There remains a paucity of methodological tools to determine the biodistribution of vaccine antigens. In response to this, we established a near-infrared (NIR) imaging method using a NIR fluorophore, ZW800-1C, conjugated with different sizes of vaccine antigens that allows for real-time monitoring of the fate of delivered vaccines in vivo. The fluorescent signal observed using the system after a model vaccine injection in mice recapitulated the size-dependent transport of the vaccine into the secondary lymphoid tissue. This methodology can be broadly applied for optimization of formulations and safety evaluation of clinical vaccines.
We recently developed a digital cytometric assay using plasmonic nanoparticles to quantify the bound ovarian cancer marker (CA125) on single cells. We demonstrate the quantification capability of our novel digital cytometry method by discriminating the different MUC16 binding levels on the PBMCs between healthy donors and EOC patients. Furthermore, we tracked the amount of surface-bound MUC16 on the EOC patient’s PBMCs over 17 months with 1-month time intervals. Initial data from our cytometric technique shows it to be a quantitative tool that can provide new information connecting leukocyte-bound MUC16 to the time course of ovarian cancer.
Our group has recently developed a method for characterizing distribution of a topical drug within skin using two-photon fluorescence lifetime imaging (FLIM) and phasor analysis. Here, we expand on this work by describing a multimodal approach for rapidly visualizing multiple components in tissue using FLIM and coherent Raman imaging (CRI). By employing a non-Euclidian FLIM phasor analysis for a three-component system informed with the vibrational signature of one of the components retrieved with CRI, we were able to semi-quantitatively describe the spatial distribution of drugs in tissue with molecular specificity and cellular resolution.
We recently proposed a method for selective visualization of topical drug distribution within human facial skin using two-photon fluorescence lifetime imaging along with non-Euclidean phasor analysis as a pharmacokinetics and pharmacodynamics imaging toolkit. In order to improve the efficacy of topical drug delivery toward the treatment of inflammatory acne, we have now developed a combination topical gel containing both minocycline and a retinoid. Since both drugs have unique fluorescence lifetimes compared to skin, we were able to selectively visualize the distribution of minocycline and the retinoid within ex vivo human facial skin while isolating the contributions of the three components.
The safety and efficacy of an investigational topical minocycline gel (BPX-01) has recently been studied in a Phase 2b trial for the treatment of acne vulgaris. As part of the drug development process, there was a need to determine if minocycline was delivered to the target tissue compartments, including the epidermis, hair follicle, and the sebaceous gland. While it was easier to demonstrate delivery on an ex vivo human skin model with an infinite dose, it was initially challenging to verify low-dose delivery with conventional fluorescence microscopy due to the high autofluorescence inherent to human skin. An integrating sphere screening approach was implemented along with conventional fluorescence microscopy to quantitatively and qualitatively assess endogenous fluorescence concurrently from numerous human facial skin specimens. Donor tissues were cut into 50-µm frozen sections, mounted onto microscope slides, and positioned on an inverted fluorescence microscope, sandwiched between the microscope’s 40x high NA objective lens and an external integrating sphere. The tissue sections were illuminated with UV excitation centered at 386 nm. For the first time, it was found that random samples from >40 human facial skin donors produced at least 5× differential in measurable autofluorescence. This observation has significant implications for the use of 2PEF microscopy and FLIM to visualize/quantify drug distribution; the endogenous autofluorescence may limit the detectability of the minocycline signature. Our studies indicated that a single daily dose of BPX-01 was detected in low autofluorescence skin specimens with FLIM, thus validating a novel imaging modality for future pharmacokinetic studies.
Oral minocycline has been the standard of care for the treatment of non-nodular moderate to severe inflammatory acne vulgaris due to its inhibitory effects on the acne-causing Propionibacterium acnes bacterium and its anti-inflammatory properties, Despite the availability of an oral dosage form since 1966, a commercial topical minocycline remains elusive because of the challenges in stabilizing the active pharmaceutical ingredient (API) in a liquid/semisolid while ensuring sufficient uptake into targeted lesions. Recently, an investigative topical minocycline gel (BPX-01) has been developed to address the unmet needs for localized and targeted delivery while minimizing the risks of systemic side effects. Earlier preclinical studies pertaining to transepidermal delivery of the API had depended on semi-infinite doses of the 1%, 2% and 4% formulations to elicit enough fluorescence yield. We have subsequently shown evidence of minocycline delivery of 1% and 4% BPX-01 into the pilosebaceous unit of ex vivo human facial skin specimens dosed with about 2.5× daily dose using two-photon excitation fluorescence microscopy. In this study, we demonstrated another novel approach to identifying minocycline fluorescence signature using fluorescence lifetime imaging microscopy (FLIM) with phasor analysis. It was found that for a single daily dose and with FLIM, minocycline was consistently noted in the epidermis and hair follicle, with some incidence in the sebaceous gland for both 1% and 2% BPX-01. These observations corroborated with the recent success of a Phase 2b dose-finding study, with 2% BPX-01 meeting the primary endpoint of lesion reduction at week 12 with statistical significance over the vehicle.
One of the primary functions of human skin is to provide a mechanical barrier and interface with the outside world, owing to its unique structure and composition. Indeed, the most superficial layer of the epidermis, the stratum corneum, is a selectively porous structure composed of thin layers of protein-rich corneocytes joined together by lipids such as ceramides. The overall impermeability of the stratum corneum is crucial for homeostasis, but also hampers the penetration of beneficial topical agents into the skin. Specifically, hydrophilic compounds typically do not permeate through the epidermis, while hydrophobic compounds can readily be delivered through lipid-containing routes such as sebaceous gland ducts or the spacing between corneocytes. A proper understanding of stratum corneum structure and composition is therefore of great benefit for the design of topical formulations in order to properly optimize the delivery of active compounds to the skin. To this aim, coherent Raman scattering imaging techniques including both coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS) microscopies can be used to study the chemical composition and structure of the stratum corneum, as these modalities are sensitive to unique vibrational modes of specific chemical groups such as lipids, proteins, and water. These metrics can further be used to measure uptake and efficacy of topical compounds in order to optimize formulation design.
Acne vulgaris is a common chronic skin disease in teenagers and young adults. Minocycline, an antibiotic, has thus far been widely utilized to treat acne, but only via oral administration. Recently, a topical minocycline gel (BPX-01) was developed to directly deliver minocycline to the epidermis and pilosebaceous unit to achieve localized treatment with lower doses of drug. In order to evaluate the effectiveness of topical drug delivery in terms of pharmacokinetics and pharmacodynamics, visualization and quantification of drug within a biological tissue is essential. As minocycline is a known fluorophore, we demonstrate a method for visualization and quantification of minocycline within human skin tissue by utilizing a phasor approach to fluorescence lifetime microscopy (FLIM). In phasor analysis of FLIM, the fluorescence decay trace from each pixel in the FLIM image is plotted as a single point in the phasor plot. Since every fluorophore has a specific decay trace, we can identify a specific molecule by its position in the phasor plot. To demonstrate the feasibility of this visualization and quantification method, the human facial skin samples treated with various concentrations of BPX-01 were investigated using the phasor approach to FLIM. The unique signature of minocycline in FLIM phasor analysis was successfully differentiated from the endogenous fluorescence of human tissue. Furthermore, by sorting the individual pixels of minocycline signature in FLIM image, the distribution of minocycline within human facial skin can be visualized and quantified. Based on these results, we believe that the visualization and quantification method using a phasor approach to FLIM can play an important role in future pharmacokinetics and pharmacodynamics analyses.
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