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Laura Contreras-Ruiz, Giovanni K. Zorzi, Jenny E. Párraga, Karyn F. Siemasko, Denis Hileeto, Margarita Calonge, Michael E. Stern, Begoña Seijo, Alejandro Sánchez, Yolanda Diebold; In Vitro and In Vivo Tolerance and Transfection Efficiency in Ocular Surface Structures of New Nanoparticles Loaded with a MUC5AC Coding Plasmid. Invest. Ophthalmol. Vis. Sci. 2011;52(14):420.
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To evaluate the in vitro and in vivo tolerance and transfection efficiency in ocular surface structures of a new nanoparticle system loaded with a plasmid coding for a modified MUC5AC protein (pMUC5AC).
Cationized gelatin and chrondroitin sulfate-based nanoparticles (NPs) were prepared by ionotropic gelation and loaded with pMUC5AC (P201031678). Epithelial cell lines from human cornea and conjunctiva were exposed to NPs for 3h. Toxicity and MUC5AC expression were evaluated after 72h by XTT, ELISA and real time RT-PCR (RT2-PCR). In vivo tolerance and transfection efficiency was studied in C57BL/6 mice. 5µl of naked plasmid, blank NPs or pMUC5AC-NPs were instilled in both eyes 4 times/day for 3 or 5 days. Control animals received no instillations. Clinical signs, corneal fluorescein staining and tear production were evaluated before and after treatment. MUC5AC expression was quantified by RT2-PCR. Ocular structures were processed for pathology study. Two-way ANOVA was done for statistical analysis.
There was no significant difference in cell viability between control and NP-exposed cells. Expression of modified MUC5AC was detected in both cell lines exposed to pMUC5AC-NPs. Neither ocular discomfort nor irritation was observed in vivo after pMUC5AC-NP treatment. pMUC5AC-NPs had no significant effect on fluorescein staining or tear production. After treatment, MUC5AC expression from the plasmid was detected in conjunctiva, but not in cornea. Ocular surface of treated mice showed normal morphology with a complete lack of inflammatory changes.
pMUC5AC-NPs were well tolerated, and induced the expression of modified MUC5AC in ocular surface structures, both in vitro and in vivo. This work is a proof-of-concept of the potential therapeutic application of a newly developed nanomedicine treatment modality.
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