Chromoblastomycosis is a chronic, neglected subcutaneous mycosis posing significant therapeutic challenges. A topical strategy combining terbinafine (TBF), an antifungal, with imiquimod (IMQ), a TLR-7/8 agonist immunomodulator, has emerged a promising alternative. However, no validated analytical method is currently available to simultaneously quantify both drugs in skin, which is crucial for novel formulation development. This study reports the development and validation of a simple HPLC method with fluorescence detection (excitation 236 nm, emission 340 nm) for the simultaneous determination of TBF and IMQ extracted from porcine skin. Separation was achieved on a C8 reversed-phase column (125 × 4.0 mm, 5 μm) using a mobile phase of methanol and water (60,40, v/v), both containing 0.1% formic acid at a flow rate of 0.8 mL/min. The method showed excellent linearity (r > 0.999) over 0.01-1.0 μg/mL for IMQ and 0.1-2.0 μg/mL for TBF. Intra- and inter-day precision demonstrated coefficients of variation below 5%, and recovery rates from skin (79-105%) confirmed accuracy. Limits of detection were 0.001 μg/mL for IMQ and 0.004 μg/mL for TBF, with quantification limits of 0.02 μg/mL and 0.16 μg/mL, respectively. This selective, sensitive, and reproducible method represents a valuable analytical tool for supporting the development and quality control of topical formulations for chromoblastomycosis and other fungal skin diseases.
Mikaella Costa de Sousa, M. P. C. Nogueira, M. Cunha-Filho et al.· Journal of chromatography. B...· 0 citations
Calcium hydroxyapatite (CaHA) is a well-established biocompatible material widely used to stimulate collagen formation in soft tissues; however, its clinical use is largely restricted to invasive deep dermal injection due to safety concerns associated with microstructured formulations, such as superficial nodule formation and vascular occlusion. Here, we report the development of a nanostructured, lactose-functionalized CaHA produced by nanospray drying, enabling safer and more versatile delivery strategies. Physicochemical properties were systematically characterized, and biological performance was evaluated in human dermal fibroblasts and co-culture with peripheral blood mononuclear cells (PBMCs). The resulting nanostructured CaHA (nanoCaHA) exhibited a submicron particle size (181.06 ± 23.83 nm), narrow polydispersity (PDI 0.23 ± 0.17), and a positive surface charge (+11.75 ± 3.63 mV), attributed to surface protonation during processing. NanoCaHA showed no cytotoxic, genotoxic, or irritant effects in vitro and induced a shift in fibroblast cell-cycle distribution consistent with enhanced proliferative activity. While both nano- and microCaHA increased total collagen production, nanoCaHA significantly upregulated COL1A1 and COL3A1 mRNA expression, particularly under PBMC co-culture conditions, outperforming a commercially available CaHA formulation. Moreover, nanoCaHA sustained collagen production for up to 5 days after treatment withdrawal. Scanning electron microscopy revealed persistent particulate structures closely associated with fibroblasts after washout, while energy-dispersive X-ray spectroscopy confirmed that these deposits were enriched in calcium and phosphorus, consistent with CaHA composition. Furthermore, skin permeation assays using porcine skin in Saarbrücken diffusion cells showed that the nanoCaHA formulation significantly increased calcium delivery into the skin (34.53 ± 6.64 μg/cm2) compared to endogenous control levels (23.43 ± 3.96 μg/cm2). Collectively, these findings demonstrate that nanoscale engineering of CaHA modulates cell-material interactions, supporting the development of safer superficial dermal and novel topical/transdermal delivery systems for regenerative aesthetics.
Elizabeth Cristina Iseke Bispo, Stefhani Martins de Barcelos, Jonad L A Contarato et al.· Journal of Biomedical Materi...· 0 citations
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