Recent progress in nanotechnology has attracted interest to a biomedical application of the carbon nanoparticle C60 fullerene (C60) due to its unique structure and versatile biological activity. In the current study the dual functionality of C60 as a photosensitizer and a drug nanocarrier was exploited to improve the efficiency of chemotherapeutic drugs towards human leukemic cells. Pristine C60 demonstrated time-dependent accumulation with predominant mitochondrial localization in leukemic cells. C60’s effects on leukemic cells irradiated with high power single chip LEDs of different wavelengths were assessed to find out the most effective photoexcitation conditions. A C60-based noncovalent nanosized system as a carrier for an optimized drug delivery to the cells was evaluated in accordance to its physicochemical properties and toxic effects. Finally, nanomolar amounts of C60-drug nanocomplexes in 1:1 and 2:1 molar ratios were explored to improve the efficiency of cell treatment, complementing it with photodynamic approach. A proposed treatment strategy was developed for C60 nanocomplexes with the common chemotherapeutic drug Doxorubicin, whose intracellular accumulation and localization, cytotoxicity and mechanism of action were investigated. The developed strategy was revealed to be transferable to an alternative potent anticancer drug – the herbal alkaloid Berberine. Hereafter, a strong synergy of treatments arising from the combination of C60-mediated drug delivery and C60 photoexcitation was revealed. Presented data indicate that a combination of chemo- and photodynamic treatments with C60-drug nanoformulations could provide a promising synergetic approach for cancer treatment. / Kürzliche Fortschritte in der Nanotechnologie haben Interesse an einer biomedizinischen Anwendung des Kohlenstoffnanopartikels C60 Fulleren (C60) aufgrund seiner einzigartigen Struktur und breiten biologischen Aktivität geweckt. In der aktuellen Studie wurde die doppelte Funktionalität von C60 als Photosensibilisator und als Wirkstoff-Nanoträger genutzt, um die Wirkung von Chemotherapeutika auf menschliche Leukämiezellen zu verbessern. C60 alleine zeigte in den Zellen eine zeitabhängige Akkumulation mit vorherrschender mitochondrialer Lokalisation. Die Wirkung von C60 auf Leukämiezellen, die mit unterschiedlicher Wellenlänge bestrahlt wurden, wurde bewertet, um die effektivsten Photoanregungsbedingungen zu finden. Die physikochemischen Eigenschaften und toxischen Wirkungen von C60 auf die Leukämiezellen wurden nach nicht kovalenter Bindung von Arzneistoffen bewertet. Schließlich wurden nanomolare Mengen von C60-Wirkstoff-Nanokomplexen in Molverhältnissen von 1:1 und 2:1 untersucht, um die Effizienz der Behandlung von Zellen zu verbessern und sie durch photodynamischen Ansatz zu ergänzen. Mit dem gängigen Chemotherapeutikum Doxorubicin wurde eine Behandlungsstrategie entwickelt und dessen intrazelluläre Akkumulation und Lokalisation, Zytotoxizität und Wirkmechanismus untersucht wurden. Es wurde gezeigt, dass die entwickelte Strategie auch auf ein alternatives Krebsmedikament übertragbar ist – das pflanzliche Alkaloid Berberin. Die erhaltenen Daten deuten darauf hin, dass eine Kombination von chemo- und photodynamischen Behandlungen mit C60-Nanokomplexen einen vielversprechenden synergetischen Ansatz für die Krebsbehandlung bieten könnte.

Recent technologies in micro-devices for investigation of functional biology in a controlled microenvironment are continually growing and evolving. In particular, electric-field mediated microfluidic platforms are evolving technologies that have significant applications in drug delivery and cell migration investigations. Although drug delivery has had several successes, in some areas, it continues to be a challenge; in recent years, the positive impact of electric fields is being explored. The primary objectives of the dissertation are to design, fabricate, and employ two novel microfluidic platforms for drug delivery and cell migration in the presence of electric fields. Description of iontophoretic carboplatin delivery into the MDA-MB-231 triple-negative breast cancer cells and investigation of neutrophil electro taxis are two main aims of the dissertation. Transdermal drug delivery systems such as iontophoresis are useful tools for delivering chemotherapeutics for tumor treatment not only because of their non-invasiveness but also due to their lower systematic toxicity compared to other drug delivery systems. While iontophoresis animal models are commonly being used for the development of new cancer therapies, there are some obstacles for precise control of the tumor microenvironment's chemoresistance and scaffold in the animal models. We employed experimental and computational approaches, the iontophoresis-on-chip and the fraction of tumor killed mathematical model, for predicting the outcome of iontophoresis treatment in a controlled microenvironment. Also, precise control over the cell electromigration is a challenging investigation which we will address in the second aim of the dissertation. Here, we developed a microfluidic platform to study the consequences of DC electric fields on neutrophil electromigration (electrotaxis), which has an application of directing neutrophils away from healthy tissue by suppressing the migration of neutrophils toward pro-inflammatory chemoattractant. / Doctor of Philosophy / Recent technologies in the micro-scale medical devices for diagnosis and treatment purposes are continually growing and evolving. Microfluidic platforms are reproducible devices with the dimensions from tens to hundreds of micrometers for manipulating and controlling fluids. In particular, electric-field mediated microfluidic platforms, are developing technologies that have significant applications in drug delivery and biological cell directional movement investigations. Although drug delivery has had several successes, in some areas, it continues to be a challenge. In recent years, the positive impact of electric fields is a significant advancement in drug delivery techniques. Transdermal drug delivery systems such as iontophoresis are useful tools for delivering chemo drugs for tumor treatment not only because of their sensitivity but also to their lower systematic toxicity compared to injection or oral drug delivery. While iontophoresis animal models are conventional for the development of new cancer therapies, there are some obstacles to precise control of the tumor scaffold in the animal models. We also developed a novel microfluidic platform to study the consequences of DC electric fields on white blood cells' (WBC) directional movement, which has an application of directing WBC away from healthy tissue by suppressing the damage of WBC accumulation in healthy organs.

Hypoxia is a characteristic pathophysiological feature of many solid tumors, which contributes significantly to resistance to chemotherapy and radiotherapy. It also induces numerous intracellular signaling pathways, which in turn trigger the upregulation of various key proteins promoting tumor cell survival, progression and metastasis. In this context, novel therapeutic approaches are urgently needed to facilitate the early detection and improve the treatment of hypoxic tumors. Focusing on the hypoxic tumor microenvironment, one can recognize that the membrane bound carbonic anhydrase IX (CA IX) isozyme represents a potential biomarker and a compelling therapeutic target for better diagnosis and management of hypoxic tumors. CA IX is significantly overexpressed under hypoxic conditions as compared to normal tissues and it assists tumor cell to maintain neutral intracellular pH values. Building on this hypothesis, we are focusing our efforts in this thesis towards the development and the optimization of drug delivery nanosystems capable of selectively targeting CA IX that is overexpressed in the hypoxic tumor niche, which in turn will enhance the early detection of hypoxic tumors as well as improve the accumulation of chemotherapeutic drugs in hypoxic cancer cells. This strategy is expected to overcome the chemoresistance associated with tumor hypoxia and minimize the systemic side effects associated with chemotherapeutic drugs administration. In chapter 2, we focused our efforts towards the development of the in vitro biological models for testing our nanoparticles. This process was achieved through screening a series of cancer cell lines for the expression of our target epitope under hypoxic conditions. We induced hypoxia either chemically, using cobalt chloride, or physicochemically, using a hypoxia chamber purged with hypoxia gas mixture containing 1% O2. Screening for CA IX overexpression under hypoxic conditions was done both in 2D monolayer cells and 3D tumor spheroids, which become naturally hypoxic due to their 3D growth. Western blot analysis was used to confirm the expression of our target protein and we have identified three cell lines with a high level of expression of CA IX under hypoxic conditions, namely HT-29 colorectal cancer, SKOV-3 ovarian cancer and MDA-MB-231 breast cancer cell lines. In chapter 3, we optimized a theranostic liposomal delivery system through the use of a combination of zwitterionic amphiphilies of different packing parameters to encapsulate a potent fluorescent carbonic anhydrase inhibitor (CAI), as a novel approach to facilitate the detection of colorectal cancer. Our main focus was to increase the aqueous concentration of poorly water-soluble CAI, to correlate its delivery efficiency with the lipid type and composition of the liposomal nanosystem, as well as to enhance the tissue permeability, allowing easy detection of small tumor polyps. Our optimized DMPC/DOPE liposomal formulation demonstrated an optimum size, high encapsulation efficiency of CAI, and a phase transition temperature below 37 ᴼC that allows efficient delivery of CAI and good tissue penetrability towards the hypoxic tumor cells overexpressing CA IX. In chapter 4, we optimized a CAI-targeted long circulating liposomal delivery system encapsulating doxorubicin. Our main focus was to enhance the accumulation of doxorubicin in hypoxic tumors through targeting CA IX protein overexpressed under hypoxic conditions. This strategy proved to enhance the internalization of the drug carrier into hypoxic cancer cells thus overcoming chemoresistance associated with hypoxia and also minimize the systemic side effects associated with the intravenous administration of non-targeted Doxil®-like formulations. In chapter 5, we optimized a pH sensitive gold nanoplatform functionalized with CAI based moieties to enhance the selective delivery of doxorubicin to hypoxic tumors in a controlled release manner. Our main focus was to combine the advantage of targeting CA IX overexpressed under hypoxic conditions with the intracellular triggered release of doxorubicin in the lysosomes inside the cell in order to enhance the delivery of doxorubicin inside the cancer cells and to overcome the chemoresistance associated with hypoxia. / Pharmaceutical Sciences

Macromolecular magnetic resonance imaging (MRI) contrast agents have unique localization and contrast enhancement properties. We have designed and studied a monodisperse paramagnetic β-cyclodextrin click cluster (Gd10) decorated with Gd-containing arms and unique contrast enhancing polymers. To synthesize Gd10, a novel alkyne-functionalized diethylenetriaminetetraacetic acid chelate was created and coupled to a per-azido-β-cyclodextrin core and chelated with Gd(III) to yield the precursor macromolecule. Luminescence measurements were carried out using an analogous structure Eu(III)-containing structure and indicated that each lanthanide has an average of 1.8 water exchange sites. Gd10 yields a high relaxivity profile (6.2 mM⁻¹ s⁻¹ per Gd(III) at 9.4 T). Gd10 shows toxicity higher than clinically used contrast agents such as Magnevist&trade in vitro in cardiomyoblast cells. No acute toxicity was observed in the rats (n = 9) and contrast enhanced image analysis indicates renal processes may be involved in clearance. The contrast enhancing polymers we developed are new macromolecular beacons that allow the delivery of nucleic acids to be visualized at different biological scales. They contain repeated oligoethyleneamines, for binding and compacting nucleic acids into nanoparticles, and Gd(III)/Eu(III) chelates. The chelated lanthanides allow the visualization of the delivery vehicle via microscopy and via magnetic resonance imaging (MRI). We demonstrate that these new delivery beacons effectively bind plasmid DNA(pDNA) and protect their cargo nucleic acids from nuclease damage. The lanthanide-chelate materials have been found to efficiently deliver pDNA into cultured cells and do not exhibit toxicity. Micrographs of cultured cells exposed to the nanoparticle complexes formed with fluorescein-labeled pDNA and the europium-chelated polymers reveal effective intracellular imaging of the delivery process. MRI of bulk cells exposed to the complexes formulated with pDNA and the gadolinium-chelated structures show bright image contrast, allowing visualization of effective intracellular delivery on the tissue-scale. Because of their versatility as imaging probes, these delivery beacons posses remarkable potential for tracking and understanding nucleic acid transfer in vitro and have promise for in vivo imaging applications. In later studies the Ln-chelating polymers were co-polymerized with dimethylgalacterate which definitively increases luciferase gene expression (up 50x enhancement) and cellular uptake (up to 2x enhancement). / Ph. D.

Yes / Therapeutic microbubbles (thMBs) contain drug-filled liposomes linked to microbubbles and targeted to vascular proteins. Upon the application of a destructive ultrasound trigger, drug uptake to tumour is improved. However, the structure of thMBs currently uses powerful non-covalent bonding of biotin with avidin-based proteins to link both the liposome to the microbubble (MB) and to bind the targeting antibody to the liposome-MB complex. This linkage is not currently FDA-approved, and therefore, an alternative, maleimide-thiol linkage, that is currently used in antibody-drug conjugates was examined. In a systematic manner, vascular endothelial growth factor receptor 2 (VEGFR2)-targeted MBs and thMBs using both types of linkages were examined for their ability to specifically bind to VEGFR2 in vitro and for their ultrasound imaging properties in vivo. Both showed equivalence in the production of the thMB structure, in vitro specificity of binding and safety profiles. In vivo imaging showed subtle differences for thMBs where biotin thMBs had a faster wash-in rate than thiol thMBs, but thiol thMBs were longer-lived. The drug delivery to tumours was also equivalent, but interestingly, thiol thMBs altered the biodistribution of delivery away from the lungs and towards the liver compared to biotin thMBs, which is an improvement in biosafety. / This research was funded by the EPSRC (EP/I000623/1, EP/L504993/1 and EP/P023266/1). S.D.E. is supported by the National Institute for Health Research infrastructure at Leeds.

Yes / Poloxamers or Pluronics®-based nanogels are one of the most used matrices for developing delivery systems. Due to their thermoresponsive and flexible mechanical properties, they allowed the incorporation of several molecules including drugs, biomacromolecules, lipid-derivatives, polymers, and metallic, polymeric, or lipid nanocarriers. The thermogelling mechanism is driven by micelles formation and their self-assembly as phase organizations (lamellar, hexagonal, cubic) in response to microenvironmental conditions such as temperature, osmolarity, and additives incorporated. Then, different biophysical techniques have been used for investigating those structural transitions from the mechanisms to the preferential component’s orientation and organization. Since the design of PL-based pharmaceutical formulations is driven by the choice of the polymer type, considering its physico-chemical properties, it is also relevant to highlight that factors inherent to the polymeric matrix can be strongly influenced by the presence of additives and how they are able to determine the nanogels biopharmaceuticals properties such as bioadhesion, drug loading, surface interaction behavior, dissolution, and release rate control. In this review, we discuss the general applicability of three of the main biophysical techniques used to characterize those systems, scattering techniques (small-angle X-ray and neutron scattering), rheology and Fourier transform infrared absorption spectroscopy (FTIR), connecting their supramolecular structure and insights for formulating effective therapeutic delivery systems. / The Sao Paulo Research Foundation - FAPESP (Grant 2019/20303-4; 2019/14773-8), National Council for Scientifc and Technological Development - CNPq (308819/2022-0), ERASMUS Program Fellowship, and The Coordination for the Improvement of Higher Education Personnel - Brazil (CAPES) - Finance Code 001.