title: Quantitative analysis of microbicide concentrations in fluids, gels and tissues using confocal Raman spectroscopy.
abstract: Topical vaginal anti-HIV microbicides are an important focus in female-based strategies to prevent the sexual transmission of HIV. Understanding microbicide pharmacokinetics is essential to development, characterization and implementation of efficacious microbicide drug delivery formulations. Current methods to measure drug concentrations in tissue (e.g., LC-MS/MS, liquid chromatography coupled with tandem mass spectrometry) are highly sensitive, but destructive and complex. This project explored the use of confocal Raman spectroscopy to detect microbicide drugs and to measure their local concentrations in fluids, drug delivery gels, and tissues. We evaluated three candidate microbicide drugs: tenofovir, Dapivirine and IQP-0528. Measurements were performed in freshly excised porcine buccal tissue specimens, gel vehicles and fluids using two Horiba Raman microscopes, one of which is confocal. Characteristic spectral peak calibrations for each drug were obtained using serial dilutions in the three matrices. These specific Raman bands demonstrated strong linear concentration dependences in the matrices and were characterized with respect to their unique vibrational signatures. At least one specific Raman feature was identified for each drug as a marker band for detection in tissue. Sensitivity of detection was evaluated in the three matrices. A specific peak was also identified for tenofovir diphosphate, the anti-HIV bioactive product of tenofovir after phosphorylation in host cells. Z-scans of drug concentrations vs. depth in excised tissue specimens, incubated under layers of tenofovir solution in a Transwell assay, showed decreasing concentration with depth from the surface into the tissue. Time-dependent concentration profiles were obtained from tissue samples incubated in the Transwell assay, for times ranging 30 minutes - 6 hours. Calibrations and measurements from tissue permeation studies for tenofovir showed good correlation with gold standard LC-MS/MS data. These results demonstrate that confocal Raman spectroscopy holds promise as a tool for practical, minimally invasive, label-free measurement of microbicide drug concentrations in fluids, gels and tissues.
authors.affiliation: Department of Biomedical Engineering, Duke University, Durham, North Carolina, United States of America.
authors.forename: Oranat
authors.initials: O
authors.lastname: Chuchuen
authors.affiliation: Department of Biomedical Engineering, Duke University, Durham, North Carolina, United States of America.
authors.forename: Marcus H
authors.initials: MH
authors.lastname: Henderson
authors.affiliation: University of North Carolina Eshelman School of Pharmacy and University of North Carolina Center for AIDS Research, University of North Carolina, Chapel Hill, North Carolina, United States of America.
authors.forename: Craig
authors.initials: C
authors.lastname: Sykes
authors.affiliation: Department of Biomedical Engineering, Duke University, Durham, North Carolina, United States of America.
authors.forename: Min Sung
authors.initials: MS
authors.lastname: Kim
authors.affiliation: University of North Carolina Eshelman School of Pharmacy and University of North Carolina Center for AIDS Research, University of North Carolina, Chapel Hill, North Carolina, United States of America ; Department of Infectious Diseases, University of North Carolina School of Medicine, Chapel Hill, North Carolina, United States of America.
authors.forename: Angela D M
authors.initials: AD
authors.lastname: Kashuba
authors.affiliation: Department of Biomedical Engineering, Duke University, Durham, North Carolina, United States of America ; Department of Obstetrics and Gynecology, Duke University, Durham, North Carolina, United States of America.
authors.forename: David F
authors.initials: DF
authors.lastname: Katz
mesh_terms: D000818:Animals; D019380:Anti-HIV Agents / Q000493:pharmacokinetics* / Q000494:pharmacology*; D005260:Female; D013059:Spectrum Analysis, Raman; D013552:Swine
chemical_list: D019380:Anti-HIV Agents