Multimodal Multispectral Optical Endoscopic Imaging for Biomedical Applications

Optical imaging is an emerging field of clinical diagnostics that can address the growing medical need for early cancer detection and diagnosis. Various human cancers are amenable to better prognosis and patient survival if found and treated during early disease onset. Besides providing wide-field, macroscopic diagnostic information similar to existing clinical imaging techniques, optical imaging modalities have the added advantage of microscopic, high resolution cellular-level imaging from in vivo tissues in real time. This comprehensive imaging approach to cancer detection and the possibility of performing an ‘optical biopsy’ without tissue removal has led to growing interest in the field with numerous techniques under investigation. Three optical techniques are discussed in this thesis, namely multispectral fluorescence imaging (MFI), hyperspectral reflectance imaging (HRI) and fluorescence confocal endomicroscopy (FCE). MFI and HRI are novel endoscopic imaging-based extensions of single point detection techniques, such as laser induced fluorescence spectroscopy and diffuse reflectance spectroscopy. This results in the acquisition of spectral data in an intuitive imaging format that allows for quantitative evaluation of tissue disease states. We demonstrate MFI and HRI on fluorophores, tissue phantoms and ex vivo tissues and present the results as an RGB colour image for more intuitive assessment. This follows dimensionality reduction of the acquired spectral data with a fixed-reference isomap diagnostic algorithm to extract only the most meaningful data parameters. FCE is a probe-based point imaging technique offering confocal detection in vivo with almost histology-grade images. We perform FCE imaging on chemotherapy-treated in vitro human ovarian cancer cells, ex vivo human cancer tissues and photosensitiser-treated in vivo murine tumours to show the enhanced detection capabilities of the technique. Finally, the three modalities are applied in combination to demonstrate an optical viewfinder approach as a possible minimally-invasive imaging method for early cancer detection and diagnosis

Multimodal optical systems for clinical oncology

This thesis presents three multimodal optical (light-based) systems designed to improve the capabilities of existing optical modalities for cancer diagnostics and theranostics. Optical diagnostic and therapeutic modalities have seen tremendous success in improving the detection, monitoring, and treatment of cancer. For example, optical spectroscopies can accurately distinguish between healthy and diseased tissues, fluorescence imaging can light up tumours for surgical guidance, and laser systems can treat many epithelial cancers. However, despite these advances, prognoses for many cancers remain poor, positive margin rates following resection remain high, and visual inspection and palpation remain crucial for tumour detection. The synergistic combination of multiple optical modalities, as presented here, offers a promising solution. The first multimodal optical system (Chapter 3) combines Raman spectroscopic diagnostics with photodynamic therapy using a custom-built multimodal optical probe. Crucially, this system demonstrates the feasibility of nanoparticle-free theranostics, which could simplify the clinical translation of cancer theranostic systems without sacrificing diagnostic or therapeutic benefit. The second system (Chapter 4) applies computer vision to Raman spectroscopic diagnostics to achieve spatial spectroscopic diagnostics. It provides an augmented reality display of the surgical field-of-view, overlaying spatially co-registered spectroscopic diagnoses onto imaging data. This enables the translation of Raman spectroscopy from a 1D technique to a 2D diagnostic modality and overcomes the trade-off between diagnostic accuracy and field-of-view that has limited optical systems to date. The final system (Chapter 5) integrates fluorescence imaging and Raman spectroscopy for fluorescence-guided spatial spectroscopic diagnostics. This facilitates macroscopic tumour identification to guide accurate spectroscopic margin delineation, enabling the spectroscopic examination of suspicious lesions across large tissue areas. Together, these multimodal optical systems demonstrate that the integration of multiple optical modalities has potential to improve patient outcomes through enhanced tumour detection and precision-targeted therapies.Open Acces

Development and Evaluation of Biocompatible Engineered Nanoparticles for Use in Ophthalmology

The synthesis and design of biocompatible nanoparticles for targeted drug delivery and bioimaging requires knowledge of both their potential toxicity and their transport. For both practical and ethical reasons, evaluating exposure via cell studies is a logical precursor to in vivo tests. As a step towards clinical trials, this work extensively investigated the toxicity of gold nanoparticles (Au NPs) and carbon dot (CD) nanoparticles as a prelude to their in vivo application, focusing specifically on ocular cells. As a further step, it also evaluated their whole-body transport in mice. The research pursued two approaches in assessing the toxicity of engineered nanoparticles and the suitability of their use in targeted delivery and bioimaging applications: (1) In vitro (using retinal pigment epithelial, corneal, and lens epithelial cells (2) In vivo (mouse whole body studies). Part. 1. In the in vitro assessments of Part 1, the biocompatibilities of spherical, rod, and cubic shaped Au NPs were compared for different exposure concentrations. Spherical Au NPs were evaluated in particular detail, and a possible toxicity mechanism was proposed, based on the findings of a colorimetric assay, electrical impedance measurements, and confocal imaging analysis. The assay measured the activity of succinate hydrogenase, a mitochondrial enzyme, while electrical impedance spectroscopy quantified the strength of cell-cell and cell-substrate attachment, a proxy of viability. Finally, confocal imaging analysis verified that the NPs were internalized and confirmed the degree of their toxicity. Collectively, the data indicated that surface area concentration was the critical toxicity parameter. Subsequently, to create biocompatible Au NPs, a unique end-thiolation of hyaluronic acid was adapted to create homogenously coated Au NPs. The end-thiolated hyaluronate (HS-HA) coating not only improved the biocompatibility of the Au NPs but also enhanced the internalization rate of the larger Au NPs, which could not enter the cells otherwise. The first part of this research also studied the synthesis of biocompatible deep red-emissive CDs for bioimaging applications. For this purpose, a central-composite design response surface methodology (CCD-RSM) was utilized. A scalable isolation-free microwave pyrolysis method for synthesizing deep red-emissive nitrogen-doped carbon dots (nCDs) from citric acid and ethylenediamine was successfully developed and optimized. The formation of C‒N and the presence of pyrrolic N content proved to be keys to creating red-emissive nCDs. Confocal images demonstrated that the nanoparticles could enter healthy corneal, retinal, and lens epithelial ocular cells, as well as cancerous Chinese Hamster Ovary cells. Part 2. Building on the results of in-vitro testing of the engineered Au NPs and nCDs, in Part 2 we developed protocols for injecting both types of NPs in-vivo. Prior to any intravenous or intravitreal injections, a preliminary study tested the ability of Au NPs to cross the tight junctions between retinal pigment epithelial cells. Transwell® permeable supports were used to simulate the blood-retinal barrier (BRB). The results showed that 20 nm Au NPs successfully crossed the permeable supports covered with confluent retinal pigment epithelial cells. Based on this finding, both intravitreal and intravenous injections of nascent and HS-HA coated Au NPs were tested. The intravitreal injections caused retinal detachment, very probably due to the mechanical intrusion of the injection needle and the volume, albeit small, of the injected NPs. Far more significant and encouraging, intravenously injected coated and uncoated NPs successfully crossed the BRB. As a result of the intravenous injections, it was observed that both coated and uncoated Au NPs were able to cross the blood-retinal barrier. As expected, the numbers of HS-HA-coated Au NPs were significantly higher in specific parts of the retina that contain more CD44 expressing cells, which have cell surface receptors for internalizing HA. Finally, based on the confocal imaging analysis, the NP concentration in each retinal layer was quantified as a function of time, post-injection. The NPs reached the retina in less than 5 minutes and reached a maximum concentration within approximately 20 minutes. Due to the enhanced retention and permeability effect of NPs, 8.5% of the uncoated and 12.1% of the HA-coated NPs that reach the retina remained after 24 hours. Next, nCDs with and without the HA coating were injected subcutaneously into post-mortem mouse and porcine eye globes. Ex-vivo porcine eye images showed that intravitreally injected nCDs had effectively diffused through the vitreous to the cornea, and post-mortem whole-body mouse images also demonstrated that the nCDs are suitable for bioimaging, excitable in the NIR region with the sensitivity of 15%. Cumulatively, our observations indicate that HA coated NPs could potentially deliver other payloads such as DNAs, mRNAs, proteins, siRNAs, and drugs into the cells which overexpress CD44 receptors, for example, cancerous and inflammatory cells, thus providing a platform for targeted treatment and imaging of many severe vision-threatening diseases and degenerative conditions

Development of Exosomal Protein Detection Assays for Cancer Diagnostics Using Nanomaterials in Conjunction with Optical Spectroscopy and Imaging

Cases of cancer are on the rise, and cancer continues to be the major cause of death in the world. It has been known for years that the survival rate and possible recovery depend on early diagnosis and personalized treatment. However, tumors are in most cases almost undetectable until cancer has already invaded the surrounding tissue and begins to metastasize to distant organs at which point the treatment is significantly less effective or completely ineffective. And even if the tumor is detected, its analysis requires a tissue biopsy, which in many instances is a risky invasive procedure that does not allow regular monitoring of the effectiveness of treatment. Therefore, any strategy for early cancer identification will be based on the correct identification of cancer detection markers found in body fluids in various forms such as proteins, RNAs, and DNA. Emerging evidence points to extracellular vesicles, more precisely their subgroup - exosomes, as an abundant source in proteins and nucleic acids that reflects the state of the parental cell. In this dissertation, we summarize the exosomal biogenesis and composition, the influence of exosomes on cancer development and progression with emphasis on breast cancer, and major analytical methods applied to exosomal protein detection. Further, we report our take on exosomal protein detection as a form of novel bulk detection and single vesicle profiling techniques, which are designed for liquid biopsy in a clinical environment. Our approaches are based on optical spectroscopy and imaging such as surface enhanced Raman scattering (SERS), fluorescence, and dark-field light scattering imaging, and are designed to operate with small amounts (8-50 L) of already diluted samples. We demonstrated the potential of 3D printing and its applicability to create a miniaturized device that made it possible to customize detection conditions for nanosized exosomes and microvolume samples. Additionally, we developed a simple, fast, and inexpensive bulk method for detection of exosomal surface proteins using quantum dots in conjunction with fluorescent spectroscopy and we demonstrated its clinical potential on detection of HER2 cancer marker in plasma samples from a breast cancer patient. Lastly, we report single vesicle technology (SVT) based on dual fluorescent and dark-field imaging to achieve protein profiles at a single exosome level. Our SVT can overcome many obstacles that bulk technologies cannot and can bring long-sought-after early cancer detection into the clinical setting

Development of Nanostructured Glucose Biosensor

With the development of nanotechnology and nanomaterials, biosensors incorporated with novel nanomaterials and nanostructures have shown significant potential in point-of-care medical devices because of their rapid interaction with target analytes and their miniaturized systems. Nanomaterials and nanostructures with special chemical, physical and biological characteristics are able to enhance biosensors’ performance in terms of sensitivity and selectivity. Therefore, my study focused on development of special nanostructures used for advanced glucose biosensor. Monitoring of blood glucose level is essential for diabetes management. However, current methods require people with diabetes to have blood test with 5-8 times per day. Compared to other methods, optical and magnetic techniques have a potential in developing minimally invasive or non-invasive, and continuous glucose monitoring nanostructured biosensors. Consequently, this thesis presented nanostructured optical and magnetic glucose biosensors by incorporating novel nanomaterials and fabricating nanostructures for the next generation of glucose biosensor in the tears. The glucose biorecognition biomolecule used in the biosensors was Concanavalin A (Con A). Con A is a lectin protein that has strong affinity to glucose. Fluorescence resonance energy transfer (FRET) technique was applied to develop optical glucose biosensors. FRET biosensor is a distance-dependent biosensor. The fluorescence emission of a donor molecule could be used to excite acceptor when the distance between donor and acceptor is close enough (\u3c 20 nm). Three different types of nanostructures were developed and used as the donors of the glucose FRET biosensors. The first type of sensor is a ZnO/quantum dots-based glucose biosensors. Hybrid ZnO nanorod array with decoration of CdSe/ZnS quantum dots were prepared and coated on silicone hydrogel which is a common materials of contact lens. The patterned nanostructured FRET sensor could quickly measure rats’ tear glucose in an extremely small amount (2 µL) of diluted tear sample. The second type of biosensor is based on upconversion nanomaterials. Upconversion NaGdF4: Yb, Er nanoparticles with diameter of about 40±5 nm have been prepared by polyol process and coated on silicone hydrogel to directly sense the tear glucose level on the rats’ eye surface. The results show that the upconversion nanomaterials based lens sensor is able to quickly measure glucose in rats’ blood samples. The third type of sensor utilizes the unique optical properties of carbon nanomaterial, fluorescent carbon dots and graphene oxide nanosheets. The carbon dots with tunable fluorescence were developed by a microwave-assisted process. The carbon dots are used as a fluorescence donor in the biosensor, the chitosan coated graphene oxide acts as the fluorescence acceptor to quench the emission of carbon quantum dots. In the presence of glucose, the emission of carbon quantum dots could be restored as a function of the concentration of glucose. Two linear relationships of the restored emission of the sensor and the concentration of glucose were observed, in the range of 0.2 mM to 1 mM, and 1 mM to 10 mM, respectively. On the other hand, a magnetoresistive (MR) nanostructured glucose biosensor has been developed by exploiting hybrid graphene nanosheets decorated with FeCo magnetic nanopartciles. The Fe3O4/silica core/shell nanoparticles are used as the magnetic label of glucose, which could bind onto the surface of FeCo/graphene nanocomposited sensor. The binding of magnetic label onto the hybrid graphene nanosheets can result in the change of the magnetoresistance. The MR signal as a function of the glucose level of diluted rat blood samples is measured in a range of 2 mM to 10 mM. In summary, novel nanomaterials and nanostructures with special fluorescent and magnetoresistive properties are fabricated for developing nanostructured glucose biosensors, which could bring alternative approaches for convenient management diabetes

Targeted Delivery Of Nrf2 Sirna Using Modular Polymeric Micellar Nanodelivery System For Efficient Target Gene Knockdown In Hepatocellular Carcinoma

Tumor selective drug delivery as well as chemotherapy associated multi drug resistance (MDR) pose tremendous hurdles for effective cancer therapy. In this regard, designing multifunctional nanocarriers loaded with drug/gene payloads and engineered with tumor targeting ligands can serve as a modular platform for targeted drug/gene delivery. In this study we undertook the synthesis of a self-assembling block copolymer constructed using poly(styrene-co-maleic anhydride, partial iso-octyl ester) (SMAPIE) and branched polyethylenimine (PEI) as building blocks and evaluated its micelle forming ability, siRNA complexation and siRNA delivery potentials. In addition, we engineered galactosamine decorated nanomicelles using modular “click” chemistry based approaches for evaluating the targeted delivery of Nrf2 siRNA to Hep G2 liver cancer cells overexpressing asialoglycoprotein receptors (ASGPRs). Our results demonstrate that the galactosamine decorated nanocarriers could effectively deliver Nrf2 siRNA into Hep G2 liver cancer cells resulting in efficient target gene knockdown, evincing its potential for targeted liver cancer therapy

Utilization of Nanoparticles for Photoacoustic Chemical Imaging

Tumors are known to have unique chemical properties, such as low pH (acidosis), high K+ (hyperkalemia), and low O2 (hypoxia). Tumor acidosis has been known to influence therapeutic activities of chemotherapeutic drugs. Another conventional cancer treatment, radiation therapy, is highly dependent on local oxygen concentrations. Hyperkalemia has been recently reported to suppress the immune response of activated T-cells. It is also believed that the spatial distribution of these analytes and its heterogeneity, are of relevance. Despite the importance of such chemical information on tumors, there are no clinically available tools for “quantitative” pH, K+, or tissue O2 imaging. Here, photoacoustic (PA) imaging is employed to provide chemical imaging of all these target analytes for cancer (pH, O2 and K+). As for pH, we report on an in vivo pH mapping nanotechnology. This subsurface chemical imaging is based on tumor-targeted, pH sensing nanoprobes and multi-wavelength photoacoustic imaging (PAI). The nanotechnology consists of an optical pH indicator, SNARF-5F, 5-(and-6)-Carboxylic Acid, encapsulated into polyacrylamide nanoparticles with surface modification for tumor targeting. Facilitated by multi-wavelength PAI plus a spectral unmixing technique, the accuracy of pH measurement inside the biological environment is not susceptible to the background optical absorption of biomolecules, i.e., hemoglobins. As a result, both the pH levels and the hemodynamic properties across the entire tumor can be quantitatively evaluated with high sensitivity and high spatial resolution in in vivo cancer models. For K+, we extend this technique to ion-selective photoacoustic optodes (ISPAOs) that serve at the same time as fluorescence-based ISOs, and apply it specifically to potassium (K+). However, unfortunately, sensors capable of providing potassium images in vivo are still a future proposition. Here, we prepared an ion-selective potassium nanosensor (NS) aimed at in vivo photoacoustic (PA) chemical imaging of the extracellular environment, while being also capable of fluorescence based intracellular ion-selective imaging. This potassium nanosensor (K+ NS) modulates its optical properties (absorbance and fluorescence) according to the potassium concentration. The K+ NS is capable of measuring potassium, in the range of 1 mM to 100 mM, with high sensitivity and selectivity, by ISPAO based measurements. Also, a near infrared dye surface modified K+ NS allows fluorescence-based potassium sensing in the range of 20 mM to 1 M. The K+ NS serves thus as both PA and fluorescence based nanosensor, with response across the biologically relevant K+ concentrations, from the extracellular 5 mM typical values (through PA imaging) to the intracellular 150 mM typical values (through fluorescence imaging). Lastly, nano-enabled tissue O2 monitoring by PA, called lifetime-based PA (PALT) imaging, was introduced and demonstrated. A known PALT oxygen indicator, Oxyphor G2, is conjugated into polyacrylamide nanoparticles, called G2-PAA NP. The oxygen sensing capability of the G2-PAA NP has been confirmed in vitro and in vivo studies. In an Appendix, we show how to monitor photodynamic therapy (PDT) using the PALT approach to measure the local oxygen depletion as a function of PDT time. Oxygen depletion during PDT is monitored using both oximeter and PALT spectroscopy in vitro. The latter is enabled by theranostic NPs of methylene blue (MB) conjugated PAA, used for both PALT and PDT. This synergistic approach has good potential for personalized medicine.PHDChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/143924/1/lechang_1.pd

Metabolic Signatures of Prostate Cancer and Renal Cell Carcinoma using High-Resolution NMR and Hyperpolarized 13C MRI

Non-invasive techniques to assess metabolic reprogramming during cancer progression can be used to improve therapeutic selection and provide an early assessment of therapeutic response or resistance in individual patients. Prior studies have shown that metabolic reprogramming plays a key role in the development of prostate cancer and renal cell carcinoma (RCC). This dissertation further elucidates the metabolic alterations that occur in treatment-resistant prostate cancer and in patient-derived models of RCC using high-resolution nuclear magnetic resonance (NMR) spectroscopy and hyperpolarized (HP) 13C magnetic resonance imaging (MRI), with the goal of identifying new non-invasive diagnostic imaging tools. Glycolysis, metabolism of pyruvate and glutamate via the tricarboxylic acid (TCA) cycle, glutaminolysis, and glutathione synthesis are upregulated in castration-resistant prostate cancer (CRPC) compared to their androgen-dependent counterparts, using human cell lines as well a treatment-driven transgenic murine model. These metabolic alterations were reversed in castration-resistant murine tumors by treatment with a secondary androgen pathway inhibitor, apalutamide, suggesting that early metabolic responses to treatment can be monitored using non-invasive imaging techniques. Furthermore, treatment-emergent small cell neuroendocrine prostate cancer, a consequence of protracted treatment with primary androgen deprivation therapy and secondary androgen pathway inhibitors, exhibits significantly upregulated glycolysis, TCA cycle metabolism of pyruvate and glutamate, and glutaminolysis, as well as significantly altered redox capacity compared to castration-resistant prostate adenocarcinoma using patient-derived xenograft models. Finally, the metabolic differences associated with the tumor microenvironment were compared between various patient-derived models of RCC, finding that RCC patient-derived xenografts (PDXs) displayed higher redox capacity and were more proliferative than cells and tissue slices derived from the PDXs and maintained ex vivo. The work presented in this dissertation suggests that a combination of HP [1-13C]pyruvate, [2-13C]pyruvate, [5-13C]glutamine, and [1-13C]dehydroascorbate can be used to distinguish advanced prostate cancer and RCC subtypes in future HP 13C MRI of patients for improved treatment selection and monitoring