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SVILUPPO DI SISTEMI NANOPARTICELLARI D'ORO PER IL DIREZIONAMENTO MULTI-MODALE AL TUMORE
No full textThe present research project was aimed at developing smart nano-systems able to selectively respond with morphological alterations to external physio-pathologic stimuli. These systems are intended for diagnostic or therapeutic applications in tumor treatment. The responsiveness of these systems is intended to improve the site-selective targeting efficiency and reduce uncontrolled disposition in healthy tissues.
Smart nano-systems have been obtained using gold nanoparticles (AuNPs) as platform. AuNPs have been surface coated with responsive polymeric components to endow stealth properties in physiologic conditions after injection in the bloodstream. The tumor interstitium is characterized by altered pH and temperature with respect to normal tissues. Thus, when the nano-system reaches the tumour, the responsive coating can undergo morphological modifications which modulate the interface properties of the nanoparticles. This will promote their interaction with the biological surfaces (i.e. cells, tissues).
In the project here discussed, different functionalization strategies of AuNPs have been investigated in order to develop nano-carriers provided with multimodal targeting to the tumor. Gold nanoparticles were surface decorated with targeting agents and thermosensitive or pH-sensitive polymers. This allowed producing nano-carriers in which bio-recognition can be controlled by temperature or pH. In physiological conditions, the polymer on particles surface can hide the targeting moiety while in the tumor tissue the thermosensitive or pH sensitive polymer chains collapse provoking the targeting agent exposition and thus promoting cellular internalization. As a result, the multimodal targeting will decrease aspecific biorecognition and increase site-specificity.
Gold nanoparticles were produced by Laser ablation technique in aqueous solution without addition of surfactants or stabilizing agents. This finely controlled method allows for obtaining AuNPs with a size of 18 nm.
The components used for gold nanoparticles surface functionalization have been thiolated since sulphidril groups promptly react with metal gold with high yield.
AuNPs have been surface decorated with biotin-SH and the 8 kDa thermosensitive polymer N-isopropylacrylamide-co-acrylamide-SH (pNIPAm-co-Am-SH), characterized by a low critical solution temperature (LCST) of 37 °C. The nanosystem obtained was tested by ELISA enzymatic assay to evaluate its ability to bind selectively avidin, chosen as model target, as a function of temperature. The results showed that the system behavior is thermally controlled since functionalized gold nanoparticles can only bind avidin when temperature is increased above the polymer LCST at which it collapses. Furthermore, the investigation highlighted the role of the biotin amount and of the biotin/polymer molar ratio on gold nanoparticles surface in affecting the system performance.
The system has been further investigated, according to the information acquired, with the biotinylated thermosensitive AuNPs. Gold nanoparticles were surface functionalized with folic acid-SH and the thermosensitive polymer pNIPAm-co-Am-SH to achieve temperature controlled targeting towards cancer cells overexpressing folate receptor (HiFR). Stability studies performed in PBS showed that thermosensitive polymer is paramount to avoid nanoparticles aggregation in the presence of salts. At temperature below polymer LCST, gold nanoparticles are very stable while above the LCST, only a mild aggregation was detected. Folated thermosensitive AuNPs have been tested in vitro with tumor cells overexpressing and non-expressing the folate receptor. The results showed that folated thermosensitive nanoparticles incubated with HiFR cell lines are internalized by cells and dispose in the citosol only at temperature above the polymer LCST.
The concept of multimodal targeting was extended to the development of pH-sensitive gold nanoparticles, using polymers able to respond with morphological alterations to environmental pH changes. Ideally, acid-sensitive polymers adequate for this purpose should be soluble and in coil form at pH 7.4 and convert into insoluble globular state, as a consequence of their protonation, in acidic tumor environment. To this aim, different pH-sensitive polymers were produced in order to have an array of materials with satisfactory pKa (in the physiopathological range) to chose from for gold nanoparticles functionalization. The materials were designed in order to endow acid sensitiveness to the decorated nanoparticles in the physic-pathological range. A 2-(methacryloyloxy)ethyl 3-chloro-4-hydroxybenzoate monomer (MOECHB) was chosen and pH responsive materials were obtained by living radical polymerization technique, namely reversible addition fragmentation chain transfer (RAFT). One homopolymer and various random and block copolymers using the 2-(methacryloyloxy)ethyl 3-chloro-4-hydroxybenzoate and hydrophilic monomers, namelly metoxy(polyetilenglycole methacrylate (mPEGMA475) or glycerol methacrylate (GMA), were synthesized. All the polymers obtained were characterised by potentiometric titration to determine the pKa value, and turbidimetric analysis to measure the cloud point. Furthermore, their pH-dependant behavior was investigated by dynamic light scattering at various pH conditions. One random and two block copolymers, composed by MOECHB and GMA, were selected for further investigation. Decreasing pH from 7.4 to 6.5, as in physiopathologic condition, they showed a conversion from the soluble to the aggregated state. One additional block copolymer, made by MOECHB and mPEGMA475, will be also evaluated: in the same physiopathologic condition, it outlined morphological modification, albeit without aggregation. The results displayed that pH responsiveness is modulated by the 2-(methacryloyloxy)ethyl 3-chloro-4-hydroxybenzoate/hydrophilic monomer molar ratio in the polymer backbone and by the polymer molecular weight.Il presente progetto di ricerca ha riguardato lo sviluppo di un nano-sistema intelligente, capace di rispondere selettivamente a stimoli esterni di tipo fisiopatologico con alterazioni morfologiche. Tali sistemi sono stati disegnati per un impiego in diagnostica o nella terapia antitumorale. La responsività di questi sistemi ha lo scopo di migliorare l’ efficienza del direzionamento selettivo e di ridurre la distribuzione aspecifica nei tessuti sani.
Sistemi intelligenti sono stati ottenuti a partire da nanoparticelle d’ oro (AuNPs), a cui sono stati associati polimeri responsivi, che conferiscono loro caratteristiche stealth in condizioni fisiologiche in seguito ad iniezione nel torrente circolatorio. E’ noto che l’ interstizio tumorale è aratterizzato da valori di pH e temperatura alterati rispetto al tessuto sano. Di conseguenza, quando il nano-sistema raggiunge il tumore, il rivestimento polimerico può2 subire modifiche morfologiche che modulano le proprietà delle nanoparticelle. Questo promuoverà la loro interazione con le superfici biologiche, quali cellule e tessuti.
Nel progetto qui discusso, sono state valutate diverse strategie di funzionalizzazione di AuNPs, allo scopo di sviluppare nano-carriers caratterizzati da un direzionamento multi-modale al tumore.
Nanoparticelle d’ oro sono state derivatizzate superficialmente con agenti di direzionamento e polimeri termosensibili o pH sensibili, affinchè il riconoscimento selettivo del tessuto tumorale da parte dei nano-carriers sia controllato dalle condizioni di temperatura o di pH circostanti. In condizioni fisiologiche i polimeri presenti sulla superficie delle AuNPs schermano l’ agente di targeting, mentre nel tessuto tumorale grazie alla temperatura o pH alterati, essi collassano determinando l’ esposizione del direzionante e promuovendo l’ endocitosi cellulare. Come conseguenza, il direzionamento multi-modale diminuirà il bio-riconoscimento aspecifico a favore invece della sito-specificità.
Le nanoparticelle d’ oro utilizzate in questo progetto sono state prodotte mediante laser ablation in soluzione acquosa, senza utilizzo di sostanze surfattanti e stabilizzanti. Attraverso questo processo controllato e riproducibile si sono ottenute dispersioni di particelle diluite, con diametro medio di 18 nm.
Per la funzionalizzazione superficiale di nanoparticelle d’ oro i materiali utilizzati sono stati tiolati, nota l’ elevata capacità di coniugazione di gruppi sulfidrilici su superfici d’ oro metallico.
Il primo sistema sviluppato in questo progetto di tesi ha riguardo l’ impiego di nanoparticelle d’ oro in grado di rispondere a stimoli termici. AuNPs sono state modificate superficialmente con biotina-SH e con un polimero termosensibile di 8 kDa, N-isopropylacrylamide-co-acrylamide-SH (pNIPAm-co-Am-SH), caratterizzato da una low critical solution temperature (LCST) di 37 °C. Il sistema ottenuto è stato testato mediante saggio enzimatico di tipo ELISA per valutarne la capacità di binding selettivo ad avidina, scelta come modello, in funzione della temperatura. I risultati hanno mostrato che il comportamento del sistema è controllato dalla temperatura, in quanto le nanoparticelle sono in grado di legare l’ avidina solo quando la temperatura è superiore all’ LCST del polimero, ovvero quando il polimero stesso collassa. Lo studio ha inoltre evidenziato che la quantità assoluta di biotina e i rapporti molari biotina/polimero sulla superficie delle nanoparticelle condizionano in modo rilevante le performance del sistema
Sulla base delle ottimizzazioni realizzate con le nanoparticelle direzionate con biotina, il sistema è stato modificato superficialmente con acido folico e lo stesso polimero termosensibile (pNIPAm-co-Am-SH), allo scopo di ottenere un direzionamento verso cellule tumorali sovraesprimenti il recettore per il folato (HiFR). Gli studi di stabilità in PBS hanno mostrato come il polimero termosensibile sia indispensabile per impedire l’ aggregazione delle particelle in presenza di sali. A temperatura inferiore all’ LCST del polimero le nanoparticelle sono stabili, ed aggregano solo marginalmente a temperatura superiore. AuNPs termosensibili e funzionalizzate con acido folico sono state testate in vitro su due diverse linee celluari, sovraesprimenti e non il recettore per l’ acido folico. Gli studi hanno mostrato che solo le particelle termosensibili modificate con acido folico e incubate con linee cellulari HiFR, a temperatura superiore all’ LCST del polimero, vengono internalizzate dalle cellule e si distribuiscono nel citosol.
Il concetto di targeting multi-modale è stato successivamente ampliato per lo sviluppo di nanoparticelle d’ oro pH-sensibili, utilizzando polimeri in grado di rispondere con cambiamenti morfologici ad alterazioni del pH ambientale.
Idealmente, polimeri acido sensibili adeguati allo scopo devono essere solubili ed in conformazione estesa a pH 7.4, ma subire una conversione allo stato globulare insolubile in seguito a protonazione nell’ ambiente acido tumorale. Per raggiungere questo obiettivo sono stati sintetizzati diversi polimeri pH-sensibili, in modo tale da disporre di una gamma di materiali caratterizzati da valori di pKa e cloud point adeguati allo scopo, tra cui poter selezionare il più1 adatto per la funzionalizzazione superficiale di nanoparticelle d’ oro. I materali sono stati disegnati per conferire alle AuNPs proprietà di acido-sensibilità nel range fisiopatologico.
E’ stato selezionato e sintetizzato un monomero acido-sensibile, 2-(metacriloilossi)etil 3-cloro-4-idrossibenzoato (MOECHB), a partire dal quale sono stati poi prodotti polimeri pH responsivi. La metodica adottata per la reazione di polimerizzazione fa parte delle cosiddette tecniche di polimerizzazioni viventi, ovvero la reversible addition fragmentation chain transfer (RAFT). Sono stati sintetizzati un omopolimero e diversi copolimeri sia random che a blocchi utilizzando MOECHB e monomeri idrofilici, nello specifico metossi(polietilenglicole metacrilato) (mPEGMA475), e glicerolo metacrilato (GMA). Tutti i polimeri ottenuti sono stati caratterizzati mediante titolazione potenziometrica per determinarne il pKa, e sottoposti ad analisi turbidimetrica per misurare il punto di intorbidimento (cloud point). Il loro comportamento pH-dipendente è stato inoltre investigato mediante analisi di dynamic light scattering (DLS) effettuate a diversi valori di pH. Sono stati selezionati un copolimero random e due copolimeri a blocchi, composti da MOECHB e GMA, per ulteriori studi di funzionalizzazione di nanoparticelle d’ oro. Infatti questi materiali hanno mostrato una conversione da una forma solubile ad una aggregata insolubile per diminuzione del pH della soluzione da 7.4 a 6.5, che mima le condizioni fisiopatologiche. Un ulteriore copolimero a blocchi di MOECHB con mPEGMA475 sarà valutato, in quanto nelle medesime condizioni ha mostrato modifiche morfologiche, sebbene non si sia osservata la formazione di prodotti insolubili.
I risultati delle analisi hanno evidenziato infine che la responsività al pH è modulata dal rapporto molare 2-(metacriloilossi)etil 3-cloro-4-idrossibenzoato/monomero idrofilico nella composizione polimerica, così come dal peso molecolare del polimero
Phosphonium polymers for gene delivery
Full text linkPhosphonium salt-containing polymers have very recently started to emerge as attractive materials for the engineering non-viral gene delivery systems. Compared to more frequently utilised ammonium-based polymers, some of these materials can enhance binding of nucleic acid at lower polymer concentration, and mediate good transfections efficiency, with low cytotoxicity. However, for years one of the main hurdles for their widespread application has been the lack of general routes for their synthesis. To date a range of polymerisation techniques have been explored, with the majority of them focussing on radical polymerisation, especially controlled radical polymerisation (CRP) techniques – ATRP, NMP and RAFT polymerisation - both by polymerisation of phosphonium monomers or by post-polymerisation modification of polymer intermediates. This review article aims at discussing key differences and similarities between phosphonium-and other analogous cations, how these affect binding to polynucleotides, and will provide an overview of the phosphonium polymer systems that have been utilised for gene delivery
pH-sensitive PEGylated liposomes for tumor cell targeting
No full textThe tumour tissue displays peculiar features, namely altered pH, temperature, redox potential and enzymatic pool, that can be exploited to yield site directed nanocarriers, namely liposomes. In particular, the pH of solid tumors is lower than in normal tissues. Therefore, it is convenient to trigger the response of adequately designed systems that can undergo morphological changes when exposed to the microenvironment alteration (1, 2).
Based on the peculiar features of tumor tissues and the drug delivery properties of liposomes, we investigated a novel class of pH-sensitive nanocarriers for anticancer drug delivery that can undergo structural changes under pathophysiological microenvironmental conditions thus providing for selective drug targeting. pH sensitive liposomes were obtained by surface decoration with stearoyl-PEG-poly-sulfadimethoxine (stearoyl-PEG-poly(SDM) that has been previously found to sense the pH decrease. Indeed, the acid character of sulfadimethoxine (pKa of 6.1) promotes the pH induced hydrophilic/hydrophobic switching as consequence of protonation, which may occur in the tumour site (3). Such physical alteration of the pH sensor stearoyl-PEG-poly-sulfadimethoxine is expected to be transferred to the surface properties of the nanocarriers, which may finally promote the interaction of the carrier with the cells or prompt the drug release
Chitosans as new tools against biofilms formation on the surface of silicone urinary catheters
No full textUrinary catheters contamination by microorganisms is a major cause of hospital acquired infections and represents a limitation for long-term use. In this work, biofilms of Klebsiella pneumoniae and Escherichia coli clinical isolates were developed on urinary catheters for 48 and 72 h in artificial urine medium (AUM) with different molecular weight chitosans (AUM-CS solutions) at pH 5.0. The number of viable bacteria was determined by standard plate count agar while crystal violet (CV) staining was carried out to assess biomass production (optical density at 570 nm) in the mentioned conditions. Re-growth of each strain was also evaluated after 24 h re-incubation of the treated catheters. Significant decreases of log CFU/catheter and biomass production were observed for all the biofilms developed in AUM-CS compared with the controls in AUM. The percentages of biofilm removal were slightly higher for E. coli biofilms (up to 90.4%) than those of K. pneumoniae (89.7%); in most cases, the complete inhibition of bacterial re-growth on treated catheter pieces was observed. Contact time influenced chitosan efficacy rather than its molecular weight or the biofilms age. The results confirmed the potentiality of chitosans as a biomacromolecule tool to contrast biofilm formation and reduce bacterial re-growth on urinary catheters
pH-Sensitive PEGylated Liposomes For Tumour Cell Targeting.
No full textIn this study we describe novel pH-sensitive PEGylated liposomes for site directed tumor targeting. pH-sensitive liposomes were obtained by including the amphiphilic molecule stearoyl-PEG-(sulfadimethoxine)11 in the liposome bilayer. At pH 6.5, the responsive nanosystem undergoes aggregation and selective association with tumor cells. The liposomes have stealth behavior at physiologic pH. Their responsiveness was found to be affected by the molar ratio of stearoyl-PEG-poly(sulfadimethoxine) assembled with the bilayer
Pullulan Bioconjugates For Tumor Targeting.
No full textA new pullulan based doxorubicin macromolecular prodrug was synthesized and tested in vitro. Folic acid conjugation conferred to the supramolecular system active targeting properties. Doxorubicin was conjugated to pullulan via pH sensitive bonds yielding a derivative stable in plasma. At pH 5.5 the drug was completely released in 96 hours. The carrier was found to be selectively toxic to folate overexpressing cell lines
Novel pH-responsive nanovectors for controlled release of ionisable drugs
No full textA family of novel ‘smart’ drug nanovectors based on 2-(methacryloyloxy)ethyl-3-chloro-4-hydroxybenzoate (MCH), a functional pH-responsive monomer, and poly(methoxy-ethylene glycol) methacrylate (PEGMA) block copolymers is presented. Modification of the monomers' relative ratios allowed facile switching from micellar (PEGMA11-b-MCH21) to polymersome-based (PEGMA11-b-MCH38) drug delivery nanocarriers. The ability of the latter to incorporate model anticancer drugs – tamoxifen, paclitaxel and doxorubicin hydrochloride – was investigated. High drug loading – up to 18 w/w% – was observed for tamoxifen, a hydrophobic drug which bears an amino group able to form ion pairs with MCH acidic functionalities. Non-loaded nanovectors were characterized (CAC, DLS and TEM), and were found to be very stable under physiological conditions (PBS pH 7.4, 37 °C), even in the presence of 10% plasma proteins for at least 48 h. Tamoxifen loaded nanocarriers showed slow drug release at pH 7.4 and faster release after exposure to weakly acidic environments, due to the loss of polymer/drug ionic interactions. Importantly, at pH 7.4 tamoxifen-loaded PEGMA11-b-MCH21 micelles were found to be less cytotoxic than free tamoxifen against MCF-7 cells, while under more acidic conditions, at pH 6.8, the opposite behaviour was observed, with a 10-fold increase in cytotoxicity for the micellar nanocarriers. The empty nanocarriers were found to be non-toxic in 48 h incubation time experiments. Pharmacokinetic studies proved the increased half-life and the slower clearance of tamoxifen after encapsulation in the micelles. These PEGMA-b-MCH based nanoassemblies could represent a novel promising delivery platform for low molecular weight ionisable drugs
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