Felix Prüfert

Polyethylene imine-6-phosphogluconic acid nanoparticles – a novel zeta potential changing system

The aim of the study was to develop nanoparticles with the ability to change their zeta potential. By covalent attachment of 6-phosphogluconic acid to polyethylene imine, a charged, enzymatically removable moiety was introduced into the polymer. The novel conjugate displayed 400 μmol phosphate per gram polymer, as determined by malachite green assay. Studies evaluating the cleavage by intestinal alkaline phosphatase revealed that 69 % of the coupled phosphate could be released from the polymer. Furthermore, nanoparticles generated by polyelectrolyte complexation technique using carboxymethyl cellulose as negatively charged component exhibited a zeta potential of -6 mV and an average particle size of 300 nm. Enzymatic cleavage of the phosphate ester moiety by isolated intestinal alkaline phosphatase on these nanoparticles caused shift of the zeta potential from negative to positive value of +3 mV whereby 58% of the total amount of phosphate were released. Studies on Caco-2 cells revealed the capability of a living system to hydrolyze the phosphate ester in the novel conjugate as well as on the nanoparticles via their intestinal alkaline phosphatase. Based on these results, polymeric nanoparticles comprising an enzymatically degradable phosphate ester moiety can provide a promising strategy for zeta potential changing systems.

Self-nanoemulsifying drug delivery systems as novel approach for pDNA drug delivery

It was the aim of this study to investigate a novel strategy for oral gene delivery utilizing a self-nanoemulsifying drug delivery system (SNEDDS). After hydrophobic ion pairing a plasmid was incorporated into SNEDDS. The mean droplet size of resulting nanoemulsions was determined to be between 45.8 and 47.5 nm. A concentration dependent cytotoxicity of the formulations was found on HEK-293 cells via MTT assay. Degradation studies via DNase I showed that incorporation into SNEDDS led to significantly, up to 8-fold prolonged resistant time against enzymatic digestion compared to naked pDNA and pDNA-lipid complexes. Transfection studies carried out revealed a significantly improved transfection compared to naked pDNA. Further, no decrease in transfection efficiency compared to transfection using Lipofectin(®) transfection reagent was observed.

Impact of lipases on the protective effect of SEDDS for incorporated peptide drugs towards intestinal peptidases

AIM The aim of this study is the development of self-emulsifying drug delivery systems (SEDDS) differing in amounts of ester substructures and to evaluate their stability in presence of pancreatic lipase and protective effect against luminal enzymatic metabolism using leuprorelin as model peptide drug. METHODS Hydrophobic leuprolide oleate was incorporated into three different SEDDS formulations and their stability towards pancreatic lipases was investigated utilizing a dynamic in vitro digestion model. Protective effect of SEDDS in respect to peptide drug stability against proteolytic enzymes, trypsin and α-chymotrypsin, was determined via HPLC. RESULTS Results of in vitro digestion demonstrated that 80% of SEDDS containing the highest amount of ester linkages was degraded within 60min. In comparison to that, SEDDS without ester bonds showed no degradation. With increasing oil droplets hydrolysis the remaining amount of peptide encapsulated into formulation decreased. Furthermore, after 180min incubation with trypsin up to 33.5% and with α-chymotrypsin up to 60.5% of leuprolide oleate was intact while leuprorelin acetate aqueous solution was completely metabolized by trypsin within 120min and by α-chymotrypsin within 5min. Protective effect in environment containing lipases was lower due to oil phase degradation, however, the amount of peptide in ester-free SEDDS was remarkably higher compared to SEDDS susceptible to lipases. CONCLUSION The present study revealed that SEDDS stable towards hydrolysis is able to exhibit a protective effect for oral peptide delivery.

Cell-penetrating self-nanoemulsifying drug delivery systems (SNEDDS) for oral gene delivery

ABSTRACT Objective: The aim of study was to investigate whether cell-penetrating peptides could amplify cellular uptake of plasmid DNA (pDNA) loaded self-nanoemulsifying drug delivery systems (SNEDDS) by mucosal epithelial cells, thereby enhancing transfection efficiency. Methods: HIV-1 Tat peptide-oleoyl conjugate (TAT-OL) was synthesized through amide bond formation between HIV-1 Tat-protein 49–57 (TAT) and oleoyl-chloride (OL). SNEDDS formulation contained 29.7% each of Cremophor EL, Capmul MCM and Crodamol, 9.9% propylene glycol and 1% TAT-OL. SNEDDS with OL instead of TAT-OL served as control. Results: Fluorescent-microscopy demonstrated 0.5% (m/v) nanoemulsions were suitable for subsequent studies. Mucus diffusion of nanoemulsion loaded with fluorescein diacetate (FDA) was 1.5-fold increased by incorporation of TAT-OL. Confocal microscopy confirmed that droplets of nanoemulsions were successfully internalized. Furthermore, quantitative analysis showed that addition of TAT-OL increases uptake of nanoemulsions by 2.3- and 2.6-folds after 2 and 4 hours of incubation, respectively. Cellular internalization pathways were found with substantial decrease in uptake in presence of indomethacin and chlorpromazine. Transfection efficiency investigated on HEK-293-cells was found to be 1.7- and 1.8-fold higher for SNEDDS loaded with TAT-OL compared to Lipofectin and control, respectively. Conclusion: In comparison to prevailing lipid and polymer-based delivery systems, these novel cell-penetrating SNEDDS likely represent most effective, simplistic and expedite dosage form for mucosal gene delivery.

Zeta potential changing phosphorylated nanocomplexes for pDNA delivery.

The objective of this study was to evaluate the suitability of a zeta potential changing system as gene delivery system. The phosphate ester bearing ligand 6-phosphogluconic acid (6-PGA) was attached to linear and branched polyethyleneimine (PEI) via a carbodiimide-mediated reaction whereby 287 μmol and 413 μmol 6-PGA could be coupled per gram polymer. Nanocomplexes of these modified polymers with pDNA showed a zeta potential of +12 mV for nanocomplexes with the linear PEI-6PGA and +16 mV in case of the branched derivative. By the addition of carboxymethylcellulose (CMC), zeta potentials of the complexes were reduced to +2.86 and +3.25, respectively. Phosphate release studies on Caco 2 cells and HEK-293 cells demonstrated the ability to cleave the phosphate ester. Compared to HEK-293 cells, enzymatic degradation of the phosphate ester in Caco 2 cells was 2.3-fold higher from nanocomplexes comprising linear PEI and 4.3-fold higher from those with branched PEI. Furthermore, incubation with alkaline phosphatase led to an increase in the zeta potential of nanocomplexes based on linear PEI-6PGA to +6.96mV and +8.26 mV in nanocomplexes comprising branched PEI-6PGA. Studying transfection efficiency in Caco 2 cells and HEK-293 cells, a higher expression of the green fluorescent protein (GFP) could be detected in HEK-293 cells. In presence of a phosphate inhibitor, transfection efficiencies were decreased in both cells lines, due to a lacking shift of the zeta potential of the tested pDNA complexes. According to these results, zeta potential changing systems seem to be a promising strategy for future gene delivery systems, as this concept allows the in situ generation of positive charges in close proximity to the cellular surface.

Zeta potential changing self-emulsifying drug delivery systems containing phosphorylated polysaccharides

Aim The aim of the study was to develop novel zeta potential changing self‐emulsifying drug delivery systems (SEDDS) containing phosphorylated polysaccharides. Methods Starch and hydroxypropyl starch (HPS) were phosphorylated by utilizing phosphorus pentoxide. The modified starches, starch phosphate (SP) and hydroxypropyl starch phosphate (HPSP), were loaded into SEDDS and investigated regarding particle size, zeta potential, stability and cell viability. The release of immobilized phosphate by intestinal alkaline phosphatase (IAP) was analyzed via malachite green assay. In parallel, the resulting shift in zeta potential of SEDDS was determined. Furthermore, Transwell chambers were applied in order to evaluate the mucus diffusion behavior of SEDDS utilizing fluorescein diacetate (FDA) as marker. Results The amount of attached phosphate for SP and HPSP revealed to be 119 &mgr;mol/g and 259 &mgr;mol/g, respectively. SEDDS consisting of 10% glycerol, 30% Capmul MCM, 30% Cremophor EL and 30% Captex 355 showed a droplet size of 39 ± 12 nm, stability over 240 min and no significant decrease in cell viability within the applied concentrations. SEDDS containing 3 mg/ml HPSP with a phosphate release of 204 &mgr;mol/g, demonstrated a shift in zeta potential from ‐6.3 mV to + 1.0 mV applying isolated IAP. Zeta potential changing SEDDS achieved a 2.5‐fold and 5.4‐fold higher amount of diffused FDA compared to the references within mucus permeation studies. Conclusion SEDDS containing HPSP represent comparable high mucus diffusion properties emphasized by a highly significant change in zeta potential. Graphical abstract Figure. No Caption available.

Synthesis and in vitro characterization of a novel S-protected thiolated alginate.

The object of this study was to synthesize and characterize a novel S-protected thiolated polymer with a high degree of modification. In this regard, an alginate-cysteine and an alginate-cysteine-2-mercaptonicotinic acid conjugate were synthesized. To achieve a high coupling rate of the thiol group bearing ligand cysteine to the polymer, the carbohydrate was activated by an oxidative ring opening with sodium periodate followed by a reductive amination to bind the primary amino group of cysteine to resulting reactive aldehyde groups. The obtained thiolated polymer displayed 1561±130μmol thiol groups per gram polymer. About one third of these thiol groups were S-protected by the implementation of a thiol bearing aromatic protection group via disulfide bond formation. Test tablets of both modified polymers showed improved stability against oxidation in aqueous environment compared to the unmodified alginate and exhibit higher water-uptake capacity. Rheological investigations revealed an increased viscosity of the S-protected thiolated polymer whereat the thiolated non S-protected polymer showed gelling properties after the addition of hydrogen peroxide. The mucoadhesive properties could be improved significantly for both derivatives and no alteration in biocompatibility tested on Caco-2 cell monolayer employing an MTT assay could be detected after modification. According to these results, both new derivatives seem promising for various applications.

Multifunctional adhesive polymers: Preactivated thiolated chitosan-EDTA conjugates

Aim The aim of this study was to synthesis preactivated thiolated chitosan‐EDTA (Ch‐EDTA‐cys‐2MNA) conjugates exhibiting in particular high mucoadhesive, cohesive and chelating properties. Methods Thiol groups were coupled with chitosan by carbodiimide reaction and further preactivated by attachment with 2‐mercaptonicotinic acid (2MNA) via disulfide bond formation. Determinations of primary amino and sulfhydryl groups were performed by TNBS and Ellman’s tests, respectively. Cytotoxicity was screened by resazurin assay in Caco‐2 cells. Mucoadhesive properties and bivalent cation binding capacity with Mg2+ and Ca2+ in comparison to chitosan‐EDTA (Ch‐EDTA) and thiolated Ch‐EDTA (Ch‐EDTA‐cys) were evaluated. Results Determination of 2MNA and total sulfhydryl groups indicated that 80% of thiol groups were preactivated. The results from cytotoxicity studies demonstrated that Ch‐EDTA‐cys and Ch‐EDTA‐cys‐2MNA were not toxic to the cells at the polymer test concentration of 0.25% (w/v) while cell viability decreased by increasing the concentration of Ch‐EDTA. Although EDTA molecule was modified by thiolation and preactivation, approximately 50% of chelating properties of the conjugates were maintained compared to Ch‐EDTA. Ch‐EDTA‐cys‐2MNA adhered on freshly excised porcine intestinal mucosa up to 6 h while Ch‐EDTA adhered for just 1 h. Conclusion According to the combination of mucoadhesive and chelating properties of the conjugates synthesized in this study, Ch‐EDTA‐cys‐2MNA might be useful for various mucosal drug delivery systems. Graphical abstract Figure. No Caption available.

Self-emulsifying drug delivery systems: Design of a novel vaginal delivery system for curcumin

Aim: The aim of this study was to develop a vaginal self‐emulsifying delivery system for curcumin being capable of spreading, of permeating the mucus gel layer and of protecting the drug being incorporated in oily nanodroplets towards mucus interactions and immobilization. Methods: The emulsifying properties of curcumin loaded SEDDS containing 30% Cremophor RH40, 20% Capmul PG‐8, 30% Captex 300, 10% DMSO and 10% tetraglycol (SEDD formulation A) as well as 25% PEG 200, 35% Cremophor RH40, 20% Captex 355, 10% Caprylic acid and 10% Tween 80 (SEDD formulation B) after diluting 1 + 2 with artificial vaginal fluid were characterized regarding droplet size and zeta potential. Collagen swelling test was used to examine the irritation potential of SEDDS. Additionally to mucus binding studies, permeation studies in the mucus were performed. Furthermore, spreading potential of the novel developed formulations was compared with a commercial available o/w cream (non‐ionic hydrophilic cream) on vaginal mucosa. Results: SEDDS displayed a mean droplet size between 38 and 141 nm and a zeta potential of −0.3 to −1.6 mV. The collagen swelling test indicated no significant irritation potential of both formulations over 24 h. An immediate interaction of unformulated curcumin with the mucus was determined, whereas both SEDDS facilitated drug permeation through the mucus layer. Formulation B showed a 2.2‐fold improved transport ratio of curcumin compared to SEDD formulation A. In comparison to the vaginal cream, SEDD formulation A and B were able to spread over the vaginal mucosa and cover the tissue to a 17.8‐ and 14.8‐fold higher extent, respectively. Conclusion: According to these results, SEDDS seems to be a promising tool for vaginal application. Graphical abstract Figure. No caption available.

Enhancing the efficiency of thiomers: Utilizing a highly mucoadhesive polymer as backbone for thiolation and preactivation

&NA; The objective of this study was to develop a novel thiomer with enhanced mucoadhesive properties using a highly mucoadhesive polymeric backbone. Fixomer™ A‐30 (poly(methacrylic acid‐co‐sodium acrylamidomethyl propane sulfonate)), exhibiting a mucoadhesive strength superior to that of all other polymers, was thiolated by conjugation with l‐cysteine and furthermore preactivated with 2‐mercaptonicotinic acid (MNA). The resulting derivatives Fix‐SH and Fix‐S‐MNA exhibited coupling rates of 755 &mgr;mol thiol groups and 304 &mgr;mol MNA per gram polymer, respectively. The mucoadhesive profile was evaluated with three different methods: tensile studies, rotating cylinder and rheological synergism. In tensile studies, a total work of adhesion of above 500 &mgr;J was determined for the unmodified polymer that increased to around 750 &mgr;J after thiolation and around 1500 &mgr;J after preactivation. The adhesion time of Fix‐SH on the rotating cylinder was 3.7‐fold and that of Fix‐S‐MNA 6.8‐fold longer compared to the unmodified polymer. A rheological synergism was observed for the unmodified polymer as well as the derivatives with a non‐significant difference for Fix‐SH but a 5.44‐fold improvement for Fix‐S‐MNA. Fix‐S‐MNA showed a significantly improved swelling behavior with a water‐uptake up to the 30‐fold of its initial weight over >50 h whereas thiolation showed only slight improvements. Derivatization had no significant influence on cell viability. According to the results, Fix‐S‐MNA seems to be a suitable polymer for mucoadhesive drug delivery systems. Graphical abstract Figure. No caption available.