Enhanced anti-HIV efficacy of indinavir after inclusion in CD4-targeted lipid nanoparticles

Abstract

Background: Combination drug therapy has reduced plasma HIV to undetectable levels; however, drug-sensitive virus persists in patients' lymphoid tissue. We have reported significant lymphoid tissue drug localization with indinavir-associated lipid nanoparticles (LNPs). Our current objective is to evaluate whether additional enhancement is achievable by targeting these particles to CD4-HIV host cells.

Methods: We characterized 2 peptide-coated (CD4-BP2 and CD4-BP4) drug-associated LNPs and demonstrated CD4-cell specificity. Drug-associated LNPs expressing polyethyleneglycol were exposed on HIV-2-infected cells under dynamic conditions that emulated lymph node physiology for 15, 30, and 60 minutes at concentrations from 0 to 25 μM and evaluated for antiviral activity and cell-associated drug concentrations. The specificity of CD4-mediated enhancement of indinavir LNPs antiviral activity was evaluated by blocking with anti-CD4 antibody.

Results: Inclusion of CD4-binding peptides on LNPs enhanced antiviral activity for all incubation conditions, compared with control particles or soluble drug (eg, 60 minutes exposure, EC50 = 0.12-0.13 vs. 0.46 μM for targeted nanoparticles vs. soluble drug). The CD4-BP4 peptide exhibited higher efficiency in eliciting antiviral activity than CD4-BP2-coated particles (EC50 = 7.5 μM vs. >25 μM at 15 minutes drug exposure). This enhancement seems to be driven by CD4 availability and cell-associated indinavir concentrations, as blocking of CD4 significantly ablated indinavir efficacy in targeted particles and indinavir concentrations reflected the observed anti-HIV activity.

Conclusions: We constructed CD4-targeted LNPs that provide selective binding and efficient delivery of indinavir to CD4-HIV host cells. Inclusion of polyethyleneglycol in LNPs would minimize immune recognition of peptides. The enhancement of anti-HIV effects is effective even under limited time exposure.

Figure 1

Concentration and time-dependent effects of CD4-targeted LNP in enhancing anti-HIV efficacy of Indinavir-associated LNP. Targeted and untargeted, Indinavir associated and empty lipid nanoparticles, and soluble Indinavir were incubated with cells for 15 min (Panel A), 30 min (Panel B), 60 min (Panel C) in quadruplicate in 96-well plates. Cells were then washed and infected at 0.1 MOI HIV-2. Unadsorbed virus was washed away and cells were incubated for 4 days at 37°C. The percentage of HIV-2 infected cells was evaluated on day 4 as described in the Materials and Methods. Data expressed were mean ± SD of quadruplicate samples from 4 experiments.

Figure 2

CD4 specific inhibition of targeted lipid nanoparticle enhancement on Indinavir anti-HIV efficacy. To determine CD4-dependent enhancement of targeted Indinavir LNP, 10⁵ CD4-positive cells infected with HIV-2₂₈₇ were preincubated with indicated CD4 antibody Sim2 for 1 hour at 37°C. Cells were then incubated for 30 minutes with 25 (Panel A), 12.5 (Panel B), and 6.25 μM (Panel C) of Indinavir in soluble and targeted LNP. The percentage of infected cells was determined as described in the Materials and Methods. Data expressed were Mean ± SD of quadruplicate samples from four experiments. Unblocked CD4-BP4-LNP, CD4-BP2-LNP, and soluble Indinavir mediated antiviral effect is shown in white bars; Antibody blocked CD4-BP4-LNP, CD4-BP2-LNP, and soluble Indinavir mediated effects are shown in black bars. *: Significantly different from unblocked treatment at p<0.05

Figure 2

CD4 specific inhibition of targeted lipid nanoparticle enhancement on Indinavir anti-HIV efficacy. To determine CD4-dependent enhancement of targeted Indinavir LNP, 10⁵ CD4-positive cells infected with HIV-2₂₈₇ were preincubated with indicated CD4 antibody Sim2 for 1 hour at 37°C. Cells were then incubated for 30 minutes with 25 (Panel A), 12.5 (Panel B), and 6.25 μM (Panel C) of Indinavir in soluble and targeted LNP. The percentage of infected cells was determined as described in the Materials and Methods. Data expressed were Mean ± SD of quadruplicate samples from four experiments. Unblocked CD4-BP4-LNP, CD4-BP2-LNP, and soluble Indinavir mediated antiviral effect is shown in white bars; Antibody blocked CD4-BP4-LNP, CD4-BP2-LNP, and soluble Indinavir mediated effects are shown in black bars. *: Significantly different from unblocked treatment at p<0.05

Figure 3

Qualitative fluorescence microscopic analysis of temperature dependent binding of targeted lipid nanoparticles or RPA-T4 antibody to CD4⁺ Molt-4 cells. 5× 10⁴ cells were incubated with 10 nmol of nanoparticles and 2μL of RPA-T4 CD4 antibody for 2 hours at 4°C, 22°C, or 37°C, washed, and observed for fluorescence. Column A: RPA-T4 Ab, B: Targeted Nanoparticles (TNP). Images are representative of at least four separate repeat experiments.

Figure 4

Proposed mechanism of targeted lipid-Indinavir nanoparticle mediated cellular accumulation and inhibition of HIV in CD4⁺ cells. It is possible that CD4 targeted peptides coated on lipid nanoparticles can bind to CD4 receptors and some particles may be endocytosed while others could release drug at the cell surface in a pH dependent manner. The fraction of release will depend on the pH at the cell surface. Endocytosed nanoparticles can release stable drug either in the endosome or lysosome. The lower pH conditions protonate Indinavir and allow release from lipid into the cytoplasm. In virally infected cells, HIV protease inhibitors will mediate antiviral effects while no effect will occur in uninfected cells lacking HIV protease. Specific proposed steps as follows: 1. Binding of Indinavir (INV)-associated targeted lipid nanoparticles (TNP) to CD4 receptors. 2. Endocytosis of targeted LNP to endosome. 3. pH dependent release of Indinavir from LNP. 4. Indinavir inhibits HIV protease and prevents virus maturation. Inset: Scheme of pH-sensitive release from lipid-Indinavir nanoparticles (Indinavir, stars). Not drawn to scale.