Takeo Urakami

Particle size-dependent triggering of accelerated blood clearance phenomenon.

A repeat-injection of polyethylene glycol-modified liposomes (PEGylated liposomes) causes a rapid clearance of them from the blood circulation in certain cases that is referred to as the accelerated blood clearance (ABC) phenomenon. In the present study, we examined whether polymeric micelles trigger ABC phenomenon or not. As a preconditioning treatment, polymeric micelles (9.7, 31.5, or 50.2 nm in diameter) or PEGylated liposomes (119, 261 or 795 nm) were preadministered into BALB/c mice. Three days after the preadministration [(3)H]-labeled PEGylated liposomes (127 nm) as a test dose were administered into the mice to determine the biodistribution of PEGylated liposomes. At 24h after the test dose was given, accelerated clearance of PEGylated liposomes from the bloodstream and significant accumulation in the liver was observed in the mice preadministered with 50.2-795 nm nanoassemblies (PEGylated liposomes or polymeric micelles). In contrast, such phenomenon was not observed with 9.7-31.5 nm polymeric micelles. The enhanced blood clearance and hepatic uptake of the test dose (ABC phenomenon) were related to the size of triggering nanoassemblies. Our study provides important information for developing both drug and gene delivery systems by means of nanocarriers.

Design of Synthetic Polymer Nanoparticles that Capture and Neutralize a Toxic Peptide

Designed polymer nanoparticles (NPs) capable of binding and neutralizing a biomacromolecular toxin are prepared. A library of copolymer NPs is synthesized from combinations of functional monomers. The binding capacity and affinity of the NPs are individually analyzed. NPs with optimized composition are capable of neutralizing the toxin even in a complex biological milieu. It is anticipated that this strategy will be a starting point for the design of synthetic alternatives to antibodies.

PET imaging of brain cancer with positron emitter-labeled liposomes

Since nanocarriers such as liposomes are known to accumulate in tumors of tumor-bearing animals, and those that have entrapped a positron emitter can be used to image a tumor by PET, we applied (18)F-labeled 100-nm-sized liposomes for the imaging of brain tumors. Polyethylene glycol (PEG)-modified liposomes, which are known to accumulate in tumors by passive targeting and those modified with Ala-Pro-Arg-Pro-Gly, which are known to home into angiogenic sites were used. Those liposomes labeled with DiI fluorescence accumulated in a glioma implanted in a rat brain 1h after the injection, although they did not accumulate in the normal brain tissues due to the protection afforded by the blood-brain barrier. Preformed liposomes were easily labeled with 1-[(18)F]fluoro-3,6-dioxatetracosane, and enabled the imaging of gliomas by PET with higher contrast than that obtained with [(18)F]deoxyfluoroglucose. In addition, the smallest tumor among those tested, having a diameter of 1mm was successfully imaged by the liposomal (18)F. Therefore, nanocarrier-based imaging of brain tumors is promising for the diagnosis of brain cancer and possible drug delivery-based therapy.

In Vivo Distribution of Liposome‐Encapsulated Hemoglobin Determined by Positron Emission Tomography

Positron emission tomography (PET) is a noninvasive imaging technology that enables the determination of biodistribution of positron emitter-labeled compounds. Lipidic nanoparticles are useful for drug delivery system (DDS), including the artificial oxygen carriers. However, there has been no appropriate method to label preformulated DDS drugs by positron emitters. We have developed a rapid and efficient labeling method for lipid nanoparticles and applied it to determine the movement of liposome-encapsulated hemoglobin (LEH). Distribution of LEH in the rat brain under ischemia was examined by a small animal PET with an enhanced resolution. While the blood flow was almost absent in the ischemic region observed by [(15)O]H(2)O imaging, distribution of (18)F-labeled LEH in the region was gradually increased during 60-min dynamic PET scanning. The results suggest that LEH deliver oxygen even into the ischemic brain from the periphery toward the core of ischemia. The real-time observation of flow pattern, deposition, and excretion of LEH in the ischemic rodent brain was possible by the new methods of positron emitter labeling and PET system with a high resolution.

Evaluation of O-[18F]fluoromethyl-D-tyrosine as a radiotracer for tumor imaging with positron emission tomography

O-[(18)F]Fluoromethyl-D-tyrosine (D-[(18)F]FMT) has been reported as a potential tumor-detecting agent for positron emission tomography (PET). However, the reason why D-[(18)F]FMT is better than L-[(18)F]FMT is unclear. To clarify this point, we examined the mechanism of their transport and their suitability for tumor detection. The stereo-selective uptake and release of enantiomerically pure D- and L-[(18)F]FMT by rat C6 glioma cells and human cervix adenocarcinoma HeLa cells were examined. The results of a competitive inhibition study using various amino acids and a selective inhibitor for transport system L suggested that D-[(18)F]FMT, as well as L-[(18)F]FMT, was transported via system L, the large neutral amino acid transporter, possibly via LAT1. The in vivo distribution of both [(18)F]FMT and [(18)F]FDG in tumor-bearing mice and rats was imaged with a high-resolution small-animal PET system. In vivo PET imaging of D-[(18)F]FMT in mouse xenograft and rat allograft tumor models showed high contrast with a low background, especially in the abdominal and brain region. The results of our in vitro and in vivo studies indicate that L-[(18)F]FMT and D-[(18)F]FMT are specifically taken up by tumor cells via system L. D-[(18)F]FMT, however, provides a better tumor-to-background contrast with a tumor/background (contralateral region) ratio of 2.741 vs. 1.878 with the L-isomer, whose difference appears to be caused by a difference in the influence of extracellular amino acids on the uptake and excretion of these two isomers in various organs. Therefore, D-[(18)F]FMT would be a more powerful tool as a tumor-detecting agent for PET, especially for the imaging of a brain cancer and an abdominal cancer.

Accumulation of macromolecules in brain parenchyma in acute phase of cerebral infarction/reperfusion

Ischemia-reperfusion injury is induced by recovery of blood flow after ischemia. This phenomenon is a main cause of ischemic brain injury. The integrity of the blood-brain barrier (BBB) fails after cerebral ischemia and reperfusion. Further elucidation of this phenomenon promotes to develop treatment strategies for ischemia-reperfusion injury. In the present study, we attempted to examine the time-dependent change of ischemia-reperfusion injury in relation to BBB disorders at acute phase in a transient middle cerebral artery occlusion (t-MCAO) model rat as a cerebral infarction and reperfusion model. Brain cell damage after the reperfusion was assessed by 2, 3, 5-triphenyltetrazolium chloride (TTC) staining. To clarify a time-dependent change of the integrity of BBB, fluorescein isothiocyanate (FITC)-dextran (150 kDa) was injected intravenously into t-MCAO rats, and time-dependent localization of FITC-dextran was monitored in ex vivo. As a result, obvious brain damage was firstly observed at 3 h after reperfusion following 1 h of MCAO. In contrast, the leakage of FITC-dextran from cerebral vessels was observed immediately after the reperfusion. The present data suggest that the integrity of BBB failed prior to the occurrence of serious brain damage induced by ischemia-reperfusion, and that macromolecules such as water-soluble polymers and proteins which cannot pass through the BBB under normal condition would reach brain parenchyma at early stage after reperfusion. These findings would be useful to establish a novel treatment strategy for reperfusion injury after cerebral infarction.

Suppression of immune response by antigen-modified liposomes encapsulating model agents: A novel strategy for the treatment of allergy

A specific antigen-sensitized animal has antigen-specific immune cells that recognize the antigen. Therefore, an antigen-modified drug carrier would be recognized by the immune cells. When such a carrier encapsulates certain drugs, these drugs should be specifically delivered to the immune cells. To examine this strategy, ovalbumin (OVA) was used as model antigen, and mice were presensitized with 100 μg of OVA with Alum. For preparing OVA-modified liposomes (OVA-lipo), OVA was incubated with DSPE-PEG-NHS and resulting DSPE-PEG-OVA was inserted into liposomes. OVA-specific IgG was produced 6-fold higher by intravenous injection of OVA-lipo thrice (10 μg as OVA in each injection) in OVA-sensitized mice, than that by the injection of control liposomes, suggesting that OVA-lipo was recognized by the antigen-specific immune cells. Moreover, intra-splenic accumulation of OVA-lipo was observed in OVA-sensitized mice, but not in naive mice. To achieve the delivery of a drug to specific immune cells, OVA-lipo encapsulated low dose of doxorubicin (DOX) as a model drug (20 μg DOX/mouse, Ca. 1 mg/kg) was injected in the sensitized mice. The injection of OVA-lipo encapsulating DOX suppressed the production of IgE against OVA, suggesting that the specific delivery of the drug to immune cells responsible for OVA recognition was achieved and that these immune cells were removed by the drug treatment. This strategy would be useful for the fundamental treatment of allergy by the use of immunosuppressing agents.

Specific delivery of an immunosuppressive drug to splenic B cells by antigen-modified liposomes and its antiallergic effect.

Abstract Background: Use of the reverse targeting drug delivery system (RT-DDS) is a new targeting strategy based on the specific delivery of drugs to immune cells in antigen-sensitized animals by using antigen-modified liposomes, and it is expected to be a curative treatment for allergic diseases. Purpose: Herein, we prepared ovalbumin (OVA)-modified liposomes encapsulating the immunosuppressive drug FK506 (OVA-LipFK) and aimed to demonstrate the delivery selectivity of the liposomes to splenic B cells, and its antiallergic effect in an OVA-sensitized allergic model mouse. Methods: Fluorescently labeled OVA-LipFK was intravenously injected into OVA-sensitized mice, and the intrasplenic localization of liposomes was observed. The antiallergic effect of OVA-LipFK in OVA-sensitized mice was examined by measuring the blood levels of OVA-specific IgE and IgG antibodies. Results and discussion: OVA-LipFK was co-localized to not only B cells but also germinal centers, in the spleen of OVA-sensitized mice. However, there was no accumulation of unmodified liposomes encapsulating FK506 (LipFK) in the splenic B-cell area. In a therapeutic study, OVA-LipFK significantly suppressed the production of both OVA-specific IgE and IgG antibodies in OVA-sensitized mice after the animals had been boosted with OVA, whereas LipFK showed little antiallergic effect. Conclusions: The present study suggested that the introduction of RT-DDS for use with immunosuppressive drugs could be useful for the treatment of allergic diseases.