Transcriptomic and proteomic profiling of bi-partite and tri-partite murine iPSC-derived neurospheroids under steady-state and inflammatory condition.

induced-pluripotent stem cell (iPSC)-derived neurospheroid (NSPH) models are an emerging in vitro toolkit to study the influence of inflammatory triggers on neurodegeneration and repair in a 3D neural environment. In contrast to their human counterpart, the absence of murine iPSC-derived NSPHs for profound characterisation and validation studies is a major experimental research gap, even though they offer the only possibility to truly compare or validate in vitro NSPH responses with in vivo brain responses. To contribute to these developments, we here describe the generation and characterisation of 5-week-old CX3CR1eGFP+/- CCR2RFP+/- murine (m)iPSC-derived bi-partite (neurons + astrocytes) and tri-partite (neurons + astrocytes + microglia) NSPH models that can be subjected to cellular activation following pro-inflammatory stimulation. First, cytokine analysis demonstrates that both bi-partite and tri-partite NSPHs can be triggered to release IL6 and CXCL10 following three days of stimulation with, respectively, TNFα + IL1ß + IFNγ and LPS + IFNγ. Additionally, immunocytochemical analysis for G3BP1 and PABPC1 revealed the development of stress granules in both bi-partite and tri-partite NSPHs after 3 days of stimulation. To further investigate the observed signs of inflammatory response and cellular stress, we performed an untargeted transcriptomic and proteomic analysis of bi- and tri-partite NSPHs under steady-state and inflammatory conditions. Here, using the combined differential gene and protein expression profiles between unstimulated and stimulated NSPHs, Ingenuity Pathway Analysis (IPA) confirms the activation of canonical pathways associated with inflammation and cellular stress in both bi-partite and tri-partite NSPHs. Moreover, our multi-omics analysis suggests a higher level of downstream inflammatory responses, impairment of homeostatic and developmental processes, as well as activation of cell death processes in stimulated tri-partite NSPHs compared to bi-partite NSPHs. Concluding, these results emphasise the advantages of including microglia in NSPH research to study inflammation-induced neurodegeneration in a 3D neural environment.

Enhanced Delivery of 5-Aminolevulinic Acid by Lecithin Invasomes in 3D Melanoma Cancer Model.

Photodynamic therapy (PDT) is a noninvasive therapeutic approach for the treatment of skin cancer and diseases. 5-Aminolevulinic acid is a prodrug clinically approved for PDT. Once internalized by cancer cells, it is rapidly metabolized to the photosensitizer protoporphyrin IX, which under the proper light irradiation, stimulates the deleterious reactive oxygen species (ROS) production and leads to cell death. The high hydrophilicity of 5-aminolevulinic acid limits its capability to cross the epidermis. Lipophilic derivatives of 5-aminolevulinic acid only partly improved skin penetration, thus making its incorporation into nanocarriers necessary. Here we have developed and characterized 5-aminolevulinic acid loaded invasomes made of egg lecithin, either 1,2-dilauroyl-sn-glycero-3-phosphocholine or 1,2-dioleoyl-sn-glycero-3-phosphocholine, and the terpene limonene. The obtained invasomes are highly thermostable and display a spherical morphology with an average size of 150 nm and an encapsulation efficiency of 80%; moreover, the ex vivo epidermis diffusion tests established that nanovesicles containing the terpene led to a much higher skin penetration (up to 80% in 3 h) compared to those without limonene and to the free fluorescent tracer (less than 50%). Finally, in vitro studies with 2D and 3D human cell models of melanoma proved the biocompatibility of invasomes, the enhanced intracellular transport of 5-aminolevulinic acid, its ability to generate ROS upon irradiation, and consequently, its antiproliferative effect. A simplified scaffold-based 3D skin model containing melanoma spheroids was also prepared. Considering the results obtained, we conclude that the lecithin invasomes loaded with 5-aminolevulinic acid have a good therapeutic potential and may represent an efficient tool that can be considered a valid alternative in the topical treatment of melanoma and other skin diseases.

Circulating extracellular vesicles expressing PD1 and PD-L1 predict response and mediate resistance to checkpoint inhibitors immunotherapy in metastatic melanoma.

BACKGROUND: The immunotherapy with immune checkpoints inhibitors (ICI) has changed the life expectancy in metastatic melanoma (MM) patients. Nevertheless, several patients do not respond hence, the identification and validation of novel biomarkers of response to ICI is of crucial importance. Circulating extracellular vesicles (EVs) such as PD-L1+ EV mediate resistance to anti-PD1, instead the role of PD1+ EV is not fully understood. METHODS: We isolated the circulating EVs from the plasma of an observational cohort study of 71 metastatic melanoma patients and correlated the amount of PD-L1+ EVs and PD1+ EVs with the response to ICI. The analysis was performed according to the origin of EVs from the tumor and the immune cells. Subsequently, we analysed the data in a validation cohort of 22 MM patients to assess the reliability of identified EV-based biomarkers. Additionally we assessed the involvement of PD1+ EVs in the seizure of nivolumab and in the perturbation of immune cells-mediated killing of melanoma spheroids. RESULTS: The level of PD-L1+ EVs released from melanoma and CD8+ T cells and that of PD1+ EVs irrespective of the cellular origin were higher in non-responders. The Kaplan-Meier curves indicated that higher levels of PD1+ EVs were significantly correlated with poorer progression-free survival (PFS) and overall survival (OS). Significant correlations were found for PD-L1+ EVs only when released from melanoma and T cells. The multivariate analysis showed that high level of PD1+ EVs, from T cells and B cells, and high level of PD-L1+ EVs from melanoma cells, are independent biomarkers of response. The reliability of PD-L1+ EVs from melanoma and PD1+ EVs from T cells in predicting PFS was confirmed in the validation cohort through the univariate Cox-hazard regression analysis. Moreover we discovered that the circulating EVs captured nivolumab and reduced the T cells trafficking and tumor spheroids killing. CONCLUSION: Our study identified circulating PD1+ EVs as driver of resistance to anti-PD1, and highlighted that the analysis of single EV population by liquid biopsy is a promising tool to stratify MM patients for immunotherapy.

Macrophage-based delivery of interleukin-13 improves functional and histopathological outcomes following spinal cord injury.

BACKGROUND: Spinal cord injury (SCI) elicits a robust neuroinflammatory reaction which, in turn, exacerbates the initial mechanical damage. Pivotal players orchestrating this response are macrophages (Mφs) and microglia. After SCI, the inflammatory environment is dominated by pro-inflammatory Mφs/microglia, which contribute to secondary cell death and prevent regeneration. Therefore, reprogramming Mφ/microglia towards a more anti-inflammatory and potentially neuroprotective phenotype has gained substantial therapeutic interest in recent years. Interleukin-13 (IL-13) is a potent inducer of such an anti-inflammatory phenotype. In this study, we used genetically modified Mφs as carriers to continuously secrete IL-13 (IL-13 Mφs) at the lesion site. METHODS: Mφs were genetically modified to secrete IL-13 (IL-13 Mφs) and were phenotypically characterized using qPCR, western blot, and ELISA. To analyze the therapeutic potential, the IL-13 Mφs were intraspinally injected at the perilesional area after hemisection SCI in female mice. Functional recovery and histopathological improvements were evaluated using the Basso Mouse Scale score and immunohistochemistry. Neuroprotective effects of IL-13 were investigated using different cell viability assays in murine and human neuroblastoma cell lines, human neurospheroids, as well as murine organotypic brain slice cultures. RESULTS: In contrast to Mφs prestimulated with recombinant IL-13, perilesional transplantation of IL-13 Mφs promoted functional recovery following SCI in mice. This improvement was accompanied by reduced lesion size and demyelinated area. The local anti-inflammatory shift induced by IL-13 Mφs resulted in reduced neuronal death and fewer contacts between dystrophic axons and Mφs/microglia, suggesting suppression of axonal dieback. Using IL-4Rα-deficient mice, we show that IL-13 signaling is required for these beneficial effects. Whereas direct neuroprotective effects of IL-13 on murine and human neuroblastoma cell lines or human neurospheroid cultures were absent, IL-13 rescued murine organotypic brain slices from cell death, probably by indirectly modulating the Mφ/microglia responses. CONCLUSIONS: Collectively, our data suggest that the IL-13-induced anti-inflammatory Mφ/microglia phenotype can preserve neuronal tissue and ameliorate axonal dieback, thereby promoting recovery after SCI.

Algal Phlorotannins as Novel Antibacterial Agents with Reference to the Antioxidant Modulation: Current Advances and Future Directions.

The increasing drug resistance of infectious microorganisms is considered a primary concern of global health care. The screening and identification of natural compounds with antibacterial properties have gained immense popularity in recent times. It has previously been shown that several bioactive compounds derived from marine algae exhibit antibacterial activity. Similarly, polyphenolic compounds are generally known to possess promising antibacterial capacity, among other capacities. Phlorotannins (PTs), an important group of algae-derived polyphenolic compounds, have been considered potent antibacterial agents both as single drug entities and in combination with commercially available antibacterial drugs. In this context, this article reviews the antibacterial properties of polyphenols in brown algae, with particular reference to PTs. Cell death through various molecular modes of action and the specific inhibition of biofilm formation by PTs were the key discussion of this review. The synergy between drugs was also discussed in light of the potential use of PTs as adjuvants in the pharmacological antibacterial treatment.

Murine induced pluripotent stem cell-derived neuroimmune cell culture models emphasize opposite immune-effector functions of interleukin 13-primed microglia and macrophages in terms of neuroimmune toxicity.

Cellular models of induced pluripotent stem cell (iPSC)-derived microglia and macrophages are an emerging toolbox to investigate neuroinflammation in vitro. We previously demonstrated that murine iPSC-microglia and iPSC-macrophages display phenotypical activation properties highly comparable to microglia and macrophages in vivo. Here we extended the characterization of iPSC-microglia and iPSC-macrophages with the analysis of their transcriptome profile. Next, these cellular models were employed to evaluate neuroimmune toxicity in vitro and to investigate the immune-modulatory properties of interleukin 13 (IL13), a cytokine known for its ability to protect against neuroinflammation-induced pathology by modulating microglia and macrophage activation. iPSC-microglia and iPSC-macrophages, in co-culture with astrocyte-committed neural stem cells (NSC), were (pre)treated with IL13 and stimulated with lipopolysaccharide (LPS) and interferon γ (IFNγ), to assess how IL13 modulates their inflammatory response. Additionally, the use of luciferase-expressing NSC (Luc-NSC) allowed real-time monitoring of immune-mediated neurotoxicity. Despite the known anti-inflammatory properties of IL13, iPSC-microglia primed with IL13 before LPS + IFNγ stimulation significantly increased NO secretion. This was associated with a marked reduction of the luminescence signal produced by Luc-NSC. Interestingly, we observed that IL13 signaling has a divergent functional outcome in microglia as compared to macrophages, as for the latter no major alterations in NO release and Luc-NSC viability were observed upon IL13 (pre)treatment. Finally, the striking IL13-induced upregulation of NO secretion by microglia under pro-inflammatory conditions was confirmed in vivo, where intracerebral delivery of IL13 increased inducible nitric oxide synthase mRNA expression. Concluding, we applied iPSC-derived neuroimmune cell culture models to identify distinct neuroimmune (toxicity) responses of microglia and macrophages to IL13-based immune modulation.

Oxidative Stress and Multi-Organel Damage Induced by Two Novel Phytocannabinoids, CBDB and CBDP, in Breast Cancer Cells.

Over the last few years, much attention has been paid to phytocannabinoids derived from Cannabis for their therapeutic potential. Δ9-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD) are the most abundant compounds of the Cannabis sativa L. plant. Recently, novel phytocannabinoids, such as cannabidibutol (CBDB) and cannabidiphorol (CBDP), have been discovered. These new molecules exhibit the same terpenophenolic core of CBD and differ only for the length of the alkyl side chain. Roles of CBD homologs in physiological and pathological processes are emerging but the exact molecular mechanisms remain to be fully elucidated. Here, we investigated the biological effects of the newly discovered CBDB or CBDP, compared to the well-known natural and synthetic CBD (nat CBD and syn CBD) in human breast carcinoma cells that express CB receptors. In detail, our data demonstrated that the treatment of cells with the novel phytocannabinoids affects cell viability, increases the production of reactive oxygen species (ROS) and activates cellular pathways related to ROS signaling, as already demonstrated for natural CBD. Moreover, we observed that the biological activity is significantly increased upon combining CBD homologs with drugs that inhibit the activity of enzymes involved in the metabolism of endocannabinoids, such as the monoacylglycerol lipase (MAGL) inhibitor, or with drugs that induces the activation of cellular stress pathways, such as the phorbol ester 12-myristate 13-acetate (PMA).

Neuroprotective modulation of microglia effector functions following priming with interleukin 4 and 13: current limitations in understanding their mode-of-action.

In recent years the long-standing theory of microglia's properties for dual polarization towards a pro- or anti-inflammatory phenotype has been deeply challenged. Furthermore, the elucidation of microglia ontogenesis exposed intrinsic differences between microglia and peripheral myeloid cells, thereby further underscoring the need to re-evaluate microglia-specific activation behavior, especially within an inflamed central nervous system (CNS) environment. This review critically summarizes recent literature on the in vitro and in vivo response of murine microglia to the immune-modulatory cytokines interleukin 4 (IL4) and interleukin 13 (IL13), i.e. those driving the so-called anti-inflammatory phenotype. Here we highlight several pivotal factors that may influence experimental outcome and/or interpretation of in vitro and in vivo studies evaluating microglia's phenotypical and functional properties upon IL4/IL13 treatment. Finally, the current therapeutic relevance of IL4/IL13-induced microglia activation in both acute and chronic CNS disorders is discussed.

Murine iPSC-derived microglia and macrophage cell culture models recapitulate distinct phenotypical and functional properties of classical and alternative neuro-immune polarisation.

The establishment and validation of reliable induced pluripotent stem cell (iPSC)-derived in vitro models to study microglia and monocyte/macrophage immune function holds great potential for fundamental and translational neuro-immunology research. In this study, we first demonstrate that ramified CX3CR1+ iPSC-microglia (cultured within a neural environment) and round-shaped CX3CR1- iPSC-macrophages can easily be differentiated from newly established murine CX3CR1eGFP/+CCR2RFP/+ iPSC lines. Furthermore, we show that obtained murine iPSC-microglia and iPSC-macrophages are distinct cell populations, even though iPSC-macrophages may upregulate CX3CR1 expression when cultured within a neural environment. Next, we characterized the phenotypical and functional properties of murine iPSC-microglia and iPSC-macrophages following classical and alternative immune polarisation. While iPSC-macrophages could easily be triggered to adopt a classically-activated or alternatively-activated phenotype following, respectively, lipopolysaccharide + interferon γ or interleukin 13 (IL13) stimulation, iPSC-microglia and iPSC-macrophages cultured within a neural environment displayed a more moderate activation profile as characterised by the absence of MHCII expression upon classical immune polarisation and the absence of Ym1 expression upon alternative immune polarisation. Finally, extending our preceding in vivo studies, this striking phenotypical divergence was also observed for resident microglia and infiltrating monocytes within highly inflammatory cortical lesions in CX3CR1eGFP/+CCR2RFP/+ mice subjected to middle cerebral arterial occlusion (MCAO) stroke and following IL13-mediated therapeutic intervention thereon. In conclusion, our study demonstrates that the applied murine iPSC-microglia and iPSC-macrophage culture models are able to recapitulate in vivo microglia and monocyte/macrophage ontogeny and corresponding phenotypical/functional properties upon classical and alternative immune polarisation, and therefore represent a valuable in vitro platform to further study and modulate microglia and (infiltrating) monocyte immune responses under neuro-inflammatory conditions within a neural environment.

Targeted intracerebral delivery of the anti-inflammatory cytokine IL13 promotes alternative activation of both microglia and macrophages after stroke.

BACKGROUND: Subtle adjustment of the activation status of CNS resident microglia and peripheral macrophages, to promote their neuroprotective and neuroregenerative functions, may facilitate research towards curing neurodegenerative disorders. In the present study, we investigated whether targeted intracerebral delivery of the anti-inflammatory cytokine interleukin (IL)13, by means of transplanting IL13-expressing mesenchymal stem cells (IL13-MSCs), can promote a phenotypic switch in both microglia and macrophages during the pro-inflammatory phase in a mouse model of ischemic stroke. METHODS: We used the CX3CR1eGFP/+ CCR2RFP/+ transgenic mouse model to separately recognize brain-resident microglia from infiltrated macrophages. Quantitative immunohistochemical analyses were applied to characterize polarization phenotypes of both cell types. RESULTS: Distinct behaviors of both cell populations were noted dependent on the anatomical site of the lesion. Immunohistochemistry revealed that mice grafted with IL13-MSCs, in contrast to non-grafted and MSC-grafted control mice, were able to drive recruited microglia and macrophages into an alternative activation state, as visualized by a significant increase of Arg-1 and a noticeable decrease of MHC-II expression at day 14 after ischemic stroke. Interestingly, both Arg-1 and MHC-II were expressed more abundantly in macrophages than in microglia, further confirming the distinct behavior of both cell populations. CONCLUSIONS: The current data highlight the importance of controlled and localized delivery of the anti-inflammatory cytokine IL13 for modulation of both microglia and macrophage responses after ischemic stroke, thereby providing pre-clinical rationale for the application of L13-MSCs in future investigations of neurodegenerative disorders.

Polymeric Nano-Micelles as Novel Cargo-Carriers for LY2157299 Liver Cancer Cells Delivery.

LY2157299 (LY), which is very small molecule bringing high cancer diffusion, is a pathway antagonist against TGFß. LY dosage can be diluted by blood plasma, can be captured by immune system or it might be dissolved during digestion in gastrointestinal tract. The aim of our study is to optimize a "nano-elastic" carrier to avoid acidic pH of gastrointestinal tract, colon alkaline pH, and anti-immune recognition. Polygalacturonic acid (PgA) is not degradable in the gastrointestinal tract due to its insolubility at acidic pH. To avoid PgA solubility in the colon, we have designed its conjugation with Polyacrylic acid (PAA). PgA-PAA conjugation has enhanced their potential use for oral and injected dosage. Following these pre-requisites, novel polymeric nano-micelles derived from PgA-PAA conjugation and loading LY2157299 are developed and characterized. Efficacy, uptake and targeting against a hepatocellular carcinoma cell line (HLF) have also been demonstrated.

In Vivo Interleukin-13-Primed Macrophages Contribute to Reduced Alloantigen-Specific T Cell Activation and Prolong Immunological Survival of Allogeneic Mesenchymal Stem Cell Implants.

Transplantation of mesenchymal stem cells (MSCs) into injured or diseased tissue-for the in situ delivery of a wide variety of MSC-secreted therapeutic proteins-is an emerging approach for the modulation of the clinical course of several diseases and traumata. From an emergency point-of-view, allogeneic MSCs have numerous advantages over patient-specific autologous MSCs since "off-the-shelf" cell preparations could be readily available for instant therapeutic intervention following acute injury. Although we confirmed the in vitro immunomodulatory capacity of allogeneic MSCs on antigen-presenting cells with standard coculture experiments, allogeneic MSC grafts were irrevocably rejected by the host's immune system upon either intramuscular or intracerebral transplantation. In an attempt to modulate MSC allograft rejection in vivo, we transduced MSCs with an interleukin-13 (IL13)-expressing lentiviral vector. Our data clearly indicate that prolonged survival of IL13-expressing allogeneic MSC grafts in muscle tissue coincided with the induction of an alternatively activated macrophage phenotype in vivo and a reduced number of alloantigen-reactive IFNγ- and/or IL2-producing CD8(+) T cells compared to nonmodified allografts. Similarly, intracerebral IL13-expressing MSC allografts also exhibited prolonged survival and induction of an alternatively activated macrophage phenotype, although a peripheral T cell component was absent. In summary, this study demonstrates that both innate and adaptive immune responses are effectively modulated in vivo by locally secreted IL13, ultimately resulting in prolonged MSC allograft survival in both muscle and brain tissue. Stem Cells 2016;34:1971-1984.

Human Hepatocarcinoma Cell Targeting by Glypican-3 Ligand Peptide Functionalized Silica Nanoparticles: Implications for Ultrasound Molecular Imaging.

Silica nanoparticles (SiNPs) are widely studied nanomaterials for their potential employment in advanced biomedical applications, such as selective molecular imaging and targeted drug delivery. SiNPs are generally low cost and highly biocompatible, can be easily functionalized with a wide variety of functional ligands, and have been demonstrated to be effective in enhancing ultrasound contrast at clinical diagnostic frequencies. Therefore, SiNPs might be used as contrast agents in echographic imaging. In this work, we have developed a SiNPs-based system for the in vitro molecular imaging of hepatocellular carcinoma cells that express high levels of glypican-3 protein (GPC-3) on their surface. In this regard, a novel GPC-3 targeting peptide was designed and conjugated to fluorescent silica nanoparticles. The physicochemical properties, acoustic behavior, and biocompatibility profile of the functionalized SiNPs were characterized; then binding and uptake of both naked and functionalized SiNPs were analyzed by laser scanning confocal microscopy and transmission electron microscopy in GPC-3 positive HepG2 cells, a human hepatocarcinoma cell line. The results obtained showed that GPC-3-functionalized fluorescent SiNPs significantly enhanced the ultrasound contrast and were effectively bound and taken up by HepG2 cells without affecting their viability.

Hybrid polymeric-protein nano-carriers (HPPNC) for targeted delivery of TGFß inhibitors to hepatocellular carcinoma cells.

TGFß1 pathway antagonists have been considered promising therapies to attenuate TGFß downstream signals in cancer cells. Inhibiting peptides, as P-17 in this study, are bound to either TGFß1 or its receptors, blocking signal transduction. However, for efficient use of these TGFß1antagonist as target therapeutic tools, improvement in their delivery is required. Here, a plasmid carrying specific shDNA (SHT-DNA), small interfering RNA (siRNA), and the peptide (P-17) were loaded separately into folic acid (FA)-functionalized nano-carriers made of Bovine Serum Albumin (BSA). The two building blocks of the carrier, (BSA and FA) were used because of the high affinity of albumin for liver and for the overexpression of folate receptors on the membrane of hepatocellular carcinoma cells. The empty and the encapsulated carriers were thoroughly investigated to characterize their structure, to evaluate the colloidal stability and the surface functionalization. The entrapment of SHT-DNA, siRNA and P-17, respectively, was demonstrated by morphological and quantitative analysis. Finally, cellular studies were performed to assess the targeting efficiency of the hybrid carriers. These vectors were used because of the high affinity of albumin for liver and for the overexpression of folate receptors on the membrane hepatocellular carcinoma cells. The empty and the encapsulated carriers were thoroughly investigated to characterize their structure, to evaluate the colloidal stability and the surface functionalization. The entrapment of SHT-DNA, siRNA and P-17, respectively, was demonstrated by morphological and quantitative analysis. A novel fabrication of Hybrid Polymeric-Protein Nano-Carriers (HPPNC) for delivering TGF ß1 inhibitors to HCC cells has been developed. SHT-DNA, siRNA and P-17 have been successfully encapsulated. TGF ß1 inhibitors-loaded HPPNC were efficiently uptaken by HLF cells.

Intracerebral transplantation of interleukin 13-producing mesenchymal stem cells limits microgliosis, oligodendrocyte loss and demyelination in the cuprizone mouse model.

BACKGROUND: Promoting the neuroprotective and repair-inducing effector functions of microglia and macrophages, by means of M2 polarisation or alternative activation, is expected to become a new therapeutic approach for central nervous system (CNS) disorders in which detrimental pro-inflammatory microglia and/or macrophages display a major contribution to the neuropathology. In this study, we present a novel in vivo approach using intracerebral grafting of mesenchymal stem cells (MSC) genetically engineered to secrete interleukin 13 (IL13-MSC). METHODS: In the first experimental setup, control MSC and IL13-MSC were grafted in the CNS of eGFP+ bone marrow chimaeric C57BL/6 mice to histologically evaluate IL13-mediated expression of several markers associated with alternative activation, including arginase1 and Ym1, on MSC graft-recognising microglia and MSC graft-infiltrating macrophages. In the second experimental setup, IL13-MSC were grafted on the right side (or on both the right and left sides) of the splenium of the corpus callosum in wild-type C57BL/6 mice and in C57BL/6 CX3CR1eGFP/+CCR2RFP/+ transgenic mice. Next, CNS inflammation and demyelination was induced by means of a cuprizone-supplemented diet. The influence of IL13-MSC grafting on neuropathological alterations was monitored by non-invasive T 2-weighted magnetic resonance imaging (MRI) and quantitative histological analyses, as compared to cuprizone-treated mice with control MSC grafts and/or cuprizone-treated mice without MSC injection. RESULTS: In the first part of this study, we demonstrate that MSC graft-associated microglia and MSC graft-infiltrating macrophages are forced into alternative activation upon grafting of IL13-MSC, but not upon grafting of control MSC. In the second part of this study, we demonstrate that grafting of IL13-MSC, in addition to the recruitment of M2 polarised macrophages, limits cuprizone-induced microgliosis, oligodendrocyte death and demyelination. Furthermore, we here demonstrate that injection of IL13-MSC at both sides of the splenium leads to a superior protective effect as compared to a single injection at one side of the splenium. CONCLUSIONS: Controlled and localised production of IL13 by means of intracerebral MSC grafting has the potential to modulate cell graft- and pathology-associated microglial/macrophage responses, and to interfere with oligodendrocyte death and demyelinating events in the cuprizone mouse model.

Synthesis and Characterization of SPIONs Encapsulating Polydopamine Nanoparticles and Their Test for Aqueous Cu2+ Ion Removal.

The removal of pollutants, such as heavy metals, aromatic compounds, dyes, pesticides and pharmaceuticals, from water is still an open challenge. Many methods have been developed and exploited for the purification of water from contaminants, including photocatalytic degradation, biological treatment, adsorption and chemical precipitation. Absorption-based techniques are still considered among the most efficient and commonly used approaches thanks to their operational simplicity. In recent years, polydopamine-coated magnetic nanoparticles have emerged for the uptake of heavy metals in water treatment, since they combine specific affinity towards pollutants and magnetic separation capacity. In this context, this work focuses on the synthesis of polydopamine (PDA)-coated Super Paramagnetic Iron Oxide Nanoparticles (PDA@SPIONs) as adsorbents for Cu2+ ions, designed to serve as functional nanostructures for the removal of Cu2+ from water by applying a magnetic field. The synthetic parameters, including the amount of SPIONs and PDA, were thoroughly investigated to define their effects on the nanostructure features and properties. Subsequently, the ability of the magnetic nanostructures to bind metal ions was assessed on Cu2+-containing solutions. A systematic investigation of the prepared functional nanostructures was carried out by means of complementary spectroscopic, morphological and magnetic techniques. Inductively coupled plasma atomic emission spectroscopy (ICP-AES) measurements were performed in order to estimate the Cu2+ binding ability. The overall results indicate that these nanostructures hold great promise for future bioremediation applications.

Poly(l-lactide-co-caprolactone-co-glycolide)-Based Nanoparticles as Delivery Platform: Effect of the Surfactants on Characteristics and Delivery Efficiency.

Polymeric nanoparticles made of the copolymer Poly(L-lactide-co-caprolactone-co-glycolide) were prepared using the solvent evaporation method. Two different surfactants, polyvinyl alcohol and dextran, and a mixture of the two were employed. The three types of nanoparticles were used as hosting carriers of two chemotherapeutic drugs, the hydrophilic doxorubicin and the hydrophobic SN-38. The morphostructural characterization showed similar features for the three types of nanoparticles, while the drug encapsulation efficiency indicated that the dextran-based systems are the most effective with both drugs. Cellular studies with breast cancer cells were performed to compare the delivery capability and the cytotoxicity profile of the three nanosystems. The results show that the unloaded nanoparticles are highly biocompatible at the administered concentrations and confirmed that dextran-coated nanoparticles are the most efficient vectors to release the two drugs, exerting cytotoxic activity. PVA, on the other hand, shows limited drug release in vitro, probably due to strong interactions with both drugs. Data also show the release is more efficient for doxorubicin than for SN-38; indeed, the doxorubicin IC50 value for the dextran-coated nanoparticles was about 35% lower than the free drug. This indicates that these nanocarriers are suitable candidates to deliver hydrophilic drugs while needing further modification to host hydrophobic molecules.

Aquaponics-Derived Tilapia Skin Collagen for Biomaterials Development.

Collagen is one of the most widely used biomaterials in health-related sectors. The industrial production of collagen mostly relies on its extraction from mammals, but several issues limited its use. In the last two decades, marine organisms attracted interest as safe, abundant, and alternative source for collagen extraction. In particular, the possibility to valorize the huge quantity of fish industry waste and byproducts as collagen source reinforced perception of fish collagen as eco-friendlier and particularly attractive in terms of profitability and cost-effectiveness. Especially fish byproducts from eco-sustainable aquaponics production allow for fish biomass with additional added value and controlled properties over time. Among fish species, Oreochromis niloticus is one of the most widely bred fish in large-scale aquaculture and aquaponics systems. In this work, type I collagen was extracted from aquaponics-raised Tilapia skin and characterized from a chemical, physical, mechanical, and biological point of view in comparison with a commercially available analog. Performed analysis confirmed that the proprietary process optimized for type I collagen extraction allowed to isolate pure native collagen and to preserve its native conformational structure. Preliminary cellular studies performed with mouse fibroblasts indicated its optimal biocompatibility. All data confirmed the eligibility of the extracted Tilapia-derived native type I collagen as a biomaterial for healthcare applications.

Therapeutic Effect of Polymeric Nanomicelles Formulation of LY2157299-Galunisertib on CCl4-Induced Liver Fibrosis in Rats.

Hepatic fibrosis (HF) is a major cause of liver-related disorders and together with cancer-associated fibroblasts can favor liver cancer development by modulating the tumor microenvironment. Advanced HF, characterized by an excess of extracellular matrix (ECM), is mediated by TGF- ß1, that activates hepatic stellate cells (HSCs) and fibroblasts. A TGF-ß1 receptor inhibitor, LY2157299 or Galunisertib (GLY), has shown promising results against chronic liver progression in animal models, and we show that it can be further improved by enhancing GLYs bioavailability through encapsulation in polymeric polygalacturonic-polyacrylic acid nanomicelles (GLY-NMs). GLY-NMs reduced HF in an in vivo rat model of liver fibrosis induced by intraperitoneal injection of CCl4 as shown by the morphological, biochemical, and molecular biology parameters of normal and fibrotic livers. Moreover, GLY-NM was able to induce recovery from HF better than free GLY. Indeed, the encapsulated drug reduces collagen deposition, hepatic stellate cells (HSCs) activation, prevents fatty degeneration and restores the correct lobular architecture of the liver as well as normalizes the serum parameters and expression of the genes involved in the onset of HF. In summary, GLY-NM improved the pharmacological activity of the free TGF- ß1 inhibitor in the in vivo HF treatment and thus is a candidate as a novel therapeutic strategy.

The Encapsulation of Citicoline within Solid Lipid Nanoparticles Enhances Its Capability to Counteract the 6-Hydroxydopamine-Induced Cytotoxicity in Human Neuroblastoma SH-SY5Y Cells.

(1) Backgrond: Considering the positive effects of citicoline (CIT) in the management of some neurodegenerative diseases, the aim of this work was to develop CIT-Loaded Solid Lipid Nanoparticles (CIT-SLNs) for enhancing the therapeutic use of CIT in parkinsonian syndrome; (2) Methods: CIT-SLNs were prepared by the melt homogenization method using the self-emulsifying lipid Gelucire® 50/13 as lipid matrix. Solid-state features on CIT-SLNs were obtained with FT-IR, thermal analysis (DSC) and X-ray powder diffraction (XRPD) studies. (3) Results: CIT-SLNs showed a mean diameter of 201 nm, -2.20 mV as zeta potential and a high percentage of entrapped CIT. DSC and XRPD analyses evidenced a greater amorphous state of CIT in CIT-SLNs. On confocal microscopy, fluorescent SLNs replacing unlabeled CIT-SLNs released the dye selectively in the cytoplasm. Biological evaluation showed that pre-treatment of SH-SY5Y dopaminergic cells with CIT-SLNs (50 µM) before the addition of 40 µM 6-hydroxydopamine (6-OHDA) to mimic Parkinson's disease's degenerative pathways counteracts the cytotoxic effects induced by the neurotoxin, increasing cell viability with the consistent maintenance of both nuclear and cell morphology. In contrast, pre-treatment with CIT 50 and 60 µM or plain SLNs for 2 h followed by 6-OHDA (40 µM) did not significantly influence cell viability. (4) Conclusions: These data suggest an enhanced protection exerted by CIT-SLNs with respect to free CIT and prompt further investigation of possible molecular mechanisms that underlie this difference.