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Breakthroughs in Medicinal Chemistry: New Targets and Mechanisms, New Drugs, New Hopes is a series of editorials which is published on a biannual basis by the Editorial Board of the Medicinal Chemistry section of the journal Molecules. In these editorials, we highlight in brief reports (of about one hundred words) a number of recently published articles that describe crucial findings, such as the discovery of novel drug targets and mechanisms of action or novel classes of drugs, which may inspire future medicinal chemistry endeavors devoted to addressing prime unmet medical needs.

Cholangiocarcinoma (CCA) remains a therapeutic challenge. The small-molecule Rigosertib can selectively synchronize cancer cells to G2/M phase improving the efficacy of radiation. In our study, we evaluated in vitro Rigosertib (gifted by Onconova Therapeutics Inc) effects on two human CCA cell lines: EGI-1 and TFK-1. Rigosertib was compared with Gemcitabine (GEM) and 5-Fluorouracil (5-FU), two antineoplastic and radio-sensitizer agents used in the treatment of CCA. Rigosertib impaired cell viability (evaluated by Tripan-blue vital count) in both cell lines in a dose- and time-dependent manner (IC50 of 100nM at 24h). GEM and 5-FU had a IC50 of 30µM and 7µM after 24h, respectively. Cell migration and invasion tests was performed by scratch wound healing and Boyden chamber assay respectively. Rigosertib caused a 50% inhibition of the EGI-1 cell migration (10µM) and invasion (100nM), while the inhibitory effects on TFK-1 cells were observed with doses of 100µM and 10µM, respectively. GEM 30µM and 5-FU 7µM had no effect on cell migration and invasion. Evaluation of cell cycle by FACS cytometry showed a G2/M arrest in both cell lines after Rigosertib 100nM for 24h. Radio-sensitizing test was performed by clonogenic survival assay after irradiation. 24h Rigosertib pre-treatment (100nM for EG-1 and 1 µM for TFK-1) when followed by 2, 4 or 6 Gy irradiation, reduced survival in both CAA cell lines when compared with radiation alone. The Rigosertib radio-sensitizer effect was similar to that seen after GEM or 5-FU 24 pre-treatment both plus irradiation. However, 48h Rigosertib pre-treatment was more effective than radiation alone as well as GEM for 48h. Our study highlights the preliminary but promising preclinical activity of Rigosertib both as antitumoral and as a radio-sensitizer agent in CCA and provides a background for further investigations., Italian Journal of Anatomy and Embryology, Vol. 122, No. 1 (Supplement) 2017

Hibiscus Sabdariffa (HS) is a plant commonly used in folk medicine (1). In recent years HS has gained great interest due to its important antioxidant, anti-inflammatory and antitumoral properties. In our work, we evaluated the in vitro anticancer effects of HS extract against two different human breast cancer cell lines: estrogen receptor (ER) positive MCF-7 cells and ER negative MDA-MB-231 cells. We tested both total extract (HSE) and one fraction obtained by ethyl acetate extraction (HSEC). MTT assay and Trypan Blue vital count showed a dose and time dependent reduction of the viability in both cell lines treated with different concentrations of HSE or HSEC compared to untreated control cells. A significantly marked reduction was observed in MCF-7 cells treated with HSEC. On the basis of our results we used the concentrations of 7.5mg/ml and 3.5mg/ml respectively for HSE and HSEC. In order to evaluate ER involvement in HS effect, we analyzed the cellular localization of the receptor (ERα isotype) by immunofluorescence experiments. Untreated MDA-MB-231 cells showed a low expression of the receptor mostly localized at the cytoplasmic level and treatment with HSE or HSEC didn’t change this state. Untreated MCF-7 cells showed a greater expression of the receptor, with nuclear and cytoplasmic localization. Following HSE or HSEC treatment ERα localization became more cytoplasmic and this effect was more evident after HSEC induction. These data were also confirmed by ERα western blot analysis. Subsequently, we studied HSE and HSEC ability to alter migration and invasion capacity of ER positive MCF-7 cells. Using a scratch wound healing assay we did not observe any change in the migration of cells compared to untreated cells. On the contrary, in a Boyden chamber invasion assay, HSE, and especially HSEC, induced reduction of MCF-7 cell invasion. In conclusion, we have demonstrated that HS is able to reduce cell viability of ER positive MCF-7 and ER negative MDA-MB-231 cells. This effect is more evident in MCF-7 cells in which ER localization and reduced cell invasion were observed. These results are more evident after HSEC treatment. Further studies will be needed to better elucidate the involved mechanisms of action., Italian Journal of Anatomy and Embryology, Vol. 121, No. 1 (Supplement) 2016

Oxygen and glucose deprivation (OGD) due to ischemic events or trauma in the brain result in neuronal loss. The therapeutic approaches available inadequate and often the outcome is unfavorable for the patient or at least unpredictable. Stem cells could be useful for the treatment of OGD injured-neurons. Mesenchymal Stem Cells (MSCs), isolated from bone marrow as well as from various tissues, have poor immunogenicity and neuroprotective properties being able to alleviate ischemic brain injuries in animal models. The Endothelial Progenitor Cells (EPCs) are present at low frequencies both in the bone marrow and in the peripheral blood. They are thought to play a role in the recovery of cerebrovasculature integrity after stroke. In the present study we evaluated the potential neuroprotective effect of human MSCs and human EPCs on rat embryonic cortical neurons injured by OGD. OGD was induced by incubating the cortical neurons in a hypoxia chamber in a 95% N2 + 5% CO2 atmosphere at 37°C without glucose. To set up the experimental protocol, OGD was maintained for 1, 2 and 3 hours. The neurons were returned in normoxic atmosphere and after 2 and 5 days neuronal survival was evaluated by MTT assay, LDH assay and viable cellular counting. The 2 hours OGD was able to reduce neuronal viability by 50% and was chosen for the subsequent experiments. To assess MSCs and EPCs neuroprotective action, after 2 hours-long OGD the neurons were 1) co-cultured with either MSCs or EPCs seeded on a cell culture insert avoiding direct contact while sharing the same medium, or 2) cultured in a medium previously conditioned by either MSCs or EPCs. Neuronal survival was evaluated by MTT assay after 2 and 5 days. Both MSCs and EPCs increased neuronal survival after ODG. The effect was observed in absence of a direct contact between MSCs or EPCs and the injured neurons, suggesting that the release of soluble factors may be involved in their neuroprotective action. In conclusion both MSCs and EPCs could represent a potential therapeutic approach for the treatment of brain ischemic injury. Further studies are needed to identify the specific molecules and pathways that play a role in the neuroprotective effect of MSCs and EPCs., Italian Journal of Anatomy and Embryology, Vol. 120, No. 1 (Supplement) 2015

Epidemiological data consistently demonstrate a reduced cancer risk associated with a polyphenols rich diet. Hibiscus sabdarifa (HS), a polyphenols rich plant widely consumed worldwide as beverage and used in folk medicine, has recently gained interest thanks to its antioxidant, anti-inflammatory and chemopreventive properties. In the present study we investigated the antitumoral potential of HS extract in two different human tumor cell lines: Multiple Myeloma cells (RPMI 8226) and Oral Squamous Cell Carcinoma cells (SCC-25). MTT assays showed that HS extract induced a dose-dependent viability reduction in both the cells lines. For the subsequent experiments we used HS at the concentration of 5 mg/ml that was the most effective in inducing cell viability reduction after 48h of treatment. Viable cell count using trypan blue staining demonstrated that the HS extract induced decrease in cell growth of both the cell lines and this was due to a reversible cytostatic rather than a cytotoxic effect. Wound-healing and cell invasion assays, respectively performed by a scratch of cell monolayer and Boyden Chamber transwell test, demonstrated that HS extract was able to reduce motility and invasiveness in both RPMI 8226 and SCC-25 cells. The chemical inhibition of ERK1/ERK2 and PI3K, with U0126 and wortmannin respectively, reduces proliferation and migration of both SSC-25 and RPMI cells and HB extract treatment played an additive action with the inhibitors. In conclusion, our results suggest that HS extract have antitumoral properties, since it proved to inhibit tumoral cell growth and cell migration and invasiveness. It is interesting to note that HS extract is effective against two very different tumor cell lines. In fact, RPMI 8226 cells are of hematopoietic origin and grow in suspension, whereas SCC-25 cells derive from epithelium and are characterized by adherent cell growth. Therefore, although further studies are needed to clarify the molecular mechanisms involved in its action, we proposed HS as a potential chemopreventive agent., Italian Journal of Anatomy and Embryology, Vol 118, No 2 (Supplement) 2013

Cholangiocarcinoma (CCA) is the second most frequent primary liver neoplasia. It mainly arises from the malignant transformation of biliary epithelial cells, although it might originate from either hepatic progenitor cells at the Hering canals or transformed hepatocytes. CCA is a highly aggressive tumor with extremely poor prognosis and limited therapeutic approaches. Baicalin (BA) is one of the main bioactive flavonoids identified in the Scutellaria Baicalensis Georgi root dried extract which is extensively used in the Chinese traditional medicine. Together with the anti-inflammatory effect, the anti-neoplastic action is the most relevant BA property demonstrated on cancer cells of different origin. Being aware of the need of new therapeutic weapons for CCA treatment, we investigated whether Baicalin could exert anti-proliferative and anti-migratory effect on EGI-1 cells, a highly metastatic CCA cell line derived from bile duct carcinoma. We first tested different BA concentrations (from 5 to 200μM) in limiting EGI-1 viability using MTT assay. After 24h and 48h treatment, 5 and 10μM BA had no effect while rising from 25μM to 200μM (i.e. 25, 50, 100 and 200μM) BA exerted a significant cell viability reduction already at 24h and increased after 48h BA exposure. This reduction well correlated with the adherent absolute cell number decrease and it cannot be due to BA induced cell cycle impairment after neither 24 nor 48h treatment. We also evaluated the anti-migratory BA potential by a wound healing assay adding different BA concentrations (5, 25, 50,100 and 200μM) to the culture medium immediately after performing a wound on confluent cell cultures. All BA concentrations but 5μM induced a significant reduction in the EGI-1 migration rate after 24h treatment. Moreover 25, 50 and 10μM BA showed similar migration inhibition extent at 24 and 48h whilst 200μM BA exerted a stronger inhibitory effect already after 24h exposure which increased with time in a significant way. Taken together our preliminary results demonstrate that BA impairs CCA cell viability and migration suggesting a promising adjuvant therapeutic use for BA as antitumoral agent., Italian Journal of Anatomy and Embryology, Vol 119, No 1 (Supplement) 2014

Neurotoxicity is a common dose-limiting side-effect of several drugs (Cavaletti et al., 2008). So far a validated test method to screen drugs neurotoxicity does not exist, therefore in this interdepartment study we have analyzed the effectiveness of a morphometric neurotoxicty assessment model. Drug neurotoxicity evaluation is based on embryonic rat dorsal root ganglia (DRG) organotypic culture. DRG primary sensory neurons are the principal target of drugs neurotoxic action. In fact, primary sensory neurons lie outside the blood-nerve barrier and are supplied by capillaries with fenestrated walls. Moreover, the axons of these cells are among the longest of the entire nervous system and, therefore, are more susceptible to any agent that interferes with the energy metabolism or the structural basis of axonal transport. In particular, in this interdepartment study, the interference of the under study neurotoxic compound with NGF-induced neurite elongation is analysed. The effectiveness and reproducibility of this model, even if commonly used to test drugs, has not yet been demonstrated. In order to assess the validity of this in vitro model, antineoplastic drugs known to be in clinical use and in animal models neurotoxic (paclitaxel and oxaliplatin) or not dangerous (cyclophosphamide and 5-Fluorouracil) have been tested. DRGs explanted from E15 rat embryos have been treated for 24h with drugs concentrations comparable to those achievable in vivo. The length of the longest neurite of each DRG has been measured by ImageJ program. Experiments have been performed by three different blinded researchers in two different laboratories. Mean and standard deviation of each experiment were obtained, subsequently the mean value and standard deviation of the three independent experiments for each researcher were calculated. Data obtained by the three researchers in two different laboratories resulted statistically comparable and no significant differences were detected (One Way Anova analysis of variance and Tukey post test; p, Italian Journal of Anatomy and Embryology, Vol 117, No 2 (Supplement) 2012

Oral Cancer (OC) is one of the most frequent cancer in Head and Neck district and Oral Squamous Cell Carcinoma (OSCC) constitutes the large majority of the neoplasia arising in oral cavity. OSCC remains a hampering matters for clinics, since the overall disease free survival has not significantly increased during the last decades and invasion to surrounding tissue and to regional lymph nodes is often reported. Therefore new strategies to prevent and inhibit OSCC growth and invasion are highly desirable and new therapeutic approaches are currently tempted also with the use of natural compounds. Myricetin (MYR) and Naringenin (NAR), two naturally occurring flavonoids, widely diffused in plants, fruits and vegetable, have recently gained consideration thanks to their anti oxidant, anti inflammatory and anti tumoral properties. In this study their potential anticancer effect has been evaluated on an OSCC cell line, SCC-25 and on spontaneously immortalized non tumoral keratinocytes, HaCaT cells. MYR and NAR induce a significant cell growth inhibition in SCC-25 cells, in addition NAR selectively affected cancer cells, since it does not impair HaCaT cell growth. Furthermore an additive effect of MYR and NAR has been highlighted. The cell proliferation inhibition is not related to apoptosis induction, as demonstrated by evaluation of phosphatidyl serine membrane translocation and dapi staining. On the contrary MYR and NAR effect depends on the cell cycle progression impairment. Wound-healing and cell invasion assays, respectively performed by cell monolayer scratch and Boyden Chamber transwell test, demonstrate that the two flavonoids are able to reduce motility and invasiveness on both SCC-25 and HaCaT cells. In conclusion the results of the present study show the anticancer potential of NAR and MYR on OSCC, since both flavonoids prevent cancer cell proliferation through a cytostatic effect, by the impairment of cell cycle progression. Moreover both the flavonoids inhibit cell migration, thus highlighting their potential effect as anti metastatic agents., Italian Journal of Anatomy and Embryology, Vol 118, No 2 (Supplement) 2013

The induction of a peripheral neuropathy is a very common side effect of many chemotherapeutic agents, including platinum compounds, and it often represents the dose limiting factor for drug clinical use. Several strategies have been suggested to reduce drug neurotoxicity without affecting the antineoplastic potential, but up to now results were not encouraging. Recently, it has been demonstrated that Mesenchymal Stem Cells (MSCs) are able to promote the survival and the maturation of untreated sensory neurons of dorsal root ganglia (DRG), which represent also the target of drug neurotoxicity. Aim of this work is to verify the neuroprotective potential of MSCs on rat DRG exposed to cisplatin (CDDP), a chemotherapeutic and neurotoxic agent. DRG post-mitotic explants from E15 rat embryos were exposed for 24 hours to different cisplatin concentrations. After 24 hours, medium was changed and DRG were directly co-cultured with human MSCs (hMSCs) or with hMSCs conditioned medium (hMSC-CM). DRG explants were photographed every day up to 1 month, and the longest neurite of each DRG was measured to evaluate neurotoxicity. DRG survival was estimated by measuring the death area percentage. The survival of CDDP-treated DRG was increased after the co-cultures with hMSCs, and both hMSCs and hMSC-CM were able to improve the neurite outgrowth of untreated and CDDP-treated DRG after 48 hours. This MSC-dependent increase of neurite length was however no longer evident at later times (1 month). This effect on neurite elongation was probably mediated by CSPG, MAG and Nogo, some proteins involved in the modulation of neurite elongation, which resulted expressed and released in the culture medium of hMSCS. Our results demonstrated a neuroprotective effect of hMSCs on CDDP toxicity and evidenced the ability of these cells to modulate neurite elongation. In this way MSCs could represent a possible mean to limit the neurotoxicity on DRG which arises after cisplatin therapy.

Nowadays obesity and its related diseases represent a major health problem with an increasing worldwide prevalence. Hyperplasia and hypertrophy of adipocytes lead to an excessive fat accumulation that is not efficiently prevented by current pharmacological treatments. So the research on anti-obesity drugs with good efficacy and tolerability able both to prevent and to reduce fat accumulation is of pivotal interest. In the present study we evaluated in vitro the effects of Valproic Acid, Berberin and Resveratrol on adipogenesis. Our experimental model was represented by human Mesenchymal Stem Cells (hMSCs), physiological precursors of adipocytes that can differentiate into adipocytes also in vitro. Preliminary cytotoxicity assays were performed in order to choose non-toxic doses of the three drugs. hMSCs were induced to adipogenic differentiation and treated with Valproic Acid, Berberin and Resveratrol at the selected doses. Controls were represented by hMSCs treated for adipogenesis in absence of the drugs. At different time points intracellular lipid droplets accumulation, a typical feature of adipogenesis, was assessed by Oil Red O staining. Valproic Acid, Berberin and Resveratrol inhibited hMSCs adipogenic differentiation in a dose dependent manner as demonstrated by the reduction of the lipid droplets accumulation. To understand the molecular mechanisms of the drugs-induced adipogenesis inhibition, we focused our attention on the effects of the drugs treatment on cell cycle progression, known to be altered by many antiadipogenic drugs, and on the MAP Kinases ERK1 and ERK2, involved in the adipogenesis control. We evaluated the expression of cyclins and CDKs by immunoblotting and flow-cytometry analyses, demonstrating that Valproic Acid, Berberin and Resveratrol interfere on cell cycle progression. The expression and the phosphorylation status of the two kinases ERK1 and ERK2 were assessed by immunoblotting demonstrating an increase of ERK1 phosphorylation (i.e. activation) in hMSCs treated with Berberin and a reduction in hMSCs treated with Valproic Acid and Resveratrol compared to control cells. No changes in phosphorylation and expression of ERK2 were observed. Our study demonstrate that Valproic Acid, Berberin and Resveratrol exert an anti-adipogenic effect in our experimental model. The mechanisms of action of these drugs involve the alteration of cell cycle progression and, at least in part, ERK1/2 modulation. However other molecular pathways are likely implicated and other studies are required to identify them., Italian Journal of Anatomy and Embryology, Vol 116, No 1 (Supplement) 2011

Introduction. Mesenchymal Stem Cells (MSCs) are adult multipotent cells able to differentiate in mesengenic (osteogenic, adipogenic, condrogenic) and non mesengenic lineages (e.g. neural) under appropriate culture conditions. MSCs represent a very promising therapeutic approach in different settings particularly for tissue repair and regeneration. The knowledge of human MSCs (hMSCs) biological properties is very important to optimize their clinical application. In view of MSCs application in neurodegenerative diseases, the neuronal differentiation potential of hMSCs has been also explored. Our preliminary data demonstrated that the neuronal markers beta III tubulin and NeuN were spontaneously expressed by a high percentage of undifferentiated hMSCs independently from serum presence and number of culture passages. The expression of neural markers by MSCs in absence of any differentiative agents is considered as a demonstration of MSC neural predisposition. The aim of this work was to evaluate if these markers, known to be neuronal ones, continued to be expressed also in hMSCs differentiated towards mesengenic lineages. Methods. hMSCs were obtained after patient consensus, from iliac crest bone marrow. In according to the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy, the isolated hMSCs were plastic-adherent, capable of extensive proliferation when maintained in standard culture conditions, lacked of hematopoietic markers expression and presented specific surface antigens. hMSCs were differentiated toward osteogenic, adipogenic and chondrogenic lineages using specific in vitro protocols. The expression of the neuronal markers beta III tubulin and NeuN were evaluated by immunofluorescence experiments at different time points depending on the differentiation protocol used. hMSCs cultured in absence of any differentiative agent represented controls. Results. In our experiments the most of hMSCs differentiated in osteogenic and adipogenic lineages expressed the neuronal markers beta III tubulin and NeuN. Unlike, chondrogenic differentiated hMSCs didn’t express these markers. Conclusions. The finding that hMSCs differentiated into adipogenic and osteogenic lineages express neuronal markers such as beta III tubulin and NeuN raises doubts about the reliability of these markers as indicators of neuronal differentiation and suggests that their expression could be an intrinsic property of a wide range of cellular types. Further studies are necessary to understand the specific biological role of of beta III tubulin and NeuN in hMSCs differentiated towards mesengenic lineages., Italian Journal of Anatomy and Embryology, Vol 116, No 1 (Supplement) 2011

Multiple sclerosis (MS) is a chronic immuno-mediated inflammatory and demyelinating disease characterised by the presence of both demyelinating lesions and axonal degeneration, which lead to the reduction of nerve conduction velocity and the development of concomitant neurological deficits. Recently, Mesenchymal Stem Cells (MSCs) have been proposed in in vivo studies as promising therapeutic treatment for MS mainly for their capacity to modulate the immune response, moreover, during our previous experiments we found that rat undifferentiated MSC promote Dorsal Root Ganglia neurons survival and maturation. The aim of this study is to verify the potential protective effect of MSCs on an in vitro model of MS represented by rat primary cultures of cortical neurons. Since glutamate excitotoxicity is an important mechanism in neurodegenerative diseases, and it induces neuronal alterations similar to those observed in advanced MS, cortical neurons cultures were treated with different concentrations of glutamate (25, 50, 100, 200 and 500 µM) for 24 hours. After the treatment cortical neurons show a suffering appearance, with damaged axons and cellular degeneration. Neuronal viability, assessed by DAPI staining and by count of viable cells, was glutamate dose-dependent. In order to evaluate the possible positive effect of MSCs on neuronal survival, both direct and indirect co-cultures of MSCs and cortical neurons were set up, and the survival after glutamate treatment analyzed. Cortical neurons treated with glutamate were still suffering after the direct co-cultures with MSCs, while in indirect co-cultures with MSCs neurons were alive, without important signs of axonal degeneration. These preliminary findings suggest that MSCs are able to reduce the cellular death induced by glutamate exposure in cortical neurons and encouraged the further study and characterization of their positive effect

Aims In order to develop therapeutic strategies to control obesity and the related pathologies, it is very important to deepen the knowledge of adipocytes biology and the mechanisms that control adipogenesis. In the present work we have studied the molecular mechanisms at the basis of the adipocytes differentiation using the human Mesenchymal Stem Cells (hMSCs), undifferentiated stem cells present in the bone marrow that are the physiological precursors of adipocytes in the organism. In particular we have focused our attention on the MAPKinases ERK1 and ERK2, that are involved in many biological and cellular processes. Methods hMSCs, obtained from iliac crest bone marrow, were induced to adipogenic differentiation by treatment with Adipogenic Induction Medium for 10 days (determination phase), then replaced with Adipogenic Maintenance Medium until the end of treatment at day 28 (terminal differentiation phase). hMSC adipogenic differentiation was evaluated by morphological and molecular techniques. Control cells were represented by hMSCs cultured in absence of adipogenic supplements. ERK1 and ERK2 expression and phosphorylation were evaluated by immunoblotting experiments. The specific ERK inhibitor U0126 was added to the adipogenic medium at different times during the adipogenic differentiation protocol. Results hMSC treated with adipogenic differentiation protocol showed lipid droplets that increased in number and size during the whole differentiation period. In treated hMSC both ERK1 and ERK2 phosphorylation was reduced in comparison to control hMSCs, but time and intensity of these reductions were different for the two isoforms. A decrease of the total amount of ERK1 was also observed. The presence of U0126 during the whole differentiation period hampered the adipogenic differentiation of hMSCs, as very few hMSCs showed the appearance of lipid droplets that were reduced both in number and size. When U0126 was administered only during the determination phase the number of hMSCs recruited in the differentiation program was reduced, while when U0126 was administered only in the terminal differentiation phase, hMSCs did not acquired a mature adipocytic phenotype. Conclusion In this work we demonstrate that ERK1 and ERK2 are important for hMSC adipogenic differentiation. Our results suggest that ERK1 and ERK2 play a key role in determining how many cells enter into the adipogenic differentiation program and acquire the phenotipycal and molecular characteristics of mature adipocytes.

Type 1 diabetes is a chronic disease often leading to systemic complications, such as peripheral neuropathy, nephropathy and cardiovascular complications. Whole pancreas as well as pancreatic islets transplantation has been proposed for the cure of type 1 diabetes, allowing a more efficient and physiological metabolic control. We investigated the effects of microencapsulated and immunoisolated islet transplantation in a model of streptozotocin-induced diabetes in 3 groups of Lewis rats: healthy controls, untreated diabetic rats and diabetic rats transplanted with microencapsulated islets into the peritoneal cavity two months after diabetes induction. Our results demonstrated that following transplantation hyperglycemic rats became normoglycemic in few days and this was accompanied by a rapid raise in body weight. Meanwhile, thermal (hot plate test) and mechanical sensitivity (Randal-Selitto paw withdrawal test measured with an analgesymeter) were increased and decreased by 180 and 40-60%, respectively. In addition, the density of footpad intraepidermal nerve fibers was significantly reduced by 20% in diabetic group and islet transplantation restored normal skin innervation. Nerve conduction velocity in the tail nerve and the Na+, K+-ATPase activity in the sciatic nerve, both reduced by about 25% in diabetic rats, were also normalized by islet transplantation. In conclusion, our data obtained in a model of type 1 diabetes, showed that transplant of microencapsulated pancreatic islets, besides controlling glycemia, arrested neuropathy worsening and was able to restore all the diabetic-induced alterations within the 2-month follow-up period after transplantation. On the basis of these encouraging results, a new experiment with 4-month long diabetes followed-up for 4 months after allogenic transplantation has been performed and the preliminary analysis confirmed the effectiveness of the procedure also in these long-term diabetic animals. In fact, effective treatment of the peripheral nervous system damage was demonstrated at the behavioral and neurophysiological levels and the viability of the transplanted cells could be demonstrated until the end of the study at the functional and morphological levels. We believe that these data can offer a solid basis for the future implementation of more sophisticated models of diabetes treatment based on cellular transplantation.

Mesoporous silica particles (MSP) are a new development in nanotechnology. Covalent modification of the surface of the silica is possible both on the internal pore and on the external particle surface. It allows the design of functional nanostructured materials with properties of organic, biological and inorganic components. Research and development are ongoing on the MSP, which have applications in catalysis, drug delivery and imaging. The most recent and interesting advancements in size, morphology control and surface functionalization of MSP have enhanced the biocompatibility of these materials with high surface areas and pore volumes. In the last 5 years several reports have demonstrated that MSP can be efficiently internalized using in vitro and animal models. The functionalization of MSP with organic moieties or other nanostructures brings controlled release and molecular recognition capabilities to these mesoporous materials for drug/gene delivery and sensing applications, respectively. In this study different MSP have been tested: silica-FITC MSP and silica-folate-FITC MSP. Folic acid was used as the targeting ligand because -folate receptor is observed to be up-regulated in various types of human cancers. We have evaluated by confocal laser scanning microscopy (CLSM) the intracellular localization of FITC-loaded MSP in two different cancer cell lines: IGROV-1 (ovarian carcinoma) and A549 (lung adenocarcinoma) characterized by a different expression of folate receptor alpha. Our results demonstrate that silica nanoparticles localize into the cytoplasm of A549 and IGROV-1 cell lines after 1, 6 and 24 hours of treatment and present a perinuclear localization. Additionally the treated cells were examined cross-sectionally in order to confirm that the MSP were indeed internalized by the cells and not simply bound on the surface membrane. Flow cytometry was used for the quantification of fraction of cells that had internalized nanoparticles. The cellular uptake of silica nanoparticles was observed with both cell lines and folate modification on the silica nanoparticles increased the particles uptake by A549 and IGROV-1 cells. The over-expression of folate receptor may facilitate the recognition of the folate-modified nanoparticles and increase the uptake through folate receptor-mediated endocytosis Electron microscopy experiments also confirmed that silica nanoparticles are able to enter into tumor cells. The particles are progressively internalized into the cell cytoplasm starting from 15 minutes after treatment and after 30 minutes are completely internalized.