The natural anionic polyelectrolyte alginate and its derivatives are of particular interest for pharmaceutical and biomedical applications. Most interesting for such applications are alginate hydrogels, which can be processed into various shapes, self-standing or at surfaces. Increasing efforts are underway to functionalize the alginate macromolecules prior to hydrogel formation in order to overcome the shortcomings of purely ionically cross-linked alginate hydrogels that are hindering the progress of several sophisticated biomedical applications. Particularly promising are derivatives of alginate, which allow simultaneous ionic and covalent cross-linking to improve the physical properties and add biological activity to the hydrogel. This review will report recent progress in alginate modification and functionalization with special focus on synthesis procedures, which completely conserve the ionic functionality of the carboxyl groups along the backbone. Recent advances in analytical techniques and instrumentation supported the goal-directed modification and functionalization.Polymers 2020, 12, 919 2 of 23 Physicochemical and Polyelectrolyte Characteristics of AlginateThe composition, internal structure, and molar mass of the alg backbone govern the functional properties [9]. The solubility of alg in water depends on the pH value and the type of the counterions of the carboxyl groups. At pH < 3, both the M-and G-structures will precipitate as alginic acid. However, alternating M-and G-structures precipitate at lower pH values compared to the alg containing Polymers 2020, 12, 919 3 of 23 more homogeneous block structures. Neutralization of the alginic acid occurs at pH > 4, where it is converted into its corresponding salt [10]. Na-alg is an example of a water-soluble alg.Na-alg behaves in aqueous solution as a typical linear homogeneously charged polyelectrolyte. The chain conformation and consequently the solution viscosity strongly depend on the ionic strength, i.e., on both the alg concentration and the simultaneous presence of low molar mass salt. The intramolecular electrostatic repulsion between the neighboring negatively charged carboxyl groups of each monomer unit forces alg molecules into an extended random coil conformation in aqueous solution [11]. This results in highly viscous solutions even at relatively low alg concentration. The dynamic viscosity increases exponentially with the molar mass, while the intrinsic flexibility of the alg chains in solution increases in the order GG < MM < MG [12]. On the other hand, the selectivity for cation-binding and gel-forming properties strongly depends on the composition and sequence. Divalent cations preferably bind to the G-blocks. The ability to form ionotropic gels is based on this selective binding of cations. Mixing solutions of Na-alg with solutions of cationic polyelectrolytes yields precipitates of polyelectrolyte complexes.The rheological behavior of Na-alg solutions is important for a number of technologies and depends on both the concentration and th...

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. ■ INTRODUCTIONProgress in therapies relying on the allo-or xenotransplantation of immobilized cells and tissue strongly depends on the quality of the immobilizing material. Currently, the translation of related therapies to the clinics, for example to treat end-stage organ failure and end-stage diseases such as cancer, diabetes mellitus, and acute liver failure, 1,2 is hindered by the lack of materials having the perfect properties. Hydrogels prepared from the biopolymer sodium alginate (Na-alg) or derivatives of it have been reported in abundant papers as particularly advantageous because they fulfill general requirements such as the spontaneous formations of hydrogels in the presence of divalent cations (e.g., Ca 2+ , Ba 2+ ) under mild conditions of temperature and pH, and high biocompatibility. 3−6 Maintaining these advantages but overcoming several drawbacks, including the lack of in vivo mechanical resistance and stability, and defects in permselectivity, 7,8 are the focus of current research. There are additional limitations caused by the dimension of the targeted final application. This calls in particular for therapies which intend the transplantation of allo-or xenogeneic cells immobilized in microspheres (MS) in order to allow for miniinvasive surgery. 9,10 The stabilization of alginate gel beads by coating the MS with polycations such as (poly(L-lysine), poly(L-ornithine), and poly(L-guanidine) was investigated, but impaired cell graft function in vivo. 11 Another strategy to improve the performance of alginate MS relies on the combination with other polymers such as poly(ethylene glycol) (P...

The controlled release of small molecular modulators of the immune response from hydrogel microspheres (MS) used for cell immobilization is an attractive approach to reduce pericapsular fibrotic overgrowth (PFO) after transplantation. Ketoprofen is a well-known nonsteroidal anti-inflammatory drug involved in the early stage inflammation cascade. PEGylated derivatives of ketoprofen, presenting either ester or amide linkage to the drug, were synthesized and conjugated to the hydroxyl groups of sodium alginate (Na-alg). Functionalized cell-free and MIN6 cells containing MS were produced from the resulting modified alginates. In vitro quantification of ketoprofen release indicated regular and sustained drug delivery over 14 days, resulting from the hydrolytic cleavage of the ester bond. The release kinetics was enhanced over the initial 7 days by the presence of MIN6 cells, probably as a result of cell esterase activity. In the presence of amide bond, traces of ketoprofen were released over 14 days due to a much slower hydrolysis kinetics. Cell-free and MIN6 cells containing MS were transplanted in immune-competent mice, either in the peritoneal cavity or under the kidney capsule, with a follow-up period of 30 days. Comparison with nonmodified Ca-alg MS transplanted in the same conditions demonstrated a clear reduction in the severity of PFO for MS functionalized with ketoprofen. Quantification of collagen deposition on MIN6 cells containing MS transplanted under the kidney capsule revealed the significant effect of ketoprofen release to decrease fibrotic tissue formation. The impact was more pronounced when the drug was covalently conjugated by an ester linkage, allowing higher concentration of the anti-inflammatory compound to be delivered at the transplantation site. The functionality of microencapsulated MIN6 cells 30 days after transplantation was confirmed by detection of insulin positive cell content.

The production of hydrogel micropsheres (MS) presenting physical, mechanical, and biological properties which can be modulated by their chemical composition is required to enlarge the panel of biomaterials for cell transplantation therapies. Functionalization of sodium alginate (Na-alg) with cross-reactive poly(ethylene glycol) (PEG) derivatives presenting terminal thiol and carbon electrophile functionalities provided novel polymers which upon simple one-step protocol formed hydrogel MS assembled by combination of Ca-alg interactions and sulfur−carbon covalent bonds. Several parameters such as the grafting degree on the alg backbone and the viscosity of the polymer solutions can be adjusted to provide optimal formulation for the capsule formation technology. Compared with pure Ca-alg MS, dual ionic−covalent MS displayed improved mechanical resistance and shape recovery performance. Importantly, under conditions which resulted in complete liquefaction of Ca-alg MS, chemically cross-linked Alg-PEG MS maintained stable spherical morphology. In addition, these hydrogels allowed excellent viability and functionality of microencapsulated Huh7 cells. After transplantation in the peritoneal cavity of immune competent mice, the dual ionic−covalent MS remained free-floating and maintained their integrity over 30 days.

Neonatal and juvenile porcine islet cell clusters (ICC) present an unlimited source for islet xenotransplantation to treat type 1 diabetes patients. We isolated ICC from pancreata of 14 days old juvenile piglets and characterized their maturation by immunofluorescence and insulin secretion assays. Multipotent mesenchymal stromal cells derived from exocrine tissue of same pancreata (pMSC) were characterized for their differentiation potential and ability to sustain ICC insulin secretion in vitro and in vivo. Isolation of ICC resulted in 142 ± 50 × 103 IEQ per pancreas. Immunofluorescence staining revealed increasing presence of insulin‐positive beta cells between day 9 and 21 in culture and insulin content per 500IEC of ICC increased progressively over time from 1178.4 ± 450 µg/L to 4479.7 ± 1954.2 µg/L from day 7 to 14, P < .001. Highest glucose‐induced insulin secretion by ICC was obtained at day 7 of culture and reached a fold increase of 2.9 ± 0.4 compared to basal. Expansion of adherent cells from the pig exocrine tissue resulted in a homogenous CD90+, CD34−, and CD45− fibroblast‐like cell population and differentiation into adipocytes and chondrocytes demonstrated their multipotency. Insulin release from ICC was increased in the presence of pMSC and dependent on cell‐cell contact (glucose‐induced fold increase: ICC alone: 1.6 ± 0.2; ICC + pMSC + contact: 3.2 ± 0.5, P = .0057; ICC + pMSC no‐contact: 1.9 ± 0.3; theophylline stimulation: alone: 5.4 ± 0.7; pMSC + contact: 8.4 ± 0.9, P = .013; pMSC no‐contact: 5.2 ± 0.7). After transplantation of encapsulated ICC using Ca2+‐alginate (alg) microcapsules into streptozotocin‐induced diabetic and immunocompetent mice, transient normalization of glycemia was obtained up to day 7 post‐transplant, whereas ICC co‐encapsulated with pMSC did not improve glycemia and showed increased pericapsular fibrosis. We conclude that pMSC derived from juvenile porcine exocrine pancreas improves insulin secretion of ICC by direct cell‐cell contact. For transplantation purposes, the use of pMSC to support beta‐cell function will depend on the development of new anti‐fibrotic polymers and/or on genetically modified pigs with lower immunogenicity.