<p>Solid organ transplantation is today an established form of treatment for end-stage organ disease. Monitoring of graft function and pharmacological therapy constitutes a maze of clinical observations and histological evaluations of biopsy specimens; with the biopsy results playing a decisive role. The aims of this doctoral research were to investigate the feasibility of detecting acute rejection of transplanted organs and monitoring the effect of anti-rejection treatment, with the use of ultrasmall superparamagnetic iron oxide particles (USPIO) and magnetic resonance (MR) imaging with a clinical MR scanner.</p><p>Allogeneic and syngeneic heterotopic heart transplantations were performed in rats. Three different-sized USPIO were given to one allogeneic and one syngeneic group. The change in MR signal intensity (SI) over time was measured. An increase in SI was interpreted as damage to micro vessels due to the pronounced inflammatory reaction caused by acute rejection, which led to leakage of USPIO into the tissue. A decrease in SI was interpreted as normal vascular structure, since USPIO normally remains in the intravascular space. The same method, using one of the previously tested USPIO, was used in a treatment study in which acute rejection in transplanted rats was induced and subsequently treated. An attempt was also made to detect presence of macrophages in an acutely rejecting graft, since this cell type plays an important role in the acute rejection process; this was done by testing the ability of macrophages to phagocytose the UPSIO compound.</p><p>In permeability studies with MR imaging all three USPIO tested discriminated between rejecting and non-rejecting grafts without any overlap of the groups. Factors that contributed to the ability to distinguish between grafts were the size and half-life of the particle. We were also able to monitor effects of anti-rejection treatment by studying the vascular permeability of USPIO and MR imaging. We did not succeed in detecting macrophages in the rejecting grafts with USPIO and MR imaging.</p><p>This thesis presents a novel approach to detection of acute rejection of transplanted organs and to monitoring the effects of anti-rejection treatment using different USPIO contrast agents and MR imaging in a clinical MR scanner.</p>

Ny kunnskap om billeddannelse av hjertet ved magnetisk resonans (MR) fremkommer i sivilingeniør Wibeke Nordhøys doktoravhandling ved Norges teknisk-naturvitenskapelige universitet (NTNU). Denne kunnskapen vil ha stor betydning for hvordan man kan finne fram til noninvasive undersøkelser, dvs. uten fysiske inngrep, av hjertemuskelens levedyktighet hos pasienter med tilstopninger i kransarterier (iskemisk hjertesykdom). Mangan som kontrastmiddel og markør for levedyktighet (viabilitet) Arbeidet har gitt ny kunnskap om mangan som intracellulær kontrastgiver og om vannutveksling i hjertemuskelen. Manganforbindelser har et stort potensial som kontrastmidler for MR av hjertet. I dyreforsøk har Nordhøy vist hvordan det magnetiske sporstoffet mangan tas opp i hjerteceller via fysiologiske ionekanaler, og hvordan det øker kontrasten i MR-bilder ved å «lyse opp hjertecellene innenfra». Hun har også gitt viktige bidrag til forståelsen av hvordan man med MR kan skille mellom vann inne i og utenfor hjertecellene. I sum betyr dette at man har kommet et skritt videre mot at MR av hjertet vil gi gradert informasjon om nettopp hjertecellenes tilstand. Avhandlingen representerer derfor et viktig norsk bidrag til den internasjonale forskning innen molekylær billeddannelse. Avhandlingen Avhandlingen har tittelen «Manganese and the heart: Intracellular MR relaxation and water exchange across the cardiac cell membrane / Mangan og hjertet: Intracellulær MR relaksasjon og vannutveksling over cellemembranen i hjertet.» Avhandlingen er blitt til innenfor et tverrfaglig samarbeid mellom biofysiker, fysiologer og kjemikere med professor Per Jynge, Institutt for sirkulasjon og bildediagnostikk, NTNU, som hovedveileder og professor Jostein Krane, Kjemisk institutt, NTNU, som medveileder. Arbeidet har vært initiert via Norges forskningsråds strategiske universitetsprogram for medisinsk teknologi (SUP-I) i Trondheim. Det er finansiert av Norges forskningsråd, Det medisinske fakultet (NTNU) og Amersham Health.

Solid organ transplantation is today an established form of treatment for end-stage organ disease. Monitoring of graft function and pharmacological therapy constitutes a maze of clinical observations and histological evaluations of biopsy specimens; with the biopsy results playing a decisive role. The aims of this doctoral research were to investigate the feasibility of detecting acute rejection of transplanted organs and monitoring the effect of anti-rejection treatment, with the use of ultrasmall superparamagnetic iron oxide particles (USPIO) and magnetic resonance (MR) imaging with a clinical MR scanner. Allogeneic and syngeneic heterotopic heart transplantations were performed in rats. Three different-sized USPIO were given to one allogeneic and one syngeneic group. The change in MR signal intensity (SI) over time was measured. An increase in SI was interpreted as damage to micro vessels due to the pronounced inflammatory reaction caused by acute rejection, which led to leakage of USPIO into the tissue. A decrease in SI was interpreted as normal vascular structure, since USPIO normally remains in the intravascular space. The same method, using one of the previously tested USPIO, was used in a treatment study in which acute rejection in transplanted rats was induced and subsequently treated. An attempt was also made to detect presence of macrophages in an acutely rejecting graft, since this cell type plays an important role in the acute rejection process; this was done by testing the ability of macrophages to phagocytose the UPSIO compound. In permeability studies with MR imaging all three USPIO tested discriminated between rejecting and non-rejecting grafts without any overlap of the groups. Factors that contributed to the ability to distinguish between grafts were the size and half-life of the particle. We were also able to monitor effects of anti-rejection treatment by studying the vascular permeability of USPIO and MR imaging. We did not succeed in detecting macrophages in the rejecting grafts with USPIO and MR imaging. This thesis presents a novel approach to detection of acute rejection of transplanted organs and to monitoring the effects of anti-rejection treatment using different USPIO contrast agents and MR imaging in a clinical MR scanner.

More than one-third of all Magnetic Resonance Imaging (MRI) scans employ image-enhancing contrast agents to increase the differential signal intensity between diseased and normal tissue. Because current clinical contrast agents exhibit low relaxivity (mM-1 s-1), low dose efficiency, and rapid secretion, we have designed a group of protein-based MRI contrast agents with multiple gadolinium binding sites. In this study, the developed purification method for Class ProCA-3 agents allows for a quick and cost-effective way to abstract up to 109 mg of pure, soluble protein from a 1L E. Coli cell pellet devoid of DNA or RNA “contamination” for extensive animal studies. Circular dichroism far-UV spectra ensure the metal stability of the agents, revealing maintenance of their native α-helical structure in the presence and absence of metal ions. Furthermore, substantial evidence supports the high dose efficiency of these agents, exhibiting up to five folds higher relaxivity than their analogous commercial competitors.

Breast cancer is the second leading cause of death of women in the United States. Improvements in screening technology have increased the breast cancer incidence rate, as smaller lesions are being detected. Due to the small size of lesions, patients can choose to receive breast conservation therapy (BCT) rather than a modified radical mastectomy. Even though the breast retains cosmesis after BCT, there is an increased risk of the patient having residual microscopic disease, known as positive margins. Patients with positive margins receive increased radiation and have an increased chance of second surgery. Pathology with hematoxylin and eosin (H&E) remains the gold standard for diagnosing margin status in patients. Intraoperative pathology has been shown to reduce the rate of positive margins in BCT. However, a minority of surgery centers have intraoperative pathology centers, limiting the number of patients that receive this standard of care. The expression profiles of surface receptors such as ErbB2 (HER2-positive) and epidermal growth factor receptor (EGFR) provide information about the aggressiveness of a particular tumor. Recent research has shown that there was elevated EGFR expression in patients with a local recurrence even though the biopsies were assessed to be disease free using standard H&E. If the physicians had known the molecular expression of these biopsies, a different treatment regimen or excision of more tissue might have prevented the recurrence. This thesis investigates targeted molecular contrast agents that enhance the visualization of molecular markers such as glucose transporters (GLUTs) and growth factor receptors in tissue specimens. First, application of 2-NBDG, a fluorescent deoxy-glucose, enhances signal in cancerous tissue with a 20-minute incubation. Then, antibody functionalized silica-gold nanoshells enhance the visualization of ErbB2 overexpression in specimens with a 5-minute incubation. To image these contrast agents in cancerous tissue, a portable, inexpensive device was developed as a tool to help physicians visualize expression of surface markers. The system visualizes absorbance from nanoshell aggregates and fluorescence in the visible and near-infrared light spectrum. This study represents the first step in the development of an intraoperative optical imaging device to enhance the visualization of molecular markers overexpressed in cancerous cells.

The main objective of this research was to develop a two-stage optical imaging platform to improve detection of cancer in Barrett’s esophagus (BE). BE caused by chronic reflux and patients with BE are at a higher risk for developing esophageal adenocarcinoma (EAC). However, neoplasia in BE is often unidentifiable under standard endoscopy, and studies have shown nearly half of early cancers can go unidentified by this method. Widefield imaging (resolves ~100 microns) allows efficient surveillance of large BE segments. Two widefield imaging techniques were identified to improve contrast between benign and abnormal lesions during an ex vivo 15 patient feasibility study. Cross-polarized imaging (CPI) reduced specular reflection and improved vascular contrast. Vital-dye fluorescence imaging (VFI) using topically-applied proflavine improved visualization of glandular pattern. Moreover, relevant pathologic features visible during VFI were seen in corresponding histology slides as well as high resolution images of the same sites. Based on these results, a cap-based Multispectral Digital Endoscope (MDE) was designed and built. The MDE can image in three different imaging modes: white light imaging, CPI, and VFI. Modifications to a Pentax EPK-i video processor and a Pentax endoscope were made to incorporate these imaging modes into one system. A 21 patient in vivo pilot study with 65 pathologically correlated sites demonstrated the feasibility of using this system in vivo; image criteria were developed to classify neoplasia with a sensitivity and specificity of 100% and 76% respectively. High resolution imaging (resolves ~2-5 micron) may verify the disease presence in suspicious areas identified using widefield techniques. 2-NBDG, a fluorescent metabolic marker, was used as to identify neoplastic biopsies. In a study with 21 patients yielding 38 pathologically correlated biopsies and 158 image sites, 2-NBDG imaging allowed classification of cancerous biopsies with a sensitivity of 96% and specificity of 90%. The unique contributions of these results is the development of a multimodal cap-based endoscopic system to identify suspicious areas in BE, and using a metabolic marker to verify the presence of disease. This application extends beyond esophageal cancer detection and may be explored for cancer detection in other organ sites characterized by columnar epithelium.

As applications of nanotechnologies for life and health sciences get booming, magnetic nanovectors undergo a considerable development. Such composite structures made from polymer spheres encapsulating magnetic nanoparticles or from a nanoparticular magnetic core surrounded by an organic coverage exhibit a combination of physical, chemical and magnetic properties very appropriate for diagnostic by imaging such as Magnetic Resonance Imaging (MRI), or for therapy: targeted pharmaceutical vectorization, therapeutic hyperthermia... When such vectors exhibit a nanometric size, intravenous injection and easy spread in the body of the patients are allowed, while effects related to the specific surface area are increased. The present doctoral work was concerned by two important families of magnetic nanovectors: - nanospheres of biocompatible polymer having loaded a radioactivable compound for tumoral therapy and having encapsulated magnetite nanoparticles for diagnostic by MRI: a system for thera-diagnostic is thus obtained.- contrast agents for MRI of vascular or cerebral inflammation, consisting of a nanometric iron oxide (maghemite or magnetite) core i.e. ultrasmall superparamagnetic iron oxide - USPIO - surrounded by an organic coverage for targeting the affected region. These USPIO were used to study inflammation in the atherotic plaque of the aorta in a murine model.Most of the time, such nanovectors are administered to the patients in liquid suspensions by intravenous injection. It is thus crucial to characterize both the collective behaviour and the individual structure of the vectors in liquid suspension. On the other hand their interactions with the targeted regions in the body have to be understood. For this purposes, a multiscale approach of the structure and properties of such nanovectors has been developed, with structural studies carried out through innovative developments based on electron microscopies down to subnanometric resolution and correlated with physical properties. To achieve characterization of nanovectors in liquid media we have developed the application of Wet-STEM, a new mode in transmission of environmental scanning electron microscopy (ESEM), to image the internal structure of the magnetic nanoparticles in liquid suspension and image calculations by Monte Carlo simulations have shown that a nanometric resolution could be theoretically achieved. By the same technique, stability or tendency to flocculation in suspensions can be evidenced with respect to the collective behavior of different nanovectors.In a second step we have investigated the interactions of the nanoparticles with targeted regions. The biodistribution and biotransformation of the USPIO contrast agents in the tissular and cellular environments were investigated at increasing spatial resolution using different techniques. The biodistribution of a MRI contrast agent grafted with a fluorophore, in ex vivo samples from atherotic aorta and spleen were revealed by biphoton microscopy with a resolution of a few hundred nanometers, down to macrophage scale. Then preparation of ex vivo samples for transmission electron microscopy (TEM) was adapted from standard protocols especially with respect to staining after inclusion in resin. This way, the first high resolution HR(TEM) images and electron diffraction patterns of crystallized USPIO contrast agents in the aorta or the spleen of an atheromatous mouse were obtained. Combining such structural studies with measurement (using a SQUID setup) of magnetic properties, a longitudinal follow-up of USPIO nanoparticles injected in mice for MRI of the atherotic plaque has been completed for USPIO particles embedded in the aorta and the spleen: the results were interpreted in terms of agglomeration of the particles with a decreasing size depending on time after injection and found consistent with a model of in vitro degradation in acidic environment proposed to mimick the lysosomal metabolism.

Fluorescence-enhanced optical imaging using near-infrared (NIR) light developed for in-vivo molecular targeting and reporting of cancer provides promising opportunities for diagnostic imaging. However, prior to the administration of unproven contrast agents, the benefits of fluorescence-enhanced optical imaging must be assessed in feasibility phantom studies. A novel intensified charge-coupled device (ICCD) imaging system has been developed to perform 3-D fluorescence tomographic imaging in the frequency-domain using near-infrared contrast agents. This study is unique since it (i) employs a large tissue-mimicking phantom (~1087 cc), which is shaped and sized to resemble a female breast and part of the extended chest wall region, and (ii) enables rapid data acquisition in the frequency-domain by using a gain-modulated ICCD camera. Diagnostic 3-D fluorescence-enhanced optical tomography is demonstrated using 0.5-1 cc single and multiple targets contrasted from their surrounding by ??M concentrations of Indocyanine green (ICG) in the breast-shaped phantom (10 cm diameter), under varying conditions of target-to-background absorption contrast ratios (1:0 and 100:1) and target depths (up to 3 cm deep). Boundary surface fluorescence measurements of referenced amplitude and phase shift were used along with the coupled diffusion equation of light propagation in order to perform 3-D image reconstructions using the approximate extended Kalman filter (AEKF) algorithm, and hence differentiate the target from the background based on fluorescent optical contrast. Detection of single and multiple targets is demonstrated under various conditions of target depths (up to 2 cm deep), absorption optical contrast ratio (1:0 and 100:1), target volumes (0.5-1 cc), and multiple targets (up to three 0.5 cc targets). The feasibility of 3-D image reconstructions from simultaneous multiple point excitation sources are presented. Preliminary lifetime imaging studies with 1:2 and 2:1 optical contrast in fluorescence lifetime of the contrast agents is also demonstrated. The specificity of the optical imager is further assessed from homogeneous phantom studies containing no fluorescently contrasted targets. While nuclear imaging currently provides clinical diagnostic opportunities using radioactive tracers, molecular targeting of tumors using non-ionizing NIR contrast agents tomographically imaged using the frequency-domain ICCD imaging system could possibly become a new method of diagnostic imaging.

Cancer has become one of the leading causes of death today. The early detection of cancer may lead to desired therapeutic management of cancer and to decrease the mortality rate through effective therapeutic strategies. Advances in materials science have enabled the use of nanoparticles for added contrast in various imaging techniques. More recently there has been much interest in the use of gold nanoparticles as optical contrast agents because of their strong absorption and scattering properties at visible and near-infrared wavelengths. Highly proliferative cancer cells overexpress molecular markers such as epidermal growth factor receptor (EGFR). When specifically targeted gold nanoparticles bind to EGFR they tend to cluster thus leading to an optical red-shift of the plasmon resonances and an increase in absorption in the red region. These changes in optical properties provide the foundation for photoacoustic imaging technique to differentiate cancer cells from surrounding benign cells. In photoacoustic imaging, contrast mechanism is based on the optical absorption properties of the tissue constituents. Studies were performed on tissue phantoms, ex-vivo and in-vivo tumor models to evaluate molecular specific photoacoustic imaging technique. The results indicate that highly sensitive and selective detection of cancer cells can be achieved by utilizing the plasmon resonance coupling effect of EGFR targeted gold nanoparticles and photoacoustic imaging. In conclusion, the combined ultrasound and photoacoustic imaging technique has the ability to image molecular signature of cancer using bioconjugated gold nanoparticles. / text

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