S-Protected Thiolated Chitosan versus Thiolated Chitosan as Cell Adhesive Biomaterials for Tissue Engineering.

Chitosan (Ch) and different Ch derivatives have been applied in tissue engineering (TE) because of their biocompatibility, favored mechanical properties, and cost-effectiveness. Most of them, however, lack cell adhesive properties that are crucial for TE. In this study, we aimed to design an S-protected thiolated Ch derivative exhibiting high cell adhesive properties serving as a scaffold for TE. 3-((2-Acetamido-3-methoxy-3-oxopropyl)dithio) propanoic acid was covalently attached to Ch via a carbodiimide-mediated reaction. Low-, medium-, and high-modified Chs (Ch-SS-1, Ch-SS-2, and Ch-SS-3) with 54, 107 and 140 µmol of ligand per gram of polymer, respectively, were tested. In parallel, three thiolated Chs, namely Ch-SH-1, Ch-SH-2, and Ch-SH-3, were prepared by conjugating N-acetyl cysteine to Ch at the same degree of modification to compare the effectiveness of disulfide versus thiol modification on cell adhesion. Ch-SS-1 showed better cell adhesion capability than Ch-SS-2 and Ch-SS-3. This can be explained by the more lipophilic surfaces of Ch-SS as a higher modification was made. Although Ch-SH-1, Ch-SH-2, and Ch-SH-3 were shown to be good substrates for cell adhesion, growth, and proliferation, Ch-SS polymers were superior to Ch-SH polymers in the formation of 3D cell cultures. Cryogels structured by Ch-SS-1 (SSg) resulted in homogeneous scaffolds with tunable pore size and mechanical properties by changing the mass ratio between Ch-SS-1 and heparin used as a cross-linker. SSg scaffolds possessing interconnected microporous structures showed good cell migration, adhesion, and proliferation. Therefore, Ch-SS can be used to construct tunable cryogel scaffolds that are suitable for 3D cell culture and TE.

Charge-Converting Nanoemulsions as Promising Retinal Drug and Gene Delivery Systems.

AIM: This study aimed to develop phosphatase-responsive ζ potential converting nanocarriers utilizing polyphosphate-coated cell-penetrating peptide (CPP)-decorated nanoemulsions (NEs) as a novel gene delivery system to retinal cells. METHODS: Poly-l-lysine (PLL) was first conjugated with oleylamine (OA) only at its carboxylic end to form the amphiphilic PLL-oleylamine (PLOA) conjugate. Afterward, NEs were loaded with PLOA prior to being coated with tripolyphosphate (TPP) to generate PLOA/TPP NEs. A plasmid containing a reporter gene for green fluorescent protein plasmid (pGFP) was complexed with cationic surfactants forming hydrophobic ion pairs that were loaded in the oily core of NEs. Phosphate removal, ζ potential conversion, and cytotoxicity of the system were evaluated. Cellular uptake and transfection efficiency were investigated in 661W photoreceptor-like cells via microscopic analysis, fluorescence spectroscopy, and flow cytometry. RESULTS: Dephosphorylation of PLOA/TPP NEs triggered by alkaline phosphatase (ALP) resulted in the exposure of positive amine groups on the surface of NE droplets and a notable conversion of the ζ potential from -22.4 to +8.5 mV. Cellular uptake of PLOA/TPP NEs performed on 661W photoreceptor-like cells showed a 3-fold increase compared to control NEs. Furthermore, PLOA/TPP NEs also showed low cytotoxicity and high transfection efficacy with ∼50% of cells transfected. CONCLUSIONS: Polyphosphate-coated CPP-decorated NEs triggered by ALP could be a promising nanosystem to efficiently deliver drugs and genetic materials to photoreceptor-like cells and other retinal cells for potential treatments of retinal diseases.

Charge converting nanostructured lipid carriers containing a cell-penetrating peptide for enhanced cellular uptake.

HYPOTHESIS: The aim of this study was the development of nanostructured lipid carriers (NLCs) decorated with a polycationic cell-penetrating peptide (CPP). A coating with polyphosphates (PP) enables charge conversion at target cells being triggered by the membrane bound enzyme intestinal alkaline phosphatase (IAP). EXPERIMENTS: The CPP, stearyl-nona-L-arginine (R9SA) was obtained by solid phase synthesis. Formed nanocarriers were characterized regarding size, polydispersity index, zeta potential and charge conversion in the presence of IAP and on Caco-2 cells. The BCS class IV drug saquinavir (SQV) was loaded into NLCs in different concentrations. Mucus diffusion ability of the NLCs was evaluated by the rotating tube method. Furthermore, cellular uptake was evaluated on Caco-2 cells and endosomal escape properties were investigated using erythrocytes. FINDINGS: All NLCs were obtained in a size range between 146 nm and 152 nm and a polydispersity index of 0.2. Incubation of PP coated PP-R9SA-NLCs with IAP led to a charge conversion from -41.8 mV to 6.4 mV (Δ48.2 mV). After four hours of incubation with IAP, phosphate release reached a plateau, indicating a faster polyphosphate cleavage than on Caco-2. Drug load and encapsulation efficiency of SQV was obtained up to 80.6% and 46.5 µg/mg. Mucus diffusion was increasing in the following rank order: R9SA-NLCs < blank NLCs < PP-R9SA-NLCs. R9SA-NLCs and PP-R9SA-NLCs increased the cellular uptake 15.6- and 13.2-fold, respectively, compared to the control NLCs. Erythrocytes interaction study revealed enhanced endosomal escape properties for R9SA-NLCs and PP-R9SA-NLCs when incubated with IAP.

Replacing PEG-surfactants in self-emulsifying drug delivery systems: Surfactants with polyhydroxy head groups for advanced cytosolic drug delivery.

AIM: Evaluation of different polyhydroxy surfaces in SEDDS to overcome the limitations associated with conventional polyethylene glycol (PEG)-based SEDDS surfaces for intracellular drug delivery. METHODS: Anionic, cationic and non-ionic polyglycerol- (PG-) and alkylpolyglucoside- (APG-) surfactant based SEDDS were developed and compared to conventional PEG-SEDDS. Particular emphasis was placed on the impact of SEDDS surface decoration on size and zeta potential, drug loading and protective effect, mucus diffusion, SEDDS-cell interaction and intracellular delivery of the model drug curcumin. RESULTS: After self-emulsification, SEDDS droplets sizes were within the range of 35-190 nm. SEDDS formulated with high amounts of long PEG-chain surfactants (>10 monomers) a charge-shielding effect was observed. Replacing PEG-surfactants with PG- and an APG-surfactant did not detrimentally affect SEDDS self-emulsification, payloads or the protection of incorporated curcumin towards oxidation. PG- and APG-SEDDS bearing multiple hydroxy functions on the surface demonstrated mucus permeation comparable to PEG-SEDDS. Steric hinderance and charge-shielding of PEG-SEDDS surface substantially reduced cellular uptake up to 50-fold and impeded endosomal escape, yielding in a 20-fold higher association of PEG-SEDDS with lysosomes. In contrast, polyhydroxy-surfaces on SEDDS promoted pronounced cellular internalisation and no lysosomal co-localisation was observed. This improved uptake resulted in an over 3-fold higher inhibition of tumor cell proliferation after cytosolic curcumin delivery. CONCLUSION: The replacement of PEG-surfactants by surfactants with polyhydroxy head groups in SEDDS is a promising approach to overcome the limitations for intracellular drug delivery associated with conventional PEGylated SEDDS surfaces.

Development and in vitro characterization of an oral self-emulsifying delivery system (SEDDS) for rutin fatty ester with high mucus permeating properties.

The aim of this study was to develop and evaluate a self-emulsifying delivery system (SEDDS) for oral rutin fatty ester administration and to improve its mucus permeating properties by the incorporation of the silicon polymer poly [dimethylsiloxane-co-(3-(2-(2-hydroxyethoxy)ethoxy)propyl]methylsiloxane] (PDMSHEPMS) in the formulation. In order to increase the lipophilicity of the flavonoid and to dissolve it in SEDDS, enzymatic acylation of rutin with lauric acid was catalyzed by lipase from Candida antarctica in acetone. Different formulations were evaluated regarding their emulsifying properties and ability to dissolve the rutin ester. Suitable SEDDS was chosen and characterized regarding droplet size, polydispersity index, and zeta potential. The rutin fatty ester was loaded into SEDDS to 7% (w/w). Different concentrations of PDMSHEPMS were incorporated in SEDDS for following mucus permeation studies. Formulation with 10% of PDMSHEPMS showed 1.9-fold increase in mucus permeation compared to the formulation without PDMSHEPMS. Furthermore, the formulation with 10% of PDMSHEPMS showed a significant increase in mucus permeation compared with rutin fatty ester without formulation. According to these results, SEDDS containing PDMSHEPMS might be a promising strategy to increase the oral bioavailability of rutin.

Synthesis and Characterization of Thiolated PVP-Iodine Complexes: Key to Highly Mucoadhesive Antimicrobial Gels.

The aim of this study was to synthesize iodine containing polymeric excipients for mucosal treatment of microbial infection exhibiting a prolonged mucosal residence time by forming an adhesive gel on the mucosal surface. In order to achieve this aim, 2-(2 acryloylamino-ethyldisulfanyl)-nicotinic acid (ACENA) was copolymerized with N-vinylpyrrolidone (NVP) to obtain thiolated polyvinylpyrrolidone (PVP) for complexation with iodine. The average molecular mass of different thiolated PVP variants was determined by size exclusion chromatography. The structure of thiolated PVP was confirmed by 1H NMR. Thiolated PVP variants were characterized for thiol content, cytotoxicity, iodine loading capacity, rheological behavior, and adhesion time on mucosa. The highest achieved degree of thiolation was 610 ± 43 µmol/g, and the maximum recorded iodine loading was 949 ± 31 µmol/g of polymer. Thiolated PVP variants (0.5% m/v) showed no toxicity after incubation on Caco-2 cells for the period of 3 and 24 h, respectively. Thiolated PVP and thiolated PVP-iodine complexes exhibited a 5.4- and 4.4-fold increased dynamic viscosity in porcine mucus in comparison to PVP and PVP-iodine complex, respectively. Compared to PVP and PVP-iodine complex thiol-functionalized PVP and PVP-iodine complexes demonstrated significantly prolonged attachment to mucosal surface over a period of 3 h. Thiol functionalized PVP proved to be a promising novel excipient for complexation with iodine and to exhibit strongly improved mucoadhesive properties.