Optimization, stabilization, and characterization of amphotericin B loaded nanostructured lipid carriers for ocular drug delivery

The current study sought to formulate, optimize, and stabilize amphotericin B (AmB) loaded PEGylated nanostructured lipid carriers (NLC) and to study its ocular biodistribution following topical instillation. AmB loaded PEGylated NLC (AmB-PEG-NLC) were fabricated by hot-melt emulsification followed by high-pressure homogenization (HPH) technique. 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)] (mPEG-2K-DSPE) was used for surface PEGylation. mPEG-DSPE with different PEG molecular weight, 1 K, 2 K, 5 K, 10 K, and 20 K, were screened for formulation stability. Furthermore, the AmB loaded PEGylated (2K) NLC (AmB-PEG2K-NLC) was optimized using Box-Behnken design with respect to the amount of AmB, castor oil, mPEG-2K-DSPE, and number of high-pressure homogenization cycles as the factors; particle size, zeta potential, PDI, entrapment efficiency, and loading efficiency as responses. Stability of the optimized AmB-PEG2K-NLC was assessed over 4 weeks, at 4 °C as well as 25 °C and effect of autoclaving was also evaluated. AmB-PEG2K-NLC were tested for their in vitro antifungal activity against Candida albicans (ATCC 90028), AmB resistant Candida albicans (ATCC 200955) and Aspergillus fumigatus (ATCC 204305). Cytotoxicity of AmB-PEG2K-NLC was studied in human retinal pigmented epithelium cells. In vivo ocular biodistribution of AmB was evaluated in rabbits, following topical application of PEGylated NLCs or marketed AmB preparations. PEGylation with mPEG-2K-DSPE prevented leaching of AmB and increased the drug load significantly. The optimized formulation was prepared with a particle size of 218 ± 5 nm; 0.3 ± 0.02 PDI, 4.6 ± 0.1% w/w drug loading, and 92.7 ± 2.5% w/w entrapment efficiency. The optimized colloidal dispersions were stable for over a month, at both 4 °C and 25 °C. AmB-PEG2K-NLCs showed significantly (p < 0.05) better antifungal activity in both wild-type and AmB resistant Candida strains and, was comparable to, or better than, commercially available parenteral AmB formulations like Fungizone™ and AmBisome®. AmB-PEG2K-NLC did not show any toxicity up to a highest concentration of 1% (v/v) (percent formulation in medium). Following topical instillation, AmB was detected in all the ocular tissues tested and statistically significant (p > 0.05) difference was not observed between the formulations tested. An optimized autoclavable and effective AmB-PEG2K-NLC ophthalmic formulation with at least one-month stability, in the reconstituted state, has been developed.