Design of chitosan and its water soluble derivatives-based drug carriers with polyelectrolyte complexes.

Chitosan, the cationic polysaccharide derived from the natural polysaccharide chitin, has been studied as a biomaterial for more than two decades. As a polycationic polymer with favorable properties, it has been widely used to form polyelectrolyte complexes with polyanions for various applications in drug delivery fields. In recent years, a growing number of studies have been focused on the preparation of polyelectrolyte complexes based on chitosan and its water soluble derivatives. They have been considered well-suited as biomaterials for a number of vital drug carriers with targeted/controlled release profiles, e.g., films, capsules, microcapsules. In this work, an overview highlights not only the favorable properties of chitosan and its water soluble derivatives but also the good performance of the polyelectrolyte complexes produced based on chitosan. Their various types of applications as drug carriers are reviewed in detail.

Microencapsulation of functional ovalbumin and bovine serum albumin with polylysine-alginate complex for sustained protein vehicle's development.

Overcoming harsh gastric environment is still a challenging to bioactive proteins, microencapsulation provides one strategy in designing this protection barrier. In this work, bovine serum albumin and ovalbumin were chosen as model proteins, while polylysine-alginate complex was fabricated for microencapsulation purpose. Both of the protein-loaded microcapsules had regular internal microstructures. The model protein's embedding increased the thermal stability of the microcapsules. Both of the protein-loaded microcapsules had a slow release rate in simulated gastric fluids (pH 3.0), while a sustained release profile in simulated intestinal fluids (pH 6.4), indicating an excellent tolerance to the acidic gastric environment. The microencapsulation process was mild and had no influence on the protein's molecular weight, while a slight peak shifting occurred in the secondary structure of the released proteins. The developed microcapsules could be explored as a kind of vehicle for bioactive proteins applied in functional foods, health care products and medical formulations.

Characterization and immunomodulatory effect of an alkali-extracted galactomannan from Morchella esculenta.

In our continuous exploration for bioactive polysaccharides, a novel polysaccharide FMP-2 was isolated and purified from the fruiting bodies of Morchella esculenta by alkali-assisted extraction. FMP-2 had an average molecular weight of 1.09 × 106 Da and contained mannose, glucuronic acid, glucose, galactose, and arabinose in a molar ratio of 4.10:0.22:1.00:5.75:0.44. The backbone of FMP-2 mainly consisted of 1,2-α-D-Galp, 1,6-α-D-Galp, and 1,4-α-D-Manp, with branches of 1,4,6-α-D-Manp and 1,2,6-α-D-Galp. FMP-2 can stimulate phagocytosis and promote the secretion of NO, ROS, and cytokines like IL-6, IL-1ß, and TNF-α in RAW264.7 cells ranging from 25 to 400 µg/mL. FMP-2 had great repairing effect on the immune injury of zebrafish induced by chloramphenicol. The phagocytosis ability of zebrafish macrophages and the proliferation of neutrophils can be greatly enhanced by polysaccharide FMP-2 with concentrations from 50 to 200 µg/mL. These findings suggest that FMP-2 might be used as a potential immunomodulator in the food and pharmaceutical industries.

Development of metformin hydrochloride loaded dissolving tablets with novel carboxymethylcellulose/poly-l-lysine/TPP complex.

Natural polymers like polysaccharides, polypeptides and their derivatives are broadly applied in drug delivery due to excellent biocompatibility and biodegradability. In this study, the dissolving tablets, formed with carboxymethylcellulose/poly-l-lysine/tripolyphosphate (CMC/PLL/TPP) complex, were prepared using metformin hydrochloride (MetHCl) as model drug. Confocal laser scanning microscopy observation manifested that FITC-labeled PLL interacted with CMC and formed a uniform interior microstructure. Scanning electron microscope images showed the drug-loaded tablets had well-formed shapes with smooth surfaces. MetHCl embedded interior the microstructures of the tablets and represented in a crystal form. Thermo-gravimetric analysis and differential scanning calorimetry indicated that the drug-loaded tablets had stable thermal properties with less moisture content (3.52%). Fourier transform infrared spectrometer confirmed that the CMC/PLL/TPP complex was fabricated via the electrostatic interactions between -NH3+, -COO- and -[P2O54-]- groups. The drug-loaded tablets had a high drug loading efficiency of 85.76% and drug encapsulation efficiency of 81.47%, and a shorter wetting time of 2.16 min in SSF (pH 6.8) and lower swelling ratio of 233.34%. The drug loaded in the samples could be released completely within 10 min in simulated saliva fluid (SSF pH 6.8), indicating a rapid drug release and dissolving profile in the environment, which could be developed for dissolving tablets.

Reduction of 5-fluorouracil-induced toxicity by Sarcodon aspratus polysaccharides in Lewis tumor-bearing mice.

5-Fluorouracil (5-Fu) is an effective anticarcinogenic agent, however, continuous use of 5-Fu may cause severe side effects. The goal of this study was to investigate the effectiveness of Sarcodon aspratus polysaccharides (SATP) in alleviating 5-Fu-induced toxicity in Lewis tumor-bearing mice. Lewis tumor-bearing mice were treated with saline, SATP, 5-Fu or 5-Fu + SATP. The results indicated that compared to the 5-Fu group, the 5-Fu + SATP group showed effective amelioration of the liver, kidney and small intestine injury caused by 5-Fu and decreases in the levels of related biochemical indicators, such as aspartate aminotransferase (AST), alanine aminotransferase (ALT) and urea nitrogen (BUN). Additionally, the combination therapy enhanced the quality of life and immune organ indexes of mice. Further mechanistic studies indicated that the 5-Fu + SATP group showed a decrease in hepatotoxicity caused by 5-Fu via a reduction in the levels of interleukin-1ß (IL-1ß), an increase in the expression of Bcl-2 and decreases in the expression of p-p38, p-JNK and Bax. Collectively, the results indicated that SATP could significantly alleviate the toxicity of 5-Fu in Lewis tumor-bearing mice and showed the hepatoprotective capability of SATP via its effect on the expression levels of inflammatory factors and components of the MAPK/P38/JNK pathway, which shows that it may be a potential adjuvant for the chemotherapeutic drug 5-Fu in cancer treatment.

Fabrication of the polyphosphates patched cellulose sulfate-chitosan hydrochloride microcapsules and as vehicles for sustained drug release.

Polyphosphates are important polyanionic electrolytes that play a major role in stabilization and consolidation of colloids surface and interior microstructures. In this study, the polyelectrolyte complexes (PEC) microcapsules (sodium cellulose sulfate-chitosan hydrochloride, sample 1), and the patched ones via sodium tripolyphosphate (sample 2), sodium pyrophosphate (sample 3) and sodium hexametaphosphate (sample 4) were fabricated under mild conditions. The effects of polyphosphates on the formation of the PEC microcapsules were investigated systematically. Scanning electron microscope (SEM) and confocal laser scanning microscope (CLSM) observation showed that both of the sample 2 and sample 3 had more compact interior microstructures with higher fluorescence intensity, compared with the sample 4 with macroporous ones and sample 1 with irregular ones. Fourier transform-infrared spectroscopy (FT-IR) and thermogravimetric analysis (TGA) showed the electrostatic interactions occurred among the -NH3+ groups, -SO3- groups, HP3O104- groups, P2O74- groups and H2PO4- groups, and the sample 2 and sample 3 had a more thermal stability comparatively. The sample microcapsules showed good capacity of drug loading and encapsulation efficiency (max. 66.9 ±â€¯4.6% and 74.2 ±â€¯5.1%). In the in vitro release studies showed that the sample 2 and sample 3 had a larger accumulative drug release rate of 5-aminosalicylic acid (5-ASA) at the same time point and released completely at 12 h; the drug release mechanisms analysis indicated that the sample 1 and sample 3 were mainly diffusion controlled, while the sample 2 and sample 4 were followed the mechanism of non-Fickian transport. Under the polyphosphate's consolidation, the PEC microcapsules fabricated with sustained drug release profiles could be used as the promising drug vehicles.

Immunomodulatory effect of a polysaccharide fraction on RAW 264.7 macrophages extracted from the wild Lactarius deliciosus.

The mushroom polysaccharides are important substances with variety of functions, especially to the human body's immunomodulation effects. In this work, a polysaccharide fraction (LDP-1) was extracted and purified from the fruiting bodies of a rare wild Lactarius deliciosus. LDP-1 with molecular weight of 9.8 × 105 Da showed an obvious immunological activity to the RAW 264.7 cells. It had no significant suppressive but promotive effects on proliferation of the macrophages. The production of nitric oxide (NO) presented a concentration-dependent manner after treated with the LDP-1, and the maximum yield of NO was 39.15 µM. LDP-1 could promote the phagocytic uptake ability of the RAW 264.7 cells significantly, and many of the antennas produced around the cells correspondingly. The cytokines of TNF-α, IL-1ß and IL-6 were secreted increasingly in a concentration-dependent manner, which were 4.83, 17.8 and 11 times than that of the control, respectively. Western blotting analysis confirmed that NF-κB levels in the nucleus were increased while cytoplasmic inhibitor of NF-κB (IκB-α) degraded after treated with the LDP-1, indicating the RAW 264.7 cells probably be stimulated by LDP-1 through activating the IκB-α-NF-κB pathway. These results demonstrated that LDP-1 could be used as a kind of immunomodulatory agent for healthcare potentially.

Effect of polysaccharide FMP-1 from Morchella esculenta on melanogenesis in B16F10 cells and zebrafish.

Polysaccharides from Morchella esculenta are known to exhibit diverse bioactivities, while an anti-melanogenesis effect has been barely addressed. Herein, the anti-melanogenesis activity of a heteropolysaccharide from M. esculenta (FMP-1) was investigated in vitro and in vivo. FMP-1 had no significant cytotoxic effect on B16F10 melanoma cells as well as zebrafish larvae, but did reduce melanin contents and tyrosinase activities in both of them. Treatment with FMP-1 also effectively suppressed the expression of melanogenesis-related proteins, including MC1R, MITF, TRP-1 and TRP-2, through decreasing the phosphorylation of cyclic adenosine monophosphate response element-binding protein (CREB). Moreover, the mitogen-activated protein kinase (MAPK) pathway was observed mediating FMP-1's inhibitory effect against melanin production. Specifically, FMP-1 treatment markedly inhibited the activation of phosphorylation of p38 mitogen-activated protein kinase. These results suggested that FMP-1's inhibitory effect against melanogenesis is mediated by the inhibition of CREB and p38 signaling pathways, thereby resulting in the downstream repression of melanogenesis-related proteins and the subsequent melanin production. These data provide insight into FMP-1's potential anti-melanogenesis effect in food and cosmetic industries.

A novel polysaccharide from the Sarcodon aspratus triggers apoptosis in Hela cells via induction of mitochondrial dysfunction.

BACKGROUND: Polysaccharides extracted from fungus that have been used widely in the food and drugs industries due to biological activities. OBJECTIVE: The objective of the present study was to investigate the tumor-suppressive activity and mechanism of a novel polysaccharide (SAP) extracted from Sarcodon aspratus. METHODS: The SAP was extracted and purified using Sepharose CL-4B gel from S. aspratus. The cytotoxicity of SAP on cell lines was determined by MTT method. Cellular migration assays were implemented by using transwell plates. The apoptosis and mitochondrial membrane potential (Δψm) of Hela cells were analyzed by flow cytometry. The western blot was used to determine the protein expression of Hela cells. RESULTS: The results showed that SAP with a molecular weight of 9.01×105 Da could significantly inhibit the growth of Hela cells in vitro. Three-dimensional cell culture (3D) and transwell assays showed that SAP restrained the multi-cellular spheroids growth and cell migration. Flow cytometry analysis revealed that SAP induced a loss of mitochondrial membrane potential (Δψm). Western blot assays indicated that SAP promoted the release of cytochrome c, increased Bax expression, down-regulated of Bcl-2 expression and activated of caspase-3 expression. CONCLUSION: This study suggested that SAP induced Hela cells apoptosis via mitochondrial dysfunction that are critical in events of caspase apoptotic pathways. The anti-tumor (Hela cells) activity of SAP recommended that S. aspratus could be used as a powerful medicinal mushroom against cancer.

Fabrication and formation studies on single-walled CA/NaCS-WSC microcapsules.

The micron-sized calcium alginate/sodium cellulose sulfate-water soluble chitosan (CA/NaCS-WSC) microcapsules were prepared by membrane emulsification method using sodium alginate (NaAlg), NaCS and WSC as raw materials. The CA/NaCS microspheres prepared dispersed well and held spherical shape with an emulsifier volume ratio of 7:3 (Span 80:Tween 80) and a concentration of cross-linking agent of 1.5% (w/v) calcium chloride and 5% (w/v) sodium chloride. The CA/NaCS-WSC microcapsules had a spherical shape with average diameter of 62.36±13.87µm. A fluorescent ring could be seen obviously on the surface of CA/NaCS-WSC microcapsules under confocal microscope, when WSC was labeled by fluorescein isothiocyanate. The discussion on the formation studies implied that Ca(2+) could diffuse into the droplets of NaAlg/NaCS forming CA/NaCS microspheres, while NaCS could react with WSC forming a polyelectrolyte complexes film. The microcapsules prepared with typical wall-capsule/core structure could be used to develop micron-sized drug delivery carriers.

Evaluation of chitosan hydrochloride-alginate as enteric micro-probiotic-carrier with dual protective barriers.

In this study, the cells-free and cells-loaded chitosan hydrochloride-alginate (CHC-Alg) microcapsules were firstly fabricated with polyelectrolyte complexes via an orifice-polymerization method. Scanning electron microscope images showed that the CHC-Alg microcapsules had a typical shell-core structure and the model probiotic cells (Bacillus licheniformis) were embedded in the core in cells-loaded microcapsules. The microcapsules prepared had good thermal stability and moisture property (3.89%). Cells survival and release studies showed that the number of probiotic cells released from the cells-loaded microcapsules (approx. 6.36logCFUml-1) was 6.19logCFUml-1 when they were performed in the simulated gastric fluid (SGF, pH 2.0) for 1h and subsequently in the simulated intestinal fluid (SIF, 0.3%) for 4h. The CHC-Alg microcapsules with favorable swelling performances were helpful to permeate the harsh acid to protect the cells in the SGF (pH 2.0). The CHC-Alg microcapsules effectively protected the model probiotic cells, which was attributed to the "dual protective barriers" of the shell-core structure, that is, the primary barrier of the Alg hydrogel layer formed with a compact polymer matrix and the secondary barrier of the PEC film formed on the surface. The microcapsules prepared could be used as an enteric micro-probiotic-carrier for designing potential probiotic delivery systems.

Effect of the cross-linking agent on performances of NaCS-CS/WSC microcapsules.

Based on the properties of oppositely charged natural polysaccharides, the polyelectrolyte complexes (PECs) prepared with chitosan-related polycationic polyelectrolytes and cellulose-related polyanionic polyelectrolytes have been widely concerned for their potential applications as micro-drug-carriers for colon. However, the poor mechanical property of the PECs becomes the obstacle encountered in practical applications. This study investigated the effect of the cross-linking agent (sodium polyphosphate, PPS) on the performances of sodium cellulose sulfate -chitosan/water soluble chitosan (NaCS-CS/WSC) microcapsules. The results revealed that PPS could penetrate through the PEC film and form tighter interior structures compared with the microcapsules without the addition of cross-linking agent. The NaCS-CS microcapsules and NaCS-WSC microcapsules with or without PPS had distinct microstructures, which could be ascribed to the different physicochemical properties of CS and WSC. During the formation process, CS can be dissolved in water under acidic conditions, while WSC can be directly dissolved and protonated in acid-free aqueous providing NH3(+) groups quickly, which resulted in the microstructure's difference. Further analysis showed the NaCS-CS-PPS microcapsules and NaCS-WSC-PPS microcapsules had lower swelling ratios due to their tighter interior microstructures that formed. The cross-linking agent had important effect on the total mass of PECs that produced; moreover, the decline of zeta potential of NaCS-CS-PPS microcapsules was lower than that of NaCS-CS microcapsules, similar trend was found in the NaCS-WSC-PPS microcapsules compared with NaCS-WSC microcapsules, indicating the PPS participated in the interactions and played a role in the microcapsules' formation process.

Novel NaCS-CS-PPS microcapsules as a potential enzyme-triggered release carrier for highly-loading 5-ASA.

In order to develop novel spherical micro-drug-carriers, an orifice-polymerization method was used to prepare spherical microcapsules which were composed of chemically crosslinked chitosan (CS) with sodium cellulose sulfate (NaCS) and sodium polyphosphate (PPS). 5-Aminosalicylic acid (5-ASA) was chosen as a model drug. The microcapsules prepared had an average diameter of 1.90 mm with loading efficiency of 60.77% and encapsulation efficiency of 90.03%. SEM results showed that the microcapsules had a double-walled capsule structure with an outer wall thickness of approximately 4.40 µm and inner wall (shell) thickness of approximately 187.14 µm. SEM transection images of the microcapsules showed that 5-ASA entrapped in the microcapsule was in a crystal form. The results of in vitro swelling/erosion and release analysis showed that the drug was preferentially and completely released in simulated colonic fluid (SCF, pH 6.4) under the mechanism of Anomalous transport. All these results indicate that the microcapsules could be a good candidate as an enzyme-triggered controlled release drug carrier.

Characterization of novel lactoferrin loaded capsules prepared with polyelectrolyte complexes.

Novel capsules loaded with lactoferrin (LF) were prepared using polyelectrolyte complexes that were formed by water soluble chitosan (WSC), sodium cellulose sulfate (NaCS) and sodium polyphosphate (PPS). Normal chitosan (soluble in acidic conditions) was chosen as a control to prepare similar capsules with NaCS and PPS. (1)H NMR and FTIR spectra analysis showed that WSC was in a form of chitosan hydrochloride which can be directly dissolved and protonated in acid-free water. SEM results showed that the capsules had a typical wall-capsule structure with a regular spherical shape and an average diameter of 1.97 mm. TGA studies revealed that the thermal stability of the capsules were enhanced and the moisture content of the drug-free/loaded capsules were 6.3% and 3.2%. SDS-PAGE results showed that the primary structures of the processed LF in the capsules were unchanged. Drug loading (LE%) and encapsulation efficiency (EE%) analysis showed that the capsules had a higher LE% (45.6%) and EE% (70.7%) than that of the control. In vitro release studies showed that the capsules had a regular and sustainable release profiles in simulated colonic fluid. All of these results indicated that the capsules prepared could be used as a candidate protein drug carrier for colon.