Sofosbuvir (SOF) and daclatasvir (DCS) are newly discovered anti-hepatitis C drugs that have direct antiviral activity. A novel and simple high-performance thin-layer chromatography (HPTLC) method was designed for simultaneous determination of SOF and DCS in miscellaneous matrices. The method adopts coupling HPTLC with dual wavelength spectrodensitometry. Consequently, this enabled sensitive, specific and cost-effective determination of the SOF-DCS mixture. The developed HPTLC procedure is based on a simple liquid–liquid extraction, enrichment of the analytes and subsequent chromatographic separation with UV detection. Separations were performed on HPTLC silica gel 60 F254 aluminum plates with a mobile phase consisting of ethyl acetate–isopropanol (85:15, v/v). Dual wavelength scanning was carried out in the absorbance mode at 265 and 311 nm for SOF and DCS, respectively. The linear ranges were 40–640 and 20–320 ng band−1 for SOF and DCS, respectively with correlation coefficients of ≥0.9997. The detection limits were 11.3 and 6.5 ng band−1 for SOF and DCS, respectively indicating high sensitivity of the proposed method. Consequently, this permits in vitro and in vivo application of the proposed method in human plasma with good percentage recovery (94.1-103.5%). Validation parameters were assessed according to ICH guidelines and US-FDA guidelines. Furthermore, the application was extended to analysis of SOF and DCS in their pharmaceutical formulations.

Poly(ethylene glycol)-b-poly(propyl methacrylate-co-methacrylic acid) (PEG-b-P(PrMA-co-MAA) can be complexed with poly(amido amine) (PAMAM) dendrimers and nucleic acids to form pH-responsive nanosized core-shell type polyion complex micelles (PICMs). These PICMs have the ability to lose their shell and release the PAMAM/nucleic acid core under mildly acidic conditions such as those encountered in the endosomal compartment. In this work, pH-sensitive PICMs composed of PEG-b-P(PrMA-co-MAA), different PAMAMs, and siRNAs were prepared and characterized. These micelles had mean diameters ranging from 50 to 100 nm depending on the structure of the polycationic component. In order to trigger PICM uptake by receptor-mediated endocytosis, the micelles were decorated with an antibody fragment directed against the transferrin receptor (anti-CD71). The targeting ligand was stably conjugated to a semi-telechelic amino-PEG-b-P(PrMA-co-MAA) via a maleimide/activated ester bifunctional linker, yielding up to 60%–80% functionalization of the maleimide groups. The cellular uptake of the micelles was assessed on human prostate cancer cells (PC-3) via flow cytometry. Native PICMs and micelles bearing a non-specific antibody fragment were taken up to the same extent with a low efficiency, whereas anti-CD71 Fab′-decorated PICMs exhibited significantly higher uptake. The capacity of the targeted, siRNA-loaded, PICMs to downregulate the expression of the Bcl-2 anti-apoptotic oncoprotein was investigated using the appropriate unmodified or 2′-modified (2′F-RNA and 2′F-ANA) siRNA sequence. Bcl-2 mRNA and protein levels were greatly reduced when the cells were transfected with anti-CD71 decorated PICMs. Optimal silencing was achieved with the chemically modified siRNA. These data suggest that combining optimized siRNA chemistry with an effective delivery system can potentiate the activity of siRNA, thereby potentially reducing the total dose of carrier required to achieve a pharmacological effect.

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