Nanoparticle-eluting stents for coronary intervention: formulation, characterization, and in vitro evaluation.

Coronary artery disease (CAD) is currently a leading cause of death worldwide. In the history of percutaneous coronary intervention for the treatment of CAD, a drug-eluting stent (DES) is recognized as a revolutionary technology that has the unique ability to significantly reduce restenosis and provide both mechanical and biological solutions simultaneously to the target lesion. The aim of the research work was to design and fabricate DES coated with a nanoparticulate drug formulation. Sirolimus, an inhibitor of the smooth muscle cell (SMC) proliferation and migration, was encapsulated in polymeric nanoparticles (NPs). The NP formulation was characterized for various physicochemical parameters. Cell viability and cell uptake studies were performed using human coronary artery smooth muscle cells (HCASMCs). The developed NP formulation showed enhanced efficacy compared to plain drug solution and exhibited time-dependent uptake into the HCASMCs. The developed NP formulation was coated on the Flexinnium™ ultra-thin cobalt-chromium alloy coronary stent platform. The NP-coated stents were characterized for morphology and residual solvent analysis. In vitro drug release was also evaluated. Ex vivo arterial permeation was carried out to evaluate the NP uptake from the surface of the stents. The characterization studies together corroborated that the developed NP coated stent can be a promising replacement of the current DESs.

Immunogenicity of virus-like Semliki Forest virus replicon particles expressing Indian HIV-1C gag, env and polRT genes.

Development of a vaccine targeting human immunodeficiency virus-1 subtype C (HIV-1C) is an important public health priority in regions with a high prevalence of the clade C virus. The present study demonstrates the immunogenicity of recombinant Semliki Forest virus (SFV)-based virus-like replicon particles (VRPs) expressing Indian HIV-1C env/gag/polRT genes. Immunization of mice with recombinant VRPs in a homologous prime-boost protocol, either individually or in combination, elicited significant antigen-specific IFN-γ T cell responses as detected by the ELISPOT assay. Additionally, Gag-specific TNF-α secreting CD8+ and CD4+ T cells and Env-specific IL-2 secreting T cells were also elicited by mice immunized with Gag and Env constructs, respectively, as estimated by intracellular cytokine staining assay. Moreover, an HIV Pol-specific TNF-α response was elicited in mice immunized with a combination of the three VRP constructs. Furthermore, HIV-1C Gag and Env-specific binding antibodies were elicited as verified by gp120 ELISA and p24 Gag ELISA, respectively. The immunogenicity of VRPs was found to be higher as compared to that of RNA replicons and VRPs may therefore be promising preventive and therapeutic candidate vaccines for the control and management of HIV/AIDS.

Nevirapine Loaded Core Shell Gold Nanoparticles by Double Emulsion Solvent Evaporation: In vitro and In vivo Evaluation.

BACKGROUND: HIV/AIDS is a macrophage resident infection localized in the reticuloendothelial system and remote locations of brain and bone marrow. We present core shell nanoparticles of gold(AuNPs) and nevirapine(NVP) for targeted delivery to the multiple HIV reservoirs. The aim of the study was to design core shell NVP loaded AuNPs with high drug loading and to evaluate biodistribution of the nanoparticles in possible HIV reservoirs in vivo. A specific objective was to assess the possible synergy of AuNPs with NVP on anti-HIV activity in vitro. METHOD: Core shell nanoparticles were prepared by double emulsion solvent evaporation method and characterized. RESULTS: Glyceryl monostearate-nevirapine-gold nanoparticles(GMS-NVP-AuNPs) revealed high entrapment efficiency (>70%), high loading (~40%), particle size <250 nm and zeta potential -35.9± 1.41mv and exhibited sustained release with good stability. Surface plasmon resonance indicated shell formation while SEM coupled EDAX confirmed the presence of Au. TEM confirmed formation of spherical core shell nanoparticles. GMS-NVP-AuNPs revealed low hemolysis (<10 %) and serum stability upto 6 h. GMS-NVP-AuNPs exhibited rapid, high and sustained accumulation in the possible HIV reservoir organs, including the major organs of liver, spleen, lymph nodes, thymus and also remote locations of brain, ovary and bone marrow. High cell viability and enhanced uptake in PBMC's and TZM-bl cells were observed. While uptake in PBMC's proposed monocytes/macrophages enabled brain delivery. GMS-NVP-AuNPs demonstrated synergistic anti-HIV activity. CONCLUSION: The superior anti-HIV activity in vitro coupled with extensive localization of the nanoparticles in multiple HIV reservoirs suggests great promise of the core shell GMS-NVP-AuNPs for improved therapy of HIV.

Immunogenicity of Semliki Forest virus based self-amplifying RNA expressing Indian HIV-1C genes in mice.

Development of recombinant vaccines is considered as a promising approach to prevent transmission and eradication of HIV/AIDS. Candidate vaccines tested so far have shown poor to modest efficacy. Self-amplifying RNAs of positive strand alphaviruses are reported to be promising vectors for development of recombinant vaccines. This study describes the construction, in vitro expression and in vivo immunogenicity of recombinant RNA vaccines developed by individually cloning gag, env and polRT genes of primary HIV-1C Indian isolates using Semliki Forest virus (SFV) vector. HIV-1C specific T cell responses were detected in mice immunized with rSFV2gen/gag RNA by IFN-γ ELISPOT assay. Furthermore, using flow cytometry based intracellular cytokine staining (ICCS) assay HIV-1C specific IL-2 responses were detected in immunized mice that were mediated by both CD4(+) and CD8(+) T cells. Mice immunized with rSFV2gen/env RNA elicited HIV-1C Env-specific antibodies as detected by gp120 ELISA. The Env, Gag and Pol (RT) RNA constructs in combination elicited better HIV-1C Env-specific humoral responses compared to mice immunized with Env RNA alone. In conclusion, rSFV2gen RNA constructs encoding HIV-1C antigens elicited clear cell mediated and humoral immune responses in mice, thus demonstrating the potential of self-amplifying rSFV2gen RNA as a promising candidate for anti-HIV vaccine development.

Association of human mannose receptor in sexual transmission of human immunodeficiency virus in serodiscordant couples.

HIV binds specifically to the human mannose receptor (hMR) on vaginal epithelial cells that are devoid of a conventional CD4 receptor. HIV binding to hMR on vaginal epithelial cells induces the production of matrix metalloproteinase 9 (MMP9) leading to degradation of the extracellular matrix, which may increase the risk of HIV entry into vaginal epithelial cells and further transmission into distal cells. Immunofluorescent localization of hMR on vaginal epithelial cells of seronegative females from the general population included the control group (n=52) and seronegative females from serodiscordant couples. There was PCR amplification of DNA from peripheral blood mononuclear cells (PBMCs) of the serodiscordant females for the CCR5 gene flanking the CCR5-Δ32 region; PCR amplification and sequencing of the C2-V3 region of HIV variants in PBMCs and sperm of the infected male partners of the serodiscordant couples; and the presence of hMR on 0-11% of the vaginal epithelial cells of seronegative females (n=39) from serodiscordant couples and 90-95% that of a control group of females (n=52). Nine of these serodiscordant females did not show a CCR5-Δ32 deletion. The translated amino acid sequence of the C2-V3 region of the env gene of HIV-1C in PBMCs (n=9) and sperm (n=5) of the male partners showed the presence of distinct variants and the variation in PBMCs and sperm of serodiscordant males was almost similar to that of infected males from concordant couples. The presence of hMR in a smaller number of vaginal epithelial cells of serodiscordant females prevented binding and HIV entry into these cells and therefore prevented sexual transmission of HIV.

Characterization of human immunodeficiency virus (HIV1C) variants in peripheral blood mononuclear cells and spermatozoa.

The presence of distinct viral variants in different cells and secretions of the same person influences the transmission of HIV as well as the response to the host defense and to therapy. Sperm-associated virus is also a risk factor for sexual transmission of HIV. Characterization of the C2-V3 region of HIV1C env gene by the Heteroduplex Mobility Assay (HMA) and sequencing demonstrated the presence of distinct variants in the peripheral blood mononuclear cells (PBMCs) and the sperm of the same individual (n = 6). The translated amino acid sequences of HIV variants in the PBMCs of all the study participants (n = 12) and spermatozoa of the six participants characterized showed the presence of distinct variants with different numbers of N-linked glycosylation (NLG) sites. Infectivity of PBMCs of these persons by co-culture with PBMCs from healthy individuals as detected by the p24 levels in the culture supernatant did not show a correlation with the blood plasma viral load. Interestingly, the infectivity of the sperm samples from four of the five individuals showed positive correlation with the viral load in seminal plasma. The study suggests the presence of distinct viral variants in the sperm and PBMCs of the same person with differential infectivity, and the NLG sites may be associated with the affinity of HIV to receptor/co-receptor usages as well as affinity toward neutralizing antibodies which may influence the risk of sperm associated virus in sexual transmission of HIV and transmit the virus further to distal cells.

CD4 independent binding of HIV gp120 to mannose receptor on human spermatozoa.

OBJECTIVE: To characterize the CD4-independent HIV-binding protein of 160 kDa on human spermatozoa. METHODS: The N-terminal amino acid sequence of the 160 kDa protein and its peptide obtained by tryptic digestion were determined. Polymerase chain reaction amplification of human testicular cDNA was performed using degenerate primers corresponding to peptide sequences of the 160 kDa protein. Localization of 160 kDa protein on sperm was performed using fluorescently labeled gp120, followed by inhibition experiments using antagonists to determine the specificity. RESULTS: The partial cDNA sequence of the 160 kDa protein demonstrated 99% identity with human macrophage mannose receptor. Sequence of testicular mannose receptor was obtained and exhibited 99% identity with that of macrophage mannose receptor. Furthermore, mannose receptor protein from sperm extract was found to have a molecular weight of 160 kDa, congruent with that of 160 kDa HIV-binding protein. gp120 binding and mannose receptor expression were localized to the equatorial segment in 10% of ejaculated sperm, which increased after capacitation. Mannan at molar excess concentrations completely inhibited gp120 binding to sperm. CONCLUSIONS: The 160 kDa, CD4-independent HIV-binding sperm protein has been identified as the human mannose receptor protein. The role of mannose receptor in HIV transmission and association with risk of sexual transmission merit further investigation.

Identification of CD4-independent HIV receptors on spermatozoa.

PROBLEM: Human immunodeficiency virus (HIV) has been demonstrated to bind and enter into the spermatozoa facilitating the transmission into urogenital cells. However, spermatozoa has been reported to be devoid of the conventional CD4 receptors for HIV. This suggests that there exists an alternate modality of HIV entry into spermatozoa using receptors other than CD4. Present communication describes the identification of HIV receptors on the spermatozoa. METHOD OF STUDY: The sperm proteins were solubilized using Triton X-100 and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Western blot analysis, using cell-free HIV or gp120 envelope glycoprotein as a probe. HIV or gp120 bound protein band was then visualized by using alkaline phosphatase (AP) labeled anti-gp120 antibody as well as by using anti-gp120 antibody and subsequently by AP-labeled anti-rabbit gamma globulin. RESULTS: The results obtained demonstrate for the first time that cell-free HIV and gp120 protein bind specifically to 160 kDa sperm protein that could be the receptor for HIV entry into spermatozoa. CONCLUSION: A 160 kDa sperm protein could be the CD4-independent HIV receptor for HIV to bind and enter into the spermatozoa. Further characterization of this 160 kDa HIV receptor on sperm will provide an insight in understanding the mechanism and probable mode of intervention or prevention of HIV transmission at the initial stage of infection.