Daisuke Kokuryo

A pH-activatable nanoparticle with signal-amplification capabilities for non-invasive imaging of tumour malignancy

Engineered nanoparticles that respond to pathophysiological parameters, such as pH or redox potential, have been developed as contrast agents for the magnetic resonance imaging (MRI) of tumours. However, beyond anatomic assessment, contrast agents that can sense these pathological parameters and rapidly amplify their magnetic resonance signals are desirable because they could potentially be used to monitor the biological processes of tumours and improve cancer diagnosis. Here, we report an MRI contrast agent that rapidly amplifies magnetic resonance signals in response to pH. We confined Mn(2+) within pH-sensitive calcium phosphate (CaP) nanoparticles comprising a poly(ethylene glycol) shell. At a low pH, such as in solid tumours, the CaP disintegrates and releases Mn(2+) ions. Binding to proteins increases the relaxivity of Mn(2+) and enhances the contrast. We show that these nanoparticles could rapidly and selectively brighten solid tumours, identify hypoxic regions within the tumour mass and detect invisible millimetre-sized metastatic tumours in the liver.

Multi-functional liposomes having temperature-triggered release and magnetic resonance imaging for tumor-specific chemotherapy.

For development of tumor-specific chemotherapy, we designed liposomes with temperature-triggered drug release and magnetic resonance imaging (MRI) functions. We prepared multi-functional liposomes by incorporating thermosensitive poly(2-ethoxy(ethoxyethyl)vinyl ether) chains with a lower critical solution temperatures around 40 °C and polyamidoamine G3 dendron-based lipids having Gd(3+) chelate residues into pegylated liposomes. These stable doxorubicin (DOX)-loaded liposomes retained DOX in their interior below physiological temperature but released DOX immediately at temperatures greater than 40 °C. They exhibited excellent ability to shorten the longitudinal proton relaxation time. When administered intravenously into colon 26 tumor-bearing mice, accumulated liposomes in tumors increased with time, reaching a constant level 8 h after administration by following T(1)-weighted MRI signal intensity in tumors. Liposome size affected the liposome accumulation efficiency in tumors: liposomes of about 100 nm diameter were accumulated more efficiently than those with about 50 nm diameter. Tumor size also affected accumulation: more efficient accumulation occurred in larger tumors. Tumor growth was strongly suppressed when liposomes loaded with DOX were administered intravenously into tumor-bearing mice and the tumor was heated mildly at 44 °C for 10 min at 8 h after administration. Multi-functional liposomes having temperature-triggered drug release and MRI functions might engender personalized chemotherapy, providing efficient patient-optimized chemotherapy.

Development of a small prototype for a proof-of-concept of OpenPET imaging

The OpenPET geometry is our new idea to visualize a physically opened space between two detector rings. In this paper, we developed the first small prototype to show a proof-of-concept of OpenPET imaging. Two detector rings of 110 mm diameter and 42 mm axial length were placed with a gap of 42 mm. The basic imaging performance was confirmed through phantom studies; the open imaging was realized at the cost of slight loss of axial resolution and 24% loss of sensitivity. For a proof-of-concept of PET image-guided radiation therapy, we carried out the in-beam tests with (11)C radioactive beam irradiation in the heavy ion medical accelerator in Chiba to visualize in situ distribution of primary particles stopped in a phantom. We showed that PET images corresponding to dose distribution were obtained. For an initial proof-of-concept of real-time multimodal imaging, we measured a tumor-inoculated mouse with (18)F-FDG, and an optical image of the mouse body surface was taken during the PET measurement by inserting a digital camera in the ring gap. We confirmed that the tumor in the gap was clearly visualized. The result also showed the extension effect of an axial field-of-view (FOV); a large axial FOV of 126 mm was obtained with the detectors that originally covered only an 84 mm axial FOV. In conclusion, our initial imaging studies showed promising performance of the OpenPET.

Hydrothermally synthesized PEGylated calcium phosphate nanoparticles incorporating Gd-DTPA for contrast enhanced MRI diagnosis of solid tumors.

Organic-inorganic hybrid nanoparticles with calcium phosphate (CaP) core and PEGylated shell were developed to incorporate magnetic resonance imaging (MRI) contrast agent diethylenetriaminepentaacetic acid gadolinium (III) (Gd-DTPA) for noninvasive diagnosis of solid tumors. A two-step preparation method was applied to elaborate hybrid nanoparticles with a z-average hydrodynamic diameter about 80nm, neutral surface ξ-potential and high colloidal stability in physiological environments by self-assembly of poly(ethylene glycol)-b-poly(aspartic acid) block copolymer, Gd-DTPA, and CaP in aqueous solution, followed with hydrothermal treatment. Incorporation into the hybrid nanoparticles allowed Gd-DTPA to show significant enhanced retention ratio in blood circulation, leading to high accumulation in tumor positions due to enhanced permeability and retention (EPR) effect. Moreover, Gd-DTPA revealed above 6 times increase of relaxivity in the nanoparticle system compared to free form, and eventually, selective and elevated contrast enhancements in the tumor positions were observed. These results indicate the high potential of Gd-DTPA-loaded PEGylated CaP nanoparticles as a novel contrast agent for noninvasive cancer diagnosis.

Optimization of self‐reference thermometry using complex field estimation

Referenceless, or self‐reference, thermometry is a technique for mapping temperature differences in the region of interest (ROI) using the baseline phase estimated by extrapolating the field in the surrounding region for estimation (RFE) and subtracting the estimated baseline from the measured field. In the present work a self‐reference technique based on complex field estimation using 2D polynomials comprising complex‐valued coefficients was proposed and optimized. Numerical simulations with a Gaussian‐profiled phase distribution demonstrated that the ROI radius had to be 2.3–2.5 times the standard deviation (SD) of the Gaussian function in order to keep the error below 8% of the peak phase change. The area ratio between the ROI and the RFE had to be larger than 2.0 to maintain the error level. Based on the simulations, and phantom and volunteer experiments, the complex‐based method with independently optimized polynomial orders for the two spatial dimensions was compared with the phase‐based method using the similar‐order optimization strategy. The complex‐based method appeared to be useful when phase unwrapping was not removed. Otherwise, the phase‐based method yielded equivalent results with less polynomial orders. Magn Reson Med, 2006.

Chemical nature and structure of organic coating of quantum dots is crucial for their application in imaging diagnostics

Background: One of the most attractive properties of quantum dots is their potential to extend the opportunities for fluorescent and multimodal imaging in vivo. The aim of the present study was to clarify whether the composition and structure of organic coating of nanoparticles are crucial for their application in vivo. Methods: We compared quantum dots coated with non-crosslinked amino-functionalized polyamidoamine (PAMAM) dendrimers, quantum dots encapsulated in crosslinked carboxyl-functionalized PAMAM dendrimers, and silica-shelled amino-functionalized quantum dots. A multimodal fluorescent and paramagnetic quantum dot probe was also developed and analyzed. The probes were applied intravenously in anesthetized animals for visualization of brain vasculature using two-photon excited fluorescent microscopy and visualization of tumors using fluorescent IVIS® imaging (Caliper Life Sciences, Hopkinton, MA) and magnetic resonance imaging. Results: Quantum dots coated with non-crosslinked dendrimers were cytotoxic. They induced side effects in vivo, including vasodilatation with a decrease in mean arterial blood pressure and heart rate. The quantum dots penetrated the vessels, which caused the quality of fluorescent imaging to deteriorate. Quantum dots encapsulated in crosslinked dendrimers had low cytotoxicity and were biocompatible. In concentrations <0.3 nmol quantum dots/kg bodyweight, these nanoparticles did not affect blood pressure and heart rate, and did not induce vasodilatation or vasoconstriction. PEGylation (PEG [polyethylene glycol]) was an indispensable step in development of a quantum dot probe for in vivo imaging, based on silica-shelled quantum dots. The non-PEGylated silica-shelled quantum dots possessed low colloidal stability in high-salt physiological fluids, accompanied by rapid aggregation in vivo. The conjugation of silica-shelled quantum dots with PEG1100 increased their stability and half-life in the circulation without significant enhancement of their size. In concentrations <2.5 nmol/kg bodyweight, these quantum dots did not affect the main physiological variables. It was possible to visualize capillaries, which makes this quantum dot probe appropriate for investigation of mediators of vasoconstriction, vasodilatation, and brain circulation in intact animals in vivo. The multimodal silica-shelled quantum dots allowed visualization of tumor tissue in an early stage of its development, using magnetic resonance imaging. Conclusion: The present study shows that the type and structure of organic/bioorganic shells of quantum dots determine their biocompatibility and are crucial for their application in imaging in vivo, due to the effects of the shell on the following properties: colloidal stability, solubility in physiological fluids, influence of the basic physiological parameters, and cytotoxicity.

SPIO-PICsome: Development of a highly sensitive and stealth-capable MRI nano-agent for tumor detection using SPIO-loaded unilamellar polyion complex vesicles (PICsomes)

Size controllable polyion complex vesicles (PICsomes), composed of biocompatible poly(ethylene glycol) (PEG) and poly(amino acid)s, have an extremely prolonged lifetime in the bloodstream that enables them to accumulate effectively in tumors via the enhanced permeability and retention (EPR) effect. The purpose of this study was to use PICsomes to synthesize a highly sensitive MRI contrast agent for more precise tumor detection. We synthesized SPIO-Cy5-PICsomes (superparamagnetic iron oxide nanoparticle-loaded Cy5-cross-linked Nano-PICsomes) and characterized them using dynamic light scattering and transmission electron microscopy in vitro and evaluated their ability to detect subcutaneously grafted tumors in vivo with MRI. The transverse relaxivity (r2) of the SPIO-Cy5-PICsomes (r2=663±28mM(-1)s(-1)) was 2.54 times higher than that of bare clinically-used SPIO. In in vivo MRI experiments on mice subcutaneously grafted with colon-26 tumor cells, the tumor signal was significantly altered at 3h after SPIO-Cy5-PICsome administration and persisted for at least 24h. Small and early-stage in vivo tumors (3days after grafting, approximately 4mm(3)) were also clearly detected with MRI. SPIO-loaded PICsomes are sensitive MRI contrast agents that can act as a powerful nanocarrier to detect small tumors for early diagnosis.

Hybrid Calcium Phosphate-Polymeric Micelles Incorporating Gadolinium Chelates for Imaging-Guided Gadolinium Neutron Capture Tumor Therapy

Gadolinium (Gd) chelates-loaded nanocarriers have high potential for achieving magnetic resonance imaging (MRI)-guided Gd neutron capture therapy (GdNCT) of tumors. Herein, we developed calcium phosphate micelles hybridized with PEG-polyanion block copolymers, and incorporated with the clinical MRI contrast agent Gd-diethylenetriaminepentaacetic acid (Gd-DTPA/CaP). The Gd-DTPA/CaP were nontoxic to cancer cells at the concentration of 100 μM based on Gd-DTPA, while over 50% of the cancer cells were killed by thermal neutron irradiation at this concentration. Moreover, the Gd-DTPA/CaP showed a dramatically increased accumulation of Gd-DTPA in tumors, leading to the selective contrast enhancement of tumor tissues for precise tumor location by MRI. The enhanced tumor-to-blood distribution ratio of Gd-DTPA/CaP resulted in the effective suppression of tumor growth without loss of body weight, indicating the potential of Gd-DTPA/CaP for safe cancer treatment.

Changes in cortical microvasculature during misery perfusion measured by two-photon laser scanning microscopy

This study aimed to examine the cortical microvessel diameter response to hypercapnia in misery perfusion using two-photon laser scanning microscopy (TPLSM). We evaluated whether the vascular response to hypercapnia could represent the cerebrovascular reserve. Cerebral blood flow (CBF) during normocapnia and hypercapnia was measured by laser-Doppler flowmetry through cranial windows in awake C57/BL6 mice before and at 1,7, 14, and 28 days after unilateral common carotid artery occlusion (UCCAO). Diameters of the cortical microvessels during normocapnia and hypercapnia were also measured by TPLSM. Cerebral blood flow and the vascular response to hypercapnia were decreased after UCCAO. Before UCCAO, vasodilation during hypercapnia was found primarily in arterioles (22.9% ± 3.5%). At 14 days after UCCAO, arterioles, capillaries, and venules were autoregulatorily dilated by 79.5% ± 19.7%, 57.2% ±32.3%, and 32.0% ± 10.8%, respectively. At the same time, the diameter response to hypercapnia in arterioles was significantly decreased to 1.9% ± 1.5%. A significant negative correlation was observed between autoregulatory vasodilation and the diameter response to hypercapnia in arterioles. Our findings indicate that arterioles play main roles in both autoregulatory vasodilation and hypercapnic vasodilation, and that the vascular response to hypercapnia can be used to estimate the cerebrovascular reserve.

Method for Target Tracking in Focused Ultrasound Surgery of Liver using Magnetic Resonance Filtered Venography

The purpose of this work is to develop a magnetic resonance (MR) technique for guiding a focal point created in focused ultrasound surgery (FUS) onto a specific target position in an abdominal organ, such as the liver, which moves and deforms with respiratory motion. The translational distance, rotational angles, and amount of expansion and contraction of the organ tissue were measured by obtaining the gravity points of the veins filtered from the sagittal, cine MR images of healthy livers during free breathing. Using the locations of the vessels at each time point, the target position at which the ultrasound focus was to be placed was estimated. In the volunteer experiments (N = 2), the lower limit of the spatial matrix dimension for delineating the veins was 128 x 128. The average displacement of the liver was 19.6 + 3.6 mm in superior-inferior (SI) direction and 3.1 + 1.4 mm in anterior-posterior (AP) direction. The deformations were 3.7 + 1.1 mm in SI direction and 3.0 + 1.2 mm in AP direction. The error between the actual and the estimated target point was 0.7 plusmn 0.5 mm in SI direction, 0.6 plusmn 0.4 mm in AP direction and 1.0 plusmn 0.5 mm in distance, and less than 2.1 mm in all the trials. These results suggested that the proposed technique is sufficient for targeting the focus on a specific tissue location and for tracking the slice slab for thermometry to cover the region of focus.