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.

Poly(2-isopropenyl-2-oxazoline) as a Versatile Functional Polymer for Biomedical Applications.

Functional polymers play an important role in various biomedical applications. From many choices, poly(2-isopropenyl-2-oxazoline) (PIPOx) represents a promising reactive polymer with great potential in various biomedical applications. PIPOx, with pendant reactive 2-oxazoline groups, can be readily prepared in a controllable manner via several controlled/living polymerization methods, such as living anionic polymerization, atom transfer radical polymerization (ATRP), reversible addition-fragmentation transfer (RAFT) or rare earth metal-mediated group transfer polymerization. The reactivity of pendant 2-oxazoline allows selective reactions with thiol and carboxylic group-containing compounds without the presence of any catalyst. Moreover, PIPOx has been demonstrated to be a non-cytotoxic polymer with immunomodulative properties. Post-polymerization functionalization of PIPOx has been used for the preparation of thermosensitive or cationic polymers, drug conjugates, hydrogels, brush-like materials, and polymer coatings available for drug and gene delivery, tissue engineering, blood-like materials, antimicrobial materials, and many others. This mini-review covers new achievements in PIPOx synthesis, reactivity, and use in biomedical applications.

Cytotoxicity and Bioimmunological Activity of Poly(2-Isopropenyl-2-oxazoline) Conjugates with Ibuprofen Using 3D Reconstructed Tissue Models.

Poly(2-isopropenyl-2-oxazoline) (PIPOx) represents a universal polymer platform with pendant 2-oxazoline groups, allowing the preparation of biomaterials for various biomedical applications. However, there is a lack of information on PIPOx concerning the effect of molar mass (Mn) on cytotoxicity and bioimmunological properties. Here, aqueous copper(0)-mediated reversible-deactivation radical polymerization (Cu0-RDPR) was used for the preparation of PIPOx with defined Mn and low dispersity. PIPOx of different Mn are used for the synthesis of conjugates with ibuprofen (5 mol %), the nonsteroidal anti-inflammatory drug. The release of ibuprofen at 37 °C and different pH values is monitored using high-performance liquid chromatography, where the rate of drug release increases with increasing pH and lower Mn. In vitro cytotoxicity and bioimmunological properties of PIPOx and drug conjugates are studied using 3D reconstructed tissue models of the human epidermis and intestinal epithelium. We demonstrate low cytotoxicity of PIPOx and conjugates with different Mn values on both 3D tissue models.

Enhancing the Functionality of Immunoisolated Human SC-ßeta Cell Clusters through Prior Resizing.

The transplantation of immunoisolated stem cell derived beta cell clusters (SC-ß) has the potential to restore physiological glycemic control in patients with type I diabetes. This strategy is attractive as it uses a renewable ß-cell source without the need for systemic immune suppression. SC-ß cells have been shown to reverse diabetes in immune compromised mice when transplanted as ≈300 µm diameter clusters into sites where they can become revascularized. However, immunoisolated SC-ß clusters are not directly revascularized and rely on slower diffusion of nutrients through a membrane. It is hypothesized that smaller SC-ß cell clusters (≈150 µm diameter), more similar to islets, will perform better within immunoisolation devices due to enhanced mass transport. To test this, SC-ß cells are resized into small clusters, encapsulated in alginate spheres, and coated with a biocompatible A10 polycation coating that resists fibrosis. After transplantation into diabetic immune competent C57BL/6 mice, the "resized" SC-ß cells plus the A10 biocompatible polycation coating induced long-term euglycemia in the mice (6 months). After retrieval, the resized A10 SC-ß cells exhibited the least amount of fibrosis and enhanced markers of ß-cell maturation. The utilization of small SC-ß cell clusters within immunoprotection devices may improve clinical translation in the future.

Synthesis and Exfoliation of Calcium Organophosphonates for Tailoring Rheological Properties of Sodium Alginate Solutions: A Path toward Polysaccharide-Based Bioink.

Layered nanoparticles with surface charge are explored as rheological modifiers for extrudable materials, utilizing their ability to induce electrostatic repulsion and create a house-of-cards structure. These nanoparticles provide mechanical support to the polymer matrix, resulting in increased viscosity and storage modulus. Moreover, their advantageous aspect ratio allows for shear-induced orientation and decreased viscosity during flow. In this work, we present a synthesis and liquid-based exfoliation procedure of phenylphosphonate-phosphate particles with enhanced ability to be intercalated by hydrophilic polymers. These layered nanoparticles are then tested as rheological modifiers of sodium alginate. The effective rheological modification is proved as the viscosity increases from 101 up to 103 Pa·s in steady state. Also, shear-thinning behavior is observed. The resulting nanocomposite hydrogels show potential as an extrudable bioink for 3D printing in tissue engineering and other biomedical applications, with good shape fidelity, nontoxicity, and satisfactory cell viability confirmed through encapsulation and printing of mouse fibroblasts.

Exchange Counterion in Polycationic Hydrogels: Tunability of Hydrophobicity, Water State, and Floating Capability for a Floating pH Device.

Smart gel materials are capable of controlling and switching swelling, water state, and wettability properties triggered by external stimuli. In this study, we fabricated a series of polyelectrolyte hydrogels bearing a 3-trimethylammoniumpropyl pendant to a methacrylamide-based backbone and examined the switchability with hydrophobic-like counteranions. The exchange between the initial chloride and camphor sulfate (CaS), dodecyl sulfate (DS), and perfluorooctanoate (PFO) counterions was investigated. The kinetics of the exchange showed that the fast exchange (within 4 h) of PFO allowed for a favorable coordination for ion pairing, resulting in a decrease in hydration. The reversibility of the exchange to the Cl- ion was only enabled for the CaS ion due to its bulkiness, while the PFO and DS hydrogels were unable to exchange, even by using tetrabutylammonium chloride, which is a structurally similar reagent, due to aggregation or the coagulates in the collapsed state of the linear tails of the counterions. The hydrogels exhibited a modulable water state and water swelling. Moreover, the hydrogels containing DS and PFO, as counterions, showed surface hydrophobic (contact angle 90°) and high hydrophobic (110°) behavior, respectively. The Raman spectrometry fluorescence with a pyrene probe indicated an increase in strong hydrogen-bonded water molecules, water confinement, and hydrophobic domains in the PFO hydrogel. Moreover, the PFO-modified hydrogel demonstrated a free-floating ability on the water surface, with a strong water repellency, showing that it has the potential to be applied in a floating pH detection device to distinguish between volatile and nonvolatile bases in a controlled manner.

Cell-Mediated Immunoreactivity of Poly(2-isopropenyl-2-oxazoline) as Promising Formulation for Immunomodulation.

Poly(2-isopropenyl-2-oxazoline) (PIPOx) represents a functional polymer with high potential for drug delivery, tissue engineering, and immunomodulation. The immunomodulatory efficiency of the PIPOx formulation has been studied in vitro following splenic cells and RAW 264.7 macrophages exposition. The cell-specific immunomodulative effect on production of Th1, Th2, Th17, and Treg signature cytokines has been demonstrated. The impact on the functionality of PIPOx-sensitized RAW 264.7 macrophages was assessed by cell phagocytosis. Time- and concentration-dependent cell internalization and intracellular organelles colocalization of fluorescently labeled PIPOx has been examined. The in vitro results demonstrated the PIPOx bioavailability and the capability of triggering immune cell responses resulting in the induced production of cell-specific signature interleukins, important prerequisite properties for future potential biomedical applications.

Effect of Dexamethasone on Thermoresponsive Behavior of Poly(2-Oxazoline) Diblock Copolymers.

Thermoresponsive polymers play an important role in designing drug delivery systems for biomedical applications. In this contribution, the effect of encapsulated hydrophobic drug dexamethasone on thermoresponsive behavior of diblock copolymers was studied. A small series of diblock copoly(2-oxazoline)s was prepared by combining thermoresponsive 2-n-propyl-2-oxazoline (nPrOx) and hydrophilic 2-methyl-2-oxazoline (MeOx) in two ratios and two polymer chain lengths. The addition of dexamethasone affected the thermoresponsive behavior of one of the copolymers, nPrOx20-MeOx180, in the aqueous medium by shifting the cloud point temperature to lower values. In addition, the formation of microparticles containing dexamethasone was observed during the heating of the samples. The morphology and number of microparticles were affected by the structure and concentration of copolymer, the drug concentration, and the temperature. The crystalline nature of formed microparticles was confirmed by polarized light microscopy, confocal Raman microscopy, and wide-angle X-ray scattering. The results demonstrate the importance of studying drug/polymer interactions for the future development of thermoresponsive drug carriers.

The Drug-Loaded Electrospun Poly(ε-Caprolactone) Mats for Therapeutic Application.

Diclofenac sodium salt (DSS)-loaded electrospun nanofiber mats on the base of poly(ε-caprolactone) (PCL) were investigated as biocompatible nanofibrous mats for medical applications with the ability to inhibit bacterial infections. The paper presents the characteristics of fibrous mats made by electrospinning and determines the effect of medicament on the fiber morphology, chemical, mechanical and thermal properties, as well as wettability. PCL and DSS-loaded PCL nanofibrous mats were characterized using scanning electron microscopy, transmission electron microscopy, attenuated total reflectance-Fourier transform infrared spectrometry, dynamic mechanical analysis, and contact angle measurements. Electron paramagnetic resonance measurements confirmed the lifetime of DSS before and after application of high voltage during the electrospinning process. In vitro biocompatibility was studied, and it was proved to be of good viability with ~92% of the diploid human cells culture line composed of lung fibroblast (MRC 5) after 48 h of incubation. Moreover, the significant activity of DSS-loaded nanofibers against cancer cells, Ca Ski and HeLa, was established as well. It was shown that 12.5% (m/V) is the minimal concentration for antibacterial activity when more than 99% of Escherichia coli (Gram-negative) and 99% of Staphylococcus aureus (Gram-positive) have been exterminated.

Diclofenac Embedded in Silk Fibroin Fibers as a Drug Delivery System.

Silk fibroin is a biocompatible, non-toxic, mechanically robust protein, and it is commonly used and studied as a material for biomedical applications. Silk fibroin also gained particular interest as a drug carrier vehicle, and numerous silk formats have been investigated for this purpose. Herein, we have prepared electrospun nanofibers from pure silk fibroin and blended silk fibroin/casein, followed by the incorporation of an anti-inflammatory drug, diclofenac. Casein serves as an excipient in pharmaceutical products and has a positive effect on the gradual release of drugs. The characteristics of the investigated composites were estimated by scanning electron microscope, transmission electron microscope, thermogravimetric analysis, and a lifetime of diclofenac by electron paramagnetic resonance analysis. The cumulative release in vitro of diclofenac sodium salt, together with the antiproliferative effect of diclofenac sodium salt-loaded silk nanofibers against the growth of two cancer cell lines, are presented and discussed.

Development of a microchannel emulsification process for pancreatic beta cell encapsulation.

In this study, we developed a high-throughput microchannel emulsification process to encapsulate pancreatic beta cells in monodisperse alginate beads. The process builds on a stirred emulsification and internal gelation method previously adapted to pancreatic cell encapsulation. Alginate bead production was achieved by flowing a 0.5-2.5% alginate solution with cells and CaCO3 across a 1-mm thick polytetrafluoroethylene plate with 700 × 200 µm rectangular straight-through channels. Alginate beads ranging from 1.5-3 mm in diameter were obtained at production rates exceeding 140 mL/hr per microchannel. Compared to the stirred emulsification process, the microchannel emulsification beads had a narrower size distribution and demonstrated enhanced compressive burst strength. Both microchannel and stirred emulsification beads exhibited homogeneous profiles of 0.7% alginate concentration using an initial alginate solution concentration of 1.5%. Encapsulated beta cell viability of 89 ± 2% based on live/dead staining was achieved by minimizing the bead residence time in the acidified organic phase fluid. Microchannel emulsification is a promising method for clinical-scale pancreatic beta cell encapsulation as well as other applications in the pharmaceutical, food, and cosmetic industries.

Sulfobetaines Meet Carboxybetaines: Modulation of Thermo- and Ion-Responsivity, Water Structure, Mechanical Properties, and Cell Adhesion.

A procedure for the preparation of copolymers bearing sulfobetaine and carboxybetaine methacrylic-based monomers by free-radical polymerization is described and discussed. A combination of monomers affects the upper critical solution temperature (UCST) in water and in the presence of a simple NaCl electrolyte while retaining the zwitterionic character. In addition, hydrogel samples were prepared and showed tunable water structure and mechanical properties. The total nonfreezable water content decreases with the amount of carboxybetaine segment in the hydrogel feed and the compression moduli were in a range of 0.7-1.6 MPa. Responses to external conditions such as temperature and ion strength were investigated and a potential application such as modulated thermal detection is proposed. The presence of the carboxylate group in the carboxybetaine segment enables a small fluorescence probe and peptide bearing RDG motif to be attached to polymer and hydrogel samples, respectively. The hydrogel samples functionalized with the RGD motif exhibit controlled cell adhesion. Such synthetic strategy based on combination of different zwitterionic segments offers a simple pathway for the development of zwitterionic materials with programmable properties.

Polyglobalide-Based Porous Networks Containing Poly(ethylene glycol) Structures Prepared by Photoinitiated Thiol-Ene Coupling.

The high interest in polymers from natural resources prompted us to investigate the use of enzymatically synthesized polyglobalide (PGL) in the preparation of polymer networks with potential applications as biomaterials for drug delivery devices. Polymer networks were obtained under mild conditions by photoinitiated thiol-ene coupling between PGL and a poly(ethylene glycol- co-thiomalate) (PEG-SH) copolymer obtained by polycondensation. The obtained polymer networks were thoroughly characterized by Raman spectroscopy, scanning electron microscopy, titration of thiol groups and elemental analysis. Our study took into consideration the synthesis parameters for the polymer networks, such as the total polymer concentration and the SH/C=C functionality molar ratio. Swelling in both THF and water was assessed, and the potential of the materials for drug delivery was determined. The scanning electron microscopy images showed that the prepared polymer networks may have different morphologies ranging from homogeneous polymer materials to macroporous structures. Additionally, the prepared materials were found to be suitable from a cytotoxicity point of view, enabling their application as biomaterials for drug delivery devices.

Structural changes in alginate-based microspheres exposed to in vivo environment as revealed by confocal Raman microscopy.

A next-generation cure for type 1 diabetes relies on immunoprotection of insulin-producing cells, which can be achieved by their encapsulation in microspheres made of non-covalently crosslinked hydrogels. Treatment success is directly related to the microsphere structure that is characterized by the localization of the polymers constituting the hydrogel material. However, due to the lack of a suitable analytical method, it is presently unknown how the microsphere structure changes in vivo, which complicates evaluation of different encapsulation approaches. Here, confocal Raman microscopy (CRM) imaging was tailored to serve as a powerful new tool for tracking structural changes in two major encapsulation designs, alginate-based microbeads and multi-component microcapsules. CRM analyses before implantation and after explantation from a mouse model revealed complete loss of the original heterogeneous structure in the alginate microbeads, making the intentionally high initial heterogeneity a questionable design choice. On the other hand, the structural heterogeneity was conserved in the microcapsules, which indicates that this design will better retain its immunoprotective properties in vivo. In another application, CRM was used for quantitative mapping of the alginate concentration throughout the microbead volume. Such data provide invaluable information about the microenvironment cells would encounter upon their encapsulation in alginate microbeads.

Bioactive polymeric formulations for wound healing.

Ricinoleic acid (RA) has potential to promote wound healing because of its analgesic and anti-inflammatory properties. This study investigates the synthesis and characterization of RA liposomes infused in a hydrogel for topical application. Lecithin liposomes containing RA were prepared and incorporated into a chitosan solution and were subsequently cross-linked with dialdehyde ß-cyclodextrin (Di-ß-CD). Chitosan/Di-ß-CD concentrations and reaction temperatures were varied to alter gelation time, water content, and mechanical properties of the hydrogel in an effort to obtain a wide range of RA release profiles. Hydrogel cross-linking was confirmed by spectroscopy, and liposome and carrier hydrogel morphology via microscopy. Chitosan, Di-ß-CD, and liposome concentrations within the formulation affected the extent of matrix swelling, mechanical strength, and pore and overall morphology. Higher cross-linking density of the hydrogel led to lower water uptake and slower release rate of RA. Optimized formulations resulted in a burst release of RA followed by a steady release pattern accounting for 80% of the encapsulated RA over a period of 48 hours. However, RA concentrations above 0.1 mg/mL were found to be cytotoxic to fibroblast cultures in vitro because of the oily nature of RA. These formulations promoted wound healing when used to treat full thickness skin wounds (2 cm2) in Wister male rats. The wound contraction rates were significantly higher compared to a commercially available topical cream after a time period of 21 days. Histopathological analysis of the RA-liposomal chitosan hydrogel group showed that the epidermis, dermis, and subcutaneous skin layers displayed an accelerated yet normal healing compared to control group.

Ex Vivo and In Vitro Studies on the Cytotoxicity and Immunomodulative Properties of Poly(2-isopropenyl-2-oxazoline) as a New Type of Biomedical Polymer.

Poly(2-alkenyl-2-oxazoline)s are promising functional polymers for a variety of biomedical applications, such as drug delivery systems, peptide conjugates, or gene delivery. In this study, poly(2-isopropenyl-2-oxazoline) (PIPOx) is prepared through free-radical polymerization initiated with azobisisobutyronitrile. Reactive 2-oxazoline units in the side chain support an addition reaction with different compounds containing a carboxylic group, which facilitates the preparation of polymers labeled with two different fluorescent dyes. The cytotoxicities of 2-oxazoline monomers, PIPOx, and fluorescently labeled PIPOx are evaluated in vitro using an 3-(4,5-Dimethyldiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and ex vivo using a cell proliferation assay with adenosine triphosphate bioluminescence. The cell uptake of labeled PIPOx is used to determine the colocalization of PIPOx with cell organelles that are part of the endocytic pathway. For the first time, it is shown that poly(2-isopropenyl-2-oxazoline) is a biocompatible material and is suitable for biomedical applications; further, its immunomodulative properties are evaluated.

In vitro study of partially hydrolyzed poly(2-ethyl-2-oxazolines) as materials for biomedical applications.

Polymers based on 2-oxazoline, such as poly(2-ethyl-2-oxazolines) (PETOx), are considered to be a type of 'pseudopeptide' with the ability to form novel biomaterials. The hydrolysis of PETOx was carried out to evaluate its use in biomedical applications. In the present work, PETOx samples with a range of molar masses were prepared by living cationic polymerization. Hydrolysis was carried out at time intervals ranging from 15 to 180 min to prepare copolymers with different amounts of ethylene imine units. (1)H NMR spectroscopy was used to identify the structure of the hydrolyzed polymers. The dependence of in vitro cell viability on the degree of hydrolysis was determined using three different model cell lines, namely, mouse embryonic 3T3 fibroblasts, pancreatic ßTC3 cells, and mouse lymphoid macrophages P388.D1. It was demonstrated that increasing the degree of hydrolysis decreased cell viability for all cell types. Fibroblast cells displayed the highest tolerance; additionally, the effect of polymer size showed no observable significance. Macrophage cells, immune system representatives, displayed the highest sensitivity to contact with hydrolyzed PETOx. The effect of polymer hydrolysis, polymer concentration and the incubation time on cell viability was experimentally observed. Confocal laser-scanning microscopy provided evidence of cellular uptake of pyrene-labeled (co)polymers.

Immunomodulatory efficiency of poly(2-oxazolines).

Poly(2-oxazolines) represent promising polymer materials for biomedical applications. The activation of mouse lymphoid macrophage line P388.D1 (clone 3124) by two selected representatives of poly(2-oxazolines), namely poly(2-ethyl-2-oxazoline) (PETOX100) and poly[2-(4-aminophenyl)-2-oxazoline-co-2-ethyl-2-oxazoline] (AEOX10), was assessed in vitro. The immunomodulatory efficacy of both polymers was evaluated via the induced release of pro-inflammatory cytokines (TNF-α, IL-1α and IL-6) and the acceleration of reactive free radicals. The present study revealed effective structure-immunomodulating associations of AEOX10 and PETOX100, which are desirable in biomedical and pharmaceutical applications of aliphatic and aromatic poly (2-oxazolines) in vivo.

Liposome-based DNA carriers may induce cellular stress response and change gene expression pattern in transfected cells.

BACKGROUND: During functional studies on the rat stress-inducible Hspa1b (hsp70.1) gene we noticed that some liposome-based DNA carriers, which are used for transfection, induce its promoter activity. This observation concerned commercial liposome formulations (LA), Lipofectin and Lipofectamine 2000. This work was aimed to understand better the mechanism of this phenomenon and its potential biological and practical consequences. RESULTS: We found that a reporter gene driven by Hspa1b promoter is activated both in the case of transient transfections and in the stably transfected cells treated with LA. Using several deletion clones containing different fragments of Hspa1b promoter, we found that the regulatory elements responsible for most efficient LA-driven inducibility were located between nucleotides -269 and +85, relative to the transcription start site. Further studies showed that the induction mechanism was independent of the classical HSE-HSF interaction that is responsible for gene activation during heat stress. Using DNA microarrays we also detected significant activation of the endogenous Hspa1b gene in cells treated with Lipofectamine 2000. Several other stress genes were also induced, along with numerous genes involved in cellular metabolism, cell cycle control and pro-apoptotic pathways. CONCLUSIONS: Our observations suggest that i) some cationic liposomes may not be suitable for functional studies on hsp promoters, ii) lipofection may cause unintended changes in global gene expression in the transfected cells.

In vitro bio-immunological and cytotoxicity studies of poly(2-oxazolines).

Poly(2-oxazolines) with varying alkyl chain lengths (e.g., methyl, ethyl, aryl) and molar masses have been tested for cell cytotoxicity in vitro. A standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used for the estimation of cell viability. Two monomers, 2-methyl-2-oxazoline and 2-ethyl-2-oxazoline, were found to provide polymers with non-cytotoxic properties. The dependence of cell viability on molar mass confirmed the expected trend; the viability increased with the higher molar mass of poly(2-ethyl-2-oxazoline) (PETOX), up to 15,000 g/mol. The results obtained for the polymers with aliphatic side chains were compared with the analogues that possessed an aromatic moiety. All results confirmed low cytotoxicity of the polymers prepared by cationic polymerization of 2-alkyl- and 2-aryl-2-oxazolines, which supports their utilization in biomedical applications. Fluorescence microscopy and steady-state fluorescence were used to observe pyrene-labeled polymer interactions with living cells. Polymer accumulated within the cells was found to be dependent on polymer concentration in media. The immunoefficiency of aromatic and aliphatic oxazoline polymers and copolymers was also studied. Phagocytic and metabolic activities of macrophages were used to assess the immunosuppressive effects of the selected copolymers for possible applications in drug delivery and immunobiology. Overall, the tested polymers demonstrated no significant influences on the cellular immunological parameters.