alginate-poly-L-lysine-alginate Microcapsules Research Articles

Programmable Electrodeposition of Janus Alginate/Poly-L-Lysine/Alginate (APA) Microcapsules for High-Resolution Cell Patterning and Compartmentalization.

Encapsulation of live cells in protective, semipermeable microcapsules is one of the kernel techniques for in vitro tissue regeneration, cell therapies, and pharmaceutical screening. Advanced fabrication techniques for cell encapsulation have been developed to meet different requirements. Existing cell encapsulation techniques place substantial constraints on the spatial patterning of live cells as well as on the compartmentalization of heterotypic cells. Alginate-Poly-L-lysine-alginate (APA) microcapsules that use sodium alginate as the polyanion and poly-L-lysine (PLL) as the polycation have been extensively employed for cell microencapsulation due to their excellent biocompatibility and biodegradability. This study proposes a novel method for developing programmable Janus APA microcapsules with variable shapes and sizes by using electrodeposition. By the versatile design of the microelectrode device, sequential electrodeposition is triggered to electro-address the cells at specific locations immobilized within a Janus APA microcapsule. The osteogenesis is evaluated by resembling cell compartmentalized and vascularized osteoblast-laden constructs. This technique allows precise spatial patterning of heterotypic cells inside the APA microcapsule, enabling the observation of cellular growth, interactions, and differentiation in a well-controlled chemical and mechanical microenvironment.

Intrapericardial Delivery of APA-Microcapsules as Promising Stem Cell Therapy Carriers in an Experimental Acute Myocardial Infarction Model

The administration of cardiosphere-derived cells (CDCs) after acute myocardial infarction (AMI) is very promising. CDC encapsulation in alginate-poly-l-lysine-alginate (APA) could increase cell survival and adherence. The intrapericardial (IP) approach potentially achieves high concentrations of the therapeutic agent in the infarcted area. We aimed to evaluate IP therapy using a saline vehicle as a control (CON), a dose of 30 × 106 CDCs (CDCs) or APA microcapsules containing 30 × 106 CDCs (APA-CDCs) at 72 h in a porcine AMI model. Magnetic resonance imaging (MRI) was used to determine the left ventricular ejection fraction (LVEF), infarct size (IS), and indexed end diastolic and systolic volumes (EDVi; ESVi) pre- and 10 weeks post-injection. Programmed electrical stimulation (PES) was performed to test arrhythmia inducibility before euthanasia. Histopathological analysis was carried out afterwards. The IP infusion was successful in all animals. At 10 weeks, MRI revealed significantly higher LVEF in the APA-CDC group compared with CON. No significant differences were observed among groups in IS, EDVi, ESVi, PES and histopathological analyses. In conclusion, the IP injection of CDCs (microencapsulated or not) was feasible and safe 72 h post-AMI in the porcine model. Moreover, CDCs APA encapsulation could have a beneficial effect on cardiac function, reflected by a higher LVEF at 10 weeks.

Benefits of cryopreservation as long-term storage method of encapsulated cardiosphere-derived cells for cardiac therapy: A biomechanical analysis

Cardiosphere-derived cells (CDCs) encapsulated within alginate-poly-L-lysine-alginate (APA) microcapsules present a promising treatment alternative for myocardial infarction. However, clinical translatability of encapsulated CDCs requires robust long-term preservation of microcapsule and cell stability, since cell culture at 37 °C for long periods prior to patient implantation involve high resource, space and manpower costs, sometimes unaffordable for clinical facilities. Cryopreservation in liquid nitrogen is a well-established procedure to easily store cells with good recovery rate, but its effects on encapsulated cells are understudied. In this work, we assess both the biological response of CDCs and the mechanical stability of microcapsules after long-term (i.e., 60 days) cryopreservation and compare them to encapsulated CDCs cultured at 37 °C. We investigate for the first time the effects of cryopreservation on stiffness and topographical features of microcapsules for cell therapy. Our results show that functionality of encapsulated CDCs is optimum during 7 days at 37 °C, while cryopreservation seems to better guarantee the stability of both CDCs and APA microcapsules properties during longer storage than 15 days. These results point out cryopreservation as a suitable technique for long-term storage of encapsulated cells to be translated from the bench to the clinic.

2288-PUB: Ameliorating Type 1 Diabetes in Mice via Implantation of APA Microencapsulated Human Stem Cell–Derived Islets

Objective: Human pluripotent stem cells derived islets (SC-islets) provide a promising alternative source for beta cell replacement therapies for type 1 diabetes, while facing immune-rejection risks. Recent studies show that alginate-poly-L-lysine-alginate (APA) microcapsules have potentials to overcome the need for immunosuppression in allogeneic transplantation. In this study, APA encapsulated SC-islets were implanted into type 1 diabetes immune-competent mice models, to explore their ability to treat diabetes. Method: First, we optimized differentiation method, SC-islets’ ratio of mature β cells and in vitro glucose response were measured; SC-islets encapsulated with APA were implanted into the intraperitoneal space of C57BL/6 STZ-induced diabetic mice (n=8), and blood glucose levels were further monitored. Result: After optimization, SC-islets mature β cells (NKX6.1+/C-peptide+) reach 43.72 ± 1.62% (previous data was 21.01± 4.07%), and C-peptide positive cells increased from 34.93 ± 10.05% to 60.86 ± 1.15%. The SC-islets also included α or Δ cells, and responded to multiple sequential high-glucose challenges (insulin secreted ranged 2.10-3.27 μIU/103 cells). Then APA encapsulated SC-islets were implanted into the intraperitoneal space of diabetes mice. The fasting blood glucose decreased to normal level (3.70 ± 0.93 mM) on day 1 post-transplant, and the blood glucose maintained normal for 11 days. Similarly, random blood glucose returned to normal levels (11.01 ± 4.63 mM) on day 7 post-transplant. Conclusion: Optimized SC-islets had a higher proportion of mature β cells and improved glucose response functions. The in vivo results showed that SC-islets in APA microcapsules can rapidly ameliorate hyperglycemia in diabetic mouse, while the duration time need further improvement through inhibiting fibrosis in alginate microspheres. Our results indicate that microencapsulated allogeneic SC-islets have potentials to treat diabetes. Disclosure L. Guo: None. Q. Pan: None. Y. Song: None. Y. Cao: None. Z. Ou: None. X. Chen: None. W. Li: None. X. Tang: None.

Type 1 Diabetes Mellitus reversal via implantation of magnetically purified microencapsulated pseudoislets

Microencapsulation of pancreatic islets for the treatment of Type I Diabetes Mellitus (T1DM) generates a high quantity of empty microcapsules, resulting in high therapeutic graft volumes that can enhance the host’s immune response. We report a 3D printed microfluidic magnetic sorting device for microcapsules purification with the objective to reduce the number of empty microcapsules prior transplantation. In this study, INS1E pseudoislets were microencapsulated within alginate (A) and alginate-poly-L-lysine-alginate (APA) microcapsules and purified through the microfluidic device. APA microcapsules demonstrated higher mechanical integrity and stability than A microcapsules, showing better pseudoislets viability and biological function. Importantly, we obtained a reduction of the graft volume of 77.5% for A microcapsules and 78.6% for APA microcapsules. After subcutaneous implantation of induced diabetic Wistar rats with magnetically purified APA microencapsulated pseudoislets, blood glucose levels were restored into normoglycemia (<200 mg/dL) for almost 17 weeks. In conclusion, our described microfluidic magnetic sorting device represents a great alternative approach for the graft volume reduction of microencapsulated pseudoislets and its application in T1DM disease.

Mechanical Characterization of APA Microcapsules by Parallel-Plate Compression

We produced microcapsules of alginate-poly(L)lysine-alginate (APA)with diameters on the order of 10 µm. To characterize their mechanical properties, we conducted an experiment on the parallel-plate compression of a microcapsule and modeled its deformation. In the modeling task, the microcapsule was assumed to be a spherical liquid-filled elastic membrane with negligible bending stiffness and permeability. The membrane thickness was estimated by applying Reissner's linear elastic theory to the experimental force-displacement relationship during loading in the small displacement region. The initial stretch of the membrane was taken into account; it was mainly caused by the osmotic pressure difference across the membrane. The initial stretch of the microcapsule was determined by fitting the calculated and experimental force-displacement relationships during loading at small to medium displacements. At large displacements, the calculated force was smaller than the experimentally measured force because of fluid permeation across the APA membrane. The calculated and experimentally imaged shapes of the deformed microcapsule were compared. The effects of varying the membrane thickness on the force-displacement and transmural pressure-displacement curves were shown, and the limitations of applying the present deformation model were examined.

Subcutaneous implantation ofmicroencapsulated cells overexpressing α-L-iduronidase for mucopolysaccharidosis type I treatment.

Mucopolysaccharidosis type I (MPS I) is caused by a deficiency of α-L-iduronidase (IDUA), resulting in accumulation of glycosaminoglycans (GAG) in lysosomes. Microencapsulation of recombinant cells is a promising gene/cell therapy approach that could overcome the limitations of the current available treatments. In the present study we produced alginate-poly-L-lysine-alginate (APA) microcapsules containing recombinant cells overexpressing IDUA, which were implanted in the subcutaneous space of MPS I mice in order to evaluate their potential effect as a treatment for this disease. APA microcapsules enclosing genetically modified Baby Hamster Kidney cells overexpressing IDUA were produced and implanted in the subcutaneous space of 4-month-old MPS I mice (Idua -/-). Treatment was performed using two cell concentrations: 8.3 × 107 and 8.3 × 106 cells/mL. Untreated MPS I and normal mice were used as controls. Microcapsules were retrieved and analyzed after treatment. Increased IDUA in the liver, kidney and heart was detected 24 h postimplantation. After 120 days, higher IDUA activity was detected in the liver, kidney and heart, in both groups, whereas GAG accumulation was reduced only in the high cell concentration group. Microcapsules analysis showed blood vessels around them, as well as inflammatory cells and a fibrotic layer. Microencapsulated cells were able to ameliorate some aspects of the disease, indicating their potential as a treatment. To achieve better performance of the microcapsules, improvements such as the modulation of inflammatory response are suggested.

Construction of doxycycline-mediated BMP-2 transgene combining with APA microcapsules for bone repair

Objectives The repairing of large segmental bone defects is difficult for clinical orthopedists at present. Gene therapy is regarded as a promising method for bone defects repair. The present study aimed to construct an effective and controllable Tet-On expression system for transferring hBMP-2 gene into bone marrow mesenchymal progenitor cells (BMSCs). Meanwhile, with combination of alginate-poly-L-lysine-alginate (APA) microencapsulation technology, we attempted to reduce the influence of immunologic rejection and examine the effect of the Tet-On expression system on osteogenesis of BMSCs. Methods The adenovirus encoding hBMP-2 (ADV-hBMP2) was constructed using the means of molecular cloning. The ADV-hBMP2 and Adeno-X Tet-On virus was respectively transfected to the HEK293 for amplification and afterward BMSCs were co-infected with the virus of ADV-hBMP2 and the Adeno-X Tet-On. The expression of hBMP-2 was measured with induction by doxycycline (DOX) at different concentration by means of RT-PCR and ELISA. Combining Tet-On expression system and APA microcapsules with the use of the pulsed high-voltage electrostatic microcapsule instrument, we examined the expression level of hBMP-2 in APA microcapsules by ELISA as well as the osteogenesis by alizarin red S staining. Key findings An effective Tet-On expression system for transferring hBMP-2 gene into BMSCs was constructed successfully. Also, the expression of hBMP-2 could be regulated by concentration of DOX. The data exhibited that BMSCs in APA microcapsules maintained the capability of proliferation and differentiation. The combination of Tet-On expression system and APA microcapsules could promote the osteogenesis of BMSCs. Conclusions According to the results, microencapsulated Tet-On expression system showed the effective characteristics of secreting hBMP-2 and enhancing osteogenesis, which would provide a promising way for bone repair.

Multifunctional hydrogel-based scaffold for improving the functionality of encapsulated therapeutic cells and reducing inflammatory response

Since the introduction of cell immunoisolation as an alternative to protect transplanted cells from host immune attack, much effort has been made to develop this technology into a realistic clinical proposal. Several promising approaches have been investigated to resolve the biotechnological and biosafety challenges related to cell microencapsulation. Here, a multifunctional hydrogel-based scaffold consisting of cell-loaded alginate-poly-l-lysine-alginate (APA) microcapsules and dexamethasone (DXM)-loaded poly(lactic-co-glycolic) acid (PLGA) microspheres embedded in alginate hydrogel is developed and evaluated. Initially, the feasibility of using an alginate hydrogel for enclosing APA microcapsules was studied in a xenogeneic approach. In addition, the performance of the local release of DXM was addressed. The in vitro studies confirmed the correct adaptation of the enclosed cells to the scaffolds in terms of metabolic activity and viability. The posterior implantation of the hydrogel-based scaffolds containing cell-loaded microcapsules revealed that the hematocrit levels were maintained high and constant, and the pericapsular overgrowth was reduced in the DXM-treated rats for at least 2months. This multifunctional scaffold might have a synergistic effect: (1) providing a physical support for APA microcapsules, facilitating administration, ensuring retention and recuperation and preventing dissemination; and (2) reducing post-transplantation inflammation and foreign body reaction, thus prolonging the lifetime of the implant by the continuous and localized release of DXM.

Behaviour and ultrastructure of human bone marrow-derived mesenchymal stem cells immobilised in alginate-poly-l-lysine-alginate microcapsules

Context: Human bone marrow mesenchymal stem cells (hBM-MSCs) show a great promise for the treatment of a variety of diseases. Despite the previous trials to encapsulate hBM-MSCs in alginate-poly-l-lysine-alginate (APA) systems, the various changes that follow immobilisation have not been ascertained yet. Objective: Determine the various consequences derived from entrapment on cell behaviour, putting special emphasis on the ultrastructure. Methods: hBM-MSCs were immobilised in APA microcapsules to further characterise their viability, metabolic activity, proliferation, VEGF-secretability, and morphology. Results: The VEGF produced by monolayer hBM-MSCs increased significantly 1 d post-encapsulation, and was maintained for at least 4 weeks. TEM imaging of cells revealed well preserved ultrastructure indicating protein synthesis and high metabolic activity. Conclusion: Although APA microencapsulation did not support 100% of fully viable hBM-MSCs for long-term cultures, it was conceived to enhance both VEGF secretion and metabolic activity while not losing their stemness characteristics.

Use of Artificial Cell Microcapsule Containing Thalidomide for Treating TNBS-induced Crohn's Disease in Mice

In this study, we examined the in-vivo characteristics of a novel microencapsulated thalidomide formulation in a murine model of experimental Crohn's disease. Crohn's disease was induced with a single intra-colonic injection of 120 mg/kg of bodyweight of 2,5,6-trinitrobenzene sulfonic acid (TNBS) dissolved in 30% ethanol in Balb/c mice. Level of tumor necrosis factor alpha (TNF-α), interleukin one beta (IL-1β), interleukin 6 (IL-6) and nitric oxide (NO) were measured in tissue homogenate. Moreover, myeloperoxidase (MPO) activity was determined to assess the extent of neutrophil infiltration. Dose response study showed that treating the mice with microencapsulated thalidomide (100 mg/kg of bodyweight) for two weeks significantly decreased the degree of intestinal inflammation related to Crohn's disease. Higher and lower doses (0, 25, 50 and 200 mg/kg of bodyweight) did not exhibit comparable effects. The present study validates the success of alginate-poly-L-lysine-alginate (APA) microcapsules containing thalidomide in reducing colonic inflammation, and proposes a potential remedy for Crohn's disease.

Artificial cell microcapsule for oral delivery of thalidomide: design, preparation, and in-vitro characterization for Crohn’s disease application

Crohn's disease is a chronic inflammatory disorder of the gut and is classified as a type of inflammatory bowel disease. The anti-inflammatory drug thalidomide has shown to be very effective against Crohn’s disease, but presents several limitations such as drowsiness, skin rash and hypertension. Therefore, development of novel delivery system is urgent and necessary. The aim of this paper is to present the formulation of Alginate- Poly-L-Lysine-Alginate (APA) microcapsules for the delivery of thalidomide to desired locations of the gastrointestinal tract. APA microcapsules were designed, prepared and characterized in-vitro for thalidomide release. Mechanical stability of capsules and the drug release profile were monitored in a simulated gastrointestinal model. Data suggest that APA microcapsules enable a slow release of thalidomide in a pH and time-dependent manner. Indeed, the characteristics of APA microcapsules make it a suitable carrier for the targeted delivery of thalidomide to specific areas of the gastrointestinal tract.

Anti-inflammatory potential of artificial microcapsules containing thalidomide for use in treating Crohn's disease

Crohn's disease is a chronic inflammatory bowel disease associated with an abnormal immune response in the gastrointestinal tract. Several studies demonstrate that thalidomide could be effective in the treatment of refractory Crohn's disease. However, its widespread use has been limited because of potential side effects. In the present study, we investigated the inhibitory activity of alginate-poly-L-lysine-alginate (APA) microcapsules containing thalidomide on Lipopolysaccharide (LPS)-induced inflammation in RAW 264.7 macrophage cells and on 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced Crohn’s disease. Results showed that APA microcapsules containing thalidomide inhibited the release of pro-inflammatory cytokines in cell supernatant following LPS activation. Moreover, treatment with microencapsulated thalidomide decreased the level of TNF-α, IL-6 and IL-1β by 49.3%, 62.3% and 54.6% respectively in TNBS-treated mice. The present project validates the efficiency of APA microcapsules in providing a targeted delivery of thalidomide for treating chronic conditions such as Crohn’s disease.

Optimization of 100 μm alginate-poly-l-lysine-alginate capsules for intravitreous administration

The field of cell microencapsulation is advancing rapidly. Particle size plays a critical role in terms of biocompatibility and limits decisively its applicability. Producing reduced size microcapsules involves broadening the possibilities to employ this technology in the treatment of many disorders. Nervous system diseases (NSD) represent a clear example of that. This work describes the feasibility of reducing the size of alginate-poly-l-lysine-alginate (APA) microcapsules up to 100μm in a highly monodisperse way using the novel Flow Focusing technique. C2C12 myoblasts genetically engineered to express the triple reporter gene thymidine kinase-green fluorescent protein-luciferase (TGL) and secrete vascular endothelial growth factor soluble receptor 2 (VEGFR2, also known as KDR) were encapsulated for further characterization. Resulting new particles were assayed in vitro to explore whether their functionality might be affected due to the physicochemical changes arising from such dramatic size reduction. Not only were negative effects at this level not noticed in terms of cell viability, cell proliferation and KDR secretion, but once again the suitability of APA microcapsules was also reinforced against other microcapsule designs. Furthermore, the fully viable and functional biosystems were successfully administered in the intravitreous space of rats, where the activity of encapsulated cells was monitoring over 3weeks.

The reconstruction of lung alveolus-like structure in collagen-matrigel/microcapsules scaffolds in vitro

This study attempted to use collagen–Matrigel as extracellular matrix (ECM) to supply cells with three-dimensional (3D) culture condition and employ alginate-poly-l-lysine-alginate (APA) microcapsules to control the formation of alveolus-like structure in vitro. We tested mice foetal pulmonary cells (FPCs) by immunohistochemistry after 2D culture. The alveolus-like structure was reconstructed by seeding FPCs in collagen–Matrigel mixed with APA microcapsules 1.5 ml. A self-made mould was used to keep the structure from contraction. Meanwhile, it provided static stretch to the structure. After 7, 14 and 21 days of culture, the alveolus-like structure was analysed histologically and immunohistochemically, or by scanning transmission electron microscopy (TEM). We also observed these structures under inverted phase contrast microscope. The expression of pro-surfactant protein C (SpC) was detected by reverse transcription-polymerase chain reaction (RT-PCR). We obtained fibroblasts, epithelial cells and alveolar type II (AE2) cells in FPCs. In the reconstructed structure, seeding cells surrounding the APA microcapsules constructed alveolus-like structures, the size of them ranges from 200 to 300 μm. In each reconstructed lung tissue sheet, microcapsules had integrity. Pan-cytokeratin, vimentin and SpC positive cells were observed in 7- and 14-day cultured structures. TEM showed lamellar bodies of AE2 cells in the reconstructed tissues whereas RT-PCR expressed SpC gene. Primary mice FPCs could form alveolus-like structures in collagen–Matrigel/APA microcapsules engineered scaffolds, which could maintain a differentiated state of AE2 cells.

Microcapsules Embedded with Three-Dimensional Fibrous Scaffolds for Cell Culture and Tissue Engineering

Aggregating into multicellular spheroids within alginate–poly-L-lysine–alginate (APA) microcapsules is important in maintaining the cellular viability and specific functions. However, in the absence of a vascular network, cells in the core of large-sized spheroids are gradually necrotic because of oxygen transfer limitations. In this study, a novel APA microcapsule embedded with three-dimensional fibrous scaffolds (called APA-FS) was proposed to eliminate cellular necrosis by regulating cells to form multi-small spheroids. HepG2 cells were embedded within the APA-FS to form spheroids and the state of these spheroids was evaluated via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide assay, glucose/lactate metabolism, live/dead staining, and hematoxylin and eosin staining. Comparing with the conventional APA microcapsules, the cells within APA-FS organized into multi-small spheroids. The size of these spheroids depended on the concentration of fibrous scaffolds embedded within the microcapsules. In the APA-FS embedded with 5% (v/v) fibrous scaffolds, the average size of cellular spheroids was controlled below 100 microm and the cellular viability was increased by 50% than the control. The results of live/dead staining and hematoxylin and eosin staining showed that the improved cellular viability might be attributed to the decreased necrosis in the core of these spheroids. The improved viability of cells demonstrated the efficiency of this technology. These findings implied that this system might provide a more suitable culture environment for a variety of tissue engineering applications.

Characterization of viability and proliferation of alginate‐poly‐L‐lysine–alginate encapsulated myoblasts using flow cytometry

Genetically modified cells encapsulated in alginate-poly-L-lysine-alginate (APA) are being developed to deliver therapeutic products to treat a variety of diseases. The characterization of the encapsulated cells thus becomes paramount. This study reports a novel method to assess the viability, granularity and proliferation of encapsulated cells based on flow cytometry. The in vitro viability of encapsulated G8 murine myoblasts secreting canine FVIII (cFVIII) measured by flow cytometry was comparable to the traditional trypan blue exclusion method and both correlated with cFVIII secretion levels. In contrast, after implantation into mice, only viability measured by flow cytometry correlated with cFVIII secretion. Further, flow cytometry analysis of encapsulated cells maintained in vitro and in vivo revealed a greater fraction of granular cells compared to free cells, suggesting that encapsulation influences the morphology (cytoplasmic composition) of cells within APA microcapsules. Interestingly, the proliferation study showed that encapsulated cells proliferate faster, on average, and were more heterogeneous in vivo compared to in vitro culture conditions, suggesting that encapsulated cell proliferation is complex and environment-dependent. In conclusion, we show that flow cytometry analysis allows for a more consistent and comprehensive examination of encapsulated cells to aid in the development of cell therapy protocols.

Microencapsulation to reduce mechanical loss of microspheres: implications in myocardial cell therapy

Previous regenerative studies have demonstrated massive cell losses after intramyocardial cellular delivery. Therefore, efforts at reducing mechanical losses may prove more successful in optimising cellular therapy. In this study, we hypothesized that escalating mesenchymal stem cells (MSCs) dose will not produce corresponding improvement in cardiac function due to washout of the small cells in microcirculation. Using microspheres similar in size to MSCs, that are encapsulated in alginate-poly-l-lysine-alginate (APA), we tested the hypothesis that size is an important factor in early losses. In experiment I, five groups of rats (n=9 each) underwent coronary ligation; group I had no treatment; the other groups received escalating 0.5 × 10(6), 1.5 × 10(6), 3 × 10(6) and 5 × 10(6) of MSCs each. Echocardiogram was performed at baseline, 2 days and 7 weeks after surgery. In experiment II, cell-sized microspheres (10 μm) were encapsulated in APA microcapsules. In group I (n=16), rats received bare microspheres, group II (n=16) microspheres within 200 μm microcapsules and in group III (n=16), microspheres within 400 μm microcapsules. After 20 min, hearts were quantified for the amount retained. Myocardial function did not improve further with escalating cell doses beyond an initial response at 1.5 × 10(6) cells. Encapsulated microspheres in 200 μm and 400 μm microcapsules demonstrated a fourfold increase in retention rate compared with 10 μm microspheres. We concluded that suboptimal functional improvement in this animal model starts at 1.5 × 10(6) cells and does not respond to escalating cell doses. Improving mechanical retention is possible by increasing the size of the injectate. Microencapsulation could be used to encapsulate donor cells and facilitate functional improvement in cellular heart failure therapy.

Adsorption of human immunoglobulin to implantable alginate‐poly‐L‐lysine microcapsules: Effect of microcapsule composition

Alginate-poly-L-lysine-alginate (APA) microcapsules continue to be the most widely studied device for the immuno-protection of transplanted therapeutic cells. Producing APA microcapsules having a reproducible and high level of biocompatibility requires an understanding of the mechanisms of the immune response towards the implants. Here, we investigate the adsorption of immunoglobulins (IgG, IgM, and IgA) onto the surface of APA microcapsules in vitro after their exposure to human serum and peritoneal fluid. Immunoglobulins (Ig) are considered to be opsonizing proteins, thus they tend to mediate inflammation when adsorbed to foreign surfaces. Ig adsorption was monitored using direct immunofluorescence. The amount of Ig adsorbed to the microcapsule surface was not significantly influenced by the guluronic acid content nor the purity level of the alginate, although microcapsules of intermediate-G purified alginate corresponded with the lowest adsorption levels. Ig adsorption was negligible when the poly-L-lysine membrane was omitted, suggesting that positive charges at the microcapsule surface are responsible for binding Ig.

Radiopaque Alginate Microcapsules for X-ray Visualization and Immunoprotection of Cellular Therapeutics

Alginate-poly-L-lysine-alginate (APA) microcapsules have been explored as vehicles for therapeutic drug and cell delivery. The permselectivity of these capsules provides a unique means of controlled drug release and immunoisolation of encapsulated cells. Immunoisolation is especially attractive as it abrogates the need for chronic immunosuppressive therapy and opens up the possibility for the delivery of numerous cell sources including xenogeneic grafts. APA microcapsules containing cellular therapeutics have proven effective in the short-term treatment of a wide range of diseases requiring enzyme or endocrine replacement therapy, including type I diabetes. If these microcapsules could be noninvasively monitored with X-ray imaging modalities (i.e., fluoroscopy, CT, and digital subtraction angiography), questions such as the ideal transplantation site, the best means of delivery, and the long-term survival of grafts could be better addressed. We have developed two novel alginate-based radiopaque microcapsule formulations containing either barium sulfate (Ba X-Caps) or bismuth sulfate (Bi X-Caps). As compared to conventional, nonradiopaque APA capsules, Ba X-Caps and Bi X-Caps containing human cadaveric islets resulted in a decrease in cellular viability of less than 5% up to 14 days after encapsulation. Both radiopaque capsules were found to be permeable to lectins < or =75 kDa, but were impermeable to lectins > or =120 kDa, thus ensuring the blockage of the penetration of antibodies while allowing free diffusion of insulin and nutrients. The glucose-responsive insulin secretion of the radiopaque encapsulated human islets was found to be unaltered compared to that of unlabeled controls, with human C-peptide levels ranging from 3.21 to 2.87 (Ba X-Caps) and 3.23 to 2.87 (Bi X-Caps) ng/islet at 7 and 14 days postencapsulation, respectively. Using fluoroscopy, both Ba X-Caps and Bi X-Caps could be readily visualized as single radiopaque entities in vitro. Furthermore, following transplantation in vivo in mice and rabbits, single capsules could be identified with no significant change in contrast for at least 2 weeks. This study represents the first attempt at making radiopaque microcapsules for X-ray guided delivery and imaging of cellular therapeutics. While human cadaveric islets were used as a proof-of-principle, these radiopaque capsules may have wide ranging therapeutic applications for a variety of cell types.