Development of Targeted Liposomal Formulation Approaches for Enhanced Colorectal Cancer Therapy

Colorectal cancer (CRC) is the 4th most commonly detected cancer in the USA. Despite promising advances, the 5-year survival rate for the metastatic disease remains dismal (40⁰C with ultrasound contrast agents and bacterial attachments can improve the real-time chemo-immunotherapy of CRC. Towards these goals, we investigated the following specific aims in murine models of colon cancer: 1) Develop echogenic-LTSL (E-LTSL) for real-time ultrasound-enhanced reporting of tumor temperature and doxorubicin delivery, 2) Utilize tumor homing Salmonella typhimurium for LTSL delivery and enhanced chemo-immunotherapy with High Intensity Focused Ultrasound (HIFU) tumor heating (~42°C), and 3) Investigate the ability of magnetic bacteria Magnetospirillim magneticum (AMB-1) to aid LTSL tumor drug delivery under magnetic guidance. Our data showed that intratumoral vascular contrast of E-LTSL as a function of temperature and doxorubicin delivery was strongly correlated, enabling robust estimation of temporal variation in colon tumor temperature and drug delivery. LTSL attachment didn’t impact Salmonella viability and improved chemo-immunotherapy outcomes in murine colon cancers by promoting the population of M1 macrophages with HIFU heating. Finally, the use of magnetic guidance for AMB-LTSL significantly reduced the colon cancer viability by enhancing cellular and tumor localizations of doxorubicin. In conclusion, we found that multifunctional LTSL formulations significantly improved the CRC treatment outcomes in murine models by aiding the real-time monitoring and removing the resistive and suppressive tumor microenvironment features

The effects of dielectric values, breast and tumor size on the detection of breast tumor

Although breast cancer is the second main cause of female deaths after lung cancer, early diagnosis plays a crucial role to diminish the death rate. Many techniques have been improved to detect the cancerous cells. At different microwave frequencies, the malignant cells indicate different electrical characteristics as compared to the normal cells. According to these frequencies, the breast tissue is more permeable than other tissues such as the brain and muscle. Due to this property of the breast tissue, microwaves can be used for the detection of breast cancer. In this study, the breast prototype was modelled using the CST STUDIO SUITE electromagnetic simulation software with respect to different breast size, tumor size and dielectric values tested at a range of the 0-3.0 GHz frequency. The objective of this paper is to investigate the effects of each factor and the interactions of factors on detecting cancer cells using the factorial analysis. The results indicate that the factors such as fat and skin permittivity, tumor and breast sizes are more effective in the detection of breast tumor. Although the effect of fibro permittivity is not significant alone, there are considerable interaction effects of a large breast size and small tumor size through low-to-high values of fibro permittivity. Furthermore, the combinations of a breast radius smaller than almost 8.5 cm with a high level tumor radius and breast radius larger than 8.5 cm with a low level tumor radius are desirable for lessening the return loss value

Mms6 expression in adipose-derived mesenchymal stem cells: development of biogenic magnetic nanoparticles and Its therapeutic potential

Magnetic nanoparticles (MNP) are attracting interest for a range of biomedical applications being used either alone or as part of cell-based therapies. An area of particular interest is magnetic nanoparticle-mediated hyperthermia (MNHT), when MNP absorb energy from alternating magnetic fields (AMF) and transform this energy into heat which results in cancer cell death. While promising, the use of MNP for diagnosis and therapy has been limited by their rapid removal from the blood and biological barriers at the tissue and cellular levels. Moreover, MNP may have adverse side effects when used clinically. To overcome these problems there is increasing interest in the development of cellbased strategies to deliver MNP. Current strategies include combining commercially available MNP with mesenchymal stem cells (MSC), as these cells can migrate to sites of tissue injury and tumor growth. However, problems with MNP cytotoxicity have hindered progress in this area and need to be overcome. Therefore, the ultimate aim of this PhD project was to find an alternative way to develop MSC that contain MNP using a genetic engineering approach by which the cells can be induced to stably produce biogenic MNP and to establish whether such an approach could be of value for MNHT for cancer treatment. To achieve this goal, the experiments conducted in this PhD project involved transfection of adipose-derived mesenchymal stem cells (AD-MSC) to introduce a synthetic magnetic gene, mms6, into the cells. The gene is originally derived from Magnetospirillum magneticum AMB-1, a genus of magnetotactic bacteria (MTB), which has unique intracellular structures called magnetosomes. The magnetosomes contain iron-rich magnetic nanoparticles that are enclosed within a lipid bilayer membrane. During the formation of magnetosome, mms6 has been known for its key role in the formation of uniform isomorphic magnetite nano-crystals and helps regulate the crystal morphology of magnetite. Due to the unique feature of this gene, therefore the novelty of this study was in introducing codon-optimised mms6 into AD-MSC, enabling the cells to produce biogenic nanoparticles. In this study, mms6 mRNA expression in AD-MSC, following transfection, was demonstrated by reverse transcription polymerase chain reaction (RT-PCR). The HisGFP tag Mms6 protein expression was demonstrated by Flow cytometry, Western blot and GFP imaging analysis revealing the expression of Mms6 protein in AD-MSC. Furthermore, for stable mms6 expression, a lentiviral transduction approach was used. AD-MSC stably expressing mms6 were then used in in vitro MNHT studies. The effect of mms6 stable expression on MSC markers of stemness and differentiation ability of AD-MSC were also investigated. The cellular ultrastructure of AD-MSC expressing mms6 was demonstrated by transmission electron microscopy (TEM), revealing the presence of nanoparticles. The magnetism of AD-MSC expressing mms6 was proved by superconducting quantum interference device (SQUID). Furthermore, as a comparison study, Ferucarbotran, chemically synthesized superparamagnetic nanoparticles, were also used to magnetize AD-MSC. Both AD-MSC expressing mms6 and Ferucarbotran-loaded AD-MSC were used in in vitro MH and magnetic resonance (MR) imaging studies. In vitro studies of MNHT were undertaken to investigate whether AD-MSC expressing mms6 and Ferucarbotran-loaded AD-MSC could have a MNHT effect when exposed to an AMF. Cell viability, cell apoptosis and HSP70 expression were assessed to investigate the MNHT effect. The results did not indicate that the AMF application on AD-MSC expressing mms6 have a MNHT effect, showing no observable difference in cell viability, cell apoptosis and HSP70 expression. In vitro MRI experiments were conducted to test whether mms6 can function as a MR reporter gene for molecular imaging. The result revealed AD-MSC expressing mms6 produced a detectable magnetic signal, revealing a promising potential of mms6 as a reporter gene for MR imaging. Overall, the results indicate that an MTB gene, mms6, can be expressed in AD-MSC without an adverse effect on important cell functions. Moreover, these results also indicate that no appreciable cell necrosis or cell apoptosis was found when AD-MSC expressing mms6 were exposed under AMF. However, this study has helped our knowledge on the biosynthesis of MNP in AD-MSC which also has potential use in MR imaging

Breast Cancer Immunotherapy Using Magnetised Oncolytic Virus

Abstract Background: Oncolytic viruses (OV) are encouraging new immunotherapies for cancer. OVs, replicate in cancer cells inducing immunogenic cell death (ICD) and activating antitumour immunity. To date, clinical use has focused on intratumoural delivery due to concerns over inadequate tumour targeting following systemic administration. We hypothesise that magnetising OVs and magnetic guidance strategies will improve their systemic delivery by protecting the viruses from inactivating immune mechanisms but at the same time promote anti-tumour immunity. Methods: To investigate this, we synthesised and characterised complexes of magnetised oncolytic herpes simplex virus (HSV1716) co-assembled with biocompatible magnetic nanoparticles (MAG) derived from magnetotactic bacteria (AMB-1) to give MAG-OV complexes. Characterization of the physical, chemical and oncolytic potential of MAG-OV was performed. The safety and efficacy of this nanomedicine in combination with magnetic guidance strategies was assessed in vivo. Results: Stable MAG-OV complexes of ~160nm diameter successfully infected human and murine breast cancer cells in a dose-dependent manner, and induced tumour oncolysis. Following MAG-OV infection a significant increase in viral replication (ICP0, gB, ICP8), ICD (HMGB1, CALR, ATP) and apoptotic (CASP 3, CASP8, FASL) signals were detected. Intravenous delivery of MAG-OV resulted in reduced tumour burden in the presence of magnetic guidance (MAG-OV 448.3mm³ vs. HSV1716 670.6mm³; p ≤ 0.05, n=6-9 mice/group) and an increase in tumour-infiltrating T-cells, NK cells and neutrophils. Furthermore, MAG-OV were protective in the presence of neutralising Abs both in vitro and in vivo. Conclusion: This study indicates that MAG are more small and uniform in size and form complexes with OV in such a way that the virus does not change its properties. MAG-OV is able to enter and replicate inside breast cancer cells, at the same time inducing tumour cell death as good as OV alone but with the addition of protecting the virus from neutralising Ab and in combination with magnetic guidance reduces tumour burden and induces anti-tumour immunity

Prokaryotic and eukaryotic expression systems for the production of recombinant proteins and nanoparticles for research and bio-industry

Gli organismi viventi sono in grado di produrre complesse strutture biologiche con funzioni specifiche per il loro metabolismo utilizzabili in settori industrali e di ricerca. In questo contesto le moderne tecnologie biotecnologiche sfruttano sistemi procariotici ed eucariotici per l'espressione di proteine ricombinanti, vaccini, anticorpi e nanoparticelle. In particolare il settore delle nanotecnologie sta conoscendo in questi ultimi anni un forte sviluppo e prodotti a base di nanoparticelle sono già stati approvati per la diagnostica e la terapia mentre altre sono attualmente in fase di sperimentazione clinica. In questa tesi, è stata analizzata la possibilità di utilizzare diversi sistemi biologici per l’ espressione di proteine ricombinanti o di nanoparticelle sfuttando sia sistemi procariotici che eucariotici. La prima parte del presente eleborato di tesi riguarda la purificazione di nanoparticelle magnetiche chiamate magnetosomi da Magnetospirillum gryphiswaldense , un batterio magnetotattico microaerofilo, nonché la successiva applicazione delle stesse come possibili agenti di contrasto in analisi NMR o come agenti terapeutici per termoterapia anti-tumorale. Nel presente elaborato di tesi è stato ottimizzato il metodo di coltura di M.gryphiswaldense, realizzando un'efficiente purificazione dei magnetosomi in lunghe catene, successivamente analizzati nelle loro caratteristiche chimico-fisiche (analisi DLS, TEM, misure di relassività e MRI) confermando la bontà come mezzi di contrasto e succesivamente impiegate in vivo come efficaci agenti terapeutici per ipertermia. Nella seconda parte della tesi invece è stato sviluppato un sistema eucariotico per la produzione di un’importante proteina di membrana, LHCSR1, coinvolta nel delicato processo di regolazione della fotoprotezione in alghe e muschi. In particolare la proteina LHCSR1 del muschio Physcomitrella patens è stata espressa in due sistemi eucariotici ,Nicotiana benthamiana e Nicotiana tabacum realizzando l’isolamento della proteina e la preliminare determinazione delle principali proprietà spettroscopiche e biochimiche.Living organisms can produce complex structures with specific functions for their metabolism that are used in a range of bio-industry and research activities. Biotechnology exploits prokaryotic and eukaryotic systems for expression of recombinant proteins, vaccines and antibodies as well as nano-structures. Indeed a number of nanoparticle-based products have been approved for diagnostics and therapeutics and more are currently under clinical trials. In this thesis work, the possibility of using different expression systems for the preparation of bio-products has been exploited. The first part of this thesis concerns the purification of magnetic nanoparticles called magnetosomes from Magnetospirillum.gryphiswaldense, a magnetotactic microaerophilic bacterium. Subsequently these nanoparticles have been tested as contrast agents in NMR analysis or as therapeutic agents for tumor thermotherapy. The chemical-physical properties of magnetosomes efficiently purified have been studied, confirming the goodness of these nanoparticles as contrast agents. Then magnetosomes has been tested in thermotherapy in vitro and in vivo against two cancer cell lines. In the second part of the thesis instead has been developed a system for the production of an important eukaryotic membrane protein, LHCSR, involved in the delicate process of regulation photoprotection in algae and mosses. In particular the protein LHCSR1 from the moss Physcomitrella patens has been efficiently expressed in two eukaryotic systems, Nicotiana benthamiana and Nicotiana tabacum realizing its isolation from thylakoid membrane and the preliminary determination of its biochemical and spectroscopic properties

Études des configurations spatio-temporelles du champ magnétique sur le contrôle des bactéries magnétotactiques

Depuis que les scientifiques se sont intéressés à travailler à l’échelle nano et micro, la création de véhicules qui puissent y travailler est devenue une nécessité. Ces véhicules sont des nano-micro robots qui doivent fonctionner dans ces milieux de manière autonome et contrôlée. L’une des plus grandes utilités de ces nano-micro robots, par exemple, est leur utilisation dans un système microvasculaire pour transporter des agents thérapeutiques vers les tumeurs cancéreuses de façon contrôlée. La technologie de fabrication des robots artificiels actuelle n’est pas en mesure de fournir ce nano-micro robot. Pour contourner cette limitation, nous avons choisi un micro robot déjà existant dans la nature. C’est la bactérie magnétotactique Magnetococcus Marinus souche MC-1, d’une taille de 2 µm de diamètre et ayant : 1) une autonomie de mouvement grâce à son propre système de propulsion fourni par deux moteurs moléculaires (flagelles), 2) une chaîne de particules nanométriques magnétiques (magnétosomes), qui permet à la bactérie de s’aligner avec le champ magnétique et de se propulser dans la direction du champ. En plus, les microrobots ont la capacité de réaliser des tâches dans l’environnement micrométrique comme : la microfabrication et le transport. L’équipe du laboratoire NanoRobotique de Polytechnique de Montréal a développé une plateforme de contrôle des bactéries magnétiques dans le but de contrôler leurs déplacements dans un système in vivo, et ainsi de transporter des agents thérapeutiques directement dans le cancer. Autrement dit, cette nouvelle plateforme permet de guider la bactérie magnétotactique vers une cible prédéfinie. L’objectif de ce mémoire de recherche est d’améliorer la modélisation du champ magnétique de cette plateforme. Cette nouvelle modélisation permettra de réduire les durées d’agrégation et de déplacement des bactéries magnétiques tout en augmentant la performance de la plateforme. D’abord, une méthode de contrôle basée sur la géométrie spatiale du champ magnétique a été développée et validée. Finalement, une étude de comportement des bactéries magnétiques exposées au champ magnétique alternatif a été effectuée afin de pouvoir développer une technique novatrice de contrôle.----------ABSTRACT Working at the nano and micro scale environment has provided scientists with an immense opportunity to explore within small and previously unreachable areas. Evidently, creation of vehicles that could facilitate such careful maneuver has gained a lot of interest. These vehicles are nanomicrorobots that perform autonomously under controlled environment. Among many research disciplines that could advance with such miniature system, drug delivery and navigation is one of the most beneficial uses for these controlled nanomicrorobots; acting as therapeutic agent carriers targeting cancerous tumors by traveling through complex microvascular structures. Current artificial robot technology lacks maturity in manufacturing mass scale nanomicrorobots. Therefore, inspired by nature, we chose special bacteria bona fide to serve as microrobots. Magnetotactic Magnetococcus Marinus strain MC-1 has: 1) an autonomy movement with its own propulsion system provided by two molecular motors (flagella) and 2) a chain of magnetic nanoparticles (magnetosomes) acting as a compass that aligns the moving bacteria in the direction of external magnetic field. These 2 µm diameter bacteria have the ability to perform as actuators, micro-fabricators and transporters. Polytechnique NanoRobotics Montreal laboratory team has developed a magnetic controller platform to control these bacteria in vivo and deliver therapeutic agents directly into the cancer tissue. In other words, this platform helps navigate the magnetotactic bacteria to the predefined target. The objective of this research thesis is to improve the magnetic field modeling of this platform. Our new proposed model will reduce the bacteria displacement and aggregation time while increasing the performance of the platform. At the beginning, a control method based on the spatial configuration of the magnetic field has been developed and validated. And at the end, a study on magnetic bacteria behavior exposed to alternating magnetic field is performed in order to develop an innovative control technique

Multicomponent magnetic nanoparticle engineering: the role of structure-property relationship in advanced applications

Combining magnetic nanomaterials with materials of other classes can produce multicomponent nanoparticles with an entire ensemble of new structures and unique, enhanced, synergetic, and/or complementary functionalities. Here we discuss the most recent developments in the synthesis of multicomponent magnetic nanoparticles, describe the resulting structures and their novel properties, and explore their application in a variety of fields, including multimodal imaging, nanomedicine, sensing, surface-enhanced Raman scattering, and heterogeneous catalysis. The current synthetic methods (usu-ally bottom-up approaches) of multicomponent nanoparticles can produce a number of tailored mor-phologies (core@shell, yolk-shell, core-satellite, Janus, nanochains, anisotropic, etc.), making them invaluable for applications in biology, medicine, chemistry, physics, and engineering. But like any new technology, their synthesis methods need to be optimized to be simple, scalable, and as environmentally friendly as possible before they can be widely adopted. In particular, the use of life cycle assessment (LCA) to guide future works toward environmental sustainability is highlighted. Overall, this review not only presents a critical and timely summary of the state-of-the-art of this burgeoning field in both fundamental and applied nanotechnology, but also addresses the challenges associated with under-standing the particular structure-property relationships of multicomponent magnetic nanoparticles.The authors thank funding from the Spanish State Research Agency (AEI) through the project PID2019-106099RB-C43/AEI/10.13039/501100011033 and from the Basque Government Industry and Education Department under the ELKARTEK, HAZITEK and PIBA (PIBA-2018-06) programs, respectively

Applications and Properties of Magnetic Nanoparticles

This Special Issue aimed to cover the new developments in the synthesis and characterization of magnetic nanoconstructs ranging from conventional metal oxide nanoparticles to novel molecule-based or hybrid multifunctional nano-objects. At the same time, the focus was on the potential of these novel magnetic nanoconstructs in several possible applications, e.g. sensing, energy storage, and nanomedicine

Design and Time-domain Analysis of Antenna Array for UWB Imaging Application.

PhDUWB technology has been developing in imaging applications. For security imaging applications, it is vital to detect and image metallic targets concealed in bag at airports, subway stations or other public environments. To reduce the cost of the deployment of X-ray machines, a novel UWB imaging system has been developed, including the design of the UWB rotating antenna array, the design of RF circuits and the implementation of the two-dimensional delay-and-sum (DAS) image reconstruction method. Two types of UWB antennas, the circular-edge antipodal Vivaldi antenna and the corrugated balanced antipodal Vivaldi antenna (BAVA) have been designed and studied in both frequency domain and time domain. Both of them can work across UWB frequency range from 3.1 GHz to 10.6 GHz, and have directional radiation patterns. The corrugated BAVA with smaller physical size has been improved to have a relative high gain around 7 dBi across the operating frequency range. It also causes less distortion to signals in the time domain. So the corrugated BAVA is used as the antenna element in the UWB rotating antenna array. The UWB rotating antenna array comprises one central transmitting antenna and four receiving antennas. The receiving antennas, which rotate around the central transmitting antenna, are placed side-by-side on a straight arm. The equivalent antenna elements in space are increased by the rotation of the antenna array. The two-dimensional image reconstruction method has been developed based on DAS algorithm. This UWB imaging system can detect and reconstruct the image of the single and pairs of metallic targets concealed in bag. The smallest single target with the size of 4 cm × 4 cm × 1 cm can be reconstructed in images at a maximum distance of 30 cm away from the system. It can achieve 6 cm in cross-range resolution and 15 cm in down-range resolution. Therefore, the feasibility of the proposed UWB imaging system has been proved