Synthesis of NanoQ, a copper-based contrast agent for high-resolution magnetic resonance imaging characterization of human thrombus.

A new site-targeted molecular imaging contrast agent based on a nanocolloidal suspension of lipid-encapsulated, organically soluble divalent copper has been developed. Concentrating a high payload of divalent copper ions per nanoparticle, this agent provides a high per-particle r1 relaxivity, allowing sensitive detection in T1-weighted magnetic resonance imaging when targeted to fibrin clots in vitro. The particle also exhibits a defined clearance and safety profile in vivo.

A fibrin-specific thrombolytic nanomedicine approach to acute ischemic stroke.

AIM: To develop a fibrin-specific urokinase nanomedicine thrombolytic agent. MATERIALS & METHODS: In vitro fibrin-clot dissolution studies were utilized to develop and characterize simultaneous coupling and loading of anti-fibrin monoclonal antibody and urokinase onto perfluorocarbon nanoparticle (NP) surface. In vivo pharmacokinetics and fibrin-specific targeting of the nanolytic agent was studied in dogs. RESULTS: Simultaneous coupling of up to 40 anti-fibrin antibodies and 400 urokinase enzymes per perfluorocarbon NP produced an effective targeted nanolytic agent with no significant surface protein-protein interference. Fibrin clot dissolution was not improved by increasing homing capacity from 10 to 40 antibodies/NP, but increasing enzymatic payload from 100 to 400/NP resulted in maximized lytic effect. Fluorescent microscopy showed that rhodamine-labeled urokinase nanoparticles densely decorated the intraluminal thrombus in canine clots in vivo analogous to the fibrin pattern, while an irrelevant-targeted agent had negligible binding. CONCLUSION: This agent offers a vascularly constrained, simple to administer, low-dose nanomedicine approach that may present an attractive alternative for treating acute stroke victims.

Conquering the dark side: colloidal iron oxide nanoparticles.

Nanomedicine approaches to atherosclerotic disease will have significant impact on the practice and outcomes of cardiovascular medicine. Iron oxide nanoparticles have been extensively used for nontargeted and targeted imaging applications based upon highly sensitive T2* imaging properties, which typically result in negative contrast effects that can only be imaged 24 or more hours after systemic administration due to persistent blood pool interference. Although recent advances involving MR pulse sequences have converted these dark contrast voxels into bright ones, the marked delays in imaging from persistent magnetic background interference and prominent dipole blooming effects of the magnetic susceptibility remain barriers to overcome. We report a T1-weighted (T1w) theranostic colloidal iron oxide nanoparticle platform, CION, which is achieved by entrapping oleate-coated magnetite particles within a cross-linked phospholipid nanoemulsion. Contrary to expectations, this formulation decreased T2 effects thus allowing positive T1w contrast detection down to low nanomolar concentrations. CION, a vascular constrained nanoplatform administered in vivo permitted T1w molecular imaging 1 h after treatment without blood pool interference, although some T2 shortening effects on blood, induced by the superparamagnetic particles, persisted. Moreover, CION was shown to encapsulate antiangiogenic drugs, like fumagillin, and retained them under prolonged dissolution, suggesting significant theranostic functionality. Overall, CION is a platform technology, developed with generally recognized as safe components, that overcomes the temporal and spatial imaging challenges associated with current iron oxide nanoparticle T2 imaging agents and which has theranostic potential in vascular diseases for detecting unstable ruptured plaque or treating atherosclerotic angiogenesis.

Detecting vascular biosignatures with a colloidal, radio-opaque polymeric nanoparticle.

A synthetic methodology for developing a polymeric nanoparticle for targeted computed tomographic (CT) imaging is revealed in this manuscript. The work describes a new class of soft type, vascularly constrained, stable colloidal radio-opaque metal-entrapped polymeric nanoparticle using organically soluble radio-opaque elements encapsulated by synthetic amphiphile. This agent offers several-fold CT signal enhancement in vitro and in vivo demonstrating detection sensitivity reaching to the low nanomolar particulate concentration range.

Sensitive and efficient detection of thrombus with fibrin-specific manganese nanocolloids.

In this work, we report novel fibrin targeted "soft-type" manganese-based contrast agents for MRI with the potential to noninvasively image intravascular thrombus which could warrant aggressive medical intervention to preclude subsequent myocardial infarction or stroke.

Molecular photoacoustic tomography with colloidal nanobeacons.

Spotting clots: Vascularly constrained colloidal gold nanobeacons (GNBs; see picture) can be used as exogenous photoacoustic contrast agents for the targeted detection of fibrin, a major biochemical feature of thrombus. Fibrin-targeted GNBs provide a more than tenfold signal enhancement in photoacoustic tomography in the near-IR wavelength window, indicating their potential for diagnostic imaging.

Textilinin-1, an alternative anti-bleeding agent to aprotinin: Importance of plasmin inhibition in controlling blood loss.

Aprotinin has been used widely in surgery as an anti-bleeding agent but is associated with a number of side effects. We report that textilinin-1, a serine protease inhibitor from Pseudonaja textilis venom with sequence relatedness to aprotinin, is a potent but reversible plasmin inhibitor and has a narrower range of protease inhibition compared to aprotinin. Like aprotinin, textilinin-1 at 5 micromol/l gave almost complete inhibition of tissue plasminogen activator-induced fibrinolysis of whole blood clots. The activated partial thromboplastin time for plasma was markedly increased by aprotinin but unaffected by textilinin-1. In a mouse tail-vein bleeding model, intravenous textilinin-1 and aprotinin caused similar decreases in blood loss but time to haemostasis in the textilinin-treated animals was significantly shorter than in aprotinin-treated mice. Based on these data, textilinin-1 merits further investigation as a therapeutic alternative to aprotinin.

High sensitivity: high-resolution SPECT-CT/MR molecular imaging of angiogenesis in the Vx2 model.

OBJECTIVES: The use of antiangiogenic therapy in conjunction with traditional chemotherapy is becoming increasingly in cancer management, but the optimal benefit of these targeted pharmaceuticals has been limited to a subset of the population treated. Improved imaging probes that permit sensitive detection and high-resolution characterization of tumor angiogenesis could improve patient risk-benefit stratification. The overarching objective of these experiments was to develop a dual modality alpha(nu)beta3-targeted nanoparticle molecular imaging agent that affords sensitive nuclear detection in conjunction with high-resolution MR characterization of tumor angiogenesis. MATERIALS AND METHODS: In part 1, New Zealand white rabbits (n = 21) bearing 14d Vx2 tumor received either alpha(nu)beta3-targeted 99mTc nanoparticles at doses of 11, 22, or 44 MBq/kg, nontargeted 99mTc nanoparticles at 22 MBq/kg, or alpha(nu)beta3-targeted 99mTc nanoparticles (22 MBq/kg) competitively inhibited with unlabeled alpha(nu)beta3-nanoparticles. All animals were imaged dynamically over 2 hours with a planar camera using a pinhole collimator. In part 2, the effectiveness of alpha(nu)beta3-targeted 99mTc nanoparticles in the Vx2 rabbit model was demonstrated using clinical SPECT-CT imaging techniques. Next, MR functionality was incorporated into alpha(nu)beta3-targeted 99mTc nanoparticles by inclusion of lipophilic gadolinium chelates into the outer phospholipid layer, and the concept of high sensitivity - high-resolution detection and characterization of tumor angiogenesis was shown using sequential SPECT-CT and MR molecular imaging with 3D neovascular mapping. RESULTS: alpha(nu)beta3-Targeted 99mTc nanoparticles at 22 MBq/kg produced the highest tumor-to-muscle contrast ratio (8.56 +/- 0.13, TMR) versus the 11 MBq/kg (7.32 +/- 0.12) and 44 MBq/kg (6.55 +/- 0.07) doses, (P < 0.05). TMR of nontargeted particles at 22.2 MBq/kg (5.48 +/- 0.09) was less (P < 0.05) than the equivalent dosage of alpha(nu)beta3-targeted 99mTc nanoparticles. Competitively inhibition of 99mTc alpha(nu)beta3-integrin-targeted nanoparticles at 22.2 MBq/kg reduced (P < 0.05) TMR (5.31 +/- 0.06) to the nontargeted control contrast level. Multislice CT imaging could not distinguish the presence of Vx2 tumor implanted in the popliteal fossa from lymph nodes in the same fossa or in the contralateral leg. However, the use of 99mTc alpha(nu)beta3-nanoparticles with SPECT-CT produced a clear neovasculature signal from the tumor that was absent in the nonimplanted hind leg. Using alpha(nu)beta3-targeted 99mTc-gadolinium nanoparticles, the sensitive detection of the Vx2 tumor was extended to allow MR molecular imaging and 3D mapping of angiogenesis in the small tumor, revealing an asymmetrically distributed, patchy neovasculature along the periphery of the cancer. CONCLUSION: Dual modality molecular imaging with alpha(nu)beta3-targeted 99mTc-gadolinium nanoparticles can afford highly sensitive and specific localization of tumor angiogenesis, which can be further characterized with high-resolution MR neovascular mapping, which may predict responsiveness to antiangiogenic therapy.

Gadolinium-modulated 19F signals from perfluorocarbon nanoparticles as a new strategy for molecular imaging.

Recent advances in the design of fluorinated nanoparticles for molecular magnetic resonance imaging (MRI) have enabled specific detection of (19)F nuclei, providing unique and quantifiable spectral signatures. However, a pressing need for signal enhancement exists because the total (19)F in imaging voxels is often limited. By directly incorporating a relaxation agent, gadolinium (Gd), into the lipid monolayer that surrounds the perfluorocarbon (PFC), a marked augmentation of the (19)F signal from 200-nm nanoparticles was achieved. This design increases the magnetic relaxation rate of the (19)F nuclei fourfold at 1.5 T and effects a 125% increase in signal--an effect that is maintained when they are targeted to human plasma clots. By varying the surface concentration of Gd, the relaxation effect can be quantitatively modulated to tailor particle properties. This novel strategy dramatically improves the sensitivity and range of (19)F MRI/MRS and forms the basis for designing contrast agents capable of sensing their surface chemistry.

Ligand-directed nanobialys as theranostic agent for drug delivery and manganese-based magnetic resonance imaging of vascular targets.

Although gadolinium has been the dominant paramagnetic metal for MR paramagnetic contrast agents, the recent association of this lanthanide with nephrogenic systemic fibrosis, an untreatable disease, has spawned renewed interest in alternative metals for MR molecular imaging. We have developed a self-assembled, manganese(III)-labeled nanobialys (1), a toroidal-shaped MR theranostic nanoparticle. In this report, Mn(III) nanobialys are characterized as MR molecular imaging agents for targeted detection of fibrin, a major biochemical feature of thrombus. A complementary ability of nanobialys to incorporate chemotherapeutic compounds with greater than 98% efficiency and to retain more than 80% of these drugs after infinite sink dissolution, point to the theranostic potential of this platform technology.

Targeted PARACEST nanoparticle contrast agent for the detection of fibrin.

A lipid-encapsulated perfluorocarbon nanoparticle molecular imaging contrast agent that utilizes a paramagnetic chemical exchange saturation transfer (PARACEST) chelate is presented. PARACEST agents are ideally suited for molecular imaging applications because one can switch the contrast on and off at will simply by adjusting the pulse sequence parameters. This obviates the need for pre- and postinjection images to define contrast agent binding. Spectroscopy (4.7T) of PARACEST nanoparticles revealed a bound water peak at 52 ppm, in agreement with results from the water-soluble chelate. Imaging of control nanoparticles showed no appreciable contrast, while PARACEST nanoparticles produced >10% signal enhancement. PARACEST nanoparticles were targeted to clots via antifibrin antibodies and produced a contrast-to-noise ratio (CNR) of 10 at the clot surface.

Resistance of porcine blood clots to lysis relates to poor activation of porcine plasminogen by tissue plasminogen activator.

In-vitro experimentation was performed on porcine and human blood to determine their comparative responsiveness to a novel fibrinolytic inhibitor and thereby assess whether the pig is a suitable animal model for subsequent in-vivo testing of this inhibitor. Thromboelastography showed the clots formed from porcine whole blood to be highly resistant to tissue plasminogen activator (t-PA)-catalyzed lysis, and this communication offers the resistance of porcine plasminogen to activation by t-PA as an explanation. Porcine blood containing 100 and 1500 IU/ml added t-PA lysed very slowly, having LY30 values of 1.9 +/- 1.4 and 2.9 +/- 1.9%, respectively. In contrast, the LY30 values for the human clots containing 100 and 1500 IU/ml t-PA were 77.1 +/- 6.3 and 93.3 +/- 1.3%, respectively. Moreover, purified porcine plasminogen was activated very slowly by added t-PA in the presence of both human and porcine fibrin. Activation of plasminogen by the endogenous activators, as measured by the euglobulin clot lysis time, was greatly prolonged for the pig (22 +/- 3 h) compared with the human (3.5 +/- 1.5 h). These results suggest caution in using the pig as an experimental model when studying the effects of various agents on fibrinolysis.

In vitro demonstration using 19F magnetic resonance to augment molecular imaging with paramagnetic perfluorocarbon nanoparticles at 1.5 Tesla.

OBJECTIVES: This study explored the use of F spectroscopy and imaging with targeted perfluorocarbon nanoparticles for the simultaneous identification of multiple bio-signatures at 1.5 T. MATERIALS AND METHODS: Two nanoparticle emulsions with perfluoro-15-crown-5-ether (CE) or perfluorooctylbromide (PFOB) cores were targeted in vitro to fibrin clot phantoms (n=12) in 4 progressive ratios using biotin-avidin interactions. The CE nanoparticles incorporated gadolinium. Fluorine images were acquired using steady-state gradient-echo techniques; spectra using volume-selective and nonselective sampling. RESULTS: On conventional T1-weighted imaging, clots with CE nanoparticles enhanced as expected, with intensity decreasing monotonically with CE concentration. All clots were visualized using wide bandwidth fluorine imaging, while restricted bandwidth excitation permitted independent imaging of CE or PFOB nanoparticles. Furthermore, F imaging and spectroscopy allowed visual and quantitative confirmation of relative perfluorocarbon nanoparticle distributions. CONCLUSIONS: F MRI/S molecular imaging of perfluorocarbon nanoparticles in vitro suggests that noninvasive phenotypic characterization of pathologic bio-signatures is feasible at clinical field strengths.

Quantitative "magnetic resonance immunohistochemistry" with ligand-targeted (19)F nanoparticles.

Unstable atherosclerotic plaques exhibit microdeposits of fibrin that may indicate the potential for a future rupture. However, current methods for evaluating the stage of an atherosclerotic lesion only involve characterizing the level of vessel stenosis, without delineating which lesions are beginning to rupture. Previous work has shown that fibrin-targeted, liquid perfluorocarbon nanoparticles, which carry a high payload of gadolinium, have a high sensitivity and specificity for detecting fibrin with clinical (1)H MRI. In this work, the perfluorocarbon content of the targeted nanoparticles is exploited for the purposes of (19)F imaging and spectroscopy to demonstrate a method for quantifiable molecular imaging of fibrin in vitro at 4.7 T. Additionally, the quantity of bound nanoparticles formulated with different perfluorocarbon species was calculated using spectroscopy. Results indicate that the high degree of nanoparticle binding to fibrin clots and the lack of background (19)F signal allow accurate quantification using spectroscopy at 4.7 T, as corroborated with proton relaxation rate measurements at 1.5 T and trace element (gadolinium) analysis. Finally, the extension of these techniques to a clinically relevant application, the evaluation of the fibrin burden within an ex vivo human carotid endarterectomy sample, demonstrates the potential use of these particles for uniquely identifying unstable atherosclerotic lesions in vivo.

"Active" refuges can inhibit the evolution of resistance in insects towards transgenic insect-resistant plants.

Negative cross-resistance (NCR) toxins that hitherto have not been thought to have practical uses may indeed be useful in the management of resistance alleles. Practical applications of NCR for pest management have been limited (i) by the scarcity of high toxicity NCR toxins among pesticides, (ii) by the lack of systematic methodologies to discover and develop such toxins, as well as (iii) by the lack of deployment tactics that would make NCR attractive. Here we present the concept that NCR toxins can improve the effectiveness of refuges in delaying the evolution of resistance by herbivorous insect pests to transgenic host plants containing insecticidal toxins. In our concept, NCR toxins are deployed in the refuge, and thus are physically separated from the transgenic plants containing the primary plant-protectant gene (PPPG) encoding an insecticidal toxin. Our models show: (i) that use of NCR toxins in the refuge dramatically delays the increase in the frequency of resistance alleles in the insect population; and (ii) that NCR toxins that are only moderately effective in killing insects resistant to the PPPG can greatly improve the durability of transgenic insecticidal toxins. Moderately toxic NCR toxins are more effective in minimizing resistance development in the field when they are deployed in the refuge than when they are pyramided with the PPPG. We explore the potential strengths and weaknesses of deploying NCR toxins in refuges.

The effect of anticoagulation with subcutaneously delivered polyethylene glycol conjugated hirudin and recombinant tissue plasminogen activator on recurrent stenosis in the rabbit double-balloon injury model.

Myointimal hyperplasia is the condition usually responsible for recurrent stenosis (restenosis) after endarterectomy, bypass grafting and angioplasty. Its cause is still not known. The present study examined whether inhibition of thrombin by tissue plasminogen activator (r-TPA) or polyethylene glycol recombinant hirudin (PEG-hirudin) could reduce restenosis in an animal model. Restenosis was induced in 20 cholesterol-fed rabbits. The right carotid artery underwent a double-balloon injury while left carotid artery acted as a control. Recombinant tissue plasminogen activator (1 mg kg(-1) s.c.) and PEG-hirudin (0.7 mg kg(-1) s.c.) were given subcutaneously with normal saline acting as a control. Blood levels of PEG-hirudin were measured by both ELISA and an Ecarin (activity) assay. Vessel dimensions were measured in histological sections, obtained from perfusion-fixed tissue, using computerised planimetry. The model reproduced many of the histological changes found in human restenosis, such as intramural thrombus, rupture of the elastic lamina, macrophage infiltration and smooth muscle migration. Reinjury caused an almost three-fold reduction in the area of the lumen (median 0.25 mm(2)) compared with uninjured vessels (median 0.72 mm(2)). The mean plasma levels of PEG-hirudin and r-tPA achieved were 291 ng/ml (S.E.M. 28 ng/ml) and 34 IU/ml (S.E.M. 12 IU/ml), respectively. PEG-hirudin significantly inhibited the effect of balloon injury on luminal area compared with saline-treated controls (0.21 versus 0.44 mm(2), respectively, P<0.05). Recombinant tPA also had a similar inhibitory affect, but this did not reach statistical significance (0.16 versus 0.44 mm(2), respectively, P>0.05). The magnitude of luminal narrowing was significantly reduced by subcutaneous injection of PEG-hirudin. Further studies are required to determine whether this effect can be enhanced by other antithrombins or improved methods of delivery.

Improved molecular imaging contrast agent for detection of human thrombus.

Molecular imaging of microthrombus within fissures of unstable atherosclerotic plaques requires sensitive detection with a thrombus-specific agent. Effective molecular imaging has been previously demonstrated with fibrin-targeted Gd-DTPA-bis-oleate (BOA) nanoparticles. In this study, the relaxivity of an improved fibrin-targeted paramagnetic formulation, Gd-DTPA-phosphatidylethanolamine (PE), was compared with Gd-DTPA-BOA at 0.05-4.7 T. Ion- and particle-based r(1) relaxivities (1.5 T) for Gd-DTPA-PE (33.7 (s*mM)(-1) and 2.48 x 10(6) (s*mM)(-1), respectively) were about twofold higher than for Gd-DTPA-BOA, perhaps due to faster water exchange with surface gadolinium. Gd-DTPA-PE nanoparticles bound to thrombus surfaces via anti-fibrin antibodies (1H10) induced 72% +/- 5% higher change in R(1) values at 1.5 T (deltaR(1) = 0.77 +/- 0.02 1/s) relative to Gd-DTPA-BOA (deltaR(1) = 0.45 +/- 0.02 1/s). These studies demonstrate marked improvement in a fibrin-specific molecular imaging agent that might allow sensitive, early detection of vascular microthrombi, the antecedent to stroke and heart attack.

A family of textilinin genes, two of which encode proteins with antihaemorrhagic properties.

Two peptides, textilinins 1 and 2, isolated from the venom of the Australian common brown snake, Pseudonaja textilis textilis, are effective in preventing blood loss. To further investigate the potential of textilinins as antihaemorrhagic agents, we cloned cDNAs encoding these proteins. The isolated full-length cDNA (430 bp in size) was shown to code for a 59 amino acid protein, corresponding in size to the native peptide, plus an additional 24 amino acid propeptide. Six such cDNAs were identified, differing in nucleotide sequence in the coding region but with an identical propeptide. All six sequences predicted peptides containing six conserved cysteines common to Kunitz-type serine protease inhibitors. When expressed as glutathione S-transferase (GST) fusion proteins and released by cleavage with thrombin, only those peptides corresponding to textilinin 1 and 2 were active in inhibiting plasmin with Ki values similar to those of their native counterparts and in binding to plasmin less tightly than aprotinin by two orders of magnitude. Similarly, in the mouse tail vein blood loss model only recombinant textilinin 1 and 2 were effective in reducing blood loss. These recombinant textilinins have potential as therapeutic agents for reducing blood loss in humans, obviating the need for reliance on aprotinin, a bovine product with possible risk of transmissible disease, and compromising the fibrinolytic system in a less irreversible manner.