Biocompatibility Of Microcapsules Research Articles

Postmodification with Polycations Enhances Key Properties of Alginate-Based Multicomponent Microcapsules.

Postmodification of alginate-based microspheres with polyelectrolytes (PEs) is commonly used in the cell encapsulation field to control microsphere stability and permeability. However, little is known about how different applied PEs shape the microsphere morphology and properties, particularly in vivo. Here, we addressed this question using model multicomponent alginate-based microcapsules postmodified with PEs of different charge and structure. We found that the postmodification can enhance or impair the mechanical resistance and biocompatibility of microcapsules implanted into a mouse model, with polycations surprisingly providing the best results. Confocal Raman microscopy and confocal laser scanning microscopy (CLSM) analyses revealed stable interpolyelectrolyte complex layers within the parent microcapsule, hindering the access of higher molar weight PEs into the microcapsule core. All microcapsules showed negative surface zeta potential, indicating that the postmodification PEs get hidden within the microcapsule membrane, which agrees with CLSM data. Human whole blood assay revealed complex behavior of microcapsules regarding their inflammatory and coagulation potential. Importantly, most of the postmodification PEs, including polycations, were found to be benign toward the encapsulated model cells.

Investigation of encapsulation of pancreatic beta cells and curcumin within alginate microcapsules

AbstractCell encapsulation is an ideal approach for the replacement of pancreatic function in Type 1 diabetes. Poor biocompatibility of microcapsules generates an inflammatory response in the implantation site and induces fibrosis infiltration, which causes microencapsulated cell death and graft failure. To prevent inflammation after implantation, composite microcapsules that exhibit anti‐inflammatory properties were designed. This study is about encapsulating beta cells and curcumin within 1.5% alginate by the jet‐breaking regime of the syringe pump. The microcapsules’ size distribution and rate of the alginate solution were characterized to find uniform particles. Micro‐size particles were attained at a rate of 25 mL/min. Uniform spherical microcapsules (200–300 μm) were created in large amounts in a short period. Microcapsule breakage was less than 3% during 7 days, which demonstrated the stability of the encapsulation method. Insulin secretion and cell viability assays were performed 1, 3, and 7 days after microencapsulation by glucose‐stimulated insulin secretion (GSIS) and 3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5 diphenyl tetrazolium bromide (MTT) assays. No significant differences in the amount of insulin secretion and beta cell viability were observed among free cells, alginate microcapsules, and curcumin‐alginate microcapsules during 7 days (p > 0.05). Therefore, the curcumin and alginate membrane did not show any harmful impacts on the function and survival of the beta cells.

Suppression of Fibrotic Reactions of Chitosan-Alginate Microcapsules Containing Porcine Islets by Dexamethasone Surface Coating.

BackgroundThe microencapsulation is an ideal solution to overcome immune rejection without immunosuppressive treatment. Poor biocompatibility and small molecular antigens secreted from encapsulated islets induce fibrosis infiltration. Therefore, the aims of this study were to improve the biocompatibility of microcapsules by dexamethasone coating and to verify its effect after xenogeneic transplantation in a streptozotocin-induced diabetes mice.MethodsDexamethasone 21-phosphate (Dexa) was dissolved in 1% chitosan and was cross-linked with the alginate microcapsule surface. Insulin secretion and viability assays were performed 14 days after microencapsulation. Dexa-containing chitosan-coated alginate (Dexa-chitosan) or alginate microencapsulated porcine islets were transplanted into diabetic mice. The fibrosis infiltration score was calculated from the harvested microcapsules. The harvested microcapsules were stained with trichrome and for insulin and macrophages.ResultsNo significant differences in glucose-stimulated insulin secretion and islet viability were noted among naked, alginate, and Dexa-chitosan microencapsulated islets. After transplantation of microencapsulated porcine islets, nonfasting blood glucose were normalized in both the Dexa-chitosan and alginate groups until 231 days. The average glucose after transplantation were lower in the Dexa-chitosan group than the alginate group. Pericapsular fibrosis and inflammatory cell infiltration of microcapsules were significantly reduced in Dexa-chitosan compared with alginate microcapsules. Dithizone and insulin were positive in Dexa-chitosan capsules. Although fibrosis and macrophage infiltration was noted on the surface, some alginate microcapsules were stained with insulin.ConclusionDexa coating on microcapsules significantly suppressed the fibrotic reaction on the capsule surface after transplantation of xenogenic islets containing microcapsules without any harmful effects on the function and survival of the islets.

Antifibrotic effect of rapamycin containing polyethylene glycol-coated alginate microcapsule in islet xenotransplantation.

Islet microencapsulation is an attractive strategy for the minimization or avoidance of life-long immunosuppression after transplantation. However, the clinical implementation of this technique is currently limited by incomplete biocompatibility. Thus, the aim of the present study was to demonstrate the improved biocompatibility of rapamycin-containing polyethylene glycol (Rapa-PEG)-coating on alginate microcapsules containing xenogeneic islets. The Rapa-PEG-coating on the alginate layer was observed using scanning electron microscopy (SEM) and the molecular cut-off weight of the microcapsules was approximately 70 kDa. The viabilities of the alginate-encapsulated and Rapa-PEG-coated alginate-encapsulated islets were lower than the viability of the naked islets just after encapsulation, but these the differences diminished over time in culture dishes. Rapa-PEG-coating on the alginate capsules effectively decreased the proliferation of macrophage cells compared to the non-coating and alginate coating of xenogeneic pancreas tissues. Glucose-stimulated insulin secretion did not significantly differ among the groups prior to transplantation. The random blood glucose levels of diabetic mice significantly improved following the transplantation of alginate-encapsulated and Rapa-PEG-coated alginate-encapsulated islets, but there were no significant differences between these two groups. However, there was a significant decrease in the number of microcapsules with fibrotic cell infiltration in the Rapa-PEG-coated alginate microcapsule group compared to the alginate microcapsule group. In conclusion, Rapa-PEG-coating might be an effective technique with which to improve the biocompatibility of microcapsules containing xenogeneic islets. Copyright © 2015 John Wiley & Sons, Ltd.

Molecular Biocompatibility Evaluation of Poly-L-Ornithine-Coated Alginate Microcapsules by Investigating mRNA Expression of Pro-Inflammatory Cytokines

Following a polyelectrolytical complex reaction, the poly-L-ornithine (PLO)-alginate microcapsules were prepared by coating PLO on calcium alginate beads which were produced by a high-voltage electrostatic droplet generator. The biocompatibility of the microcapsules at the molecular level was evaluated through investigating the mRNA expression of pro-inflammatory cytokines; that is, the effect of the PLO coating of alginate beads on the mRNA expression of TNF-α, IL-1β, and IL-6 were measured using the RT-PCR method. The resulting PLO-coated alginate microcapsules have a smooth surface with a mean diameter of 309µm. The molecular biocompatibility studies show that coating microcapsules with PLO has no significant effect on the biocompatibility of alginate microcapsules (p>0.05), and both alginate microcapsules and PLO-coated microcapsules are significantly different from the positive control (p<0.05); however, both are also capable of causing an inflammatory response at a molecular level since both are significantly different from the blank control (p<0.05). Furthermore, with the increase in concentration of microcapsules or co-cultured time, part of the mRNA expression of cytokines is significantly increased. The results also demonstrate that the method used in this study, co-incubating the microcapsules with macrophages and measuring the mRNA expression of cytokines by RT-PCR, may be a useful method for evaluating the biocompatibility of coating materials of microcapsules.

Understanding the biocompatibility of microcapsules designed for islet cell transplantation

| 233 (score 1.02 ± 0.03). This difference in biocompatibility was significant (p < 0.001). Yet, alginate hydrophilicity was similar, as water droplets on dry films had contact angles of 38.7 ± 1.7° and 39.0 ± 1.3° for Alg I and Alg II, respectively. When PLL was added to the capsule membrane, the immune response was more severe (p < 0.01), as cell adhesion scores increased to 1.11 ± 0.11 for Alg I and to 1.55 ± 0.07 for Alg II. Similarly, the cell adhesion score increased to 0.49 ± 0.06 when PLO was added to Alg I capsules, indicating a non-significant increase in capsule immunogenicity. A comparison of contact angles of water on dry films containing polycations was not possible due to irregular surfaces and complete wetting. We are currently measuring the contact angles of air bubbles on hydrated films. conclusions: The guluronic acid content of alginate has a significant effect on its biocompatibility in vivo. Incorporating polycations into the membranes of alginate microcapsules lowers their biocompatibility. So far, these results cannot be explained by the hydrophilicity of the samples. Transplantation islet and pancreas No conflict of interest

Optimizing the Islet Encapsulation System : Investigating Wettability as a Measure of Microcapsule Biocompatibility

Optimizing the Islet Encapsulation System : Investigating Wettability as a Measure of Microcapsule Biocompatibility

Chemistry and the biological response against immunoisolating alginate–polycation capsules of different composition

Implantation of microencapsulated cells has been proposed as a therapy for a wide variety of diseases. An absolute requirement is that the applied microcapsules have an optimal biocompatibility. The alginate-poly-L-lysine system is the most commonly applied system but is still suffering from tissue responses provoked by the capsule materials. In the present study, we investigate the biocompatibility of microcapsules elaborated with two commonly applied alginates, i.e. an intermediate-G alginate and a high-G alginate. These alginates were coated with poly-L-lysine (PLL), poly-D-lysine (PDL) and poly-L-ornithine (PLO). The main objective of this study is to determine the interaction of each alginate matrix with the different polycations and the potential impact of these interactions in the modulation of the host's immune response. To address these issues the different types of microcapsules were implanted into the peritoneal cavity of rats for I month. After this period the microcapsules were recovered and they were evaluated by different techniques. Monochromatised X-ray photoelectron spectroscopy (XPS) was performance and the degree of capsular recovery, overgrowth on each capsule, and the cellular composition of the overgrowth were evaluated by histology. Our results illustrate that the different observed immune responses are the consequence of the variations in the interactions between the polycations and alginates rather than to the alginates themselves. Our results suggest that PLL is the best option available and that we should avoid using PLO and PDL in its present form since it is our goals to produce capsules that lack overgrowth and do not induce an immunological response as such.

Comparison of two types of alginate microcapsules on stability and biocompatibility in vitro and in vivo

Transplantation of encapsulated living cells is a promising approach for the treatment of a wide variety of diseases, especially diabetes. Range-scale application of the technique, however, is hampered by insufficient stability of the capsules. It is difficult to find the optimal membrane to meet all the properties required for cell transplantation. To overcome these difficulties, it is necessary to compare characteristics such as mechanical strength, cell proliferation and biocompatibility of different membranes. We prepared Ca–alginate–poly-L-lysine–alginate (APA) and Ba–alginate–poly-L-lysine–alginate (BPA) microcapsules using the electrostatic droplet method. The integrity of the microcapsules was measured by suspending them in a saline buffer and shaking at 150 rpm for 48 h. The microcapsules were cultured in simulated body fluid to analyze the osmotic pressure stability and implanted in the leg muscle pouch of SD rats to test in vivo transplantation stability. The microcapsules were implanted in the intraperitoneal cavity; then the biocompatibility of microcapsules was identified through analyzing fibrosis formation of microcapsules. The proliferation of cells (Cos-7 and HL-60) cultured in the microcapsules was measured by MTT assay. After 48 h shaking at 150 rpm, the percentages of intact microcapsules of BPA and APA microcapsules were 98.5 ± 0.248% and 95.7 ± 0.221% (p < 0.05), respectively. The intact percentages of APA and BPA microcapsules were 96.9% and 97.7%, respectively, after being soaked in SBF at 37 °C for 15 days. The empty APA and BPA microcapsules were not adhered to the muscle and there was light cellular overgrowth. There is no difference on biocompatibility in implantation into peritoneal cavities. After the cells were cultured in microcapsules, A490 nm of the 8th week was significantly higher than that of 1 day, and the 4th week was at the peak of the cell proliferation curve. After culture for 2 to 6 weeks, spheroids started to develop gradually within the beads. The mechanical strength of BPA microcapsules was higher than that of APA microcapsules. However, there was no difference between the two kinds of capsules in biocompatibility. Microencapsulation did not affect cell proliferation or increase the quantity of cells. In conclusion, BPA microcapsules were more suitable for transplantation in vivo.

Quantitative analysis of fibrosis formation on the microcapsule surface with the use of picro‐sirius red staining, polarized light microscopy, and digital image analysis

Transplantation of microencapsulated cells shows potential to treat a variety of diseases. To improve the quality of microcapsules, efficient methods need to be established and standardized for the evaluation of biocompatibility of microcapsules. To this end, a microscopical method was developed to analyze quantitatively the degree of fibrotic overgrowth (FO), which serves as the index of biocompatibility. In this method, sections were stained with picro-sirius red and illuminated with a polarized light microscope. The images were digitized with the use of a computer-video system in which collagenous content was depicted by gray levels 1 to 255. Both the area of collagen and the thickness of FO were examined. The ratio of Type I/Type III collagen and the thickness were used as indexes of the degree of FO. The data showed that the ratio of Type I/Type III collagen and the thickness increased with time, and FO was not significant 4 weeks after transplantation. In comparison, this new method is more efficient to evaluate FO.

Effect of Microcapsule Composition and Short-Term Immunosuppression on Intraportal Biocompatibility

With higher nutrient and oxygen supply and close contact to blood, the portal vein is a possible alternative to the peritoneal cavity for transplantation of encapsulated cells. Data regarding intraportal biocompatibility of microcapsules are lacking. Microcapsules were built from five alginate types differing in their molar mass and mannuronic/guluronic acid ratios by complex formation with divalent cations (barium or calcium) or mixtures of divalent cations and polycations. They were injected in the portal vein of rats, and cellular and fibrotic pericapsular infiltration thickness was measured 3 and 7 days after implantation. Overgrowth was characterized using various stainings or immunohistochemistry (hematoxylin and eosin, Giemsa, ED-1 for monocyte/macrophage, alpha-actin for myofibroblasts, CD31 for endothelial cells). The impact of short-term immunosuppression (gadolinium-chloride IV 20 mg/kg/day on days--1 and 4 as well as 10 days of rapamycin PO 1 mg/kg/day, tacrolimus PO 3 mg/kg/day, or combinations of rapamycin/tacrolimus or gadolinium/tacrolimus) was further assessed 3, 7, and 42 days after implantation. Overall, overgrowth increased from day 3 to day 7 (p < 0.05). Three and 7 days after implantation, polycation-containing microcapsules induced more reaction than microbeads (p < 0.0001 and p < 0.01). Considering polycation-free beads, barium-alginate induced the weakest reaction. Biocompatibility of microbeads was independent of mannuronic/guluronic acid ratio and molar mass of the alginate. Infiltration was mainly a monocyte/macrophage-rich foreign body reaction, but an eosinophil-containing immunoallergic reaction was also observed. Short-term immunosuppression significantly reduced infiltration in all conditions and up to 42 days after implantation. Biocompatibility after intraportal infusion was best for barium-alginate microbeads and poorest for polycation-containing microcapsules. Short- and long-term overgrowth could be significantly reduced by short-term immunosuppression.

Effect of poly‐L‐lysine coating on macrophage activation by alginate‐based microcapsules: Assessment using a new in vitro method

The characteristics of the microcapsule surface, which interacts directly with the host macrophages, may have a role in the biocompatibility of alginate-poly-L-lysine (PLL)-alginate (APA) microcapsule. The objectives of the study were: 1) to develop and validate a simple, rapid, and sensitive in vitro method for assessing microcapsule biocompatibility, based on microcapsule coincubation with macrophages and measurement, by reverse transcriptase-polymerase chain reaction, of cytokine mRNA expression, and 2) to evaluate the effect of alginate purification and PLL coating on macrophage activation. The mRNA expression of tumor necrosis factor-alpha and interleukin-1beta was significantly higher when macrophages were coincubated with beads made with nonpurified compared with purified alginate (p<0.01, p<0.05, respectively) and negative control (p<0.001) or with APA microcapsules compared with non-PLL-coated alginate beads and negative control (p<0.001). The mRNA expression of interleukin-6 differed significantly only when APA microcapsules were compared with a negative control (p<0.05). These results confirm that alginate purification improves microcapsule biocompatibility, and suggest that PLL is not completely covered and/or neutralized by the second alginate incubation and thus has a role in the host macrophage activation. The assay is sensitive to both alginate contaminants and microcapsule surface characteristics and may be a useful tool for the development of biocompatible microcapsules.

The role of capsule composition and biologic responses in the function of transplanted microencapsulated islets of Langerhans.

The role of capsule composition and biologic responses in the function of transplanted microencapsulated islets of langerhans

Biocompatibility of microcapsules for cell immobilization elaborated with different type of alginates

The biocompatibility of alginate-PLL-alginate (APA) microcapsules has been evaluated with respect to impurity levels. The impurity content of three different alginates (a raw high M-alginate, a raw high G-alginate and a purified high G-alginate) has been determined and the in vivo antigenic response of APA beads made with each alginate assessed. Results show that purification of the alginate not only reduces the total amount of impurities (63% less in polyphenols, 91.45% less in endotoxins and 68.5% less in protein in relation to raw high M-alginate), but also avoids an antibody response when microcapsules of this material are implanted in mice. In contrast, raw alginates produced a detectable antibody response though the differences in their impurity content. Consequently, this work revealed that purity of the alginate rather than their chemical composition, is probably of greater importance in determining microcapsule biocompatibility.

Considerations for successful transplantation of encapsulated pancreatic islets.

Encapsulation of pancreatic islets allows for transplantion in the absence of immunosuppression. The technology is based on the principle that transplanted tissue is protected for the host immune system by an artificial membrane. Encapsulation offers a solution to the shortage of donors in clinical islet transplantation because it allows animal islets or insulin-producing cells engineered from stem cells to be used. During the past two decades three major approaches to encapsulation have been studied. These include intravascular macrocapsules, which are anastomosed to the vascular system as AV shunt; extravascular macrocapsules, which are mostly diffusion chambers transplanted at different sites; and extravascular microcapsules transplanted in the peritoneal cavity. The advantages and pitfalls of these three approaches are discussed and compared in the light of their applicability to clinical islet transplantation. All systems have been shown to be successful in preclinical studies but not all approaches meet the technical or physiological requirements for application in human beings. The extravascular approach has advantages over the intravascular because since it is associated with less complications such as thrombosis and infection. Microcapsules, due to their spatial characteristics, have a better diffusion capacity than macrocapsules. Recent progress in biocompatibility of microcapsules has brought this technology close to clinical application. Critical issues such as limitations in the functional performance and survival are being discussed. The latest results show that both issues can be solved by the transplantation of microencapsulated islets close to blood vessels in prevascularized solid supports.

Alginate-poly-L-lysine microcapsule biocompatibility: a novel RT-PCR method for cytokine gene expression analysis in pericapsular infiltrates.

Transplantation of microencapsulated islets of Langerhans is impaired by a pericapsular host reaction that eventually induces graft failure. We are studying the role of cytokines in the pathogenesis of this reaction, using the model of alginate-polylysine microcapsule implantation in rat epididymal fat pads. The objectives were: (1) to develop a method to measure, by semiquantitative PCR, TGF-beta1 gene expression in fat pad pericapsular infiltrates, and (2) to use this method to evaluate TGF-beta1 gene expression 14 days after microcapsule implantation. TGF-beta1 mRNA level was significantly higher in pericapsular infiltrate cells than in nonimplanted tissue cells and saline-injected tissue cells (p < 0.0001 and p < 0.01, respectively). There was no significant difference between the TGF-beta1 mRNA levels of the two types of controls (p = 0.0945). These results suggest that TGF-beta1 plays a role in the pathogenesis of the pericapsular reaction. The method developed can be used to study the role of other fibrogenic cytokines potentially involved. This will shed light on the mechanisms underlying the pericapsular reaction and will serve as a basis for the development of strategies to control this reaction.

Surface grafting of poly(ethylene glycol) onto poly(acrylamide-co-vinyl amine) cross-linked films under mild conditions

Poly(ethylene glycol) (PEG) was grafted onto poly(acrylamide-co-vinyl amine) (poly(AM-co-VA)) film using tresylated PEG (TPEG) at 37°C in aqueous buffers (pH 7.4) with a view to surface-modifying microencapsulated mammalian cells. Poly(AM-co-VA) film was synthesized by Hofmann degradation of a cross-linked poly(acrylamide) film. Conversion to vinyl amine on the surface of the film was approximately 50%, but bulk conversion was not observed; surface specificity was thought to be the result of cleavage of aminated polymer chains at the surface due to chain scission. Reaction between primary amine and TPEG gave a graft yield of 2 mol% (based on XPS) with respect to available surface amine groups, equivalent to 54 mol% ethylene oxide based on monomer units. Physical adsorption of non-activated polymer was done under identical conditions as a control and the difference in oxygen content was significant compared to TPEG. The type of buffer agent and buffer concentration did not influence graft yields. This graft reaction, which was completed in as little as 2 h was considered to be mild enough to be used for a surface modification of microcapsules containing cells without affecting their viability. Such a surface modification technique may prove to be a useful means of enhancing the biocompatibility of microcapsules (or any tissue engineering construct) even after cell encapsulation or seeding.

The rat epididymal fat pad as an implantation site for the study of microcapsule biocompatibility: validation of the method.

The study of microcapsule biocompatibility is hindered by their uneven distribution and low recovery when implanted into the peritoneum. We evaluated the use of the rat epididymal fat pad as a microcapsule implantation site for biocompatibility studies. The recovery rate of microcapsules containing 85Sr-labeled microspheres was 99.6 +/- 0.75%. Microcapsules made from the same batch of nonpurified alginate, were injected into both fat pads of male Lewis rats (n = 18) and retrieved 14 days later. A semiquantitative fibrosis score scaled from 0 to 3.0 showed that the pericapsular reaction was uniform throughout a fat pad, and that the results of the two fat pads were equivalent because the null hypothesis of inequivalence was rejected (P < .001). Thus, this method can be used to compare the biocompatibility of microcapsule of differing compositions.

Interfacial photopolymerization of poly(ethylene glycol)-based hydrogels upon alginate-poly( l-lysine) microcapsules for enhanced biocompatibility

The biocompatibility of microcapsules made by the co-acervation of alginate and poly( l-lysine) (PLL) was enhanced by coating the surface of these microcapsules with a poly(ethylene glycol) (PEG)-based hydrogel. The hydrogel was formed by an interfacial photopolymerization technique using visible light from an argon ion laser. The light absorbing chromophore, eosin Y, was immobilized on the microcapsule surface. This restricted the formation of the PEG hydrogel to the surface of the microcapsule. The presence of the PEG gel on the surface was confirmed by fluorescent dextran entrapment, by direct visualization after dissolution of the underlying membrane and by electron spectroscopy for chemical analysis. The biological response of such microcapsules was evaluated by intraperitoneal implantation in mice. The PEG-coated microcapsules were found to be less inflammatory and were seen not to elicit a fibrotic response, as was the case with alginate-PLL microcapsules.