Hyaluronidase-sensitive nanoparticle templates for triggered release of HIV/AIDS microbicide in vitro

Abstract

This study was designed to test the hypothesis that a triggered release of a topical microbicide (tenofovir) from hyaluronic acid nanoparticles (HA-NPs) can be achieved under the influence of hyaluronidase (HAase) enzyme. A fractional factorial experimental design was used to examine the factors [molar concentrations of adipic acid dihydrazide (X1) and 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (X2), volume of acetone (X3) and reaction time (X4)] influencing the responses, Y1; particle mean diameter: PMD (nanometers: nm), Y2; polydispersity index: PDI and Y3; zeta (ζ) potential: (millivolts). The amide bond formation between HA and ADH after cross-linking was confirmed by FT-IR and (13)C-NMR analyses. These NPs were also characterized for cytotoxicity on a human vaginal epithelial cell line and L. crispatus. When formulated with factors X1; 2.49 mM, X2; 9.96 mM, X3; 60 mL, X4; 6 h, HA-NPs exhibited a spherical shape with PMD, PDI, ζ potential, encapsulation efficiency, and drug loading of 70.6 ± 4.1 nm, 0.07 ± 0.02, -38.2 ± 2.8 mV, 51.8 ± 2.4% w/w and 26.1 ± 1.2% w/w, respectively, (n = 3). Unlike for HA based gel, HAase significantly triggered the drug release and HA degradation from the NPs after 24 h (~90% w/w and 65% w/w, respectively); whereas, in its absence, these values were ~39% w/w and 26% w/w, respectively. The NPs were non-cytotoxic to human vaginal VK2/E6E7, End1/E6E7 cells and Lactobacillus crispatus. These data highlight the potential of HAase-sensitive HA-NPs templates for the controlled and vaginal delivery of anti-HIV/AIDS microbicides.

Fig. 1

Pareto charts showing the standardized effects of factors (as defined in the above Table I) and their interactions for responses. a Y ₁ particle mean diameter (PMD, nanometer). b Y ₂ polydispersity index (PDI). Bars extending past the vertical lines indicates the values reaching statistical significance (α = 0.05). Asterisk (*) indicates the significant effect (p < 0.05) of factors on the responses. c Prediction profiler and desirability plot showing the effect of factors, X₁, X₂, X₃, and X₄, on the responses, Y₁ and Y₂. The desirability value of 0.91 corresponds to the optimized HA nano formulation (F13) with the coded values of X₁ = −1, X₂ = +1, X₃ = +1, and X₄ = +1. The coded values listed above correspond with the actual values of X₁ = 2.49 mM, X₂ = 9.96 mM, X₃ = 60 mL, and X₄ = 6 h

Fig. 2

Percent cumulative HA degradation (% w/w) either in presence or absence of HAase at pH 7.1. a Native HA. b F13. c F1 and d F7 nanoformulations. Results are given as mean ± SD, (n = 3)

Fig. 3

Percent cumulative drug release (% w/w) profile either in presence or absence of HAase at pH 7.1. a HA-gel and TFV control, b F13, c F1, and d F7 nanoformulations. Results are given as mean ± SD, (n = 3)

Fig. 4

a Surface morphology: scale bar 500 nm, and size distribution analysis of native HA-NPs (F13) by TEM and DLS analysis, respectively. b Surface morphology of formulation F13 in the absence of HAase, after day 1: scale bar, 500 nm, and day 3: scale bar, 200 nm. c Surface morphology of formulation F13 in the presence of HAase, after day 1: scale bar, 500 nm and day 3: scale bar 200 nm

Fig. 5

Cytotoxicity assays: Effect of HA-NPs; F1, F7, and F13 at the concentration of 1 mg/mL for each treatment. a Effect on the viability of VK2/E6E7 and End1/E6E7 cell lines (MTS assay). b Effect on the lactate dehydrogenase release from VK2/E6E7 and End1/E6E7 cell lines (LDH assay). c Effect on the viability of L. crispatus (Lactobacillus viability assay). Results are given after 48 h of incubation as mean ± SD, n = 3. Asterisk (*) indicates the significant difference (p < 0.05), compared with the positive control (1% Triton X)