Enhanced Mucoadhesion of Thiolated ß-Cyclodextrin by S-Protection with 2-Mercaptoethanesulfonic Acid.

This study aimed at designing an S-protected thiolated ß-cyclodextrin (ß-CD) exhibiting enhanced mucoadhesive properties. The native ß-CD was thiolated with phosphorus pentasulfide resulting in a thiolated ß-CD (ß-CD-SH) and subsequently S-protected with 2-mercaptoethanesulfonate (MESNA) to form ß-CD-SS-MESNA. The structure of the novel excipient was confirmed by 1H NMR and Fourier-transform infrared spectroscopy. The sulfhydryl content of ß-CD-SH, determined by Ellman's test, was 2281.00 ± 147 µmol/g, and it was decreased to 45.93 ± 19.40 µmol/g by S-protection. Due to thiolation and S-protection, the viscosity of the mixture of mucus with ß-CD-SH and ß-CD-SS-MESNA increased 1.8 and 4.1-fold, compared to native ß-CD, respectively. The unprotected ß-CD-SH diffused to a lesser extent into the mucus than native ß-CD, while S-protected ß-CD-SS-MESNA showed the highest mucodiffusion among the applied CDs. A 1.5- and 3.0-fold higher cellular uptake of ß-CD-SH and ß-CD-SS-MESNA, compared to the native one, was established on Caco-2 cell line by flow cytometry, respectively, causing slightly decreased cell viability. On account of the enhanced mucoadhesion, this higher cellular uptake does not affect the application potential of ß-CD-SS-MESNA as an oral drug delivery system since the carrier remains in the mucus and does not reach the underlying epithelial layer. According to these results, the S-protection of ß-CD-SH with MESNA promotes improved mucodiffusion, strong mucoadhesion, and prolonged mucosal residence time.

PEG vs. zwitterions: How these surface decorations determine cellular uptake of lipid-based nanocarriers.

AIM: To evaluate the impact of polyethylene glycol (PEG) and zwitterionic surface decoration of lipid-based nanocarriers (NC) on cellular uptake. METHODS: Anionic, neutral and cationic zwitterionic lipid-based NCs based on lecithin were compared with conventional PEGylated lipid-based NCs regarding stability in biorelevant fluids, interaction with endosome mimicking membranes, cytocompatibility, cellular uptake and permeation across intestinal mucosa. RESULTS: PEGylated and zwitterionic lipid-based NCs exhibited a droplet size between 100 and 125 nm with a narrow size distribution. For the PEGylated and zwitterionic lipid-based NCs only minor alterations in size and PDI in fasted state intestinal fluid and mucus containing buffer were observed, demonstrating similar bioinert properties. Erythrocytes interaction studies revealed enhanced endosomal escape properties for zwitterionic lipid-based NCs compared to PEGylated lipid-based NCs. For the zwitterionic lipid-based NCs negligible cytotoxicity on Caco-2 and HEK cells, even in the highest tested concentration of 1 % (v/v) was recorded. The PEGylated lipid-based NCs showed a cell survival of ≥75 % for concentrations ≤0.05 % on Caco-2 and HEK cells, which was considered as non-toxic. For the zwitterionic lipid-based NCs up to 60-fold higher cellular uptake on Caco-2 cells was determined compared to PEGylated lipid-based NCs. For the cationic zwitterionic lipid-based NCs the highest cellular uptake with 58.5 % and 40.0 % in Caco-2 and HEK cells, respectively, was determined. The results were confirmed visually by life cell imaging. Ex-vivo permeation experiments using rat intestinal mucosa demonstrated up to 8.6-fold enhanced permeation of the lipophilic marker coumarin-6 in zwitterionic lipid-based NCs compared to the control. Up to 6.9-fold enhanced permeation of coumarin-6 in neutral zwitterionic lipid-based NCs compared to the PEGylated counterpart was recorded. CONCLUSION: The replacement of PEG surfactants with zwitterionic surfactants is a promising approach to overcome the drawbacks of conventional PEGylated lipid-based NCs regarding intracellular drug delivery.

Phosphatase-degradable nanoparticles: A game-changing approach for the delivery of antifungal proteins.

HYPOTHESIS: Polyphosphate nanoparticles as phosphatase-degradable carriers for Penicillium chrysogenum antifungal protein (PAF) can enhance the antifungal activity of the protein against Candida albicans biofilm. EXPERIMENTS: PAF-polyphosphate (PP) nanoparticles (PAF-PP NPs) were obtained through ionic gelation. The resulting NPs were characterized in terms of their particle size, size distribution and zeta potential. Cell viability and hemolysis studies were carried out in vitro on human foreskin fibroblasts (Hs 68 cells) and human erythrocytes, respectively. Enzymatic degradation of NPs was investigated by monitoring release of free monophosphates in the presence of isolated as well as C. albicans-derived phosphatases. In parallel, shift in zeta potential of PAF-PP NPs as a response to phosphatase stimuli was determined. Diffusion of PAF and PAF-PP NPs through C. albicans biofilm matrix was analysed by fluorescence correlation spectroscopy (FCS). Antifungal synergy was evaluated on C. albicans biofilm by determining the colony forming units (CFU). FINDINGS: PAF-PP NPs were obtained with a mean size of 300.9 ± 4.6 nm and a zeta potential of -11.2 ± 2.8 mV. In vitro toxicity assessments revealed that PAF-PP NPs were highly tolerable by Hs 68 cells and human erythrocytes similar to PAF. Within 24 h, 21.9 ± 0.4 µM of monophosphate was released upon incubation of PAF-PP NPs having final PAF concentration of 156 µg/ml with isolated phosphatase (2 U/ml) leading to a shift in zeta potential up to -0.7 ± 0.3 mV. This monophosphate release from PAF-PP NPs was also observed in the presence of C. albicans-derived extracellular phosphatases. The diffusivity of PAF-PP NPs within 48 h old C. albicans biofilm matrix was similar to that of PAF. PAF-PP NPs enhanced antifungal activity of PAF against C. albicans biofilm decreasing the survival of the pathogen up to 7-fold in comparison to naked PAF. In conclusion, phosphatase-degradable PAF-PP NPs hold promise as nanocarriers to augment the antifungal activity of PAF and enable its efficient delivery to C. albicans cells for the potential treatment of Candida infections.

Thiolated α-cyclodextrin: The likely smallest drug carrier providing enhanced cellular uptake and endosomal escape.

This study aimed to evaluate the effect of thiolated α-cyclodextrin (α-CD-SH) on the cellular uptake of its payload. For this purpose, α-CD was thiolated using phosphorous pentasulfide. Thiolated α-CD was characterized by FT-IR and 1H NMR spectroscopy, differential scanning calorimetry (DSC), and powder X-ray diffractometry (PXRD). Cytotoxicity of α-CD-SH was evaluated on Caco-2, HEK 293, and MC3T3 cells. Dilauryl fluorescein (DLF) and coumarin-6 (Cou) serving as surrogates for a pharmaceutical payload were incorporated in α-CD-SH, and cellular uptake was analyzed by flow cytometry and confocal microscopy. Endosomal escape was investigated by confocal microscopy and hemolysis assay. Results showed no cytotoxic effect within 3 h, while dose-dependent cytotoxicity was observed within 24 h. The cellular uptake of DLF and Cou was up to 20- and 11-fold enhanced by α-CD-SH compared to native α-CD, respectively. Furthermore, α-CD-SH provided an endosomal escape. According to these results, α-CD-SH is a promising carrier to shuttle drugs into the cytoplasm of target cells.