A novel vascular homing peptide strategy to selectively enhance pulmonary drug efficacy in pulmonary arterial hypertension.

A major limitation in the pharmacological treatment of pulmonary arterial hypertension (PAH) is the lack of pulmonary vascular selectivity. Recent studies have identified a tissue-penetrating homing peptide, CARSKNKDC (CAR), which specifically homes to hypertensive pulmonary arteries but not to normal pulmonary vessels or other tissues. Some tissue-penetrating vascular homing peptides have a unique ability to facilitate transport of co-administered drugs into the targeted cells/tissues without requiring physical conjugation of the drug to the peptide (bystander effect). We tested the hypothesis that co-administered CAR would selectively enhance the pulmonary vascular effects of i.v. vasodilators in Sugen5416/hypoxia/normoxia-exposed PAH rats. Systemically administered CAR was predominantly detected in cells of remodeled pulmonary arteries. Intravenously co-administered CAR enhanced pulmonary, but not systemic, effects of the vasodilators, fasudil and imatinib, in PAH rats. CAR increased lung tissue imatinib concentration in isolated PAH lungs without increasing pulmonary vascular permeability. Sublingual CAR was also effective in selectively enhancing the pulmonary vasodilation by imatinib and sildenafil. Our results suggest a new paradigm in the treatment of PAH, using an i.v./sublingual tissue-penetrating homing peptide to selectively augment pulmonary vascular effects of nonselective drugs without the potentially problematic conjugation process. CAR may be particularly useful as an add-on therapy to selectively enhance the pulmonary vascular efficacy of any ongoing drug treatment in patients with PAH.

A highly selective KIT inhibitor MOD000001 suppresses IgE-mediated mast cell activation.

Background: The KIT receptor tyrosine kinase and its ligand, stem cell factor (SCF), control proliferation and survival of mast cells. Thus, targeting KIT signaling may show promise for the treatment of allergic diseases involving mast cells. Recently, we discovered a new compound, MOD000001, as a potential small-molecule KIT kinase inhibitor by using an in silico approach. Objective: We sought to determine whether MOD000001 is highly selective to KIT, inhibits KIT signaling in mast cells, and affects IgE-mediated mast cell activation. Methods: The interaction of MOD000001 with 468 human kinases and its inhibitory activity against KIT were profiled and evaluated by using KINOMEscan (Discover X/Eurofins Corporation, Fremont, Calif) and cell-free kinase assays, respectively. The effects of MOD000001 on SCF-dependent signaling were examined by using primary mouse and human mast cells. The effects of MOD000001 on SCF-induced degranulation and passive cutaneous anaphylaxis reaction were examined in mice. Results: MOD000001 interacted with KIT and inhibited KIT kinase activity with high selectivity. MOD000001 suppressed SCF-induced KIT signaling in mouse and human mast cells and in mice. Passive cutaneous anaphylaxis reaction was suppressed in mice treated with MOD000001 both for a short-term (1 week) and for a long-term (7 weeks). Mice treated with MOD000001 for a long-term, but not for a short-term, showed skin mast cell reduction. Conclusions: MOD000001 is a highly selective KIT inhibitor that can suppress IgE-mediated mast cell activation in vivo. MOD000001 may do so by reducing tissue mast cell numbers or by other unknown mechanisms. The findings suggest potential benefits of MOD000001 for allergic diseases involving IgE-mediated mast cell activation.

Peptide-directed highly selective targeting of pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a disorder of the pulmonary vasculature associated with elevated pulmonary vascular resistance. Despite recent advances in the treatment of PAH, with eight approved clinical therapies and additional therapies undergoing clinical trials, PAH remains a serious life-threatening condition. The lack of pulmonary vascular selectivity and associated systemic adverse effects of these therapies remain the main obstacles to successful treatment. Peptide-mediated drug delivery that specifically targets the vasculature of PAH lungs may offer a solution to the lack of drug selectivity. Herein, we show highly selective targeting of rat PAH lesions by a novel cyclic peptide, CARSKNKDC (CAR). Intravenous administration of CAR peptide resulted in intense accumulation of the peptide in monocrotaline-induced and SU5416/hypoxia-induced hypertensive lungs but not in healthy lungs or other organs of PAH rats. CAR homed to all layers of remodeled pulmonary arteries, ie, endothelium, neointima, medial smooth muscle, and adventitia, in the hypertensive lungs. CAR also homed to capillary vessels and accumulated in the interstitial space of the PAH lungs, manifesting its extravasation activity. These results demonstrated the remarkable ability of CAR to selectively target PAH lung vasculature and effectively penetrate and spread throughout the diseased lung tissue. These results suggest the clinical utility of CAR in the targeted delivery of therapeutic compounds and imaging probes to PAH lungs.

Folate receptor-targeted aggregation-enhanced near-IR emitting silica nanoprobe for one-photon in vivo and two-photon ex vivo fluorescence bioimaging.

A two-photon absorbing (2PA) and aggregation-enhanced near-infrared (NIR) emitting pyran derivative, encapsulated in and stabilized by silica nanoparticles (SiNPs), is reported as a nanoprobe for two-photon fluorescence microscopy (2PFM) bioimaging that overcomes the fluorescence quenching associated with high chromophore loading. The new SiNP probe exhibited aggregate-enhanced emission producing nearly twice as strong a signal as the unaggregated dye, a 3-fold increase in two-photon absorption relative to the DFP in solution, and approximately 4-fold increase in photostability. The surface of the nanoparticles was functionalized with a folic acid (FA) derivative for folate-mediated delivery of the nanoprobe for 2PFM bioimaging. Surface modification of SiNPs with the FA derivative was supported by zeta potential variation and (1)H NMR spectral characterization of the SiNPs as a function of surface modification. In vitro studies using HeLa cells expressing a folate receptor (FR) indicated specific cellular uptake of the functionalized nanoparticles. The nanoprobe was demonstrated for FR-targeted one-photon in vivo imaging of HeLa tumor xenograft in mice upon intravenous injection of the probe. The FR-targeting nanoprobe not only exhibited highly selective tumor targeting but also readily extravasated from tumor vessels, penetrated into the tumor parenchyma, and was internalized by the tumor cells. Two-photon fluorescence microscopy bioimaging provided three-dimensional (3D) cellular-level resolution imaging up to 350 µm deep in the HeLa tumor.

Two-photon fluorescence vascular bioimaging with new bioconjugate probes selective toward the vascular endothelial growth factor receptor 2.

We report the synthesis and characterization of two amine reactive fluorescent dyes with efficient two-photon absorption (2PA) properties and high fluorescence quantum yields. Bioconjugation of these dyes with the DC-101 antibody proved to be useful for selectively imaging the vascular endothelial growth factor receptor 2 (VEGFR-2) in cells expressing this receptor in vitro and in "whole" mounted excised tumors (ex vivo) by two-photon fluorescence microscopy (2PFM). The penetration depths reached within the tumors by 2PFM was over 800 µm. In addition, the concentration of dye required for incubation of these bioconjugates was in the picomolar domain, the probes possessed very good photostability, and the 2PFM setup did not require any additional means of increasing the collection efficiencies of fluorescent photons to achieve the relatively deep tissue imaging that was realized, due, in large part, to the favorable photophysical properties of the new probes.

Recognition, neutralization, and clearance of target peptides in the bloodstream of living mice by molecularly imprinted polymer nanoparticles: a plastic antibody.

We report that simple, synthetic organic polymer nanoparticles (NPs) can capture and clear a target peptide toxin in the bloodstream of living mice. The protein-sized polymer nanoparticles, with a binding affinity and selectivity comparable to those of natural antibodies, were prepared by combining a functional monomer optimization strategy with molecular-imprinting nanoparticle synthesis. As a result of binding and removal of melittin by NPs in vivo, the mortality and peripheral toxic symptoms due to melittin were significantly diminished. In vivo imaging of the polymer nanoparticles (or "plastic antibodies") established that the NPs accelerate clearance of the peptide from blood and accumulate in the liver. Coupled with their biocompatibility and nontoxic characteristics, plastic antibodies offer the potential for neutralizing a wide range of biomacromolecules in vivo.

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.

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.

Novel amphiphilic probes for [18F]-radiolabeling preformed liposomes and determination of liposomal trafficking by positron emission tomography.

Positron-emission tomography (PET) is a noninvasive real-time functional imaging system and is expected to be useful for the development of new drug candidates in clinical trials. For its application with preformulated liposomes, we devised an optimized [18F]-compound and developed a direct liposome modification method that we termed the "solid-phase transition method". We were successful in using 1-[18F]fluoro-3,6-dioxatetracosane ([18F]7a) for in vivo trafficking of liposomes. This method might be a useful tool in preclinical and clinical studies of lipidic particle-related drugs.

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

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.

Correction: Deep Vascular Imaging in Wounds by Two-Photon Fluorescence Microscopy.

[This corrects the article DOI: 10.1371/journal.pone.0067559.].

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.

Deep vascular imaging in wounds by two-photon fluorescence microscopy.

Deep imaging within tissue (over 300 µm) at micrometer resolution has become possible with the advent of two-photon fluorescence microscopy (2PFM). The advantages of 2PFM have been used to interrogate endogenous and exogenous fluorophores in the skin. Herein, we employed the integrin (cell-adhesion proteins expressed by invading angiogenic blood vessels) targeting characteristics of a two-photon absorbing fluorescent probe to image new vasculature and fibroblasts up to ≈ 1600 µm within wound (neodermis)/granulation tissue in lesions made on the skin of mice. Reconstruction revealed three dimensional (3D) architecture of the vascular plexus forming at the regenerating wound tissue and the presence of a fibroblast bed surrounding the capillaries. Biologically crucial events, such as angiogenesis for wound healing, may be illustrated and analyzed in 3D on the whole organ level, providing novel tools for biomedical applications.

Small molecule fluorophore and copolymer RGD peptide conjugates for ex vivo two-photon fluorescence tumor vasculature imaging.

We report the use of small molecule and block copolymer RGD peptide conjugates for deep ex vivo imaging of tumor vasculature in "whole" excised tumors using two-photon fluorescence microscopy (2PFM). The fluorescent probes were administered to mice via tail-vein injection, after which the tumors were excised, fixed, and imaged without further sample preparation. Both RGD conjugates demonstrated specific targeting to tumor blood vessels, and this selectivity imparted excellent contrast in 2PFM micrographs that captured high-resolution 3-D images of the tumor vasculature up to depths of 830 µm in Lewis Lung Carcinoma (LLC) tumors. 2PFM ex vivo fluorescence micrographs clearly revealed tumor vessels, while differences in the sensitivity of tumor vessel imaging were apparent between the small molecule and block copolymer conjugates. Both the small molecule and polymer-based two-photon absorbing probe conjugate are valuable for deep tissue tumor microvasculature imaging.

Small GTPase R-Ras regulates integrity and functionality of tumor blood vessels.

We show that R-Ras, a small GTPase of the Ras family, is essential for the establishment of mature, functional blood vessels in tumors. The genetic disruption of R-Ras severely impaired the maturation processes of tumor vessels in mice. Conversely, the gain of function of R-Ras improved vessel structure and blood perfusion and blocked plasma leakage by enhanced endothelial barrier function and pericyte association with nascent blood vessels. Thus, R-Ras promotes normalization of the tumor vasculature. These findings identify R-Ras as a critical regulator of vessel integrity and function during tumor vascularization.

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.

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.

Evaluation of O-[(18)F]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.

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.