable to form a ternary guest-host complex .
[081] (3) comprises a first polymer covalently linked to a plurality of first guest molecules of cucurbituril, in which the first guest molecule added to the cucurbituril is suitable to form a binary host-host complex. Optionally, the composition further comprises a second polymer covalently attached to one or more second guest molecules of cucurbituril, in which the second guest molecule joined to the cucurbituril are suitable to form a binary host-host complex.
[082] In one embodiment, the water content of the hydrogel is at least 90%, by weight, at least 95%, by weight, at least 97%, by weight, at least 98%, by weight, at least 99% at least 99.5% by weight. When the water content was present in these quantities, the present inventors found that hydrogels can be diluted, for example, with a volume of water equivalent to the volume of the hydrogel, with only a slight reduction in mechanical properties.
[083] In one embodiment, the total amount of polymer present in the hydrogel is at most 20%, by weight, at most 10%, by weight, at most 7.0%, by weight, at most 5.0% , by weight, maximum 2.5%, by weight, maximum 2.0%, by weight, maximum 1.5%, by weight, maximum 1.0%, by weight, maximum 0.5% , by weight or at most 0,4% by weight.
[084] In one embodiment, the total amount of polymer present in the hydrogel is at least 0.05%, by weight, at least 0.1%, by weight, at least 0.2%, by weight, at least 0.3% by weight.
[085] In one embodiment, the total amount of polymer present in the hydrogel is in a range where the minimum and maximum amounts are selected from the previous modalities. For example, the total amount of polymer present is in the range of 0.3 to 2.0% by weight.
[086] In an alternative embodiment, a polymer present in the hydrogel is present in the amounts or ranges indicated above.
[087] When only one polymer is present in the hydrogel, the total amount of polymer present in the hydrogel refers to the amount of that polymer. When two or more polymers are present in the hydrogel, the total amount is the sum of the quantities of each of the two or more polymers present.
[088] In some embodiments, the hydrogel comprises a first polymer and a second polymer, such as a composition comprising cucurbituril and (1) as discussed above.
[089] The two polymeric systems provide scope to make relatively easy changes to the hydrogel structure by varying the ratio between the polymers and / or and the cucurbituryl.
[090] In one embodiment, the amount of weight of the first polymer in the hydrogel is substantially equal to the amount of weight of the second polymer in the hydrogel.
[091] In one embodiment, the amount of weight of the second polymer in the hydrogel is at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times or at least 20 times the amount weight of the first polymer in the hydrogel.
[092] In one embodiment, the molar amount of the second polymer in the hydrogel is substantially equal to the molar amount of the first polymer in the hydrogel.
[093] In one embodiment, the molar amount of the second polymer in the hydrogel is at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, at least 20 times or at least 25 times the molar amount of the first polymer in the hydrogel.
[094] In alternative embodiments, the first polymer may be present in excess weight and / or molar excess of the second polymer in the amounts specified above.
[095] During the preparation of the hydrogel, the concentration and volumes of polymer in the aqueous preparation mixtures can be selected in order to provide the desired amounts of the first and second polymers in the hydrogel product. Therefore, the amount of weights mentioned above can refer to the amounts of weight in the aqueous compositions comprising the first and second polymers, from which the network is obtained or obtainable.
[096] The previous references to the amount of polymer present in the hydrogel refer to the amount of polymer including its guest functionality.
[097] In one embodiment, the total amount of cucurbituryl present in the hydrogel is equal to the amount, by weight, of a polymer present in the hydrogel. For example, when a polymer is present in 0.1% by weight, cucurbituryl can also be present in 0.1% by weight. For convenience, it can be referred to as 1 equivalent.
[098] In one embodiment, cucurbituryl is present at least 0.9, at least 0.8, at least 0.5, at least 0.2 or at least 0.1 equivalent to a polymer present in the hydrogel.
[099] In one embodiment, cucurbiturila is present at most 10, at most 5, at most 4, at most 3 or at most 2 equivalent to a polymer present in the hydrogel.
[0100] In one modality, the amount of cucurbituril present in the hydrogel is in a range where the minimum and maximum quantities are selected from the previous modalities. For example, the amount of cucurbiturile present is in the range of 0.5 to 2 equivalents, for example, 0.7 to 1.5 equivalents.
[0101] In one embodiment, a cucurbituril is present in about 1 equivalent.
[0102] When two polymers are present, for example, a first and a second polymer, the amount of cucurbituril can refer to the first or the second polymer.
[0103] In one embodiment, the amount of crosslinking in the hydrogel is a maximum of 10%, a maximum of 8% or a maximum of 5%.
[0104] In one embodiment, the amount of crosslinking in the hydrogel is at least 0.1%, at least 0.5%, at least 1% or at least 2%.
[0105] In one modality, the amount of crosslinking is in a range where the minimum and maximum quantities are selected from the previous modes. For example, the amount of crosslinking is in the range of 1 to 8%.
[0106] Crosslinking refers to the% of monomeric units that participate in the formation of crosslinking and is determined by the addition of molar equivalent of cucurbituril with reference to the functionality of a polymer. Therefore, the addition of 0.5 equivalent of cucurbituryl with respect to a polymer having 10% functionality present, provides a network having a crosslinking value of 5%. Therefore, 5% of all monomers available in the polymer participate in a crosslinking. Therefore, the crosslinking value assumes that all available polymeric guests participate in a complex with cucurbiturila.
[0107] When two polymers are present, for example, a first and a second polymer, the crosslinking value can be expressed with reference to the first or second polymer.
[0108] In one embodiment, the hydrogel has a complex viscosity in a voltage amplitude sweep measurement of at least 5, at least 7, or at least 10 Pa s.
[0109] In one embodiment, the hydrogel has a complex viscosity in a voltage amplitude sweep measurement of a maximum of 1,000, a maximum of 500, or a maximum of 100 Pa s.
[0110] In one mode, the complex viscosity value is that recorded in a voltage in the range of 9 to 90 Pa s or in the range of 9 to 500 Pa s.
[0111] The complex viscosity value can be the value recorded at 37 ° C from a voltage amplitude sweep measurement and is the value adopted for a voltage value in the range of 0.1 to 10%.
[0112] In one embodiment, the complex viscosity value in a voltage amplitude sweep measurement is substantially the same over the voltage range of 0.1 to 100%, 0.1 to 10% or 1 to 10%.
[0113] In one embodiment, the hydrogel has a complex viscosity in a frequency sweep measurement of at least 10, at least 100, at least 200 or at least 300 Pa s.
[0114] The complex viscosity value can be the value recorded at 37 ° C from a frequency sweep measurement and is the value adopted for a frequency value in the range of 0.1 to 10 rad / s, preferably , the value recorded in 0.1 rad / s.
[0115] In one embodiment, the complex viscosity values for the hydrogel decrease substantially linearly with increasing frequency in the frequency sweep measurement.
[0116] In one embodiment, the hydrogel has a viscosity of at least 60, at least 70, at least 80, at least 90 or at least 100 Pa s.
[0117] In one embodiment, the hydrogel has a viscosity of at most 2,000, at most 3,000, or at most 5,000 Pa s.
[0118] In one embodiment, the hydrogel has a viscosity in the range of 90 to 2,000 Pa s.
[0119] The viscosity value can be the value recorded at low shear rates, for example, at a shear rate in the range of 0.1 to 0.5 1 / s, for example, 0.1 to 0.3 1 / s. The viscosity value can be the value recorded at 25 ° C in a stable shear measurement.
[0120] In one embodiment, the hydrogel exhibits a shearing behavior in the range of 0.3 to 10 1 / s or in the range of 1 to 10 1 / s. therefore, the viscosity of the hydrogel decreases in this range of shear values. In contrast, prior art hydrogels do not exhibit such behavior. Preferably, the viscosity of the prior art hydrogel remains substantially constant in these ranges, and exhibits a rapid reduction in viscosity at high shear rates, for example, at shear rates in excess of 30 l / s.
[0121] The storage module value (G ') of the hydrogel can be the value recorded at 37 ° C from a voltage amplitude sweep measurement and is the value adopted for a voltage value in the range of 0.1 to 100%, for example, in the range of 0.1 to 10%.
[0122] In one embodiment, the hydrogel has a storage module (from a voltage amplitude sweep measurement) of at least 5 Pa, at least 10 Pa, at least 20 Pa, at least 50 Pa, or at least minus 100 Pa.
[0123] In one embodiment, the hydrogel has a storage module (from a voltage amplitude sweep measurement) of a maximum of 2,000 Pa, a maximum of 1,500 Pa, or a maximum of 1,000 Pa.
[0124] In one mode, the hydrogel has a storage module (from a voltage amplitude sweep measurement) in a range where the minimum and maximum quantities are selected from the previous modes. For example, the storage module is in the range of 10 to 1,000 Pa, for example. 50 to 1,000 Pa.
[0125] Alternatively, the hydrogel storage module value can be the value recorded at 37 ° C from a frequency sweep measurement and is the value adopted for a frequency value in the range of 0.1 to 100 rad / s, for example, in the range of 0.1 to 10 rad / s.
[0126] In one embodiment, the hydrogel has a storage module (from a frequency sweep measurement) of at least 4 Pa, at least 9 Pa, at least 10 Pa, or at least 20 Pa.
[0127] In one embodiment, the hydrogel has a storage module (from a frequency sweep measurement) of a maximum of 500, a maximum of 800 Pa, or a maximum of 1,000 Pa.
[0128] In one mode, the hydrogel has a storage module (from a frequency sweep measurement) in a range where the minimum and maximum quantities are selected from the previous modalities. For example, the storage module is in the range of 4 to 1,000 Pa, for example, 10 to 1,000 Pa.
[0129] The loss modulus value (G '') of the hydrogel can be the value recorded at 37 ° C from a voltage amplitude sweep measurement and is the value adopted for a voltage value in the range of 0 , 1 to 100%, for example, in the range of 0.1 to 10%.
[0130] In one embodiment, the hydrogel has a loss module (from a voltage amplitude sweep measurement) of at least 5 Pa, at least 10 Pa or at least 20 Pa.
[0131] In one embodiment, the hydrogel has a loss module (from a voltage amplitude sweep measurement) of a maximum of 200 Pa, a maximum of 500 Pa, or a maximum of 1,000 Pa.
[0132] In one mode, the hydrogel has a loss module (from a voltage amplitude sweep measurement) in a range where the minimum and maximum amounts are selected from the previous modes. For example, the loss module is in the range of 10 to 1,000 Pa, for example, 20 to 1,000 Pa.
[0133] Alternatively, the hydrogel loss modulus value can be the value recorded at 37 ° C from a frequency sweep measurement and is the value adopted for a frequency value in the range of 0.1 to 100 rad / s, for example, in the range of 0.1 to 10 rad / s.
[0134] In one embodiment, the hydrogel has a loss module (from a frequency sweep measurement) of at least 1 Pa, at least 5 Pa, at least 10 Pa, or at least 20 Pa.
[0135] In one mode, the hydrogel has a loss module (from a frequency sweep measurement) of a maximum of 200 Pa, a maximum of 500 Pa, or a maximum of 1,000 Pa.
[0136] In one mode, the hydrogel has a loss module (from a frequency sweep measurement) in a range where the minimum and maximum quantities are selected from the previous modalities. For example, the loss module is in the range of 1 to 1,000 Pa, for example, 10 to 500 Pa.
[0137] In one embodiment, the storage module value and / or the loss module value are substantially the same over the voltage range of 0.1 to 100%, 0.1 to 10% or 1 to 10% .
[0138] The present inventors have found that the hydrogels of the invention have an extremely wide linear viscoelastic region. Only where the total polymer content in the hydrogel is high (for example, 1.5% by weight, or greater) does the hydrogel begin to show a deviation from linear viscoelasticity, for example, at a stress value of 10 % or greater.
[0139] In one embodiment, the loss module is not greater than the storage module for any frequency value in the range of 0.1 to 100 rad / s for a hydrogel analyzed by measuring frequency sweep at 37 ° C. Therefore, the storage module for hydrogels is dominant.
[0140] In one mode, changes in storage and loss values with a change in frequency (in a frequency sweep experiment) are substantially the same. Therefore, the storage and loss modules can be said to be parallel. The parallel nature of the modulus values is apparent in a frequency sweep experiment, where the voltage (as%) and the modulus (as Pa) are both expressed on a logarithmic scale. As shown in Figure 2 of the present case.
[0141] When a voltage amplitude sweep measurement is recorded, the frequency can be adjusted to 10 rad / s.
[0142] When a frequency sweep measurement is recorded, the voltage amplitude can be adjusted to 5% voltage. Alternatively, the voltage amplitude can be adjusted to 10% voltage. Those skilled in the art will choose a stress value that is appropriate for the material under research. The well-versed individual will establish that the stress values are selected so that frequency sweeps are carried out in the linear viscoelastic regions for the material.
[0143] The tan δ value of the hydrogel is recorded at 37 ° C from a frequency sweep measurement and is the value adopted for a frequency value in the range of 1 to 100 rad / s.
[0144] In one embodiment, the hydrogel has a tan δ value (from a frequency sweep measurement) of at least 0.1, at least 0.2, or at least 0.3.
[0145] In one mode, the hydrogel has a tan δ value (from a frequency sweep measurement) of a maximum of 0.4, a maximum of 0.5 or a maximum of 1.0.
[0146] In one mode, the hydrogel has a tan δ value (from a frequency sweep measurement) in a range where the minimum and maximum quantities are selected from the previous modalities. For example, the tan δ value is in the range of 0.1 to 0.5, for example, 0.2 to 0.5.
[0147] Alternatively, the tan δ value can be the value recorded at 37 ° C from a voltage amplitude sweep measurement and is the value adopted for a voltage value in the range of 0.1 to 10%.
[0148] In one embodiment, the hydrogel has a tan δ value (from a voltage amplitude sweep measurement) of at least 0.1, at least 0.2 or at least 0.4.
[0149] In one mode, the hydrogel has a tan δ value (from a voltage amplitude sweep measurement) of a maximum of 0.5, a maximum of 0.1, or a maximum of 2.0.
[0150] In one modality, the hydrogel has a tan δ value (from a voltage amplitude sweep measurement) in a range where the minimum and maximum quantities are selected from the previous modalities. For example, the tan δ value is in the range of 0.1 to 0.5, for example, 0.2 to 0.4.
[0151] In one embodiment, the tan δ value is substantially the same over the voltage range of 0.1 to 100%, 0.1 to 10% or 1 to 10%.
[0152] The inventors found that the hydrogels of the invention are highly elastic and recorded tan δ values of approximately 0.3, as measured from a frequency sweep measurement.
[0153] The hydrogels of the invention can be heated without a significant loss of mechanical integrity.
[0154] In one embodiment, a hydrogel heated to a temperature of 30 ° C, 40 ° C, 50 ° C, or 60 ° C has a complex viscosity of 1 Pa s or greater.
[0155] In one embodiment, a hydrogel heated to a temperature of 30 ° C, 40 ° C, 50 ° C, or 60 ° C has a complex viscosity of 2 Pa s or greater.
[0156] In one embodiment, a hydrogel heated to a temperature of 30 ° C, 40 ° C, 50 ° C, or 60 ° C has a tan δ value of 0.30 or less.
[0157] In one embodiment, a hydrogel heated to a temperature of 30 ° C, 40 ° C, 50 ° C, or 60 ° C has a tan δ value of 0.35 or less.
[0158] In one embodiment, a hydrogel heated to a temperature of 30 ° C, 40 ° C, 50 ° C, or 60 ° C has a tan δ value of 0.40 or less.
[0159] The complex viscosity and tan δ values presented above can be recorded in a dynamic oscillatory temperature sweep test.
[0160] As the temperature of the hydrogel increases, the properties of bulky material generally decrease, as the association complex increases, for example, for a network based on ternary cucurburyl complexes, the association constant decreases.
[0161] The hydrogels of the present invention have excellent reforming characteristics when deformed. A hydrogel that is exposed to a high shear rate of magnitude, for example, where V is 500 s-1, gets together quickly and completely. Therefore, the original viscosity properties of the original hydrogel can be obtained again when the hydrogel is assembled after the disturbance.
[0162] In one embodiment, a rheological property of the hydrogel remains substantially the same after at least one cycle of deformation and assembly, like two cycles of deformation and assembly. The rheological property can be one or more properties selected from the group consisting of complex viscosity, storage modules, loss modules, and tan δ.
[0163] The hydrogel's response to the deformation cycle demonstrates the resistance of a cucurbituril based network to reversibly form strong hydrogel structures.
[0164] The physical properties of a hydrogel, as described above, can refer to a hydrogel that does not hold a component. In other embodiments, the properties may refer to the hydrogel while maintaining a component, although it is less preferred.
[0165] In one embodiment, the hydrogel has a correlation extension in the range of 150 to 250 A, such as 170 to 230 A, such as 180 to 220 A. These values can be obtained by a combination of Debye-Bueche and Ornstein models -Zerniche, as described in this document.
[0166] In one embodiment, the extent of correlation of the frozen hydro-gel structure, -, is in the range 500 to 1000 A, such as 600 to 900 A. These values can be obtained from the results of a SANS analysis , as described below.
[0167] The correlation extension values typically differ from those reported elsewhere for other polymeric gel structures. However, this difference is believed to occur as a result of the extraordinarily low amounts of total polymer that are typically used in the hydrogels of the present invention. The correlation extension values are consistent with the calculated frame size, which is based on the distances between the functionalities of guest molecules over a polymer.
[0168] In one embodiment, the hydrogel is transparent to light having a wavelength in the visible range, for example, a wavelength in the range of 380 to 740 nm.
[0169] In general, the shape, dimensions and volume of the hydrogel are not particularly limited.
[0170] In one embodiment, the hydrogel has a greater cross-section of maximum 100 cm, maximum 50 cm, maximum 35 cm, maximum 10 cm.
[0171] In one embodiment, the hydrogel has a smaller cross-section of at least 100 nm, at least 1.0 μm, at least 10 μm, at least 100 μm, at least 1 mm, or at least 10 mm.
[0172] In one modality, the hydrogel has a cross section where the minimum and maximum values are selected from the previous modalities. For example, the hydrogel has a cross section in the range of 1 mm to 10 cm.
[0173] The dimensions and shape of the hydrogel can be dictated by the dimensions of the container in which the hydrogel is formed. For example, a hydrogel having a larger cross-section of about 1 cm is obtainable from a complexable composition held in a bottle, such as a 15 x 45 mm bottle. Examples of hydrogels formed in these flasks are exemplified in this document.
[0174] In one embodiment, a hydrogel of the invention is obtainable or obtained by the methods for the preparation of hydrogels as described in the present document.
[0175] The hydrogel is not a supramolecular capsule. These capsules have a shell that consists of a crosslinked supramolecular network. A capsule has a substantial internal cavity that is free from the cross-linked supramolecular network. The hydrogels of the invention do not take the form of a capsule. The hydrogels of the invention have an extensive network of inter- and intra-bonded polymers. This network does not provide substantial internal cavities that are exempt from the reticulated supramolecular network. The hydrogels of the invention can be further distinguished from supramolecular capsules by virtue of their beneficial rheological properties, such as those properties described above. Complex
[0176] The hydrogel consists of a network that is held in place by a supra-molecular handcuff. The complex that forms this supramolecular cuff is based on a cucurbiturila hosting a guest (binary complex) or two guests (ternary complex). Cucurbituril forms a non-covalent bond to each guest. The present inventors have established that cucurbituryl complexes are readily formed and provide robust non-covalent bonds between polymer building blocks. The complex formation is tolerant to many functionalities within the polymers. One of the present inventors has demonstrated that polymeric networks, including a basic hydrogel, can be prepared using a cucurbituryl cuff. However, to date, the formation of cucurbituril-based hydrogels having useful physical characteristics has not been described. The formation of cucurbituril-based hydrogels that are capable of maintaining a component has also apparently not been described.
[0177] As noted earlier, the cucurbituryl complex with one or two guests is the non-covalent bond that binds and / or interconnects the polymers to form a supramolecular network of material.
[0178] In one embodiment, the hydrogel is a network having a plurality of complexes, each complex comprising cucurbituril hosting a first guest molecule and a second guest molecule. The first and second guest molecules are covalently attached to a first polymer, or to a first polymer and a second polymer.
[0179] When the complex comprises two guests within the cucurbituryl cavity, the association constant, Ka, for such a complex is at least 103 M-2, at least 104 M-2, at least 105 M-2, at least 106 M-2, at least 107 M-2, at least 108 M-2, at least 109 M-2, at least 1010 M-2, at least 1011 M-2, or at least 1012 M-2.
[0180] When a cucurbiturila hosts two guest molecules, the guest molecules can be the same or different. A cucurbituryl that is capable of hosting two guest molecules may also be able to form a stable complexobinary with a single guest. It is believed that the formation of a ternary guest-host complex proceeds through an intermediate binary complex. Within a hydrogel of the invention, Ester can present a binary complex formed between a guest molecule and a cucurbituryl. The binary complex can be considered as a partially formed ternary complex that has still formed a non-covalent bond to another host molecule.
[0181] In one embodiment, the hydrogel is a network having a plurality of complexes, where each complex comprises cucurbituril hosting a guest molecule, and each cucurbituril is covalently linked to at least one other cucurbituril. The guest molecules are covalently attached to a first polymer, or to a first polymer and a second polymer.
[0182] When the complex comprises a guest within the cucurbituryl cavity, the association constant, Ka, for such a complex is at least 103 M1, at least 104 M-1, at least 105 M-1, at least 106 M- 1, at least 107 M-1, at least 108 M-1, at least 109 M-1, at least 1010 M-1, at least 1011 M-1, or at least 1012 M-1.
[0183] In one modality, the guest is a compound capable of forming a complex that has an association constant in the range of 104 to 107 M-1.
[0184] In one embodiment, the formation of the complex is reversible. The separation of a guest from the cucurbituryl host, to thereby separate a bond or crosslink with a polymer, can be referred to as decompression.
[0185] Complex decompression to separate the guest or guests may occur in response to an external stimulus, including, for example, a competing guest, light, an oxidizing or reducing agent, electrochemical potential, and temperature changes , among others. Such decomplexing can be induced to provide additional or larger pores in the hydrogel through which a component that is retained in the hydrogel can pass. Decomplexing can also be used to break the entire network and cause the hydrogel to break.
[0186] As described in this document, a competitive guest for use in decomplexing a CB-based network [8] is 2,6-dihydroxy naphthalene or toluene. The competing guest can be used in excess of the quantity (molar quantity) of guest molecules present in the polymers of the network. In a modality, the competitive guest has a greater association constant than that of a guest in the complex.
[0187] In other modalities, the decompression of the network, and therefore of the hydrogel, is achieved by oxidizing or reducing a guest in a complex. The change in the oxidation state of a guest can be achieved using an oxidizing or reducing chemical agent, or the application of an electrochemical potential. As described in this document, a complex that comprises a virogen, such as a methyl viologene, can be decompiled by treatment with a reducing agent, such as a dithionite.
[0188] In one mode, the decompression reaction is reversible. Therefore, a hydrogel can be converted to an uncomplexed low viscosity form, then returned to a high viscosity hydrogel form, as appropriate, which can be the same or different from the original hydrogel. Network structure
[0189] As noted earlier, the hydrogels of the present invention are based on a network that is formed from the complexation of cucurbituril, as a host, next to a guest (binary complex) or two guests (ternary complex). The guest or guests are covalently linked to the polymers, which provide the raw network structure. The nature of the network depends on the form of complex used (binary or ternary), which successively is based on the cucurbitura and the employed guests. The number and nature of the polymer are also relevant.
[0190] Two types of networks are provided. The first type is based on the formation of a plurality of ternary complexes, each complex comprising a cucurbituryl host with a first guest molecule and a second guest molecule. The second type is based on the formation of a plurality of binary complexes, each complex comprising a host of cucurbituryl with a first guest molecule. In this second type, each cucurbituril is covalently linked to at least one other cucurbituril. These types of network can be combined into a hydrogel of the invention.
[0191] When a polymer is endowed with a plurality of guest molecules, all guest molecules do not need to participate in a complex with cucurbituril. When the network is based on the connection between ternary structures, a guest molecule of a building block (polymer) can be in a binary complex with a cucurbituryl. The binary complex can be considered as a partially formed ternary complex that has not yet been combined with an additional guest molecule to generate the ternary form.
[0192] Throughout the description, references are made to a polymer, a first polymer and a second polymer. It is established that such a reference consists of a reference to a collection of individual polymers, etc. which are the building blocks (polymers). When a reference is intended for an individual polymeric molecule, the term "unique" is used in reference to the building blocks, for example, a first unique polymer.
[0193] The networks described below are the basic networks that are obtainable from the described compositions. It is established that the present inventions extend to more complex networks that are obtainable from compositions that comprise additional polymers. Network of ternary complexes based on cucurbiturila
[0194] This network is obtainable from the complexation of a first guest molecule and a second guest molecule together to a cu-curbituryl host. The guest molecules can be provided in one or two (or more) polymers as described below. As described in this document, a network can be formed using only one polymer. This polymer can have guest molecules that are different (the first and second guests are not the same) or the same (the first and second guests are the same).
[0195] In one embodiment, a network is obtainable or obtained from a complex composition comprising a cucurbituryl, a first polymer covalently linked to a plurality of first cucurbituryl guest molecules and a second polymer covalently linked to a plurality of second guest molecules of cucurbituril, in which a first guest-molecule and a second guest molecule together with cucurbituril are suitable to form a ternary guest-host complex.
[0196] The ternary complex serves to non-covalently bond the first and second polymers. A single first polymer can form a plurality of non-covalent bonds to a plurality of second polymers. Similarly, a second single polymer can form a plurality of non-covalent bonds to a plurality of first polymers. In this way, a network of material is established.
[0197] It should be noted that, in some modalities, the first and second guest molecules can be identical. Therefore, the first and second polymers may differ in their composition. In some embodiments, the first and second polymers can be identical. In this case, the first and second guest molecules are different.
[0198] Below is a schematic structure of a basic network formed between cucurbituril, a first unique polymer and two second unique polymers. In the schemes included in this text, the guest molecules are depicted as rectangles that are covalently linked (vertical line) to a building block (horizontal line). The vertical line can describe a direct covalent bond or a binder to the polymer.
[0199] In the scheme below, some of the first guest molecules (non-hatched rectangles) of the first polymer are in complex with cucurbituryl hosts (barrels) and the second guest molecules (hatched rectangles) of the second polymers.

[0200] It appears that not all guest molecules present need to participate in a complex in the final network. Each of the first and second polymers can form complexes with other second and first polymers, respectively. The guest molecules are hatched for ease of understanding. However, as explained, the guest molecules of the first and second building blocks can be the same.
[0201] In an alternative embodiment, a network is obtainable or obtained from the complexation of a composition comprising a cucurbituril and a first polymer covalently linked to a plurality of first cucurbituril host molecules and to a plurality of second cucurbituril guest molecules , in which a first and a second guest molecule together with cu-curbituryl are suitable to form a ternary guest-host complex. As noted earlier, the first and second guest molecules can be the same or different.
[0202] The ternary complex serves to non-covalently bond and / or interconnect the first polymer. A single first polymer can form a plurality of non-covalent bonds to a plurality of other first polymers. In addition, or alternatively, a first unique polymer can form a plurality of non-covalent interconnections with itself, to thereby crosslink the first unique building block.
[0203] As before, the first and second guest molecules can be identical.
[0204] Below is a schematic structure of a basic network formed between cucurbituril and two first unique polymers, each having a plurality of first and second guest molecules. Some of the first guest molecules (non-hatched rectangles) of the first polymer are in the complex with cucurbituryl hosts (barrels) and the second guest molecules (hatched rectangles) of another first polymer. It can be seen from the illustrated network that a first building block can form intramolecular complexes, thus reticulating a first unique polymer.

[0205] It appears that not all guest molecules present need to participate in a complex in the final network. Each of the first building blocks can form complexes with another first polymer, or with other parts of the same polymer. As explained, the first and second guest molecules can be the same.
[0206] Optionally, the composition further comprises a second polymer covalently attached to one or more third cucurbituryl guest molecules, one or more fourth cucurbituryl guest molecules or both, where a third and fourth molecule together with cucurbituril are suitable to form a ternary host-host complex, or first and fourth guest molecules together with cucurbituril are suitable for forming a ternary guest-host complex, or second and third guest molecules together with cucurbituryl are suitable to form a ternary guest-host complex.
[0207] When the second polymer is provided with a plurality of third and fourth guest molecules, the ternary complex serves to bond and / or interconnect non-covalently the second polymer. A second single polymer can form a plurality of non-covalent bonds to a plurality of other second polymers. Additionally, or alternatively, a second single polymer can form one or more non-covalent interconnections with itself, to thereby crosslink the second single polymer.
[0208] The third and fourth guest molecules may be suitable to form complexes with the first and second guest molecules of the first polymer. In one embodiment, the first and third guest molecules are the same. In one embodiment, the second and fourth guest molecules are the same. In this document, the ternary complex serves to non-covalently bond the first and second polymers, for example, through a complex of the first and fourth guest molecules and / or through a complex of the second and third guest molecules.
[0209] Therefore, a single first polymer can form a plurality of non-covalent bonds to a plurality of second polymers. Similarly, a second single polymer can form a plurality of non-covalent bonds to a plurality of first polymers. In this way, a network of material is established. Polymers can also form intermolecular non-covalent bonds as previously described.
[0210] When a second polymer is covalently attached to one or more third guest molecules or one or more fourth guest molecules, the first and fourth molecules together with the cucurbituryl are suitable to form a ternary guest-host complex, and the second and third molecules together with cucurbituril are suitable to form a ternary host-host complex. Therefore, the ternary complex serves to non-covalently bond the second polymer to the first polymer.
[0211] Below is a schematic structure of a basic network formed between cucurbiturila, three first unique building blocks, each having a plurality of first and second guest molecules, and two building blocks, each having a plurality of third and fourth guest molecules. Some of the first guest molecules (non-hatched rectangles) of the first building block are in complex with the hosts of cucurburil (barrels) and the second guest molecules (hatched rectangles) of another first building block. Some of the third guest molecules (partially hatched rectangles) of the second building block are in complex with the hosts of cucurbituril (barrels) and fourth guest molecules (traced them) of another second building block. A first guest molecule from the first building block is in complex with a cucurbituryl host and a fourth guest molecule (dashed rectangles) from a second building block. A second guest molecule from the first building block is in complex with a host of cucurbituryl and a third guest molecule from a second building block.

[0212] The first and third guest molecules can be the same. The second and fourth guest molecules can be the same. Network of binary complexes based on a plurality of covalently linked cucurbiturilas
[0213] This network is obtainable from the complexation of a first guest molecule with a cucurbituryl host, and that host is co-valently linked to one or more other cucurbiturils. The guest molecules can be provided in one, or two (or more) polymers as described in this document.
[0214] Covalently linked cucurbiturils serve to link polymer molecules through the plurality of complexes that are formed within each of the covalently linked cucurbiturils.
[0215] Below is a schematic structure of a basic network formed between a plurality of covalently linked cucurbiturils and two unique first polymers, each having a plurality of first guest molecules. Some of the first guest molecules (non-hatched rectangles) from each of the first unique polymers are in a binary complex with cucurbituril (barrel) hosts. The cucurbiturilas are covalently linked, in order to form a connection between each of the first building blocks. Cucurbiturils can be covalently linked through a polymer.

[0216] It appears that not all guest molecules present need to participate in a complex in the final network. Each of the first unique building blocks can form complexes with other first polymers, respectively, or can form an intramolecular crosslink with another portion of the same polymer. As explained, the guest molecules of the first and second polymers can be the same. In the previous scheme, one of the first polymers can be replaced by a second polymer that is covalently attached to a second host molecule. The second guest molecule is one capable of forming a binary complex with cucurbituryl. The second guest molecule can be the same as the first guest molecule.
[0217] In the scheme, two cucurbiturilas are shown linked together. The present invention encompasses the use of systems where more than two cucurbiturils are connected together. For example, a number of cucurbiturils may be pendant to a polymer, such as a polymer described herein. Network of ternary complexes based on a plurality of covalently linked cucurbiturilas
[0218] It is clear from the description of the previous networks that each of the cucurbituryl hosts in the plurality of covalently linked cucurbiturils may be suitable to form ternary complexes. Therefore, one can use the plurality of covalently linked cucurbiturils instead of the cucurbiturila described for use in the network of ternary complexes based on cucurbituril.
[0219] Below is a structural scheme of a basic network formed between a plurality of covalently linked cucurbiturils, two first unique polymers, each having a plurality of first guest molecules, and two second unique polymers, each having a plurality of second guest molecules. Some of the first guest molecules (non-hatched rectangles) of the first polymer are in a tertiary complex with a host of cucurbituril (barrel) and the second guest molecules (hatched rectangles) of the second polymer. The cucurbiturils are bonded to thereby form a bond between each of the first and second polymers.

[0220] As before, the first and second guest molecules can be the same. Each of the first and second polymers can form complexes with other second and first polymers, respectively. Other permutations are possible, for example, when the plurality of covalently linked cucurbiturils has more than two cucurbiturils. Other networks
[0221] The basic networks of the invention which are obtained or obtainable from the described compositions have been described above. It will be clear to a person skilled in the art that the described compositions may include additional building blocks, for example, third and fourth polymers, each attached to one or more guest molecules of cucurbituryl. The present invention also addresses networks that comprise a mixture of any of the networks described above. These are obtainable from compositions comprising an appropriate selection of cucurbituryl, covalently bonded cucurbiturils, first polymer and second polymer as appropriate.
[0222] The invention also relates to a network that comprises different cucurbiturilas. Different cucurbiturilas can be chosen in order to obtain a network that is based on ternary and binary complexes. Different cucurbiturils can be chosen in order to generate networks that result from the selective complexation of each cucurbituril to different guest molecules, which can be present in the same or different polymers. Cucurbiturila
[0223] The present invention provides the use of cucurbiturils as a supramolecular handcuff to bind and / or crosslink polymers. Cucurbituril can be used to form ternary complexes with first and second guest molecules present in one or more polymers. The formation of these complexes links individual polymeric molecules to thereby form a network of material. Together with water, this network forms the hydrogel.
[0224] Recent studies have shown that cucurbituryl compounds have high biocompatibility in vitro and in vivo and have extremely low toxicity (see Uzunova et al. Org. Biomol. Chem. 2010, 8, 2037-2042). Therefore, when used together with non-toxic polymeric components, the present hydrogels are also suitable for use in biological systems.
[0225] In one embodiment, cucurbituril is capable of forming a ternary complex. For example, CB [8], is capable of forming a ternary complex, as well as compounds CB [9], CB [10], CB [11] and CB [12].
[0226] In one embodiment, cucurbituril is capable of forming a binary complex. For example, CB [7], is capable of forming a ternary complex, as well as CB [8] with the appropriate guest molecule.
[0227] In one embodiment, cucurbituryl is a compound CB [8], CB [9], CB [10], CB [11] or CB [12].
[0228] In one embodiment, cucurbituril is a CB compound [8].
[0229] References to a cucurbituryl compound are references to variants and derivatives thereof.
[0230] In one embodiment, the cucurbituryl compound has a solubility of at least 0.01 mg / ml, at least 0.02 mg / ml, at least 0.05 mg / ml, or at least 0.10 mg / ml ml.
[0231] In one embodiment, solubility refers to aqueous solubility.
[0232] Cucurbit [8] urine (CB [8]; CAS 259886-51-6) is a barrel-shaped container molecule that has eight repeat units of glycoluryl and an internal cavity size of 479A3 (see structure below) ). CB [8] is readily synthesized using standard techniques and is commercially available (eg Sigma-Aldrich, Missouri, USA).

[0233] In other aspects of the invention, CB variants [8] are provided and used in the methods described in this document.
[0234] A variant of CB [8] may include a structure having one or more repeating units that are structurally analogous to glycoluryl. The repeating unit may include an ethylurea unit. When all units are ethylurea units, the variant is a hemicucurbituryl. The variant may be a hemicucurbit [12] urine (shown below, see also Lagona et al. Angew. Chem. Int. Ed. 2005, 44, 4844).

[0235] In other aspects of the invention, cucurbituryl derivatives are proportional and used in the methods described in this document. A derivative of a cucurbituryl consists of a structure having one, two, three, four or more substituted glycoluryl units. A substituted cucurbituryl compound can be represented by the structure below:
where: n is an integer at least equal to 5; and for each unit of glycoluryl each X is O, S or NR3, and
[0236] -R1 and -R2 are independently selected from -H and from the following optionally substituted groups: -R3, -OH, -OR3, -COOH, -COOR3, - NH2, -NHR3 and -N (R3) 2 where -R3 is independently selected from C1-20 alkyl, C6-20 carbonaryl, and C5-20 heteroaryl, or where -R1 and / or -R2 is -N (R3) 2, both -R3 together form a C5 heterocyclic ring -7; or together -R1 and -R2 are C4-6 alkylene forming a C6-8 carbocyclic ring next to the uracil frame.
[0237] In one embodiment, one of the glycoluryl units consists of a glycoluryl unit. Therefore, -R1 and -R2 are independently -H to n-1 of the glycoluryl units.
[0238] In one embodiment, n is 5, 6, 7, 8, 9, 10, 11 or 12.
[0239] In one mode, n is 5, 6, 7, 8, 10 or 12.
[0240] In one mode, n is 8, 10 or 12.
[0241] In one mode, n is 8.
[0242] In one mode, n is 7.
[0243] In one modality, each X is O.
[0244] In one modality, each X is S.
[0245] In one mode, R1 and R2 are independently H.
[0246] In one embodiment, for each unit one between R1 and R2 is H and the other is independently selected from -H and from the following optionally substituted groups: -R3, -OH, -OR3, -COOH, -COOR3, - NH2, -NHR3 and -N (R3) 2. In one embodiment, for a unit one between R1 and R2 is H and the other is independently selected from -H and from the following optionally substituted groups: -R3, -OH, -OR3, -COOH, -COOR3, -NH2, - NHR3 and -N (R3) 2. In this modality, the remaining glycoluryl units are such that R1 and R2 are independently H.
[0247] Preferably, -R3 is C1-20 alkyl, most preferably C1-6 alkyl. The C1-20 alkyl group can be linear and / or saturated. Each -R3 group can be independently unsubstituted or substituted. Preferred substituents are selected from: -R4, -OH, -OR4, -SH, -SR4, -COOH, -COOR4, -NH2, -NHR4 and -N (R4) 2, where -R4 is selected from C1-20 alkyl, C6-20 carboaryl, and C5-20 heteroaryl. Substituents can be independently selected from - COOH and -COOR4.
[0248] In some modalities, -R4 is not equal to -R3. In some embodiments, -R4 is preferably unsubstituted.
[0249] When -R1 and / or -R2 is -OR3, -NHR3 or -N (R3) 2, then -R3 is preferably C1-6alkyl. In some embodiments, -R3 is replaced by a substitute -OR4, -NHR4 or -N (R4) 2. Each -R4 is C1-6 alkyl and is preferably substituted.
[0250] In some embodiments of the invention, the use of a plurality of covalently linked cucurbiturils is provided. These covalently linked cucurbiturils are suitable for forming networks based on the complexation of cucurbituril with guest molecules of a building block (polymer). The complexes formed can be ternary or binary complexes.
[0251] One cucurbituryl can be covalently linked to the other cucurbituryl through a linker group that is a substituent at the R1 or R2 position on one of the glycoluryl units in the cucurbituryl as represented in the structure shown below. There are no particular limitations on the covalent bond between cuorbiturils. The binder can be in the form of a simple alkylene group, a polyoxyalkylene group or a polymer, such as a polymer described in this document (except the guest functionality). When the linker is a polymeric molecule, cucurbituryls can be pendent to such a polymer. When cucurbituryls are covalently linked by a polymer, that polymer may have the characteristics of the polymers described in this document. For example, the polymer can have a high molecular weight, and the polymer can be hydrophilic. These preferences are presented below with respect to polymers having a guest functionality. Polymers
[0252] The hydrogel network of the invention is formed by complexing one or more polymers with a cucurbituryl handcuff. Each polymer is endowed with a suitable guest functionality to interact with the cucurbityl host.
[0253] Some of the present inventors have previously described hydrogels that are networks formed from the complexation of CB [8] with a properly functionalized polystyrene-based polymer and a properly functionalized polyacrylamide-based polymer (Appel et al. J. Am. Chem. Soc. 2010, 132, 14251-14260). The molecular weight of each polymer is relatively low, and the polydispersity of one of the polymers, typically the polystyrene-based polymer, is relatively high. Additionally, the functionality of the polymers is high.
[0254] The present hydrogels are distinguishable by this previous study, at least for the reason that one of the polymers has a high molecular weight and / or a low functionality. In some embodiments, the polydispersity of all polymers in the network is low. In other embodiments, the polymers present in the hydrogel comprise monomers having a hydroxyl functionality.
[0255] Cucurbituril is used as a supramolecular handcuff to join one or more polymers together. The formation of a cucurbituril complex with suitable guest components that are attached to the polymers forms a network of material. The complex non-covalently cross-links one polymer or non-covalently bonds one polymer to another.
[0256] It is established that the polymer is an entity that serves to provide a structure to the formed network. The polymer also serves as the link between a plurality of guest molecules, and can therefore also be referred to as a linker. In some embodiments, a polymer is provided for the purpose of introducing a desirable physical or chemical characteristic into the formed network. A polymer can include functionality to assist in the detection and characterization of the network, or to assist in the detection and characterization of a component that is retained in the hydrogel. These polymers do not necessarily need to participate in a crosslinking.
[0257] A polymer can be selected for its molecular weight, polydispersion, solubility, and / or its mechanical and physical characteristics.
[0258] A polymer, like a first polymer, is covalently linked to a plurality of cucurbituryl guest molecules. Therefore, a building block (polymer) will bind non-covalently to a plurality of cucurbiturils, and these cucurbiturils will bind non-covalently to other polymers, to thereby generate a network of material.
[0259] A polymer, such as a first polymer or a second polymer, can be covalently attached to a plurality of cu-curbituryl guest molecules. In one embodiment, a polymer is covalently bonded to at least 3, at least 4, at least 5, at least 20, at least 50, at least 100, at least 500, at least 1,000, at least 2,000, at least 5,000 or at least 10,000 guest molecules of cucurbituryl.
[0260] In certain modalities, polymers covalently linked to one or more guest molecules of cucurbituril may be used. However, these polymers are used only in combination with other polymers that are covalently bound to at least two cucurbitilyl guest molecules.
[0261] The number of guest molecules present in a particular polymeric molecule can be expressed as the percentage of monomers present in the polymer that are bound to the guest molecules as a total of all the monomers present in the polymeric molecule. This can be referred to as the percentage of functionality.
[0262] In one embodiment, the functionality of a polymeric molecule is at least 0.1%, at least 0.2%, at least 0.5% or at least 1.0%.
[0263] In one embodiment, the functionality of a polymeric molecule is at most 20%, at most 15%, at most 10%, at most 7%, at most 5%, or at most 2%.
[0264] In one mode, the functionality is in a range where the minimum and maximum quantities are selected from the previous modes. For example, the functionality is in the range of 1 to 10%.
[0265] The percentage of functionality can be determined from NMR measurements of protons from a polymer sample.
[0266] In one embodiment, the network comprises a first polymer and a second polymer.
[0267] One or more added polymers can be provided, each having guest molecules, in the hydrogel. Such additional polymers can be provided to further adjust the mechanical and physical properties of the hydrogel.
[0268] In one embodiment, the first polymer is covalently linked to a plurality of first guest molecules and the second polymer is covalently linked to a plurality of second guest molecules. The hydrogel network is formed by connecting the first polymer to the second polymer through non-covalent interactions of cucurbituryl with the first and second guest molecules. This is a network based on ternary complexation. Optionally, the first and second polymers can be attached to additional guest molecules.
[0269] In an alternative embodiment, the hydrogel comprises a first polymer. This first polymer is covalently linked to a plurality of first and second guest molecules. The hydrogel network can be formed by linking and cross-linking the first polymer through non-covalent interactions. This is a network based on ternary complexation.
[0270] In another embodiment, the hydrogel comprises a first polymer. This first polymer is covalently linked to a plurality of first guests. The hydrogel network can be formed by bonding and cross-linking the first polymer through non-covalent interactions. This is a network based on binary complexation. Optionally, a second polymer can also be provided that is covalently attached to one or more second guest molecules.
[0271] Advantageously, a polymer can be endowed with certain functionalities to assist the formation of the hydrogel, or to improve its physical or chemical properties. This functionality can be provided as a multifunctionality.
[0272] In one embodiment, the polymer is equipped with features to change, or, preferably, improve, the interaction of the network with water. The functionality may take the form of a solubilizing group, particularly an aqueous solubilizing group, such as a group comprising a polyethylene glycol functionality. Other examples include groups that comprise amino, hydroxy, thiol, and carboxy functionality. In one embodiment, each polymer, such as one or both the first polymer and the second polymer, may have hydroxy functionality.
[0273] In one embodiment, the polymer is endowed with functionality to assist in the detection or analysis of the polymer, and to assist in the detection or analysis of the formed network. Advantageously, this functionality can also assist in the detection of material encapsulated in the hydrogel. The functionality can take the form of a detectable label, such as a fluorescent label.
[0274] A polymer is attached to a guest molecule or guest molecules of cucurbituryl by covalent bonds. The covalent bond can be a carbon-carbon bond, a carbon-nitrogen bond, or a carbon-oxygen bond, among others. The bond may form part of a bonding group, such as an ester or an amide, and / or part of a group comprising an alkylene or an alkoxylene functionality.
[0275] Each guest molecule can be attached to the polymer using routine chemical bonding techniques. For example, guest molecules can be attached to the polymer by: alkylating a polymer carrying an appropriate leaving group, esterification reactions; amidation reactions; ether-forming reactions; olefin cross metathesis; or reactions initiated by small guest molecules in which a polymeric chain developed from an initiating guest molecule. Suitably functionalized polymers can also be prepared from suitably functionalized monomers, which can be used in a polymerizable composition optionally together with other monomers.
[0276] Examples of linkers to form a connection between a polymer and a guest molecule are described in this document.
[0277] In one embodiment, the network comprises a polymer having a molecular weight (Pm) of 50 kDa or greater, 100 kDa or greater, 200 kDa or greater, 500 kDa or greater, 1.0 MDa or greater, 1.5 MDa or greater, 2.0 MDa or greater, or 3.0 MDa or greater.
[0278] In one embodiment, the polymeric molecule has a molecular weight in the range of 50 kDa to 4.0 MDa, such as from 500 kDa to 4.0 MDa, such as from 1.0 MDa to 4.0 MDa.
[0279] The molecular weight can refer to the average numerical molecular weight or the average weight molecular weight. The average numerical molecular weight or the average molecular weight of a polymer can be determined by conventional techniques.
[0280] When the network comprises first and second polymers, optionally with additional polymers, one of the polymers has a molecular weight selected from the values or ranges given above. Alternatively, both the first and the second polymers, optionally, as well as the additional polymers present, have a molecular weight selected from the values or ranges given above.
[0281] In one embodiment, a polymer is a synthetic polydispersed polymer. A polydispersed polymer comprises polymeric molecules having a molecular weight range. The polydispersity index (PDI) (average molecular weight divided by the average numerical molecular weight) of a polydispersed polymer is greater than 1, and can be in the range of 5 to 20. The polydispersion of a polymeric molecule can be determined by conventional techniques such as gel permeation or size exclusion chromatography.
[0282] Polymers having a relatively low polydispersity are particularly suitable for use in the present invention. These polymers can have a polydispersity in the selected range from 1 to 5, 1 to 3, 1 to 2, or 1 to 1.5. Such polymers can be referred to as polymers with low dispersion or mono-dispersed in view of their relatively low dispersion. In one embodiment, a polymer has a polydispersity index in the range of 1 to 1.5, such as 1 to 1.4, such as 1 to 1.3.
[0283] The network can comprise a polymer having a PDI value selected from the ranges given above. When there are two or more polymers present in the network, each polymer can have a PDI value selected from the ranges given above.
[0284] The use of polymeric molecules with low dispersion or monodispersed is particularly attractive, since the reactivity of individual molecules is relatively uniform, and the products that result from their use can also be physically and chemically relatively uniform, and they can be relatively low or monodisperse. Methods for preparing low dispersion or monodispersed polymers are well known in the art, and include polymerization reactions based on radical initiated polymerization, including RAFT polymerization (reversible chain transfer by addition-fragmentation) (see, for example) , Chiefari et al. Macromolecules 1998, 31, 5559).
[0285] In one embodiment, a polymer is hydrophilic.
[0286] In one embodiment, a polymer is soluble in water.
[0287] In one embodiment, a polymer has a solubility of at least 0.5 mg / ml, at least 1 mg / ml, at least 5 mg / ml or at least 10 mg / ml, at least 20 mg / ml, at least 50 mg / ml or at least 100 mg / ml.
[0288] Many polymers are known in the art and can be used to produce a mesh material as described in this document. The choice of polymer will depend on the particular application of the network. Suitable polymeric molecules include natural polymers, such as proteins, oligopeptides, nucleic acids, glycosaminoglycans or polysaccharides (including cellulose and related forms, such as guar, chitosan, agarose, and alginate and their functionalized derivatives), or synthetic polymers, such as polyethylene glycol ( PEG), cis-1,4-polyisoprene (PI), polyl (meth) acrylate, polystyrene, polyacrylamide, and polyvinyl alcohol. The polymer can be a homopolymer. Alternatively, the polymer may be a copolymer where the different monomer units are uniformly arranged, alternatively, in blocks or in another arrangement.
[0289] The polymeric molecule can comprise two or more natural and / or synthetic polymers. These polymers can be arranged in a linear architecture, cyclic architecture, comb or graft architecture, branched (hyper) architecture or star architecture.
[0290] In one embodiment, the polymer is a biopolymer.
[0291] Suitable polymeric molecules include those polymeric molecules having hydrophilic characteristics. Therefore, a part of the polymer, being that part refers, among others, to a monomeric unit, the main chain itself, a side chain or a grafted polymer, is hydrophilic. In one embodiment, the polymeric molecule is capable of forming hydrogen bonds in a polar solvent, such as water. The polymeric molecule is soluble in water to form a continuous phase.
[0292] Some of the exemplary polymers given above can be provided with adequate functionality in order to provide hydrophilic characteristics to such polymer. Some of the present inventors have previously described the use of functionalized polystyrene and a polystyrene copolymer in the preparation of a hydrogel material (see Appel et al. J. Am. Chem. Soc. 2010, 132, 14251-14260). The polystyrene copolymer is a copolymer with acrylamide, which includes hydroxyl functionality.
[0293] In one embodiment, a polymer is endowed with a plurality of monomer units having a functionality selected from hydroxyl, amino, starch, carboxy, and sulfonate, including their salt forms. The amino group can be a tertiary or quaternary amino group. The amide group can be a tertiary amide.
[0294] In one embodiment, a polymer is a polymer having a plurality of monomeric units containing hydroxyl. A monomeric unit containing hydroxyl may have one or more hydroxyl groups, such as one, two or three hydroxyl groups.
[0295] In one embodiment, a polymer is a polymer having a plurality of monomer units containing alkyl ether.
[0296] A monomeric unit can have both hydroxyl groups and alkyl ether groups present. Additionally or alternatively, a polymer can comprise monomer units having hydroxyl groups, monomer units having alkyl ether groups and / or units having both hydroxyl groups and alkyl ether groups.
[0297] The alkyl ether can be a C1-6 alkyl ether.
[0298] In one embodiment, substantially all monomer units in the polymer have a hydroxyl group or an alkyl ether group.
[0299] In one embodiment, the molar fraction of monomeric units containing hydroxyl or alkyl ether in a polymer is at least 0.50, at least 0.60, at least 0.70, at least 0.80, at least 0, 90, or at least 0.95. The molar fraction refers to the number of monomeric units having a hydroxyl or ether functionality as a fraction of the total number of monomeric units in the polymer. The molar fraction can be obtained using standard analytical techniques, for example, NMR spectroscopy.
[0300] In one embodiment, a polymer in the network is a polymeric polyol.
[0301] In one embodiment, a polymer has hydroxyl functionality present in each monomer unit.
[0302] In one embodiment, when there are two or more polymers in the network, each polymer is a polymeric polyol. In contrast, the networks described in previous studies by the present inventors include only a hydroxyl-containing polymer, and, more often, no hydroxyl-containing polymer.
[0303] In one embodiment, a polymer comprises a plurality of monomer units derived from vinyl alcohol.
[0304] In one embodiment, a polymer is or comprises a poly (vinyl) alcohol (PVA). In this modality, the guest molecules can be covalently linked to the polymeric main chain through hydroxy functionality. PVA is readily available from commercial sources, in a variety of different different average molecular weights. PVA can be functionalized as appropriate to include adequate guest functionality.
[0305] In one embodiment, a polymer in the network comprises a plurality of monomeric saccharide units.
[0306] In one embodiment, a polymer is or comprises a polysaccharide. The polysaccharide can be cellulose or a related form, such as alkylated cellulose, guar, chitosan, agarose, and alginate and its functionalized derivatives. In this embodiment, the guest molecules can be covalently linked to the polymeric main chain through the hydroxy functionality of the polymer, typically, the hydroxy ring functionality.
[0307] In one embodiment, the polysaccharide is cellulose or a functionalized derivative.
[0308] In one embodiment, the polysaccharide is hydroxyethyl cellulose.
[0309] In one embodiment, in addition to or as an alternative to the polysaccharides described above, the polysaccharide is carboxymethyl cellulose or hyaluronic acid.
[0310] In one embodiment, the network comprises first and second polymers, in which the first polymer is a poly (vinyl) alcohol and the second polymer is a polysaccharide.
[0311] In one embodiment, the network comprises only a first polymer, and the first polymer is a polysaccharide, for example, a cellulose, such as hydroxyethyl cellulose, carboxymethyl cellulose or hyaluronic acid.
[0312] The polymers in the network are functionalized with guest molecules that are suitable for interacting with cucurbituril. In some embodiments, a first polymer is endowed with a plurality of first guests in which the first guest is suitable to form a ternary complex next to the cucurbituril and a second guest of a second polymeric molecule. In another modality, the polymer is endowed with a plurality of first and second guests, in which the first guest is suitable to form a ternary complex next to the cucurbituila and a second guest of the same first polymer (crosslinking) or a second guest of another first polymeric molecule. In this modality, the first and second guests can be the same or different.
[0313] A guest molecule is covalently connected to the polymer. The guest molecule can be connected directly to the functionality present in the polymer, or the guest molecule can be connected indirectly through a ligand to the functionality.
[0314] For example, the hydroxyl, amino and carboxyl functionality present in a polymer can serve as binding sites for the host molecule or ligand.
[0315] Polymers for use in the present invention can be obtained from commercial sources, and these polymers can be properly functionalized with guest molecules using standard organic chemistry techniques. In some embodiments, the polymer is a biopolymer, which can be sourced from renewable sources.
[0316] An example of the functionalization of a polysaccharide and a polyvinyl alcohol is described in this document. As examples of the polysaccharide, the use of carboxymethyl cellulose and hyaluronic acid is shown.
[0317] In other embodiments, a polymer having an adequate cucurbituryl guest-functionality may be prepared directly from a polymerizable composition comprising monomers having adequate guest functionality and non-functionalized monomers. The number of guests in the final product can be varied by appropriate changes in the monomer concentration and other reaction conditions, as will appear to an individual skilled in the art. The polymerization conditions and polymerization reagents can be selected in order to provide a desired molecular weight, polydispersity, solubility, and / or mechanical and physical parameters.
[0318] A polymer described in this document can be further functionalized to provide useful properties to the polymer and the resulting hydrogel. For example, the polymer may comprise a detectable label to assist in the detection and analysis of the polymer in a hydrogel.
[0319] In one embodiment, polymers having adequate guest functionality of cucurbituril are non-toxic. Toxicity can be determined by exposing a cell to an aqueous mixture, including a solution, of a functionalized polymer as described in this document, and by monitoring cell viability for a period of time, for example, 1, 2 , 5, 10 or 30 days.
[0320] In one embodiment, a functionalized polymer, or a mixture of functionalized polymers, can be considered non-toxic if 60% or more, 70% or more, 80% or more, or 90% or more of the cell population viable during the period of time for analysis.
[0321] The toxicity study can be repeated at different concentrations of the functionalized polymer in the aqueous mixture. For example, the total concentration of the polymer in the aqueous mixture can be 0.01% by weight, 0.02% by weight, 0.04% by weight, 0.1% by weight, 0.3%, by weight, 0.5% by weight, 1.0% by weight, 2.0% by weight, or 5% by weight.
[0322] Toxicity may be cell viability as recorded in one of the above concentrations, for a specific period of time, such as those mentioned above.
[0323] In a toxicity test, all polymers used in the hydrogel can be tested together in a combined test mixture. Therefore, the concentration of the polymer mentioned above can be the total combined concentration of the polymers in such a test mixture. Cucurbiturila guest
[0324] As noted earlier, the guest is a compound that is capable of forming a guest-host complex with a cucurbituril. Therefore, the term "complexation" refers to the establishment of the guest-host complex.
[0325] In some embodiments of the invention, the host-host complex is a ternary complex comprising the cucurbituryl host and a first guest molecule and a second molecule. Typically, these complexes are based on CB [8] and variants and derivatives thereof.
[0326] In particular, any compound having adequate binding affinity can be used in the methods of the present invention. The compound used can be selected based on the size of the portions imagined to interact with the cucurbituril cavity. The size of these portions may be large enough to allow complexation with only large forms of cucurburyl.
[0327] The host molecules of cucurbituril are notorious in the art. Examples of guest compounds for use include those described in WO 2009/071899, Jiao et al. (Jiao et al. Org. Lett. 2011, 13, 3044), Jiao et al. (Jiao et al. J. Am. Chem. Soc. 2010, 132, 15734), Rauwald et al. (Rauwald et al. J. Phys. Chem. 2010, 114, 8606) and WO 2011/077099.
[0328] The present inventors have studied the complexation of guest molecules when they are attached to a polymer and when these molecules are released. The use of isothermal calorimetry demonstrated that the attachment of a guest molecule to a polymer does not result in a reduction in the guest's binding constant. therefore, there are no observable effects in bonding from the stereo impediment of polymers.
[0329] In one embodiment of the invention, cucurbituryl is CB [8] and the host molecules are formed to form a ternary complex with that host. In one embodiment, a guest is a guest rich in electrons and a guest molecule is deficient in electrons.
[0330] A cucurbituryl guest molecule can be derived, or contain, a structure from the table below:




[0331] where the structure can be a salt, including protonated forms, when appropriate. In one embodiment, the guest molecules are guest molecules for CB [8].
[0332] In one embodiment, the guest molecule consists of, or is derived from, or contains, an A1-A43, A46 or B1-B4 structure, in the table above.
[0333] In one embodiment, the guest molecule consists of, or is derived from, or contains, an A1, A2, or A13 structure in the table above.
[0334] In one embodiment, the guest molecule consists of, or is derived from, or contains, a B1 structure.
[0335] In addition or alternatively to the guests in the table above, the following guest molecules can be selected:

[0336] In one embodiment, the guest molecule consists of, or is derived from, or contains, an A47 or A 48 structure
[0337] Additionally, the host molecule consists of, or is derived from, or contains, adamantane, ferrocene or cyclooctane (including bicyclo [2,2,2] octane). These are described by Moghaddam et al. (see J. Am. Chem. Soc. 2011, 133, 3570).
[0338] Other guest molecules suitable for use include pyrene, di-benzofuran and fluorine, and derivatives thereof. The derivative can be a compound where the aromatic ring atom is replaced by a hetero atom, such as nitrogen. Additionally or alternatively, the derivative can be a compound that is replaced by a ring atom with a group such as halogen, alkyl, hydroxy, amino, alkoxy or the like.
[0339] In some embodiments, the first and second guest molecules form a pair that can interact within the cucurbituryl cavity to form a stable ternary host-guest complex. Any pair of guest that fits inside the cavity of the cucurbiturila can be used. In some embodiments, the pair of guest molecules can form a charge transfer pair that comprises an electron-rich compound and an electron-deficient compound. One of the first and second guest molecules acts as an electron acceptor and the other as an electron donor in the CT pair. For example, the first guest ramolecule may be an electron deficient molecule that acts as an electron acceptor and the second guest molecule may be an electron-rich molecule that acts as an electron donor or vice versa. In one embodiment, cucurbituril is CB [8].
[0340] Suitable electron acceptors include 4,4'-bipyridinium derivatives, for example, N, N'-dimethyldipyridyliuethylene, and other related acceptors, such as those based on diazapyrenes and diazafenanthrenes. Viologen compounds including alkyl viologens are particularly suitable for use in the present invention. Examples of alkyl viologene compounds include N, N'-dimethyl-4,4'-bipyridinium salts (also known as Paraquat).
[0341] Suitable electron donors include electron-rich aromatic molecules, for example, 1,2-dihydroxybenzene, 1,3-dihydroxybenzene, 1,4-dihydroxybenzene, tetratiafulvalene, naphthalenes, such as 2,6-naphthalene dihydroxy and 2-naphthol, indoles and sesamol (3,4-methylenedioxyphenol). Polycyclic aromatic compounds in general can be used as suitable electron donors in the present invention. Examples of such compounds include anthracene and naphtacene.
[0342] In one mode, the guest is anthracene. In one embodiment, the guest is cinnamic acid.
[0343] Amino acids, such as tryptophan, tyrosine and phenylalanine may be suitable for use as electron donors. Peptide sequences that comprise these amino acids at their terminations can be used. For example, a donor comprising an amino acid sequence N-WGG-C, N-GGW-C or N-GWG-C can be used.
[0344] In one embodiment, the guest is tryptophan or phenylalanine. In one embodiment, the guest is phenylalanine.
[0345] In some embodiments, the guest molecules are a pair of compounds, for example, first and second guest molecules, where one among the pair is a compound A as shown in the table above (for example, A1, A2, A3 etc. ), and the other is a compound B as shown in the table above (for example, B1, B2, B3 etc.). In one embodiment, compound A is selected from A1-A43 and A46. In one embodiment, compound B is B1.
[0346] Other suitable guest molecules include peptides, such as WGG (Bush, M. E. et al J. Am. Chem. Soc. 2005, 127, 14511-14517).
[0347] An electron-rich guest molecule can be correlated with any electron-deficient guest molecule [8]. Examples of suitable pairs of guest molecules, for example, first and second guest molecules, for use as described in this document may include: Viologenium and naphthol; viologene and dihydroxybenzene; viologenium and tetratiafulvaleno; viologenium and indole; methylviologenium and naphthol; methylviologenium and dihydroxybenzene; methylviologen and tetratiafulvalene; methylviologenium and indole; N, N'-dimethyldipyridylium ethylene and naphthol; N, N'-dimethyldipyridylium ethylene and dihydroxybenzene; N, N'-dimethyldipyridylium ethylene and tetratiafulvalene; N, N'-dimethyldipyridylium ethylene and indole; 2,7-dimethyldiazapyrenium and naphthol; 2,7-dimethyldiazapyrenium and dihydroxybenzene; 2,7-dimethyldiazapyrenium and tetratiafulvalene; and 2,7-dimethyldiazapyrenium and indole.
[0348] In particular, suitable pairs of guest molecules for use as described herein may include 2-naphthol and methyl viologene, 2,6-dihydroxy naphthalene and methyl viologene and tetratiafulvalene AND methyl viologene.
[0349] In one embodiment, the guest pair is 2-naphthol and methyl viologene.
[0350] In one embodiment, the guest pair is a reference to a pair of guest molecules suitable for forming a ternary complex with CB [8].
[0351] In one embodiment, the guest molecule is preferably a liquid-doionic. Typically, these guests are suitable to form a complex with CB [7]. However, they can also form complexes with CB [8] in a com-plexobinary, or in a ternary complex next to another small guest molecule or solvent (see Jiao et al. Org. Lett. 2011, 13, 3044).
[0352] The ionic liquid typically comprises a cationic organic nitrogen heterocycle, which can be an aromatic nitrogen heterocycle (a heteroaryl) or a non-aromatic nitrogen heterocycle. The ionic liquid also typically comprises a counter-anion to the cationic organic nitrogen heterocycle. The heteroaryl nitrogen group is preferably a C510 heteroaryl nitrogen group, most preferably a C5-6 heteroaryl nitrogen group, where the subscript refers to the total number of atoms in the ring or rings, including carbon atoms and nitrogen. The non-aromatic nitrogen heterocycle is preferably a C5-6 nitrogen heterocycle, where the subscript refers to the total number of atoms in the ring or rings, including carbon and nitrogen atoms. A nitrogen atom in the nitrogen heterocycle ring is quaternized.
[0353] The counter-anion can be a halide, preferably a bromide. Other suitable anions for use are those that result in a complex that is soluble in water.
[0354] The guest is preferably a compound, including a salt, which comprises one of the groups selected below from the list consisting of: portion of imidazolium; pyridinium portion; quinoline portion; pyrimidinium portion; pyrrole portion; and portion of quaternary pyrrolidine.
[0355] Preferably, the guest comprises a portion of imidazolium. An especially preferred guest is 1-alkyl-3-alkylimidazolium, where alkyl groups are optionally substituted.
[0356] 1-alkyl-3-alkylimidazolium compounds, where alkyl groups are unsubstituted, are especially suitable for forming a complex with CB [7].
[0357] 1-alkyl-3-alkylimidazolium compounds, where alkyl groups are unsubstituted, are especially suitable for forming a complex with CB [6]
[0358] 1-alkyl-3-alkylimidazolium compounds, where an alkyl group is substituted by aryl (preferably naphthyl), are especially suitable to form a complex with CB [8].
[0359] The 1-alkyl and 3-alkyl substituents can be the same or different. Preferably, they are different.
[0360] In one embodiment, the 3-alkyl substituent is methyl, and is preferably unsubstituted.
[0361] In one embodiment, the 1-alkyl substituent is ethyl or butyl, and is preferably unsubstituted.
[0362] In one embodiment, the optional substituent is aryl, preferably C5-10 aryl. Aryl includes carboaryl and heteroaryl. Aryl groups include phenyl, naphthyl and quinolinyl.
[0363] In one embodiment, the alkyl groups described herein are linear alkyl groups.
[0364] Each alkyl group is independently a C1-6 alkyl group, preferably a C1-4 alkyl group.
[0365] The aryl substituent may be another 1-alkyl-3-substituted-imidazolium moiety (where the alkyl group is attached to the 3-position of the ring).
[0366] In another embodiment, the compound preferably comprises a portion of pyridinium.
[0367] The liquid ionic molecules described above are particularly useful for forming binary host-host complexes. Complexes that comprise two liquid ionic molecules as guests in a cucurbituryl host are also covered by the present invention.
[0368] A cucurbiturila may be able to form a binary complex and a ternary complex. For example, CB [6] compounds were previously noted to form ternary complexes with short-chain 1-alkyl-3-methylimidazolium molecules, while longer-chain 1-alkyl-3-methylimidazolium molecules form complexes binaries with the host of cucurbiturila.
[0369] The preferred guests for use in the present invention are of the form H + X-, where H + is one of the following cations,

[0370] and X-is a suitable counter-anion, as previously defined. A preferred counter-anion is a halide anion, preferably Br-.
[0371] In a preferred embodiment, cation A or cation B can be used to form a complex with CB [7] or CB [6].
[0372] In a preferred embodiment, cation D or cation E can be used to form a complex with CB [8].
[0373] Cations A and B can be referred to as 1-ethyl-3-methylimidazolium and 1-butyl-3-methylimidazolium, respectively.
[0374] Cations D and E can be referred to as 1-naphthalenylmethyl-3-methylimidazolium, where D is 1-naphthalen-2-ylmethyl-3-methylimidazolium and E is 1-naphthalen-1-ylmethyl-3-methylimidazole.
[0375] Alternatively or in addition, the guest compounds can be an imidazolium salt of formula (I):
where X-is a counter-anion;
[0376] R1 is independently selected from H and saturated C1-6 alkyl;
[0377] R2 is independently C1-10 alkyl which may optionally contain one or more double or triple bonds, and may be optionally interrupted by a heteroatom selected from -O-, -S-, -NH-, and -B-, and can be optionally-replaced.
[0378] In one embodiment, X-is independently selected from the group consisting of Cl-, Br-, I-, BF4-, PF6-, OH-, SH-, HSO4-, HCO3-, NTf2, C2N5O4, AlCl4-, Fe3Cl12, NO3-, NMeS2-, MeSO3-, SbF6-, PrCB11H11-, AuCl4-, HF2-, NO2-, Ag (CN) 2-, and NiCl4-. In one embodiment, X-is selected from Cl-, Br-, and I-.
[0379] In one embodiment, R1is selected from H and linear saturated C1-6 alkyl.
[0380] In one embodiment, R2 is linear C1-10 alkyl, which may optionally contain one or more double bonds, and may be optionally interrupted by a heteroatom selected from -O-, -S-, -NH-, and -B-, and can be optionally-replaced.
[0381] In one embodiment, R2 is linear C1-10 alkyl, which may optionally contain one or more double bonds, and may be optionally substituted.
[0382] In one embodiment, when a double or triple bond is present, it can be conjugated to the imidazole portion. Alternatively, the double or triple bond may not be conjugated to the imidazole portion.
[0383] In one embodiment, optional substituents are independently selected from the group consisting of halo, optionally-substituted C5-20 aryl, -OR3, -OCOR3, = O, -SR3, = S, -BR3, -NR3R4 , -NR3COR3, - N (R3) CONR3R4, -COOR3, -C (O) R3, -C (= O) SR3, -CONR3R4, -C (S) R3, -C (= S) SR3, and - C (= S) NR3R4,
[0384] where each R3 and R4 is independently selected from H and C1-6 alkyl, C5-20 aryl and optionally substituted C5-20 saturated C1-6 alkylene.
[0385] or R3 and R4 can together form an optionally saturated 5, 6 or 7 membered heterocyclic ring that is optionally substituted by a group - R3.
[0386] In one embodiment, optional substituents are independently selected from the group consisting of halo, optionally substituted C5-20 aryl, -OR3, -OCOR3, -NR3R4, -NR3COR3, -N (R3) CONR3R4, - COOR3, - C (O) R3, and -CONR3R4, where R3 and R4 are defined as above.
[0387] Each C5-20 aryl group can be independently selected from a C6-20 carboaryl group or a C5-20 heteroaryl group.
[0388] Examples of C6-20 carboaryl groups include phenyl and naphthyl.
[0389] Examples of C5-20 heteroaryl groups include pyrrole (azole) (C5), pyridine (azine) (C6), furan (oxola) (C5), thiophene (thiol) (C5), oxazole (C5), thiazole (C5), imidazole (1,3-diazola) (C5), pyrazola (1,2-diazola) (C5), pyridazine (1,2-diazine) (C6), and pyrimidine (1,3-diazine) (C6) (for example, cytosine, thymine, uracil).
[0390] Each C5-20 aryl is preferably selected from optionally substituted phenyl, naphthyl and imidazolium.
[0391] Each C5-20 aryl group is optionally substituted. Optional substituents are independently selected from halo, C1-6 alkyl, -OR3, - OCOR3, -NR3R4, -NR3COR3, -N (R3) CONR3R4, -COOR3, -C (O) R3, and -CONR3R4, where R3 and R4 are defined as above.
[0392] In one embodiment, each C5-20 aryl group is optionally substituted by C1-6 alkyl.
[0393] When the C5-20 aryl group is an imidazole, it is preferably substituted in nitrogen by an R1 group (thus forming a quaternary nitrogen).
[0394] The compound of formula (I) comprises a portion of imidazolium having a substituent R2 in position 1 and a substituent R1 in position 3. In a further aspect of the invention, the compound of formula (I) can be optionally substituted in position 2, 4 or 5 by a group RA, where RA has the same meaning as R1.
[0395] The previous modalities can be combined in any combination, as appropriate. Alternative hosts and guests
[0396] The hydrogels described here can also be prepared using alternative host compounds. Therefore, cucurbituryl can be replaced by a host that is capable of forming ternary or binary complexes, as described above. Alternatively, the hosts described below can be used in addition to a cucurbituryl host in the hydrogels described herein.
[0397] In some embodiments, a host is selected from cyclodextrin, calix [n] arene, and crown ether, and one or more building blocks have a host-host functionality suitable for cyclodextrin, calix [n] arene, or crown ether, respectively.
[0398] In one embodiment, the host is cyclodextrin and one or more building blocks have adequate cyclodextrin guest functionality.
[0399] The host can form a binary complex with a guest. In such cases, the host will be covalently linked to one or more other guest molecules to allow the formation of cross-links between the building blocks.
[0400] In one embodiment, a host is cyclodextrin. Cyclodextrin compounds are readily available from commercial sources. Many com-post guests for use with cyclodextrin are also known.
[0401] Cyclodextrin consists of cyclic oligomers with a non-symmetrical barrel shape of D-glucopyranose. Typically, cyclodextrin is capable of hosting hydrophobic uncharged guests. For example, guests include those molecules having hydrocarbon and aromatic functionality, such as adamantane, azobenzene, and stylbene derivatives. Other guest molecules for cyclodextrin include biomolecules, such as xylose, tryptophan, estriol, sterone and estradiol.
[0402] In one embodiment, the cyclodextrin is an a-, β- or y-cyclodextrin. In one embodiment, the cyclodextrin is a β- or y-cyclodextrin. Larger guests are typically used in conjunction with a y-cyclodextrin.
[0403] Cyclodextrin has a toroidal geometry, with the secondary hydroxyl groups of D-glycopyranosis located in the largest opening, and the primary hydroxyl groups in the smallest opening. One or more of the hydroxy groups, which can be secondary or primary hydroxy groups, can be functionalized. Typically, the primary hydroxyl groups are functionalized. In one embodiment, references to a cyclodextrin compound are references to derivatives thereof. For example, one or two primary hydroxyl groups in the cyclodextrin are functionalized with an alkylamine-containing substitute. In another example, one, two or three hydroxyl groups in each D-glycopyranose unit is replaced by an alkyl ester group, for example, a methoxy group. A plurality of covalently linked cyclodextrins can be connected via hydroxyl groups.
[0404] Examples of non-functionalized and functionalized cyclodextrins are shown in Graph 1 by Rekharsky et al. (Chem. Rev. 1998, 98, 1875), and examples of compounds for use as guests are presented in Tables 1 to 3 and in Graph 2. Rekharsky et al. is incorporated herein by way of reference.
[0405] In preparation methods, cyclodextrin can be present in the second phase, for example, in an aqueous phase, as described in this document.
[0406] In one embodiment, the host is calix [n] arene. Calix [n] arene compounds are readily available from commercial sources, or can be prepared by condensation of phenol, resorcinol and pyrogallol aldehydes, for example, formaldehyde.
[0407] Many guest compounds for use with calix [n] arenes are known. Typically, calix [n] arene is capable of hosting amino-containing molecules. Piperidine-based compounds and amino-functionalized cyclohexyl compounds can be used as guests. Additional examples of guests include atro-pina, critanda, phenol blue, and anthrol blue among others.
[0408] Examples of non-functionalized and functionalized cyclodextrins are shown in Graph 1 by Danil de Namor et al. (Chem. Rev. 1998, 98, 2495-2525), which is hereby incorporated by reference. Examples of compounds for use as guests are presented in Tables 2, 3, 5 and 10 by Danil de Namor et al.
[0409] In one embodiment, the calix [n] arene is a calix [4] arene, calix [5] arene or calix [6] arene. In one embodiment, the calix [n] arene is a calix [4] arene.
[0410] Calix [n] properly functionalized arenes can be prepared using appropriately functionalized aryl hydroxide aldehydes. For example, the hydroxyl group can be replaced by a group containing alkyl ether or a group containing ethylene glycol. A plurality of calix [n] covalently bonded arenes can be connected via hydroxyl groups.
[0411] In preparation methods, calix [n] arene can be present in the second phase, for example, in an aqueous phase or in a water immiscible phase, as described in this document.
[0412] In one embodiment, the host is a crown ether. Crown ether compounds are readily available from commercial sources or can be readily prepared.
[0413] Many guest compounds for use with crown ether are also known. For example, cationic hosts, such as amino- and pyridinium-functionalized molecules, may be suitable guest molecules.
[0414] Examples of non-functionalized and functionalized cyclodextrins are presented in Gokel et al. (Chem. Rev. 2004, 104, 2723-2750), which is incorporated herein by reference. Examples of compounds for use as guests are described throughout the text.
[0415] In one embodiment, the crown ether is selected from groups consisting of 18-crown-6, dibenzo-18-crown-6, diaza-18-crown-6 and 21-crown-7. In the present invention, larger crown ethers are preferred. Smaller crown ethers may be able to bind only small metal ions. Larger crown ethers are able to link functional groups and molecules.
[0416] In some modalities, the host is a guest having a crown ether and calix [n] arene functionality. These hosts are referred to as calix [n] crowns.
[0417] In preparation methods, the crown ether may be present in the second phase, for example, in a water immiscible phase, as described in this document.
[0418] Other guest-host relationships may be used as will appear to an individual skilled in the art. Other host-host complexes for use in the present invention include those highlighted by Dsouza et al. (Chem. Rev. 2011, 111, 7941-7980) which is incorporated herein by reference, and in particular those hosts shown in Schemes 6 and 7, which include cucurbituril, cildoextrin, and calixeran, as well as AVCyc cyclophane , calixpiridine C4P and SQAM Escarimide.
[0419] The use of cyclodextrin is preferred over crown ether and calix [n] arene hosts. Component
[0420] The hydrogel of the invention can be used to maintain a component. In one aspect of the invention, a hydrogel is provided which comprises a component. The hydrogel is suitable for storing a component, and that component can later be released as needed in a chosen location.
[0421] In one aspect, a hydrogel is provided having a cross-linked supramolecular network obtainable from the complexation of an aqueous composition comprising cucurbituril and one or more polymers having a suitable cucurbituryl guest functionality, in which the hydrogel maintains a component. A polymer of the aqueous composition can have a molecular weight of 50 kDa or greater.
[0422] It is established that a reference to a component retained by the hydrogel is not a reference to a solvent molecule. For example, the component is water or an organic solvent. Therefore, a component is provided in addition to the solvent that may be present in the hydrogel.
[0423] It is also established that a reference to a component is not a reference to a cucurbituril or a polymer used in the preparation of the hydrogel, or an intermediate product formed from the complexation of cucurbituril with a properly functionalized polymer. Otherwise, the component is not particularly limited.
[0424] In one embodiment, the encapsulated component has a molecular weight of at least 100, at least 200, at least 300, at least 1,000, at least 5,000 (1k), at least 10,000 (10k), at least 15,000 (15k) ), at least 20,000 (20k), at least 50,000 (50k), at least 100,000 (100k) or at least 200,000 (200k).
[0425] The present inventors have found that the hydrogels of the invention can maintain and profitably distribute a component, such as a bioactive component, to a location. The component's activity can be maintained during its incorporation into a hydrogel, during its storage in the hydrogel and after its subsequent distribution to the desired location.
[0426] In one embodiment, the component is a therapeutic compound.
[0427] In one embodiment, the component is or comprises a biological molecule, such as a polynucleotide (for example, DNA and RNA), a polypeptide or a polysaccharide.
[0428] In one embodiment, the component is a polymeric molecule, including biological indopolymers, like those biological molecules mentioned above.
[0429] In one embodiment, the component is a cell.
[0430] In one embodiment, the component has a detectable label. The detectable label can be used to quantify and / or locate the component. The label can be used to determine the amount of component contained in the hydrogel.
[0431] In one embodiment, the detectable label is a luminescent label. In one embodiment, the detectable label is a fluorescent label or a phosphorescent label.
[0432] In one embodiment, the detectable label is a visible label.
[0433] In one embodiment, the fluorescent label is a rhodamine or fluorescein label.
[0434] In one embodiment, the component is a polypeptide, such as a protein. The protein can be a serum albumin or lysozyme. Examples of this protein include bovine serum albumin.
[0435] In one embodiment, the component is a particle. The particle can be a metal particle.
[0436] In one embodiment, the component is selected from the group consisting of toxic molecules (such as nerve agents and heavy metals), hormones, herbicides, pesticides, antibodies, pathogens (such as viruses), adjuvants, gels, nanoparticles ( including metallic or non-metallic particles), polymers (including synthetic and natural polymers), catalysts (organic, inorganic, and organometallic), adhesives and sealants.
[0437] The presence of a component in the hydrogel can be determined using appropriate analytical techniques that are able to distinguish the material network and the component. These techniques are notorious for individuals skilled in the art. Methods for preparing hydrogels
[0438] Polymers for use in the invention are functionalized with one or more guest molecules to form a non-covalent interaction with a cucurbituryl. The hydrogels of the present invention are simple and quick to prepare. The components of the hydrogel, one or more properly functionalized polymers and a cucurbituril, can be mixed together, in appropriate concentrations, in water. Typically, the supramolecular network and, therefore, the hydrogel are formed in seconds.
[0439] In one aspect of the invention, a method for preparing a hydrogel is provided, the method comprising the step of combining in a watery mixture a cucurbituryl, a first polymer having a plurality of first guest molecules, and a second polymer having a plurality of second guest molecules, to thereby generate a hydrogel.
[0440] In one embodiment, the hydrogel is prepared from the complexation of (a) an aqueous composition comprising cucurbituryl and (1) or (2); or (b) a composition comprising a plurality of covalently linked cucurbiturils and (1), (2) or (3). Each (1), (2) and (3) is discussed above in the Hydrogel section.
[0441] The composition is prepared by combining in an aqueous mixture the cucurbiturils and (1) or (2), or by combining in the aqueous mixture the plurality of covalently linked cucurbiturils and (1), (2) or (3 ).
[0442] The relative amounts of cucurbituryl or plurality of cucurbiturils covalently linked to (1), (2) or (3) can be appropriately selected to produce a hydrogel having the desired relative amounts of these components. The relative quantities of components used in the mixture before mixing will result in a hydrogel having the same relative quantities of components, since all components of the mixture are incorporated into the hydrogel. The amounts of water, cucurbituryl and polymer present are described above in the Hydrogel section.
[0443] In the methods of the invention, at least one cucurbituril and a suitably functionalized polymer are brought into contact only when it is necessary to prepare the hydrogel. The hydrogels of the present inventions are formed quickly, typically, in seconds under ambient conditions, when the complexing components are combined in a complexable aqueous mixture. Therefore, the individual components of the complexable mixture can be stored separately, optionally as an aqueous mixture, until needed. When necessary, the components can be joined to form the hydrogel.
[0444] In one embodiment, a hydrogel can be formed using the steps as described above, and the resulting hydrogel diluted with water, to thereby obtain a hydrogel having a higher water content (lower polymer content). This dilution step can be carried out in order, for example, to fine-tune the hydrogel's mechanical properties. After adding water, the mixture can be stirred.
[0445] The formation of the hydrogel may be apparent to the visible eye, and simple tests such as flask tests (whereby a flask containing material is simply placed upright) are useful indicators of hydrogel formation. To fully analyze the hydrogel, more rigorous analysis steps can be performed during hydrogel formation and / or after hydrogel formation. Suitable methods for analyzing hydrogels of the invention are described below.
[0446] Hydrogels with a component are also described. The component can be introduced into a hydrogel during hydrogel formation, or the component can be added to a preformed hydrogel, which is then interrupted to allow incorporation of the component. In particular, the hydrogel's desreological properties can be analyzed and are useful for characterization.
[0447] When the component is incorporated into the hydrogel formation, the component simply needs to be mixed with the hydrogel components before hydrogel formation. The supramolecular network is formed around the component to thereby provide a hydrogel while maintaining a component.
[0448] Therefore, in another aspect of the invention, a method is provided for preparing a hydrogel while maintaining a component, the method comprising the step of bringing into contact in an aqueous solution a mixture of cucurbituril, a component, and one or more polymers having an adequate guest function of cu-curbituryl, to thereby generate a hydrogel while maintaining a component.
[0449] In one embodiment, the method comprises the step of combining a cucurbituryl in an aqueous solution, a first polymer having a plurality of first guest molecules, a second polymer having a plurality of second guest molecules, and a component for thereby , generate a hydrogel while maintaining a component.
[0450] Alternatively, a method for preparing a hydrogel while maintaining a component comprises the steps of providing a hydrogel of the invention and stirring that hydrogel in the presence of a component, to thereby incorporate the component into the hydrogel. The stirring step can be a mechanical stirring or interruption of the hydrogel.
[0451] The complexes of the present invention are reversible, and a complex that is interrupted is capable of reforming. Hydrogel analysis
[0452] The hydrogel material can be analyzed using techniques familiar to those skilled in the art.
[0453] During a hydrogel formation process, the formation of a complex can be monitored by analyzing a color change in the reaction mixture. The formation of a complex, such as a ternary complex, can be associated with the formation of charge transfer bands that are associated with a complex format. For example, guests of viologenium and naphthyl together can form a ternary complex with CB [8] in water. The formation of this complex is apparent from the orange color resulting from the mixture, which derives from the formation of charge transfer between guests of viologenium and naphthyl.
[0454] For example, a scanning electron microscopy (SEM) is described. This is useful for analyzing and measuring the shape and pore size in the hydrogel. Typically, a hydrogel sample for SEM analysis is dried and lyophilized prior to such analysis. For hydrogels having a very high water content (for example, having a water content of not more than 0.95% by weight), SEM techniques may not be appropriate, as the hydrogel is seen to disintegrate during sample preparation . For these hydrogels, other analytical techniques may be more appropriate, such as the techniques described below.
[0455] Neutron scattering at low angle (SANS) can also be used to analyze the hydrogel microstructure. Correlation lengths can be established from SANS measurements, as described in detail in this document.
[0456] Standard rheological techniques can be employed to establish the storage module, loss module, complex viscosity, elasticity and tan δ of the hydrogel. For example, voltage amplitude sweep measurement and frequency sweep measurement can be adopted as part of the dynamic oscillatory rheological characterization of hydrogels. These techniques are as described in detail in this document, and the rheological properties of the hydrogel are defined in detail in the Hydrogel section above.
[0457] The formation of a hydrogel material can also be clearly visible from the reaction mixture. The formation of a gel from an aqueous mixture may be apparent from an inverted flask test, and the like. Use of hydrogel
[0458] The hydrogels described here can be used as materials in medical applications, due to their low toxicity and high water content. The hydrogels of the invention, when properly loaded with a component, can be used to deliver that component to a target location.
[0459] The present inventors have established that the components retained in the hydrogel of the invention can be released from the hydrogel at a chosen location. Therefore, a method is provided to distribute a component to a location, the method comprising the steps of:
[0460] (i) providing a hydrogel while maintaining a component, as described in this document;
[0461] (ii) making the hydrogel available to a target location;
[0462] (iii) releasing the component from the hydrogel.
[0463] In one embodiment, the target site is an in vivo site. Therefore, the hydrogel can be placed in a target location or in an individual. The individual may be a mammal, such as a human or a rodent, such as a rat or mouse.
[0464] In this modality, the component can be a therapeutic compound for use in the treatment or prophylaxis of a disease. The hydrogel comprising the therapeutic compound is suitable for use in methods of treating the body of a human or animal.
[0465] In other embodiments, the hydrogel is suitable for delivering a component to a location that is ex vivo, or in vitro.
[0466] In one embodiment, the hydrogel is capable of releasing at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of a component retained over a defined period of time. These 5 amounts can be molar amounts or weight amounts, depending on the techniques used to measure the release.
[0467] In one embodiment, the hydrogel is capable of releasing substantially all of the component retained for a defined period of time.
[0468] In one embodiment, the time period can be a period of at least 1 day, at least 2 days, at least 5 days, at least 20 days, at least 40 days, or at least 90 days.
[0469] The period of time can be a period of a maximum of 100 days, a maximum of 150 days, or a maximum of 200 days.
[0470] In one mode, the time period is selected from a range where maximum and minimum values are selected from previous values. For example, the time period can be a value in the range of 20 to 150 days.
[0471] The time for release can start from the time in which the hydrogel holding the component is prepared. Alternatively, the time period can start from the time when the hydrogel becomes available at a target location. In an additional modality, the time period can start from the time in which the hydrogel network is interrupted, for example, through exposure to a competitive guest, reducing agent or other external stimulus.
[0472] As noted earlier, the hydrogels described here are capable of maintaining a component. This component can be released from the hydrogel as and when needed. The present inventors have found that changes to a hydrogel composition can be used to change the timing of component release from a hydrogel. Here, timing can refer to the rate at which the component is released, and additionally, timing can refer to the change in that rate over time.
[0473] The present inventors have found that the hydrogels of the invention allow the release of the component for a period of hours, days, weeks or months. The inventors have found that the material can be released at a substantially constant rate during such a period of time. This can be referred to as a sustained release of the component from the hydrogel.
[0474] Typically, a sustained release of the component is obtainable when the total amount of polymer present in the hydrogel is 1.0%, in feet, or greater, 1.1% by weight, or greater, 1.2%, in weight, or greater, or 1.5%, by weight, or greater.
[0475] Typically, a sustained release of the component is obtainable when the component has a molecular weight of at least 20,000, or at least 50,000.
[0476] The inventors have established that the component can also be released with changes in the release rate over time. In a fashion, a proportion of the component can be released over a first period of time at a first release rate, followed by the release of the component over a second period of time at a second rate. In one embodiment, the first release rate is greater than the second release rate. When the first release rate is higher, the release can be referred to as a quick release. The first and second time periods can be periods of hours, days, weeks or months.
[0477] Typically, a quick release of the component is obtainable when the total amount of polymer present in the hydrogel is less than 1.0% by weight, 0.9% by weight or less than 0.7% by weight , or less, or 0.5% by weight, or less.
[0478] Typically, a sustained release of the component is obtainable when the component has a maximum molecular weight of 15,000 or a maximum of 10,000.
[0479] In one embodiment, a component is released without the application of an external stimulus to the hydrogel. Therefore, a hydrogel can be placed in a desired location and the component is simply allowed to leach from the hydrogel.
[0480] In other modalities, the release of the component can be associated with at least partial decompression of the network. This decompression can be initiated by applying an external stimulus from the hydrogel. Examples of decompression techniques are described above in the Complex section, and include the use of competitive guest molecules to interrupt the network, and the oxidation or reduction of a guest molecule, for example, using an oxidizing or reducing agent, as appropriate. Covalent bonds and crosslinks
[0481] As previously described, the hydrogel network includes polymers that are linked or cross-linked by non-covalent bonding. For example, a host, such as CB [8], can be used as a "handcuff" to maintain guest first and second molecules from the same or different polymers in a ternary complex.
[0482] Alternatively or in addition to non-covalent bonds, the polymers can be linked or cross-linked by covalent bond. The presence of covalent bonds in a network can provide a hydrogel having greater resistance in relation to those networks that are formed only from non-covalent interactions. In one embodiment, covalent bonds are formed between the guest molecules of the polymers.
[0483] The present inventors have found that the formation of covalent bonds between the building blocks can be achieved through a guest-host intermediary. Therefore, in a first step, a supramolecular polymer can be formed when a ternary complex having a host non-covalently maintains the first and second guest molecules from the same or different polymers. The first and second guest molecules are allowed to react, to thereby form a covalent bond linking the polymers. A supramolecular polymer is a polymer where two polymers are held together by a non-covalent complex.
[0484] In preferred embodiments of the invention, the host in the ternary complex has a cavity consisting of a channel through the molecule. There, guest molecules can enter the cavity from one of a plurality of channel openings. For example, cucurbituryl compounds, such as CB [8], have two openings to a central cavity and each opening is accessible.
[0485] Hosts having a through channel can accommodate two guests in a ternary complex in a head-tail or head-head arrangement. In the head-to-head arrangement, the two guests occupy the same opening. In the head-tail arrangement, the two guests entered different openings in the host.
[0486] In one mode, guests are kept in a head-tail arrangement within the host's cavity. Next, the formation of a covalent bond between the guests traps the host in the joined polymers. The host can continue to connect non-covalently to the guest formed by the reaction of the first and second guests.
[0487] Polymers, like the first and second polymer guests, react in response to an external stimulus, such as light, heat or a change in pH. In one embodiment, the reaction is initiated by irradiation of light, for example, irradiation of UV light.
[0488] The first and second guests can participate in a pericyclic reaction, in order to form a covalent bond.
[0489] The first and second guest molecules can participate in a cycloaddition reaction, to thereby form a covalent bond. For example, the cycloaddition reaction can be a cycloaddition reaction [4 + 4] or [2 + 2].
[0490] In one aspect, a method of covalently bonding or crosslinking a polymer is provided, the method comprising the steps of:
[0491] (i) providing a non-covalently bonded polymer or polymers, wherein the non-covalent bond is formed from a ternary complex of a host maintaining the first and second guest molecules provided in the polymer or polymers;
[0492] (ii) allowing the polymer or polymers to react, to thereby form a covalent bond linking the polymer or polymers.
[0493] Therefore, in one aspect, a method is provided for preparing a hydrogel having a crosslinked supramolecular network, in which the hydrogel is formed from the covalent crosslinking of a polymer and / or the covalent bonding of a polymer to another polymer , the method comprising the steps of:
[0494] (i) providing a hydrogel having a crosslinked supramolecular network that is obtainable from the ternary complexation of an aqueous composition comprising a host, such as cucurbituryl, and one or more polymers having suitable guest functionality, such as functionality cucurbituril guest;
[0495] (ii) allowing the polymer or polymers to react, to thereby form a covalent bond linking the polymer or polymers.
[0496] In one embodiment, step (i) provides a hydrogel having a crosslinked supramolecular network obtainable from the complexation of an aqueous composition comprising cucurbituril and one or more polymers having a suitable cucurbituryl guest function, in which one or each polymer has a molecular weight of 50 kDa or greater.
[0497] In step (ii), it is not necessary for all polymers, such as the first and / or second polymers, to react. The product can retain some ternary complexes when the host maintains the first and second guest molecules.
[0498] In one embodiment, the first and second guest molecules are kept in a head-tail arrangement in the host's cavity.
[0499] In one embodiment, the first and second guest molecules are capable of participating in a cycloaddition reaction.
[0500] In one embodiment, the first and second guest molecules are the same.
[0501] In one embodiment, the hydrogel provided in (i) is obtainable from the ternary complexation of an aqueous composition comprising a host, such as cucurbituril, and a polymer, which has first and second guest molecules, which can be the same or different.
[0502] In one embodiment, each of the first and second guest molecules includes an anthracene compound. As shown, two anthracene-containing guest molecules maintained by a host in a ternary complex can be subjected to a cycloaddition reaction, to thereby form a covalent bond between the guest molecules. The product formed from the reaction of the first and second guest molecules can be referred to as the addition product.
[0503] In one embodiment, each of the first and second guest molecules includes a cinnamic acid compound.
[0504] The addition product can become a guest that is kept non-covalently in a binary complex with the host. Therefore, the addition product can be retained within the host cavity.
[0505] It will be assessed that the addition product and the host can separate (dissociate). This does not result in the loss of structural integrity to the network. The formation of the covalent bond between the first and second guest molecules provides a bond between polymers. Therefore, the host is no longer needed to bind polymers.
[0506] In practice, the dissociation and movement of the host from the addition product may be limited. The formation of the addition product effectively contains the host in the crosslinked polymers, and its movement can be limited or prevented by structural and functional resources of the addition product, or other resources of the polymer.
[0507] The formation of a covalent bond between the first and second guest molecules produces a unique guest, and a resulting complex can be referred to as a binary complex.
[0508] It is not necessary for the first and second covalently linked guest molecules to have a high constant of association. Once the covalent bond is made, there is no need for the host to bond non-covalently to the addition product: the covalent bond provides a structural link between the polymers that will not dissociate, and it is no longer necessary for the host maintain the integrity of the bond between the polymers.
[0509] In one embodiment, the reaction is a reaction initiated by light or heat.
[0510] The light can refer to UV or visible light. Heat refers to a reaction temperature that is above the reaction temperature for the preparation of the reticulated supramolecular network. Heat can refer to a reaction temperature higher than the ambient temperature. Heat can refer to a reaction temperature of 50 ° C or higher, 60 ° C or higher, or 70 ° C or higher.
[0511] The network is formed from the covalent crosslinking of a polymer and / or the covalent connection of one polymer to another polymer, thus forming the network. Other preferences
[0512] Each and every compatible combination of the modalities described above is explicitly revealed, as if each and every combination were individually and explicitly mentioned.
[0513] Various additional aspects and modalities of the present invention will become apparent to those skilled in the art in view of the present disclosure.
[0514] "and / or" according to the use in question must be taken as specific disclosure of each of the two specific resources or components with or without the other. For example, "A and / or B" should be taken as a specific disclosure for each (i) A, (ii) B and (iii) A and B, as if each were individually presented.
[0515] Except where the context indicates otherwise, the resource descriptions and definitions presented above are not limited to any particular aspect or modality of the invention and apply equally to all aspects and modalities described.
[0516] Certain aspects and modalities of the invention will now be illustrated by way of example and with reference to the figures described above. Experiments and results
[0517] 1H NMR spectra (400 MHz) were recorded using a Bruker Avance QNP 400. Chemical shifts are recorded in ppm (δ) in D2O with an internal reference set to δ 4.79 ppm. An ATR FT-IR spectroscopy was performed using a Perkin-Elmer Spectrum 100 series FT-IR spectrometer equipped with a universal ATR sampling accessory. UV-VIS studies were conducted on a Varian Cary 4000 UV-Vis spectrophotometer. A gel permeation chromatography (GPC) in water (H2O) was performed on a Shodex glucose column with a prominent Shimadzu SPD-M20A diode array detector, an Optilab refractive index detector and a dispersion detector dynamic light (both Wyatt). The samples were filtered through 0.2 mm PVDF filters before injection using a flow rate of 0.6 ml / min.
[0518] ITC titration experiments were performed in a VP-ITC available from Microcal Inc. at 25 ° C in 10 mM sodium phosphate buffer (pH = 7). In a typical experiment, the host was in the sample cell at a concentration of 0.1 mM, and the guest was in the syringe at a concentration 10 times higher. In the case of functional polymers, the concentration used is determined from the concentration of functional monomeric units in solution and not from the concentration of polymer. A titration consisted of 29 consecutive injections of 2-10 mL with intervals of at least 300 s between injections. The first data point was removed from the data set before the curve fit. The dilution heats were verified by titration as being well beyond saturation or by titration of the guest in a buffer solution and subtracted from the standard enthalpies, but relatively small in all cases. The data were analyzed using the Origin 7.0 software, using a set of site models.
[0519] A rheological characterization was performed using an ARES-LC controlled voltage rheometer equipped with a water bath set to 25 ° C. The voltage sweep measurements (dynamic oscillatory voltage amplitude) were performed at a frequency of 10 rad / s. Frequency sweep measurements were performed at a voltage range of 5%. The temperature scan was performed on a temperature ramp from 25 to 75 ° C at a rate of 10 ° C / min and performed at a voltage of 5% and 10 rad / s. All measurements were performed using a 25 mm parallel plate geometry adjusted to a span height of 0.75 mm and analyzed using the TA Instruments TA Orchestrator software.
[0520] Low-angle scattering measurements of neutrons were carried out on D11 at the Institut Laue Langevin (ILL) (Grenoble, France). A wavelength (À) of 10 A and any of the two configurations were used to cover a q range from 4.4 x 10-3 A-1 to 3.1 x 10-1 A-1, where q is the module scattering vector. The samples were measured in 1 mm quartz cells using D2O as a solvent and the data were recorded in a thermostatically controlled rack at 25 ° C. The scattering from each sample was corrected for electronic background, dead time detector, scattering from the empty cell and sample transmission. The intensity was converted to the cross-section of differential scattering in absolute units (cm-1) using scattering from a water sample. Data reduction was performed using the Lamp, Large Array Manipulation Program software (http://www.ill.fr/data_treat/lamp/lamp.html; D. Richard, M. Ferrand and GJ Kearley, J. Neutron Research 4 , 33-39, 1996).
[0521] Scanning electron microscopy (SEM) images were obtained using a Leo 1530 variable pressure SEM and an InLens detector. SEM samples were prepared by direct freezing of supramolecular hydrogels in liquid nitrogen followed by lyophilization. The resulting cryo-dry materials were depicted in images after ion bombardment. It was not possible to capture SEM images of cryo-dried samples with lower loading (<0.5% by weight) since the hydrogel structures did not survive the lyophilization process and collapsed due to their high water content.
[0522] Hydroxyethylcellulose (HEC) was purchased from Aldrich and dried overnight in a vacuum oven at 105 ° C. Poly (vinyl) alcohol (PVA, 98% hydrolyzed) was purchased from Aldrich, dissolved in water by 5% by weight, precipitated from a 1: 1 solution of acetone and methanol and dried overnight. another at 60 ° C. MVNCO and cucurbit [8] urine were prepared according to literature procedures (Biedermann et al. Macromolecules 2011, 44, 4828-4835; Kim et al. J. Am. Chem. Soc. 2000, 122, 540-541) . All other materials were purchased from Aldrich and used as received. General synthetic protocol
[0523] A cellulose-based framework, hydroxyethyl cellulose (HEC), was easily functionalized using commercially available 2-naphthyl isocyanate (Np) in a single step reaction performed at room temperature in N-methylpyrrolidone using dibutiltine dilaurate ( TDL) as a catalyst (as previously described). A viologene unit containing a reactive isocyanate group, prepared according to an easy preparation in the literature (Biedermann et al. Macromolecules 2011, 44, 4828-4835), was conjugated to commercially available poly (vinyl) alcohol (PVA) under conditions similar. These synthetic protocols are easy, fast and easily scaled. Synthesis of HEC-Np
[0524] HEC (1.00 g) was dissolved in N-methylpyrrolidone (NMP, 150 ml) at 110 ° C. The solution was cooled to room temperature and Np-NCO (29.7 mg, 0.18 mmol) and dibutyltin dilaurate (3 drops) were added and the mixture allowed to stir at room temperature overnight. The functional polymer was then purified by precipitation from acetone, filtered, and dried overnight under vacuum at 60 ° C (1.01 g, 98%). 1H-NMR spectroscopy (MeOD, 500 MHz) d (ppm) = 7.99-7.29 (7H, br, Np-H), 4.60-2.75 (455H, br, cellulose backbone). Elementary: Found C, 47.14; H, 6.93; N, 0.68. C85.5H144.2O60.6N1 required C, 47.63; H, 6.74; N, 0.65. FT-IR (ATR) n = 3410 (br), 2950 (br), 2910 (br), 1395, 1075 (s) cm-1. GPC (H2O): Mn (PDI) = 3.4 MDa (1.25).
Synthesis of PVA-MV
[0525] PVA (1 g, Pm 195 kDa) was dissolved in N-methylpyrrolidone (NMP, 60 mL) and MV-NCO (0.63 g, 1.13 mmol) was added together with dibutiltin dilaurate (3 drops ) and stirred overnight at room temperature. The functional polymer was then purified by precipitation from ethyl acetate, filtered, and dried overnight under vacuum at 60 ° C (1.55 g, 95%). 1H-NMR spectroscopy (D2O, 500 MHz) d (ppm) = 9.18-8.88 (4H, br, MV aryl-H), 8.60-8.33 (4H, br, MV aryl-H) , 4.51-4.45 (2H, br, MV-CH2), 4.38 (3H, s, MV-CH3), 4.20-4.05 (3H, br, MV-CH2-CH2-OCN - and -NCO-CH from the main chain), 3.21-3.08 (4H, br, -CH2- NCO-), 1.95-1.32 (48H, br, polymer main chain and binder hexamethylene). Elementary: Found C, 49.77; H, 7.66; N, 3.24. C61H106O23N4B2F8 required C, 50.98; H, 7.43; N, 3.90. FT-IR (ATR) n = 3320 (br), 2920 (br), 2900 (br), 1715, 1690, 1580, 1450, 1290, 1060 (s), 820 cm-1. GPC (H2O): Mn (PDI) = 1.5 MDa (1.26).
General hydrogel preparation
[0526] Hydrogels were first prepared by dissolving HEC-Np (5 mg) in water (0.5 mL) with stirring and gentle heating. PVA-MV (0.1 mg) and CB [8] (0.1 mg) were then dissolved in water (0.5 ml) with some sonication (less than 5 minutes). The solutions were then mixed and stirred for approximately 1 s before hydrogel formation.
[0527] Isothermal titration calorimetry (ITC) was used for the quantitative study of the respective binding thermodynamics for HEC-Np and PVA-MV with CB [8] (see Reczek et al. J. Am. Chem. Soc. 2009, 131, 2408-2415; Appel et al. J. Am. Chem. Soc. 2010, 132, 14251-14260; Biedermann et al. Macromolecules 2011, 44, 4828-4835; Heitmann et al. J. Am. Chem 2006, 128, 12574-12581). The complete thermodynamic data for the connection of the second guest based on the concentration of functional units (that is, not on the polymeric concentration) are shown in Figure 5. ITC measurements were performed in PVA-MV with a monovalent small molecule 2-naphthol with HEC-Np in the presence of CB [8] in order to identify the effect on binding resulting from both the steric impediment and the explicit structure of the polymeric main chains. Similarly, measurements were performed on HEC-Np with a monovalent small molecule (M2V) viologene. No significant difference in binding constants was observed between the polymeric entities and their corresponding small molecules, identifying that there are no observable effects on binding from steric hindrance. Table 1 - Thermodynamic data for connection of second guest of HEC-N and PVA-MV
a Mean values measured from at least three ITC experiments at 25 ° C in 10 mM PBS buffer at pH 7.0. b Gibbs free energy values calculated from Ka values. c Entropic contributions to ΔG calculated from Ka and ΔH values
[0528] Simple mixing of a solution of HEC-Np (0.5% by weight) with a solution of PVA-MV (0.1% by weight) containing a 1: 1 loading of MV: CB [8 ] (PVA-MV @ CB [8]) instantly produced a slightly orange transparent hydrogel. The orange color is inherent in the MV: Np: CB [8] ternary complex and is a product of the charge-transfer complex between the MV and Np portions within the CB cavity [8]. There is a clear dependence on hydrogel formation in the presence of all three components of the ternary complex since only the system with Np, MV and CB [8] forms hydrogels. An absence of any of the components, or the addition of CB [7] (whose cavity is only large enough to encapsulate MV alone) instead of CB [8] does not produce hydrogels.
[0529] The frequency-dependent rheological characterization of a PVA-MV @ CB [8] titration in an aqueous solution of HEC-Np (0.5% by weight) is shown in Figure 2 (upper row, extreme end to right). The addition of only 0.05%, by weight, of PVA-MV @ CB [8] is required for hydrogel formation and the relative fillings of HEC-Np to PVA-MV @ CB [8] can produce materials with a wide range of mechanical properties. The voltage-dependent oscillatory rheology (Figure 2, upper row, extreme left) shows an extremely wide linear viscoelastic region, indicating that these materials have a wide processing region. Only at a load greater (1.5% by weight) than a deviation from linear viscoelasticity is observed since a rupture of the hydrogel structure at stress amplitudes greater than 10% produces a great reduction in oscillatory shear modules and complex viscosity.
[0530] The frequency dependence of the oscillatory shear modules for storage and loss (G 'and G' ', respectively) clearly identifies the hydrogel-like behavior since the two are linear and parallel and G' is dominant throughout the observed frequency range (Figure 2, top row, extreme right). In general, these hydrogels are soft (G '= 0.5 kPa at 1.5% by weight of HEC-Np loading) and exhibit linear' shear thinning 'behavior, even though the range of materials produced be highly elastic (tan δ = 0.3). Highly elastic materials (tan δ = 0.26) are obtained even at an extremely high water content (99.7%). In addition, viscosity and mechanical properties can be adjusted to two orders of magnitude (Figure 2, upper, middle and right tiers, respectively). The temperature-dependent rheological behavior was characterized by up to 75 ° C (Figure 2, bottom row, left) and shows a reduction in material properties as part of the mechanical integrity is lost (Tan δ = 0.26 to 0.4) . The association constant of the cross-links of the ternary complex decreases with temperature links, leading intuitively to a reduction in the properties of bulk material.
[0531] Pitch rate measurements were performed in order to research the recovery of hydrogel material properties after deformation. A high magnitude shear rate (V = 500 s-1) was applied to disrupt the hydrogel structure, followed by a low magnitude shear rate (V = 0.05 s-1) to monitor the rate and extent to recover the properties of bulk material. Figure 2f clearly demonstrates the complete and exceptionally rapid recovery of viscosity. The rate and extent of recovery remain unchanged for several cycles of rupture and reform, highlighting the reversible nature of the non-covalently cross-linked hydrogel structure.
[0532] So, in summary, the voltage-dependent oscillatory rheology (see also Figure 9a) exhibits an extremely wide linear viscoelastic region and only at a higher load (1.5% by weight) than a deviation from viscoelasticity linear is observed. The frequency dependence of the oscillatory shear modules for storage and loss (G 'and G' ', respectively) clearly identifies the hydrogel-like behavior since the two are linear and parallel and G' is dominant throughout the observed frequency range (Figure 9c). In general, these hydrogels are soft (G '= 0.5 kPa at 1.5% by weight of HEC-Np loading) and exhibit linear' thinning under shearing 'behavior, even though they are highly elastic (tan δ = 0.3).
[0533] The structure of hydrogels formed in 0.5% by weight of HEC-Np loading was characterized by scanning electron microscopy (SEM) and neutron scattering at low angle (SANS). Figure 3 shows a great dependence on the microstructure observed for the two cryo-dried and lyophilized samples in the relative loading of HEC-Np and PVA-MV @ CB [8]. The higher load of PVA-MV @ CB [8] shown in Figure 3a with the same load of HEC-Np (0.5% by weight) produces much smaller pores than the analog hydrogel in Figure 3b. This is presumably due to the higher crosslink density and is in line with previously observed trends (Appel et al. J. Am. Chem. Soc. 2010, 132, 14251-14260).
[0534] SEM measurements were also captured for a hydrogel comprising 1.5% by weight of 0.3% HEC-Np by weight of PVA-MV and 0.3 eq. CB [8] (see Figure 10). SANS data analysis
[0535] The SANS experiments were conducted using the D11 instrument in the Institut Laue-Langevin high flow reactor in Grenoble, France. Neutrons are sensitive to the nm range of length scales, so a more refined image of the molecular structure of the hydrogel can be obtained in relation to that probed by SEM. The scattering data for the hydrogels is superimposed, showing no major changes in the nanostructure with the amount of PVA-MV @ CB [8]. The data can be appropriately described by a combination of the Debye-Bueche and Ornstein-Zerniche models that are widely used to consider the spread of gels and polymeric solutions (Benguigui et al. Euro. Phys. JB 1999, 11, 439-444; Horkay et al. Polymer 2005, 46, 4242-4247). The extent of correlation E, could be adjusted to values around 200 A (± 30 A), which is preferably compared to the values reported for polymeric gels, probably considering the exceptionally low loading of polymer material compared to previously reported systems. , also according to the weft size calculated based on the distances between the guest portions along a well solvated extended polymeric chain. The correlation extent of the frozen structure, -, could adopt a wide range of values, between 500 and 1000 A. A highly excess scattering was observed at low q, which followed a Porod law q-4, typical of an acute interface , and suggesting the presence of very large inhomogeneities in the sample. Therefore, scattering may not be fully described by the Debye-Bueche and Ornstein-Zerniche models and a power law (q-4) was necessary to consider the complete scattering curve. These inhomogeneities are probably the result of the insoluble pulp of the cellulose since the samples were not filtered before the scattering measurements.
[0536] For gels composed of flexible polymer chains, the spreading intensity is generally described by the combination of two terms, a com-ponentedinamic and a static component (see Horkay et al. Macromolecules 1991, 24, 2896-2902; Pezron et al Polymer 1991, 32, 3201-3210). The dynamic term follows an Ornstein-Zernike law, regarding the semi-diluted polymeric solutions in a good solvent, leading to a Lorentzian form of the scattering function given by:

[0537] where IL (0) is the structural factor extrapolated to zero qe E, a thermal correlation term, which can be assimilated to a 'weft size' of the gel network. The second term arises from frozen concentration fluctuations causing an excess spread in low q and is described by the Debye-Bueche term:

[0538] IDB (0) is the structural factor extrapolated at q = 0 and - the size of inhomogeneities. This term considers a medium of two densities with an acute interface. The combination of the Ornstein-Zernike (Lorentzian) and Debye-Bueche models provides the spreading intensity I (q) as follows: / (< ) = I ( ) L + J (<7) DS (3) Ability to respond to hydrogel stimuli
[0539] Hydrogels prepared according to the previous preparation method were placed in a small bottle. In the case of liquids, such as toluene or hexane, a volume approximately equivalent to the top of the hydrogel was added and the bilayer system vortexed for 10 seconds. In the case of solids, such as 2,6-dihydroxy naphthalene and sodium dithionite, an excess (typically 3 equivalents) was added as a solid to the top of the hydrogel and the system mixed with a vortex for 10 seconds.
[0540] The ternary complex CB [8], in addition to providing a means for the preparation of self-assembling hydrogels, also provides an adjustable response capacity inherent to stimuli to the resulting materials. These hydroels are highly sensitive to specific external stimuli, including second competitive guests and reduction conditions. The addition of an excess of a competitive guest per mixture, i.e. 2,6-dihydroxy naphthalene or an equivalent volume of a solvent, such as toluene, leads to the dissociation of the polymeric network and the complete loss of aggregate mechanics. In the case of toluene, the partitioning of the toluene from the aqueous layer after laying causes hydrogel reform. On the other hand, when hexane is added instead of toluene, no change in hydrogel properties has been observed (hexane is not a suitable second guest for the CB ternary complex [8]). In addition, dilution with an equivalent volume of H2O reduced the mechanical properties only slightly (see Figure 2).
[0541] Another important advantage of this system is the reduction of MV electrons, which reversibly disrupts the ternary complex due to the specific complex formation of 2: 1 MV +: CB [8] (see Lee et al. Chem. Commun. 2002, 2692 -2693; Coulston et al. Chem. Commun. 2011, 47, 164-166). Therefore, the addition of sodium dithionite (a good reducing agent for MV) produces a low viscosity solution. Toxicity studies
[0542] NIH 3T3 cells and cell media were cultured in DMEM containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin. The cells were developed as a monolayer and passed through the confluence using trypsin (0.5%, w / v in PBS). The cells were collected from the culture by incubating in trypsin solution for 10 minutes. The cells were centrifuged and the supernatant discarded. DMEM supplemented with serum (3 ml) was added to neutralize any residual trypsin. The cells were resuspended in DMEM supplemented with serum at a concentration of 2 x 104 cells / mL. The cells were grown at 37 ° C and 5% CO2.
[0543] The toxicities of the hydrogel constituent polymers were assessed by determining their ability to affect the proliferation and viability of 3T3 cells cultured in DMEM. The polymers were incubated in 24-well multiplates in 1 x 104 cells per well for 24 hours at 37 ° C in 500 μL of medium. Different cell viability was assessed using the MTT assay on 3T3 cell lines. Here, 10 mL of filtered MTT stock solution sterilized in PBS (5 mg / mL) was added to each well, reaching a final MTT concentration of 0.5 mg / mL. After 5 hours, the unreacted dye was removed by aspiration. The formazan crystals were dissolved in DMSO (100 mL per well), and the absorbance was measured using a microplate reader at a wavelength of 570 nm. Cell viability (%) = [A] test / [A] control x 100%, where [A] test is the absorbance in the wells with polymers and [A] control is the absorbance in the control wells. All experiments were conducted with six repetitions and averaged. The control group consists of cells incubated without polymers and cultured in DMEM.
[0544] In vitro cytotoxicity studies are of paramount importance when considering any biomedical application for these hydrogels and were performed using 3T3 cells. Toxicity studies were performed using only the polymeric constituents in order to maximize availability to the cells and limit the increases in viscosity due to the formation of hydrogel in the presence of CB [8]. A recent in vivo and in vitro toxicity study of cucurbit [n] urils demonstrated the bio-compatibility and extremely low toxicity of macrocyclic hosts (Uzunova et al. Org. Biomol. Chem. 2010, 8, 2037-2042). The cytotoxicity of the polymers was tested in various concentrations ranging from 0.1 to 50 mg / mL and the quantification of the cytotoxic response was performed using the MTT assay. In general, polymers do not show significant toxicity (Figure 11). In addition, the cytotoxicity of the leachable products of the copolymer gel was assessed by incubating the gel in the cell culture medium for a period of 30 days at 37 ° C to simulate the actual conditions of use. The quantification of the cytotoxic response occurred through the MTT assay. The aqueous extracts of the polymers do not show significant cytotoxicity in relation to 3T3 cells, regardless of the incubation length. There has been concern about the use of TDL as a catalyst in the polymer preparation described herein, particularly when TDL is a known cytotoxic chemical. However, it has been shown that at very low concentrations (1 ppm), TDL does not obtain a cytotoxic response against the fibroblast cells of L929 mice (Loh et al. Biomaterials 2007, 28, 4113-4123). From these studies, the polymeric constituents are not cytotoxic and the resulting hydrogels are expected to be safe for bio-medical applications. Studies of protein release from hydrogels
[0545] The aqueous solutions of 1 and 3% by weight of HEC-Np were mixed and allowed to equilibrate overnight at room temperature. Appropriate amounts of lysozyme or BSA solutions were loaded to a predetermined concentration of lysozyme or BSA in the polymeric solution. The aqueous solution of PVA-MV (0.2 and 0.6% by weight) and CB [8] (0.2 and 0.6% by weight) was then prepared. In a typical example, 0.5 ml of a polymeric solution of HEC-Np loaded with protein was injected into a sample vial and 0.5 ml of a solution of PVA-MV @ CB [8] was added and the mixture stirred for approximately 1 s until the hydrogel is formed. The sample bottle was then placed in 7 mL of phosphate buffer release solutions in a test tube, which was incubated and shaken at 50 rpm in a water bath equilibrated at 37 ° C. The buffer solutions were replaced with new solutions at predetermined time intervals, and the experiments were carried out in triplicate. The collected buffer solutions were lyophilized and kept at -80 ° C for further analysis. Lysozyme and BSA levels were determined using the Pierce BCA Protein Assay kit. Lysozyme E BSA quantization was based on a calibration curve, obtained using the new lysozyme and BSA standards, in the range of 20 to 2,000 mg / mL (see Loh et al. J. Mater. Chem. 2011, 21, 2246 -2254).
[0546] The low polymer concentration and high water content of the gels of the invention are highly attractive for biomedical applications due to improved biocompatibility. Several methods for the preparation of protein loaded samples have been studied. Two therapeutic model protein treatments were chosen for this study, Serum Bovine Albumin (BSA) and Lysozyme, due to their differences in size, providing information on the release of materials over a wide range of molecular sizes. Since the hydrogels self-assemble quickly at room temperature, it was possible to dissolve the protein with one of the polymeric solutions before mixing and hydrogel formation in situ. However, it was determined that the protein could be easily mixed with preformed hydrogel simply by mechanically stirring the protein as a solid in the hydrogel. Protein-loaded gels were formed and incubated at 37 ° C. This form of protein loading method minimizes the risk of protein denaturation as it does not expose the protein to high temperatures or organic solvents during the formulation process. In this study, two factors that affect the release of protein were studied, (1) the effect of polymer concentration (0.5% by weight, versus 1.5% by weight) and (2) the effect of molecular weights of protein (BSA, Pm: 67,000 g mol-1 against lysozyme, Pm: 14,000 g mol-1).
[0547] The proteins were released continuously and their release profiles are shown in Figure 12. For all curves except 0.5% by weight of BSA-loaded gel, an initial rapid release occurs before constant release is observed. When the polymer concentration is high, the protein release rate is reduced and a more sustained release is observed. The rapid release of 0.5% by weight of gel released approximately 50% of the BSA protein within the first week, while the 1.5% by weight of gel managed to suppress this effect by approximately 10%. In addition, BSA is a protein with a molecular weight of about 67,000 g mol-1 while lysozyme has a molecular weight of about 14,000 g mol-1. For BSA, the protein release rate is slower compared to lysozyme and a more sustained release is observed. The quick release is also effectively suppressed and the duration of the sustained release is extremely promising. Previously, the extremally sustained protein release profile of up to 80 days was demonstrated by the polyl thermogels (PEG / PPG / PHB urethane) reported by Loh et al. (Loh et al. Biomaterials 2007, 28, 4113-4123). When the protein is large, the release rate becomes correspondingly slower as the mobility of the protein from the gel is reduced. The release profile of all polymers can be adjusted to the following RitgerPeppas equation for drug release in the range of M t / M ~ = 0.6 (see Ritger et al. J. Controlled Release 1987, 5, 23; Ritger et J. Controlled Release 1987, 5, 37).
Protein bioactivity studies
[0548] BSA esterase activity was determined by following the formation of p-nitrophenol from the synthetic substrate of p-nitrophenyl acetate at 400 nm using a spectrophotometer. The reaction mixtures contained 50 μM of p-nitrophenyl acetate and 20 μM of proteins in 0.1 M of phosphate buffer, pH 7.4 at 37 ° C. A molar extinction coefficient for p-nitrophenol of ε = 17,700 M-1 cm-1 was used for all calculations.
[0549] The activity of lysozyme released from the gel was determined using an EnzChek Lysozyme Assay kit (RTM) (Molecular Probes, E-22013). The experimental protocols were performed according to the instructions provided in the kit.
[0550] The preservation of BSA activity is of paramount importance for any biological application involving gels. The biological activities of the BSA and Lysozyme materials when administered from the hydrogel are shown in Figure 13. The evaluation was carried out using well-established activity tests. The control experiments were carried out, in this way, the proteins were kept in solutions buffered by the analogous time frames throughout the administration. It is observed that BSA maintains more than 80% of its original activity when released from the gel even after 50 days, while only 2% of the activity is retained without a gel encapsulation. These results imply that BSA maintains most of its activity when retained in a gel structure, which is a reaction of the retention of the native structure by BSA. Additional experimental results - Vinylbenzene polymers
[0551] The additional experimental study described here shows that the use of a polymer having a higher molecular weight, such as greater than 50 kDa, can be used to generate hydrogels.
[0552] The guest portions used in the polymers are the natural and commercially available amino acids phenylalanine and tryptophan. The use of these amino acids reduces the potential toxicity profile of the system, but also simplifies the hydrogel from a three-component system to a two-component system (see Figure 14). The notorious stimulus-responsive nature of the ternary complex and the ease of synthesis of the various components makes this system well suited for a variety of important biomedical and industrial applications.
[0553] The analytical and preparation techniques described above in relation to cellulose polymers have also been used to analyze and prepare the vinylbenzene polymers described below.
[0554] The study below shows that amino acids can be used as guests in a host, such as cucurbituril.
[0555] Water-soluble styrenic monomers were copolymerized with aromatic amino acid monomers synthetically derived from phenylalanine and tryptophan. The resulting polymers are shown to form dynamic, self-healing, physically cross-linked hydrogels through the recognition and binding of amino acids to cucurbit [8] urine. The study is described in detail below. The polymers used in this study had molecular weights of 10.9 kDa and 12.1 kDa. It was found that the hydrogels produced have rheological properties where it is observed that the loss module (G '') dominates the storage module (G ') at higher frequencies. The hydrogels of the present invention typically employ a polymer having a molecular weight that is 50 kDa or greater. It was found that the use of these polymers is associated with a hydrogel product having a dominant storage module (G ') over the loss module (G' ') for any frequency value in the range of 0.1 to 100 rad / s.
[0556] The polymers used in this study had a molecular weight less than 50 kDa. As described in this document, hydrogels resulting from complexing polymers do not have the desired rheological characteristics. As such, the experiments can be compared to other examples, which refer to hydrogels that are formed from polymers with molecular weights of 50 kDa or greater. However, it will be appreciated that the hydrogels described in these examples are capable of maintaining an encapsulant and, therefore, have a general use in accordance with the broader aspects of the present invention. Design and synthesis of functional polymers
[0557] Polymers derived from (vinylbenzyl) trimethylammonium chloride are rigid and highly soluble in water due to their cationic charge, making this monomer ideal for copolymerization with guest-functional monomers. Polymeric rigidity is particularly important in order to enhance the hydrogel strength by limiting the intramolecular bonding of the amino acid units in the same polymer chain. Rigid polymers are ideal as they promote the formation of an intermolecular complex, leading to stronger materials.
[0558] For the purpose of copolymerization, a compatible amino acid monomer was also required to guarantee the random distribution of the functional units. Therefore, the synthesis of an amino acid monomer derived from styrene was adopted, as shown below. The coupling of the activated amino acids Boc-L-phenylalanine N-hydroxy succinimide ester and Boc-L-tryptophan N-hydroxy succinimide ester with (4-vinylbenzyl) amine in the presence of triethylamine produced the Boc-protected amino acid monomers ( StPhe, 3a and StTrp, 3b) in good yields a.

[0559] With the monomers StPhe and StTrp under control, 'traditional' free radical copolymerization with trimethylammonium (vinylbenzyl) chloride was performed using cyanopentanoic azobis acid (ACPA). After acid treatment for Boc deprotection, cationic styrenic amino acid copolymers (5a, StPhe-StAm and 5b, StTrp-StAm) were provided as HCl salts, which were highly soluble in water and easily purified by dialysis. The NMR analysis of protons of these copolymers determined that 7% of the monomers were functional with amino acids in both phenylalanine and tryptophan cases (data not shown). Comparing the integration of the aromatic signal (7.5-6.0 ppm) with the integration of the trimethylammonium singlet (2.7 ppm), the excess of aromatic protons in the polymer was determined, which is directly correlated to the number of monomers functional guest incorporated in the final polymers.
Synthesis of tert-butyl (1-oxo-3-phenyl-1 - ((4-vinylbenzyl) amino) propan-2-yl) carbamate (StPhe)
[0560] (4-vinylbenzyl) amine (0.500 g, 3.75 mmol) was added dropwise to a solution of Boc-Phe-OSu (1.770 g, 4.88 mmol) in dichloromethane (DCM, 10 mL) at 0 ° C. Then, triethylamine (0.988 ml, 7.50 mmol) was added dropwise to the cooled solution and the reaction was allowed to warm to room temperature and stirred for 24 hours. The reaction was cooled with a saturated sodium carbonate solution and the product extracted with dichloromethane (DCM) (x 3). The combined organic extracts were dried over magnesium sulfate, filtered and concentrated in vacuo. The crude residue was dissolved again in a 50:50 mixture of hexane and ethyl acetate and washed through a pad of silica. Removal of the solvent in vacuo produced the title compound as a soft amorphous solid which was dried under high vacuum overnight (1.324 g, 93%).
[0561] 1H-NMR spectroscopy (CDCl3, 500 MHz) d (ppm) = 7.35-6.95 (9H, m, Ar-H), 6.73-6.63 (1H, dd, J = 17 , 5 Hz, 10.7 Hz, alkene-H), 6.10-6.02 (1H, br s, N-H), 5.78-5.68 (1H, dd, J = 17.5 Hz , 0.8 Hz, H-alkene), 5.28-5.20 (1H, dd, J = 10.7 Hz, 0.8 Hz, H-alkene), 5.10-4.95 (1H, br s, NH), 4.40-4.37 (3H, m, CH2, CH), 3.16-2.99 (2H, m, CH2), 1.39 (9H, s, CH3). 13C-NMR spectroscopy (CDCl3, 500 MHz) d (ppm) = 170.98 (CO), 155.37 (CO), 137.19 (ArC), 136.87 (ArC), 136.63 (ArC), 136.32 (CH), 129.33 (ArCH), 128.72 (ArCH), 127.86 (ArCH), 126.95 (ArCH), 126.41 (ArCH), 113.95 (CH2), 56 , 08 (CH), 43.19 (CH2), 38.53 (CH2), 28.24 (CH3). Elementary: Found C, 72.39; H, 7.51; N, 7.22. C23H26O3N2 calculated C, 72.60; H, 7.42; N, 7.36. FT-IR (ATR) n = 3339 (m), 2983 (m), 1678 (s), 1658 (s), 1517 (s) cm-1. HRMS: Found 381.2178 [C23H26O3N2] + calculated 381.2190. Synthesis of poly (2-amino-3-phenyl-N- (4-vinylbenzyl) propanamide (vinylbenzyl) trimethylammonium chloride) (StPhe-StAm)
[0562] StPhe (0.498 g, 1.31 mmol), (vinylbenzyl) trimethylammonium chloride (2,500 g, 11.81 mmol) and 4,4'-azobis (4-cyanopentanoic acid) (ACPA, 36.4 mg) they were dissolved in methanol (MeOH, 7 mL) and degassed with nitrogen for 30 minutes. The reaction was heated to 70 ° C and stirred for 24 hours. The product was precipitated with diethyl ether and collected by vacuum filtration. The crude residue was dissolved again in MeOH (20 ml) and 4 N of hydrogen chloride in dioxane solution (20 ml) added by dripping. The reaction was stirred for 8 hours and the unprotected polymer was precipitated with diethyl ether. The crude residue was purified by dialysis in relation to water for 24 hours and the product lyophilized to produce a white amorphous solid (1,774 g, 62%) that had 7% StPhe and 93% StAm.
[0563] FT-IR (ATR) n = 3374 (br), 3014 (m), 2923 (m), 1680 (m), 1614 (m), 1479 (s) cm-1. GPC (H2O): Mn = 10.9 kDa, PDI = 2.2. Synthesis of tert-butyl (3- (1H-indol-3-yl) -1-oxo-1 - (((4-vinylbenzyl) amino) propan-2-yl) carbamate (StTrp)
[0564] (4-vinylbenzyl) amine (0.500 g, 3.75 mmol) was added dropwise to a solution of Boc-Phe-OSu (1.960 g, 4.88 mmol) in dichloromethane (DCM, 10 mL) at 0 ° C. Then, triethylamine (0.988 ml, 7.50 mmol) was added dropwise to the cooled solution and the reaction was allowed to warm to room temperature and stirred for 24 hours. The reaction was cooled with a saturated solution of sodium carbonate and the product extracted with DCM (x 3). The combined organic extracts were dried over magnesium sulfate, filtered and concentrated in vacuo. The crude residue was dissolved again in a 50:50 mixture of hexane and ethyl acetate and washed through a pad of silica. Removal of the solvent in vacuo produced the title compound as a white amorphous solid which was dried under a high vacuum overnight (1.541 g, 98%).
[0565] 1H-NMR spectroscopy (CDCl3, 500 MHz) d (ppm) = 8.07 (1H, s, ArH), 7.70-7.62 (1H, d, J = 7.8 Hz, Ar- H), 7.39-7.31 (1H, d, J = 8.1 Hz, Ar-H), 7.307.20 (1H, m, Ar-H), 7.20-7.15 (1H, ddd, J = 8.1, 6.9, 1.1 Hz, Ar-H), 7.15-7.10 (1H, ddd, J = 8.1, 6.9, 1.1 Hz, Ar -H), 7.00-6.85 (3H, m, Ar-H), 6.71-6.61 (1H, dd, J = 17.7, 10.9 Hz), 6.08-5 , 90 (1H, br s, NH), 5.73-5.65 (1H, dd, J = 17.7, 0.8 Hz, H-alkene), 5.27-5.20 (1H, dd , J = 10.9, 0.8 Hz, H-alkene), 5.27-5.10 (1H, br s, NH), 4.51- 4.37 (1H, br s, CH), 4 , 32-4.18 (2H, m, CH2), 3.39-3.25 (1H, dd, J = 14.3, 5.2 Hz, HC-H), 3.25-3.10 ( 1H, dd, J = 14.3, 7.5 Hz, HCH), 1.41 (9H, s). 13C-NMR spectroscopy (CDCl3, 500 MHz) d (ppm) = 171.52 (CO), 155.45 (CO), 136.74 (ArC), 136.20 (CH2), 136.05 (ArC), 128.02 (ACH), 127.82 (ArC), 127.37 (ArCH), 126.32 (ArCH), 123.19 (ArCH), 123.03 (ArC), 122.35 (ArCH), 119 , 86 (ArCH), 113.92 (CH), 111.18 (ArCH), 110.67 (ArC), 55.31 (CH), 43.21 (CH2), 28.43 (CH2), 28, 27 (CH3). Elementary: Found C, 69.27; H, 6.98; N, 9.44. C25H29O3N3 calculated C, 71.57; H, 6.97; N, 10.02. FT-IR (ATR) n = 3310 (br), 2978 (m), 2930 (m), 1693 (s), 1655 (s), 1494 (s) cm-1. HRMS: Found 420.2303 [C25H30O3N3] + calculated 420.2287. Synthesis of poly (2-amino-3- (3H-indol-3-yl) -N- (4-vinylbenzyl) propanamide-co- (vinylbenzyl) trimethylammonium) chloride (StTrp-StAm)
[0566] StTrp (0.550 g, 1.31 mmol), (vinylbenzyl) trimethylammonium chloride (2,500 g, 11.81 mmol) and ACPA (36.4 mg) were dissolved in MeOH (7 mL) and degassed with nitrogen for 30 minutes. The reaction was heated to 70 ° C and stirred for 24 hours. The product was precipitated with diethyl ether and collected by vacuum filtration. The crude residue was dissolved again in MeOH (20 ml) and 4N of hydrogen chloride in a solution of dioxane (20 ml) added by dripping. The reaction was stirred for 8 hours and the unprotected polymer was precipitated with diethyl ether. The crude residue was purified by dialysis in relation to water for 24 hours and the product lyophilized to produce a white amorphous solid (1.752 g, 60%).
[0567] FT-IR (ATR) n = 3373 (br), 3012 (br), 2921 (br), 1679 (m), 1614 (m), 1478 (s) cm-1. GPC (H2O): Mn = 12.1 kDa, PDI = 2.4. Hydrogel preparation
[0568] Polymeric solutions (20% w / v) were prepared and diluted with equivalent volumes of CB solutions [8] of various concentrations resulting in a final polymer concentration of 10% w / v. The combined solutions were gently heated and stirred for a few seconds before allowing to cool to room temperature, so the hydrogel could settle. Phenylalanine and tryptophan bond within the CB cavity [8] in a 2: 1 manner (see Heitmann et al. J. Am. Chem. Soc. 2006, 128, 12574-12581). Therefore, 0.5 equivalent of CB [8] theoretically produces 100% crosslinking of aromatic amino acid units. Initial CB [8] titrations in polymeric solutions exemplified this well. The polymeric solutions containing 20% by weight of the functional polymer were diluted with equivalent volumes of solutions containing several equivalents of CB [8], gently heated and stirred. As predicted, the addition of CB [8] resulted in large increases in viscosity which was easily observed by inverted flask tests (images not shown). It was observed that without CB [8] the polymeric solution remains transparent and the colorless solution flows. With 0.35 equivalent of CB [8], the solution is much more viscous, but retains the same flow. The addition of> 0: 5 equivalent of CB [8] caused the solution flow to slow down dramatically when flipping the bottle. The addition of 1 equivalent of CB [8] could theoretically favor 100% of the formation of amino acid and CB complexes [8] 1: 1.
[0569] Since strong hydrogels are still formed, this is clearly not the case. As a control, 0.5 equivalent of CB [7] was also added to a polymeric solution. CB [7] is large enough to connect only one Phe / Trp unit, thus unable to promote crosslinking. With the addition of CB [7], no changes in viscosity were observed, therefore, the 1: 1 binding of Phe or Trp to a CB [n] molecule is not constructive for gel formation. As a second non-functional control, poly [(vinylbenzyl) trimethylammonium chloride] (p-StAm) was also synthesized. When adding CB [8] to a 10% by weight control StAm polymer solution, no visible change in viscosity was observed. Therefore, the formation of hydrogel arises exclusively from the 2: 1 bond of the charged amino acids within the CB cavity [8] and the cationic polymeric main chain is not involved in the crosslinking process. Rheological characterization of supramolecular hydrogels
[0570] Supramolecular hydrogels were successfully designed and prepared and rheological characterization was performed to quantify their mechanical properties. The rheological analysis was carried out on supramolecular hydrogels prepared by mixing solutions 5a and 5b with various molar equivalents of CB [8] (in relation to the functionalized amino acid units, for example, 0, 0.05, 0.15 , 0.35, 0.50, 0.70 equivalents) corresponding to various percentages of theoretical percentages. Proceeding beyond the theoretical limit of 100% crosslinking (from 0.5 equivalent of CB [8] to 0.7 equivalent) two results are possible: a) The gel properties, including viscosity, decrease. In the case of phenylalanine, 20% of the amino acid units would be linked in a 1: 1 manner and only 80% of the amino acid units in a 2: 1 manner leading to crosslinking. This occurs due to the first link constant being equivalent to the second. In the case of tryptophan, the second binding constant is weaker than the first and therefore 40% of the amino acids would be linked in a 1: 1 way and 60% in a 2: 1 way (Heitmann et al. J Am. Chem. Soc. 2006, 128, 12574-12581). Therefore, at 0.7 equivalent of CB [8] the actual crosslinking would be 80% or 60% depending on the amino acids used. b) As a 2: 1 bond is more favorable than a 1: 1 bond (this is a higher Keq value for the ternary complex compared to the ternary complex) the excess CB [8] suspended in the solution would not prevent crosslinking as the case 'a', but instead increases viscosity by acting as a solid viscosity modulator. It is also possible that CB [8] in excess in the solution promotes the formation of the ternary complex, simultaneously implying 100% crosslinking and reducing the apparent rate of dissociation of the components. Oscillatory measurements
[0571] Stress-dependent oscillatory shear measurements at 20 ° C were first performed in order to determine the linear viscoelastic properties of the material. During the experiment, all hydrogels were shown to have a wide viscoelastic window and no linearity deviations were observed even at the highest oscillation voltage, except for the StTrp-StAm gels containing 0.7 equivalent (see Figures 18a and 18b). Both materials regardless of CB concentration [8] retained their broad viscoelastic regions. It was observed that, with an increasing CB [8] concentration, the plateau module of the materials increased, the guest portions. Interestingly, the modules for StPhe-StAm are distinctly larger than those of StTrp-StAm gels by an order of magnitude.
[0572] Frequency-dependent oscillatory rheological measurements were also performed on the materials in a linear viscoelastic regime (Figures 15A and 15B). As for voltage-dependent oscillatory measurements, the modules of the StPhe-StAm gels have a greater magnitude than those of the StTrp-StAm materials. It is also beneficial to note that StPhe-StAm gels become elastically active at lower angular frequencies than StTrp-StAm gels. In both cases, the more equivalents of CB [8] added caused not only an increase in storage modules and loss of gel, but also a reduction in the angular frequency at which the storage module (G ') becomes dominant over to the loss module (G ”). With more CB [8] present, at any time there will be more crosslinking between the polymers, allowing the material to behave more elasticly as the supramolecular network is subjected to a penalty for the breakdown of higher energy.
[0573] Since the crossing point occurs at lower angular frequencies with a higher CB [8] concentration, it can be deduced that a greater degree of cross-linking has been achieved and that higher CB [8] concentrations (even beyond 0.5 equivalent) imposes a 2: 1 complexation. This leads to the conclusion that the balance of ternary complex formation does not extend completely towards 2: 1 complex formation. Preferably, it is thought that in this case the balance between the free amino acid units and CB [8] and their respective 1: 1 binary complex extends to the left. By increasing the CB concentration [8], a 1: 1 complex is imposed by the Le Chateliers principle, which then proceeds to form a ternary complex that is energetically favorable. Stable shear measurements
[0574] Stable shear rheological measurements were performed on both StPhe-StAm and StTrp-StAm gels to determine the mechanical effect of varying amounts of CB [8] (Figures 16A and 16B). In both cases, the zero shear viscosity of the materials increased with CB [8] equivalents added, even beyond 0.5 equivalent. Initially, the gels behaved like Newtonian materials without changes in viscosity. At high shear rates some gels with a higher CB [8] loading exhibited a slight degree of thickening under shear as the shear rate increased. All materials exhibited thinning behavior under shear under high shear conditions.
[0575] Considering only the zero shear viscosities, with an increase in the addition of CB [8] both materials StPhe-StAm and StTrp-StAm exhibited an increase as would be expected (Figure 7). While initially at low CB [8] concentrations both materials have a very low and similar viscosity, since in addition to 0.15 equivalent of CB [8], the zero shear viscosity of StPhe-StAm gel increases at a very high rate bigger. This shows that the ternary Phe2 CB complex [8] is much stronger than the Trp2-CB complex [8]. Figure 17 also exemplifies the CB equivalents [8] needed to induce gelation. Extrapolating lines of best fit for each material show gelling points so that the two materials are distinctly different. The StPhe-StAm polymer requires only 0.305 equivalent of CB [8] for gelation to begin while the StTrp gel requires 0.5 equivalent. For the StTrp-StAm polymer, the viscosity is not dramatically changed with the CB [8] concentration until it exceeds the 100% theoretical cross-linking point. This is responsible for the difference in amino acid association constants with CB [8] and how it can affect the present dynamic balance. Additional experimental results - polymers of hyaluronic acid and cellulose
[0576] HA and cellulose polymers were prepared having guest compounds of phenylalanine. The resulting polymers are shown to form physically dynamic and self-healing cross-linked hydrogels through the recognition and binding of the amino acid to the cucurbit [8] urine.
[0577] The molecular weights of the polymers were greater than 50 kDa. The experiments described below show that a hydrogel can be prepared from a biological polymer, such as a polysaccharide polymer, using biological molecules. Therefore, the hydrogel formed has a predominant biological component. Preparation of Azide Phe

[0578] Sodium azide (11 g, 169 mmol) and 2-bromoethylamine hydrobromide (10 g, 49.0 mmol) were dissolved in water (150 mL) and heated at 75 ° C for 24 hours. 50 ml of a 10% NaOH solution was added and the product extracted in diethyl ether, dried over magnesium sulfate and filtered. The product was concentrated to dryness in vacuo to produce 1.69 g of a yellow oil: 2-azidoethylamine.
[0579] The yellow oil was added to a solution of Boc-Phe-OSu (7.115 g, 19.7 mmol), in DMF (200 mL) and cooled in an ice bath. Triethylamine (2.72 ml) was added dropwise and the reaction stirred for 12 hours before cooling with water. The product was extracted into ethyl acetate and concentrated to dryness. The crude residue was dissolved in 20 ml of 2 M HCl in diethyl ether and stirred for 4 hours. The resulting precipitate was filtered and washed with additional diethyl ether yielding the product (phe azide) as a white solid as an HCl salt. Preparation of cellulose polymer - CMC-Phe polymer

[0580] 200 mg of sodium carboxymethyl cellulose (CMC) were dissolved in pH 4 buffer (16 mL). To this solution were added propargyl amine (172 µL, 2.68 mmol), N-hydroxy succinimide (NHS, 187 mg, 1.625 mmol) and N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride (EDC, 312 mg, 1.625 mmol). The reaction mixture was stirred for 24 hours and the intermediate product purified by brine dialysis (24 hours), then water (48 hours).
[0581] The intermediate polymer was isolated by lyophilization. The amorphous product was dissolved again in a 1: 1 mixture of water and ethanol (20 ml). Azide-Phe (73.25 mg, 0.245 mmol) was then added by sodium ascorbate (16.3 mg, 0.0823 mmol) and copper (II) sulfate pentahydrate (21 mg, 0.0823 mmol). The reaction mixture was stirred overnight and the product was purified by dialysis in a solution of sodium hydrogencarbonate (48 hours) and then water (48 hours). The product was isolated by lyophilization.
[0582] The Pm of the polymer was approximately equal to 700 kDa. Preparation of cellulose polymer - HA-Phe polymer

[0583] The polymer was prepared in the same way as the cellulose polymer above, except that carboxymethyl cellulose was replaced by hyaluronic acid (200 mg).
[0584] The Pm of the polymer was approximately equal to 200 kDa. Hydrogel preparation
[0585] The CMC-Phe and HA-Phe polymer hydrogels described above were prepared in a similar manner to the vinylbenzene polymer hydrogels (also described above).
[0586] The CMC-Phe hydrogel was prepared from an aqueous solution comprising 2% w / v of the cellulose polymer and 0.5 equivalent of CB [8]. The formation of the hydrogel was apparent from an inverted flask test (images not shown). In the absence of CB [8], an aqueous solution comprising 2% w / v of the cellulose polymer does not form a hydrogel.
[0587] The HA-Phe hydrogel was prepared from an aqueous solution comprising 2% w / v of the HA-Phe polymer and 0.5 equivalent of CB [8]. The formation of the hydrogel was apparent from an inverted flask test (images not shown). In the absence of CB [8], an aqueous solution comprising 2% w / v of the HA polymer does not form a hydrogel. When CB [7] is used instead of CB [8] (in similar equivalence), the HA polymer does not form a hydrogel. Additional experimental results - anthracene hydrogels
[0588] Cucurbit [8] urine (CB [8]) can accommodate up to two aromatic guest molecules simultaneously in its cavity as shown in Figure 19 (b), to form homoternary complexes CB [8] - (guest) 2 1: 2 with mono-cationic guests or heteroternary complexes 1: 1: 1 with a dicationic guest and a neutral guest. Pre-arranging two portions of anthracene (see Figure 19 (a)) in the CB cavity [8] in a π-π face-to-face pile dramatically increases the rate of photodimerization between the two anthracenes and can be used to bond and cross-link the polymers photochemically. This will be discussed and exemplified below.
[0589] Anthracene molecules were designed to carry a positive charge directly adjacent to the aromatic nucleus in order to allow strong complexation with CB [8]. For steric reasons, substitution of the 9 position of anthracene would prevent the ability to form ternary complexes with CB [8]. Therefore, anthracene derivatives that carry a substituent in position 2 were used. Commercially available 2-amino-anthracene was subjected to reductive amination with paraformaldehyde and sodium cyanoborohydride which proceeded smoothly to produce N, N'-dimethyl-2-amino-anthracene in good yield and high purity without the need for further purification. Subsequently, quaternary ammonium salts were obtained using potent alkylating reagents, such as methyl iodide (to produce 1a) and propagyl bromide (to produce a “clickable” precursor at 1b and 1c) with moderate yields.
[0590] However, the purification (filtration) and ion exchange steps to produce the chloride salt required only minimal effort. The labeling of macromolecules with such portions of anthracene was achieved through accelerated cycloaddition reactions by copper from readily available terminal group functional azidopoly (ethylene glycol) (PEG) and functional side chain azidohydroxyethyl cellulose followed by purification through dialysis. Host-guest complexation and anthracene photodimerization 1a
[0591] Initially, the binding characteristics of small molecules 1a with CB [8] were studied by 1H NMR, UV / vis and fluorescence spectroscopy, ESI-MS and isothermal titration calorimetry (ITC). In agreement with the literature reports of other homoternary complexes CB [8] - (guest) 2 1: 2 (Jiao et al. J. Am. Chem. Soc., 2010, 132, 15734; Liu et al. Chem. Eur J., 2011, 17, 9930), the characteristic displacements of the aromatic proton peaks were observed in the 1H NMR spectrum by adding CB [8]. UV / vis titration experiments (not shown) provided evidence of a 1: 2 complex stoichiometry, see Figure 19 (b). Additionally, a strong excimer band emerged in the 1st emission spectra when CB [8] was added, which is indicative of a π-π stack face-to-face with portions of anthracene in the host cavity (Liu et al Chem. Eur. J., 2011, 17, 9930). The dominant species in the ESI-MS spectra can be attributed to the CB [8] - 1a2 complex, which further confirms the formation of a 1: 2 homologous complex suggested by CB [8] with 1a.
[0592] Furthermore, in ITC experiments, the inflection point of the isotherm was observed in a 1: 2 ratio between CB [8] and 1a, in agreement with the complex 1: 2 stoichiometry proposed in solution. The general aqueous ternary binding constant Ka (ternary) = (1.0 ± 0.5) x 1012 M-2 is essentially identical to Ka (ternary) of a recently reported anthracene pyridinium guest. A devolution of Ka (ternary) in the individual binding constants Ka (1) and Ka (2), although somehow numerically uncertain (Heitmann et al. J. Am. Chem. Soc., 2006, 128, 12574), Ka (1) = (4 ± 1) x 104 M-1 and Ka (2) = (2 ± 1) x 107 M-1, clearly shows strong positive cooperativeness, in contrast to previous reports from structurally similar guests (Heitmann et al. J. Am. Chem. Soc., 2006, 128, 12574; Jiao et al. J. Am. Chem. Soc., 2010, 132, 15734). Finally, the binding of 1a to CB [7], a smaller member of the cucurbit [n] urine family that can form only 1: 1 complexes with aromatic guests (Kim et al. J. Am. Chem. Soc., 2000, 122, 540; Lagona et al. Angew. Chem. Int. Ed., 2005, 44, 4844) was also studied with the aforementioned techniques and with emphasis on the non-observance of an excimer band in the emission spectrum of the aqueous solution of CB [7] and 1a.
[0593] As it was established that CB [8] stacks π-π efficiently the 1st anthracene units in their cavity, a study was carried out on the dimerization of these portions under photoiradiation. It was previously demonstrated by Inoue et al. that the photodimerization of anthracene carboxylic acids and their attached g-cyclodextrin esters in the presence of CB [8] produced a completely different product distribution than in the absence of the CB host [8] (Yang et al. J. Am Chem, Soc., 2008, 130, 8574). From the reported connection constants, Ka (1) = 2.4 x 105 M-1 and Ka (2) = 1.4 x 104 M-1, it is unlikely that the host CB [8] will quantitatively pre-organize both neutral or negatively charged anthracene molecules under the experimental conditions used (50 μM CB [8] and anthracene species) and much more likely that the 1: 1 complex is the predominant species in the solution. In addition, no accelerations in photodimerization rates have been reported in the presence of CB [8], in contrast to previous reports where cyclodextrins were employed as hosts (Tamaki Chem. Lett., 1984, 53; Nakamura et al. J Am. Chem. Soc., 2003, 125, 966). More recently, a π-π cell dimer of neutral covalently bonded anthracene was used by the same group and photodimerization in the presence of CB [8] resulted in values and and impressively high asymmetric dimers. It is expected to shorten the photodimerization time for anthracene derivatives, previously reported by taking approximately 1 hour to achieve a complete conversion through the non-covalent model effect that results from the cooperative bonding of positively charged anthracene derivatives with CB [ 8] (Yang et al. J. Am. Chem. Soc., 2008, 130, 8574). In fact, photoiradiation of a diluted aqueous solution of 1a (10 μM) and 0.5 equiv. CB [8] with a 350 nm light source led to a rapid reduction in the absorbance of the bands centered around 254 nm and 366 nm with an isosynthetic point at 222 nm, achieving a complete conversion within 3 minutes (Figure 20 ( The)).
[0594] In addition, the intensity of fluorescence emission has decreased through photo-radiation. Both UV / vis and kinetic fluorescence data produced an identical rate constant of 2 x 10-2 s-1 from mono-exponential adjustments. Control experiments carried out under identical conditions with the presence of 1.0 equiv. of CB [7], or in the absence of any host, resulted in a conversion of only 10% in the same time scale and the rate constants in both cases were of an order of magnitude lower than when photodimerization was performed in the presence CB [8] (see Table 1). The special UV / vis resources of the product formed when 1a was photo-radiated in the absence or presence of the CB host [8] were almost identical, suggesting that in both cases structurally similar products were formed. The further confirmation for the proposed photodimerization reaction came from ESI-MS measurements of the mixture of 1a and CB [8] treated with UV light.
a Photoreaction rates (350 nm irradiation) were determined from mono-exponential adjustments of absorbance at 254 nm vs. irradiation time. Sample volume and geometry, identical cuvette and light source were used in all cases. b Secondary reactions occur
[0595] After 15 minutes of photo-radiation, only one species was observed in the ESI-MS spectrum, it had the correct m / z value and the charge state was identified as the [CB [8] -2a] 2+ complex; Additional peaks including the binary complex 1: 1 [CB [8] - 1a] + were present before photoiradiation in ESI-MS, but were not observed after photoiradiation. Subsequently, acetonitrile was added to the ESI-MS samples in order to release the anthracene dimer from the ternary CB [8] complex. After photoiradiation, the species at m / z 236 Da had a +2 charge, which is characteristic of photodimer 2a whereas before photoiradiation, the monomeric 1a with a +1 charge was observed at the same m / z value.
[0596] Structural information was obtained from 1H NMR experiments (CB [8]: 1a = 1: 2; 500 μM in 1a). After 15 minutes of photo-radiation, a complete depletion of the proton signals corresponding to the anthracene reagents in the CB cavity [8] was observed with the appearance of new peaks that can be attributed to an anthracene cyclodimer [4 + 4] ( 2a) as seen in the 1H NMR spectrum in Figure 20 (b). However, in the absence of the CB host [8], reagent conversion of only 50% was obtained even after three hours of exposure to UV light. It is also important to note that a greater number of species was formed when 1a was photo-radiated in the absence of the CB host [8]. A subsequent NMR analysis of the non-complexed photoreaction products further evidenced this finding. From the 1H and 13C NMR spectra of the products, it is clear that a photoreaction type [4 + 4] of the anthracene portions occurred, for example, protons 9- and 10-anthryl and the carbons displaced to the upper field in the peak region aliphatic by photo-radiation. In principle, four different regioisomers could result as racemic mixtures upon dimerization of 1a. The analysis of the 1H and 13C NMR spectra revealed that an approximately equimolar mixture of two regioisomers was formed in the presence of CB [8], while in the absence of the host all four regioisomers were observed.
[0597] The attempted structural attribution of these products by NOESY and COZY NMR was inconclusive, however, it is reasonable to assume that the NMR peaks of groups N (CH3) 3 are more displaced to the lower field for head-to-head dimers than to head-dimers. tail due to charge buildup on one side of the molecule. Under this premise, a comparison of all NMR spectra is concluded that only head-tail dimers were produced for CB-mediated photodimerization [8].
[0598] The head-tail arrangement of two 1a molecules in the CB cavity [8] is also energetically preferred due to the minimized charge repulsion and the maximized cation-n interactions of the quaternary ammonium groups with the cartridges in both CB portals [8], and as such, it is more plausible that the head-tail model of two anthracene monomers results in the preferential formation of the head-tail photodimer. Therefore, from the combination of all ex-experimental observations, it can be concluded that the non-covalent union of two portions of small molecule anthra- cene carrying positive charges with CB [8] accelerates the anthracene and photodimerization reaction reduces the number of regioisomers and secondary products. Host-guest complexation and photodimerization of an anthracene-labeled PEG polymer
[0599] In an attempt to explore the photodimerization of anthracene [4 + 4] for the binding of polymeric entities, the PEG labeled with anthra- cene terminal group (1b) with a molecular weight of 2.4 kDa was synthesized. The spectroscopic signatures observed for the titration of CB [8] to an aqueous solution of polymer 1b were quite similar to its small molecule analog 1a, for example, an excimer band around 500 nm in emission, a isosynthetic point at 259 nm in UV / vis, and the upper field shifts characteristic of aromatic protons in the 1H NMR spectrum suggested that CB [8] can join two polymer entities even at considerably low concentrations (10 μM in 1b) . The ternary bond constant, Ka (ternary) = (2.2 ± 1.0) * 1010 M-2 for polymer 1b was found by ITC measurement to be two orders of magnitude less than for small molecule 1a, but still large enough to allow the formation of an almost quantitative ternary complex in the μM concentration regime.
[0600] The photoiradiation of the CB [8] - 1b2 complex with a 350 nm light source was again accompanied by a reduction in fluorescence intensity, a reduction in absorbance at 254 nm and 366 nm and the appearance of an isosynthetic point in 226 nm, suggesting that the photoreaction of CB [8] '1a2 and CB [8] - 1b2 ternary complexes produced structurally similar products. The CB [8] mediated photo-dimerization rate of 1b is 9 * 10-3 s-1, approximately twice as slow as for small molecule 1a at the same concentration (see Table 1).
[0601] Additional structural verification was obtained by ESI-MS experiments. Unfortunately, ESI-MS signals for 1b (and its CB complex [8]) could not be seen in suitable aqueous solutions, so a large excess of acetonitrile needed to be added (1:10) before injection. As previously mentioned, the decompression of CB meetings [8] occurs in mixtures of H2O: acetonitrile. Consequently, the peaks corresponding to the monomeric polymer chains 1b were observed before photo-radiation (results not shown). After 15 minutes of treatment with 350 nm light in the presence of CB [8], the charge, attributed by the isotropic spacing, of the species at the same m / z value doubled, confirming that photodimer 2b was indeed present (results not shown). The isotropically assigned loads were in accordance with those obtained from the peak-to-peak distance between the polymer chains that consist of monomeric units N and (N + 1). For example, ethylene oxide has a monomeric mass M (ethylene oxide) = 44 Da and, therefore, a difference m / z of 44 Da and 22 Da before and after photo-radiation in the presence of CB [8] produces z charges = 1 and z = 2, respectively. A quantitative analysis of conversion 1b to photodimerized polymer 2b is not possible using ESI-MS since the signal intensity is highly dependent on the ionization efficiency, and therefore on the charge and length of the polymer chains, both of which have been doubled through photodimerization. Therefore, the conversion of photodimerization was monitored by 1H NMR experiments and it was found to be quantitative within 15 minutes of photo-radiation when 0.5 equiv. host CB [8] was present (500 μM in 1b).
[0602] Additionally, a shift in retention time was observed in gel permeation chromatography (GPC) experiments, after photo-radiation of the mixture of 1b and CB [8], suggesting that a covalent bond was formed between the groups anthracene terminals of two polymer chains. Before photoiradiation, the CB-mediated non-covalent ternary complex [8] was not strong enough to withstand the separation forces on the GPC columns (at a flow rate of 0.6 mL / min) and resulted in decomplexing the individual components, that is, the GPC chromatograms of 1b alone, and the CB [8] -1b2 complex was almost identical. Secondary photochemical reactions in the absence of the CB host [8]
[0603] Surprisingly, the rate of reagent consumption in the absence of the CB host [8] was similar for small molecule 1a and for polymer 1b, see Table 1, since a biomolecular cycloaddition must be sensitive to the rate of diffusion of reagents. However, the relatively fast reagent consumption of 1b in the absence of CB [8] is the result of competitive side reactions in addition to anthracene dimerization. For example, although the absorbance at 254 nm was reduced by photo-radiation, there was an increase in absorbance in the region of 265-400 nm when the sample was irradiated in the absence of CB [8], which is in opposition to the findings mentioned above for 1a, CB [8] -1a2 and CB [8] -1b2. Additionally, reversing the order of photoiradiation and CB addition [8] resulted in considerably different absorption spectra, suggesting that different chromophoric species are formed by irradiation in the absence and presence of CB [8].
[0604] The exposure to UV light of an aqueous solution of 1b led to the emergence of an emission band around 525 nm while the irradiation of the solutions of 1a, CB [8] -1a2 and CB [8] - 1b2 was accompanied by a reduction in the emission intensity. Additionally, this red-shifted emission band did not disappear if CB [8] was added after exposure to UV light. Structural information from the ESI-MS experiments provided evidence that photoiradiation of 1b in the absence and presence of CB [8] led to completely different photoreaction products. In fact, no evidence for a 1b dimer could be found in the ESI-MS spectrum of a photoiradiated 1b solution, however, strong signals were found that could be attributed to a species [HO- (CH2CH2- O) nCH3 + Na ] + monomethyl ether of poly (ethylene glycol) terminated with degraded hydroxyl. In addition, the degraded PEG showed a large polydispersity although the starting material 1b had a much lower molecular weight distribution. Therefore, it must be concluded that 1b photoiradiation in the absence of the host is accompanied by a hydrolytic cleavage of PEG chains at random positions. The cleavage of the anthracene-polymer bond was also witnessed in 1H NMR experiments, revealing that only a small fraction of the polymer chains carried an aromatic end group after exposure to UV light. As additional evidence, the anthracene by-product precipitated as a red solid from the aqueous solution after 1b photo-radiation in the absence of a CB host [8]. Furthermore, the residual aromatic peaks remained in the lower field (7.0-9.0 ppm), even after the subsequent addition of CB [8], and therefore were distinctly different from those of the covalent anthracene dimers that were -wed in the presence of CB [8].
[0605] Non-functionalized PEG does not absorb light at 350 nm and was found to be stable under photoiradiation in a control experiment, so the 1b degradation process is most likely initiated by the anthrax end group. A transfer of photoelectrons (PET) from the triazole unit of 1b to the cationic portion of anthracene followed by thermal redox or radical reactions is a plausible mechanism for cleavage of the anthracene portion and decomposition of the main polymer chain by photoiradiation. It is worth mentioning that these secondary reactions were probably not the result of a photo-oxidation with dioxigen (O2) since the exposure to UV light of 1b degassed aqueous solutions caused similar spectral changes at comparable rates. From a synthetic point of view, it is of utmost importance that the complexation of CB [8] of the cationic portions of anthracene completely changed the trajectory of the photochemical reaction from a degradation reaction in the absence of anthene dimerization. desired in the presence of the host CB [8]. Gel formation and photochemical cross-linking
[0606] In order to modify the material properties and explore the anthracene dimerization findings, a hydroxyethyl cellulose (HEC) side chain functionalization with the anthracene portions was performed to induce supramolecular gelation through homoternary complexation by adding CB [8] followed by photo-crosslinking. The formation of supramolecular gels through the formation of a non-covalent 1: 1: 1 ternary complex with CB [8] has been demonstrated (Appel et al. J. Am. Chem. Soc., 2010, 132, 14251; Appel et al J. Am. Chem. Soc., 2012, 134, 11767). It would be advantageous for certain applications if covalent cross-links could be introduced after the polymer has self-assembled in a network in order to increase the mechanical stability of the polymer and slow down gel erosion. Here only two, instead of three, components are needed to activate the gelation: HEC labeled with anthracene and CB [8], see Figure 21 (b) for a schematic representation. Figure 21 (a) provides a pictorial view of the gels before and after photo-radiation. A 1.0% solution by weight of 1c in water is slightly viscous and exhibits "blue" fluorescence under UV light, which is indicative of single anthracene units.
[0607] However, when CB [8] was added (0.5 equiv. Per portion of anthracene), the fluorescence color changed from blue to green, representative of the emission of anthracene excimer from complex 2 : 1 with CB [8] (second bottle from the right). The solution also became much more gelatinous, but it did not form a self-sufficient gel. However, photoiradiation for 15 minutes with a 350 nm light source resulted in a cross-linked polymer to the point where a self-sufficient solid remained, suggesting that a network of covalently cross-linked polymer was formed (the flask on the right in Figure 21 (The)). In the absence of the CB host [8], photo-crosslinking did not occur to any noticeable extent, leaving the viscosity of a 1.0% solution by weight of modified HEC polymer unchanged (second flask from left ), even if the sample is photo-radiated for one hour. To quantify the mechanical strength of the materials, rheological experiments were conducted.
[0608] Mechanical tests of hydrogels demonstrated a considerable improvement in the properties of the materials through the addition of CB [8] and the subsequent heat treatment with UV light, which were much superior to that in the absence of the host.
[0609] The frequency-dependent rheological characterizations performed in the linear viscoelastic region are shown in Figure 22a. By adding CB [8] to a 1.0% solution by weight of 1c, the oscillatory shear modules for storage (G ') and loss (G' ') increased by three to four orders of magnitude (comparing Figure 22a and Figure 23 (b)). The frequency response of CB crosslinked material 1c [8] is characteristic of a highly elastic soft hydrogel, since the curves of the storage and loss modules are preferably linear and parallel to tan d = G '' / G '< 0.15. “Hardening”, that is, an additional increase in the G 'and G' 'values by a factor of three occurred by treating the gel with UV light (350 nm, 15 minutes) while the tan value d <0, 15 remained low, see Figure 22a.
[0610] In contrast, in the absence of the CB host [8], changes in the rheological characteristics of aqueous 1c solutions were hardly observed with exposure to UV light, maintaining a behavior similar to typical fluid (see Figure 23 ). In addition, non-covalently bonded and photo-crosslinked hydrogels exhibited a wide linear viscoelastic region (see Figure 24) with their structure breaking at stress amplitudes above 10% while the other supramolecular hydrogels typically disintegrated at much lower stresses ( see Appel et al., J. Chem. Soc. Rev. 2012, 41, 6195 and Mynar et al. Nature 2008, 451, 895).
[0611] It is worth noting that the treatment with UV light has further improved the resistance to gel tension. The photo-crosslinking effect of anthracene portions in the presence of CB [8] was also tested by stable shear measurements, which exhibited an increase in the viscosity (h) of the hydrogel by a factor of 80 through photoiradiation, depicted in Figure 22b , while solution 1c in the absence of CB [8] kept its viscosity low (Figure 23 (c)). When CB addition and photoiradiation experiments [8] were performed under a high dilution of 1c solution (60 μg / mL), spectroscopic techniques, such as UV / vis and fluorescence, could be used.
[0612] The titration of CB [8] in an aqueous solution of 1c showed essentially the same characteristics observed for the complexation of CB [8] with 1a and 1b (data not shown). Most importantly, maximum photodimerization occurred within 5 minutes of photo-radiation in the presence of CB [8], as determined by UV / vis (Fura. 25 (a)) and fluorescence spectroscopy (Figure 22d), which is similar to the results for the photo-radiation of CB complexes [8] with 1a and 1b. Not surprisingly, the rate of reagent consumption was also very high in the absence of the CB host [8] (Table 1) since multiple anthracene side chains of 1c are covalently maintained in strict spatial proximity. However, in the absence of CB [8], there was again evidence for the occurrence of photochemical secondary reactions as the red-shifted emission and absorption bands emerged through photoiradiation (see Figure S25 (b)). Consequently, the host-guest complexation of the anthracene portions with CB [8] not only induces the formation of a non-covalent network and accelerates the subsequent photochemical dimerization, but also prevents the chromophore from being led to undesirable side reactions that can result in deterioration of material and therefore prevent the gelation process.
[0613] Furthermore, the CB complexation [8] suppressed the photochemical side reactions including the degradation of the main polymer chains that were readily observed by irradiation in the absence of the CB host [8].
[0614] The rheological experiments were carried out as previously described. REFERENCES
[0615] All documents mentioned in this specification are to be found here incorporated in two totals for reference. Appel et al. J. Am. Chem. Soc. 2010, 132, 14251-14260 Appel et al. J. Am. Chem. Soc., 2012, 134, 11767 Appel et al., J. Chem. Soc. Rev. 2012, 41, 6195 Benguigui et al. Euro. Phys. J. B. 1999, 11, 439-444 Biedermann et al. Macromolecules 2011, 44, 4828-4835 Coulston et al. Chem. Commun. 2011, 47, 164-166 Danil de Namor et al. Chem. Rev. 1998, 98, 2495 Dsouza et al. Chem. Rev. 2011, 111, 7941 Esposito et al. Int. J. Pharm. 1996, 142, 923 Estroff et al. Chem. Rev. 2004, 104, 1201-1217 Gokel et al. Chem. Rev. 2004, 104, 2723 Greef et al. Chem. Rev. 2009, 109, 5687-5754 Greenfield et al. Langmuir 2010, 26, 3641-3647 Hartgerink et al. Science 2001, 294, 1684-1688 Heitmann et al. J. Am. Chem. Soc. 2006, 128, 12574-12581 Horkay et al. Macromolecules 1991, 24, 2896-2902 Horkay et al. Polymer 2005, 46, 4242-4247 Hunt, J. et al. Adv. Mater. 2011, 23, 2327-2331 Jiao et al. J. Am. Chem. Soc., 2010, 132, 15734 Jiao et al. Org. Lett. 2011, 13, 3044 Katakam et al. Int. J. Pharm. 1997, 152, 53-58 Kim et al. J. Am. Chem. Soc. 2000, 122, 540-541 Koopmans et al. Macromolecules 2008, 41, 7418-7422 Kretschmann et al. Angew. Chem. Int. Edit. 2006, 45, 4361-4365 Lagona et al. Angew. Chem. Int. Ed. 2005, 44, 4844 Large Array Manipulation Program (http://www.ill.fr/data treat / lamp / lamp.html; D. Richard, M. Ferrand and GJ Kearley, J. Neutron Research 1996, 4, 33-39) Lee et al. Chem. Commun. 2002, 2692-2693 Li et al. Biomacromolecules 2005, 6, 2740-2747 Liu et al. Chem. Eur. J., 2011, 17, 9930 Loh et al. Biomacromolecules 2007, 8, 585-593 Loh et al. Biomaterials 2007, 28, 4113-4123 Loh et al. Biomaterials 2008, 29, 2164-2172 Loh et al. Biomaterials 2008, 29, 3185-194 Loh et al. J. Control. Release 2010, 143, 175-182 Loh et al. J. Mater. Chem. 2011, 21, 2246-2254 Loh et al. J. Phys. Chem. B 2009, 113, 11822-11830 Loh et al. Macromol. Symp. 2010, 296, 161-169 Lutolf. Nat. Mat. 2009, 8, 451-453 Mynar et al. Nature 2008, 451, 895 Nakamura et al. J. Am. Chem. Soc., 2003, 125, 966 Nochi et al. Nat. Mat. 2010, 9, 572-578 Peppas et al. Annu. Rev. Biomed. Eng. 2 2000, 9-29 Pezron et al. Polymer 1991, 32, 3201-3210 Reczek et al. J. Am. Chem. Soc. 2009, 131, 2408-2415 Rekharsky et al. Chem. Rev. 1998, 98, 1875 Ritger et al. J. Controlled Release 1987, 5, 23 Ritger et al. J. Controlled Release 1987, 5, 37 Sijbesma et al. Science 1997, 278, 1601-1604 Staats et al. Nat. Mat. 2010, 9, 537-538 Tamaki Chem. Lett., 1984, 53 Uzunova et al. Org. Biomol. Chem. 2010, 8, 2037-2042 Van Tomme et al. Biomaterials 2005, 26, 2129-2135 Wang et al. Nature 2010, 463, 339-343 WO 2009/071899 WO 2011/077099 Wojtecki et al. Nat. Mat. 2010, 10, 14-27 Wu et al. Langmuir 2008, 24, 10306-10312 Yang et al. J. Am. Chem. Soc., 2008, 130, 8574

权利要求:
Claims (17)
[0001]
1. Hydrogel CHARACTERIZED by the fact that it has a crosslinked supramolecular network obtainable from the complexation of an aqueous composition comprising a host and one or more polymers having an adequate guest functionality, in which one or each polymer has a molecular weight equal to 50 kDa or greater and the host is cucurbituril and the one or more polymers has a suitable guest functionality for cucurbituril.
[0002]
2. Hydrogel CHARACTERIZED by the fact that it has a crosslinked supramolecular network obtainable from the complexation of an aqueous composition comprising a host and one or more polymers having an adequate guest functionality, in which the hydrogel maintains a component, and the host is cucurbituril and the one or more polymers has a suitable guest functionality for cucurbituril, in which one or each polymer has a molecular weight of 50 kDa or more.
[0003]
3. Hydrogel, according to claim 1 or 2, CHARACTERIZED by the fact that the host is CB [8] and the one or more polymers that have adequate guest functionality for CB [8].
[0004]
Hydrogel, according to any one of claims 1 to 3, CHARACTERIZED by the fact that: i) the one or each polymer has a molecular weight of 200 kDa or greater; ii) the water content of the hydrogel is at least 95% by weight; iii) a polymer is a hydrophilic polymer; iv) the hydrogel has a dominant storage module (G ') in relation to the loss module (G' ') over all voltage values in the range of 0.1 to 100 rad / s, as measured by measurement of frequency scanning at 37 ° C in a voltage range of 5 or 10% voltage; v) the hydrogel has a viscosity of at least 60 Pa.s at a shear rate in the range of 0.1 to 0.5 1 / s, as measured by stationary shear measurement at 25 ° C.
[0005]
Hydrogel according to any one of claims 1 to 4, CHARACTERIZED by the fact that the network is obtainable from the complexation of (a) an aqueous composition comprising cucurbituril and (1) or (2); or (b) a composition comprising a plurality of covalently linked cucurbiturils and (1), (2) or (3), and (1) comprising a first polymer covalently linked to a plurality of first cucurbituryl guest molecules and a second polymer covalently attached to a plurality of second cucurbituryl guest molecules, wherein a first guest molecule and a second guest molecule together with cucurbituryl are suitable to form a ternary guest-host complex; (2) comprises a first polymer covalently linked to a plurality of first guest molecules of cucurbituril and to a plurality of second guest molecules of cucurbituril, in which a first and second guest molecule together with cucurbituril are suitable to form a ternary guest-host complex and optionally the composition further comprises one or more fourth guest molecules of cucurbituril or both, wherein a third and fourth molecule together with cucurbituril are suitable to form a ternary guest-host complex, and / or the first and fourth molecules together with the cucurbituril are suitable to form a ternary guest-host complex, and / or the second and third molecules together with the cucurbituril are suitable to form a ternary guest-host complex; and (3) comprises a first polymer covalently linked to a plurality of first guest molecules of cucurbituril, wherein the first guest molecule together with cucurbituryl are suitable to form a binary host-host complex, and optionally the composition further comprises a second polymer covalently attached to one or more second cucurbituryl guest molecules, in which the second guest molecule together with the cu-curbituryl are suitable to form a binary guest-host complex.
[0006]
6. Hydrogel according to claim 5, CHARACTERIZED by the fact that the aqueous composition comprises cucurbituril and (2), so that the first and second guest molecules of the first polymer are the same.
[0007]
7. Hydrogel, according to claim 6, CHARACTERIZED by the fact that the first and second guest molecules are the same, and are selected from phenylalanine and tryptophan, so that the first and second guest molecules are phenylalanine.
[0008]
8. Hydrogel according to claim 6 or 7, CHARACTERIZED by the fact that the first polymer is a biopolymer.
[0009]
9. Hydrogel according to claim 6 or 7, CHARACTERIZED by the fact that the first polymer has hydroxy functionality.
[0010]
Hydrogel according to any one of claims 7 to 9, CHARACTERIZED by the fact that the first polymer is or comprises a polysaccharide and, optionally, the polysaccharide is a cellulose or a functionalized derivative thereof, so that the polysaccharide is carboxymethyl cellulose, hyaluronic acid, hydroxyethyl cellulose, cellulose, guar, chitosan, agarose or alginate.
[0011]
11. Hydrogel according to claim 5, CHARACTERIZED by the fact that the aqueous composition comprises cucurbituril and (1), so that one or both of the first polymer and the second polymer has a hydroxy functionality, so that both the first polymer as the second polymer has hydroxy functionality.
[0012]
Hydrogel, according to claim 11, CHARACTERIZED by the fact that: i) the first polymer is or comprises poly (vinyl) alcohol; ii) the second polymer is or comprises a polysaccharide, and the polysaccharide is optionally a cellulose or a functionalized derivative thereof, so that the polysaccharide is carboxymethyl cellulose, hyaluronic acid, hydroxyethyl cellulose, cellulose, guar, chitosan, agarose or alginate .
[0013]
13. Hydrogel according to claim 11 or 12, CHARACTERIZED by the fact that the first and second guest molecules of the first polymer are the same and are optionally selected from phenylalanine and tryptophan, so that the first and second guest molecules are each phenylalanine.
[0014]
14. Hydrogel, according to claim 1 or 2, CHARACTERIZED by the fact that one or each polymer has an average numerical molecular weight of 50 kDa or greater.
[0015]
15. Method for the preparation of a hydrogel, as defined in claim 1, CHARACTERIZED by the fact that the method comprises the step of putting in contact, in an aqueous solution, a mixture of a host and one or more polymers having a functionality of suitable guest, to thereby form a cross-linked supramolecular network, in which one or each polymer has a molecular weight of 50 kDa or greater and the aqueous solution comprises a cucurbituryl host, such as CB [8].
[0016]
16. Method for preparing a hydrogel while maintaining a component, as defined in claim 2, CHARACTERIZED by the fact that the method is selected independently from methods A or B: (A) the method that comprises the step of putting in contact, in an aqueous solution, a mixture of a host, a component and one or more polymers having adequate guest functionality, to thereby generate a hydrogel while maintaining a component, wherein the aqueous composition comprises a cucurbituryl host and one or each polymer has a molecular weight of 50 kDa or greater; and (B) the method comprising the steps of providing a hydrogel, the hydrogel having a crosslinked supramolecular network obtainable from the complexation of an aqueous composition comprising a host and one or more polymers having adequate guest functionality, and stirring that hydrogel in the presence of a component, to thereby incorporate the component into the hydrogel, wherein the aqueous composition comprises a cucurbituryl host and one or each polymer has a molecular weight of 50 kDa or greater.
[0017]
17. Hydrogel CHARACTERIZED by the fact that it maintains a component, as defined in claim 2, for use in a method of releasing a component to a location, the method comprising the steps of: (i) providing a hydrogel while maintaining a component, as defined in claim 2; (ii) making the hydrogel available at a target location; (iii) releasing the hydrogel component.

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同族专利:

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公开号 | 公开日

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ES2759112T3|2020-05-07|

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ZA201406007B|2020-03-25|

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BR112014020450A8|2018-01-16|

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WO2013124654A1|2013-08-29|

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CN104245801A|2014-12-24|

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EP2817359A1|2014-12-31|

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US20150110772A1|2015-04-23|

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CN104245801B|2020-06-23|

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BR112014020450A2|2017-06-20|

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EP2817359B9|2020-03-25|

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EP2817359B1|2019-09-11|

引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

---

DE19825486C2|1998-06-08|2000-07-06|Stockhausen Chem Fab Gmbh|Water-absorbing polymers with supramolecular cavity molecules, process for their preparation and their use|

---

GB0723714D0|2007-12-04|2008-01-16|Cambridge Entpr Ltd|Supramolecular handcuffs in polymeric architecture|

---

GB0922623D0|2009-12-23|2010-02-10|Cambridge Entpr Ltd|Methods for the purification of cucurbituril|

---

CN103827178B|2011-07-26|2016-10-05|剑桥实业有限公司|Supermolecule capsule|

---

US9827321B2|2012-08-14|2017-11-28|The Trustees Of The University Of Pennsylvania|Stabilizing shear-thinning hydrogels|GB201301648D0|2013-01-30|2013-03-13|Cambridge Entpr Ltd|Nested supramolecular capsules|

---

US10258698B2|2013-03-14|2019-04-16|Modernatx, Inc.|Formulation and delivery of modified nucleoside, nucleotide, and nucleic acid compositions|

---

EP3041938A1|2013-09-03|2016-07-13|Moderna Therapeutics, Inc.|Circular polynucleotides|

---

AU2014315287A1|2013-09-03|2015-03-12|Moderna Therapeutics, Inc.|Chimeric polynucleotides|

---

WO2015153217A1|2014-04-02|2015-10-08|Dow Global Technologies Llc|Cellulose ethers having 1,2,3-triazolyl substituents|

---

CN103962112B|2014-05-13|2016-06-29|东南大学|Preparation method for the photoresponse intelligent gel microsphere of three-dimensional cell cultivation|

---

US10323104B2|2014-08-02|2019-06-18|Lg Chem, Ltd.|Dye complex, photoconversion film and electronic element including same|

---

WO2016049360A1|2014-09-24|2016-03-31|Massachusetts Institute Of Technology|Shear-thinning self-healing networks|

---

CN105061775B|2015-08-09|2017-08-01|大连理工大学|It is a kind of that the method for preparing new fluorescent organic solid material is assembled by Cucurbituril anion|

---

CN105112050B|2015-10-15|2017-03-08|南开大学|A kind of adjustable fluorescent material of solvent and its preparation method of pseudopolyrotaxane hydrogel|

---

WO2017096367A1|2015-12-04|2017-06-08|Colorado State University Research Foundation|Thermoplastic elastomer hydrogels|

---

CN105837744A|2016-06-12|2016-08-10|江苏华昌织物有限公司|Preparation method of double sensitive polymer hydrogel based on crown ether|

---

US10125228B2|2016-08-22|2018-11-13|Colorado State University Research Foundation|Phototunable thermoplastic elastomer hydrogel networks|

---

US10590257B2|2016-09-26|2020-03-17|The Board Of Trustees Of The Leland Stanford Junior University|Biomimetic, moldable, self-assembled cellulose silica-based trimeric hydrogels and their use as viscosity modifying carriers in industrial applications|

---

US20190263973A1|2016-10-14|2019-08-29|Cambridge Enterprise Limited|Cucurbituril-based hydrogels|

---

CN107033281B|2017-05-05|2019-01-15|南开大学|A kind of fluorescence hydrogel and preparation method thereof that swelling behavior is excellent|

---

CN109206575A|2017-06-30|2019-01-15|翁秋梅|A kind of hybrid cross-linked dynamic aggregation object|

---

CN109206576A|2017-06-30|2019-01-15|翁秋梅|A kind of hybrid cross-linked dynamic aggregation object|

---

CN109208110B|2017-07-01|2020-06-09|中国石油化工股份有限公司|Spider silk-like polymer fiber based on non-spherical porous particles and preparation method thereof|

---

CN107936262B|2017-12-07|2020-07-28|西北师范大学|Preparation and application of supramolecular polymer framework material|

---

CN108610615B|2018-05-08|2019-07-09|西南交通大学|A kind of supramolecular hydrogel and preparation method thereof|

---

CN109081927B|2018-06-25|2020-07-03|天津科技大学|Preparation method of hydrogel|

---

CN109160995B|2018-08-19|2021-02-23|南京理工大学|Column [5] arene self-assembly elastomer material and preparation method thereof|

---

WO2020072495A1|2018-10-01|2020-04-09|The Board Of Trustees Of The Leland Stanford Junior University|Injectable hydrogels for controlled release of immunomodulatory compounds|

---

CN109705372B|2018-12-29|2021-08-13|广东工业大学|Supramolecular material, hydrogel and preparation method thereof|

---

CN110628039B|2019-09-27|2021-06-08|西北师范大学|Supramolecular polymer hydrogel based on bipod gelator and application thereof|

---

CN111057245B|2019-12-09|2021-05-14|山西大学|Metal supramolecular polymer network based on pillared aromatic hydrocarbon and preparation method thereof|

---

CN111378146A|2020-01-17|2020-07-07|中国科学院长春应用化学研究所|Polymer, nanogel for carrying protein drug and application of nanogel|

---

CN111484634A|2020-04-27|2020-08-04|哈尔滨工业大学|Self-healing multi-bridged network chitosan-derived hydrogel and preparation method thereof|

---

CN112375185A|2020-11-27|2021-02-19|中国石油大学|Small-molecule thick oil viscosity reduction polymer and preparation method thereof|

法律状态:
2018-01-16| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

---

2018-03-27| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

---

2018-03-27| B15K| Others concerning applications: alteration of classification|Ipc: C08J 3/075 (2006.01), A61K 47/00 (2006.01), C08G 8 |

---

2018-09-25| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |

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2020-03-17| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

---

2020-09-01| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

---

2020-12-08| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 20/02/2013, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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GBGB1202834.6A|GB201202834D0|2012-02-20|2012-02-20|Cucurbituril-based hydrogels|

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GB1202834.6|2012-02-20|

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GB1301648.0|2013-01-30|

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GBGB1301648.0A|GB201301648D0|2013-01-30|2013-01-30|Nested supramolecular capsules|

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PCT/GB2013/050414|WO2013124654A1|2012-02-20|2013-02-20|Cucurbituril-based hydrogels|

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