Free DOX and chitosan-N-arginine conjugate stabilized indocyanine green nanoparticles for combined chemophotothermal therapy.

Indocyanine green (ICG) is a FDA-approved near-infrared (NIR) cyanine dye used in medical diagnostics. However, the utility of ICG remains limited by its unstable optical property, and concentration-dependent aggregation and precipitation. A chitosan-arginine conjugate (CS-N-Arg) was developed to increase the stability of ICG in physiological buffer saline via formation of strong electrostatic interactions between ICG and CS-N-Arg. The CS-N-Arg/ICG complex prevented ICG from aggregation and precipitation, thus it could serve as a theranostic nanomaterial for image-guided photothermal cancer therapy. The CS-N-Arg/ICG NPs showed excellent photostability, clear fluorescent images, and rapid temperature rise under laser irradiation. Cell viability assay indicated that CS-N-Arg/ICG NPs could efficiently suppress the growth of doxorubicin (DOX) resistant breast cancer cell (MCF-7/ADR cells) under NIR photothermal treatments. In combination of DOX with CS-N-Arg/ICG NPs, a combined effect was observed in MCF-7/ADR breast cancer cells due to dual hyperthermia and chemical therapeutic effects. The present observations suggest that CS-N-Arg/ICG NPs can effectively deliver ICG molecules to MCF-7/ADR breast cancer cells and overcome DOX resistance in the cells by hyperthermia.

Preparation of fucoidan-shelled and genipin-crosslinked chitosan beads for antibacterial application.

In this study, a fucoidan-shelled chitosan bead was developed with the purpose of oral delivery of berberine to inhibit the growth of bacteria. The cross-linking level and swelling property of the beads were affected by the pH value and the composition of the genipin/fucoidan combined gelling agent. The drug release of the berberine-loaded beads was faster in simulated gastric fluid (pH 1.2) than those in simulated intestinal fluid (pH 7.4). Furthermore, a nanoparticles/beads complex system was developed by incorporation of berberine-loaded chitosan/fucoidan nanoparticles in the fucoidan-shelled chitosan beads. The nanoparticles/beads complex served as a drug carrier to delay the berberine release in simulated gastric fluid, with an estimated lag time of 2 h. Our results showed that the berberine-loaded beads and nanoparticles/beads complex could effectively inhibit the growth inhibition of common clinical pathogens, such as Staphylococcus aureus and Escherichia coli, and have the advantage of continually releasing berberine to inhibit the growth of the bacteria over 24 h.

Hydrogel microspheres for stabilization of an antioxidant enzyme: effect of emulsion cross-linking of a dual polysaccharide system on the protection of enzyme activity.

Catalase is an antioxidant enzyme abundant in natural resources. However, the enzyme is usually inactivated by gastric acid and digestive enzymes after oral ingestion. In this study, carboxymethyl chitosan (CM-chitosan) and hyaluronic acid (HA) conjugate hydrogel microspheres have been prepared by an emulsion cross-linking technique to retain the activity of catalase in simulated gastrointestinal (GI) fluids. Cross-linking reduced the swelling capability and increased the resistance toward hyaluronidase digestion of prepared HA-CM-chitosan hydrogel microspheres. Catalase entrapped in the hydrogel microspheres exhibited superior stability over a wide pH range (pH 2.0 and 6.0-8.0) as compared to the native enzyme. The entrapped catalase was also protected against degradation by digestive enzymes. Following the treatments, the catalase-loaded microspheres, in contrast to native catalase, could effectively decrease the intracellular H2O2 level and protect HT-29 colonic epithelial cells against H2O2-induced oxidative damage to preserve cell viability. These results suggested that the HA-CM-chitosan hydrogel microspheres can be used for entrapment, protection and intestinal delivery of catalase for H2O2 scavenging.

Nanoparticle-induced tight-junction opening for the transport of an anti-angiogenic sulfated polysaccharide across Caco-2 cell monolayers.

Fucoidan has the ability to inhibit angiogenesis by human umbilical vein endothelial cells (HUVECs). However, a major clinical limitation is its poor oral availability because fucoidan is a hydrophilic macromolecule. In this study, an oversulfation reaction of fucoidan has been performed to enhance its anti-angiogenic activities. The synthesized, oversulfated fucoidan (OFD) was characterized by Fourier transform infrared spectroscopy. The oversulfate content of OFD was estimated to be 41.7% by using a BaCl2 gelatin method. Nanoparticles (NPs) composed of chitosan (CS) and OFD were prepared by a polycation-polyanion complex method. The mean particle sizes of prepared CS/OFD NPs were in the range of 172-265nm with a negative or positive surface charge, depending on the relative concentrations of CS to OFD used. The self-assembled NPs with pH-sensitive characteristics could be used as a pH-switched nanocarrier for oral delivery of the antiangiogenic macromolecule, OFD, in response to simulated gastrointestinal (GI) tract media. Evaluation of test NPs in enhancing the intestinal paracellular transport of OFD suggested that the NPs with a positive surface charge could transiently open the tight junctions between Caco-2 cells and thus increase the paracellular permeability. Tight-junction opening and restoration were examined by monitoring the redistribution of ZO-1 tight-junction proteins using confocal laser scanning microscopy (CLSM). The transported OFD significantly inhibits the tube formation of HUVECs via competitive binding of OFD and basic fibroblast growth factor (bFGF) to bFGF receptors (bFGFRs).

Preparation and characterization of dexamethasone-immobilized chitosan scaffold.

Chitosan film (CSF), 2-dimensional scaffold, was modified with dexamethasone (DEX) in the present work via amino-alcohol forming reaction between amino group of chitosan and carbonyl group of DEX. Successful immobilization was identified by ATR-FTIR spectroscopy and, the immobilized amounts were determined by weighting methods (WM) and integrated area analysis method (AM) of HPLC. For experiments of cell culture of osteogenic differentiation, our results showed that DEX immobilization has more efficiency than the other group (such as both groups of CSF and CSF with free DEX, CSF/fDEX), which was demonstrated as indicated by cell image analysis and ALP activity assay. The results show, as AD-MSCs cultured on DEX-CSF, its shape of cell transformed to polygonal or cubical. The ALP activity assay indicates that of DEX-CSF increase about 3.5 folds than that of CSF's. The results suggest that immobilization of DEX can make chitosan scaffolds to induce differentiation of AD-MSCs toward osteoblastic lineage for bone tissue engineering application.

Tripolyphosphate cross-linked macromolecular composites for the growth of shape- and size-controlled apatites.

Bioactive composites that enable the formation of calcium phosphates have received increased attention over the last decade, in the development of osteoconductive biomaterials for orthopaedic applications. In this work, tripolyphosphate (TPP)-cross-linked chitosan/gelatin composites (TPP-CG) were prepared for the growth of shape- and size-controlled calcium phosphates on/in the composites. The mineralization pattern of the composites, after soaking in the Ca(OH)(2) aqueous solution, clearly demonstrated oriented, needle-like nanocrystallites of calcium phosphates in the matrix with especially high Ca/P molar ratio (3.98) as detected by energy dispersive X-ray spectroscopy (EDX) analysis. Subsequent to mineralization in a simulated body fluid (SBF), the mineralized composites showed micro-scaled spherical aggregates deposited on the surface and granule-like nanocrystallites grew in the matrix. The Ca/P molar ratio (1.72) and X-ray diffraction pattern of the nanocrystallites grown in the composites were similar to those of hydroxyapatite (HAp). Osteoblastic differentiation of ROS cells cultured on the mineralized composites allowed an enhanced expression of the chosen osteogenic marker (alkaline phosphatase, ALPase). These results indicated that the composites mineralized with micro- and nano-scaled calcium phosphates with various structural features make them attractive for bone tissue engineering applications.

Stimuli-responsive materials prepared from carboxymethyl chitosan and poly(γ-glutamic acid) for protein delivery.

The development of stimuli-responsive materials in response to the molecules involved in biological processes has gained increased attentions. In this work, carboxymethyl chitosan (CM-chitosan) and poly(γ-glutamic acid) (pGlu) were reacted with a naturally occurring compound, genipin, leading to the formation of genipin-crosslinked CM-chitosan/pGlu conjugates with fluorescence emissions. The genipin-conjugated polymers were sensitive to the oxidation product of glucose, gluconic acid and hydrogen peroxide (H2O2). Fluorescence emissions of the polymers were quenched by gluconic acid and H2O2. An increase in the hydrodynamic diameter together with the quenching of fluorescence indicated that the genipin-conjugated polymers were self-aggregated into nanoparticles, in response to the stimulus of gluconic acid (but not for H2O2). Bovine serum albumin (BSA) could be loaded in the self-aggregated nanoparticles, and the incorporated BSA slowly released from the nanoparticles under hyper-gluconic acid conditions. This material is hence proposed as a stimuli-responsive material for optical sensing and protein delivery purposes.

Heparinized chitosan/poly(γ-glutamic acid) nanoparticles for multi-functional delivery of fibroblast growth factor and heparin.

To improve blood supply following ischemic injury, angiogenic factors such as fibroblast growth factor (bFGF) that stimulate new blood vessel formation have been used for therapeutic angiogenesis in ischemic tissues. In this study, heparin-functionalized chitosan (CS)/poly(γ-glutamic acid) (γ-PGA) nanoparticles (HP-CS/γ-PGA nanoparticles) were prepared for multi-functional delivery of basic fibroblast growth factor (bFGF) and heparin. The mean particle sizes and bFGF loading efficiency increased with the increase of functionalized heparin contents. The HP-CS/γ-PGA nanoparticles were pH-sensitive that could sustain bFGF release at pH 񥌈 6.7 (simulate the pH of ischemia tissue) and were rapidly disintegrated at pH 7.4 (simulate the pH of repaired tissue). Sustained release of bFGF from the nanoparticles enhanced the proliferation of human foreskin fibroblast cells (HFF) and angiogenic tube formation by human umbilical vein endothelial cells (HUVEC), suggesting the retaining of bFGF mitogenic activity. Heparin, a traditionally used anticoagulant, could release from the disintegrated nanoparticles to maintain the anti-factor Xa activity in blood plasma, after increasing the pH value from 6.6 to 7.4. The nanocarriers for multi-functional delivery of bFGF and heparin developed in this study may be a potential therapeutic method for enhancing ischemic tissue regeneration and preventing blood vessel rethrombosis.

Novel technology for the preparation of self-assembled catechin/gelatin nanoparticles and their characterization.

In this study, self-assembled tea catechin/gelatin nanoparticles were prepared by directly mixing the catechins and gelatin solutions. The mean particle sizes were almost less than 200 nm, and the zeta potential values were negatively charged. FT-IR spectral analysis indicated that hydrogen bonding between aliphatic and aromatic hydroxyl groups, respectively, on gelatin and catechins is responsible for the self-assembly of nanoparticles. Free radical (DPPH* and ABTS*(+)) scavenging assays showed that tea catechins could be protected by the nanoparticles and that the antioxidant activity of tea catechins was almost retained after three weeks of storage. The tea catechin/gelatin nanoparticles exhibited 28-41% inhibition to trypsin against the degradation of gelatin. This result suggested that the tea catechin/gelatin nanoparticles might be a useful antioxidant carrier because catechins and gelatin were, respectively, protected from oxidation and enzymatic digestion.

Thiol-modified chitosan sulfate nanoparticles for protection and release of basic fibroblast growth factor.

A series of chitosan (CS) derivatives, the 6-O-carboxymethylchitosan (6-O-CC), 2-N sulfated 6-O-carboxymethylchitosan (N-SOCC) and the 2-N and 3,6-O sulfated 6-O-carboxymethyl chitosan (N,O-SOCC) were synthesized in this study. The chemical structures and the degrees of substituted carboxymethyl and sulfate groups of the synthesized compounds were respectively determined by FT-IR spectra and elemental analysis. N,O-SOCC displayed the highest protective efficiency for basic fibroblast growth factor (bFGF) as examined by the L929 fibroblast culture test and docking simulation. N,O-SOCC-4-thio-butylamidine (TBA) conjugates prepared by modification of N,O-SOCC with 2-iminothiolane were in situ cross-linkable. The degrees of thiol substitution of the 2-iminothiolane modified N,O-SOCC polymers were determined to be in the ranges of 45.9 +/- 3.7 and 415.6 +/- 12.5 micromol SH/g SOCC by quantifying the amount of thiol groups on the thiolated polymers with Ellman's reagent. The 2-iminothiolane modified N,O-SOCC and CS complex could be used for preparing nanoparticles by a polyelectrolyte self-assembly method, and the release of bFGF from the nanoparticles was successfully controlled. L929 fibroblast culture tests showed that the thiol modified N,O-SOCC/CS nanoparticles could effectively protect bFGF from inactivation over a 120 h period. The results of this study suggest that the thiol modified N,O-SOCC/CS nanoparticles may be useful as novel materials for specific delivery of bFGF with mitogenic activity.

Synthesis of a novel glycoconjugated chitosan and preparation of its derived nanoparticles for targeting HepG2 cells.

In the study, a novel chitosan (CS) derivative conjugated with multiple galactose residues in an antennary fashion (Gal-m-CS) was synthesized. A galactosylated CS (Gal-CS) was also prepared by directly coupling lactobionic acid on CS. Using an iontropic gelation method, CS and the synthesized Gal-CS and Gal-m-CS were used to prepare nanoparticles (CS, Gal-CS, and Gal-m-CS NPs) for targeting hepatoma cells. TEM examinations showed that the morphology of all three types of NPs was spherical in shape. No aggregation or precipitation of NPs in an aqueous environment was observed during storage for all studied groups, as a result of the electrostatic repulsion between the positively charged NPs. Little fluorescence was observed in HepG2 cells after incubation with the FITC-labeled CS NPs. The intensity of fluorescence observed in HepG2 cells incubated with the Gal-m-CS NPs was stronger than that incubated with the Gal-CS NPs. These results indicated that the prepared Gal-m-CS NPs had the highest specific interaction with HepG2 cells among all studied groups, via the ligand-receptor-mediated recognition.