Laponite nanoclays for the sustained delivery of therapeutic proteins.

Protein therapeutics hold immense promise for treating a wide array of diseases. However, their efficacy is often compromised by rapid degradation and clearance. The synthetic smectite clay Laponite emerges as a promising candidate for their sustained delivery. Despite its unique properties allow to load and release proteins mitigating burst release and extending their effects, precise control over Laponite-protein interactions remains challenging since it depends on a complex interplay of factors whose implication is not fully understood yet. The aim of this review article is to shed light on this issue, providing a comprehensive discussion of the factors influencing protein loading and release, including the physicochemical properties of the nanoclay and proteins, pH, dispersion buffer, clay/protein concentration and Laponite degradation. Furthermore, we thoroughly revise the array of bioactive proteins that have been delivered from formulations containing the nanoclay, highlighting Laponite-polymer nanocomposite hydrogels, a promising avenue currently under extensive investigation.

Modulating the immune system towards a functional chronic wound healing: A biomaterials and Nanomedicine perspective.

Chronic non-healing wounds persist as a substantial burden for healthcare systems, influenced by factors such as aging, diabetes, and obesity. In contrast to the traditionally pro-regenerative emphasis of therapies, the recognition of the immune system integral role in wound healing has significantly grown, instigating an approach shift towards immunological processes. Thus, this review explores the wound healing process, highlighting the engagement of the immune system, and delving into the behaviors of innate and adaptive immune cells in chronic wound scenarios. Moreover, the article investigates biomaterial-based strategies for the modulation of the immune system, elucidating how the adjustment of their physicochemical properties or their synergistic combination with other agents such as drugs, proteins or mesenchymal stromal cells can effectively modulate the behaviors of different immune cells. Finally this review explores various strategies based on synthetic and biological nanostructures, including extracellular vesicles, to finely tune the immune system as natural immunomodulators or therapeutic nanocarriers with promising biophysical properties.

PLGA-PEI nanoparticle covered with poly(I:C) for personalised cancer immunotherapy.

Melanoma is the main cause of death among skin cancers and its incidence worldwide has been experiencing an appalling increase. However, traditional treatments lack effectiveness in advanced or metastatic patients. Immunotherapy, meanwhile, has been shown to be an effective treatment option, but the rate of cancers responding remains far from ideal. Here we have developed a personalized neoantigen peptide-based cancer vaccine by encapsulating patient derived melanoma neoantigens in polyethylenimine (PEI)-functionalised poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) and coating them with polyinosinic:polycytidylic acid (poly(I:C)). We found that PLGA NPs can be effectively modified to be coated with the immunoadjuvant poly(I:C), as well as to encapsulate neoantigens. In addition, we found that both dendritic cells (DCs) and lymphocytes were effectively stimulated. Moreover, the developed NP was found to have a better immune activation profile than NP without poly(I:C) or without antigen. Our results demonstrate that the developed vaccine has a high capacity to activate the immune system, efficiently maturing DCs to present the antigen of choice and promoting the activity of lymphocytes to exert their cytotoxic function. Therefore, the immune response generated is optimal and specific for the elimination of melanoma tumour cells.

Genetically engineered loaded extracellular vesicles for drug delivery.

The use of extracellular vesicles (EVs) for drug delivery is being widely explored by scientists from several research fields. To fully exploit their therapeutic potential, multiple methods for loading EVs have been developed. Although exogenous methods have been extensively utilized, in recent years the endogenous method has gained significant attention. This approach, based on parental cell genetic engineering, is suitable for loading large therapeutic biomolecules such as proteins and nucleic acids. We review the most commonly used EV loading methods and emphasize the inherent advantages of the endogenous method over the others. We also examine the most recent advances and applications of this innovative approach to inform on the diverse therapeutic opportunities that lie ahead in the field of EV-based therapies.

3D-printed Laponite/Alginate hydrogel-based suppositories for versatile drug loading and release.

Traditional approaches to solid rectal therapies have halted progress, leading to a continual decline in the use of conventional suppositories. Additive manufacturing techniques have been recently explored as a suitable innovative tool for suppository fabrication. However, little advancement has been made in composition materials for 3D-printed suppository (3DPS) manufacturing and still, conventional vehicles are often used for construct fabrication, hindering the growth in the field. As a novelty, this study unveils a ground-breaking Laponite-alginate hydrogel-based 3DPS. Interestingly, this study proposes a novel approach for loading drugs into the 3DPS employing for the first time the post-printing loading. Thus, a passive loading strategy of molecular models is developed, demonstrating the versatility and capacity to load molecules of different charges and molecular sizes within the matrix systems. This novel strategy allows adapting the load of a wide range of drugs into a single ink, which simplifies and speeds up the 3DPS technological development process for drugs with different physico-chemical properties. Additionally, in this research, a displacement strategy of the three-dimensional Laponite matrices is developed in order to enhance the drug release capacity through the 3DPS and their disintegration capacity, resulting in a significant improvement of the drug diffusion through the hydrogel matrix and a rapid disintegration of the 3DPS. Finally, our study demonstrates that the obtained 3DPS have a suitable in vivo behavior, being non-obstructive and allowing the normal motility of the rats intestine.

Soy protein/ß-chitin sponge-like scaffolds laden with human mesenchymal stromal cells from hair follicle or adipose tissue promote diabetic chronic wound healing.

Chronic wounds are a worldwide problem that affect >40 million people every year. The constant inflammatory status accompanied by prolonged bacterial infections reduce patient's quality of life and life expectancy drastically. An important cell type involved in the wound healing process are mesenchymal stromal cells (MSCs) due to their long-term demonstrated immunomodulatory and pro-regenerative capacity. Thus, in this work, we leveraged and compared the therapeutic properties of MSCs derived from both adipose tissue and hair follicle, which we combined with sponge-like scaffolds (SLS) made of valorized soy protein and ß-chitin. In this regard, the combination of these cells with biomaterials permitted us to obtain a multifunctional therapy that allowed high cell retention and growing rates while maintaining adequate cell-viability for several days. Furthermore, this combined therapy demonstrated to increase fibroblasts and keratinocytes migration, promote human umbilical vein endothelial cells angiogenesis and protect fibroblasts from highly proteolytic environments. Finally, this combined therapy demonstrated to be highly effective in reducing wound healing time in vivo with only one treatment change during all the experimental procedure, also promoting a more functional and native-like healed skin.

Agar/gelatin hydro-film containing EGF and Aloe vera for effective wound healing.

In the current study, we produced a hydro-film dressing for the treatment of chronic wounds. The hydro-film structure was composed of gelatin cross-linked with citric acid, agar and Aloe vera extract (AV); additionally epidermal growth factor (EGF) was loaded to promote wound healing. Due to the excellent hydrogel-forming ability of gelatin, the obtained hydro-film was able to swell 884 ± 36% of its dry weight, which could help controlling wound moisture. To improve gelatin mechanical properties, polymer chains were cross-linked with citric acid and agar, reaching an ultimate tensile strength that was in the highest range of human skin. In addition, it showed a slow degradation profile that resulted in a remaining weight of 28 ± 8% at day 28. Regarding, biological activity, the addition of AV and citric acid provided the ability to reduce human macrophage activation, which could help reverse the permanent inflammatory state of chronic wounds. Moreover, loaded EGF, together with the structural AV of the hydro-film, promoted human keratinocyte and fibroblast migration, respectively. Furthermore, the hydro-films presented excellent fibroblast adhesiveness, so they could be useful as provisional matrices for cell migration. Accordingly, these hydro-films showed suitable physicochemical characteristics and biological activity for chronic wound healing applications.

Targeting the central nervous system: From synthetic nanoparticles to extracellular vesicles-Focus on Alzheimer's and Parkinson's disease.

Neurodegenerative diseases (NDs) such as Alzheimer's disease (AD) and Parkinson's disease (PD) are an accelerating global health problem as life expectancy rises worldwide. Despite their significant burden in public health systems to date, the existing treatments only manage the symptoms without slowing down disease progression. Thus, the ongoing neurodegenerative process remains untreated. Moreover, the stronghold of the brain-the blood-brain barrier (BBB)-prevents drug penetrance and dwindles effective treatments. In the last years, nanotechnology-based drug delivery systems (DDS) have become a promising approach to target and treat these disorders related to the central nervous system (CNS). PLGA based nanoparticles (NPs) were the first employed DDS for effective drug delivery. However, the poor drug loading capacity and localized immunogenicity prompted the scientific community to move to another DDS such as lipid-based NPs. Despite the lipid NPs' safety and effectiveness, their off-target accumulation together with the denominated CARPA (complement activation-related pseudo allergy) reaction has limited their complete clinical translation. Recently, biological NPs naturally secreted by cells, termed as extracellular vesicles (EVs) have emerged as promising more complex biocompatible DDS. In addition, EVs act as dual players in NDs treatment, as a "cell free" therapy themselves, as well as new biological NPs with numerous characteristics that qualify them as promising carriers over synthetic DDS. The present review aims to display advantages, drawbacks, current limitations and future prospective of the previously cited synthetic and biological DDS to enter the brain and treat one of 21st century most challenging diseases, NDs. This article is categorized under: Therapeutic Approaches and Drug Discovery > Nanomedicine for Neurological Disease.

Extracellular vesicles released by hair follicle and adipose mesenchymal stromal cells induce comparable neuroprotective and anti-inflammatory effects in primary neuronal and microglial cultures.

BACKGROUND AIMS: Despite intensive research, to date, there is no effective treatment for neurodegenerative diseases. Among the different therapeutic approaches, recently, the use of extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) has gained attention. METHODS: In the present work, we focused on medium/large extracellular vesicles (m/lEVs) derived from hair follicle--derived (HF) MSCs, comparing their potential neuroprotective and anti-inflammatory effect against adipose tissue (AT)-MSC-derived m/lEVs. RESULTS: The obtained m/lEVs were similar in size with comparable expression of surface protein markers. The neuroprotective effect of both HF-m/lEVs and AT-m/lEVs was statistically significant in dopaminergic primary cell cultures, increasing cell viability after the incubation with 6-hidroxydopamine neurotoxin. Moreover, the administration of HF-m/lEVs and AT-m/lEVs counteracted the lipopolysaccharide-induced inflammation in primary microglial cell cultures, decreasing the levels of pro-inflammatory cytokines, tumor necrosis factor-α and interleukin-1ß. CONCLUSIONS: Taken together, HF-m/lEVs demonstrated comparable potential with that of AT-m/lEVs as multifaceted biopharmaceuticals for neurodegenerative disease treatment.

High resolution and fidelity 3D printing of Laponite and alginate ink hydrogels for tunable biomedical applications.

The formulation of hydrogels that meet the necessary flow characteristics for their extrusion-based 3D printing while providing good printability, resolution, accuracy and stability, requires long development processes. This work presents the technological development of a hydrogel-based ink of Laponite and alginate and evaluates its printing capacity. As a novelty, this article reports a standardizable protocol to quantitatively define the best printing parameters for the development of novel inks, providing new printability evaluation parameters such as the Printing Accuracy Escalation Index. As a result, this research develops a printable Laponite-Alginate hydrogel that presents printability characteristics. This ink is employed for the reproducible manufacture of 3D printed scaffolds with versatile and complex straight or curved printing patterns for a better adaptation to different final applications. Obtained constructs prove to be stable over time thanks to the optimization of a curing process. In addition, the study of the swelling and degradation behavior of the Laponite and alginate 3D printed scaffolds in different culture media allows the prediction of their behavior in future in vitro or in vivo developments. Finally, this study demonstrates the absence of cytotoxicity of the printed formulations, hence, setting the stage for their use in the field of biomedicine.

Tumour-derived extracellular vesicle based vaccines for melanoma treatment.

The interest of extracellular vesicles (EVs) in cancer immunotherapy is increasing every day. EVs are lipid bilayer vesicles released by most cells, which contain the molecular signature of their parent cell. Melanoma-derived EVs present antigens specific to this aggressive type of cancer, but they also exert immunomodulatory and pro-metastatic activity. Until now, most reviews focus on the immunoevasive characteristics of tumour-derived EVs, but do not help to overcome the issues related to them. In this review, we describe isolation methods of EVs from melanoma patients and most interesting markers to oversee their effect if they are used as antigen carriers. We also discuss the methods developed so far to overcome the lack of immunogenicity of melanoma-derived EVs, which includes EV modification or adjuvant co-administration. In summary, we conclude that EVs can be an interesting antigen source for immunotherapy development once EV obtaining is optimised and the understanding of the mechanisms behind their multiple effects is further understood.

Hybrid 3D Printed and Electrospun Multi-Scale Hierarchical Polycaprolactone Scaffolds to Induce Bone Differentiation.

Complex scaffolds composed of micro- and nano-structures are a key target in tissue engineering and the combination of sequential 3D printing and electrospinning enables the fabrication of these multi-scale structures. In this work, dual 3D printed and electrospun polycaprolactone (PCL) scaffolds with multiple mesh layers were successfully prepared. The scaffold macro- and micro-porosity were assessed by optical and scanning electron microscopy, showing that electrospun fibers formed aligned meshes within the pores of the scaffold. Consequently, the hydrophilicity of the scaffold increased with time, enhancing cell adhesion and growth. Additionally, compression tests in back and forth cycles demonstrated a good shape recovery behavior of the scaffolds. Biological results indicated that hybrid PCL scaffolds are biocompatible and enable a correct cell culture over time. Moreover, MC3T3-E1 preosteoblast culture on the scaffolds promoted the mineralization, increased the alkaline phosphatase (ALP) activity and upregulated the expression of early and late osteogenic markers, namely ALP and osteopontin (OPN), respectively. These results demonstrate that the sequential combination of 3D printing and electrospinning provides a facile method of incorporating fibers within a 3D printed scaffold, becoming a promising approach towards multi-scale hierarchical scaffolds capable of guiding the osteogenic differentiation.

Impresión 3D de medicamentos / 3D Printing of medicines

La industria farmacéutica está en continua búsqueda de nuevas tecnologías que permitan mejorar las formas de dosificación de las que se dispone, siendo uno de los objetivos el aumento de la adherencia a los tratamientos por parte de los pacientes. En este sentido, la impresión en 3 dimensiones (3DP) es una emergente técnica de fabricación aditiva que ha comenzado a abarcar muchos sectores industriales e influir directa e indirectamente en la calidad de vida de los individuos. Tanto es así, que la 3DP se postula como una de las técnicas que podría contribuir a que se produzca un gran cambio en el sector farmacéutico, permitiendo la personalización de los tratamientos de los pacientes, mejorando la biodisponibilidad de fármacos que presentan problemas de disolución o combinando toda la medicación de un paciente en una sola forma farmacéutica de toma diaria (polypill), entre otros. Esta nueva técnica de producción va a diferir enormemente de las clásicas formas de fabricación farmacéuticas y, en los próximos años puede suponer una transformación revolucionaria en la práctica farmacéutica. (AU)

The pharmaceutical industry is continually searching for new technologies to improve the characteristics of current medicines. One of the objectives is the increase of adherence to the treatments by patients. Simultaneously, 3-dimensional printing (3DP) is an emerging additive technique that is reaching many sectors of industry and influencing directly and indirectly the quality of life of patients. In this sense, 3DP postulates to be one of the technologies that contribute to the pharmaceutical development, allowing the personalized medicine in patients, improving the bioavailability of drugs with dissolution problems or combining all the medication of the patients in a single tablet (polypill), among others. This new technique will differ greatly from the traditional pharmaceutical manufacturing and in the coming years it may involve a revolutionary transformation in pharmaceutical practice. (AU)

Cell-based dressings: A journey through chronic wound management.

The field of regenerative medicine has undergone a paradigm shift in recent decades thanks to the emergence of novel therapies based on the use of living organisms. The development of cell-based strategies has become a trend for the treatment of different conditions and pathologies. In this sense, the need for more adequate, biomimetic and well-planned treatments for chronic wounds has found different and innovative strategies, based on the combination of cells with dressings, which seek to revolutionize the wound healing management. Therefore, the objective of this review is to analyze the current state and the latest advances in the research of cell-based dressings for chronic wounds, ranging from traditional and "second generation" bioengineered living skin equivalents to mesenchymal stem cell dressings; the latter include biopolymeric porous scaffolds, electrospun nanofiber meshes, hydrogels and 3D printed bio-printed dressings. Finally, this review updates the completed and ongoing clinical trials in this field and encourages researchers to rethink these new approaches, manufacturing processes and mechanisms of action, as well as their administration strategies and timings.

Clinical progress in MSC-based therapies for the management of severe COVID-19.

Considering the high impact that severe Coronavirus disease 2019 (COVID-19) cases still pose on public health and their complex pharmacological management, the search for new therapeutic alternatives is essential. Mesenchymal stromal cells (MSCs) could be promising candidates as they present important immunomodulatory and anti-inflammatory properties that can combat the acute severe respiratory distress syndrome (ARDS) and the cytokine storm occurring in COVID-19, two processes that are mainly driven by an immunological misbalance. In this review, we provide a comprehensive overview of the intricate inflammatory process derived from the immune dysregulation that occurs in COVID-19, discussing the potential that the cytokines and growth factors that constitute the MSC-derived secretome present to treat the disease. Moreover, we revise the latest clinical progress made in the field, discussing the most important findings of the clinical trials conducted to date, which follow 2 different approaches: MSC-based cell therapy or the administration of the secretome by itself, as a cell-free therapy.

Green hemostatic sponge-like scaffold composed of soy protein and chitin for the treatment of epistaxis.

Epistaxis is one of the most common otorhinolaryngology emergencies worldwide. Although there are currently several treatments available, they present several disadvantages. This, in addition to the increasing social need of being environmentally respectful, led us to investigate whether a sponge-like scaffold (SP-CH) produced from natural by-products of the food industry - soy protein and ß-chitin - can be employed as a nasal pack for the treatment of epistaxis. To evaluate the potential of our material as a nasal pack, it was compared with two of the most commonly used nasal packs in the clinic: a basic gauze and the gold standard Merocel®. Our SP-CH presented great physicochemical and mechanical properties, lost weight in aqueous medium, and could even partially degrade when incubated in blood. It was shown to be both biocompatible and hemocompatible in vitro, clearing up any doubt about its safety. It showed increased blood clotting capacity in vitro, as well as increased capacity to bind both red blood cells and platelets, compared to the standard gauze and Merocel®. Finally, a rat-tail amputation model revealed that our SP-CH could even reduce bleeding time in vivo. This work, carried out from a circular economy approach, demonstrates that a green strategy can be followed to manufacture nasal packs using valorized by-products of the food industry, with equal or even better hemostatic properties than the gold standard in the clinic.

Bioactive and degradable hydrogel based on human platelet-rich plasma fibrin matrix combined with oxidized alginate in a diabetic mice wound healing model.

In the present study we developed an injectable, bioactive and degradable hydrogel composed of alginate at 2.5% oxidation degree and calcium-activated platelet rich plasma (PRP) for wound healing applications (PRP-HG-2.5%). The alginate gives mechanical support to the hydrogel while the activated PRP provides growth factors that enhance wound healing and fibrin which creates an adequate microenvironment for cell migration and proliferation. The rheological and mechanical properties of the hydrogel were characterized. Further characterization revealed that PRP-HG-2.5% showed a faster hydrolitic degradation rate than unmodified alginate and a similar platelet derived growth factor (PDGF-BB) release profile. In vitro efficacy studies, carried out in human fibroblasts and keratinocytes, showed that PRP-HG-2.5% was not cytotoxic and that it was able to promote cell adhesion and proliferation. Thereafter, in an in vivo full thickness wound healing study conducted in diabetic mice, no differences were found among PRP-HG-2.5% and its counterpart without PRP, likely due to the xenogeneic origin of the PRP. This hypothesis was validated in vitro, since a cytotoxic effect was observed after human PRP application to mouse fibroblasts. Therefore, PRP-HG-2.5% might be a promising strategy for chronic woundstreatment, although its effectiveness should be evaluated in a more reliable preclinical model.

Extracellular vesicles from hair follicle-derived mesenchymal stromal cells: isolation, characterization and therapeutic potential for chronic wound healing.

BACKGROUND: Mesenchymal stromal cells (MSCs) and their extracellular vesicles (MSC-EVs) have demonstrated to elicit immunomodulatory and pro-regenerative properties that are beneficial for the treatment of chronic wounds. Thanks to different mediators, MSC-EVs have shown to play an important role in the proliferation, migration and cell survival of different skin cell populations. However, there is still a big bid to achieve the most effective, suitable and available source of MSC-EVs. METHODS: We isolated, characterized and compared medium-large EVs (m-lEVs) and small EVs (sEVs) obtained from hair follicle-derived MSCs (HF-MSCs) against the gold standard in regenerative medicine, EVs isolated from adipose tissue-derived MSCs (AT-MSCs). RESULTS: We demonstrated that HF-EVs, as well as AT-EVs, expressed typical MSC-EVs markers (CD9, CD44, CD63, CD81 and CD105) among other different functional markers. We showed that both cell types were able to increase human dermal fibroblasts (HDFs) proliferation and migration. Moreover, both MSC-EVs were able to increase angiogenesis in human umbilical vein endothelial cells (HUVECs) and protect HDFs exposed to a hyperglycemic environment from oxidative stress and cytotoxicity. CONCLUSIONS: Taken together, HF-EVs demonstrated to exhibit comparable potential to that of AT-EVs as promising candidates in the treatment of chronic wounds.

Human Hair Follicle-Derived Mesenchymal Stromal Cells from the Lower Dermal Sheath as a Competitive Alternative for Immunomodulation.

Mesenchymal stromal cells (MSCs) have unique immunomodulatory capacities. We investigated hair follicle-derived MSCs (HF-MSCs) from the dermal sheath, which are advantageous as an alternative source because of their relatively painless and minimally risky extraction procedure. These cells expressed neural markers upon isolation and maintained stemness for a minimum of 10 passages. Furthermore, HF-MSCs showed responsiveness to pro-inflammatory environments by expressing type-II major histocompatibility complex antigens (MHC)-II to a lesser extent than adipose tissue-derived MSCs (AT-MSCs). HF-MSCs effectively inhibited the proliferation of peripheral blood mononuclear cells equivalently to AT-MSCs. Additionally, HF-MSCs promoted the induction of CD4+CD25+FOXP3+ regulatory T cells to the same extent as AT-MSCs. Finally, HF-MSCs, more so than AT-MSCs, skewed M0 and M1 macrophages towards M2 phenotypes, with upregulation of typical M2 markers CD163 and CD206 and downregulation of M1 markers such as CD64, CD86, and MHC-II. Thus, we conclude that HF-MSCs are a promising source for immunomodulation.

Mesenchymal stromal cells encapsulated in licensing hydrogels exert delocalized systemic protection against ulcerative colitis via subcutaneous xenotransplantation.

The ability of mesenchymal stromal cells (MSCs) to release a plethora of immunomodulatory factors makes them valuable candidates to overcome inflammatory bowel diseases (IBD). However, this cell therapy approach is still limited by major issues derived from nude MSC-administration, including a rapid loss of their immunomodulatory phenotype that impairs factor secretion, low persistence and impossibility to retrieve the cells in case of adverse effects. Here, we designed a licensing hydrogel system to address these limitations and thus, obtain a continuous delivery of bioactive factors. IFNγ-loaded heparin-coated beads were included in injectable in situ crosslinking alginate hydrogels, providing a 3D microenvironment that ensured continuous inflammatory licensing, cell persistence and implant retrievability. Licensing-hydrogel encapsulated human MSCs (hMSCs) were subcutaneously xenotransplanted in an acute mouse model of ulcerative colitis. Results showed that encapsulated hMSCs exerted a delocalized systemic protection, not presenting significant differences to healthy mice in the disease activity index, colon weight/length ratio and histological score. At day 7, cells were easily retrieved and ex vivo assays showed fully viable hMSCs that retained an immunomodulatory phenotype, as they continued secreting factors including PGE2 and Gal-9. Our data demonstrate the capacity of licensing hydrogel-encapsulated hMSCs to limit the in vivo progression of IBD.