Huizhen Zheng

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.

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.

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 25u2009μm, restricted many more autoinducers (AIs) than did small aggregates (SA), with a size of approximately 10u2009μm, 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.

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.

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.

Fabrication of stable galactosylated alginate microcapsules via covalent coupling onto hydroxyl groups for hepatocytes applications

Galactose moieties are covalently coupled with sodium alginate to enhance liver-specific functions in microcapsules owing to the specific interaction between the galactose moieties and the asialoglycoprotein receptors (ASGPRs) of hepatocytes. In this study, galactosylated alginate (L-NH2-OH-alginate) based microcapsules with desirable stability and a suitable 3D microenvironment are designed and fabricated for primary hepatocyte applications. The designed L-NH2-OH-alginate is fabricated via the application of ethylenediamine grafted lactobionic acid (L-NH2) onto the hydroxyl groups of sodium alginate so that the negatively charged carboxyl groups intact in L-NH2-OH-alginate can effectively bond with Ca2+ to form a stable three-dimensional gel network; a subsequent reaction with polycations forms a stable membrane of microcapsules. As a result, L-NH2-OH-alginate based microcapsules exhibit an excellent mechanical stability. Moreover, with a higher degree of substitution in L-NH2-OH-alginate (DS 0.41), the hepatocytes entrapped in L-NH2-OH-alginate microcapsules exhibit better viability and well-maintained liver-specific functions.

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.

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.

In situ grafting MPEG on the surface of cell-loaded microcapsules for protein repellency

ABSTRACT The protein repelled alginate-graft-BAT/chitosan/MPEG-norbornene (ABCPN) hydrogel microcapsules were achieved by copper-free click reaction between azides from BAT and alkylenes from norbornene. The MPEG modified polyelectrolyte microcapsules showed significant resistance to immune protein adsorption and good biocompatibility in vivo. Moreover, the mild reaction condition made it feasible that the microcapsules could be formed and modified in situ even when live cells were encapsulated, and precluded the damage cause by other violent modifications methods to transplanted cells or tissues. GRAPHICAL ABSTRACT

An improved model for exploring the effect of physicochemical properties of alginate-based microcapsules on their fibrosis formation in vivo

In vivo microcapsule implantation suffers from varying degrees of inflammatory responses. Considering the complications of the implantation process and the individual variation in laboratory animals, a feasible model, constructed in vitro by culturing fibroblasts on the surfaces of different types of microcapsules, is established in the present study. A high consistency is shown between the cell adhesion on beads or microcapsules based on the developed model in vitro and their fibrosis formation after implantation in vivo, which can be used to evaluate the biocompatibility of the microcapsules. Moreover, the relationship between the surface properties of the microcapsules and the cell adhesion behavior was explored using this developed model. It was found that cell adhesion was aggravated with chitosan reaction, but was reduced with alginate neutralization. This was closely related to the variation trend of surface charges, but not surface roughness and the stiffness of alginate-based microcapsules during the process of chitosan coating and alginate neutralization. This model could therefore provide a rapid evaluation and guidance to tailor the optimal surface properties for long-term transplantation.