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.

Design of charge converting lipid nanoparticles via a microfluidic coating technique.

It was the aim of this study to design charge converting lipid nanoparticles (LNP) via a microfluidic mixing technique used for the preparation and coating of LNP. LNP consisting of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, N-(carbonyl-methoxypolyethyleneglycol-2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (MPEG-2000-DSPE), and various cationic surfactants were prepared at diverging flow rate ratios (FRR) via microfluidic mixing. Utilizing a second chip in the microfluidic set-up, LNP were coated with polyoxyethylene (9) nonylphenol monophosphate ester (PNPP). LNP were examined for their stability in different physiologically relevant media as well as for hemolytic and cytotoxic effects. Finally, phosphate release and charge conversion of PNPP-coated LNP were evaluated after incubation with alkaline phosphatase and on Caco2-cells. LNP produced at an FRR of 5:1 exhibited a size between 80 and 150 nm and a positive zeta potential. Coating with PNPP within the second chip led to LNP exhibiting a negative zeta potential. After incubation with 1 U/ml alkaline phosphatase for 4 h, zeta potential of the LNP containing 1,2-dioleoyloxy-3-trimethylammonium-propane chloride (DOTAP) as cationic component shifted from - 35 mV to approximately + 5 mV. LNP prepared with other cationic surfactants remained slightly negative after enzymatic phosphate cleavage. Manufacturing of LNP containing PNPP and DOTAP via connection of two chips in a microfluidic instrument proves to show efficient change in zeta potential from negative to positive after incubation with alkaline phosphatase.

Comparative Analysis of PEG-Free and PEG-Based Self-Emulsifying Drug Delivery Systems for Enhanced Oral Bioavailability of Therapeutic (Poly) Peptides.

This study aims to compare the potential of Polyethylene glycol (PEG-free and PEG-based self-emulsifying drug delivery systems (SEDDS) for the oral administration of insulin glargine (IG). Hydrophobic ion pairs (HIPs) of IG are formed using various counterions. HIPs are assessed for log P octanol/water and dissociation behavior. They are incorporated into SEDDS based on polyglycerol (PG) and zwitterionic surfactant (ZW) using response surface methodology and compared to conventional PEG-SEDDS in size, stability, and log D SEDDS/release medium . Oral IG bioavailability in PG/ZW-SEDDS and PEG-SEDDS is evaluated in rats. Among the various counterions studied, IG-BIS (bis(isotridecyl)sulfosuccinate) HIPs demonstrated the highest log P and an improved dissociation profile. PG/ZW-SEDDS and PEG-SEDDS have similar ≈40 nm sizes and are stable over 24 h. Both formulations have log D > 4 in water and >2 in 50 mM phosphate buffer pH 6.8. PG/ZW-SEDDS yielded an oral bioavailability of 2.13 ± 0.66% for IG, while the employment of PEG-SEDDS resulted in an oral bioavailability of 1.15 ± 0.35%. This study highlights the prospective utilization of PEG-free SEDDS involving the concurrent application of PG and ZW surfactants, an alternative to conventional PEG surfactants, for improved oral therapeutic (poly) peptide delivery.

Inhibition of P-glycoprotein-mediated efflux by thiolated cyclodextrins.

Overcoming P-glycoprotein (P-gp)-mediated efflux poses a significant challenge for the pharmaceutical industry. This study investigates the potential of thiolated ß-cyclodextrins (ß-CD-SHs) as inhibitors of P-gp-mediated efflux in Caco-2 cells. Through a series of transport assays, intracellular accumulation, and efflux of the P-gp substrates Rhodamine 123 (Rh123) and Calcein-AM with and without co-administration of ß-CD-SHs were assessed. The results revealed that the cellular uptake of Rh123 and Calcein-AM were enhanced up to 7- and 3-fold, compared to the control, respectively. In efflux studies an up to 2.5-fold reduction of the Rh123 efflux was reached compared the control, indicating a substantial decrease of Rh123 efflux by ß-CD-SHs. Furthermore, it was observed that ß-CD-SHs led to a decrease in the reactivity of fluorescence-labeled anti-P-gp, suggesting additional effects on the conformation of P-gp. Overall, this study demonstrates the potential of ß-CD-SHs as effective modulator of P-gp-mediated drug efflux in Caco-2 cells.

Power-Up for Mucoadhesiveness: Two Generations of Thiolated Surfactants for Enhanced Sticky Nanoemulsions.

Nanoemulsions can be tuned toward enhanced gastro-intestinal retention time by incorporating thiolated surfactants into their surface. Tailoring the chemical reactivity of the thiol headgroup has major influence on mucoadhesive features of the nanoemulsion. Two generations of thiolated surfactants were synthetically derived from PEG-40-stearate featuring either a free thiol group or an S-protected thiol group. The surfactants were characterized regarding critical micelle concentration (CMC), hemolytic activity, and cytotoxicity. Subsequently, they were incorporated into nanoemulsions and the resulting nanoemulsions were characterized regarding particle size, polydispersity index (PDI), zeta potential, and time-dependent stability. Afterward, mucosal interactions as well as mucoadhesion on porcine intestinal mucosa were investigated. Successful synthesis of Cysteine-PEG-40-stearate (CYS-PEG-40-stearate) and MNA-Cysteine-PEG-40-stearate (MNA-CYS-PEG-40-stearate) was confirmed by 1H NMR spectroscopy. Both chemical modifications led to slightly elevated CMC values while preserving low cytotoxicity and hemotoxicity. Incorporation into nanoemulsions had minor influence on overall physical particle characteristics, while interactions with mucus and mucoadhesiveness of the nanoemulsions were drastically improved resulting in the rank order PEG-40-stearate < CYS-PEG-40-stearate < MNA-CYS-PEG-40-stearate. Accordingly, thiolated surfactants, especially S-protected derivatives, are versatile tools to generate highly mucoadhesive nanoemulsions.

Three generations of thiolated cyclodextrins: A direct comparison of their mucus permeating and mucoadhesive properties.

AIM: This study aims to compare the mucus permeating and mucoadhesive properties of three generations of thiolated cyclodextrins (CDs). METHODS: Free thiol groups of thiolated γ-CDs (CD-SH) were S-protected with 2-mercaptonicotinic acid (MNA), leading to a second generation of thiolated CDs (CD-SS-MNA) and with 2 kDa polyethylene glycol (PEG) bearing a terminal thiol group leading to a third generation of thiolated CDs (CD-SS-PEG). The structure of these thiolated CDs was confirmed and characterized by FT-IR, 1H NMR and colorimetric assays. Thiolated CDs were evaluated regarding viscosity, mucus diffusion, and mucoadhesion. RESULTS: The viscosity of the mixture of CD-SH, CD-SS-MNA, or CD-SS-PEG with mucus increased up to 11-, 16-, and 14.1-fold compared to unmodified CD within 3 hours, respectively. Mucus diffusion increased in the following rank order: unprotected CD-SH < CD-SS-MNA < CD-SS-PEG. The residence time of CD-SH, CD-SS-MNA, and CD-SS-PEG on porcine intestine was up to 9.6-, 12.55-, and 11.2-fold prolonged compared to native CD, respectively. CONCLUSION: According to these results, S-protection of thiolated CDs can be a promising approach to improve their mucus permeating and mucoadhesive properties. STATEMENT OF SIGNIFICANCE: Three generations of thiolated cyclodextrins (CDs) with different types of thiol ligands have been synthesized to improve mucus interaction. 1st generation of thiolated CDs was synthesized by converting hydroxyl groups into thiols by reaction with Thiourea. For 2nd generation, free thiol groups were S-protected by reaction with 2-mercaptonicotinic acid (MNA), resulting in high reactive disulfide bonds. For 3rd generation, terminally thiolated short PEG chains (2 kDa) were used for S-protection of thiolated CDs. Mucus penetrating properties were found to be increased as follows: 1st generation < 2nd generation < 3rd generation. Furthermore, mucoadhesive properties were improved in the following rank order: 1st generation < 3rd generation < 2nd generation. This study suggests that the S-protection of thiolated CDs can enhance mucus penetrating and mucoadhesive properties.

Peptide Antibiotic-Polyphosphate Nanoparticles: A Promising Strategy to Overcome the Enzymatic and Mucus Barrier of the Intestine.

The aim of this study was to develop peptide antibiotic-polyphosphate nanoparticles that are able to overcome the enzymatic and mucus barriers providing a targeted drug release directly on the intestinal epithelium. Polymyxin B-polyphosphate nanoparticles (PMB-PP NPs) were formed via ionic gelation between the cationic peptide and the anionic polyphosphate (PP). The resulting NPs were characterized by particle size, polydispersity index (PDI), zeta potential, and cytotoxicity on Caco-2 cells. The protective effect of these NPs for incorporated PMB was evaluated via enzymatic degradation studies with lipase. Moreover, mucus diffusion of NPs was investigated with porcine intestinal mucus. Isolated intestinal alkaline phosphatase (IAP) was employed to trigger the degradation of NPs and consequent drug release. PMB-PP NPs exhibited an average size of 197.13 ± 14.13 nm, a PDI of 0.36, a zeta potential of -11.1 ± 3.4 mV and a concentration and time-dependent toxicity. They provided entire protection toward enzymatic degradation and exhibited significantly (p < 0.05) higher mucus permeating properties than PMB. When incubated with isolated IAP for 4 h, monophosphate and PMB were constantly released from PMB-PP NPs and zeta potential raised up to -1.9 ± 0.61 mV. According to these findings, PMB-PP NPs are promising delivery systems to protect cationic peptide antibiotics against enzymatic degradation, to overcome the mucus barrier and to provide drug release directly at the epithelium.

Thiolated cyclodextrins: A comparative study of their mucoadhesive properties.

AIM: The aim of this study was the comparison of the mucoadhesive properties of nonionic, negatively, and positively charged thiolated cyclodextrins (CDs), including α-, ß-, and γ-CDs of low and high degree of thiolation. METHODS: Native α-, ß-, and γ-CDs were thiolated with phosphorous pentasulfide in sulfolane (CD-SH) (i), via reductive amination with cysteamine after oxidative ring opening (CD-Cya) (ii), and via esterification with mercaptosuccinic acid (CD-MSA) (iii). These thiolated CDs were characterized via 1H NMR and Ellman's test. Cytotoxicity was determined via resazurin and hemolysis assay. Mucoadhesive properties were evaluated via rheological studies with freshly isolated porcine mucus, as well as residence time studies on porcine small intestinal mucosa. RESULTS: The structure of thiolated CDs was confirmed via 1H NMR. The degree of thiolation was in the range of 594-1034 µmol/g for low and 1360-3379 µmol/g for high CD-SH, whereas thiolated CD-Cya and thiolated CD-MSA exhibited a degree of thiolation of 1142-3242 µmol/g and 243-1227 µmol/g, respectively. Just cationic CDs showed cytotoxicity. Nonionic highly thiolated α-CD-SH, α-CD-Cya, and α-CD-MSA exhibited with mucus 5.6-, 15.7- and 2.8-fold improved dynamic viscosity, while improvement was 7.7-, 6.1-, and 5.4-fold for the corresponding thiolated ß-CDs and 12.3-, 15.4- and 17.8-fold for the corresponding thiolated γ-CDs compared with native CDs, respectively. A prolonged mucosal residence time following the rank order γ > ß > α was observed for all thiolated CDs, whereby γ-CD-Cya, nonionic highly thiolated ß-CD-SH and α-CD-Cya showed the highest mucoadhesive properties. CONCLUSION: A high degree of thiolation and the introduction of cationic charges are mainly responsible for high mucoadhesive properties of CDs.

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.

Per-thiolated cyclodextrins: Nanosized drug carriers providing a prolonged gastrointestinal residence time.

Oral delivery is one of the most advantageous routes for drug administration, but due to the short gastrointestinal (GI) residence time, the systemic uptake of poorly absorbed drugs is too low to reach the desired therapeutic effect. In order to prolong the GI residence time of orally given drugs, we synthesized per-thiolated ß-cyclodextrin (CD) as mucoadhesive drug carrier. Due to thiolation, the mucoadhesive properties of CD on porcine intestinal mucosa were increased 2-fold. In vivo studies showed 4 h after oral administration, a 19.4-fold, 2.1- fold, and 4.5-fold higher quantity of per-thiolated ß-CD vs. unmodified ß-CD in the stomach, duodenum/jejunum, and the ileum of rat model, respectively. Eight hours after oral administration, still, 60 % of per-thiolated CD, but no native CD remained in the GI tract. These results provide evidence that due to thiolation of ß-CD, GI-residence time can be essentially prolonged.

Oral delivery of therapeutic peptides and proteins: Technology landscape of lipid-based nanocarriers.

The oral administration of therapeutic peptides and proteins is favoured from a patient and commercial point of view. In order to reach the systemic circulation after oral administration, these drugs have to overcome numerous barriers including the enzymatic, sulfhydryl, mucus and epithelial barrier. The development of oral formulations for therapeutic peptides and proteins is therefore necessary. Among the most promising formulation approaches are lipid-based nanocarriers such as oil-in-water nanoemulsions, self-emulsifying drug delivery systems (SEDDS), solid lipid nanoparticles (SLN), nanostructured lipid carriers (NLC), liposomes and micelles. As the lipophilic character of therapeutic peptides and proteins can be tremendously increased such as by the formation of hydrophobic ion pairs (HIP) with hydrophobic counter ions, they can be incorporated in the lipophilic phase of these carriers. Since gastrointestinal (GI) peptidases as well as sulfhydryl compounds such as glutathione and dietary proteins are too hydrophilic to enter the lipophilic phase of these carriers, the incorporated therapeutic peptide or protein is protected towards enzymatic degradation as well as unintended thiol/disulfide exchange reactions. Stability of lipid-based nanocarriers towards lipases can be provided by the use to excipients that are not or just poorly degraded by these enzymes. Nanocarriers with a size <200 nm and a mucoinert surface such as PEG or zwitterionic surfaces exhibit high mucus permeating properties. Having reached the underlying absorption membrane, lipid-based nanocarriers enable paracellular and lymphatic drug uptake, induce endocytosis and transcytosis or simply fuse with the cell membrane releasing their payload into the systemic circulation. Numerous in vivo studies provide evidence for the potential of these delivery systems. Within this review we provide an overview about the different barriers for oral peptide and protein delivery, highlight the progress made on lipid-based nanocarriers in order to overcome them and discuss strengths and weaknesses of these delivery systems in comparison to other technologies.