archives@tulane.edu / Preeclampsia is a condition that occurs during pregnancy and affects both the mother and fetus. It is the leading cause of fetal and maternal mortality, affecting up to 8% of pregnancies. The disorder is diagnosed after the new onset of maternal high blood pressure and proteinuria. There is currently no cure for preeclampsia, other than delivery of the baby (and placenta), which often has to occur pre-term. Abnormal placental development that results in placental ischemia is a precursor for the development of preeclampsia in the mother. Current clinical imaging systems have been unable to accurately characterize placental function enough to make diagnoses. Therefore, there is a need to further study placental ischemia so that better diagnostic tools and therapies can be developed. Spectral photoacoustic (PA) imaging of placental oxygen saturation is a promising approach to studying placental ischemia. PA imaging uses nanosecond light pulses to excite endogenous or exogenous chromophores in biological tissue. When a chromophore undergoes a thermo-elastic expansion after absorbing light, a pressure wave is released within the tissue. The pressure wave resulting from the chromophore’s expansion are received by an ultrasound transducer. Our endogenous chromophore of interest is hemoglobin, an oxygen carrier protein in the blood. Hemoglobin and deoxyhemoglobin have distinctly different absorption spectra, which allows for the estimation of sO2 in vivo. One aim of this work is to use photoacoustic imaging to study altered levels of induced placental ischemia. Exogenous contrasts, such as perfluorocarbon microbubbles are commonly used in ultrasound because of their acoustic impedance mismatch with surrounding tissues. Perfluorocarbon nanodroplets are their liquid counterpart and offer longer stability in vivo. Additionally, they can be targeted and phase-changed into gas by a surrounding change in pressure, offering multimodal use. We use a modified perfluorocarbon construct loaded with ICG as an exogenous contrast agent. In this work, we will first investigate the feasibility of nanodroplets as oxygen delivery agents to blood. Then, we explore the utility of oxygen-loaded nanodroplets targeted to the placenta as a potential theranostic for preeclampsia. / 1 / Megan Escott

Abstract Samson Dada and Dr. Hua Mei, Department of Chemistry, College of Arts and Sciences, East Tennessee State University, Johnson City, TN Our overall research goal is to synthesize a water-soluble, bio-absorbable theranostic nanoparticle (NP) that will improve diagnostic and therapeutic efficacy for cancer. Such theranostic nanoparticles are composed of carbon dots (CDs), conjugated with a targeting agent through a non-cleavable peptide bond; and an anticancer drug Doxorubicin (DOX) using an acid-labile hydrazine linkage for targeted delivery and bio-imaging functions. Recent studies have shown that Carbon dots (CDs) are of interest in biological applications due to their unique properties such as inherent fluorescence, extremely high biocompatibility, and facile synthetic route. The large surface area and multiple surface functionalities make CDs versatile platforms to conjugate with other moieties, including therapeutic agents or targeting agents. The target agents, such as folic acid (FA), are proposed to be permanently linked with CDs to improve the target specificity of the tumor cells. Folic acid is used as a targeting agent as it is a water-soluble, low molecular weight vitamin as it plays an essential role in cell survival and binds with high affinity to the folate receptor (FR) – a membrane-anchored protein that is a cancer biomarker. The multimodal nano-platforms of CDs can also facilitate the delivery the anticancer drugs. The anticancer drug is attached by a cleavable linker that can release the drug inside the tumor cell. We will use the cytotoxic chemotherapeutic agent doxorubicin (DOX) as an example. One series of CDs, FA-CD and FA-CD-DOX, are successfully prepared in the lab. The UV-vis and Fluorescence spectra of the sample was investigated and compared. The concentration of each part in nanoparticles are calculated. The final Drug Load Content (DLC) and Drug Load Efficiency (DLE) are also calculated and compared with the literature. Another series of FA-CD-DOX will be prepared and compared. The characterization of the diagnostic and therapeutic potential of the NP particles will be carried out in the pharmaceutical department.

Abstract Our overall research goal is to synthesize a water-soluble, bio-absorbable theranostic nanoparticle (NP) that will improve diagnostic and therapeutic efficacy for cancer. Such theranostic nanoparticles are composed of carbon dots (CDs), conjugated with a targeting agent through a non-cleavable peptide bond; and an anticancer drug Doxorubicin (DOX) using an acid-labile hydrazine linkage for targeted delivery and bio-imaging functions. Recent studies have shown that Carbon dots (CDs) are of interest in biological applications due to their unique properties such as inherent fluorescence, extremely high biocompatibility, and facile synthetic route. The large surface area and multiple surface functionalities make CDs versatile platforms to conjugate with other moieties, including therapeutic agents or targeting agents. The target agents, such as folic acid (FA), are proposed to be permanently linked with CDs to improve the target specificity of the tumor cells. Folic acid is used as a targeting agent as it is a water-soluble, low molecular weight vitamin as it plays an essential role in cell survival and binds with high affinity to the folate receptor (FR) – a membrane-anchored protein that is a cancer biomarker. The multimodal nano-platforms of CDs can also facilitate the delivery the anticancer drugs. The anticancer drug is attached by a cleavable linker that can release the drug inside the tumor cell. We will use the cytotoxic chemotherapeutic agent doxorubicin (DOX) as an example. One series of CDs, FA-CD and FA-CD-DOX, are successfully prepared in the lab. The UV-vis and Fluorescence spectra of the sample was investigated and compared. The concentration of each part in nanoparticles are calculated. The final Drug Load Content (DLC) and Drug Load Efficiency (DLE) are also calculated and compared with the literature. Another series of FA-CD-DOX will be prepared and compared. The characterization of the diagnostic and therapeutic potential of the NP particles will be carried out in the pharmaceutical department.

This thesis is centered on the Gadonanotubes (GNTs), an ultra-high-performance magnetic resonance imaging (MRI) contrast agent material discovered in our laboratories in 2005. The GNTs are a new paradigm in MRI contrast agent design with small clusters of Gd3+ ions within ultra-short carbon nanocapsules (ca. 50 nm) cut from full-length single-walled carbon nanotubes. Here, the factors underlying the performance efficacy of the GNTs have been investigated for the first time by variable-field (-50,000 Oe to 50,000 Oe at 2K) and variable-temperature (2K to RT at 100 Oe) magnetic susceptibility measurements using a Magnetic Property Measurement System (MPMS, based on a SQUID magnetometer). Additionally, experiments focused on the effects of hydroxylation of the GNTs’ exterior surface regarding water-solubility are examined. Finally, the use of the GNTs as potential replacements for traditional metal-chelating/sequestering agents is explored. More specifically, the internal Gd3+-ion clusters of the GNTs have been radiolabeled: (1) with 153Gd3+ ions to test Gd3+-ion stability to simulated biological challenge, (2) with 225Ac3+ ions to generate a new concept for a GNT-based agent for α-radiotherapy, and finally (3) with 64Cu2+ ions to produce the first bimodal MRI/PET (PET = positron emission tomography) imaging agent derived from the GNTs.

Introduction Irradiation of gold with protons provides access to no-carrier-added 197mHg and 197Hg. Interests in these radionuclides were awakened by the unique chemical and physical properties of mercury and its compounds combined with convenient nuclear properties like suitable half life (197mHg: T1/2 = 23.8 h, 197Hg: T1/2 = 64.14 h), low energy gamma radiations for imaging, Auger – and conversion electrons for therapy. The high thermal conductivity of gold enables high current irradiations and the monoisotopic natural abundance of 197Au supersedes expensive enrichment of the target material. The 197Au(p,n)197(m)Hg reaction was applied until now only for beam monitoring1, stacked foil meas-urements2 or very small scale tracer production. Material and Methods The irradiations were performed at a Cyclone 18/9 (IBA, Louvain la Neuve, Belgium). Its beam-line was sealed with a 1.0 mm vacuum foil (high purity aluminum, 99.999 %) from Goodfellow (Huntingdon, England). High purity gold disks (23 mm diameter, 2 mm thickness, 99.999% pure, 1 ppm Cu) as target material were purchased from ESPI (Ashland, USA). Gold foils as alternative gold targets (12.5×12.5 mm, 0.25 mm thickness, 99.99+ %, 1 ppm Cu) between an aluminum disk (22 mm diameter, 1 mm thickness, 99.0 %, hard) and an aluminum lid (23 mm diameter, 99.0 %, hard) were purchased from Goodfellow (Huntingdon, England). Hydrochloric acid (30%) and nitric acid (65%) were purchased from Roth (Karlsruhe, Germany) in Rotipuran® Ultra quality. Deionized water with > 18 MΩcm resistivity was prepared by a Milli-Q® system (Millipore, Molsheim, France). For separation of target material and side products a liquid-liquid extraction method (Gold was extracted with methyl isobutyl ketone (MIBK) from 2 M HCl target solution) and an ion exchange method (cation exchange resin (Dowex50W-x8, 100–200 mesh, H+ form) were applied. Results and Conclusion No-carrier-added 197(m)Hg was produced from gold via the 197Au(p,n)197(m)Hg reaction at proton energies of 10 MeV in sufficient quantity and quality for imaging studies. Two different methods were studied for the separation of Hg radionuclides generated from Au targets. The results demonstrate the possibility to produce 197(m)Hg from gold at low proton energies. Combined with the presented radiochemical separation methods, the 197Au(p,n) reaction could be the basis for repeatable production of 197(m)Hg for imaging and therapy research on sufficient activity level.

abstract: In recent years, the field of nanomedicine has progressed at an astonishing rate, particularly with respect to applications in cancer treatment and molecular imaging. Although organic systems have been the frontrunners, inorganic systems have also begun to show promise, especially those based upon silica and magnetic nanoparticles (NPs). Many of these systems are being designed for simultaneous therapeutic and diagnostic capabilities, thus coining the term, theranostics. A unique class of inorganic systems that shows great promise as theranostics is that of layered double hydroxides (LDH). By synthesis of a core/shell structures, e.g. a gold nanoparticle (NP) core and LDH shell, the multifunctional theranostic may be developed without a drastic increase in the structural complexity. To demonstrate initial proof-of-concept of a potential (inorganic) theranostic platform, a Au-core/LDH-shell nanovector has been synthesized and characterized. The LDH shell was heterogeneously nucleated and grown on the surface of silica coated gold NPs via a coprecipitation method. Polyethylene glycol (PEG) was introduced in the initial synthesis steps to improve crystallinity and colloidal stability. Additionally, during synthesis, fluorescein isothiocyanate (FITC) was intercalated into the interlayer spacing of the LDH. In contrast to the PEG stabilization, a post synthesis citric acid treatment was used as a method to control the size and short-term stability. The heterogeneous core-shell system was characterized with scanning electron microscopy (SEM), energy dispersive x-ray spectroscopy (EDX), dynamic light scattering (DLS), and powder x-ray diffraction (PXRD). A preliminary in vitro study carried out with the assistance of Dr. Kaushal Rege's group at Arizona State University was to demonstrate the endocytosis capability of homogeneously-grown LDH NPs. The DLS measurements of the core-shell NPs indicated an average particle size of 212nm. The PXRD analysis showed that PEG greatly improved the crystallinity of the system while simultaneously preventing aggregation of the NPs. The preliminary in vitro fluorescence microscopy revealed a moderate uptake of homogeneous LDH NPs into the cells. / Dissertation/Thesis / M.S. Materials Science and Engineering 2011

Cancer mortality is progression-dependent thus its treatment relies on effective therapy and monitoring of responses. Nanoparticles have long been used to improve the therapeutic index of drugs by facilitating their transit to the target site at higher concentrations than free drugs, whilst protecting healthy tissues from an often potent and cytotoxic payload. Through the EPR (enhanced permeability and retention) effect, injected, PEGylated nanoparticles preferentially accumulate in tumour tissue deeming them eminently suitable for cancer intervention for delivery of both therapeutic and contrast agents The development of theranostic liposomal systems comprising both imaging and therapeutic capabilities exploits the facets of liposomes, and forms an elegant strategy to address major problems which hinder effective cancer therapy. Liposomes can be tailored to be thermosensitive in a low hyperthermic range of ~42°C, above physiological temperature but below that which can induce tissue damage. This allows the use of heating as an external triggering modality to induce targeted drug release. Throughout the course of this work, the photoacoustic contrast agent ICG was successfully incorporated into PEGylated doxorubicin-encapsulating liposomes, marrying two FDA approved entities. The project commenced with the development of the basic liposomal-DOX. Differing lipid compositions of varying fluidities were tested against those which have been previously established. These compositions carried a range of phase transition temperatures, above which the liposomes release the encapsulated DOX. This study concluded with the generation of a library of liposomes with differing release kinetics at 42°C in simulated physiological conditions. The second section of the project investigated the methodology behind the incorporation of ICG into the liposomal bilayers. The lipid composition used for the study was based on the DOXIL® formulation, due to its robust structure and establishment in the field of cancer therapy. The protocols used varied on the basis of chronology in regards to the liposome preparation protocol. The film insertion method incorporated the ICG in initial lipid film generation. The freeze fracture protocol introduced the ICG during lipid film hydration. The post insertion protocol introduced ICG in the final stages of DOX loading. The downsizing protocol was also varied between extrusion and sonication. Through varying of the protocols and downsizing methodology, it was possible to incorporate differing ICG concentrations and attain differing levels of structural stability. The most successful liposome was then tested for its imaging potential in vivo through a photoacoustic imaging modality namely multispectral optoacoustic tomography. This validated accumulation of the liposomes at the tumour site along with co-localisation of both drug and dye. The project culminated in the combination of the two studies, producing a thermosensitive theranostic ICG labelled liposomal doxorubicin system. The system showed improved blood stability than the current clinical systems, and demonstrated imaging potential through IVIS based fluorescence imaging. Through exploitation of the photothermal effects of ICG within a thermosensitive lipid vesicle, it was also possible to induce drug release through irradiation with a non-thermal near-infrared laser. This shows promise for future therapy, allowing simultaneous imaging, optimum release induction and monitoring response to therapy, in a cheap, effective and time-efficient manner.

Les composés fluorés sont très utilisés dans les agents de contraste ultrasonore (ACU) pour faciliter le diagnostic de nombreuses maladies par imagerie en temps réel. Tous les ACU commerciaux sont des microbulles de gaz perfluoré stabilisé par une monocouche de phospholipides, protéines ou tensioactifs. Cependant, l'application théranostique (de la contraction de thérapeutique et de diagnostic) de ces matériaux est sévèrement limitée par (i) la faible stabilité du composé fluoré, (ii) leur taille micrométrique et (iii) le manque de compartiments efficaces pour l'encapsulation d’un principe actif. Nous avons proposé deux stratégies différentes pour améliorer la stabilité du cœur fluoré et fournir simultanément des interfaces fonctionnelles pour l'encapsulation d’un principe actif.La première approche a consisté à intercaler le chitosane avec des phospholipides (DSPC) pour augmenter la stabilité de microvésicules contenant du gaz fluoré décafluorobutane (C4F10). L'affinité du DSPC et du chitosane a été révélée par des techniques de caractérisation de surface et par microscopie à fluorescence. Les microvésicules contenant du chitosane ont présenté des signaux intenses de la composante gazeuse en résonance magnétique nucléaire du fluor (RMN 19F) et en échographie in vitro après 48 h, deux fois plus longtemps que les échantillons sans chitosane. Le chitosane permet ainsi d’augmenter la stabilité des microvésicules et constitue une plateforme appropriée pour l'encapsulation de médicaments. La coque de chitosane-phospholipide pourrait donc améliorer le potentiel théranostique de ces microvésicules. Cependant, l'utilisation d'un coeur gazeux a rendu la stabilisation de vésicules submicrométriques difficile. Par conséquent, la deuxième stratégie s’est focalisée sur le développement d'un agent théranostique à l'échelle nanométrique en piégeant un coeur fluoré liquide de perfluorohexane (PFH; C6F14) dans une enveloppe polymère rigide de polylactide (PLA). Pour améliorer l'interaction des polymères biodégradables avec les perfluorocarbones, nous avons synthétisé des polymères PLA contenant cinq longueurs différentes de groupes terminaux fluorés (de C3F7 à C13F27) par polymérisation par ouverture de cycle du D,L-lactide. Les mesures de temps de relaxation spin-spin 19F ont démontré la présence d’interactions fluorophiles intenses entre les chaînons fluorés et le PFH. Les polymères ont ensuite été formulés en nanocapsules (NCs) sphériques de 150 nm de diamètre, comme vérifié par microscopie électronique en transmission. La RMN 19F a montré que l'efficacité d'encapsulation du PFH dans les capsules est doublée grâce à l’utilisation des polymères fluorés comparé aux dérivés non fluorés. Par conséquent, la réponse acoustique des NCs a été multipliée par dix avec les deux modes d'imagerie fondamentale et harmonique. En outre, l’utilisation d’ultrasons focalisés a permis la vaporisation acoustique de gouttelettes de PFH, confirmée par l’observation de morphologies fragmentées ou perturbées dans de nombreux échantillons. Les effets des groupes terminaux fluorés ont été davantage explorés par une évaluation morphologique des microcapsules (MCs) produites avec les polymères. Finalement, les NCs et MCs présentent un potentiel théranostique intéressant, puisqu’elles permettent d'effectuer un diagnostic assisté par ultrasons et de libérer potentiellement un principe actif lorsqu'elles sont soumises à des pressions acoustiques élevées. / Fluorinated materials are intensively used as ultrasound contrast agents (UCA) to facilitate the diagnosis of many diseases by real-time imaging. All the commercially available UCAs are microbubbles constituted by a perfluorinated gaseous-core stabilized by a monolayer of phospholipids, proteins or surfactants. Unfortunately, the theranostic application (i.e. therapeutic and diagnostic ability) of such materials are severely limited by the (i) poor stability of the fluorinated component, (ii) inherent micrometer size range and (iii) lack of effective compartments for drug accumulation. To overcome these limitations, we proposed two different strategies to improve the persistence of the fluorinated core and simultaneously provide functional interfaces for drug encapsulation.The first approach involves intercalating chitosan with phospholipids (DSPC) to increase the stability of microvesicles containing the fluorinated gas decafluorobutane (C4F10). The affinity of DSPC and chitosan was disclosed by surface sensitive techniques and fluorescence microscopy. 19F nuclear magnetic resonance (19F-NMR) and in vitro ultrasound of chitosan-coated microvesicles exhibited intense signals of the gaseous-component after 48 h, twice as long compared to plain samples. Altogether, chitosan increased the stability of microvesicles and is a suitable platform for drug accumulation. As a result, the chitosan-phospholipid shell may enhance the theranostic potential of related microvesicles. However, the use of a fluorinated gas-core imposed an important restriction to stabilize sub-micrometric vesicles. Therefore, the second strategy was focused in developing a theranostic agent at the nanoscale by entrapping a liquid fluorinated core of perfluorohexane (PFH; C6F14) into a rigid polymeric shell of polylactide (PLA). To enhance the interaction of biodegradable polymers with perfluorocarbons, we synthesized PLA polymers containing five distinct lengths of fluorinated end-groups (from C3F7 until C13F27) by ring-opening polymerization of D,L-lactide. A greater extent of fluorous interactions was indicated by 19F spin-spin relaxation time and, subsequently, all the block copolymers were formulated into spherical nanocapsules (NC) with average diameter of 150 nm as verified by transmission electron microscopy. 19F-NMR showed that NC produced with fluorinated polymers increased two-fold the encapsulation efficiency of PFH compared with non-fluorinated derivatives. As a result, the NC echogenicity increased 10-fold for both fundamental and harmonic ultrasound imaging modalities. In addition, acoustic drop vaporization of PFH was successfully attained by focused ultrasound as observed by fragmented or disrupted morphologies in many samples. Effects of the fluorinated end-groups were further explored by a morphological evaluation of microcapsules (MC) produced with the polymers. Finally, both NC and MC present an interesting theranostic potential, being able to perform ultrasound-assisted diagnosis and potentially release drug contents when irradiated by high acoustic pressures.

Indiana University-Purdue University Indianapolis (IUPUI) / Glioblastoma (GBM) is a deadly, malignant brain tumor with a poor long-term prognosis. The current median survival is approximately fifteen to seventeen months with the standard of care therapy which includes surgery, radiation, and chemotherapy. An important factor contributing to recurrence of GBM is high resistance of GBM cancer stem cells (CSCs), for which a systemically delivered single drug approach will be unlikely to produce a viable cure. Therefore, multi-drug therapies are needed. Currently, only temozolomide (TMZ), which is a DNA alkylator, affects overall survival in GBM patients. CSCs regenerate rapidly and over-express a methyl transferase which overrides the DNA-alkylating mechanism of TMZ, leading to drug resistance. Idasanutlin (RG7388, R05503781) is a potent, selective MDM2 antagonist that additively kills GBM CSCs when combined with TMZ. By harnessing the strengths of nanotechnology, therapy can be combined with diagnostics in a truly theranostic manner for enhancing personalized medicine against GBM. The goal of this thesis was to develop a multi-drug therapy using multi-functional nanoparticles (NPs) that preferentially target the GBM CSC subpopulation and provide in vivo preclinical imaging capability. Polymer-micellar NPs composed of poly(styrene-b-ethylene oxide) (PS-b-PEO) and poly(lactic-co-glycolic) acid (PLGA) were developed investigating both single and double emulsion fabrication techniques as well as combinations of TMZ and RG7388. The NPs were covalently bound to a 15 base-pair CD133 aptamer in order to target a specific epitope on the CD133 antigen expressed on the surface of GBM CSC subpopulation. For theranostic functionality, the NPs were also labelled with a positron emission tomography (PET) radiotracer, zirconium-89 (89Zr). The NPs maintained a small size of less than 100 nm, a relatively neutral charge and exhibited the ability to produce a cytotoxic effect on CSCs. There was a slight increase in killing with the aptamer-bound NPs compared to those without a targeting agent. This work has provided a potentially therapeutic option for GBM specific for CSC targeting and future in vivo biodistribution studies.

Immune checkpoint blockade antibodies have promising clinical applications but suffer from disadvantages such as severe toxicities and moderate patient-response rates. None of the current delivery strategies, including local administration aiming to avoid systemic toxicities, can sustainably supply drugs over the course of weeks; adjustment of drug dose, either to lower systemic toxicities or to augment therapeutic response, is not possible. Herein, an implantable miniaturized device has been developed using electrode-embedded optical fibers with both local delivery and measurement capabilities over the course of a few weeks. The combination of local immune checkpoint blockade antibodies delivery via this device with photodynamic therapy elicits a sustained anti-tumor immunity in multiple tumor models. Named Implantable Miniature Optical Fiber Device (IMOD), this device uses tumor impedance measurement for timely presentation of treatment outcomes, and allows modifications to the delivered drugs and their concentrations, rendering IMOD as outstandingly valuable for on-demand delivery of potent immunotherapeutics without exacerbating toxicities. Rigorous studies performed using IMOD are presented and discussed in the follow chapters, followed by exploration of proposed work to expand the breadth of functions offered by this implantable biomedical platform. / Doctor of Philosophy / Aside from efficient energy and data transfer, optical fibers today are used in varying fields including optogenetics and neuroscience. However, merging fiber optics with therapeutics against cancer has rarely been reported. We establish a versatile polymer/drug integrated optical fiber for both diagnosis and treatment of cancers, with minimum mechanical invasiveness. Release profiles of polymer/drug nanoparticles loaded onto our fibers, regardless of their hydrophilicity, can be adjusted to accommodate both short-term and long-term delivery specifications. This enhances intratumoral drug accumulation with minimal systemic toxicity, thus overcoming the dosing obstacle. The optical fibers are also ideal to be utilized during photodynamic therapy (PDT), since photosensitizers can be easily incorporated and activated by near-infrared light traveling through the fibers. Hollow channel within the optical fiber allows for repetitive on-demand delivery of immune checkpoint inhibitors to surrounding tumor tissue, thus stimulating and reactivating cytotoxic and helper T cells. The synergistic combination of PDT and immunotherapy can potentially boost the tumor-targeted treatment outcome by numerous folds. Lastly, our optical fibers are adaptable to integrate biosensing functionality. Devices are built upon the optical fibers to monitor treatment outcome along tumor regression. Our data establishes a correlation between tumor impedance and tumor volumes, thus allowing us to track tumor progression and treatment response towards administered treatments.