2.6 In vitro efficacy of PLGA and PLCL fibers against HIV-1 infection

HIV pseudovirus assays were used to assess the efficacy of TDF released from EFs against HIV infection in vitro. TZM-bl cells were infected with Env-pseudotype HIV, kindly provided by both Dr. Nobuyuki Matoba (University of Louisville) and the NIH ARRRP. To produce and propagate HIV Env-pseudovirus, HEK293T/17 cells were transfected with two plasmids, one containing an Env-defective HIV genome and a plasmid solely expressing Env. Transfection was facilitated with the use of FuGENE (Promega). HEK293T cells were allowed to incubate for 48 hr, after which viral particles were collected and titered using the 50% Tissue Culture Infectious Doses assay (TCID₅₀). Viral particles were stored at −80°C until use [41].

To determine the in vitro efficacy of PLGA and PLCL TDF EFs against Env-pseudotype HIV infection, TZM-bl cells were seeded in 96-well plates at 100,000 cells/well in 100 μL of DMEM. Fifty microliters of fiber eluate media (DMEM 10% FBS) collected from time points: 1 and 24 hr; week 1 (release from days 0–7); week 2 (release from days 8–14), week 3 (release from days 15–21), and week 4 (release from days 22–28) were diluted by a maximum of 5 orders of magnitude from collected eluate (1:100,000 maximum dilution). Eluate dilutions were added to cells in triplicate, and 50 μL of diluted virus stock (1:8) was subsequently added to each well. The administered virus dose resulted in relative luminescence units (RLU) of at least twenty times that of background observed in untreated/uninfected cells, yielding an average of 100,000 RLUs in our experiments. Experimental controls included untreated/uninfected cells, untreated/infected cells, and blank fiber eluate-treated/infected cells. For wells containing untreated/uninfected and untreated/infected cell controls, 100 μL DMEM was added to the wells; for infected cells with blank fiber eluate, 50 μL DMEM was added to 50 μL blank fiber eluate, resulting in a final volume of 200 μL for all wells. After infection, plates were incubated 48 hr at 37°C, and 100 μL of media was subsequently removed from each well (post-incubation), and replaced with 100 μL of Bright Glo Reagent (Promega). Cells were incubated at room temperature for another 5 minutes and the luminescence of each well was read at an integration time of 1 sec and a gain of 135 (Synergy HT luminometer). The amount of virus inhibition was determined by normalizing the RLUs of treated/infected cells to untreated/infected cells. Additionally, all RLU values were corrected by subtracting the RLU of untreated/uninfected cells. IC50 values were determined using GraphPad 6.0 sigmoidal regression analysis. Unless otherwise noted, all experiments were run with three or more replicates per treatment group. Statistical significance between the IC50s was determined using one-way ANOVA with the Bonferroni post hoc t-test (p < 0.05).