Novel targeted lipid prodrugs to improve cellular absorption of acyclovir and topical delivery to the eye via nanomicellar approach

Biotinylated lipid prodrugs of acyclovir (ACV) were designed to target the sodium dependent multivitamin transporter (SMVT) on the intestine and cornea to facilitate ACV cellular transport. All the prodrugs were screened for their uptake across MDCK-MDR1, Caco-2, human corneal epithelial cells (HCEC) and interaction with SMVT on freshly excised rabbit corneas. Further confirmation of interaction of biotinylated lipid prodrugs with SMVT was evaluated by docking analysis. The cytotoxicity of the prodrugs was evaluated on MDCK-MDR1, Caco-2, HCEC and rabbit primary corneal epithelial cells (rPCEC). The enzymatic stability of all the prodrugs was studied in various ocular tissue homogenates. Finally, the in vitro antiviral activity of the prodrugs was studied against HSV-1, HSV-2, HCMV and EBV viruses. Uptake of biotinylated lipid prodrugs (B-R-ACV and B-12HS-ACV) was significantly higher than B-ACV, R-ACV, 12HS-ACV and ACV in all cell lines studied. Transepithelial transport studies across rabbit cornea showed lower [ 3 H] biotin permeability in the presence of biotin and biotinylated prodrugs, indicating recognition of the prodrugs by SMVT on cornea. Average Vina scores obtained from docking studies further confirm that biotinylated lipid prodrugs of ACV possess higher affinity to SMVT over B-ACV. None of the prodrugs exhibited any cytotoxicity on MDCK-MDR1, Caco-2, HCEC and rPCEC iv cells which suggest that these compounds were safe and non-toxic. B-R-ACV and B12HS-ACV were found to be relatively more stable in all ocular tissue homogenates in comparison to non-lipidated but biotinylated prodrug, B-ACV. B-R-ACV and B-12HSACV were found to possess excellent antiviral activity against HSV-1 and HSV-2. Therefore, biotinylated lipid prodrugs of ACV appeared to possess enhanced affinity towards SMVT. Synergistic improvement in cellular uptake is probably due to recognition of the prodrugs by SMVT on the intestine/cornea and lipid mediated transcellular diffusion. These compounds displayed excellent tissue stability and minimal/no cytotoxicity. Also, these novel compounds exhibited excellent antiviral activity against HSV-1, HSV-2 and EBV. Micellar carriers composed of two non-ionic surfactants, Vitamin E TPGS stabilized with octoxynol-40 in a defined ratio were investigated for the delivery of biotinylated lipid prodrug of ACV to the eye. Mixed nanomicelles were characterized by their hydrodynamic diameter, zeta-potential, polydispersity index and surface morphology. Furthermore, in vitro biocompatibility studies such as cytotoxicity assessment, pro-inflammatory gene and cytokine expression analyses were carried out by MTT assay and real time PCR analysis, respectively. The average micelle size was 10.46 nm with a PDI of 0.086 for unloaded micelles, and 10.78 nm with a PDI of 0.075 for prodrug loaded micelles. TEM analysis confirmed that the micelles were spherical and homogenous, and devoid of aggregation. Unloaded and loaded micelles did not differ in terms of morphology. The particle sizes visualized by TEM were very similar to the size obtained by DLS. The formulations did not exhibit any cytotoxic or inflammatory effects on HCEC cells.