Figure 1.
Schematic of Transwell assay for measuring Tenofovir transport into and through an excised tissue specimen.
Circular porcine buccal tissue specimens were maintained in Transwell supports, under which was a bath of PBS to hydrate the tissue. Matrigel was used to create a gelatinous seal around tissue edges to prevent leakage. An isotonic solution of 1% Tenofovir in PBS was applied to the apical surface. The assay was maintained at 37°C and 5% CO2 in a Heracell incubator for 30 min to 6 h.
Figure 2.
Raman spectrum of 1%w/w Tenofovir in water.
1% Tenofovir solution was prepared in alkalinized water (50 mM NaOH). The Raman spectrum of the Tenofovir solution was acquired using a Horiba LabRam ARAMIS Raman microscope with an excitation wavelength of 633 nm, 100μm slit width and a 10x objective lens. The acquisition time was 60s with 3 accumulations. Data were normalized to the area under the curve.
Figure 3.
Raman spectra for clinical 1% Tenofovir (TFV) gel.
Fresh porcine buccal tissue specimens (7-mm diameter, 1-mm thickness) were incubated by fully submerging them in 1% Tenofovir test gel (solid line) and HEC placebo gel (dotted line) for 6 h at 37°C and 5% CO2 in a Heracell incubator. The recorded Raman spectra demonstrated that the Tenofovir band was clearly distinguishable from Raman peaks due to the gel and tissue. β, bending; ν, stretching ; τ, twisting; r, rocking.
Figure 4.
Relationships of signal intensity vs.
Tenofovir concentration in fluid, clinical gel, and tissue homogenate.
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.
Figure 5.
Calibration sensitivity for detection of Tenofovir, IQP-0528 and Dapivirine in fluid, clinical gel and tissue.
The slopes, i.e. the incremental changes in intensity per change in concentration, of the linear dilution response curves represent the sensitivities of measurement. Response differences were observed for the three drugs in the different matrices. A significant difference in sensitivities among the drugs was observed (P<0.0001, two-way ANOVA with Fisher’s PLSD), and these relative sensitivities of drugs were dependent on the matrix in which they were measured (drug*matrix interaction, P <0.01). Fluids gave higher sensitivities than other matrices due to less background scattering. Dapivirine tended to have higher sensitivity than other microbicide drugs. The data shown represent the mean ± standard error of 3-4 replicates.
Figure 6.
Chemical structures of Tenofovir, IQP-0528, and Dapivirine.
Figure 7.
MTT assay of porcine tissue specimens fully submerged in 1% Tenofovir clinical gel at 37 °C.
Freshly excised tissue specimens were incubated while fully submerged in 1% Tenofovir gel and maintained at 5% CO2 and 37 °C for different amounts of time. After incubation, the tissue specimens were removed from the gel, washed five times, and subjected to the MTT assay. Results were computed as a tetrazolium reductase index (TR index; absorbance per mg tissue) by dividing the absorbance the formazan product at 570 nm by the dry weight of the tissue specimen. Control samples were fresh untreated tissue specimens (1.6 h post mortem). To obtain deactivated (dead) samples, tissue specimens were boiled in water for 2h to inactivate enzyme activity. Data shown represent mean ±SE of 3-4 replicates.
Figure 8.
time in tissue and the top fluid compartment in the Transwell assay.
Transwell assay experiments were performed to characterize the transport of Tenofovir into and through tissue after microbicide application. The Transwell setup was maintained at 37°C and 5% CO2 in a Heracell incubator for different amounts of time, ranging from 30 min to 6 h. After incubation, the tissue (7-mm diameter, 1-mm thickness) and fluids were isolated and stored at −80°C. Thawed tissue specimens and fluids were scanned. Quantification of Tenofovir concentrations was performed by interpolation, referencing to calibration curves of Tenofovir in tissue homogenates and fluids (shown in Figure 4). Each data point gives the median, and the smallest and largest measurements in the 3 independent experiments (n=3).
Figure 9.
Tenofovir concentrations (measured vs. computed) in the bottom fluid compartment in the Transwell assay.
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).
Figure 10.
Z-scanning Raman spectra for Tenofovir penetrating excised tissue in the Transwell assay.
7 porcine buccal tissue specimens were incubated under an isotonic solution of 1% Tenofovir in PBS for 6 h in the Transwell assay. After incubation, the tissue was stored at −80°C overnight to stop the drug permeation process. Thawed tissue specimens were subjected to confocal Raman scans at different depths beneath the tissue surface to quantify Tenofovir distribution within the tissue specimens. The Raman spectra were acquired at different z-axis depths using a Horiba Xplora confocal Raman microscope with an excitation wavelength of 785 nm and an Olympus 50x long-working distance objective lens (N.A. =0.5). The acquisition time was 240 s for each spectrum. All spectra were normalized with respect to the tissue Raman band at 643 cm-1 and offset vertically for clarity of presentation.
Figure 11.
depth in tissue in the
Transwell assay.
Confocal Raman scans were performed on tissue specimens that had been incubated under a fluid layer (containing 1% Tenofovir) in the Transwell assay for 6 h (Figure 10). After baseline correction, the intensity of the Tenofovir Raman band was normalized with respect to the intensity of the tissue band at 643 cm-1. Normalized intensities were background-subtracted and fitted into a respective calibration curve of Tenofovir in tissue homogenate to deduce the concentration vs. depth profiles. The Raman measurements showed a decline in concentration as the depth into the tissue increased. Data shown give mean ± standard error of the mean (n=7).
Figure 12.
Comparative measurement of Tenofovir concentration using Raman vs.
LC-MS/MS in solution and tissue.
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).
Figure 13.
Agreement of Raman measurements of Tenofovir Concentration (Horiba instrument) with gold-standard
measurements by LC-MS/MS.
Serial dilutions of Tenofovir in Ringer's solution were equilibrated with freshly excised tissue specimens for 24 h at 4 °C. The Tenofovir concentration in fluid before (initial) and after (final) incubation, and the final concentration in tissue, were evaluated by both techniques: (a) the correlations between Tenofovir concentration values in fluid and tissue obtained from the Raman spectroscopic method vs. those derived from the validated LC-MS/MS technique. The dashed line indicates a straight 45-degree line (y=x) passing through the origin of the axes. (b) Bland-Altman analysis for determining the levels of agreement between the two measurement methods of Tenofovir concentrations in fluid and tissue. This gives absolute differences between the two measurement methods in relation to their average values. Solid lines represent mean differences, and dashed lines indicate the 95% confidence intervals.