A pH-sensitive stearoyl-PEG-poly(methacryloyl sulfadimethoxine)-decorated liposome system for protein delivery: an application for bladder cancer treatment

Stealth pH-responsive liposomes for the delivery of therapeutic proteins to the bladder epithelium were prepared using methoxy-poly(ethylene glycol)5kDa-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (mPEG5kDa-DSPE) and stearoyl-poly(ethylene glycol)-poly(methacryloyl sulfadimethoxine) copolymer (stearoyl-PEG-polySDM), which possesses an apparent pKa of 7.2. Liposomes of 0.2:0.6:100, 0.5:1.5:100 and 1:3:100 mPEG5kDa-DSPE/stearoyl-PEG-polySDM/(soybean phosphatidylcholine + cholesterol) molar ratios were loaded with bovine serum albumin (BSA) as a protein model. The loading capacity was 1.3% w/w BSA/lipid. At pH 7.4, all liposome formulations displayed a negative zeta-potential and were stable for several days. By pH decrease or addition to mouse urine, the zeta potential strongly decreased, and the liposomes underwent a rapid size increase and aggregation. Photon correlation spectroscopy (PCS) and transmission electron microscopy (TEM) analyses showed that the extent of the aggregation depended on the stearoyl-PEG-polySDM/lipid molar ratio. Cytofluorimetric analysis and confocal microscopy showed that at pH 6.5, the incubation of MB49 mouse bladder cancer cells and macrophages with fluorescein isothiocyanate-labelled-BSA (FITC-BSA) loaded and N-(Lissamine Rhodamine B sulfonyl)-1, 2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine triethylammonium salt (rhodamine-DHPE) labelled 1:3:100 mPEG5kDa-DSPE/stearoyl-PEG-polySDM/lipid molar ratio liposomes resulted in a time-dependent liposome association with the cells. At pH 7.4, the association of BSA-loaded liposomes with the MB49 cells and macrophages was remarkably lower than at pH 6.5. Confocal images of bladder sections revealed that 2 h after the instillation, liposomes at pH 7.4 and control non-responsive liposomes at pH 7.4 or 6.5 did not associate nor delivered FITC-BSA to the bladder epithelium. On the contrary, the pH-responsive liposome formulation set at pH 6.5 and soon administered to mice by bladder instillation showed that, 2 h after administration, the pH-responsive liposomes efficiently delivered the loaded FITC-BSA to the bladder epitheliu

Physical PEGylation to Prevent Insulin Fibrillation

Insulin is one of the most marketed therapeutic proteins worldwide. However, its formulation suffers from fibrillation, which affects the long-term storage limiting the development of novel devices for sustained delivery including portable infusion devices. We have investigated the effect of physical PEGylation on structural and colloidal stability of insulin by using 2 PEGylating agents terminating with polycyclic hydrophobic moieties, cholane and cholesterol: mPEG5kDa-cholane and mPEG5kDa-cholesterol, respectively. Microcalorimetric analyses showed that mPEG5kDa-cholane and mPEG5kDa-cholesterol efficiently bind insulin with binding constants (Ka) of 3.98 104 and 1.14 105 M-1, respectively. At room temperature, the 2 PEGylating agents yielded comparable structural stabilization of \u3b1-helix conformation and decreased dimerization of insulin. However, melting studies showed that mPEG5kDa-cholesterol has superior stabilizing effect of the protein conformation than mPEG5kDa-cholane. Furthermore, the fibrillation study showed that at a 1:1 and 1:5 insulin/polymer molar ratios, mPEG5kDa-cholesterol delays insulin fibrillation 40% and 26% more efficiently, respectively, as compared to mPEG5kDa-cholane which was confirmed by transmission electron microscopy imaging. Insulin was released from the mPEG5kDa-cholane and mPEG5kDa-cholesterol assemblies with comparable kinetic profiles. The physical PEGylation has a beneficial effect on the stabilization and shielding of the insulin structure into the monomeric form, which is not prone to fibrillation and aggregation

Influence of folate-targeted gold nanoparticles on subcellular localization and distribution into lysosomes

The cell interaction, mechanism of cell entry and intracellular fate of surface decorated nanoparticles are known to be affected by the surface density of targeting agents. However, the correlation between nanoparticles multivalency and kinetics of the cell uptake process and disposition of intracellular compartments is complicated and dependent on a number of physicochemical and biological parameters, including the ligand, nanoparticle composition and colloidal properties, features of targeted cells, etc. Here, we have carried out an in-depth investigation on the impact of increasing folic acid density on the kinetic uptake process and endocytic route of folate (FA)-targeted fluorescently labelled gold nanoparticles (AuNPs). A set of AuNPs (15 nm mean size) produced by the Turkevich method was decorated with 0–100 FA-PEG3.5kDa-SH molecules/particle, and the surface was saturated with about 500 rhodamine-PEG2kDa-SH fluorescent probes. In vitro studies carried out using folate receptor overexpressing KB cells (KBFR-high) showed that the cell internalization progressively increased with the ligand surface density, reaching a plateau at 50:1 FA-PEG3.5kDa-SH/particle ratio. Pulse-chase experiments showed that higher FA density (50 FA-PEG3.5kDa-SH molecules/particle) induces more efficient particle internalization and trafficking to lysosomes, reaching the maximum concentration in lysosomes at 2 h, than the lower FA density of 10 FA-PEG3.5kDa-SH molecules/particle. Pharmacological inhibition of endocytic pathways and TEM analysis showed that particles with high folate density are internalized predominantly by a clathrin-independent process

Stimuli‐Responsive Multifunctional Nanomedicine for Enhanced Glioblastoma Chemotherapy Augments Multistage Blood‐to‐Brain Trafficking and Tumor Targeting

Minimal therapeutic advances have been achieved over the past two decades for glioblastoma (GBM), which remains an unmet clinical need. Here, hypothesis‐driven stimuli‐responsive nanoparticles (NPs) for docetaxel (DTX) delivery to GBM are reported, with multifunctional features that circumvent insufficient blood‐brain barrier (BBB) trafficking and lack of GBM targeting—two major hurdles for anti‐GBM therapies. NPs are dual‐surface tailored with a i) brain‐targeted acid‐responsive Angiopep‐2 moiety that triggers NP structural rearrangement within BBB endosomal vesicles, and ii) L‐Histidine moiety that provides NP preferential accumulation into GBM cells post‐BBB crossing. In tumor invasive margin patient cells, the stimuli‐responsive multifunctional NPs target GBM cells, enhance cell uptake by 12‐fold, and induce three times higher cytotoxicity in 2D and 3D cell models. Moreover, the in vitro BBB permeability is increased by threefold. A biodistribution in vivo trial confirms a threefold enhancement of NP accumulation into the brain. Last, the in vivo antitumor efficacy is validated in GBM orthotopic models following intratumoral and intravenous administration. Median survival and number of long‐term survivors are increased by 50%. Altogether, a preclinical proof of concept supports these stimuli‐responsive multifunctional NPs as an effective anti‐GBM multistage chemotherapeutic strategy, with ability to respond to multiple fronts of the GBM microenvironment

Star-like guanidyl-oligosaccharidic polymer as a new nanoplatform for sirna delivery towards cancer cells

Gene therapy is defined as the treatment or prevention of human diseases by introduction of new genetic material, such as therapeutic oligonucleotides (e.g. siRNA, miRNA, etc.), into specific cells.1 In the last few decades the research community has invested substantial resources in the delivery of therapeutic oligonucleotides (ONs). Unfortunately, due to their anionic nature, high molecular weight and structural fragility, ON administration suffers from poor bioavailability and biological efficiency. In fact, these macromolecules may undergo fast degradation by serum nucleases, negligible transmembrane transport, off-target profile and also elicit immunogenic response.2 So far, cationic nanocarriers (including polymers and lipids) represent the most promising strategies to protect and deliver ONs. In this work, we have designed a novel cationic polymer for ON (i.e. siRNA) complexation driven by coulombic interactions. Starting from an oligosaccharide (maltotriose) we synthesize through a multistep protocol a star-like molecule (Agm6-M-COOH) using Atom Transfer Radical Polymerization (ATRP) which was then conjugated to polyethylene glycol (PEG) to improve the biocompatibility of the system (Agm6-M-PEG). Complexes, here referred as polyplexes, were prepared by self-assembly of the cationic polymer with a model ON. The obtained polyplexes have been found to improve the pharmacokinetic and pharmacodynamics parameters of ONs generating a valuable tool for cancer treatment. Electrophoresis gel shift assay was used to investigate polyplexes formation at different nitrogen/phosphate (N/P) ratios starting from 0.5 up to 5. A complete complexation of the siRNA was achieved at the N/P ratio of 3. The interaction thermodynamic between the cationic polymer and ONs was explored by Isothermal Titration Calorimetry (ITC). The polyplexes were characterized by Dynamic Light Scattering (DLS) showing a mean size in the range of 50-75 nm and a surface charge in the range from -5 mV to +11 mV depending on the N/P ratio. A narrow polydispersity index (PDI) was observed confirming that polyplexes formulations were monodispersed. Polyplexes formed at N/P ratio 3 and 5 showed the most performing physico-chemical features and were thus further characterized and tested in vitro. Morphological analysis of these formulations were performed by Transmission Electron Microscopy (TEM) showing a rod-shape particles. Moreover, polyplexes showed also a satisfactory serum stability and remarkable siRNA protection from nucleases. The strength and the stability of the polymer/siRNA interaction was confirmed by the heparin displacement test performed by electrophoresis. In fact, 2.5 IU/mL heparin concentration, about 16 fold higher compared to the heparin concentration in blood (0.15 IU/mL4) was required to achieve total siRNA displacement from polyplexes. Preliminary in vitro studies were performed on human MCF-7 breast adenocarcinoma cells, human KB cervical carcinoma cells and murine MC3T3-E1 embryonic fibroblast showing a good biocompatibility and cell uptake of the polyplexes. In addition, the different composition and structure of the polyplexes was also found to affect the cell up-take: polyplexes with smaller aspect ratio were taken up more efficiently than polyplexes with larger aspect ratio. The hemolytic activity was also assessed by measuring red blood cells (RBCs) lysis and negligible hemolysis was observed for both the formulations tested. To enhance the site-selectivity of the system, targeted polyplexes towards tumor cells were formulated. To this aim, the cationic polymer was functionalized at one end-chain with folic acid (Agm6-M-PEG-FA). The silencing activity of polyplexes decorated with different amount of folic acid was evaluated and polyplexes formed with 25 %w/w of Agm6-M-PEG-FA were found possesses the better activity. In this contest, a comparative study between folate and non-folate polyplexes was performed. Aiming at identify the best in vitro and in vivo model, several cell lines were screened for the expression of the folate receptor finally selecting the human MDA-MB-231 breast adenocarcinoma cells. Silencing and toxicity studies of polyplexes were then performed on HeLa and MDA-MB-231 cells. For this purpose, siRac1 was used as a therapeutic siRNA for its ability to inhibit cell migration, metastatization and cell invasion3. The folate and non-folate polyplexes were found highly biocompatible (cell viability >80% for all the concentration tested assessed by MTT assay) and immuno-compatible (assessed by immune stimulation). Folate-targeted polyplexes showed a gene silencing about two folds higher compared to non-folate polyplexes (86% and 53%, respectively). The ability of the polyplexes to inhibit cell migration was assessed by wound healing assay. The gap closure was observed after 12 hours and the results showed a 40% inhibition of the cell migration when cells were treated with 500 nM siRNA equivalent dose for folate polyplexes. On the contrary, non-folate polyplexes inhibited cell migration of only 10%. Despite the different silencing activity and inhibition of cell migration, no relevant differences in the cell uptake were observed between the targeted and non-targeted system by confocal analysis. Intracellular trafficking studies were then performed. Folate-targeted polyplexes were found in the cytoplasm and did not co-localize with early endosomes and lysosomes. Conversely, non-folate polyplexes fully co-localized with early endosomes. The cell internalization mechanism was also evaluated by immunostaining the caveolin-1 and clathrin heavy chain, responsible for the caveolae and clathrin-mediated uptake, respectively. Caveolae- mediated internalization was observed for folate-targeted polyplexes while a clathrin-mediated mechanism was assessed for non-targeted polyplexes. These results highlighted the folic acid involvement on the caveolae mediated uptake which was found to enhance the gene silencing activity. Finally, the systemic toxicity of folate and non-folate polyplexes using a siRNA concentration ranging from 1 to 6 mg/kg was evaluated on Nu/Nu female mice by stepwise escalation dose administered by intravenous injection until the maximum tolerated dose for short period. No toxicity was found for all the doses used for both formulations. Finally, folate polyplexes accumulation in the tumor was investigated observing a ~500-fold and ~65-fold increase of Rac1 siRNA tumor accumulation in comparison with saline or scramble siRNA (siEGFP) treatment, respectively. It was possible to detect a linear correlation between the size of the tumor and the polyplexes accumulation. Histologic studies confirmed the accumulation in the tumor tissue.La terapia genica viene definita come il trattamento o la prevenzione di malattie umane attraverso l’introduzione all’interno di cellule specifiche di nuovo materiale genetico quali gli oligonucleotidi terapeutici (siRNA, miRNA etc.)1. Negli ultimi decenni, molti ricercatori hanno investito nello sviluppo in sistemi di delivery di oligonucleotidi terapeutici (ONs). Tuttavia, la somministrazione di ONs risulta difficile e si osserva una bassa biodisponibilità ed efficacia biologica data la loro natura anionica, l’elevato peso molecolare e le fragilità strutturali. Queste macromolecole, subiscono una rapida degradazione da parte delle nucleasi del siero, possiedono un trascurabile trasporto transmembrana, possono accumularsi in maniera aspecifica e stimolare il sistema immunitario2. Nanocarrier cationici (ad esempio polimeri e lipidi) rappresentano una delle più promettenti strategie per proteggere e trasportare ONs all’interno di cellule malate. In questo lavoro, è stato disegnato un nuovo un nuovo polimero cationico per la complessazione di siRNA basata su interazioni di tipo colombiano (attrazione di carica). Partendo da un oligosaccaride (il maltotriosio) una molecola dalla struttura a stella è stata sintetizzata (Agm6-M-COOH) tramite un protocollo multi-step utilizzando una polimerizzazione a trasferimento atomico radicale (ATRP). A questa molecola è stata quindi coniugata una catena di glicole polietilenico (PEG) al fine di aumentare la biocompatibilità del sistema (Agm6-M-PEG). I complessi, che normalmente vengono chiamati “poliplessi”, sono stati preparati per auto assemblaggio tra il polimero cationico e ONs. I poliplessi hanno dimostrato migliorare i parametri farmacocinetici e farmacodinamici degli ON generando un interessante modello per la terapia antitumorale. I saggi elettroforetici sono stati impiegati per valutare la formazione dei poliplessi a diversi rapporti azoto/fosforo (N/P ratio) partendo da 0.5 fino a 5. La completa complessazione del siRNA è stata ottenuta a valori di N/P ratio pari a 3. Le interazioni termodinamiche tra il polimero cationico e ONs sono state esplorate attraverso la calorimetria a titolazione isotermica (Isothermal Titration Calorimetry, ITC). I poliplessi sono stati caratterizzati tramite Dynamic Light Scattering (DLS) mostrando un dimensione media nel range di 50-75 nm e una carica superficiale in un intervallo di valori da -5 mV a +11 mV in funzione del rapporto N/P. Un basso indice di polidispersione è stato osservato, confermando che le formulazioni sono monodisperse. I poliplessi aventi 3 e 5 N/P ratio hanno mostrato possedere le migliori caratteristiche fisico-chimiche e sono stati ulteriormente caratterizzati e testati in vitro. Le analisi morfologiche delle formulazioni sono state effettuate attraverso la microscopia elettronica a trasmissione. Da tale analisi si è osservato che le particelle possiedono una struttura a bastoncello. I poliplessi inoltre hanno evidenziato la capacità di proteggere siRNA dalle nucleasi e una buona stabilità in siero. La forza dell’interazione tra il polimero e il siRNA e la stabilità dei poliplessi è stata ulteriormente confermata via elettroforesi attraverso il test di spiazzamento con eparina. Infatti, il totale spiazzamento di siRNA dal complesso avviene solo a concentrazioni di eparina pari a 2.5 IU/mL, circa 16 volte superiori rispetto a quelle presenti nel torrente ematico (0.15 IU/mL). Studi preliminari in vitro sono stati effettuati su cellule MCF-7 di adenocarcinoma mammario umano, cellule KB di carcinoma della cervice uterina umana e cellule MC3T3-E1 di fibroblasti di embrioni murini e hanno mostrato una buona biocompatibilità e uptake cellulare. Inoltre, è stato osservato come la differente composizione e struttura dei poliplessi influenzi l’uptake cellulare: i poliplessi con un aspect ratio più piccolo sono internalizzati più efficientemente rispetto ai poliplessi che presentano un aspect ratio maggiore. L’attività emolitica dei poliplessi è stata presa in considerazione misurando l’effetto dei poliplessi sui globuli rossi del sangue. Dai test efettuati è emerso un trascurabile effetto emolitico per entrambe le formulazioni in analisi. Al fine di aumentare la sito-selettività del sistema, i poliplessi sono stati successivamente formulati al fine di essere direzionati contro cellule tumorali. Per questa ragione, il polimero cationico è stato funzionalizzato alla fine della catena con acido folico (Agm6-M-PEG-FA). L’attività di silencing dei poliplessi decorati con diverse quantità di acido folico è stata valutata utilizzando siRac1. Dallo studio è emerso che i poliplessi costituiti con il 25% w/w di Agm6-M-PEG-FA sono risultati i più efficienti nella riduzione dell’espressione di Rac1. In questo contesto, uno studio comparativo tra poliplessi aventi 25%w/w di polimero derivatizzato con acido folico e poliplessi senza acido folico è stato intrapreso. Al fine di individuare il miglior modello in vitro e in vivo, è stato fatto uno screening di diverse linee cellulari per valutare l’espressione del recettore dell’acido folico e le cellule MDA-MB-231 di adenocarcinoma mammario sono state selezionate come quelle a maggior espressione. Studi di silenziamento e tossicità da parte dei poliplessi sono stati effettuati. Per questo motivo, siRac1 è stato utilizzato come siRNA terapeutico per le sue capacità di inibire la migrazione cellulare, la formazione di metastasi e l’invasione cellulare3. I poliplessi con e senza acido folico hanno mostrato un’elevata biocompatibilità (maggiore dell’80% per tutte le concentrazioni testate con il saggio MTT) e una elevata immuno-compatibilità (valutata attraverso stimolazione del sistema immunitario). I poliplessi direzionati con acido folico hanno mostrato un miglior gene silencing di circa due volte superiore rispetto ai poliplessi non direzionati (86% e 53% rispettivamente). L’abilità dei poliplessi di inibire la migrazione cellulare è stata osservata attraverso il saggio di wound healing. La chiusura del “taglio” è stata osservata dopo 12 ore e i risultati hanno mostrato un’inibizione del 40% della migrazione cellulare dopo trattamento con una dose equivalente di siRNA pari a 500 nM di poliplessi decorati con acido folico. Al contrario, poliplessi non decorati inibiscono la migrazione solo del 15%. Nonostante la differente attività di silencing e di inibizione della migrazione cellulare, non sono state osservate differenze di internalizzazione tra i poliplessi con e senza acido folico con analisi confocale e Imagestream. Pertanto, studi di traffiking intracellulare sono stati eseguiti. Poliplessi portanti acido folico sono stati osservati nel citoplasma e nessuna co-localizzazione con endosomi e lisosomi è stata osservata. Al contrario, poliplessi non portanti acido folico sono stati visualizzati all’interno di entrambi i compartimenti cellulari. I meccanismi di internalizzazione cellulare sono stati valutati attraverso la marcatura di caveolin-1 e di clathrin heavy chain attraverso anticorpi. L’internalizzazione caveolae-mediata è stata osservata per i poliplessi con acido folico mentre per i poliplessi senza acido folico un meccanismo di endocitosi mediato da clatrine è stato osservato. Questi risultati evidenziano un coinvolgimento dell’acido folico nel processo di uptake mediato da caveolae che è stato visto incrementare l’attività di silenziamento genico. La tossicità sistemica dei poliplessi con e senza acido folico è stata studiata su femmine di topo Nu/Nu usando dosi equivalenti di siRNA nell’intervallo di 1-6 mg/kg. L’obiettivo di questo test è di raggiungere la massima dose tollerata attraverso una crescente somministrazione di siRNA. Tuttavia nessuna tossicità è stata ottenuta per entrambe le formulazioni. Infine, è stato studiato in vivo l’accumulo dei poliplessi con acido folico. Da questo studio si è osservato un accumulo di Rac1 rispetto alla soluzione salina o a un siRNA non codificante (siEGFP) di circa 500 e 65 volte rispettivamente. Inoltre è stato possibile individuare una correlazione tra le dimensioni del tumore e l’accumulo dei poliplessi. Studi istologici hanno confermato l’accumulo dei poliplessi nel tessunto tumorale

Star-like guanidyl-oligosaccharidic polymer as a new nanoplatform for sirna delivery towards cancer cells

Gene therapy is defined as the treatment or prevention of human diseases by introduction of new genetic material, such as therapeutic oligonucleotides (e.g. siRNA, miRNA, etc.), into specific cells.1 In the last few decades the research community has invested substantial resources in the delivery of therapeutic oligonucleotides (ONs). Unfortunately, due to their anionic nature, high molecular weight and structural fragility, ON administration suffers from poor bioavailability and biological efficiency. In fact, these macromolecules may undergo fast degradation by serum nucleases, negligible transmembrane transport, off-target profile and also elicit immunogenic response.2 So far, cationic nanocarriers (including polymers and lipids) represent the most promising strategies to protect and deliver ONs. In this work, we have designed a novel cationic polymer for ON (i.e. siRNA) complexation driven by coulombic interactions. Starting from an oligosaccharide (maltotriose) we synthesize through a multistep protocol a star-like molecule (Agm6-M-COOH) using Atom Transfer Radical Polymerization (ATRP) which was then conjugated to polyethylene glycol (PEG) to improve the biocompatibility of the system (Agm6-M-PEG). Complexes, here referred as polyplexes, were prepared by self-assembly of the cationic polymer with a model ON. The obtained polyplexes have been found to improve the pharmacokinetic and pharmacodynamics parameters of ONs generating a valuable tool for cancer treatment. Electrophoresis gel shift assay was used to investigate polyplexes formation at different nitrogen/phosphate (N/P) ratios starting from 0.5 up to 5. A complete complexation of the siRNA was achieved at the N/P ratio of 3. The interaction thermodynamic between the cationic polymer and ONs was explored by Isothermal Titration Calorimetry (ITC). The polyplexes were characterized by Dynamic Light Scattering (DLS) showing a mean size in the range of 50-75 nm and a surface charge in the range from -5 mV to +11 mV depending on the N/P ratio. A narrow polydispersity index (PDI) was observed confirming that polyplexes formulations were monodispersed. Polyplexes formed at N/P ratio 3 and 5 showed the most performing physico-chemical features and were thus further characterized and tested in vitro. Morphological analysis of these formulations were performed by Transmission Electron Microscopy (TEM) showing a rod-shape particles. Moreover, polyplexes showed also a satisfactory serum stability and remarkable siRNA protection from nucleases. The strength and the stability of the polymer/siRNA interaction was confirmed by the heparin displacement test performed by electrophoresis. In fact, 2.5 IU/mL heparin concentration, about 16 fold higher compared to the heparin concentration in blood (0.15 IU/mL4) was required to achieve total siRNA displacement from polyplexes. Preliminary in vitro studies were performed on human MCF-7 breast adenocarcinoma cells, human KB cervical carcinoma cells and murine MC3T3-E1 embryonic fibroblast showing a good biocompatibility and cell uptake of the polyplexes. In addition, the different composition and structure of the polyplexes was also found to affect the cell up-take: polyplexes with smaller aspect ratio were taken up more efficiently than polyplexes with larger aspect ratio. The hemolytic activity was also assessed by measuring red blood cells (RBCs) lysis and negligible hemolysis was observed for both the formulations tested. To enhance the site-selectivity of the system, targeted polyplexes towards tumor cells were formulated. To this aim, the cationic polymer was functionalized at one end-chain with folic acid (Agm6-M-PEG-FA). The silencing activity of polyplexes decorated with different amount of folic acid was evaluated and polyplexes formed with 25 %w/w of Agm6-M-PEG-FA were found possesses the better activity. In this contest, a comparative study between folate and non-folate polyplexes was performed. Aiming at identify the best in vitro and in vivo model, several cell lines were screened for the expression of the folate receptor finally selecting the human MDA-MB-231 breast adenocarcinoma cells. Silencing and toxicity studies of polyplexes were then performed on HeLa and MDA-MB-231 cells. For this purpose, siRac1 was used as a therapeutic siRNA for its ability to inhibit cell migration, metastatization and cell invasion3. The folate and non-folate polyplexes were found highly biocompatible (cell viability >80% for all the concentration tested assessed by MTT assay) and immuno-compatible (assessed by immune stimulation). Folate-targeted polyplexes showed a gene silencing about two folds higher compared to non-folate polyplexes (86% and 53%, respectively). The ability of the polyplexes to inhibit cell migration was assessed by wound healing assay. The gap closure was observed after 12 hours and the results showed a 40% inhibition of the cell migration when cells were treated with 500 nM siRNA equivalent dose for folate polyplexes. On the contrary, non-folate polyplexes inhibited cell migration of only 10%. Despite the different silencing activity and inhibition of cell migration, no relevant differences in the cell uptake were observed between the targeted and non-targeted system by confocal analysis. Intracellular trafficking studies were then performed. Folate-targeted polyplexes were found in the cytoplasm and did not co-localize with early endosomes and lysosomes. Conversely, non-folate polyplexes fully co-localized with early endosomes. The cell internalization mechanism was also evaluated by immunostaining the caveolin-1 and clathrin heavy chain, responsible for the caveolae and clathrin-mediated uptake, respectively. Caveolae- mediated internalization was observed for folate-targeted polyplexes while a clathrin-mediated mechanism was assessed for non-targeted polyplexes. These results highlighted the folic acid involvement on the caveolae mediated uptake which was found to enhance the gene silencing activity. Finally, the systemic toxicity of folate and non-folate polyplexes using a siRNA concentration ranging from 1 to 6 mg/kg was evaluated on Nu/Nu female mice by stepwise escalation dose administered by intravenous injection until the maximum tolerated dose for short period. No toxicity was found for all the doses used for both formulations. Finally, folate polyplexes accumulation in the tumor was investigated observing a ~500-fold and ~65-fold increase of Rac1 siRNA tumor accumulation in comparison with saline or scramble siRNA (siEGFP) treatment, respectively. It was possible to detect a linear correlation between the size of the tumor and the polyplexes accumulation. Histologic studies confirmed the accumulation in the tumor tissue

Immunotherapy for glioblastoma: the promise of combination strategies

Glioblastoma (GBM) treatment has remained almost unchanged for more than 20 years. The current standard of care involves surgical resection (if possible) followed by concomitant radiotherapy and chemotherapy. In recent years, immunotherapy strategies have revolutionized the treatment of many cancers, increasing the hope for GBM therapy. However, mostly due to the high, multifactorial immunosuppression occurring in the microenvironment, the poor knowledge of the neuroimmune system and the presence of the blood−brain barrier, the efficacy of immunotherapy in GBM is still low. Recently, new strategies for GBM treatments have employed immunotherapy combinations and have provided encouraging results in both preclinical and clinical studies. The lessons learned from clinical trials highlight the importance of tackling different arms of immunity. In this review, we aim to summarize the preclinical evidence regarding combination immunotherapy in terms of immune and survival benefits for GBM management. The outcomes of recent studies assessing the combination of different classes of immunotherapeutic agents (e.g., immune checkpoint blockade and vaccines) will be discussed. Finally, future strategies to ameliorate the efficacy of immunotherapy and facilitate clinical translation will be provided to address the unmet medical needs of GBM

Synergistic effect of doxorubicin lauroyl hydrazone derivative delivered by α-tocopherol succinate micelles for the treatment of glioblastoma

We hypothesized that tocopherol succinate (TOS) and D-α-tocopherol polyethylene2000 succinate (TPGS(2000)) micelles could work as a drug delivery system while enhancing the anti-cancer efficacy of doxorubicin lauryl hydrazone derivative (DOXC(12)) for the treatment of glioblastoma. The DOXC(12)-TOS-TPGS(2000) micelles were formulated with synthesized DOXC(12) and TPGS(2000). They showed a high drug loading of hydrophobic DOXC(12) (29%), a size of <100 nm and a pH sensitive drug release behaviour. In vitro, fast uptake of DOXC(12)-TOS-TPGS(2000) micelles by GL261 cells was observed. For cytotoxicity, DOXC(12)-TOS-TPGS(2000) micelles were evaluated on two glioblastoma cell lines and showed synergism between DOXC(12) and TOS-TPGS(2000.) The higher cytotoxicity of DOXC(12)-TOS-TPGS(2000) micelles was mainly caused by necrosis. The DOXC(12)-TOS-TPGS(2000) micelles seem to be a promising delivery system for enhancing the anticancer efficacy of doxorubicin in glioblastoma (GBM)

Rationally designed drug delivery systems for the local treatment of resected glioblastoma.

Glioblastoma (GBM) is a particularly aggressive brain cancer associated with high recurrence and poor prognosis. The standard of care, surgical resection followed by concomitant radio- and chemotherapy, leads to low survival rates. The local delivery of active agents within the tumor resection cavity has emerged as an attractive means to initiate oncological treatment immediately post-surgery. This complementary approach bypasses the blood-brain barrier, increases the local concentration at the tumor site while reducing or avoiding systemic side effects. This review will provide a global overview on the local treatment for GBM with an emphasis on the lessons learned from past clinical trials. The main parameters to be considered to rationally design fit-of-purpose biomaterials and develop drug delivery systems for local administration in the GBM resection cavity to prevent the tumor recurrence will be described. The intracavitary local treatment of GBM should i) use materials that facilitate translation to the clinic; ii) be characterized by easy GMP effective scaling up and easy-handling application by the neurosurgeons; iii) be adaptable to fill the tumor-resected niche, mold to the resection cavity or adhere to the exposed brain parenchyma; iv) be biocompatible and possess mechanical properties compatible with the brain; v) deliver a therapeutic dose of rationally-designed or repurposed drug compound(s) into the GBM infiltrative margin. Proof of concept with high translational potential will be provided. Finally, future perspectives to facilitate the clinical translation of the local perisurgical treatment of GBM will be discussed