STRATEGIE DI DIREZIONAMENTO ALL’EPATOCARCINOMA DI FARMACI ANTITUMORALI MEDIANTE SISTEMI NANOPARTICELLARI E DI VISUALIZZAZIONE IN CELLULE TUMORALI DELLA PROTEINA DI TRASLOCAZIONE MITOCONDRIALE TSPO.

Lo scopo di questo lavoro di tesi è stato quello di realizzare nuovi sistemi nanoparticellari per il direzionamento di farmaci o di agenti per l’imaging, potenzialmente utilizzabili per la terapia e/o per la diagnosi dell’epatocarcinoma (HCC), in particolare per quelle forme caratterizzate dall’overespressione del recettore di membrana degli epatociti ASGP-R o del recettore mitocondriale TSPO. In particolare, nel capitolo 2 sono state descritte la sintesi, la caratterizzazione chimico fisica, la capacità di internalizzazione cellulare e l’efficacia antitumorale di un nuovo sistema nanoparticellare, costituito da un dendrimero a struttura poli-amido-aminica (PAMAM) di quarta generazione, opportunamente funzionalizzato con acido lactobionico per il riconoscimento selettivo del recettore ASGP-R e marcato con fluoresceina isotiocianato, in grado di veicolare nel core idrofobico, il farmaco attualmente più utilizzato nella terapia dell’ HCC, il Sorafenib. Come evidenziato dai saggi di citotossicità, di recovery ed annexina V, condotti su due linee cellulari di tumore epatico umane (HepG-2 ed HLE), il sorafenib incluso nel dendrimero, non perde la sua efficacia, al contrario, è in grado di produrre un effetto più duraturo nel tempo rispetto al sorafenib somministrato tal quale. In aggiunta, come prevedibile, si è registrato un aumento della solubilità del sorafenib incluso nel dendrimero. Questi risultati consentono di considerare questo nuovo dendrimero ASGP-R-direzionato, come un vettore adatto per il rilascio selettivo di sorafenib nel citoplasma delle cellule di cancro al fegato overesprimenti tale recettore. Nel capitolo 3 sono state descritte delle nuove nanoformulazioni rappresentate da micelle fosfolipidiche modificate con polietilenglicole e caricate con SPIONs e sorafenib. Esse sono state accuratamente preparate e caratterizzate mediante tecniche complementari, risultando dei nanovettori efficaci per il drug delivery, con una buona stabilità in ambiente acquoso ed un opportuno carico di farmaco. È stata effettuata anche un’analisi magnetica delle strutture ottenute, che ha permesso di determinare la giusta quantità di SPIONs da incorporare nelle micelle. È stato specificamente progettato e realizzato un sistema in vitro per dimostrare che le micelle inglobanti le SPIONs erano efficacemente trattenute dal campo magnetico generato dal sistema nelle condizioni di flusso tipicamente presenti nel fegato umano. Gli esperimenti condotti su linee cellulari di tumore epatico (HepG-2) hanno dimostrato che questa nuova piattaforma di distribuzione del farmaco, è in grado di migliorare l'efficacia antitumorale del farmaco quando magneticamente direzionato. I suddetti nanovettori magnetici rappresentano pertanto dei candidati promettenti per il direzionamento verso specifici siti di tumore epatico, dove il rilascio selettivo di sorafenib risulta in grado di migliorare il suo profilo di efficacia e sicurezza. Nei capitoli 4 e 5 sono stati invece descritti due nuovi sistemi nanoparticellari, in grado di legare selettivamente il recettore mitocondriale TSPO e potenzialmente utilizzabili per l’imaging in vitro di tale recettore. I mitocondri rappresentano un bersaglio subcellulare interessante date le innumerevoli funzioni svolte per garantire la vitalità cellulare, quali ad esempio il metabolismo del calcio, controllo dello stress ossidativo, e la regolazione dei processi apoptotici. Tuttavia, il targeting mitocondriale è stato un settore trascurato finora. In particolare il sistema descritto nel capitolo 4 è costituito da un dendrimero PAMAM di quarta generazione, funzionalizzato con un ligando altamente affine e selettivo per il, suddetto recettore a struttura 2-fenil-imidazo[1,2-a]piridinacetammidica sintetizzato nei nostri laboratori, e con fluoresceina isotiocianato (FITC) come fluoroforo organico. Queste nuove nano-piattaforme hanno caratteristiche uniche, infatti, vengono preparati con un livello di controllo non raggiungibile con la maggior parte dei polimeri lineari, portando a macromolecole globulari quasi monodisperse, con un gran numero di gruppi periferici. Di conseguenza, rappresentano un candidato ideale come vettore per il delivery, e per studiare gli effetti di dimensioni del polimero, carica, composizione e architettura, sulle proprietà biologicamente rilevanti quali le interazioni con il doppio strato lipidico, citotossicità, internalizzazione cellulare e interazioni con gli scomparti e organelli subcellulari. Gli studi di uptake cellulare condotti su cellule overesprimenti il recettore TSPO (cellule C6 di glioma di ratto), anche sotto l’influenza di specifici inibitori di endocitosi, mediante esperimenti di frazionamento cellulare e analisi di co-localizzazione (mediante microscopia CAT(Confocale-AFM-TIRF)), hanno evidenziato che questo nuovo nanosistema viene rapidamente internalizzato nelle cellule e soprattutto è in grado di direzionarsi selettivamente sul target mitocondriale. Inoltre, le limitazioni ascrivibili all’utilizzo di FITC come fluoroforo organico, sono state superate con la realizzazione di una nuova nanostruttura ibrida, descritta accuratamente nel capitolo 5, basata su QDs fluorescenti opportunamente funzionalizzati per il riconoscimento del recettore TSPO, con lo stesso ligando altamente affine e selettivo utilizzato per il dendrimero descritto nel capitolo precedente. In particolare, la progettazione razionale di nanoparticelle di QDs (QD@SiO2 NPs) rivestite di silice ammino-funzionalizzata, ha permesso lo sviluppo di una nanopiattafomra versatile per la funzionalizzazione con il ligando del TSPO. La stabilità colloidale e le proprietà ottiche di questo nuovo nanomateriale sono state accuratamente valutate. Gli esperimenti di frazionamento cellulare e l’analisi di microscopia confocale condotti sulle cellule C6 overesprimenti il recettore TSPO, hanno confermato il grande potenziale di questo nanosistema multifunzinale come agente selettivo per l’imaging mitocondriale in vitro.The purpose of the present work was to provide new nanoparticles for the delivery of drugs or imaging agents, potentially useful for the therapy and/or for diagnosis of hepatocellular carcinoma (HCC), in particular for those forms characterized by overexpression of asialoglycoprotein receptor (ASGP-R) or of Translocator Protein mitochondrial receptor TSPO. In particular, in Chapter 2 are reported the synthesis, the physical-chemical characterization, the cellular internalization ability and the antitumor efficacy of a new nanoparticle system, consisting of a poly-amido-amine dendrimer (PAMAM), suitably functionalized with lactobionic acid for the selective recognition of the ASGP-R receptor and labeled with fluorescein isothiocyanate, able to convey in the hydrophobic core, the drug Sorafenib, currently used in most of the 'HCC therapy. As shown by cytotoxicity assays, recovery and annexin V, conducted on two human liver cancer cell lines (HepG-2 and HLE), sorafenib included in the dendrimer, does not lose its effectiveness, on the contrary, is able to produce a more lasting effect over time compared to sorafenib administered alone. In addition, as expected, there was an increase in the solubility of sorafenib included in the dendrimer. These results allow to consider this new dendrimer ASGP-R-targeted, as a nanovector suitable for the selective release of sorafenib in the cytoplasm of liver cancer cells overexpressing this receptor. In Chapter 3 is reported a new nanoformulations represented by phospholipid micelles modified with polyethylene glycol and loaded with SPIONs and sorafenib. They have been carefully prepared and characterized by means of complementary techniques. The nanocarriers resulted effective for drug delivery, with a good stability in an aqueous environment and a suitable drug loading. It was also carried out an analysis of the obtained magnetic structures, which allowed to determine the correct amount of SPIONs to be incorporated in the micelles. An in vitro system has been specifically designed and produced to show that the micelles incorporating SPIONs were effectively retained by the magnetic field generated by the system under the conditions typically found in the human liver flow. The experiments conducted on liver cancer cell lines (HepG-2) have shown that this new delivery nanoplatform of drug, is able to improve the antitumor efficacy of the drug when magnetically directed. These magnetic nanovectors therefore represent promising candidates for targeting of specific hepatic tumor sites, where the selective release of sorafenib is able to improve its efficacy and safety profile. In chapters 4 and 5 are instead described two new nanoparticellar systems, able to selectively bind the mitochondrial receptor TSPO and potentially useful for the in vitro imaging of this receptor. Mitochondria represent an subcellular interesting target, given the innumerable functions performed by this organelle to ensure cell viability, such as the calcium metabolism, the control of oxidative stress, and the regulation of apoptotic processes. However, the mitochondrial targeting has been a neglected area so far. In particular the system described in chapter 4 consists of a PAMAM dendrimer of fourth generation, functionalized with an highly affine selective ligand of TSPO with a structure 2-phenyl-imidazo [1,2-a] piridinacetammidica synthesized in our laboratories, and fluorescein isothiocyanate (FITC) as an organic fluorophore. These new nano-platforms have unique characteristics, in fact, are prepared with a level of control not achievable with most of the linear polymers, leading to globular macromolecules almost monodisperse, with a large number of peripheral groups. Consequently, they represent an ideal candidate as a nanovector for the delivery, and to study the effects of the polymer size, charge, composition and architecture, on the biologically relevant properties such as interactions with the lipid bilayer, cytotoxicity, cell internalization and interactions with subcellular compartments and organelles. The cellular uptake studies conducted on cells overexpressing the receptor TSPO (C6 rat glioma cells), also under the influence of specific inhibitors of endocytosis, by cell fractionation experiments and analysis of co-localization (by microscopy CAT (Confocale- AFM-TIRF)), have shown that this new nanosystem is rapidly internalized into the cells and especially is able to target selectively the mitochondria. Furthermore, the limitations attributable to the use of FITC as organic fluorophore, have been overcome with the realization of a new hybrid nanostructure, thoroughly described in chapter 5, based on fluorescent QDs suitably functionalized for the recognition of TSPO receptor, with the same highly affine and selective ligand used for the dendrimer described in the previous chapter. In particular, the rational design of QDs nanoparticles (QD@SiO2NPs) coated with amino-functionalized silica, has allowed the development of a versatile nanoplatform for the functionalization with the TSPO ligand. The colloidal stability and the optical properties of this new nanomaterial have been carefully evaluated. The cell fractionation experiments and confocal microscopy analysis conducted on C6 cells overexpressing the receptor, confirmed the great potential of this multifunctional nanosystem as a selective agent for mitochondrial imaging in vitro

The mitochondrial citrate carrier: A new player in inflammation

The mitochondrial CIC (citrate carrier) catalyses the efflux of citrate from the mitochondrial matrix in exchange for cytosolic malate. In the present paper we show that CIC mRNA and protein markedly increase in lipopolysaccharide-activated immune cells. Moreover, CIC gene silencing and CIC activity inhibition significantly reduce production of NO, reactive oxygen species and prostaglandins. These results demonstrate for the first time that CIC has a critical role in inflammation

Investigating alkyl nitrates as nitric oxide releasing precursors of multitarget acetylcholinesterase-monoamine oxidase B inhibitors

Herein we envisaged the possibility of exploiting alkyl nitrates as precursors of alcohol-bearing dual inhibitors targeting acetylcholinesterase (AChE) and monoamine oxidase B (MAO B), key enzymes in neurodegenerative syndromes such as Alzheimer's disease (AD), through biotransformation unmasking an alcoholic function upon nitric oxide (NO) release. The cooperation to neuroprotection of low fluxes of NO and target enzymes’ inhibition by the alcohol metabolites might return a multitargeting effect. The in vitro screening towards ChEs and MAOs of a collection of 21 primary alcohols disclosed a subset of dual inhibitors, among which three diverse chemotypes were selected to study the corresponding nitrates. Nitrate 14 proved to be a brain permeant, potent AChE-MAO B inhibitor by itself. Moreover, it protected human SH-SY5Y lines against rotenone and hydrogen peroxide with a poor inherent cytotoxicity and showed a slow conversion profile to its alcohol metabolite 9d that still behaved as bimodal and neuroprotective molecule

Potential predictive role of chemotherapy-induced changes of soluble CD40 ligand in untreated advanced pancreatic ductal adenocarcinoma

Pancreas ductal adenocarcinoma lacks predictive biomarkers. CD40 is a member of the tumor necrosis factor superfamily. CD40-sCD40L interaction is considered to contribute to the promotion of tumor cell growth and angiogenesis. The aim of the present study was to investigate the role of serum sCD40L as a predictor in metastatic pancreatic cancer. We evaluated 27 consecutive pancreatic cancer patients treated with FOLFIRINOX (21 patients) or gemcitabine plus nab-paclitaxel combination (six patients). The sCD40L level was measured in serum by enzyme-linked immunosorbent assay at baseline, at first evaluation (all patients), and at time to progression (18 patients). The radiological response was evaluated according to the Response Evaluation Criteria in Solid Tumors, Version 1.1. The Wilcoxon signed-rank test was used to compare pre-post treatment sCD40L levels with respect to clinical response, while Pearson's correlation coefficient was used for the correlation between sCD40L and CA19.9 pre- and post-treatment. The Kruskal-Wallis test was also conducted for further comparisons. We observed a statistically significant reduction in the sCD40L level after 3 months of treatment in patients with partial response (11,718.05\ub17,097.13 pg/mL vs 4,689.42\ub15,409.96 pg/mL; P<0.01). Conversely, in patients with progressive disease, the biomarker statistically increased in the same time (9,351.51\ub17,356.91 pg/mL vs 22,282.92\ub111,629.35 pg/mL; P<0.01). This trend of sCD40L was confirmed in 18 patients at time to progression after the first evaluation. No differences were recorded within the stable disease group. Moreover, there was a positive correlation between the sCD40L and CA19.9 pre-post treatment variation percentage (Pearson's correlation coefficient =0.52; P<0.05). Our data suggest a possible predictive role of sCD40L in pancreatic cancer patients, similar to CA19.9

Grape seed extracts modify the outcome of oxaliplatin in colon cancer cells by interfering with cellular mechanisms of drug cytotoxicity

Grape seed extracts are commonly utilized as dietary supplements for their antioxidant properties, even from cancer patients. However, whether these natural extracts interfere with chemotherapeutics utilized in colon cancer treatment is still poorly investigated. The cytotoxicity of extracts from Italia and Palieri cultivars either alone or in combination with oxaliplatin was evaluated in colon cancer cells. Grape seed extracts displayed anti-proliferative activity depending on the concentration utilized through apoptosis induction. In combination, they affected the activation of Erk1/2 and counteracted the intrinsic and the extrinsic pathway of apoptosis, the DNA damage and the generation of ROS induced by oxaliplatin. Noteworthy grape seed extracts strongly enhanced the uptake of oxaliplatin into all cells, by affecting the cell transport system of platinum. The addition of these natural extracts to oxaliplatin strongly reduced the cellular response to oxaliplatin and allowed a huge accumulation of platinum into cells. Here, we shed light on the chemical biology underlying the combination of grape seed extracts and oxaliplatin, demonstrating that they might be detrimental to oxaliplatin effectiveness in colon cancer therap

Spray-dried mucoadhesives for intravesical drug delivery using N-acetylcysteine- and glutathione-glycol chitosan conjugates

This work describes N-acetylcysteine (NAC)- and glutathione (GSH)-glycol chitosan (GC) polymer conjugates engineered as potential platform useful to formulate micro-(MP) and nano-(NP) particles via spray-drying techniques. These conjugates are mucoadhesive over the range of urine pH, 5.0-7.0, which makes them advantageous for intravesical drug delivery and treatment of local bladder diseases. NAC- and GSH-GC conjugates were generated with a synthetic approach optimizing reaction times and purification in order to minimize the oxidation of thiol groups. In this way, the resulting amount of free thiol groups immobilized per gram of NAC-and GSH-GC conjugates was 6.3 and 3.6 mmol, respectively. These polymers were completely characterized by molecular weight, surface sulfur content, solubility at different pH values, substitution and swelling degree. Mucoadhesion properties were evaluated in artificial urine by turbidimetric and zeta (zeta)-potential measurements demonstrating good mucoadhesion properties, in particular for NAC-GC at pH 5.0. Starting from the thiolated polymers, MP and NP were prepared using both the Bidchi B-191 and Nano Buchi B-90 spray dryers, respectively. The resulting two formulations were evaluated for yield, size, oxidation of thiol groups and ex-vivo mucoadhesion. The new spray drying technique provided NP of suitable size (<1 mu m) for catheter administration, low degree of oxidation, and sufficient mucoadhesion property with 9% and 18% of GSH- and NAC-GC based NP retained on pig mucosa bladder after 3 h of exposure, respectively

Microfluidic preparation and in vitro evaluation of iRGD-functionalized solid lipid nanoparticles for targeted delivery of paclitaxel to tumor cells

Solid lipid nanoparticles (SLNs) can combine the advantages of different colloidal carriers and prevent some of their disadvantages. The production of nanoparticles by means of microfluidics represents a successful platform for industrial scale-up of nanoparticle manufacture in a reproducible way. The realisation of a microfluidic technique to obtain SLNs in a continuous and reproducible manner encouraged us to create surface functionalised SLNs for targeted drug release using the same procedure. A tumor homing peptide, iRGD, owning a cryptic C-end Rule (CendR) motif is responsible for neuropilin-1 (NRP-1) binding and for triggering extravasation and tumor penetration of the peptide. In this study, the Paclitaxel loaded-SLNs produced by microfluidics were functionalized with the iRGD peptide. The SLNs proved to be stable in aqueous medium andwere characterized by a Z-average under 150 nm, a polydispersity index below 0.2, a zeta-potential between -20 and -35 mV and a drug encapsulation efficiency around 40%. Moreover, in vitro cytotoxic effects and cellular uptake have been assessed using 2D and 3D tumour models of U87 glioblastoma cell lines. Overall, these results demonstrate that the surface functionalization of SLNs with iRGD allow better cellular uptake and cytotoxicity ability.Peer reviewe

Induced expression of P-gp and BCRP transporters on brain endothelial cells using transferrin functionalized nanostructured lipid carriers:A first step of a potential strategy for the treatment of Alzheimer's disease

P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP) are two transporters expressed in human neural stem/progenitor cells and at the Blood-Brain Barrier (BBB) level with decreased activity in the early stage of Alzheimer's disease (AD). Both proteins, have a protective role for the embryonic stem cells in the early developmental step, maintaining them in an undifferentiated state, and limit the access of exogenous and endogenous agents to the brain. Recently, MC111 selected from a P-gp/BCRP ligands library was investigated as multitarget strategy for AD treatment, considering its ability to induce the expression and activity of both proteins. However, MC111 clinical use could be limited for the ubiquitous physiological expression of efflux transporters and its moderate toxicity towards endothelial cells. Therefore, a selective MC111 delivery system based on nanostructured lipid carriers (NLC) functionalized with transferrin were developed. The results proved the formation of NLC with average size about 120 nm and high drug encapsulation efficiency (EE% greater than 50). In vitro studies on hCMEC/D3 cells revealed that the MC111 was selectively released by NLC at BBB level and then inducing the activity and expression of BCRP and P-gp, involved in the clearance of amyloid p peptide on brain endothelial cells

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 identifcation 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 identifed 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 signifcantly correlated with poorer progression-free survival (PFS) and overall survival (OS). Signifcant 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 confrmed 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 trafcking and tumor spheroids killing. Conclusion: Our study identifed 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

The Interaction between Reactive Peritoneal Mesothelial Cells and Tumor Cells via Extracellular Vesicles Facilitates Colorectal Cancer Dissemination

Simple SummaryEmerging evidence has suggested that cancer-derived extracellular vesicles (EVs) have a crucial role in mediating directional metastasis to the peritoneal surface in colorectal cancer (CRC). We investigated the EV-mediated crosstalk between tumor and mesothelial cells which may drive remodeling of the premetastatic niche to allow tumor spread to the peritoneal surface. Our findings demonstrated that cancer-derived EVs triggered apoptosis and reduced mesothelial cell invasiveness and mesothelial-to-mesenchymal transition. On the other hand, mesothelial cells actively supported tumor invasion by releasing EVs, which induced upregulation of the major pro-invasive system in tumor cells. For the first time, we provide evidence of EV-driven mechanisms of CRC progression in patient-derived models, highlighting the crucial role of EVs in the reprogramming of mesothelial and tumor cells to establish the metastatic process.Advanced colorectal cancer (CRC) is highly metastatic and often results in peritoneal dissemination. The extracellular vesicles (EVs) released by cancer cells in the microenvironment are important mediators of tumor metastasis. We investigated the contribution of EV-mediated interaction between peritoneal mesothelial cells (MCs) and CRC cells in generating a pro-metastatic environment in the peritoneal cavity. Peritoneal MCs isolated from peritoneal lavage fluids displayed high CD44 expression, substantial mesothelial-to-mesenchymal transition (MMT) and released EVs that both directed tumor invasion and caused reprogramming of secretory profiles by increasing TGF-beta 1 and uPA/uPAR expression and MMP-2/9 activation in tumor cells. Notably, the EVs released by tumor cells induced apoptosis by activating caspase-3, peritoneal MC senescence, and MMT, thereby augmenting the tumor-promoting potential of these cells in the peritoneal cavity. By using pantoprazole, we reduced the biogenesis of EVs and their pro-tumor functions. In conclusion, our findings provided evidence of underlying mechanisms of CRC dissemination driven by the interaction of peritoneal MCs and tumor cells via the EVs released in the peritoneal cavity, which may have important implications for the clinical management of patients