In these days, nanomaterials are applied in a variety of biomedical applications including magnetic resonance imaging (MRI), cell imaging, drug delivery, and cell separation. Most MRI contrast agents affect the longitudinal relaxation time (T1) and transverse relaxation time (T2) of water protons in the tissue and result in increased positive or negative contrast. Here, we report the optimization of r1 (1/T1) or r2 (1/T2) relaxivity dynamics with diameter controlled gadolinium oxide nanocrystals (2~22 nm) and iron based magnetic nanocrystals (4 ~33 nm). The r1 and r2 MR relaxivity values of hydrated nanocrystals were optimized and examined depending on their core diameter, surface coating, and compositions; the high r1 value of gadolinium oxide was 40-60 S-1mM-1, which is 10-15 fold higher than that of commercial Gd (III) chelates (4.3~4.6 S-1mM-1). Moreover, in vitro toxicological studies revealed that polymer coated nanocrystals suspensions had no significant effect on human dermal fibroblast (HDF) cells even at high concentration. Towards multimodal imaging or multifunctional ability, we developed the iron oxide/QDs complexes, which consist of cores of iron oxide that act as nucleation sites for fluorescent QDs. The choice of variable QDs helped to visualize and remove large iron oxide materials in a magnetic separation. Additionally, diluted materials concentrated on the magnet could be fluorescently detected even at very low concentration. The designed MRI or multifunctional nanomaterials will give great and powerful uses in biomedical applications.

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Doctor of Philosophy / Department of Chemistry / Santosh Aryal / Nanotechnology offers exciting options for the site-selective delivery of chemotherapeutics and diagnostic agents using nanoparticles. Varieties of organic and inorganic nanomaterials have been explored extensively as a delivery system either in the form of drug carriers or imaging agents. Successful stories include the clinical translation of anticancer nanomedicines such as PEGylated liposomal doxorubicin (DOXIL®), albumin-bound paclitaxel (Abraxane®), and polymeric micelle loaded paclitaxel (Genexol®), which are currently used in the clinic as one of the first lines for cancer chemotherapies. These conventional nanomedicines rely on passive-drug targeting taking advantage of leaky tumor vasculature, called the Enhanced Permeability and Retention (EPR) effect. However, delivering biologically active components selectively to the diseased cell, for example, cancer, is highly challenging due to the biological barriers in the body including blood pool cells/proteins, heterogeneous microenvironment, and intracellular degradation. Therefore, the goal of this dissertation is to develop nanoplatforms that can deliver the agents of interest in targeted fashion to cancer while bypassing or collaborating with the biological barriers. The design consideration of these nanoplatforms centralizes on using simple chemical reactions and cell biology to engineer nanoparticles. The presented nanoparticles were extensively studied and evaluated for their biological functions using in vitro and in vivo models. These nanoconstructs described herein address current limitations of conventional nanomedicine such as (1) the lack of understanding of the interaction of nanoparticle and biological system, and (2) the lack of an effective targeting strategy to deliver drugs to the cancer cell in the tumors. The significant findings of each system will be highlighted and discussed throughout this dissertation. Results obtained highlight key findings such as NP intracellular fate, maximized tumor accumulation, and unique pharmacokinetics could open the avenues for systemic investigations for personalized medicine and lay the foundation for nanomedicine design to accelerate clinical translation.

Magnetic resonance imaging (MRI) is the leading imaging technique for disease diagnostics. MRI contrast agents facilitate MRI technique to obtain tissue-specific image with improved sensitivity and signal-to-noise ratio. However, the applications of current MRI contrast agents are hampered by their uncontrolled blood circulation time, low relaxivity, and low specificity. To address such need, I have developed a series of analitical methods to determine and evaluate the strong metal binding affinity and metal selectivity of developed protein-based contrast agents (ProCAs). In addition, we have successed designed contrast agents ProCA3 series based on key determinats for metal binding sites and relaxivity. We have dementrated that one of the ProCA3 variants, ProCA32, has a high Gd3+ affinity less than 10-21 M and high metal selectivity with relxivity of more than 30 mM-1s-1 per Gd and 60 mM-1s-1 per particle. Moreover, we have demonstrated that ProCA3 variants have proper blood circulation time, high relaxivity, high metal selectivity and low toxicity, which facilitate MR imaging of multiple organs, such as liver, kidney, and blood vessels, as well as tumors. ProCA32 is also able to image liver metastases a tumor size less than 0.25 mm, which is more than fourty times more sensitive than that of clinical diagnostics of liver metastases using MRI and our developed methodology. We have further created ProCA3 variants with targeting peptide moieties such as ProCA3.bomb or ProCA3.affi to against cancer biomarkers such as GRPR and HER2 with capability to imaging tumor biomarker expressions in vivo at molecular level. We have shown that ProCA3 has an excellent safety profile and pharmacokinetics for MRI in animals. With our additional effect in protein expression, modification, and scale up production of these developed protein contrast agents, ProCA3 is expected to be a promising MRI contrast for the diagnostics for disease, such as metastatic tumor and blood vessel abnormalities, and tumor biomarkers.

Texaphyrins represent the vanguard of experimental anticancer drugs and also symbolize a well-known example of expanded porphyrins, a class of oligopyrrolic macrocycles with tumor localization properties and powerful metal chelating properties. Chapter 1 of this thesis describes the unique structural characteristics of this complex synthetic molecule along with the biological relevance and scientific justifications for studying its anticancer properties and powerful MRI contrast ability. This Chapter also serves to underscore the need to improve further and refine the efficacy of texaphyrins as compounds that may be applied in the struggle against cancer. Chapter 2 details the synthesis of bismuth(III) and lead(II)-texaphyrin complexes that could potentially find use as [alpha]-core emitters for radiotherapy. In principle, porphyrins would ostensibly appear to be ideal ligands for use in radiotherapy due to their tumor-localizing ability. However, Bi(III)- and Pb(II)-porphyrin complexes are extremely rare, most reflecting the vastly challenging synthesis of these compounds as well as their general lack of stability. These limitations provided an incentive for us to use texaphyrins as more versatile ligands to prepare and fully characterize stable bismuth(III) and lead(II) complexes. To be of interest in future medical applications, we needed to prepare these complexes quickly as compared to the relevant time scales set by the half-lives of the isotopes targeted for use in radiotherapy. This goal was successfully realized. As mentioned above, texaphyrin is able to form stable complexes with a large variety of metals particularly in the lanthanide series. Gadolinium(III) complexes of texaphyrin have been studied in considerable detail. Chapter 3 details the synthesis and conjugation methods used to develop a texaphyrin conjugated dual mode nanoparticle contrast agent. This project has been done in collaboration with the group of Prof. Jinwoo Cheon (Yonsei University, Seoul, Korea), who demonstrated fascinating results with the texaphyrin functionalized nanoparticles. Not only do these conjugates act as improved magnetic resonance contrast agents displaying enhanced signals in both the T1 and T2 MRI modes, but also serve to sensitize apoptotic hyperthermia. It is this latter, double effector feature, that has been most extensively studied to date. Chapter 4 of this dissertation describes work done in close collaboration with Dr. Natalie Barkey and Dr. David Morse (Moffitt Cancer Center, Tampa, FL) where a gadolinium texaphyrin complex was developed that is able to target the melanocortin 1 receptor (MC1R) when encapsulated in a micellar system. As detailed in this Chapter, these collaborateurs demonstrated that these gadolinium-texaphyrin micelles are able to target MC1R-expressing xenograft tumors in vivo. This work relied on the supply of a new set of texaphyrin derivatives that were prepared and characterized as part of this dissertation work Chapter 5 of this disseration introduces sapphyrins, another class of expanded porphyrins with tumor selectivity. This project is based on the hypothesis that a direct linkage of sapphyrin with an anticancer agent based on ruthenium(II) could improve the efficacy of both compounds. Since sapphyrins exhibit limited ability to form stable complexes with transition metals, an appended 1,10-phenanthroline unit was chosen as an efficient N-donor aromatic ligand for ruthenium(II). Therefore, extensive synthetic efforts were made to form this sapphyrin-1,10-phenanthroline construct in an effort to stabilize a mixed sapphyrin-metallo-phenanthroline complex. Finally, Chapter 6 of this dissertation demonstrates the author's efforts to synthesize a planar rosarin species. Non-aromatic and non-planar rosarins have been known for over two decades. Through structural modification of the compound, namely through linking of both [Beta] positions on the bipyrrole unit, a new planar rosarin species has been synthesized exhibiting Hückel antiaromaticity. / text

Temporal and spatial molecular imaging of disease biomarkers using non-invasive MRI with high resolution is largely limited by lack of MRI contrast agents with high sensitivity, high specificity, optimized biodistribution and pharmacokinetics. In this dissertation, I report my Ph. D. work on the development of protein-based MRI contrast agents (ProCAs) specifically targeting different cancer biomarkers, such as grastrin-releasing peptide receptor (GRPR), prostate specific membrane antigen (PSMA), and vascular endothelial growth factor receptor-2 (VEGFR-2). Similar to non-targeted ProCAs, these biomarker-targeted ProCAs exhibit 5 - 10 times higher r1 and r2 relaxivites than that of clinical MRI contrast agents. In addition, these biomarker-targeted ProCAs have high Gd3+ binding affinities and metal selectivities. The highest binding affinity of the three GRPR-targeted contrast reagents obtained by grafting a GRPR ligand binding moiety into ProCA32 for GRPR is 2.7 x 10-9 M. We further demonstrate that GRPR-targeted ProCAs were able to semi-quantitatively evaluate GRPR expression levels in xenograft mice model by MRI. In addition, we have also created a PSMA-targeted ProCA which has a binding affinity to PSMA biomarker of 5.2 x 10-7 M. Further, we developed VEGFR-targeted contrast agent which is able to image VEGFR2 in mice models using T1-weighted and T2-weighted sequences. Moreover, the relaxivities and coordination water numbers of ProCAs can be tuned by protein design of ProCA4. Since disease biomarkers are expressed in various tumors and diseases, our results may have strong preclinical and clinical implications for the diagnosis and therapeutics of cancer and other type of diseases.

In this dissertation, research efforts mainly focused on exploring the applications of superparamagnetic iron oxide nanoparticles (SPIONs) in MR imaging and nanocatalysis via surface functionalization. A dopamine-based surface-functionalization strategy was established. The Simanek dendrons (G1 to G3), oligonucleotides and amino acids were loaded onto SPION surfaces via this approach to develop pH-sensitive MRI contrast agents, specific-DNA MR probes and a biomimetic hydrolysis catalyst. Dendron-SPION conjugates (G1 to G3) have good aqueous solubilities and high transverse relaxivities (>300 s-1*mM-1). They also showed interesting strong pH-sensitive R2 and R2* relaxivities, which were governed by the clustering states of dendron-SPIONs in different pH environments. Values of R2m and R2* m/R2m varied by over an order of magnitude around pH 5. The efficient cell-uptake (~3 million/cell) and low cytotoxicity of G1 to G3-SPIONs were demonstrated on HeLa cell cultures. The strong R2* effects were observed indicating the SPION clustering in HeLa cells. Two SPION-oligonuleotide conjugates were synthesized by coupling two half-match oligonucleotides onto domapine-capped SPIONs via SPDP linkers. They served as MR probes to detect a single-strand DNA with the same sequence to miRNA-21 based on the change of R2 values due to the DNA-bridged SPION clustering. The detection limit of the DNA could reach to 16.5 nM. A biomimetic hydrolysis nanocatalyst (i.e., Fe2O3-Asp-His complex) was developed by loading Asp and His-dopamine derivatives onto SPIONs. Paraoxon and nitrophenyl acetate were hydrolyzed under a mild condition (neutral pH, 37 °C) catalyzed by the Fe2O3-Asp-His complex. The two amino acids Asp and His cooperated with each other on the SPION surfaces to catalyze hydrolysis reactions. This catalyst could be recycled by a magnet and reused for four times without a significant loss of catalytic activity.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第19738号 / 工博第4193号 / 新制||工||1647(附属図書館) / 32774 / 京都大学大学院工学研究科物質エネルギー化学専攻 / (主査)教授 近藤 輝幸, 教授 辻 康之, 教授 大江 浩一 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

Magnetic nanoparticles are of great interest for a wide range of applications. This work has focused on three primary forms of iron based nanoparticles and combinations thereof: α-iron, iron oxide, and iron carbide or cementite. The synthesis of several core-shell particles including cementite-iron oxide, α-iron-cementite, and α-iron-iron oxide was accomplished through reverse micelle routes and high temperature decomposition of iron pentacarbonyl in various media. Structural analysis to confirm the structures was performed using extended x-ray absorption fine structure (EXAFS) techniques. A rapid characterization technique was developed utilizing a correlation between Fourier transform infrared spectroscopy and EXAFS to determine the full metal cation distribution between the octahedral and tetrahedral sites in manganese zinc ferrite (MZFO). This method was then used to show that the initial Fe3+ to Fe2+ ratio in MZFO synthesis could be used to design a desired cation distribution and affected the zinc incorporation levels into the resultant ferrite. Functionalization of nanoparticles for aqueous dispersions and ferrofluids has varying degrees of importance, depending on the application. In applications such as magnetic resonance imaging (MRI) where the targets are biological systems, it was important to produce solutions that will not aggregate in the high magnetic field of the MRI. It was also vital to characterize decomposition mechanisms and products that would be presented to the body after use as a contrast agent. This work has provided insight into both the preparation of magnetic samples for MRI applications and implications of the biocompatibility of reactive and decomposition products. Three successful methods of forming dispersions that would not aggregate in the high magnetic field of the MRI were comprised of cysteine/polyethylene glycol (PEG), PEG based ferrofluids, and dopamine/PEG. The dopamine functionalization however showed reactivity with the iron/iron oxide nanoparticles and led to the formation of the cytotoxic dopamine quinone and resulted in the destruction of the nanoparticles. Using all three types of dispersions to compare the iron based nanomaterials, the MRI measurements concluded with the iron oxide ferrofluid yielding the highest R2 enhancement.

Optical modulation of the physical properties of materials is important for future development of optical memories and switches, optoelectronics, and smart surfaces. Incorporation of an optically bistable photochromic compound into an electronically bifunctional material is a promising strategy for a development of photoswitchable materials. Photochromic spirooxazine ligands undergo light-induced ring-opening and closure between the closed-spirooxazine (SO) and open-photomerocynanine (PMC) forms. The structural reorganization leads to accompanying changes in electronic structure which can lead to a change in the oxidation/reduction potentials and spin state of a bound metal center. Changes in the ligand field about a metal center in turn can lead to “non-classical” photoinduced magnetic (PIM) effects. The “non-classical” PIM effect is an effect that occurs through ligand-centered processes via the metal center, rather than direct excitation at the metal center. The structural change of the photochromic compounds also results in a change in the frontier orbital energies and donor-acceptor character, which may lead to optically-gated charge-transfer and energy-transfer processes. In this dissertation, the structural factors that govern thermal relaxation of spirooxazines, as optical control units, was investigated toward controlling the photostationary states of this important class of photochromes. The electronic structure of the PMC form of azahomoadamantyl-based spirooxazines was found to control the thermal coloration/decoloration rates of photochromic spirooxazines. A significant charge-separated character of the PMC form was correlated with the slow thermal coloration/decoloration rates in spirooxazines. This concept was then extended to an investigation of the effect of Lewis-acidic metal complexation. Solution study of the charge-separated character of the PMC form via metal complexation of the photochromic spirooxazines supported the correlation between the charge-separated character of the PMC form and the rate of the thermal coloration/decoloration. The studies provide a potential pathway for modulating PMC thermal relaxation rates through optimization of the structure of the spirooxazines and metal complexation. The studies were then extended to an investigation of the photomodulation of charge-transfer processes in cobalt multinuclear clusters by photoisomerization of photochromic spirooxazines. Incorporation of optically bistable phenanthroline-spirooxazine ligands into a magnetically bistable cobalt-dioxolene valence tautomeric cluster resulted in large magnetic moments in the solid and solution states. This study suggests that the redox-isomeric behavior of the cobalt dioxolenes can be coupled to isomerization of the photochromic ligand in the solution state when the π-acceptor ability of the photochromic ligands align with the direction of charge transfer of the cobalt dioxolene components. The potential of these cobalt multinuclear clusters to enhance the relaxivity of water in MRI for biological imaging was investigated. A cobalt tetranuclear cluster was prepared and found to exhibit high magnetic moments in solution at room temperature, and large relaxivities relative to commercially available gadolinium based MRI contrast agents. Lastly, the photomodulation of ionic doping of graphene organic field-effect transistors (OFETs) by photochromic spirooxazines was investigated. The electron donor or acceptor nature of the photochromic isomers modulates the direction and magnitude of ionic doping of graphene, and in turn the gate voltages of graphene OFETs, leading to optical modulation of OFET gate voltages for data processing and memory technologies. / Graduate / 2020-02-08