Confocal fluorescence microscopy: An ultra-sensitive tool used to evaluate intracellular antiretroviral nano-drug delivery in HeLa cells

In the last decade, confocal fluorescence microscopy has emerged as an ultra-sensitive tool for real-time study of nanoparticles (NPs) fate at the cellular-level. According to WHO 2007 report, Human Immunodeficiency Virus/Acquired Immunodeficiency Syndrome (HIV/AIDS) is still one of the world’s major health threats by claiming approximately 7,000 new infections daily worldwide. Although combination antiretroviral drugs (cARV) therapy has improved the life-expectancy of HIV-infected patients, routine use of high doses of cARVhas serious health consequences and requires complete adherence to the regimen for success. Thus, our research goal is to fabricate long-acting novel cARV loaded poly(lactide-co-glycolic acid) (PLGA) nanoparticles (cARV-NPs) as drug delivery system. However, important aspects of cARV-NPs that require special emphasis are their cellular-uptake, potency, and sustained drug release efficiency over-time. In this article, ultra-sensitive confocal microscopy is been used to evaluate the uptake and sustained drug release kinetics of cARV-NPs in HeLa cells. To evaluate with the above goal, instead of cARV-drug, Rhodamine6G dye (fluorescent dye) loaded NPs (Rho6G NPs) have been formulated. To correlate the Rhodamin6G release kinetics with the ARV release from NPs, a parallel HPLC study was also performed. The results obtained indicate that Rho6G NPs were efficiently taken up at low concentration (/ml) and that release was sustained for a minimum of 4 days of treatment. Therefore, high drug assimilation and sustained release properties of PLGA-NPs make them an attractive vehicle for cARV nano-drug delivery with the potential to reduce drug dosage as well as the number of drug administrations per month

Confocal fluorescence microscopy: An ultra-sensitive tool used to evaluate intracellular antiretroviral nano-drug delivery in HeLa cells

In the last decade, confocal fluorescence microscopy has emerged as an ultra-sensitive tool for real-time study of nanoparticles (NPs) fate at the cellular-level. According to WHO 2007 report, Human Immunodeficiency Virus/Acquired Immunodeficiency Syndrome (HIV/AIDS) is still one of the world’s major health threats by claiming approximately 7,000 new infections daily worldwide. Although combination antiretroviral drugs (cARV) therapy has improved the life-expectancy of HIV-infected patients, routine use of high doses of cARVhas serious health consequences and requires complete adherence to the regimen for success. Thus, our research goal is to fabricate long-acting novel cARV loaded poly(lactide-co-glycolic acid) (PLGA) nanoparticles (cARV-NPs) as drug delivery system. However, important aspects of cARV-NPs that require special emphasis are their cellular-uptake, potency, and sustained drug release efficiency over-time. In this article, ultra-sensitive confocal microscopy is been used to evaluate the uptake and sustained drug release kinetics of cARV-NPs in HeLa cells. To evaluate with the above goal, instead of cARV-drug, Rhodamine6G dye (fluorescent dye) loaded NPs (Rho6G NPs) have been formulated. To correlate the Rhodamin6G release kinetics with the ARV release from NPs, a parallel HPLC study was also performed. The results obtained indicate that Rho6G NPs were efficiently taken up at low concentration (/ml) and that release was sustained for a minimum of 4 days of treatment. Therefore, high drug assimilation and sustained release properties of PLGA-NPs make them an attractive vehicle for cARV nano-drug delivery with the potential to reduce drug dosage as well as the number of drug administrations per month

Thermosensitive Gel Containing Cellulose Acetate Phthalate-Efavirenz Combination Nanoparticles for Prevention of HIV-1 Infection

The objective of this investigation was to develop and evaluate a nano-microbicide containing a combination of cellulose acetate phthalate (HIV-1 entry inhibitor) and efavirenz (anti-HIV agent) for HIV prophylaxis. Cellulose acetate phthalate-efavirenz combination nanoparticles (CAP-EFV-NPs) were fabricated by the nanoprecipitation method and were characterized for particle size, zeta potential and encapsulation efficiency of efavirenz. CAP-EFV-NPs were incorporated into a thermosensitive gel (CAP-EFV-NP-Gel). CAP-EFV-NPs, CAP-EFV-NP-Gel and efavirenz solution were evaluated for cytotoxicity to HeLa cells and for in vitro short-term (1-day) and long-term (3-day) prophylaxis against HIV-1 infection in TZM-bl cells. CAP-EFV-NPs had size \u3c 100 nm, negative surface charge and encapsulation efficiency of efavirenz was \u3e 98%. CAP-EFV-NPs and CAP-EFV-NP-Gel were significantly less toxic (P \u3c 0 01) to HeLa cells as compared to efavirenz solution. CAP-EFV-NPs showed significantly higher prophylactic activity (P \u3c 0 01) against HIV-1 infection to TZM-bl cells as compared to efavirenz solution and blank CAP nanoparticles. CAP-EFV-NP-Gel can be a promising nano-microbicide for long-term HIV prophylaxis

Peripheral nerve induces macrophage neurotrophic activities: regulation of neuronal process outgrowth, intracellular signaling and synaptic function.

Abstract Rat cortical neurons cultured in conditioned media from human monocyte-derived macrophages (MDM) show increased neuronal protein synthesis, neurite outgrowth, mitogen-activating protein kinase activity, and synaptic function. Neurotrophic properties of human MDMconditioned media are significantly enhanced by human peripheral nerve and to a more limited extent by CD40 ligand pre-stimulation. Such positive effects of MDM secretions on neuronal function parallel the secretion of brain-derived neurotrophic factor (BDNF). MDM activation cues may serve to balance toxic activities produced during neurodegenerative diseases and thus, under certain circumstances, mitigate neuronal degeneration.

Correction: Nanoformulations of Rilpivirine for Topical Pericoital and Systemic Coitus-Independent Administration Efficiently Prevent HIV Transmission

Vaginal HIV transmission accounts for the majority of new infections worldwide. Currently, multiple efforts to prevent HIV transmission are based on pre-exposure prophylaxis with various antiretroviral drugs. Here, we describe two novel nanoformulations of the reverse transcriptase inhibitor rilpivirine for pericoital and coitus-independent HIV prevention. Topically applied rilpivirine, encapsulated in PLGA nanoparticles, was delivered in a thermosensitive gel, which becomes solid at body temperature. PLGA nanoparticles with encapsulated rilpivirine coated the reproductive tract and offered significant protection to BLT humanized mice from a vaginal high-dose HIV-1 challenge. A different nanosuspension of crystalline rilpivirine (RPV LA), administered intramuscularly, protected BLT mice from a single vaginal high-dose HIV-1 challenge one week after drug administration. Using transmitted/founder viruses, which were previously shown to establish de novo infection in humans, we demonstrated that RPV LA offers significant protection from two consecutive high-dose HIV-1 challenges one and four weeks after drug administration. In this experiment, we also showed that, in certain cases, even in the presence of drug, HIV infection could occur without overt or detectable systemic replication until levels of drug were reduced. We also showed that infection in the presence of drug can result in acquisition of multiple viruses after subsequent exposures. These observations have important implications for the implementation of long-acting antiretroviral formulations for HIV prevention. They provide first evidence that occult infections can occur, despite the presence of sustained levels of antiretroviral drugs. Together, our results demonstrate that topically- or systemically administered rilpivirine offers significant coitus-dependent or coitus-independent protection from HIV infection

Combination antiretroviral drugs in PLGA nanoparticle for HIV-1

<p>Abstract</p> <p>Background</p> <p>Combination antiretroviral (AR) therapy continues to be the mainstay for HIV treatment. However, antiretroviral drug nonadherence can lead to the development of resistance and treatment failure. We have designed nanoparticles (NP) that contain three AR drugs and characterized the size, shape, and surface charge. Additionally, we investigated the <it>in vitro </it>release of the AR drugs from the NP using peripheral blood mononuclear cells (PBMCs).</p> <p>Methods</p> <p>Poly-(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) containing ritonavir (RTV), lopinavir (LPV), and efavirenz (EFV) were fabricated using multiple emulsion-solvent evaporation procedure. The nanoparticles were characterized by electron microscopy and zeta potential for size, shape, and charge. The intracellular concentration of AR drugs was determined over 28 days from NPs incubated with PBMCs. Macrophages were imaged by fluorescent microscopy and flow cytometry after incubation with fluorescent NPs. Finally, macrophage cytotoxicity was determined by MTT assay.</p> <p>Results</p> <p>Nanoparticle size averaged 262 ± 83.9 nm and zeta potential -11.4 ± 2.4. AR loading averaged 4% (w/v). Antiretroviral drug levels were determined in PBMCs after 100 μg of NP in 75 μL PBS was added to media. Intracellular peak AR levels from NPs (day 4) were RTV 2.5 ± 1.1; LPV 4.1 ± 2.0; and EFV 10.6 ± 2.7 μg and continued until day 28 (all AR ≥ 0.9 μg). Free drugs (25 μg of each drug in 25 μL ethanol) added to PBMCs served as control were eliminated by 2 days. Fluorescence microscopy and flow cytometry demonstrated phagocytosis of NP into monocytes-derived macrophages (MDMs). Cellular MTT assay performed on MDMs demonstrated that NPs are not significantly cytotoxic.</p> <p>Conclusion</p> <p>These results demonstrated AR NPs could be fabricated containing three antiretroviral drugs (RTV, LPV, EFV). Sustained release of AR from PLGA NP show high drug levels in PBMCs until day 28 without cytotoxicity.</p

The Entner-Doudoroff Pathway in \u3ci\u3eEscherichia coli\u3c/i\u3e Is Induced for Oxidative Glucose Metabolism via Pyrroloquinoline Quinone-Dependent Glucose Dehydrogenase

The Entner-Doudoroff pathway was shown to be induced for oxidative glucose metabolism when Escherichia coli was provided with the periplasmic glucose dehydrogenase cofactor pyrroloquinoline quinone (PQQ). Induction of the Entner-Doudoroff pathway by glucose plus PQQ was established both genetically and biochemically and was shown to occur in glucose transport mutants, as well as in wild-type E. coli. These data complete the body of evidence that proves the existence of a pathway for oxidative glucose metabolism in E. coli. PQQ-dependent oxidative glucose metabolism provides a metabolic branch point in the periplasm; the choices are either oxidation to gluconate followed by induction of the Entner-Doudoroff pathway or phosphotransferase- mediated transport. The oxidative glucose pathway might be important for survival of enteric bacteria in aerobic, low-phosphate, aquatic environments

Thermosensitive Gel Containing Cellulose Acetate Phthalate-Efavirenz Combination Nanoparticles for Prevention of HIV-1 Infection

The objective of this investigation was to develop and evaluate a nano-microbicide containing a combination of cellulose acetate phthalate (HIV-1 entry inhibitor) and efavirenz (anti-HIV agent) for HIV prophylaxis. Cellulose acetate phthalate-efavirenz combination nanoparticles (CAP-EFV-NPs) were fabricated by the nanoprecipitation method and were characterized for particle size, zeta potential and encapsulation efficiency of efavirenz. CAP-EFV-NPs were incorporated into a thermosensitive gel (CAP-EFV-NP-Gel). CAP-EFV-NPs, CAP-EFV-NP-Gel and efavirenz solution were evaluated for cytotoxicity to HeLa cells and for in vitro short-term (1-day) and long-term (3-day) prophylaxis against HIV-1 infection in TZM-bl cells. CAP-EFV-NPs had size \u3c 100 nm, negative surface charge and encapsulation efficiency of efavirenz was \u3e 98%. CAP-EFV-NPs and CAP-EFV-NP-Gel were significantly less toxic (P \u3c 0 01) to HeLa cells as compared to efavirenz solution. CAP-EFV-NPs showed significantly higher prophylactic activity (P \u3c 0 01) against HIV-1 infection to TZM-bl cells as compared to efavirenz solution and blank CAP nanoparticles. CAP-EFV-NP-Gel can be a promising nano-microbicide for long-term HIV prophylaxis

Targeted Immuno-Antiretroviral to Promote Dual Protection against HIV: A Proof-of-Concept Study

The C&ndash;C motif chemokine receptor-5 (CCR5) expression on the T-cell surface is the prime barrier to HIV/AIDS eradication, as it promotes both active human immunodeficiency virus (HIV)-infection and latency; however, antiretrovirals (ARVs) suppress plasma viral loads to non-detectable levels. Keeping this in mind, we strategically designed a targeted ARVs-loaded nanoformulation that targets CCR5 expressing T-cells (e.g., CD4+ cells). Conceptually, CCR5-blocking and targeted ARV delivery would be a dual protection strategy to prevent HIV infection. For targeting CCR5+ T-cells, the nanoformulation was surface conjugated with anti-CCR5 monoclonal antibodies (CCR5 mAb) and loaded with dolutegravir+tenofovir alafenamide (D+T) ARVs to block HIV replication. The result demonstrated that the targeted-ARV nanoparticle&rsquo;s multimeric CCR5 binding property improved its antigen-binding affinity, prolonged receptor binding, and ARV intracellular retention. Further, nanoformulation demonstrated high binding affinity to CCR5 expressing CD4+ cells, monocytes, and other CCR5+ T-cells. Finally, the short-term pre-exposure prophylaxis study demonstrated that prolonged CCR5 blockage and ARV presence further induced a &ldquo;protective immune phenotype&rdquo; with a boosted T-helper (Th), temporary memory (TM), and effector (E) sub-population. The proof-of-concept study that the targeted-ARV nanoformulation dual-action mechanism could provide a multifactorial solution toward achieving HIV &ldquo;functional cure.&rdquo