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.

Novel thiazolopyridine derivatives of diflapolin as dual sEH/FLAP inhibitors with improved solubility.

Inflammatory responses are orchestrated by a plethora of lipid mediators, and perturbations of their biosynthesis or degradation hinder resolution and lead to uncontrolled inflammation, which contributes to diverse pathologies. Small molecules that induce a switch from pro-inflammatory to anti-inflammatory lipid mediators are considered valuable for the treatment of chronic inflammatory diseases. Commonly used non-steroidal anti-inflammatory drugs (NSAIDs) are afflicted with side effects caused by the inhibition of beneficial prostanoid formation and redirection of arachidonic acid (AA) into alternative pathways. Multi-target inhibitors like diflapolin, the first dual inhibitor of soluble epoxide hydrolase (sEH) and 5-lipoxygenase-activating protein (FLAP), promise improved efficacy and safety but are confronted by poor solubility and bioavailability. Four series of derivatives bearing isomeric thiazolopyridines as bioisosteric replacement of the benzothiazole core and two series additionally containing mono- or diaza-isosteres of the phenylene spacer were designed and synthesized to improve solubility. The combination of thiazolo[5,4-b]pyridine, a pyridinylen spacer and a 3,5-Cl2-substituted terminal phenyl ring (46a) enhances solubility and FLAP antagonism, while preserving sEH inhibition. Moreover, the thiazolo[4,5-c]pyridine derivative 41b, although being a less potent sEH/FLAP inhibitor, additionally decreases thromboxane production in activated human peripheral blood mononuclear cells. We conclude that the introduction of nitrogen, depending on the position, not only enhances solubility and FLAP antagonism (46a), but also represents a valid strategy to expand the scope of application towards inhibition of thromboxane biosynthesis.

Synthesis and structure-activity relationships for some novel diflapolin derivatives with benzimidazole subunit.

A series of derivatives of the potent dual soluble epoxide hydrolase (sEH)/5-lipoxygenase-activating protein (FLAP) inhibitor diflapolin was designed, synthesised, and characterised. These novel compounds, which contain a benzimidazole subunit were evaluated for their inhibitory activity against sEH and FLAP. Molecular modelling tools were applied to analyse structure-activity relationships (SAR) on both targets and to predict solubility and gastrointestinal (GI) absorption. The most promising dual inhibitors of these series are 5a, 6b, and 6c.

Antifungal Siderophore Conjugates for Theranostic Applications in Invasive Pulmonary Aspergillosis Using Low-Molecular TAFC Scaffolds.

Invasive pulmonary aspergillosis (IPA) is a life-threatening form of fungal infection, primarily in immunocompromised patients and associated with significant mortality. Diagnostic procedures are often invasive and/or time consuming and existing antifungals can be constrained by dose-limiting toxicity and drug interaction. In this study, we modified triacetylfusarinine C (TAFC), the main siderophore produced by the opportunistic pathogen Aspergillus fumigatus (A. fumigatus), with antifungal molecules to perform antifungal susceptibility tests and molecular imaging. A variation of small organic molecules (eflornithine, fludioxonil, thiomersal, fluoroorotic acid (FOA), cyanine 5 (Cy5) with antifungal activity were coupled to diacetylfusarinine C (DAFC), resulting in a "Trojan horse" to deliver antifungal compounds specifically into A. fumigatus hyphae by the major facilitator transporter MirB. Radioactive labeling with gallium-68 allowed us to perform in vitro characterization (distribution coefficient, stability, uptake assay) as well as biodistribution experiments and PET/CT imaging in an IPA rat infection model. Compounds chelated with stable gallium were used for antifungal susceptibility tests. [Ga]DAFC-fludioxonil, -FOA, and -Cy5 revealed a MirB-dependent active uptake with fungal growth inhibition at 16 µg/mL after 24 h. Visualization of an A. fumigatus infection in lungs of a rat was possible with gallium-68-labeled compounds using PET/CT. Heterogeneous biodistribution patterns revealed the immense influence of the antifungal moiety conjugated to DAFC. Overall, novel antifungal siderophore conjugates with promising fungal growth inhibition and the possibility to perform PET imaging combine both therapeutic and diagnostic potential in a theranostic compound for IPA caused by A. fumigatus.

Imine bond formation as a tool for incorporation of amikacin in self-emulsifying drug delivery systems (SEDDS).

AIM: The aim was to develop a self-emulsifying drug delivery system (SEDDS) for amikacin via imine bond formation with hydrophobic aldehydes. METHODS: Trans-2, cis-6-nonadienal, trans-cinnamaldehyde, citral and benzaldehyde were conjugated to amikacin at pH 8.5. Based on results of precipitation efficiency, Fourier-transform infrared spectroscopy (FTIR) and NMR analysis, amikacin-trans-cinnamaldehyde conjugates were further characterized regarding log Poctanol/water via HPLC. The release of amikacin from the amikacin-trans-cinnamaldehyde conjugates was examined through in vitro incubation with bovine serum albumin (BSA). SEDDS containing the amikacin-trans-cinnamaldehyde conjugates were tested regarding mean droplet size (MDS), polydispersity index (PDI), log DSEDDS/release medium and cell viability. RESULTS: Trans-cinnamaldehyde formed the most hydrophobic conjugates with amikacin whereas benzaldehyde did not form hydrophobic conjugates at all. Imine bond formation was confirmed by FTIR and NMR analysis. The highest increase in log P was achieved for the amikacin-trans-cinnamaldehyde conjugate in a molar ratio of 1:5, shifting from -8.58 up to 1.59. Incubation of this conjugate with BSA led to the formation of BSA-trans-cinnamaldehyde releasing in turn amikacin. SEDDS based on Capmul MCM, Cremophor EL and propylene glycol containing the conjugate demonstrated a MDS of 61.4 nm and PDI of 0.265. Log DSEDDS/release medium was calculated to be 3.38. Cell viability studies showed very good tolerability of conjugate loaded SEDDS in concentrations of 0.1% - 0.5%. CONCLUSION: Imine bond formation of amikacin with trans-cinnamaldehyde and the incorporation of the resulting conjugate into SEDDS represents a promising strategy for oral delivery of amikacin.

Lysine-Based Biodegradable Surfactants: Increasing the Lipophilicity of Insulin by Hydrophobic Ion Paring.

AIM: The aim of this study was to evaluate biodegradable cationic surfactants based on lysine. METHODS: Lysine was esterified with cholesterol, oleyl alcohol and 1-decanol resulting in cholesteryl lysinate (CL), oleyl lysinate (OL) and decyl lysinate (DL). Esters were investigated regarding their log Dn-octanol/water, critical micelle concentration (CMC) and biodegradability. Hemolytic potential of CL, OL, DL and the already established hexadecyl lysinate (HL) was determined and complexes with insulin (INS) were formed by hydrophobic ion pairing (HIP). Lipophilic characteristics of ion-pairs were examined by analyzing their log Pn-butanol/water. RESULTS: Successful synthesis of CL, OL and DL was confirmed by IR, NMR and MS. Log D analysis revealed amphiphilic properties for the esters and a CMC of 0.01 mM, 2.0 mM and 6.0 mM was found for CL, OL and DL, respectively. Biodegradability was proven, as over 99% of OL and DL were degraded by isolated enzymes within 30 min and after 3 h 97% of CL was cleaved by membrane bound enzymes. OL as well as DL displayed no hemolytic effect and for CL cytotoxicity was significantly reduced in comparison to HL. INS/CL complex exhibited highest lipophilicity. CONCLUSION: Cholesterol-amino acid based surfactants seem to be promising agents for HIP.

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.

Thiolated PVP-Amphotericin B Complexes: An Innovative Approach toward Highly Mucoadhesive Gels for Mucosal Leishmaniasis Treatment.

The aim of this study was to synthesize polymeric excipients that can form mucoadhesive hydrogels containing amphotericin B (AmB) for the treatment of mucosal leishmaniasis. 2-(2-Acryloylaminoethyldisulfanyl)-nicotinic acid (ACENA) was copolymerized with N-vinyl pyrrolidone to obtain thiolated polyvinylpyrrolidone (PVP) that was then complexed with AmB to improve its solubility. The resulting structure of thiolated PVP was evaluated by 1H nuclear magnetic resonance to confirm S-protected thiol groups, and the average molecular mass was determined by size exclusion chromatography. Moreover, variants of thiolated PVP-AmB were studied for the thiol content, amount of complexed AmB, cytotoxicity, mucoadhesive properties, and antileishmaniasis activity. The highest achieved degree of thiolation was 772 ± 24.64 µmol/g, and the amount of complexed AmB was 27.05 ± 0.31 µmol per g of polymer. Thiolated PVP and thiolated PVP-AmB variants (0.5% m/v) showed no cytotoxicity, whereas the equivalent concentration of free AmB reduced Caco-2 cell viability to 70% within 24 h. Thiol-functionalized PVP and PVP-AmB complexes displayed 7.66- and 7.20-fold higher adhesion to the mucosal surface in comparison to unmodified PVP and PVP-AmB, respectively. In addition, variants of thiolated PVP-AmB complexes displayed 100% antileishmaniasis activity in comparison to the 80% killing efficiency of Fungizone, which has been applied in the equivalent AmB concentration of 0.2 µg/mL. Thiol-functionalized PVP proved to be a promising novel excipient for the delivery of AmB providing enhanced solubility and improved mucoadhesive properties which are beneficial for the treatment of mucosal leishmaniasis.

Dual Inhibitory Action of a Novel AKR1C3 Inhibitor on Both Full-Length AR and the Variant AR-V7 in Enzalutamide Resistant Metastatic Castration Resistant Prostate Cancer.

The expanded use of second-generation antiandrogens revolutionized the treatment landscape of progressed prostate cancer. However, resistances to these novel drugs are already the next obstacle to be solved. Various previous studies depicted an involvement of the enzyme AKR1C3 in the process of castration resistance as well as in the resistance to 2nd generation antiandrogens like enzalutamide. In our study, we examined the potential of natural AKR1C3 inhibitors in various prostate cancer cell lines and a three-dimensional co-culture spheroid model consisting of cancer cells and cancer-associated fibroblasts (CAFs) mimicking enzalutamide resistant prostate cancer. One of our compounds, named MF-15, expressed strong antineoplastic effects especially in cell culture models with significant enzalutamide resistance. Furthermore, MF-15 exhibited a strong effect on androgen receptor (AR) signaling, including significant inhibition of AR activity, downregulation of androgen-regulated genes, lower prostate specific antigen (PSA) production, and decreased AR and AKR1C3 expression, indicating a bi-functional effect. Even more important, we demonstrated a persisting inhibition of AR activity in the presence of AR-V7 and further showed that MF-15 non-competitively binds within the DNA binding domain of the AR. The data suggest MF-15 as useful drug to overcome enzalutamide resistance.

Self-Emulsifying Drug Delivery Systems: Hydrophobic Drug Polymer Complexes Provide a Sustained Release in Vitro.

The aim of this study was to develop hydrophobic ionic drug polymer complexes in order to provide sustained drug release from self-emulsifying drug delivery systems (SEDDS). Captopril (CTL) was used as an anionic model drug to form ionic complexes with the cationic polymers Eudragit RS, RL, and E. Complexes of polymer to CTL charge ratio 1:1, 2:1, and 4:1 were incorporated in two SEDDS, namely FA which was 40% Kolliphor RH 40, 20% Kolliphor EL, and 40% castor oil and FB, which was 40% Kolliphor RH 40, 30% glycerol, 15% Kolliphor EL, and 15% castor oil. Blank and complex loaded SEDDS were characterized regarding their droplet size, polydispersity index (PDI), and zeta potential. Resazurin assay was performed on Caco-2 cells to evaluate the biocompatibility of SEDDS. Release of CTL from SEDDS was determined in release medium containing 0.2 mg/mL of 5,5'-dithiobis(2-nitrobenzoic acid) (DNTB) allowing quantification of free drug released into solution via a thiol/disulfide exchange reaction between CTL and DNTB forming a yellow dye. The droplet size of SEDDS FA and SEDDS FB were in the range of 100 ± 20 nm and 40 ± 10 nm, respectively, with a PDI < 0.5. The zeta potential of SEDDS FA and SEDDS FB increased after the incorporation of complexes. Cell viability remained above 80% after incubation with SEDDS FA and SEDDS FB in a concentration of 1% and 3% for 4 h. Without any polymer, CTL was entirely released from both SEDDS within seconds. In contrast, the higher the cationic lipophilic polymer to CTL ratio in SEDDS, the more sustained was the release of CTL. Among the polymers which were evaluated, Eudragit RL provided the most sustained release. SEDDS FA containing Eudragit RL and CTL in a ratio of 1:1 released 64.78 ± 8.28% of CTL, whereas SEDDS FB containing the same complex showed a release of 91.85 ± 1.17% within 1 h. Due to the formation of lipophilic ionic polymer complexes a sustained drug release from oily droplets formed by SEDDS can be achieved. Taking into account that drugs are otherwise instantly released from SEDDS, results of this study might open the door for numerous additional applications of SEDDS for which a sustained drug release is essential.

Less Reactive Thiol Ligands: Key towards Highly Mucoadhesive Drug Delivery Systems.

As less reactive s-protected thiomers can likely interpenetrate the mucus gel layer to a higher extent before getting immobilized via disulfide bond formation with mucins, it was the aim of this study to develop a novel type of s-protected thiomer based on the less reactive substructure cysteine-N-acetyl cysteine (Cys-NAC) in order to obtain improved mucoadhesive properties. For this purpose, two types of s-protected thiomers, polyacrylic acid-cysteine-mercaptonicotinic acid (PAA-Cys-MNA) and polyacrylic acid-cysteine-N-acetyl cysteine (PAA-Cys-NAC), were synthesized and characterized by Fourier-transform infrared spectroscopy (FT-IR) and the quantification of attached disulfide ligands. The viscosity of both products was measured in the presence of NAC and mucus. Both thiomers were also evaluated regarding swelling behavior, tensile studies and retention time on the porcine intestinal mucosa. The FT-IR spectra confirmed the successful attachment of Cys-MNA and Cys-NAC ligands to PAA. The number of attached sulfhydryl groups was in the range of 660-683 µmol/g. The viscosity of both s-protected thiomers increased due to the addition of increasing amounts of NAC. The viscosity of the mucus increased in the presence of 1% PAA-Cys-MNA and PAA-Cys-NAC 5.6- and 10.9-fold, respectively, in comparison to only 1% PAA. Both s-protected thiomers showed higher water uptake than unmodified PAA. The maximum detachment force (MDF) and the total work of adhesion (TWA) increased in the case of PAA-Cys-MNA up to 1.4- and 1.6-fold and up to 2.4- and 2.8-fold in the case of PAA-Cys-NAC. The retention of PAA, PAA-Cys-MNA, and PAA-Cys-NAC on porcine intestinal mucosa was 25%, 49%, and 76% within 3 h, respectively. The results of this study provide evidence that less reactive s-protected thiomers exhibit higher mucoadhesive properties than highly reactive s-protected thiomers.

S-protected thiolated hyaluronic acid: In-situ crosslinking hydrogels for 3D cell culture scaffold.

The purpose of this study was to synthesize S-protected thiolated hyaluronic acid (HA) and to evaluate its potential for 3D cell culture scaffold. S-protected thiolated HA was synthesized by the covalent attachment of N-acetyl-S-((3-((2,5-dioxopyrrolidin-1-yl)oxy)-3-oxopropyl)thio)cysteine hydrazide ligand to the HA. Hydrogels were characterized for texture, swelling behavior and rheological properties. Furthermore, the potential of S-protected thiolated HA hydrogels as a scaffold for tissue engineering was evaluated by cell proliferation studies with Caco-2 and NIH 3T3 cells. It showed enhanced cohesion upon addition of N-acetyl cysteine (NAC). Dynamic viscosity of S-protected thiolated HA hydrogel was increased up to 19.5-fold by addition of NAC and 10.1-fold after mixing with mucus. Furthermore, Caco-2 and NIH 3T3 cells encapsulated into hydrogels proliferated in-vitro. As this novel S-protected thiolated HA is stable towards oxidation and forms highly cohesive gels when getting into contact with endogenous thiols due to disulfide-crosslinking, it is a promising tool for 3D cell culture scaffold.

Characterization of an amino acid based biodegradable surfactant facilitating the incorporation of DNA into lipophilic delivery systems.

HYPOTHESIS: Lysine based cationic surfactants are well-tolerated tools for hydrophobic ion pairing (HIP) with DNA and its incorporation into lipophilic delivery systems. EXPERIMENTS: Di-Boc-lysine was esterified with 1-hexadecanol and the Boc-residues were cleaved off resulting in hexadecyl lysinate (HL). Subsequently, its Log POctanol/water and the critical micelle concentration (CMC) were determined. Degradability was evaluated utilizing trypsin and pancreas lipase as well as Caco-2 cells. Afterwards, the viability of Caco-2 cells upon incubation with HL was investigated. Finally, HL was ion-paired with plasmid DNA (pDNA, 6159 bp) and the obtained complex was incorporated into self-emulsifying drug delivery systems (SEDDS) for transfection studies on HEK-293 cells. FINDINGS: HL was synthesized with a yield of 53% and subsequent characterization revealed a Log PWater/Octanol of 0.05 and a CMC of 2.7 mM. Enzymatic degradation studies showed rapid degradation of HL by isolated enzymes and Caco-2 cells and cell viability experiments revealed no toxic effect of HL even in a concentration of 250 µg·ml-1 within 24 h. HIP with pDNA was the most efficient in a molar ratio of 6159:1 (HL:pDNA) equalling a charge ratio of 1:1. Formed complexes could be incorporated into SEDDS facilitating successful transfection of HEK-293 cells.

Tetradeca-thiolated cyclodextrins: Highly mucoadhesive and in-situ gelling oligomers with prolonged mucosal adhesion.

The purpose of this study was to synthesize a highly mucoadhesive tetradeca-thiolated ß-cyclodextrin (ß-CD) by replacement of all primary OH groups at C-6 position and all secondary OH groups at C-2 position of ß-CD backbone viaSH groups and to evaluate its rheological and mucoadhesive properties in-vitro. Primary and secondary OH groups of ß-CD were substituted by SH groups using a microwave-assisted method. The structure of tetradeca-thiolated ß-CD was confirmed by FTIR and 1H NMR spectroscopy. The modified ß-CD was evaluated for SH content, thiol stability towards oxidation and cytotoxicity. Moreover, the viscoelastic behavior of the modified oligomer was investigated via rheological studies with porcine intestinal mucus and fibrous structural protein keratin, whereas mucoadhesive properties were evaluated using different porcine mucosae. Tetradeca-thiolated ß-CD oligomer displayed 8144 ± 317 µmol thiol groups per gram. These thiol groups displaying a pKa value of 8.2 were stable at pH 4 but prone to oxidation at higher pH values. The newly synthesized thiolated CD did not show any cytotoxicity to Caco-2 cells at a concentration of 0.5% (m/v) within 24 h. Due to the addition of 0.5 and 2% (m/v) tetradeca-thiolated ß-CD to mucus and keratin, the dynamic viscosity was increased up to 7.6- and 5.9- fold, respectively, within 4 h at 37 °C. Moreover, in-vitro mucoadhesion studies of tetradeca-thiolated CD showed 78.6-, 60.3-, 62.3- and 49.3- fold improved mucoadhesion on intestinal, buccal, bladder and vaginal mucosa as compared to unmodified ß-CD, respectively. According to these results, tetradeca-thiolated ß-CD might be a promising auxiliary agent to provide a prolonged residence time of drug delivery systems on different mucosal surfaces.

Per-6-Thiolated Cyclodextrins: A Novel Type of Permeation Enhancing Excipients for BCS Class IV Drugs.

The purpose of the study was to develop a per-6-thiolated α-cyclodextrin (α-CD) by substituting all primary hydroxyl groups of α-CD with thiol groups and to assess its solubility-improving and permeation-enhancing properties for a BCS Class IV drug in vitro as well as in vivo. The primary hydroxyl groups of α-CD were replaced by iodine, followed by substitution with -SH groups. The structure of per-6-thiolated α-CD was approved by FT-IR and 1H NMR spectroscopy. The per-6-thiolated was characterized for thiol content, -SH stability, cytotoxicity, and solubility-improving properties by using the model BCS Class IV drug furosemide (FUR). The mucoadhesive properties of the thiolated oligomer were investigated via viscoelastic measurements with porcine mucus, whereas permeation-enhancing features were evaluated on the Caco-2 cell monolayer and rat gut mucosa. Furthermore, oral bioavailability studies were performed in rats. The per-6-thiolated α-CD oligomer displayed 4244 ± 402 µmol/g thiol groups. These -SH groups were stable at pH ≤ 4, exhibiting a pKa value of 8.1, but subject to oxidation at higher pH. Per-6-thiolated α-CD was not cytotoxic to Caco-2 cells in 0.5% (m/v) concentration within 24 h. It improved the solubility of FUR in the same manner as unmodified α-CD. The addition of per-6-thiolated α-CD (0.5% m/v) increased the mucus viscosity up to 5.8-fold at 37 °C within 4 h. Because of the incorporation in per-6-thiolated α-CD, the apparent permeability coefficient (Papp) of FUR was 6.87-fold improved on the Caco-2 cell monolayer and 6.55-fold on the intestinal mucosa. Moreover, in vivo studies showed a 4.9-fold improved oral bioavailability of FUR due to the incorporation in per-6-thiolated α-CD. These results indicate that per-6-thiolated α-CD would be a promising auxiliary agent for the mucosal delivery of, in particular, BCS Class IV drugs.

Grafting of wool fibers through disulfide bonds: An advanced application of S-protected thiolated starch.

The purpose of this study is to develop a potential pathway for grafting polymers onto wool fibers based on thiol-disulfide exchange reactions. S-protected thiolated starch (PTS) was synthesized by coupling 3-(2-pyridyldithio) propanoic acid to starch through esterification, resulting in 417.3 ± 15.1 µmol ligand binding to 1 g of starch. PTS was labelled with fluorescein isothiocyanate (FITC) prior to grafting. Wool fibers were preactivated by raising the amount of thiol groups utilizing mild reducing agents. The highest degree of preactivation on the surface of wool fibers was achieved by a 0.2% (w/v) sodium borohydride and 1.5% (w/v) sodium bisulfite mixture pH 5.0 resulting in 182.6 ± 8.7 µmol thiol groups per gram of fibers. Different incubation times and ratios between FITC-labelled PTS and wool fibers were investigated. A graft yield of 58.5% was achieved at a ratio of 1:1.5 (w/w) between wool fibers and FITC-labelled PTS within 18 h of incubation. Successful coating of PTS on wool fibers was confirmed by confocal imaging, scanning electron microscopy and FT-IR. Mechanical properties of grafted wool fibers were tested regarding elongation and tensile strength. These results provide evidence for the potential of S-protected thiolated starch as a superior coating material for wool fibers.

Imine bond formation: A novel concept to incorporate peptide drugs in self-emulsifying drug delivery systems (SEDDS).

HYPOTHESIS: Because of its hydrophilic character the peptide drug Polymyxin B (PMB) cannot be incorporated in lipophilic nanocarrier systems such as self-emulsifying drug delivery systems (SEDDS) for oral administration. Due to the formation of imine conjugates between the primary amino groups of PMB and the carbonyl group of cinnamaldehyde, however, drug lipophilicity might be sufficiently raised for incorporation in SEDDS. METHODS: Imine bonds were formed between the primary amino groups of PMB and the carbonyl group of cinnamaldehyde. PMB-cinnamaldehyde conjugate was characterized regarding degree of substitution, log P and release of PMB due to interaction with bovine serum albumin (BSA), SEDDS loading and cell viability. RESULTS: 87.1% of primary amines formed imines with cinnamaldehyde. Log P was increased 69.183 - folds. BSA triggered release of PMB was 45.2%, 64.9% and 80.6% within 16 h. Log DSEDDS/Release medium of PMB-cinnamaldehyde conjugate was 3.4. CONCLUSION: According to these findings, the concept of imine bond formation with cinnamaldehyde can be considered as a novel concept for increasing lipophilicity of the hydrophilic antibiotic peptide PMB.

Arginine-based cationic surfactants: Biodegradable auxiliary agents for the formation of hydrophobic ion pairs with hydrophilic macromolecular drugs.

WORKING HYPOTHESIS: It was the hypothesis of this study that esters of arginine (Arg) with medium and long chain aliphatic alcohols are biodegradable and less cytotoxic than well-established cationic surfactants being used for hydrophobic ion pairing (HIP) with hydrophilic macromolecular drugs. EXPERIMENTS: Arg was linked to nonan-1-ol and hexadecan-1-ol (C9 and C16) via an ester linkage. The newly formed Arg-nonyl ester (ANE) and Arg-hexadecanoyl ester (AHE) surfactants were evaluated regarding critical micelle concentration (CMC) using pyrene fluorescent method, cytotoxicity on human colorectal adenocarcinoma-derived cells (Caco-2) and biodegradability at the concentrations of 2.5 and 5 mg/mL using 2500 Nα-benzoyl-l-arginine ethyl ester hydrochloride (BAEE) units/mL of trypsin. Furthermore, in order to evaluate their potential for HIP, heparin and daptomycin were used as model polysaccharide and peptide drugs, respectively. FINDINGS: Chemical structures of ANE and AHE surfactants were confirmed by FTIR, 1H NMR, and LC-MS. CMC of ANE was 7.5 mM and CMC of AHE was 2 mM. Arg-surfactants were not cytotoxic below their CMC. At CMC and above CMC, ANE was significantly (P < 0.05) more cytotoxic than AHE. ANE in both concentrations was degraded ˃98% within 48 h. The degradation of AHE at lower concentration was ˃97% and about 50% at higher concentration. Arg-surfactants were able to efficiently precipitate heparin and daptomycin from corresponding aqueous solutions. CONCLUSION: Arg-surfactants being biodegradable and less toxic seems to be a promising alternative to well-established cationic surfactants for the formation of hydrophobic ion pairs (HIPs) with hydrophilic macromolecular drugs.

Chitosan based micelle with zeta potential changing property for effective mucosal drug delivery.

Mucus permeation, mucoadhesion and cell membrane interaction are properties that a drug carrier needs to deliver macromolecule compounds through the mucus barrier and inside epithelial cells effectively. Herein, we prepared micelles from phosphorylated chitosan-stearic acid conjugates (CSSAP) possessing those properties. Their zeta potential can be shifted from negative to neutral once contacting with alkaline phosphatase (ALP). CSSAP micelles showed effective mucus permeation and cell association, thus could be used as a promising platform for mucosal drug delivery. CSSAP was obtained via two modifications: alkylation of CS with stearic acid (termed CSSA) followed by phosphorylation utilizing phosphorus pentoxide. CSSAP had critical micelle concentration value of 76 µg/mL and phosphate content of 1066 µmol/g polymer. Micelle hydrodynamic size was 50-60 nm. Upon contacting with ALP, the polymeric micelles showed a phosphate release of 626 µmol/g polymer (~60%) and a zeta potential shift from -20 to -9 mV within 30 min. They exhibited 6-times higher mucus permeation capacity than positively charged CSSA micelles. CSSAP micelles association to Caco-2 and HEK 293 cells depended on the ALP activity. On Caco-2 cells, cell association rate after 3 h was 2-times higher compared to association rate in the presence of 0.5% phosphatase inhibitors.

Modifying the Siderophore Triacetylfusarinine C for Molecular Imaging of Fungal Infection.

PURPOSE: Aspergillus fumigatus produces the siderophore triacetylfusarinine C (TAFC) for iron acquisition which is essential for its virulence. Therefore, TAFC is a specific marker for invasive aspergillosis. We have shown previously that positron emission tomography (PET) imaging with [68Ga]TAFC exhibited excellent targeting properties in an A. fumigatus rat infection model. In this study, we aimed to prepare TAFC analogs modifying fusarinine C (FSC) by acylation with different carbon chain lengths as well as with charged substituents and investigated the influence of introduced substituents on preservation of TAFC characteristics in vitro and in vivo. PROCEDURES: Fifteen TAFC derivatives were prepared and labeled with gallium-68. In vitro uptake assays were carried out in A. fumigatus under iron-replete as well as iron-depleted conditions and distribution coefficient was determined. Based on these assays, three compounds, [68Ga]tripropanoyl(FSC) ([68Ga]TPFC), [68Ga]diacetylbutanoyl(FSC) ([68Ga]DABuFC), and [68Ga]trisuccinyl(FSC) ([68Ga]FSC(suc)3), with high, medium, and low in vitro uptake in fungal cultures, were selected for further evaluation. Stability and protein binding were evaluated and in vivo imaging performed in the A. fumigatus rat infection model. RESULTS: In vitro uptake studies using A. fumigatus revealed specific uptake of mono- and trisubstituted TAFC derivatives at RT. Lipophilicities as expressed by logD were 0.34 to - 3.80. The selected compounds displayed low protein binding and were stable in PBS and serum. Biodistribution and image contrast in PET/X-ray computed tomography of [68Ga]TPFC and [68Ga]DABuFC were comparable to [68Ga]TAFC, whereas no uptake in the infected region was observed with [68Ga]FSC(suc)3. CONCLUSIONS: Our studies show the possibility to modify TAFC without losing its properties and specific recognition by A. fumigatus. This opens also new ways for multimodality imaging or theranostics of fungal infection by introducing functionalities such as fluorescent dyes or antifungal moieties.