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[FP-SA-39] Corneal Transplantation: Material and Techniques
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Apr 05 (Sat)
15:30 - 17:00
Room 12 - Tokyo International Forum 5F G510
External Eye Disease, Cornea, Eye Banking
Chair)Rafael Barraquer、Chair)Petja Vassileva、Chair)Junko Hori


Duration 5min, Q&A 3min

Biosynthetic Corneal Substitutes with an Anti-Infective Peptide Release System

Oleksiy Buznyk
Oleksiy Buznyk Nataliya Pasyechnikova Stanislav Iakymenko Mohammad Islam May Griffith

To develop corneal substitutes (CS) that will serve a dual purpose of: 1) promoting corneal regeneration, and 2) delivering anti-infective peptides (AIPs).

 Nano and microparticles fabrication: tetraethylorthosilicate, Triton, acetic acid and cyclohexane for silica nanoparticles; chitosan and tripolyphosphate for chitosan microparticles; sodium alginate and calcium chloride for alginate microparticles.
 Corneal substitutes fabrication: type I porcine atelocollagen, poly(ethylene glycol) diacrylate, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, ammonium persulfate, N,N,N,N-tetramethyl ethylene diamine, N-hydroxyl-succinimide, 2-methacryloyloxyethyl phosphorylcholine and MES.
 ELISA: antigen coating buffer, LL37 mouse monoclonal antibody (D-5), goat anti-mouse HRP conjugated secondary antibody and tetramethyl-benzidine liquid substrate.
Cell proliferation assay: human corneal epithelial cells (HCEC), WST-1 reagent and keratinocyte serum free medium (KSFM) with supplements.
Anti-viral activity of released peptide: kidney epithelial cells from African green monkey (Vero), type I herpes simplex virus laboratory strain F, KSFM and Dulbecco's modified Eagle medium, anti-HSV-1,2 (biotin) rabbit polyclonal antibody, 4,6-diamidino-2-phenylindole (DAPI) and Alexa Fluor 647 IgG (mouse anti-biotin secondary antibody).

AIP LL37 (cathelicidin) was encapsulated within a range of different particles: chitosan, alginate and silica particles. Particle encapsulated AIP LL37 were then introduced in CS during their fabrication. CS were fabricated from interpenetrating networks of type I collagen and 2-methacryloyloxy-ethylphosphorylcholine (coll-MPC). Efficacy of encapsulation and LL37 release from particles within CS were assessed using ELISA. Particle morphology was studied by transmission electron and light microscopy. Cytotoxicity of LL37-releasing implants was assessed using WST-1 based colorimetric assay. The anti-viral properties of CS were tested against type 1 herpes simplex virus (HSV-1) using plaque formation assay and immunofluorescent analysis.

Results and Conclusion
Encapsulation efficacy of LL37 was best within silica nanoparticles (SiNPs) of 54.4%. The resulting SiNPs were 10-20 nm in size. Sustained release of SiNP encapsulated LL37 from within CS occurred up to day 21. Release from control CS (with incorporated free LL37) occurred as a burst on day 1. WST-1 colorimetric assay showed no increased cytotoxicity of CS with incorporated release system compared to clear CS. CS with SiNP encapsulated LL37 showed good anti-viral activity both when put on HSV-1 infected HCEC and when the implant was put on HCEC before adding the virus.
We have shown that CS providing sustained release of AIP can be fabricated. The released LL37 in this case was non-cytotoxic but were able to block HSV-1 activity. In the future, such composite implants may be useful in corneal transplantation in HSV-infected patients.

[ Keyword ]
corneal substitute / LL37 / herpes simplex / keratitis / leukoma

[ Conflict of Interest ]

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