Thèses sur le sujet « Nanoparticles, Breast Cancer, BOS »

A common feature of nanoparticles (NPs) is their large surface area to volume ratio that makes them an attractive tools for the development of drug delivery systems, improving several properties of ‘‘free’’ drugs: solubility, pharmacokinetic profile and biodistribution. During my PhD, I studied different nanovehicles loaded with “free” hydrophobic drugs against three “over-proliferative” diseases to evaluate the efficacy and safety of nanodrugs. In the first year, I focused the anti-proliferative effect of ASC-J9, a promise in the treatment of prostate cancer. ASC-J9 is a very hydrophobic molecule, which limits drug administration. ASC-J9 was loaded on PLGA NPs, in order to improve the solubility of the molecule and applied to an estrogen-dependent breast cancer. My results showed a cellular growth inhibition associated with a specific G2/M cell cycle block, which led to apoptosis. In the second year, I worked in collaboration with group of IRCCS San Matteo (PV). In a previous work (2014), Cova et al evaluated the effectiveness of gold NPs functionalized with anti-CD44 monoclonal antibody loaded with everolimus (GNP-HCe), prepared by our laboratory, in inhibiting mesenchymal cells (MCs), the primarily responsible for the bronchiolitis obliterans syndrome (BOS). Considering that BOS is due to an alteration of inflammatory process the aim is determined the effect of GNP-HCe on macrophages, neutrophils and lymphocytes with in vitro assays assessing cytokine secretion, cell apoptosis and ROS production. Next, we administered GNP-HCe by aerosolization in normal mice evaluating the localization and toxicity on lungs and peripheral organs. GNP-HCe were able to reduce ROS and not to enhance the activity of three major components of immune response. The in vivo experiments confirmed that inhaled NPs did not raise the inflammatory response, because the bronchoalveolar lavage of mice did not show significant increase of IL-8 cytokine. This study discloses the possibility to plan a new pharmacological treatment for patients affected by pulmonary fibroproliferative disorders directly targeting MCs without altering the immune response. During the last year, I focused on triple negative breast cancer (TNBC) representing only the 15% of breast cancers, but the most aggressive phenotype. TNBCs lack three specific molecular markers (ER, PR and HER-2), which makes the current chemotherapeutic approaches mostly ineffective. TNBC cells show strong drug resistance due to the overexpression of P-glycoprotein (Pgp), MDR-associated proteins (MRP1), and breast cancer resistance protein (BCRP) that actively efflux the drug outside the cells conferring resistance to treatment. Curcumin possesses a wide range of pharmacological activities; one is the capacity to modulate the MDR activity in different type of cancer cells, affecting the expression and the functionality of MDR proteins. There are no data in literature on TNBC cells, so, we aim to evaluate modulation of Pgp, MRP1 and BCRP expression and their functionality in two different TNBC cell lines (MDA-MB-468 and MDA-MB-231) after treatment with curcumin. One of the problems is the lipophilic nature of this molecule; thus, to improve the drug solubility, we encapsulated curcumin inside H-ferritin nanocages (HFn). We have demonstrated that HFn recognize transferrin receptor 1 (TfR1), overexpressed by TNBC cells, with high binding capacity. The HFn-TfR1 interaction mediates the internalization of HFn, which disassembles into subunits inside the cytoplasm, allowing the intracellular release of drugs. Work is in progress to assess the effect of HFn-curcumin on TNBCs

The early identification of the insurgence of a malignant cancer and the selective targeting of the tumor with specific drugs are still an open frontier for cancer diagnosis and treatment. The ultimate goal is to improve the therapy efficiency and to reduce the side effects usually encountered with conventional chemotherapy. Worldwide, mammary carcinoma represents the second most recurrent type of malignant tumor in adult women and the fifth cause of death among cancer types. In the context of this thesis, I have designed and developed multifunctional hybrid nanoparticles consisting of an inorganic iron oxide core, useful as source of signal for magnetic resonance imaging (MRI), and an organic shell, including bioactive ligands for the pharmacological effect combined with specific cell targeting, and a molecular dye as fluorescence signal emitter. The nanoparticle characteristics were optimized in terms of size, morphology, surface charge, stability, fluorescence emission and capability to enhance the MRI contrast. In addition, specific biomolecular ligands based on anti-HER-2 monoclonal antibody have been developed and novel strategies for their conjugation to nanoparticles were explored. The resulting hybrid nanocomplexes were tested both in vitro and in vivo to evaluate their toxicity, endocytosis, degradation pathways, and the efficient recognition of cell-surface biomarkers. Next, these nanoparticles proved to be highly effective in selectively targeting breast cancer cells in transplanted mice bearing HER-2-positive tumors. A multifaceted bioanalytical approach, combining fluorescence, magnetic relaxivity, transmission electron microscopy, and histological experiments in vivo and ex vivo, has demonstrated that these nanoprobes prevalently accumulated at the tumor by an active targeting route. The nanoparticles were endocytosed by the tumor cells following a lysosomal pathway of degradation, while did not result in permanent damage of healthy tissues. The principal outcome of this work was the development of a versatile and reliable biotechnological platform based on finely structured, multifunctional nanosized probes useful for the interrogation of biological systems.

Breast cancer is the second most common type of cancer after lung cancer and the fifth most common cause of cancer death. It is by far the most widespread cancer amongst women, with an incidence rate of more than twice that of colorectal cancer and cervical cancer and about three times that of lung cancer. In this thesis, for the first time, a novel nanoparticle for effective drug delivery using the antibody trastuzumab and genipin crosslinking agent was designed and evaluated for its use in breast cancer. In vitro studies were also conducted to compare different crosslinkers in the preparation method of the drug loaded nanoparticles. In addition the efficacy of noscapine drug loaded nanoparticles were evaluated in breast cancer cell lines. Results showed that novel nanoparticles containing targeting antibody trastuzumab can be an alternate method for breast cancer treatment and other biomedical applications. This work highlights the potential of novel antibody targeting nanoparticles crosslinked with genipin or glutaraldehyde as a drug delivery system for breast cancer therapy as they can effectively deliver drugs such as noscapine. Further in-vivo studies however are required to evaluate the full potentials.
Suivant le cancer des poumons, le cancer du sein est le deuxième type de cancer le plus répandu et commun à travers le monde. C'est la cinquième cause de décès du à un cancer. Certes, il est le cancer le plus commun parmi les femmes, ayant un double taux d'incidence à ceux du cancer du colon et cancer du col de l'utérus, et triple à celui des poumons. Dans cette dissertation, une drogue carrière qui combine les anticorps trastuzumab et la drogue noscapine, a été conçue et évaluée pour être utilisée dans le traitement du cancer du sein. Des études « in vitro » ont étés conduites pour comparer différents crosslinkers dans la méthode de préparation des nanoparticules chargées de cette drogue. De plus, l'efficacité de nanoparticules chargées de drogues a été évaluée au niveau des cellules du cancer de sein. L'objectif de cette étude est le développement d'une procédure coacervation pour la préparation et la caractérisation in vitro des nanoparticules HSA sous un aspect d'une taille contrôlée, en combinaison d'une distribution mono dispersée. Leur propriétés sont donc étudiés pour la délivrance optimale, prudente et efficace de drogues anti-cancer. Les résultats prouvent que les nanoparticules chargées de noscapine qui contiennent l'anticorps trastuzumab peuvent être une méthode alternative pour le traitement du cancer du sein ainsi que d'autres applications biomédicales. Des études "in-vivo" plus profondes sont recommanés pour évaluer leur potentiel maximum. Ce travail mets en valeur le potentiel des trastuzumab-modifié nanoparticules charges de noscapine reliés avec du genipin ou du glutaraldehyde comme un system de drogue conçu pour la thérapie du cancer du sein. fr

Background Axillary nodal status is the single most important prognostic factor in breast cancer diagnosis. If cancerous cells are present, the sentinel lymph node (SLN) is the axillary lymph node that is most likely to contain metastatic disease. In early stage breast cancer, the SLN is localised (then surgically removed for pathological analysis) using a radioisotope and/or a blue dye injected into the breast Super-paramagnetic iron oxide (SPIO) nanoparticles are novel agents that, when injected, could potentially both localise and characterise the SLN using MRI such that surgical SLN biopsy is no longer required. Aims To evaluate axillary SLN localisation after SPIO injection with, pre-operatively, axillary MRI and, intra-operatively, with a hand held magnetometer and to characterise SLN SPIO uptake using ex-vivo MRI. Methods From November 2009 - March 2011, 51 patients with early stage breast cancer underwent SLN biopsy following a subcutaneous injection of SPIO in addition to the standard injection of radioisotope (Tc99M) and blue dye. SPIO injection technique was refined during the trial with an initial dose of 2mls and then 4mls in 8 and then 43 women respectively. Pre-operative axillary in vivo MRI (1.5T) was carried out on 14 women and ex vivo high resolution MRI (9.4T) on 36 nodes. During surgery, an SLN was defined as either "hot", "blue", "palpable" or "SPIO detected". Axillary clearance was carried out for SLN-positive disease. Results In total, 11 of the 51 patients had positive SLNs. On pre-operative axillary MRI, SPIO uptake was noted in at least one node in all 14 patients. A total of 35 nodes were identified. Uptake of SPIO in the SLN was seen at a minimum of 12mins post injection. Involved SLNs were not differentiated from normal SLNs following morphological characterisation or based on loss of T2 signal within the individual SLN. At SLN biopsy, 134 hot, blue, palpable or SPIO-containing nodes were identified in 51 patients. The magnometer identified 92 SPIO-containing nodes in 51 (84%) patients. One node in one patient was not identified using the combined technique but was found to contain SPIO. Of the 16 hot, blue or palpable involved nodes in 11 patients, 9 contained SPIO. In summary, the SPIO SLN localisation rate and FNR in patients was 84% and 16% respectively. Ex vivo SLN MRI demonstrated SPIO uptake in all 35 SLNs preferential to the sinuses and sub-capsular spaces. Of the 3 involved nodes, areas of metastasis did not take up SPIO, whereas in normal areas of the node, SPIO was positively identified. Conclusion In our study, subcutaneous SPIO, a novel SLN-localising agent, was taken up by axillary nodes and identified on pre-operative axillary MRI. Node positive SLNs were identified on ex vivo MRI, but SPIO did not demonstrate sufficient accuracy at SLN localisation for routine clinical use.

Introduction: The HER2 receptor overexpression is normally associated to aggressive and infiltrating breast cancer (BC) phenotype with propensity to spread into metastases. Nowadays, the detection of HER2 in primary tumor lesions and in their metastases is based on invasive methods as well therapy clinical outcomes are not satisfactory yet. Recent advances in nanotechnology have led to the development of nanoparticles able to host various functionalities for specific targeting and to be loaded with therapeutic molecules, making possible the simultaneous diagnosis and treatment of human cancers (theranostic). In the present Thesis study, was evaluated the potential use of targeted silica nanoparticles (SiNPs) as theranostic agent for HER2+ breast cancer. Methods: SiNPs were engineered with anti-HER2 monoclonal antibody Trastuzumab, in the form of half-chain (Hc-TZ), and radiolabeled employing 99mTc for in vivo SPECT imaging detection of HER2+ BC lesions. Subsequently, SiNPs were loaded with doxorubicin (DOX) for treatment evaluation. Experimental design was divided in three main tasks. First, we evaluated the contribution offered by active targeting (Hc-TZ) to the selective distribution of SiNPs in solid HER2 positive BC lesions. To this aim, both Hc-TZ conjugated (SiNP-TZ) and non-conjugated (SiNP) nanosilica shells were radiolabeled with 99mTc-Tricarbonyl complex, through nitrilotriacetic acid (NTA) linker procedure, and their distribution kinetics evaluated in vitro and ex vivo in ad hoc cancer models. Nanoparticles were simultaneous filled with a fluorescent dye and their uptake were also assessed by FACS analysis and fluorescence microscopy. In the second experimental step, nanoparticles were also engineered with several amount of Hc-TZ (SiNPs to Hc-TZ ratio, 1:2 and 1:8 respectively), and were 99mTc-labeled at histidine residues of the antibody chain for ex vivo/in vivo biodistribution evaluation. Finally, SiNP-TZ were loaded with DOX and in vitro/in vivo DOX distribution in HER2 positive models was evaluated using confocal microscopy and Optical Imaging, in comparison to liposomal doxorubicin (Caelyx). The treatment efficacy of DOX-SiNP-TZ (1:8 Hc-TZ) versus Caelyx was evaluated in vivo for six weeks of treatment, also using PET molecular imaging ([18F]FDG) approach. Results: In vitro assays showed a higher fluorescence signal (FICT) in SK-BR-3 compared to MDA-MB-468 cells, exclusively for targeted SiNP-NTA-TZ/SiNP-TZ with an increase over time. Ex vivo biodistribution of 99mTc-labelled nanoparticles via NTA, at 4 h post-injection of SiNP-NTA-TZ and/or non-targeted (SiNP-NTA), exhibited values of 3.53 and 1.69 in tumor (tumor to muscle ratio) respectively, with a rapid reduction over time for targeted nanoparticles. These results indicated the presence of an antibody-receptor mediated tumor uptake of SiNP-NTA-TZ, with a faster washout of nanoparticles radiolabeled shell. In the second set of experiments, performed with 99mTc-SiNP-TZ labelled on TZ half chain, uptake was confirmed at 4 h p.i. for SiNP-TZ (1:8 Hc-TZ) with similar results to SiNP-TZ (1:2 Hc-TZ). Instead, was showed a progressive retention of radioactivity until 24 h p.i., confirming the presence of radiolabeled Hc-TZ to the tumor also at latter times, with improved results for SiNP-TZ (1:8 Hc-TZ), also in terms of radiochemical yield. Doxorubicin loaded SiNP-TZ (1:8 Hc-TZ) showed in vivo similar delivery results in comparison to Caelyx at 6 h p.i., meanwhile at the end of treatment tumor volume reduction resulted significant improved by targeted nanoparticles administration. Conclusion: Results of this Thesis study, demonstrated a promising specificity and treatment efficacy of the silica nanoparticles-based system SiNP-TZ, encouraging its potential use as theranostic agent for HER2+ breast cancer lesions.

Magister Scientiae - MSc
In the present study we report the synthesis and characterization of iron oxide magnetic nanoparticles doped with zinc in an attempt to enhance the magnetic properties. The nanoparticles were prepared via the co-precipitation route and capped with 3-phosphonopropionic acid (3-PPA). The amount of zinc dopant was varied to yield nanoparticles with the general formula ZnxFe3-xO4 (x=0, 0.1, 0.2, 0.3, 0.4). Characterization was carried out using high resolution transmission electron microscopy (HRTEM), X-ray diffraction spectroscopy (XRD), energy-dispersive X-ray spectroscopy (EDS), Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA) and superconducting quantum interference device (SQUID) analysis. Results from HRTEM, XRD and SQUID confirm that doping took place and x=0.2 was found to be the doping limit for these nanoparticles with a maximum size of 10.73 nm and saturation magnetization of 73.37 emu/g. The EDS further confirmed successful doping with zinc, while FTIR and TGA confirmed successful capping with 3-PPA. Despite agglomeration at all doping levels, these nanoparticles show great potential for application in breast cancer diagnostic studies.

Cancer is a leading cause of disease worldwide and breast cancer, which exists in four major molecular subtypes, is the second most common cause of cancer mortality. Although there are many therapeutic options, chemotherapy is still subject to failures, due to (1) the low selectivity of drugs, which requires high doses with side effects and the risk of recurrence and (2) the development of resistance by different mechanisms that cells put in place to defend themselves from the action of drugs. In my PhD project I exploited the unique features of a nanoparticle based on recombinant heavy-chain ferritin cages (HFn) for the targeted delivery of various active molecules to improve the treatment efficacy in breast cancer. My work is divided into three main subprojects, all sharing the use of HFn. Initially, I exploited HFn nanocages for the encapsulation of a chemotherapeutic drug, doxorubicin (DOX). HFn-DOX acts as a “Trojan Horse”: nanoparticles are internalized in cancer cells faster and more efficiently compared to free DOX, then promptly translocated into the nucleus with a self-triggered mechanism, thus promoting a fast and massive delivery of the drug inside the nuclear compartment, strongly affecting viability and circumventing MDR mechanisms. Then, I tested HFn-DOX on an aggressive breast cancer model, in vitro and in vivo, under a Low Dose Nanometronomic regimen (LDNM). Metronomic HFn-DOX strongly improved the antitumor potential of DOX chemotherapy arresting the tumor progression. Such effect is attributable to multiple nanodrug actions, including inhibition of tumor angiogenesis and avoidance of chemoresistance. Moreover, metronomic HFn-DOX drastically reduced cardiotoxicity. In the second project, curcumin was encapsulated in HFn (CFn) and used to treat triple negative breast cancer (TNBC) cell lines. Curcumin is a natural anti tumor compound, but is rapidly degraded and scantily bioavailable. CFn had instead good stability and solubility and was able to enhance the sensitization of TNBC cells to DOX treatment. Finally, HFn was used as a vehicle to transport anti-microRNAs, since miR21 plays a role in the development of resistance against Trastuzumab (TZ), the treatment of choice for HER2 positive breast cancer. The major limiting factor in gene therapy is the ability to specifically deliver nucleotide sequences: however, anti-miR21 cross-linked to HFn, was released into the cytoplasm. Based on our results, ferritin is an effective system for the delivery of anti-tumor molecules, promoting their chemotherapic action and/or overcoming the problem of resistance that limits the effectiveness of many therapies.
Cancer is a leading cause of disease worldwide and breast cancer, which exists in four major molecular subtypes, is the second most common cause of cancer mortality. Although there are many therapeutic options, chemotherapy is still subject to failures, due to (1) the low selectivity of drugs, which requires high doses with side effects and the risk of recurrence and (2) the development of resistance by different mechanisms that cells put in place to defend themselves from the action of drugs. In my PhD project I exploited the unique features of a nanoparticle based on recombinant heavy-chain ferritin cages (HFn) for the targeted delivery of various active molecules to improve the treatment efficacy in breast cancer. My work is divided into three main subprojects, all sharing the use of HFn. Initially, I exploited HFn nanocages for the encapsulation of a chemotherapeutic drug, doxorubicin (DOX). HFn-DOX acts as a “Trojan Horse”: nanoparticles are internalized in cancer cells faster and more efficiently compared to free DOX, then promptly translocated into the nucleus with a self-triggered mechanism, thus promoting a fast and massive delivery of the drug inside the nuclear compartment, strongly affecting viability and circumventing MDR mechanisms. Then, I tested HFn-DOX on an aggressive breast cancer model, in vitro and in vivo, under a Low Dose Nanometronomic regimen (LDNM). Metronomic HFn-DOX strongly improved the antitumor potential of DOX chemotherapy arresting the tumor progression. Such effect is attributable to multiple nanodrug actions, including inhibition of tumor angiogenesis and avoidance of chemoresistance. Moreover, metronomic HFn-DOX drastically reduced cardiotoxicity. In the second project, curcumin was encapsulated in HFn (CFn) and used to treat triple negative breast cancer (TNBC) cell lines. Curcumin is a natural anti tumor compound, but is rapidly degraded and scantily bioavailable. CFn had instead good stability and solubility and was able to enhance the sensitization of TNBC cells to DOX treatment. Finally, HFn was used as a vehicle to transport anti-microRNAs, since miR21 plays a role in the development of resistance against Trastuzumab (TZ), the treatment of choice for HER2 positive breast cancer. The major limiting factor in gene therapy is the ability to specifically deliver nucleotide sequences: however, anti-miR21 cross-linked to HFn, was released into the cytoplasm. Based on our results, ferritin is an effective system for the delivery of anti-tumor molecules, promoting their chemotherapic action and/or overcoming the problem of resistance that limits the effectiveness of many therapies.

Superparamagnetic iron oxide nanoparticles (SPIONs) have gained signif- icant interest over the past decades because of their wide range of appli- cations. In biomedicine, SPIONs had been used extensively in the past as MRI contrast agents but they are currently being investigated for hyperther- mia therapies, magnetic manipulation and as part of diagnostic devices. The main aim of this study is to develop a method to synthesise positive MRI enhancing iron oxide nanoparticles (T1) and use these as the diagnos- tic component to produce a theranostic (therapeutic and diagnostic) agent. An aqueous flow-based synthesis method was assembled and tested producing 25nm iron oxide nanoparticles with T1 enhancement, CMDxUS- PIONs. The devised synthetic method enabled the fabrication of the desired nanoparticles without the use of organic solvents and at higher outputs than previously reported with flow-based methods, with 12 clinical doses being produced per hour using a lab-scale system. Surface functionalisation of CMDxUSPIONs with aptamers for active targeting was accomplished using a recently discovered anti-annexin 2A aptamer (ACE4). Particle uptake results show that ACE4-CMDxUSPIONs presented at least a two-fold increase in cell uptake when compared to un- modified CMDxUSPIONs. Further modifications of CMDxUSPIONs included drug-loading with cisplatin. Cisplatin loaded CMDxUSPIONs (CPt-CMDxUSPIONs) were achieved following pre-optimisation using a Design of Experiments ap- proach. The drug-loaded CPt-CMDxUSPIONs containing 0.64mg of CPt/mg of iron were able to retain both the activity of free cisplatin (in vitro) and the imaging capabilities of CMDxUSPIONs. In vivo experiments using a 4T1 mammary carcinoma cell line in Balb/C mice showed that CPt-CMDxUSPIONs were trackable in a 1T preclinical MRI and therapeutically, CPt-CMDxUSPIONs reduced tumour size and minimised cisplatin’s nephro- toxicity. Moreover, preclinical MRI scans show that it is possible to use CMDxUSPIONs as reporters for nanoparticle uptake detected by a T1-T2 signal switch in MRI. Iron oxide based theranostic systems reported in this thesis could play an important role in future cancer treatments by enabling a personalised therapy approach and modifying current chemotherapeutic drug biodistribu- tion as a carrier. The facile synthesis together with the addition of therapeu- tic agents to iron oxide nanoparticles demonstrates that the production of an experimental theranostic is possible.

Pattern Recognition Receptors (PRRs) are a group of evolutionarily conserved and germline-encoded cellular receptors of the innate immune system that are responsible for recognizing and responding to the entirety of the pathogens a host encounters. The ingenuity of the innate immune system is that with a comparatively miniscule pool of receptors, these receptors are capable of responding to a diverse and large array of pathogens and damage signals. Two highly relevant subsets of PRRs include nucleotide binding domain leucine rich repeat containing (NOD-like) receptors (NLRs) and Toll-like receptors (TLRs). Both NLRs and TLRs have been implicated in several diseases, including autoimmune disorders, inflammatory conditions, and cancer. Mice lacking a specific NLR, NLRP1, are more susceptible to chemically induced colitis and colitis-associated tumorigenesis. We investigated whether the absence of NLRP1 in the gastrointestinal tract influenced the composition of the microbiome, and whether it was responsible for the predisposition of these animals to colitis-associated cancer. By carefully controlling for non-genotype influences, we found that in fact maternal and housing factors were greater predictors over genotype of gut flora composition. This study concluded with a clearer understanding of NLRP1. We next investigated the effectiveness of a novel tumor ablation therapy, termed High-Frequency Irreversible Electroporation (H-FIRE) in a murine model of triple negative breast cancer. The chosen 4T1 model closely mimics aggressive human metastatic triple negative breast cancer, and metastasizes to the same organs. After ablation of the primary mammary tumor, we saw significant improvements in disease burden and metastases, both of which were accompanied by PRR activation within the tumor microenvironment, implicating PRRs in the successful treatment outcome following H-FIRE ablation. Lastly, we generated novel CRISPR-Cas9 plasmids to genetically manipulate the Tlr4 gene of wild type C57Bl/6 mice in order to recapitulate the LPS-hyporesponsive TLR4 protein of C3H/HeJ mice. This proof-of-concept study successfully demonstrated that PRRs can be targets for gene editing purposes, and that nanoparticle delivery leads to enhanced and improved delivery. Collectively, this work attempts to better appreciate the role of PRRs in understanding, treating, and targeting cancer.
Doctor of Philosophy

Dendritic cells play a pivotal role in antigen presentation and adaptive immune response activation. Studies on mice models reported that tumoral antigens-loaded DCs can induce therapeutic and protective anti-tumour immune responses, both in vitro and in vivo. These results provide an evidence of the possible use of DCs as vaccines. However, the efficacy of such therapeutic vaccination was recently questioned due to the limited degree of actual tumour regression in some clinical trials. Thus, it is necessary to better characterize immunological and clinical parameters of DCs immunization to increase the therapeutic efficay of the vaccines. In vivo imaging tecniques, as magnetic resonance imaging (MRI) and single photon emission tomography (SPET), are used to study cell trafficking mechanisms in vivo and state anti-tumoral treatments efficacy, allowing an accurate assessment. In this work we initially set the optimal culture conditions to obtain functional immature DCs. Evaluations based on DCs number, cell viability and immature DCs percentage led to establish day 6 as the one in which the highest number of functionally active immature DCs can be achieved. We then set a DCs labelling protocol with commercial SPIO (Endorem®) evaluating Iron content using Perl’s staining and relassometry. Time and dose-response kinetic evaluations allowed to define as optimal labelling conditions the use of 200 μg Iron of Endorem®/ ml of culture for 16 hours. Cell viability and phenotypic markers analysis proved that SPIO labelling had no influence on immature DCs viability or activation status. Images acquired by transmission electron microscopy showed that Endorem® were inside cells, in particular inside lysosomes, and were taken up by endocytosis mechanisms. We then focused on DCs antigens loading. Tumoral antigens were obtained lysing neoplastic masses extracted from a mammary tumour mouse model (MMTV-Ha-Ras). DCs pulsed with different antigen concentrations resulted mature by means of high levels of activation and maturation markers. Moreover, such cells were able to stimulate T lymphocytes extracted from MMTV-Ha-Ras spleens. 3H-thymidine and CFSE proliferation assays, together with IFN-γ production measured by ELISA, showed an increase in T lymphocytes activation when co-cultured with antigen-pulsed DCs. Cytokines production assessed by flow cytometry and ELISA allowed us to verify that tumoral antigen-pulsed DCs were able to elicit a specific T-lymphocyte response toward a Th1 type. Indeed, high amounts of IFN-γ were produced by T lymphocytes, high levels of IL-12 were produced by DCs and low levels of IL-10 were detectable. In vitro migration showed that tumoral antigen-pulsed DCs displayed migratory abilities toward MIP3β and 6Ckine chemockines, responsible in vivo of mature DCs attraction. Flow cytometric analysis confirmed that migrated cells displayed a mature phenotype and expressed high levels of CCR7. We then performed in vivo experiments in MMTV-Ha-Ras mice bearing neoplastic lesions and in control mice without tumours. MRI showed that Endorem®-labelled DCs pulsed with tumour antigens are able to migrate to the draining lymph site in vivo. Histological analysis on ex vivo lymph nodes showed the presence of Iron-labelled cells in the cortical area. Immunohistochemistry reveals co-localization of Iron-containing cells with CD208-labelled DCs, but not with macrophages. In vivo SPET migration of antigen-pulsed DCs labelled with 111In-oxine showed specific radiotracer presence in the lymph nodes omolateral to injection site; γ-counter analysis of ex-vivo organs confirmed such results. Taken together, these results suggest that this protocol could candidate for experimental use on animal models for in vivo tumour immunotherapy efficacy imaging.

The first project focused on the preparation, characterization, and application of dual emission quantum dot encoded mesoporous silica microparticles. The quantum dots were added in precisely controlled ratios and were stably encapsulated within the pores of the silica. Several experiments were performed to test the superior stability of the quantum dot-silica composites over dye-loaded silica particles. The composite particles exhibited very high fluorescence, were functionalized with antibodies, and were used as signal transducers for the detection of a protein expressed by breast cancer cells. The second project focused in more detail on the detection capabilities of the quantum dot-silica composites. Three different types of quantum dot-silica composites were prepared. Each type was loaded with a separate type of quantum dot with distinct emission wavelengths and was functionalized with separate antibodies for detection of three different breast cancer biomarkers. These three composite sensors were used together for the simultaneous detection of each of the breast cancer markers. The initial strategy utilized the direct detection method in which the antigen is nonspecifically adsorbed to a glass plate. An improved second strategy was more sensitive and used a capture antibody which was covalently bound to a glass plate to immobilize the antigen. The third project focused on the preparation and application of magnetic, fluorescent human serum albumin nanoparticle composites. A fluorescent drug analogue and iron oxide nanoparticles were encapsulated into 100 nm human serum albumin nanoparticles. The advantage of these composite particles is that they could be used as a theranostic tool which could target, detect, and treat diseased tissue in a single application. Release of the drug analogue from the nanocomposites was achieved by addition of proteolytic enzymes that are expressed or overexpressed in cancer cells. The temporal release of the fluorescent drug analogue was measured as a function of enzyme concentration. The amount of drug released was directly proportional to enzyme concentration.

Magister Scientiae (Medical Bioscience) - MSc(MBS)
Despite the advances in therapies such as chemotherapy and radiotherapy, tumours have been shown to be resistant to the treatments. Gold nanoparticles (AuNPs) have been recognized as effective radiosensitizers of low energy (e.g. 200–500 kV) X-rays, leading to the emission of Auger electrons that cause highly localised ionizing damage to cells. Spherical AuNPs were synthesised via the reduction of the chloroaurate ions by sodium citrate. Characterisation of AuNPs involved UV-visible spectrophotometry, zeta (Z) potential, dynamic light scattering (DLS) and polydispersity index (PDI) measurements for determination of surface plasmon resonance (SPR), surface charge and stability, as well as transmission electron microscopy (TEM) for hydrodynamic core sizes, size distribution width and shape of AuNPs. Both the 5 and 10 nm AuNPs were found to be anionic with λmax absorbance of 525 nm and uniform size distribution. DLS measurement at 38.12 nm and 48.50 nm, respectively for 5 nm and 10 nm AuNPs, points to aggregation of the AuNPs. However, TEM measurements confirmed the core size of the 10 nm AuNPs. Non-malignant Chinese hamster ovary (CHO-K1), brain endothelial (BEnd5), breast (MCF-10A), isolated human lymphocytes and malignant breast (MCF-7) cell lines were treated with 50 μg/ml of AuNPs, and irradiated with either 1, 2 or 4 Gy X-rays or 1 or 2 Gy p(66)/Be neutron radiation. The γ-H2AX foci assay, cytokinesis-block micronucleus assay, MTT assay and fluorescence-activated cell sorting (FACS) was used to determine that amount of double stranded breaks (DSBs) in isolated lymphocytes, the presence and number of micronuclei (MNi) within binucleated cells (BNCs), cell viability and cell cycle progression, respectively. Preliminary experiments that established the reliability of the study regarding the induction of DNA damage after the bombardment of AuNPs by scattered low kV X-rays, were carried out on lymphocytes. Combined treatment (AuNPs and radiation) resulted in more endogenous foci in comparison to lymphocytes that were treated with AuNPs only. The CHO-K1 and MCF-7 cells showed higher MNi frequencies after the combination treatment of AuNPs and radiation compared to the number of MNi in samples exposed to AuNPs and radiation separately. The AuNPs alone influenced the cellular kinetics of all cell types. Interaction indices, which is the enhancement factor of AuNPs in combination with radiation, for AuNPs and 6 MV 2 Gy X-rays of 1.6 to 1.7 and 1.3 to 1.4 have respectively been determined for CHO-K1 and MCF-7 cells, whilst that for the other cell types used in the study were not different from Unity. As expected, the interaction indices between AuNPs and p(66)/Be neutrons was lower than the interaction indices after 2 Gy X-rays, as p(66)/Be neutrons interact only with the nuclei of the AuNP's atoms and the X-ray photons interact with the orbital electrons of the atoms of the AuNPs leading to Auger electron emission. The cell viability assay showed that 50 μg/ml of AuNPs had an inhibitory effect on cellular proliferation, in all four cell linnes whereas the lower concentrations (2.5, 5 and 10 μg/ml) had no effect. Results in this study, revealed an increase in the accumulation of CHO-K1 an MCF-7 cells in the G₂/M phase of the cell cycle after being treated with AuNPs followed by X-ray radiation, suggesting that the cells have possibly been sensitised to the damaging effects of radiation. Further studies are required to quantify internalised AuNPs and to then link the possible concentration differences of the AuNPs to differences in radiation damage effects observed for the different cell types.

Nanotechnology has recently emerged as a strong contributor toward research efforts to develop targeted systems of drug delivery in cancer therapy. Our work investigates the therapeutic potential of Targeted Charge-Reversal Nanoparticles (TCRNs), a novel nanoparticle with in vitro evidence of nuclear drug delivery. Using M12 prostate cancer cells, MDA-MB-231 breast cancer cells, and modified derivatives of these cell lines, we investigated the ability of Folic Acid-tagged TCRNs to deliver Nile Red and Dimethyl Indole Redfluorescent (DiR) fluorescent dyes to the nucleus of cells using confocal microscopy and in vivo biphontonic imaging using Xenogen® Technology. Confocal imaging with the SCP28 derivative of MDA-MB-231 cells shows nuclear association of the TCRNs over time, although specific nuclear deposition was unclear. Biophotonic imaging with M12 and SCP28 xenograft tumors in athymic nude mice shows retention of TCRNs in animals out to 7 days with minimal localization of TCRNs to tumor tissues. Our findings suggest that further characterization and manipulation of both the cells and the nanoparticle is necessary in order to make definitive claims regarding the TCRN’s ability to deliver fluorescent dyes, and eventually therapeutic compounds, to the nucleus of cells.

Les macrophages (Mɸ) infiltrés dans les tumeurs orchestrent les différentes étapes du développement tumoral. De par leur capacité à internaliser les nanoparticules (NPs) et leur plasticité phénotypique, ils sont impliqués dans l’efficacité thérapeutique des actifs vectorisés par un rôle de réservoir de NPs ou une modulation de leur réponse envers les cellules néoplasiques. Le calcitriol, métabolite actif de la vitamine D, possède des activités à la fois anticancéreuse et immunomodulatrice. Sa vectorisation via des NPs est une approche thérapeutique intéressante pour potentialiser ses activités tout en limitant les effets secondaires s’opposant à son utilisation clinique dans le cadre de la chimiothérapie. Une étude de formulation a permis de développer des NPs à base d’acide poly(D,L)lactique et de triglycérides (ratio 1:2) d’une taille de 200 nm et présentant une libération prolongée du calcitriol. Des études in vitro menées sur les cellules de cancer du sein MCF-7 ont permis de mettre en évidence l’avantage d’une libération prolongée du calcitriol vis-à-vis de son activité antiproliférative aboutissant à une réduction de 65% de la viabilité cellulaire après 10 jours par rapport au contrôle, non observable avec le calcitriol libre. La participation active des M? à l’activité cytotoxique du calcitriol sur les lignées cellulaires de cancer du sein MCF-7 et de leucémie MV4-11 a aussi été mise en évidence par un modèle de co-culture in vitro. En effet, les NPs de calcitriol, après internalisation par les Mɸ, provoquent une action cytotoxique prolongée contre les cellules MCF-7 en co-culture au bout de 10 jours avec seulement 20% de cellules viables vs 70% en l’absence de Mɸ
Tumor associated macrophages (Mɸ) orchestrate the different stages of tumor development. They are able to internalize nanoparticles (NPs) and are known for their phenotypic plasticity, which make them interesting targets for cancer treatment through the storage of NPs or a modulation of their activity towards the neoplastic cells. Calcitriol, the active metabolite of vitamin D, exerts both anticancer and immunomodulatory activities. Its vectorization via NPs is an interesting therapeutic approach to potentiate its activities while limiting its side effects, which hamper its current clinical use in chemotherapy. We developed poly (D, L) lactic acid and triglyceride-based NPs (1:2 ratio) measuring 200 nm and exhibiting a sustained release of calcitriol. In vitro studies, performed on breast cancer cells (MCF-7), showed the advantages of a sustained release of calcitriol regarding its antiproliferative activity with a 65%-decrease in cell viability after 10 days compared to unexposed cells, while it was unobservable for free calcitriol. The implication of Mɸ in the cytotoxic activity of calcitriol towards MCF-7 cells and MV4-11 cells (leukemia) cells has been demonstrated using an in vitro co-culture model. Calcitriol-NPs showed a sustained cytotoxic activity towards MCF-7 cells in co-cultures after 10 days, through their uptake by Mɸ, with a decrease in cell viability of 80% vs 30% in mono-cultures

Cancer progression and physiological changes within the cells are accompanied by alterations in the biophysical properties. Therefore, the cell biophysical properties can serve as promising markers for cancer detection and physiological activities. To aid in the investigation of the biophysical markers of cells, a microfluidic chip has been developed which consists of a constriction channel and embedded microelectrodes. Single-cell impedance magnitudes at four frequencies and entry and travel times are measured simultaneously during their transit through the constriction channel. This microchip provides a high-throughput, label-free, automated assay to define biophysical signatures of malignant cells and monitor the therapeutic efficacy of drugs. Here, we monitored the dynamic cellular biophysical markers in response to sphingosine kinase inhibitors (SphKIs), and compared the effectiveness of drug delivery using Poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) loaded with SphKIs versus conventional delivery. Cells treated with SphKIs showed significantly higher impedance magnitudes at all four frequencies. The bioelectrical parameters extracted using a model also revealed that the highly aggressive breast cells treated with SphKIs shifted electrically towards that of a less malignant phenotype; SphKI-treated cells exhibited an increase in cell-channel interface resistance and a significant decrease in specific membrane capacitance. Furthermore, SphKI-treated cells became slightly more deformable as measured by a decrease in their channel entry and travel times. We observed no significant difference in the bioelectrical changes produced by SphKI delivered conventionally or with NPs. However, NPs-packaged delivery of SphKI decreased the cell deformability. In summary, the results showed that while the bioelectrical properties of the cells were dominantly affected by SphKIs, the biomechanical properties were mainly changed by the NPs.
Master of Science

Gangliosides, sphingolipids containing sialic acid residue(s), are components of the external layer of cell plasma membranes where they are inserted with the hydrophilic head group turned towards the extra cellular environment. In this strategic position, gangliosides contribute to very special cell processes as development, differentiation and oncogenic transformation. Analysis of the tumour associated ganglioside component profile may aid in the characterization of tumour cells and to establish the degree of malignant transformation. Furthermore, gangliosides that become prominently exposed on tumour cell during oncogenic dedifferentiation may be used as targets for specific immunohistological identification and possible therapeutic approaches. In humans the main sialic acid is N-acetyl-neuraminic acid (Neu5Ac), whereas N-glycolyl neuraminic acid (Neu5Gc) is not expressed in normal human tissues because a species-specific genetic mutation abrogate its biosynthesis. However breast cancer cells contain ganglioside GM3 carryng not only Neu5Ac but also Neu5Gc which resulted highly immunogenic and can be considered a good target for immunotherapy development. Some laboratories produced murine or human monoclonal antibodies (MAbs) against GM3(NeuGc) ganglioside antigen but the low specificity of these monoclonal antibodies made them only partially useful for the cancer diagnosis and therapy. The aim of this thesis was to synthesize the oligosaccaridic chain of GM3(NeuGc) and obtain an idiotopic antigen that mimic the oligosaccharide of GM3(NeuGc). The first part of the research involved the synthesis of the oligosaccharide α-Neu5Gc-(2-3)-β-Gal-(1-4)-β-Glc: starting from the natural GM3, which contains the Neu5Ac, we removed the acetyl group from the sialic acid and then synthesized the GM3(NeuGc). Later we removed the ceramide portion in order to obtain the free oligo-saccharide chain of GM3(NeuGc). Because the it is known that the oligosaccharidic molecules alone are not capable to induce an antibody response enough specific and efficient, we decided to synthesized a complex between the prepared oligosaccharide and gold nanoparticles. For this purpose we performed a two-phase synthesis: a thiol ligand (thio-acetyl-nonanole) is first tied with the free oligo-saccharide chain of GM3(NeuGc) and then strongly bound to colloidal gold thus obtaining the gold-glyconanoparticles.

The ability of engineered cerium oxide nanoparticles to confer radioprotection was examined. Rat astrocytes were treated with cerium oxide nanoparticles to a final concentration of 10 nanomolar, irradiated with a single 10 Gy dose of ionizing radiation and cell death was evaluated by propidium iodine uptake at 24 and 48 hours after radiation insult. Treatment of rat astrocytes with nanoceria resulted in an approximate 3-fold decrease in radiation induced death. These results suggest that the nanoceria are conferring protection from radiation induced cell death. Further experiments with human cells were conducted. Human normal and tumor cells (MCF-7 and CRL8798) were treated with the same dosage of cerium oxide nanoparticles, irradiated and evaluated for cell survival. Treatment of normal cells (MCF-7) conferred nearly 99% protection from radiation-induced cell death while the same concentration of nanoceria showed almost no protection in tumor cells (CRL8798). TUNEL analysis results of similarly treated cells demonstrated that nanoceria reduced radiation-induced cell death by 3-fold in normal breast cells but not in MCF-7 tumor cell lines when cultured under the same conditions. We concluded that cerium oxide nanoparticles confer radioprotection in a normal human breast line (CRL 8798) but not in a human breast tumor line (MCF-7). It is hoped that the outcome of this study will guide future endeavors toward a better elucidation of the molecular pathways involved in the protection of cells with nanoceria against radiation-induced cell death, as well as the minimization of the bystander effect in radiation therapy.
M.S.
Masters
Molecular Biology and Microbiology
Burnett College of Biomedical Sciences
Molecular and Microbiology

Bone metastasis represents a common and terminal outcome for patients suffering from advanced breast and prostate cancer. The underlying molecular mechanisms are still not fully understood and to date no curative treatments are available. In this PhD project, a humanized mouse model was, for the first time, explored as drug response tool, using new and already clinically approved targeted treatments against cancer bone metastasis. The outcomes and observations presented, show that further humanization of the model will allow for the in-depth dissection of molecular differences between the murine and human bone-microenvironment in the context of drug response.

Breast cancer is considered the second most commonly diagnosed type of cancer across the world. The common modes of treatment are limited by severe side-effects that hinder the efficacy of the drugs, compromise the patients' quality of life and often lead to other disorders. One of the main focuses of nanobiotechnology research is to develop novel anti-cancer drug delivery systems that improve the drug efficacy, limit harmful side effects and also allow for the delivery of developing therapeutics that are rapidly degraded in circulation, such as small interfering RNA (siRNA). Nano-carriers are helpful particularly in anti-cancer drug delivery due to the Enhanced Permeability and Retention (EPR) effect. In the current research study, we developed and investigated the use of surface modified HSA nanoparticles for the delivery of anti-cancer therapeutics in breast cancer applications. Results showed formation of modified HSA nanoparticles of sizes below 150 nm and contained a positive surface charge. The cellular uptake of the nanoparticles was higher in coated particles (average: ~70%) than uncoated particles. Furthermore, the cytotoxicity assessment of modified HSA nanoparticles suggested that empty particles are biocompatible and non-toxic to cells. Therefore, the presented PEI-enhanced and TAT-coated HSA nanoparticles form an appealing delivery system for anti-cancer therapeutics with a potential for clinical application.
Le cancer du sein est considéré comme le deuxième type de cancer le plus couramment diagnostiqué à travers le monde. La plupart des traitements sont characterisés par des effets secondaires nocifs qui limitent l'efficacité des médicaments, compromettent la qualité de vie des patients et conduisent souvent à d'autres troubles nocifs. L'un des principaux axes de recherche en nanobiotechnologie est de développer un nouveaux système de délivrance qui permet d'améliorer l'efficacité du médicament, de limiter les effets secondaires nocifs et aussi de permettre la livraison de molecules qui sont rapidement dégradées dans la circulation, tels que les petits ARN interférents (siRNA). Les nano-transporteurs sont utiles en particulier dans l'administration de médicaments anticancerigenes en raison de leur perméabilité accrue et de leur conservation (EPR). Dans l'étude de la recherche actuelle, nous avons développé et étudié l'utilisation de nanoparticules HSA à surface modifiée pour la livraison de médicaments anticancéreux dans les applications de cancer du sein. Les résultats ont montré la formation de nanoparticules HSA de tailles modifiées en dessous de 150 nm contenant une charge de surface positive. L'absorption cellulaire des nanoparticules est plus élevée dans les particules enrobées (moyenne: ~ 70%) que les particules non enrobée. Par ailleurs, l'évaluation de la cytotoxicité des nanoparticules HSA modifiées a suggéré que les particules vides sont biocompatibles et non toxiques pour les cellules. Par conséquent, les nanoparticules HSA revêtues de TAT et PEI-améliorée forment un système de prestation idéale pour les thérapies anti-cancereuses avec un potentiel d'application clinique.

Les possibilités de combinaison thérapeutiques offertes par les nanoparticules ont ouvert un nouveau champ d’investigation pour la recherche sur le cancer. Dans ce projet de recherche, des nanoparticules dérivées de la protéine de capside du virus de la mosaïque du tabac (TMV) ont été utilisées afin de transporter différents peptides thérapeutiques ciblant le récepteur neuropiline-1. Cette stratégie a permis de solubiliser un peptide fortement hydrophobe ayant préalablement démontré son efficacité anticancéreuse sur des lignées de cancer du sein humain et de glioblastome. Les résultats obtenus ont également permis de démontrer la possibilité de combiner différents peptides thérapeutiques via l’auto-assemblage de la protéine de capside du TMV
Nanoparticles play an ever increase role in carrying therapeutic compounds in the cancer field. In this research project, the coat protein of Tobacco mosaic virus (TMV) was used as nanocarrier to solubilize a hydrophobic peptide interfering with the transmembrane domain of neuropilin-1. The nanoparticles created have conserved the antiangiogenic and antimigratory effect of the therapeutic peptide. This strategy was also used to create nanoparticles carrying a peptide targeting the ectodomain of neuropilin-1. The two types of nanoparticles were then assembled through auto-assembling ability of the coat protein. These nanoparticles also exhibit antiangiogenic ability thus, confirming the validity of this approach to combine therapeutic peptides

La chimiothérapie systémique par voie intraveineuse, essentiellement réservée aux cancers avancés, n'est pas ciblée sur la tumeur, il est très difficile d’atteindre des niveaux thérapeutiques en intra tumoral, et ses effets secondaires et sa toxicité sont doses-limitantes.La chimiothérapie localisée pourrait permettre :1) la stabilisation des molécules médicamenteuses incorporées une seule administration médicamenteuse,2) une libération prolongée et contrôlée du médicament pour assurer une diffusion adéquate et l'absorption par les cellules cancéreuses sur plusieurs cycles de division cellulaire 3) le chargement de molécules de chimiothérapie insolubles dans l’eau, 4) l’apport direct au site de la maladie, 6) des effets secondaires diminués en évitant la circulation systémique,7) des résections chirurgicales moindres en traitant les marges de la tumeur. Nous nous sommes plus particulièrement intéressés aux cancers gynécologiques. Nous avons étudié les effets pharmacologiques et cliniques de microsphères chargées en doxorubicine (Doxo) sur un modèle de carcinose péritonéale et de tumeur de glande mammaire, et étudié le profil de diffusion ganglionnaire de divers implants non chargés. 12 jours après injection laparoscopique de tumeurs VX2 sur les ligaments larges droits et gauches de lapines WNZ 12 une injection laparoscopique de 0,5 ml de microsphères chargées ou non de Doxo (respectivement DM, groupe 1 et BM, groupe 2) a été réalisée de façon aléatoire d’un côté ou de l’autre, en sous péritonéal, au site tumoral. 7 jours après les ligaments larges, l’utérus, les ovaires, les orifices de trocarts, les intestins, la vessie, le foie et les poumons ont été examinés en macroscopie et microscopie. Le volume tumoral était plus faible dans le groupe 1 (3,6 ± 3,2 cm) par rapport au groupe 2 (8,9 ± 5,4 cm) (MW, p = 0,0179). La nécrose a été observée autour de toutes les DM, sans nécrose autour des BM. La concentration de Doxo était de 2,1 ± 2,7 uM aux limites tumorales, au-dessus du niveau thérapeutique de 1,0 uM. Sur un autre modèle, 19 jours après injection locale de suspensions tumorales de VX2 sur la deuxième glande mammaire de lapines WNZ chaque glande a été aléatoirement traitée par injection locale de 0,5 ml de microsphères chargées ou non de Doxo (HSDOXO, Groupe1, et HS, groupe 2).Pour les tumeurs de moins de 5 cm3 ou 2 cm de diamètre avant traitement, le volume final était plus faible dans le groupe 1 par rapport à groupe2 (respectivement p<0.008 et p<0.3, MW)et la croissance tumorale a été diminuée après injection de HSDOXO par rapport à HS. En microscopie une nécrose tissulaire a été observée autour des HSDOXO en extratumoral, sans nécrose autour des HS.Nous avons enfin étudié la diffusion de particules de diverses tailles, non chargées, au ganglion sentinelle d’une tumeur de glande mammaire . Les animaux ont été répartis en trois groupes de trois, chacun d'eux recevant des particules de 100 nM, 1 uM ou 10 uM. Cinq jours après traitement, l'intensité de fluorescence a été évaluée par lampe UV. Le ganglion sentinelle a été disséqué selon la technique du bleu, avant curage complet. Les premiers résultats montrent la capture de particules de 1 et 100µm par les ganglions tumoraux mais aussi dans les ganglions sains, ce qui permettrait d’envisager un traitement ganglionnaire préventif et curatif.De plus en plus de tumeurs seront décelées au stade local. Par ailleurs l'identification des phénotypes génomiques permettra un traitement personnalisé « à la carte ». On pourrait envisager un dispositif de délivrance programmable traitant tous les aspects de la maladie, de l'inhibition de la croissance tumorale et de l'angiogenèse à la promotion de la cicatrisation des tissus normaux
Systemic chemotherapy is mainly reserved for advanced cancers, is not targeted to the tumor, it is very difficult to achieve intratumoral therapeutic levels and its side effects and toxicity are dose-limiting.Local chemotherapy may have several advantages:1) stabilization of embedded drug molecules and preservation of anticancer activity,2) controlled and prolonged drug release to ensure adequate diffusion and uptake into cancer cells over many cycles of tumor cell division, 3) loading and release of water-insoluble chemotherapeutics, 4) direct delivery to the site of disease, 5) one-time administration of the drug, 6) diminished side effects due to the avoidance of systemic circulation of chemotherapeutic drugs.We were particularly interested in gynecological cancers. We studied the pharmacological and clinical effects of doxorubicin-loaded microspheres (Doxo) in a model of peritoneal carcinomatosis,a model of mammary gland tumor, and studied the diffusion profile of various micro and nanoparticles in tumoral and non tumoral lymph nodes.12 days after laparoscopic injection of VX2 tumors on the right and left broad ligament of WNZ rabbits laparoscopic injection of 0.5 ml of microspheres loaded or not with Doxo (DM or Group 1, BM Group 2 respectively) was conducted randomly to one side or another, at the sub peritoneal tumor site. 7 days after the broad ligaments, uterus, ovaries, trocars, bowels, bladder, liver and lungs were examined macroscopically and microscopically. The tumor volume was lower in group 1 (3.6 ± 3.2 cm) compared with group 2 (8.9 ± 5.4 cm) (MW, p = 0.0179). Necrosis was observed around all DM without necrosis around the BM. Doxo concentration was 2.1 ± 2.7 µM at the tumor margins, above the therapeutic level of 1.0 uM.On another model, 19 days after local injection of VX2 tumor suspensions in the second mammary gland of WNZ rabbits each gland was randomly treated by local injection of 0.5 ml of microspheres loaded or not with Doxo (HSDOXO, Group1, and HS Group 2).For tumors less than 5 cm3 or 2 cm in diameter before treatment, the final volume was lower in group 1 compared to Group 2 (p <0.008 and p <0.3, MW) and tumor growth was reduced after HSDOXO injection compared to HS. Microscopic tissue necrosis was observed around extratumoral HSDOXO without necrosis around the HS.We finally studied the diffusion of unloaded particles of various sizes on the lymph nodes of a mammary gland tumor. The animals were divided into three groups of three, each receiving particles of 100 nm, 1 µm or 10 µm. Five days after treatment, the fluorescence intensity was measured by UV lamp. The sentinel lymph node was dissected according to the technique of blue dye.The first results show the capture of 1 μm and 100μm particles by the tumoral and non tumoral lymph nodes, which would consider a preventive and curative treatment of the nodes.Since more and more tumors are detected at the local stage and with the identification of genomic phenotypes, a personalized local chemotherapy could be the next step of cancer therapy. One could imagine a programmable controlled drug delivery device dealing with all aspects of the disease, inhibition of tumor growth and angiogenesis, while promoting the healing of normal tissues

[ES] La presente tesis doctoral titulada "Mesoporous silica and gold-based nanodevices: new controlled delivery platforms for biomedical applications" se centra en el diseño, síntesis, caracterización y evaluación de distintos nanodispositivos híbridos orgánico-inorgánicos. En concreto, se utilizan como soporte nanopartículas mesoporosas de sílice y nanopartículas de oro para su aplicación biomédica, en concreto en el campo del cáncer de mama. En el primer capítulo se introduce el marco general en el que se engloban los estudios realizados. Se presentan los conceptos relacionados con nanotecnología y nanomedicina, así como la interacción de las nanopartículas a nivel biológico con el organismo y las células. Finalmente, se introducen conceptos básicos del cáncer de mama y la aplicación de nanomateriales como terapia. A continuación, en el segundo capítulo, se exponen los objetivos de la presente tesis doctoral que son abordados en los siguientes capítulos experimentales. En el tercer capítulo se describe el primer nanomaterial para la liberación controlada de dos inhibidores (navitoclax y S63845) de las proteínas anti- apoptóticas de la familia Bcl-2. Este sistema se ha diseñado con el objetivo de superar la resistencia a navitoclax en un modelo celular de cáncer de mama triple negativo. En concreto, se han preparado nanopartículas mesoporosas de sílice cargadas con navitoclax y S63845, y funcionalizadas con un aptámero dirigido a la proteína de superficie MUC1, que actúa como puerta molecular. En este trabajo hemos demostrado que las nanopartículas diseñadas son internalizadas preferentemente por células tumorales de cáncer de mama. También hemos demostrado la capacidad de las nanopartículas de revertir la resistencia a navitoclax en un modelo celular de cáncer de mama triple negativo. Además, ponemos de manifiesto la disminución del principal efecto adverso (trombocitopenia) asociado a la administración del navitoclax en su formulación libre, gracias a la encapsulación en las nanopartículas. En el capítulo cuatro se presenta un sistema sensible a pH para la liberación controlada de un cargo fluorescente y la maquinaria de edición génica basada en el sistema CRISPR/Cas9, dirigido a la edición del gen codificante de la proteína fluorescente verde (GFP, del inglés gren fluorescent protein). El nanodispositivo está constituido por nanopartículas mesoporosas de sílice cargadas con rodamina B, funcionalizadas con polietilenimina y revestidas con el plásmido codificante del sistema CRISPR/Cas9. En este trabajo se ha demostrado el escape lisosomal de las nanopartículas, mediado por el efecto esponja de protones de la PEI. Asimismo, mostramos un nanodispositivo pionero en su campo, basado en nanopartículas mesoporosas de sílice, capaz de realizar la doble función de llevar a cabo la edición del gen codificante de GFP y la liberación exitosa del cargo fluorescente. En el quinto, y último, capítulo experimental se propone una nueva aproximación para realizar una terapia enzimática prodroga empleando nanopartículas de oro como transportadores enzimáticos. En este caso, se aborda la funcionalización de nanopartículas de oro con la enzima peroxidasa de rábano (HRP, del inglés horseradish peroxidase), capaz de transformar la prodroga inocua ácido indol-3-acético en especies radicales que resultan tóxicas para las células tumorales. En este capítulo se ha demostrado el efecto terapéutico del nanodispositivo en combinación con la prodroga en modelos celulares de cáncer de mama de los subtipos luminal A y triple negativo. Además, se ha confirmado la eficacia terapéutica del sistema en esferoides tumorales formados por células de cáncer de mama triple negativo. Por último, se presentan en el capítulo seis las conclusiones extraídas del desarrollo de esta tesis doctoral. Los resultados obtenidos en este trabajo contribuirán al desarrollo de nuevos nanomateriales inteligentes con aplicación en diversas áreas de la nanomedicina.
[CA] La present tesi doctoral titulada "Mesoporous silica and gold-based nanodevices: new controlled delivery platforms for biomedical applications" se centra en el disseny, síntesi, caracterització i avaluació de diferents nanodispositius híbrids orgànic-inorgànics. En concret, s'utilitzen com a suport nanopartícules mesoporoses de sílice i nanopartícules d'or per a la seua aplicació biomèdica, en concret en el camp del càncer de mama. En el primer capítol s'introdueix el marc general en el qual s'engloben els estudis realitzats. Es presenten els conceptes relacionats amb la nanotecnologia i nanomedicina, així com la interacció de les nanopartícules a nivell biològic amb l'organisme i les cèl·lules. Finalment, s'introdueixen conceptes bàsics del càncer de mama i l'aplicació de nanomaterials com a teràpia. A continuació, en el segon capítol, s'exposen els objectius de la present tesi doctoral que són abordats en els següents capítols experimentals. En el tercer capítol es descriu el primer nanomaterial utilitzat per a l'alliberament controlat de dos inhibidors (navitoclax i S63845) de les proteïnes anti-apoptòtiques de la família Bcl-2. Aquest sistema s'ha dissenyat amb l'objectiu de superar la resistència a navitoclax en un model cel·lular de càncer de mama triple negatiu. En concret, s'han preparat nanopartícules mesoporoses de sílice carregades amb navitoclax i S63845, i funcionalitzades amb un aptàmer dirigit a la proteïna de superfície MUC1, que actua com a porta molecular. En aquest treball hem demostrat que les nanopartícules dissenyades són internalitzades preferentment per cèl·lules tumorals de càncer de mama. També hem demostrat la capacitat de les nanopartícules de revertir la resistència a navitoclax en un model cel·lular de càncer de mama triple negatiu. A més, posem de manifest la disminució del principal efecte advers (trombocitopènia) associat a l'administració del navitoclax en la seua formulació lliure, gràcies a l'encapsulació en les nanopartícules. En el capítol quatre es presenta un sistema sensible a pH per a l'alliberament controlat d'una càrrega fluorescent i la maquinària d'edició gènica basada en el sistema CRISPR/Cas9, dirigit a l'edició gènica del gen codificant de la proteïna fluorescent verda (GFP, del anglés gren fluorescent protein). El nanodispositiu està constituït per nanopartícules mesoporoses de sílice carregades amb rodamina B, funcionalitzades amb polietilenimina i revestides amb el plàsmid codificant del sistema CRISPR/Cas9. En aquest treball s'ha demostrat la fuga lisosomal de les nanopartícules, mediat per l'efecte esponja de protons de la PEI. Així mateix, vam mostrar un nanodispositiu pioner en el seu camp, basat en nanopartícules mesoporoses de sílice, capaç de realitzar la doble funció de dur a terme l'edició del gen codificant de la GFP i l'alliberament exitós de la càrrega fluorescent. En el cinqué i últim capítol experimental es proposa una nova aproximació per a realitzar una teràpia enzimàtica prodroga emprant nanopartícules d'or com a transportadors enzimàtics. En aquest cas, s'aborda la funcionalització de nanopartícules d'or amb l'enzim peroxidasa de rave (HRP, del anglés horseradish peroxidase), capaç de transformar la prodroga innòcua àcid indol-3-acètic en espècies radicals que resulten tòxiques per a les cèl·lules tumorals. En aquest capítol s'ha demostrat l'efecte terapèutic del nanodispositiu en combinació amb la prodroga en models cel·lulars de càncer de mama dels subtipus luminal A i triple negatiu. A més, s'ha confirmat l'eficàcia terapèutica del sistema en esferoides tumorals formats per cèl·lules de càncer de mama triple negatiu. Finalment, en el capítol sis es presenten les conclusions extretes del desenvolupament d'aquesta tesi doctoral. Els resultats obtinguts en aquesta tesi contriburan al desenvolupament de nous nanomaterials intel·ligents amb aplicació en diverses àrees de la nanomedicina.
[EN] This Ph.D. thesis entitled "Mesoporous silica and gold-based nanodevices: new controlled delivery platforms for biomedical applications" is focused on the design, synthesis, characterisation, and evaluation of several hybrid organic-inorganic nanomaterials. We have developed mesoporous silica nanoparticles and gold nanoparticles for biomedical applications, specifically in the breast cancer area. The first chapter includes an overview of the concepts related to the research performed. Introductory notions about nanotechnology and biomedicine are presented, as well as the basis of the interactions of nanoparticles with biological systems. Finally, breast cancer disease and the application of nanomaterials as therapy are described. Next, in the second chapter, the objectives addressed in the following experimental chapters are displayed. In the third chapter, we present the first nanomaterial for the controlled delivery of two inhibitors (navitoclax and S63845) of the Bcl-2 anti-apoptotic proteins. This nanosystem has been designed to overcome navitoclax resistance in a triple-negative breast cancer cellular model. We have prepared mesoporous silica nanoparticles loaded with navitoclax and S63845 and functionalised with an aptamer targeting MUC1 surface protein as a molecular gate. In this work, the specific targeting of the nanodevice to breast cancer cells has been demonstrated. The ability to overcome navitoclax resistance has been shown in navitoclax-resistant triple-negative breast cancer cells. Furthermore, navitoclax encapsulation in the nanoparticles has proved to reduce the main adverse effect (thrombocytopenia) associated with free formulated drug administration. In the fourth chapter, we describe a pH-responsive nanosystem for the controlled co-delivery of a fluorescent cargo and the genome-editing machinery based on CRISPR/Cas9, which targets the green fluorescent protein (GFP) coding gene. The nanodevice consists of mesoporous silica nanoparticles loaded with rhodamine B, functionalised with polyethyleneimine, and capped with the CRISPR/Cas9 plasmid. In the present work, we have shown the lysosomal scape capacity of the nanodevice enhanced by the proton sponge effect of PEI. We have also demonstrated a pioneering mesoporous silica-based nanodevice efficient in the simultaneous genome editing of the GFP gene (as a model gene) and the successful release of a fluorescent cargo (as a model drug). In the fifth and last experimental chapter, we propose a new approximation to develop enzyme prodrug therapy using gold nanoparticles as enzyme carriers. In this case, we use gold nanoparticles functionalised with the enzyme horseradish peroxidase (HRP), which transforms the non-toxic prodrug indol-3-acetic acid into radical species toxic to tumour cells. In this chapter, the therapeutic effect of the nanodevice in combination with the prodrug has been demonstrated in two breast cancer cell subtypes (luminal A and triple-negative breast cancers). Also, the therapeutic effect of the material has been corroborated in multicellular tumour spheroid-like cultures formed by triple-negative breast cancer cells. Finally, in the sixth chapter, the conclusions derived from the presented studies and the general conclusions of this Ph.D. thesis are released. The obtained results will promote the development of new smart nanomaterials with diverse biomedical applications.
Gema Vivo-Llorca thanks the Generalitat Valenciana for her fellowship ACIF/2017/072. Vicente Candela-Noguera thanks the Spanish Government for his fellowship FPU15/02753. We would like to thank Servier for the workart used in the figures of this manuscript (Servier Medical Art https://smart.servier.com/). We thank the Spanish Government (project RTI2018-100910-B-C41 (MCUI/AEI/FEDER, UE); SAF2017-84689-R-B (MCUI/AEI/FEDER, UE)) and the Generalitat Valenciana (project PROMETEO/2018/024 and PROMETEO/2019/065) for support.
Vivo Llorca, G. (2021). Mesoporous silica and gold-based nanodevices: new controlled delivery platforms for biomedical applications [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/172713
TESIS

L’utilisation de nanoparticules de haut numéro atomique pour potentialiser les effets de la radiothérapie fait l’objet de nombreux travaux de recherches. Historiquement, l’intérêt porté à ces nanoparticules repose sur un principe d’action physique : en densifiant la matière, les nanoparticules localisées dans une tumeur augmentent localement la probabilité d’interaction des rayonnements ionisants avec celle-ci. L’augmentation de la dose délivrée localement serait alors associée à un stress oxydatif et à une augmentation des lésions de l’ADN. Ainsi la radio-sensibilisation liée aux nanoparticules permettrait d’une part de lutter contre la radio-résistance et d’autre part d’améliorer l’index thérapeutique. La radiothérapie constitue l’un des piliers du traitement du cancer du sein. Néanmoins, les tumeurs mammaires sont hétérogènes et plusieurs sous populations coexistent avec des sensibilités différentes aux traitements. Les cellules souches cancéreuses (CSC) et le processus de transition épithélio-mésenchymateuses (TEM) contribuent principalement à cette hétérogénéité et confèrent aux cellules tumorales des propriétés de plasticité, de tumorogénicité et de résistance à la mort cellulaire. Dans le cas particulier du cancer du sein, les cellules issues de la TEM et les cellules souches cancéreuses partagent un même phénotype surfacique caractérisé par la perte d’expression de la protéine membranaire CD24 et la surexpression de CD44. Ce travail de thèse vise à évaluer l’efficacité de nanoparticules métalliques de nature (or, platine), de taille (5 à 35 nm) et de charges (positives, négatives) différentes comme agents potentialisateurs des effets de l’irradiation sur des modèles de tumeurs mammaires aux statuts épithélio-mésenchymateux différents. Face à la difficulté à mettre en évidence un effet biologique significatif du double traitement in vitro, j’ai cherché à comprendre quels paramètres physiques, chimiques et biologiques sont nécessaires pour observer l’effet radiosensibilisant. L’un des principaux enjeux a été de parvenir à une bonne diffusion des nanoparticules au sein des tumeurs. Pour comprendre les mécanismes, nous avons comparé la réponse transcriptomique de cellules cancéreuses mammaires, au fond génétique commun mais de phénotype épithélial ou mésenchymateux (triées selon leur niveau d’expression CD24/CD44), exposées à un groupe de nanoparticules de composition physico-chimique différente de manière à s’affranchir d’un l’effet qui serait spécifique à une nanoparticule. L’analyse du transcriptome souligne l’importance des structures membranaires et extracellulaires dans la reconnaissance et l’interaction préférentielle des nanoparticules avec les cellules mésenchymateuses. Ces résultats sont d’autant plus importants que l’identification de gènes et de mécanismes clés favorisant l’accumulation de nanoparticules dans les cellules cancéreuses les plus résistantes aux traitements devrait permettre d’améliorer la conception de nouvelles nanoparticules à haut potentiel thérapeutique
The use of high-Z nanoparticles to enhance radiotherapy effects has gained momentum over the last decade. Historically, as nanoparticles increase tumor density, they were thought to improve radiation dose by locally increasing the probability of interactions with ionizing radiations. Local dose enhancement is then associated with increased oxidative stress and DNA damage. Therefore, radiosensitization with nanoparticles could impair radioresistance as well as improve therapeutic index. Radiotherapy is a cornerstone of breast cancer treatment. However, mammary tumors are heterogeneous and comprise distinct populations of cancer cells that respond differently to treatments. Cancer stem cells (CSC) and epithelial to mesenchymal transition (EMT) are major factors contributing to cancer cells plasticity, tumor heterogeneity, and escape from programmed cell death (apoptosis). In breast cancer, both CSC and cells undergoing EMT are characterized by the expression of two surface markers CD24 and CD44 (CD24-/low, CD44 high). This work aims to evaluate the efficiency of high-Z nanoparticles of different nature (gold, platinum), different size (from 5 to 35 nm) and different surface charge (positive and negative) as potent radiosensitizer on several breast cancer models of different epithelial or mesenchymal state. As no significant change could initially be observed in vitro following the combination of nanoparticles with radiation compared to radiation alone, I gain insight on the influence of physical, chemical and biological parameters required for characterizing radio-enhancement. Among them, I focused on improving the diffusion of nanoparticles and their internalization in tumor cells. I showed that nanoparticles uptake by breast cancer cells was depending on their mesenchymal state: nanoparticle internalization by cancer cells is dramatically increased in mesenchymal-like cancer cells compared to epithelial-like cells across a panel of several breast cancer cell lines. Importantly this discrepancy was not affected by the charge, size or surface chemistry of the nanoparticles themselves. This strongly suggests a cell-dependent mechanism, in opposition to the current paradigm that nanoparticles uptake is mainly governed by their inherent physical/chemical properties. This study emphasized the importance of membrane and extracellular structures in nanoparticle recognition and preferential interaction with cells. Our results are of peculiar interests as the identification of genes or mechanisms facilitating nanoparticles accumulation into radioresistant cancer cells could further conception of promising therapeutic nanoparticles

O câncer de mama constitui o segundo tipo de câncer mais frequente no mundo e o mais comum entre as mulheres, representando uma das principais causas de morte. O tamoxifeno é um fármaco antiestrogênico utilizado para o tratamento deste tipo de câncer desde 1971 e ainda é o mais utilizado nos casos de tumores mamários que expressam receptores de estrógeno. Apesar de apresentar resultados significativamente positivos, seu efeito antiestrogênico não se restringe apenas ao sítio tumoral causando, com isso, efeitos colaterais graves que podem deixar sequelas. A proposta deste trabalho foi desenvolver sistemas de liberação nanoparticulados à base de PLA e PLA-PEG para veiculação do tamoxifeno, como uma estratégia para o potencial aumento da segurança e da eficácia deste fármaco através de um possível direcionamento passivo ao sítio de ação, devido à permeabilidade vascular aumentada destas regiões tumorais. As nanopartículas foram preparadas pela técnica de nanoprecipitação e apresentaram diâmetro médio inferior a 200 nm para a maioria das formulações. Foram avaliados três estabilizantes, o poloxamer 407, o poloxamer 188 e o polissorbato 80, este último proporcionou maior eficiência de encapsulação, 86,7% e 100%, nas nanopartículas de PLA e PLA-PEG, respectivamente. Quanto à composição das nanopartículas de PLA-PEG, o polímero utilizado inicialmente (PLA(1000)-PEG(750)) apresentou distribuição de tamanho heterogênea, perfil multimodal e alto índice de polidispersividade. Assim, este polímero foi substituído pelo PLA(5000)-PEG(1000), que apresentou distribuição de tamanho uniforme, perfil monomodal e baixo índice de polidispersividade. A caracterização por microscopia eletrônica de varredura comprovou a homogeneidade no tamanho de partícula, mostrando seu formato esférico. As análises de espectrofotometria no infravermelho e calorimetria diferencial exploratória sugeriram que não ocorreu nenhum tipo de interação ou reação entre o fármaco e os demais componentes das formulações. Dois métodos analíticos para a determinação do tamoxifeno foram validados com sucesso por CLAE e espectroscopia UV-vis. O perfil de liberação in vitro do tamoxifeno a partir das nanopartículas de PLA apresentou característica sustentada e alcançou 50% em 180 h, tendo sido totalmente liberado após 288 h. Já as nanopartículas de PLA(5000)-PEG(1000) liberaram apenas 16,9% do fármaco após 216 h. A liberação do fármaco a partir das nanopartículas foi muito mais lenta comparada ao tamoxifeno não encapsulado, evidenciando a vantagem da incorporação do fármaco em nanopartículas compostas por PLA e PLA-PEG. No estudo do perfil de concentração plasmática em ratas Wistar, não foi possível detectar o fármaco e seu principal metabólito pelo método por CLAE desenvolvido, sugerindo que os sistemas nanoparticulados tenham extravasado rapidamente para os órgãos.
Breast cancer is the second most frequent type of cancer in the world and it is the most common among women, representing a major cause of death. Tamoxifen is an antiestrogen drug used in the treatment of this type of cancer since 1971 and it is the most employed drug in the treatment of breast cancer subtypes that expresses estrogen receptors. Despite presenting significantly positive results, its antiestrogen effect is not restricted to the tumour site, causing, as consequence, severe side effects. The purpose of this work was to develop nanostructured drug delivery systems based on PLA and PLA-PEG loaded with tamoxifen, as a strategy to potentially increase the safety and efficacy of this drug through a possible passive accumulation the site of action, due to the enhanced vascular permeability of tumour sites. Nanoparticles were prepared by the nanoprecipitation technique and presented average diameter smaller than 200 nm for the majority of the formulations. Three stabilizing adjuvants were analysed, poloxamer 407, poloxamer 188 and polysorbate 80 and the last one yielded the highest encapsulation efficiency, 86.7% and 100%, for the PLA and PLA-PEG nanoparticles, respectively. Regarding the PLA-PEG nanoparticles composition, the first polymer employed was (PLA(1000)-PEG(750)), which presented heterogeneous particle size distribution, multimodal profile and high polydispersity index. So, it was replaced by PLA(5000)-PEG(1000), which exhibited uniform particle size distribution, monomodal profile and low polydispersity index. The characterization by scanning electron microscopy confirmed the homogeneity of particles size, evidencing their spherical shape. Infrared spectrophotometry and differential scanning calorimetry analysis suggested that any interaction or reaction had occurred between the drug and the other components of the formulations. Two analytical methods for tamoxifen quantification were successfully validated by HPLC and UV-vis spectroscopy. In vitro tamoxifen release profile from PLA nanoparticles presented sustained release and reached 50% in 180 h, being completely released after 288 h, whereas PLA(5000)-PEG(1000) nanoparticles released only 16.9% of tamoxifen after 216 h. Drug release from nanoparticles was much slower compared to the non-encapsulated tamoxifen, showing the advantage of nanoparticles composed of PLA and PLA-PEG. In the plasmatic concentration profile study carried out in Wistar rats, it was not possible to detect tamoxifen or its main metabolite by the HPLC method, suggesting that nanoparticles quickly extravased to organs.

Les dificultats existents en l’administració de certs fàrmacs, que es tradueix en una considerable reducció de la seva eficàcia terapèutica, ha portat a l’exploració d’un nou camp en la recerca de fàrmacs, l’ús de polímers per a transportar fàrmacs. Aquests polímers es presenten com a vehicles transportadors que aporten protecció al fàrmac, evitant la seva degradació, i permeten la seva distribució dirigida fins la diana terapèutica, disminuint així els efectes secundaris. Una combinació adequada del polímer transportador amb el fàrmac, permet l’alliberament d’aquest en el teixit on ha de desenvolupar el seu efecte terapèutic. Tot i així, per tal de garantir l’èxit d’aquests sistemes de distribució de fàrmacs, aquests han de complir una sèrie de requisits pel que fa a la mida, càrrega superficial, composició, capacitat d’encapsular i d’alliberar un fàrmac, funcionalització i biocompatibilitat. En aquest treball, s’ha explorat la fabricació de diversos sistemes de distribució de fàrmacs per tal d’aportar coneixement sobre la modificació d’aquests polímers, que permetin obtenir plataformes de distribució de fàrmacs que reuneixi els requisits prèviament esmentats. Per una banda, s’ha obtingut un sistema termosensible i versàtil a través d’una estratègia de core-shell, que permet ajustar la seva mida i el seu comportament termosensible, com també la seva modificació superficial mitjançant un mètode fàcil i ràpid basat en una química clàssica. Per altra banda, la preparació de sistemes polimersòmics s’ha explorat per polimerització de tipus RAFT, és a dir, s’empra una química més sofisticada, que permet la síntesi de copolímers de multibloc amfifílics i auto-ensamblables, des de dos fins a cinc blocs, de manera controlada, obtenint polímers de pes molecular determinada amb distribucions de pes molecular molt estretes. De manera similar a l’anterior sistema, la modulació de la proporció entre blocs i del nombre de blocs permet el control de la mida de les nanoestructures formades i de la seva capacitat d’encapsular fàrmacs. Finalment, els sistemes polimersòmics desenvolupats s’han comparat amb un sistema de distribució de fàrmacs molt ben establert, com ara els liposomes, pel que fa a funcionalització, encapsulació i alliberament de fàrmacs, com a potencials sistemes de distribució de fàrmacs per al tractament de metàstasis de càncer de mama al cervell a través d’una estratègia de doble funcionalització, per tal d’avaluar la idoneïtat del sistema desenvolupat en aquest treball.
Las dificultades existentes en la administración de ciertos fármacos, que se traduce en una considerable reducción de su eficacia terapéutica, ha llevado a la exploración de un nuevo campo en el desarrollo de fármacos, el uso de polímeros como transportadores de estos. Estos polímeros se presentan como vehículos transportadores que aportan protección al fármaco, evitando así su degradación i permitiendo su distribución dirigida hasta la diana terapéutica, disminuyendo a su vez los efectos secundarios. Una combinación adecuada del polímero transportador con el fármaco, permite la liberación de este en el tejido dónde debe desarrollar su efecto terapéutico. Aun así, con tal de garantizar el éxito de estos sistemas de distribución de fármacos, estos deben cumplir una serie de requisitos por lo que respecta a tamaño, carga superficial, composición, capacidad de encapsular i liberar un fármaco, funcionalización i biocompatibilidad. En este trabajo, se ha explorado la fabricación de varios sistemas de distribución de fármacos con la finalidad de aportar conocimiento sobre la modificación de estos polímeros, que permitan obtener plataformas de distribución de fármacos que reúnan los requisitos previamente mencionados. Por un lado, se ha obtenido un sistema termosensible i versátil a través de una estrategia de core-shell, que permite ajustar su tamaño i su comportamiento termosensible, como también su modificación superficial mediante un método fácil i rápido basado en química clásica. Por otro lado, la preparación de sistemas polimersómicos se ha explorado mediante polimerización RAFT, es decir, utilizando una química más sofisticada, que permite la síntesis de copolímeros de multibloque amfifílicos i auto-ensamblables, desde dos a cinco bloques, de manera controlada, obteniendo polímeros de peso molecular determinado con distribución de peso molecular muy estrecha. De manera similar al anterior sistema, la modulación de la proporción entre bloques i del número de bloques permite el control del tamaño de las nanoestructuras formadas i de su capacidad de encapsular fármacos. Finalmente, los sistemas polimersómicos desarrollados se han comparado con un sistema de distribución de fármacos muy bien establecido, como son los liposomas, por lo que respecta a su funcionalización, encapsulación i liberación de fármacos, como potenciales sistemas de distribución de fármacos para el tratamiento de metástasis de cáncer de mama al cerebro a través de una estrategia de doble funcionalización, con tal de evaluar la idoneidad del sistema desarrollado en este trabajo.
The existing difficulties in the delivery of certain drugs, having a direct influence on their therapeutic efficiency, has lead to the exploration of a new field in pharmaceuticals, the use of polymers as drug carriers. Polymers are presented as carrier vehicles, which provide drug protection preventing its degradation and targeted delivery to the site of action diminishing side effects. An appropriate combination of the drug and the polymer allows the release of the drug in the tissue where it has to develop its therapeutic effect. However, in order to ensure the success of these drug delivery systems, they must fulfil a list of requirements according to size, surface charge, composition, drug loading capacity and release, targetability and biocompatibility. In this work, the fabrication of diverse drug delivery systems has been explored in order to provide know-how regarding polymers’ tunability to achieve delivery platforms that fulfil the aforementioned requirements. On one hand, a versatile thermo-responsive delivery system has been obtained trough a core-shell approach, allowing the tailoring of its size and thermosensitivity, while providing a simple and fast method to decorate its surface by means of classic chemistry. On the other hand, the preparation of polymersomic systems was explored by RAFT polymerization, a more sophisticated chemistry, which allowed the synthesis of self-assembling amphiphilic multiblock copolymers, ranging from diblock to pentablock, in a controlled manner, obtaining predetermined molecular weight polymers with narrow molecular weight distributions. Similarly to the previous system, the tunability of blocks ratio and number allowed the control over nanostructures size and loading capacity. Finally, polymersomes have been compared with a very well established delivery system, such as liposomes, in terms of targeting and drug loading and release, as potential drug delivery systems to breast cancer metastasis in the brain through a dual-targeting approach, in order to evaluate the suitability of the system developed in this work.

Research Doctorate - Doctor of Philosophy (PhD)
Albumin can promote intratumoural drug accumulation and uptake due to its interaction with albumin-binding proteins in tumour microenvironment, therefore albumin has emerged as an attractive drug carrier for cancer diagnosis and treatment. Tropomyosin receptor kinase A (TrkA) is regarded as one of the potential therapeutic targets in oncology and, in breast cancer, is associated with the stimulation of tumour growth and metastasis. By taking advantage of these two perspectives, in this project, we have developed novel albumin-based nanoparticles (NPs) to enable targeted delivery of TrkA inhibitors for breast cancer therapy. In the first part of this project, novel albumin hybrid NPs (Alb-HNPs) loaded with a selective TrkA inhibitor GNF-5837 were prepared and evaluated for antineoplastic efficacy in a panel of breast cancer cell lines. The nanomedicines (GNF-Alb-HNPs) were formed through a unique polyelectrolyte complexation process where albumin and GNF-5837 were encapsulated by a stabilising layer of oppositely charged chitosan and dextran sulphate polysaccharides. GNF-Alb-HNPs showed a particle size of ∼150 nm and excellent colloidal stability, which makes them ideal for passive targeting to tumours through the enhanced permeability and retention (EPR) effect. GNF-Alb-HNPs were shown to specifically inhibit TrkA phosphorylation and downstream mitogen-activated protein kinase (MAPK) signalling in MDA-MB-231 breast cancer cells, resulting in anti-proliferative and pro-apoptotic effects. Compared with the free GNF-5837, the GNF-Alb-HNPs not only exhibited an enhanced anti-proliferative and anti-invasive effects but also significantly increased the apoptosis of cancer cells. In the second part of this project, we aimed to develop on-demand Alb-HNPs that can respond to the tumour microenvironment and facilitate deep tumour penetration. Gelatine-albumin hybrid NPs (Gel-Alb HNPs) were developed for the delivery of the TrkA inhibitor GNF-5837, where the overexpression of matrix metalloproteinase (MMP) enzymes in the tumour can trigger site-specific release of the small drug-bound albumins for efficient uptake by cancer cells. The nanomedicines (Gel-Alb-GNF HNPs) were prepared using a pH-controlled complexation process from the pre-synthesised cationic gelatine, dextran sulphate and albumin-bound GNF-5837. Gel-Alb-GNF HNPs had a particle size of ~130 nm with narrow size distribution (polydispersity index: 0.15). They displayed excellent colloidal stability but disassembled in the presence of MMP-2 which is elevated in the extracellular matrix of tumours and can degrade cationic gelatine. Gel-Alb-GNF HNPs were shown to significantly inhibit malignant TrkA phosphorylation and downstream MAPK or Akt signalling in breast cancer cells but markedly increased their caspase-dependant apoptosis. Moreover, the migration and invasion activities of cancer cells were dramatically suppressed and the inhibitory effects were more prominent with Gel-Alb-GNF HNPS than the GNF-Alb-HNPs. Overall, results suggest that both Alb-HNPs and Gel-Alb HNPs are able to markedly improve the efficacy and specificity of encapsulated TrkA inhibitor GNF-5837 for breast cancer therapy. As TrkA receptor has been implicated in chemosensitisation as well as neuropathic pain, it is anticipated that these novel therapeutic approaches will be adaptable for the treatment of chemotherapy resistance and cancer associated pain.

A significant challenge in the surgical resection of tumors is accurate identification of tumor margins. Current methods for margin detection are time-intensive and often result in incomplete tumor excision and recurrence of disease. The objective of this project was to develop a near-infrared narrowband imaging (NIR NBI) system to image tumor and its margins in real-time during surgery utilizing the contrast provided by gold nanoparticles (GNPs). NIR NBI images narrow wavelength bands to enhance contrast from plasmonic particles in a widefield, portable and non-contact device that is clinically compatible for real-time tumor margin demarcation. GNPs have recently gained significant traction as nanovectors for combined imaging and photothermal therapy of tumors. Delivered systemically, GNPs preferentially accumulate at the tumor site via the enhanced permeability and retention effect, and when irradiated with NIR light, produce sufficient heat to treat tumor tissue. The NIR NBI system consists of 1) two LED's: green (530 nm) and NIR (780 nm) LED for illuminating the blood vessels and GNP, respectively, 2) a filter wheel for wavelength selection, and 3) a CCD to collect reflected light from the sample. The NIR NBI system acquires and processes images at a rate of at least 6 frames per second. We have developed custom control software with a graphical user interface that handles both image acquisition and processing/display in real-time. We used mice with a subcutaneous tumor xenograft model that received intravenous administration and topical administration of gold nanoshells and gold nanorods. We determined the GNP's distribution and accumulation pattern within tumors using NIR NBI. Ex vivo NIR NBI of tumor xenografts accumulated with GNPs delivered systemically, demonstrated a highly heterogeneous distribution of GNP within the tumor with higher accumulation at the cortex. GNPs were observed in unique patterns surrounding the perivascular region. The GNPs clearly defined the tumor while surrounding normal tissue did not indicate the presence of particles. In addition, we present results from NBI of tumors that received topical delivery of conjugated GNPs. We determined that tumor labeling using topical delivery approach resulted in a more homogenous distribution of GNPs compared to the systemic delivery approach. Finally, we present results from the on-going in vivo tumor margin imaging studies using NIR NBI. Our results demonstrate the feasibility of NIR NBI in demarcating tumor margins during surgical resection and potentially guiding photo-thermal ablation of tumors.
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碩士
國立交通大學
分子醫學與生物工程研究所
108
This study designed the various functional magnetic nanoparticles for molecular image of breast cancer and liver cancer. We synthesized superparamagnetic iron oxide nanoparticles conjugated specific antibody and modified fluorescent dyes. The experiments were proved that the nanoparticles had highly stability and selectivity. Moreover, the nanoparticles can not only be used as a contrast agent for MRI, but also can be used as a fluorescent probe for biological imaging. In our experiments there were three anti-bodies were selected for breast cancer and liver cancer which named CXCR2, PD-L1, and ISX. We used these antibodies to synthesize nanoparticles named MnMEIO-CXCR2-CyTE777-mPEG, SPIO-PD-L1-CyTE777-mPEG, and MnMEIO-ISX-CyTE777-mPEG. All of these nanoparticles were confirmed by UV/Vis absorption spectroscopy. We confirmed the particles’ size and ζ potential by DLS. In the stability study, we confirmed that the hydrodynamic size of the product did not change much at the pH range of 2 to 10. In the storage condition study, we confirmed that the particles’ size was very stable whatever they store in 25 ℃ or 4 ℃. In the in vitro experiments, cell viability tests were performed on cells with overexpression and low expression. The results confirmed that the nanoparticles had less cytotoxicity and could be accepted by the organism without affecting cell viability. The confocal microscopy confirmed that the nanoparticles had highly selectivity for targeting. In the part of the animal experiment which is continued the experiment before. The mice with overexpression and low expression tumors were injected SPIO-PD-L1-CyTE777-mPEG or MnMEIO-ISX-CyTE777-mPEG through the tail vein. Then, using IVIS system to confirm the in vivo molecular imaging. The result was confirmed that the nanoparticles can highly target to overexpression cell. Finally, in the MRI image, the SPIO-PD-L1-CyTE777-mPEG can achieve a negative enhancement of -30.58%, while the MnMEIO-ISX-CyTE777-mPEG can achieve a negative enhancement of -37.86%.

Tese no âmbito do Doutoramento em Ciências Farmacêuticas, ramo de Farmacologia e Farmacoterapia, apresentada à Faculdade de Farmácia da Universidade de Coimbra.
Breast cancer is a global public health problem, being one of the most frequently diagnosed neoplasms and the most common cause of cancer death in women. Recent studies suggest that environmental exposures to certain chemicals may increase the risk of developing breast cancer. As an example, 7,12-Dimethylbenzanthracene (DMBA) appears, which is a chemical present in tobacco smoke, considered a potential etiological factor of malignant neoplasms. First-line treatments for breast cancer (surgery or chemotherapy or a combination of both) are generally invasive and often associated with serious side effects and are recognized as highly comorbid strategies. Consequently, new approaches are needed with a view to a more targeted treatment to the tumor and minimally invasive to the patient. This doctoral thesis proposes the development of an experimental model in female Sprague-Dawley rats and its full characterization using histological, hematological, biochemical and epigenetic techniques. Subsequently, a proof-of-concept is proposed using photothermal therapy coupled with conjugated gold nanoparticles. In this work, it was observed that DMBA was a selective breast cancer inducer and very useful for the study of breast cancer, with 100% incidence in the tested animal model, inducing an average of 4.7 tumors per animal. In this study, no metastases were found in other organs or tissues. Epigenetic analysis showed that tumors classified with the worst prognosis were hypomethylated. Finally, treatment with gold and laser nanoparticles showed changes in terms of tumor histology, confirming, in most cases, the presence of necrosis. Thus, these results could be considered as very promising in therapy for superficial breast cancer. As a future, it is important to define the tumor's eligibility and specific efficiency criteria to better evaluate its application in the treatment of breast cancer in other species. O cancro de mama é um problema global de Saúde Pública, sendo uma das neoplasias mais frequentemente diagnosticadas e a causa mais comum de morte por cancro em mulheres. Estudos recentes sugerem que exposições ambientaisacertos produtos químicos podem aumentar o risco de desenvolver cancro de mama. Como exemplo,surge o7,12-Dimetilbenzantraceno (DMBA) que é um produto químico presente nofumo dotabaco, considerado um potencial fator etiológico de neoplasias malignas. Os tratamentos de primeira linha para o cancro de mama tais como cirurgia ou quimioterapia,ou combinação de ambos,são geralmente invasivos e frequentemente associados a efeitos secundários graves e reconhecidoscomo estratégias com alta comorbidade. Consequentementesão necessáriasnovas abordagens visando um tratamento mais direcionado ao tumor e, certamente, menos invasivopara o doente, comparativamente aos tradicionais. Esta tese de doutoramento propõe o desenvolvimento de um modelo experimental em ratos fêmea Sprague-Dawley e asua caracterização recorrendo a técnicas histológicas, hematológicas, bioquímicas e epigenéticas. Posteriormente, propõe-seumaprova de conceito recorrendo auma terapia fototérmica acopladaa nanopartículas de ouro funcionalizadas. Neste trabalho observou-se que oagente DMBA foi um indutor de cancro de mama seletivo e muito útil para o estudo docancro de mama, com 100% de incidênciano modelo animal testado,induzindo uma média de 4,7 tumores por animal. Neste trabalho não foram encontradas metástases em outros órgãos ou tecidos. A análise epigenética demostrou que os tumores classificados com pior prognóstico eram hipometilados. Finalmente, otratamento com as nanopartículas de ouro e laser apresentou alterações em termos de histologia tumoral, confirmando, na maioria dos casos, presença de necrose.Assim, estesresultados poderão ser considerados como muito promissores na terapia para o cancro de mamasuperficial. Como futuro interessa definir a elegibilidade do tumor e critérios específicos de eficiência para avaliar melhorasua aplicação no tratamento do cancro de mama em outras espécies.

碩士
國立中央大學
生物醫學工程研究所
103
The biodegradable indocyanine green (ICG)-loaded poly(lactic-co-glycolic acid) nanoparticles (IPNPs) have been successfully established by using solvent evaporation method. Furthermore we conjugated the functional molecules like Polyethylene glycol (PEG) and human epithelial receptor-2 (HER-2) antibody onto the IPNPs surface by utiliznig the EDC/NHS crosslinking method to fabricate the HER-2 targeted ICG-loaded PLGA nanoparticles (HIPNPs) for phototherapy of breast cancer cells. The ICG encapsulation efficiency was about 70 % determined from the UV-Vis spectrophotometry. The mean size and surface charge of the HIPNPs were 302 ± 1.8 nm and -15 ± 0.15mV through dynamic light scattering and zeta potential analyzer, respectively. Based on the analysis of UV-Vis spectrophotometric absorbances, the encapsulated ICG modestly disintegrated 18% and 63% while ICG freely distributed in water dramatically degraded 60% and 95% at 4 and 37℃ for 48 h, respectively, manifesting the ICG molecules in HIPNPs can be protected by PLGA matrix. In addition, the bulk temperature dramatically increased 45℃ and the singlet oxygen released in the presence of HIPNPs (ICG concentration = 25 μM) under 808 nm-laser exposure with intensity of 6 W/cm2 for 5 min has successfully proved the photothermal and photodynamic function of ICG. Finally through the cellular assay using MCF-7 (HER-2 negative), and MDA-MB-453 (HER-2 positive), we found HIPNPs can specifically target to the HER-2 protein and didn’t have biotoxicity. Even, the nmber of survival MDA-MB-453 was significantly low by treating HIPNPs with identical laser exposure, identifying the effectiveness of phototherapy of HIPNPs. We demonstrated the developed HIPNPs enable to provide the theranostic efficacy for breast cancer cells

Cerium oxide nanoparticles have been proposed as an anticancer agent, thanks to their ability of tuning the redox activity in accordance to different conditions, which lead to selective roles on healthy and cancer cells. Recent evidence suggested the ability of these nanoparticles to be toxic against cancer cells, while confer protection from oxidative stress, toward healthy cells. The main focus of this study was to determine the ultrastructural effects of cerium oxide nanoparticles over multiple incubation time of 1, 3, and 7 days on breast healthy and cancer cells. Cellular characterizations were carried out using electron microscopes, both transmission and scanning electron microscopes, while the viability assessments were performed by propidium iodide and trypan blue viability assays. The obtained results of the viability assays and electron microscopy suggested higher toxic effects on the cancer cell line viability by using a nanoceria dose of 300 μg/mL after 1 day of treatment. Such effects were shown to be preserved at 3 days, and in a longer time point of 7 days. On the contrary, the healthy cells underwent less effects on their viability at time point of 1 and 7 days. The 3 days treatment demonstrated a reduction on the number of cells that did not correlate with an increase of the dead cells, which suggested a possible initial decrease of the cell growth rate, which could be due to the high intracellular loading of nanoparticles. To conclude, the overall result of this experiment suggested that 300 μg/mL of CeO2 nanoparticles is the most suitable dose, within the range and the time point tested, which induces long-lasting cytotoxic effects in breast cancer cells, without harming the normal cells, as highlighted by the viability assays and ultrastructural characterization of electron microscopy analysis.

碩士
國立中興大學
化學工程學系所
107
Although there are many different kinds of treatment for breast cancer clinically, patients’ prognosis is always dismal since breast cancer is pretty easy to metastasize to other places and is very easy to relapse. Nowadays, immunotherapy is emerging because of its low side effect and long-term efficacy. With a view to curing breast cancer, we can not noly focus on cancer itself, but also concentrate on tumor microenvironment, so we prepare the polymeric nanoparticles that have tumor-associated macrophage-reeducating function and load with photosensitizer indocyanine green (ICG) and immune-adjuvant imiquimod (R837) together. We utilize graft copolymer hyaluronic acid-g-ploy(lactic-co-glycolic acid) (HA-g-PLGA), additional hydrophobic copolymer poly(lactic-co-glycolic acid) (PLGA), surfactant D-α-tocopherol polyethylene glycol 1000 succinate (TPGS), ICG and R837 to form nanoparticles (ICG/R837 loaded HA-g-PLGA NPs) in aqueous solution by using nanoprecipitation method. The hydrodynamic diameter of the nanoparticles is 161.6 ± 4.1 nm. The loading content of ICG and R837 is 3.1 ± 0.4 and 3.7 ± 0.3 wt%, respectively. According to our experiments, we deduce that our ICG/R837 loaded HA-g-PLGA NPs can skew tumor-associated macrophage from M2 type to M1 type attributing to low molecule weight hyaluronic acid as well as R837. We prove that loading photosensitizer ICG can be triggered by near-infrared laser to produce hyperthermia that can kill cancer cells through necrosis as well as produce tumor-associated antigens. The tumor associated antigens can be uptaken by macrophages and dendritic cells and they can present to T cells; on the other hand, R837 can make more dendritic cells mature to recruit T cells, but they still required to be verified.

Tese de Doutoramento em Genética Molecular Comparativa
As nanopartículas de lípidos sólidos (SLN, do inglês “solid lipid nanoparticles”) são transportadores coloidais de fármacos, constituídos por uma matriz de lípidos sólidos à temperatura corporal e à temperatura ambiente, estabilizados por agentes tensioactivos apropriados. No âmbito da presente tese, estes sistemas foram desenvolvidos com objectivo de administração de fármacos pouco solúveis em água, e para facilitar a administração direccionada a células de cancro. O objectivo deste trabalho consistiu em explorar o potencial das SLN no tratamento da invasão celular de cancro de mama, nomeadamente das células HER2/neu positivas. Foram desenvolvida partículas cuja composição consistiu no Imwitor 900K ou Compritol 888 ATO (como lípidos sólidos), no cetyltrimethylammonium bromide (CTAB) como lípido catiónico/tensioactivo e no Lutrol F68 ou Miranol C32 Ultra como agente tensioactivo. Como método de produção, foi utilizada a homogenização a alta pressão ou a alta velocidade, seguindo-se a optimização das respectivas formulações, as quais foram utilizadas para os estudos posteriores. As SLN foram caracterizadas em termos de distribuição de tamanho médio das partículas, cristalinidade de matriz lipídica sólida e estabilidade durante armazenamento. Foram obtidas SLN com tamanho médio das partículas entre 115 nm e 334 nm e d0.90 inferior a 1 μm. O estado sólido das mesmas foi confirmado por calorimetria diferencial de varrimento e por difracção de raios X. Todas as formulações apresentaram estabilidade adequada ao longo de 5 semanas, quer à temperatura ambiente, quer a 4 °C. Apesar da liofilização com o crioprotector trealose, a estabilidade das SLN não liofilizadas revelou-se bastante superior. A formulação designada como cSLN-C manteve-se estável durante um período mínimo de 12 semanas. As SLN são, em geral, consideradas como transportadores coloidais com baixa toxicidade. Mesmo assim, o efeito das SLN per si tem importância na interpretação da interacção de formulações que contém um fármaco ou um anticorpo com as células. As SLN desenvolvidas neste trabalho não apresentaram toxicidade na concentração de 0,01 mg/ml. Utilizando a concentração de 0,1 mg/ml a viabilidade celular diminuiu dependendo da linha celular utilizada e tempo de exposição. A dose 1,0 mg/ml foi tóxica nas linhas celulares seleccionadas para este trabalho. Dentro destas, MCF-7 (carcinoma de mama, receptor de estrogénio positivo, HER2-neu negativo) foram as mais susceptíveis aos danos causados pelas SLN, as BT-474 (carcinoma mamário, HER2-neu positivo), HepG2 (hepatocarcinoma) e Caco-2 (cólon adenocarcinoma) foram menos susceptíveis em ordem decrescente. A toxicidade das SLN foi causada por disrupção de integridade das membranas celulares. Danos em ácido deoxiribonucléico (ADN) foram detectados por ensaio cometa. Foram reportados poucos danos – quando comparado com controlo sem tratamento (não significativo nas concentrações não tóxicas). Também foram detectados danos em purinas, que não causaram quebras de ADN. Alguns sinais de stress oxidativo foram detectados em células HepG2: a fluorescência de diacetato de diclorofluoresceina (DCFDA) encontrou-se aumentada relativamente aos controlos sem tratamento e aos positivos, verificou-se um aumento da actividade da enzima superóxido dismutase e uma diminuição da actividade de glutationa reductase. Apesar destes sinais de existência de stress oxidativo, os lípidos membranares não foram afectados (determinação como substâncias reactivas ao ácido tiobarbitúrico, TBARS). Estes resultados estão em concordância com poucos danos detectados em ADN (relativamente ao controlo sem tratamento). Os danos causados por stress oxidativo podem ocorrer em células com capacidade antioxidante inferior à das células HepG2. A capacidade de indução de stress oxidativo pode, hipoteticamente, ser vantajosa em veiculação de fármacos quimioterapêuticos, cujo mecanismo de acção exige existência de radicais livres, e pode, parcialmente, contribuir para a melhoria de eficácia destes medicamentos, quando veiculadas em SLN in vitro e in vivo. A Curcumina foi seleccionada como fármaco-modelo com potencial actividade antineoplásica. A baixa solubilidade aquosa, instabilidade em pH alcalino e fotossensibilidade são propriedades que fazem da curcumina um fármaco ideal para a encapsulação em SLN. Contudo, a solubilidade em vários lípidos foi igual ou inferior a 1 %. A baixa solubilidade em lípidos influenciou a capacidade de carga. Em combinação com as limitações atribuídas à toxicidade das SLN, apenas pode ser administrada 10 μg/ml (27 μM) no máximo, uma dose que é insuficiente para observar os efeitos anticancerígenos da curcumina. Um anticorpo anti- HER2/neu foi colocado na superfície das SLN utilizando a interacção streptavidina-biotina. O efeito de complexo anticorpo-SLN foi governado pela toxicidade das próprias SLN. A conjugação com o anticorpo melhorou significativamente a internalização de complexos nas células de cancro mamário. O efeito foi mais marcado em células BT474, HER2/neu positivas. O tratamento com complexo SLN-anticorpo causou uma diminuição de viabilidade celular das linhas de cancro de mama superior ao efeito das partículas isoladas ou do anticorpo isolado.
Solid lipid nanoparticles (SLN) are colloidal carriers consisting of lipid cores that are solid at body and room temperature dispersed in aqueous phase and stabilized by suitable surfactant. They were developed to improve drug delivery of drugs that are poorly soluble in water and to enable targeted delivery to cancer cells. The aim of this work was to explore the potential of SLN in treatment of breast cancer cell invasion, namely HER2/neu positive breast cancer cells. A series of SLN composed of Imwitor 900K or Compritol 888 ATO as solid lipid, cetyltrimethylammonium bromide (CTAB) as cationic lipid/surfactant and Lutrol F68 or Miranol C32 Ultra as surfactants was developed. Optimized high shear homogenisation of high pressure homogenisation were used as preparation methods. SLN were characterized in terms of particle size distibution, lipid core crystalinity and storage stability. SLN with mean particle size between 115 nm and 334 nm and d0.50 below 0.5 μm were obtained; the crystalline state of lipid cores was confirmed by differential scanning calorimetry and X-ray diffraction. All SLN were stable for at least 4 weeks at room temperature and 4 °C, which was superior to stability of the same SLN freezedryed with trehalose. Compritol-composed SLN were stable over 12 weeks. SLN are in general considered as safe colloidal carriers, their effect on living cells however cannot be neglected when interpreting the studies of interaction of drug-loaded and/or targeted SLN with cells. SLN developed in this work were non-toxic to living cells at a dose 0.01 mg/ml; cell viability was reduced to various extent at 0.1 mg/ml depending on cell line and time of exposure and at 1.0 mg/ml the SLN were toxic to the selected cell lines. Among the used cell lines, MCF-7 cells (breast carcinoma, estrogen receptor positive, Her2/neu negative) were the most susceptible to our SLN, followed by BT-474 (breast carcinoma, HER2/neu positive), HepG2 (hepatocarcinoma) and Caco-2 (colorectal carcinoma) cells. Toxicity of SLN was caused mostly by disruption of membrane integrity. DNA damage was examined by comet assay and was detected in a limited extent, compared to untreated controls (not significant at non-toxic concentrations). Damage to purine bases that did not directly lead to DNA strand breaks was also detected. Some signs of oxidative stress was detected in HepG2 cells: dichlorofluorescein-diacetate (DCFDA) assay revealed increase in free radicals content compared to untreated and positive controls, activity of superoxid dismutase was found increased and activity of glutathion reductase was drastically decreased. Despite these signs of oxidative stress, membrane lipids were not affected – as determined by thiobarbituric acid reactive species (TBARS) determination. This finding is in line with only slightly increased DNA strand breaks (compared to untreated control). Damage caused by oxidative stress after SLN exposure may however occur in cells with lower antioxidant capacity than HepG2 cells. The capacity to induce oxidative stress can hypotethically be beneficial for delivery of chemotherapeutic drugs – that require some free radical increase for their action – and may partly explain many reports on SLN improving efficiency of chemotherapeutics in vitro and in vivo. Curcumin was selected as model drug with potential chemotherapeutic effect. Its low solubility, instability at alkaline pH and light make it an ideal candidate for encapsulation into SLN. Unfortunately, its solubility in solid lipids was limited to 1% (w/w) and to lipid mixtures containing either monoacylglycerides or polyethylenglycol. This affected the resulting drug loading, which together with limitations by SLN toxicity only enabled use of dose equal or lower than 10 μg/ml (27μM) of curcumin – i.e. doses lower than those at which anticancer effects were observed. An antiHER2/neu antibody was attached to SLN surface via streptavidin-biotin binding. The effect of targeted complex was influenced mostly by the toxicity of SLN alone, but at non-toxic dose of SLN a synergistic effect between SLN and the antibody was observed. The antibody improved significantly cell internalization into breast cancer cells, mostly in HER2/neu positive BT-474 cells but to some extent also in MCF-7 cells. Exposure to targeted SLN leads to cell viabilities lower than when exposed to antibody alone or SLN alone.

Yes
The delivery of anticancer agents to their subcellular sites of action is a significant challenge for effective cancer therapy. Peptides, which are integral to several oncogenic pathways, have significant potential to be utilised as cancer therapeutics due to their selectivity, high potency and lack of normal cell toxicity. Novel Ras protein-Regulator of chromosome condensation 1 (Ran-RCC1) inhibitory peptides designed to interact with Ran, a novel therapeutic target in breast cancer, were delivered by entrapment into polyethylene glycol-poly (lactic-co-glycolic acid) PEG-PLGA polymeric nanoparticles (NPs). A modified double emulsion solvent evaporation technique was used to optimise the physicochemical properties of these peptide-loaded biodegradable NPs. The anti-cancer activity of peptide-loaded NPs was studied in vitro using Ran-expressing metastatic breast (MDA-MB-231) and lung cancer (A549) cell lines, and in vivo using Solid Ehrlich Carcinoma-bearing mice. The anti-metastatic activity of peptide-loaded NPs was investigated using migration, invasion and colony formation assays in vitro. A PEG-PLGA-nanoparticle encapsulating N-terminal peptide showed a pronounced antitumor and anti-metastatic action in lung and breast cancer cells in vitro and caused a significant reduction of tumor volume and associated tumor growth inhibition of breast cancer model in vivo. These findings suggest that the novel inhibitory peptides encapsulated into PEGylated PLGA NPs are delivered effectively to interact and deactivate Ran. This novel Ran-targeting peptide construct shows significant potential for therapy of breast cancer and other cancers mediated by Ran overexpression.

碩士
國立中興大學
生醫工程研究所
104
Lymphatic metastasis is the most common form of metastasis and is one of the key factors in tumor staging and therapy in patients with breast cancer. In this study, lymphatic endothelial cells (SVEC4-10) and human breast cancer cells (MCF-7) were respectively cultured with polymer precursor solution containing Fe3O4 magnetic nanoparticles for in vitro observing the hyperthermia effect on cell growth. 3D polymer scaffold was synthesized by photopolymerization of poly(ethylene glycol) diacrylate (PEGDA)-based precursor solution under a visible light exposure for mimicking real living environment. Photosensitive PEGDA and poly(ethylene glycol) (PEG) were mixed at 5:1 ratio to form 3D polymer precursor solution. In order to control the temperature and the condition of hyperthermia, Fe3O4 magnetic nanoparticles with varied concentrations, including 50, 80, 100, 150 ppm, were added to PEGDA-based hydrogels, respectively. After that, Fe3O4 mixed PEGDA-based hydrogels were exposed under 808-nm laser light for 10 min, the changes of temperature were recorded in search of a suitable temperature used in hyperthermia treatment. SVEC4-10 and MCF-7 cells were respectively mixed in 80-ppm Fe3O4 precursor solution for the investigation of hyperthermia effect at 40.9 oC. The distribution of SVEC4-10 cells and MCF-7 cells in 3D culture environment was visualized by the fluorescent labeling of rhodamine–phalloidin (red) and DAPI (blue) on day 1, 3, 5 of culture. MCF-7 cells significantly decreased after 5 days of culture under hyperthermia condition at 42.9 oC. However, SVEC4-10 cells normally grew after 5 days of cell culture in PEGDA-based hydrogel. In addition, it is possible to consolidate 3D polymer scaffold to regenerate the lymphatic tissue. Our results showed Fe3O4 magnetic nanoparticles were biocompatible to cells and suitable to create a hyperthermia environment for effectively clinical treatment for breast cancer without damage of lymphatic endothelial growth.

Broad distribution and activity limit the utility of anti-cancer compounds by causing unacceptable systemic toxicity and narrow therapeutic indices. To improve tumour accumulation, drug-loaded macromolecular assemblies have been designed to replace conventional surfactant-based formulations. Their nanoscale size enhances tumour accumulation via hyperpermeable vasculature and reduced lymphatic drainage. Incorporating targeting ligands introduces cell specificity through receptor-specific binding and uptake, enabling drugs to reach intracellular targets. In this work, the targeting properties of polymer nanoparticle micelles of poly(2-methyl-2-carboxytrimethylene carbonate-co-D,L-lactide)-graft-poly(ethylene glycol)-furan (poly(TMCC-co-LA)-g-PEG) were verified using in vitro and in vivo models of breast cancer. To select a relevant mouse model, the vascular and lymphovascular properties of two tumour xenograft models were compared. Greater accumulation of a model nanocarrier was observed in orthotopic mammary fat pad (MFP) tumours than size matched ectopic subcutaneous tumours, suggesting that the organ environment influenced the underlying pathophysiology. Immunostaining revealed greater vascular thickness, density and size, and thinner basement membranes in MFP tumours, likely contributing to greater blood perfusion and vascular permeability. Based on these observations, MFP tumour-bearing mice were used to characterize the pharmacokinetics and biodistribution of a taxol drug, docetaxel, encapsulated in poly(TMCC-co-LA)-g-PEG nanoparticles. The nanoparticle formulation demonstrated longer docetaxel circulation in plasma compared to the conventional surfactant-based formulation. As a result, greater docetaxel retention was uniquely measured in tumour tissue, extending exposure of tumour cells to the active compound and suggesting potential for increased anti-cancer efficacy. Furthermore, active targeting of antibody-modified nanoparticles to live cells was shown to be selective and receptor-specific. Binding isotherms were used to quantify the impact of antibody density on binding strength. The equilibrium binding constant increased linearly with the average number of antibodies per particle, which is consistent with a single antibody-antigen interaction per particle. This mechanistic understanding enables binding behaviour to be adjusted in a predictive manner and guides rational nanoparticle design. These studies validate poly(TMCC-co-LA)-g-PEG nanoparticles as a platform for targeted delivery to cancer on both a tissue and cellular level, forming a compelling justification for further pre-clinical evaluation of this system for safety and efficacy in vivo.

碩士
國立中正大學
生命科學系生物醫學研究所
106
According to the National Institutes of Health, female breast cancer accounts for the first place in all new cancer cases, and the median age at death is 68 years old. Current breast cancer therapies include chemical treatment and surgery, which cause many side effects. Photodynamic therapy (PDT) is a minimally invasive therapeutic approach with low systemic toxicity and fewer side effects. In this study, we synthesized nanoparticles by cross-linking between cationic chitosan and anionic tripolyphosphate. In addition, water-insoluble pheophorbide a, a natural breakdown of chlorophyll, was encapsulated within HER2-antibody conjugated chitosan/TPP nanoparticles. The synthesized nanoparticles can target tumor cells with overexpression of epidermal growth factor receptor 2 , the targeting can enhance the efficacy of PDT and avoid normal cell damage.The results showed that the confugated of HER2 antibody on the nanoparticles increased the effect of photodynamic therapy and avoided the damage of normal cells. The cell viability of PDT with antibody conjugated chitosan/TPP nanoparticles, encapsulate pheophorbide a, treated epidermal growth factor receptor 2 overexpressing cell line MDAMB453, MDAMB361, and breast epidermal cell line H184B5F5/M10, the IC50 60 nM, 60 nM, 160 nM. we successfully used antibody-conjugated nanoparticles, and the target increased photodynamic therapy in epidermal growth factor receptor 2 overexpressing breast cancer. The efficacy is expected to be applied to the clinical treatment of cancer.

Tese de doutoramento em Ciências Farmacêuticas, no ramo de Tecnologia Farmacêutica, apresentada à Faculdade de Farmácia da Universidade de Coimbra
Breast cancer remains a major public health care burden, with tremendous impact on society. Therapeutic intervention is often undermined by the intrinsic heterotypic nature of tumors, in which a multitude of cell types intertwine to foster new biological features that support tumor development. Among them, resistance to cornerstone chemotherapies remains pivotal. It has been postulated that cancer stem cells (CSC), a sub-population of stem-like cancer cells exhibiting self-renewal capability and high tumorigenic capacity, have a central role in tumor development, metastization, recurrence as well as drug resistance. In addition, the recent acknowledgement that CSC can originate from non-stem cancer cells (non-SCC) highlighted the need to develop strategies targeting both cell sub-populations. It has been recognized that success requires the identification of compounds that, when combined, lead to synergistic tumor inhibition. However, in vivo application of such protocols is dependent on the ability to deliver the appropriate drug ratio at the tumor level. In this respect, nanotechnology-based delivery platforms, like liposomes, offer an elegant solution for the in vivo translation of such strategy. Modifying drugs’ pharmacokinetics by the co-encapsulation into liposomes enables one to achieve the synchronous temporal and spatial delivery of a drug combination at tumor site. Additionally, the coating of nanoparticles with ligands targeting specific overexpressed receptors would enable the precise delivery of drug combinations into particular cellular sub-populations, such as the CSC, ultimately enabling a gain in terms of efficacy while simultaneously decreasing systemic toxicity. In the present work, it is described the development of a PEGylated liposomal formulation co-encapsulating a combination of doxorubicin (inner aqueous core) and the pro-apoptotic C6-ceramide (liposomal membrane bilayer), capable to target, by a ligand coupled at its surface, both putative breast CSC and non-SCC, besides other tumor cells. The ligand - F3 peptide - enables the specific binding to nucleolin (NCL), a protein overexpressed by cancer cells and endothelial cells of tumor angiogenic blood vessels, promoting active nanoparticle internalization. In addition, a pH-sensitive triggered release mechanism enabling burst release of the cargo upon intracellular delivery, upon endosomal acidification, has also been included. Drug screening has demonstrated that a combination of doxorubicin (DXR):C6-ceramide (C6-Cer) at 1:2 molar ratio interacted synergistically against drug resistant/triple negative MDA-MB-231 breast cancer cells, as well as drug sensitive MDA-MB-435S melanoma cells. F3 peptide-targeted liposomes encapsulating the DXR:C6-Cer 1:2 molar ratio performed similarly as targeted liposomal DXR, encapsulating twice the amount of DXR. Importantly, F3-targeted liposomes encapsulating DXR:C6-Cer 1:2 molar ratio enabled a cell death above 90% at 24 h of incubation against both DXR-resistant and sensitive cells, unattainable by the F3 peptide-targeted liposomal doxorubicin. Furthermore, a F3-targeted formulation encapsulating a mildly additive/antagonistic DXR:C6-Cer 1:1 molar ratio enabled an effect above 90% for an incubation period as short as 4 h, suggesting that delivery route, at the cell level, may shift the nature of drug interaction. Such activity induced a marked cell and nucleus swelling at similar extent, consistent with necrotic cell death. Moreover, it was demonstrated that F3 peptide-targeted liposomes associated with both breast non-SCC and putative CSC, but in higher extent with the latter (2.6- and 3.2-fold for triple negative MDA-MB-231 and luminal-like MCF-7 cells, respectively), in an energy-dependent process. Increased mRNA levels of NANOG and OCT4 transcription factors, paralleled by NCL, were found in putative breast CSC as compared to non-SCC, from triple negative breast cancer cells. Additionally, using mouse embryonic stem cells as stemness bona fide model, it was shown that both NCL mRNA levels and cellular association of F3 peptide-targeted liposomes were dependent on stemness status. In addition, it was demonstrated that triple negative breast NCL+ cells were more tumorigenic than NCL- cells, paralleling putative breast CSC behavior. Furthermore, F3 peptide-targeted triggered-release liposomes promoted the efficient and simultaneous delivery of DXR:C6-Cer combinations into triple negative breast CSCs, enabling extensive cell death. Altogether, the results presented in chapters 2 and 3 of this thesis demonstrated that F3 peptide-targeted intracellular delivery of different DXR:C6-Cer ratios, with diverse drug interactions, enabled a significant increase of efficacy against chemotherapy resistant cells. Additionally, the results suggested a clear link between NCL expression (including cell membrane NCL) and the stem cell-like phenotype, namely in triple negative breast cancer, enabling the simultaneous intracellular delivery of drug combinations-containing liposomes functionalized with the F3 peptide into both CSC and non-SCC. Provided the necessary accessibility to the CSC niche, this technology, combined with the established NCL-mediated targeting of tumor angiogenic blood vessels, has the potential to simultaneously debulk multiple cellular compartments of the tumor microenvironment, while decreasing tumor recurrence and systemic toxicity, ultimately providing long-term disease free survival.
O cancro da mama representa um enorme problema de saúde pública com grande impacto na sociedade. A abordagem terapêutica é muitas vezes comprometida pela natureza heterotípica intrínseca dos tumores nos quais diferentes tipos celulares interagem conduzindo o ganho de novas funções biológicas que suportam o desenvolvimento do tumor. Entre outras, a resistência a quimioterapia permanece uma questão central. As células estaminais cancerígenas (CSC) representam uma subpopulação celular com características estaminais com elevada capacidade de renovação e elevado potencial tumorigénico. Estas têm um papel fundamental no desenvolvimento tumoral, metastatização, recorrência, assim como na resistência a fármacos. O facto reconhecido recentemente de que as células cancerígenas não-estaminais (non-SCC) podem dar origem a CSC sublinha a necessidade de se encontrarem estratégias terapêuticas direcionadas simultaneamente a estas subpopulações. O sucesso de uma intervenção terapêutica eficaz poderá estar dependente da identificação de combinações de fármacos capazes de inibir sinergicamente o crescimento tumoral. No entanto, a aplicação de protocolos desta natureza in vivo é dependente da entrega, ao nível do tumor, do rácio de fármacos adequado. Nesse sentido, plataformas nanotecnológicas de entrega de fármacos, como os lipossomas, representam uma abordagem adequada para a translação in vivo daquela estratégia. A alteração da farmacocinética, através da co-encapsulação em lipossomas, permite a entrega da combinação de fármacos de forma sincronizada, espacial e temporalmente, ao nível do tumor. A funcionalização destas nanopartículas com ligandos direcionados a recetores específicos poderia permitir a entrega de uma combinação de fármacos a subpopulações celulares particulares, como as CSC, levando a um aumento da eficácia e, simultaneamente, a uma diminuição de toxicidade sistémica. No âmbito do presente trabalho, é descrito o desenvolvimento de lipossomas PEGuilados, encapsulando uma combinação de doxorrubicina (núcleo aquoso) e C6-ceramida (bicamada lipídica) direcionados, através de um ligando à superfície, às CSC e non-SCC da mama, para além de outras células tumorais. O ligando – peptídeo F3 – reconhece especificamente a nucleolina (NCL), uma proteína abundante em células cancerígenas e células endoteliais de vasos angiogénicos de tumores, levando à internalização ativa da nanopartícula. Foi ainda incluindo um mecanismo de libertação de fármacos sensível ao pH, ativado após internalização seguida de acidificação dos endossomas. Após screening, demonstrou-se que a combinação doxorrubicina (DXR):C6-ceramide (C6-Cer) no rácio molar de 1:2 interagiu sinergicamente contra células cancerígenas da mama MDA-MB-231 (resistentes/triplas negativas), assim como em células de melanoma (MDA-MB-435S) sensíveis a fármacos. Os lipossomas direcionados pelo peptídeo F3, co-encapsulando o rácio molar 1:2 da combinação DXR:C6-Cer foram semelhantes, em termos de eficácia, à DXR lipossomal direcionada pelo mesmo peptídeo e encapsulando o dobro da quantidade desta. Não menos importante, os lipossomas direcionados pelo peptídeo F3 contendo a combinação DXR:C6-Cer 1:2 levaram a uma morte celular acima de 90% após 24 h de incubação em ambas a linhas, o que não se verificou para a lipossomas direcionados contendo apenas DXR. Por outro lado, lipossomas direcionados pelo peptídeo F3, encapsulando a combinação DXR:C6-Cer no ratio molar 1:1 (aditivo/antagonista) conduziram a uma morte celular superior a 90% para um período de incubação de 4 h, sugerindo que a natureza da interação entre fármacos pode mudar com a via de entrada na célula. Tal atividade levou a um aumento do tamanho celular e nuclear, consistente com morte celular por necrose. Adicionalmente foi demonstrado que os lipossomas direcionados pelo peptídeo F3 associavam ativamente com ambas non-SCC e CSC da mama, em maior extensão com estas últimas (2,6 e 3,6 vezes para as linhas MDA-MB-231 e MCF-7 (tipo luminal), respetivamente). Também foram encontrados níveis de mRNA dos fatores de transcrição NANOG e OCT4 aumentados, à semelhança da NCL, nas CSC triplas negativas quando comparadas com non-SCC. Usando células estaminais embrionárias de murganho como modelo bona fide de propriedades estaminais, foi demonstrado que o nível de mRNA da NCL assim como a associação celular de lipossomas direcionados pelo peptídeo F3 era dependente do estado estaminal. Demonstrou-se ainda que células cancerígenas da mama NCL+, triplas negativas, eram mais tumorigénicas do que células NCL-, um comportamento semelhante às CSC. Em paralelo, mostrou-se que os lipossomas direcionados pelo peptídeo F3 promoveram uma entrega eficiente da combinação DXR:C6-Cer em CSC da mama triplas negativas, levando a uma extensa morte celular. Em suma, os resultados apresentados nos capítulos 2 e 3 desta tese demonstraram que a entrega intracelular, direcionada pelo peptídeo F3, de diferentes ratios DXR:C6-Cer conduziu a um aumento relevante da eficácia contra células resistentes à quimioterapia. Ainda, os dados sugeriram uma ligação clara entre a expressão de NCL e o fenótipo estaminal, nomeadamente em cancro da mama triplo negativo, permitindo a entrega intracelular de lipossomas direcionados pelo peptídeo F3 encapsulando combinações de fármacos, a ambas CSC e non-SCC. Assegurada a acessibilidade ao nicho das CSC, esta tecnologia, aliada ao direcionamento para os vasos angiogénicos dos tumores mediado pela NCL já descrito, tem o potencial de atacar múltiplos compartimentos celulares do microambiente tumoral, levando a uma diminuição da recorrência e da toxicidade, potencialmente providenciando um aumento da esperança de sobrevivência a longo termo de doentes com tumores de mama.
FCT - SFRH/BD/64243/2009

碩士
中原大學
奈米科技碩士學位學程
97
Cancer multidrug resistance is associated with plasma membrane expression of P-glycoprotein which results to a reduction of the intracellular drug concentration after activation of P-glycoprotein efflux. In this study, an AB2 type amphiphilic block copolymer was synthesized from biodegradable methoxy poly (ethylene glycol) (mPEG) and poly (ε-caprolactone) (PCL) to encapsulate the antitumor drug, Doxorubicin (DOX). The initiator mPEG was first esterified with 2,2-bis (hydroxymethyl) butyric acid. Removal of the protective group by acidic resin afforded a copolymer with two hydroxyl chain terminals which was subsequently used in a ring-opening polymerization of ε-caprolactone. The AB2 copolymer mPEG-(PCL)2 was characterized by Fourier transform infrared spectroscopy and 1H nuclear magnetic resonance spectroscopy. By increasing the 2,2-bis (hydroxymethyl) butyric acid content in the copolymer, the melting point can decrease from 62.29 oC to 60.73 oC. The molecular weight of the copolymer was calculated as 15,245 g/mol by gel permeation chromatography and 19,312 g/mol by 1H nuclear magnetic resonance. The amphiphilic block copolymer self-assembled into micelles and the critical micelle concentration was 43.7×10-3 mg/mL. The particle size of the empty micelle was measured by dynamic light scattering and found to be 95.1 nm. After loading with doxorubicin, the particle size was 21.4 nm. The drug encapsulation was measured at 22.29% by UV-vis spectrophotometer. The in vitro release study indicated that 50% of the drug was released from micelles at pH 5 acetate buffer and 40 % at pH 7.4 phosphate buffer in the duration of 48 hours. Incubation of macrophage cells with the empty micelles resulted in 0.420 O.D./mg protein and 0.413-0.472 O.D./mg protein NO production for the control and micelle, respectively. This indicated that the micelles could avoid recognition by macrophage cells. Rhodamine 123 assay by flow cytometry and western blot were used to monitor the relative P-glycoprotein expression in human breast cancer cell lines MCF-7/WT and MCF-7/ADR. The results showed that the fluorescence of rhodamine 123 was stronger in MCF-7/WT than MCF-7/ADR. Western blot analysis detected a single band for P-glycoprotein at 170 kDa. The IC50 value of drug-loaded micelle for MCF-7/WT and MCF-7/ADR were 0.285 μg/mL and 7.476 μg/mL, respectively. These values are 7.9-fold higher than the IC50 of free drug for MCF-7/WT and MCF-7/ADR at 0.036 μg/mL and 0.937 μg/mL, respectively. This can be due to the slow release rate of DOX from the micelles. The cell uptake study by confocal laser scanning microscopy (CLSM) showed that drug-loaded micelles accumulated mostly in the cytoplasm instead of the nuclei through endocytosis. In contrast, free drug diffused throughout the cell. In addition the number of cell uptake under the same fluorescence intensity was measured to be 83.16 % and 50.91 % for drug-loaded micelles and free drug in MCF-7/ADR cells. In conclusion, the AB2 copolymer was able to overcome multidrug resistance of breast cancer cells as it can accumulate more in MCF-7/ADR cells compared to free drug. Future in vivo studies could focus on how the drug delivery system can inhibit the tumor growth and prolong the survival rate.