Enhanced quorum sensing capacity via regulating microenvironment to facilitate stress resistance of probiotic in alginate-based microcapsules.

Alginate-based microcapsule has becoming a promising carrier for probiotic encapsulation due to the improved stress resistant ability. Besides the physical protection of microcapsules, bacterial quorum sensing (QS) is another prominent factor affecting microbial stress resistance in microcapsules. In the present study, Vibrio harveyi cells were entrapped and proliferated into cell aggregates in alginate-based microcapsules. The microenvironment composed of cells and biomacromolecules was regulated by the diameter, alginate concentration and core state of microcapsule. Then the effect of microenvironment on bacterial QS capacity was investigated, including bioluminescence, autoinducers (AIs) production and QS related genes expression. The highest diameter of 1200 µm and highest alginate concentration of 2.0 % w/v under the investigation range presented strongest QS capacity, and the maintenance of hydrogel core could enhance bacterial QS. Moreover, the mechanism analysis revealed that the formed biofilm on the surface of cell aggregates hampered the outward transfer of AIs, and the local AIs inside the cell aggregates induced stronger bacteria QS by close-range interaction. As a whole, these findings are helpful to guide the technological development and optimization of microencapsulated probiotics with stronger stress resistance, and the potential application in food, dairy, wastewater treatment and biosensor.

Preparation of Cationic Amphiphilic Nanoparticles with Modified Chitosan Derivatives for Doxorubicin Delivery.

Polymeric micelle-like nanoparticles have demonstrated effectiveness for the delivery of some poorly soluble or hydrophobic anticancer drugs. In this study, a hydrophobic moiety, deoxycholic acid (DCA) was first bonded on a polysaccharide, chitosan (CS), for the preparation of amphiphilic chitosan (CS-DCA), which was further modified with a cationic glycidyltrimethylammounium chloride (GTMAC) to form a novel soluble chitosan derivative (HT-CS-DCA). The cationic amphiphilic HT-CS-DCA was easily self-assembled to micelle-like nanoparticles about 200 nm with narrow size distribution (PDI 0.08-0.18). The zeta potential of nanoparticles was in the range of 14 to 24 mV, indicating higher positive charges. Then, doxorubicin (DOX), an anticancer drug with poor solubility, was entrapped into HT-CS-DCA nanoparticles. The DOX release test was performed in PBS (pH 7.4) at 37 °C, and the results showed that there was no significant burst release in the first two hours, and the cumulative release increased steadily and slowly in the following hours. HT-CS-DCA nanoparticles loaded with DOX could easily enter into MCF-7 cells, as observed by a confocal microscope. As a result, DOX-loaded HT-CS-DCA nanoparticles demonstrated a significant inhibition activity on MCF-7 growth without obvious cellular toxicity in comparison with blank nanoparticles. Therefore, the anticancer efficacy of these cationic HT-CS-DCA nanoparticles showed great promise for the delivery of DOX in cancer therapy.

Two types of polyelectrolyte multilayers hydrogel membrane based on chitosan and alginate with different self-assembled process for control L929 cell behavior.

Controlling the adhesion of mammalian cells at the interface between materials and biological environments is a real challenge when designing materials for tissue engineering applications. The surface properties of implanted materials are known to have a significant impact on cell adhesion. Herein, two types of polyelectrolyte multilayers (PEMs) hydrogel membrane based on marine-derived polysaccharides of chitosan (CHI) and alginate (ALG) biopolymers were fabricated by the layer-by-layer (LbL) technique using simple approach involving the change in assemble sequence of chitosan with different degree of deacetylation (DD). The effect of PEMs formation on the surface properties and their effects on the adhesion of mouse fibroblast cell (L929) of the two membranes were studied. The results showed that the formations of ALG/CHI membranes were related to the rigidity and conformations of chitosan molecules. The adhesion of L929 cell was strongly depended on the surface roughness rather than stiffness. The surface of PEMs can be strongly cytophilic (cell adhesive, terminated with chitosan (C1)) or strongly cytophobic (cell resistant, terminated with chitosan (C2)). The results obtained indicate that ALG/CHI PEMs with different surface morphology and roughness could be used in vitro to manipulate cell behaviors to improve upon the design of tissue-engineered membranes.

An improved pH-responsive carrier based on EDTA-Ca-alginate for oral delivery of Lactobacillus rhamnosus ATCC 53103.

A pH-responsive carrier based on an ethylenediaminetetraacetic-calcium-alginate (EDTA-Ca-Alg) system was developed by controlling the release of Ca2+. The system remained in the solution state at neutral pH since EDTA completely chelated the Ca2+. In contrast, a hydrogel immediately formed when the pH was below 4.0, which triggered the in situ release of Ca2+ from the EDTA-Ca compound and led to alginate-Ca binding. Taking advantage of the pH sensitivity, we prepared hydrogel microspheres with uniform size to entrap Lactobacillus rhamnosus ATCC 53103 through emulsification. In an acidic environment, the hydrogel structure remained compact with negligible pores to protect L. rhamnosus ATCC 53103. However, in a neutral intestinal environment, the hydrogel structure gradually disassembled because of the Ca2+ release from the hydrogel, which caused cell release. Therefore, a pH-responsive carrier was developed for the protection and the controlled release of cells in gastrointestinal tract, thus providing potential for oral delivery of probiotics.

A crucial role for spatial distribution in bacterial quorum sensing.

Quorum sensing (QS) is a process that enables bacteria to communicate using secreted signaling molecules, and then makes a population of bacteria to regulate gene expression collectively and control behavior on a community-wide scale. Theoretical studies of efficiency sensing have suggested that both mass-transfer performance in the local environment and the spatial distribution of cells are key factors affecting QS. Here, an experimental model based on hydrogel microcapsules with a three-dimensional structure was established to investigate the influence of the spatial distribution of cells on bacterial QS. Vibrio harveyi cells formed different spatial distributions in the microcapsules, i.e., they formed cell aggregates with different structures and sizes. The cell aggregates displayed stronger QS than did unaggregated cells even when equal numbers of cells were present. Large aggregates (LA) of cells, with a size of approximately 25 µm, restricted many more autoinducers (AIs) than did small aggregates (SA), with a size of approximately 10 µm, thus demonstrating that aggregate size significantly affects QS. These findings provide a powerful demonstration of the fact that the spatial distribution of cells plays a crucial role in bacterial QS.

Alginate-based microcapsules with galactosylated chitosan internal for primary hepatocyte applications.

Alginate-galactosylated chitosan/polylysine (AGCP) microcapsules with excellent stability and high permeability were developed and employed in primary hepatocyte applications. The galactosylated chitosan (GC), synthesized via the covalent coupling of lactobionic acid (LA) with low molecular weight and water-soluble chitosan (CS), was ingeniously introduced into the core of alginate microcapsules by regulating the pH of gelling bath. The internal GC of the microcapsules simultaneously provided a large number of binding sites for the hepatocytes and further promoted the hepatocyte-matrix interactions via the recognition of asialoglycoprotein receptors (ASGPRs) on the hepatocyte surface, and afforded the AGCP microcapsules an excellent stability via the electrostatic interactions with alginate. As a consequence, primary hepatocytes in AGCP microcapsules demonstrated enhanced viability, urea synthesis, albumin secretion, and P-450 enzyme activity, showing great prospects for hepatocyte applications in microcapsule system.

Controlling Gel Structure to Modulate Cell Adhesion and Spreading on the Surface of Microcapsules.

The surface properties of implanted materials or devices play critical roles in modulating cell behavior. However, the surface properties usually affect cell behaviors synergetically so that it is still difficult to separately investigate the influence of a single property on cell behavior in practical applications. In this study, alginate-chitosan (AC) microcapsules with a dense or loose gel structure were fabricated to understand the effect of gel structure on cell behavior. Cells preferentially adhered and spread on the loose gel structure microcapsules rather than on the dense ones. The two types of microcapsules exhibited nearly identical surface positive charges, roughness, stiffness, and hydrophilicity; thus, the result suggested that the gel structure was the principal factor affecting cell behavior. X-ray photoelectron spectroscopy analyses demonstrated that the overall percentage of positively charged amino groups was similar on both microcapsules. The different gel structures led to different states and distributions of the positively charged amino groups of chitosan, so we conclude that the loose gel structure facilitated greater cell adhesion and spreading mainly because more protonated amino groups remained unbound and exposed on the surface of these microcapsules.

Culture of low density E. coli cells in alginate-chitosan microcapsules facilitates stress resistance by up-regulating luxS/AI-2 system.

Entrapped low density cells with culture (ELDCwc) have been proved as a more effective way than direct entrapped high density cells (dEHDC) and free cells to protect probiotics from harsh environment, that is, to improve their stress resistance. The aim of this study was to investigate whether bacterial quorum sensing (QS) facilitated the stress resistance of Escherichia coli in microcapsules by detecting the expression of luxS/AI-2 system. As a result, both the expression of luxS gene and the concentration of autoinducer-2 (AI-2, QS signal molecule) have been discovered higher in ELDCwc than in dEHDC and free cells. Besides that, the luxS mutant E. coli strain was used as a negative control of QS to verify the influence of QS on bacterial stress resistance in microcapsules. The significantly decreased viability of luxS mutant strain in simulated gastric fluid also indicated that the QS played a critical role in protecting microorganisms from severe environment.

Insight of In Vitro Small-Interfering RNA Release From Chitosan Nanoparticles Under Enzymolysis With Förster Resonance Energy Transfer Analysis.

Small-interfering RNA (siRNA)-mediated gene silencing with the aid of chitosan (CS)-based carriers has shown efficient and reliable outcome in vitro, but the gene silencing efficiency in vivo is still limited. It is of great importance to balance the protection and release of siRNA from nanoparticles (NPs) so as to achieve high efficiency. However, siRNA release profile from CS/siRNA NPs has been rarely concerned. Here, Förster resonance energy transfer technique was adopted for in vitro investigation of siRNA release from CS NPs in lysozyme-contained buffer. The results clearly showed that siRNA molecules experienced a fast and short release phase under lysozyme competition to both CS and siRNA, and then a slow and long release under lysozyme degradation on CS. Moreover, lysozyme competition played more important role than enzymolysis on trigging siRNA release. This preliminary study of siRNA release is the first step to get insight of in vivo siRNA release mechanism from CS/siRNA NPs, which will be helpful to adjust the design of CS/siRNA NPs for balancing the protection and release of siRNA molecules.

The cause and influence of sequentially assembling higher and lower deacetylated chitosans on the membrane formation of microcapsule.

Alginate-chitosan (AC) microcapsules with desired strength and biocompatibility are preferred in cell-based therapy. Sequential assembly of higher and lower deacetylated chitosans (C1 and C2 ) on alginate has produced AC1 C2 microcapsule with improved membrane strength and biocompatibility. In this article, the assembly and complexation processes of two cationic chitosans on anionic alginate were concerned, and the cause and influence of sequentially assembling chitosans on AC1 C2 microcapsules membrane formation were evaluated. It was found that C1 complexation was the key factor for deciding the membrane thickness of AC1 C2 microcapsule. Specifically, the binding amount of C2 positively related to the binding amount of C1 , which suggested the first layer by C1 complexation on alginate had no obvious resistance on the sequential cationic C2 complexation. Further analyses demonstrated that outward migration of alginate molecules and inward diffusion of both chitosans under electrostatic interaction contributed to the sequential coating of C2 on first C1 layer. Moreover, C2 complexation through the surface to inner layer of membrane helped smoothen the first layer by C1 complexation that displayed a synergy role on the formation of AC1 C2 microcapsule membrane. Therefore, the two chitosans played different roles and synergistically contributed to membrane properties that can be easily regulated with membrane complexation time.

Improved quorum sensing capacity by culturing Vibrio harveyi in microcapsules.

Microcapsule entrapped low density cells with culture (ELDCwc), different from free cell culture, conferred stronger stress resistance and improved cell viability of microorganisms. In this paper, the quorum sensing (QS) system of Vibrio harveyi was used to investigate changes when cells were cultured in microcapsules. Cells in ELDCwc group grew into cell aggregates, which facilitated cell-cell communication and led to increased bioluminescence intensity. Moreover, the luxS-AI-2 system, a well-studied QS signal pathway, was detected as both luxS gene and the AI-2 signaling molecule, and the results were analyzed with respect to QS capacity of unit cell. The V. harveyi of ELDCwc also showed higher relative gene expression and stronger quorum sensing capacity when compared with free cells. In conclusion, the confined microcapsule space can promote the cell aggregates formation, reduce cell-cell communication distance and increase local concentration of signal molecule, which are beneficial to bacterial QS.

Improved probiotic viability in stress environments with post-culture of alginate-chitosan microencapsulated low density cells.

In this study, probiotics (Saccharomyces cerevisiae Y235) were entrapped in alginate-chitosan microcapsules by emulsification/internal gelation technique. Two different encapsulation patterns were established as directly entrapped high density cells (dEHDC) and entrapped low density cells with culture (ELDCwc). The performance of microencapsulated cells, with free cells (FC) as control, was investigated against sequential stress environments of freeze-drying, storage, and simulated gastrointestinal fluids. After being freeze-dried without cryoprotectant, the survival rate of ELDCwc (14.33%) was significantly higher than 10.00% of dEHDC, and 0.05% of FC. The lower temperature (-20°C) and ELDCwc pattern were beneficial for keeping viable cells at 7.00 logCFU g(-1) after 6 months. Furthermore, the ELDCwc microcapsule maintained viable cells of 6.29 logCFU g(-1) after incubation in SGF and SIF. These studies demonstrated that the pattern of entrapped low density cells with culture was an effective and superior technique of resisting harmful stress environments.

Improving stability and biocompatibility of alginate/chitosan microcapsule by fabricating bi-functional membrane.

Cell encapsulation technology holds promise for the cell-based therapy. But poor mechanical strength and biocompatibility of microcapsule membrane are still obstacles for the clinical applications. A novel strategy is presented to prepare AC1 C2 A microcapsules with bi-functional membrane (that is, both desirable biocompatibility and membrane stability) by sequentially complexing chitosans with higher deacetylation degree (C1) and lower deacetylation degree (C2) on alginate (A) gel beads. Both in vitro and in vivo evaluation of AC1C2 A microcapsules demonstrate higher membrane stability and less cell adhesion, because the introduction of C2 increases membrane strength and decreases surface roughness. Moreover, diffusion test of AC1C2 A microcapsules displays no inward permeation of IgG protein suggesting good immunoisolation function. The results demonstrate that AC1C2 A microcapsules with bi-functional membrane could be a promising candidate for microencapsulated cell implantation with cost effective usage of naturally biocompatible polysaccharides.

Microencapsulated probiotics using emulsification technique coupled with internal or external gelation process.

Alginate-chitosan microcapsules containing probiotics (Yeast, Y235) were prepared by emulsification/external gelation and emulsification/internal gelation techniques respectively. The gel beads by external gelation showed asymmetrical structure, but those by internal gelation showed symmetrical structure in morphology. The cell viability was approximately 80% for these two techniques. However, during cell culture process, emulsification/internal gelation microcapsules showed higher cell growth and lower cell leakage. Moreover, the survival rate of entrapped low density cells with culture (ELDCwc) increased obviously than that directly entrapped high density cells (dEHDC) and free cells when keeping in simulated gastrointestinal conditions. It indicated the growth process of cells in microcapsule was important and beneficial to keep enough active probiotics under harmful environment stress. Therefore, the emulsification/internal gelation technique was the preferred method for application in food or biotechnological industries.

Enhancement of surface graft density of MPEG on alginate/chitosan hydrogel microcapsules for protein repellency.

Alginate/chitosan/alginate (ACA) hydrogel microcapsules were modified with methoxy poly(ethylene glycol) (MPEG) to improve protein repellency and biocompatibility. Increased MPEG surface graft density (n(S)) on hydrogel microcapsules was achieved by controlling the grafting parameters including the buffer layer substrate, membrane thickness, and grafting method. X-ray photoelectron spectroscopy (XPS) model was employed to quantitatively analyze n(S) on this three-dimensional (3D) hydrogel network structure. Our results indicated that neutralizing with alginate, increasing membrane thickness, and in situ covalent grafting could increase n(S) effectively. ACAC(PEG) was more promising than ACC(PEG) in protein repellency because alginate supplied more -COO(-) negative binding sites and prevented MPEG from diffusing. The n(S) increased with membrane thickness, showing better protein repellency. Moreover, the in situ covalent grafting provided an effective way to enhance n(S), and 1.00 ± 0.03 chains/nm(2) was achieved, exhibiting almost complete immunity to protein adsorption. This antifouling hydrogel biomaterial is expected to be useful in transplantation in vivo.

Synthesis and characterization of amphiphilic glycidol-chitosan-deoxycholic acid nanoparticles as a drug carrier for doxorubicin.

Novel amphiphilic chitosan derivatives (glycidol-chitosan-deoxycholic acid, G-CS-DCA) were synthesized by grafting hydrophobic moieties, deoxycholic acid (DCA), and hydrophilic moieties, glycidol, with the purpose of preparing carriers for poorly soluble drugs. Based on self-assembly, G-CS-DCA can form nanoparticles with size ranging from 160 to 210 nm, and G-CS-DCA nanoparticles maintained stable structure for about 3 months when stored in PBS (pH 7.4) at room temperature. The critical aggregation concentration decreased from 0.043 mg/mL to 0.013 mg/mL with the increase of degree of substitution (DS) of DCA. Doxorubicin (DOX) could be easily encapsulated into G-CS-DCA nanoparticles and keep a sustained release manner without burst release when exposed to PBS (pH 7.4) at 37 °C. Antitumor efficacy results showed that DOX-G-CS-DCA have significant antitumor activity when MCF-7 cells were incubated with different concentration of DOX-G-CS-DCA nanoparticles. The fluorescence imaging results indicated DOX-G-CS-DCA nanoparticles could easily be uptaken by MCF-7 cells. These results suggested that G-CS-DCA nanoparticles may be a promising carrier for DOX delivery in cancer therapy.

Preparation and characterization of nanoaggregates self-assembled by lithocholic acid and N-trimethyl modified chitosan derivatives.

A novel kind of chitosan derivatives (CS-LA-TM) were synthesized by grafting hydrophobic molecules of lithocholic acid (LA) and quaternization. CS-LA-TM and micelle-like self-aggregates were characterized by FTIR, 1H NMR, fluorescence spectroscopy, dynamic light scattering, and transmission electron microscopy (TEM). The critical micelles concentration (CMC) ranging from 0.009 mg/mL to 0.030 mg/mL decreased with increasing of the degree of substitution (DS) of LA, pH of medium but with decreasing of the degree of quaternization (DQ) of amino groups. The TEM images demonstrated that spherical CS-LA-TM nanoaggregates with uniform size were formed by self-assembly. The sizes (100-200 nm) of CS-LAs-TMs nanoaggregates increased with increasing of DS, DQ, and can be easily controlled by pH of medium, which may confer the nanoaggregates potential as delivery systems for anticancer drugs, or DNA and siRNA.