The Potential of the Fibronectin Inhibitor Arg-Gly-Asp-Ser in the Development of Therapies for Glioblastoma.

Glioblastoma (GBM) is the most lethal and common malignant primary brain tumor in adults. An important feature that supports GBM aggressiveness is the unique composition of its extracellular matrix (ECM). Particularly, fibronectin plays an important role in cancer cell adhesion, differentiation, proliferation, and chemoresistance. Thus, herein, a hydrogel with mechanical properties compatible with the brain and the ability to disrupt the dynamic and reciprocal interaction between fibronectin and tumor cells was produced. High-molecular-weight hyaluronic acid (HMW-HA) functionalized with the inhibitory fibronectin peptide Arg-Gly-Asp-Ser (RGDS) was used to produce the polymeric matrix. Liposomes encapsulating doxorubicin (DOX) were also included in the hydrogel to kill GBM cells. The resulting hydrogel containing liposomes with therapeutic DOX concentrations presented rheological properties like a healthy brain. In vitro assays demonstrated that unmodified HMW-HA hydrogels only caused GBM cell killing after DOX incorporation. Conversely, RGDS-functionalized hydrogels displayed per se cytotoxicity. As GBM cells produce several proteolytic enzymes capable of disrupting the peptide-HA bond, we selected MMP-2 to illustrate this phenomenon. Therefore, RGDS internalization can induce GBM cell apoptosis. Importantly, RGDS-functionalized hydrogel incorporating DOX efficiently damaged GBM cells without affecting astrocyte viability, proving its safety. Overall, the results demonstrate the potential of the RGDS-functionalized hydrogel to develop safe and effective GBM treatments.

Diaphragmatic hernia repair porcine model to compare the performance of biodegradable membranes against Gore-Tex®.

BACKGROUND: Patch repair of congenital diaphragmatic hernia (CDH) using Gore-Tex® is associated with infection, adhesions, hernia recurrence, long-term musculoskeletal sequels and poor tissue regeneration. To overcome these limitations, the performance of two novel biodegradable membranes was tested to repair CDH in a growing pig model. METHODS: Twelve male pigs were randomly assigned to 3 different groups of 4 animals each, determined by the type of patch used during thoracoscopic diaphragmatic hernia repair (Gore-Tex®, polycaprolactone electrospun membrane-PCLem, and decellularized human chorion membrane-dHCM). After 7 weeks, all animals were euthanized, followed by necropsy for diaphragmatic evaluation and histological analysis. RESULTS: Thoracoscopic defect creation and diaphragmatic repair were performed without any technical difficulty in all groups. However, hernia recurrence rate was 0% in Gore-Tex®, 50% in PCLem and 100% in dHCM groups. At euthanasia, Gore-Tex® patches appeared virtually unchanged and covered with a fibrotic capsule, while PCLem and dHCM patches were replaced by either floppy connective tissue or vascularized and floppy regenerated membranous tissue, respectively. CONCLUSION: Gore-Tex® was associated with a higher survival rate and lower recurrence. Nevertheless, the proposed biodegradable membranes were associated with better tissue integration when compared with Gore-Tex®.

Intracellular Trafficking of Size-Tuned Nanoparticles for Drug Delivery.

Polymeric nanoparticles (NPs) are widely used as drug delivery systems in nanomedicine. Despite their widespread application, a comprehensive understanding of their intracellular trafficking remains elusive. In the present study, we focused on exploring the impact of a 20 nm difference in size on NP performance, including drug delivery capabilities and intracellular trafficking. For that, poly(ethylene glycol) methyl ether-block-poly(lactide-co-glycolide) (PLGA-PEG) NPs with sizes of 50 and 70 nm were precisely tailored. To assess their prowess in encapsulating and releasing therapeutic agents, we have employed doxorubicin (Dox), a well-established anticancer drug widely utilized in clinical settings, as a model drug. Then, the beneficial effect of the developed nanoformulations was evaluated in breast cancer cells. Finally, we performed a semiquantitative analysis of both NPs' uptake and intracellular localization by immunostaining lysosomes, early endosomes, and recycling endosomes. The results show that the smaller NPs (50 nm) were able to reduce the metabolic activity of cancer cells more efficiently than NPs of 70 nm, in a time and concentration-dependent manner. These findings are corroborated by intracellular trafficking studies that reveal an earlier and higher uptake of NPs, with 50 nm compared to the 70 nm ones, by the breast cancer cells. Consequently, this study demonstrates that NP size, even in small increments, has an important impact on their therapeutic effect.

Synthesis and Anti-Inflammatory Evaluation of a Library of Chiral Derivatives of Xanthones Conjugated with Proteinogenic Amino Acids.

In recent decades, the relationship between drug chirality and biological activity has been assuming enormous importance in medicinal chemistry. Particularly, chiral derivatives of xanthones (CDXs) have interesting biological activities, including enantioselective anti-inflammatory activity. Herein, the synthesis of a library of CDXs is described, by coupling a carboxyxanthone (1) with both enantiomers of proteinogenic amino esters as chiral building blocks (2-31), following the chiral pool strategy. The coupling reactions were performed at room temperature with good yields (from 44 to 99.9%) and very high enantiomeric purity, with most of them presenting an enantiomeric ratio close to 100%. To afford the respective amino acid derivatives (32-61), the ester group of the CDXs was hydrolyzed in mild alkaline conditions. Consequently, in this work, sixty new derivatives of CDXs were synthetized. The cytocompatibility and anti-inflammatory activity in the presence of M1 macrophages were studied for forty-four of the new synthesized CDXs. A significant decrease in the levels of a proinflammatory cytokine targeted in the treatment of several inflammatory diseases, namely interleukin 6 (IL-6), was achieved in the presence of many CDXs. The amino ester of L-tyrosine (X1AELT) was the most effective in reducing IL-6 production (52.2 ± 13.2%) by LPS-stimulated macrophages. Moreover, it was ≈1.2 times better than the D-enantiomer. Indeed, enantioselectivity was observed for the majority of the tested compounds. Thus, their evaluation as promising anti-inflammatory drugs should be considered.

Metronidazole Delivery Nanosystem Able To Reduce the Pathogenicity of Bacteria in Colorectal Infection.

Metronidazole (MTZ) is a drug potentially used for the treatment of intestinal infections, namely, the ones caused by colorectal surgery. The traditional routes of administration decrease its local effectiveness and present off-site effects. To circumvent such limitations, herein a drug delivery system (DDS) based on MTZ-loaded nanoparticles (NPs) immobilized at the surface of electrospun fibrous meshes is proposed. MTZ at different concentrations (1, 2, 5, and 10 mg mL-1) was loaded into chitosan-sodium tripolyphosphate NPs. The MTZ loaded into NPs at the highest concentration showed a quick release in the first 12 h, followed by a gradual release. This DDS was not toxic to human colonic cells. When tested against different bacterial strains, a significant reduction of Escherichia coli and Staphylococcus aureus was observed, but no effect was found against Enterococcus faecalis. Therefore, this DDS offers high potential to locally prevent the occurrence of infections after colorectal anastomosis.

Microfluidic-driven mixing of high molecular weight polymeric complexes for precise nanoparticle downsizing.

Chitosan (CHIT) and hyaluronic acid (HA) are two polysaccharides (PSs) with high value in several biomedical applications. In this study, we present a microfluidic method to synthetize CHIT-HA NPs to overcome the disadvantages of the dropwise approach generally used for nanoprecipitation of polyelectrolyte complexes. The proposed microfluidic approach enables to generate monodisperse suspensions of NPs with ≈100 nm of size compared to the dropwise method that generated ≈2 times bigger NPs. Finally, we evaluated the potential of obtained NPs in an inflammatory scenario. The treatment with NPs led to the reduction of the main inflammatory molecules produced by macrophages (PGE2, IL-6, IL-8, MCAF and TNF-α) and fibroblasts (IL-1 α, PGE2, TNF-α) stimulated with lipopolysaccharide or conditioned medium, respectively. This study demonstrates that our approach can be used to enhance the synthesis of nanocarriers based on bioactive macromolecules.

Microfluidic mixing system for precise PLGA-PEG nanoparticles size control.

In this study, a microfluidic device was employed to produce polymeric nanoparticles (NPs) with well-controlled sizes. The influence of several parameters in the synthesis process, namely, polymer concentration, flow rate and flow rate ratio between the aqueous and organic solutions was investigated. To evaluate the NPs size effect, three diameters were selected (30, 50 and 70 nm). Their cytocompatibility was demonstrated on endothelial cells and macrophages. Additionally, their efficacy to act as drug carriers was assessed in an in vitro inflammatory scenario. NPs loaded and released diclofenac (DCF) in a size-dependent profile (smaller sizes presented lower DCF content and higher release rate). Moreover, 30 nm NPs were the most effective in reducing prostaglandin E2 concentration. Therefore, this study demonstrates that microfluidics can generate stable NPs with controlled sizes, high monodispersity and enhanced batch-to-batch reproducibility. Indeed, NPs size is a crucial parameter for drug encapsulation, release and overall biological efficacy.

On the Bioactivity of Echinacea purpurea Extracts to Modulate the Production of Inflammatory Mediators.

Inflammatory diseases are the focus of several clinical studies, due to limitations and serious side effects of available therapies. Plant-based drugs (e.g., salicylic acid, morphine) have become landmarks in the pharmaceutical field. Therefore, we investigated the immunomodulatory effects of flowers, leaves, and roots from Echinacea purpurea. Ethanolic (EE) and dichloromethanolic extracts (DE) were obtained using the Accelerated Solvent Extractor and aqueous extracts (AE) were prepared under stirring. Their chemical fingerprint was evaluated by liquid chromatography-high resolution mass spectrometry (LC-HRMS). The pro- and anti-inflammatory effects, as well as the reduction in intracellular reactive oxygen and nitrogen species (ROS/RNS), of the different extracts were evaluated using non-stimulated and lipopolysaccharide-stimulated macrophages. Interestingly, AE were able to stimulate macrophages to produce pro-inflammatory cytokines (tumor necrosis factor -TNF-α, interleukin -IL-1ß, and IL-6), and to generate ROS/RNS. Conversely, under an inflammatory scenario, all extracts reduced the amount of pro-inflammatory mediators. DE, alkylamides-enriched extracts, showed the strongest anti-inflammatory activity. Moreover, E. purpurea extracts demonstrated generally a more robust anti-inflammatory activity than clinically used anti-inflammatory drugs (dexamethasone, diclofenac, salicylic acid, and celecoxib). Therefore, E. purpurea extracts may be used to develop new effective therapeutic formulations for disorders in which the immune system is either overactive or impaired.

Cellular Uptake of Three Different Nanoparticles in an Inflammatory Arthritis Scenario versus Normal Conditions.

Nanoparticles (NPs) have wide potential applications in the biomedical field. To promote targeted and controlled delivery of encapsulated drugs, it is fundamentally important to understand the factors regulating NP uptake by different cells. Thus, the goal of the present study is to assess the internalization rates of different NPs under normal and proinflammatory states in primary human articular chondrocytes (hACs), human umbilical vein endothelial cells (EA), and human monocytes (THP-1). Here, we compared chitosan-hyaluronic acid (Ch-HA) polymeric NPs, methoxypolyethylene glycol amine-glutathione-palmitic acid (mPEG-GSHn-PA) micelles, and cholesterol/l-α-phosphatidylcholine/DSPE-PEG-Mal (Chol/EPC/DSPE-PEG-Mal) unilamellar liposomes (LUVs). Our results reveal the importance of surface charge and chemistry in determining the levels of NP internalization. Under normal conditions, the cellular uptake was ≈30% for Ch-HA NPs and ≈100% for mPEG-GSHn-PA micelles and Chol/EPC/DSPE-PEG-Mal LUVs. A proinflammatory cell state promoted a higher uptake of the Ch-HA NPs by EA cells (93% after 24 h). Since the therapeutic efficacy of the NP-loaded cargo is dependent on trafficking routes after cellular internalization, we tested their internalization pathways. Accordingly, caveolae-mediated endocytosis or energy-independent non-endocytic pathways, which circumvent lysosomal degradation, were accomplished in hACs and EA by LUVs and in M1 polarized macrophages by micelles. The present outcomes highlight the importance of considering cellular uptake and internalization pathways by the target cell when designing functional NPs for therapeutic applications.

A New Chalcone Derivative with Promising Antiproliferative and Anti-Invasion Activities in Glioblastoma Cells.

Glioblastoma (GBM) is the most common and most deadly primary malignant brain tumor. Current therapies are not effective, the average survival of GBM patients after diagnosis being limited to few months. Therefore, the discovery of new treatments for this highly aggressive brain cancer is urgently needed. Chalcones are synthetic and naturally occurring compounds that have been widely investigated as anticancer agents. In this work, three chalcone derivatives were tested regarding their inhibitory activity and selectivity towards GBM cell lines (human and mouse) and a non-cancerous mouse brain cell line. The chalcone 1 showed the most potent and selective cytotoxic effects in the GBM cell lines, being further investigated regarding its ability to reduce critical hallmark features of GBM and to induce apoptosis and cell cycle arrest. This derivative showed to successfully reduce the invasion and proliferation capacity of tumor cells, both key targets for cancer treatment. Moreover, to overcome potential systemic side effects and its poor water solubility, this compound was encapsulated into liposomes. Therapeutic concentrations were incorporated retaining the potent in vitro growth inhibitory effect of the selected compound. In conclusion, our results demonstrated that this new formulation can be a promising starting point for the discovery of new and more effective drug treatments for GBM.

Fibronectin Bound to a Fibrous Substrate Has Chondrogenic Induction Properties.

Articular cartilage is an avascular tissue characterized by a dense and specific extracellular matrix (ECM). Fibronectin (FN) is a key constituent of the pericellular ECM, assembled into a fibrillar matrix through a cell-mediated process, being implicated in chondrogenic events. In this study, we evaluate the chondrogenic potential of FN bound to the surface of an electrospun nanofibrous mesh (NFM). For that, an anti-FN antibody was immobilized at the surface of NFMs, rendering them capable of selectively binding endogenous FN (eFN) from blood plasma. The chondrogenic potential of bound eFN was further assessed by culturing human bone marrow-derived mesenchymal stem cells (hBM-MSCs) for 28 days, in a basal growth medium. The biological results indicate that NFMs functionalized with eFN were able to successfully induce the chondrogenesis of hBM-MSCs, as demonstrated by the high expression of SOX9, Aggrecan, and Collagen type II. Therefore, biofunctionalized nanofibrous substrates comprising eFN significantly enhance the efficacy of a cartilage tissue-engineering strategy.

Fucoidan Immobilized at the Surface of a Fibrous Mesh Presents Toxic Effects over Melanoma Cells, But Not over Noncancer Skin Cells.

The use of fucoidan, a marine-origin bioactive polymer, is herein proposed as a component of an innovative and effective strategy against melanoma, one of the most aggressive skin cancers. First, fucoidan antitumor activity, in its soluble form, was assessed presenting increased cytotoxicity over melanoma cells when compared to human dermal fibroblasts and keratinocytes. After this antitumor activity validation and trying to develop a more targeted and local strategy aiming to diminish the cytotoxic effects over noncancer cells, fucoidan was immobilized at the surface of an electrospun nanofiber mesh (NFM_Fu), envisioning the development of a therapeutic patch. The maximum immobilization concentration was 1.2 mg mL-1, determined by the Toluidine Blue Assay and confirmed by XPS. Furthermore, NFM_Fu is more hydrophilic than NFM, presenting a contact angle of 36°, lower than the 121° of the control condition. NFM_Fu was able to reduce human melanoma cell viability by 50% without affecting human dermal fibroblasts and keratinocytes. Taken together, these results set the basis for a valuable approach for melanoma treatment.

Tubular Fibrous Scaffolds Functionalized with Tropoelastin as a Small-Diameter Vascular Graft.

Cardiovascular disorders are a healthcare problem in today's society. The clinically available synthetic vascular grafts are thrombogenic and could induce intimal hyperplasia. Rapid endothelialization and matched mechanical properties are two major requirements to be considered when designing functional vascular grafts. Herein, an electrospun tubular fibrous (eTF) scaffold was biofunctionalized with tropoelastin at the luminal surface. The luminal surface functionalization was confirmed by an increase of the zeta potential and by the insertion of NH2 groups. Tropoelastin was immobilized via its -NH2 or -COOH groups at the activated or aminolysed eTF scaffolds, respectively, to study the effect of exposed functional groups on human endothelial cells (ECs) behavior. Tensile properties demonstrated that functionalized eTF scaffolds presented strength and stiffness within the range of those of native blood vessels. Tropoelastin immobilized on activated eTF scaffolds promoted higher metabolic activity and proliferation of ECs, whereas when immobilized on aminolysed eTF scaffolds, significantly higher protein synthesis was observed. These biofunctional eTF scaffolds are a promising small-diameter vascular graft that promote rapid endothelialization and have compatible mechanical properties.

Fibronectin-Functionalized Fibrous Meshes as a Substrate to Support Cultures of Thymic Epithelial Cells.

Thymic epithelial cells (TECs) are the main regulators of T lymphocyte development and selection, requiring a three-dimensional (3D) environment to properly perform these biological functions. The aim of this work was to develop a 3D culture substrate that allows the survival and proliferation of TECs. Thus, electrospun fibrous meshes (eFMs) were functionalized with fibronectin, one of the major extracellular matrix (ECM) proteins of the thymus. For that, highly porous eFMs were activated using oxygen plasma treatment followed by amine insertion, which allows the immobilization of fibronectin through EDC/NHS chemistry. The medullary TECs presented increased proliferation, viability, and protein synthesis when cultured on fibronectin-functionalized eFMs (FN-eFMs). These cells showed a spread morphology, with increased migration toward the inner layers of FN-eFMs and the production of thymic ECM proteins, such as collagen type IV and laminin. These results suggest that FN-eFMs are an effective substrate for supporting thymic cell cultures.

In Vivo Evaluation of the Biocompatibility of Biomaterial Device.

Biomaterials are widely used to produce devices for regenerative medicine. After its implantation, an interaction between the host immune system and the implanted biomaterial occurs, leading to biomaterial-specific cellular and tissue responses. These responses may include inflammatory, wound healing responses, immunological and foreign-body reactions, and even fibrous encapsulation of the implanted biomaterial device. In fact, the cellular and molecular events that regulate the success of the implant and tissue regeneration are played at the interface between the foreign body and the host inflammation, determined by innate and adaptive immune responses. This chapter focuses on host responses that must be taken into consideration in determining the biocompatibility of biomaterial devices when implanted in vivo of animal models.

Application of Gellan Gum-Based Scaffold for Regenerative Medicine.

Gellan gum (GG) is a linear microbial exopolysaccharide which is derived naturally by the fermentation process of Pseudomonas elodea. Application of GG in tissue engineering and regeneration medicine (TERM) is already over 10 years and has shown great potential. Although this biomaterial has many advantages such as biocompatibility, biodegradability, nontoxic in nature, and physical stability in the presence of cations, a variety of modification methods have been suggested due to some disadvantages such as mechanical properties, high gelation temperature, and lack of attachment sites. In this review, the application of GG-based scaffold for tissue engineering and approaches to improve GG properties are discussed. Furthermore, a recent trend and future perspective of GG-based scaffold are highlighted.

Bone Regeneration Using Duck's Feet-Derived Collagen Scaffold as an Alternative Collagen Source.

Collagen is an important component that makes 25-35% of our body proteins. Over the past decades, tissue engineers have been designing collagen-based biocompatible materials and studying their applications in different fields. Collagen obtained from cattle and pigs has been mainly used until now, but collagen derived from fish and other livestock has attracted more attention since the outbreak of mad cow disease, and they are also used as a raw material for cosmetics and foods. Due to the zoonotic infection using collagen derived from pigs and cattle, their application in developing biomaterials is limited; hence, the development of new animal-derived collagen is required. In addition, there is a religion (Islam, Hinduism, and Judaism) limited to export raw materials and products derived from cattle and pig. Hence, high-value collagen that is universally accessible in the world market is required. Therefore, in this review, we have dealt with the use of duck's feet-derived collagen (DC) as an emerging alternative to solve this problem and also presenting few original investigated bone regeneration results performed using DC.

The Role of Natural-Based Biomaterials in Advanced Therapies for Autoimmune Diseases.

Autoimmune diseases (ADs) constitute a heterogeneous group of more than 100 pathophysiological conditions in which an immune response against the self is observed. The incidence and prevalence of these chronic diseases are increasing with inherently high social and economic impacts. The currently available therapies generally focus on reducing the activity of the immune system and, therefore, can present severe side effects such as enhanced patient susceptibility to opportunistic infections. Advanced therapies emerged as promising treatments and with real curative potential for ADs. Additionally, the use of natural polymers to engineer gene therapies, cell therapies and/or tissue-engineered medicinal products presents specific advantages. Natural polymers present higher affinity with biological systems than synthetic polymers, and frequently have a chemical structure and motifs similar to those existing in the extracellular matrix of the tissues. They also have good biological performance, making them very strong candidates for advanced therapy medicinal products. This review discusses the therapeutic advances and provides demonstrative examples of the role of natural-based biomaterials for the development of advanced therapies for ADs.

Micro/Nano Scaffolds for Osteochondral Tissue Engineering.

To develop an osteochondral tissue regeneration strategy it is extremely important to take into account the multiscale organization of the natural extracellular matrix. The structure and gradients of organic and inorganic components present in the cartilage and bone tissues must be considered together. Another critical aspect is an efficient interface between both tissues. So far, most of the approaches were focused on the development of multilayer or stratified scaffolds which resemble the structural composition of bone and cartilage, not considering in detail a transitional interface layer. Typically, those scaffolds have been produced by the combined use of two or more processing techniques (microtechnologies and nanotechnologies) and materials (organic and inorganic). A significant number of works was focused on either cartilage or bone, but there is a growing interest in the development of the osteochondral interface and in tissue engineering models of composite constructs that can mimic the cartilage/bone tissues. The few works that give attention to the interface between cartilage and bone, as well as to the biochemical gradients observed at the osteochondral unit, are also herein described.

The Use of Electrospinning Technique on Osteochondral Tissue Engineering.

Electrospinning, an electrostatic fiber fabrication technique, has attracted significant interest in recent years due to its versatility and ability to produce highly tunable nanofibrous meshes. These nanofibrous meshes have been investigated as promising tissue engineering scaffolds since they mimic the scale and morphology of the native extracellular matrix. The sub-micron diameter of fibers produced by this process presents various advantages like the high surface area to volume ratio, tunable porosity, and the ability to manipulate the nanofiber composition in order to get desired properties and functionality. Electrospun fibers can be oriented or arranged randomly, giving control over both mechanical properties and the biological response to the fibrous scaffold. Moreover, bioactive molecules can be integrated with the electrospun nanofibrous scaffolds in order to improve the cellular response. This chapter presents an overview of the developments on electrospun polymer nanofibers including processing, structure, and their applications in the field of osteochondral tissue engineering.