Surface Modification of Liposomes Using IR700 Enables Efficient Controlled Contents Release Triggered by Near-IR Light.

Stimuli-responsive liposomes are promising drug carriers for cancer treatment because they enable controlled drug release and the maintenance of desired drug concentrations in tumor tissue. In particular, near-IR (NIR) light is a useful stimulus for triggering drug release from liposomes based on its advantages such as deep tissue penetration and safety. Previously, we found that a silicon phthalocyanine derivative, IR700, conjugated to antibodies, can induce the rupture of the cell membrane following irradiation by NIR light. Based on this finding, we constructed IR700-modified liposomes (IR700 liposomes) and evaluated their drug release properties triggered by NIR light. IR700 liposomes released substantial amounts of encapsulated calcein following irradiation by NIR light. Drug release was substantially suppressed by the addition of sodium azide, suggesting that liposomal membrane permeabilization was mediated by singlet oxygen generated from IR700. Moreover, calcein release from IR700 liposomes triggered by NIR light was promoted under conditions of deoxygenation and the presence of electron donors. Thus, membrane disruption should be induced by the physical change of IR700 from highly hydrophilic to hydrophobic as we previously described, although singlet oxygen can cause a certain level of membrane disruption under normoxia. We also observed that doxorubicin-encapsulated IR700 liposomes exhibited significant cytotoxic effects against CT-26 murine colon carcinoma cells following NIR light exposure. These results indicate that IR700 liposomes can efficiently release anti-cancer drugs following NIR light irradiation even under hypoxic conditions and, therefore, they would be useful for cancer treatment.

Attenuated polyethylene glycol immunogenicity and overcoming accelerated blood clearance of a fully PEGylated dendrimer.

Polyethylene glycol (PEG) is a popular biocompatible polymer and PEGylated nanoparticles passively accumulate in tumor tissues because of their enhanced permeability and retention effects. Recently, the anti-PEG immunity of PEGylated nanoparticles has become an issue that needs to be solved for their clinical applications. Dendrimers are highly branched and well-defined polymers with many terminal groups, which act as potent drug carriers. In this study, we examined the pharmacokinetics, biodistribution, anti-PEG immunity, and tumor accumulation of a fully PEGylated polyamidoamine (PAMAM) dendrimer after the first and second injections and compared them to those of a PEGylated liposome with the same lipid component as Doxil®. The PEGylated dendrimer showed greater blood circulation than that of the PEGylated liposome after the first and second injections in rats. In mice injected with the PEGylated dendrimer, much less anti-PEG immunoglobulin M (IgM) was generated than that in mice injected with the PEGylated liposome. The PEGylated dendrimer accumulated in the tumor after both the first and second injections. Our results indicated that the PEGylated dendrimer with a small size and high PEG density showed attenuated anti-PEG immunity and overcame the accelerated blood clearance phenomenon, which is useful for drug delivery systems for cancer treatment.

Dendrimer-based MRI contrast agents: the effects of PEGylation on relaxivity and pharmacokinetics.

Polyethylene glycol (PEG) surface modification can make nanomaterials highly hydrophilic, reducing their sequestration in the reticuloendothelial system. In this study, polyamidoamine (PAMAM) dendrimers bearing gadolinium (Gd) chelates were PEGylated with different PEG-chain lengths, and the effects on paramagnetic and pharmacokinetic properties were evaluated. Specifically, Gd chelate-bearing PAMAM dendrimers (generations 4 and 5; G4 and G5) were conjugated with two different PEG chains (2 kDa and 5 kDa; 2k and 5k). Long PEG chains (5k) on the smaller (G4) dendrimer resulted in reduced relaxivity compared to non-PEGylated dendrimers, whereas short PEG chains (2k) on a larger (G5) dendrimer produced relaxivities comparable to non-PEGylated G4 dendrimers. The relaxivity of all PEGylated or lysine-conjugated dendrimers increased at higher temperature, whereas that of intact G4 Gd-PAMAM dendrimer decreased. All PEGylated dendrimers had minimal liver and kidney uptake and remained in circulation for at least 1 hour. Thus, surface-PEGylated Gd-PAMAM dendrimers showed decreased plasma clearance and prolonged retention in the blood pool. Shorter PEG, higher generation conjugates led to higher relaxivity. FROM THE CLINICAL EDITOR: In this study, polyamidoamine dendrimers bearing gadolinium (Gd) chelates were PEGylated with different PEG-chain lengths, and the effects on paramagnetic and pharmacokinetic properties were evaluated.

X-ray computed tomography contrast agents prepared by seeded growth of gold nanoparticles in PEGylated dendrimer.

Gold nanoparticles (Au NPs) are a potential x-ray computed tomography (CT) contrast agent. A biocompatible and bioinactive surface is necessary for application of gold nanoparticle to CT imaging. Polyethylene glycol (PEG)-attached dendrimers have been used as a drug carrier with long blood circulation. In this study, the Au NPs were grown in the PEGylated dendrimer to produce a CT contrast agent. The Au NPs were grown by adding gold ions and ascorbic acid at various equivalents to the Au NP-encapsulated dendrimer solution. Both size and surface plasmon absorption of the grown Au NPs increased with adding a large number of gold ions. The x-ray attenuation of the Au NPs also increased after the seeded growth. The Au NPs grown in the PEG-attached dendrimer at the maximum under our conditions exhibited a similar CT value to a commercial iodine agent, iopamidol, in vitro. The Au NP-loaded PEGylated dendrimer and iopamidol were injected into mice and CT images were obtained at different times. The Au NP-loaded PEGylated dendrimer achieved a blood pool imaging, which was greater than a commercial iodine agent. Even though iopamidol was excreted rapidly, the PEGylated dendrimer loading the grown Au NP was accumulated in the liver.

Macrophage-targeted, enzyme-triggered fluorescence switch-on system for detection of embolism-vulnerable atherosclerotic plaques.

The development of atherosclerotic plaques is a critical step that can result in an arterial embolism. Therefore, detection of these vulnerable plaques is of clinical significance for the diagnosis of atherosclerosis. However, there are few imaging systems able to detect such plaques easily. In this study, we designed a new platform for near-infrared fluorescence (NIRF) imaging of macrophages in atherosclerotic plaques, one using both a liposomal DDS and an activatable fluorescent probe, and evaluated the utility of this imaging for the diagnosis of atherosclerosis. We first synthesized a fluorescent switch-on probe, Peptide-ICG2, which is optically silent under normal conditions but activated in the presence of the lysosomal enzyme, cathepsin B. To achieve macrophage-specific fluorescence activation, we encapsulated Peptide-ICG2 into phosphatidylserine-containing liposome (P-ICG2-PS-Lip), since the accumulation of phosphatidylserine receptor-bearing macrophages is characteristic of embolism-vulnerable plaques. The experiments using macrophage-like RAW264 cells in culture showed that P-ICG2-PS-Lip was selectively taken up into the cells and that significant fluorescence of the probe was observed. For NIRF imaging of the atherosclerotic plaques, P-ICG2-PS-Lip was intravenously injected into ApoE-knockout atherosclerotic model mice or WHHL rabbits, and the fluorescence at the aortae was imaged. The results indicated that ICG fluorescence could be successfully observed at the plaques on the artery walls. The results of the present study thus suggest that NIRF imaging using P-ICG2-PS-Lip would be useful for detecting embolism-vulnerable atherosclerotic plaques.

PEG modification on (111)In-labeled phosphatidyl serine liposomes for imaging of atherosclerotic plaques.

INTRODUCTION: Previously, we reported a probe for imaging of atherosclerotic plaques: (111)In-labeled liposomes. Liposomes were modified with phosphatidylserine (PS) because macrophages recognize PS and phagocytize apoptotic cells in plaques. PS modification was successful and we could visualize atherosclerotic plaques by single-photon emission computed tomography (SPECT). However, too-rapid blood clearance reduced accumulation of PS-liposomes in plaques in vivo. Therefore, in the present study, PS-liposomes were modified with polyethylene glycol (PEG) to retard the rate of blood clearance. METHODS: PS-liposomes (size, 100 nm or 200 nm) were PEGylated with PEG2000 or PEG5000 at 1 or 5 mol%, and radiolabeled with (111)In. For the study of uptake in vitro, liposomes were incubated with mouse peritoneal macrophages. Biodistribution studies in vivo were carried out in ddY mice. En face autoradiograms were obtained with apoE(-/-) mice upon intravenous injection of (111)In-liposomes. RESULTS: Uptake was decreased significantly at 5 mol% PEGylation in 100-nm PS-liposomes (*P<0.05 vs. 0 mol%). All the PEGylated liposomes tested showed significantly lower uptake than the non-PEGylated control in 200-nm liposomes. In vivo results showed slower blood clearance in PEGylated liposomes. Autoradiograms in apoE(-/-) mice were well matched with Oil Red O staining. Additionally, 200-nm PS-liposomes modified with 5%PEG2000 ([(111)In]5%PEG2000PS200) showed the highest uptake to the region in vivo. CONCLUSIONS: As expected, PEGylation retarded the rate of blood clearance. In addition, it affected liposome uptake by macrophages in vitro. These results suggest that the balance between the rate of blood clearance and macrophage recognition is important, and [(111)In]5%PEG2000PS200 showed the best results in our investigation.

Prolonged local retention of subcutaneously injected polymers monitored by noninvasive SPECT imaging.

Polymers are widely applied to drug delivery systems because polymers are generally excreted from the body more slowly than small molecules. Subcutaneous injection is one plausible means of administration. In this study, the in vivo behaviors of subcutaneously injected polymers, linear poly(glutamic acid) (Poly-Glu), acetylated dendrimer (Ac-den) and collagen peptide-conjugated dendrimer (CP-den), were investigated. Single photon emission computed tomography (SPECT) imaging was used to noninvasively monitor the in vivo behaviors. Diethylenetriaminepentaacetic acid (DTPA) was conjugated to these polymers, which were labeled with radioactive (111)In. These (111)In-DTPA-bearing polymers (Poly-Glu-DTPA, Ac-den-DTPA and CP-den-DTPA) and unconjugated DTPA were subcutaneously injected into tumor-bearing mice, which were subjected to SPECT imaging. These (111)In-DTPA-bearing polymers were largely retained at the injection site for at least 1 day, whereas the unconjugated DTPA was rapidly cleared from the whole body through excretion. Poly-Glu-DTPA and Ac-den-DTPA were partly accumulated in the kidney (and the liver), but the CP-den-DTPA was not. However, these (111)In-DTPA-bearing polymers were accumulated in the liver and the kidney following intravenous administration. These results indicate that the subcutaneously injected polymers did not largely gain substantial access to the systemic circulation, which is useful for a depot of drug around the injection site.

Development of 111In-labeled liposomes for vulnerable atherosclerotic plaque imaging.

UNLABELLED: Macrophage infiltration is a common characteristic feature of atherosclerotic-vulnerable plaques. Macrophages recognize phosphatidylserine (PS) exposed on the surface of apoptotic cells, which triggers the engulfment of the apoptotic cells by macrophages through phagocytosis. In this study, we prepared radiolabeled PS liposomes for detection of vulnerable plaques. METHODS: PS liposomes were prepared by lipid film hydration. Phosphatidylcholine (PC) liposomes were prepared as controls. Liposomes (100 or 200 nm) were generated by an extruder to produce PS100, PS200, PC100, and PC200 liposomes. These were then radiolabeled by encapsulating (111)In-nitrilotriacetic acid using an active-loading method. (111)In liposomes were incubated with cultured macrophages for 2 h, and the uptake level was measured. For biodistribution studies, the (111)In liposomes were injected intravenously into ddY mice. In addition, the (111)In liposomes were injected into apolipoprotein E-deficient (apoE-/-) mice, and the aortas were harvested for autoradiography and oil red O staining. For SPECT imaging, (111)In liposomes were injected intravenously into Watanabe heritable hyperlipidemic rabbits and scanned 48 h after injection. RESULTS: The radiochemical yields were greater than 95% for all the prepared (111)In liposomes. The level of in vitro uptake by macrophages was 60.5, 14.7, 32.0, and 14.4 percentage injected dose per milligram of protein for (111)In-PS100, (111)In-PC100, (111)In-PS200, and (111)In-PC200, respectively. In biodistribution studies, high spleen uptake was seen with PC liposomes. Liver uptake was high for all liposomes but was lowest with (111)In-PS200. The blood half-lives were 3.2, 22.0, 3.6, and 7.4 min for (111)In-PS100, (111)In-PC100, (111)In-PS200, and (111)In-PC200, respectively. The distribution of (111)In-labeled PS liposomes into atherosclerotic regions determined by autoradiography was well matched with the results of oil red O staining in apoE-/- mice. The target-to-nontarget ratios were 2.62, 2.23, 3.27, and 2.51 for (111)In-PS100, (111)In-PC100, (111)In-PS200, and (111)In-PC200, respectively. The aorta was successfully visualized by SPECT at 48 h after (111)In-labeled PS liposome injection; however, high liver uptake was also observed. DISCUSSION: From the in vitro uptake study, it has been demonstrated that macrophage targeting was accomplished by PS modification. Also, an atherosclerotic region was successfully detected by (111)In-PS200 in apoE-/- mice and Watanabe heritable hyperlipidemic rabbits in vivo. Liposome modification to obtain slower blood clearance and lower liver uptake would be required to improve the SPECT images.