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  • 1.
    Ahn, Jae-Il
    et al.
    Ottawa Hospital, Canada .
    Kuffova, Lucia
    University of Aberdeen, Scotland .
    Merrett, Kimberley
    Ottawa Hospital, Canada .
    Mitra, Debbie
    Ottawa Hospital, Canada .
    Forrester, John V.
    University of Aberdeen, Scotland .
    Li, Fengfu
    Ottawa Hospital, Canada .
    Griffith, May
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Health Sciences. Ottawa Hospital, Canada .
    Crosslinked collagen hydrogels as corneal implants: Effects of sterically bulky vs. non-bulky carbodiimides as crosslinkers2013In: Acta Biomaterialia, ISSN 1742-7061, E-ISSN 1878-7568, Vol. 9, no 8, p. 7796-7805Article in journal (Refereed)
    Abstract [en]

    We have previously shown that recombinant human collagen can be crosslinked with N-(3-dimethylaminopropyl)-N-ethylcarbodiimide (EDC) to fabricate transparent hydrogels possessing the shape and dimensions of the human cornea. These corneal implants have been tested in a Phase I human clinical study. Although these hydrogels successfully promoted corneal tissue and nerve regeneration, the gelling kinetics were difficult to control during the manufacture of the implants. An alternative carbodiimide capable of producing hydrogels of similar characteristics as EDC in terms of strength and biocompatibility, but with a longer gelation time would be a desirable alternative. Here, we compared the crosslinking kinetics and properties of hydrogels crosslinked with a sterically bulky carbodiimide, N-Cyclohexyl-N-(2-morpholinoethyl) carbodiimide metho-p-toluenesulfonate (CMC), with that of EDC. CMC crosslinking was possible at ambient temperature whereas the EDC reaction was too rapid to control and had to be carried out at low temperatures. The highest tensile strength obtained using optimized formulations were equivalent, although CMC crosslinked hydrogels were found to be stiffer. The collagenase resistance of CMC crosslinked hydrogels was superior to that of EDC crosslinked hydrogels while biocompatibility was similar. We are also able to substitute porcine collagen with recombinant human collagen and show that the in vivo performance of both resulting hydrogels as full-thickness corneal implants is comparable in a mouse model of an orthotopic corneal graft. In conclusion, CMC is a viable alternative to EDC as a crosslinker for collagen-based biomaterials for use as corneal implants, and potentially for use in other tissue engineering applications.

  • 2.
    Guex, Anne Geraldine
    et al.
    Imperial Coll London, England.
    Puetzer, Jennifer L.
    Imperial Coll London, England.
    Armgarth, Astrid
    Imperial Coll London, England.
    Littmann, Elena
    Imperial Coll London, England.
    Stavrinidou, Eleni
    Linköping University, Department of Science and Technology, Physics and Electronics. Linköping University, Faculty of Science & Engineering. Imperial Coll London, England.
    Giannelis, Emmanuel P.
    Cornell University, NY 14853 USA.
    Malliaras, George G.
    Ecole National Super Mines, France.
    Stevens, Molly M.
    Imperial Coll London, England.
    Highly porous scaffolds of PEDOT:PSS for bone tissue engineering2017In: Acta Biomaterialia, ISSN 1742-7061, E-ISSN 1878-7568, Vol. 62, p. 91-101Article in journal (Refereed)
    Abstract [en]

    Conjugated polymers have been increasingly considered for the design of conductive materials in the field of regenerative medicine. However, optimal scaffold properties addressing the complexity of the desired tissue still need to be developed. The focus of this study lies in the development and evaluation of a conductive scaffold for bone tissue engineering. In this study PEDOT:PSS scaffolds were designed and evaluated in vitro using MC3T3-E1 osteogenic precursor cells, and the cells were assessed for distinct differentiation stages and the expression of an osteogenic phenotype. Ice-templated PEDOT:PSS scaffolds presented high pore interconnectivity with a median pore diameter of 53.6 +/- 5.9 mu m and a total pore surface area of 7.72 +/- 1.7 m(2).g(-1). The electrical conductivity, based on I-V curves, was measured to be 140 mu S.cm(-1) with a reduced, but stable conductivity of 6.1 mu S.cm(-1) after 28 days in cell culture media. MC3T3-E1 gene expression levels of ALPL, COL1A1 and RUNX2 were significantly enhanced after 4 weeks, in line with increased extracellular matrix mineralisation, and osteocalcin deposition. These results demonstrate that a porous material, based purely on PEDOT:PSS, is suitable as a scaffold for bone tissue engineering and thus represents a promising candidate for regenerative medicine. Statement of Significance Tissue engineering approaches have been increasingly considered for the repair of non-union fractions, craniofacial reconstruction or large bone defect replacements. The design of complex biomaterials and successful engineering of 3-dimensional tissue constructs is of paramount importance to meet this clinical need. Conductive scaffolds, based on conjugated polymers, present interesting candidates to address the piezoelectric properties of bone tissue and to induce enhanced osteogenesis upon implantation. However, conductive scaffolds have not been investigated in vitro in great measure. To this end, we have developed a highly porous, electrically conductive scaffold based on PEDOT:PSS, and provide evidence that this purely synthetic material is a promising candidate for bone tissue engineering. (C) 2017 Acta Materialia Inc. Published by Elsevier Ltd.

  • 3.
    Hayes, Sally
    et al.
    Cardiff University, Wales; Cardiff University, Wales.
    Lewis, Phillip
    Cardiff University, Wales; Cardiff University, Wales.
    Islam, Mohammad Mirazul
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Medicine and Health Sciences. Karolinska Institute, Sweden.
    Doutch, James
    Diamond Light Source, England.
    Sorensen, Thomas
    Diamond Light Source, England.
    White, Tomas
    Cardiff University, Wales; Cardiff University, Wales.
    Griffith, May
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Medicine and Health Sciences. Karolinska Institute, Sweden.
    Meek, Keith M.
    Cardiff University, Wales; Cardiff University, Wales.
    The structural and optical properties of type III human collagen biosynthetic corneal substitutes2015In: Acta Biomaterialia, ISSN 1742-7061, E-ISSN 1878-7568, Vol. 25, p. 121-130Article in journal (Refereed)
    Abstract [en]

    The structural and optical properties of clinically biocompatible, cell-free hydrogels comprised of synthetically cross-linked and moulded recombinant human collagen type III (RHCIII) with and without the incorporation of 2-methacryloyloxyethyl phosphorylcholine (MPC) were assessed using transmission electron microscopy (TEM), X-ray scattering, spectroscopy and refractometry. These findings were examined alongside similarly obtained data from 21 human donor corneas. TEM demonstrated the presence of loosely bundled aggregates of fine collagen filaments within both RHCIII and RHCIII-MPC implants, which X-ray scattering showed to lack D-banding and be preferentially aligned in a uniaxial orientation throughout. This arrangement differs from the predominantly biaxial alignment of collagen fibrils that exists in the human cornea. By virtue of their high water content (90%), very fine collagen filaments (2-9 nm) and lack of cells, the collagen hydrogels were found to transmit almost all incident light in the visible spectrum. They also transmitted a large proportion of UV light compared to the cornea which acts as an effective UV filter. Patients implanted with these hydrogels should be cautious about UV exposure prior to regrowth of the epithelium and in-growth of corneal cells into the implants. (C) 2015 Acta Materialia Inc. Published by Elsevier Ltd.

  • 4.
    Islam, Mohammad M.
    et al.
    Swedish Medical Nanoscience Center, Karolinska Institutet, Stockholm, Sweden.
    Cėpla, Vytautas
    Center for Physical Sciences and Technology, Vilnius, Lithuania.
    He, Chaoliang
    Ottawa Hospital Research Institute, Ontario, Canada.
    Edin, Joel
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Health Sciences.
    Rakickas, Tomas
    Center for Physical Sciences and Technology, Vilnius, Lithuania.
    Kobuch, Karin
    Technische Universität München, Germany.
    Ruželė, Živilė
    Center for Physical Sciences and Technology, Vilnius, Lithuania.
    Jackson, Bruce W.
    Ottawa Hospital Research Institute, Ontario, Canada.
    Rafat, Mehrdad
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Department of Biomedical Engineering. Linköping University, Faculty of Health Sciences. Ottawa Hospital Research Institute, Ontario, Canada.
    Lohmann, Chris P.
    Technische Universität München, Germany.
    Valiokas, Ramūnas
    Center for Physical Sciences and Technology, Vilnius, Lithuania.
    Griffith, May
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Health Sciences. Swedish Medical Nanoscience Center, Karolinska Institutet, Stockholm, Sweden; Ottawa Hospital Research Institute, Ontario, Canada.
    Functional fabrication of recombinant human collagen–phosphorylcholine hydrogels for regenerative medicine applications2015In: Acta Biomaterialia, ISSN 1742-7061, E-ISSN 1878-7568, Vol. 12, p. 70-80Article in journal (Refereed)
    Abstract [en]

    The implant-host interface is a critical element in guiding tissue or organ regeneration. We previously developed hydrogels comprising interpenetrating networks of recombinant human collagen type III and 2-methacryloyloxyethyl phosphorylcholine (RHCIII-MPC) as substitutes of the corneal extracellular matrix that promote endogenous regeneration of corneal tissue. To render them functional for clinical application, we have now optimized their composition and thereby enhanced their mechanical properties. We have demonstrated that such optimized RHCIII-MPC hydrogels are suitable for precision femtosecond laser cutting to produce complementing implants and host surgical beds for subsequent tissue welding. This avoids the tissue damage and inflammation associated with manual surgical techniques, thereby leading to more efficient healing. Although we previously demonstrated in clinical testing that RHCIII-based implants stimulated cornea regeneration in patients, the rate of epithelial cell coverage of the implants needs improvement, e.g. modification of the implant surface. We now show that our 500 μm thick RHCIII-MPC constructs comprising over 85% water, are suitable for microcontact printing with fibronectin. The resulting fibronectin micropatterns promote cell adhesion, as compared to the bare RHCIII-MPC hydrogel. Interestingly, a pattern of 30 μm wide fibronectin stripes enhanced cell attachment and showed highest mitotic rates, an effect that potentially can be utilized for faster integration of the implant. We have therefore shown that laboratory-produced mimics of naturally occurring collagen and phospholipids can be fabricated into robust hydrogels that can be laser profiled and patterned to enhance their potential function as artificial substitutes of donor human corneas.

  • 5.
    Jangamreddy, Jaganmohan
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Medicine and Health Sciences. LV Prasad Eye Inst, India.
    Haagdorens, Michel K. C.
    Antwerp Univ Hosp, Belgium; Univ Antwerp, Belgium.
    Mirazul Islam, Mohammad Mirazul
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Medicine and Health Sciences.
    Lewis, Philip
    Cardiff Univ, Wales.
    Samanta, Ayan
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Medicine and Health Sciences.
    Fagerholm, Per
    Linköping University, Department of Clinical and Experimental Medicine, Division of Neuro and Inflammation Science. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Anaesthetics, Operations and Specialty Surgery Center, Department of Ophthalmology in Linköping.
    Liszka, Aneta
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Medicine and Health Sciences.
    Kozak Ljunggren, Monika
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Medicine and Health Sciences.
    Buznyk, Oleksiy
    Linköping University, Department of Clinical and Experimental Medicine, Division of Neuro and Inflammation Science. Linköping University, Faculty of Medicine and Health Sciences.
    Alarcon, Emilio I.
    Univ Ottawa, Canada.
    Zakaria, Nadia
    Antwerp Univ Hosp, Belgium; Univ Antwerp, Belgium.
    Meek, Keith M.
    Cardiff Univ, Wales.
    Griffith, May
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Medicine and Health Sciences. Univ Montreal, Canada; Univ Montreal, Canada.
    Short peptide analogs as alternatives to collagen in pro-regenerative corneal implants2018In: Acta Biomaterialia, ISSN 1742-7061, E-ISSN 1878-7568, Vol. 69, p. 120-130Article in journal (Refereed)
    Abstract [en]

    Short collagen-like peptides (CLPs) are being proposed as alternatives to full-length collagen for use in tissue engineering, on their own as soft hydrogels, or conjugated to synthetic polymer for mechanical strength. However, despite intended clinical use, little is known about their safety and efficacy, mechanism of action or degree of similarity to the full-length counterparts they mimic. Here, we show the functional equivalence of a CLP conjugated to polyethylene glycol (CLP-PEG) to full-length recombinant human collagen in vitro and in promoting stable regeneration of corneal tissue and nerves in a pre- clinicalmini-pig model. We also show that these peptide analogs exerted their pro-regeneration effects through stimulating extracellular vesicle production by host cells. Our results support future use of CLP-PEG implants for corneal regeneration, suggesting the feasibility of these or similar peptide analogs in clinical application in the eye and other tissues. Statement of significance Although biomaterials comprising full-length recombinant human collagen and extracted animal collagen have been evaluated and used clinically, these macromolecules provide only a limited number of functional groups amenable to chemical modification or crosslinking and are demanding to process. Synthetic, customizable analogs that are functionally equivalent, and can be readily scaled-up are therefore very desirable for pre-clinical to clinical translation. Here, we demonstrate, using cornea regeneration as our test bed, that collagen-like-peptides conjugated to multifunctional polyethylene glycol (CLP-PEG) when grafted into mini-pigs as corneal implants were functionally equivalent to recombinant human collagen-based implants that were successfully tested in patients. We also show for the first time that these materials affected regeneration through stimulation of extracellular vesicle production by endogenous host cells that have migrated into the CLP-PEG scaffolds. (C) 2018 Acta Materialia Inc. Published by Elsevier Ltd.

  • 6.
    Lademann, J.
    et al.
    Charite University of Medical Berlin, Germany.
    Richter, H.
    Charite University of Medical Berlin, Germany.
    Knorr, F.
    Charite University of Medical Berlin, Germany.
    Patzelt, A.
    Charite University of Medical Berlin, Germany.
    Darvin, M. E.
    Charite University of Medical Berlin, Germany.
    Ruehl, E.
    Free University of Berlin, Germany.
    Cheung, Kwan Yee
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Medicine and Health Sciences.
    Lai, K. K.
    Hong Kong University of Science and Technology, Peoples R China.
    Renneberg, R.
    Hong Kong University of Science and Technology, Peoples R China.
    Mak, Wing Cheung
    Linköping University, Department of Physics, Chemistry and Biology, Biosensors and Bioelectronics. Linköping University, Faculty of Science & Engineering. Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Medicine and Health Sciences.
    Triggered release of model drug from AuNP-doped BSA nanocarriers in hair follicles using IRA radiation2016In: Acta Biomaterialia, ISSN 1742-7061, E-ISSN 1878-7568, Vol. 30, p. 388-396Article in journal (Refereed)
    Abstract [en]

    Recent advances in the field of dermatotherapy have resulted in research efforts focusing on the use of particle-based drug delivery systems for the stimuli-responsive release of drugs in the skin and skin appendages, i.e. hair follicles and sebaceous glands. However, effective and innocuous trigger mechanisms which result in the release of the drugs from the nanocarriers upon reaching the target structures are still lacking. For the first time, the present study demonstrated the photo-activated release of the model drug fluorescein isothiocyanate (FITC) from topically applied gold nanoparticle-doped bovine serum albumin (AuNPs-doped BSA) particles (approx. 545 nm) using water-filtered infrared A (IRA) radiation in the hair follicles of an ex vivo porcine skin model. The IRA radiation-induced plasmonic heating of the AuNPs results in the partial decomposition or opening of the albumin particles and release the model drug, while control particles without AuNPs show insignificant release. The results demonstrate the feasibility of using IRA radiation to induce release of encapsulated drugs from plasmonic nanocarriers for the targeting of follicular structures. However, the risk of radiation-induced skin damage subsequent to repeated applications of high infrared dosages may be significant. Future studies should aim at determining the suitability of lower infrared A dosages, such as for medical treatment regimens which may necessitate repeated exposure to therapeutics. Statement of significance Follicular targeting using nanocarriers is of increasing importance in the prophylaxis and treatment of dermatological or other diseases. For the first time, the present study demonstrated the photo activated release of the model drug fluorescein isothiocyanate (FITC) from topically applied gold nanoparticle-doped bovine serum albumin (AuNPs-doped BSA) particles using water-filtered infrared A (IRA) radiation in the hair follicles of an ex vivo porcine skin model. The results demonstrate the feasibility of using wIRA radiation to induce release of encapsulated drugs for the targeting of follicular structures, and provide a new vision on the development of optically addressable delivery systems for controlled release of drugs in the skin and skin appendages, i.e. hair follicles and sebaceous glands. (C) 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  • 7.
    Qu, Jing
    et al.
    University of Delaware, DE 19716 USA.
    Ouyang, Liangqi
    Linköping University, Department of Physics, Chemistry and Biology, Biomolecular and Organic Electronics. Linköping University, Faculty of Science & Engineering. University of Delaware, DE 19716 USA.
    Kuo, Chin-chen
    University of Delaware, DE 19716 USA.
    Martin, David C.
    University of Delaware, DE 19716 USA.
    Stiffness, strength and adhesion characterization of electrochemically deposited conjugated polymer films2016In: Acta Biomaterialia, ISSN 1742-7061, E-ISSN 1878-7568, Vol. 31, p. 114-121Article in journal (Refereed)
    Abstract [en]

    Conjugated polymers such as poly(3,4-ethylenedioxythiphene) (PEDOT) are of interest for a variety of applications including interfaces between electronic biomedical devices and living tissue. The mechanical properties, strength, and adhesion of these materials to solid substrates are all vital for long-term applications. We have been developing methods to quantify the mechanical properties of conjugated polymer thin films. In this paper, the stiffness, strength and the interfacial shear strength (adhesion) of electrochemically deposited PEDOT and PEDOT-co-1,3,5-tri[2-(3,4-ethylene dioxythienyl)]-benzene (EPh) were studied. The estimated Youngs modulus of the PEDOT films was 2.6 +/- 1.4 GPa, and the strain to failure was around 2%. The tensile strength was measured to be 56 +/- 27 MPa. The effective interfacial shear strength was estimated with a shear-lag model by measuring the crack spacing as a function of film thickness. For PEDOT on gold/palladium-coated hydrocarbon film substrates an interfacial shear strength of 0.7 +/- 0.3 MPa was determined. The addition of 5 mole% of a tri-functional EDOT crosslinker (EPh) increased the tensile strength of the films to 283 +/- 67 MPa, while the strain to failure remained about the same (2%). The effective interfacial shear strength was increased to 2.4 +/- 0.6 MPa. Statement of significance This paper describes methods for estimating the ultimate mechanical properties of electrochemically deposited conjugated polymer (here PEDOT and PEDOT copolymers) films. Of particular interest and novelty is our implementation of a cracking test to quantify the shear strength of the PEDOT thin films on these solid substrates. There is considerable interest in these materials as interfaces between biomedical devices and living tissue, however potential mechanisms and modes of failure are areas of continuing concern, and establishing methods to quantify the strengths of these interfaces are therefore of particular current interest. We are confident that these results will be useful to the broader biological materials community and are worthy of broader dissemination. (C) 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  • 8.
    Rajendran, Vijayalakshmi
    et al.
    University of Aberdeen, Scotland.
    Netukova, Magdalena
    Charles University of Prague, Czech Republic.
    Griffith, May
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Medicine and Health Sciences. University of Montreal, Canada.
    Forrester, John V.
    University of Aberdeen, Scotland; University of Western Australia, Australia; Lions Eye Institute, Australia.
    Kuffova, Lucia
    University of Aberdeen, Scotland.
    Mesenchymal stem cell therapy for retro-corneal membrane - A clinical challenge in full-thickness transplantation of biosynthetic corneal equivalents2017In: Acta Biomaterialia, ISSN 1742-7061, E-ISSN 1878-7568, Vol. 64, p. 346-356Article in journal (Refereed)
    Abstract [en]

    Artificial corneas (keratoprostheses) and biosynthetic collagen-based corneal equivalents are surgical implants designed to ease the global burden of corneal blindness. However, keratoprostheses in many cases fail due to development of fibrous retro-corneal membranes (RCM). Fibrous membranes which develop in the anterior chamber after prosthesis implantation do so on a matrix of fibrin. This study investigated fibrin deposition and RCM formation after full-thickness collagen-based hydrogel implants and compared them with syngeneic and allogeneic corneal grafts in mice. Fibrin cleared from the anterior chamber within 14 days in both allo- and syn-grafts but, persisted in hydrogel implants and developed into dense retro-corneal membrane (RCM) which were heavily infiltrated by activated myofibroblasts. In contrast, the number of CD11 b(+) macrophages infiltrating the initial deposition of fibrin in the anterior chamber (AC) after hydrogel implantation was markedly reduced compared to syn- and allo-grafts. Inoculation of mesenchymal stem cells prior to collagen gel implant promoted clearance of gel associated fibrin from the anterior chamber. We propose that a failure of macrophage-mediated clearance of fibrin may be the cause of RCM formation after collagen-based hydrogel implants and that mesenchymal stem cell therapy promotes clearance of fibrin and prevents RCM formation. Statement of Significance The manuscript addresses the potential value of bone marrow-derived mesenchymal stem cell therapy for retro-corneal membrane (RCM) formation in full-thickness transplantation of biosynthetic corneal equivalents. This work reports the pathophysiological changes in the anterior chamber of the mouse eye following full-thickness recombinant human cross-linked collagen-based hydrogel implants in which persistent fibrin promotes the development of dense RCM. Furthermore, pre-treatment with mesenchymal stem cells reduces RCM formation and enhances corneal transparency. (C) 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  • 9.
    Wassmer, Sarah
    et al.
    Ottawa Hospital Research Institute, Vision Sciences Program, Ottawa, Canada.
    Rafat, Mehrdad
    Ottawa Hospital Research Institute, Vision Sciences Program, Ottawa, ON, Canada; University of Ottawa, Department of Cellular and Molecular Medicine, Ottawa, ON, Canada.
    Fong, Wai Gin
    Ottawa Hospital Research Institute, Vision Sciences Program, Ottawa, Canada.
    Baker, Adam N.
    Ottawa Hospital Research Institute, Vision Sciences Program, Ottawa, Canada.
    Tsilfidis, Catherine
    Ottawa Hospital Research Institute, Vision Sciences Program, Ottawa, Canada; University of Ottawa, Department of Cellular and Molecular Medicine, Ottawa, ON, Canada; University of Ottawa, Department of Ophthalmology, Ottawa, ON, Canada.
    Chitosan microparticles for delivery of proteins to the retina2013In: Acta Biomaterialia, ISSN 1742-7061, E-ISSN 1878-7568, Vol. 9, no 8, p. 7855-7864Article in journal (Refereed)
    Abstract [en]

    Chitosan microparticles (CMPs) have previously been developed for topical applications to the eye, but their safety and efficacy in delivering proteins to the retina have not been adequately evaluated. This study examines the release kinetics of CMPs in vitro, and assesses their biocompatibility and cytotoxicity on retinal cells in vitro and in vivo. Two proteins were used in the encapsulation and release studies: BSA (bovine serum albumin) and tat-EGFP (enhanced green fluorescent protein fused to the transactivator of transcription peptide). Not surprisingly, the in vitro release kinetics were dependent on the protein encapsulated, with BSA showing higher release than tat-EGFP. CMPs containing encapsulated tat-EGFP were tested for cellular toxicity in photoreceptor-derived 661W cells. They showed no signs of in vitro cell toxicity at a low concentration (up to 1 mg ml 1), but at a higher concentration of 10 mg ml1 they were associated with cytotoxic effects. In vivo, CMPs injected into the subretinal space were found beneath the photoreceptor layer of the retina, and persisted for at least 8 weeks. Similar to the in vitro studies, the lower concentration of CMPs was generally well tolerated, but the higher concentration resulted in cytotoxic effects and in reduced retinal function, as assessed by electroretinogram amplitudes. Overall, this study suggests that CMPs are effective long-term delivery agents to the retina, but the concentration of chitosan may affect cytotoxicity.

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