Tomohiro Asai

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.

T cell-independent B cell response is responsible for ABC phenomenon induced by repeated injection of PEGylated liposomes

Repeated injection of polyethyleneglycol-modified (PEGylated) liposomes causes a rapid clearance of them from the bloodstream, this phenomenon is called accelerated blood clearance (ABC). In the present study, we focused on the immune system responsible for the ABC phenomenon. PEGylated liposomes were preadministered to BALB/c mice and [(3)H]-labeled ones were then administered to them 3 days after the preadministration. Consistent with our previous results, the preadministration with PEGylated liposomes triggered the rapid clearance of [(3)H]-labeled PEGylated liposomes from the bloodstream, but that with PEGylated liposomes encapsulating doxorubicin (Dox) did not. In addition, we found that the ABC phenomenon was observed when a mixture of free Dox and PEGylated liposomes was preadministered. These data indicate that immune cells responsible for the ABC phenomenon might be selectively damaged by the Dox encapsulated in PEGylated liposomes. The ABC phenomenon was also observed in BALB/c nu/nu mice, but not in BALB/c SCID mice. The amount of anti-PEG IgM antibody induced by the stimulation with the PEGylated liposomes was significantly increased in the BALB/c nu/nu mice, but not in the BALB/c SCID ones. These data indicate that a T cell-independent B cell response would play a significant role in the ABC phenomenon. Furthermore, the present study suggests that PEGylated liposomes might be recognized by B cells as a thymus-independent type 2 (TI-2) antigen. The present study provides important information for the future development of liposomal medicines.

Prolonged targeting of ischemic/reperfused myocardium by liposomal adenosine augments cardioprotection in rats.

OBJECTIVESnThe purpose of this study was to investigate whether liposomal adenosine has stronger cardioprotective effects and fewer side effects than free adenosine.nnnBACKGROUNDnLiposomes are nanoparticles that can deliver various agents to target tissues and delay degradation of these agents. Liposomes coated with polyethylene glycol (PEG) prolong the residence time of drugs in the blood. Although adenosine reduces the myocardial infarct (MI) size in clinical trials, it also causes hypotension and bradycardia.nnnMETHODSnWe prepared PEGylated liposomal adenosine (mean diameter 134 +/- 21 nm) by the hydration method. In rats, we evaluated the myocardial accumulation of liposomes and MI size at 3 h after 30 min of ischemia followed by reperfusion.nnnRESULTSnThe electron microscopy and ex vivo bioluminescence imaging showed the specific accumulation of liposomes in ischemic/reperfused myocardium. Investigation of radioisotope-labeled adenosine encapsulated in PEGylated liposomes revealed a prolonged blood residence time. An intravenous infusion of PEGylated liposomal adenosine (450 microg/kg/min) had a weaker effect on blood pressure and heart rate than the corresponding dose of free adenosine. An intravenous infusion of PEGylated liposomal adenosine (450 microg/kg/min) for 10 min from 5 min before the onset of reperfusion significantly reduced MI size (29.5 +/- 6.5%) compared with an infusion of saline (53.2 +/- 3.5%, p < 0.05). The antagonist of adenosine A(1), A(2a), A(2b), or A(3) subtype receptor blocked cardioprotection observed in the PEGylated liposomal adenosine-treated group.nnnCONCLUSIONSnAn infusion as PEGylated liposomes augmented the cardioprotective effects of adenosine against ischemia/reperfusion injury and reduced its unfavorable hemodynamic effects. Liposomes are promising for developing new treatments for acute MI.

Cancer antineovascular therapy with liposome drug delivery systems targeted to BiP/GRP78

Angiogenesis is crucial for tumor growth and hematogenous metastasis. Specifically expressed and functional protein molecules in angiogenic endothelial cells, especially on the plasma membrane, may be molecular targets for antiangiogenic drugs and drug delivery systems (DDS) in cancer therapy. To discover such target molecules, we performed subcellular proteome analysis of human umbilical vein endothelial cells (HUVECs) treated with or without vascular endothelial growth factor (VEGF) using 2‐dimensional difference in‐gel electrophoresis (2D‐DIGE) and matrix‐assisted laser desorption/ionization tandem time‐of‐flight mass spectrometry (MALDI‐TOF/TOF‐MS). Among the identified proteins, BiP/GRP78, a molecular chaperone, was highly expressed in the membrane/organelle fraction of HUVECs after VEGF treatment. The involvement of BiP in VEGF‐induced angiogenesis was examined by RNA interference. BiP knockdown significantly suppressed VEGF‐induced endothelial cell proliferation and VEGF‐induced phosphorylation of extracellular‐regulated kinase 1/2, phospholipase C‐γ, and VEGF receptor‐2 in HUVECs. Cell surface biotinylation analysis revealed that the cell surface expression of BiP was elevated in VEGF‐activated HUVECs. Aiming to apply BiP to a target molecule in liposomal DDS, we developed liposomes modified with the WIFPWIQL peptide, which has been shown to bind to BiP, and investigated its potential for cancer therapy. The WIFPWIQL‐modified liposomes (WIFPWIQL liposomes) were significantly taken up by VEGF‐activated HUVECs as compared to peptide‐unmodified liposomes. WIFPWIQL liposomes appeared to accumulate in tumor endothelial cells in vivo. WIFPWIQL liposomes containing doxorubicin significantly suppressed tumor growth and prolonged the survival of colon26 NL‐17 carcinoma cell‐bearing mice. In summary, BiP may regulate VEGF‐induced endothelial cell proliferation through VEGFR‐2‐mediated signaling and be an effective target molecule for cancer antineovascular therapy.

A novel DDS strategy, “dual-targeting”, and its application for antineovascular therapy

Dual-targeting liposomes modified with Ala-Pro-Arg-Pro-Gly (APRPG) and Gly-Asn-Gly-Arg-Gly (GNGRG) peptides were developed. They remarkably associated to growing human umbilical vein endothelial cells (HUVECs) compared with single-targeting liposomes modified with APRPG or GNGRG. Doxorubicin (DOX) encapsulated in the dual-targeting liposomes significantly suppressed the growth of HUVECs compared with that in single-targeting liposomes. The dual-targeting liposomes containing DOX strongly suppressed tumor growth in Colon26 NL-17 carcinoma-bearing mice. Confocal microscopic data indicated that this anticancer effect was brought by the association of these liposomes to angiogenic vessels in the tumor. These findings suggest that dual-targeting would be a hopeful method for targeting therapies.

Development of double-stranded siRNA labeling method using positron emitter and its in vivo trafficking analyzed by positron emission tomography.

Pharmacokinetic study of small interfering RNA (siRNA) is an important issue for the development of siRNAs for use as a medicine. For this purpose, a novel and favorable positron emitter-labeled siRNA was prepared by amino group-modification using N-succinimidyl 4-[fluorine-18] fluorobenzoate ([(18)F]SFB), and real-time analysis of siRNA trafficking was performed by using positron emission tomography (PET). Naked [(18)F]-labeled siRNA or cationic liposome/[(18)F]-labeled siRNA complexes were administered to mice, and differential biodistribution of the label was imaged by PET. The former was cleared quite rapidly from the bloodstream and excreted from the kidneys; but in contrast, the latter tended to accumulate in the lungs. We also confirmed the biodistribution of fluorescence-labeled naked siRNA and cationic liposome/siRNA complexes by use of a near-infrared fluorescence imaging system. As a result, a similar biodistribution was observed, although quantitative data were obtained only by planar positron imaging system (PPIS) analysis but not by fluorescence in vivo imaging. Our results indicate that PET imaging of siRNA provides important information for the development of siRNA medicines.

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.

Antiangiogenic cancer therapy using tumor vasculature-targeted liposomes encapsulating 3-(3,5-dimethyl-1H-pyrrol-2-ylmethylene)-1,3-dihydro-indol-2-one, SU5416

Previously, we identified angiogenic vessel-homing peptide Ala-Pro-Arg-Pro-Gly (APRPG), and showed that APRPG-modified liposomes could selectively target to tumor neovasculature. Here, we designed an APRPG-modified liposome encapsulating SU5416, an angiogenesis inhibitor, to overcome the solubility problem, and to enhance the antiangiogenic activity of SU5416. Liposomal SU5416 appeared to have the appropriate characteristics, such as particle size and stability in serum. It showed a significantly lower hemoglobin release than SU5416 dissolved in a Cremophor EL-containing solvent. Compared with peptide-unmodified liposomal SU5416, the APRPG-modified liposomal SU5416 significantly suppressed tumor growth and with no remarkable side effects. Thus, targeted delivery of antiangiogenic drugs with tumor vasculature-targeted liposomes may be useful for antiangiogenic cancer therapy.

Disappearance of the angiogenic potential of endothelial cells caused by Argonaute2 knockdown

Argonaute2 (Ago2), a component protein of RNA-induced silencing complex, plays a central role in RNA interference. We focused on the involvement of Ago2 in angiogenesis. Human umbilical vein endothelial cells (HUVECs) stimulated with several growth factors such as vascular endothelial growth factor were used for angiogenesis assays. We applied polycation liposomes for transfection of small interfering RNA (siRNA) to determine the biological effects of siRNA for Ago2 (siAgo2) on HUVECs. The proliferation study indicated that siAgo2 significantly suppressed the growth of HUVECs compared with control siRNA. TUNEL staining showed a certain population of HUVECs treated with siAgo2 underwent apoptosis. Furthermore, the treatment with siAgo2 suppressed the tube formation of HUVECs and significantly reduced the length of the tubes. These present data demonstrate that siAgo2 inhibited indispensable events of angiogenesis in vitro. This is the first report suggesting that Ago2 is required for angiogenesis.

Antineovascular therapy with angiogenic vessel-targeted polyethyleneglycol-shielded liposomal DPP-CNDAC.

Causing damage to angiogenic vessels is a promising approach for cancer chemotherapy. The present study is a codification of a designed liposomal drug delivery system (DDS) for antineovascular therapy (ANET) with 2′‐C‐cyano‐2′‐deoxy‐1‐β‐D‐arabino‐pentofuranosylcytosine (CNDAC). The authors have previously reported that liposomalized 5′‐O‐dipalmitoylphosphatidyl CNDAC (DPP–CNDAC), a phospholipid derivative of the novel antitumor nucleoside CNDAC, is quite useful for ANET. DPP–CNDAC liposomes modified with APRPG, a peptide having affinity toward angiogenic vessels, efficiently suppressed tumor growth by damaging angiogenic endothelial cells. In the present study, the authors masked the hydrophilic moiety of DPP–CNDAC, namely, CNDAC, on the liposomal surface with APRPG–polyethyleneglycol (PEG) conjugate to improve the availability of DPP–CNDAC liposomes. The use of the APRPG–PEG conjugate attenuated the negative ζ‐potential of the DPP–CNDAC liposomes and reduced the agglutinability of them in the presence of serum. These effects improved the blood level of DPP–CNDAC liposomes in colon 26 NL‐17 tumor‐bearing BALB/c male mice, resulting in enhanced accumulation of them in the tumor. Laser scanning microscopic observations indicated that APRPG–PEG‐modified DPP–CNDAC liposomes (LipCNDAC/APRPG–PEG) colocalized with angiogenic vessels and strongly induced apoptosis of tumor cells, whereas PEG‐modified DPP–CNDAC liposomes (LipCNDAC/PEG) did not. In fact, LipCNDAC/APRPG–PEG suppressed the tumor growth more strongly compared to LipCNDAC/PEG and increased significantly the life span of the mice. The present study is a good example of an effective liposomal DDS for ANET that is characterized by: (i) phospholipid derivatization of a certain anticancer drug to suit the liposomal formulation; (ii) PEG‐shielding for masking undesirable properties of the drug on the liposomal surface; and (iii) active targeting to angiogenic endothelial cells using a specific probe. (Cancer Sci 2008; 99: 1029–1033)