Long acting Efavirenz and HIV-1 fusion inhibitor peptide co-loaded polymer-lipid hybrid nanoparticles (PLN): statistical optimization, cellular uptake and in vivo biodistribution.

The objective of the present study was to develop long-acting Efavirenz-Enfuvirtide co-loaded polymer-lipid hybrid nanoparticles (PLN) with improved intracellular delivery to target T- and macrophage cells located in multiple HIV sanctuaries. Box-Behnken design was utilized to optimize three high-risk factors namely, Efavirenz amount, sonication time for primary emulsion, and sonication time for aqueous nano-dispersion obtained from preliminary studies. Lyophilized Efavirenz-Enfuvirtide co-loaded PLN using trehalose elicited spherical morphology, drug amorphization on incorporation, and absence of drug-excipient interaction. In vitro release studies revealed extended-release of both the drugs from PLN with differential release profile. Efavirenz-Enfuvirtide co-loaded PLN exhibited low hemolytic, platelet and leukocyte aggregation as well as low cytotoxicity in Jurkat E6.1 T and U937 macrophage cells. Circular dichroism spectra confirmed the presence of an α-helix form of Enfuvirtide after encapsulation in PLN. Coumarin-6 loaded PLN exhibited enhanced cellular uptake in Jurkat E6.1 T and U937 macrophage cells in comparison to free coumarin-6 as evidenced by fluorescent microscopy and flow cytometry. In-vivo biodistribution studies after intravenous administration of near-infrared dye (DIR) loaded PLN (surrogate for Efavirenz-Enfuvirtide PLN) revealed non-uniform distribution within 2h in order of spleen ≥ liver > lymph node > thymus > lungs >female reproductive tract (FRT)> heart > kidneys > brain. While, subcutaneous administration caused non-uniform biodistribution after 3 days eliciting long-acting slow release from injection site depot until day 5 in infection spread site (lymph nodes and female reproductive tract), reservoir sites (liver and spleen) and difficult-to-access site (brain). Furthermore, it presents a vital illustration of available tissue specific drug concentration prediction from simulated surrogate PLN. Keywords: Fusion inhibitor peptide; polymer-lipid hybrid nanoparticle (PLN); long-acting; simulation; Jurkat E 6.1 cells; U937 macrophage.