Guided nanoparticles for tumor imaging and therapy

L\u2019immunoterapia passiva basata sull\u2019utilizzo di anticorpi monoclonali ha recentemente migliorato la risposta terapeutica e la sopravvivenza in neoplasie ematologiche e solide. Alcuni pazienti non manifestano per\uf2 risposta a questi trattamenti ed inoltre possono altresi\u2019 insorgere meccanismi di resistenza; per questi motivi sono attualmente in studio clinico numerosi coniugati di anticorpi a molecole citotossiche o radionuclidi. Poich\ue9 nella terapia delle neoplasie la prognosi \ue8 fortemente influenzata dalla possibilit\ue0 di una diagnosi precoce vi \ue8 la necessit\ue0 di metodiche diagnostiche ad alta sensibilit\ue0 e specificit\ue0; sono percio\u2019 motivo di interesse nuovi protocolli basati sull\u2019immuno-imaging. Nell\u2019ottica quindi di implementare il trasporto a livello delle lesioni tumorali di elevate quantit\ue0 di farmaco e di molecole trackers, le nanotecnologie possono offrire valide soluzioni. I nanosistemi presentano la caratteristica di concentrarsi a livello delle lesioni neoplasiche grazie all\u2019 \u201dEPR Effect\u201d; l\u2019accumulo e la ritenzione a livello delle lesioni puo\u2019 essere implementata coniugando le nanoparticelle con molecole veicolanti quali anticorpi o fattori di crescita. Combinando quindi la nanomedicina con la tecnologia degli anticorpi ricombinanti \ue8 possibile incrementare notevolmente la specificit\ue0 dei trattamenti antitumorali, permettendo quindi anche la diminuzione delle dosi utilizzate e aumentare la sensibilit\ue0 dei sistemi diagnostici attualmente in uso (MRI). Il presente lavoro di tesi, svolto in collaborazione con il gruppo del Prof. Meneghetti (Universit\ue0 di Padova) e con i gruppi del Prof. Mancin e del Prof Papini (Universit\ue0 di Padova) nell\u2019ambito del progetto europeo Nanophoto, descrive la generazione e caratterizzazione di diverse tipologie di nanocomposti e il loro possibile impiego nell\u2019imaging e nella terapia antitumorale. La prima parte dello studio \ue8 stata incentrata sulla sintesi e caratterizzazione di nanoparticelle metalliche, oro e ossido di Fe, non guidate. L\u2019oro \ue8 un materiale utilizzato in medicina per la sua biocompatibilit\ue0 e per le sue particolari propriet\ue0 chimiche e ottiche. Tra queste \ue8 interessante la possibilit\ue0 di produrre segnale Raman, che pu\uf2 essere amplificato piu\u2019 di 108 volte se caricate con molecole SERS. Abbiamo dimostrato che \ue8 possibile quantificare mediante spettroscopia Raman la quantit\ue0 di NPs incorporata dalle cellule. Questo risultato pone le basi per un sistema ultrasensibile di rivelazione della quantit\ue0 di nanosistemi veicolati a livello delle lesioni neoplastiche. Mediante l\u2019applicazione di nanoparticelle di FeOX (nanoparticelle magnetiche) abbiamo evidenziato che: a) \ue8 possibile utilizzate le NP magnetiche per il sorting di popolazioni cellulari modificate (vd. Cellule caricate con FeOX-NPs adsorbite con SiRNA o cDNA) b) \ue8 possibile, mediante campi magnetici esterni, direzionare ai siti di interesse specifiche popolazioni cellulari (vd. Cellule T anti-tumore o macrofagi, caricati con FeOX-NPs). La seconda parte dello studio si \ue8 focalizzata sull\u2019analisi del targeting di nanoparticelle d\u2019oro e di silice, coniugate ad anticorpi monoclonali specifici per TAA, al fine di implementarne l\u2019accumulo a livello del sito d\u2019interesse. La specificit\ue0 di legame e l\u2019internalizzazione di questi nanocomposti \ue8 stata dimostrata \u201cin vitro\u201d e \u201cex vivo\u201d utilizzando cellule antigene positive e negative e tessuti espiantati da modelli murini singenici (topi C57/B16 iniettati sc con cellule B16 trasfettate con gli Ags di interesse). E\u2019 stato inoltre valutato \u201cin vitro\u201d il potenziale terapeutico sia di nanoparticelle d\u2019oro caricate con un agente chemioterapico, la doxorubicina, sia di nanoparticelle di silice caricate con l\u2019agente fotosensibilizzante Foscan\uf0d2. In entrambi i casi, i risultati ottenuti sono promettenti; si \ue8 evidenziata, infatti, una tossicit\ue0 del 60% e 40% maggiore rispetto alle NP non caricate, utilizzando rispettivamente NP caricate con Doxorubicina e Foscan. Abbiamo inoltre dimostrato che \ue8 possibile rilevare singole cellule Ag positive ed inoltre \ue8 possibile sviluppare protocolli di imaging multi-target, mediante spettroscopia Raman e nanosistemi d\u2019oro veicolati con diversi mAbs e caricati con molecole SERS.The passive immunotherapy based on the use of monoclonal antibodies has recently improved the therapeutic response and survival in hematologic and solid tumor. However, some patients show no response to these treatments, moreover a resistance mechanism can rise up. For these reasons many conjugates of antibodies and radionuclides or cytotoxic molecules are currently in clinical trials. As in the cancer treatment, the prognosis is strongly influenced by the possibility of an early diagnosis; there is the neccesity of diagnostic methods with high sensitivity and specificity. Therefore in the last years new protocols based on immuno-imaging are object of interest. In order to increase the transport of a large amount of drug and tracker molecules at tumor lesions site, nanotechnology can offer effective solutions. The nanosystems have the characteristic to focus in neoplastic lesions thanks to the EPR effect; the accumulation and the retention in the injury sites can be implemented by conjugating nanoparticles with targeting moieties such as antibodies or growth factors. Combining nanomedicine together with recombinant antibody technology, it is possible to increase the specificity of cancer treatments greatly, thus allowing the decrease of therapeutic doses and increasing the sensitivity of diagnostic tools currently used (MRI). This thesis work was done in collaboration with the reaserch group of Prof. Meneghetti (University of Padua) and the groups of Prof. Mancin and Prof. Papini (University of Padua) in the context of the European project \u201cNanophoto\u201d. The work describes the production and characterization of different types of nanoconjugates and their potential use in imaging and cancer therapy. The first part of the study was focused on the synthesis and characterization of metal nanoparticles, gold and iron oxide, not targeted. Gold is a material used in medicine thanks to its biocompatibility and its unique chemical and optical properties. Among these, the ability to produce Raman signal is interesting, this can be amplified more than 108 times by using SERS dies. We have shown that by Raman spectroscopy it is possible to quantify the amount of NPs uptaken by the cells. This result provides the basis for an ultrasensitive method that can detect the amount of nanocompound conveyed at neoplatic lesions. By applying FeOX-MNPs (magnetic nanoparticles) we have shown that: a) magnetic NPs can be used to sort modified cell populations (see cells loaded with FeOX-MNPs coated with siRNA or cDNA) b) it is possible to direct into the site of interest specific cell populations by external magnetic field (see anti tumor T cells or macrophage, loaded with FeOX MNPs). The second part of the study was focused on the analysis of the targeting of gold and silica nanoparticles, conjugated to monoclonal antibodies specific for TAAs, in order to increase the accumulation in the site of interest. The binding specificity and internalization of these nanocompounds has been demonstrated \u201cin vitro\u201d and \u201cex vivo\u201d using antigen positive and negative cells and tissue harvested from syngenic mouse models (C57/B16 mice injected sc with B16 cells trasfected with the Ags of interest). It was also assessed \u201cin vitro\u201d the therapeutic potential of both gold nanoparticles, loaded with a chemoterapeutic agent, doxorubicin, and silica nanoparticles loaded with the photosensitiser Foscan\uae. In both cases the results are promising: it is highlighted a toxicity of 60% and 40% greater than not loaded NPs, using NPs loaded with doxorubicin and Foscan\uae respectively. We have also proved that it is possible to detect single antigen positive cells and develope multi-target methods, by using Raman spectroscopy and gold nanoparticles conjugated with different mAbs and loaded with SERS dies

SERS labels for quantitative assays: application to the quantification of gold nanoparticles uptaken by macrophage cells

Labels based on noble metal nanoparticles and surface enhanced Raman scattering (SERS) opened new opportunities for the ultrasensitive detection of analytes. To date, however, SERS labels were mostly used for qualitative analysis, while leaving largely unexploited their potential for ultrasensitive quantitative assays. Here we synthesized SERS labels based on gold nanoparticles (AuNPs) obtained by laser ablation synthesis in solution and we developed a general method for the correlation of the SERS label concentration with the intensity of the Raman signal. We successfully used this method for the quantification of the number of AuNPs uptaken by PMA differentiated U937 macrophage cells. Our work shows that quantitative ultrasensitive assays by SERS labels are possible and points out some issues that must be considered when performing this type of analysis

Targeted Silica Nanoparticles for PhotoDynamic Therapy (PDT)

In the last decade, engineered nanoparticles have become an important class of new materials with several properties that make them very attractive in a variety of fields. In particular, nanotechnology has been applied to diverse medical and biological applications ranging from biomarkers and molecular diagnostics to drug discovery and drug delivery. The importance of such applications pushed the National Institute of Health (USA) to introduce the term \u201cnanomedicine\u201d to describe the nanotechnologies applied to human diseases[1]. The peculiar properties of silica nanoparticles make them well suited to the development of smart nanomaterials for medicine and particularly for Photo Dynamic Therapy (PDT) and imaging

SERS IMAGING OF CANCER CELLS WITH ANTIBODY FUNCTIONALIZED GOLD NANOPARTICLES

Targeting cancer cells with antibodies is a powerful methodology for both tumor imaging and drug delivery. We show how surface enhanced resonance Raman scattering (SERRS) signals of single cells can be obtained engineering gold nanoparticles synthesized by laser ablation. In particular we will use an antibody (D2/B) recognizing the prostate specific membrane antigen (PSMA), overexpressed both in prostate tumors in patients and on LNCaP prostate cancer cells (our \u201cin vitro\u201d tumor model), and Texas Red as SERRS reporter. High intensity signals are observed for LNCaP cells incubated with the targeted nanoparticles and comparison with DU145 cells, which lack PSMA, shows the high targeting specificity of the prepared nanoparticles

Characterization of the Aroma Profile of Commercial Prosecco Sparkling Wines

In this work, the aromatic characterization of commercially available Prosecco wines with a price range between EUR 7 and 13 was carried out. These wines came from three different areas of origin: Valdobbiadene, Asolo and Treviso. Seventy volatile compounds were identified and quantified in the wines. Quantitatively, the wines were mainly characterized by compounds of fermentation origin (alcohols, acids, esters), and C6 -alcohols, and to a lesser extent, terpenes, low molecular weight volatile sulfur compounds (VSC), and benzenoids. To determine their impact on the aroma of Prosecco wine, the respective OAVs were calculated. The molecules with higher OAV were ethyl hexanoate, isoamyl acetate, and β-damascenone. More generally, esters, responsible for fruity notes, seemed to play a major role in the aroma of Prosecco wine. Investigation into the possible effect of different production zones indicated 16 significantly different compounds accounting for differences between the various areas of origin of the wines, being mostly VSC, esters and C6 -alcohols. A sensory evaluation through a sorting task highlighted the formation of clusters; wine samples were divided into two main groups partially attributable to the areas of origin. From a chemical point of view, cluster A was richer in esters, while cluster B had, on average, higher concentrations of compounds associated with wine aging such as cyclic terpenes, norisoprenoids (TDN and vitispirane), and VSC

Influence of Different Modalities of Grape Withering on Volatile Compounds of Young and Aged Corvina Wines

Withering is a practice traditionally used in various regions to produce sweet or dry wines. During withering there is an increase in sugar content but also a modification in volatile compound profiles. Controlling metabolic changes through the dehydration process to obtain wines with desired characteristics is therefore a challenging opportunity. The effects of two different withering technologies, post-harvest or on-vine with blocked sap vessel flow, on the volatile profile of young and aged Corvina red wines was investigated. The results showed that modulation of wine aroma due to the withering process is associated with fermentative metabolites, such as esters, higher alcohols, and acids, as well as grape-related compounds such as C6 alcohols, terpenes and norisoprenoids. Significant differences were also found by comparing the two withering techniques. Post-harvest in a traditional "fruttaio" warehouse wines showed higher content of ethyl acetate, ethyl butanoate, \u3b2-citronellol and 3-oxo-\u3b1-ionol, whereas post-harvest withering on-vine increased \u3b2-damascenone in wines. The type of withering technique has an influence on the evolution of some aroma compounds during the aging of wine, among them linalool, (E)-1-(2,3,6-trimethylphenyl)buta-1,3-diene (TPB), n-hexyl acetate, ethyl acetate, ethyl 3-methylbutanoate, 3-oxo-\u3b1-ionol and \u3b2-damascenone

Investigation of Properties of Anti-Prostate Specific Membrane Antigen-targeted Gold Nanoparticles as Drug Carriers in Tumor Therapy

Specific targeted delivery and control drug release are desirable properties of a drug for tumor therapy. Nanomedicine, the science that studies the application of nanotechnology to disease treatment, might be of help. Targeted nanoparticles (NP) for their size and structure are able to enhance the accumulation in the tumor of encapsulated or linked / adsorbed molecules (gene, drug). We have therefore investigated the binding properties of gold-NPs (20 nm) conjugated to D2/B, a mAb recognizing the prostate specific membrane antigen (PMSA). PSMA for its wide distribution in prostate tumor (about 70-80% of patients are PSMA+), is an important biomarker in the management of this malignancy using targeted drugs. Gold-NPs have been synthesized by laser ablation, mixed with a reporter solution (Texas red) and treated with a thiol-PEG solution. Derivatization of PEG-COOH with EDC/Sulfo NHS has been used to covalently link the mAb to the NPs. D2/B-NP binding to LNCaP (PSMA+) cells has been assessed by cytometry. We have measured a MFI (mean fluorescence value) of 1,383 for D2/B-NP whereas the MFI of the negative control (CTRL-) was 68. The binding specificity was confirmed on PSMA– Jurkat cells (MFI of 121 and 101 for D2/B-NP and CTRL-, respectively). Binding and internalization of D2/B-NP in LNCaP cells were assayed by confocal microscopy; detection of D2/B-NP by surface-enhanced Raman scattering also revealed the selective binding of the NPs to Ag+ cells. The results of specific delivery and internalization support our idea to use mAb anti-PSMA targeted NPs to enhance the transport of toxic drugs in the tumo

Tumor Targeting with Guided Silica Nanopartilces for Prostate Cancer Therapy

Nanotechnology offers unique approaches to probe and control a variety of biological and medical processes that occur at nanometer lenght scales, and is expected to have a revolutionary impact on biology and medicine. Nanoparticles (NPs) have been recently developed as one of the most coming out technological tools in cancer diagnosis and therapy. Thanks to their advantageous chemical-physical properties, the nanoparticles are able to accumulate in the tumor (EPR effect \u201cEnhaced permeability and retention effect). This tumor distribution is enhaced when the nanoparticles are functionalized with targeting moieties as monoclonal antibody, or peptide. Furthermore guided nanopartilces increase the accumulation of encapsulated molecules (drug or tracker) in the tumor sites for therapy and diagnosis application. Specific targeted delivery and control drug release are desiderable properties of a drug for tumor therapy. The aim of this project is to use a monoclonal antibody (mAb) direct against a tumor associate antigen (TAA) : PSMA ( prostate specific membrane antigen) in order to veiculate specifically silica NPs loaded with mTHPC, a photosensitizing reagent, that once admistrated is later activeted by light to induce production of cytotoxic specie leading to tumor cell destruction