Gene Transfer by DNA/mannosylated Chitosan Complexes into Mouse Peritoneal Macrophages

Chitosan is a biodegradable and biocompatible polymer and is useful as a non-viral vector for gene delivery. In order to deliver pDNA/chitosan complex into macrophages expressing a mannose receptor, mannose-modified chitosan (man-chitosan) was employed. The cellular uptake of pDNA/man-chitosan complexes through mannose recognition was then observed. The pDNA/man-chitosan complexes showed no significant cytotoxicity in mouse peritoneal macrophages, while pDNA/man-PEI complexes showed strong cytotoxicity. The pDNA/man-chitosan complexes showed much higher transfection efficiency than pDNA/chitosan complexes in mouse peritoneal macrophages. Observation with a confocal laser microscope suggested differences in the cellular uptake mechanism between pDNA/chitosan complexes and pDNA/man-chitosan complexes. Mannose receptor-mediated gene transfer thus enhances the transfection efficiency of pDNA/chitosan complexes.     

Trypsin immobilization by direct adsorption on metal ion chelated macroporous chitosan-silica gel beads

Silica gel bead coated with macroporous chitosan layer (CTS-SiO₂) was prepared, and the metal immobilized affinity chromatographic (IMAC) adsorbents could be obtained by chelating Cu²⁺, Zn²⁺, Ni²⁺ ions, respectively on CTS-SiO₂, and trypsin could be adsorbed on the IMAC adsorbent through metal–protein interaction forces. Batch adsorption experiments show that adsorption capacity for trypsin on these IMAC adsorbent variated with change of pH. The maximal adsorption reached when the solution was in near neutral pH in all three IMAC adsorbents. Adsorption isothermal curve indicated that maximal adsorption capacity could be found in the Cu²⁺-CTS-SiO₂ with the value of 4980 ± 125 IU g⁻¹ of the adsorbent, while the maximal adsorption capacity for trypsin on Zn²⁺ and Ni²⁺ loaded adsorbent was 3762 ± 68 IU g⁻¹ and 2636 ± 53 IU g⁻¹, respectively. Trypsin immobilized on the IMAC beads could not be desorbed by water, buffer and salt solution if the pH was kept in the range of 5–10, and could be easily desorbed from the IMAC beads by acidic solution and metal chelating species such as EDTA and imidazole. The effect of chelated metal ions species on CTS-SiO₂ beads on the activity and stability of immobilized trypsin was also evaluated and discussed. Trypsin adsorbed on Zn-IMAC beads retained highest amount of activity, about 78% of total activity could be retained. Although the Cu-IMAC showed highest affinity for trypsin, only 25.4% of the calculated activity was found on the beads, while the activity recovery found on Ni-IMAC beads was about 37.1%. A remarkable difference on stability of trypsin immobilized on three kinds of metal ion chelated beads during storage period was also found. Activity of trypsin on Cu-IMAC decreased to 24% of its initial activity after 1-week storage at 4 °C, while about 80% activity was retained on both Ni-IMAC and Zn-IMAC beads. Trypsin immobilized on Zn-CTS-SiO₂ could effectively digest BSA revealed by HPLC peptide mapping.     

Synthesis, characterization and thermal sensitivity of chitosan-based graft copolymers

Novel chitosan-based graft copolymers (CECTS-g-PDMA) were synthesized through homogeneous graft copolymerization of (N,N-dimethylamino)ethyl methacrylate (DMA) onto N-carboxyethylchitosan (CECTS) in aqueous solution by using ammonium persulfate (APS) as the initiator. The effect of polymerization variables, including initiator concentration, monomer concentration, reaction time and temperature, on grafting percentage was studied. XRD, FTIR, DSC and TGA were used to characterize the graft copolymers. Surface-tension measurements, turbidity measurements and temperature-variable (1)H NMR analysis were combined to investigate the thermal sensitivity of CECTS-g-PDMAs in aqueous solution.     

Combining submerged electrospray and UV photopolymerization for production of synthetic hydrogel microspheres for cell encapsulation

Microencapsulation within hydrogel microspheres holds much promise for drug and cell delivery applications. Synthetic hydrogels have many advantages over more commonly used natural materials such as alginate, however their use has been limited due to a lack of appropriate methods for manufacturing these microspheres under conditions compatible with sensitive proteins or cells. This study investigated the effect of flow rate and voltage on size and uniformity of the hydrogel microspheres produced via submerged electrospray combined with UV photopolymerization. In addition, the mechanical properties and cell survival within microspheres was studied. A poly(vinyl alcohol) (PVA) macromer solution was sprayed in sunflower oil under flow rates between 1-100 μL/min and voltages 0-10 kV. The modes of spraying observed were similar to those previously reported for electrospraying in air. Spheres produced were smaller for lower flow rates and higher voltages and mean size could be tailored from 50 to 1,500 μm. The microspheres exhibited a smooth, spherical morphology, did not aggregate and the compressive modulus of the spheres (350 kPa) was equivalent to bulk PVA (312 kPa). Finally, L929 fibroblasts were encapsulated within PVA microspheres and showed viability >90% after 24 h. This process shows great promise for the production of synthetic hydrogel microspheres, and specifically supports encapsulation of cells.     

Preparation and characterization of nano-hydroxyapatite/chitosan cross-linking composite membrane intended for tissue engineering

In this paper, a series of nano-hydroxyapatite(n-HA)/chitosan cross-linking composite membranes (n-HA; 0, 5, 10, 15, 20 and 30 wt%) were successfully developed by a simple casting/solvent evaporation method. n-HA with size about 20 nm in vertical diameter and about 100 nm in horizontal diameter was successfully synthesized by a hydro-thermal precipitation method, and then dispersed into chitosan/genipin solution with the aid of continuous ultrasound to develop n-HA/chitosan cross-linking composite membranes. The detailed characterizations including Fourier transform infrared spectroscopy (FTIR), X-ray diffractometer (XRD), thermogravimetric analysis (TGA), scanning electron microscopy (SEM), water adsorption and tensile test were performed. With the analysis of FTIR spectra and TGA spectra, it suggested that there was existence of possible interactions between polymer and n-HA. Meanwhile, the n-HA content was greatly effected on the morphology as well as the tensile property of composite membrane. In vitro cytotoxicity test suggested that the developed n-HA/chitosan cross-linking composite membrane was non-cytotoxicity against L929 cells after 24 h's incubation might be suitable for further in vivo application.     

Determination of the degree of N-acetylation (DA) of chitin and chitosan in the presence of water by first derivative ATR FTIR spectroscopy

A new method for the determination of the degree of N-acetylation (DA) of chitin and chitosan is described using first derivative diamond ATR FTIR spectroscopy. Applying the derivative values of the amide III band at 1327 cm⁻¹ and the CH deformation band of the N-acetyl group at 1383 cm⁻¹ as measure of the N-acetyl content of the sample in relation to the derivative value of the bridge oxygen vibration at 1163 cm⁻¹ as internal standard, a linear correlation to the results of first derivative UV spectroscopy was obtained and confirmed by elemental analysis and Raman spectroscopy. The described method allows the determination of the degree of N-acetylation of chitosan and chitin in the presence of water thus making drying procedures unnecessary.     

Physicochemical and structural characteristics of chitosan nanopowders prepared by ultrafine milling

Two different molecular weights of chitosan were pulverized to nanopowders by ultrafine milling. The nanopowders were characterized by viscometry small angle X-ray scattering (SAXS), transmission electron microscopy (TEM), X-ray diffraction (XRD), thermogravimetric analysis (TGA), FT-IR spectroscopy and UV-vis spectroscopy. Our results showed that ultrafine milling effectively reduced the particle size of chitosan to a nanoscale. The viscosity average molecular weight (Mv) of chitosan was decreased by the milling treatment. The crystalline structure of chitosan was destroyed by the milling since the nanopowder exhibited an amorphous XRD pattern. In addition, thermal stability of the low molecular weight chitosan was decreased after the milling treatment. FT-IR and UV-vis spectra showed that the milling process did not cause significant changes in the chemical structure of chitosan.     

Aggregation of some water-soluble derivatives of chitin in aqueous solutions: Role of the degree of acetylation and effect of hydrogen bond breaker

By dynamic light scattering in combination with fluorescence spectroscopy and TEM it was shown that aggregation in aqueous solutions is inherent not only to chitosan, but also to two other water-soluble derivatives of chitin: O-carboxymethylchitin and di-N,N-carboxymethylchitosan. Aggregation is observed even for the samples without N-acetyl-d-glucosamine units, which remain upon incomplete chemical modification of chitin, indicating that specific interactions between residual chitin repeat units cannot be the main reason for the aggregation. At the same time, 7 M urea weakens the aggregation, thus testifying that hydrogen bonding and/or hydrophobic interactions are partially responsible for this phenomenon. The incomplete disruption of aggregates in 7 M urea may arise from crystallization of junction zones between different macromolecules, which makes some hydrogen bonds inaccessible for urea or too stable for breaking by this agent.