Relationships of signal intensity vs.

<div><p><b>Tenofovir concentration in fluid, clinical gel, and tissue homogenate</b>. </p> <p>Serial dilutions of Tenofovir, ranging from 0.01 to 1%w/w, were prepared in alkalinized water (50mM NaOH), gel, and porcine buccal tissue homogenates. A strong linear dilution response (R² ≥ 0.99, P <0.05) for concentrations of Tenofovir in solutions, gels, and tissue homogenates was observed. A saturation effect was not seen at the higher concentrations. This shows that Raman spectrometry is able to respond linearly to the physiologically relevant concentrations of drug. This linear dependence, in reverse, shows that Raman spectrometry can be used in practice to deduce the concentration of microbicide from the intensity of Raman peaks. Also, Tenofovir showed relatively higher signals in clear fluids and gels than in strongly scattering (opaque) tissues. The data shown represent the mean of 3-4 replicates.</p></div

Quantitation of Residual Tenofovir (TFV) on Gel Applicators.

1<p>Manually handled, gel expelled in trash, n = 2.</p>2<p>Unopened applicator, removed from foil packet only, n = 2.</p>3<p>BLD  =  Below level of detection.</p><p>Quantitation of Residual Tenofovir (TFV) on Gel Applicators.</p

Lack of Bacterial Marker DNA Amplification in Semen or Sperm DNA.

<p>30 and 90 ng, respectively of sperm DNA (lanes 1–2) and semen DNA (lanes 3–4) were used as template DNA for the expanded multiplex PCR. No bacterial markers were amplified. Only the Y-chromosomal DNA markers, TSPY4 and SRY, along with the internal control gene, amelogenin, were amplified. Vaginal swab DNA showing <i>L. jensenii</i> amplification was used as a positive control (lane 5). Lane 6 is a PCR negative control.</p

Tenofovir concentrations (measured vs. computed) in the bottom fluid compartment in the Transwell assay.

<p>A mass balance computation calculated the difference between the initial amount of Tenofovir in the fluid overlayer and the subsequent time-dependent sum of the total amounts of drug in the top layer and tissue. The good agreement between this mass balance prediction and the actual values measured supports the accuracy of the measurements. The box-and-whisker plot shows smallest value (lower bar), lower quartile (bottom of box), median (line through box), upper quartile (top of box), and largest observation (upper bar). The concentrations in the bottom fluid compartment from 0 to 9 h were not measurable (i.e., they were below the lower detection limit of the Raman quantification). </p

Hydroxyethylcellulose (HEC) Placebo Gel Does Not Interfere with DNA Biomarker Detection.

<p>A) Different patterns of DNA biomarkers were still detected in the presence of vaginally expelled HEC gel (Lane 1). These patterns matched the corresponding patterns observed from vaginal swab DNA of those same women (Lane 3). DNA from sham applicators manually handled by the women (Lane 2) and No DNA PCR (Lane 4) controls were negative. B) Exposure of HEC gel did not interfere with the DNA biomarkers before (Lane 1) or after semen exposure (Lane 2). No DNA PCR negative control is included (Lane 3).</p

Chemical structures of Tenofovir, IQP-0528, and Dapivirine.

<p>Chemical structures of Tenofovir, IQP-0528, and Dapivirine.</p

Vaginal Bacteria Primers for Multiplex PCR.

<p>Vaginal Bacteria Primers for Multiplex PCR.</p

Comparative measurement of Tenofovir concentration using Raman vs.

<div><p><b>LC-MS/MS in solution and tissue</b>. </p> <p>Freshly excised porcine buccal tissue specimens were incubated in serially diluted concentrations of Tenofovir in Ringer's solution and allowed to equilibrate for 24 h. After incubation, the tissue specimens and the surrounding fluid were collected and stored at −80°C overnight for analysis by both techniques: (a) The initial serially-diluted concentrations of Tenofovir in Ringer's solution measured by both techniques were plotted against its known concentrations in solution. These two techniques produced slopes that did not differ significantly (ANCOVA, P > 0.05); (b) the final concentrations of Tenofovir within tissue specimens measured by both techniques were plotted against concentrations of Tenofovir solution in equilibrium with tissue. ANCOVA also showed that the slopes obtained by both techniques were not significantly different (P > 0.05). </p></div

Hydroxyethylcellulose (HEC) Placebo Gel Does Not Interfere with CK4 Biomarker Detection.

<p>HEC gel applicators were inserted and gel expelled into the vaginas of three different women. The applicators were swabbed for analysis of CK4 expression in isolated vaginal cells. While the observed particles above appear to be associated with the presence of gel, the cells from all three women still demonstrated positive CK4 expression. Normal rabbit IgG negative control (inset pictures).</p

DNA and CK4 Biomarkers on Applicators Stored for 30 days.

<p>After 30 days of storage at room temperature, applicators from two women (#1 and #2) were swabbed and DNA/cells isolated. CK4 expression was positive (A) while amelogenin and bacterial markers were amplified by the multiplex PCR (B) confirming stability of the biomarkers over the course of 30 days. Vaginal swab DNA from woman #1(“VS”) is included as a positive control to confirm that identical amplified bacterial species were found on the applicator #1 (Lane 1). A no DNA PCR control is also included (“neg”); Normal rabbit IgG negative control (inset pictures).</p