PURPOSE: Developing and testing of microbicides for pre-exposure prophylaxis and post-exposure protection from HIV are on the list of major HIV/AIDS research priorities. To improve solubility and bioavailability of highly potent anti-retroviral drugs, we explored the use of a nanoparticle (NP) for formulating a combination of two water-insoluble HIV inhibitors. METHODS: The combination of a non-nucleoside HIV reverse transcriptase inhibitor (NNRTI), Efavirenz (EFV), and an inhibitor of HIV integrase, Elvitegravir (ELV) was stabilized with a graft copolymer of methoxypolyethylene glycol-polylysine with a hydrophobic core (HC) composed of fatty acids (HC-PGC). Formulations were tested in TZM-bl cells infected either with wild-type HIV-1IIIB, or drug-resistant HIV-1 strains. In vivo testing of double-labeled NP formulations was performed in female rats after a topical intravaginal administration using SPECT/CT imaging and fluorescence microscopy. RESULTS: We observed a formation of stable 23-30 nm NP with very low cytotoxicity when EFV and ELV were combined with HC-PGC at a 1:10 weight ratio. For NP containing ELV and EFV (at 1:1 by weight) we observed a remarkable improvement of EC50 of EFV by 20 times in the case of A17 strain. In vivo imaging and biodistribution showed in vivo presence of NP components at 24 and 48 h after administration, respectively. CONCLUSIONS: insoluble orthogonal inhibitors of HIV-1 life cycle may be formulated into the non-aggregating ultrasmall NP which are highly efficient against NNRTI-resistant HIV-1 variant.