Potential antiaging activity of secretome gel of human Wharton's jelly mesenchymal stem cells (hWJ-MSCs) in UV-induced mice models.

Objectives: Skin aging is a degenerative process that can be induced by UV irradiation. UV radiation can produce reactive oxidate stress which causes premature aging. This study aims to examine the antiaging potential of secretome gel (SC) from human Wharton Jelly Mesenchymal Stem Cells (hWJ-MSCs) in a UVB-induced mice model. Materials and Methods: The secretome was obtained from hWJ-MSCs and made in gel form. Male mice were radiated by UVB for 15 min twice daily for 14 days. The gel was topically applied to the mice's dorsal skin. Two treatments of secretome gel: secretome 1 is applied once and secretome 2 is applied twice daily after UVB radiation. TGF-ß1, IL-10, and IL-18 gene expression was determined using RT-PCR. Hematoxylin Eosin staining was used to observe the inflammation and collagen density of skin tissue. An immunohistochemistry assay was used to analyze the protein expression of P53, COL4A1, MMP-2, and MMP-13. The data were statistically analyzed using the ANOVA test followed by the Tukey post hoc test (P<0.05). Results: UVB induction caused loss of collagen, increasing inflammation and high expression of aging mediators. SC increased the gene expression of TGF-ß1 and IL-10 and decreased IL-18 gene expression. Histopathological tests showed that SG increased collagen density, lowered inflammation, and repaired cell damage in skin tissue. Immunohistochemistry test showed that SC decreased MMP-2, MMP-13, and P53 expression, in contrast, increased COL4A1. Conclusion: The secretome gel of hWJ-MSCs showed antiaging activities with potential for preventing and curing skin aging.

Synthesis and Characterization of a Cationic Thiomer Based on Ethyl Cellulose for Realization of Mucoadhesive Tablets and Nanoparticles.

Purpose: Ethyl cellulose (EC) based nanoparticles are being extensively studied for their ability to achieve prolonged drug release and improve drug stability. Within this study, the thiolation of unmodified EC using cysteamine as a ligand was carried out to design nanoparticles with mucoadhesive properties and comparatively strong lipophilic properties. Methods: The thiolation was performed via oxidation and reductive amination, whereas the nanoparticles were generated via the ionic gelation followed by the precipitation method. Results: The number of free thiol groups on EC-cysteamine was in the range of 210-261 µmol per gram of polymer. Tablets based on EC-cysteamine demonstrated mucoadhesive properties 16.7-fold improved compared with those comprising unmodified EC. The mean diameter of the particles was in the range of 94-123 nm and the zeta potential was determined to be -7.97 to -14.70 mV. The nanoparticles remained attached to porcine intestinal mucosa for up to 36% after 3 h of incubation. The formation of nanoparticles improved the stability of EC-cysteamine conjugate against cellulase and provided a zero-order release. Moreover, both EC-cysteamine and the nanoparticles did not show any pronounced cytotoxicity. Conclusion: Accordingly, EC-cysteamine nanoparticles could be a specific type of nanoparticulate delivery system with mucoadhesive properties. The amount of free thiol groups within EC-cysteamine nanoparticles together with their lipophilic properties could be further modified and modulated for a desired release behavior.

Development, Characterization and Pharmacokinetic Profile of Chitosan-Sodium Tripolyphosphate Nanoparticles Based Drug Delivery Systems for Curcumin.

Purpose: This study aimed to provide the method of preparation, characterization of curcumin-loaded chitosan-sodium tripolyphosphate (NaTPP) nanoparticle, and evaluate its pharmacokinetic profiles. Methods: Curcumin-loaded chitosan-NaTPP nanoparticles were synthesized using ionic gelation methods. Curcumin was dissolved using surfactants and cosurfactants. Chitosan polymer was then mixed in the curcumin solution and dripped with NaTPP solution until nanoparticle formation. The mucoadhesive study was evaluated by measuring the fluorescence of curcumin within the prepared nanoparticles. The pharmacokinetic profiles of curcumin particles and nanoparticles were then assessed in rats by administering a single oral dose of 100 mg/kg BW. Blood samples were taken from nine predetermined time points, and curcumin plasma concentrations were then analyzed using UPLC-MS/MS. Results: The particle size of the curcumin nanoparticles obtained were 11.5 nm. Entrapment efficiency (EE) of curcumin nanoparticles were exceeding 99.97%, and drug loading capacity (DLC) was 11.34%. The mucoadhesive properties of the nanoparticles were superior to that of curcumin particles. Pharmacokinetic evaluation in rats revealed that curcumin nanoparticles resulted in an increase of area under the curve (AUC), maximum concentration (Cmax), earlier time to reach maximum concentration (Tmax), and lower clearance (CL). Conclusion: Curcumin-loaded chitosan-NaTPP nanoparticles is an effective formulation to improve curcumin plasma concentrations. Thus, enable its applications for the treatment of various diseases.

Curcumin Nanoparticle Enhances the Anticancer Effect of Cisplatin by Inhibiting PI3K/AKT and JAK/STAT3 Pathway in Rat Ovarian Carcinoma Induced by DMBA.

Cisplatin has been used for decades for the treatment of ovarian cancer. However, despite its potent anticancer effect, cisplatin's efficacy as a single agent was inadequate in patients with advanced stage. Curcumin has been shown to sensitize cisplatin activity in several cancer models. However, the low bioavailability of curcumin has limited its anticancer potential. Hence, nano-formulation of curcumin was developed to increase its therapeutic efficacy in ovarian cancer. The objective of this study was to investigate the mechanism of curcumin nanoparticles given in combination with cisplatin in rat ovarian carcinoma induced by dimethylbenz(a)anthracene (DMBA). The administration of cisplatin and nanocurcumin resulted in a significant reduction in ovarian tumor volume and weight. Furthermore, there were reduction in expressions of Ki67, TGF-ß, PI3K, and Akt phosphorylation. Co-treatment of cisplatin and nanocurcumin also reduced JAK expression, STAT3 phosphorylation, and reduced IL-6 concentrations. Altogether, nanocurcumin, given as a co-treatment with cisplatin has therapeutic potential in ovarian cancer models by inhibiting proliferation through downregulation of PI3K/Akt and JAK/STAT3 signaling pathways.

Comparative Effect of Curcumin and Nanocurcumin on Nephroprotection at Cisplatin-induced Rats.

BACKGROUND: Cisplatin is a first-line chemotherapeutic agent for various solid tumors including ovarian and breast cancer. Thereby, it has been proven effective as an antineoplastic agent, but its clinically use is limited because of its nephrotoxicity side effect. AIMS AND OBJECTIVES: This study aimed to investigate curcumin as a renoprotector agent against cisplatin nephrotoxicity. MATERIALS AND METHODS: The samples used were curcumin and its nanoparticles formulated using ionic gelation method. The nephrotoxicity was investigated through several parameters such as serum creatinine, blood urea nitrogen, serum albumin, kidney weight ratio, and histopathology. These parameters were tested on rats and divided into the following four groups: normal group, negative control group that administered cisplatin with doses amount of 7 mg/kg body weight (BW) intraperitoneally, nanocurcumin group (cisplatin + nanocurcumin) and curcumin group (cisplatin + curcumin). The agents were administered at a dose of 100 mg/kg BW every day in 9 days before cisplatin administration. The sample of blood serum and kidneys organ were taken 48h after cisplatin administration. RESULTS: The negative control group showed a significant increase in serum creatinine, blood urea nitrogen, and kidney weight ratio, whereas it showed a significant decrease in serum albumin. The administration of sample agents showed a significant decrease in serum creatinine, blood urea nitrogen, and kidney weight ratio and an increase in the albumin level as compared to negative control group. CONCLUSION: Nanocurcumin showed significant improvement in kidneys more than curcumin. In contrast, histopathological examination verified the necrosis in negative control group, suggesting the renoprotection effect of nanocurcumin against nephrotoxicity on cisplatin-induced rats.

Synthesis and characterization of thiolated ß-cyclodextrin as a novel mucoadhesive excipient for intra-oral drug delivery.

The objective of the present study was to synthesize and characterize cysteamine conjugated ß-cyclodextrin (ß-CD-Cys) as a novel mucoadhesive oligomeric excipient for intra-oral drug delivery. ß-CD-Cys conjugates were obtained in a two-step synthetic pathway, whereby, vicinal diol groups of the oligomer were oxidized using increasing concentrations of sodium-per-iodate (NaIO4), prior to the covalent coupling of cysteamine via reductive amination. Quantification of immobilized thiol groups through Ellman's test revealed 561.56 ± 81 µmol/g, 1054.26 ± 131 µmol/g and 1783.92 ± 201 µmol/g of free thiol groups attached to the oligomer backbone depending on the extent of oxidation. ß-CD-Cys conjugates at concentrations of 0.5% (m/v) showed no toxic effects on Caco-2 cells within 72 h. Furthermore, ß-CD-Cys conjugates displayed a 4-fold improved water solubility compared to the parent oligomer. ß-CD-Cys conjugates (ß-CD-Cys561, ß-CD-Cys1054 and ß-CD-Cys1783) showed 2.86-, 15.09- and 49.08-fold improved retention time on porcine intestinal mucosa and 9.66-, 16.43- and 34.51-fold improved on the porcine buccal mucosa, respectively. Formation of inclusion complexes of miconazole nitrate and ß-CD-Cys1054 resulted in 150-fold increased solubility of miconazole nitrate. According to these results, it seems that ß-CD-Cys conjugates might provide a new promising tool for delivery of poorly water soluble therapeutic agents, such as miconazole nitrate for intra-oral delivery.

Development and in vitro evaluation of slippery nanoparticles for enhanced diffusion through native mucus.

AIM: The aim of this study was to investigate the mucus-penetrating properties of neutral nanoparticles comprising poly(acrylic acid) (PAA) and poly(allylamine) (PAM). MATERIALS & METHODS: PAA and PAM nanoparticles were prepared on the basis of ionic interactions between the two polymers. Nanoparticles were characterized by particle size as well as surface charge. The cytotoxicity was examined via resazurin and lactate dehydrogenase assays. Using a modified Ussing chamber with mucus, the diffusion properties of obtained neutral nanoparticles were compared with control particles. RESULTS: The obtained PAA-PAM nanoparticles demonstrated no significant cytotoxicity and displayed smooth and spherical surfaces, a particle size range of 200 nm and ζ-potential of 0.9 mV. The diffusion efficiency of neutral nanoparticles was 2.5- and 1.8-fold higher than PAM and PAA nanoparticles, respectively. CONCLUSION: Taking enhanced mucus-penetrating properties into account, neutral nanoparticles were shown to be very promising in drug delivery via mucus membranes of different cavities.

Efficient MRI labeling of endothelial progenitor cells: design of thiolated surface stabilized superparamagnetic iron oxide nanoparticles.

The aim of this study was to design thiolated surface stabilized superparamagnetic iron oxide nanoparticles (TSS-SPIONs) for efficient internalization with high MRI sensitivity. TSS-SPIONs were developed by chelation between thiolated chitosan-thioglycolic acid (chitosan-TGA) hydrogel and iron ions (Fe(2+)/Fe(3+)). Likely, unmodified chitosan hydrogel SPIONs (UC-SPIONs) and uncoated SPIONs were used as control. Moreover, TSS-SPIONs were investigated regarding to their iron core size, hydrodynamic diameter, zeta potential, iron contents, molar relaxivities (r1 and r2), and cellular internalization. TSS-SPIONs demonstrated an iron oxide core diameter (crystallite size by XRD) of 3.1 ± 0.02 nm, a hydrodynamic diameter of 94 ± 20 nm, a zeta potential of +21 ± 5 mV, and an iron content of 3.6 ± 0.9 mg/mL. In addition, internalization of TSS-SPIONs into human endothelial progenitor cells (EPC) from umbilical cord blood was more than threefold and 17-fold higher in contrast to UC-SPIONs and SPIONs, respectively. With twofold lower incubation iron concentration of TSS-SPIONs, more than threefold higher internalization was achieved as compared to Resovist®. Also, cell viability of more than 90% was observed in the presence of TSS-SPIONs after 24h. The molar MR relaxivities (r2) value at 1.5 T was threefold higher than that of Resovist® and demonstrated that TSS-SPIONs have the potential as very effective T2 contrast-enhancement agent. According to these findings, TSS-SPIONs with efficient internalization, lower cytotoxicity, and high MRI sensitivity seem to be promising for cell tracking.

Enzymatic degradation of thiolated chitosan.

The objective of this study was to evaluate the biodegradability of thiolated chitosans in comparison to unmodified chitosan. Mediated by carbodiimide, thioglycolic acid (TGA) and mercaptonicotinic acid (MNA) were covalently attached to chitosan via formation an amide bond. Applying two different concentrations of carbodiimide 50 and 100 mM, two chitosan TGA conjugates (TGA A and TGA B) were obtained. According to chitosan solution (3% m/v) thiomer solutions were prepared and chitosanolytic enzyme solutions were added. Lysozyme, pectinase and cellulase were examined in chitosan degrading activity. The enzymatic degradability of these thiomers was investigated by viscosity measurements with a plate-plate viscometer. The obtained chitosan TGA conjugate A displayed 267.7 µmol and conjugate B displayed 116.3 µmol of immobilized thiol groups. With 325.4 µmol immobilized thiol groups, chitosan MNA conjugate displayed the most content of thiol groups. In rheological studies subsequently the modification proved that chitosan TGA conjugates with a higher coupling rate of thiol groups were not only degraded to a lesser extent by 20.9-26.4% but also more slowly. Chitosan mercaptonicotinic acid was degraded by 31.4-50.1% depending the investigated enzyme and even faster than unmodified chitosan. According to these results the biodegradability can be influenced by various modifications of the polymer which showed in particular that the rate of biodegradation is increased when MNA is the ligand, whereas the degradation is hampered when TGA is used as ligand for chitosan.

HEC-cysteamine particles: influence of particle size, zeta potential, morphology and sulfhydryl groups on permeation enhancing properties.

Within this study, the influence of particle size and zeta potential of hydroxyethyl cellulose-cysteamine particles on permeation enhancing properties was investigated. Particles were prepared by four different methods namely ionic gelation, spray drying, air jet milling and grinding. Particles prepared by grinding were additionally air jet milled. All particles were characterized in terms of particle size and zeta potential. The transport of fluorescein isothiocyanate-dextran 4 (FD4) across Caco-2 cell monolayers in the presence of these particles and the decrease in transepithelial electrical resistance (TEER) was evaluated. The cytotoxic effect of the particles was investigated using resazurin assay. Nanoparticles displaying a zeta potential of 3.3 ± 1.3 mV showed the highest enhancement of FD4 transport among all particles with a 5.83-fold improvement compared to buffer only. Due to the larger particle size, particles generated by grinding exhibited a lower capability in opening of tight junctions compared to smaller particles generated by air jet milling. In addition, the results of the transport studies were supported by the decrease in the TEER. All particle formulations tested were comparatively non-cytotoxic. Accordingly, the zeta potential and particle size showed a significant impact on the opening of tight junctions and hence could play an important role in the design of hydroxyethyl cellulose (HEC)-cysteamine-based nano- and micro-particles as drug delivery systems.

Thiolated hydroxyethyl cellulose: design and in vitro evaluation of mucoadhesive and permeation enhancing nanoparticles.

Within this study, HEC-cysteamine nanoparticles with free thiol groups in the range of 117-1548 µmol/g were designed and characterized. Nanoparticles were generated via ionic gelation of the cationic polymer with tripolyphosphate (TPP) followed by covalent crosslinking via disulfide bond formation using H2O2 as oxidant. The mean diameter of the particles was in the range of 270-360 nm, and zeta potential was determined to be +4 to +10 mV. Nanoparticles were evaluated in terms of mucoadhesive, permeation enhancing, and biocompatible properties as well as biodegradability. The particles remained attached to porcine intestinal mucosa up to 70% after 3h of incubation. The more nanoparticles were oxidized; however, the less were their mucoadhesive properties. Nanoparticles applied in a concentration of 0.5% (m/v) with the highest content of free thiol groups improved the transport of fluorescein isothiocyanate dextran 4 (FD4) across Caco-2 cell monolayer 3.94-fold in comparison with control (buffer). In addition, the transport of FD4 was even 1.84-fold enhanced in the presence of 0.5% (m/v) nanoparticles with the lowest free thiol group content. The higher the disulfide bond content within nanoparticles was, to a lower degree nanoparticles were hydrolyzed by cellulase. None of these nanoparticles showed pronounced cytotoxicity. Accordingly, HEC-cysteamine could be a promising excipient for nanoparticulate delivery systems for poorly absorbed drugs.

Preactivated thiomers: permeation enhancing properties.

The study was aimed to prepare a series of poly(acrylic acid)-cysteine-2-mercaptonicotinic acid conjugates (preactivated thiomers) and to evaluate the influence of molecular mass or degree of preactivation with 2-mercaptonicotinic acid (2MNA) on their permeation enhancing properties. Preactivated thiomers with different molecular mass and different degree of preactivation were synthesized and categorized on the basis of their molecular mass and degree of preactivation as PAA(100)-Cys-2MNA (h), PAA(250)-Cys-2MNA (h), PAA(450)-Cys-2MNA (h), PAA(450)-Cys-2MNA (m) and PAA(450)-Cys-2MNA (l). In vitro permeation studies, the permeation enhancement ability for preactivated thiomers was ranked as PAA(450)-Cys-2MNA (h)>PAA(250)-Cys-2MNA (h)>PAA(100)-Cys-2MNA (h) on both Caco-2 cell monolayers and rat intestinal mucosa. Comparing the influence of degree of preactivation with 2MNA on permeation enhancement, the following order PAA(450)-Cys-2MNA (h)>PAA(450)-Cys-2MNA (m)≈PAA(450)-Cys-2MNA (l) on Caco-2 cell monolayers and PAA(450)-Cys-2MNA (m)>PAA(450)-Cys-2MNA (h)>PAA(450)-Cys-2MNA (l) on intestinal mucosa was observed. The P(app) of sodium fluorescein was 5.08-fold improved on Caco-2 cell monolayers for PAA(450)-Cys-2MNA (h) and 2.46-fold improved on intestinal mucosa for PAA(450)-Cys-2MNA (m), respectively, in comparison to sodium fluorescein in buffer only. These results indicated that preactivated thiomers could be considered as a promising macromolecular permeation enhancing polymer for non-invasive drug administration.

S-protected thiolated chitosan for oral delivery of hydrophilic macromolecules: evaluation of permeation enhancing and efflux pump inhibitory properties.

The objective of this study was the investigation of permeation enhancing and P-glycoprotein (P-gp) inhibition effects of a novel thiolated chitosan, the so-named S-protected thiolated chitosan. Mediated by a carbodiimide, increasing amounts of thioglycolic acid (TGA) were covalently bound to chitosan (CS) in the first step of modification. In the second step, these thiol groups of thiolated chitosan were protected by disulfide bond formation with the thiolated aromatic residue 6-mercaptonicotinamide (6-MNA). Mucoadhesive properties of all conjugates were evaluated in vitro on porcine intestinal mucosa based on tensile strength investigations. Permeation enhancing effects were evaluated ex vivo using rat intestinal mucosa and in vitro via Caco-2 cells using the hydrophilic macromolecule FD(4) as the model drug. Caco-2 cells were further used to show P-gp inhibition effects by using Rho-123 as P-gp substrate. Apparent permeability coefficients (P(app)) were calculated and compared to values obtained from each buffer control. Three different thiolated chitosans were generated in the first step of modification, which displayed increasing amounts of covalently attached free thiol groups on the polymer backbone. In the second modification step, more than 50% of these free thiol groups were covalently linked with 6-MNA. Within 3 h of permeation studies on excised rat intestine, P(app) values of all S-protected chitosans were at least 1.3-fold higher compared to those of corresponding thiomers and more than twice as high as that of unmodified chitosan. Additional permeation studies on Caco-2 cells confirmed these results. Because of the chemical modification and higher amount of reactive thiol groups, all S-protected thiolated chitosans exhibit at least 1.4-fold pronounced P-gp inhibition effects in contrast to their corresponding thiomers. These features approve S-protected thiolated chitosan as a promising excipient for various drug delivery systems providing improved permeation enhancing and efflux inhibition effects.

Thiolated chitosan nanoparticles for the nasal administration of leuprolide: bioavailability and pharmacokinetic characterization.

The purpose of this study was to develop thiolated nanoparticles to enhance the bioavailability for the nasal application of leuprolide. Thiolated chitosan-thioglycolic acid (chitosan-TGA) and unmodified chitosan nanoparticles (NPs) were developed via ionic gelation with tripolyphosphate (TPP). Leuprolide was incorporated during the formulation process of NPs. The thiolated (chitosan-TGA) NPs had a mean size of 252 ± 82 nm, a zeta potential of +10.9 ± 4 mV, and payload of leuprolide was 12 ± 2.8. Sustained release of leuprolide from thiolated NPs was demonstrated over 6h, which might be attributed to inter- and/or intramolecular disulfide formation within the NPs network. Ciliary beat frequency (CBF) study demonstrated that thiolated NPs can be considered as suitable additives for nasal drug delivery systems. Compared to leuprolide solution, unmodified NPs and thiolated NPs provoked increased leuprolide transport through porcine nasal mucosa by 2.0 and 5.2 folds, respectively. The results of a pharmacokinetic study in male Sprague-Dawley rats showed improved transport of leuprolide from thiolated NPs as compared to leuprolide solution. Thiolated NPs had a 6.9-fold increase in area under the curve, more than 4-fold increase in elimination half-life, and a ∼3.8-fold increase in maximum plasma concentration compared to nasal solution alone. The relative nasal bioavailability (versus s.c. injection) of leuprolide thiolated NPs calculated on the basis of AUC((0-6)) was about 19.6% as compared to leuprolide solution 2.8%. The enhanced bioavailability of leuprolide is likely due to facilitated transport by thiolated NPs rather than improved release.

Development and in vivo characterization of a novel peptide drug delivery system providing extended plasma half life.

It was the aim of this study to develop a sustained parenteral peptide (DALCE) delivery system by the immobilization of DALCE to thiolated carboxymethyl dextran-cysteine (CMD-Cys) via disulfide bond formation. The resulting CMD-Cys-DALCE conjugate displayed a 22.6±7.9% (m/m) of DALCE (mean±S.D.; n=3). The conjugation of DALCE with CMD-Cys was confirmed by FTIR-ATR spectroscopy. In vitro release studies of conjugate CMD-Cys-DALCE in the presence of 2 µM/ml reduced glutathione (GSH) being also available in the plasma showed a sustained peptide release over a time period of 8 h, because of thiol/disulfide exchange reactions. For in vivo pharmacokinetic study, DALCE and CMD-Cys-DALCE were administered intravenously to male Sprague-Dawley rats at a dose of 1mg/kg. The AUC(0-8) (ng.min/ml) was determined to be 268848±924 and 40019±495 for CMD-Cys-DALCE and DALCE, respectively. The mean residence time (MRT) was determined to be 256±8 and 53.1±9.5 min for CMD-Cys-DALCE and for DALCE, respectively. CMD-Cys-DALCE showed a more than 5-fold increased elimination half-life (p<0.01), 3-fold decreased volume of distribution (p<0.01) and a 6.7-fold decreased plasma clearance rate (p<0.01) compared to DALCE. According to these findings, CMD-Cys-DALCE seems to act as prodrug by improving half-life and decreasing plasma clearance.

Synergistic effects of conjugating cell penetrating peptides and thiomers on non-viral transfection efficiency.

Nanoparticles generated by complex coacervation of plasmid DNA (pDNA) and modified chitosans namely chitosan-thioglycolic acid (TGA) conjugate and chitosan-HIV-1 Tat peptide conjugate were evaluated as gene delivery systems. In order to optimize transfection efficiency, chitosan-HIV-1 Tat peptide conjugate was combined with chitosan-TGA before its complexation with pDNA. Particle size and zeta potential measurements were performed to characterize the generated nanoparticles. The nanoparticles transfection efficiencies were assessed by exploitation of the green fluorescent protein (GFP) reporter gene. HEK293 cells were incubated for 24 h with the nanoparticles and the GFP positive cells were observed by fluorescence microscopy. The nanoparticles in the size range of 200-300 nm could transfect HEK293 cells as a model cell line with different transfection efficiencies. Unlike chitosan-TGA, chitosan-HIV-1 Tat peptide led to increased zeta potential of nanoparticles as compared to unmodified chitosan. The transfection efficiency of the nanoparticles generated by combination of chitosan-HIV-1 Tat peptide with chitosan-TGA was comparatively higher than that of the nanoparticles generated by either chitosan-TGA or the combination of chitosan-HIV-1 Tat peptide with unmodified chitosan. After 72 h of incubation, the combination of chitosan-HIV-1 Tat peptide with chitosan-TGA was found to be 7.12- and 67.37 times more efficient than unmodified chitosan and pDNA alone, respectively and showed a synergistic effect in transfection of pDNA into the cells. Moreover, none of the nanoparticles showed any severe cytotoxicity. Accordingly, this strategy might result in a potent carrier for gene delivery.

HEC-cysteamine conjugates: influence of degree of thiolation on efflux pump inhibitory and permeation enhancing properties.

Within the present study hydroxyethyl cellulose-cysteamine conjugates are investigated regarding biocompatibility, in situ gelling, permeation enhancing and efflux pump inhibitory properties. For this purpose, a series of concentrations of sodium periodate was prepared to oxidize HEC leading to ring opening of glucose subunits. The resulting polymers showing varying degrees of oxidation (DO) were then conjugated with cysteamine stabilized via reductive amination. Consequently, HEC-cysteamine conjugates with increasing degree in thiolation were obtained. Since the conjugates are positively charged, potency of cytotoxicity was tested by resazurin assay. In situ gelling properties of the conjugates were studied to investigate change of their viscosity due to inter- and/or intramolecular crosslinking via disulfide bonds. The influence of the presence of the conjugates on transport of rhodamine 123 and fluoresceinisothiocyanate-dextran 4 (FD4) representing model compounds for P-glycoprotein (P-gp) inhibition and permeation enhancing studies, respectively, across Caco-2 cell monolayers was determined. The conjugates showed a degree of thiolation in the range of 316-2158 µmol/g. Within 30 min, dynamic viscosity of the conjugate with the lowest degree of thiolation 0.5% (m/v) increased up to 300-fold. The conjugates showed a degree of thiolation-dependent increase in cytotoxicity but they all were found comparatively low cytotoxic. The addition of the conjugate with thiol group content of 1670 µmol/g resulted in the highest improvement in the transport of both rhodamine 123 and FD4 as compared to buffer control. Accordingly, the degree of thiolation strongly influences the properties of the conjugates and the modulation of the degree of thiolation could be exploited for development of various drug delivery systems.

Poly(acrylic acid)-cysteine for oral vitamin B12 delivery.

The aim of this study was to investigate the potential of poly(acrylic acid)-cysteine (PAA-cys) solution and microparticles to enhance the transport of vitamin B12 (VB 12) across Caco-2 cell monolayer and rat intestinal mucosa. Thiolated PAA was synthesized by covalent attachment of L-cysteine. Microparticles were prepared by spray-drying and characterized regarding their size, morphology, thiol group content, VB 12 payload and release, swelling behavior, mucoadhesion, permeation-enhancing effect, and cytotoxicity. Particles with a mean diameter of 2.452±2.26 µm, a payload of 1.11±0.72%, and 190.2±8.85 µmol of free thiol groups per gram were prepared. Swelling behavior studies revealed that the stability of thiolated particles was improved compared with unmodified ones. Of the total VB 12 loaded, 95±0.12% was released within 3 h from thiolated particles. PAA-cys particles exhibited 2.24-fold higher mucoadhesive properties compared with unmodified particles. Permeation experiments with Caco-2 cells proved that permeability of VB 12 with PAA-cys solution and particles was 3.8- and 3.6-fold higher than control, respectively, and with rat intestinal mucosa it was 4.8- and 4.4-fold higher than control, respectively. Negligible cytotoxicity was assessed. PAA-cys is a promising excipient for oral delivery of VB 12 as a solution and as microparticles.

Design and synthesis of a novel cationic thiolated polymer.

The purpose of this study was to design and characterize a novel cationic thiolated polymer. In this regard a hydroxyethylcellulose-cysteamine conjugate (HEC-cysteamine) was synthesized. Oxidative ring opening with periodate and reductive amination with cysteamine were performed in order to immobilize free thiol groups to HEC. The resulting HEC-cysteamine displayed 2035 ± 162 µmol immobilized free thiol groups and 185 ± 64 µmol disulfide bonds per gram of polymer being soluble in both acidic and basic conditions. Unlike the unmodified HEC, in case of HEC-cysteamine, a three-fold increase in the viscosity was observed when equal volumes of the polymer were mixed with mucin solution. Tablets based on HEC-cysteamine remained attached on freshly excised porcine mucosa for 8 0h and displayed increased disintegration time of 2h. Swelling behavior of HEC-cysteamine tablets in 0.1M phosphate buffer pH 6.8 indicated swelling ratio of 19 within 8h. In contrast, tablets comprising unmodified HEC detached from the mucosa within few seconds and immediately disintegrated. In addition, they did not exhibit swelling behavior. The transport of rhodamine 123 across freshly excised rat intestine enhanced by a value of approximately 1.6-fold (p-value = 0.0024) in the presence of 0.5% (m/v) HEC-cysteamine as compared to buffer control. Result from cytotoxicity test of HEC-cysteamine applied to Caco-2 cells in concentration of 0.5% (m/v) revealed 82.4 ± 4.60% cell viability. According to these results, HEC-cysteamine seems to be a promising polymer for various pharmaceutical applications especially for intestinal drug delivery.

Uptake of phenothiazines by the harvested chylomicrons ex vivo model: influence of self-nanoemulsifying formulation design.

The aim of this study was to examine the potential of self-nanoemulsifying drug delivery systems (SNEDDS) on the uptake of the lipophilic and poorly water soluble phenothiazines thioridazine and chlorpromazine with the isolated plasma derived chylomicron (CM) ex vivo model. The multi-component delivery systems were optimized by evaluating their ability to self-emulsify when introduced to an aqueous medium under gentle agitation. The uptake of phenothiazines by isolated plasma derived chylomicrons was investigated with short chain triglyceride (SCT) SNEDDS, medium chain triglyceride (MCT) SNEDDS, and long chain triglyceride (LCT) SNEDDS. SNEDDS were also evaluated for their stabilities, dispersibilities, percentage transmittances and by particle size analyses. For thioridazine a 5.6-fold and for chlorpromazine a 3.7-fold higher CM uptake could be observed using a LCT-SNEDDS formulation compared to the drugs without formulation. In contrast, ex vivo uptake by isolated CM was not significantly increased by SNEDDS formulations based on MCT and SCT. Compared with isolated CM, the CM sizes were increased 2.5-fold in LCT-SNEDDS, whereas in MCT-SNEDDS or SCT-SNEDDS only a small, non-significant (P<0.05) increase in CM size was observed. These results show that distinct SNEDDS formulations containing phenothiazines are efficiently uptaken by plasma derived chylomicrons ex vivo.