A multilayered hydrogel film system based on hyaluronic acid-cysteamine (HA-Cym) and polyvinylalcohol (PVA) was fabricated. It contained a drug-impermeable backing layer, a supporting layer preventing direct contact between the loaded drug and the backing layer, a drug-loading layer and a mucoadhesive layer. Scanning electron microscopy demonstrated the presence of the distinct layers. The composition and preparation procedure of the films influenced their mucoadhesion, swelling, in vitro release of insulin and loaded insulin stability. Vacuum drying and crosslinked PVA with glutaraldehyde might reduce mucoadhesion, and they partially decreased the bioactivity of loaded insulin. Lyophilized hydrogel film with uncrosslinked PVA as a mucoadhesive layer possessed high mucoadhesion and showed no influence on the bioactivity of loaded insulin. The application of vacuum-dried PVA-crosslinked HA-Cym/PVA hydrogel film as a drug-impermeable backing layer would provide a controllable unidirectional insulin release. Therefore, such a multilayered hydrogel film system could be a promising mucoadhesive delivery system for controlled macromolecular drug release.

Trimethyl chitosan–cysteine conjugate (TMC–Cys) was evaluated as non-viral gene carriers to combine the advantages of TMC and thiolated chitosan. TMC–Cys with various molecular weights (30, 100, and 200kDa) and quaternization degrees (15 and 30%) was allowed to form polyelectrolyte nanocomplexes with plasmid encoding enhanced green fluorescence protein (pEGFP), which demonstrated preferable diameters of below 200nm and zeta potentials of +15 to +20mV. Cell binding and uptake of TMC–Cys/pEGFP nanocomplexes (TMC–Cys NC) were enhanced 2.4–3.0 and 1.4–3.0 folds, respectively, compared to TMC/pEGFP nanocomplexes (TMC NC). pEGFP could be easily released from TMC–Cys NC at the intracellular glutathione concentration, which promoted its nuclear transport and accumulation. Consequently, TMC–Cys NC showed a 1.4 to 3.2-fold increase in the transfection efficiency in HEK293 cells as compared to TMC NC and the optimal TMC–Cys(100,30) NC showed a 1.5-fold enhancement than Lipofectamine2000. Such results were further confirmed by in vivo transfection with a 2.3-fold and 4.1-fold higher transfection efficiency of TMC–Cys(100,30) NC than TMC(100,30) NC and Lipofectamine2000, respectively. Therefore, TMC–Cys/DNA nanocomplexes could be a promising gene delivery system with in vitro and in vivo superiority to Lipofectamine2000.

Superporous hydrogel containing poly(acrylic acid-co-acrylamide)/O-carboxymethyl chitosan interpenetrating polymer networks (SPH-IPN) was evaluated as the oral delivery vehicle for insulin, emphasizing on the effect of polymer integrity on insulin absorption mechanisms. The integral SPH-IPN (I-SPH-IPN) and powdered SPH-IPN (P-SPH-IPN) exhibited potent and equivalent in vitro enzymatic inhibition capacities, which were attributed to both enzyme incorporation and Ca2+ deprivation. Nevertheless, I-SPH-IPN showed marked superiority to P-SPH-IPN in in vivo enzymatic inhibition. Through reversible opening of epithelial tight junctions, I-SPH-IPN notably enhanced paracellular permeability of insulin in Caco-2 cell monolayers and excised rat intestine by 4.9 and 4.2 folds, respectively, wherein I-SPH-IPN outperformed P-SPH-IPN by 2.5 and 2.3 folds, respectively. Besides, orally delivered I-SPH-IPN could retain in rat intestine for more than 8 h while P-SPH-IPN was quickly eliminated, suggesting better retentive properties of I-SPH-IPN. Such results were further confirmed by in vivo assessment in that oral administration of insulin-loaded I-SPH-IPN yielded notable insulin absorption and hypoglycemic effect, while P-SPH-IPN was ineffective. Finally, an oral acute and sub-acute toxicity study in mice confirmed biocompatibility of SPH-IPN. Therefore, the detailed mechanism assessment confirmed that I-SPH-IPN was an effective and safe peroral carrier for protein drugs.

The superporous hydrogel containing poly(acrylic acid-co-acrylamide)/O-carboxymethyl chitosan (O-CMC) interpenetrating polymer networks (SPH-IPN) that had been developed as an oral delivery vehicle for protein drugs was subject to cytotoxicity and genotoxicity testing, thus evaluating its biological safety in use. In a battery of cytotoxicity assays on RBL-2H3 and Caco-2 cells, the SPH-IPN caused minimal damage towards cell viability, lysosomal activity, and metabolic activity following both direct and indirect treatment. The SPH-IPN did not induce cell apoptosis or DNA breakage in the above cell lines; it did not increase micronucleus (MN) incidence in mouse bone marrow, either. Therefore, the SPH-IPN was preliminarily considered to be biocompatible and might be a safe carrier for protein drugs. In addition, using the HPLC method, residual acrylic acid, acrylamide, and glutaraldehyde in the SPH-IPN were quantified to be 1.4, 2.0, and below 0.2 ppm, respectively. Lack of these low molecular monomers and crosslinker that were mainly responsible for the toxicity provided evidence for the good biocompatibility of the SPH-IPN.

Trimethyl chitosan-cysteine conjugate (TMC-Cys) was synthesized in an attempt to combine the mucoadhesion and the permeation enhancing effects of TMC and thiolated polymers related to different mechanisms for oral absorption. TMC-Cys with various molecular weights (30, 200, and 500 kDa) and quaternization degrees (15 and 30%) was allowed to form polyelectrolyte nanoparticles with insulin through self-assembly, which demonstrated particle size of 100–200 nm, zeta potential of +12 to +18 mV, and high encapsulation efficiency. TMC-Cys/insulin nanoparticles (TMC-Cys NP) showed a 2.1–4.7-fold increase in mucoadhesion compared to TMC/insulin nanoparticles (TMC NP), which might be partly attributed to disulfide formation between TMC-Cys and mucin as evidenced by DSC measurement. Compared to insulin solution and TMC NP, TMC-Cys NP induced increased insulin transport through rat intestine by 3.3–11.7 and 1.7–2.6 folds, promoted Caco-2 cell internalization by 7.5–12.7 and 1.7–3.0 folds, and augmented uptake in Peyer's patches by 14.7–20.9 and 1.7–5.0 folds, respectively. Such results were further confirmed by in vivo experiment with the optimal TMC-Cys NP. Biocompatibility assessment revealed lack of toxicity of TMC-Cys NP. Therefore, self-assembled nanoparticles between TMC-Cys and protein drugs could be an effective and safe oral delivery system.

The swelling of a superporous hydrogel containing poly(acrylic acid-co-acrylamide)/O-carboxymethyl chitosan interpenetrating polymer networks (SPH-IPN) was sensitive toward the pH, ionic strength, and temperature stimuli. With insulin as a model drug, polymer-protein interaction was detected, and it was physical rather than covalent. Freezing water was the majority of the imbibed water in the swollen SPH-IPNs, and the water-retention ability of the polymer against compression and time of exposure at 37 degrees C was improved as the amount of the O-carboxymethyl chitosan network increased. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay on AD293 and RBL-2H3 cells and an in situ lactate dehydrogenase assay and morphological study on rat intestine confirmed that the SPH-IPNs had satisfactory biocompatibility. These pronounced properties suggested that the SPH-IPNs could be developed as an attractive peroral delivery vehicle for peptide and protein drugs. (C) 2008 Wiley Periodicals, Inc.

In this investigation, superporous hydrogels containing poly (acrylic acid-co-acrylamide)/O-carboxymethyl chitosan (O-CMC) full-interpenetrating polymer networks (SPH-IPNs) were evaluated for their potentials in effective insulin absorption via the oral route. Insulin release from the SPH-IPNs exhibited sensitivity towards pH and ionic strength. After drug loading and release, the circular dichroism (CD) spectra revealed that conformation of insulin had no significant alteration and bioactivity of insulin was well preserved according to hypoglycaemic effect in mice. Through their abilities to bind Ca2+ and to entrap the enzymes, SPH-IPNs could partly inactivate trypsin and α-chymotrypsin, and SPH-IPN with higher O-CMC/monomer ratio appeared more potent. Swollen SPH-IPNs could attach mechanically and muco-adhere to the intestinal wall, thus achieving improved retentive properties compared to commonly used muco-adhesive excipient Carbopol® 934. Transport of insulin across rat intestine and colon ex vivo was enhanced around two- to three-fold after application of the SPH-IPN. Insulin-loaded SPH-IPN showed significant hypoglycaemic effects following oral administration to healthy rats, achieving a 4.1% pharmacological availability compared to subcutaneous insulin injection. These pronounced properties demonstrated that the SPH-IPN would be a promising peroral carrier for insulin and other peptide drugs.

The chemical compositions, antimicrobial activities, antioxidant activities and cytotoxicities of the essential oils isolated from the root of Kadsura longepedunculata Finet et Gagnep (KLREO) and the fruit of Schisandra sphenanthera Rehd. et Wills. (SSFEO) were investigated.The analyses of gas chromatography-mass spectrometry (GC-MS) showed that cadinane type compounds and their derivatives were rich in both oils (54.2% and 39.7%, respectively) and delta-cadinene was the major component of both oils (13.8% and 25.6%, respectively). The antimicrobial activities of both oils were evaluated against five microorganisms with the disc diffusion and the broth micro-dilution method. Results showed that Gram-positive bacteria were more sensitive to both oils than Gram-negative bacteria and the yeast. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the oil of KLREO were lower than those of SSFEO, indicating that the former possessed slightly stronger antibacterial capability than the latter. The reducing power and lipid peroxidation assays were employed to study the potential antioxidant activities of both oils. Both oils remarkably decreased the content of malondialdehyde (MDA) in rat liver homogenate in a dose dependent manner. The antioxidant activities of KLREO appeared to be more potent than that of SSFEO. The oils of KLREO and SSFEO exhibited concentration-dependent cytotoxicities and were proved to be toxic to HepG2 cells with IC(50) of 147 and 189 mug/ml, respectively.

Several novel functionalized graft copolymer nanoparticles consisting of chitosan (CS) and the monomer methyl methacrylate (MMA), N-dimethylaminoethyl methacrylate hydrochloride (DMAEMC), and N-trimethylaminoethyl methacrylate chloride (TMAEMC), which show a higher solubility than chitosan in a broader pH range, have been prepared by free radical polymerization. The nanoparticles were characterized in terms of particle size, zeta potential, TEM, and FT-IR. These nanoparticles were 150-280 nm in size and carried obvious positive surface charges. Protein-loaded nanoparticles were prepared, and their maximal encapsulation efficiency was up to 100%. In vitro release showed that these nanoparticles provided an initial burst release followed by a slowly sustained release for more than 24 h. These graft copolymer nanoparticles enhanced the absorption and improved the bioavailability of insulin via the gastrointestinal (GI) tract of normal male Sprague-Dawley (SD) strain rats to a greater extent than that of the phosphate buffer solution (PBS) of insulin.