SUPRAMOLECULAR CAPSULES, METHOD FOR PREPARING SUCH CAPSULES AND NON-THERAPEUTIC METHOD OF DISTRIBUTI

专利摘要:
supramolecular capsules. it is a capsule having a shell of material consisting of a supramolecular lattice network. the network is formed from a host-guest complexation of cucurbituril (the host) and one or more building blocks comprising appropriate guest functionality. the complex non-covalently cross-links the building block and/or non-covalently links the building block to another building block, thereby forming the supramolecular cross-linked network. the capsules are obtained or obtainable by complexing a composition comprising cucurbituril and one or more building blocks having a suitable cucurbituril visitor functionality to thereby form a supramolecular cross-linked network.
公开号:BR112014001868B1
申请号:R112014001868-5
申请日:2012-07-25
公开日:2022-01-11
发明作者:Oren Alexander Scherman；Roger Coulston；Christopher Abell；Jing Zhang
申请人:Cambridge Enterprise Limited；
IPC主号:

专利说明:

PRIORITY
[001]This application claims priority to document GB 1112893.1 filed on July 26, 2011 and to document GB 1202127.5 filed on February 08, 2012, the contents of both being incorporated herein in their entirety by reference. FIELD OF THE INVENTION
[002] The present invention relates to capsules, particularly microcapsules, based on a cucurbituril lattice, and to methods for preparing such capsules, and their use in methods for delivering encapsulated components. FUNDAMENTALS
[003]The microencapsulation of a component by self-assembling hollow microspheres is one of the important aspects of nanotechnology and materials science. Control over the shape and composition of the support structure, parameters that influence material properties, is important for many applications, such as diagnosis, drug delivery, electronic displays, and catalysis (see Ke et al. Angew. Chem 2011, 123, 3073; De Cock et al. Angew. Chem. Int. Ed. 2010, 49, 6954; Yang et al. Angew. Chem. 2011, 123, 497; Comiskey et al. Nature 1998, 394, 253 ; Peyratout et al. Angew. Chem. Int. Ed. 2004, 43, 3762 ). Preparation of conventional polymeric microcapsules proceeds through a layer-by-layer (LbL) scheme, where a solid support is coated by the sequential addition of a series of oppositely charged polyelectrolyte layers (see Caruso et al. Science 1998, 282, 1111). ; Donath et al. Angew. Chem. Int. Ed. 1998, 37, 2201 ). This strategy provides a uniform material but suffers from reduced encapsulation efficiencies due to the solid model. An alternative method uses colloidal emulsion modeling where liquid-liquid interfaces drive self-assembly of shell components (see Cui et al. Adv. Funct. Mater. 2010, 20, 1625). However, it is difficult to control the monodispersion and material diversity of the resulting microcapsules, thus limiting their functionality in drug delivery and uptake applications.
[004]In contrast, microfluidic droplets, a subset of colloidal emulsion, showed great potential for microcapsule fabrication (see Gunther et al. Lab Chip 2006, 6, 1487; Huebner et al. Lab Chip 2008, 8, 1244; Theberge et al. Angew. Chem. Int. Ed. 2010, 49, 5846 ). These narrowly distributed droplets (polydispersion index < 2%) can be generated at extremely high frequency with economical use of reagents (see Xu et al. AIChE Journal 2006, 52, 3005). Initial efforts to prepare capsules based on microdroplet-assisted manufacturing focused on phase separation using dual emulsion and liquid crystal core modeling (see Utada et al. Science 2005, 308, 537; Priest et al. Lab Chip 2008, 8 , 2182). The formation of polymeric capsule walls has also been described in an approach involving microfluidic device surface treatment and rapid polymerization techniques (see Zhou et al. Electrophoresis 2009, 31, 2; Abraham et al. Advanced Materials 2008, 20, 2177) . The wall is formed as the solvent evaporates from the organic solvent droplets formed. Metallic organic framework capsules have also been recently reported (see Ameloot et al. Nat. Chem. 2011, 3, 382). With current ionic or covalent crosslinking strategies, however, the primary challenge in capsule manufacturing is in the simultaneous production of uniform capsules with high charge-loading efficiencies and easy incorporation and diverse functionality into the capsule shell.
[005]Now, the present inventors have established a capsule based on a cucurbituril-based host-visitor network. Designing microstructures using multivalence and cooperativity through molecular recognition provides an unparalleled opportunity in the fabrication of microcapsules with adaptive interactions and functionality. However, efforts to prepare microcapsules using a supramolecular host-visitor approach as described herein are lacking (see De Cock et al. Angew. Chem. Int. Ed. 2010, 49, 6954).
[006] Previous disclosures include a colloidal microcapsule comprising β-cyclodextrin and modified gold nanoparticles (AuNPs) prepared via emulsion modeling (Patra et al., Langmuir 2009, 25, 13852), and a microcapsule comprising polymers functionalized with cyclodextrin and ferrocene prepared using a LbL synthesis ( Wang et al., Chemistry of Materials 2008, 20, 4194 ). SUMMARY OF THE INVENTION
[007] The present invention generally provides capsules having a shell of material consisting of a supramolecular lattice network. The network is formed from a host-guest complexation of cucurbituril (the host) and one or more building blocks comprising suitable guest functionality. The complex noncovalently cross-links the building block and/or noncovalently links the building block to another building block, thus forming the network.
[008] In a general aspect, the present invention provides a capsule having a shell obtainable from the complexation of cucurbiturils with suitable visiting molecules.
[009] In a first aspect of the invention, there is provided a capsule having a shell which is obtainable from the complexation of a composition comprising cucurbituril and one or more building blocks having a cucurbituril visitor functionality suitable therefor. form a supramolecular lattice network.
[010] In one embodiment, the shell is obtainable from the complexation of (a) a composition comprising cucurbituril and (1) or (2); or (b) a composition comprising a plurality of covalently linked cucurbituryls and (1), (2) or (3).
[011] In one embodiment, the shell is obtainable from complexing a composition comprising cucurbituril and (1) or (2).
[012] In one embodiment, the shell is obtainable from complexing a composition comprising cucurbituril and (1). (1) comprises a first building block covalently linked to a plurality of first visiting cucurbituril molecules and a second building block covalently linked to a plurality of second visiting cucurbituril molecules, wherein a first visiting molecule and a second visiting molecule together with cucurbituril are suitable to form a ternary visitor-host complex. (2) comprises a first building block covalently linked to a plurality of first visiting cucurbituril molecules and a plurality of second visiting cucurbituril molecules, wherein a first and second visiting molecules together with cucurbituril are suitable to form a visitor- ternary host. Optionally, the composition further comprises a second building block covalently linked to one or more third cucurbituril visiting molecules, one or more fourth cucurbituryl visiting molecules, or both, wherein a third and fourth molecules together with cucurbituril are suitable for form a ternary visitor-host complex, and/or the first and fourth molecules together with cucurbituril are suitable to form a ternary visitor-host complex, and/or the second and third molecules together with cucurbituril are suitable to form a visitor complex -ternary host; (3) comprises a first building block covalently linked to a plurality of first visiting molecules of cucurbituril, wherein the first visiting molecules together with cucurbituril are suitable to form a binary visitor-host complex. Optionally, the composition further comprises a second building block covalently linked to one or more second visitor molecules of cucurbituril, wherein the second visitor molecules together with cucurbituril are suitable to form a binary visitor-host complex.
[013] In one embodiment, cucurbituril is selected from CB[8] and variants and derivatives thereof.
[014] In one embodiment, cucurbituril forms a ternary complex with suitable visiting molecules, for example with first and second visiting molecules.
[015] In one embodiment, the capsule is a microcapsule.
[016] In one embodiment, the capsule encapsulates a component.
[017] In a second aspect of the invention, there is provided a method for preparing a capsule having a shell, such as the capsule of the first aspect of the invention, the method comprising the step of: (1) placing a stream of a first phase and a flow of a second phase in contact in a channel, to thereby generate in the channel a dispersion of discrete regions, preferably droplets, of the second phase in the first phase, the second phase comprising cucurbituril and one or more building blocks having cucurbituril visitor functionality suitable for forming a supramolecular lattice network, to thereby form a capsule shell within the discrete region, the first and second phases being immiscible.
[018] In one embodiment, the second phase comprises (a) a cucurbituril and (1) or (2); or (b) a plurality of covalently linked cucurbituryls and (1), (2) or (3).
[019] In one embodiment, one between the first and second phases is an aqueous phase and the other phase is a water-immiscible phase.
[020] In one embodiment, the second phase is an aqueous phase. The first phase is a water-immiscible phase, for example an oil phase.
[021] In one embodiment, the first phase is an aqueous phase. The second phase is a water-immiscible phase, for example an oil phase.
[022] In one embodiment, the method further comprises the step of (ii) collecting the flow from the channel, to thereby obtain a droplet, which contains a capsule.
[023] In one embodiment, the method comprises step (ii) above and (iii) optionally drying the capsule obtained in step (ii).
[024] In one embodiment, the channel is a microfluidic channel.
[025] In one embodiment, the second phase flow is brought into contact with the first phase flow substantially perpendicular to the first phase. In this embodiment, the channel structure can be a T-junction geometry.
[026] In one embodiment, the second phase flow further comprises a component for encapsulation, and step (i) provides a capsule having a shell that encapsulates the component.
[027] In a third aspect of the invention, there is provided a capsule obtained or obtainable by the method of the second aspect of the invention.
[028] In a fourth aspect of the invention, there is provided a method of delivering a component to a location, the method comprising the steps of: (i) providing a capsule having a shell that encapsulates a component; (ii) delivering the capsule to a target site; (iii) release the component from the enclosure.
[029] In an alternative aspect of the invention, there is provided a capsule having a shell that is obtainable from complexing a composition comprising a host and one or more building blocks having suitable host-visitor functionality to thereby , form a supramolecular network.
[030] In one embodiment, the host is selected from cyclodextrin, calix[n]arene, crown ether and cucurbituryl, and one or more building blocks having a suitable guest-host functionality for cyclodextrin, calix [n]arene, crown ether or cucurbituril host. In one embodiment, references in the above first to fourth aspects to cucurbituril and a cucurbituril host may be interpreted as referring to an alternate host and a suitable visitor to such a host. SUMMARY OF THE FIGURES
[031]Figure 1(a) is a schematic representation of the microdroplet generation process using a microfluidic flow concentrating device, which consists of a continuous oily phase perpendicular to a combination of aqueous solutions of CB[8] 1, MV2+ -AuNP 2, and Np-pol 3 as the dispersed phase. (b) Microscopic image and schematic representation of the flow concentration region, with a downstream mixing channel allowing for judicious mixing of reagents online. (c) The high monodispersity of microfluidic droplets is demonstrated by their narrow size distribution.
[032]Figure 2 (a) Brightfield images of the late stage of the capsule formation process as water evaporates, rendering a collapsed microcapsule. Scale bar = 5 μm. (b) Light microscopic image of the exploded capsules showing the capsule shell residues. Scale bar = 10 μm. (c) SEM image of a dry and at least partially collapsed capsule. Scale bar = 2 μm. (d) TEM image of the microcapsule shell, showing 5 nm AuNPs dispersed in a polymer mesh. Scale bar = 10 nm. (e) Schematic representation of the proposed microcapsule formation process from the initial droplet (with diameter d) to the dehydrated stable capsule (with diameter d'). The crosslinking structure of 1 and 2 for the capsule material is also proposed.
[033]Figure 3 (a) Chemical structure and schematic representation of NP-RD-pol 4. (b) LSCM image of droplets containing aqueous solutions of Np-RD-pol, CB[8] and MV-AuNP, and the profile of fluorescence intensity. Scale bar = 40 μm. (c) LSCM image of a droplet (46 μm in diameter) containing aqueous solutions of Np-RD-pol, CB[8], MV-AuNP and FITC-dextran and the corresponding fluorescence intensity profile. Scale bar = 7.5 μm. (d) LSCM image of a droplet (23 μm in diameter) containing aqueous solutions of Np-Rd-pol, CB[8], MV-AuNP and FITC-dextran and the corresponding fluorescence intensity profile. Scale bar = 10 μm.
[034]Figure 4 are brightfield and fluorescence images of dry microcapsules containing FITC-dextran before and after rehydration, showing (a) microcapsule wall expansion accompanied by leakage of FITC-dextran (10 kDa) , (b) retention of FITC-dextran (500 kDa), and (c) partial permeability of FITC-dextran (70 kDa) to microcapsules containing a doubly concentrated CB[8] crosslinker. Scale bars = 20 μm.
[035]Figure 5 (a) Schematic representation of the proposed effect of MV2+ reduction on the CB[8]:MV2+-AuNP:Np-pol ternary complex, and the resulting formation of the 2:1 complex (MV+,-AuNP)2: CB[8]. (b) Fluorescence images of the process of disintegration of the microcapsule wall material in an aqueous solution of Na2S2O4 and in H2O for 12 hours in the N2 environment at 25°C. Scale bars = 5 μm.
[036]Figure 6 (a) Schematic representations of microcapsules with and without MV2+-AuNPs (5 nm and 20 nm). For a negative control, MV2+-pol 5 was used instead of AuNPs. (b) SERS spectra of empty microcapsules consisting of MV2+-pol, 5 nm MV2+-AuNP, and 20 nm MV2+-AuNP, showing the characteristic peaks for CB[8] and MV2+ (indicated by arrows). (c) SERS spectra of FITC-dextran-encapsulated microcapsules consisting of MV2+-pol and 20 nm MV2+-AuNP, showing characteristic peaks for FITC (indicated by arrows) in addition to the capsule shell materials. All spectra were obtained using a 633 nm excitation laser line. (d) SERS mapping of the microcapsule, showing the location of the SERS signal for CB[8] and MV2+.
[037] Figure 7 is the excitation spectrum of Np-RD-pol and the emission spectra excited at 514 nm and 544 nm.
[038]Figure 8 shows the variation in mean droplet diameter as a function of the Qoil/Qaq ratio, of various aqueous streams of Qaq = 80 μL/h, 100 μL/h, 120 μL/h using a T-junction and a channel 40 μm wide (solid line), and various aqueous streams of Qaq = 40 μL/h, 60 μL/h, 80 μL/h using a T-junction and a channel 20 μm wide (line dashed).
[039] Figure 9 shows the variation in mean droplet diameter as a function of the ratio of oily flow to aqueous flow, and as a function of the ratio of individual aqueous flow rates.
[040] Figure 10 is the LSCM image of a droplet containing aqueous solutions of Np-RD-pol, CB[8], MV-AuNP and E. coli cells expressing GFP and the corresponding fluorescence intensity profile. DETAILED DESCRIPTION OF THE INVENTION
[041] The present inventors have established that capsules can be prepared having a shell that is obtainable from supramolecular complexation of cucurbituril with building blocks covalently linked to appropriate cucurbituril visiting molecules.
[042] Capsules are formed using fluidic droplet generation techniques, among others. The ability of cucurbituril and building blocks to form a shell is surprising given the previously reported behavior of such materials.
[043] A previous study by one of the present inventors found that cucurbituril can be used to form a supramolecular cross-linked network through host-visitor complexation (see Appel et al. J. Am. Chem. Soc. 2010, 132, 14251) . The network is based on the supramolecular organization of a ternary complex of CB[8] together with a polymer functionalized with methyl viologen (MV) and a polymer functionalized with naphthol (Np). However, the networks described herein are in the form of supramolecular hydrogels. Capsules are not described or suggested.
[044]The hydrogels are prepared by sonication of the MV-functionalized polymer together with CB[8], followed by the addition of the Np-functionalized polymer, with a subsequent short mixing step.
[045] Therefore, the finding by the present inventors that cucurbituril can be mixed together with the building blocks connected to appropriate visiting molecules to thereby produce a shell of material was unexpected. The capsule is obtainable through the use of fluidic droplet preparation techniques and bulk droplet generation techniques. The former is particularly beneficial in that it generates droplets having a very low size distribution, which results in capsules having a very low size distribution. Furthermore, the methods of the invention allow a strict control of the formation of the product capsule. Simple changes in the fluidic droplet preparation technique, such as changes in flow rates, can be used to control the size of the capsule obtained, the size of the pores in the shell, and the shell thickness, among others.
[046] The capsules of the invention are shown to be robust, and are capable of withstanding temperatures of at least 100°C. Capsules also maintain their integrity under reduced pressure.
[047] The capsules of the invention are suitable for encapsulating a component. Using the fluidic droplet preparation techniques described herein, a capsule shell can be constructed in the presence of the component to be encapsulated. Therefore, in a procedure, the wrapper can be formed and the component encapsulated. Therefore, advantageously, the capsule can be constructed without the need for a subsequent passive diffusion step after construction of the capsule. Additionally, the encapsulation method allows for high rates of material incorporation into the capsule, and material scrap is thereby minimized.
[048]Now, the invention will be described in greater detail with reference to each feature of the invention. capsules
[049] A capsule of the invention comprises a shell of material. The material is the supramolecular complex which is formed from the complexation of cucurbituril with building blocks covalently linked to the appropriate cucurbituril visitor molecule. The housing defines an internal space, which may be referred to as a hollow space, which is suitable for holding a component. Therefore, in one embodiment, the capsules of the invention extend to those capsules that encapsulate a component within the shell. The housing can form a barrier that limits or prevents the release of encapsulated material.
[050] The component may be releasable from the capsule through pores that are present in the shell. In some embodiments, the pores are small enough to prevent the component from being released. Therefore, the mesh constituting the housing can be at least partially dismantled, thus allowing the release of material from within the housing. Additional pores can be generated by the application of an external stimulus to the sheath. In this case, pores can be generated through a disruption of the cucurbituril visitor complex. Therefore, this decomplexation creates pores through which encapsulated components can be released from within the housing. In some embodiments of the invention, the shell material may be subsequently reformed by reassembly of the shell components.
[051] In one embodiment, the capsule holds water within the shell. The water may be an aqueous solution comprising one or more of the reagents that serve for use in preparing the supramolecular shell, i.e., unreacted reagents. In one embodiment, the aqueous solution comprises cucurbituryl and/or (1) or (2) or (b) a plurality of covalently linked cucurbituryls and/or (1), (2) or (3). Within the shell may also be present a network that is formed from the complexation of the reagents that were used to generate the shell.
[052] Within the housing, an encapsulated material can be provided, which may be provided in addition to water and reagents that serve for use in the supramolecular assembly of the housing.
[053] When the capsule is said to encapsulate a component, it is understood that this encapsulated component may be present within the internal space defined by the casing. In one embodiment, the encapsulant is also present, at least partially, within the pores of the shell.
[054] The presence of a component within the shell and/or within the shell pores can be determined using appropriate analytical techniques that are capable of distinguishing the shell material and the encapsulant. For example, each between the shell material and the component may have a detectable marker or suitable functionality that is independently detectable (orthogonal) to the marker or functionality of the other. In one embodiment, each between the housing and the component has an orthogonal fluorescent marker. For example, one has a rhodamine label and the other has a fluorescein label. Confocal laser scanning microscopy techniques can be used to independently detect the fluorescence of each marker, thereby localizing each shell and encapsulant. When the component signals are located at the same point as the housing signals, it is understood that the component resides within a pore of the housing.
[055]The generic shape of the housing, and therefore the shape of the capsule, is not particularly limited. However, in practice, the shape of the capsule may be dictated by its method of preparation. In the preparation methods described herein, a capsule shell can be prepared using fluidic droplet formation techniques. Typically, the shell material is formed at the boundary of a discrete (or discontinuous) phase into a continuous phase. For example, one phase may be an aqueous phase, and the other may be a water-immiscible phase. The discrete region may be a droplet, having a substantially spherical shape. Therefore, the formed shell is also substantially spherical.
[056] In certain embodiments, a capsule can be obtained when the shell has a substantially spherical shape. This capsule can be subjected to a drying step, which reduces the amount of solvent (e.g. water) in and around the capsule. As a result of this step, the capsule shrinks in size. First, the housing maintains a substantially spherical shape. After further drying, the capsule sphere may collapse partially or completely into itself. The structural integrity of the capsule is maintained and the shell simply changes to accommodate changes in internal volume. Therefore, the capsules of the invention include those capsules where the shell is an at least partially collapsed sphere.
[057] Given the formation of the capsule shell at the boundary of the discrete region (eg a droplet), references to the dimensions of a droplet can also be taken as references to the capsule dimension. The capsule shell can form prior to a drying step.
[058] The inventors have established that capsules which have contracted, for example, by desolvation, can subsequently be returned to their original substantially spherical shape, for example, by resolving the capsule.
[059] The shape of a capsule can be determined by simply observing the capsule formed using a microscope, such as a bright field microscope, scanning electron microscope or transmission electron microscope. Where the shell material comprises a marker, detection of the marker through the shell will reveal the shape of the capsule. For example, where the marker is a fluorescent marker, the confocal laser scanning microscope can be used to locate the shell material and its shape.
[060]Capsule size is not particularly limited. In one embodiment, the capsule is a microcapsule and/or a nanocapsule.
[061] In one embodiment, each capsule has an average size of at least 0.1, 0.2, 0.5, 0.7, 1, 5, 10, 20, 30, 40, 50, 100, or 200 μm in diameter.
[062] In one embodiment, each capsule has an average size of at most 400, 200, 100, 75 or 50 μm in diameter.
[063] In one embodiment, the capsule size is in a range where the minimum and maximum diameters are selected from the previous embodiments. For example, the capsule size is in the range of 10 to 100 μm in diameter.
[064]Average size refers to the numerical average of diameters measured for a sample of capsules. Typically, at least 5 capsules in the sample are measured. A cross-sectional measurement is taken from the outermost edges of the housing.
[065]The cross-section of a capsule can be determined using a simple microscopic analysis of the capsules formed. For example, the formed capsules can be placed on a microscope slide and the capsules analyzed. Alternatively, capsule size can be measured during the preparation process, for example, as capsules are formed in a channel of a fluidic device (i.e., in-line).
[066] Cross-sectional measurement can also be obtained using techniques related to detecting a detectable marker or functionality present within the enclosure material. As mentioned above in connection with the detection and location of the encapsulated component, the shell material may comprise a fluorescent marker which can be detected by laser scanning confocal microscopy techniques. The presence of multiple markers in and around the capsule shell allows the cross-sectional shape to be determined, and the largest cross-section measured.
[067] In the preparation method described herein, a capsule is prepared using a fluidic droplet generation technique. The capsule shell is formed into a droplet, which is created in a channel of a fluid droplet generating device, at the boundary of the aqueous phase of the droplet with the continuous phase. Therefore, the size of the capsule is substantially equal to that of the droplet.
[068] The present inventors have established that the capsules of the invention can be prepared with a low size distribution. This is particularly advantageous as a large number of capsules can be prepared, each with predictable physical and chemical characteristics.
[069] In one embodiment, the capsule diameter has a relative standard deviation (RPR) of at most 0.5%, at most 1%, at most 1.5%, at most 2%, at most 4%, in the maximum 5%, maximum 7%, or maximum 10%.
[070]The relative standard deviation is calculated from the standard deviation divided by the numerical mean and multiplied by 100. Capsule size refers to the largest cross-section of the capsule, in any section. The cross-section of a substantially spherical capsule is the diameter.
[071]The enclosure defines an internal cavity that is suitable for encapsulating a component. The size of the internal space will generally correspond to the size of the capsule itself. Therefore, the dimension, eg diameter, of the inner space can be selected from any of the diameter values given above for the housing itself.
[072] When the capsule size is measured, the diameter refers to the distance from the outermost edge to the outermost edge of the shell material from two opposite points as mentioned above. When the size of the inner space is measured, the diameter refers to the distance from the innermost edge to the innermost edge of the wrapping material from two opposite points.
[073] The inventors have established techniques that allow the outer and inner edges of the casing to be determined. For example, the presence of a detectable marker within the shell material allows the outermost edges and innermost edges of the shell to be determined. If these edges can be detected, the shell thickness can be determined.
[074] Typically, the diameter as measured from the outermost edge to the outermost edge is not significantly different from the diameter as measured from the innermost edge to the innermost edge. The difference is the thickness of the housing at the two opposite points.
[075] In one embodiment, the wrapper has a thickness of at least 0.02, at least 0.05, at least 0.1, at least 0.5, at least 1.0, at least 2.0, or at least minus 5.0 μm.
[076]As previously noted, the casing has pores. In one embodiment, the pores may be of a size to allow material to pass therethrough. For example, components encapsulated within the capsule may pass through pores in the shell to be released from the capsule. Conversely, the pores may be large enough to allow the components to pass into the inner space of the housing, and thus become encapsulated. This can be referred to as a passive diffusion encapsulation step. This technique can be used to provide a capsule having an encapsulant therein. As described herein, the present inventors have provided alternative methods for encapsulating material in the shell preparation step. These methods allow for more efficient loading of the capsule with material as the material is fully encapsulated within the shell.
[077] In one embodiment, the pores may be of a size that is so small as to allow material to pass through. For example, components encapsulated within the capsule can be prevented from passing through the pores of the housing, and therefore cannot be released from the capsule. This material can be released from the capsule, for example, by breaking down the cucurbituril complexes that maintain the shell. In this way, the rupture of the casing creates larger pores through which the material can pass.
[078] It is believed that the pore size can be increased by solvating a previously desolved capsule. As the capsule contracts, the porosity of the capsule may decrease as the shell material bends, thus at least partially blocking some of the pores.
[079]The size of a pore can be experimentally estimated using a range of encapsulated components having a different cross-section, such as a different diameter. The cross section may be known or may be predicted based on an understanding of the likely configuration of the component. Pore size can be determined based on which components are released from the capsule and which are not.
[080] The cross-section, typically the diameter, of a component can be predicted based on the radius of gyration calculated for each encapsulated component. These calculations are best suited for determining the size of small globular particles, and can be used in relation to polymeric systems such as polypeptides, polynucleotides, and polysaccharides. Methods for calculating the radius of gyration are described in Andrieux et al. Analytical Chemistry 2002, 74, 5217, which is incorporated herein by reference.
[081] A capsule comprising an encapsulated component can be prepared using the methods described herein. Once the capsule (with encapsulant) is prepared, the capsule and its aqueous surroundings can be analyzed for loss of material from the inside of the shell outwards to the outer aqueous phase. Encapsulated compounds may have an analytical label to aid detection. Suitable labels include fluorescent labels that are detectable using standard fluorescence microscopy techniques.
[082] In one embodiment, dextran compounds of different molecular weight can be used as test compounds to determine the pore size of a formed capsule. The dextran can be labeled, and preferably with a fluorescent label.
[083] Dextran compounds of different molecular weight are readily available from commercial sources, including, for example, Sigma Aldrich. Dextrans having an average molecular weight of 1,000 to 500,000 are available. Dextran with a molecular weight of 70 kDa has a swivel radius of approximately 8 nm, while dextran with a molecular weight of 150 kDa has a swivel radius of approximately 11 nm (see Granath Journal of Colloid Science 1958, 13, 308 ). Dextran compounds having a fluorescent label, such as fluorescein isothiocyanate, are also available from commercial sources, including, again, Sigma Aldrich.
[084] In one embodiment, the pore size is a maximum of 20, a maximum of 15, a maximum of 10, a maximum of 5, a maximum of 1 or a maximum of 0.5 μm.
[085] In one embodiment, the pore size is a maximum of 500, a maximum of 200, a maximum of 100, a maximum of 50, or a maximum of 20 nm.
[086] In one embodiment, the pore size is at least 0.5, at least 1, or at least 5 nm.
[087] In one embodiment, the pore size is in a range where the minimum and maximum pore sizes are selected from the previous embodiments. For example, the pore size is in the range of 1 to 20 nm.
[088]As an alternative to dextran, protein standards can be used. As an alternative to the previously described labeled compounds, it is also possible to detect the compound released from the capsule using spectroscopy, or protein gel electrophoresis (for protein standards).
[089]Surface area, porosity and pore size can also be determined experimentally using BET gas absorption techniques.
[090]As expected, the pore size of the shell is influenced by the amount of cucurbituril present in the complexable composition from which the capsule can be prepared. Increasing the amount of cucurbituryl present in the complexable composition is believed to increase the amount of crosslinking with the network, thus reducing the pore size in the formed shell material.
[091] The capsule shell may comprise one or more layers of material. The layers of material can be linked, for example, by a supramolecular cucurbituryl ternary complex with a first visiting molecule present in one layer and a second visiting molecule present in a second layer. Additionally, or alternatively, the layers of material can be linked by a first building block having a plurality of visiting molecules, where a visiting molecule forms a ternary complex with a cucurbituryl and another visiting molecule present in a first layer, and another visiting molecule. forms a ternary complex with a cucurbituril and another visitor molecule present in a second layer. In these embodiments, the enclosure can be seen as a mesh extending in three dimensions. While the housing may have a depth of material, such as a thickness described herein, it is understood that the formation of the housing will nevertheless provide an internal space in which a component can reside. Therefore, the present invention is not intended to encompass particles having no internal space.
[092] Alternatively, the capsule shell may comprise a plurality of concentric layers of mesh material which are not interconnected. In either embodiment, reference to capsule size refers to the cross-section of the outermost shell.
[093]As discussed above, the housing material may include detectable markers or detectable features.
[094]A detectable functionality is a functionality of a capsule shell component having a characteristic that is detectable with respect to characteristics that are present in other components of the capsule, or even other functionality of the same component. Detectable functionality can refer to a particular chemical group that produces a unique signal, for example in IR, UV-VIS, NMR or Raman analysis. The functionality may be a radioactive element.
[095] Typically, a part of the casing material or encapsulant is provided with a detectable marker, since the introduction of a chosen marker allows the use of techniques that are more appropriate for the property to be measured. Building blocks having detectable fluorescent labels are described. Also described are building blocks that are capable of providing an enhanced surface resonance effect.
[096]The enclosure may have additional functionality on its inner and/or outer surfaces. Building blocks are described having functionality to improve solubility, assist detection, reactive functionality for future wrapping, and catalysis, among others.
[097] The capsule shell of the invention is stable and can be stored without loss of shell structure. Therefore, the integrity of the shell allows the capsule to be used as a storage container for an encapsulant. The capsules of the invention are thermally stable and the shell is known to maintain its integrity at least up to 100°C. The capsules of the invention are also stable at reduced pressures (i.e. below ambient pressure). The casing is known to maintain its integrity to at least 20 Pa.
[098] The capsules of the invention have a long shelf life. The present inventors have confirmed that structural integrity is maintained for at least 10 months.
[099]The structural integrity of the casing is due, in part, to the strength of the cucurbituril visitor-host complex, which will be described in more detail below. Additional or alternative capsule features
[0100]Capsule shell has pores. Porosity is adjustable by appropriate changes in the stoichiometry of the reagents used to form the capsule. Increasing the crosslinking between the building blocks will reduce the pore size in the capsule. Alternatively, the building blocks may be selected to provide a shell material that has increased or reduced porosity. When a relatively small size encapsulant needs to be encapsulated, the capsule is prepared with relatively small diameter pores, to thereby limit or prevent leakage of the encapsulant out of the shell. When a relatively large encapsulant needs to be encapsulated, the pore size can be larger.
[0101]As noted earlier, the housing may have additional functionality on its inner and/or outer surfaces. In some embodiments, the functionality is provided for further chemical functionalization of the capsule shell, for example, as a reaction site for binding to a compound having a particularly desirable reactivity.
[0102] In one embodiment, the shell has a chemical functional group available for reaction on the outer and/or inner surface of the capsule. The chemical functional group is selected from the group consisting of hydroxyl, amine (preferably primary and secondary amine), carboxy, thiol, ester, thioester, carbonate, urethane, and thiourea.
[0103] In one embodiment, the shell is bonded to a functional compound.
[0104] In one embodiment, the functional compound is an analytical marker to aid in the detection and quantification of the capsule. This is described in the previous section.
[0105] The functional compound may be catalytic (including enzymatic), anti-fungal, herbicide, or antigenic.
[0106]The functional compound may have surface adhesion properties. This functionality can be used to attach the capsule to a surface, either covalently or non-covalently.
[0107]Functional compounds may be able to bind (or sequester) a compound or ion. This functionality can be of assistance in purification, such as filtration, and for the capture of toxic and non-toxic elements and compounds.
[0108] In one embodiment, the functional compound is a biomolecule.
[0109] In one embodiment, the functional compound is a polypeptide, a polysaccharide, a polynucleotide, or a lipid.
[0110]Examples of polypeptides include enzymes, antibodies, hormones, and receptors.
[0111]Function can be introduced into the housing by the appropriate choice of a building block material. Therefore, when the building block is a polymer, suitable functionality can be incorporated into the polymer's monomers, such monomer being present in the polymer backbone, or in a side chain. When the building block is a particle, the surface of such a particle can be suitably functionalized.
[0112]When a functional molecule is present on a surface of the shell, this molecule can be added after the capsule is formed. Functional molecules can be attached to the shell using a chemical functional group that has been introduced for this purpose.
[0113]In principle, cucurbituril may have a functionality that is available for reaction. However, this may be less preferred.
[0114] When necessary, appropriate protection groups can be used to protect functionality during the capsule preparation procedure. Protection groups can be removed later as and when needed. Complex
[0115]The capsule shell comprises a mesh that is held together by a supramolecular cuff. The complex that forms this supramolecular handcuff is based on a cucurbituryl that hosts one visitor (binary complex) or two visitors (ternary complex). Cucurbituril forms a non-covalent bond to each visitor. The present inventors have established that cucurbituril complexes are readily formed and provide robust non-covalent bonds between building blocks. Complex formation is tolerant of many functionality within the building blocks. One of the present inventors has demonstrated that polymer networks can be prepared using a cucurbituryl cuff. However, until now, the formation of precise polymer structures, such as capsules, using cucurbituril has been described.
[0116] In one embodiment, the shell is a network having a plurality of complexes, each complex comprising cucurbituryl that hosts a first visiting molecule and a second visiting molecule. The first and second visiting molecules are covalently linked to a first building block, or to a first building block and a second building block.
[0117]When the complex comprises two visitors within the cucurbituril 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.
[0118]When a cucurbituryl hosts two visiting molecules, the visiting molecules may be the same or they may be different. A cucurbituril that is capable of hosting two visiting molecules may also be able to form a stable binary complex with a single visitor. It is believed that the formation of a ternary visitor-host complex proceeds through an intermediate binary complex. Within the shell, a binary complex formed between a visiting molecule and a cucurbituril may be present. The binary complex can be considered as a partially formed ternary complex that has not yet formed a non-covalent bond to the other visiting molecule.
[0119] In one embodiment, the shell is a network having a plurality of complexes, wherein each complex comprises cucurbituryl hosting a visiting molecule, and each cucurbituryl is covalently linked to at least one other cucurbituryl. Visitor molecules are covalently linked to a first building block, or to a first building block and a second building block.
[0120]When the complex comprises a visitor within the cucurbituril cavity, the association constant, Ka, for such a complex is at least 103 M1, of at least 104 M-1, of at least 105 M-1, of 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.
[0121] In one embodiment, the visitor is a compound capable of forming a complex that has an association constant in the range of 104 to 107 M-1.
[0122]In one embodiment, the formation of the complex is reversible. Decomplexing the complex to separate the visitor or visitors may occur in response to an external stimulus, including, for example, a competing visitor compound. Such decomplexation can be induced in order to provide additional or larger pores in the capsule through which an encapsulated material can pass.
[0123]As noted earlier in relation to the capsule shell, the cucurbituril complex with one or two visitors is the non-covalent bond that links and/or interconnects the building blocks to form a supramolecular network of material. The complex is thermally stable and does not separate under reduced pressure as explained for the shell. Network
[0124] The formation of a supramolecular complex serves to link and/or interconnect the building blocks, thus forming a network of material. This is the capsule shell.
[0125]Two types of network are provided. The first type is based on the formation of a plurality of ternary complexes, each complex comprising a cucurbituril host with a first visiting molecule and a second visiting molecule. The second type is based on the formation of a plurality of binary complexes, each complex comprising a cucurbituril host with a first visiting molecule. In this second type, each cucurbituryl is covalently linked to at least one other cucurbituryl. These types of nets can be combined with an enclosure.
[0126]When a building block is provided with a plurality of visiting molecules, all the visiting molecules do not need to participate in a complex with cucurbituril. When the lattice is based on a link between ternary structures, a visiting molecule from a building block can be in a binary complex with a cucurbituryl. The binary complex can be thought of as a partially formed ternary complex that has not yet been combined with an additional visiting molecule to generate the ternary form.
[0127] Throughout the description, references are made to a building block, a first building block, and a second building block. It is understood that a reference to this is a reference to a collection of individual molecules, particles, polymers, etc. which are the building blocks. When a reference is intended for an individual building block molecule, particle, etc. the term “single” is used in reference to building blocks, for example a single first building block.
[0128]The networks described below are the basic networks that are obtainable from the compositions described. It is understood that the present invention extends to more complex networks obtainable from compositions comprising additional building blocks. Cucurbituril-based ternary complex network
[0129]This network is obtainable by assembling a first visiting molecule and a second visiting molecule together with a cucurbiturilla host. Visitor molecules can be provided in one or two (or more) building blocks as described below.
[0130] In one embodiment, a network is obtainable or obtained from complexing a composition comprising a cucurbituryl, a first building block covalently linked to a plurality of first visiting cucurbituryl molecules, and a second building block covalently linked to a plurality of second visitor molecules of cucurbituril, wherein a first visitor molecule and a second visitor molecule together with cucurbituril are suitable to form a ternary visitor-host complex.
[0131]The ternary complex serves to non-covalently link the first and second building blocks. A single first building block may form a plurality of non-covalent bonds to a plurality of second building blocks. Similarly, a single second building block may form a plurality of non-covalent bonds to a plurality of first building blocks. In this way, a material network is established.
[0132] It is noted that in some embodiments, the first and second visiting molecules may be identical. Therefore, the first and second building blocks may differ in their composition. In some embodiments, the first and second building blocks may be identical. In this case, the first and second visiting molecules are different.
[0133]Shown below is a schematic structure of a basic network formed between cucurbituril, a single first building block and two Punic second building blocks. In the schematic diagrams included in this text, visiting molecules are depicted as rectangles that are covalently bonded (vertical line) to a building block (horizontal line). The vertical line may depict a direct covalent bond or a ligand to the building block. The building block can be a polymeric molecule, a particle or the like, as described herein.
[0134]In the schematic diagram below, some of the first visiting molecules (shaded rectangles) from the first building block are in the complex with cucurbituril hosts (barrels) and the second visiting molecules (shaded rectangles) from the second building blocks.

[0135] It is apparent that not all the visiting molecules present participate in a complex in the final network. Each of the first and second building blocks can form complexes with other second and first building blocks, respectively. Visitor molecules are shaded for ease of understanding. However, as explained herein, the visiting molecules of the first and second building blocks may be the same.
[0136] In an alternative embodiment, a network is obtainable or obtained from complexing a composition comprising a cucurbituryl and a first building block covalently linked to a plurality of first visiting cucurbituril molecules and a plurality of second visiting molecules of cucurbituril. cucurbituril, in which a first and second visitor molecule together with cucurbituril are suitable to form a ternary visitor-host complex.
[0137]The ternary complex serves to link and/or non-covalently interconnect the first building block. A single first building block may form a plurality of non-covalent bonds to a plurality of other first building blocks. Additionally, or alternatively, a single first building block may form a plurality of non-covalent interconnects with itself to thereby cross-link the single first building block.
[0138]As before, the first and second visiting molecules may be identical.
[0139] Shown below is a schematic structure of a basic network formed between cucurbituril and two unique first building blocks each having a plurality of first and second visiting molecules. Some of the first visiting molecules (unshaded rectangles) from the first building block are in the complex with cucurbituryl hosts (barrels) and second visiting molecules (shaded rectangles) from another first building block. It can be seen from the illustrated lattice that a first building block can form intramolecular complexes, thus crosslinking a single first building block.

[0140] It becomes apparent that not all visiting molecules present need to participate in a complex in the final network. Each of the first building blocks can form complexes with other first building blocks, or with other parts of the same building block. As explained herein, the first and second visiting molecules may be the same.
[0141]Optionally, the composition further comprises a second building block covalently linked to one or more third visiting cucurbituril molecules, one or more fourth visiting cucurbituril molecules, or both, wherein a third and fourth molecules together with cucurbituril are suitable to form a ternary visitor-host complex, or the first and fourth visitor molecules together with cucurbituril are suitable to form a ternary visitor-host complex, or the second and third visitor molecules together with cucurbituril are suitable to form a visitor complex - ternary host.
[0142]When the second building block is provided with a plurality of third and fourth visiting molecules, the ternary complex serves to non-covalently link and/or interconnect the second building block. A single second building block may form a plurality of non-covalent bonds to a plurality of other second building blocks. Additionally, or alternatively, a single second building block may form one or more non-covalent interconnects with itself to thereby cross-link the single second building block.
[0143]The third and fourth visiting molecules may be suitable to form complexes with the first and second visiting molecules of the first building block. In one embodiment, the first and third visiting molecules are the same. In one embodiment, the second and fourth visiting molecules are the same. In the present document, the ternary complex serves to non-covalently link the first and second building blocks, for example, through a complex of the first and fourth visiting molecules and/or through a complex of the second and third visiting molecules.
[0144]Therefore, a single first building block can form a plurality of non-covalent bonds to a plurality of second building blocks. Similarly, a single second building block may form a plurality of non-covalent bonds to a plurality of first building blocks. In this way, a material network is established. Building blocks can also form non-covalent intermolecular bonds as described previously.
[0145]When a second building block is covalently linked to one or more third visiting molecules or one or more fourth visiting molecules, the first and fourth molecules together with cucurbituril are suitable to form a ternary visitor-host complex, and the second and third molecules together with cucurbituril are suitable to form a ternary visitor-host complex. Therefore, the ternary complex serves to non-covalently link the second building block to the first building block.
[0146] Shown below is a schematic structure of a basic network formed between cucurbituril, three single first building blocks each having a plurality of first and second visiting molecules, and two second building blocks each having a plurality of third and fourth visiting molecules. Some of the first visiting molecules (unshaded rectangles) from the first building block are in the complex with cucurbituryl hosts (barrels) and the second visiting molecules (shaded rectangles) from another first building block. Some of the third visiting molecules (partially shaded rectangles) from the second building block are in the complex with cucurbituril hosts (barrels) and the fourth visiting molecules (dashed rectangles) from another second building block. A first visiting molecule from the first building block is in the complex with a cucurbituryl host and a fourth visiting molecule (dashed rectangles) from a second building block. A second visiting molecule from the first building block is in the complex with a cucurbituril host and a third visiting molecule from a second building block.

[0147]The first and third visiting molecules may be the same. The second and fourth visiting molecules may be the same.
[0148]A second building block can be covalently linked to a visitor molecule (which can be a third or fourth visitor molecule). In this embodiment, the second building block is not capable of forming a plurality of links to other building blocks. As such, the building block would not contribute to the formation of crosslinks within the network. However, the second building block may be provided in order to introduce into the network a particular physical or chemical characteristic that is possessed by the second building block. For example, the second building block may comprise a detectable label or a functional group, such as a solubilizing group. Therefore, the incorporation of the second building block into the network allows modification of the physical or chemical characteristics of the overall network.
[0149]Shown below is a schematic structure of a basic network formed between cucurbituril, two unique first building blocks each having a plurality of first and second visiting molecules, and also including a single second building block, which is covalently linked to a fourth visiting molecule, and a detectable label. Some of the first visiting molecules (unshaded rectangles) from the first building block are in the complex with cucurbituryl hosts (barrels) and second visiting molecules (shaded rectangles) from another first building block. A first visiting molecule from the first building block is in the complex with a cucurbituril host and a fourth visiting molecule. The detectable marker (partially shaded circle) may be provided to allow identification of the resulting network.
Binary complex network based on a plurality of covalently linked cucurbituryls
[0150]This network is obtainable from the assembly of a first visitor molecule together with a cucurbituryl host, which host is covalently linked to one or more other cucurbituryls. Visitor molecules may be provided in one, or two (or more) building blocks as described herein.
[0151] The covalently linked cucurbituryls serve to link the building block molecules through the plurality of complexes that are formed within each of the covalently linked cucurbituryls.
[0152] Shown below is a schematic structure of a basic network formed between a plurality of covalently linked cucurbituryls and two single first building blocks each having a plurality of visiting first molecules. Some of the first visiting molecules (unshaded rectangles) of every single first building block are in a binary complex with cucurbituril (barrel) hosts. The cucurbituryls are linked, to thereby form a link between each of the first building blocks.

[0153] It becomes apparent that not all visiting molecules present need to participate in a complex in the final network. Each of the single first building blocks may form complexes with other first building blocks respectively, or may form an intramolecular cross-link with another portion of the same building block. As explained herein, the visiting molecules of the first and second building blocks may be the same. In the above schematic structure, one of the first building blocks can be replaced by a second building block which is covalently linked to a second visiting molecule. The second visiting molecule is the one capable of forming a binary complex with cucurbituril. The second visiting molecule may be the same as the first visiting molecule.
[0154]In the schematic structure, two cucurbituryls are shown linked together. The present invention encompasses the use of systems where more than two cucurbiturillas are linked together. For example, multiple cucurbituryls can be dangling from a polymer molecule. Network of ternary complexes based on a plurality of covalently linked cucurbituryls
[0155] It will be apparent from the description of the networks above that each of the cucurbituryl hosts in the plurality of covalently linked cucurbituryls may be suitable for forming ternary complexes. Therefore, the plurality of covalently linked cucurbituryls can be used instead of the cucurbituryl described for use in the cucurbituril-based ternary complex network.
[0156] Shown below is a schematic structure of a basic network formed between a plurality of covalently linked cucurbituryls, two unique first building blocks each having a plurality of first visiting molecules, and two unique second building blocks each having a plurality of second visiting molecules. Some of the first visiting molecules (unshaded rectangles) from the first building block are in a tertiary complex with a cucurbituril host (barrel) and the second visiting molecules (shaded rectangles) from the second building block. The cucurbititurils are bonded, to thereby form a bond between each of the first and second building blocks.

[0157]As before, the first and second visiting molecules may be the same. Each of the first and second building blocks can form complexes with other second and first building blocks respectively. Other permutations are possible, for example, where the plurality of covalently linked cucurbituryls has more than two cucurbituryls. other networks
[0158] Below are described basic networks of the invention that are obtained or obtainable from the described compositions. It will be clear to one skilled in the art that the described compositions may include additional building blocks, for example third and fourth building blocks, each linked to one or more cucurbituril visiting molecules. The present invention also covers capsules where the shell comprises a mixture of any of the above-described networks. These are obtainable from compositions comprising an appropriate selection of cucurbituryl, covalently linked cucurbituryls, first building block and second building block as appropriate.
[0159] The invention also relates to a capsule having a shell consisting of a network comprising different cucurbituryls. Different cucurbiturils can be chosen in order to obtain a network that is based on ternary and binary complexes. Different cucurbituryls can be chosen in order to generate networks that result from the selective complexation of each cucurbituryl to different visiting molecules, which may be present in the same or different building blocks. cucurbituril
[0160] The present invention provides the use of cucurbituril as a supramolecular shackle to link and/or cross-link building blocks. Cucurbituril can be used to form ternary complexes with first and second visiting molecules present in one or more building blocks. The formation of these complexes links individual building blocks to thereby form a network of material. This net is the shell of the capsule.
[0161] In addition, or alternatively, a plurality of covalently linked cucurbituryls are provided and each cucurbituryl can be used to form binary complexes with a visiting molecule present in one or more building blocks. The formation of a binary complex with each of the covalently bound cucurbituryls thereby forms a network of material. This net is the shell of the capsule.
[0162] In one embodiment, cucurbituril is capable of forming a ternary complex. For example, CB[8], is capable of forming a ternary complex.
[0163] In one embodiment, cucurbituril is capable of forming a binary complex. For example, CB[7], is capable of forming a binary complex.
[0164] In one embodiment, cucurbituril is capable of forming ternary and binary complexes. For example, CB[8] is capable of forming a ternary complex or a binary complex, depending on the nature of the visitor.
[0165]In one embodiment, cucurbituril is a compound CB[5], CB[6], CB[7], CB[8], CB[9], CB[10], CB[11] or CB[12 ].
[0166]In one embodiment, cucurbituril is a CB[6], CB[7], or CB[8] compound.
[0167]In one embodiment, cucurbituril is a CB[8] compound.
[0168] In one embodiment, references to a cucurbituril compound are references to variants and derivatives thereof.
[0169]Cucurbituril compounds differ in their solubility in water. Capsule preparation methods can be adapted to take this solubility into account, as described below. Therefore, the choice of cucurbituril compound is not limited by its aqueous solubility.
[0170] In one embodiment, the cucurbituril 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.
[0171] In one embodiment, solubility refers to aqueous solubility (ie, an aqueous phase).
[0172] In one embodiment, solubility refers to solubility in a water-immiscible phase, such as an oil phase or an organic phase.
[0173]Cucurbit[8]uryl (CB[8]; CAS 259886-51-6) is a barrel-shaped container molecule that has eight repeating glycoluril units 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, MO, USA).

[0174]In other aspects of the invention, CB[8] variants are provided and find use in the methods described herein.
[0175]A CB[8] variant may include a structure having one or more repeat units that are structurally analogous to glycoluril. The repeating unit may include an ethyl urea unit. Where all units are ethyl urea units, the variant is a hemicucurbituryl. The variant may be a hemicucurbit[12]uryl (shown below, see also Lagona et al. Angew. Chem. Int. Ed. 2005, 44, 4844).

[0176]In other aspects of the invention, the cucurbituril derivatives are provided and find use in the methods described herein. A derivative of a cucurbituryl is a structure having one, two, three, four or more substituted glycoluril 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 glycoluril unit each X is O, S or NR3 , and -R1 and -R2 are independently selected from -H and the following optionally substituted substituted groups: -R3 , -OH, -OR3 , -COOH, -COOR3, -NH2, -NHR3 and -N(R3)2 where -R3 is independently selected from C1-20 alkyl, C6-20 carboaryl, and C5-20 heteroaryl, or where -R1 and/or -R2 is -N(R3)2, both -R3 together form a C5-7 heterocyclic ring; or together -R1 and -R2 are C4-6 alkylene forming a C6-8 carbocyclic ring together with the uracil backbone.
[0177] In one embodiment, one of the glycoluril units is a substituted glycoluril unit. Therefore, -R1 and -R2 are independently -H for n-1 of the glycoluril units
[0178] In one embodiment, n is 5, 6, 7, 8, 9, 10, 11 or 12.
[0179] In one embodiment, n is 5, 6, 7, 8, 10 or 12.
[0180]In one embodiment, n is 8.
[0181] In one embodiment, each X is O.
[0182] In one embodiment, each X is S.
[0183]In one embodiment, R1 and R2 are independently H.
[0184] In one embodiment, for each unit of one R1 and R2 is H and the other is independently selected from -H and the following optionally substituted groups: -R3, -OH, -OR3, -COOH, -COOR3 , -NH2 , -NHR3 and -N(R3)2. In one embodiment, for one unit of one R1 and R2 is H and the other is independently selected from -H and the following optionally substituted groups: -R3, -OH, -OR3, -COOH, -COOR3, -NH2 , -NHR3 and -N(R3)2. In this embodiment, the remaining glycoluril units are such that R1 and R2 are independently H.
[0185] Preferably, -R3 is C1-20 alkyl, most preferably C1-6 alkyl. The C1-20 alkyl group may be linear and/or saturated. Each -R3 group may 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 from C1-20 alkyl, C6-20 carboaryl, and C5-20 heteroaryl. The substituents may be independently selected from -COOH and -COOR4.
[0186]In some embodiments, -R4 is not equal to -R3. In some embodiments, -R4 is preferably unsubstituted.
[0187]Where -R1 and/or -R2 is -OR3, -NHR3 or -N(R3)2, then -R3 is preferably C1-6 alkyl. In some embodiments, -R3 is substituted with a substituent -OR4, -NHR4, or -N(R4)2. Each -R4 is C1-6 alkyl and is preferably substituted.
[0188] In some embodiments of the invention, the use of a plurality of covalently linked cucurbituryls is provided. These covalently linked cucurbituryls are suitable for forming networks based on the complexation of cucurbituril with visiting molecules of a building block. The complexes formed can be ternary or binary complexes.
[0189]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 glycoluril units on the cucurbituryl as depicted in the structure shown above. There are no particular limitations to the covalent bonding between cucurbituryls. The linker may be in the form of a single alkylene group, a polyoxyalkylene group, or a polymer, such as a polymer molecule described herein for use in the building block. When the linker is a polymeric molecule, the cucurbituryls may be pendant to such a polymer. building block
[0190]Cucurbituril is used as a supramolecular handcuff to join one or more building blocks. The formation of a cucurbituril complex with suitable visitor components that are linked to the building blocks forms a material network. This material is the capsule shell. The complex non-covalently cross-links the building block or non-covalently links the building block to another building block.
[0191] It is understood from this that a building block is an entity that serves to provide structure to the network formed. The building block also serves as the link between a plurality of visiting molecules, and can therefore be referred to as a ligand. In some some embodiments, a building block is provided for the purpose of introducing a desirable physical or chemical characteristic into the network formed. As mentioned above in relation to the network, a building block may include functionality to aid in the detection and characterization of the enclosure. These building blocks do not necessarily need to participate in a crosslink.
[0192] A building block, such as a first building block, can be covalently linked to a plurality of cucurbituril visiting molecules. Therefore, a building block will non-covalently bond to a plurality of cucurbituryls, such cucurbituryls to non-covalently bond to other building blocks, to thereby generate a network of material.
[0193] A building block, such as a first building block or a second building block, can be covalently linked to a plurality of cucurbituril visiting molecules. In one embodiment, a building block is covalently bonded to at least 3, at least 4, at least 5, at least 10, 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 cucurbituril visiting molecules.
[0194] In certain embodiments, the building blocks may be used covalently linked to one or more cucurbituril visiting molecules. However, such building blocks are only used in combination with other building blocks that are covalently linked to at least two cucurbituril visiting molecules.
[0195] In one embodiment, a first building block is provided covalently linked to a plurality of first visiting molecules and a second building block covalently linked to a plurality of second visiting molecules. Each of the first and second building blocks can be covalently linked to at least the number of visiting molecules described above.
[0196] In one embodiment, a first building block is provided covalently linked to a plurality of first visiting molecules and covalently linked to a plurality of second visiting molecules.
[0197]The first building block can be covalently linked to at least a number of visitor molecules described above, such numbers may independently refer to the number of first visiting molecules and the number of second visiting molecules.
[0198] In one embodiment, a second building block is provided covalently linked to one or more third guest molecules and/or covalently linked to one or more fourth guest molecules. In one embodiment, the second building block is covalently linked to at least a number of visiting molecules described above, such numbers may independently refer to the number of third visiting molecules and the number of fourth visiting molecules. This second building block can be used together with the first building block described in the previous paragraph.
[0199] Throughout the description, references are made to the first and second building blocks. In some embodiments, the first and second building blocks can be distinguished from each other due to differences, at least, in the structure of the building blocks themselves. In some add embodiments, the structures of the first and second building blocks are the same. In this case, the building blocks can be distinguished from each other due to differences at least in the visiting molecules that are covalently bound to each other between the first and second visiting molecules. Therefore, the terms first and second are meant to communicate a difference between the first building block together with its visiting molecules and the second building block together with its visiting molecules.
[0200]Building blocks are not particularly limited, and the building block includes compounds and particles, and can encompass assemblies of these. Visitor molecules are covalently bound to some portion of the building block.
[0201] In its simplest form, a building block is a ligand for connecting visiting molecules.
[0202] In one embodiment, the building block is a polymeric molecule or a particle.
[0203] Advantageously, a building block can be provided with certain functionality to assist in the formation of the capsule shell, or improve its physical or chemical properties.
[0204] In one embodiment, the building block is provided with functionality to alter, or, preferably, improve, water solubility. The functionality may take the form of a solubilizing group, such as a group comprising a polyethylene glycol functionality. Other examples include groups that comprise an amino, hydroxy, thiol, and carboxy functionality.
[0205] In one embodiment, the building block is provided with functionality to assist in detecting or analyzing the building block, and assisting in detecting or analyzing the formed shell. Advantageously, this functionality can also assist in detecting material encapsulated within the housing. The functionality may take the form of a detectable label, such as a fluorescent label.
[0206] In one embodiment, the building block is provided with reactive functionality for use in further elaboration of the shell material. Reactive functionality can be shielded to the wrapper that forms reactions, then later unshielded to reveal the functionality. The functionality can be a group comprising an amino, hydroxy, thiol, and carboxy functionality.
[0207]When the building block is endowed with a reactive functionality, this functionality may be suitable for linking the building block (and therefore the formed capsule) to a surface.
[0208] In one embodiment, the building block is provided with a catalyst for further use in catalyzing a reaction at or near the surface of the shell. The catalyst may be provided on the inner or outer edges of the housing to thereby catalyze the inner and/or outer reactions.
[0209] In one embodiment, the building block is chosen for its ability to influence the optical-electronic properties of the encapsulant. Additionally or alternatively, the building block may be chosen for its ability to be influenced by the encapsulant. The building block may be suitable for transferring signals from the encapsulant to the external environment.
[0210] In one embodiment, a building block is capable of providing an accentuated surface resonance effect.
[0211] When functionality is provided, it can be located on the outside, on the inside and/or inside the housing. Therefore, functionality may be provided in connection with improvements related to the environment outside the enclosure, within the internal space (the space to hold an encapsulant) of the enclosure, and/or within the enclosure (within the net of enclosure material).
[0212]For the purposes of the methods described herein, the building block, together with the visiting molecules to which it is covalently bound, must be soluble, for example, in the second phase.
[0213] In one embodiment, the building block 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.
[0214] In one embodiment, solubility refers to aqueous solubility (ie, an aqueous phase).
[0215] In one embodiment, solubility refers to solubility in a water-immiscible phase, such as an oil phase or an organic phase.
[0216] A building block is linked to a cucurbituril visitor or visitor molecules by covalent bonds. The covalent bond can be a carbon-carbon bond, a carbon-nitrogen bond, a carbon-oxygen bond. The bond may be part of a linking group, such as an ester or an amide, and/or part of a group comprising an alkylene or alkoxylene functionality.
[0217]Each visiting molecule can be linked to the building block using routine chemical bonding techniques. For example, visiting molecules can be linked to the building block by: alkylating a building block carrying a leaving group; esterification reactions; amidation reactions; ether forming reactions; olefin cross metathesis; or small visitor-initiated reactions in which a polymer chain is developed out of an initiating visitor molecule.
[0218] In one embodiment, the average molecular weight of a building block, optionally along with any visiting molecules, is at least 1,000, at least 5,000, at least 10,000, or at least 20,000.
[0219]In one embodiment, the average molecular weight of a building block, optionally together with any visiting molecules, is at most 30,000, at most 50,000, at most 100,000, at most 200,000, at most 500,000, at most 1,000,000 , or a maximum of 2,000,000.
[0220]The average molecular weight can refer to the number average molecular weight or the weight average molecular weight.
[0221]In one embodiment, the average molecular weight of a building block is in a range where minimum and maximum amounts are selected from the above embodiments. For example, the average molecular weight is in the range of 1,000 to 100,000.
[0222] In one embodiment, a building block is capable of providing an accentuated surface resonance effect. Typically, this ability is provided by a particle, and more particularly, a metal-containing particle. Such particles are those described herein. Most suitable are those particles that are capable of providing a surface accentuated effect for surface accentuated Raman spectroscopy.
[0223]Described below are building blocks that are based on polymeric molecules and particles, including nanoparticles.
[0224] In one embodiment, when the network is obtainable from a composition comprising first and second building blocks, the first building block is a polymeric molecule and the second building block is a polymeric particle or molecule. In one embodiment, when the network is obtainable from a composition comprising first and second building blocks, the first building block is a polymeric molecule and the second building block is a particle.
[0225] In one embodiment, when the network is obtainable from a composition comprising a first, the first building block is a polymeric molecule. polymeric molecule
[0226] In one embodiment, a building block is a polymeric molecule.
[0227] Polymeric compounds that are covalently linked to cucurbituril visiting molecules are known from WO 2009/071899, which is incorporated herein by reference.
[0228] Polymer molecules comprise a plurality of repeating structural units (monomers) that are connected by covalent bonds. Polymer molecules can comprise a single type of monomer (homopolymers), or more than one type of monomer (copolymers). Polymer molecules can be linear or branched. When the polymer molecule is a copolymer, it may be a random, alternative, periodic, statistical, or block polymer, or a mixture thereof. The copolymer may also be a graft polymer.
[0229] In one embodiment, the polymeric molecule has 2, 3, 4 or 5 repeating units. For reasons of convenience, such a polymer may be referred to as an oligomer.
[0230] In other embodiments, the polymer molecule has at least 4, at least 8, at least 15, at least 100, or at least 1,000 monomer units. The number of units can be an average number of units.
[0231] In other embodiments, the polymer molecule has an average number of monomer units in a selected range of 10 to 200, 50 to 200, 50 to 150, or 75 to 125.
[0232]The number of visiting molecules per polymer molecule building block is as prepared above. Alternatively, the number of visiting molecules can be expressed as the percentage of monomers present in the polymer that are attached to the visiting molecules as a total of all monomers present in the polymer molecule. This can be referred to as a percentage of functionality.
[0233] In one embodiment, the functionality of a polymeric molecule is at least 1%, at least 2%, or at least 5%.
[0234] In one embodiment, the functionality of a polymer molecule is at most 50%, at most 40%, at most 20%, at most 15, or at most 10%.
[0235]In one mode, the functionality is in a range where the minimum and maximum amounts are selected from the above modes. For example, functionality is in the range of 5 to 40%.
[0236]Percent of functionality can be determined from proton NMR measurements of a polymer sample.
[0237] In one embodiment, the polymer molecule has a molecular weight (Mw) greater than 500, greater than 1000, greater than 2000, greater than 3000, or greater than 4000. The molecular weight may be the weight average molecular weight or the weight number average molecular. The number and weight average molecular weights of a polymer can be determined by conventional techniques.
[0238] In one embodiment, the polymer is a synthetic polydisperse polymer. A polydisperse polymer comprises polymeric molecules having a range of molecular weights. The polydispersity index (PDI) (weight average molecular weight divided by number average molecular weight) of a polydisperse polymer is greater than 1, and can be in the range of 5 to 20. The polydispersity of a polymer molecule can be determined by conventional techniques such as gel penetration or size exclusion chromatography.
[0239] Suitable for use in the present invention are polymer molecules having a relatively low polydispersity. These polymer molecules may have a polydispersity in the selected range of 1 to 5, 1 to 3, or 1 to 2. These polymers may be referred to as low-dispersed or monodispersed in view of their relatively low dispersion.
[0240] The use of low-dispersion or monodisperse polymer molecules is particularly attractive, as 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 dispersed or monodispersed. Methods for preparing low dispersed or monodisperse polymers are well known in the art, and include radical-initiated polymerization-based polymerization reactions, including RAFT (Reversible Addition Fragmentation Chain Transfer) polymerization (see, for example, , Chiefari et al. Macromolecules 1998, 31, 5559 ). An exemplary synthesis of a polymer having a low dispersion is also provided herein.
[0241] Many polymeric molecules are known in the art and can be used to produce a shell material as described herein. The choice of polymer molecule will depend on the particular application of the capsule. 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), poly(meth)acrylate, polystyrene, polyacrylamide, and polyvinyl alcohol. The polymer may be a homopolymer or a copolymer.
[0242]The polymeric molecule may comprise two or more natural and/or synthetic polymers. These polymers can be arranged in a linear architecture, cyclic architecture, comb or graft architecture, (hyper)branched architecture or star architecture.
[0243] Suitable polymeric molecules include those polymeric molecules having hydrophilic characteristics. Therefore, a part of the polymer, which part refers to, among others, a monomer 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 polymer molecule is soluble in water to form a continuous phase.
[0244] In one embodiment, the polymer molecule is amphiphilic.
[0245] When two or more building blocks are provided, such as a first and a second building block, each building block can be independently selected from the polymer molecules described above. In one embodiment, the first and second building blocks are different. In one embodiment, the first and second building blocks are the same. In the latter case, the building blocks themselves are different only with respect to the visiting molecules that are covalently linked together.
[0246] In one embodiment, the polymer molecule is or comprises a polymer of poly(meth)acrylate, polystyrene and/or poly(meth)acrylamide.
[0247] In one embodiment, the polymer is or comprises a poly(meth)acrylate polymer, which may be or comprise a polyacrylate polymer.
[0248]The acrylate functionality of the (meth)acrylate may be the site to connect the desired functionality, for example, to connect a solubilization group or a detectable label.
[0249] In one embodiment, the polymeric molecule is obtained or obtainable from a polymerizable composition comprising: (i) monomer, such as a (meth)acrylate or a styrene, which is linked to a cucurbituril visitor molecule; and, optionally, further comprises: (ii) a monomer, such as a (meth)acrylate or a styrene, which is linked to a detectable label; and/or (iii) a monomer, such as a (meth)acrylate or a styrene, which is attached to a solubilizing group, such as an aqueous solubilizing group.
[0250] In one embodiment, each monomer is a (meth)acrylate monomer.
[0251] In one embodiment, each monomer is a styrene monomer.
[0252] When (i) it is present with other components, such as (ii) or (iii), it is present in the polymerizable composition in at least 1, at least 5, at least 10 or at least 20 mol %.
[0253]When (i) it is present with other components, such as (ii) or (iii), it is present in the polymerizable composition at a maximum of 90, a maximum of 50, a maximum of 40 or at least 30% by mol.
[0254] In one embodiment, the amount of (i) present is in a range where the minimum and maximum amounts are selected from the previous modalities. For example, the amount present in the range of 10 to 50 % by mol.
[0255] In one embodiment, (i) is present at a level sufficient to provide a polymeric molecule having a plurality of cucurbituryl visitor molecules bound to each single polymer molecule.
[0256] In one embodiment, (i) is present at a level sufficient to provide a polymeric molecule having unique visiting cucurbituryl molecules bound to each single polymer molecule.
[0257] In one embodiment, (i) is present at a level sufficient to provide a polymeric molecule having the % functionality described above.
[0258] When (ii) is present, it is present in the polymerizable composition in at least 0.5, at least 1, or at least 2% by mol.
[0259]When (ii) is present, it is present in the polymerizable composition in a maximum of 20, a maximum of 10, or a maximum of 5% by mol.
[0260] In one embodiment, the amount of (ii) present is in a range where the minimum and maximum amounts are selected from the above modalities. For example, the amount present in the range of 1 to 5% by mol.
[0261] When (iii) is present, it is present in the polymerizable composition in at least 0.5, at least 1, at least 2, at least 5, at least 10, at least 20, or at least 50% in mole
[0262]When (iii) is present, it is present in the polymerizable composition in n at most 90, at most 80, or at most 70% by mol.
[0263] In one embodiment, the amount of (iii) present is in a range where the minimum and maximum amounts are selected from the above modalities. For example, the amount present in the range of 10 to 80% by mol.
[0264]When a reference is made to % by mol, this is a reference to the amount of a component present in relation to the total amount, in moles, of (i), and (ii) and (iii), when present, and any other polymerizable monomers, when present. Said component may be one of (i), (ii), (iii), or any other polymerizable monomers.
[0265] In one embodiment, the composition further comprises one or more additional (meth)acrylate monomers. A monomer may be a (meth)acrylate monomer. One or more monomers may be a (meth)acrylate monomer which is substituted on the ester group.
[0266]When a reference is made to % by mol, this is a reference to the amount of a component present in relation to the total amount, in moles, of (i), and (ii) and (iii), when present, and any other polymerizable monomers, when present. Said component may be one of (i), (ii), (iii), or any other polymerizable monomers. Said component may be a chain transfer agent or a radical initiator, as described above.
[0267]The term “fixed” refers to the connection of the acrylate (ester) group or the styrene phenyl group, either directly or indirectly to the specified group. Where an indirect connection exists, it is understood that a linking group may form a connection between the acrylate and the specified group. In one embodiment, the linker may comprise a (poly)ethylene glycol (PEG) group.
[0268] In one embodiment, the detectable label is a fluorescent label. The fluorescent label may be a fluorescein or rhodamine label. The “color” of the marker is not particularly restricted, and green, red, yellow, cyan and orange markers are suitable for use.
[0269] In one embodiment, the aqueous solubilizing group is a PEG group. The PEG group may have at least 2, 3, 4, 5 or 10 repeating ethylene glycol units. The PEG group can have a maximum of 50, 40, 20, or 15 repeating ethylene glycol units.
[0270] In one embodiment, the aqueous solubilizing group is or comprises amino, hydroxy, carboxy, or sulfonic acid.
[0271] In one embodiment, the amino group is a quaternary amino group, for example, a trimethylamino group.
[0272] In one embodiment, the composition further comprises a chain transfer agent.
[0273] In one embodiment, the chain transfer agent is a thiocarbonylthio compound.
[0274] When a chain transfer agent is present, it is present in the polymerizable composition in at least 0.1, at least 0.5, or at least 1 mol %.
[0275] When a chain transfer agent is present, it is present in the polymerizable composition in a maximum of 10, a maximum of 5, or a maximum of 2 mol %.
[0276] In one embodiment, the amount of a chain transfer agent present is in a range where the minimum and maximum amounts are selected from the above embodiments. For example, the amount present in the range of 0.5 to 2 % by mol.
[0277] In one embodiment, the composition further comprises a radical initiator.
[0278] When a radical initiator is present, it is present in the polymerizable composition in at least 0.01, at least 0.05, at least 0.1 mol %.
[0279] When a radical initiator is present, it is present in the polymerizable composition at a maximum of 5, at a maximum of 2, at a maximum of 1, or at a maximum of 0.5% by mol.
[0280] In one embodiment, the amount of a radical initiator present is in a range where the minimum and maximum amounts are selected from the previous embodiments. For example, the amount present in the range of 0.1 to 0.5 % by mol.
[0281] In one embodiment, the radical initiator is selected from the group consisting of AIBN (azobisisobutyronitrile), ACPA (4,4'-azobis(4-cyanopentanoic acid)) and ACVA (4,4'-Azobis acid (4-cyanovaleric).
[0282] In one embodiment, the polymeric molecule is obtained or obtainable from the polymerization of a composition comprising (i) and optionally (ii) and/or (iii) using the chain transfer agent and/or an initiator of radical described.
[0283] In one embodiment, the polymeric molecule is obtainable or obtained from a composition described herein using a radical polymerization process.
[0284] In one embodiment, the polymerization reaction is carried out at elevated temperature. The reaction can be carried out at a temperature of at least 30, at least 40 or at least 50°C.
[0285] The reaction can be carried out at a temperature of a maximum of 100, a maximum of 90 or a maximum of 80°C.
[0286] In one embodiment, the polymerization reaction is carried out in an organic solvent. The organic solvent may be an ether solvent, for example 1,4-dioxane, or an alkyl alcohol solvent, for example ethanol. The polymerization reaction can be carried out at reflux temperature.
[0287]The concentration of the polymerizable mixture in the organic solvent may be at most 5.0, at most 2.0, or at most 1.5 M.
[0288]The concentration of the polymerizable mixture in the organic solvent may be at least 0.05, at least 0.1, at least 0.5M, or at least 1.0M.
[0289] In one embodiment, the concentration is in a range where the minimum and maximum amounts are selected from the above modalities. For example, the concentration is in the range of 1.0 to 2.0 M.
[0290] In one embodiment, the polymerization reaction is carried out for at least 1, at least 5, or at least 10 hours.
[0291] In one embodiment, the polymerization reaction is carried out for a maximum of 72, or a maximum of 48 hours.
[0292] The polymerization reaction can be stopped using techniques familiar to those skilled in the art. Steps may include dilution of reaction mixture and/or temperature reduction.
[0293] In one embodiment, the polymerization reaction is carried out for a time sufficient to obtain a polymeric molecule having a molecular weight as described herein.
[0294] In one embodiment, the polymerization reaction is carried out for a time sufficient to obtain a polymeric molecule having a plurality of visiting molecules.
[0295] In one embodiment, the polymerization reaction is carried out for a time sufficient to obtain a polymeric molecule having a visiting molecule.
[0296] The concentration of the polymerizable mixture refers to the total amount of monomer present (which includes (i), and (ii) and (iii), when present, and any other polymerizable monomers, when present) in moles, in unit volume of organic solvent (i.e. per litre).
[0297] In one embodiment, the polymer may be formed as a particle. Particle
[0298] In one embodiment, the building block is a particle. The type of particle for use in the present invention is not particularly limited.
[0299] In one embodiment, the particle is a first building block and the particle is linked to a plurality of visiting cucurbituril molecules.
[0300] In one embodiment, the particle is a second building block and the particle is linked to one or more visiting cucurbituril molecules.
[0301] In one embodiment, the particle is a second building block and the particle is linked to a plurality of visiting cucurbituril molecules.
[0302] Typically, the particle has a size that is one, two, three or four magnitudes smaller than the size of the capsule.
[0303] In one embodiment, the particle is a nanoparticle. A nanoparticle has an average size of at least 1, at least 5, or at least 10 nm in diameter. A nanoparticle has an average size of at most 900, at most 500, at most 200, or at most 100 nm in diameter.
[0304] In one embodiment, the nanoparticle has an average size in the range of 1 to 100 nm or 5 to 60 nm in diameter.
[0305]The mean refers to the numerical mean. The diameter of a particle can be measured using microscopic techniques, including TEM.
[0306] In one embodiment, the particles have a relative standard deviation (RSD) of at most 0.5%, at most 1%, at most 1.5%, at most 2%, at most 4%, at most 5 %, maximum 7%, maximum 10%, maximum 15%, maximum 20% or maximum 25%.
[0307] In one embodiment, the particle has a hydrodynamic diameter of at least 1, at least 5, or at least 10 nM in diameter.
[0308] In one embodiment, the particle has a hydrodynamic diameter of at most 900, at most 500, at most 200, or at most 100 nM in diameter.
[0309]The hydrodynamic diameter can refer to the numerical average or the volumetric average. The hydrodynamic diameter can be determined from dynamic light scattering (DLS) measurements of a particle sample.
[0310] In one embodiment, the particle is a metal particle.
[0311] In one embodiment, the particle is a transition metal particle.
[0312] In one embodiment, the particle is a noble metal particle.
[0313] In one embodiment, the particle is or comprises copper, ruthenium, palladium, platinum, titanium, zinc oxide, gold or silver, or mixtures thereof.
[0314] In one embodiment, the particle is or comprises a gold particle, a silver particle, or a mixture thereof.
[0315] In one embodiment, the particle is a gold particle or a silver particle, or a mixture thereof.
[0316] In one embodiment, the particle is a gold nanoparticle (AuNP).
[0317] In one embodiment, the particle is or comprises silica or calcium carbonate.
[0318] In one embodiment, the particle is a quantum dot.
[0319] In one embodiment, the particle is or comprises a polymer. The polymer may be a polystyrene polymer or a polyacrylamide polymer. The polymer may be a biological polymer including, for example, a polypeptide or a polynucleotide.
[0320] In one embodiment, the particle comprises a material suitable for use in surface enhanced Raman spectroscopy (SERS). Particles of gold and/or silver and/or other transition metals are suitable for such use.
[0321]Gold and silver particles can be prepared using techniques known in the art. Examples of preparations include those described by Coulston et al. (Chem. Commun. 2011, 47, 164) Martin et al. (Martin et al. Langmuir 2010, 26, 7410) and Frens (Frens Nature Phys. Sci. 1973, 241, 20), which are incorporated herein in their entirety by reference.
[0322] The particle is bound to one or more visiting molecules, as appropriate. Typically, when the particle is a first building block, it is provided with at least a plurality of visiting molecules. When the particle is a second building block, it is provided in one or more visiting molecules.
[0323] In one embodiment, a visiting molecule may be covalently linked to a particle via a linking group. The linking group may be a spacer element to provide a distance between the visiting molecule and the particle volume. The binder may include functionality to enhance the water solubility of the combined building block and visitor molecule construct. The binder is provided with functionality to allow connection to the surface of the particle. For example, when the particle is a gold particle, the ligand has a thiol functionality to form a gold-sulfur bond.
[0324]Alternatively, a visiting molecule can be attached directly to the particle surface, through a suitable functionality. For example, when the particle is a gold particle, the visiting molecule can be attached to the gold surface via a thiol functionality of the visiting molecule.
[0325] In one embodiment, the particle comprises solubilizing groups such that the particle, along with its visiting molecules, is either water-soluble or is soluble in a water-immiscible phase.
[0326]The solubilizing groups are attached to the surface of the particle. The solubilizing group may be covalently attached to the particle through suitable functionality. When the particle is a gold particle, the solubilizing group is attached via a sulfur bond to the gold surface.
[0327] The solubilizing group may be, or comprise, polyethylene glycol or amine, hydroxy, carboxy or a thiol functionality.
[0328] In one embodiment, the building block is obtained or obtainable from a composition comprising: (iv) a gold particle; (v)) a visiting molecule next to a linking group having a thiol functionality; and (vi)) a solubilizing molecule having a thiol functionality; and optionally further comprising (iv) an additional visiting molecule, together with a linking group having a thiol functionality.
[0329] In one embodiment, the amount of visiting molecule present in the composition is at least 1, at least 5, at least 10, or at least 15 mol %.
[0330] In one embodiment, the amount of visitor molecule present in the composition is a maximum of 80, a maximum of 50, or a maximum of 25 % in mol.
[0331]A reference to % by mol is a reference to the amount of visitor molecule present as a percentage of the total amount of (ii) and (iii), and (iv) when present, in the composition.
[0332] The amount of (ii) present in the composition may be such as to allow the preparation of a particle building block having a plurality of visiting molecules. cucurbituril visitor
[0333] As noted earlier, the visitor is a compound that is capable of forming a visitor-host complex with a cucurbituril. Therefore, the term “complexation” refers to the establishment of the visitor-host complex.
[0334] In some embodiments of the invention, the visitor-host complex is a ternary complex comprising the cucurbituril host and a first visitor molecule and a second molecule. Typically, these complexes are based on CB[8] and variants and derivatives thereof.
[0335] In some embodiments of the invention, the visitor-host complex is a binary complex comprising the cucurbituril host and a first visitor molecule. Typically, these complexes are based on CB[5] or CB[7], and variants of derivatives thereof. In the present invention, binary complexes are obtainable from a plurality of covalently linked cucurbituryls. CB[8], and variants of derivatives thereof, can also form binary complexes.
[0336] Primarily, any compound having a suitable 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 that are thought to interact with the cucurbituril cavity. The size of these portions may be large enough to allow complexation only with larger cucurbituryl forms.
[0337]The term selective can be used to refer to the amount of visitor-host complex formed when cucurbituryl (the first cucurbituryl) and a second cucurbituryl are present in a mixture with a particular visiting molecule or molecules. The visitor-host complex formed between the first cucurbituril and the host (in a binary complex) or hosts (in a ternary complex) can be at least 60 mol%, at least 70 mol%, at least 80 mol%, at least 90 mol %, at least 95 mol %, at least 97 mol %, at least 98 mol %, or at least 99 mol %, of the total amount of visitor-host complex formed (for example, taking consideration is given to the amount of visitor-host complex formed between the second cucurbituril and the host or hosts, if any).
[0338] In one embodiment, the visitor-host complex formed from the (first) cucurbituril and the visitor or visitors has a binding affinity that is at least 100 times greater than the binding affinity of a visitor-host complex formed at from the second cucurbiturilla of the visitor or visitors. Preferably, the binding affinity is at least 103, at least 104, at least 105, at least 106, or at least 107 greater.
[0339] Cucurbituril visiting molecules are well known in the art. Examples of visiting 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 ) and Rauwald et al. (Rauwald et al. J. Phys. Chem. 2010, 114, 8606 ).
[0340]Described below are visiting molecules that are suitable for use in forming a capsule shell. These visiting molecules can be connected to a building block using standard synthetic techniques.
[0341]A cucurbituril visitor molecule can be derived from, or contain, a structure from the table below:



where the structure may be a salt, including protonated forms where appropriate. In one embodiment, the visiting molecules are visiting molecules for CB[8].
[0342] In one embodiment, the visiting molecule is, or is derived from, or contains an A1-A43, A46, or B1-B4 structure, in the table above.
[0343] In one embodiment, the visiting molecule is, or is derived from, or contains an A1, A2, or A13 structure in the table above.
[0344] In one embodiment, the visiting molecule is, or is derived from, or contains a B1 structure.
[0345]Additionally, the visiting molecule is 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).
[0346] In some embodiments, the first and second visitor molecules form a pair that can interact with the cucurbituril cavity to form a stable ternary host-visitor complex. Any host pair that fits into the cucurbituril cavity can be used. In some embodiments, the pair of visiting molecules can form a charge transfer pair comprising an electron-rich compound and an electron-deficient compound. The first and second visiting molecules act as an electron acceptor and the other as an electron donor in the CT pair. For example, the first visiting molecule might be an electron-deficient molecule that acts as an electron acceptor, and the second visiting molecule might be an electron-rich molecule that acts as an electron donor, or vice versa. In one embodiment, cucurbituril is CB[8].
[0347] Suitable electron acceptors include 4,4'-bipyridinium derivatives, for example, N,N'-dimethyldipyridyliumyl ethylene, and other related acceptors, such as those based on diazapyrenes and diazaphenanthrenes. Viologen compounds that include alkyl viologens are particularly suitable for use in the present invention. Examples of alkyl viologen compounds include N,N'-dimethyl-4,4'-bipyridinium salts (also known as Paraquat).
[0348] Suitable electron donors include electron-rich aromatic molecules, for example 1,2-dihydroxy benzene, 1,3-dihydroxy benzene, 1,4-dihydroxy benzene, tetrathiafulvalene, naphthalenes such as 2,6-dihydroxy naphthalene and 2-naphthol, indoles and sesamol (3,4-methylene dioxyphenol). Polycyclic aromatic compounds in general may find use as electron donors in the present invention. Examples of such compounds include anthracene and naphthacene.
[0349]Amino acids such as tryptophan, tyrosine and phenylalanine may be suitable for use as electron donors. The peptide sequences comprising these amino acids at their termini 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.
[0350] In some embodiments, the visiting molecules consist of a pair of compounds, e.g., first and second visiting molecules, where one of the pairs is a compound A as shown in the previous table (e.g., A1, A2, A3, etc. ), and the other pair is a compound B as shown in the table above (e.g. B1, B2, B3 etc.). In one embodiment, compound A is selected from A1-A43 and A46. In one embodiment, compound B is B1.
[0351] Other suitable visiting molecules include peptides such as WGG (Bush, M.E. et al J. Am. Chem. Soc. 2005, 127, 14511-14517).
[0352]An electron-rich visitor molecule can be correlated with any electron-deficient CB[8] visitor molecule. Examples of suitable pairs of visiting molecules, eg first and second visiting molecules, for use as described herein may include: viologen and naphthol; viologen and dihydroxy benzene; viologen and tetrathiafulvalene; viologen and indole; methyl viologen and naphthol; methyl viologen and dihydroxy benzene; methyl viologen and tetrathiafulvalene; methyl viologen and indole; N,N'-dimethyl dipyridyliumyl ethylene and naphthol; N,N'-dimethyl dipyridyliumyl ethylene and dihydroxy benzene; N,N'-dimethyl dipyridyliumyl ethylene and tetrathiafulvalene; N,N'-dimethyl dipyridyliumyl ethylene and indole; 2,7-dimethyl diazapyrene and naphthol; 2,8-dimethyl diazapyrene and dihydroxy benzene; 2,9-dimethyl diazapyrene and tetrathiafulvalene; and 2,7-dimethyl diazapyrene and indole.
[0353] In particular, suitable pairs of visiting molecules for use as described herein may include 2-naphthol and methyl viologen, 2,6-dihydroxy naphthalene and methyl viologen, and tetrathiafulvalene and methyl viologen.
[0354] In one embodiment, the neighboring pair is 2-naphthol and methyl viologen.
[0355]In one embodiment, the neighbor pair is a reference to a pair of visiting molecules suitable for forming a ternary complex with CB[8].
[0356] In one embodiment, the visiting molecule is preferably an ionic liquid. Typically, these visitors are suited to form a complex with CB[7]. However, they can also form complexes with CB[8] in a binary complex, or in a ternary complex together with another small visiting molecule or solvent (see Jiao et al. Org. Lett. 2011, 13, 3044).
[0357] The ionic liquid typically comprises a cationic organic nitrogen heterocycle, which may 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 nitrogen heteroaryl group is preferably a C5-10 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 and nitrogen atoms. The 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 ring of the nitrogen heterocycle is quaternized.
[0358] The counter-anion may be a halide, preferably a bromide. Other counter-anions suitable for use are those that result in a complex that is soluble in water.
[0359] The visitor is preferably a compound, including a salt, comprising one of the following groups selected from the list consisting of: imidazolium moiety; pyridinium moiety; quinolinium moiety; pyrimidinium moiety; pyrrole moiety; and quaternary pyrrolidine moiety.
[0360] Preferably, the visitor comprises a portion of imidazolium. An especially preferred visitor is 1-alkyl-3-alkylimidazolium, where the alkyl groups are optionally substituted.
[0361]1-Alkyl-3-alkylimidazolium compounds, where the alkyl groups are unsubstituted, are especially suitable for complexing with CB[7].
[0362] 1-Alkyl-3-alkylimidazolium compounds, where the alkyl groups are unsubstituted, are especially suitable for complexing with CB[6]
[0363] 1-Alkyl-3-alkylimidazolium compounds, where an alkyl group is replaced by aryl (preferably naphthyl), are especially suitable for complexing with CB[8].
[0364] The 1-alkyl and 3-alkyl substituents may be the same or different. Preferably, they are different.
[0365] In one embodiment, the 3-alkyl substituent is methyl, and preferably is unsubstituted.
[0366] In one embodiment, the 1-alkyl substituent is ethyl or butyl, and preferably is unsubstituted.
[0367] In one embodiment, the optional substituent is aryl, preferably C5-10 aryl. Aryl includes carboaryl and heteroaryl. Aryl groups include phenyl, naphthyl and quinolinyl.
[0368] In one embodiment, the alkyl groups described herein are linear alkyl groups.
[0369]Each alkyl group is independently a C1-6 alkyl group, preferably a C1-4 alkyl group.
[0370]The aryl substituent may be another portion of substituted 1-alkyl-3-imidazolium (where the alkyl group is attached to the 3-position of the ring).
[0371] In another embodiment, the compound preferably comprises a pyridinium moiety.
[0372]The ionic liquid molecules described above are particularly useful for forming binary visitor-host complexes. Complexes comprising two ionic liquid molecules as visitors within a cucurbituril host are also encompassed by the present invention.
[0373]A cucurbituril may be able to form both binary and ternary complexes. For example, it was previously noted that CB[6] compounds form ternary complexes with short-chain 1-alkyl-3-methylimidazolium visiting molecules, while longer-chain 1-alkyl-3-methylimidazolium visiting molecules form binary complexes. with the cucurbituril host.
[0374]Preferred visitors for use in the present invention are of the form H+X-, where H+ is one of the following cations,

and X- is a suitable counter-anion as defined above. A preferred counter-anion is a halide anion, preferably Br-.
[0375]In a preferred embodiment, cation A or cation B can be used to form a complex with CB[7] or CB[6].
[0376]In a preferred embodiment, the D cation or E cation can be used to form a complex with CB[8].
[0377]Cations A and B may be referred to as 1-ethyl-3-methylimidazolium and 1-butyl-3-methylimidazolium, respectively.
[0378]The D and E cations may 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-methylimidazolium.
[0379]Alternatively or additionally, the neighboring compounds may be an imidazolium salt of formula (I):
wherein X- is a counter-anion; R1 is independently selected from H and saturated C1-6 alkyl; 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 may optionally be substituted.
[0380] 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-.
[0381] In one embodiment, R1 is selected from H and linear saturated C1-6 alkyl.
[0382] In one embodiment, R2 is C1-10 alkyl, which may optionally contain one or more double bonds, and may optionally be interrupted by a heteroatom selected from -O-, -S-, -NH-, and -B-, and may optionally be substituted.
[0383] In one embodiment, R2 is linear C1-10 alkyl, which may optionally contain one or more double bonds, and may be optionally substituted.
[0384] In one embodiment, where a double or triple bond is present, it may be conjugated to the imidazolium moiety. Alternatively, the double or triple bond may not be conjugated to the imidazolium moiety.
[0385] In one embodiment, the 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, where each R3 and R4 is independently selected from H and optionally substituted saturated C1-6 alkyl, C5-20 aryl and C1-6 aryl C5-20-alkylene. or R3 and R4 may together form an optionally saturated 5-, 6- or 7-membered heterocycle ring that is optionally substituted with a -R3 group.
[0386] In one embodiment, the 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 may 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 (thiole) (C5), oxazole (C5), thiazole (C5), imidazole (1,3-diazole) (C5), pyrazole (1,2-diazole) (C5), pyridazine (1,2-diazine) (C6), and pyrimidine (1,3- diazine) (C6) (e.g. 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 with C1-6 alkyl.
[0393]When the C5-20 aryl group is an imidazolium, it is preferably substituted in nitrogen by an R1 group (thus forming a quaternary nitrogen).
[0394] The compound of formula (I) comprises an imidazolium moiety having an R2 substituent at the 1-position and an R1 substituent at the 3-position. In a further aspect of the invention, the compound of formula (I) may be optionally substituted at the position 2, 4 or 5 by a group RA, where RA has the same meaning as R1.
[0395] The above modalities are combinable in any combination as appropriate. encapsulant
[0396]The capsule of the invention can be used to encapsulate a component (the encapsulant). In one embodiment, a capsule is provided which comprises an encapsulant. The capsule is suitable for storing a component, and this component can later be released at a chosen location.
[0397] It is understood that a reference to an encapsulated component is not a reference to a solvent molecule. For example, the encapsulated component is not water, it is not an oil or an organic solvent. It is also understood that a reference to an encapsulated component is not a reference to a cucurbituryl or a building block for use in preparing the capsule shell. Otherwise, the component is not particularly limited.
[0398] Therefore, the encapsulant is a component of the capsule that is provided in addition to the solvent that may be present within the shell.
[0399] In the methods of the invention, the capsule shell is prepared from a composition comprising a cucurbituril and one or more building blocks, as appropriate. Not all of the cucurbituril and one or more building blocks can react to form a shell material. Additionally, cucurbituril and one or more building blocks may react to form a network, but this network may not be included in the shell that forms the capsule. Such unreacted or partially reacted reagents may be contained within the shell, and may be contained in addition to the encapsulant. Therefore, the encapsulant is a component of the capsule that is provided in addition to the unreacted or partially reacted reactants and products that may be present within the shell.
[0400] In one embodiment, the encapsulating 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.
[0401] In one embodiment, solubility refers to aqueous solubility (ie, an aqueous phase).
[0402] In one embodiment, solubility refers to the solubility in an oil phase or an organic phase.
[0403] The capsules of the invention can be used to encapsulate a wide range of components
[0404] 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 50,000 (50k) ), at least 100,000 (100k) or at least 200,000 (200k).
[0405] In one embodiment, the encapsulant is a therapeutic compound.
[0406] In one embodiment, the encapsulant is a biological molecule, such as a polynucleotide (eg, DNA and RNA), a polypeptide, or a polysaccharide.
[0407] In one embodiment, the encapsulant is a polymeric molecule, including biological polymers, such as those aforementioned polymers.
[0408]In one embodiment, the encapsulant is a cell.
[0409] In one embodiment, the encapsulant is an ink.
[0410] In one embodiment, the encapsulant is a carbon nanotube.
[0411] In one embodiment, the encapsulant is a particle. The particle may be a metallic particle.
[0412]Capsule size is selected to accommodate encapsulant size. Therefore, the inner diameter (the distance from the innermost wall to the innermost wall) is greater than the largest dimension of the encapsulant.
[0413]In one embodiment, the encapsulant has a detectable label. The detectable label can be used to quantify and/or locate the encapsulant. The marker can be used to determine the amount of encapsulant contained with the capsule.
[0414]In one embodiment, the detectable marker is a luminescent marker. In one embodiment, the detectable label is a fluorescent label or a phosphorescent label.
[0415]In one embodiment, the detectable marker is visible.
[0416]In one embodiment, the fluorescent label is a rhodamine or fluorescein label.
[0417] In one embodiment, the capsule of the invention is for use as a reactor. The capsule preparation method as described herein brings together the reagents, which are supplied in separate second-phase substreams, and are brought into contact at substantially the same time as the second phases contact the first phase. A shell of material is formed at the interface of the discrete regions, and this shell contains the reactants that may be allowed to react. Locating reagents within a discrete region allows control over reaction timings.
[0418] When the capsule serves for use as a microreactor, it is understood that the composition of the inner space of the shell will change over time as the reactants react to form a product, along with associated by-products, if any. As will become apparent, the amount of reactant will be reduced as the reaction progresses. Additional and alternative encapsulants
[0419] In addition, or as alternatives to the above-mentioned encapsulants, the encapsulant may be selected from one or more of the encapsulants discussed below. In one embodiment, the molecular weight preferences given above apply to such encapsulants.
[0420] In one embodiment, the encapsulant 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.
[0421] A pathogen is an agent that is capable of causing disease in a host. The pathogen can be a virus, a bacterium, a fungus, or a prion.
[0422]In one embodiment, the encapsulant is a virus.
[0423]The virus may be a virus selected from a family selected from the group consisting of adenoviridae (e.g. adenovirus), herpesviridae (e.g. Herpes simplex, type 1 and type 2, and Epstein-barr) , papillomaviridae (eg, human papillomavirus), hepadnaviridae (eg, Hepatitis B), flaviviridae (eg, Hepatitis C, yellow fever, dengue, West Nile virus), retroviridae (eg, immunodeficiency virus (HIV) )), orthomyxoviridae (eg, influenza), paramyxoviridae (eg, measles, mumps), rhabdoviridae (eg, rabies), and reoviridae (eg, rotavirus).
[0424] In one embodiment, the encapsulant is a microorganism.
[0425] As noted earlier, in one embodiment, the encapsulant is a cell. The cell may be a prokaryotic or eukaryotic cell.
[0426]The cell may be a mammalian cell, such as a human cell, a rodent cell (e.g. a guinea pig, a hamster, a mouse, a mouse), a lagomorph cell (e.g. a rabbit), a bird cell (e.g. a bird), a canine cell (e.g. a dog), a feline cell (e.g. a cat), an equine cell (e.g. a horse) , a porcine cell (e.g. a pig), a sheep cell (e.g. a sheep), a bovine cell (e.g. a cow), an ape cell (e.g. a monkey or primate) , a monkey cell (eg, tamarin, baboon), a primate cell (eg, gorilla, chimpanzee, orangutan, gibbon), or an ornithorhynchidae cell (eg, platypus).
[0427]The cell may be a tumor cell, which may be a benign or malignant tumor cell.
[0428]Examples of eukaryotic cells include epithelial, endoterial, neural, skeletal, and fibroblast cells, among others.
[0429] In one embodiment, the encapsulant is a bacterium, such as a gram positive bacterium and a gram negative bacterium.
[0430]Examples of gram positive bacteria include Corynebacterium, Mycobacterium, Nocardia, Streptomyces, Staphylococcus (such as S. aureus), Streptococcus (such as S. pneumoniae), Enterococcus (such as E. faecium), Bacillus, Clostridium (such as C. diff. ) and Listeria.
[0431]Examples of gram negative bacteria include Hemophilus, Klebsiella, Legionella, Pseudomonas, Escherichia (as E. coli), Proteus, Enterobacter, Serratia, Helicobacter (as Helicobacter pylori), and Salmonella.
[0432] In one embodiment, the encapsulant is an antibody.
[0433]The term “antibody” is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g. bispecific antibodies), and antibody fragments as long as they exhibit the desired biological activity . Antibodies can be murine, human, humanized, chimeric, or derived from other species. An antibody is a protein generated by the immune system that is able to recognize and bind to a specific antigen. A target antigen usually has multiple binding sites, also called epitopes, recognized by CDRs on multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Therefore, an antigen may have more than one corresponding antibody. An antibody includes a full-length immunoglobulin molecule or an immunologically active portion of a full-length immunoglobulin molecule, i.e., a molecule that contains an antigen-binding site that immunospecifically binds to an antigen on a target of interest or part thereof, such targets include, but are not limited to, cancer cell or cells that produce autoimmune antibodies associated with an autoimmune disease. The immunoglobulin can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass of immunoglobulin molecule. Immunoglobulins can be derived from any species, including human, murine, or rabbit origin.
[0434]The "antibody fragments" comprise a portion of a full-length antibody, usually the antigen binding or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; divalent scFv antibodies ("diabodies"); linear antibodies; fragments produced by a Fab expression library, anti-idiotypic antibodies (anti-Id), CDR (complementary determination region), and epitope-binding fragments of any of the foregoing that immunospecifically bind to cancer cell antigens, viral antigens or microbial antigens, single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.
[0435]The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, that is, the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minute amounts. Monoclonal antibodies are highly specific, being directed against a single binding site. Additionally, in contrast to polyclonal antibody preparations that include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they can be synthesized uncontaminated by other antibodies. The "monoclonal" modifier indicates the character of the antibody being obtained from a substantially homogeneous population of antibodies, and should not be construed as requiring the production of antibodies by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method, or they may be made by recombinant DNA methods. Monoclonal antibodies can also be isolated from phage antibody libraries.
[0436] Specifically, monoclonal antibodies include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species belonging to a class or subclass of antibody. in particular, while the remainder of the chain is identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another class or subclass of antibody, as well as fragments of such antibodies, provided that they exhibit a desired biological activity
[0437] In one embodiment, the antibody is an antibody-drug conjugate (ADC).
[0438]The antibody may be suitably labeled for detection and analysis, either while held in the capsule, for later use when the antibody is released.
[0439] In one embodiment, the encapsulant is a hormone. The hormone may be a peptide hormone, such as insulin or growth hormone, or a lipid hormone, such as a steroid hormone, for example, prostaglandin and estrogen.
[0440] In one embodiment, the encapsulant is a polypeptide. In one embodiment, the polypeptide is a protein. In one embodiment, the protein has catalytic activity, for example, having ligase, isomerase, lyase, hydrolase, transferase, or oxidoreductase activity.
[0441] In one embodiment, the encapsulant is a polymer. In some embodiments, the capsule shell of the present invention includes a building block that is a functionalized polymer. When such a building block is present, a polymer which is an encapsulant differs from the building block. In one embodiment, the encapsulating polymer is not suitable for forming a non-covalent bond with a cucurbituryl.
[0442] In one embodiment, the encapsulant is a metallic particle.
[0443] In one embodiment, the nanoparticle is or comprises a noble metal.
[0444] In one embodiment, the nanoparticle is or comprises a transition metal.
[0445] In some embodiments, the capsule shell of the present invention includes a building block that is a functionalized particle. When such a building block is present, a particle that is an encapsulant differs from the building block. In one embodiment, the encapsulant particle is not suitable for forming a non-covalent bond with a cucurbituryl.
[0446] In one embodiment, the nanoparticle is a gold nanoparticle (AuNP) or a silver nanoparticle (AgNP), or a nanoparticle comprising both silver and gold.
[0447] In general, the particle is substantially spherical. However, particles having other shapes may be used if appropriate or desirable.
[0448] In one embodiment, the nanoparticle has a diameter of at most 500 nm, at most 200 nm, at most 150 nm, at most 100 nm, at most 80 nm, or at most 70 nm.
[0449] In one embodiment, the nanoparticle has a diameter of at least 1 nm, at least 2 nm, at least 5 nm, at least 10 nm, at least 15 nm, at least 20 nm, at least 30 nm, or at least 30 nm minus 40 nm.
[0450] In one embodiment, the particle diameter is in a range where the minimum and maximum rates are selected from the previous embodiments. For example, the diameter is in the range of 1 to 100 nm, or, for example, in the range of 10 to 100 nm. For example, the diameter is in the range of 2 to 500 nm
[0451] In one embodiment, the nanoparticle has a diameter of about 20 nm.
[0452]The mean refers to the numerical mean. The diameter of a particle can be measured using microscopic techniques, including TEM.
[0453] The particles used in the present invention are sustainably monodispersed or have a very low dispersion. In one embodiment, the particles have a relative standard deviation (RSD) of at most 0.5%, at most 1%, at most 1.5%, at most 2%, at most 4%, at most 5%, at most maximum 7%, maximum 10%, maximum 15%, maximum 20% or maximum 25%.
[0454] In one embodiment, the particle has a hydrodynamic diameter of at least 5 nm, at least 10 nm, at least 15 nm, at least 20 nm, at least 30 nm, at least 40 nm.
[0455] In one embodiment, the particle has a hydrodynamic diameter of at most 500 nm, at most 200 nm, at most 150 nm, at most 100 nm, at most 80 nm, or at most 70 nm.
[0456]The hydrodynamic diameter can refer to the numerical average or the volumetric average. The hydrodynamic diameter can be determined from dynamic light scattering (DLS) measurements of a particle sample.
[0457]Particle size and particle composition can be selected to provide a more appropriate or beneficial enhanced surface effect.
[0458]Gold and silver particles can be prepared using techniques known in the art. Examples of preparations include those described by Coulston (Coulston et al Chem. Commun. 2011, 47, 164) and Martin (Martin et al. Langmuir 2010, 26, 7410) and Frens (Frens Nature Phys. Sci. 1973, 241, 20 ), which are incorporated herein in their entirety by reference.
[0459] In one embodiment, the encapsulant is a polymer. In one embodiment, the polymer is not a polymer that is present as a building block in the capsule shell. Otherwise, the polymer is not particularly limited. Additional and alternative encapsulants
[0460] In addition, or as alternatives to the above-mentioned encapsulants and the above-mentioned additional and alternative encapsulants, the encapsulant may be selected from one or more additional encapsulants discussed below. In one embodiment, the molecular weight preferences given above apply to such encapsulants.
[0461] In one embodiment, the encapsulant is a fragrance compound or a fragrance composition. A fragrance compound or composition has odorant properties suitable for use in a perfume.
[0462] In one embodiment, the encapsulant is a flavoring compound or flavoring composition. A flavoring may be or include a flavor enhancer, such as a sweetener.
[0463] In one embodiment, the encapsulant is an oil, such as an essential oil. Examples of essential oils include those obtained or obtainable from lime orange, mint, lemon and clove, among others.
[0464] In one embodiment, the encapsulant is itself a vehicle for maintaining an encapsulant. For example, the encapsulant can be a liposome, micelle, or vesicle. The liposome, micelle, or vesicle may retain an encapsulant, such as one of the encapsulants described herein. Properly loaded liposomes, micelles or vesicles can be prepared using standard techniques known in the art. The loaded liposome, micelle or vesicle can then be encapsulated in the supramolecular capsules of the invention using the methods described herein. Methods for preparing capsules
[0465] In a second aspect of the invention, there is provided a method for preparing a capsule having a shell, such as the capsule of the first aspect of the invention, the method comprising the steps of: (i) placing a stream of a first phase and a flow of a second phase in contact with a channel to thereby generate in the channel a dispersion of discrete regions, preferably droplets, of the second phase in the first phase, wherein the second phase comprises cucurbituril and one or more building blocks having suitable cucurbituril visitor functionality to form a supramolecular lattice network to thereby form a capsule shell within the discrete region wherein the first and second phases are immiscible.
[0466] In one embodiment, the second phase comprises (a) a cucurbituryl and (1) or (2); or (b) a plurality of covalently linked cucurbituryls and (1), (2) or (3) to thereby form a capsule shell within the discrete region wherein the first and second phases are immiscible.
[0467] In one embodiment, the second phase comprises a cucurbituril and (1) or (2).
[0468] In one embodiment, the second phase comprises a cucurbituril and (1).
[0469] In the method of the invention a second phase dispersion is created within the continuous first phase. In one embodiment, one of the first and second phases is an aqueous phase and the other phase is a water-immiscible phase.
[0470] In one embodiment, the second phase is an aqueous phase. The first phase is a water-immiscible phase, for example an oil phase.
[0471] In one embodiment, the first phase is an aqueous phase. The second phase is a water-immiscible phase, for example an oil phase.
[0472] In one embodiment, the method further comprises the step of (ii) collecting the flow from the channel to thereby obtain a droplet, within which a capsule is located.
[0473] In one embodiment, the method comprises step (ii) above and (iii) optionally drying the capsule obtained in step (ii). The drying step refers to the desolvation of the droplet and the capsule. Where the second phase is an aqueous phase, the drying step is a dehydration.
[0474] In one embodiment, the second (dispersed) phase stream is a stream generated by combining a plurality of sub streams, where each sub stream comprises a reagent for use in preparing the shell. Therefore, a substream may comprise one cucurbituryl (as in case (a)) or the plurality of covalently linked cucurbituryls (as in case (b)). Additional subflows may comprise a first building block and a second building block (as in the case of composition (1)). The first and second building blocks can be contained within the subflow or different subflows.
[0475]In one embodiment, the cucurbituril and building block (or blocks) are provided in separate substreams.
[0476] In one embodiment, a subflow is provided for each reagent for use in preparing the shell. In this embodiment, it is possible to independently change the flow rate of each subflow, thereby independently changing the final concentration of a particular reactant in the discrete region formed. The ability to independently change the flow rate and therefore the reagent concentration allows for control over the structure of the formed shell. Therefore, pore size and shell thickness can be controlled by appropriate changes in underflow rates.
[0477]Subflows can be brought into contact before contacting the first phase flow. In this arrangement, multiple subflows can be contacted at the same time, or they can be contacted in a sequence. Alternatively, the subflows can be brought into contact at substantially the same time as the second phases are brought into contact with the first phase flow.
[0478] In order to minimize the formation of an unstructured aggregation, which includes, for example, a hydrogel, within the second phase the cucurbituryl or the plurality of covalently bound cucurbituryls is brought into contact with the building blocks immediately before or substantially at the same time as the second phases are brought into contact with the flow of the first phase.
[0479] In one embodiment, the second phase flow is brought into contact with the first phase flow substantially perpendicular to the first phase. In this embodiment, the channel structure may be a T-junction geometry. The channel path may follow the flow path of the first stage, in which case the second flow will be substantially perpendicular to the resulting combined flow in the channel. Alternatively, the channel path may follow the second phase flow path, in which case the first phase flow will be substantially perpendicular to the resulting combined flow in the channel.
[0480]Methods utilizing a T-junction geometry provide discrete regions, typically droplets, of the aqueous phase in the oil phase as a result of the shear forces induced within the two-phase system.
[0481]In one embodiment, an additional flow from the first phase is provided. The first-phase flows are brought into contact with each side of the second-phase flow in a channel, and the phase flow is then passed through a region of the reduced cross-sectional channel (an orifice) to thereby generate , a discrete region, preferably a droplet, of the second phase in the channel. Such methods that have one internal second-phase flow and two external first-phase flows are referred to as flow focusing configurations.
[0482]Methods using flow focusing techniques provide discrete regions, typically droplets, of the second phase in the first phase as a result of the external first phase applying pressure and viscous stresses to the internal second phase thereby generating a flow narrow of this phase. This narrow flow then separates into discrete regions, typically droplets, at the orifice or shortly after the combined flow has passed through the orifice.
[0483] In one embodiment, the discrete region is a droplet.
[0484]In one embodiment, the discrete region is a nugget.
[0485]After the discrete region is formed in the channel, the discrete region can be passed along the channel to a collection area. The residence time of the discrete region in the channel is not particularly limited. In one embodiment, the residence time is sufficient to allow the shell to form.
[0486] As the discrete region is passed along the channel it may undergo a mixing stage whereby the components of the discrete region are more evenly distributed around this discrete region. In one embodiment, the channel comprises a winding region. The winding region may take the form of a substantially sigmoid path through which the discrete region is passed.
[0487] In one embodiment, the second phase further comprises a component for encapsulation, and step (i) provides a capsule that encapsulates the component.
[0488] The first phase and the second phase can contact in a simple T-junction. The second phase may be formed from the combination of separate streams of cucurbituril and (1), (2) or (3) as appropriate. Where there are more than two components, these components can be brought into contact simultaneously or sequentially.
[0489]These flows may contact before contact with the first phase. Alternatively, they can be contacted simultaneously with the first phase.
[0490]The second phase discrete regions are generated in the channel as the immiscible first phase breaks through the second phase. The shear frequency is dependent on the flow rate ratio of the two phases.
[0491]In one embodiment, the flow rate is selected in order to provide an adjusted number of droplets per unit of time (droplets per second).
[0492]Droplets can be prepared at a rate of max 10,000, max 5,000, max 1,000 or max 500 Hz.
[0493] The droplets may be prepared at a rate of at least 1, at least 10, at least 50, at least 100, or at least 200 Hz.
[0494]In one embodiment, the droplets may be prepared at a rate that is in a range where the minimum and maximum rates are selected from the above modalities. For example, the rate is in the range 100 to 500 Hz.
[0495] In one embodiment, the method comprises the step of (iii) drying the capsule. The drying step at least partially removes the solvent (which may be water or organic solvent) from the capsule and may be referred to as desolvation.
[0496] There are no particular limitations on the method for drying the capsules. In one embodiment, the capsules obtained may simply be allowed to stand at ambient conditions, and the solvent allowed to evaporate.
[0497] In one embodiment, the method comprises steps (ii) and (iii) and, optionally, step (iv), whose step is a washing step, whereby the capsules obtained are washed with a solvent. The purpose of the washing step may be to remove the surfactant (where used) or any other component used in the casing step. Step (iv) can be performed as an intermediate step between steps (ii) and (iii)
[0498] In one embodiment, the method comprises the step of (iii) drying the capsule and (v) subsequently resolving the capsule. The resolving step can be performed minutes, hours, days, weeks or months after step (iii) is completed.
[0499] In one embodiment, a reference to a droplet size is also a direct reference to a capsule size. A droplet is a droplet formed in a channel of a fluidic device or a droplet that is collected from the channel of such a device. Size refers to a droplet that has not been subjected to a desolvation step.
[0500]Capsules may be desolvated for storage and subsequently resolved for use. In the resolving step, the capsule is brought into contact with the solvent to thereby resolve the capsule.
[0501]A capsule directly formed after preparation is substantially spherical. Capsule desolvation can result in capsule collapse as the spherical edge becomes distorted. The wrapping material appears to bend in a random way.
[0502] In the preparation method described herein, a droplet is formed and a capsule shell forms at the droplet interface. The formed droplet may be subjected to a desolvation step, thereby resulting in the shrinkage of the capsule shell. In one embodiment, capsule size refers to the size of a capsule that has undergone a dehydration step.
[0503]Capsule shell may have pores. Such pores may be of a suitable size to allow an encapsulated molecule to pass through the shell to thereby be released from the capsule.
[0504] The flow rate of the first phase and/or the second phase can be varied to allow the preparation of droplets, and therefore capsules, of a desired size. As the flow rate of the first phase is increased relative to the second phase, the average droplet size decreases, and the size of the capsule formed also decreases.
[0505]Typically, the flow rate of the first stage is at least 1.5, 2, 3, 4, 5 or 10 times that of the second stage.
[0506] In one embodiment, the first and second phase flow rates are selected in order to provide droplets that have a desired mean diameter.
[0507]The average particle size can be determined from measurements of a sample of droplets collected from the flow channel using simple microscopy techniques.
[0508] In one embodiment, each droplet is a microdroplet.
[0509] In one embodiment, each droplet is a nanodroplet.
[0510] In one embodiment, the average droplet size is at least 1, 5, 10, 20, 30, or 40 μm in diameter.
[0511] In one embodiment, the average droplet size is a maximum of 400, 200, 100, 75 or 50 μm in diameter.
[0512]In one embodiment, the average droplet size is in a range where the minimum and maximum rates are selected from the above modalities. For example, the average size is in the range of 1 to 100 μm.
[0513]The droplet formed from the fluidic preparation has a narrow size distribution. This can be empirically measured by observing the packaging of the collected droplets. A nearly hexagonal packing arrangement of the collected droplets is indicative of a low monodispersity value (see, for example, L.J. De Cock et al. Angew. Chem. Int. Ed. 2010, 49, 6954 ).
[0514] In one embodiment, the droplets have a relative standard deviation (RSD) of a maximum of 1.5%, a maximum of 2%, a maximum of 4%, a maximum of 5%, a maximum of 7%, or a maximum of 10% .
[0515]The concentration of one or more components in the second phase can be changed. Changes in concentration can alter the physical and chemical properties of the shell material subsequently formed.
[0516] In one embodiment, the concentration of cucurbituril may be altered in order to alter the degree of binding and/or cross-linking in the network formed. An increase in bonding or cross-linking is associated with a decrease in pore size in the material.
[0517] In one embodiment, the cucurbituril concentration and the building block present in the second phase can be changed in order to change the shell thickness.
[0518] In one embodiment, the concentration of cucurbituril in the second phase is at least 0.05, at least 0.1, at least 0.2, at least 0.3, at least 0.5, at least 1.0, at least 5.0 or at least 10 μM.
[0519] In one embodiment, the concentration of cucurbituril in the second phase is at most 500, at most 200, at most 100, at most 75, at most 50 μM.
[0520] In one embodiment, the concentration of cucurbituril in the second phase is in a range where the minimum and maximum rates are selected from the above modalities. For example, the concentration of cucurbituril in the second phase is in the range of 0.1 to 100 μM.
[0521] In one embodiment, the concentration of a building block in the second phase is at least 0.05, at least 0.1, at least 0.2, at least 0.3, at least 0.5, at least least 1.0, at least 5.0 or at least 10 μM.
[0522] In one embodiment, the concentration of a building block in the second phase is at most 500, at most 200, at most 100, at most 75, at most 50 μM.
[0523]In one embodiment, the concentration of a building block in the second phase is in a range where the minimum and maximum rates are selected from the above modalities. For example, the concentration of a building block in the second phase is in the range of 0.1 to 100 μM.
[0524]The reference to a building block may refer to a first building block or a second building block as described herein.
[0525]In one embodiment, the ratio of the cucurbituril concentration to the building block concentration in the second phase is selected from the group consisting of: 1:1, 1:2, 2:3, 1:3, 15:85, 7.5:92.5 and 5:95.
[0526]Where two or more building blocks are present, the building block concentration may refer to the combined concentration of all building blocks present.
[0527] In one embodiment, the concentration of cucurbituril and building block refers to the concentration in the second phase after any subflows, where present, have been brought together.
[0528]Alternatively, the concentration of cucurbituril and building block refers to the concentration within a substream, prior to the coming together of the substreams to produce the second phase. In this embodiment, it will be appreciated that the final concentration of a particular reagent in the second stage will be less than the concentration of that reagent in the downstream. The final concentration of a particular reactant in the final combined second phase is determined by the flow rate of the substream relative to the flow rate of one or more substreams with which it is combined. The flow rate ratio will therefore influence the final concentration of a reactant.
[0529] In one embodiment, the concentration of a component in the second phase may be 5%, 7.5%, 10%, 15%, 20%, 25%, 30%, 33%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 66%, 70%, 75%, 80%, 85% 90%, or 95% of the concentration of this component in the substream.
[0530] In one embodiment, the second phase is generated from the combination of three separate subflows. These streams independently comprise a cucurbituryl, a first building block and a second building block. Where the flow rates are equal, it will be appreciated that the concentration of each cucurbitituril, first building block and second building block in the combined aqueous phase will be a third concentration of each of the reactants within the respective substreams.
[0531]In one embodiment, the ratio of flow rates to subflows of cucurbituril, first building block and second building block is selected from the group consisting of: 1:1:1, 2:2:1 , 1:1:2, 15:15:70, 7.5:7.5:85, and 5:5:90.
[0532] In this embodiment, the first building block may comprise a polymeric molecule and the second building block may comprise a particle.
[0533] In one embodiment, the methods of the invention are carried out at room temperature.
[0534] In one embodiment, the methods of the invention are carried out at about 5, 10, 15, 20, 25, or greater than 25°C. Device
[0535] The methods of the present invention require that a second-phase flow and a first-phase flow, which is immiscible with the second phase, be brought together in a channel to thereby generate a second-phase dispersion in the first. phase. Methods for generating a first-phase and second-phase stream are well known in the art.
[0536] In one embodiment, each stream (a stream or a sub stream) can be generated from a syringe under the control of a programmable syringe pump. Each syringe is loaded with an appropriate aqueous solution or water-immiscible phase.
[0537] In the method of the invention, droplets can be collected only when the flows meet the required flow rate.
[0538]The channel on which the first-phase and second-phase flows come into contact is not particularly limited.
[0539] In one embodiment, the channel is a microfluidic channel.
[0540]In one embodiment, the channel has a greater cross-section of a maximum of 1000, a maximum of 500, a maximum of 200, a maximum of 100 or a maximum of 50 μm.
[0541] In one embodiment, the channel has a major cross-section of at least 0.1, at least 1, at least 10, or at least 20 μm.
[0542]The channel may be provided on an appropriate substrate. The substrate is one that will not react with the components of the complex composition.
[0543]The substrate may be a PDMS-based substrate.
[0544] The preparation of substrates for use in fluidic flow techniques is well known to those skilled in the art. Examples in the art include the preparation described by Yang et al. (Yang et al. Lab Chip 2009, 9, 961), which is incorporated in this document. Second level
[0545]The second phase is immiscible with the first phase. The second phase can be referred to as a dispersed phase, particularly as it has come into contact with the first phase and has been separated into discrete regions, such as droplets.
[0546] In one embodiment, one of the first or second phase is an aqueous phase. Therefore, the other first or second phase is immiscible in water.
[0547] However, it is not essential that one of the phases be an aqueous phase, and those familiar with fluidic techniques will recognize that other combinations of immiscible phases can be used. For example, the use of an organic chloroform phase together with a polyvinyl alcohol phase has been described (see Yang et al. Lab Chip 2009, 9, 961).
[0548] Typically, the second phase is a phase which is suitable to contain, for example, dissolve, one or more between the cucurbituril and the building blocks, and the encapsulant, where present.
[0549] In one embodiment, the second phase is chosen for its ability to dissolve cucurbituril.
[0550]Cucurbituril compounds differ in their solubility in water. Preparation methods can be adapted to take this solubility into account. As described herein, the methods of the invention require the use of a second phase comprising cucurbituril. The second phase can be selected as a phase that is suitable for dissolving cucurbituryl. Where the cucurbituril compound is water soluble, the second phase may be an aqueous phase. Where the cucurbituryl compound has low or no solubility in water, the second phase may be a water-immiscible phase, such as an oil phase or an organic phase.
[0551] In one embodiment, the flow rate of the second phase is max 1000, max 500, max 250, or max 100 μL/min.
[0552] In one embodiment, the flow rate of the second phase is at least 0.05, at least 0.1, at least 0.5, at least 1, at least 5, at least 10, or at least 50 μL /min
[0553]In one embodiment, the flow rate of the second phase is in a range where the minimum and maximum rates are selected from the above modes. For example, the second phase flow rate is in the range of 0.1 to 100 μL/min.
[0554]The second phase flow rate refers to the flow rate of this phase before the phase contacts the first phase.
[0555]Where the second flow is a combination of two or more subflows that are brought into contact, the second phase flow rate refers to the numerical sum of the flow rates of each of the subflows. The flow rate refers to the combined flow rate of the subflows when they are brought into contact, which can be before or substantially at the same time that the flows make contact with the first phase. First phase
[0556] The first phase comprises a component that is immiscible with the second phase. The first phase can be referred to as a continuous or carrier phase.
[0557]In one embodiment, the flow rate of the first stage is max 1000, max 500, or max 250 μL/min.
[0558] In one embodiment, the flow rate of the first stage is at least 10, at least 50, or at least 100 μL/min.
[0559]In one embodiment, the flow rate of the first stage is in a range where the minimum and maximum rates are selected from the above modalities. For example, the flow rate of the first stage is in the range of 100 to 250 μL/min.
[0560]First phase flow rate refers to the flow rate of that phase before the phase contacts the second phase.
[0561]Where a flow focusing technique is used to develop discrete regions of a second phase, the flow rates of the first two phases can be equal.
[0562]The first phase may additionally comprise a surfactant. The surfactant is provided in the first stage in order to stabilize the macroemulsion that is formed in fluidic preparation methods. The step of forming the discrete region (such as a droplet) may require the presence of a surfactant. Furthermore, the presence of a surfactant is useful in limiting or preventing the coalescence of collected droplets.
[0563] The surfactant chosen is not particularly limited, and encompasses any surfactant that is capable of promoting and/or stabilizing the formation of discrete regions, such as droplets, from the second phase to the first phase.
[0564] Surfactants suitable for use in the present invention include those described by Holtze et al. Lab Chip 2008, 8, 1632 . Typically, such surfactants comprise an oligomeric perfluorinated polyether (PFPE) bonded to a polyethylene glycol. Such surfactants are especially useful for stabilizing fluorocarbon oil-in-water emulsions.
[0565] The surfactant is present in a maximum of 0.1%, a maximum of 0.2%, a maximum of 0.5%, a maximum of 0.75%, a maximum of 1%, a maximum of 2%, a maximum of 5% by weight of the full phase.
[0566] The surfactant is present at least 0.05% or at least 0.07% by weight of the total phase.
[0567]Where the first phase is an aqueous phase, the surfactant may be polyvinyl alcohol.
[0568] In one embodiment, the first phase has a solubility in the second phase of at most 50, at most 20, at most 10, or at most 5 ppmw.
[0569] In one embodiment, the second phase has a solubility in the first phase of at most 50, at most 20, at most 10, or at most 5 ppmw. aqueous phase
[0570] The present invention requires the use of an aqueous phase as a continuous or dispersed phase in the methods of the invention. Methods for preparing suitable aqueous solutions comprising the appropriate components will be apparent to those skilled in the art. phase immiscible in water
[0571] The present invention requires the use of a phase that is immiscible in water. This phase can be an oil-based phase (oil phase) or an organic solvent-based phase (organic phase), or a combination of the two.
[0572] In one embodiment, the water-immiscible phase is a liquid phase.
[0573] In another embodiment, the water immiscible phase is a gas phase. Typically, this embodiment is suitable where the water-immiscible phase is the first phase.
[0574] The oil phase has an oil as a main component. Oil is a liquid at room temperature.
[0575]Oil is inert. That is, it does not react with cucurbituril to form a complex, or any other product. Oil does not react with any building blocks present. Oil does not react with the housing.
[0576] In one embodiment, the oil is a hydrocarbon based oil.
[0577] In one embodiment, the oil is a mineral oil.
[0578] In one embodiment, the oil is a fluorinated hydrocarbon oil.
[0579] In one embodiment, the oil is a perfluorinated oil. An example of a perfluorinated oil for use in the invention is FC-40 (Fluoroinert available from 3M).
[0580] In one embodiment, the oil is a silicone oil.
[0581] In one embodiment, the water-immiscible phase has an organic solvent as a main component. For example, the organic solvent is selected from chloroform and octane. Capsule with encapsulant
[0582] The methods of the invention are suitable for incorporating a material into a capsule. Therefore, the capsule produced comprises an encapsulated material (an encapsulant).
[0583] In a further aspect of the invention there is provided a method for preparing a capsule having a shell, wherein the capsule encapsulates a component, the method comprising the steps of: (i) contacting a stream of a first phase and a flow of a second phase in a channel to thereby generate a discrete region, preferably a second phase droplet in the channel, wherein the second phase comprises (a) a cucurbituryl, a component and (1 or 2); or (b) a plurality of covalently linked cucurbituryls, a component and (1), (2) or (3) to thereby form a capsule shell within the discrete region, wherein the capsule encapsulates the component and the first and second phases are immiscible.
[0584] The method of the invention is particularly attractive as it allows all components present in the second phase flow to be encapsulated within the capsule shell. Capsule shell formation occurs at the droplet boundary at the interface with the first phase. The entire component is therefore encapsulated within the formed housing. The present method, therefore, provides an efficient method for incorporating the component into a capsule.
[0585]In one embodiment, the component to be encapsulated is provided in a subflow. This substream is brought into contact with one or more other substreams comprising reagents for preparing the shell, for example cucurbituril and one or more building blocks. The subflow comprising the component to be encapsulated can be brought into contact with another subflow prior to contacting the flow of the first phase. Alternatively, the sub-stream comprising the component to be encapsulated may be brought into contact with another sub-stream at substantially the same time as the first phase is brought into contact with the second-phase flow.
[0586] Providing a substream for the component to be encapsulated separate from a substream comprising a material for the capsule shell allows the concentration of the component in the discrete region formed (such as a droplet) to be controlled, and the amount encapsulant end present within the housing is also controlled.
[0587] In one embodiment, the method is a method for preparing a capsule that encapsulates a plurality of components. In this embodiment, the aqueous phase comprises at least a first component to be encapsulated and a second component to be encapsulated. The plurality of components may be provided in separate substreams which come into contact prior to contacting the first phase or substantially at the same time as the second phases are brought into contact with the flow of the first phase.
[0588] In one embodiment, the concentration of a component to be encapsulated in the second phase is at least 0.01, at least 0.02, at least 0.05, at least 0.1, at least 0.2, at least 0.3, at least 0.5, at least 1.0, or at least 5.0 µM.
[0589] In one embodiment, the concentration of a component to be encapsulated in the second phase is at most 500, at most 200, at most 100, at most 75, at most 50, or at most 10 μM.
[0590] In one embodiment, the concentration of a component to be encapsulated in the second phase is in a range where the minimum and maximum rates are selected from the above modalities. For example, the concentration of a component to be encapsulated in the second phase is in the range of 0.02 to 50 μM.
[0591] In one embodiment, the concentration of the component to be encapsulated refers to the concentration in the second phase after any underflows, where present, have been gathered.
[0592]Alternatively, where the aqueous stream is a combination of two or more sub streams that are brought into contact, the concentration of the component to be encapsulated refers to the concentration within a sub stream, before the sub streams come together to produce the second phase. In this modality, it will be evaluated that the final concentration of the encapsulant in the second phase will be lower than the concentration of this reagent in the substream. The final concentration of encapsulant in the final combined second phase is determined by the flow rate of the substream relative to the flow rate of one or more substreams with which it is combined. The flow rate ratio will therefore influence the final concentration of a reactant.
[0593] In one embodiment, the concentration of a component in the aqueous phase may be 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% 90%, or 95% of the concentration of this component in the substream.
[0594]Where an additional encapsulating subflow is provided along with the subflows of each cucurbituryl and one or more building block flows, the flow rates for each cucurbituryl and one or more building blocks may be changed to account for the effect of dilution to provide an additional underflow to the encapsulant.
[0595] The present invention provides a capsule that is obtainable or obtainable from any of the methods described herein. The capsule may comprise an encapsulated component, which may also be prepared using the methods described herein. capsule analysis
[0596] In the above sections, the analysis of the housing material, housing format, housing dimensions are described. For example, the capsule can be analyzed by simple brightfield microscopy to determine the shape of the capsule shell. The images obtained can also be used to determine the cross-section, typically the diameter, of the capsule shell.
[0597]The capsule shell can also be analyzed for shape, cross-section and thickness using scanning electron microscopy and transmission electron microscopy. The latter is particularly useful for studying envelope network compositions. For example, where the network comprises a polymeric composite building block and a nanoparticle building block, the nanoparticles can be seen as dispersed throughout a polymeric material. This dispersion is the result of the complexation and interconnection of nanoparticles and polymeric molecules with cucurbituril.
[0598] The present inventors have generally incorporated a detectable marker into the shell material and encapsulant, to then allow each to be located and defined. When this marker is a fluorescent marker, it can be detected by laser scanning fluorescent microscopy, for example.
[0599] The present inventors have also generated capsules that have building blocks that are capable of providing a surface-augmented resonance effect. In particular, this effect is provided by particles, such as metal nanoparticles, which are used as building blocks.
[0600] The presence of building blocks within the shell that are capable of providing a surface-augmented resonance effect can be advantageously used to analyze the shell itself, or the encapsulated material. A building block can be selected to provide the highest suitably useful boost. For example, a larger nanoparticle building block, for example a 5 nm diameter nanoparticle, can provide a greater magnification than a 2 nm diameter nanoparticle.
[0601]In one embodiment, the surface-augmented spectroscopy is a surface-augmented Raman spectroscopy (SERS).
[0602] In one embodiment, the surface-augmented spectroscopy is anti-Stokes Coherent Raman spectroscopy (CARS).
[0603] In one embodiment, surface-augmented spectroscopy is photoluminescent spectroscopy.
[0604]In one embodiment, the surface-augmented spectroscopy is infrared spectroscopy.
[0605]The present inventors have demonstrated that surface-augmented resonance spectroscopy can be used to confirm the presence of an encapsulant within the capsule. For example, SERS can be used to identify the presence of a biological molecule, such as dextran, by identifying characteristic signals in the Raman spectrum of dextran.
[0606]The intensity of the peaks can be used to quantify the amount of encapsulant present within the capsule.
[0607]Surface augmented spectroscopic techniques are well known to those skilled in the art, and suitable techniques are described in detail here. use of capsules
[0608]Capsules as described herein are suitable for use as material encapsulants. This material can be stored inside the capsule and released from the capsule as required.
[0609] In one embodiment, a capsule of the invention is provided which comprises an encapsulated component.
[0610] In one aspect, the present invention provides a method of applying a component to a location, the method comprising the steps of: (i) providing a capsule of the invention, which comprises an encapsulated component; (ii) applying the capsule to a site; and (iii) allow the release of the encapsulated component of the capsule in place.
[0611]In one embodiment, the location is in vivo.
[0612]In one embodiment, the location is ex vivo.
[0613]In one embodiment, the release of the encapsulated component occurs in response to an external stimulus.
[0614] In one embodiment, the external stimulus is selected from the group consisting of a visiting compound, the competitor, light, oxidizing agent, and reducing agent.
[0615]In one embodiment, the release of the encapsulated component occurs in response to a change in local conditions.
[0616] In one embodiment, the change in local conditions may be a change in pH, a change in temperature, a change in oxidation level, a change in concentration, or the emergence of a reactive chemical entity.
[0617] In one embodiment, the release of the encapsulant is accomplished by disrupting the complex formed between the cucurbituril and the visiting molecule or molecules. In one embodiment, a compound covalently linked to a competing visiting molecule is provided at the delivery site. The competing visitor molecule displaces a visiting molecule from a building block and then disrupts the network that forms the capsule shell. This rupture can cause pores to appear in the casing, through which the encapsulated compound can pass and be released. In one embodiment, the competing visiting molecule causes extensive disruption of the capsule shell.
[0618] In one embodiment, the release of the encapsulant is accomplished by breaking the complex using light, for example, incident laser light. In their experiments to determine the surface-enhanced spectroscopic properties of the capsules of the invention (e.g., those capsules that contain particles), the present inventors have found that exposing the capsule to laser light results in at least partial loss of capsule integrity. The inventors have discovered that SERS analysis can still be conducted on the capsules of the invention. When the power of incident laser light is increased and/or the time of exposure of the capsules to laser light is increased, the capsules are observed to degrade. The degrading effect can be influences from the shell components, for example the nature of the building blocks. For example, small gold nanoparticles (eg 5 nm in diameter) are known to absorb rather than scatter incident laser light. This absorbed light can radiate out as heat, this can have the effect of disrupting the local network of complexes.
[0619]In one embodiment, the capsule encapsulates two or more components.
[0620]When there are two or more components, the components can later be released simultaneously or sequentially.
[0621]In one embodiment, a first component is released. A second component is released after the first component.
[0622] In one embodiment, the capsule of the invention is suitable for connection to a surface. For example, the capsule may be provided with functionality that is suitable for forming a bond, such as a covalent bond, to the surface. This functionality can be contained within a building block that is a component of the wrapper. Capsules may be disposed on a surface to provide a matrix. Alternative visitor-host supramolecular complexes
[0623]The capsules described herein possess a shell that is obtainable from supramolecular complexation of cucurbituril with building blocks covalently linked to the appropriate cucurbituril visitor molecules.
[0624] In a further general aspect of the invention, the present invention provides capsules that have a shell that is obtainable from supramolecular complexation with a host with building blocks covalently linked to the appropriate host visiting molecules.
[0625] As noted above, the host may be cucurbituryl and the visitor may be a cucurbituril visitor molecule. Other visitor-host complexes can be used, alternatively to the cucurbituril visitor-host complex or in addition to the cucurbituril visitor-host complex.
[0626] Therefore, the present invention encompasses the use of a visitor who is capable of non-covalently hosting one or two visitors, thereby, to cross-link the building blocks to which the visitors are covalently linked.
[0627] The use of cucurbituril is preferred due to the high binding constants that are available and the ease with which the complexes, and capsules, can be assembled.
[0628]A reference to cucurbituril in the present application may therefore be adopted as a reference to an alternate host. A reference to a cucurbituril visitor molecule can therefore be adopted as a reference to an alternative host visitor molecule. The preferences set forth in the sections pertaining to the capsule, complex, building block, method of preparation, and capsule use apply to the visitor and additional and alternate hosts described herein, with appropriate adaptations of characteristics as necessary. Therefore, the inventors believe that the technical methods described here are generally applicable to other visitor-host systems.
[0629]An alternate host may be able to form a ternary complex. When the complex comprises two visitors within a visitor cavity, the association constant, Ka, of that 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. In this embodiment, the shell is a network that has a plurality of complexes, each complex comprising a host that hosts a first visiting molecule and a second visiting molecule. The first and second visiting molecules are covalently linked to a first building block, or to a first building block and a second building block.
[0630]An alternate host may be able to form a binary complex. When the complex comprises a visitor within a visitor cavity, the association constant, Ka, of that complex is at least 103 M-1, 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. In this embodiment, the envelope is a network that has a plurality of complexes, where each complex comprises a host that hosts a visiting molecule, and each host is covalently linked to at least one other host. Visitor molecules are covalently linked to a first building block, or to a first building block and a second building block.
[0631]In one embodiment, the host is selected from cyclodextrin, calix[n]arene, crown ether, and cucurbituril, and one or more building blocks have adequate host-guest functionality for cyclodextrin, calix [n]arene, crown ether or cucurbituril host respectively.
[0632]In one embodiment, the host is selected from cyclodextrin, calix[n]arene, and crown ether, and one or more building blocks have adequate host-visitor functionality for the cyclodextrin, calix[n] arene, or crown ether respectively.
[0633]In one embodiment, the host is cyclodextrin and one or more building blocks have adequate cyclodextrin visitor functionality.
[0634]The host can form a binary complex with a visitor. In such cases, the host will be covalently linked to one or more other visiting molecules to allow crosslinks to form between the building blocks.
[0635] In one embodiment, the host is cyclodextrin. Cyclodextrin compounds are readily available from commercial sources. Many visiting compounds for use with cyclodextrin are also known.
[0636]Cyclodextrin consists of barrel-shaped non-symmetrical cyclic oligomers of D-glucopyranose. Typically, cyclodextrin is capable of hosting uncharged hydrophobic visitors. For example, visitors include those molecules that have hydrocarbon and aromatic functionalities such as adamantane, azobenzene, and stilbene derivatives. Other cyclodextrin visitor molecules include biomolecules such as xylose, tryptophan, estriol, sterone, and estradiol.
[0637] In one embodiment, the cyclodextrin is an a-, β- or y-cyclodextrin. In one embodiment, the cyclodextrin is a β- or y-cyclodextrin. Typically, larger visitors are used together with a y-cyclodextrin.
[0638]Cyclodextrin has a toroid geometry, with the secondary hydroxyl groups of D-glucopyranose located in the larger opening, and the primary hydroxyl groups in the smaller opening. One or more hydroxyl groups, which may be secondary or primary hydroxyl groups, may be functionalized. Typically, 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 of the cyclodextrin are functionalized with an alkylamine-containing subsistent. In another example, one, two or three hydroxyl groups within each D-glucopyranose unit are replaced by an alkyl ether group, for example a methoxy group. A plurality of covalently linked cyclodextrins may be connected through hydroxyl groups.
[0639]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 visitors are shown in Tables 1 to 3 and Graph 2. Rekharsky et al. is incorporated by reference herein.
[0640] In the preparation methods, the cyclodextrin may be present in the second phase, for example, in an aqueous phase, as described herein.
[0641]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.
[0642]Many visiting 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 may be used as visitors. Additional examples of visitors include atropine, critting, blue phenol, and blue anthrole among others.
[0643]Examples of non-functionalized and functionalized cyclodextrins are presented in Graph 1 by Danil de Namor et al. (Chem. Rev. 1998, 98, 24952525), which is incorporated herein by reference. Examples of compounds for use as visitors are presented in Tables 2, 3, 5 and 10 of Danil de Namor et al.
[0644]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.
[0645]Properly functionalized calix[n]arenes can be prepared through the use of properly functionalized hydroxy aryl aldehydes. For example, the hydroxyl group may be substituted with an alkyl ether-containing group or an ethylene glycol-containing group. A plurality of covalently bonded calix[n]arenes can be connected through the hydroxyl groups.
[0646]In the preparation methods, the calix[n]arene may be present in the second phase, for example, in an aqueous phase or a water-immiscible phase, as described herein.
[0647] In one embodiment, the host is a crown ether. Crown ether compounds are readily available from commercial sources or can be easily prepared.
[0648]Many visiting compounds for use with crown ether are also known. For example, cationic visitors such as functionalized amino and pyridinium molecules may be suitable visitor molecules.
[0649]Examples of non-functionalized and functionalized cyclodextrins are presented throughout Gokel et al. (Chem. Rev. 2004, 104, 2723-2750), which is incorporated herein by reference. Examples of compounds for use as visitors are described throughout the text.
[0650] In one embodiment, the crown ether is selected from the groups consisting of 18-crown-6, dibenzo-18-crown-6, diaza-18-crown-6 and 21-crown-7. In the present invention, major crown ethers are preferred. Smaller crown ethers may be able to bind only small metal ions. Larger crown ethers are capable of binding functional groups and molecules.
[0651]In some embodiments, the host is a visitor who has crown ether and calix[n]arene functionality. These hosts are referred to as calix[n]crowns.
[0652]In the preparation methods, the crown ether may be present in the second phase, for example in a water-immiscible phase, as described herein.
[0653]Other visitor-host relationships may be used as will be apparent to one skilled in the art. Other visitor-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, cyldoextrin, and calixeran as well as cyclophane AVCyc, calixpyridine C4P and SQAM squarimide.
[0654]The use of cyclodextrin is preferred over crown ether and calix[n]arene hosts. other preferences
[0655]Each and every compatible combination of the modalities described above is explicitly described, as if each combination were individually and explicitly mentioned.
[0656] Several additional aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present description. “and/or” when used herein shall be taken as the specific description of each of the two specified features or components with or without the other. For example, “A and/or B” should be adopted as the specific description of each (i) A, (ii) B, and (iii) A and B, just as if each were presented individually here.
[0657]Except where the context dictates otherwise, the descriptions and definitions of features set forth above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments that are described.
[0658] Certain aspects and embodiments of the invention will now be illustrated by way of example and with reference to the figures described above. Experiments and Results
[0659]IH NMR spectra (400 MHz) were recorded using a Bruker Avance QNP 400. Chemical shifts are recorded in ppm (δ) in CDCl3 with the internal reference set to d 7.26 ppm or MeOD with the internal reference adjusted for d 3.31 ppm. Chemical shifts are recorded in ppm (δ) in CDCl3 with the internal reference set to d 77.16 ppm and d 49.00 ppm, respectively. ATR FT-IR spectroscopy was performed using a Perkin-Elmer Spectrum 100 series FT-IR spectrometer equipped with an ATR universal sampling accessory. UV Visible Studies were performed on a Varian Cary 4000 UV-Vis spectrophotometer. High resolution mass spectra were recorded on a Bruker BioASpex II 4.7e FT-ICR mass spectrometer by Waters ZQ liquid chromatography mass spectrometry. All starting materials were purchased from Alfa Aesar and Sigma Aldrich and used as received, unless otherwise stated. CB[8] was prepared as previously documented by Kim (see Kim et al. J. Am. Chem. Soc. 2000, 122, 540). MV2+-AuNP (5 nm) and Np-pol were synthesized according to literature methods (see Coulston et al. Chem. Commun. 2011, 47, 164), while MV2+-pol was prepared as previously reported (see Appel et al. al. J. Am. Chem. Soc. 2010, 132, 14251 ). The entire aqueous phase was dissolved in deionized water treated with a Milli-QTM reagent system which guarantees a resistivity of >15 MQcm-1. General strategy for capsule formation
[0660] The general strategy for preparing capsules is described below. Detailed experimental details are provided in subsequent sections.
[0661]In a typical microcapsule preparation procedure, microdroplets were first generated using a simple T-junction geometry (see Xu et al. AIChE Journal 2006, 52, 3005 and Thorsen et al. Phys. Rev. Lett. 2001 , 86, 4163) as schematically depicted in Figure 1a. In this experiment, the first phase is an oily phase comprising a surfactant, and the second phase is an aqueous phase. The oily carrier phase was directed to the device perpendicular to the dispersed aqueous phase, which comprises three inlets for the CB[8] 1 solution, the MV2+-AuNP 2 solution, and the Np-pol 3 solution. Droplets are generated as the immiscible oil phase cuts through the water phase at a frequency that is dependent on the flow rate ratio of the two phases. Immediately after generation, the droplets pass through a spiral channel that is designed for complete mixing of the three reactants in line, as shown in Figure 1b. With an oil:water flow rate ratio of 2:1 (where the aqueous phase flow rate refers to the combined flow rate of the three substreams), the droplets were generated at a frequency of 300 Hz and exhibit a high level of monodispersity when collected on a microscope slide, as indicated by its limited size distribution and a low coefficient of variation of 1.3% (Figure 1c).
[0662]The formation of microcapsules was observed after dehydration of the microdroplets. The process was captured in Figure 2a where in the final stage of droplet shrinkage, microcapsule formation was visible as the spherical shape of the droplet was distorted. This one appears to be bent at the edge at random, apparently deformed. The nature of this capsule-like structure was further verified when the burst capsules were observed after induction of the osmotic force mechanism of rehydration (Figure 2b). Scanning electron microscopy (SEM) image was also obtained (Figure 2c), showing a highly wrinkled, randomly bent structure that looks like a hollow capsule that has been deformed due to lack of internal support. The capsule shell consists of a network of MV2+-AuNPs and polymers, as shown in the transmission electron microscopy (TEM) image (Figure 2d), whereby individual MV2+-AuNPs were interconnected through a network of presumably Np polymers. . It is likely that capsule shells consist of multiple layers, as the MV2+-AuNPs appear to be superimposed on each other. The process of this supramolecular microcapsule formation is schematically represented as in Figure 2e. Although the aqueous mixture of CB[8], MV2+-AuNP, and Np-pol is initially confined to the droplets, it is believed that the deposition of the cross-linked supramolecular composite at the oil-water interface is assisted not only by the driving force of evaporation of water, as well as interfacial energy stabilization through colloidal nanoparticles (see Patra et al. Chemistry - An Asian journal 2010, 5, 2442). Once formed, these microcapsules are highly resistant to heat (100°C) and reduced pressure, presumably due to the highly stable backbone of 1:1:1 CB[8] ternary complexes. Microcapsule formation was not observed when CB[8] is replaced by CB[7], which is unable to form ternary complexes, or when AuNPs are not functionalized with the MV2+ ligand (see below).
[0663]The interfacial molding effect was further investigated by incorporating a fluorophore into the polymer so that the polymer distribution can be visualized during capsule formation. As shown in Figure 3a, rhodamine-6G functionality was incorporated into the polymer containing PEG and NP functionality (NP-RD-pol 4) via RAFT polymerization (Supplementary Information). The aqueous NP-RD-pol solution was then injected into the droplet generation device along with the aqueous solution of the other two microcapsule components as described above. Droplets were conveyed and collected by a PDMS reservoir mounted on a glass slide and fluorescence images of the aqueous droplets in oil were recorded using a laser scanning confocal microscope (LSCM) with a 63x oil immersion objective. Figure 3b shows a clearly defined layer of rhodamine fluorescence confined at the water-oil interface of a droplet, indicative of polymer distribution during microcapsule formation. This observation is due to the hydrophobic interaction between the fluoro oil and the polymer, and is consistent with the previously reported interfacial molded synthesis of polymeric microcapsules (see Abraham et al. Advanced Materials 2008, 20, 2177 and Yang et al. Lab Chip 2009, 9, 961).
[0664] The fact that a hollow capsule can be easily fabricated through microdroplet-assisted encapsulation, in turn, results in the notion that water-soluble fillers can be loaded into these microcapsules with 100% efficiency, since loading does not relies on passive diffusion post-microcapsule synthesis (see Peyratout et al. Angew. Chem. Int. Ed. 2004, 43, 3762; Abraham et al. Advanced Materials 2008, 20, 2177; An et al. Biomacromolecules 2006, 7 , 580). The hypothesis was tested by loading fluorescein isothiocyanate-labeled dextran (FITC-dextran) in the capsules. Dextrans, because of their high biocompatibility and versatility, have been widely exploited for the delivery of drugs, proteins, and imaging agents (see Mehvar Journal of Controlled Release 2000, 69, 1). Using the current microfluidic system, loading of FITC-dextran was carried out simply by incorporating a FITC-dextran solution into one of the aqueous inlets. The microdroplets generated in this way contained 25% vIv of FITC-dextran in addition to the mixture of CB[8], MV2+-AuNP, and Np-RD-pol.
[0665]LSCM images were recorded and analyzed as before and shown in Figure 3c. The initial 46 μm diameter droplet exhibited a defined layer of rhodamine fluorescence at the water-oil interface, while the center of the capsule was uniformly filled with FITC fluorescence from the FITC-dextran. By examining the fluorescence intensity graph, one can clearly observe that outer rhodamine is distributed on the outer edge of inner-FITC, showing that the "shell" is indeed formed out of the "charge". As the droplets dehydrate over time, the material becomes more concentrated and so the fluorescence intensity increases. The same 23 μm diameter droplet, with its LSCM image and recorded fluorescence intensity profile (Figure 3d), showed a smaller visible boundary between the inner FITC and outer rhodamine. Indeed, FITC-interior appears to have begun to become a part of the shell wall, most likely through the interstitial sites of the capsule shell. This observation provides the basis for the SERS analysis of this material, which will be elaborated later. Overall, these LSCM results clearly demonstrated the hollow nature of these supramolecular microcapsules that exhibit quantitative loading efficiency with supramolecularly constructed capsule shells easily subjective to various functionalities. General strategy for forming capsules comprising an encapsulate
[0666] The permeability of polymeric microcapsules is generally studied using commercially available FITC-dextrans of varying molecular weight as a model filler, due to their simplicity in chemical composition and uniformity in format (see An et al. Biomacromolecules 2006, 7, 580; Hermanson et al. Physical Chemistry Chemical Physics 2007, 9, 6442 ). The permeability of the present microcapsules was studied by encapsulating an aqueous solution of the FITC-dextran followed by complete dehydration, and then rehydrating the capsules while monitoring the distribution of FITC using a fluorescence microscope. As shown in Figure 4a, dried microcapsules containing FITC-dextran exhibit bright fluorescence despite the wrinkled surface. Permeation of the fluorescent cargo was initiated by re-dispersing the capsules in water. When 10 kDa of microcapsules containing FITC-dextran are hydrated, the capsule wall expands to resume the initial spherical outline, while the previously confined FITC fluorescence is spilled into the outer aqueous phase (Figure 4a). In contrast, when 500 kDa of FITC-dextran was encapsulated in the capsules, upon rehydration, the fluorescence was strictly localized inside the capsules without any permeation (Figure 4b). This result reveals the porous nature of the microcapsule shell, which has a certain limit for a particular sized load. This is in agreement with many previously reported polymeric and polyelectrolyte microcapsules (see An et al. Biomacromolecules 2006, 7, 580; Cavalieri et al. ACS Nano 2009, 3, 234; Ameloot et al. Nat. Chem. 2011, 3, 382 ; ). Despite swelling and expansion of the capsule wall to some extent, these capsules are stable in water for at least 48 h.
[0667] In addition to the FITC-dextrans mentioned above, another molecular weight fluorescent probe was also tested to further explain the permeability of the microcapsules. The results are summarized in Table 1a, where a permeability of "0" denotes complete blocking of the dextrans by the microcapsule shell, and "100%" means that the capsules are completely permeable. Each result was observed in more than 20 capsules. For that particular microcapsule manufactured from a given ratio of components, any dextrans with a molecular weight of 70 kDa and below are able to spread out of the capsules with ease while any dextrans with a molecular weight of 150 kDa and above are completely trapped by the capsule shell. Since the dextran chain conformation can be observed as a small globular particle in solution, the turning radius (RE) of FITC-dextran can provide a satisfactory assessment of pore size (see Andrieux et al. Analytical Chemistry 2002, 74 , 5217). FITC-dextran with a molecular weight of 70 kDa has a gyrating radius of approximately 8 nm, while its 150 kDa counterpart has a radius of 11 nm (see Granath Journal of Colloid Science 1958, 13, 308). With the change in permeability as a function of the molecular weight of FITC-dextran, this indicates that the microcapsule is permeable to macromolecules with a size smaller than 11 nm.
[0668]Table 1 has data on the qualitative permeability variation of (a) microcapsules containing MV2+-AuNP, CB[8] and Np-pol with a molar ratio of 2:1:2 for MV2+:CB[8]:Np , and (b) microcapsules containing MV2+-AuNP, CB[8] and Np-pol with a 1:1:1 molar ratio for MV2+:CB[8]:Np, as a function of the molecular weight (MW) of the probe fluorescent.

[0669]The relationship between this degree of capsule shell permeability and the extent of crosslinking between MV2+-AuNP and Np-pol, the CB[8] pathway was also investigated. For this purpose, a different microcapsule was manufactured containing twice the amount of CB[8], simply by varying the flow rate of the reactants and then the percentage of the component. This is believed to increase the degree of cross-linking, consequently, as more cross-links are provided before all binding sites are saturated. Permeability studies were then performed by encapsulating and diffusing FITC-dextrans with varying molecular weight in water. The results are summarized in Table 1b, showing that these capsules are completely permeable to FITC-dextran with a molecular weight of 40 kDa and are impermeable to those with a molecular weight of 70 kDa and above. Compared to their counterpart's permeability cutoff with only 50% CB[8] in the mix, these capsules are permeable to macromolecules with a size smaller than 8 nm. This demonstrates the ease of adjusting the pore size of these supramolecular microcapsules by varying the ratio of capsule shell components, providing great potential for the pore size to be customized depending on the properties of the charge to be released. Additional comments on the general strategy for forming capsules that comprise an encapsulant
[0670] The present inventors have also confirmed that the methods of the present invention can be used to prepare a capsule that encapsulates a microorganism, and, in particular, a cell. In the examples described above for the preparation of a dextran-containing capsule, the dextran-containing phase was adapted so that a bacterial cell suspension was used in place of the dextran. In this case, an E. coli suspension of green fluorescent protein (GFP) expression was used. A capsule containing E. coli was obtained and analyzed by LSCM. The LCSM image and intensity profile are shown in Figure 10. General methods for determining accentuated resonance effects on capsule surface
[0671] The potential of using these AuNP-containing microcapsules as an innovative type of plasmonic material for surface-enhanced Raman spectroscopy was also investigated. Since dehydration proceeds during capsule formation, a small amount of the encapsulated material appears to be located in the pores of the shell, these capsules would have the potential to accentuate not only the Raman signals from the capsule shell material itself, but also possibly any material that has been encapsulated by the microcapsule. This hypothesis was investigated by preparing different microcapsules containing 5 nm and 20 nm MV2+-AuNPs and with a multivalent methyl viologen-functional polymer (MV2+-pol 5) (Figure 6a). In both examples of AuNP-containing capsules, when the sample was excited using a 633 nm laser line, the characteristic SERS signals for CB[8] and MV were acquired: CNC strain and CH2 chain angular strain for CB[8] (830 cm-1), and CC ring stretch for MV2+ (1630 cm-1), CN ring stretch for MV2+ (1560 cm-1), and CC inter-ring stretch for MV2+ (peaks of 1308 cm-1) -1) (see Forster et al. Journal of Raman Spectroscopy 1982,12, 36) (Figure 6b). However, the signal intensity of capsules containing 5nm MV2+-AuNPs is almost negligible compared to that of capsules containing 20nm MV2+-AuNPs. This is in agreement with the fact that the SERS field accentuation is dependent on the distance between NPs and hence on the size of NPs (see Anema et al. Annual Review of Analytical Chemistry 2011). SERS mapping of a microcapsule showed that SERS signals were uniformly localized only to the capsule area (Figure 6c). A true indication of the utility of our microcapsule fabrication method, a negative control microcapsule was also synthesized by replacing the MV2+-AuNPs with a multivalent MV2+-polymer 5 to produce a polymer-polymer host-visit microcapsule. For this system, no SERS signal was recorded (Figure 6c). These results demonstrate that these supramolecular microcapsules can be used as an effective plasmonic material that produces strong SERS signals for compounds among the AuNPs.
[0672]To investigate the possibility of detecting encapsulated materials, FITC-dextran was loaded into capsules containing MV2+-AuNPs. Significant Raman enhancement from FITC was observed in addition to that from CB[8] and MV: 1186, 1232 and 1400 cm-1 (Figure 6d). A similar dependence of the degree of enhancement on the size of AuNPs was observed where strong signals were measured for capsules containing the 20 nm MV2+-AuNPs, a negligible enhancement for those containing the 5 nm MV2+-AuNPs, while no signal was recorded when no AuNP was incorporated into the wrapper. This can also be attributed to the highly absorbing nature of the smaller-sized MV2+-AuNPs (see Link et al. The Journal of Physical Chemistry B 1999, 103, 4212), where light is more efficiently converted to heat and therefore providing weak SERS signals. AuNPs with a diameter of 20 nm, conversely, have a stronger plasmon resonance (see Kelly et al. Journal of Physical Chemistry B 2002, 107, 668) and therefore produce strong SERS signals. This result indisputably demonstrated that the porous shell of this supramolecular microcapsule can be used as a SERS substrate for the detection of encapsulated materials. Synthesis of 30 nm MV-AuNPs
[0673]AuNPs were synthesized using a Modified Literature Method (see Martin et al. Langmuir 2010, 26, 7410). Briefly, to an aqueous solution (200 mL) of gold(III) chloride trihydrate (24 mg, 0.06 mmol) was added sodium borohydrate (3.4 mg, 0.09 mmol) at 50% (v/v) of aqueous ethanol (5 mL) and the resulting solution immediately cooled in an ice bath for 30 seconds. The solution turned red in color and was stored in the refrigerator (approximately 4°C) until future use (Note: AuNPs were used within 2 to 3 days of preparation). Stock solutions (5 mL) for each of the thiols EG3 S1 (4.72 mg, 1.98 x 10-2 mmol) and MV2+ S2 (8.5 mg, 1.98 x 10-2 mmol) were prepared in water (5 mL). Aliquots of the stock solutions (MV2+ S2: 0.45 mL, MV2+ 20%, approximately 1170 MV ligands per AuNP; EG3 S1: 1.8 mL) were pooled and quickly added to a swirling solution (10 mL) of the synthesized AuNPs. . The mixture was then incubated for 48 hours. The MV2+-AuNPs were purified using a centrifuge (120 seconds, 12,100 g) and washed with Milli-Q water. In the final wash, the AuNPs were concentrated to 1 mL and used directly to prepare the microcapsules.
[0674]Average diameter: 32 nm ± 5 nm, (TEM), λmax 529 nm.
Synthesis of 50 nm MV-AuNPs
[0675] The synthesis of citrate-stabilized AuNPs followed a standard literature method (see G. Frens Nature Phys. Sci. 1973, 241, 20). The AuNPs stabilized in citrate (10 mL) were added dropwise to an aqueous solution (10 mL) of CTAB (3.6 mg, 9.8 μmol). The AuNPs were then washed using a centrifuge (120 seconds, 12,100g) and Milli-Q water to remove any excess binders. Stock solutions (5 mL) for each of the thiols EG3 S1 (4.72 mg, 1.98 x 10-2 mmol) and MV2+ S2 (8.5 mg, 1.98 x 10-2 mmol) were prepared in water (5 mL). Aliquots of the stock solutions (MV2+ S2: 0.45 mL, 20% MV, approximately 7340 MV ligands per AuNP; EG3 S1: 1.8 mL) were combined and then quickly added to a swirl solution (10 mL) of AuNPs stabilized in CTAB. The mixture was then placed on a shaker for 48 hours at 150 rpm. The 50 mm d MV2+-AuNPs were then isolated from the excess ligands with two washes using a centrifuge (1 x 60 s, 12,100 g and 1 x 45 s, 12,100 g) and Milli-Q water. In the final wash, the AuNPs were concentrated to 1 mL and used directly to prepare the microcapsules. Mean diameter: 52 nm ± 7 nm (TEM), Amax 547 nm.
General synthesis of Np and rhodamine functional polymer
[0676]Based on RAFT polymerization (see Chiefari et al. Macromolecules 1998, 31, 5559), S1 (6.3 mg, 0.026 mmol), S2 (0.50 g, 1.05 mmol), S3 (0. 18 g, 0.52 mmol), 54 (34.0 mg, 0.052 mmol), 4,4-azobis(4-cyanopentanoic acid (ACPA, 1.4 mg, 0.0052 mmol), and dioxane (1.0 mL) were added to a Schlenk tube and the mixture was deeply degassed using three freeze-pump-thaw cycles. The mixture was subsequently immersed in a thermostated oil bath at 70°C for 10 hours. The polymerization was quenched using liquid nitrogen followed by dilution with THF before the solution was added dropwise to diethyl ether. The polymer was dissolved in water and then placed in a dialysis tubing (cut NMWCO 2000 Da) and dialyzed against water by more than 48 hours with 3 water changes. The aqueous solution was then freeze-dried to yield polymer 5 as a pink oil (0.46 g, 68%).1H-NM Spectroscopy R (D2O, 500 MHz, 298.5 K) δ = 7.41, 7.18, 6.98, 4.11, 3.60, 3.31, 0.91, 0.35ppm, FT-IR ( ATR) v = 2867.61, 1730.65, 1629.16, 1600.63, 1511.08, 1452.77, 1390.05, 1349.85, 1257.03, 1218.03, 1099.26, 1038, 26, 947.86, 844.85, 751.49 cm -1 , GPC (DMF): Mn = 25.7 kDa (PDI) = 1.18. Fluorescence spectrum: λex max (H2O) = 566 nm , λem max (H2O) = 580 nm.

[0677]The excitation spectrum of isolated Np-RD-pol is shown in Figure 8. Microfluidic device
[0678] The detailed soft lithography procedure for standard fabrication was as reported by Duffy (see Duffy, et al. Anal. Chem. 1998, 70, 4974). The microfluidic device was fabricated by covering the pattern with a vigorously stirred mixture of polydimethyl siloxane (PDMS) and its curing agent (10% pIp) (Sylgard (RTM) 184, Dow Coring). The PDMS was allowed to solidify at 70°C overnight before being peeled, while inlets and outlets were generated using a biopsy punch (Kai Medical, ID 1.0 mm). Confined microfluidic channels were formed by attaching the molded PDMS replica onto microscope slides (Thermo Scientific) after exposure to an oxygen plasma (Femto, 40 kHz, 100 W, Diener Electronic). Microchannels from sealed devices were quickly rinsed with Aquapel (Duxback™) prior to rinsing with Flourinert FC-40 (3M). Fabrication and characterization of microcapsules
[0679] The fluorous oil phase and the appropriate dispersed phase solution were loaded into syringes (Hamilton, Gasligh (RTM)) with needles (Becton Dickinson, PrecisionGlide (RTM)) attached. Needles were equipped with polyethylene tubing (Portex (RTM) Fine Bore, 0.38 mm ID, 1.09 mm 00). The syringes were mounted on syringe pumps (Harvard PHD 2000 Infusion), while the other end of the tubing was inserted into the appropriate ports on the PDMS device. The devices were imaged in real time using an inverted microscope (IX71, Olympus) connected to a Phantom fast camera (V72, Vision Research). Still images and videos were recorded and analyzed using Phantom software. Droplet formation was initiated as oil was first pumped into the device at 200 μL/h to fill the appropriate channels. The aqueous dispersed phase was then pumped into the device at 10-90 μL/h depending on the individual experiment. The fluorous surfactant (1% pv) was dissolved in FC-40 oil and used as the carrier phase. In a typical experiment, the concentrations of the stock solutions of the reagents were 0.3 to 80 μM, yielding the final concentration Cn of a given reagent in further dilute droplets based on its initial concentration (C0), its flow rate Qn, and the total flow rate of all aqueous streams (QT) once encapsulated in droplets:

[0680]After formation, droplets were collected in the downstream PDMS reservoir or transferred to a microscope slide (76 x 26 mm, 1.2 mm, Menzel-Glaser). Upon collection, the droplets were allowed to dehydrate over time for complete microcapsule formation. Detailed capsule preparation example
[0681] In a standard capsule fabrication experiment, an oil phase was introduced into the channel of the microfluidic device described above at a flow rate of 200 μL/h, such flow rate being held constant. The aqueous phases were introduced into the channel at a total flow rate of 100 μL/h, where the individual flow rates were 50 μL/h for CB[8] 1 (40 μM), 25 μL/h for MV2+-AuNP 2 (0.4 μM), and 25 μL/h for Np-pol 3 (2.3 μM). This combination of flow rates in the 40 μm T-junction geometry (cross section) generates discrete aqueous droplets in oil with a diameter of approximately 60 μm. Upon drying, the resulting stable individual capsules retain a diameter of approximately 25 μm. A sample of the microcapsules generated using the above parameters was collected and the size (diameter) of the droplets was measured:

[0682]The size of the droplets and hence that of the capsules are subject to change based on the geometry of the T-junction and the ratio between the continuous phase flow rate and the total dispersed phase flow rate (see, for example, Garstecki et al.). The effect of these factors is investigated and summarized in Figure 8.
[0683]However, varying the flow rates of individual aqueous streams will not change the resulting droplet size as long as the total flow rate of the aqueous streams remains at the same rate as the oil phase. The graph below shows that while the overall droplet size varies depending on the ratio between the oil flow rate and the combined aqueous flow rate, the variation in the ratio between the flow rates of individual aqueous streams does not result in any significant change in the resulting distribution of droplet size. The raw data is summarized in the table below.
Confocal laser scanning microscopy experiments and analysis
[0684]Sample preparation initially involved collecting aqueous droplets in FC40 oil into a PDMS reservoir mounted on a microscope slide (22 x 50 mm, 0.17 mm, MenzelGlaser). The sample was imaged in the reservoir at different time intervals to capture the capsules at different stages of dehydration. LSCM measurements were performed using a Leica TCS SP5 confocal microscope using a 63x objective lens (NA = 1.4, Leica HCX PL APO Lambda blue) with water or oil (Leica Type F Immersion Fluid, n23 = 1.518) used as the immersion medium depending on the experiment. Samples were illuminated with 488 nm or 544 nm laser lines to excite FITC-dextran and rhodamine-containing polymer, respectively. FITC-dextran emission, peaking at 520 nm (product data sheet) and Np-RD-pol emission, peaking at 582 nm (shown in Figure 7), were collected by emission band steps of 550 to 540 nm and 560 to 650 nm, respectively. Fluorescence images were analyzed and intensity profiles obtained using Leica LAS AF 2.3.6 software. electron microscopy
[0685]For a scanning electron microscopy (SEM), samples were prepared by dehydrating the capsules in a reservoir before transferring them to a flask and washing them with fresh FC-40 oil twice by centrifugation. An oily suspension of dry capsules was deposited on a silica wafer followed by a gentle blow of nitrogen to remove excess oil. Measurements were taken and images recorded using a Leo 1530 variable pressure SEM and an InLens detector. For transmission electron microscopy (TEM), a similar sample preparation was performed by applying several drops of the oily suspension from the dry capsules onto a carbon-coated TEM sample grid (400 mesh). Excess oil was removed by drying first at room temperature and then in an oven (100 °C). Measurements and images were obtained using a TEM JEOL 2000FX under an accelerating voltage of 200 kV. Andor camera adjustment and fluorescence permeability analysis
[0686]Fluorescence images of microcapsules were recorded using an EM-CCO camera (Xion+, Andor Technologies model A247 together with Pixelinkand) connected to an inverted microscope (IX 71, Olympus) operating in epifluorescence mode, mounted with a automatic microscope stage (ProScan II, Prior Scientific). A mercury lamp (U-LH100HG, Olympus) was installed for extended spectrum illumination with FITC filters and dichroic lamps (BrightLine©, Semrock) fitted to separate fluorescence excitation and emission light. A computer controlled shutter was added to the excitation path to reduce the time during which the specimen was excited to minimize photobleaching. The camera, stage, and shutter were controlled by standard written software (LabVIEW 8.2, National Instruments), which was used to record and analyze the brightfield and fluorescence images.
[0687]The permeability of microcapsules was analyzed by encapsulating FITC-dextrans of various molecular weights. Encapsulation was generated by introducing a separate stream of an aqueous FITC-dextran solution (1-10 μM) online, either by adding a separate inlet channel to the device, or by incorporating the FITC-dextran solution with a existing aqueous solution. Flow rates were correspondingly adjusted to generate a range of FITC-dextran concentrations in the microcapsule before concentration for optimal imaging quality was achieved. The droplets were then allowed to dehydrate on glass slides completely as described previously and their brightfield and fluorescent images were captured. The dry capsules were re-dispersed in H2O by covering the sample with a drop of water and a microscope slide. Another set of brightfield and fluorescent images were captured immediately after rehydration, while capsule permeability was judged by the distribution of FITC fluorescence in relation to the location of the capsules.
[0688] To demonstrate the active and responsive release of FITC-dextran reduction, capsules were fabricated containing FITC-dextran (500 kDa) and dried on glass slides as described previously. The sample was placed in a transparent chamber sealed with parafilm. Nitrogen was directed into the chamber to create an oxygen-free environment. The chamber was then mounted on the Andor microscope, and a few drops of excess aqueous sodium dithionite solution (Na2S2O4) were applied to the dry capsules using a syringe. The fluorescent images of the capsules were captured every 30 minutes for 10 hours. SERS measurements
[0689]All SERS experiments were performed on a Renishaw InVia Raman confocal microscope with a 100x objective lens (NA = 0.85) in reverse scatter geometry. Microcapsule samples for SERS were prepared by collecting and drying the droplets on a glass slide. SERS spectra were acquired using the 633 nm or 785 nm laser line typically with an incident power of 0.015 mW and 0.20 mW, respectively, with an acquisition time ranging between 1 and 20 seconds. SERS image maps were collected using a Streamline® scan and lasted from 2 to 20 minutes depending on sample-specific SERS enhancements and acquisition conditions (including area) with typical pixel contact times of 1 to 20 seconds. All measurements were performed at room temperature. REFERENCES
[0690]All documents mentioned in this specification are hereby incorporated in their entirety by way of reference. Abraham et al. Advanced Materials 2008, 20, 2177 Ameloot et al. Nat. Chem. 2011, 3, 382 An et al. Biomacromolecules 2006, 7, 580 Andrieux et al. Analytical Chemistry 2002, 74, 5217 Anema et al. Annual Review of Analytical Chemistry 2011 Appel et al. J. Am. Chem. Soc. 2010, 132, 14251 Bush, M.E. et al. J. Am. Chem. Soc. 2005, 127, 14511 Caruso et al. Science 1998, 282, 1111 Cavalieri et al. ACS Nano 2009, 3, 234 Chiefari et al. Macromolecules 1998, 31, 5559 Comiskey et al. Nature 1998, 394, 253 Coulston et al. Chem. common 2011, 47, 164 Cui et al. Adv. Funct. mother 2010, 20, 1625 De Cock et al. Angew. Chem. Int. Ed. 2010, 49, 6954 Donath et al. Angew. Chem. Int. Ed. 1998, 37, 2201 Duffy, et al. Anal. Chem. 1998, 70, 4974 Forster et al. Journal of Raman Spectroscopy 1982,12, 36 Frens Nature Phys. Sci. 1973, 241, 20 Garstecki et al. Lab Chip 2006, 6, 437 Granath Journal of Colloid Science 1958, 13, 308 Gunther et al. Lab Chip 2006, 6, 1487 Hermanson et al. Physical Chemistry Chemical Physics 2007, 9, 6442 Huebner et al. Lab Chip 2008, 8, 1244 Holtze et al. Lab Chip 2008, 8, 1632 Jiao et al. J. Am. Chem. Soc. 2010, 132, 15734 Jiao et al. Org. Lett. 2011, 13, 3044 Kelly et al. Journal of Physical Chemistry B 2002, 107, 668 Kim et al. J. Am. Chem. Soc. 2000, 122, 540 Lagona et al. Angew. Chem. Int. Ed. 2005, 44, 4844 Link et al. The Journal of Physical Chemistry B 1999, 103, 4212 Martin et al. Langmuir 2010, 26, 7410 Mehvar Journal of Controlled Release 2000, 69, 1 Moghaddam et al. J. Am. Chem. Soc. 2011, 133, 3570 Patra et al., Langmuir 2009, 25, 13852 Patra et al. Chemistry - An Asian journal 2010, 5, 2442 Peyratout et al. Angew. Chem. Int. Ed. 2004, 43, 3762 Priest et al. Lab Chip 2008, 8, 2182 Rauwald et al. J. Phys. Chem. 2010, 114, 8606 Theberge et al. Angew. Chem. Int. Ed. 2010, 49, 5846 Thorsen et al. Phys. Rev. Lett. 2001, 86, 4163 Utada et al. Science 2005, 308, 537 Wang et al., Chemistry of Materials 2008, 20, 419 WO 2009/071899 . Xu et al. AIChE Journal 2006, 52, 3005 Yang et al. Lab Chip 2009, 9, 961 Yang et al. Angew. Chem. 2011, 123, 497 Zhou et al. Electrophoresis 2009, 31, 2 Additionally Coulston et al Chem. common 2011, 47, 164 Danil de Namor et al., Chem. Rev. 1998, 98, 2495-2525 Dsouza et al. Chem. Rev. 2011, 111, 7941-7980 Frens Nature Phys. Sci. 1973, 241, 20 Gokel et al., Chem. Rev. 2004, 104, 2723-2750 Martin et al. Langmuir 2010, 26, 7410 Rekharsky et al. Chem. Rev. 1998, 98, 1875

权利要求:
Claims (14)
[0001]
1. Capsule, CHARACTERIZED in that it has a shell that is a complex of a host and one or more building blocks having a suitable host-visitor functionality to thereby form a supramolecular lattice network, in which the host is cucurbituryl , and the one or more building blocks having suitable guest-host functionality for the cucurbituril host, wherein a first building block is a polymeric molecule, preferably the polymeric molecule is or comprises a poly(met) polymer. acrylate, polystyrene and/or poly(meth)acrylamide, and optionally the polymer molecule comprises a detectable label.
[0002]
2. Capsule, according to claim 1, CHARACTERIZED in that the shell is a complex of (a) a composition comprising cucurbituryl and (1) or (2); or (b) a composition comprising a plurality of covalently linked cucurbituryls and (1), (2) or (3), wherein: (1) it comprises a first building block covalently linked to a plurality of first visiting molecules of cucurbituril and a second building block covalently linked to a plurality of second visitor molecules of cucurbituril, wherein a first visitor molecule and a second visitor molecule together with cucurbituril are suitable to form a ternary visitor-host complex; (2) comprises a first building block covalently linked to a plurality of first visiting cucurbituril molecules and a plurality of second visiting cucurbituril molecules, wherein a first and second visiting molecule together with cucurbituril are suitable to form a visitor- ternary host, and optionally the composition further comprises a second building block covalently linked to one or more third cucurbituril visiting molecules, one or more fourth cucurbituril visiting molecules, or both, wherein a third and fourth molecules together with cucurbituril are suitable for forming a ternary visitor-host complex, and/or the first and fourth molecules together with cucurbituril are suitable for forming a ternary visitor-host complex, and/or the second and third molecules together with cucurbituril are suitable for forming a ternary visitor-host complex; and (3) comprises a first building block covalently linked to a plurality of first visiting molecules of cucurbituril, wherein the first visiting molecule together with cucurbituril are suitable to form a binary visitor-host complex, optionally the composition further comprises a second building block covalently linked to one or more second visiting cucurbituril molecules, wherein the second visiting molecule together with cucurbituril are suitable to form a binary visitor-host complex.
[0003]
3. Capsule according to claim 2, CHARACTERIZED in that the shell is a complex of a composition comprising cucurbituril and (1) or (2).
[0004]
4. Capsule according to any one of claims 1 to 3, CHARACTERIZED by the fact that cucurbituril is selected from CB[8] and variants and derivatives thereof, in which variants and derivatives thereof are selected from a compound where one or more repeat units in CD[8] is an ethylurea unit, or the compound is represented by the structure below:
[0005]
5. Capsule according to claim 4, CHARACTERIZED in that cucurbituril forms a ternary complex with a first visiting molecule and a second visiting molecule, and the first and second visiting molecules are selected from the following pairs: viologen and naphthol; viologen and dihydroxy benzene; viologen and tetrathiafulvalene; viologen and indole; methyl viologen and naphthol; methyl viologen and dihydroxy benzene; methyl viologen and tetrathiafulvalene; methyl viologen and indole; N,N'-dimethyl dipyridyliumyl ethylene and naphthol; N,N'-dimethyl dipyridyliumyl ethylene and dihydroxy benzene; N,N'-dimethyl dipyridyliumyl ethylene and tetrathiafulvalene; N,N'-dimethyl dipyridyliumyl ethylene and indole; 2,7-dimethyl diazapyrene and naphthol; 2,7-dimethyl diazapyrene and dihydroxy benzene; 2,7-dimethyl diazapyrene and tetrathiafulvalene; and 2,7-dimethyl diazapyrene and indole.
[0006]
6. Capsule according to any one of claims 1 to 5, CHARACTERIZED in that the second building block, when present, is a polymeric particle or molecule, and optionally the particle is or comprises gold or silver or mixtures thereof .
[0007]
7. Capsule according to any one of claims 1 to 6, CHARACTERIZED by the fact that: (i) the capsule size is in the range of 0.5 μm to 400 μm, preferably from 10 μm to 100 μm in diameter; and/or (ii) the capsule diameter has a relative standard deviation (RSD) of at most 10%; and/or (iii) the pore size of the shell is at least 0.5 nm and is preferably in the range 1 to 20 nm.
[0008]
8. Capsule, according to any one of claims 1 to 7, CHARACTERIZED in that the capsule encapsulates a component, preferably the component is a biological molecule, preferably the biological molecule is a polynucleotide, a polypeptide or a polysaccharide, for example the polynucleotide is either DNA or RNA.
[0009]
9. Capsule according to claim 8, CHARACTERIZED in that the component is a fragrance compound or composition or a flavoring compound or composition.
[0010]
10. Capsule, according to claim 8, CHARACTERIZED in that the component is a herbicide, pesticide or a catalyst.
[0011]
11. Method for preparing a capsule having a shell, as defined in any one of claims 1 to 10, CHARACTERIZED in that it comprises the step of: (1) contacting a flow of a first phase and a flow of a second phase phase in a channel, to thereby generate in the channel a dispersion of discrete regions, preferably droplets, of the second phase in the first phase, wherein the second phase comprises cucurbituril and one or more building blocks having suitable visitor functionality for cucurbituril to forming a supramolecular lattice network, to thereby form a capsule shell within the discrete region where the first and second phases are immiscible; and optionally wherein the second phase further comprises a component for encapsulation, and step (i) provides a capsule having a shell which encapsulates the component.
[0012]
12. Method, according to claim 11, CHARACTERIZED in that it further comprises the step of (ii) collecting the flow from the channel, to thereby obtain a droplet, which contains a capsule, optionally comprises , further, the step of drying the capsule obtained in step (ii).
[0013]
13. A non-therapeutic method of delivering a component to a site, CHARACTERIZING the fact that it comprises the steps of: (i) providing a capsule having a shell that encapsulates a component, as defined in any one of claims 8 to 10; (ii) delivering the capsule to a target location; (iii) releasing the component from the housing, preferably wherein the step of releasing the component from the housing is in response to a change in local conditions, preferably a change in concentration.
[0014]
14. Capsule, according to claim 8, CHARACTERIZED by the fact that it is for use as a medicine.

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WO2013014452A1|2013-01-31|

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

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

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2019-05-28| B06T| Formal requirements before examination [chapter 6.20 patent gazette]|

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2021-11-03| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2022-01-11| 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 25/07/2012, 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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GB1112893.1|2011-07-26|

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GBGB1112893.1A|GB201112893D0|2011-07-26|2011-07-26|Supramolecular Capsules|

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GBGB1202127.5A|GB201202127D0|2012-02-08|2012-02-08|Supramolecular capsules|

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GB1202127.5|2012-02-08|

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PCT/GB2012/051787|WO2013014452A1|2011-07-26|2012-07-25|Supramolecular capsules|

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