Purpose: Lymphoid tissue and cells are one of the main reservoirs of HIV. This HIV reservoir has been shown to cause latent recurrence and HIV resistance acceleration during the cessation of ART, and is difficult to eradicate due to poor accumulation of the majority of drugs used for antiretroviral therapy (ART). Thus, optimal ART regimens require better drug penetration into lymphoid tissue and accumulation there with limited toxicities to other organs and tissues. In this study, our goal is to develop a new nanoemulsion system for improved antiretroviral treatment of lymphoid tissue, and evaluate their potential for lymphocyte targeting in vitro. As lymphocytes were reported to over-express folate receptors after HIV activation, our key hypothesis is that folate-decoration on an ART nanoemulsion can significantly enhance the ART uptake into these activated lymphocytes or any other cell models that simulate the folate receptor overexpression.
Methods: Folate-modified nanoemulsion (FA-NE) was prepared based on standard emulsification-solvent evaporation method using a combination of lipids, phospholipids and PEGylated phospholipids. For folate receptor targeting, some PEGylated phospholipids were substituted with folate-conjugated equivalents. Rilpivirine (RPV), a poorly water-soluble second-generation non-nucleoside reverse transcriptase inhibitor, was encapsulated into FA-NE as an antiretroviral agent. Size, polydispersity index (PDI) and dispersion stability were characterized using dynamic light scattering system. Drug encapsulation and release were quantified using HPLC detected at 280 nm. To study the affinity of FA-NE for folate-receptors, uptake studies of labeled FA-NE by a number of cell lines with different folate-receptor expression levels (e.g. ES2, SKOV3, OVCAR3) were performed. Western blot assays were conducted to verify the folate receptor levels of the cell lines so semi-quantitative correlation between the cellular uptake and the folate receptor level can be drawn. To evaluate in vitro toxicity of FA-NE-RPV, MTT assay was performed.
Results: FA-NE of RPV was successfully prepared with average size in the range of 100-130 nm (diameter) and PDI around 0.3. The new nanoformulation demonstrated good dispersion stability, as indicated by the steady size and PDI values of the nanoemulsion after incubation in both PBS and serum-enriched medium at 37°C for 7 days. In general, drug encapsulation efficiency of RPV in FA-NE was shown to be over 50%. FA-NE-RPV did not cause significant reduction in the viability on human cell lines, indicating low inherent toxicity of the nanoformulation. The uptake of FA-NE-RPV into OVCAR3, a folate-receptor over-expressing cell line, was considerably more efficient than the other cell lines that expressed lower levels of folate receptor. The cellular uptake studies also indicated positive correlation between the folate level on FA-NE-RPV and the uptake rate into OVCAR3 cells.
Conclusion: A new folate-receptor targeting nanoemulsion of RPV has been developed. This nanoemulsion was shown to be stable, efficient and non-toxic to human cells, and demonstrated selectivity to target cells with high expression levels of folate receptor. This new nanoformulation shows the potential to deliver antiretroviral agents into HIV-activated lymphocytes to achieve more efficient eradication of this normally drug-resistant HIV reservoir.