Antiviral therapy using nucleoside reverse transcriptase inhibitors (NRTIs) is neurotoxic and has low efficiency in eradication of HIV-1 harbored in central nervous system (CNS). Previously, we reported that active 5′-triphosphates of NRTIs encapsulated in cationic nanogels (nano-NRTIs) suppress HIV-1 activity more efficiently than NRTIs and exhibit reduced mitochondrial toxicity1,2. Here, we demonstrated low neurotoxicity and excellent antiviral activity of nano-NRTIs decorated with the peptide (AP) binding brain-specific apolipoprotein E receptor. Nano-NRTIs induced lower levels of apoptosis and formation of reactive oxygen species, a major cause of neuron death, than free NRTIs. Optimization of size, surface decoration with AP significantly increased brain accumulation of nano-NRTIs. The efficient CNS delivery of nano-NRTIs resulted in up to 10-fold suppression of retroviral activity and reduced virus-associated inflammation in humanized mouse model of HIV-1 infection in the brain. Our data provide proof of the advanced efficacy of nano-NRTIs as safer alternative of current antiviral drugs.
Background Macrophages serve as depot for HIV-1 in the central nervous system (CNS). To efficiently target macrophages, we developed nanocarriers for potential brain delivery of activated nucleoside reverse transcriptase inhibitors (Nano-NRTI). Methods Nanogel carriers consisting of PEG- or Pluronic-PEI biodegradable networks, star PEG-PEI, or PAMAM-PEI-PEG dendritic networks, as well as nanogels decorated with multiple ApoE peptide molecules, specifically binding to the apolipoprotein E receptor, were synthesized and evaluated. Nano-NRTIs were obtained by mixing aqueous solutions of triphosphates (AZTTP or ddITP) and nanocarriers followed by freeze-drying. Intracellular accumulation, cytotoxicity, and antiviral activity of Nano-NRTIs were monitored in monocyte-derived macrophages (MDMs). HIV-1ADA viral activity in infected MDMs was measured by micro-RT assay following the treatment with Nano-NRTIs. Mitochondrial DNA (mtDNA) depletion in MDMs and human HepG2 cells was assessed by quantitative PCR (qPCR). Results Nanogels were efficiently captured by MDMs and demonstrated low cytotoxicity, not affecting viral activity without drugs. All Nano-NRTIs demonstrated high efficacy of HIV-1 inhibition at drug levels as low as 1 μmol/L, representing from 4.9 to 14-fold decrease in effective drug concentrations (EC90) as compared to NRTIs, while cytotoxicity effects (IC50) started at 200 times higher concentrations. Nanocarriers with core-shell structure and decorated with vector peptides (e.g. brain-targeting ApoE peptide) displayed the highest antiviral efficacy. The mtDNA depletion, a major cause of NRTI neurotoxicity, was reduced 3-fold compared to NRTIs at application of selected Nano-NRTIs. Conclusions Nano-NRTIs demonstrated a promising antiviral efficacy in MDMs and showed strong potential as nanocarriers for delivery of antiviral drugs to brain-harboring macrophages.
Purpose-To enhance transfection efficacy of pDNA through the application of multifunctional peptide-PEG-tris-acridine conjugates (pPAC) and the formation of biodegradable core-shell polyplexes for gene delivery to the blood-brain barrier (BBB).Methods-pPAC-mediated transfection was compositionally optimized in mouse BBB cells (bEnd.3). Cellular uptake and trafficking, and brain accumulation of pDNA was evaluated by fluorescent imaging and histochemistry. We constructed anti-MRP4 siRNA-producing vectors and evaluated the efficacy of MRP4 down-regulation of MRP4 by Western blot and qPCR, and its effect on the uptake of 3 H-AZT, an MRP4 substrate.Results-A core-shell gene delivery system (GDS) was assembled from pDNA and pPAC, carrying multifunctional peptides with NLS, TAT, and brain-specific BH, or ApoE sequences, and biodegradable pLPEI polyamine. This GDS demonstrated better cellular and nuclear accumulation, and a 25-fold higher transfection efficacy in slow-dividing bEnd.3 cells compared to ExGen500. Inclusion of brain-targeting pPAC enhanced in vivo accumulation of functional pDNA in brain capillaries. Treatment by encapsulated anti-MRP4 siRNA-producing pDNA caused transient down-regulation of MRP4, and, after intravenous injection in Balb/c mice, enhanced AZT uptake in the brain by 230-270%.Conclusions-The pPAC represent novel efficient components of GDS that could find various gene therapy applications, including genetic modulation of the BBB.
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