Effects of excipients on the interactions of self-emulsifying drug delivery systems with human blood plasma and plasma membranes.

Due to its versatility in formulation and manufacturing, self-emulsifying drug delivery systems (SEDDS) can be used to design parenteral formulations. Therefore, it is necessary to understand the effects of excipients on the behavior of SEDDS formulations upon parenteral administration, particularly their interactions with blood plasma and cell membranes. In this study, we prepared three neutrally charged SEDDS formulations composed of medium-chain triglycerides as the oil phase, polyoxyl-35 castor oil (EL35) and polyethylene glycol (15)-hydroxystearate (HS15) as the nonionic surfactants, medium-chain mono- and diglycerides as the co-surfactant, and propylene glycol as the co-solvent. The cationic surfactant, didodecyldimethylammonium bromide (DDA), and the anionic surfactant, sodium deoxycholate (DEO), were added to the neutral SEDDS preconcentrates to obtain cationic and anionic SEDDS, respectively. SEDDS were incubated with human blood plasma and recovered by size exclusion chromatography. Data showed that SEDDS emulsion droplets can bind plasma protein to different extents depending on their surface charge and surfactant used. At pH 7.4, the least protein binding was observed with anionic SEDDS. Positive charges increased protein binding. SEDDS stabilized by HS15 can adsorb more plasma protein and induce more plasma membrane disruption activity than SEDDS stabilized by EL35. These effects were more pronounced with the HS15 + DDA combination. The addition of DDA and DEO to SEDDS increased plasma membrane disruption (PMD) activities, and DDA (1% w/w) was more active than DEO (2% w/w). PMD activities of SEDDS were concentration-dependent and vanished at appropriate dilution ratios.

Polyaminated pullulan, a new biodegradable and cationic pullulan derivative for mucosal drug delivery.

AIM: To prepare new polycationic pullulan derivatives exhibiting highly mucoadhesive and sustained drug release properties. METHODS: Hydroxy groups of pullulan were activated with mesyl chloride followed by conjugation with low-molecular weight polyamines. Pullulan-tris(2-aminoethyl)amine (Pul-TAEA) and pullulan-polyethyleneimine (Pul-PEI) were evaluated regarding swelling behaviour, mucoadhesive properties and potential to control drug release. RESULTS: Pul-TAEA and Pul-PEI exhibited excellent swelling properties at pH 6.8 showing 240- and 370-fold increase in weight. Compared to unmodified pullulan, Pul-TAEA and Pul-PEI displayed 5- and 13.3-fold increased dynamic viscosity in mucus. Mucoadhesion studies of Pul-TAEA and Pul-PEI on intestinal mucosa showed a 6- and 37.8-fold increase in tensile strength, and a 72- and 120-fold increase in mucoadhesion time compared to unmodified pullulan, respectively. Due to additional ionic interactions between cationic groups on polyaminated pullulans and an anionic model drug, a sustained drug release was achieved. CONCLUSIONS: Polyaminated pullulans are promising novel mucoadhesive excipients for mucosal drug delivery.

Thiolated pectins: In vitro and ex vivo evaluation of three generations of thiomers.

In recent decades, three generations of thiomers have been developed with the main purpose of obtaining enhanced interactions with mucosal tissues. Therefore, many different types of thiolated ligands have been generated and attached to polymeric backbones. The aim of this study was to synthesize all three generations of thiomers and to directly compare their properties regarding mucus penetration and mucoadhesion. Starting from pectin, the unprotected thiomer pectin-cysteine (Pec-Cys), the preactivated S-protected thiomer pectin-cysteine-mercaptonicotinic acid (Pec-Cys-MNA) and the less reactive S-protected thiomer pectin-cysteine-glutathione (Pec-Cys-GSH) were synthesized and characterised by FT-IR, NMR, and colorimetric studies. The polymers were evaluated regarding their cytotoxicity, swelling behaviour, viscosity after mixing with mucus, mucus diffusion, penetration into mucosa, and mucoadhesion. The amount of the three ligands (Cys, Cys-MNA and Cys-GSH) bound to the polymer was determined to be in the range of 193-196 µmol/g. All polymers showed no cytotoxicity. Viscosity of the mixture of Pec-Cys-MNA and Pec-Cys-GSH with mucus increased 21.5- and 26.7-fold, respectively, compared to the unmodified polymer within 3 hours. Swelling, mucoadhesion, interpenetration and mucus diffusion were increased in the following rank order: Pec-Cys < Pec-Cys-MNA < Pec-Cys-GSH. Results of mucoadhesion study indicated a 7.4 and 8.1-fold increase of Pec-Cys-MNA and Pec-Cys-GSH, respectively, compared to the unmodified polymer. As the less reactive S-protected thiomer exhibited higher mucoadhesive properties than the other thiomers, this study provides evidence for the superior mucoadhesion of 3rd generation thiomers. STATEMENT OF SIGNIFICANCE: Three generations of thiolated polymers have been developed bearing different types of thiol ligands with the main purpose of enhancing mucus interactions. In this study, all generations were synthesized on the polymeric backbone of pectin for the first time to directly compare their mucus penetrating and mucoadhesive properties. 1st generation exhibited covalently bound L-cysteine moieties. For 2nd generation, thiols of cysteines were S-protected with 2-mercaptonicotinic acid (MNA), resulting in high reactive disulfide bonds. 3rd generation was synthesized by a thiol/disulfide exchange of glutathione with MNA, producing a less reactive disulfide bond. Mucus penetrating and mucoadhesive properties were found to be increased as follows: 1st generation < 2nd generation < 3rd generation. According to these results, the thiomer of 3rd generation represents a promising excipient with strong mucoadhesion.

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.

Mucolytic self-emulsifying drug delivery systems (SEDDS) containing a hydrophobic ion-pair of proteinase.

AIM: The aim of this study was to form hydrophobic ion-pairs of proteinase with cationic surfactants and to incorporate them into self-emulsifying drug delivery systems (SEDDS) to improve their mucus permeating properties. METHODS: Proteinase was ion-paired with benzalkonium chloride (BAK), hexadecylpyridinium chloride (HDP), alkyltrimethylammonium bromide (ATA) and hexadecyltrimethylammonium bromide (HDT) at pH 8.5-9.0, and subsequently incorporated into SEDDS consisting of Cremophor EL, propylene glycol, and Capmul 808-G (40/20/40). Mucus permeation of SEDDS containing proteinase complexes was evaluated via rotating tube technique and cell-free Transwell® insert system. Additionally, enzymatic activity of proteinase complexes as well as their potential cytotoxicity was evaluated. RESULTS: Among all tested hydrophobic ion-pairs, proteinase/BAK showed highest potential. Mucus diffusion of SEDDS containing proteinase/BAK complex yielded in 2.3-fold and 2.5-fold higher mucus permeability with respect to blank SEDDS at Transwell® insert system and rotating tube technique, respectively. Furthermore, proteinase/BAK complex maintained the highest enzymatic activity of 50.5 ± 5.6% compared to free proteinase. At a SEDDS concentration as low as 0.006% cell viability was just 80%. The addition of proteinase complexes to SEDDS increased cytotoxicity on Caco-2 cells in a concentration-dependent manner. CONCLUSION: SEDDS loaded with proteinase/BAK complexes are promising nanocarriers because of enhanced mucus permeating properties.

Cellular uptake of self-emulsifying drug-delivery systems: polyethylene glycol versus polyglycerol surface.

Aim: Comparison of the impact of polyethylene glycol (PEG) and polyglycerol (PG) surface decoration on self-emulsifying drug delivery system (SEDDS)-membrane interaction and cellular uptake. Materials & methods: PEG-, PEG/PG- and PG-SEDDS were assessed regarding their self-emulsifying properties, surface charge, bile salt fusibility, cellular uptake and interaction with endosome-mimicking membranes. Results: SEDDS exhibited droplet sizes between 150 and 175 nm, a narrow size distribution and self-emulsified within 7 min. Higher PEG-surfactant amounts in SEDDS resulted in charge-shielding and thus in a decrease of ζ potential up to Δ11 mV. The inert PEG-surface hampered bile salt fusion and interfered SEDDS-cell interaction. By reducing the PEG-surfactant amount to 10%, cellular uptake increased twofold compared with PEG-SEDDS containing 40% PEG-surfactant. PG-SEDDS containing no PEG-surfactants showed a threefold increased cellular uptake. Furthermore, complete replacement of PEG-surfactants by PG-surfactants led to enhanced cellular interaction and improved disruption endosome-like membranes. Conclusion: PG-surfactants demonstrated high potential to address PEG-surface associated drawbacks in SEDDS.

Synthesis and in vitro characterization of a preactivated thiolated acrylic acid/acrylamide-methylpropane sulfonic acid copolymer as a mucoadhesive sprayable polymer.

AIM: Development of a preactivated thiomer as sprayable excipient for mucoadhesive formulations. METHODS: CG4500 (acrylic acid/acrylamide-methyl propane sulfonic acid copolymer) was thiolated by conjugation with L-cysteine and preactivated by further modification with 2-mercaptonicotinic acid (MNA) in a two-step synthesis and characterized regarding degree of modification and cytotoxicity on Caco-2 cells. The mucoadhesive properties of this novel thiomer were evaluated via rheological synergism, tensile and mucosal residence time studies. Furthermore, the sprayability of the thiomer was evaluated. RESULTS: The newly synthesized derivatives CG4500-SH and CG4500-S-S-MNA showed mean coupling rates of 651 µmol thiol groups and 264 µmol MNA per gram polymer, respectively. Even for the unmodified polymer a rheological synergism was observed with isolated porcine intestinal mucus, which was 2.81-fold higher in case of the preactivated thiomer. Mucoadhesion studies on freshly excised porcine intestinal mucosa confirmed these results via a 2.43-fold higher total work of adhesion and a 2.31-fold higher mucosal residence time of the preactivated thiomer. In sprayability tests it was shown that solutions of the preactivated thiomer could be sprayed in concentrations up to 12% (m/V). CONCLUSION: The novel polymer CG4500-S-S-MNA is a promising sprayable excipient for mucoadhesive formulations.

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.

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.

S-protected gellan gum: Decisive approach towards mucoadhesive antimicrobial vaginal films.

The aim of this study was to synthesize novel polymeric excipients forming mucoadhesive films for treatment of vaginal microbial infections. 2-(2-Amino ethyldisulfanyl) nicotinic acid was conjugated with gellan gum via amide bond formation. The structure of the resulting S-protected gellan gum was confirmed by 1H NMR. S-protected gellan gum variants were characterized for thiol content, cytotoxicity, rheological behaviour and film forming capability. Depending on the added amount of AMENA degree of thiolation was 81 ±â€¯13 (S-GG 81) and 174 ±â€¯16 (S-GG 174) µmol/g, respectively. Vaginal films were casted from S-protected gellan gum variants and studied for adherence to vaginal mucosa, drug release and antimicrobial activity. S-protected gellan gum remained biocompatible showing >87% cell viability. S-GG 81 and S-GG 174 exhibited 1.84- and 4.3-fold increased dynamic viscosity in porcine mucus in comparison to unmodified gellan gum, respectively. Compared to gellan gum films, thiol functionalized gellan gum films showed 3-fold improved adhesion on mucosal surface over a period of 3 h along with significant antimicrobial activity. Moreover, S-protected gellan gum provided a sustained release of metronidazole. According to these results, S-protected gellan gum proved to be a promising novel excipient for casting vaginal films, exhibiting strongly improved mucoadhesive and antimicrobial properties.

Host and viral features of human dengue cases shape the population of infected and infectious Aedes aegypti mosquitoes.

Dengue is the most prevalent arboviral disease of humans. The host and virus variables associated with dengue virus (DENV) transmission from symptomatic dengue cases (n = 208) to Aedes aegypti mosquitoes during 407 independent exposure events was defined. The 50% mosquito infectious dose for each of DENV-1-4 ranged from 6.29 to 7.52 log10 RNA copies/mL of plasma. Increasing day of illness, declining viremia, and rising antibody titers were independently associated with reduced risk of DENV transmission. High early DENV plasma viremia levels in patients were a marker of the duration of human infectiousness, and blood meals containing high concentrations of DENV were positively associated with the prevalence of infectious mosquitoes 14 d after blood feeding. Ambulatory dengue cases had lower viremia levels compared with hospitalized dengue cases but nonetheless at levels predicted to be infectious to mosquitoes. These data define serotype-specific viremia levels that vaccines or drugs must inhibit to prevent DENV transmission.