JP2007517077A - Ophthalmic hydrogel nanocomposites - Google Patents

Abstract

  The present invention relates to a reversible hydrogel system. In particular, the hydrogel of the present invention comprises a copolymer that can be a hydrogel in the oxidized state and a solution in the reduced state. The copolymer solution can be oxidized to produce a hydrogel, and the hydrogel can be reduced to produce a copolymer solution. Reversible nanogels can also be produced from dilute solutions of copolymers. When a hydrogel is formed including nanoparticles embedded in it, a nanocomposite is produced whose refractive index and modulus can be controlled by varying the amount of nanoparticles and the polymer concentration of the hydrogel, respectively.

Description

  This application is a continuation-in-part of pending US patent application Ser. No. 10/706081. This application also claims priority from US Provisional Application No. 499887 and US Provisional Patent Application No. 556492.

  The present invention relates to a hydrogel system comprising nanoparticles or nanophases. In particular, the hydrogel of the present invention comprises a copolymer solution containing nanoparticles, which can generate a hydrogel triggered by changes in oxidation state, light frequency and intensity, or mechanical stress. For example, the copolymer solution can be oxidized to produce a hydrogel; and the hydrogel can be reduced to a copolymer solution. Reversibility can also be achieved using photoaddition chemistry or using polymers that are sensitive to mechanical stress (physical gels that cause viscosity reduction due to shear). The above method principles can also be used to generate reversible nanogels from dilute solutions of copolymers. When a hydrogel is formed including nanoparticles embedded in it, a nanocomposite is produced whose refractive index and modulus can be controlled by varying the amount of nanoparticles and the polymer concentration of the hydrogel, respectively.

  The cloudy part of the cataract is usually the cloudy part or cloudy part of the clear eye lens. As turbidity increases, light rays pass through the lens, preventing it from focusing on the retina (the photosensitive tissue covering the interior behind the eye). Early lens changes or opacity may not cause inconvenience in vision, but if the lens continues to change, blurred vision, sensitivity to light and shine, myopia progression, and / or astigmatism in either eye Some special symptoms may appear, including.

  Once the turbid areas of cataracts are created, there is no therapeutic agent, eye drops, training, or glasses to eliminate them. If a person cannot see enough to perform normal daily activities, surgery is necessary to remove the cloudy part of the cataract and restore normal vision.

  In modern cataract opacity surgery, the opacity is removed from the lens through the opening of the lens capsule. Using a surgical microscope, the eye and then the lens capsule is cut small. Using a microsurgical instrument, the cloudy lens is first shredded and then aspirated through the eyes. The membrane behind the lens (called the posterior capsule) is left intact. In this case, the focus of the visual system is restored, usually by far vision, by replacing it with a prefabricated permanent transparent plastic intraocular lens (IOL) (popular in the early 1980s). To do.

  Prior to IOL advancement, cataract patients were forced to wear thick “coke bottle” glasses or contact lenses after surgery. Unfortunately, vision is not very good with thick glasses, and thick contact lenses are not a good option. The IOL invention solved this problem.

  Intraocular lenses can be divided into two main groups: non-foldable and foldable. The first intraocular lens was made from a hard plastic (non-foldable) material, so that it could only be introduced into the eye by an incision of the same size as the lens diameter. In order to reduce eye trauma in cataract surgery, it is desirable to make the incision in which the surgical procedure is performed as small as possible. The foldable lens is made of acrylic or silicone and can be rolled and placed in a very small tube. The tube is inserted through a very small incision that is less than 3.2 mm in length. Once in the eye, IOL gradually spreads.

  Before cataract surgery is performed, the corneal curvature and axial length of the patient's eyes are measured to determine the appropriate focus for the inserted IOL. Using a complex formula to calculate the prescription correction magnification of a lens, IOL not only replaces the need for thick glasses, but it can also correct refractive errors present in the eye.

  Standard IOLs with various focal lengths are available, but these distances are determined by the lens. For this reason, unlike the natural lens of the eye, a standard IOL cannot change focus. Thus, patients who have to rely on standard IOLs lose their ability to adjust after surgery. The IOL is usually selected so that a suitable distance is visible. However, if the distance is visible, the vicinity may be blurred, and the patient may need to use reading glasses after cataract surgery.

  To remedy this problem, bifocal and multifocal IOLs were developed. They reduce or even eliminate the need for reading glasses, but these IOLs result in reduced contrast sensitivity and a subjective experience of halos around the light source.

  Thus, there is a need for a material that can make reading glasses unnecessary after cataract surgery because it moves like the natural lens of the eye. Such a material must be able to change its refractive power by changing its shape in the eye. In addition to being used as an IOL in cataract surgery, such materials are also used to treat other refractive errors, including presbyopia (physiological loss of eye accommodation with age). You will get.

  Injectable, in situ generated gels have several possible uses in medicine (eg, in intraocular lenses, as vitreous substitutes, and as drug delivery devices). In general, gels generated in situ are cavities that are minimally invasive, can be easily delivered, are inherently or possible, while conforming to various shapes that would otherwise be difficult to make in advance. There is an advantage that can be satisfied. The mechanism of gelation can be physical (temperature change, hydrogen bonding, hydrophobic interaction) or chemical (ion bond or covalent bond formation). Usually, physical cross-linking is more unstable than chemical cross-linking. In situ gelation produces covalently bonded networks by radical polymerization and can be initiated by the absorption of heat, chemical initiators, or photons. However, radical polymerization is rarely quantitative, and the resulting gel usually contains significant amounts of unreacted monomers, initiators, and accelerators, any or all of which are toxic, The reaction itself can be very exothermic. Particularly demanding for eye applications, the requirement is very close to room temperature and a narrow reaction temperature range, optically transparent materials, very low chemical and phototoxicity, and wet and oxygenated , Also includes long-term stability in light-rich environments. An object of the present invention to generate gels in situ is to develop new surrogate vitreous and injectable intraocular lens materials.

  Adjustment is a dynamic process in which the refractive power of the visual system, mainly the lens, is automatically adjusted and focused on the retina. This ability is usually drastically reduced by the 30s of life and is almost completely lost by the 60s of life due to progressive changes in lens volume and elasticity, to subjects closer to the length of the arm It becomes impossible to focus (a state called presbyopia). It is also possible to restore the presbyopic patient's accommodation by sucking out the contents of the capsule bag and filling it with the right volume of the right material. The development of a surgical procedure that empties the capsular bag through a small opening and the identification of a suitable material that fills the sac again is sought. Such materials include restoration of accommodation, smaller scleral incisions smaller than those currently required for semi-rigid substitute lenses, improved physiological positioning of intraocular lenses, and reduced secondary turbidity rates. And preferably have several advantages.

  Physical and chemical crosslinks that produce gels within the sac are sought. For example, Kessler (Experiments in refilling the lens. Arch. Ophthalmol. 71: 412-417, 1964) is a gel that is physically cross-linked using Carquille immersion oil, silicone fluid, and damar gum. Was produced in the eyes of rabbits. Parel et al. (Phaco-Ersatz: Cataract surgery designed to preserve accommodation.Graefes Arch. Clin. Exp. Ophthalmol. 224: 165-173, 1986), in this case for several hours at room temperature. A filler-free divinylmethylcyclosiloxane elastomer that is normally cured within was utilized. Nishi et al. (Accommodation amplitude after lens refilling with injectable silicone by sealing the capsule with a plug in primates.Arch. Ophthalmol. 116: 1358-1361, 1998) uses polymethyldisiloxane containing hydrogen polysiloxane as a crosslinking agent. It was. Other researchers have reported endocapsular polymerization in which a mixture containing monomers is injected and photopolymerized in situ to form a gel. Jacqueline et al. (Injectable intraocular lens materials based upon hydrogels, Biomacromolecules 2: 628-634, 2001) recently reported photopolymerization of acrylate-modified N-vinylpyrrolidone / vinyl alcohol copolymer in the sac using an acrylamide photoinitiator. It was confirmed that the dimensions of some compositions were stable and optically transparent. However, this system is not practical due to the toxicity of the unreacted monomer and the exothermic nature of the polymerization reaction. Furthermore, in all the above cases, the mechanical properties of the refill material were not studied. Since none of these chemically cross-linked gels are reversible, the chances of lens recovery are entirely up to the result.

In our previous work, we synthesized polyethylene glycol acrylate as a typical macromonomer, evaluated its properties, and used it to perform intracapsular polymerization with simultaneous gelation. The degree of conversion during the polymerization was about 95%, as is usually the case with many free radical reactions. To address the problem of residual monomer toxicity, we quantitatively explored the relationship between structure and toxicity and confirmed the following: 1) In general, acrylates were more toxic than methacrylates; 2) which Also, the hydrophobic monomer was more toxic than the hydrophilic monomer in this type; and 3) the toxic effect was probably due to residual monomer crossing the lipid bilayer, and then with intracellular protein and DNA by Michael addition reaction. By being able to react. We have also observed that acrylates or methacrylates containing hydrophilic hydrogels are unstable to hydrolysis in tissue culture media. It is our continuing intention to establish and develop new technologies that deepen our understanding of the use of polymers in ophthalmology, especially when they affect accommodation and presbyopia.
U.S. Patent Application No. 10/706081 US Provisional Patent Application No. 499887 U.S. Provisional Patent Application No. 556492 Kessler, Experiments in refilling the lens. Arch. Ophthalmol. 71: 412-417, 1964 Parel et al., Phaco-Ersatz: Cataract surgery designed to preserve accommodation.Graefes Arch. Clin. Exp. Ophthalmol. 224: 165-173, 1986 Nishi et al., Accommodation amplitude after lens refilling with injectable silicone by sealing the capsule with a plug in primates. Arch. Ophthalmol. 116: 1358-1361, 1998 Jacqueline et al., Injectable intraocular lens materials based upon hydrogels, Biomacromolecules 2: 628-634, 2001 Gupta et al., Laser Raman spectroscopy of polyacrylamide. J. Polym. Sci., Polym. Phys. Edn. 19: 353-360, 1981 Hisano et al., Entrapment of islets into reversible disulfide hydrogels. J Biomed Mater Res. 40: 115-123, 1998 Ellman, Arch. Biochem. Biophys., 1959, 82: 70-77 Mori et al., J. Am. Chem. Soc., 2003, 125: 3712

  However, it is difficult to control the refractive index of the hydrogel without significantly changing the modulus of the material. Traditionally, the refractive index of hydrogels is often changed by using different polymer concentrations. However, this also results in changing the modulus of the material. Therefore, there remains a need for alternatives that can control the refractive index and modulus of a material almost independently.

  In one embodiment, the present invention reversibly converts between sol-gel phases by oxidation / reduction, by irradiation with light of different wavelengths, or by applying shear in the case of physical gels. A reversible hydrogel system is provided. The hydrogel can be reversed to form a solution; the solution can also be converted by a suitable trigger to become a hydrogel. Thus, the system is reversibly converted between the hydrogel and the solution.

  The chemically reversible redox hydrogel system of the present invention includes a copolymer formed by polymerizing a monomer with a crosslinker. The crosslinker provides disulfide bonds within the copolymer molecule to form a hydrogel. When the hydrogel is reduced, the disulfide bonds are broken to yield a soluble copolymer solution. On the other hand, the copolymer solution can be oxidized (by oxygenation reaction, disulfide exchange reaction, or photooxidation in the presence of riboflavin and oxygen) to form disulfide bonds and regenerate hydrogel. Oxidation can be performed at a physiological pH of about 7.0 to about 7.4. Gels can also be generated by incorporating photosensitive groups that undergo 2 + 2 photoaddition or groups that photochemically react with thiols (eg, thiol-acrylamide reactions). Hydrophobic hydrogels or associative hydrogels also exhibit such reversibility to shear forces, i.e., they are like solutions when subjected to shear and are free of shear forces or When it becomes smaller than a specific yield stress, it behaves as a gel.

  In another embodiment, the present invention provides a method for producing a nanogel (hydrogel nanoparticle) comprising a disulfide bridge. Preferably, the nanogel is made from a reversible hydrogel system that is reversibly converted between the hydrogel state and the solution by oxidation / reduction or by light of different wavelengths. The hydrogel can be reduced to a solution; and the solution can be oxidized to a hydrogel. This system is thus reversibly converted between the hydrogel and the solution. Nanogels consist of copolymerizing monomers with a crosslinking agent to form a crosslinked hydrogel; reducing the crosslinked hydrogel to a copolymer solution; diluting the copolymer solution to a diluted copolymer solution; and then Oxidizing a diluted copolymer solution to produce a nanogel; Thiol-containing nanogels can also incorporate metal particles (eg, gold) therein. Furthermore, all of the above basic concepts used to produce hydrogels can potentially be used to produce nanogels by first diluting the copolymer solution to its critical concentration.

In yet another embodiment, the present invention combines a reversible hydrogel and nanoparticles to produce a hydrogel nanocomposite for use as an injectable, adjustable intraocular lens. The nanocomposites of the present invention comprise nanoparticles dispersed in a polymer hydrogel formulation, and the refractive index and modulus of the material are controlled using two variables (i.e., the concentration of nanoparticles in the hydrogel and the copolymer concentration). Can be done. The refractive index can be controlled by changing the amount of nanoparticles and the modulus can be controlled by changing the polymer concentration in the hydrogel. Most importantly, the nanoparticle preferably has a particle size of less than about 150 nm, most preferably about 3-20 nm, and it must be non-scattering. It is very important that the nanoparticles are of such dimensions that do not disperse or scatter visible light. The nature of the nanoparticle is not so important, as long as it is dispersible in aqueous media, does not scatter visible light, and remains stable with the polymer formulation, nanogel, protein, silica, gold, silver, TiO ₂ , Transition metals, ceramics, or combinations thereof.

I. Reversible Hydrogel System The hydrogel system of the present invention comprises a copolymer that is a hydrogel in one state and a solution in another state. This copolymer is preferably obtained by copolymerizing a monomer and a crosslinking agent. The cross-linking agent cross-links between molecules to form a hydrogel. Monomers are acrylamide, N-ornithine acrylamide, N- (2-hydroxypropyl) acrylamide, hydroxy-ethyl acrylate, hydroxyethyl methacrylate, N-vinyl pyrrolidone, N-phenyl acrylamide, dimethylaminopropyl methacrylamide, acrylic acid, benzyl methacryl It can be an amide, methylthioethylacrylamide, or a combination thereof. Functionalized macromonomers or polymers (eg, polyethylene glycol acrylate, polyethylene glycol methacrylate, polyvinyl alcohol, etc.) that can be modified or derivatized to incorporate disulfide groups or reversible crosslinks may also be suitable for the present invention. Preferred polymer systems have a semi-flexible or rigid water-soluble polymer backbone such as polyacrylic acid, polystyrene sulfonic acid, collagen, polysaccharides.

  The polymer is preferably a crosslinkable group capable of forming covalent bonds within the polymer or with other polymers when in aqueous solution (after macromer gelation depending on heat or photochemistry, Alternatively, the polymer may be crosslinked to form a gel). Chemical or ionic crosslinkable groups known in the art may be provided on the macromer. Preferred crosslinkable groups are unsaturated groups (including vinyl, allyl, cinnamate, acrylate, diacrylate, oligoacrylate, methacrylate, dimethacrylate, oligomethacrylate, acrylamide) or biologically acceptable Other photopolymerizable groups. The cross-linking agent is preferably a disulfide linker, such as N, N′-bis (acryloyl) cystamine (BAC). Other useful cross-linking agents include, but are not limited to, methylene bisacrylamide, methylene bismethacrylamide, esters of unsaturated mono- or polycarboxylic acids and polyols, such as diacrylates or triacrylates (e.g., butanediol diacids). Acrylate, butanediol dimethacrylate, ethylene glycol diacrylate, ethylene glycol dimethacrylate, or trimethylolpropane triacrylate), allyl compounds (e.g., allyl (meth) acrylate, triallyl cyanurate, diallyl maleate, polyallyl ester, tetra Allyloxyethane, triallylamine, tetraallylethylenediamine, pentaerythritol triallyl ester, or allyl ester of phosphoric acid), and vinyl compounds (For example, vinyl acrylate, divinyl adipate, divinyl benzene, and vinyl phosphonic acid derivatives). Other irreversible linkers may be included in the polymer to produce branching.

  Photochemically reversible linkers suitable for the present invention can include, but are not limited to, stilbene, azo, and cinnamoyl derivatives. Usually, due to the photochemically reversible linker, gelation of the copolymer occurs at a specific wavelength and liquefaction of the copolymer occurs at a different wavelength. For example, the copolymer solution produces a hydrogel upon exposure to a first wavelength, and the hydrogel returns to the copolymer solution upon exposure to a second wavelength.

  FIG. 1 schematically illustrates hydrogel formation, copolymer solubilization, and hydrogel regeneration. Monomer and linker copolymerization forms a crosslinked hydrogel. The polymerization is initiated by a combination of a water soluble or monomer soluble initiator or redox initiator. Examples of water-soluble initiators are sodium, potassium and ammonium salts of peroxodisulfate, hydrogen peroxide, tert-butyl peroxide, tert-butyl hydroperoxide, potassium peroxodiphosphate, tert-butyl peroxypivalate, cumyl hydro Peroxide, isopropylbenzyl monohydroperoxide and azobisisobutyronitrile. Examples of monomer soluble initiators are diacetyl peroxydicarbonate, dicyclohexyl peroxydicarbonate and dibenzoyl peroxide. Usually, the initiator is used in an amount of 0.01 to 0.5% by weight, based on the total weight of the monomers. The initiator combination as a combination with the reducing agent (s) can be used as a redox initiator. Suitable reducing agents include, but are not limited to, alkali metal and ammonia sulfites and bisulfites (e.g., sodium sulfite), zinc formaldehyde sulfoxylate or derivatives of sulfoxylic acid such as alkali metal salts (e.g., Sodium hydroxymethanesulfonate), and ascorbic acid. Preferably, the amount of reducing agent is 0.01 to 0.5% by weight, based on the total weight of monomers.

  Once the copolymer is formed, it is preferably washed to completely remove unreacted monomer and crosslinker. The washing step is particularly preferred for monomers that are toxic when used in humans. For example, acrylamide is known to be a carcinogen and neurotoxin, but its polymer, polyacrylamide, is harmless. Thus, it is highly desirable that unreacted acrylamide be thoroughly washed from the hydrogel after acrylamide polymerization. After removal of unreacted monomer and crosslinker, the copolymer is further swollen with a liquid (preferably water) to make the water content desired.

  This hydrogel can be liquefied by breaking of the cross-links into a copolymer solution. In the case of disulfide bonds, liquefaction can be performed by chemically reducing the hydrogel and reducing the disulfide bonds to thiols. Preferably, the reduction is performed in the presence of a reducing agent such as dithiothreitol (DTT). Other reducing agents include, but are not limited to, 2-mercaptoethanol, dithioerythritol, cysteine, butanethiol, sodium borohydride, cyanoborohydride, mercaptoethylamine, ethylmaleimide, and tri (2-carboxyethyl) There may be phosphine hydrochloride (TCEP.HCl). The reducing agent is selected based on the nature of the crosslinking. For disulfide bonds, DTT is the preferred reducing agent. Once reduced, the hydrogel liquefies and becomes a copolymer solution. In the case of a copolymer having a disulfide bond, a water-soluble thiol-containing copolymer is obtained by reduction.

  The copolymer solution can be diluted, concentrated and / or dried as desired. For storage, the copolymer is preferably precipitated from the solution, for example with methanol, filtered and dried. Other methods are also suitable, including lyophilization. The stored copolymer solids are later dissolved in the solution to the desired concentration for use.

  The hydrogel can be regenerated from the copolymer solution by regenerating crosslinks within the copolymer molecule. In the case of disulfide bonds, the regelation can be carried out by oxidation of the copolymer solution, preferably in the presence of an oxidant (preferably atmospheric oxygen). Atmospheric oxygen is preferred, but other oxidizing agents such as dithiodipropionic acid (DTDP), cystamine, 2-hydroxyethyl disulfide, hydrogen peroxide, organic peracids, peroxycarbonates, ammonium sulfate peroxide Benzoyl peroxide, perborate and the like can also be used. More importantly, however, preferred oxidants should not have any significant toxicity to humans and / or animals.

  For polymers with photochemically dependent linkers, such as derivatives of stilbene, azo, cinnamoyl, regelation is performed by exposing the copolymer solution to light of the appropriate wavelength. Thus, liquefaction of the reversible group can be carried out by exposing the gel to light of an appropriate wavelength, usually a wavelength different from the gelation wavelength.

  Note that photochemically dependent and oxidation dependent linkers are not exclusive. Both types of linkers may be used on the same polymer to achieve the desired result. For example, polymers containing both types of linkers can be prepared and washed using oxidation / reduction. However, once the lens is formed, photochemically dependent linkers can also be activated, resulting in a more permanent and stable hydrogel. In this embodiment, a reversible linker may be used with an irreversible linker. For example, the polymer can use an oxidation / reduction reversible linker and a linker that relies on irreversible photochemistry (eg, thiol-acrylamide and / or thiol-acrylate).

II. Hydrogels on nanogels can also be used to make nanogels whose particle size depends on the molecular weight of the copolymer. The method of the present invention teaches the production of nanogels having a diameter of less than 150 nm, preferably about 3-20 nm and a refractive index similar to that of a natural lens. The chemistry involved in the preparation of the reversible hydrogel in Section I above is suitable for the production of nanogels. Nanogels are the copolymerization of monomers with a crosslinking agent to form a crosslinked hydrogel; reducing or irradiating the crosslinked hydrogel to form a copolymer solution: diluting the copolymer solution into a diluted copolymer solution; And oxidizing the diluted copolymer solution into a nanogel. Thus, the method of producing the nanogel is substantially the same as that of the reversible hydrogel, except that the nanogel is produced from a dilute copolymer solution. By diluting is meant that the concentration of the copolymer solution is less than 1 percent (w / v), preferably less than 0.5 percent (w / v), and most preferably less than 0.01 percent (w / v). Thus, when the copolymer concentration is dilute, the nanogel is produced by oxidation and / or irradiation of the copolymer solution, and when the copolymer concentration is dense, the hydrogel is produced by oxidation and / or irradiation of the copolymer solution. Because dilute copolymer solutions result in very little intermolecular interactions, crosslinks are formed within the molecule, thus producing a nanogel. On the other hand, if the copolymer solution is thicker, intermolecular crosslinking becomes dominant and a hydrogel is formed.

  In one embodiment of the invention, the thiol-containing nanogel particles can also encapsulate metal particles (eg, gold). In this case, the metal particles act spontaneously by their tendency towards their thiol groups. Without metal, the solution is oxidized or irradiated to intramolecular crosslinks to form nanogels. However, during nanogel production, metal particles are trapped within the nanogel. If the concentration of metal particles is sufficiently low, it is possible to bind one metal particle to one nanogel particle. Depending on the metal, crosslinking can also occur between the metal particles and the nanogel. For example, gold can crosslink with -SH groups of the copolymer upon oxidation.

III. Hydrogel nanocomposites These hydrogel materials exhibit a modulus similar to that of natural lens, but the refractive index of hydrogel is usually less than that of natural lens material. Usually, the refractive index increases in proportion to the polymer concentration, but the modulus increases exponentially. Thus, it is almost impossible to obtain a material with a high refractive index and a low modulus with a typical hydrogel. Applicants have found that nanocomposites of hydrogels and nanoparticles can achieve a high refractive index (RI) and low modulus similar to natural lenses. The nanocomposite system also exhibits accommodation properties similar to natural lenses, preferably within about 1 second, more preferably within about 50-250 milliseconds.

  The hydrogel nanocomposite of the present invention comprises nanoparticles dispersed in a reversible hydrogel matrix, and by changing two variables (ie, the concentration of nanoparticles in the hydrogel and the concentration of copolymer in the hydrogel) There is an advantage in that the refractive index and the modulus can be controlled. RI can be controlled by changing the concentration of nanoparticles in the hydrogel, and the modulus can be controlled by changing the polymer concentration in the hydrogel. This is particularly true in the case of nanoparticles that do not interact. However, if the nanoparticles interact with the copolymer backbone, a system may be obtained in which the modulus increases with the nanoparticles (or RI) depending on the crosslink density and particle size.

The nanoparticles preferably have a particle size of less than about 150 nm, most preferably about 3-20 nm. It is very important that the nanoparticles are of such a size that does not disperse or scatter visible light. Nanoparticles are preferably, but not limited to, polymer nanogels (see Section II above), proteins, as long as they are dispersed in an aqueous medium and remain stable with the polymer formulation, and preferably do not interact with the polymer backbone. , Silica, metal (eg, gold, silver, and transition metals), TiO ₂ , ceramic, or combinations thereof.

  The reversible hydrogel already described in Section I is a preferred matrix for the nanocomposite. However, other hydrogels are also suitable for the present invention.

  To produce the nanocomposite, the nanoparticles are added to the liquefied copolymer solution and stirred to form a uniform dispersion. This dispersion is then oxidized and / or irradiated to produce the nanocomposite of the present invention.

  When nanogels are used as nanoparticles, it is preferred that the nanogel and the reversible hydrogel contain different crosslinkable groups. For example, if the hydrogel uses an oxidation / reduction linker, the nanogel preferably uses a photochemically dependent linker and vice versa. More preferably, the nanogel comprises both oxidation / reduction and photochemical dependent crosslinking groups to further improve stability and performance.

  In a preferred embodiment, the nanoparticles should be selected so that cross-linking between the hydrogel matrix and the nanoparticles is minimized. In this case, if there is no cross-linking between the matrix and the nanoparticles, the RI and modulus of the nanocomposite can be controlled substantially independently. Specifically, RI can be controlled by adjusting the concentration of nanoparticles in the hydrogel, and the modulus can be controlled by adjusting the copolymer concentration in the hydrogel. The greater the level of interaction and / or crosslinking between the hydrogel matrix and the nanoparticles, the less likely it is that the RI and modulus of the nanocomposite can be independently controlled.

  To be a surrogate lens, the nanocomposite should achieve an RI of about 1.40 to 1.41 and a modulus of about 1,000 to 1,500 Pascals.

  Without further explanation, one of ordinary skill in the art, using the preceding description and the following examples, will be able to make and utilize the composites of the present invention and implement the claimed methods. The following examples are given to illustrate the present invention. It should be understood that the invention is not limited to the specific conditions or details described in this example.

(Example 1)
Acrylamide / BAC Hydrogel Experimental Method Synthesis of Polyacrylamide / BAC Hydrogel
In 25% ethanol (25:75 = ethanol: water v / v), 5% (w / w) hydrogels of various compositions, acrylamide (Aam) and BAC, 98/2, 96/4 , And 94/6 acrylic molar ratio. Prior to the start of polymerization, nitrogen was bubbled through the solution for about 30 minutes to expel any dissolved oxygen. The reaction was started by adding 2.1% (w / w) TEMED and 0.4% (w / w) APS and continued for 15 hours at 25 ° C. Aqueous ethanol was used as the solvent due to the limited solubility of BAC in water. The resulting gel was removed from the beaker, swollen in 500 mL of water for 2 days, crushed into small pieces, and washed with distilled water. Gels from the above copolymer composition show that they contain disulfide (-SS-) bonds by incorporating 2, 4, and 6 acryl mol% BAC, respectively, ABSS2, ABSS4, and Named ABSS6.

Reduced liquefaction of swollen hydrogel containing no monomer The gel was crushed into small pieces and liquefied by adding DTT at 10 mol / (1 mol of BAC) at pH 7.0. Reductions were performed on ABSS2, ABSS4, and ABSS6 for 2, 4, and 6 hours, respectively, with nitrogen bubbling through the solution while stirring. After complete dissolution of the gel, the solution was acidified to pH 3 using 10% (V / V) HCl and precipitated into excess methanol with vigorous stirring. The precipitated -SH polymer was filtered, dried under vacuum and stored under reduced pressure until needed. The soluble polymers above from ABSS2, ABSS4, and ABSS6 were named ABSH2, ABSH4, and ABSH6, respectively, indicating that they now contain -SH groups instead of disulfide bonds.

Characterization of soluble copolymers The thiol (-SH) content of each copolymer was determined using the Ellman reagent. Briefly, 50 μL of 0.5% (w / v) copolymer solution (pH 4, nitrogen bubbling) was added to 50 μL of 0.01 M Ellman reagent (0.1 M phosphate buffer, pH 8.0), 500 μL. It was added to a mixture of 0.1 M phosphate buffer (pH 8) and 450 μL distilled water. The absorbance of the resulting solution at 412 nm (using a Beckman DU54 spectrophotometer) was determined 5 minutes after mixing. The concentration of -SH in each ABSH polymer was calculated using a molar extinction coefficient of 13,600 M ^-1 cm ^-1 .

The molecular weight of the polymer after reduction was determined using a Viscotek HPLC-GPC system (Houston, TX) equipped with a static light scattering and refractive index detector coupled in tandem with a viscosity detector. The stationary phase consists of two columns of G6000PWXL and G4000PWXI (Tosoh Biosep, Montgomeryville, PA) connected in series, and the mobile phase is 20 mM Bis-Tris buffer (pH 6.0, 0.1% azide). Sodium). Samples were prepared with water (pH 4, saturated with N ₂ ) at a concentration of 0.5% (w / v). Calibrated using polyethylene glycol standards (Viscotek, Houston, TX) with a molecular weight (Mw) of 1000 to 950,000.

The presence of thiol groups in the soluble polymer and their disappearance upon regelation was investigated by Raman spectroscopy (Kaiser Holoprobe Series 5000 Raman spectrophotometer, operated at an argon laser wavelength of 514 nm). The polymer solution in the vial was directly irradiated with a laser beam, and a spectrum was obtained with a resolution of 2 cm ⁻¹ . Using a custom sample holder on a commercially available platform, reproducibility of the sample position relative to the laser focus was ensured. Spectra were analyzed using the GRAMS / 32 software package (Galactic Industries Corporation, Salem, NH).

  Regelation of the copolymer solution was performed using DTDP (details are given below), after which the gel was allowed to swell for 2 days. After the swollen gel was washed several times with water, it was used in a Raman experiment to observe the disappearance of the -SH group peak and the appearance of a peak corresponding to the formation of a disulfide (-S-S-) bond.

Regelation of aqueous copolymer solution Three different concentrations (% w / v) of polymer solution in pH 4 water saturated with nitrogen were prepared from each of the polymers after reduction: 10.0, 12.5, and 15.0 from ABSH2. %; From ABSH4, 5.0, 7.5, and 10.0%; from ABSH6, 2.0, 3.0, 4.0%. Polymer solutions (1 mL each) were placed in a test tube and the pH of the solution was adjusted to approximately 7.4 using a calculated amount of 10 M NaOH, after which the required amount of DTDP (0.5 M, pH 7) was added and stirred vigorously. . Based on the -SH content of each ABSH polymer solution, an equimolar amount of DTDP was added. Gelation was visually observed by tilting the test tube. To evaluate the ease of injection and the uniformity of the gel within the sac, a special mold that mimics the natural porcine lens was used.

  A cylindrical gel was prepared using a Teflon (registered trademark) frame. The static modulus of the regelled sample (as a cylinder or thin disk) was determined from stress / strain experiments using a dynamic viscoelastic analyzer (Perkin Elmer DMA7e, Norwalk, USA) and the analysis was performed after addition of DTDP. Completed in about an hour. Static stress scans were performed from 0 to 25 mN at a rate of 5 mN / min at 25 ° C. The preparation of copolymer hydrogels and their reduction and regelation from water-soluble copolymers is schematically illustrated in FIG.

Intracapsular gelation
Copolymer solutions (% w / v) of ABSH2 to 10.0 and 12.5%; ABSH4 to 5.0 and 7.5%; and ABSH6 to 2.0%; were evaluated for intracapsular gelation, respectively. Newly extracted pig eyes were purchased from a local slaughterhouse immediately after slaughter. Usually, each eye was stabilized on a styrofoam plate to remove the cornea and iris. A 1.0-1.2 mm diameter capsulotomy was performed near the equator on the anterior capsule using an Ellman Surgitron tool (Edmonton, NJ). The sac was then emptied using a Storz Phacoemulsification ultrasound device (Premiere type, Bausch and Lomb, St. Louis, Missouri). DTDP was added to the copolymer solution just before injection and mixed well. In a typical case, 800 μL of 5% (w / v) ABSH4 solution (adjusted to pH 7.4 using 10 M NaOH) is placed in a test tube and 43 μL of DTDP (0.5 M, pH 7.0) is added. In addition, the mixture was thoroughly mixed with a vortex stirrer for 10 seconds, and sucked into a syringe with an injection needle having an outer diameter of 1.0 mm (the tip of which was attached to a plastic conical micro tip). Carefully, the copolymer was quickly poured into the bottom of the sac, thus filling the sac without foam and keeping it closed for the next 2-3 minutes. Gelation usually occurred within 3 minutes. The operation method of hydrogel injection and regelation is schematically shown in FIG.

Results Synthesis of poly (AAm-co-BAC) hydrogels Three hydrogels with different compositions were prepared from acrylamide and BAC at 2, 4, and 6 acrylmol% BAC relative to acrylamide. As expected, increasing BAC resulted in a gel with better structural integrity. The gel with higher amounts of BAC was slightly less transparent. ABSS2 did not produce a stable gel but was a highly viscous solution. However, higher concentrations resulted in stable gels (> 15%).

Synthesis and Characterization of ABSH Copolymers A key step in obtaining water-soluble copolymers (ABSH) from cross-linked gels (ABSH) is the disulfide bond (-SS-) to thiol (-SH) group, as shown in Figure 2. A complete reduction was included. Reduction of the gel with DTT almost completely reduced the disulfide bond (as indicated by the -SH content values in Table 1). Nitrogen bubbling through the solution significantly accelerated gel dissolution. As shown in Table 1, the -SH content of the water-soluble copolymer (ABSH) is proportional to the BAC concentration used for the copolymerization.

  FIG. 3 shows the GPC curve of the ABSH copolymer observed by three detectors of HPLC-GPC. As shown by curve a in FIG. 3, the molecular weight increases and the molecular weight distribution becomes wider as the BAC content in the copolymerization increases. The results of the weight average molecular weight (Mw) of the ABSH polymer are shown in Table 1.

The thiol and its disappearance upon regelation (simultaneously accompanied by the formation of disulfide (-SS-) bonds) were confirmed by Raman spectroscopy. FIG. 4 shows the Raman spectrum of ABSH6 (4 w / v% aqueous solution) and the corresponding regenerated gel RABSS6 (where “R” indicates a gel regenerated from ABSH6). The characteristic absorption at 2580 cm ⁻¹ corresponds to —SH stretching vibration (ν _SH ) and is observed in the curve b in FIG. 4, which disappeared completely by gelation. Absorption corresponding to -SS ^- stretching (ν _ss ) usually appears at about 510 cm ^-1 unless it overlaps with other vibrations. Since the polymer is mostly polyacrylamide, as can be seen in curve c in FIG. 4, ν _ss is reduced due to interference from the wide asymmetry of the polyacrylamide centered at 485 cm ⁻¹ -CC-skeletal bending peak. It is difficult to recognize clearly with ν _ss . This problem was solved by subtracting the polyacrylamide spectrum (curve a, FIG. 4, without BAC, prepared using the same procedure) from the RABSS6 spectrum. As shown in the inset of FIG. 2, ν _ss was clearly recognized as a shoulder peak at 505 cm ⁻¹ .

As shown in the inset of Fig. 4 as well as -SH and -SS-vibrations, after drawing the spectrum of polyacrylamide, -CS-stretching from the -CSH group of ABSH6 and -CSSC- of RABSS6 (ν _Cs ) was also clearly observed at 662 cm ^-1 and 621 cm ^-1 respectively. In addition to the characteristic absorption above, the other absorption peaks shown in FIG. 4 correspond to the Raman spectrum of polyacrylamide (Gupta et al. Laser Raman spectroscopy of polyacrylamide. J. Polym. Sci., Polym. Phys. Edn. 19: 353-360, 1981).

Regelation and mechanical properties
Regelation of ABSH polymers can be performed by air oxidation of thiols or by thiol disulfide exchange reaction (Figure 5) (Figure 5). The rate of one-electron transfer from ABS ^- ions (from ABSH) to oxygen determines the rate of thiol air oxidation. This reaction rate increases with increasing pH. At pH 7.4, gelation usually occurred within 12 hours. Hisano et al. (Entrapment of islets into reversible disulfide hydrogels. J Biomed Mater Res. 40: 115-123, 1998) is a thiolated acrylamide polymer of the same type, at pH 8.8 it produces gels, but with air oxidation Reported that it took about 6 hours. Unlike air oxidation, the thiol disulfide exchange reaction gelled within a few minutes at a pH between 7.0 and 7.5 (closer to physiological pH). Because the gel time was so short (all less than 5 minutes), regelation experiments could be performed on several different concentrations of ABSH polymer. The ABSH polymer concentration and the static modulus of the regelled specimen are listed in Table 2. The modulus of the gel produced at the same concentration increased with increasing molecular weight and -SH content. All regenerated gels were clear.

  In this experiment, gelation occurred in less than 30 seconds with a 15% solution of ABSH2, a 10% solution of ABSH4, and 3 and 4% solutions of ABSH6. However, due to the high viscosity and quick reaction, no intracapsular gelation was attempted using these samples. Instead, air oxidation was the preferred method. Lower concentrations (these are not included in Table 2) were not appropriate to produce stable gels. Overall, the rate of gelation was a function of oxidant, pH, and light.

Intracapsular gelation The suitability of an aqueous copolymer solution (ABSH) for intracapsular gelation was illustrated in a porcine lens capsule from which the contents had previously been removed. As already described, the very fast regelation prevented testing at several polymer concentrations. Intracapsular gelation was performed using freshly prepared 10.0 and 12.5% solutions of ABSH2, 5.0 and 7.5% solutions of ABSH4, and 2.0% solutions of ABSH6. In all these cases, regelation occurred within 5 minutes. Thanks to the original high viscosity, which increases with the addition of DTDP, there was no leakage during refilling. The procedure for in-vitro refilling surgery for intracapsular gelation is shown schematically in FIG. Here, a hole is made in the cornea and the contents of the lens are removed by phagofragmentation to empty the sac. The empty sac was then refilled with the appropriate ABSH solution and regelled in situ.

  FIG. 7a shows a representative sample of pig eyes when intracapsular gelation was performed with a 10% (w / v) solution of ABSH4. When viewed through the lens, the object appears clearly undistorted. FIG. 7b shows a regelled lens separated from within the porcine lens capsule. The injection frame shaped into the lens shape also confirmed the formation of a uniform and transparent gel (Fig. 7c).

Discussion The main purpose of this experiment was to demonstrate the feasibility of using a thiol-containing copolymer as an injectable precursor for chemical cross-linking in vivo under physiological conditions (room temperature In the presence of oxygen, near neutral pH). It is possible to produce an optically transparent gel (whose modulus was close to that of the young lens material (about 1000 Pa)) in the sac from which the contents had been previously removed. Gelling chemistry uses smooth oxidation of pendant thiols that disulfide by slow air oxidation or fast exchange reactions mediated by a suitable non-toxic disulfide reagent. Such systems do not contain toxic monomers, do not contain thermal reactions near living tissue, are leak-free, and can be controlled by suitable accelerators and photons that can coexist with living organisms. It has.

  Here, polyacrylamide is used as a framework or skeletal model and can be replaced by any polymer chain. By incorporating hydrophobic groups, the properties of the copolymer solution, i.e. viscosity and / or thixotropy, can be significantly increased. Furthermore, the thiol may be pendant or at the end of a chain in a multi-armed polymer. This chemistry is also applicable to thiol-containing silicones (with exceptionally high oxygen permeability). Because hydrophilic and water-soluble acrylates are usually biodegradable and not suitable for long-term use as a substitute vitreous or intraocular lens material, acrylamide derivatives are generally due to their relatively large hydrolytic stability. Selected. Another unique advantage of this system is that it initially removes the heat and removes monomers and other toxic chemicals (if not removed, in vivo polymerization is severely limited) Problem) is performed smoothly. Reduction with hydrogel disulfide bond DTT to obtain water-soluble ABSH copolymers is an important step that is influenced not only by the redox potential of the reducing agent but also by concentration, time, pH, and atmospheric nitrogen. is there. Considering these factors, it can be seen that using 10 molar excess of DTT and stirring under nitrogen is the most suitable technique to obtain a copolymer for intracapsular regelation. The use of acidic methanol (pH 3) during polymer precipitation is extremely important to keep the thiol in the reduced state during the next treatment. Otherwise, the copolymer is only partially soluble. The sample was kept under vacuum until dried and next use. As can be seen from the -SH content in Table 1, disulfide bonds can be reduced almost quantitatively. The gel can be regenerated either simply by air oxidation or by a thiol-disulfide exchange reaction by adding DTDP. -SH content, concentration, and molecular weight of the copolymer affected the regelation properties and the resulting modulus of the gel, but from Table 2 it can be seen that this material can be Clearly it becomes unsuitable for gelation. In general, the modulus of the hydrogel increases with increasing -SH and copolymer concentration, and the hydrogel remains optically clear. Cystamine and 2-hydroxyethyl disulfide can also be used for regelation, but DTDP is less toxic than any of these.

  In situ capsular hydrogel generation using reversible disulfide chemistry is a promising technology not only for the development of injectable intraocular lenses, but also for use as a vitreous substitute and as a topical treatment . Unlike in situ polymerization and gelation, the reversible hydrogel systems described herein involve only in situ gelation with no appreciable change in temperature. Because the copolymer does not contain monomer and is injected at a concentration with a viscous consistency, monomer toxicity and leakage are avoided. The regelation time can be easily manipulated using DTDP, oxygen, pH, and / or photons.

(Example 2)
Acrylamide / BAC / N-phenylacrylamide hydrogel (hydrophobic hydrogel)
Copolymerization of acrylamide (AAm), bisacryloylcystamine (BAC), and N-phenylacrylamide (NPA) in 5% (w / w) in 25% ethanol (25: 75 = ethanol: water v / v). ) With an acrylic molar ratio of 94/4/2. Nitrogen was bubbled through the solution for about 30 minutes to remove any dissolved oxygen prior to the start of the polymerization. The reaction was started by adding 2.1% (w / w) theotramethylenediamine and 0.4% (w / w) ammonium persulfate and continued for 15 hours at 25 ° C. Aqueous ethanol was used as the solvent due to the limited solubility of BAC in water. The resulting gel was removed from the beaker, swollen in 500 mL of water for 2 days, crushed into small pieces, and washed with distilled water. The gel from the above copolymer composition was named AB4N2SS, indicating that it contained disulfide (-SS-) bonds and 2 acrylic mole% NPA by incorporating 4 acrylic mole% BAC.

  Liquefaction of the crushed gel (AB4N2SS) was performed by adding dithiothreitol (DTT) (10 mol / 1 mol of BAC used) to the crushed hydrogel. This reduction was carried out at pH 7.0 for 4 hours, during which time nitrogen was bubbled through the solution with stirring. After complete solubilization, the copolymer solution was acidified to pH 4 using 10% (v / v) HCl and precipitated in methanol (pH 4) with vigorous stirring. The precipitated -SH copolymer was filtered, dried under vacuum and always stored under reduced pressure. The thiol-containing water-soluble copolymer obtained above from AB4N2SS was named AB4N2SH.

A 5% (w / v) solution of AB4N2SH was originally prepared with about pH 4 water (saturated with N ₂ ) and after complete dissolution, the pH was adjusted to 7 using 7 μL of 5M NaOH. did. Next, 162 μL of 0.5 M DTDP (pH = 7) was added to regenerate the hydrogel. The total volume of the composition was 3 ml. Similarly, AB4N2SH prepared 7%, 9%, and 11% (w / v) solutions and used to generate hydrogels. These hydrogels were examined to determine the modulus value. The polymer solution (9%, 11%) shows a `` honey-like '' consistency when injected by syringe, shear thinning due to shear, almost instantaneously as a physical gel without leakage in the pig sac Gelled. This physical gel was then converted to a chemical gel.

(Example 3)
Hydrogel as Substitute Vitreous Body A copolymer (AB4SH) was prepared from a hydrogel obtained by polymerizing acrylamide and 4 acrylic mol% bisacryloylcystamine (BAC). The detailed experimental procedure was the same as that described in Example 1.

A 7% (w / v) solution of ABSH4, originally prepared with about pH 4 water (saturated with N ₂ ), completely dissolved, then adjusted to pH 7 with 15 μL of 1M NaOH did. 62 μL of 0.5 M DTDP (pH = 7) was then added. The total volume of the composition was 1 ml, which was injected into the vitreous cavity of the human cadaver eye from which the internal use had been previously removed. Since the in-situ gel reached equilibrium with residual water in the vitreous cavity, the final composition of the gel in the cavity was substantially less than 7% (FIG. 8). However, in general, a gel containing BAC at a higher concentration may be a lower concentration because it gels, and is preferable as a vitreous substitute.

  In this study, acrylamide is used as the monomer to be copolymerized with BAC, but other acrylamides or vinyl monomers can also be used. This technique of introducing pendant thiols into the polymer can be used in conjunction with an appropriate choice of the main polymer to design the gel for a specific end use. Although much effort has been expended to develop hydrogels that can coexist with living organisms, this reversible hydrogel system has not been studied for in situ therapeutic applications. Overall, this information indicates that the system is novel.

(Example 4)
Nanogel Experimental Method Preparation of Copolymer Hydrogel A polyacrylamide / BAC hydrogel was prepared as in Example 1 above. The resulting polymer was named AB6SH. In the preparation of other types of copolymer hydrogels that follow, N-phenylacrylamide, dimethylaminopropylmethacrylamide (DA), acrylic acid (AA), to prepare thiol copolymers with hydrophobic, positive and negative characteristics Used additionally. The preparation of the copolymers and their composition are reported in Table 4.

Characterization of the soluble copolymer The thiol (-SH) content present in the copolymer was determined using Ellman analysis (Ellman, Arch. Biochem. Biophys., 1959, 82: 70-77). Two series connected columns of polymer after reduction (ABSH) using Viscotek HPLC-GPC system (Houston, Texas, USA) G6000PWXL and G4000PWXL (Tosoh Biosep, Montgomery Ville, PA, USA) Was determined using. The mobile phase was 20 mM Bis-Tris buffer (pH 6.0, 0.1% sodium azide). Samples were prepared with water (pH 4, saturated with N ₂ ) at a concentration of 0.5% (w / v). Calibrated using polyethylene glycol standards (Viscotek, Houston, TX, USA) with a molecular weight (Mw) of 1000 to 950,000.

Nanogel Preparation A large volume of 0.1% w / v thiol copolymer solution was prepared with pH 4 water. A small amount of 1M NaOH was used to adjust the pH of the solution to 7, and bubbled with air for 3 days. The absence of -SH was confirmed by Ellman analysis and the solution was concentrated to 25% w / w.

Crystalline solution preparation Pig eyes were obtained from a local slaughterhouse and the lens was cut out. The lens out of the sac was placed in buffer (50 mM Tris, 50 mM NaCl, 1 mM EDTA, 1 mM DTT, and 0.1% Na azide), homogenized, and centrifuged at 12,000 RPM for 30 minutes. About 1.2 g of the soluble fraction was applied to a 5 × 90 cm Sephacryl S-300 column (Pharmacia), and the fraction was collected every 10 minutes at a flow rate of 1.3 mL / min. Absorbance was measured at 280 nm using an ISCO model UA-5 monitor. Fractions were collected in the respective peaks of α, β _high , β _low , and γ crystallin and concentrated using an Amicon DC2 concentrator. The separated and concentrated material was dialyzed against distilled water, lyophilized and stored at -20 ° C until use. The standard buffer used to resuspend the sample was 20 mM Tris, 0.1% sodium azide, pH 7.6.

Rheological experiments
The viscoelastic behavior of a 4% w / w solution of β _high -crystallin (CBH) and NP-AB6SS was investigated using a Vilastic-3 Rheometer (Vilastic Scientific, Austin, TX). Each sample was measured in a cylindrical tube having a diameter of 0.04953 cm and a length of 6.278 cm at 22 ° C. and a frequency of 2 Hz. Measurements were performed over a shear rate range of 2 / sec to 900 / sec for fluid samples and 0.2 / sec to 40 / sec for gel samples. For each sample, the measurement was performed when the measuring tube was first filled and after the measuring tube was filled a second time.

Refractive Index Measurement The refractive index of all nanoparticles and crystallin solutions at various concentrations was measured at 25 ° C. using an Abbe refractometer (ATAGO Abbe Refractometer 1T / 4T, Kirkland, WA, USA).

Results and Discussion Preparation and characterization of thiol copolymers
A hydrogel was obtained by copolymerization of AAm and BAC. An important step in obtaining the desired water-soluble copolymer (AB6SH) from the cross-linked gel (AB6SS) is the complete attachment of all disulfide bonds (-SS-) in the gel to the -SH group, as shown in Figure 2. Including a significant reduction. Similar to the preparation of AB6SH, other polymers were prepared using various monomers listed in Table 4.

As clearly shown by the measurement of -SH, the reduction of the gel with DTT almost completely reduced the disulfide bond. Ellman analysis showed that the -SH content was 5.1 × 10 ⁻⁴ and 7.9 × 10 ⁻⁴ mol / g for polymers containing 4 and 6 acrylic mol% BAC, respectively. The calculated values are 5.4 × 10 ⁻⁴ and 8.0 × 10 ⁻⁴ mol / g. In general, the molecular weight distribution of the polymer was wide. The weight average molecular weights (Mw) were 9.1, 3.0, 4.3, and 1.75 × 10 ⁵ Da for AB6SH, AB4N4SH, AB4N4AA2SH, and AB4N4DA2SH, respectively.

Nanogel Preparation Nanoparticles (NP-AB6SS) were prepared by intramolecular crosslinking between -SH groups at very dilute concentrations using a polymer containing pendant -SH groups (AB6SH). Although very dilute concentrations favored the generation of nanoparticles by intramolecular crosslinking alone, at the concentration investigated in this experiment (0.1% w / v), some nanoparticles still formed between molecules. It was shown that Well-defined nanoparticles will be prepared with ultra-dilute concentration and control of the molecular weight distribution of the polymer. The generation of nanoparticles by intramolecular crosslinking is shown in FIG. Similarly, nanogels were prepared from other thiol polymers.

Refractive Index of Nanogel and Lens Crystallin The refractive index (RI) values for the disulfide-containing nanogel and lens crystallin protein molecules are shown in FIG. The RI value of nanogel is similar to that of crystallin.

Rheological measurements of nanogels and crystallins
The viscoelastic properties of the three polymer samples were compared to that of the β _high crystallin (CBH) sample at the same concentration (4% w / w). All three samples (AB6SH, RAB6SH, NPAB6SS) were derived from the same polymer.

  AB6SH is a copolymer thiol obtained from a hydrogel prepared by polymerizing acrylamide and BAC at an acrylic molar ratio of 94/6. RAB6SS is a regelled hydrogel sample from AB6SH polymer. NP-AB6SS is a nanogel sample prepared from the same AB6SH polymer as described in the experimental section. The viscoelastic properties of the nanogel (NP-AB6SS) were very similar to those of crystallin, as can be seen in FIG.

Conclusion Thiol-containing polyacrylamide copolymers were prepared from hydrogels obtained by polymerization of acrylamide and other monomers using the disulfide-containing crosslinker bisacrylcystamine. Using these thiol polymers, nanogels were prepared by intramolecular crosslinking between thiol groups. The refractive index and viscoelasticity of these nanogels were similar to that of β _high lens crystallin. The subtle differences in viscosity are thought to be due to the larger hydrodynamic volumes of the nanogels and their polydispersity.

(Example 5)
Hydrogel Nanocomposite Experimental Method Preparation of poly (AAm-co-BAC) hydrogel and its reduction Polyacrylamide / BAC hydrogel was prepared as in Example 1 above. The polymer obtained was named AB4SH.

Characterization of soluble polymer The content of thiol (-SH) present in the copolymer (AB4SH) was determined using Ellman analysis. Using a Viscotek HPLC-GPC system (Houston, Texas, USA) and two serially connected columns of G6000PWXL and G4000PWXL (Tosoh Biosep, Montgomery Ville, Pennsylvania, USA) Asked. The mobile phase was 20 mM Bis-Tris buffer (pH 6.0, 0.1% sodium azide). Samples were prepared with water (pH 4, saturated with N ₂ ) at a concentration of 0.5% (w / v). Calibrated using polyethylene glycol standards (Viscotek, Houston, TX, USA) with a molecular weight (Mw) of 1000 to 950,000.

Preparation of nanoparticles
a) From AB4SH thiol polymer: A large amount of 0.1% w / v AB4SH polymer solution was prepared with pH 4 water. A small amount of 1M NaOH was used to adjust the pH of the solution to 7, and bubbled with air for 3 days. After confirming the absence of -SH by Ellman analysis, the solution was concentrated to 25% w / w. The nanoparticle (solution) was named NP-AB4SS.
b) From silica: Water-soluble silica nanoparticles were prepared as reported by Mori et al. (J. Am. Chem. Soc., 2003, 125: 3712). The particle size was reported to be about 3 nm in diameter and was not further characterized here in this report. 65% w / w stock solutions were prepared and used in nanocomposites at various concentrations.
c) From bovine serum albumin (BSA): A 30% w / w solution of BSA in buffer (20 mM Bis-Tris, pH 6.0, 0.1% NaN ₃ ) was prepared. This solution was used in hydrogel nanocomposite materials at various concentrations.

Nanocomposite Preparation First, a 15% w / w solution of AB4SH was prepared in water saturated with nitrogen at pH4. This solution was mixed with various nanoparticle solutions at various concentrations. The concentration of AB4SH was kept constant at 5% w / w in all compositions. The concentration of nanoparticles was varied from 0 to 36% w / w as shown in Table 5. The pH of the composite was adjusted to about 7 using a small amount of 1M NaOH just prior to gelation. The nanocomposite was gelled with dithiopropionic acid (DTDP) in an equimolar amount with the thiol content of the AB4SH polymer. The composition and concentration of the polymer and nanoparticles are shown in Table 5.

Measurement of the refractive index and modulus of the nanocomposite The regelation of the composite material was carried out in a cylindrical Teflon frame (diameter 10 mm, height 5 mm). The mechanical properties of these cylindrical samples were determined by compression between parallel plates using a dynamic viscoelastic analyzer (DMA7e, Perkin Elmer, Norwalk, Connecticut, USA). The refractive index of the regelled composite material was determined using an Abbe refractometer (ATAGO Abbe Refractometer 1T / 4T, Kirkland, WA, USA).

Results and discussion
Preparation and characterization of AB4SH copolymer
Hydrogel was obtained by copolymerization of AAm and BAC. The key step in obtaining the desired water-soluble copolymer (AB4SH) from the cross-linked gel (AB4SS) is to the -SH group of all disulfide bonds (-SS-) in the gel, as shown in Figure 2. Includes a complete reduction of.

As clearly shown by the measurement of -SH, the reduction of the gel with DTT almost completely reduced the disulfide bond. Ellman analysis showed that the -SH content was 5.1 x ^10-4 mol / g. The calculated value is 5.4 × 10 ⁻⁴ mol / g. Molecular weight distribution analysis of AB4SH showed a polydispersity of 3.4 and a wide distribution, and a weight average molecular weight (Mw) of 3.8 × 10 ⁵ Da.

Nanoparticle Preparation Nanoparticles were prepared by intramolecular crosslinking between —SH groups at a very dilute concentration using a polymer containing pendant —SH groups (AB4SH). A very dilute concentration was advantageous for the formation of nanoparticles by intramolecular crosslinking alone, but at the concentration investigated in this experiment (0.1% w / v), some nanoparticles were formed between molecules. It was shown that Well-defined nanoparticles will be prepared with ultra-dilute concentration and control of the molecular weight distribution of the polymer. The generation of nanoparticles by intramolecular crosslinking is shown in FIG. Concentrated nanoparticle solution (25% w / v) was used to prepare nanocomposites at various concentrations.

  Silica nanoparticles were prepared by an addition reaction between glycidol and aminopropyltriethoxysilane followed by acidic condensation of the addition product by the sol-gel method reported by Mori et al. The particle size was reported to be about 3 nm. Due to the relatively high reactivity of the hydroxyl groups of each silicon atom and the very small diameter, these particles are well dispersed and behave like molecules in water. Very high concentration (65% w / w) solutions were prepared and used in nanocomposite hydrogel compositions at various concentrations. The preparation of silica nanoparticles is shown in FIG.

  In order to compare and confirm the idea of the present invention with a very high concentration of biomolecule solution, BSA (30% w / w) was used as the nanoparticle solution in the nanocomposite.

Refractive index and modulus of nanocomposites The refractive index (RI) and modulus values are shown in the table for all nanocomposites. As the concentration of nanoparticles increases, the RI increases for all composites. For composites containing NP-AB4SS, the modulus value also increases. NP-AB4SS was prepared from AB4SH, and NP-AB4SS was stable if not placed in a reducing atmosphere. However, when mixed with AB4SH in the composite, a thiol-disulfide exchange reaction occurs, so the disulfide bond of NP-AB4SS is broken and incorporated into the AB4SH network during gelation, resulting in a high modulus value. became. In the other two nanocomposites, both BSA and silica nanoparticles did not react with AB4SH polymer, but increased network defects and hence lower modulus values.

Conclusion A polyacrylamide / BAC hydrogel was prepared and reduced to obtain a water-soluble copolymer with pendant thiol (-SH) groups. Using this polymer, hydrogel nanocomposites were prepared with three different types of nanoparticles and regelled by thiol-disulfide exchange reaction. Nanocomposites containing nanoparticles that did not react with the thiol polymer resulted in hydrogel nanocomposites with high refractive index and low modulus. It appears that systems in which nanoparticles and hydrogel are activated by different mechanisms will solve this problem. For example, a system in which the nanoparticles are activated with light and the hydrogel is activated with pH, or vice versa, will not cause a thiol reaction between the hydrogel and the nanoparticles.

  While specific embodiments of the presently preferred invention have been described in detail herein, variations and modifications of the various embodiments shown and described herein depart from the spirit and scope of the invention. It will be apparent to those skilled in the field relevant to the present invention that this can be done. Thus, it is considered that the present invention should be limited only to the extent required by the appended claims and the applicable rules of law.

1 shows a general method for producing a reversible hydrogel system. An outline of the preparation of a polyacrylamide / BAC reversible hydrogel system is shown. Shown are HPLC-GPC triple detector chromatograms for ABSH2 (continuous line), ABSH4 (----), and ABSH6 (......) polymers. Signals are from a) RI, b) viscosity, and c) light scattering detector. a) 4% (w / v) aqueous solution of ABSH6 before gelation; b) 4% (w / v) aqueous solution of ABSH6 after gelation; and c) 5% (w / v) aqueous solution (BAC) of polyacrylamide. Without and prepared using the same experimental conditions); The inset shows an enlarged portion of b) and c) after a) has been subtracted. Figure 2 shows a) re-gelation by oxidation of a thiol-containing ABSH polymer by a) air oxidation at alkaline pH; and b) thiol disulfide exchange reaction. Summary of surgical procedure to generate hydrogel in the sac: a) perforation of the cornea and retraction of the iris followed by removal of the lens contents; b) refilling the empty capsular bag with reversible hydrogel material solution; and c ) In-situ regelation of reversible hydrogel materials. a) in porcine lens capsule; b) isolated from lens capsule; c) prepared in mold; 10 w / v% ABSH4 regelled sample. 1a shows a vitreous substitute in a human cadaver eye observed after excision of the sclera, RPE, and cornea; and b) an eye cut along the visual axis. Figure 3 schematically shows the generation of nanoparticles from an ABSH polymer by intramolecular crosslinking between -SH groups. Refractive index values of disulfide-containing nanogel and porcine lens crystallin protein are shown. Shows viscoelasticity of synthetic polymer and crystallin. Figure 3 schematically shows the preparation of silica nanoparticles by a) addition and b) acidic condensation reaction.

Claims (104)

a) providing a monomer;
b) copolymerizing the monomer with a crosslinking agent to form a crosslinked hydrogel;
c) liquefying the crosslinked hydrogel to form a copolymer solution;
d) diluting the copolymer solution to form a diluted copolymer solution; and
e) gelling the diluted copolymer solution to produce hydrogel nanoparticles;
The nanogel manufacturing method containing this.   The monomer is acrylamide, N-ornithine acrylamide, N- (2-hydroxypropyl) acrylamide, hydroxy-ethyl acrylate, hydroxyethyl methacrylate, polyethylene glycol acrylate, polyethylene glycol methacrylate, N-vinyl pyrrolidone, N-phenyl acrylamide, dimethylamino 2. The method of claim 1 selected from the group consisting of propyl methacrylamide, acrylic acid, benzyl methacrylamide, methylthioethyl acrylamide, and combinations thereof.   The crosslinking agent is N, N′-bis (acryloyl) cystamine, vinyl group, allyl group, cinnamate, acrylate, diacrylate, oligoacrylate, methacrylate, dimethacrylate, oligomethacrylate, methylenebisacrylamide, methylenebismethacrylamide, 2. The saturated mono- or polycarboxylic acid and polyol ester, trimethylolpropane triacrylate, allyl compound, vinyl compound, stilbene derivative, azo derivative, cinnamoyl derivative, and combinations thereof according to claim 1. Method.   2. The method according to claim 1, wherein the cross-linking agent is a disulfide linker.   The method of claim 1, wherein step c) is performed with a reducing agent.   6. The method of claim 5, wherein the reducing agent is selected from the group consisting of 2-mercaptoethanol, dithiothreitol, dithioerythritol, cysteine, butanethiol, sodium borohydride, and cyanoborohydride.   The method according to claim 1, wherein step e) is performed with atmospheric oxygen.   The process according to claim 1, wherein step e) is carried out at neutral pH.   The method of claim 1, further comprising concentrating and lyophilizing the hydrogel nanoparticles.   2. The method of claim 1, wherein the nanogel is hydrophobic.   2. The method of claim 1, wherein the nanogel is hydrophilic.   2. The method of claim 1, wherein the nanogel is anionic.   2. The method of claim 1, wherein the nanogel is cationic.   The method according to claim 1, wherein immediately after step b), the crosslinked hydrogel is washed to remove all unreacted monomers.   The method of claim 1, wherein step d) comprises diluting the copolymer solution to a concentration of less than 0.1 percent (w / v).   The method of claim 1, wherein the liquefaction and gelation steps are performed by reduction and oxidation, respectively.   The method according to claim 1, wherein the liquefaction and gelation steps are performed by irradiation at different wavelengths.   The method of claim 1, wherein metal nanoparticles are added to the diluted copolymer solution prior to performing step e).   The method of claim 18, wherein the metal is gold.   A hydrogel nanocomposite comprising a hydrogel with nanoparticles dispersed therein.   21. The hydrogel nanocomposite according to claim 20, wherein the hydrogel is a reversible hydrogel. Wherein the nanoparticles, nanogels, proteins, silica, gold, silver, TiO _2, transition metals, ceramics or hydrogel nanocomposite of claim 20 which is selected from the group consisting of.   21. The hydrogel nanocomposite according to claim 20, wherein the nanoparticles do not disperse or scatter visible light.   21. The hydrogel nanocomposite of claim 20, wherein the nanoparticles have a particle size of less than about 150 nm.   21. The hydrogel nanocomposite of claim 20, wherein the nanoparticles have a particle size of about 3-20 nm.   21. The hydrogel nanocomposite of claim 20, wherein the hydrogel is reversible and comprises a copolymer (a hydrogel in the oxidized state and a solution in the reduced state).   27. The hydrogel nanocomposite of claim 26, wherein the copolymer is a polymer produced by polymerization of a monomer and a crosslinker, or derived to include reversible crosslinking.   The monomer is acrylamide, N-ornithine acrylamide, N- (2-hydroxypropyl) acrylamide, hydroxy-ethyl acrylate, hydroxyethyl methacrylate, polyethylene glycol acrylate, polyethylene glycol methacrylate, N-vinyl pyrrolidone, N-phenyl acrylamide, dimethylamino 27. The hydrogel nanocomposite of claim 26, selected from the group consisting of propyl methacrylamide, acrylic acid, benzyl methacrylamide, and methylthioethyl acrylamide.   The crosslinking agent is N, N′-bis (acryloyl) cystamine, vinyl group, allyl group, cinnamate, acrylate, diacrylate, oligoacrylate, methacrylate, dimethacrylate, oligomethacrylate, methylenebisacrylamide, methylenebismethacrylamide, 27. The ester of claim 26 selected from the group consisting of saturated mono- or polycarboxylic acid and polyol esters, trimethylolpropane triacrylate, allyl compounds, vinyl compounds, stilbene derivatives, azo derivatives, cinnamoyl derivatives, and combinations thereof. Hydrogel nanocomposite.   27. The hydrogel nanocomposite according to claim 26, wherein the crosslinking agent is a disulfide linker.   21. The hydrogel nanocomposite according to claim 20, wherein the hydrogel can be reduced to form a solution.   32. The hydrogel nanocomposite according to claim 31, wherein the hydrogel can be reduced by the addition of a reducing agent.   32. The hydrogel nanocomposite according to claim 31, wherein the solution can be oxidized to regenerate the hydrogel.   34. The hydrogel nanocomposite of claim 33, wherein the solution is capable of being oxidized by atmospheric oxygen, or light and riboflavin.   21. The hydrogel of claim 20, wherein the hydrogel comprises a copolymer that produces a hydrogel when exposed to light of a first wavelength and that produces a solution when exposed to light of a second wavelength. Gel nanocomposite.   21. The hydrogel nanocomposite according to claim 20, wherein the refractive index of the hydrogel nanocomposite can be changed by changing the concentration of nanoparticles in the hydrogel. Wherein the nanoparticles, nanogels, proteins, silica, metal (e.g., gold, silver, and transition metals), hydrogel nanocomposite of claim 20 which is selected from the group consisting of TiO _2, a ceramic, or a combination thereof, . a) providing a liquid reversible hydrogel;
b) adding nanoparticles to the liquid reversible hydrogel to form a dispersion; and
c) gelling the dispersion;
A method for producing a hydrogel nanocomposite comprising: Wherein the nanoparticles, nanogels, proteins, silica, gold, silver, TiO _2, transition metals, ceramics or method according to claim 38 which is selected from the group consisting of.   40. The method of claim 38, wherein the nanoparticles do not disperse or scatter visible light.   40. The method of claim 38, wherein the nanoparticles have a particle size of less than about 150 nm.   40. The method of claim 38, wherein the nanoparticles have a particle size of about 3-20 nm.   39. The method of claim 38, wherein the hydrogel is reversible and comprises a copolymer (a hydrogel in the oxidized state and a solution in the reduced state).   44. The method of claim 43, wherein the copolymer is a polymer produced by polymerization of a monomer and a crosslinker or derivatized to include reversible crosslinking.   The monomer is acrylamide, N-ornithine acrylamide, N- (2-hydroxypropyl) acrylamide, hydroxy-ethyl acrylate, hydroxyethyl methacrylate, polyethylene glycol acrylate, polyethylene glycol methacrylate, N-vinyl pyrrolidone, N-phenyl acrylamide, dimethylamino 45. The method of claim 44, selected from the group consisting of propyl methacrylamide, acrylic acid, benzyl methacrylamide, and methylthioethyl acrylamide.   45. The method of claim 44, wherein the cross-linking agent is N, N′-bis (acryloyl) cystamine.   45. The method of claim 44, wherein the cross-linking agent is a disulfide linker.   39. The method of claim 38, wherein the hydrogel comprises a copolymer that forms a hydrogel when exposed to light of a first wavelength and forms a solution when exposed to light of a second wavelength. .   40. The method of claim 38, wherein the refractive index of the hydrogel nanocomposite can be changed by changing the concentration of nanoparticles in the hydrogel. Wherein the nanoparticles, nanogels, proteins, silica, metal (e.g., gold, silver, and transition metals) The method of claim 38, TiO _2, is selected from ceramic or combinations thereof.   21. An adjustable intraocular lens produced by in situ gelation of the hydrogel nanocomposite of claim 20.   52. The adjustable intraocular lens according to claim 51, wherein the hydrogel is a reversible hydrogel. Wherein the nanoparticles, nanogels, proteins, silica, gold, silver, TiO _2, transition metals, ceramics or adjustable intraocular lens according to claim 51 which is selected from the group consisting of.   52. The adjustable intraocular lens according to claim 51, wherein the nanoparticles do not disperse or scatter visible light.   52. The adjustable intraocular lens according to claim 51, wherein the nanoparticles have a particle size of less than about 150 nm.   52. The adjustable intraocular lens according to claim 51, wherein the nanoparticles have a particle size of about 3-20 nm.   52. The adjustable intraocular lens according to claim 51, wherein the hydrogel is reversible and comprises a copolymer (hydrogel in the oxidized state and solution in the reduced state).   58. The adjustable intraocular lens according to claim 57, wherein the copolymer is a polymer produced by polymerization of a monomer and a crosslinker or derived to include reversible crosslinks.   The monomer is acrylamide, N-ornithine acrylamide, N- (2-hydroxypropyl) acrylamide, hydroxy-ethyl acrylate, hydroxyethyl methacrylate, polyethylene glycol acrylate, polyethylene glycol methacrylate, N-vinyl pyrrolidone, N-phenyl acrylamide, dimethylamino 59. The adjustable intraocular lens according to claim 58, selected from the group consisting of propyl methacrylamide, acrylic acid, benzyl methacrylamide, and methylthioethyl acrylamide.   The crosslinking agent is N, N′-bis (acryloyl) cystamine, vinyl group, allyl group, cinnamate, acrylate, diacrylate, oligoacrylate, methacrylate, dimethacrylate, oligomethacrylate, methylenebisacrylamide, methylenebismethacrylamide, 59. The ester of claim 58, selected from the group consisting of esters of saturated mono- or polycarboxylic acids and polyols, trimethylolpropane triacrylate, allyl compounds, vinyl compounds, stilbene derivatives, azo derivatives, cinnamoyl derivatives, and combinations thereof. Adjustable intraocular lens.   59. The adjustable intraocular lens according to claim 58, wherein the cross-linking agent is a disulfide linker.   52. The adjustable intraocular lens according to claim 51, wherein the hydrogel can be reduced to produce a solution.   64. The adjustable intraocular lens according to claim 62, wherein the hydrogel can be reduced by the addition of a reducing agent.   63. The adjustable intraocular lens according to claim 62, wherein the solution can be oxidized to regenerate the hydrogel.   65. The adjustable intraocular lens according to claim 64, wherein the solution can be oxidized by atmospheric oxygen, or light and riboflavin.   52. The conditioning of claim 51, wherein the hydrogel comprises a copolymer that produces a hydrogel when exposed to light of a first wavelength and a solution when exposed to light of a second wavelength. Possible intraocular lens.   52. The adjustable intraocular lens according to claim 51, wherein the refractive index can be changed by changing the concentration of nanoparticles in the hydrogel. 52. The adjustable intraocular according to claim 51, wherein the nanoparticles are selected from the group consisting of nanogels, proteins, silica, metals (eg, gold, silver, and transition metals), TiO ₂ , ceramics, or combinations thereof. lens. a) providing a liquid reversible hydrogel;
b) adding nanoparticles to the liquid reversible hydrogel to form a dispersion;
c) introducing the dispersion into the sac; and
c) allowing the dispersion to gel within the sac;
A method of generating a hydrogel in situ in the eye, comprising:   70. The method of claim 69, wherein the hydrogel is a reversible hydrogel. Wherein the nanoparticles, nanogels, proteins, silica, gold, silver, TiO _2, transition metals, ceramics or method according to claim 69 which is selected from the group consisting of.   70. The method of claim 69, wherein the nanoparticles do not disperse or scatter visible light.   70. The method of claim 69, wherein the nanoparticles have a particle size of less than about 150 nm.   70. The method of claim 69, wherein the nanoparticles have a particle size of about 3-20 nm.   70. The method of claim 69, wherein the hydrogel is reversible and comprises a copolymer (a hydrogel in the oxidized state and a solution in the reduced state).   76. The method of claim 75, wherein the copolymer is a polymer produced by polymerization of a monomer and a crosslinker or derivatized to include reversible crosslinking.   The monomer is acrylamide, N-ornithine acrylamide, N- (2-hydroxypropyl) acrylamide, hydroxy-ethyl acrylate, hydroxyethyl methacrylate, polyethylene glycol acrylate, polyethylene glycol methacrylate, N-vinyl pyrrolidone, N-phenyl acrylamide, dimethylamino 77. The method of claim 76, selected from the group consisting of propyl methacrylamide, acrylic acid, benzyl methacrylamide, and methylthioethyl acrylamide.   The crosslinking agent is N, N′-bis (acryloyl) cystamine, vinyl group, allyl group, cinnamate, acrylate, diacrylate, oligoacrylate, methacrylate, dimethacrylate, oligomethacrylate, methylenebisacrylamide, methylenebismethacrylamide, 77. The ester of claim 76 selected from the group consisting of esters of saturated mono- or polycarboxylic acids and polyols, trimethylolpropane triacrylate, allyl compounds, vinyl compounds, stilbene derivatives, azo derivatives, cinnamoyl derivatives, and combinations thereof. Method.   77. The method of claim 76, wherein the cross-linking agent is a disulfide linker.   70. The method of claim 69, wherein the hydrogel can be reduced to produce a solution.   81. The method of claim 80, wherein the hydrogel can be reduced by the addition of a reducing agent.   81. The method of claim 80, wherein the solution can be oxidized to regenerate the hydrogel.   84. The method of claim 82, wherein the solution can be oxidized by atmospheric oxygen, or light and riboflavin.   70. The method of claim 69, wherein the hydrogel comprises a copolymer that forms a hydrogel when exposed to light of a first wavelength and forms a solution when exposed to light of a second wavelength. .   70. The method of claim 69, wherein the refractive index can be changed by changing the concentration of nanoparticles in the hydrogel. Wherein the nanoparticles, nanogels, proteins, silica, metal (e.g., gold, silver, and transition metals) The method of claim 69, TiO _2, is selected from ceramic or combinations thereof. a) preparing a lens from the animal's eye;
b) removing the contents of the lens and leaving the capsule intact;
c) providing a liquid reversible hydrogel;
d) adding nanoparticles to the liquid reversible hydrogel to form a dispersion;
e) introducing the dispersion into the sac; and
d) allowing the dispersion to gel within the sac;
A method for producing an artificial crystalline lens.   90. The method of claim 87, wherein the hydrogel is a reversible hydrogel. Wherein the nanoparticles, nanogels, proteins, silica, gold, silver, TiO _2, transition metals, ceramics or method according to claim 87 which is selected from the group consisting of.   90. The method of claim 87, wherein the nanoparticles do not disperse or scatter visible light.   90. The method of claim 87, wherein the nanoparticles have a particle size less than about 150 nm.   90. The method of claim 87, wherein the nanoparticles have a particle size of about 3-20 nm.   90. The method of claim 87, wherein the hydrogel is reversible and comprises a copolymer (a hydrogel in the oxidized state and a solution in the reduced state).   94. The method of claim 93, wherein the copolymer is a polymer produced by polymerization of a monomer and a crosslinker or derivatized to include reversible crosslinking.   The monomer is acrylamide, N-ornithine acrylamide, N- (2-hydroxypropyl) acrylamide, hydroxy-ethyl acrylate, hydroxyethyl methacrylate, polyethylene glycol acrylate, polyethylene glycol methacrylate, N-vinyl pyrrolidone, N-phenyl acrylamide, dimethylamino 95. The method of claim 94, selected from the group consisting of propyl methacrylamide, acrylic acid, benzyl methacrylamide, and methylthioethyl acrylamide.   The crosslinking agent is N, N′-bis (acryloyl) cystamine, vinyl group, allyl group, cinnamate, acrylate, diacrylate, oligoacrylate, methacrylate, dimethacrylate, oligomethacrylate, methylenebisacrylamide, methylenebismethacrylamide, 95. The ester of claim 94 selected from the group consisting of esters of saturated mono- or polycarboxylic acids and polyols, trimethylolpropane triacrylate, allyl compounds, vinyl compounds, stilbene derivatives, azo derivatives, cinnamoyl derivatives, and combinations thereof. Method.   95. The method of claim 94, wherein the crosslinking agent is a disulfide linker.   90. The method of claim 87, wherein the hydrogel can be reduced to produce a solution.   99. The method of claim 98, wherein the hydrogel can be reduced by the addition of a reducing agent.   99. The method of claim 98, wherein the solution can be oxidized to regenerate the hydrogel.   101. The method of claim 100, wherein the solution can be oxidized by atmospheric oxygen, or light and riboflavin.   88. The method of claim 87, wherein the hydrogel comprises a copolymer that produces a hydrogel when exposed to light of a first wavelength and that produces a solution when exposed to light of a second wavelength. .   88. The method of claim 87, wherein the refractive index can be changed by changing the concentration of nanoparticles in the hydrogel. Wherein the nanoparticles, nanogels, proteins, silica, metal (e.g., gold, silver, and transition metals) The method of claim 87, TiO _2, is selected from ceramic or combinations thereof.

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