Chitosan-maleic acid conjugate as potential excipient candidate for oral drug delivery?

Aim: To develop and evaluate chitosan-maleic acid conjugate. Methods: Maleic anhydride was attached to chitosan backbone via amide bond formation resulting in chitosan-maleic acid. After characterization of the product via 1H nuclear magnetic resonance, attenuated total reflectance-Fourier transform IR spectroscopy and 2,4,6-trinitrobenzenesulfonic acid assay, examination of mucoadhesion assessment was carried out. Results: The conjugate presented 44.91% modification and no toxicity could be observed after 1 day of incubation. Mucoadhesive properties exhibited 40.97-fold, 13.31-fold and 9.07-fold increase in elastic modulus, dynamic viscosity and viscous modulus, respectively. Moreover, detachment time was increased in 44.44-fold. Conclusion: Chitosan-maleic acid demonstrated enhanced in mucoadhesive properties resulting in biocompatibility. Therefore, potent candidates as polymeric excipients for oral drug delivery could be developed over corresponding chitosan.

Biodegradable arginine based steroid-surfactants: Cationic green agents for hydrophobic ion-pairing.

The aim of this study was to evaluate the safety and efficacy for hydrophobic ion-pairing of surfactants based on arginine (Arg). The prepared Arg-cholesteryl ester (ACE) and Arg-diosgenyl ester (ADE) were characterized regarding solubility, pKa, critical micellar concentration (CMC), biodegradability as well as membrane- and aquatic toxicity using DOTAP as reference. The ability for hydrophobic ion-pairing was evaluated and the lipophilicity of formed complexes was determined. NMR, FT-IR and MS confirmed successful synthesis of Arg-surfactants. The slightly soluble single-charged Arg-surfactants (pH < pKa3 (ACE = 10.42 ± 0.52; ADE = 10.38 ± 0.27)) showed CMCs of 27.17 µM for ACE and 35.67 µM for ADE. CMCs of the sparingly soluble double-charged species (pH < pKa2 (ACE = 5.30 ± 0.20; ADE = 5.55 ± 0.06)) were determined at concentrations of ≥ 250 µM for ACE and ≥ 850 µM for ADE. The enzymatic- and environmental biodegradability was proven by an entire cleavage of Arg-surfactants within 24 h, whereas DOTAP remained stable. Arg-surfactants exhibited lower membrane- (> 2-fold) and aquatic toxicity (> 15-fold) than DOTAP. The complexes formed with Arg-surfactants and insulin showed higher lipophilicity than the DOTAP-complex. According to these results, Arg-surfactants might be a promising safe tool for the delivery of peptide drugs.

Entirely S-protected thiolated hydroxyethylcellulose: Design of a dual cross-linking approach for hydrogels.

AIM: The aim of this study was the synthesis and evaluation of entirely S-protected thiolated hydroxyethylcellulose (HEC) with low and high viscosity, as well as thiolated poly-L-lysine (poly-L-Lys) used as dual-acting ionic as well as thiol-disulfide exchange mediated cross-linking hydrogel. METHODS: Bis(mercaptosuccinic acid) was covalently attached to low and high viscous HECs via Fisher esterification, obtaining S-protected polymers. Poly-L-Lys-cysteine was synthesized via amidation of poly-L-Lys-HBr with cysteine (Cys). Thiolated polymers were examined in terms of cytotoxicity and rheological behavior of hydrogels containing these thiomers was evaluated with a cone-plate rheometer. RESULTS: Thiomers showed less cytotoxicity compared to the corresponding unmodified polymers. Rheological studies showed that cross-linking occurred between the two polymers via thiol-disulfide exchange reactions facilitated by the complementary charges. Employing poly-L-Lys-Cys in a concentration of either 0.5 or 5% (m/v) resulted in a 34.5-fold or 17.3-fold as well as a 53.6-fold or 29.6-fold improvement in dynamic viscosity within 5 min at 37 °C on S-protected thiolated low and high viscous HEC, compared to the corresponding unmodified HECs, respectively. CONCLUSION: By the combination of anionic S-protected thiolated polymers with a cationic thiolated polymer, dual-acting hydrogels exhibiting a time dependent increase in viscosity can be designed.

Zeta potential changing nanoemulsions based on a simple zwitterion.

HYPOTHESIS: Simple zwitterions used as auxiliary agents might have the potential to change the zeta potential of oil-in-water (o/w) nanoemulsions on the mucosa. EXPERIMENTS: The zwitterion phosphorylated tyramine (p-Tyr) was synthesized by phosphorylation of Boc-tyramine (Boc-Tyr) using phosphoryl chloride (POCl3). It was incorporated with 2% (m/v) in a self-emulsifying lipophilic phase comprising Captex 35, Cremophor EL, Capmul MCM and glycerol 85 at a ratio of 30:30:30:10 v/v. Phosphate release and resulting change in zeta potential were evaluated by incubating p-Tyr containing nanoemulsion with isolated intestinal alkaline phosphatase (AP). Mucus permeating behavior was evaluated across mucus obtained from porcine small intestinal mucosa. Subsequently, cellular uptake studies were accomplished on Caco-2 cells. FINDINGS: The p-Tyr loaded nanoemulsion exhibited a mean droplet size of 43 ± 1.7 nm and zeta potential of -8.40 mV. Phosphate moieties were rapidly cleaved from p-Tyr loaded nanoemulsions after incubation with isolated AP resulting in a shift in zeta potential from -8.40 mV to +1.2 mV. p-Tyr loaded nanoemulsion revealed a significantly (p ≤ 0.001) improved mucus permeation compared to the same nanoemulsion having been pre-treated with AP. Cellular uptake of the zeta potential changing oily droplets was 2.4-fold improved. Phosphorylated zwitterions seem to be an alternative to cationic surfactants and considered as promising auxiliary agents for zeta potential changing nanoemulsions.

Thiolated chitosans: Are Cys-Cys ligands key to the next generation?

The potential of Cys-Cys ligands for the development of a novel type of S-protected thiomers was evaluated. S-protected thiomers chitosan-N-acetylcysteine-mercaptonicotinamide (CS-NAC-MNA) and chitosan-N-acetylcysteine-N-acetylcysteine (CS-NAC-NAC) were synthesized and characterized. Viscosity of polymers in presence of various concentrations of S-amino acids was monitored. Mucoadhesive properties were evaluated. FT-IR characterization confirmed the covalent attachment of NAC-MNA and NAC-NAC. Attached sulfhydryl groups were found in the range of 550 µmol/g. In the presence of amino acids bearing a free thiol group viscosity of both polymers increased. This increase in viscosity depended on the amount of added free thiols. Maximum force required to detach CS-NAC-MNA and CS-NAC-NAC from porcine intestinal mucosa was 1.4- and 2.7-fold higher than that required for chitosan, respectively. CS-NAC-MNA adhered up to 3 h, whereas CS-NAC-NAC adhered even for 8 h on this mucosa. Accordingly, the Cys-Cys substructure could be identified as highly potent ligand for the design of mucoadhesive polymers.

Lipophilic Arginine Esters: The Gateway to Preservatives without Side Effects.

This study hypothesized that long carbon chain cationic arginine (Arg) esters can be considered as toxicologically harmless preservatives. Arg-esters with C18 and C24 carbon chains, namely, arginine-oleate (Arg-OL) and arginine-decyltetradecanoate (Arg-DT), were synthesized. Structures were confirmed by FT-IR, 1H NMR, and mass spectroscopy. Both Arg-esters were tested regarding hydrophobicity in terms of log Poctanol/water, critical micelle concentration (CMC), biodegradability, cytotoxicity, hemolysis, and antimicrobial activity against Escherichiacoli (E. coli), Staphylococcusaureus (S. aureus), Bacillussubtilis (B. subtilis), and Enterococcusfaecalis (E. faecalis). Log Poctanol/water of arginine was raised from -1.9 to 0.3 and 0.6 due to the attachment of C18 and C24 carbon chains, respectively. The critical micelle concentration of Arg-OL and Arg-DT was 0.52 and 0.013 mM, respectively. Both Arg-esters were biodegradable by porcine pancreatic lipase. In comparison to the well-established antimicrobials, benzalkonium chloride (BAC) and cetrimide, Arg-esters showed significantly less cytotoxic and hemolytic activity. Both esters exhibited pronounced antimicrobial properties against Gram-positive and Gram-negative bacteria comparable to that of BAC and cetrimide. The minimum inhibitory concentration (MIC) of Arg-esters was <50 µg mL-1 against all tested microbes. Overall, results showed a high potential of Arg-esters with long carbon chains as toxicologically harmless novel preservatives.

Phosphorylated PEG-emulsifier: Powerful tool for development of zeta potential changing self-emulsifying drug delivery systems (SEDDS).

AIM: It was the aim of this study to synthesize a phosphorylated emulsifier possessing a PEG-linker for establishment of a potent zeta potential changing system in self-emulsifying drug delivery systems (SEDDS). METHODS: N,N'-Bis(polyoxyethylene)oleylamine (POA) was phosphorylated utilizing pyrophosphoric acid. Successful synthesis of POA bisphosphate (POAP) was confirmed by NMR and HR CS MAS. After incorporation of 1% POAP into SEDDS (Kolliphor RH 40, Capmul PG-8, Labrafac Lipophile WL 1349, Labrafac PG; 30/20/20/30, v/v), according emulsions were incubated with intestinal alkaline phosphatase (IAP) and the zeta potential was measured. Additionally, the amount of released phosphate upon incubation with IAP or on Caco-2 cells was quantified by malachite green assay. Finally, cell viability studies on Caco-2 cells were performed and mucus permeation properties with and without IAP preincubation were assessed. RESULTS: POAP was synthesized as brown viscous liquid with a yield of 36% and could be incorporated into SEDDS. By incubation with IAP a zeta potential shift from -15.1 to 6.5 mV was observed. A corresponding phosphate release in presence of isolated IAP as well as on Caco-2 cells was found. Assessment of the cytotoxic potential revealed no significant alteration in the safety profile of SEDDS by incorporation of POAP. Mucus permeation studies exposed a 2-fold higher permeation of fluorescein diacetate (FDA) having been embedded in SEDDS loaded with POAP in comparison to blank formulation and 3-fold higher permeability than for emulsions having been preincubated with phosphatase. CONCLUSION: The novel phosphorylated surfactant exhibiting a PEG-linker facilitated a potent zeta potential change of SEDDS.

Fluorination as tool to improve bioanalytical sensitivity and COX-2-selective antitumor activity of cobalt alkyne complexes.

The cobalt alkyne complex [(prop-2-ynyl)-2-acetoxybenzoate]dicobalthexacarbonyl (Co-ASS) is an auspicious lead, which exhibits its anticancer activity mainly by inhibition of both cyclooxygenases (COX-1 and COX-2). Since COX-2 participates in carcinogenesis, a selective inhibition of that isoenzyme is aimed. To study if fluorination increases the COX-2/COX-1 inhibition ratio of the lead, substitution was respectively performed in the positions 3, 4, 5, and 6 of the aromatic moiety. The complexes 3/4/5F-Co-ASS and to a much lower extent also 6F-Co-ASS showed cytotoxic, antimetabolic, and apoptotic effects in COX-1/2-positive HT-29 and MDA-MB-231 cells and remarkably less activity in the COX-1/2-negative MCF-7 cell line. The metabolic activity in MCF-7 cells was even unaffected up to a concentration of 40 µM. With exception of 6F-Co-ASS, the complexes strongly reduced the PGE2 synthesis in HT-29 cells and all complexes inhibited COX-2 more effectively than COX-1 in an assay at isolated enzymes. These findings point to an interference in the COX cascade as part of the mode of antitumor action. The limited cellular effects of 6F-Co-ASS are related to its poor uptake as determined by HR CS AAS/MAS. Moreover, the cellular uptake studies confirm fluorination as beneficial tool for bioanalytical labeling. The higher quantification of fluorine by HR CS MAS makes this method about 5-fold more sensitive than HR CS AAS measuring cobalt. As a further positive result, 3/4/5/6-Co-ASS demonstrated high selectivity to tumor cells due to lack of antimetabolic activity against the non-tumorigenic bone marrow stromal cell line HS-5.

Zeta potential changing self-emulsifying drug delivery systems: A promising strategy to sequentially overcome mucus and epithelial barrier.

AIM: The aim of the present study was to develop zeta potential changing self-emulsifying drug delivery systems (SEDDS) via a flip-flop mechanism in order to improve their mucus permeating and cellular uptake properties. METHODS: Phosphorylated serine-oleylamine (p-Ser-OA) conjugates were synthesized and incorporated into SEDDS at a concentration of 1% (v/v). Cytotoxic potential of p-Ser-OA and p-Ser-OA loaded SEDDS was investigated on Caco-2 cells. Phosphate release was evaluated using isolated as well as cell-associated intestinal alkaline phosphatase (AP). In parallel, change in zeta potential and amino group concentration on the surface of SEDDS was determined. Furthermore, mucus permeation and cellular uptake studies were performed. RESULTS: p-Ser-OA was synthesized by covalent attachment of serine (Ser) to oleylamine (OA) via a carbodiimide-mediated reaction followed by phosphorylation using phosphorous pentoxide (P2O5) and phosphoric acid (H3PO4). The chemical structure of p-Ser-OA was confirmed via FT-IR, 1H NMR, 13C NMR, 31P NMR and mass spectroscopic analysis. p-Ser-OA loaded SEDDS exhibited a droplet size and zeta potential of 46.42 ±â€¯0.35 nm and -11.53 mV, respectively. A significant amount of phosphate was released after incubation with isolated as well as cell-associated AP within 6 h and zeta potential raised up to -2.04 mV. p-Ser-OA loaded SEDDS showed improved mucus permeation in comparison to p-Ser-OA loaded SEDDS treated with AP. Moreover, cellular uptake increased almost 2-fold after phosphate cleavage using AP. CONCLUSION: Findings of this study show that SEDDS changing their zeta potential via a flip-flop mechanism exhibit both high mucus permeating and high cellular uptake properties.