A Transferrin conjugated nanoemulsion system for brain delivery of antiretroviral therapy

Abstract

HIV- associated neurocognitive disorder (HAND), also known as HIV encephalopathy and AIDS dementia, is one of the critical complications of HIV infection that causes severe morbidity and even shortens survival. This complication is challenging to treat because most of the antiretroviral therapeutic (ART) agents cannot achieve the desired therapeutic levels in the central nervous system (CNS) because they cannot efficiently cross the blood-brain barrier (BBB). The goal of this study is to develop a new transferrin conjugated nanoemulsion system (Tf-NE) for antiretroviral medication delivery and evaluate its potential to cross the BBB. Nanoemulsions were prepared based on the solvent evaporation sonication method using lipids and phospholipids. To achieve brain delivery, holo-transferrin was conjugated to DSPE-PEG (2000)-Maleimide by NHS ester crosslinking reaction. Darunavir (DRV) was encapsulated into Tf-NE as an antiretroviral agent. Size, polydispersity index, and dispersion stability were characterized using dynamic light scattering (DLS) system. Morphology of the Tf-NE was investigated using transmission electron microscopy (TEM). Differential scanning calorimetry was performed to study the extent of drug solubilization inside the nanoemulsion system. To evaluate the in vitro toxicity of Tf-NE-DRV, MTT assay was performed by using the human brain endothelial capillary hCMEC/D3 cells and 293T cells. Cell uptake and drug transport assays were also conducted to investigate the in vitro activity of Tf-NE-DRV. In vitro efficacy studies, ex vivo imaging studies and in vivo biodistribution studies were performed to investigate the brain targeting function of Tf-NE-DRV. Tf-NE-DRN was prepared 100-130nm in diameter with polydispersity index smaller than 0.3. This nanoemulsion system was stable in serum-enriched medium and PBS at 37 ℃ for 5 days. All the therapeutic compounds were well-dispersed inside the oil core of Tf-NE-DRV. Within the therapeutic concentration range, Tf-NE-DRV did not cause a significant reduction in the cell viability, indicating low toxicity of the formulation. Considerable uptake of Tf-NE-DRV into the BBB model of hCMEC/D3 cells was observed. Tf-NE-DRV can maintain the same therapeutic function as the free drug form of darunavir in vitro. Imaging and biodistribution results revealed the formulation was superior to the free drug and able to transport the drug across the BBB in vivo to reach the therapeutic level. We successfully developed a biocompatible nanoemulsion system that can effectively penetrate the BBB. This Tf-NE-DRV system shows the clinical potential to deliver antiretroviral agents into the CNS system to achieve improved treatment of HAND.