Biomacromolecules 2005, 6, 204-211
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Refilling of Ocular Lens Capsule with Copolymeric Hydrogel Containing Reversible Disulfide Hyder A. Aliyar,† Paul D. Hamilton,‡ and Nathan Ravi*,†,‡.§ Department of Surgery, Veteran Affairs Medical Center, Saint Louis, Missouri, and Department of Ophthalmology and Visual Sciences and Department of Chemical Engineering, Washington University, Saint Louis, Missouri Received July 24, 2004; Revised Manuscript Received September 30, 2004
Polymer solutions can fill any potential irregular cavity using minimally invasive techniques and thus have potential uses in ophthalmology. We prepared acrylamide hydrogels containing disulfide bonds by free radical polymerization in aqueous ethanol. The hydrogels were liquefied using dithiothreitol to yield watersoluble acrylamide copolymers containing pendant thiol (-SH) groups. The weight average molecular weights of the copolymers ranged from 1.43 × 105 to 9.22 × 105 daltons by GPC. Ellman’s analysis and Raman spectroscopy confirmed the presence of -SH. The aqueous solutions of these purified thiol-containing copolymers were oxidized with 3,3′-dithiodipropionic acid or air to reform the hydrogels. The moduli of the reformed hydrogels ranged from 0.27 to 1.1 kPa depending on concentration and thiol content. Rapid endocapsular gelation yielded optically clear gel within the lens capsular bag. This technique now enables us to validate methods to determine the biomechanics of the lens and its role in accommodation. Introduction Polymer solutions, which can form a gel when placed in a body cavity or tissue with latent space, have potential uses in ophthalmology as intra-ocular lenses,1-5 vitreous substitutes,6,7 and drug delivery devices.8 Such in situ forming gels have the advantage of being easily deliverable by minimally invasive techniques and are capable of filling native or potential cavities by conforming to different shapes, which may be difficult to prefabricate. The mechanism of gelation of the polymer solution, once injected into the body, may be physical (changes in temperature, hydrogen bonding, or hydrophobic interactions) or chemical (ionic or covalent bond formation). Usually, physically cross-linked networks are less mechanically and dimensionally stable than their covalently cross-linked counterparts. In situ gelation resulting in a covalently cross-linked network is most commonly accomplished by free-radical polymerization initiated by free radicals generated by heat, chemical reactions, or photon absorption. For ophthalmic applications, the requirements for an in situ gel-forming system are stringent, including a narrow range of temperatures very close to ambient, optically clear material, very low chemical and phototoxicity, and longterm stability in a wet, oxygen- and photon-rich environment. Our interest in forming in situ gels is aimed at developing new injectable hydrogel materials for ophthalmic applica* To whom correspondence may be addressed. Washington University, Department of Ophthalmology and Visual Sciences, 660 South Euclid Avenue, Box # 8096, Saint Louis, MO 63110. E-mail:
[email protected]. † Department of Ophthalmology and Visual Sciences, Washington University. ‡Veteran Affairs Medical Center. § Department of Chemical Engineering, Washington University.
tions, particularly to validate novel techniques for determining the biomechanics of the lens and its role in accommodation.9-11 Accommodation is a dynamic process by which the refractive power of the lens increases, enabling one to see small objects clearly or to see print that is within arm’s reach (Figure 1). The closest distance that an eye can focus, when fully accommodated, is referred to as the “near point”. Both the speed at which the eye focuses and the near point increase with age; usually around 40 years of age one becomes presbyopic.12-14 Currently, presbyopia is inadequately alleviated by a variety of external prostheses, such as bifocals and monovision contact lenses. The pathophysiology of presbyopia is multifactorial, involving age-related anatomical and physiological changes in the choroid, ciliary body, vitreous, lens capsule, and lens fibers.14-19 Although the exact extent to which each of these tissues contributes to presbyopia is unknown, it is commonly accepted that age-related changes in the lens might play a major role in presbyopia. The human lens increases in size (volume) and modulus with advancing age.20 The volume changes of the lens can be determined with sufficient accuracy both in vivo as well as ex vivo; however, determining the viscoelastic properties of the lens is not straightforward, even ex vivo. Several novel techniques have been attempted, but not validated.21,22 To develop a validated technique, one needs a “phantom” material with mechanical properties that closely encompass those of the lens. The material should be acquiescent enough to be formed into different standard shapes (cylinder, disk, or cubes) as well as into the shape of the lens, preferably within the evacuated lens capsular bag. Refilling the evacuated lens capsular bag through a very small capsular opening (300 nm) microheterogeneity or phase-separation. Cystamine and 2-hydroxyethyl disulfide have also been used for re-gelation, but DTDP is less toxic.34 This technique of endocapsular hydrogel formation, with the present composition, is well suited for use as a validation material to investigate the mechanisms of accommodation and presbyopia.9 Unlike endocapsular polymerization, the present reversible hydrogel system involves only the endocapsular gelation, thus circumventing monomer toxicity and heat of polymerization. As the copolymer was prepared from monomer-free gels and injected at the desired concentration, monomer toxicity and leakage were also avoided. Even though only a minimally toxic disulfide, DTDP, was used for re-gelation, its cytotoxicity with hydrogel material needs to be investigated. The stress-strain (viscoelastic) characteristics (Figure 6) of the lens capsule-hydrogel (after endocapsular gelation) composite were comparable to that of the natural lens as observed by the preliminary experiments conducted using the uniaxial mechanical stretcher. In the current study, acrylamide was used as a monomer to be copolymerized with BAC, but other acrylamides or vinyl monomers can also be used. These options will offer different types of copolymers with pendant thiols, which can be investigated to obtain the appropriate properties for reformed hydrogels. Although much effort has been made to develop biocompatible hydrogels, this reversible hydrogel system has not been investigated for refilling materials through endocapsular gelation. Its refractive index is too low to be of value as an accommodation intra-ocular lens. Appropriate optical modifications have to be made to be of value as an injectable intraocular lenses.5 Our next-generation material has been designed and synthesized and is currently being characterized. Preliminary results of these materials indicate the mechanical property to range from 0.35 to 0.75 kPa, and the refractive index to range from 1.35 to 1.43 similar to that of the human lens.45 Their use as vitreous substitutes have also been recently presented.46 Collectively, these observations indicate that this system merits further research for use as an injectable accommodative intraocular lens, vitreous substitute, and as drug delivery formulations. Conclusion Polymer solutions, which can form a gel when placed in a body cavity or tissue with latent space, have potential uses in ophthalmology as intra-ocular lenses, vitreous substitutes, and drug-delivery devices. Such in situ forming gels have the advantage of being easily delivered using minimally invasive techniques. To summarize the work described in this article, poly(AAm-co-BAC) hydrogels of varying BAC were prepared. The disulfide cross-linked hydrogels were reduced to obtain water-soluble copolymers with pendant thiol (-SH) groups. The copolymers were characterized for their structure, molecular weight, and -SH content. The aqueous solutions of the above water-soluble copolymers were re-gelled through a thiol-disulfide exchange reaction using dithiodipropionic acid. The polymer concentration,
Aliyar et al.
-SH content, and molecular weight play important roles in the re-gelation and mechanical characteristics of the reformed hydrogels. A nonexothermic, monomer (toxic)-free, leakfree in situ endocapsular gelation was demonstrated to form the hydrogel within 5 min at pH 7 in the preevacuated porcine lens capsular bag. To enhance the utilization of these materials as mechanistic probes for the investigation of lenticular presbyopia and for their potential application as endocapsular refilling materials, further investigation is required on their swelling and biological characteristics. Acknowledgment. This research was supported by VA Merit Review Grant to N.R. This work was supported by awards to the Department of Ophthalmology and Visual Sciences from Research to Prevent Blindness, Inc., and the NIH (P30 EY 02687) Core grant. References and Notes (1) Parel, J. M.; Gelender, H.; Trefers, W. F.; Norton, E. W. D. Graefes Arch. Clin. Exp. Ophthalmol. 1986, 224, 165. (2) Blaydes, J. E. DeV. Opthalmol. 1989, 18, 107. (3) Nishi, O.; Nishi, K. Arch. Ophthalmol. 1998, 116, 1358. (4) Hettlich, H. J.; Lucke, K.; Asiyo-Vogel, M. N.; Schulte, M.; Vogel, A. J. Cataract Refract. Surg. 1994, 20, 115. (5) Jacqueline, H. G.; Folkert, J. B.; Henk, J. H.; Keith, A. D.; Kenn, A. H.; Steven, A. K.; Sverker, N. Biomacromolecules 2001, 2, 628. (6) Chirila, T. V.; Tahija, S.; Hong, Y.; Vijayasekaran, S.; Contable, I. J. J. Biomater. Appl. 1994, 9, 121. (7) Hogen-Esch, T. E.; Shah, K. R.; Fitzgerald, C. R. J. Biomed. Mater. Res. 1976, 10, 975. (8) Hatefi, A.; Amsden, B. J. Controlled Release 2002, 80, 9. (9) Murthy, K. S.; Ravi, N. Curr. Eye. Res. 2001, 22, 384. (10) Ravi, N.; Mitra, A.; Zhang, L.; Szabo, B. The effect of hydrophobicity on the viscoelastic creep characteristics of poly(ethylene glycol)acrylate hydrogels. In Polymer gels: Fundamental and applications; Bohidar, H. B., Dublin, P., Osada, Y., Ed.; ACS symposium series 833; American Chemical Society: Washington, DC, 2002; p 233. (11) Murthy, K. S.; Ravi, N. Polym. Prepr (ACS DiV. Polym.Chem.) 1999, 40, 630. (12) Glasser, A.; Campbell, M. C. W. Vis. Res. 1999, 39, 1991. (13) Beers, A. P.; Van der Heijde, G. L. Optom. Vis. Sci. 1996, 73, 235. (14) Weale, R. Vis. Res. 1999, 39, 1263. (15) Koretz, J. F.; Cook, C. A.; Kaufman, P. L. InVest. Ophthalmol. Vis. Sci. 1997, 38, 569. (16) Fransworth, P. N.; Shyne, S. E. Exp. Eye. Res. 1979, 28, 291. (17) Koretz, J. F.; Handelman, G. H. Math. Model. 1986, 38, 209. (18) Glasser, A.; Campbell, M. C. W. Vis. Res. 1998, 38, 209. (19) Koretz, J. F.; Kaufman, P. L.; Neider, M. W.; Goeckner, P. A. Vis. Res. 1989, 29, 1685. (20) Al-Ghoul, K. J.; Nordgren, R. K.; Kuszak, A. J.; Freel, C. D.; Costello, M. J.; Kuszak, J. R. Exp. Eye. Res. 2001, 72, 199. (21) Fisher, R. F. J. Physiol. (London) 1973, 228, 765. (22) van Alphen, G. W.; Graebel, W. P. Vis. Res. 1991, 31, 1417. (23) Levin, M. L.; Wyatt, K. D. J. Cataract Refract. Surg. 1990, 16, 96. (24) Mackool, R. J. J. Cataract Refract. Surg. 1991, 17, 221. (25) Okihiro, N.; Kayo, N. J. Cataract Refract. Surg. 1990, 16, 757. (26) Tsutomu, H.; Takako, H. J. Cataract Refract. Surg. 1987, 13, 441. (27) Koopmans, S. A.; Terwee, T.; Barkhof, J.; Haitjema, H. J.; Kooijman, A. C. InVest. Ophthalmol. Vis. Sci. 2003, 44, 250. (28) Kessler, J. Arch. Ophthalmol. 1964, 71, 412. (29) Gindi, J. J.; Wan, W. L.; Schanzlin, D. J. Cataract 1985, 2, 6. (30) Chirila, T. V. J. Cataract Refract. Surg. 1994, 20, 675. (31) Jacqueline, H. G.; Coenraad, J. S.; Ralph, V. C.; Folkert, J. B.; Sverker, N.; Albert, J. P. Biomacromolecules 2003, 4, 608. (32) Hansen, J. N. Anal. Biochem. 1980, 76, 37. (33) Lee, H.; Park, T. G. Polym. J. 1998, 30, 976. (34) Hisano, N.; Morikawa, N.; Iwata, H.; Ikada, Y. J. Biomed. Mater. Res. 1998, 40, 115. (35) Ellman, G. L. Arch. Biochem. Biophys. 1959, 82, 70. (36) Michail, H.; Hamilton, P. D.; Ravi, N. In The Meeting of the Association for Research in Vision and Ophthalmology, Abstract # 250, Fort Lauderdale, FL, 2003.
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