Multipotent mesenchymal stromal cells enhance insulin secretion from human islets via N-cadherin interaction and prolong function of transplanted encapsulated islets in mice.

BACKGROUND: Multipotent mesenchymal stromal cells (MSC) enhance viability and function of islets of Langerhans. We aimed to examine the interactions between human MSC and human islets of Langerhans that influence the function of islets. METHODS: Human MSC and human islets (or pseudoislets, obtained after digestion and reaggregation of islet cells) were cocultured with or without cellular contact and glucose-stimulated insulin secretion assays were performed to assess cell function. The expression of several adhesion molecules, notably ICAM-1 and N-cadherin on islets and MSC, was investigated by qPCR. The role of N-cadherin was analyzed by adding an anti-N-cadherin antibody in islets cultured with or without MSC for 24 h followed by insulin measurements in static incubation assays. Islets and MSC were coencapsulated in new hydrogel microspheres composed of calcium alginate and covalently crosslinked polyethylene glycol. Encapsulated cells were transplanted intraperitoneally in streptozotocin-induced diabetic mice and glycemia was monitored. Islet function was evaluated by the intraperitoneal glucose tolerance test. RESULTS: In vitro, free islets and pseudoislets cocultured in contact with MSC showed a significantly increased insulin secretion when compared to islets or pseudoislets cultured alone or cocultured without cell-to-cell contact with MSC (p < 0.05). The expression of ICAM-1 and N-cadherin was present on islets and MSC. Blocking N-cadherin prevented the enhanced insulin secretion by islets cultured in contact with MSC whereas it did not affect insulin secretion by islets cultured alone. Upon transplantation in diabetic mice, islets microencapsulated together with MSC showed significantly prolonged normoglycemia when compared with islets alone (median 69 and 39 days, respectively, p < 0.01). The intraperitoneal glucose tolerance test revealed an improved glycemic response in mice treated with islets microencapsulated together with MSC compared to mice transplanted with islets alone (p < 0.001). CONCLUSIONS: MSC improve survival and function of islets of Langerhans by cell-to-cell contact mediated by the adhesion molecule N-cadherin.

Synthesis Strategies to Extend the Variety of Alginate-Based Hybrid Hydrogels for Cell Microencapsulation.

The production of hydrogel microspheres (MS) for cell immobilization, maintaining the favorable properties of alginate gels but presenting enhanced performance in terms of in vivo durability and physical properties, is desirable to extend the therapeutic potential of cell transplantation. A novel type of hydrogel MS was produced by straightforward functionalization of sodium alginate (Na-alg) with heterotelechelic poly(ethylene glycol) (PEG) derivatives equipped with either end thiol or 1,2-dithiolane moieties. Activation of the hydroxyl moieties of the alginate backbone in the form of imidazolide intermediate allowed for fast conjugation to PEG oligomers through a covalent carbamate linkage. Evaluation of the modified alginates for the preparation of MS combining fast ionic gelation ability of the alginate carboxylate groups and slow covalent cross-linking provided by the PEG-end functionalities highlighted the influence of the chemical composition of the PEG-grafting units on the physical characteristics of the MS. The mechanical properties of the MS (resistance and shape recovery) and durability of PEG-grafted alginates in physiological environment can be adjusted by varying the nature of the end functionalities and the length of the PEG chains. In vitro cell microencapsulation studies and preliminary in vivo assessment suggested the potential of these hydrogels for cell transplantation applications.

Microencapsulation of Hepatocytes and Mesenchymal Stem Cells for Therapeutic Applications.

Encapsulated hepatocyte transplantation and encapsulated mesenchymal stem cell transplantation are newly developed potential treatments for acute and chronic liver diseases, respectively. Cells are microencapsulated in biocompatible semipermeable alginate-based hydrogels. Microspheres protect cells against antibodies and immune cells, while allowing nutrients, small/medium size proteins and drugs to diffuse inside and outside the polymer matrix. Microencapsulated cells are assessed in vitro and designed for experimental transplantation and for future clinical applications.Here, we describe the protocol for microencapsulation of hepatocytes and mesenchymal stem cells within hybrid poly(ethylene glycol)-alginate hydrogels.

Microencapsulated human mesenchymal stem cells decrease liver fibrosis in mice.

BACKGROUND & AIMS: Mesenchymal stem cell (MSC) transplantation was shown to be effective for the treatment of liver fibrosis, but the mechanisms of action are not yet fully understood. We transplanted encapsulated human MSCs in two mouse models of liver fibrosis to determine the mechanisms behind the protective effect. METHODS: Human bone marrow-derived MSCs were microencapsulated in novel alginate-polyethylene glycol microspheres. In vitro, we analyzed the effect of MSC-conditioned medium on the activation of hepatic stellate cells and the viability, proliferation, cytokine secretion, and differentiation capacity of encapsulated MSCs. The level of fibrosis induced by bile duct ligation (BDL) or carbon tetrachloride (CCl4) was assessed after intraperitoneal transplantation of encapsulated MSCs, encapsulated human fibroblasts, and empty microspheres. RESULTS: MSC-conditioned medium inhibited hepatic stellate cell activation and release of MSC secreted anti-apoptotic (IL-6, IGFBP-2) and anti-inflammatory (IL-1Ra) cytokines. Viability, proliferation, and cytokine secretion of microencapsulated MSCs were similar to those of non-encapsulated MSCs. Within the microspheres, MSCs maintained their capacity to differentiate into adipocytes, chondrocytes, and osteocytes. 23% (5/22) of the MSC clones were able to produce anti-inflammatory IL-1Ra in vitro. Microencapsulated MSCs significantly delayed the development of BDL- and CCl4-induced liver fibrosis. Fibroblasts had an intermediate effect against CCl4-induced fibrosis. Mice transplanted with encapsulated MSCs showed lower mRNA levels of collagen type I, whereas levels of matrix metalloproteinase 9 were significantly higher. Human IL-1Ra was detected in the serum of 36% (4/11) of the mice transplanted with microencapsulated MSCs. CONCLUSIONS: MSC-derived soluble molecules are responsible for an anti-fibrotic effect in experimental liver fibrosis.

Arginine-based biodegradable ether-ester polymers with low cytotoxicity as potential gene carriers.

The success of gene therapy depends on safe and effective gene carriers. Despite being widely used, synthetic vectors based on poly(ethylenimine) (PEI), poly(l-lysine) (PLL), or poly(l-arginine) (poly-Arg) are not yet fully satisfactory. Thus, both improvement of established carriers and creation of new synthetic vectors are necessary. A series of biodegradable arginine-based ether-ester polycations was developed, which consists of three main classes: amides, urethanes, and ureas. Compared to that of PEI, PLL, and poly-Arg, much lower cytotoxicity was achieved for the new cationic arginine-based ether-ester polymers. Even at polycation concentrations up to 2 mg/mL, no significant negative effect on cell viability was observed upon exposure of several cell lines (murine mammary carcinoma, human cervical adenocarcinoma, murine melanoma, and mouse fibroblast) to the new polymers. Interaction with plasmid DNA yielded compact and stable complexes. The results demonstrate the potential of arginine-based ether-ester polycations as nonviral carriers for gene therapy applications.

Survival of free and encapsulated human and rat islet xenografts transplanted into the mouse bone marrow.

Bone marrow was recently proposed as an alternative and potentially immune-privileged site for pancreatic islet transplantation. The aim of the present study was to assess the survival and rejection mechanisms of free and encapsulated xenogeneic islets transplanted into the medullary cavity of the femur, or under the kidney capsule of streptozotocin-induced diabetic C57BL/6 mice. The median survival of free rat islets transplanted into the bone marrow or under the kidney capsule was 9 and 14 days, respectively, whereas that of free human islets was shorter, 7 days (bone marrow) and 10 days (kidney capsule). Infiltrating CD8+ T cells and redistributed CD4+ T cells, and macrophages were detected around the transplanted islets in bone sections. Recipient mouse splenocytes proliferated in response to donor rat stimulator cells. One month after transplantation under both kidney capsule or into bone marrow, encapsulated rat islets had induced a similar degree of fibrotic reaction and still contained insulin positive cells. In conclusion, we successfully established a small animal model for xenogeneic islet transplantation into the bone marrow. The rejection of xenogeneic islets was associated with local and systemic T cell responses and macrophage recruitment. Although there was no evidence for immune-privilege, the bone marrow may represent a feasible site for encapsulated xenogeneic islet transplantation.

Alginate-Poly(ethylene glycol) Hybrid Microspheres for Primary Cell Microencapsulation.

The progress of medical therapies, which rely on the transplantation of microencapsulated living cells, depends on the quality of the encapsulating material. Such material has to be biocompatible, and the microencapsulation process must be simple and not harm the cells. Alginate-poly(ethylene glycol) hybrid microspheres (alg-PEG-M) were produced by combining ionotropic gelation of sodium alginate (Na-alg) using calcium ions with covalent crosslinking of vinyl sulfone-terminated multi-arm poly(ethylene glycol) (PEG-VS). In a one-step microsphere formation process, fast ionotropic gelation yields spherical calcium alginate gel beads, which serve as a matrix for simultaneously but slowly occurring covalent cross-linking of the PEG-VS molecules. The feasibility of cell microencapsulation was studied using primary human foreskin fibroblasts (EDX cells) as a model. The use of cell culture media as polymer solvent, gelation bath, and storage medium did not negatively affect the alg-PEG-M properties. Microencapsulated EDX cells maintained their viability and proliferated. This study demonstrates the feasibility of primary cell microencapsulation within the novel microsphere type alg-PEG-M, serves as reference for future therapy development, and confirms the suitability of EDX cells as control model.

Preparation of well-defined ibuprofen prodrug micelles by RAFT polymerization.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used to treat acute pain, fever, and inflammation and are being explored in a new indication in cancer. Side effects associated with long-term use of NSAIDs such as gastrointestinal damage and elevated risk of stroke, however, can limit their use and exploration in new indications. Here we report a facile method to prepare well-defined amphiphilic diblock copolymer NSAID prodrugs by direct reversible addition-fragmentation transfer (RAFT) polymerization of the acrylamide derivative of ibuprofen (IBU), a widely used NSAID. The synthesis and self-assembling behavior of amphiphilic diblock copolymers (PEG-PIBU) having a hydrophilic poly(ethylene glycol) block and a hydrophobic IBU-bearing prodrug block were investigated. Release profiles of IBU from the micelles by hydrolysis were evaluated. Furthermore, the antiproliferative action of the IBU-containing micelles in human cervical carcinoma (HeLa) and murine melanoma (B16-F10) cells was assessed.

Hyaluronic acid (HA) presentation as a tool to modulate and control the receptor-mediated uptake of HA-coated nanoparticles.

The natural turnover of free hyaluronic acid (HA) is predominantly based on its CD44-mediated internalisation in leukocytes. In a phagocytic cell model (RAW 264.7 murine macrophages) we here provide conclusive evidence that this receptor-mediated mechanism endocytosis is responsible also of the uptake of materials where HA is used as a coating agent, in this case chitosan/triphosphate nanoparticles on whose surface HA is electrostatically adsorbed. Alginate-coated nanoparticles were used as a control and they appeared to undergo a qualitatively similar endocytic process, which was mediated by a different scavenging receptor yet to be identified. In this general picture, an important, modulating role appears to be played by how receptors can cluster around individual nanoparticles. The CD44 slow representation (24-48 h) enforces a limit in the amount of available HA internalisation receptors; therefore a higher affinity, and hence a higher degree of clustering, would yield a lower number of internalised nanoparticles. HA presentation can be varied by acting on nanoparticle structure/morphology, and our data suggest that a better presentation may be linked to both higher affinity and lower capacity/uptake rate. Paradoxically, this result would suggest that particles with a lower affinity for CD44 may allow a more efficient HA-mediated delivery of payloads.

Encapsulation of Huh-7 cells within alginate-poly(ethylene glycol) hybrid microspheres.

Novel calcium alginate poly(ethylene glycol) hybrid microspheres (Ca-alg-PEG) were developed and evaluated as potentially suitable materials for cell microencapsulation. Grafting 5-13% of the backbone units of sodium alginate (Na-alg) with α-amine-ω-thiol PEG maintained the gelling capacity in presence of calcium ions, while thiol end groups allowed for preparing chemically crosslinked hydrogel via spontaneous disulfide bond formation. The combination of these two gelling mechanisms yielded Ca-alg-PEG. Human hepatocellular carcinoma cells (Huh-7) were encapsulated in Ca-alg-PEG and calcium alginate beads (Ca-alg), and cultured for 2 weeks under agitation conditions. Immediately after completion of the microencapsulation, the cell viability was 60% and similar in Ca-alg-PEG and Ca-alg. The proliferation of Huh-7 encapsulated in Ca-alg-PEG was slightly higher than in Ca-alg. Accelerated proliferation after 2 weeks was observed for the encapsulation in Ca-alg-PEG. The production of albumin confirmed the functionality of the encapsulated Huh-7 cells. The study confirms the suitability of Ca-alg-PEG and the one-step technology for cell microencapsulation.

Cell response to the exposure to chitosan-TPP//alginate nanogels.

Hydrophilic nanocarriers formed by electrostatic interaction of chitosan with oppositely charged macromolecules have a high potential as vectors in biomedical and pharmaceutical applications. However, comprehensive information about the fate of such nanomaterials in biological environment is lacking. We used chitosan from both animal and fungal sources to form well-characterized chitosan-pentasodium triphosphate (TPP)//alginate nanogels suitable for comparative studies. Upon exposure of human colon cancer cells (HT29 and CaCo2), breast cancer cells (MDA-MB-231 and MCF-7), glioblastoma cells (LN229), lung cancer cells (A549), and brain-derived endothelial cells (HCEC) to chitosan-(TPP)//alginate nanogels, cell type-, nanogel dosage-, and exposure time-dependent responses are observed. Comparing chitosan-TPP//alginate nanogels prepared from either animal or fungal source in terms of nanogel formation, cell uptake, reactive oxygen species production, and metabolic cell activity, no significant differences become obvious. The results identify fungal chitosan as an alternative to animal chitosan in particular if biomedical/pharmaceutical applications are intended.

Carbon monoxide-releasing micelles for immunotherapy.

With the discovery of important biological roles of carbon monoxide (CO), the use of this gas as a therapeutic agent has attracted attention. However, the medical application of this gas has been hampered by the complexity of the administration method. To overcome this problem, several transition-metal carbonyl complexes, such as Ru(CO)(3)Cl(glycinate), [Ru(CO)(3)Cl(2)](2), and Fe(η(4)-2-pyrone)(CO)(3), have been used as CO-releasing molecules both in vitro and in vivo. We sought to develop micellar forms of metal carbonyl complexes that would display slowed diffusion in tissues and thus better ability to target distal tissue drainage sites. Specifically, we aimed to develop a new CO-delivery system using a polymeric micelle having a Ru(CO)(3)Cl(amino acidate) structure as a CO-releasing segment. The CO-releasing micelles were prepared from triblock copolymers composed of a hydrophilic poly(ethylene glycol) block, a poly(ornithine acrylamide) block bearing Ru(CO)(3)Cl(ornithinate) moieties, and a hydrophobic poly(n-butylacrylamide) block. The polymers formed spherical micelles in the range of 30-40 nm in hydrodynamic diameter. Further characterization revealed the high CO-loading capacity of the micelles. CO-release studies showed that the micelles were stable in physiological buffer and serum and released CO in response to thiol-containing compounds such as cysteine. The CO release of the micelles was slower than that of Ru(CO)(3)Cl(glycinate). In addition, the CO-releasing micelles efficiently attenuated the lipopolysaccharide-induced NF-κB activation of human monocytes, while Ru(CO)(3)Cl(glycinate) did not show any beneficial effects. Moreover, cell viability assays revealed that the micelles significantly reduced the cytotoxicity of the Ru(CO)(3)Cl(amino acidate) moiety. This novel CO-delivery system based on CO-releasing micelles may be useful for therapeutic applications of CO.

Chitosan-based nanogels for selective delivery of photosensitizers to macrophages and improved retention in and therapy of articular joints.

Macrophages play key roles in inflammatory disorders. Therefore, they are targets of treatments aiming at their local destruction in inflammation sites. However, injection of low molecular mass therapeutics, including photosensitizers, in inflamed joints results in their rapid efflux out of the joints, and poor therapeutic index. To improve selective uptake and increase retention of therapeutics in inflamed tissues, hydrophilic nanogels based on chitosan, of which surface was decorated with hyaluronate and which were loaded with one of three different anionic photosensitizers were developed. Optimal uptake of these functionalized nanogels by murine RAW 264.7 or human THP-1 macrophages as models was achieved after <4h incubation, whereas only negligible uptake by murine fibroblasts used as control cells was observed. The uptake by cells and the intracellular localization of the photosensitizers, of the fluorescein-tagged chitosan and of the rhodamine-tagged hyaluronate were confirmed by fluorescence microscopy. Photodynamic experiments revealed good cell photocytotoxicity of the photosensitizers entrapped in the nanogels. In a mouse model of rheumatoid arthritis, injection of free photosensitizers resulted in their rapid clearance from the joints, while nanogel-encapsulated photosensitizers were retained in the inflamed joints over a longer period of time. The photodynamic treatment of the inflamed joints resulted in a reduction of inflammation comparable to a standard corticoid treatment. Thus, hyaluronate-chitosan nanogels encapsulating therapeutic agents are promising materials for the targeted delivery to macrophages and long-term retention of therapeutics in leaky inflamed articular joints.