Molecular imaging is an emerging imaging principle which can visually represent the biological processes both spatially and temporally down to the sub-cellular level in vivo. The outcome of this research is expected to have a profound impact on facilitating the early diagnosis of diseases, accelerating the development of new drugs, and improving the efficacy of therapy. In general, molecular imaging highly relies on probes to sense the occurrence of molecular biological events, and to generate signals which could be picked up by diagnostic imaging modalities. The advances in the design of molecular probes not only have equipped traditional anatomical medical imaging with new capabilities but also, in some cases, stimulated developments of new imaging modalities and renaissance of existing medical imaging modalities. One of these is photoacoustic imaging, which as an emerging medical imaging modality, unites the merits from both optical imaging and ultrasound imaging. It shares with optical imaging, that it uses non-ionizing radiation and provides higher contrast and higher sensitivity than ultrasound imaging. Unlike optical imaging, which requires ballistic photons for imaging, photoacoustic imaging requires only diffusive photons to excite the ultrasound signal from the imaging target; therefore, it is capable of imaging much deeper into the tissue. In combination with molecular probes, photoacoustic molecular imaging has been demonstrated by several research groups using various photoacoustic molecular probes. However, the molecular probes used for most of these studies were contrast agents simply adopted from other optical imaging modalities. Our research on photoacoustic contrast agents indicated that the mechanism of photoacoustic signal generation from nanometer-sized contrast agents is distinct from that of optically homogeneous materials, such as tissue. We have discovered that, the amplitude of the photoacoustic signal generated from nano-contrast agents depends not only on the optical absorption of the particles, but more importantly, on the dynamic process of the heat conduction from the nanoparticles to the ambient, and the thermal properties of the surrounding materials. Based on our finding, we explored and further improved the photoacoustic response of the nanoparticles by exploiting the heat conduction process between the nanoparticle and its surrounding materials and by manipulating the excitations. This research allows to create optimized molecular specific contrast enhanced photothermal stable probes which can aid photoacoustic imaging and image guided photothermal cancer therapy. / text

Gold nanoparticles, a class of plasmonic nanoparticle, have increasingly been explored as an imaging and therapeutic agent to treat cancer due to their characteristic surface plasmon resonance phenomenon and penchant for tumor accumulation. Photothermal therapy has been shown as a promising cancer treatment by delivering heat specifically to the tumor site via gold nanoparticles. In this study, we demonstrate that gold nanorod (GNR)-mediated photothermal therapy can be more effective through the understanding of cell death mechanisms. By targeting GNRs to various cellular localizations, we explored the association of GNR localization with cell death pathway response to photothermal therapy. Furthermore, we compared the 2D monolayer experiments with 3D in vitro tumor models, multicellular tumor spheroids (MCTS), to mimic the structure of in vivo tumors. With MCTS, we evaluated the cell death response with GNRs distributed only on the periphery, as seen in typical in vivo studies, and distributed evenly throughout the tumor. We demonstrated that GNR localization influences the cell death response to photothermal therapy by showing the power threshold necessary to induce significant apoptotic and necrotic increases was lower for internalized GNRs than membrane-bound GNRs. Furthermore, apoptosis was found to increase with increasing laser power until the necrotic threshold and decreased above it, as necrosis became the dominant cell death pathway response. A similar trend was revealed with the 3D MCTS; however, the overall cell death percentages were lower, most likely due to the upregulated cell repair response and varied GNR distributions due to the presence of cell-cell and cell-matrix interactions. Furthermore, the uniformly distributed GNRs induced more apoptosis and necrosis than GNRs located in the MCTS periphery. In conclusion, we quantitatively analyzed the cell death pathway response to GNR-mediated photothermal therapy to establish that it has some dependence on GNR localization and distribution to gain a more thorough understanding of this response for photothermal therapy optimization. / text

Sarcomas are rare mesenchymally-derived tumors with limited treatment options. Tyrosine kinases may serve as potential targets for sarcoma therapy because many are mutated or overexpressed in sarcomas and cell lines. One potential molecular target for sarcoma treatment is the Src tyrosine kinase. Three independently synthesized Src kinase inhibitors were evaluated in human sarcoma cell lines. Of the three, dasatinib, provided promising results as a potential sarcoma therapy. Until this study, dasatinib activity had not been characterized in sarcoma cells. Based on our previous findings of Src activation in human sarcomas, we evaluated the effects of dasatinib in twelve sarcoma cell lines. Dasatinib inhibited Src activity and downstream signaling at nanomolar concentrations. Inhibition of Src signaling was accompanied by blockade of cell migration and invasion. Moreover, apoptosis was induced in a subset of bone sarcomas at nanomolar concentrations of dasatinib. Inhibition of Src protein expression by siRNA also induced apoptosis, indicating that these bone sarcoma cell lines are dependent on Src activity for survival. These results demonstrate that dasatinib inhibits migration and invasion of diverse sarcoma cell types, and selectively blocks the survival of bone sarcoma cells. Therefore dasatinib may provide therapeutic benefit by preventing the growth and metastasis of sarcomas. Microarray analysis of the sarcoma cell lines lead to the identification of a molecular signature that successfully predicts response to dasatinib by induction of apoptosis. Components of this molecular signature are expressed in primary human sarcomas. Furthermore, expression of the molecular signature in sarcomas can be utilized to cluster tumors based on theoretical response to dasatinib. While the prediction of response in tumors is theoretical, there is encouraging evidence to support further endeavors into validating the potential of this molecular signature to predict response in patients.Together, these studies reveal that, in cell lines, both constitutive Src activation and the presence of a molecular signature that predicts response to dasatinib are important parameters to consider when selecting dasatinib as a treatment for. Furthermore, novel therapeutic approaches that inhibit Src signaling may selectively induce apoptosis in tumor cells and sensitize to chemotherapy those tumors that contain the relevant molecular signature.

Tumour cell dissemination is a major issue with the treatment of cancer, thus new therapeutic strategies which can control this process are needed. Antagonism of integrins highly expressed in tumours is one potential strategy. The integrins are transmembrane glycoprotein adhesive receptors. Two of the integrins, αVβ3 and αIIbβ3, are highly expressed in a number of tumours and induce bi-directional signalling through their interaction with extracellular matrix proteins, and growth factor receptors. Through this signalling they play an important role in a number of cellular processes that are involved in tumour dissemination such as tumour growth, migration, invasion, metastasis and angiogenesis. Dual αIIbβ3 and αVβ3 integrin antagonism will have a direct effect on β3-expressing tumour cells that leads to the inhibition of cell migration and dissemination. Furthermore, through targeting tumour cell interaction with endothelial cells and platelets, this will also lead to inhibition of angiogenesis and metastasis. The aim of this project was to characterise the expression of αVβ3 and αIIbβ3 integrin in a panel of tumour cell lines and in human tumour xenograft samples, and to develop and utilise cell-based models to investigate potential novel β3 antagonists. The expression of αV and β3 subunits was detected in xenograft tissue using immunoblotting techniques. A panel of cell lines of different tumour types including melanoma, prostate, breast, colon and non small cell lung carcinoma was then characterised for αVβ3 and αIIbβ3 integrin expression using immunoblotting and immunocytochemistry. Melanoma cell lines demonstrated the strongest αVβ3 expression. No αIIbβ3 integrin expression was seen in any of the cell lines evaluated. A selection of cell lines with varying αVβ3 expression were then used to develop a functional test for cell migration, the scratch wound healing assay. Migration of tumour cells that expressed αVβ3 integrin was inhibited by the known β3 antagonists, cRGDfV peptide and LM609 antibody. A panel of 12 potential novel β3 integrin antagonists was screened for cytotoxicity and activity in the validated scratch assay. ICT9055 was the most effective antagonist in inhibition of M14 cell migration as determined by the scratch assay, with an IC₅₀ of < 0.1 μM. Therefore the work presented in this thesis has established models and tools for evaluating potential novel β3 integrin antagonists, and identified a promising molecule to progress for further preclinical evaluation.

Despite advances in technology and understanding of biological systems in the past two decades, modern drug discovery is still a lengthy, expensive, difficult and inefficient process with low rate of new therapeutic discovery. The search for new effective drugs remains a somewhat empirical process. There is compelling need for a more fundamental, mechanistic understanding of human cancers and anticancer drugs to design more appropriate drugs. Radiotherapy is still the major therapy of cancer. It uses high-energy ionizing radiation such as x-rays and charged particle beams to destroy cancer cells. DNA is well known to be the principal biological target of radiotherapy, but the molecular mechanism of ionizing radiation induced DNA damage was elusive. The conventional thought of the ???OH radical as the major origin for ionizing radiation induced DNA damage is questionable. Although various strategies and types of compounds have been designed and developed as potential radiosensitizers to enhance the radiosensitizing efficiency of radiotherapy, none of them have been approved for clinical use. The general outcomes of clinical trials have been disappointing. This thesis presents an innovative molecular-mechanism-based drug discovery project to develop novel drugs for effective radiotherapy of cancer through the emerging femtomedicine approach. Its ultimate goal is to develop more effective radiosensitizers, based on our unique molecular understandings of ionizing radiation induced DNA damage and halopyrimidines as a family of potential radiosensitizers. Direct, real-time observation of molecular reactions is of significant importance in diverse fields from chemistry and biology, environmental sciences to medicine. Femtosecond time-resolved laser spectroscopy (fs-TRLS) is a very powerful, direct technique for real-time observation of molecular reactions. Its key strength lies in short duration laser flashes of a time scale at which reactions actually happen - femtoseconds (fs) (1fs = 10???15 second). Since the late 1980s, its application to study chemical and biological systems led to the births of new subfields of science, called femtochemistry and femtobiology. Recently, femtomedicine has been proposed as a new transdisciplinary frontier to integrate ultrafast laser techniques with biomedical methods for advances in fundamental understandings and treatments of major human diseases. This the remarkable opportunity afforded through real-time observation of biochemical reactions at the molecular level. Femtomedicine holds the promise of advances in the radiotherapy of cancer. Several important findings were made in this thesis. First, our results of careful and high-quality fs-TRLS measurements have resolved the long existing controversies about the physical nature and lifetimes of a novel ultrashort-lived electron species (epre???) generated in radiolysis of water. These results have not only resolved the large discrepancies existing in the literature but provided new insights into electron hydration dynamics in bulk water. Such information is important for quantitative understanding and modeling of the role of non-equilibrium epre??? in electron-driven reactions in diverse environmental and biological systems, from radiation chemistry and radiation biology to atmospheric ozone depletion. Second, our fs-TRLS results have unraveled how epre??? plays a crucial role in ionizing radiation induced DNA damage. We found that among DNA bases, only T and especially G are vulnerable to a dissociative electron transfer (DET) reaction with epre??? leading to bond breaks, while the electron can be stably trapped at C and especially A to form stable anions. The results not only challenge the conventional notion that damage to the genome by ionizing radiation is mainly induced by the oxidizing ???OH radical, but provide a deeper fundamental understanding of the molecular mechanism of the DNA damage caused by a reductive agent (epre???). Our findings have led to a new molecular mechanism of reductive DNA damage. Third, halopyrimidines, especially BrdU and IdU, have passed Phase I to II clinical trials as potential hypoxic radiosensitizers, but the outcome of Phase III clinical trials was disappointing. Our results of fs-TRLS studies have provided a new molecular mechanism of action of halopyrimidines (XdUs, X=F, Cl, Br and I) in liquid water under ionizing radiation. We found that it is the ultrashort-lived epre???, rather than the long-lived ehyd???, that is responsible for DET reactions of XdUs. This reaction leads to the formation of the reactive dU??? radical, which then causes DNA strand breaks and cancer cell death. Our results have challenged a long accepted mechanism that long-lived ehyd??? would be responsible for the radical formation from halogenated molecules. Furthermore, we found that the DET reaction efficacy leading to the formation of the reactive dU??? radical is in the order of FdU << CldU < BrdU < IdU. Thus, only BrdU and IdU could be explored as potential radiosensitizers, in agreement with the results of bioactivity tests and clinical trials. Fourth, our fs-TRLS studies have provided a molecular mechanism for the DNA sequence selectivity of BrdU and IdU in radiosensitization. We found the DET reactions of BrdU/ IdU with dAMP*??? and dGMP*??? formed by attachment of epre??? generated by radiolysis of water in aqueous BrdU-dAMP/dGMP and IdU-dAMP/dGMP complexes under ionizing radiation. This new mechanistic insight into the interaction of BrdU and IdU with DNA provides clues to improve the halogen familty as potential radiosensitizers and to develop more effective radiosensitizers for clinical applications. Fifth, based on our molecular mechanistic understandings of DNA damage induced by ionizing radiation and halopyrimidines as potential radiosensitizers, we develop more effective new radisensitizing drug candidates through the femtomedicine approach. We have performed a fs-TRLS study of the DET reaction of a candidate compound (RS-1) with epre???, and found that the DET reaction of epre??? with RS-1 is much stronger than that of IdU (and certainly BrdU and CldU). Moreover, we have tested the radiosensitizing effect of RS-1 against human cervical cancer (HeLa) cells exposed to various doses of x-ray irradiation through DNA damage measurements by gel electrophoresis and cell viability/death assays by MTT. Our results have confirmed that RS-1 can largely enhance the radiosensitivity of treated human cervical cancer (HeLa) cells to x-ray (ionizing) radiation. It is clearly demonstrated that RS-1 has a much better radiosensitizing effect than IdU. Although these are just preliminary results, our results have shown promise of developing more effective radiosensitizers. In summary, our studies have demonstrated the potential of femtomedicine as an exciting new frontier to bring breakthroughs in understanding fundamental biological processes and to provide an efficient and economical strategy for development of new anticancer drugs.

Current diagnostic and therapeutic nanocarriers, including liposomes, micelles, and polymeric- and protein-based nanoparticles, are designed to have key functional properties such as: (i) longevity in the bloodstream, leading to accumulation of therapeutic cargos in neoplastic areas with leaky vasculatures; (ii) targeting of specific pathological sites through surface modification with targeting ligands; (iii) stimuli-responsive characteristics for controlled drug release under specific conditions. While some of these drug delivery systems have advanced into clinical stages, other nanocarriers remain under development to overcome issues with effective delivery such as lack of target-ability and fast clearance from circulation. Self-assembling peptides have recently shown great potential as nanocarrier materials for drug and gene delivery, owing to their safety, efficiency, and targeting capabilities. An amino acid pairing strategy enables us to design self/co-assembling peptides with multiple functionalities to fulfill drug delivery requirements. This thesis focuses on functionalization and characterization of self/co-assembling peptides as nanocarriers for hydrophobic anticancer drug delivery. Diethylene glycol (DEG) conjugation and protein binding are the two modification strategies used in this thesis to impart longevity and target-ability upon the peptide-based delivery system. The studies include: (i) characterization of self-assembling properties of the diethylene glycol (DEG)-conjugated amino acid pairing peptide AAP8, (ii) investigation of the self/co-assembling features of a model ionic-complementary peptide (EAR8-II) in complex with the hydrophobic drug pirarubicin, and the anticancer activity of the complex, (iii) the interactions between peptide-drug complexes and serum proteins from the thermodynamic viewpoint, (iv) quantification of the effect of protein binding to the peptide-based delivery system on immune responses and biocompatibility, and (v) exploration of the targeting capability of albumin-bound peptide-drug complexes towards lung cancer cells. Uncontrollable aggregation of AAP8 was the first issue to address in order to develop a promising platform for the peptide-based delivery system. Diethylene glycol (DEG), a short segment of polyethylene glycol (PEG), was conjugated to AAP8 either at one or both terminals, and then self-assembling and drug encapsulation properties of both functionalized AAP8s were characterized to evaluate the effect of DEG-modification. The results illustrated a significant reduction in uncontrollable aggregation, and the formation of uniform fibular nanostructures. In addition, DEG conjugation provided the peptide with safer features towards immune cells by reducing cellular toxicity to macrophages. Moreover, DEG-functionalization improved hydrophobic drug stabilization, as demonstrated by sustained cytotoxic efficacy against lung carcinoma cells over a relatively long time compared to the non-functionalized AAP8. Protein binding strategy was the second approach to utilize the peptide-based delivery system with more biocompatibility and target-ability features. EAR8-II was studied as a model ionic-complementary peptide with high capability of pirarubicin encapsulation and anticancer activities against different cancer cells. Albumin as a most abundant protein in serum was selected to assess its binding affinity to the delivery system, and evaluate its binding effect on immune responses and anticancer activities. The results showed a central role of albumin in the in vitro delivery of peptide-drug complexes to target lung cancer cells based on the following characteristics: (a) Non-covalent binding of albumin to the complex through hydrogen bonding and Van der Waals interactions. The interaction was confirmed by physicochemical methods such as fluorescence quenching and isothermal titration calorimeter (ITC). (b) Shielding properties of albumin for the complex against macrophages and blood components (erythrocytes and complement protein C5b-9). In the presence of albumin, phagocytosis and cytokine expression level of macrophages and hemolytic activity of the peptide-drug complex reduced significantly due to the smaller particle size of the albumin-bound complexes compared to unprotected ones. (c) Targeting the lung cancer cells, possibly because of the inhibition of the albumin-binding protein SPARC (secreted protein, acidic and rich in cysteine). SPARC is a glycoprotein over expressed in lung cancer cells with high affinity to albumin. The results from in vitro SPARC expression in A549 cells, a type of human non-small cell lung carcinoma (NSCLC), showed a significant drop by the albumin-bound complex at the mRNA level evaluated by qRT-PCR. This effect can be explained by transporting the albumin-bound complex into the cell surface, binding to the SPARC proteins, and so inhibiting the SPARC expressions. This work lays out a foundation for modification and characterization of the self/co-assembly peptide-based nanocarriers for hydrophobic anticancer drug delivery, especially to improve longevity and target-ability properties.

This thesis describes observations, techniques and strategies, which contribute towards the development of nanoparticle based detection and treatment of cancer. Quantum dots and biorecognition molecules were studied towards applications in detection and microgels were used in the rational design of a targeted drug delivery vehicle. The fluorescence intensity of quantum dots was examined in buffers commonly used in molecular biology. The fluorescence intensity of ZnS-capped CdSe quantum dots (QDs) was found to vary significantly, depending on the amount of ZnS capping on the QDs or the concentration, pH and type of buffer the QDs were in. Since fluorescence cannot reliably be used to quantify QDs, an alternative quantification method was developed, which does not rely on their fluorescence. This method employs phage display to identify nanoparticle-specific bacteriophage which were then applied in an assay to quantify QDs in environments where absorbance or fluorescence spectroscopy are ineffective. Biorecognition molecules, which can direct nanoparticles to a molecular target, were also identified through phage display. Phage display on whole cells was used to identify a peptide, which was conjugated with QDs to stain HeLa (cervical cancer) cells. A high-throughput phage display screening strategy was also developed, which could enable the simultaneous identification of multiple biorecognition molecules from a single library. QD-encoded microbead barcodes were conjugated to protein targets and then used to screen a phage display library. The beads and the binding phage were then separated using flow cytometry and fluorescence assisted cell sorting. Finally, biorecognition molecules were combined with nanoparticles to create drug delivery vehicles, which were designed to protect, deliver and then release chemotherapeutic drugs through an intracellular pH trigger. PolyNIPAAm and chitosan hydrogels, under 200 nm in diameter, were loaded with chemotherapeutic drugs, conjugated to transferrin and tested in vitro on HeLa cells. These projects demonstrate the great potential in this growing field as well as some of the many challenges that have yet to be overcome.

This thesis describes observations, techniques and strategies, which contribute towards the development of nanoparticle based detection and treatment of cancer. Quantum dots and biorecognition molecules were studied towards applications in detection and microgels were used in the rational design of a targeted drug delivery vehicle. The fluorescence intensity of quantum dots was examined in buffers commonly used in molecular biology. The fluorescence intensity of ZnS-capped CdSe quantum dots (QDs) was found to vary significantly, depending on the amount of ZnS capping on the QDs or the concentration, pH and type of buffer the QDs were in. Since fluorescence cannot reliably be used to quantify QDs, an alternative quantification method was developed, which does not rely on their fluorescence. This method employs phage display to identify nanoparticle-specific bacteriophage which were then applied in an assay to quantify QDs in environments where absorbance or fluorescence spectroscopy are ineffective. Biorecognition molecules, which can direct nanoparticles to a molecular target, were also identified through phage display. Phage display on whole cells was used to identify a peptide, which was conjugated with QDs to stain HeLa (cervical cancer) cells. A high-throughput phage display screening strategy was also developed, which could enable the simultaneous identification of multiple biorecognition molecules from a single library. QD-encoded microbead barcodes were conjugated to protein targets and then used to screen a phage display library. The beads and the binding phage were then separated using flow cytometry and fluorescence assisted cell sorting. Finally, biorecognition molecules were combined with nanoparticles to create drug delivery vehicles, which were designed to protect, deliver and then release chemotherapeutic drugs through an intracellular pH trigger. PolyNIPAAm and chitosan hydrogels, under 200 nm in diameter, were loaded with chemotherapeutic drugs, conjugated to transferrin and tested in vitro on HeLa cells. These projects demonstrate the great potential in this growing field as well as some of the many challenges that have yet to be overcome.

A basic issue for translational genomics is to model gene interactions via gene regulatory networks (GRNs) and thereby provide an informatics environment to derive and study effective interventions eradicating the tumor. In this dissertation, we present two different approaches to intervention methods in cancer-related GRNs. Decisions regarding possible interventions are assumed to be made at every state transition of the network. To account for dosing constraints, a model for the sequence of treatment windows is considered, where treatments are allowed only at the beginning of each treatment cycle followed by a recovery phase. Due to biological variabilities within tumor cells, the action period of an antitumor drug can vary among a population of patients. That is, a treatment typically has a random duration of action. We propose a unified approach to such intervention models for any Markovian GRN governing the tumor. To accomplish this, we place the problem in the general framework of partially controlled decision intervals with infinite horizon discounting cost. We present a methodology to devise optimal intervention policies for synthetically generated gene regulatory networks as well as a mutated mammalian cell-cycle network. As a different approach, we view the phenotype as a characterization of the long- run behavior of the Markovian GRN and desire interventions that optimally move the probability mass from undesirable to desirable states. We employ a linear programming approach to formulate the maximal shift problem, that is, optimization is directly based on the amount of shift. Moreover, the same basic linear programming structure is used for a constrained optimization, where there is a limit on the amount of mass that may be shifted to states that are not directly undesirable relative to the pathology of interest, but which bear some perceived risk. We demonstrate the performance of optimal policies on synthetic networks as well as two real GRNs derived from the metastatic melanoma and mammalian cell cycle. These methods, as any effective cancer treatment must, aim to carry out their actions rapidly and with high efficiency such that a very large percentage of tumor cells die or shift into a state where they stop proliferating.

The coupling efficiency of carbon nanotubes with near infrared laser radiation at 940nm wavelength was investigated. Nanotubes treated with different post processing methods were irradiated at different laser power intensities as dry samples and suspensions in water or ethanol. The interaction with the laser beam was measured and quantified based on the temperature increase in the samples as well as the amount of energy transmitted through them. Parallel experiments using carbon black revealed better performance of carbon nanotubes in terms of coupling efficiency and heat dissipation to their surroundings. It was found that most of the incident radiation on an individual carbon nanotube is absorbed, resulting in extreme local temperature increases proportional to the laser intensity, which can lead to instant tube oxidation in air. Such high heats are efficiently transferred to the material in immediate contact with the nanotubes, increasing its temperature very rapidly. The most intriguing results were obtained in the presence of water where the observations suggested, disintegration of carbon nanotubes with each laser pulse. It is shown that extremely high local temperatures vaporise the water in the immediate vicinity of a carbon nanotube and result in a water-gas reaction. It is further postulated that such effects can be achieved with laser beams at power intensities near the skin tissue's safe exposure thresholds, and therefore can potentially be used as a method of removing nanotubes from living tissue. This has advantages in providing an exit route for nanotubes whether introduced on purpose for reasons of medicine or therapy, or possibly, as a result of inadvertent exposure. Further studies on laser heating and transmission through different dry samples, highlighted that more crystalline structures such as that of a heat-treated nanotube, are more effective in causing extinction of the laser beam and a reduction in the transmitted beam intensity, however the tubes with more defects or with a length comparable to the radiation wavelength are very effective in converting the absorbed laser energy to heat. This effect is exacerbated when the laser beam is polarised parallel to the long axis of the carbon nanotubes. These heating effects were exploited to create welds in high density polyethylene using through transmission laser welding. The resultant welds showed better than or equal mechanical performance to welds made using industrial absorbers such as carbon black or Clearweld®.