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    • 专利摘要:
    Hydrogel coatings based on vinyl-lactams. The invention relates to a material formed by a polymeric substrate and a hydrogel based on vinyl lactams and ionic methacrylates. The invention also relates to the process for obtaining the aforementioned material and its application for the cell culture/engineering of cellular monolayers, as well as for the preparation of 3D scaffolds and for the manufacture of thermosensitive mechanical actuators. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2678773A1
    申请号:ES201730039
    申请日:2017-01-16
    公开日:2018-08-17
    发明作者:Alberto Gallardo Ruiz；Juan RODRÍGUEZ HERNÁNDEZ；Helmut Reinecke；Carlos Elvira Pujalte；Carolina GARCÍA SÁNCHEZ；Maria Eugenia PÉREZ-OJEDA RODRÍGUEZ；Enrique MARTÍNEZ CAMPOS；Ana María SANTOS COQUILLAT；Ana CIVANTOS FERNÁNDEZ
    申请人:Consejo Superior de Investigaciones Cientificas CSIC；Universidad Complutense de Madrid；
    IPC主号:
    专利说明:

    The invention relates to a material comprising a polymeric substrate and a hydrogel-like coating based on vinyl-Iactams and ionic methacrylates, where the coating has been anchored to the polymeric substrate. An interface has formed between the substrate and the hydrogel as a result of the diffusion of hydrogel precursor molecules in the outer layers of the polymeric substrate. By controlling the experimental parameters, the hydrogel formed on the substrate can either remain intact or partially detach leaving the hybrid interface, that is, giving rise to a functionalized substrate that can be structured on said surface by wrinkle formation. This invention also relates to the application of these materials as supports for cell culture and engineering of cell sheets, preparation of 3D scaffolding and the manufacture of thermosensitive mechanical actuators.
    STATE OF ART
    The use of polymeric materials such as thermosensitive substrates based on poly-N-isopropylacrylamide (pN / PAm), capable of first housing cells until confluence and secondly allowing the detachment of cells or cell monolayers by thermal stimulation (temperature drop) has emerged as a potentially viable approach in cellular manipulation. These types of heat-sensitive substrates are commercial today (UpCeIl ™). Once the cell growth has reached the desired level of confluence, a monolayer or cell sheet can be detached simply by lowering the temperature at room temperature (i.e., below the lower critical temperature of dissolution or lower critical / s). / ution temperature, LSCT). This approach of cellular detachment is a form of non-destructive and more moderate cellular collection than traditional methods, which almost inevitably require the use of aggressive proteolytic enzymes (usually tri psina) or cell scraping. These techniques can damage the harvested cells as they can cause cell membrane disruption and destroy the extracellular matrix. This disruption constitutes a significant deficiency in these cell disaggregation approaches, and therefore, alternative and more moderate takeoff methods are desirable.
    Therefore, the development of other alternatives for such gentle detachment of cells and cell sheets remains attractive. An alternative thermosensitive polymer to pNIPAm is polyvinylcaprolactam (pVCL), which has an LCST similar to that of pNIPAm (also in a physiologically relevant temperature range) and cytocompatibility. However, few efforts have been devoted to the preparation of useful supports in cell collection on a pVCL basis. Lee et al. (Acta biomateriafia; 2013; 9 (8): 7691-7698) described the preparation of thin films of pVCL (about 50 nm) on woven Nylon substrates, using chemical vapor deposition techniques, and obtained partial cell detachment. Yang et al. They described another example (Polymer ChemistfY 2015; 6 (18): 3431-3442) in which they prepared VCL independent hydrogels with a zwitterionic methacrylate, which were able to detach cells by temperature drop.
    Recently, hydrogel-type supports for cellular manipulation based on VP, a component similar to VCL but not thermosensitive, have been described. Specifically, it has been described the preparation of a family of non-thermosensitive hydrogels derived from VP and with a double pseudo-network structure (pseudo-DN) capable of harboring cells until confluence and subsequently allowing rapid detachment or transplantation of the cell sheet by a simple mechanical agitation, without the need for a superstratum (Journal 01 Materials Chemistry B; 2014; 2 (24): 3839-3848). The term double pseudo-network refers to the structural tendency of these networks to form double networks (DNs), which are defined as interpenetrated networks (lPNs) consisting of two networks with high asymmetry in cross-linking density. The mentioned hydrogels are constituted by VP and different ionic methacrylates M: anionic (M-S03-), cationic (MM), zwi Sulfobetaine type (M-N '"- S03-), phosphorylcholine-like zwilterion (M-P03-- N '"), or pseudo-zwitterionic formulations (stoichiometric amounts of MS03-and M-N'"). All these hydrogels were robust despite their high water content (about 90% water in the equilibrium state) It was found that all ionic hydrogels were superior to the control without ionic component (without M), which is in accordance with the known non-stick and anti-fouling nature of PVP and other neutral and water soluble polymers (PEO, etc.). The chemical and topographic supertitial modifications of mechanically robust polymers can modulate their interaction with the environment while maintaining the mechanical properties of the block.This fact seems to be especially relevant in fields such as tribology or medicine. , in which resistant materials with supertitial properties (both chemical and topographic) are desired. On the one hand, the functionalization of hydrophobic supports to provide them with hydrophilic surface characteristics is currently a requirement for certain biomedical applications. On the other, wrinkled surfaces have found application in multiple areas such as their use as templates to create orderly surface formations, in the manufacture of flexible electronic components, or in the design of surfaces with wettability and controlled adhesion / friction properties.
    In addition, it is important to note that supporting said hydrogels in the form of a coating on mechanically robust materials, such as Nylon or polycarbonate, can significantly improve the handling of the active hydrogel layer, since the hydrogel support can be stapled, sewn, cast, etc., and therefore may allow complex constructs to be formed. The hydrogel can even be dried and rehydrated, which is advantageous in terms of storage under clean conditions and delivery. Finally, these platforms may be of interest in fields that require engineering of cell sheets, such as tissue transplantation in burned or cornea areas, examples in which the tissue regeneration process can be improved.
    DESCRIPTION OF THE INVENTION
    A first aspect of the present invention relates to a product that includes:
    a) A polymeric substrate and
    b) A hydrogel based on vinyl-Iactam monomers, ionic methacrylates and at
    less a cross-linking agent, characterized in that there is an interface between the substrate and the hydrogel, formed by a gradient of both semi-interpenetrated, net and substrate, which in case of detachment functionalizes the upper layers of the substrate.
    In a preferred embodiment the invention relates to a product as defined above containing at least two crosslinking agents.
    In a preferred embodiment, the polymeric substrate is selected from polystyrene, methyl polymethacrylate, nylon, polycarbonate, lactic polyacid or polycaprolactone. In a more preferred embodiment, the polymeric substrate is nylon or polycarbonate. In an even more preferred embodiment, the polymeric substrate is nylon.
    In another preferred embodiment, the vinyl-Iactam is selected from vinyl-caprolactam or vinyl-pyrrolidone. In a more preferred embodiment, the vinyl-Iactam is vinylcaprolactam.
    In another preferred embodiment, the vinyl-Iaclama can be combined with different functional monomers (ionic, non-ionic, zwitterionic).
    In a preferred embodiment, the ionic methacrylate is a cationic methacrylate selected from [2- (Methacryloxy) alkyl] trimethylammonium salts, dimethyl methacrylates and diethylaminoethyl.
    In another preferred embodiment, the ionic methacrylate is a zwitterionic methacrylate selected from 2-methacryloxyethyl phosphorylcholine, [3 (methacrylamino) propyl] dimethyl (3-sulfopropyl) ammonium hydroxide.
    In another preferred embodiment, the ionic methacrylate is an anionic methacrylate selected from sulfoalkyl methacrylate salts.
    In another preferred embodiment, the ionic methacrylate is a mixture of anionic methacrylate selected from salts of sulfoalkyl methacrylates and cationic methacrylate selected from [2- (Methacryloxy) alkyl] trimethylammonium salts.
    In a preferred embodiment, the crosslinking agents are selected from ethylene glycol di (meth) acrylate, triethylene glycol di (meth) acrylate, tetraethylene glycol glycol di (meth) acrylate, trimethylolpropane tri (meth) acrylate, bisphenol A di (meth) acrylate, ethoxylate bisphenol A di (meth) acrylate, pentaerythritol tri-, and tetra (meth) acrylate, tetramethylene di (meth) acrylate, methylene bisacrylamide, methacryloxyethyl vinyl carbonate, triallylcyanurate, methacryloxyethyl vinyl urea, divinyl benzene, diallyl methacrylate, diallyl methacrylate, diallyl methacrylate, diallyl methacrylate, diallyl methacrylate, diallyl methacrylate, diallyl methacrylate, diallyl methacrylate, diallyl methacrylate, diallyl methacrylate, diallyl methacrylate polysiloxanilbisalkyl (meth) acrylate, polyethylene glycol di (meth) acrylate, vinyl methacrylate, divinyl adipate, divinyl pyrrolidone derivatives or combinations of the foregoing. In a more preferred embodiment, two crosslinking agents are used, for example ethylene glycol di (meth) acrylate and 3,3'- (propyl) -di-1-vinyl-2-pyrrolidone.
    Another aspect of the invention relates to a process for obtaining the product described above, where the process comprises at least the following steps:
    a) mixture of the hydrogel precursor monomers, the vinyl derivative
    lactam, ionic methacrylate, and at least two crosslinking agents with
    a photoinitiator and solvent,
    b) deposition of the mixture of (a) on the surface of the polymeric substrate e
    induction of photopolymerization under UV radiation and
    e) swelling of the product obtained in (b) by immersion in water or
    in ethanol
    In a preferred embodiment, the polymeric substrate is selected from polystyrene, methyl polymethacrylate, nylon, polycarbonate, lactic polyacid or polycaprolactone. In a more preferred example, the polymeric substrate is nylon or polycarbonate.
    In another preferred embodiment, the vinyl lactam is selected from vinyl caprolactam or vinyl pyrrolidone. In a more preferred example, vinyl-Iactam is vinyl-caprolactam.
    In another preferred embodiment, the ionic methacrylate is a cationic methacrylate selected from [2- (Methacryloxy) alkyl] trimethylammonium salts.
    In another preferred embodiment, the ionic methacrylate is a zwitterionic methacrylate selected from methacryloxyxyethyl phosphorylcholine, [3 (methacrylamino) propyl] dimethyl (3-sulfopropyl) ammonium hydroxide.
    In another preferred embodiment, the ionic methacrylate is an anionic methacrylate selected from sulfoalkyl methacrylate salts.
    In another preferred embodiment, the ionic methacrylate is a mixture of anionic methacrylate selected from salts of sulfoalkyl methacrylates and cationic methacrylate selected from [2- (Methacryloxy) alkyl] trimethylammonium salts.
    In a preferred embodiment, the crosslinking agents are selected from ethylene glycol di (meth) acrylate, triethylene glycol di (meth) acrylate, tetraethylene glycol glycol di (meth) acrylate, trimethylolpropane tri (meth) acrylate, bisphenol A di (meth) acrylate, ethoxylate bisphenol A di (meth) acrylate, pentaerythritol tri-, and tetra (meth) acrylate, tetramethylene di (meth) acrylate, methylene bisacrylamide, methacryloxyethyl vinyl carbonate, triallylcyanurate, methacryloxyethyl vinyl urea, divinyl benzene, diallyl methacrylate, diallyl methacrylate, diallyl methacrylate, diallyl methacrylate, diallyl methacrylate, diallyl methacrylate, diallyl methacrylate, diallyl methacrylate, diallyl methacrylate, diallyl methacrylate, diallyl methacrylate polysiloxanilbisalkyl (meth) acrylate, polyethylene glycol di (meth) acrylate, vinyl methacrylate, divinyl adipate, divinyl pyrrolidone derivatives or combinations of the foregoing.
    In a more preferred embodiment, two crosslinking agents are used, for example ethylene glycol di (meth) acrylate and 3,3'- (propyl) -di-1-vinyl-2-pyrrolidone.
    In another preferred embodiment, the UV radiation is maintained between 10 and 60 minutes. In a more preferred example, UV radiation is maintained 40 minutes.
    Another aspect of the invention relates to the use of the material described above to obtain materials for cell culture.
    Another aspect of the invention relates to the use of the material described above for coating 3D objects. Preferably it refers to the use of the material described above for coating 3D objects for the manufacture of scaffolding.
    Another aspect of the invention relates to the use of the material described above for the manufacture of thermosensitive mechanical actuators.
    Unless stated otherwise, all technical and scientific terms used herein have the same meaning commonly understood by the person skilled in the art to which this invention belongs. Methods and materials similar or equivalent to those described herein can be used in practice in the present invention. Throughout the description and the claims, the word "understand" and its variations are not intended to exclude other technical characteristics, additives, components or steps. Additional objects, advantages and features of the invention will become apparent to those skilled in the art upon examination of the description or can be learned through the practice of the invention. The following examples and illustrations are provided by way of illustration and are not intended to be limiting of the present invention.
    BRIEF DESCRIPTION OF THE ILLUSTRATIONS
    FIG. 1. The photographs indicate the optical changes associated with VPTT (volume phase transition temperature). At temperatures above the VPTT (left image), the polymer network is collapsed and has a white appearance. Upon cooling first to the VPTT (central image) and later to temperatures below the VPTT (right image) the white color disappeared and a transparent coating is observed indicating a polymerization of the polymer network.
    FIG. 2. Evaluation of monolayer formation in hydrogel coated nylon. On cooling from 37 to 20 oC (below VPTT) the monolayer separates (a) and the cells are transplanted on a culture plate. As evidenced at 44h (b) and 144h (e), a complete monolayer can be formed from the transplanted cells.
    FIG. 3. Assembly for the manufacturing of modified pe surfaces with VP-based hydrogels, together with optical profilometry images illustrating original PC films and a wrinkled PC surface obtained after the photopolymerization of a precursor solution (HYD _1) placed on the surface of pe.
    FIG. 4. Evolution of the PC surface topography that leads to the formation of wrinkles as a function of the time elapsed between the contact of the HYD_1 solution with the substrate pe and the start of the photopolymerization stage: (a) O min, (b) 5 min, (e) 10 min, (D) 20 min and (e) 30 min WAMP: amplitude of wrinkles, Wp: period of wrinkles.
    FIG. 5. (a) Morphological evolution of wrinkles depending on the solvent mixture used (Image size: 350 IJm x 250 IJm). Variation of the wrinkle period (b) and its amplitude (e) as a function of the exposure time of the monomer solution to the substrate of pe. Table 1 gives the references of HYD.
    FIG. 6. (a) Measures of static contact angle and (b) forward-reverse contact angle for 7 cycles in a treated and untreated pe. (C) Optical profilometry image of a water droplet that advances in the wet-dry interface. The surface of the wrinkled pe was obtained after photopolymerization of a precursor solution HYD_1 placed on the surface of the pe with an elapsed time of 10 minutes between the contact and UV irradiation
    FIG. 7. Above: Representation of the relationship of intensities between the signals at 1676
    cm-1 observed in the hydrogel and the band at 1615 cm-1 assigned to the PC based on the depth of HYD_2 (a-e) and HYD_1 (f-j). Below: Raman spectrum evolution as a function of the depth of HYD_2 (k) and HYD_1 (1).
    FIG. 8. Formation of hydrogels based on VP and situations observed after the swelling of the hydrogel. Mainly, two different possibilities were observed: (a) separation of the non-integrated regions of the hydrogels leaving a thin layer of hydrogel above the surface and (b) the hydrogels remain partially or totally anchored to the surface. (c) Optical images and 3D optical images illustrative of a PC surface after treatment (left) and the complementary hydrogel surface obtained upon drying (right).
    FIG. 9. Optical images of the surface of a 3 mm diameter cylinder after treatment with the solution that includes vinyl pyrrolidone and potassium sulfopropyl methacrylate (6: 1 molar ratio), as well as crosslinking agents of methacrylic and vinyl type. The cylinder was immersed in the solution described above and after a certain period of time (0.15 or 30 min), the entire system was exposed to UV radiation for 30 minutes. Next, the cylinder was immersed in ethanol and the hydrogel separated from the surface of the cylinder leaving a wrinkled surface with a thin surface layer of hydrogel.
    EXAMPLES
    1. Obtaining the hydrogel coating
    The hydrogels were synthesized by conventional radical polymerization in a single stage using Milli-Q water, alcohols (ethanol or methanol) or mixtures of water and alcohol as solvents. Briefly, a solution of vinyl pyrrolidone (VP) or vinylcaprolactam (VCL) (6 mol IL) and ionic methacrylate (1 or 0.5 mol II of potassium sulfopropyl methacrylate (M-SO) ·), chloride of (2- (methacryloxy) ) ethyl] trimethylammonium ((M-N '), [3- (methacrylamino) propyl) dimethyl (3-sulfopropyl) ammonium hydroxide (MN - + - S03t 2-methacryloyloxyethyl phosphorylcholine (M-P03 · -N "), or a mixture of MS03 · and MN "Y ethylene91icol (e1) and 3,3'- (propyl) -di-1-vinyl-2-pyrrolidone (e2) dimethylacrylate crosslinking agents were added at 2 and 0.1 mol % (versus total monomer content) Hydroxy-cyclohexyl phenyl ketone (HCPK) was used as a photoinitiator (0.5% by weight).
    The reaction mixtures were bubbled with NzY and transferred to molds by means of a syringe. For the preparation of the coated materials, polycarbonate molds 5 were used! Polypropylene or Nylon! polypropylene, among other combinations. The molds were separated by silicone spacers (0.05 to 1.5 mm thick). The photopolymerization was carried out for 40 minutes under UV radiation (A = 365 nm) in a UVP ultraviolet lamp (CL-tOOOL model, 230V). A summary of the types of hydrogels prepared is given in Table 1. The supports 10 coated with the hydrogel were recovered from the molds by removing the polypropylene shell and allowed to swell in ethanol or Milli-Q water until equilibrium was reached. Subsequently, they were thoroughly washed with water to remove any soluble material. The samples were stored in ethanol at 4 oC until it was necessary for experimentation. 24 hours before the different experiments, the samples are
    15 transferred back to Milli-Q water and washed several times to completely remove ethanol.
    2. Coating of Nylon substrates with VeL-based hydrogels. Use as support for cell culture.
    The hydrogel prepared using only VCL and ethylene glycol (C1) dimethacrylate and 3,3 '(propyl) -di-1-vinyl-2-pyrrolidone (C2) as crosslinking agents (i.e., without M), has been chosen to carry out preliminary studies on the coating of hydrophobic surfaces with thermosensitive hydrogels.
    Photopolymerization of the VCL-based formulation on this material (Nylon) produced a hydrogel with an interface integrated into the surface of the support as shown in Figure 1. This hydrogel was stable and did not separate or break due to manipulation or changes of temperature when submerged in aqueous medium. Even more, the hydrogel layer can be dried and rehydrated perfectly.
    This coated Nylon was evaluated as a cell culture support using C166-GFP endothelial cells (Figure 2). A cell monolayer formed after a few days and this monolayer could be completely separated by temperature decrease (Figure 2 (a.) After transplanting the recovered monolayer onto a tissue culture plate, the cells were able to grow and form a monolayer again after a few days.
    All hydrogels were sterilized with a 70% ethanol solution rinsing six times for 10 minutes each. They were then washed with PBS six times, exposed to UV radiation for 30 minutes on each side of the hydrogel and washed twice with Dulbecco-modified Eagle's medium (DMEM) with high glucose content. To simulate the temperature of the culture conditions (37 ° C), above the VPTT, a hot plate with a constant temperature was used for the process of cutting the material obtaining 2 cm2 samples that fit the 24-well plates. After cutting, the samples were left overnight incubated with DMEM 10% fetal bovine serum (FBS) and 1% antibiotics (100 IU mi of penicillin and 100 IJg! Mi of streptomycin sulfate, SigmaAldrieh, SI. Louis, MO).
    C166-GFP (ATCC® CRL-2583 ™) is a mouse endothelial cell line transfected with green fluorescent protein (GFP). The culture conditions are Dulbecco MEM (06429) supplemented with 10% FBS plus 100 IU mL penicillin and 100 IJg I streptomycin sulfate adding 0.2 mg I mi of G-418 antibiotic to the culture medium for selection of retained GFP cells. The cells were seeded in the networks with a density of 3 x 10 4 I cells and incubated at 37 ° C with 5% COz. The cells were monitored using an inverted fluorescence microscope with an FITC filter (1. "/ A, m 488/568 nm).
    The hydrogels supporting the cell cultures were turned around and placed in new TCP wells. Subsequently, medium cold was added to each well to reach T :::; 27 oC. A temperature probe was used to monitor this process.
    After 45 minutes, the hydrogels were removed and the samples were re-incubated at 37 ° C with CO2. The transplanted cells were observed daily using an inverted fluorescence microscope and micrographs were taken. The Trypan Blue test was performed following the manufacturer's instructions.
    The metabolic activity of cell transplants was measured by Alamar Blue assay following the manufacturer's instructions. This method is non-toxic, scalable and uses the natural reducing power of living cells, generating a quantitative measure of cell viability and cytotoxicity. In summary, dye was added
    10 Alamar Blue (10% of the culture volume) to each well, containing live cells seeded on films, and incubated for 90 minutes. The tests were performed, in each type of sample, in triplicate. The fluorescence (Aex 1 Aem 535/590 nm) of each well was measured using a plate reader.
    15 3. Coating polycarbonate substrates with VP based hydrogels. As a starting point in all cases a previously optimized VP-based formulation was used: water as solvent, VP and M-SOJ-in concentrations of 6 and 1 mollL respectively, two cross-linking agents (called C2 and C1) with molar percentages (vs. monomers) of 2.0 and 0.1%, respectively.
    20 Hydrogels were synthesized as described above. A summary of the types of hydrogels prepared is given in Table 1.
    Table 1. Photosensitive precursor solutions were used comprising: 6 moles of VP (1,265 mL), 2 moles of M-SO, '(492 mg), 0.1 mol% of C2 (3.1 mg) and
    25 different amounts of C1 and solvents (mixture of water and ethanol). The contact times used before the photopolymerization were O, 5, 10, 20 and 30 minutes_ The polypropylene caps were removed after the curing and the nets formed on the PC substrates were allowed to swell in ethanol until equilibrium was reached. . Subsequently, the coated pe substrates were washed
    Name HYD_lHYD_2HYD_3HYD_4HYD_5HYD_6HYDJ
    Cl (mol%) 2.02.02.02.00.51.04, 0
    Solvent EtOH--0, 2450.490---
    (me) Water0.7351,0000.4900.2450.7350.7350.735
    5 thoroughly with ethanol and water to remove any soluble material and finally dried for analysis.
    Transversal profiles and 3D images of wrinkled surfaces were characterized by a Zeta-20 True Color 3D Zeta Instruments profilometer. 10 Static contact angle measurements were made using a contact angle goniometer (Tetha instruments, KSV) with the sessile drop method. In addition to the static contact angle values, forward and reverse contact angle values were carried out. A motorized syringe was set at a specific speed to control the volumetric flow rate of the liquid to or from the sessile drop. The mechanism pushed the plunger of the syringe during the advance procedure and pulled it during the withdrawal procedure, resulting in an increase and decrease in the size of the drop, respectively. The images of the drop in growth and shrinkage were recorded by the computer, typically at a rate of one image every 1 s. In this study, the forward and reverse processes were repeated at
    20 minus 7 times, taking the system to 7 cycles.
    The chemical composition and depth profiles of polymeric films are
    determined using integrated Raman Confocal Microscopy with microscopy of
    Atomic force (AFM) in a CRM-Alpha 300 RA microscope equipped with Nd laser:
    25 YAG (maximum power of 50 mW of power at 532 nm). Raman spectra were acquired point by point every 100 nm.
    The precursor formulation that has been labeled HYo_1 has been shown to be capable of inducing surface microstructuring in PC substrates, which
    30 obtained in a single stage by UV initiated polymerization of the monomer solution Hyo_1 deposited on a PC substrate (using the assembly depicted in Figure 3), followed by separation of the hydrogel by swelling in ethanol.
    The HYD_1 solution was confined between a transparent cover and the polycarbonate substrate using a spacer. The hydrogel, resulting after a waiting time of 10 minutes followed by the UV-vis photopolymerization step, was immersed in an EtOH solution. Upon swelling, the hydrogel separates from the PC support leaving a substrate in which the topography has been significantly modified. Figure 3 shows illustrative images of 3D optical profilometry of an untreated PC surface and another after the UV photopolymerization stage. In contrast to the untreated flat PC surface, the surface of films obtained after hydrogel removal shows randomly distributed wrinkles. In particular, under the conditions described above, wrinkles with homogeneous dimensions (around -30 IJm in wavelength and amplitudes of around -9 IJm) were observed over the entire surface. This particular surface topography on the PC surface must be related to hydrogel formation and post-polymerization evolution after ethanol immersion. Note that the control experiments carried out without the small percentage of 0.1 mol% of the C2 divinyl compound did not cause a homogeneous swelling of hydrogel, or wrinkles. C2 plays a key role not only for network properties, but also because of its possible participation in the links between SPM-rich chains and VP-rich chains. It has been described that analogous hetero-junctions improve the mechanical properties of double networks. In addition, an increase of C1 to 4% molar leads to a partial detachment or a complete anchoring of the hydrogel on the PC. This influence of C1 on the evolution of the hydrogel will be addressed later.
    The hypothesis is raised here that the wrinkle formation of Figure 3 is related in some way to a PC swelling and, therefore, to a penetration of the monomer mixture in the outer PC layers. The release of the hydrogel after swelling in ethanol would reveal wrinkles. To address this hypothesis, the influence on the surface wrinkling of different parameters that may influence the proposed diffusion of monomers in pe, such as the type of solvent or the exposure time between the substrate and the photopolymerizable solution prior to UV irradiation In addition, the chemical composition of the surface has been analyzed; First, the possible changes in surface wettability were analyzed using forward and reverse contact angle measurements. Second, the possible changes in the chemical composition of the surface were investigated by confocal Raman microspectroscopy.
    To analyze the influence of the contact time, the mixture of HYD _1 manomeres was contacted with the PC surface and the delay time between the established contact and the start of the irradiation time with UV light between 0 and 30 min was varied (Note that the sample represented in Figure 3 was obtained after a delay time of 10 minutes). As seen in Figure 4, surface topography gradually varies from a fairly flat substrate to a wrinkled surface increasing contact time. Short contact times produced only a slight increase in surface roughness (Figure 4 (a ». However, surface topography changes significantly keeping the precursor solution in contact with the substrate for 5 minutes or more. In this situation (5 min ), the surface resulting from complete hydrogel detachment revealed the formation of wrinkles with periods around -20 ~ m and amplitudes below 3 ~ m (Figure 4 (b)). In the period of time observed, that is, from O up to 30 minutes, a gradual increase in wrinkle dimensions was observed, as a result, wrinkles with periods between 19 IJm and 40 IJm and amplitudes between 2.6 IJm and 17 IJm can be easily prepared by increasing the time between the contact of the precursor solution and the UV curing.
    In addition to the contact time, the nature and ratio of the solvents used for the photosensitive mixture can have a strong influence on the PC's surface swelling process. For Hyo_1 a small and optimized amount of water was used as solvent. To address this issue, samples Hyo_2 to HYo_4 have been studied in Table 1, in which the quantity and nature of the solvent have been varied.
    The dimensions of the wrinkles (amplitude and period) varied clearly depending on the solvent used. Figure 5 shows 3D optical profilometry images and two additional graphs that represent the variation of wrinkle characteristics as a function of contact time for different precursor solutions. Using the Hyo_2 solution, with a larger amount of water than in the case of Hyo_1, the wrinkles observed are clearly smaller (than those observed using Hyo_1). On the other hand, the partial replacement of water with EtOH results in wrinkles with larger dimensions. In addition, an increase in the amount of EtOH in the solvent mixture resulted in wrinkles with greater periods and amplitudes. Therefore, beyond the addition of water, the incorporation of an additional solvent with greater affinity to the substrate allowed us to precisely adjust the resulting wrinkle dimensions. In particular, as shown in Figure 5, structured rough surfaces with periods between 10 and 100 IJm and amplitudes ranging from 1-20 IJm were directly obtained.
    Regarding the chemical nature of the surface, static contact angle measurements carried out in both unmodified planar PC and modified PC, indicated an increase in surface wettability, that is, the treated surface becomes more hydrophilic ( Figure 6 (a ». For this study, a sample of type HYD_1 with 10 minutes of contact time was selected. Measurements of forward and reverse angles showed significant surface changes. Figure 6 (b) shows the angles of forward and reverse contact of 7 cycles for treated and untreated substrates The precursor substrate, ie pure PC, has feed angles of approximately 90-93 ° and recoil contact angle values of 15-18 °. values remain constant during all cycles scanned, however, for the surfaces treated with the photosensitive mixture and after the release of hydrogel, the contact angle of ava nce has significant differences between the first and subsequent cycles. While in the first cycle the angle of advance has values around 82 °, the measured values for the following cycles are around 10-12 °. This observation indicates that in the first cycle the treated interface requires a first wetting to become highly hydrophilic. Most likely, part of the hydrogel formed remains anchored to the hydrated interface during the following cycles. The water front feed during feed angle measurements has been represented using an optical profilometer (Figure 6 (e)). The water droplet dips the valleys formed by wrinkles and advances forming a thin layer of water. Likewise, as expected, the recoil contact angle in the treated films is very low with values below 10 °.
    The contact angle experiments show the formation of a hydrophilic surface layer, but do not provide any information on the chemical composition of the surface and the depth profile of the treatment. Information on these two aspects was obtained by Raman Confocal. Prior to the investigation of the modified substrates, the differences between the Raman spectra of the PVP-based hydrogel and the PC substrate were evaluated. By comparison of these two spectra we have observed several characteristic signals. First, the signal found at 1675 cm-1 corresponds to the C = Q groups of the PVP-based hydrogel. However, the carbonyl functional groups present in the PC provide a Raman signal at 1613 cm-1_ In addition, the bands found at 1495, 1457, 1425 cm_1 correspond to the methylene deformation of the main chain of the PVP material_ Finally, the band at 944 cm-1 corresponds to the breathing mode (breathing mode) of the pyrrolidone ring and those observed at 860 and 768 cm-1 to the ring modes.
    The formation of an upper layer in which the chemical composition is a mixture of PC and hydrogel in VP base can be clearly seen in the Raman spectrum observed for the treated pe substrate. Another interesting feature of Raman Confocal microspectroscopy is related to the possibility of obtaining depth profiles that show the variation of the chemical composition from the surface to the interior of the PC. For this analysis, the top of a wrinkle hill was used as a reference and the Raman spectra were recorded at different depths of up to 30 IJm. Raman spectra of HYD_1 obtained at different depths at different contact times, show a gradual variation of the spectra of a pure hydrogel composed mainly of
    PVP, pure PC at a depth of 30 IJm. By normalizing the signal to 1615 cm-assigned to the C = O of the PC, a gradual decrease can be easily observed for example of the bands at 1495, 1457, 1425 cm-1 due to the deformation of the main chain methylene or the band at 1678 cm- 'due to C = Q of the hydrogel formed. As a result, a comparison of the 1676 cm-1 bands of the hydrogel and the characteristic 1615 cm-1 band of pe allows to estimate the variation of the chemical composition and the depth of the modified layer. As shown in Figure 7, the representation of the relationship between the two signals allows the construction of transversal profiles that indicate the extent of the surface modification. An increase in the contact time of the light-curing solution on the substrate before the UV-vis irradiation stage, leads to an increase in the wrinkle dimensional characteristics.
    As the contact time increases, the monomer mixture penetrates deeper into the PC, greater swelling occurs and surface instabilities appear. Light-curing forms the VP-based network (in fact, the integrated external layers of hydrogel l PC form a semi-interpenetrated structure) and "freezes" the surface deformation. The release of the hydrogel, finally, reveals the wrinkles at the interface. According to the observations described above using the 3D optical profilometer, the size of the wrinkles increases as the modified layer also increases. Therefore, the wrinkle formation process is directly related to the degree of swelling.
    Figure 7 also shows the relevance of monomer concentration when comparing HYD_1 with HYD_2. While HYD_1 is prepared using 0.735 ml of water, HYD_2 contains 1 ml of water (it is less concentrated). First, as depicted in the cross sections and summarized in the graphs (Figure 7 (k) Y (1)), the profiles indicate a greater diffusion of the hydrogel precursor components as the monomer concentration increases. Considering that HYD_2 exhibits penetration profiles of monomers with thicknesses below 15 J..Im, HYD_1 showed that the affected surface region is greater than 25 J..Im thick.
    In the previous scenario of gradient swelling and surface deformation, wrinkles become visible if the hydrogel is able to separate at the deformed interface. Based on related literature, the release of the hydrogel may be related to the tensions originated on the surface after the swelling of the hydrogel and is influenced by the number of anchor points established between the hydrogel and the PC substrate. This number of anchor points is strongly related to the degree of crosslinking. As mentioned earlier, an increase in C1 leads to a partial detachment or a complete anchoring of the hydrogel to the PC substrate (depending on the contact time). A value of C1 between 0.5 and 1% molar resulted, however, in a complete detachment of the hydrogel from the PC, which leads to the wrinkling described herein. Interestingly, the surface of this hydrogel (Figure 8 (c)) also exhibits the formation of wrinkles, which are complementary to the wrinkles observed on the surface of the PC. A hypothetical model has been developed according to these results (see Figure 8). Networks of higher cross-linking density are capable of absorbing a limited amount of solvent and therefore have reduced swelling and lower tensions related to the swelling phenomenon. The number of anchor points between the interface and the hydrogel also increases. Therefore, a critical degree of crosslinking is proposed, below which there is a break in the interface and a hydrogel detachment. This is due to the small number of anchor points and the greater extent of stresses during swelling, compared to the degrees of cross-linking above the critical point, which are able to keep the entire hydrogel anchored.
    In conclusion, it can be established that the selected mixture of monomers I crosslinking agents, upon contact with the PC, diffuses and swells the polymer surface. As a result, after swelling of the hydrogel formed in
    EtOH or water, a controlled release of the hydrogel occurs, leaving athin layer of hydrogel on the PC surface. This thin layer of hydrogel isconsequence of the initial diffusion and swelling process of the PC surfaceby hydrogel precursors. The diffusion observed has two consequences5 simultaneous. First, the surface chemical composition of the PC is altered andObtain a surface with greater hydrophilicity. Second, the diffusion ePC surface swelling induces surface instabilities thatThey eventually lead to the formation of wrinkled surfaces. Curiously, throughthe modification of the composition of the precursor solution, as well as the time of
    10 contact, a reasonable control is obtained on the characteristics of wrinkles (period and amplitude).
    4. Coating of non-flat substrates.
    15 The piece, in this case a 3 mm diameter cylinder, was immersed in a mixture solution of vinyl pyrrolidone, sulfopropyl methacrylate (in 6/1 molar ratio) and cross-linking agents dimethacrylate and divinyl type, and irradiated with UV light for 30 min The initiation of the photopolymerization process takes place instantaneously (previous contact time O min), as well as after 15 and
    20 after 30 min. As seen in Figure 9, the roughness increases with contact time, and wrinkle formation already occurs for short contact times (prior to photopolymerization).
    权利要求:
    Claims (25)
    [1]
    one. A product comprising: a) a polymeric substrate and b) a hydrogel based on vinyl-Iactam monomers, ionic methacrylates and the
    less a cross-linking agent, characterized in that there is an interface between the substrate and the hydrogel, formed by a gradient of both semi-interpenetrated, net and substrate, which in case of detachment functionalizes the upper layers of the substrate.
    [2]
    2. Product according to claim 1, wherein the polymeric substrate is selected from polystyrene, methyl polymethacrylate, nylon, polycarbonate, lactic polyacid or polycaprolactone.
    [3]
    3. Product according to claim 2, wherein the polymeric substrate is nylon.
    [4]
    Four. Product according to claims 1 and 2, wherein the vinyl-Iactam is selected vinyl-caprolactam or vinyl-pyrrolidone.
    [5]
    5. Product according to claim 4, wherein the vinyl-Iactam is vinyl-caprolactam.
    [6]
    6. Product according to claims 1 to 3, wherein the ionic methacrylate is a cationic methacrylate selected from salts of [2 (Methacryloxy) alkyl] trimethylammonium, dimethyl and diethylaminoethyl methacrylates.
    [7]
    7. Product according to claims 1 to 3, wherein the ionic methacrylate is a zwitterionic methacrylate selected from methacryloxyethyl phosphorylcholine, [3- (methacrylamino) propyl] dimethyl (3-sulfopropyl) ammonium methacrylate zwitterionic methacrylate hydroxide.
    [8]
    8. Product according to claims 1 to 3, wherein the ionic methacrylate is an anionic methacrylate selected from sulfoalkyl methacrylate salts.
    [9]
    9. Product according to claims 1 to 3, wherein the ionic methacrylate is a mixture of anionic methacrylate selected from salts of sulfoalkyl methacrylates and cationic methacrylate selected from salts of [2 (Metacri loiloxy) alq ui II] tri meti la mon io .
    [10]
    10. Product according to claims 1 to 6, wherein the crosslinking agents are selected from ethylene glycol di (meth) acrylate, triethylene glycol di (meth) acrylate, tetraethylene glycol glycol di (meth) acrylate, trimethylolpropane tri (meth) acrylate, bisphenol A di (meth) acrylate, ethoxylate bisphenol A di (meth) acrylate, pentaerythritol tri-, and tetra (meth) acrylate, tetramethylene di (meth) acrylate, methylene bisacrylamide, methacryloxyethyl vinyl carbonate, triallylcyanurate, methacryloxyethyl vinyl biene urea, dividene diallyne urea, dividene diallyne diane, dialkyl urea, divine , allyl methacrylate, diallyl phthalate, polysiloxanylbisalkyl (meth) acrylate, polyethylene glycol di (meth) acrylate, vinyl methacrylate, divinyl adipate, divinyl pyrrolidone derivatives or combinations of the foregoing.
    [11]
    eleven. Product according to claim 10, wherein the crosslinking agents are ethylene glycol di (meth) acrylate and 3,3'- (propyl) -di-1-vinyl-2-pyrrolidone.
    [12]
    12. A process for obtaining the product according to claims 1 to 11, wherein the process comprises at least the following steps:
    a) mixing the hydrogel precursor monomers, the vinillactam derivative, the ionic methacrylate, and at least two cross-linking agents with a photoinitiator and solvent,
    b) deposition of the mixture of (a) on the surface of the polymeric substrate and induction of light curing under UV radiation and c) swelling of the product obtained in (b) by immersion in water or ethanol.
    [13]
    13. A process according to claim 12, wherein the polymeric substrate is selected from polystyrene, methyl polymethacrylate, nylon, polycarbonate, lactic polyacid or polycaprolactone.
    [14]
    14. A process according to claim 13, wherein the polymeric substrate is nylon or polycarbonate.
    [15]
    fifteen. A process according to claims 12 to 14, wherein the vinyl-Iactam is selected vinyl-caprolactam or vinyl-pyrrolidone.
    [16]
    16. A process according to claim 15, wherein the vinyl-Iactam is vinylcaprolactam.
    [17]
    17. A process according to claims 12 to 16, wherein the ionic methacrylate is a cationic methacrylate selected from salts of [2 (Methacryloxy) alkyl II] tri metiium monium.
    [18]
    18. A process according to claims 12 to 16, wherein the ionic methacrylate is a zwitterionic methacrylate selected from methacryloxyethyl phosphorylcholine, [3- (methacrylamino) propyl] dimethyl (3-sulfopropyl) ammonium methacrylate hydroxide.
    [19]
    19. A process according to claims 12 to 16, wherein the ionic methacrylate is an anionic methacrylate selected from sulfoalkyl methacrylate salts.
    [20]
    twenty. A process according to claims 12 to 19, wherein the crosslinking agents are selected from ethylene glycol di (meth) acrylate, triethylene glycol di (meth) acrylate, tetraethylene glycol glycol di (meth) acrylate, trimethylolpropane tri (meth) acrylate, bisphenol A di (meth) acrylate, ethoxylate bisphenol A di (meth) acrylate, pentaerythritol tri-, and tetra (meth) acrylate, tetramethylene di (meth) acrylate, methylene bisacrylamide, methacryloxyethyl vinyl carbonate, triallylcyanurate, methacryloxyethyl vinyl vinylane, vinyl urea, vinyl urea conate, allyl methacrylate, diallyl phthalate, polysiloxanylbisalkyl (meth) acrylate, polyethylene glycol di (meth) acrylate, vinyl methacrylate, divinyl adipate, divinyl pyrrolidone derivatives or combinations of the foregoing.
    [21]
    twenty-one. A process according to claim 20, wherein the crosslinking agents are ethylene glycol di (meth) acrylate and 3,3'- (propyl) -di-1-vinyl-2-pyrrolidone.
    [22]
    22 A process according to any of claims 12 to 21, wherein the UV radiation is maintained between 10 and 60 minutes.
    [23]
    2. 3. Use of the material according to any of claims 1 to 11, to obtain materials for cell cultures.
    [24]
    24. Use of the material according to any of claims 1 to 11, for coating 3D parts.
    [25]
    25. Use of the material according to any of claims 1 to 11, for the manufacture of thermosensitive mechanical actuators.
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    同族专利:
    公开号 | 公开日
    US20200002494A1|2020-01-02|
    WO2018130739A1|2018-07-19|
    EP3569623A1|2019-11-20|
    EP3569623A4|2020-10-21|
    ES2678773B1|2019-06-12|
    US10988591B2|2021-04-27|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    ES2726602A1|2018-04-06|2019-10-08|Consejo Superior Investigacion|VINYL-CAPROLACTAMA BASED HYDROGELS |US3532679A|1969-04-07|1970-10-06|Robert Steckler|Hydrogels from cross-linked polymers of n-vinyl lactams and alkyl acrylates|
    US4058491A|1975-02-11|1977-11-15|Plastomedical Sciences, Inc.|Cationic hydrogels based on heterocyclic N-vinyl monomers|
    WO2009099539A2|2008-01-30|2009-08-13|Corning Incorporated|acrylate surfaces for cell culture, methods of making and using the surfaces|
    ES2526469B1|2013-06-07|2015-10-26|Consejo Superior De Investigaciones Científicas - 67%|MULTICOMPONENT HYDROGELS BASED ON VINILPIRROLIDONA AND ITS APPLICATION IN TISSUE ENGINEERING AND / OR REGENERATIVE MEDICINE|
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    ES201730039A|ES2678773B1|2017-01-16|2017-01-16|HYDROGEL-TYPE COATINGS IN BASE VINYL-LACTAMAS|ES201730039A| ES2678773B1|2017-01-16|2017-01-16|HYDROGEL-TYPE COATINGS IN BASE VINYL-LACTAMAS|
    EP18738443.3A| EP3569623A4|2017-01-16|2018-01-16|Vinyl-lactam-based hydrogel coatings|
    PCT/ES2018/070029| WO2018130739A1|2017-01-16|2018-01-16|Vinyl-lactam-based hydrogel coatings|
    US16/512,572| US10988591B2|2017-01-16|2019-07-16|Vinyl-lactam-based hydrogel coatings|
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