Engineering alginate-based injectable hydrogels combined with bioactive polymers for targeted plasma-derived oxidative stress delivery in osteosarcoma therapy.

Reactive Oxygen and Nitrogen Species (RONS) in biological systems display hormetic effects, capable of either promoting cell regenerative effects or inducing cell death. Recently, hydrogels have emerged as a promising delivery platform for RONS generated from Cold Atmospheric Plasmas (CAP), known as Plasma-Treated Hydrogels (PTH). PTH have been proposed as an alternative therapy to conventional cancer treatments, offering reduced side effects through the controlled and localized delivery of plasma-derived RONS. In this work, we have developed alginate-based PTH with dual therapeutic action provided by plasma-derived RONS acting as selective anticancer agents for osteosarcoma treatment, and biomolecules (hyaluronic acid and gelatin) to promote stem cell-mediated bone regeneration. For this purpose, we designed a novel manufacturing process to maximize the load of plasma-derived RONS within the PTH. Then, we assessed the PTH bioactivity on osteosarcoma MG-63 cells, and human mesenchymal stem cells (hMSCs). The results showed that the PTH composed of 0.25 % alginate +1 % hyaluronic acid is the most promising formulation in osteosarcoma treatment, showing a dual-action bioactivity as a selective cytotoxic anticancer agent, and as promoter of the proliferation and osteogenic differentiation of hMSCs. These findings provide strong evidence of the significant potential of PTH in the oncological field.

Does non-thermal plasma modify biopolymers in solution? A chemical and mechanistic study for alginate.

In the last decades, non-thermal plasma has been extensively investigated as a relevant tool for various biomedical applications, ranging from tissue decontamination to regeneration and from skin treatment to tumor therapies. This high versatility is due to the different kinds and amount of reactive oxygen and nitrogen species that can be generated during a plasma treatment and put in contact with the biological target. Some recent studies report that solutions of biopolymers with the ability to generate hydrogels, when treated with plasma, can enhance the generation of reactive species and influence their stability, resulting thus in the ideal media for indirect treatments of biological targets. The direct effects of the plasma treatment on the structure of biopolymers in water solution, as well as the chemical mechanisms responsible for the enhanced generation of RONS, are not yet fully understood. In this study, we aim at filling this gap by investigating, on the one hand, the nature and extent of the modifications induced by plasma treatment in alginate solutions, and, on the other hand, at using this information to explain the mechanisms responsible for the enhanced generation of reactive species as a consequence of the treatment. The approach we use is twofold: (i) investigating the effects of plasma treatment on alginate solutions, by size exclusion chromatography, rheology and scanning electron microscopy and (ii) study of a molecular model (glucuronate) sharing its chemical structure, by chromatography coupled with mass spectrometry and by molecular dynamics simulations. Our results point out the active role of the biopolymer chemistry during direct plasma treatment. Short-lived reactive species, such as OH radicals and O atoms, can modify the polymer structure, affecting its functional groups and causing partial fragmentation. Some of these chemical modifications, like the generation of organic peroxide, are likely responsible for the secondary generation of long-lived reactive species such as hydrogen peroxide and nitrite ions. This is relevant in view of using biocompatible hydrogels as vehicles for storage and delivery reactive species for targeted therapies.

Current State of Cold Atmospheric Plasma and Cancer-Immunity Cycle: Therapeutic Relevance and Overcoming Clinical Limitations Using Hydrogels.

Cold atmospheric plasma (CAP) is a partially ionized gas that gains attention as a well-tolerated cancer treatment that can enhance anti-tumor immune responses, which are important for durable therapeutic effects. This review offers a comprehensive and critical summary on the current understanding of mechanisms in which CAP can assist anti-tumor immunity: induction of immunogenic cell death, oxidative post-translational modifications of the tumor and its microenvironment, epigenetic regulation of aberrant gene expression, and enhancement of immune cell functions. This should provide a rationale for the effective and meaningful clinical implementation of CAP. As discussed here, despite its potential, CAP faces different clinical limitations associated with the current CAP treatment modalities: direct exposure of cancerous cells to plasma, and indirect treatment through injection of plasma-treated liquids in the tumor. To this end, a novel modality is proposed: plasma-treated hydrogels (PTHs) that can not only help overcome some of the clinical limitations but also offer a convenient platform for combining CAP with existing drugs to improve therapeutic responses and contribute to the clinical translation of CAP. Finally, by integrating expertise in biomaterials and plasma medicine, practical considerations and prospective for the development of PTHs are offered.

Collagen-Sealed Polyester Vascular Prostheses Functionalized by Polycatecholamine Coatings.

Collagen-sealed polyester (PET) prostheses are commonly used in reconstructive vascular surgery due to their self-sealing properties. To prevent post-surgical infection, different modification methods have been tested but so far none have showed long-term satisfactory efficiency. For this reason, in the present study, a commercial collagen-sealed PET prosthesis was coated by a highly adhesive poly (L-DOPA) layer maintaining the sealing protein without losing the original properties and functionality. This modified (as proven by SEM, FTIR, XPS and contact angle) graft exhibited comparable wettability and elasticity as pristine commercial graft, as well as reduced hemolysis-inducing effect, lowered toxicity against human endothelial cells and reduced toxicity in Danio rerio model. Poly (L-DOPA)-coated grafts were shown to bind six times more aminoglycoside antibiotic (gentamicin) than pristine graft. Poly (L-DOPA)-coated antibiotic-bound prostheses exhibited an improved antibacterial activity (bacterial growth inhibition and anti-adhesive capacity) in comparison with pristine antibiotic-bound graft. Overall, poly (L-DOPA)-coatings deposited on PET vascular grafts can effectively functionalize collagen-sealed prostheses without the loss of protein sealing layer and allow for antibiotics incorporation to provide higher safety in biomedical applications.

Polycatecholamine and gentamicin as modifiers for antibacterial and blood-biocompatible polyester vascular prostheses.

Polyester (PET) prostheses are commonly used in reconstructive vascular surgery. The most serious complication after implantation is early or late infection of the graft. Therefore, there is high demand to protect prosthesis against bacterial adhesion and biofilm development. For this reason, in this work PET prostheses were first coated by highly adhesive polycatecholamine layer. The grafts were then coupled with gentamicin and studied in relation to morphological and structural properties, biological safety (contact with blood, reaction of vascular endothelial cells (HUVEC), Danio rerio fish), drug release and antibacterial activity. Among two tested catecholamine monomers, L-DOPA was found to be more effective precursor in this process than dopamine. For L-DOPA, assistance of Cu2+, Mg2+ and Na+ ions seems to increase the amount of further immobilized drug. Coated prostheses exhibited greater human endothelial cell proliferation increase and lower cytotoxic effect than uncoated. The modification reduced the hemolysis observed for pristine commercial graft and limited the rate of abnormalities in D. rerio larvae, confirming the safety of the proposed modification. The coating allowed to double the amount of immobilized antibiotic in comparison with uncoated graft which resulted in increased antibacterial activity and reduced bacterial adhesion against 4 bacterial strains prevalent in biomaterials infections. Overall, poly(L-DOPA)-coatings deposited on PET vascular grafts can effectively functionalize these prostheses for higher safety in biomedical applications.

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.

Characterization of encapsulated porcine cardiosphere-derived cells embedded in 3D alginate matrices.

Myocardial infarction is caused by an interruption of coronary blood flow, leading to one of the main death causes worldwide. Current therapeutic approaches are palliative and not able to solve the loss of cardiac tissue. Cardiosphere derived cells (CDCs) reduce scarring, and increase viable myocardium, with safety and adequate biodistribution, but show a low rate engraftment and survival after implantation. In order to solve the low retention, we propose the encapsulation of CDCs within three-dimensional alginate-poly-L-lysine-alginate matrix as therapy for cardiac regeneration. In this work, we demonstrate the encapsulation of CDCs in alginate matrix, with no decrease in viability over a month, and showing the preservation of CDCs phenotype, differentiation potential, gene expression profile and growth factor release after encapsulation, moving a step forward to clinical translation of CDCs therapy in regeneration in heart failure.

Plasma-Conditioned Liquids as Anticancer Therapies In Vivo: Current State and Future Directions.

Plasma-conditioned liquids (PCL) are gaining increasing attention in the medical field, especially in oncology, and translation to the clinics is advancing on a good path. This emerging technology involving cold plasmas has great potential as a therapeutic approach in cancer diseases, as PCL have been shown to selectively kill cancer cells by triggering apoptotic mechanisms without damaging healthy cells. In this context, PCL can be injected near the tumor or intratumorally, thereby allowing the treatment of malignant tumors located in internal organs that are not accessible for direct cold atmospheric plasma (CAP) treatment. Therefore, PCL constitutes a very interesting and minimally invasive alternative to direct CAP treatment in cancer therapy, avoiding surgeries and allowing multiple local administrations. As the field advances, it is progressively moving to the evaluation of the therapeutic effects of PCL in in vivo scenarios. Exciting developments are pushing forward the clinical translation of this novel therapy. However, there is still room for research, as the quantification and identification of reactive oxygen and nitrogen species (RONS) in in vivo conditions is not yet clarified, dosage regimens are highly variable among studies, and other more relevant in vivo models could be used. In this context, this work aims to present a critical review of the state of the field of PCL as anticancer agents applied in in vivo studies.

Force Spectroscopy Imaging and Constriction Assays Reveal the Effects of Graphene Oxide on the Mechanical Properties of Alginate Microcapsules.

Microencapsulation of cells in hydrogel-based porous matrices is an approach that has demonstrated great success in regenerative cell therapy. These microcapsules work by concealing the exogenous cells and materials in a robust biomaterial that prevents their recognition by the immune system. A vast number of formulations and additives are continuously being tested to optimize cell viability and mechanical properties of the hydrogel. Determining the effects of new microcapsule additives is a lengthy process that usually requires extensive in vitro and in vivo testing. In this paper, we developed a workflow using nanoindentation (i.e., indentation with a nanoprobe in an atomic force microscope) and a custom-built microfluidic constriction device to characterize the effect of graphene oxide (GO) on three microcapsule formulations. With our workflow, we determined that GO modifies the microcapsule stiffness and surface properties in a formulation-dependent manner. Our results also suggest, for the first time, that GO alters the conformation of the microcapsule hydrogel and its interaction with subsequent coatings. Overall, our workflow can infer the effects of new additives on microcapsule surfaces. Thus, our workflow can contribute to diminishing the time required for the validation of new microcapsule formulations and accelerate their clinical translation.

BSA- and Elastin-Coated GO, but Not Collagen-Coated GO, Enhance the Biological Performance of Alginate Hydrogels.

The use of embedded cells within alginate matrices is a developing technique with great clinical applications in cell-based therapies. However, one feature that needs additional investigation is the improvement of alginate-cells viability, which could be achieved by integrating other materials with alginate to improve its surface properties. In recent years, the field of nanotechnology has shown the many properties of a huge number of materials. Graphene oxide (GO), for instance, seems to be a good choice for improving alginate cell viability and functionality. We previously observed that GO, coated with fetal bovine serum (FBS) within alginate hydrogels, improves the viability of embedded myoblasts. In the current research, we aim to study several proteins, specifically bovine serum albumin (BSA), type I collagen and elastin, to discern their impact on the previously observed improvement on embedded myoblasts within alginate hydrogels containing GO coated with FBS. Thus, we describe the mechanisms of the formation of BSA, collagen and elastin protein layers on the GO surface, showing a high adsorption by BSA and elastin, and a decreasing GO impedance and capacitance. Moreover, we described a better cell viability and protein release from embedded cells within hydrogels containing protein-coated GO. We conclude that these hybrid hydrogels could provide a step forward in regenerative medicine.

Immobilization of INS1E Insulin-Producing Cells Within Injectable Alginate Hydrogels.

Alginate has demonstrated high applicability as a matrix-forming biomaterial for cell immobilization due to its ability to make hydrogels combined with cells in a rapid and non-toxic manner in physiological conditions, while showing excellent biocompatibility, preserving immobilized cell viability and function. Moreover, depending on its application, alginate hydrogel physicochemical properties such as porosity, stiffness, gelation time, and injectability can be tuned. This technology has been applied to several cell types that are able to produce therapeutic factors. In particular, alginate has been the most commonly used material in pancreatic islet entrapment for type 1 diabetes mellitus treatment. This chapter compiles information regarding the alginate handling, and we describe the most important steps and recommendations to immobilize insulin-producing cells within a tuned injectable alginate hydrogel using a syringe-based mixing system, detailing how to assess the viability and the biological functionality of the embedded cells.

Review of Advanced Hydrogel-Based Cell Encapsulation Systems for Insulin Delivery in Type 1 Diabetes Mellitus.

: Type 1 Diabetes Mellitus (T1DM) is characterized by the autoimmune destruction of ß-cells in the pancreatic islets. In this regard, islet transplantation aims for the replacement of the damaged ß-cells through minimally invasive surgical procedures, thereby being the most suitable strategy to cure T1DM. Unfortunately, this procedure still has limitations for its widespread clinical application, including the need for long-term immunosuppression, the lack of pancreas donors and the loss of a large percentage of islets after transplantation. To overcome the aforementioned issues, islets can be encapsulated within hydrogel-like biomaterials to diminish the loss of islets, to protect the islets resulting in a reduction or elimination of immunosuppression and to enable the use of other insulin-producing cell sources. This review aims to provide an update on the different hydrogel-based encapsulation strategies of insulin-producing cells, highlighting the advantages and drawbacks for a successful clinical application.

3D printed polyamide macroencapsulation devices combined with alginate hydrogels for insulin-producing cell-based therapies.

Cell macroencapsulation has shown a great potential overcoming the low survival of the transplanted pancreatic islets in the Type 1 Diabetes Mellitus (T1DM) treatment, as it avoids the need for lifelong immunosuppression. It is still not completely known how these devices interact with the host immune system when implanted. However, their surface properties seem to be crucial factors for a successful implant. In this context, the hydrophilicity and porosity of the surface of the macrocapsules are two of the most important properties that can affect the functionality of the graft; hydrophilicity defines the interactions with the host's immune cells, while the porosity determines the biosafety of the device while conditioning the oxygen, nutrients and insulin diffusion. Here, we report a novel ß-cell macroencapsulation system that combines an injectable alginate hydrogel with an external 3D-printed implantable device. This external macrocapsule protects the inner hydrogel containing cells, while allowing the precise location of the implant in the body. In addition, it would allow the easy extraction of the grafted cells in the case the implant fails or the renewal of the therapeutic cells is required. This study evaluates the biological effect of the macroencapsulation devices' surface properties (hydrophilicity and porosity). We studied two different pore sizes and hydrophilicities in four different devices containing rat INS1E ß-cells embedded in alginate hydrogels. All the devices showed great biocompatibility, although the hydrophilic ones exhibited higher fibroblast adhesion, which could potentially enhance the fibrotic response when implanted. Importantly, INS1E cells did not escape from the devices, denoting high biosafety. Cells grown within all devices and maintained their insulin secretory function. However, the hydrophobic device with a smaller pore size showed better cell viability values and, therefore, it might be the best candidate for the development of a safe ß-cell replacement therapy in T1DM.

Hyaluronic Acid Promotes Differentiation of Mesenchymal Stem Cells from Different Sources toward Pancreatic Progenitors within Three-Dimensional Alginate Matrixes.

Islet transplantation has shown to be a successful alternative in type 1 diabetes treatment, but donor scarcity precludes its worldwide clinical translation. Stem cells are an unlimited source that could circumvent the lack of donors if complete differentiation into insulin-producing cells (IPCs) could be accomplished. We have performed the differentiation of mesenchymal stem cells (MSCs) from different sources into IPCs within three-dimensional (3D) alginate matrixes. We quantified an increased insulin release at the final stage of differentiation compared to undifferentiated MSCs, which is more pronounced in IPCs differentiated from pancreatic-derived MSCs tissues. Moreover, the addition of hyaluronic acid (HA) in alginate microcapsules enhanced, even more, the insulin release from the final IPCs, independent of the MSC source. We can conclude that MSCs can be differentiated into IPCs within alginate microcapsules, enhancing insulin release when HA is present in the 3D alginate matrixes.

Hyaluronic acid enhances cell survival of encapsulated insulin-producing cells in alginate-based microcapsules.

Pancreatic islet transplantation has proved to be a promising therapy for T1DM, in spite of the chronic immunosuppression required. Although cell microencapsulation technology represents an alternative to circumvent the immune system rejection of transplanted pancreatic islets, the environment provided by classical alginate microcapsules does not mimic the natural ECM, affecting the islet survival. Since hyaluronic acid, one of the major components of pancreatic ECM, is involved in cell adhesion and viability, we assessed the beneficial outcomes on encapsulated insulin-producing cells by the HA inclusion in alginate matrices. In this manuscript we describe how alginate-HA hybrid microcapsules enhance the viability of encapsulated cells, reducing early apoptosis percentage and decreasing membrane damage. A stable insulin production was maintained in encapsulated cells, not altering the response to a glucose stimulus. Therefore, we can conclude that the inclusion of HA within alginate microcapsules is beneficial for encapsulated insulin-producing cells, representing a step forward in the clinical translation of microcapsules technology for the treatment of T1DM.

3D Printed porous polyamide macrocapsule combined with alginate microcapsules for safer cell-based therapies.

Cell microencapsulation is an attractive strategy for cell-based therapies that allows the implantation of genetically engineered cells and the continuous delivery of de novo produced therapeutic products. However, the establishment of a way to retrieve the implanted encapsulated cells in case the treatment needs to be halted or when cells need to be renewed is still a big challenge. The combination of micro and macroencapsulation approaches could provide the requirements to achieve a proper immunoisolation, while maintaining the cells localized into the body. We present the development and characterization of a porous implantable macrocapsule device for the loading of microencapsulated cells. The device was fabricated in polyamide by selective laser sintering (SLS), with controlled porosity defined by the design and the sintering conditions. Two types of microencapsulated cells were tested in order to evaluate the suitability of this device; erythropoietin (EPO) producing C2C12 myoblasts and Vascular Endothelial Growth Factor (VEGF) producing BHK fibroblasts. Results showed that, even if the metabolic activity of these cells decreased over time, the levels of therapeutic protein that were produced and, importantly, released to the media were stable.

Current advanced therapy cell-based medicinal products for type-1-diabetes treatment.

In the XXI century diabetes mellitus has become one of the main threats to human health with higher incidence in regions such as Europe and North America. Type 1 diabetes mellitus (T1DM) occurs as a consequence of the immune-mediated destruction of insulin producing ß-cells located in the endocrine part of the pancreas, the islets of Langerhans. The administration of exogenous insulin through daily injections is the most prominent treatment for T1DM but its administration is frequently associated to failure in glucose metabolism control, finally leading to hyperglycemia episodes. Other approaches have been developed in the past decades, such as whole pancreas and islet allotransplantation, but they are restricted to patients who exhibit frequent episodes of hypoglycemia or renal failure because the lack of donors and islet survival. Moreover, patients transplanted with either whole pancreas or islets require of immune suppression to avoid the rejection of the transplant. Currently, advanced therapy medicinal products (ATMP), such as implantable devices, have been developed in order to reduce immune rejection response while increasing cell survival. To overcome these issues, ATMPs must promote vascularization, guaranteeing the nutritional contribution, while providing O2 until vasculature can surround the device. Moreover, it should help in the immune-protection to avoid acute and chronic rejection. The transplanted cells or islets should be embedded within biomaterials with tunable properties like injectability, stiffness and porosity mimicking natural ECM structural characteristics. And finally, an infinitive cell source that solves the donor scarcity should be found such as insulin producing cells derived from mesenchymal stem cells (MSCs), embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Several companies have registered their ATMPs and future studies envision new prototypes. In this review, we will discuss the mechanisms and etiology of diabetes, comparing the clinical trials in the last decades in order to define the main characteristics for future ATMPs.

Tunable injectable alginate-based hydrogel for cell therapy in Type 1 Diabetes Mellitus.

Islet transplantation has the potential of reestablishing naturally-regulated insulin production in Type 1 diabetic patients. Nevertheless, this procedure is limited due to the low islet survival after transplantation and the lifelong immunosuppression to avoid rejection. Islet embedding within a biocompatible matrix provides mechanical protection and a physical barrier against the immune system thus, increasing islet survival. Alginate is the preferred biomaterial used for embedding insulin-producing cells because of its biocompatibility, low toxicity and ease of gelation. However, alginate gelation is poorly controlled, affecting its physicochemical properties as an injectable biomaterial. Including different concentrations of the phosphate salt Na2HPO4 in alginate hydrogels, we can modulate their gelation time, tuning their physicochemical properties like stiffness and porosity while maintaining an appropriate injectability. Moreover, these hydrogels showed good biocompatibility when embedding a rat insulinoma cell line, especially at low Na2HPO4 concentrations, indicating that these hydrogels have potential as injectable biomaterials for Type 1 Diabetes Mellitus treatment.

Alginate Microcapsules Incorporating Hyaluronic Acid Recreate Closer in Vivo Environment for Mesenchymal Stem Cells.

The potential clinical application of alginate cell microencapsulation has advanced enormously during the past decade. However, the 3D environment created by alginate beads does not mimic the natural extracellular matrix surrounding cells in vivo, responsible of cell survival and functionality. As one of the most frequent macromolecules present in the extracellular matrix is hyaluronic acid, we have formed hybrid beads with alginate and hyaluronic acid recreating a closer in vivo cell environment. Our results show that 1% alginate-0.25% hyaluronic acid microcapsules retain 1.5% alginate physicochemical properties. Moreover, mesenchymal stem cells encapsulated in these hybrid beads show enhanced viability therapeutic protein release and mesenchymal stem cells' potential to differentiate into chondrogenic lineage. Although future studies with additional proteins need to be done in order to approach even more the extracellular matrix features, we have shown that hyaluronic acid protects alginate encapsulated mesenchymal stem cells by providing a niche-like environment and remaining them competent as a sustainable drug delivery system.