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.

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.

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.

Computational and Experimental Evaluation of the Immune Response of Neoantigens for Personalized Vaccine Design.

In the last few years, the importance of neoantigens in the development of personalized antitumor vaccines has increased remarkably. In order to study whether bioinformatic tools are effective in detecting neoantigens that generate an immune response, DNA samples from patients with cutaneous melanoma in different stages were obtained, resulting in a total of 6048 potential neoantigens gathered. Thereafter, the immunological responses generated by some of those neoantigens ex vivo were tested, using a vaccine designed by a new optimization approach and encapsulated in nanoparticles. Our bioinformatic analysis indicated that no differences were found between the number of neoantigens and that of non-mutated sequences detected as potential binders by IEDB tools. However, those tools were able to highlight neoantigens over non-mutated peptides in HLA-II recognition (p-value 0.03). However, neither HLA-I binding affinity (p-value 0.08) nor Class I immunogenicity values (p-value 0.96) indicated significant differences for the latter parameters. Subsequently, the new vaccine, using aggregative functions and combinatorial optimization, was designed. The six best neoantigens were selected and formulated into two nanoparticles, with which the immune response ex vivo was evaluated, demonstrating a specific activation of the immune response. This study reinforces the use of bioinformatic tools in vaccine development, as their usefulness is proven both in silico and ex vivo.

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.

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.

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.

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.

GSE4-loaded nanoparticles a potential therapy for lung fibrosis that enhances pneumocyte growth, reduces apoptosis and DNA damage.

Idiopathic pulmonary fibrosis is a lethal lung fibrotic disease, associated with aging with a mean survival of 2-5 years and no curative treatment. The GSE4 peptide is able to rescue cells from senescence, DNA and oxidative damage, inflammation, and induces telomerase activity. Here, we investigated the protective effect of GSE4 expression in vitro in rat alveolar epithelial cells (AECs), and in vivo in a bleomycin model of lung fibrosis. Bleomycin-injured rat AECs, expressing GSE4 or treated with GSE4-PLGA/PEI nanoparticles showed an increase of telomerase activity, decreased DNA damage, and decreased expression of IL6 and cleaved-caspase 3. In addition, these cells showed an inhibition in expression of fibrotic markers induced by TGF-ß such as collagen-I and III among others. Furthermore, treatment with GSE4-PLGA/PEI nanoparticles in a rat model of bleomycin-induced fibrosis, increased telomerase activity and decreased DNA damage in proSP-C cells. Both in preventive and therapeutic protocols GSE4-PLGA/PEI nanoparticles prevented and attenuated lung damage monitored by SPECT-CT and inhibited collagen deposition. Lungs of rats treated with bleomycin and GSE4-PLGA/PEI nanoparticles showed reduced expression of α-SMA and pro-inflammatory cytokines, increased number of pro-SPC-multicellular structures and increased DNA synthesis in proSP-C cells, indicating therapeutic efficacy of GSE4-nanoparticles in experimental lung fibrosis and a possible curative treatment for lung fibrotic patients.

Latest advances to enhance the therapeutic potential of mesenchymal stromal cells for the treatment of immune-mediated diseases.

Mesenchymal stromal cells (MSCs) present the capacity to secrete multiple immunomodulatory factors in response to their microenvironment. This property grants them a golden status among the novel alternatives to treat multiple diseases in which there is an unneeded or exaggerated immune response. However, important challenges still make difficult the clinical implementation of MSC-based therapies, being one of the most remarkable the lack of efficacy due to their transient immunomodulatory effects. To overcome this issue and boost the regulatory potential of MSCs, multiple strategies are currently being explored. Some of them consist of ex vivo pre-conditioning MSCs prior to their administration, including exposure to pro-inflammatory cytokines or to low oxygen concentrations. However, currently, alternative strategies that do not require such ex vivo manipulation are gaining special attention. Among them, the recreation of a three dimensional (3D) environment is remarkable. This approach has been reported to not only boost the immunomodulatory potential of MSCs but also increase their in vivo persistence and viability. The present work revises the therapeutic potential of MSCs, highlighting their immunomodulatory activity as a potential treatment for diseases caused by an exacerbated or unnecessary immune response. Moreover, it offers an updated vision of the most widely employed pre-conditioning strategies and 3D systems intended to enhance MSC-mediated immunomodulation, to conclude discussing the major challenges still to overcome in the field.

3D encapsulation and inflammatory licensing of mesenchymal stromal cells alter the expression of common reference genes used in real-time RT-qPCR.

Human mesenchymal stromal cells (hMSCs) hold great promise in the treatment of inflammatory and immune diseases, due to their immunomodulatory capacity. Their therapeutic activity is often assessed measuring levels of expression of immunomodulatory genes such as indoleamine 2,3-dioxygenase 1 (IDO1) and real-time RT-qPCR is most predominantly the method of choice due to its high sensitivity and relative simplicity. Currently, multiple strategies are explored to promote hMSC-mediated immunomodulation, overlooking the effects they pose in the expression of genes commonly used as internal calibrators in real-time RT-qPCR analyses. However, variations in their expression could introduce significant errors in the evaluation of the therapeutic potential of hMSCs. This work investigates, for the first time, how some of these strategies - 3D encapsulation, the mechanical properties of the 3D matrix and inflammatory licensing - influence the expression of common reference genes in hMSCs. Both 3D encapsulation and inflammatory licensing alter significantly the expression of ß-actin (ACTB) and Ubiquitin C (UBC), respectively. Using them as normalization factors leads to an erroneous assessment of IDO1 mRNA levels, therefore resulting in over or underestimation of the therapeutic potential of hMSCs. In contrast, the range of mechanical properties of the matrix encapsulating the cells did not significantly affect the expression of any of the reference genes studied. Moreover, we identify RPS13 and RPL30 as reference genes of choice under these particular experimental conditions. These results demonstrate the vital importance of validating the expression of reference genes to correctly assess the therapeutic potential of hMSCs by real-time RT-qPCR.

Multifunctional biomimetic hydrogel systems to boost the immunomodulatory potential of mesenchymal stromal cells.

Mesenchymal stromal cells (MSCs) hold great therapeutic potential, in part because of their immunomodulatory properties. However, these properties can be transient and depend on multiple factors. Here, we developed a multifunctional hydrogel system to synergistically enhance the immunomodulatory properties of MSCs, using a combination of sustained inflammatory licensing and three-dimensional (3D) encapsulation in hydrogels with tunable mechanical properties. The immunomodulatory extracellular matrix hydrogels (iECM) consist of an interpenetrating network of click functionalized-alginate and fibrillar collagen, in which interferon γ (IFN-γ) loaded heparin-coated beads are incorporated. The 3D microenvironment significantly enhanced the expression of a wide panel of pivotal immunomodulatory genes in bone marrow-derived primary human MSCs (hMSCs), compared to two-dimensional (2D) tissue culture. Moreover, the inclusion of IFN-γ loaded heparin-coated beads prolonged the expression of key regulatory genes upregulated upon licensing, including indoleamine 2,3-dioxygenase 1 (IDO1) and galectin-9 (GAL9). At a protein level, iECM hydrogels enhanced the secretion of the licensing responsive factor Gal-9 by hMSCs. Its presence in hydrogel conditioned media confirmed the correct release and diffusion of the factors secreted by hMSCs from the system. Furthermore, co-culture of iECM-encapsulated hMSCs and activated human T cells resulted in suppressed proliferation, demonstrating direct regulation on immune cells. These data highlight the potential of iECM hydrogels to enhance the immunomodulatory properties of hMSCs in cell therapies.

Mesenchymal stromal cell based therapies for the treatment of immune disorders: recent milestones and future challenges.

Introduction: Mesenchymal stromal cells (MSCs) present unique immunomodulatory properties that make them promising candidates for the treatment of inflammatory and immune disorders. MSC-mediated immunomodulation is a complex combination of mechanisms, in which the secretome plays a fundamental role. The plethora of bioactive molecules MSCs produce, such as indoleamine 2,3-dioxygenase (IDO) or prostaglandin E2 (PGE2), efficiently regulates innate and adaptive immunity. As a result, MSCs have been extensively employed in preclinical studies, leading to the conduction of multiple clinical trials.Areas covered: This review summarizes the effects of some of the key biomolecules in the MSC secretome and the advances in preclinical studies exploring the treatment of disorders including graft-versus-host disease (GvHD) or inflammatory bowel disease (IBD). Further, late-stage clinical trials and the first MSC-based therapies that recently obtained regulatory approval are discussed.Expert opinion: Extensive research supports the potential of MSC-based immunomodulatory therapies. However, to establish the bases for clinical translation, the future of study lies in the standardization of protocols and in the development of strategies that boost the therapeutic properties of MSCs.

The role of osmolarity adjusting agents in the regulation of encapsulated cell behavior to provide a safer and more predictable delivery of therapeutics.

Transplantation of cells within alginate microspheres has been extensively studied for sustained drug delivery. However, the lack of control over cell behavior represents a major concern regarding the efficacy and the safety of the therapy. Here, we demonstrated that when formulating the biosystem, an adequate selection of osmolarity adjusting agents significantly contributes to the regulation of cell responses. Our data showed that these agents interact in the capsule formation process, influencing the alginate crosslinking degree. Therefore, when selecting inert or electrolyte-based osmolarity adjusting agents to encapsulate D1 multipotent mesenchymal stromal cells (MSCs), alginate microcapsules with differing mechanical properties were obtained. Since mechanical forces acting on cells influence their behavior, contrasting cell responses were observed both, in vitro and in vivo. When employing mannitol as an inert osmolarity adjusting agent, microcapsules presented a more permissive matrix, allowing a tumoral-like behavior. This resulted in the formation of enormous cell-aggregates that presented necrotic cores and protruding peripheral cells, rendering the therapy unpredictable, dysfunctional, and unsafe. Conversely, the use of electrolyte osmolarity adjusting agents, including calcium or sodium, provided the capsule with a suitable crosslinking degree that established a tight control over cell proliferation and enabled an adequate therapeutic regimen in vivo. The crucial impact of these agents was confirmed when gene expression studies reported pivotal divergences not only in proliferative pathways, but also in genes involved in survival, migration, and differentiation. Altogether, our results prove osmolarity adjusting agents as an effective tool to regulate cell behavior and obtain safer and more predictable therapies.

LL37 loaded nanostructured lipid carriers (NLC): A new strategy for the topical treatment of chronic wounds.

The LL37 is a human antimicrobial peptide which not only has a broad spectrum of antimicrobial activity, but it has also been proved to modulate wound healing by participating in angiogenesis, epithelial cell migration and proliferation, and immune response. In this work, LL37 has been encapsulated in nanostructured lipid carriers (NLCs), produced by the melt-emulsification method, in order to improve its effectiveness. The characterisation of the NLC-LL37 showed a mean size of 270nm, a zeta potential of -26mV and an encapsulation efficiency of 96.4%. The cytotoxicity assay performed in Human Foreskin Fibroblasts demonstrated that the NLC-LL37 did not affect cell viability. Moreover, the in vitro bioactivityassay evidenced that the peptide remained active after the encapsulation, since the NLC-LL37 reversed the activation of the macrophages induced by LPS in the same way as the LL37 in solution. In addition, the in vitro antimicrobial assay revealed the NLC-LL37 activity against Escherichia coli. The effectiveness of the nanoparticles was assessed in a full thickness wound model indb/dbmice. The data demonstrated that NLC-LL37 significantly improved healing compared to the same concentration of the LL37 solution in terms of wound closure, reepithelisation grade and restoration of the inflammatory process. Overall, these findings suggest a promising potential of the NLC-LL37 formulation for chronic wound healing.

The topical administration of rhEGF-loaded nanostructured lipid carriers (rhEGF-NLC) improves healing in a porcine full-thickness excisional wound model.

The development of an effective treatment able to reduce the healing time of chronic wounds is a major health care need. In this regard, our research group has recently demonstrated the in vivo effectiveness of the topical administration of rhEGF-loaded lipid nanoparticles in healing-impaired db/db mice. Here we report the effectiveness of rhEGF-NLC (rhEGF loaded nanostructured lipid carriers) in a more relevant preclinical model of wound healing, the porcine full-thickness excisional wound model. The rhEGF-NLC showed a particle size of around 335nm, negative surface charge (-27mV) and a high encapsulation efficiency of 94%. rhEGF plasma levels were almost undetectable, suggesting that no systemic absorption occurred, which may minimise potential side effects and improve treatment safety. In vivo healing experiments carried out in large white pigs demonstrated that 20µg of rhEGF-NLC topically administered twice a week increased the wound closure and percentage of healed wounds by day 25, compared with the same number of intralesional administrations of 75µg free rhEGF and empty NLC. Moreover, rhEGF-NLC improved the wound healing quality expressed in terms of number of arranged microvasculature, fibroblast migration and proliferation, collagen deposition and evolution of the inflammatory response. Overall, these findings demonstrated that topically administered rhEGF-NLC may generate de novo intact skin after full thickness injury in a porcine model, thereby confirming their potential clinical application for the treatment of chronic wounds.

Increased antiparkinson efficacy of the combined administration of VEGF- and GDNF-loaded nanospheres in a partial lesion model of Parkinson's disease.

Current research efforts are focused on the application of growth factors, such as glial cell line-derived neurotrophic factor (GDNF) and vascular endothelial growth factor (VEGF), as neuroregenerative approaches that will prevent the neurodegenerative process in Parkinson's disease. Continuing a previous work published by our research group, and with the aim to overcome different limitations related to growth factor administration, VEGF and GDNF were encapsulated in poly(lactic-co-glycolic acid) nanospheres (NS). This strategy facilitates the combined administration of the VEGF and GDNF into the brain of 6-hydroxydopamine (6-OHDA) partially lesioned rats, resulting in a continuous and simultaneous drug release. The NS particle size was about 200 nm and the simultaneous addition of VEGF NS and GDNF NS resulted in significant protection of the PC-12 cell line against 6-OHDA in vitro. Once the poly(lactic-co-glycolic acid) NS were implanted into the striatum of 6-OHDA partially lesioned rats, the amphetamine rotation behavior test was carried out over 10 weeks, in order to check for in vivo efficacy. The results showed that VEGF NS and GDNF NS significantly decreased the number of amphetamine-induced rotations at the end of the study. In addition, tyrosine hydroxylase immunohistochemical analysis in the striatum and the external substantia nigra confirmed a significant enhancement of neurons in the VEGF NS and GDNF NS treatment group. The synergistic effect of VEGF NS and GDNF NS allows for a reduction of the dose by half, and may be a valuable neurogenerative/neuroreparative approach for treating Parkinson's disease.

rhEGF-loaded PLGA-Alginate microspheres enhance the healing of full-thickness excisional wounds in diabetised Wistar rats.

Diabetic foot ulcers (DFUs) represent a major clinical challenge in the ageing population. To address this problem, rhEGF-loaded Poly-Lactic-co-Glycolic-Acid (PLGA)-Alginate microspheres (MS) were prepared by a modified w/o/w-double-emulsion/solvent evaporation method. Different formulations were evaluated with the aim of optimising MSs properties by adding NaCl to the surfactant solution and/or the solvent removal phase and adding alginate as a second polymer. The characterisation of the developed MS showed that alginate incorporation increased the encapsulation efficiency (EE) and NaCl besides increasing the EE also became the particle surface smooth and regular. Once the MS were optimised, the target loading of rhEGF was increased to 1% (PLGA-Alginate MS), and particles were sterilised by gamma radiation to provide the correct dosage for in vivo studies. In vitro cell culture assays demonstrated that neither the microencapsulation nor the sterilisation process affected rhEGF bioactivity or rhEGF wound contraction. Finally, the MS were evaluated in vivo for treatment of the full-thickness wound model in diabetised Wistar rats. rhEGF MS treated animals showed a statistically significant decrease of the wound area by days 7 and 11, a complete re-epithelisation by day 11 and an earlier resolution of the inflammatory process. Overall, these findings demonstrate the promising potential of rhEGF-loaded MS (PLGA-Alginate MS) to promote faster and more effective wound healing, and suggest its possible application in DFU treatment.

VEGF-releasing biodegradable nanospheres administered by craniotomy: a novel therapeutic approach in the APP/Ps1 mouse model of Alzheimer's disease.

This study attempts to develop a novel nanotechnology-based strategy to deliver vascular endothelial growth factor (VEGF) to the brain, as a possible therapeutic approach for AD. For this purpose, VEGF was encapsulated in biodegradable poly(lactic-co-glycolic acid) (PLGA) nanospheres (VEGF-NS). The nanosphere particle size was about 200 nm, with a narrow size distribution, and the zeta potential around -30 mV. The encapsulation efficiency of VEGF was 44.06±5.61%, showing a biphasic release profile in vitro. The biological activity and neuroprotective effect of encapsulated VEGF were investigated in neuronal cell cultures, confirming the neuronal proliferative effect and the protection against Aß42 induced neurotoxicity. In vivo studies were carried out in amyloid precursor protein/presenilin-1 (APP/Ps1) mice administering VEGF-NS through minimally invasive craniotomy. The results obtained showed that VEGF-NS were able to improve behavioral deficits, decrease Aß deposits and promote angiogenesis, as well as reduce neuronal loss and cerebrovascular abnormalities. Furthermore, their ability to protect neuronal cultures against neuroinflammation induced by LPS provides new insight for future therapeutic approaches in other neurodegenerative disorders.

In vivo administration of VEGF- and GDNF-releasing biodegradable polymeric microspheres in a severe lesion model of Parkinson's disease.

In this work, the neuroregenerative potentials of microencapsulated VEGF, GDNF and their combination on a severely lesioned rat model were compared with the aim of developing a new strategy to treat advanced stages of Parkinson's disease. Both neurotrophic factors were separately encapsulated into polymeric microspheres (MSs) to obtain a continuous drug release over time. The regenerative effects of these growth factors were evaluated using a rotation behaviour test and quantified by the number of surviving TH+cells. The biological activities of encapsulated vascular endothelial growth factor (VEGF) and glial cell line-derived neurotrophic factor (GDNF) were investigated in HUVEC and PC12 cells, respectively. The treatment of 6-OHDA-lesioned rats with GDNF microspheres and with both VEGF and GDNF microspheres resulted in improved results in the rotation behaviour test. Both groups also showed higher levels of neuroregeneration/neuroreparation in the substantia nigra than the control group did. These results were confirmed by the pronounced TH+neuron recovery in the group receiving VEGF+GDNF-MS, demonstrating regenerative effects.