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.

Synthesis and evaluation of sulfosuccinate-based surfactants as counterions for hydrophobic ion pairing.

Hydrophobic ion pairing is a promising strategy to raise the lipophilic character of therapeutic peptides and proteins. In past studies, docusate, an all-purpose surfactant with a dialkyl sulfosuccinate structure, showed highest potential as hydrophobic counterion. Being originally not purposed for hydrophobic ion pairing, it is likely still far away from the perfect counterion. Thus, within this study, docusate analogues with various linear and branched alkyl residues were synthesized to derive systematic insights into which hydrophobic tail is most advantageous for hydrophobic ion pairing, as well as to identify lead counterions that form complexes with superior hydrophobicity. The successful synthesis of the target compounds was confirmed by FT-IR, 1H-NMR, and 13C-NMR. In a screening with the model protein hemoglobin, monostearyl sulfosuccinate, dioleyl sulfosuccinate, and bis(isotridecyl) sulfosuccinate were identified as lead counterions. Their potential was further evaluated with the peptides and proteins vancomycin, insulin, and horseradish peroxidase. Dioleyl sulfosuccinate and bis(isotridecyl) sulfosuccinate significantly increased the hydrophobicity of the tested peptides and proteins determined as logP or lipophilicity determined as solubility in 1-octanol, respectively, in comparison to the gold standard docusate. Dioleyl sulfosuccinate provided an up to 8.3-fold higher partition coefficient and up to 26.5-fold higher solubility in 1-octanol than docusate, whereas bis(isotridecyl) sulfosuccinate resulted in an up to 6.7-fold improvement in the partition coefficient and up to 44.0-fold higher solubility in 1-octanol. The conjugation of highly lipophilic alkyl tails to the polar sulfosuccinate head group allows the design of promising counterions for hydrophobic ion pairing. STATEMENT OF SIGNIFICANCE: Hydrophobic ion pairing enables efficient incorporation of hydrophilic molecules into lipid-based formulations by forming complexes with hydrophobic counterions. Docusate, a sulfosuccinate with two branched alkyl tails, has shown highest potential as anionic hydrophobic counterion. As it was originally not purposed for hydrophobic ion pairing, its structure is likely still far away from the perfect counterion. To improve its properties, analogues of docusate with various alkyl tails were synthesized in the present study. The investigation of different alkyl residues allowed to derive systematic insights into which tail structures are most favorable for hydrophobic ion pairing. Moreover, the lead counterions dioleyl sulfosuccinate and bis(isotridecyl) sulfosuccinate bearing highly lipophilic alkyl tails provided a significant improvement in the hydrophobicity of the resulting complexes.

Size shifting of solid lipid nanoparticle system triggered by alkaline phosphatase for site specific mucosal drug delivery.

We aim to prepare a size-shifting nanocarrier for site-targeting mucosal drug delivery that can penetrate through mucus gel layer and remain close to the absorption membrane. As nanocarriers can be engineered to penetrate mucus but they can also back diffuse into outer mucus regions, a size shifting to micron range once they have reached the absorption membrane would prevent back-diffusion effect and extend drug release over a long period of time. For this purpose, we loaded solid lipid nanoparticles (SLN) with a phosphate ester surfactant and octadecylamine. Alkaline phosphatase (AP), a membrane bound enzyme was for the first time utilized as an in situ partner for triggering the size conversion at epithelial cell surface. Having the size of ~120 nm, SLN with hydrophilic and phosphate-decorated shells were shown to penetrate through mucus gel and form aggregates above cell layer surface. Aggregates of 5-8 µm were formed due to interparticle interactions induced by enzymatic phosphate removal after ~30 min in contact with isolated AP. The developed SLN system could be a potential tool for mucosal drug delivery to AP-expressing tissues like colon, lung, cervix, vagina and some mucus-secreting tumors.

Polyphosphate coatings: A promising strategy to overcome the polycation dilemma.

AIM: It was the aim of this study to develop a zeta potential changing drug delivery system by decorating lipid-based nanocarriers with a polycationic cell penetrating peptide (CPP) and subsequently masking these cationic substructures with polyphosphates. METHODS: In order to anchor the CPP poly-l-lysine (PLL) on the surface of the oily droplets of an o/w nanoemulsion, stearic acid was covalently attached to the peptide. The resulting CPP-decorated oily droplets were coated with phytic acid and tripolyphosphate. The elimination of these polyphosphates due to cleavage by alkaline phosphatase was monitored by the release of monophosphate from the surface of the nanocarriers, by the change in zeta potential and by cellular uptake studies on Caco-2 cells. RESULTS: Polyphosphate coated PLL-decorated nanocarriers exhibited a pronounced conversion of zeta potential from -14.1 mV to +4.2 mV in case of tripolyphosphate coated nanocarriers and from -9.9 mV to -2.6 mV in case of phytic acid coated nanocarriers. The cellular uptake on Caco-2 cells of the polyphosphate coated nanocarriers was 4-fold improved compared to the control nanocarriers. Furthermore, confocal images showed that the majority of nanodroplets distributed in cytoplasm not being internalized into lysosomes. CONCLUSION: Polyphosphate coating of CPP-decorated nanocarriers seems to be a promising and simple strategy to overcome the polycation dilemma.