Immuno Magnetic Thermosensitive Liposomes For Cancer Therapy

The present work describes the encapsulation of the drug doxorubicin (DOX) in immuno paramagnetic thermosensitive liposomes. DOX is the most common chemotherapeutic agent for the treatment of a variety of carcinomas. However, the pure drug has high cytotoxicity and therefore requires a targeted and biocompatible delivery system. The introduction includes concepts, modalities, and functionalities of the project. First, a detailed description of the cell type (triple-negative breast cancer) is given. Furthermore, the importance of liposomal doxorubicin is explained and the current state of research is shown. The importance of modification to achieve thermosensitive properties and the procedure for co-encapsulation with Gd chelate to achieve paramagnetic properties is also discussed. In addition, the first part describes the surface modification with ADAM8 antibodies, which leads to improved targeting. The second part of the thesis covers the different materials and methods used in this paper. The production of the liposomes LipTS, LipTS-GD, LipTS-GD-CY, LipTS-GD-CY-MAB and the loading of DOX using an ammonium sulfate gradient method were described in detail. The results part deals with the physicochemical characterization using dynamic light scattering and laser Doppler velocimetry, which confirmed a uniform monodisperse distribution of the liposomes. These properties facilitate the approach of liposomes to target cancer cells. The influence of lipid composition of liposomes, co-encapsulation with Gd chelate and surface modification of liposomes was evaluated and described accordingly. The size and structure of the individual liposomal formulations were determined by atomic force microscopy and transmission electron microscopy. Morphological examination of the liposomes confirmed agreement with the sizes obtained by dynamic light scattering. Temperature-dependent AFM images showed an intact liposome structure at 37 °C, whereas heating by UHF-MRI led to a lipid film indicating the destruction of the lipid bilayer. Furthermore, TEM images showed the morphological properties of the liposomes and gave a more precise indication of how Gd-chelate accumulates within the liposomes. Liposomes with Gd-chelate showed well-separated vesicles, suggesting that Gd- chelate is deposited in the lipid bilayer of the liposomes. Gd was encapsulated in the hydrophilic core whereas chelate was extended into the lipid bilayer. By differential scanning calorimetry and drug release, the heat-sensitive functionality of the liposomes could be determined. Liposomes showed a beginning of phase transition temperature at about 38 °C, which can be achieved by UHF-MRI exposure. The maximum phase transition temperature in the case of LipTS-GD and LipTS-GD-CY-MAB was 42 °C and 40 °C, respectively. A proof of concept study for the thermosensitive properties of liposomes and a time-dependent DOX release profile in hyperthermia was performed. Gd-chelate is encapsulated in both LipTS-GD and LipTS-GD-CY-MAB and led to paramagnetic properties of the liposomes. This facilitates imaging mediated DOX delivery and diagnosis of the solid tumor and metastatic cells. The change in relaxation rate R1 of liposomes was quantified before and after heating above Tm (T> Tm). The relaxivity of the liposomes was obtained from the adapted slope of the relaxation rate against the Gd concentration. Remarkably, the relaxation rate and relaxivity increased after heating the liposomes above Tm (T> Tm), suggesting that the liposomes opened, released Gd chelate, and the exchange of water molecules became faster and more practicable. Toxicity studies describe the different mechanisms for induced DOX toxicity. The increased cytotoxic effect at elevated temperatures showed that the induced toxicity is thermally dependent, i.e. DOX was released from the liposomes. The high viability of the cells at 37 °C indicates that the liposomes were intact at normal physiological temperatures. Under UHF-MRI treatment, cell toxicity due to elevated temperature was observed. The cellular uptake of liposomes under UHF-MRI was followed by a confocal laser scanning microscope. An increase in fluorescence intensity was observed after UHF-MRI exposure. The study of the uptake pathway showed that the majority of liposomes were mainly uptake by clathrin-mediated endocytosis. In addition, the liposomes were modified with anti-ADAM8 antibodies (MAB 1031) to allow targeted delivery. The cellular binding capabilities of surface-modified and non-modified liposomes were tested on cells that had ADAM8 overexpression and on ADAM8 knockdown cells. Surface-modified liposomes showed a significant increase in binding ability, indicating significant targeting against cells that overexpress ADAM8 on their surface. In addition, cells with knockdown ADAM8 could not bind a significant amount of modified liposomes. The biocompatibility of liposomes was assessed using a hemolysis test, which showed neglected hemolytic potential and an activated thromboplastin time (aPTT), where liposomes showed minimal interference with blood clotting. Hemocompatibility studies may help to understand the correlation between in vitro and in vivo. The chorioallantois model was used in ovo to evaluate systematic biocompatibility in an alternative animal model. In the toxicity test, liposomes were injected intravenously into the chicken embryo. The liposomes showed a neglectable harmful effect on embryo survival. While free DOX has a detrimental effect on the survival of chicken embryos, this confirms the safety profile of liposomes compared to free DOX. LipTS-GD-CY-MAB were injected into the vascular system of the chicken embryo on egg development day 11 and scanned under UHF-MRI to evaluate the magnetic properties of the liposomes in a biological system with T2-weighted images (3D). The liposomal formulation had distinct magnetic properties under UHF MRI and the chick survived the scan. In summary, immunomagnetic heat-sensitive liposomes are a novel drug for the treatment of TNBC. It is used both for the diagnosis and therapy of solid and metastasizing tumors without side effects on the neighboring tissue. Furthermore, a tumor in the CAM model will be established. Thereafter, the selective targeting of the liposomes will be visualized and quantitated using fluorescence and UHF-MRI. Liposomes are yet to be tested on mice as a xenograft triple-negative breast cancer model, in which further investigation on the effect of DOX-LipTS-GD-CY-MAB is evaluated. On one hand, the liposomes will be evaluated regarding their targetability and their selective binding. On the other hand, the triggered release of DOX from the liposomes after UHF-MRI exposure will be quantitated, as well as evaluate the DOX-Liposomes therapeutic effect on the tumor

MESOSCOPIC LIGHT SCATTERING APPROACH FOR STRUCTURAL DISORDER ANALYSIS OF BIOLOGICAL CELLS: APPLICATION IN CANCER DIAGNOSTICS

Optical techniques are often used to study of biological cells and tissues to gain valuable information about them. Recently, the mesoscopic physics based light scattering techniques have provided unprecedented insight into the physical properties of biological systems. In particular, the mesoscopic light transport and light localization approaches allow to measure and quantify nano to micron scale structural alterations in the biological system. The applications of these techniques have been foreseen in efficient diseases diagnostics and therapeutic studies. Genesis and progression of diseases such as cancer is known to accompany with structural alterations in the building blocks of cells, such as DNA, proteins, lipids, etc. In that context, this dissertation presents a detailed study on quantification of structural changes in the cancer cells, by employing the mesoscopic physics based light transport and light localization analysis. Two different techniques, namely the partial wave spectroscopy (PWS) (light transport) and inverse participation ratio (IPR) (light localization), are implemented to image and quantify structural disorder developed as a result of alteration in the cellular structure caused by cancer diseases. The PWS and IPR techniques were used to quantify structural disorder, represented as disorder strength, and thus differentiate normal from cancer cells in several human breast, brain and prostate cell lines. Additionally, the effect of drug resistance developed by the prostate cancer cells, on prolonged chemotherapy treatment, on the structural disorders of the cells was also analyzed. Results show that the cancer cells have higher structural disorder compared to the normal cells and that the degree of structural disorder is correlated with the aggressiveness/metastatic potential of the cancer cells. The results with drug study suggest that the cancer cells which develop resistance to the chemotherapy become more aggressive. Further, the results of this study strongly indicate that the parameter disorder strength can acts as an efficient biomarker/numerical index to asses hierarchy of cancer as well as evaluate efficiency of drug treatment processe

Quantitative mRNA detection with advanced nonlinear microscopy

Cell-specific information on quantity and localization of key mRNA transcripts in single-cell level are critical to the assessment of cancer risk, therapy efficacy, and effective prevention strategies. While current techniques are not capable to visualize single mRNA transcript beyond the diffraction limit. In this thesis, two nonlinear technologies, second harmonic super-resolution microscopy (SHaSM) and transient absorption microscopy (TAM), are developed to detect and quantify single Human edimer receptor 2 (Her2) mRNA transcripts. The SHaSM is used to detect single mRNA transcript beyond the diffraction limit, while the TAM is employed to detect mRNA without the interference of fluorescence background. The thesis presents the fundamental study on the probes used in SHaSM, the concept and instrumental layout of the two technologies, and the detection as well as quantification of mRNA transcript in cells and tissues by super resolution microscopy and background-free detection microscopy. The first part of my dissertation focuses on the introduction of available mRNA detection methods and nonlinear imaging techniques. In chapter 2, I mainly characterize the SHG emission behavior of individual BTO nanocrystals via time-resolved single molecule spectroscopy, correlation spectroscopy, and confocal microscopy. High-intensity stable emission is collected from individual BTO nanocrystals with a high signal-to-noise ratio; the polar-dependent emission behavior of individual BTO NCs was also investigated theoretically and experimentally; and the dynamics of individual BTO in turbid medium is studied by an improved autocorrelation spectroscopy. The third chapter develops a novel second harmonic super-resolution microscopy (SHaSM), which is capable of detecting individual BTO nanocrystals with the lateral resolution as high as 30 nm. Motivated by the capability of SHaSM to visualize single BTO nanocrystals beyond the diffraction limit, we develop a dimer configuration of BTO nanocrystals for detecting single mRNA transcript beyond the diffraction limit. We validate our SHaSM to resolve single mRNA transcript first in vitro. Preformed BTO dimers are detected and differentiated by the SHaSM and by the SEM as the control. Expression level and localization patterns of Her2 mRNA transcript in single SKBR3, MCF7, and HeLa cell are investigated with the SHaSM. SHaSM can successfully differentiate the Her2 mRNA from the nonspecific BTO monomers, and identify more than one transcript in a diffraction-limited spot for SKBR3 cells. Quantification results agree well with the theoretical estimation and the RNA FISH results, and in addition it shows that the SHaSM has more accurate quantification when detecting over-expressed mRNA transcript. Furthermore we applied the SHG probes and SHaSM to study the heterogeneity of Her2 mRNA transcript in breast cancer tissues. High-specific binding of the SHG probes is observed and high penetration detection can be realized. In addition to the SHaSM, I also develop a background-free method to detect and quantify mRNA transcript. A femto-second transient absorption microscopy (TAM) is developed in the lab. It starts with the theoretical description of the TAM process, and then introduce the fundamental optical properties of the gold nanoparticles in TAM. By chemically treating the gold nanoparticles and conjugating with ODN probes, the gold nanoparticles hybridize to the mRNA molecules and are visualized in the TAM, together with label-free images of cells obtained in the SRS microscopy. mRNA is quantified with single copy sensitivity and is validated by the FISH approach. Super resolution microscopy of Her2 mRNA transcript in single cells will provide more accurate quantification in single cells; what\u27s more, it can be potentially employed to investigate the dynamics of single mRNA transcript beyond the diffraction limit, which is extremely significant in basic biology. TAM microscopy promotes the detection of mRNA transcript at a high speed without fluorescence background, which can be further utilized to investigate the dynamics of RNA regulation. Both these two methods will promote our understandings of the expression level and localization patterns of mRNA transcript in single cells, provide a route to employ mRNA transcript as a marker or indicator for cancer diagnosis and therapy

Coupling Scanning Electrochemical Microscopy and 3D Modelling to Probe Membrane Permeability of Human Bladder Cancer (T24) Cells

Scanning electrochemical microscopy (SECM) scans a biased ultramicroelectrode (≤ 25 µm) probe over a sample to characterize topography, physical properties and chemical reactivity. In this dissertation, SECM was used to investigate the metal-induced changes in membrane response of single live human bladder cancer cells (T24). SECM imaging was coupled to 3D finite element method (FEM) simulations which were the first of their kind, providing advanced quantification of sample traits under conditions not previously usable. The effects of Cd2+ on T24 cell membrane permeability were examined. Experimental depth-scan imaging was coupled with full 3D FEM simulations, eliminating many limitations of previous 2D-axially symmetric models. Hundreds of probe approach curves (PACs) can now be extracted from depth-images and theoretically fit to quantify membrane permeability at any location across the cell surface (Chapter 2). SECM was utilized to examine the membrane response of T24 cells following exposure to toxic dichromate (Cr(VI)). Two electrochemical mediators were examined, the membrane permeable ferrocenemethanol (FcCH2OH) and impermeable ferrocenecarboxylate (FcCOO‑). Cr(VI) induced permeability change was observed with both mediators and compared (Chapter 3). Chronic Cr(VI) induced cell stress, was then examined. Similar permeability curve shape was observed, with shifts in response time based on concentration of Cr(VI) stressor (Chapter 4). Trace essential metals such as Cr(III) are essential in low concentrations but toxic in high concentration. Membrane-response was investigated by SECM, using both FcCH2OH and FcCOO- redox mediators. Theoretical SECM depth-scans were produced using 3D FEM simulations, and used to quantify cell membrane permeability (Chapter 5). Complex close-proximity cell clusters were experimentally imaged by SECM 3D scanning mode. Tailored 3D model geometries were created, generating complimentary theoretical maps of the experimental cell clusters. The simulations were capable of providing a strong theoretical fit to the experimental results. Limits of cell proximity for SECM characterization were determined based on the probe size (Chapter 6). Nanoscale SECM imaging of single live cells were performed using a laser-pulled 130 nm radius Pt disk electrode. A tailored 3D model was created, from which cell topography was accurately characterized using membrane-impermeable Ru(NH3)63+, and cell membrane permeability was quantified with FcCH2OH (Chapter 7)

High-throughput Droplet Barcoding and Automated Image Analysis in Microfluidic Droplet Trapping Array

Molecularly-targeted therapeutics and personalized medicine have dramatically increased the median survival rate of patients suffering from cancer. However, cellular heterogeneity and the personalized nature of cancer have resulted in the limited success of single drug treatments which has led to the use of multiple therapeutic combinations. This has required the development of new analytical methods capable of multiplexed high-throughput screening (HTS) technologies necessary to identify is single or multi-agent therapies are effective in ex vivo samples like liquid biopsies. Droplet microfluidic devices have garnered significant interest to facilitate high-throughput, single cell analysis of heterogeneous populations. However, these devices are still limited in their ability to assess multiple input conditions such as combinations of multiple drugs or different doses of the same drug. Moreover, HTS approaches need to be coupled with automated image analysis metrics capable of rapidly processing raw data and quantifying it in an efficient manner. The goal of this work is to address these two areas of need by developing a new method to track different inputs in a droplet microfluidic trapping array coupled with automated image analysis of single cell behavior. The first part of this study highlights the use of rare-earth (RE)-doped luminescent nanoparticles (NP) as novel method to track input conditions in droplets in a microfluidic device. The second part of the work deals with the development of an algorithm called FluoroCellTrack to efficiently analyze single cell data from high-throughput experiments in the droplet microfluidic trapping array. The β-hexagonal NaYF4 nanoparticles used for droplet tracking were doped with a rare-earth emitter with unique spectral properties that do not overlap with established fluorophores like GFP and Rhodamine. In this study, we employed europium as the dopants which has a luminescence emission spectrum in the red region upon UV excitation. We demonstrated that the RE-doped nanoparticles are biologically inert and spectrally independent with common fluorophores and fluorescent stains. This work provided a foundation for future applications using the combination of NPs and microfluidics for multiplexed droplet tracking to quantify tumor heterogeneity and assess the effectiveness of combinatorial therapies. To perform HTS of single cells, a Python algorithm (FluoroCellTrack) was developed to: (i) automatically distinguish droplets from cells, (ii) count cells in each droplet, (iii) quantify cell viability, and (iv) identify input conditions using the RE-doped nanoparticles. The performance of FluoroCellTrack was compared to manual image analysis with a difference in intracellular quantification of ~2% coupled with a decrease in analysis time ofquantification, droplet barcoding and biomarker detection