Fabrication of phospholipid-xanthan microcapsules by combining microfluidics with self-assembly.

We report the synthesis of an amphiphilic polysaccharide, a phospholipid (1,2-dioleoyl-sn-glycero-phosphoetilamine, DOPE) conjugated with the anionic xanthan gum, and its ability to spontaneously self-assemble under mild aqueous conditions. This work also aimed to apply a microfluidic platform that can precisely fabricate microsized and monodispersed capsules for cell encapsulation. Stable hollow capsular structures were obtained by the generation of homogeneous spherical droplets of the self-assembled polymer in the microfluidic device through the formation of a water-in-oil emulsion, followed by the stabilization of the polymer aggregates in a separate collection vessel containing phosphate-buffered saline (physiological ionic strength and pH). The properties (size, morphology, permeability) and performance (stability) of the obtained microcapsules were studied, as well their ability to support the viability, function and proliferation of encapsulated cells. ATDC5 cells were encapsulated within the capsules and shown to remain viable, evidencing increased cellular metabolic activity over 21 days of in vitro culture. By combining microfluidic droplet generation and self-assembly of xanthan-DOPE, we were able to fabricate microcapsules that provided an adequate environment for cells to survive and proliferate.

Injectable biodegradable starch/chitosan delivery system for the sustained release of gentamicin to treat bone infections.

Starch-conjugated chitosan microparticles were produced aimed to be used as a carrier for the long term sustained/controlled release of antibiotic drugs to control bone infection. The microparticles were prepared by a reductive alkylation crosslinking method. The obtained microparticles showed a spherical shape, with a slightly rough and porous surface, and a size range of 80-150µm. Gentamicin was entrapped into the starch-conjugated chitosan microparticles and its release profile was studied in vitro. Increasing concentrations of gentamicin (from 50 to 150mg/mL) led to a decrease in the encapsulation efficiency (from 67 to 55%), while drug loading increased from 4 to 27%. A sustained release of gentamicin was observed over a period of 30 days. The release kinetics could be controlled using an ionic crosslinker agent. In addition, a bacterial inhibition test on Staphylococcus aureus shows a diameter of the sample inhibition zone ranging from 12 to 17mm (70-100% of relative activity).

Growth of a bonelike apatite on chitosan microparticles after a calcium silicate treatment.

Bioactive chitosan microparticles can be prepared successfully by treating them with a calcium silicate solution and then subsequently soaking them in simulated body fluid (SBF). Such a combination enables the development of bioactive microparticles that can be used for several applications in the medical field, including injectable biomaterial systems and tissue engineering carrier systems. Chitosan microparticles, 0.6microm in average size, were soaked either for 12h in fresh calcium silicate solution (condition I) or for 1h in calcium silicate solution that had been aged for 24h before use (condition II). Afterwards, they were dried in air at 60 degrees C for 24h. The samples were then soaked in SBF for 1, 3 and 7 days. After the condition I calcium silicate treatment and the subsequent soaking in SBF, the microparticles formed a dense apatite layer after only 7 days of immersion, which is believed to be due to the formation of silanol (Si-OH) groups effective for apatite formation. For condition II, the microparticles successfully formed an apatite layer on their surfaces in SBF within only 1 day of immersion.

Starch-chitosan hydrogels prepared by reductive alkylation cross-linking.

Starch-chitosan hydrogels were produced by oxidation of soluble starch to produce polyaldehyde and subsequently cross-linked with chitosan via reductive alkylation. The swelling ratio of starch-chitosan membranes was increased gradually with increasing starch ratio, but it was always lower than the native chitosan. In dry state, starch-chitosan membranes with low starch ratio (0.16, 0.38) showed similar tensile strength values to those of native chitosan while these values decreased with increasing starch ratios (0.73-1.36). Membranes in physiological buffer solution (PBS) gave a tensile modulus between 2.8 and 1.0 MPa, decreasing with increasing starch ratio (0.16-1.36 (Wstarch/Wchitosan)). When the membranes were incubated in PBS only, a moderate weight loss was observed for the first two weeks. Original weights of low starch weight ratio membranes (0-0.38) were at near 85%, while high ratio samples (0.73-1.55) were kept around 70% after three months. However, for the membranes incubated in alpha-amylase solution, very fast weight loss was observed. For low starch ratio membranes (0.16, 0.38, 0.73), the residual original weights were measured to be 11%, 6%, 20%, while for high ratio membranes (1.04 and 1.36) these were 45% and 30%, respectively, after two months of enzyme incubation. Scanning electron microscopy analysis of alpha-amylase degraded membranes exhibited rough surface morphology.

Multichannel mould processing of 3D structures from microporous coralline hydroxyapatite granules and chitosan support materials for guided tissue regeneration/engineering.

A three-dimensional composite material was produced from microporous coralline origin hydroxyapatite (HA) microgranules, chitosan fibers and chitosan membrane. Cylindrical HA microgranules were oriented along channel direction within multichannel mould space and aligned particles were supported with fibers and a chitosan membrane. The positive replica of mould channels was clasp fixed to produce thicker scaffolds. Light microphotographs of the developed complex structure showed good adhesion between the HA particles, the fibers and the supporting membrane. The composite material showed 88% (w/w) swelling in one hour and preserved the complex structure of the original material upon long-term incubation in physiological medium. MEM extract test of HA chitosan complex showed no cell growth inhibition and cell viability assay (MTS) indicated over 90% cell viability.

Poly(hydroxybutyrate-co-hydroxyvalerate) nanocapsules as enzyme carriers for cancer therapy: an in vitro study.

In the present paper, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) nanocapsules were prepared by a double emulsion-solvent evaporation procedure (w/o/ w) for the encapsulation of model enzymes (L-asparaginase, catalase, glucose oxidase) and bovine serum albumin. To increase the encapsulation efficiency and activity of the encapsulated enzyme, numerous modifications were made in the compositions of the phases of double emulsion. For the preparation of low molecular weight PHBV, the polymer was treated with sodium borohydride. A 14-fold decrease in molecular weight (from 297000 to 21000) was observed upon 4 h of incubation. Although the amount of encapsulated protein was not increased, the enzyme activity increased upon use of low molecular weight PHBV, indicating that these nanocapsules have a higher permeability to solutes (reactants and products). The adjustment of the second water phase to the isoelectric point of the proteins significantly increased the encapsulation yields of catalase, L-asparaginase and BSA. Likewise, polyethylene glycol coupling significantly increased the entrapment efficiency as well as the activity of catalase and L-asparaginase. A combination of the various optimum preparation conditions further increased the encapsulated catalase activity (about six-fold) in comparison to the initial basic conditions (with no modification and no isoelectric point adjustment).

In vivo half life of nanoencapsulated L-asparaginase.

In the present study, antileukemic enzyme L-asparaginase (ASNase) was encapsulated into poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) nanocapsules in order to decrease the immunogenicity and toxicity of the enzyme and to increase its in vivo half life in mice. Nanocapsules were prepared by water-in-oil-in-water approach and each phase was changed systematically. By changing the pH of the w(2) phase to the isolelectric point of L-ASNase, the encapsulation efficiency was increased from 23.7% to 28.0%. Also, modification of ASNase with PEG(2) increased the encapsulation efficiency from 23.7% to 27.9% and protected the enzyme against denaturation. Combination of the various optima enabled a substantial increase in the activity (0.074-0.429 U/mg nanocapsule). The enzyme activity in the blood due to unmodified PHBV nanocapsules dropped to 38% of its initial value 4 h after injection. When the same sample was tested for the enzyme content in the circulation by using the radio-labeled enzyme a much lower enzyme (30% of initial) could be detected after a shorter time (3 h). The PHBV nanocapsules with heparin conjugated on their surface had a longer presence in the circulation than unmodified PHBV nanocapsules. After 6 h, around 50% of the enzyme was still present in the blood. Radioactivity measurements using the same sample showed a sharp decrease in enzyme amount in the circulation in the early stages. However, radioactivity was still detectable at the eighth hour. No adverse effects and symptoms of anaphylaxis were observed upon injection of encapsulated ASNase-PHBV nanocapsules to mice i.v. through the tail vein.