Sustained release nanoparticles containing acyclovir prodrugs for ocular herpes simplex keratitis and characterization of folate transport proteins in a corneal epithelial cell line

Abstract

Ocular herpes is a persistent viral infection caused by the herpes simplex virus-1. It is one of the most common infectious diseases causing corneal blindness in the United States. In this study, polymeric nanoparticles of stereoisomeric di-peptide prodrugs of acyclovir (L-valine- L-valine-ACV, L-valine-D-valine-ACV, D-valine-L-valine-ACV, and D-valine-D-valine-ACV) were developed and characterized for the treatment of ocular herpes keratitis. L-valine-Lvaline- ACV and L-valine-D-valine-ACV were determined to be optimum in terms of enzymatic stability, uptake and cytotoxicity. Uptake and docking results indicated that Lvaline in the terminal position increases the affinity of prodrug to the peptide transporter protein. Entrapment efficiency values of L-valine-L-valine-ACV and L-valine-D-valine-ACV were optimal with PLGA 75:25 and PLGA 65:35 polymers, respectively. In vitro release of prodrugs from nanoparticles exhibited a biphasic release pattern with initial burst phase followed by sustained release. Dispersion of nanoparticles in thermosensitive gels eliminated the burst release phase. Novel nanoparticulate systems of dipeptide prodrugs of ACV suspended in thermosensitive gels may provide sustained delivery following topical administration. The uptake of nanoparticles into corneal cells can be enhanced by attachment of cell-specific ligands such as folate and biotin. Folate carrier-mediated system is widely used in the targeted delivery of drugs. Thus Staten's Seruminstitut rabbit corneal (SIRC) epithelial cell line was investigated for the expression of folate transport proteins that can be utilized for targeted drug delivery of folate-conjugated nanoparticles and prodrugs to cornea. Linear increase in [3H] Folic acid uptake was observed over 30min. The process followed saturation kinetics with apparent Km of 14.2 nM, Vmax of 1.5x10-5 micro.moles/min/mg protein and Kd of 2.1x10-6 min.-1 Uptake was inhibited in the presence of structural analogs (cold folic acid, MTF and MTX) but structurally unrelated vitamins did not show any effect. RT-PCR and Western blot analysis confirmed the presence of folate receptor-α (FR-α) and proton-coupled folate transporter (PCFT). This work demonstrated the functional and molecular presence of FR-α and PCFT in SIRC cells that can be utilized for enhanced uptake of folate conjugated nanoparticles and prodrugs. In a different study the differential expression of FR-α, sodium dependent multivitamin transporter (SMVT) and amino acid transporter B (0, +) in retinoblastoma (Y-79) and retinal pigment epithelial (ARPE-19) cells. Higher expression of FR-α, SMVT and B (0, +) at mRNA level was observed in cancerous Y- 79 cells compared to normal ARPE-19 cells.