Subtle clotting that occurs on the luminal surface of atherosclerotic plaques presents a novel target for nanoparticle-based diagnostics and therapeutics. We have developed modular multifunctional micelles that contain a targeting element, a fluorophore, and, when desired, a drug component in the same particle. Targeting atherosclerotic plaques in ApoE-null mice fed a high-fat diet was accomplished with the pentapeptide cysteine-arginine-glutamic acid-lysine-alanine, which binds to clotted plasma proteins. The fluorescent micelles bind to the entire surface of the plaque, and notably, concentrate at the shoulders of the plaque, a location that is prone to rupture. We also show that the targeted micelles deliver an increased concentration of the anticoagulant drug hirulog to the plaque compared with untargeted micelles.cysteine-arginine-glutamic acid-lysine-alanine ͉ hirulog ͉ plaque ͉ imaging ͉ nanoparicles C ardiovascular disease affects 1 in 3 people in the United States during their lifetime, and accounts for nearly a third of the deaths that occur each year (1). Atherosclerosis is one of the leading causes of cardiovascular disease, and it results in raised plaques in the arterial wall that can occlude the vascular lumen and block blood flow through the vessel. Recently, it has become clear that not all plaques are the same. Those susceptible to rupture, fissuring, and subsequent thrombosis are most frequently the cause of acute coronary syndromes and death (2).Rupture of an atherosclerotic plaque exposes collagen and other plaque components to the bloodstream. This rupture initiates hemostasis in the blood vessel and leads to activation of thrombin and a thrombus to form at the site of rupture. Elevated levels of activated thrombin bound to the vessel wall have been observed up to 72 h after vascular injury (3). These elevated thrombin levels not only induce clot formation but also have been implicated in the progression of atherosclerosis by causing smooth muscle cells to bind circulating low density lipoprotein (4). Subtle clotting in plaques is also indicated by deposition of fibrin(ogen) both inside and on the surface of atherosclerotic plaques, which has been well documented since the 1940s (5-7).Fibrin-containing blood clots have been extensively used as a target for site-specific delivery of imaging agents and anticlotting agents to thrombi (8-10). Delivering anticoagulants into vessels where clotting is taking place has been shown to be effective at reducing the formation and expansion of clots, and it also decreases the risk of systemic side effects (11,12). Antibodies and peptides that bind to molecular markers specifically expressed on atherosclerotic plaques have shown promise for plaque imaging in vivo (13-16), but clotting on the plaque has not been used as a target. We reasoned that the fibrin deposited on plaques could serve as a target for delivering diagnostic and therapeutic compounds to plaques.We chose the clot-binding peptide cysteine-arginine-glutamic acid-lysine-alanine (CREKA) to te...

The tumor-homing pentapeptide CREKA (Cys-Arg-Glu-Lys-Ala) specifically homes to tumors by binding to fibrin and fibrinassociated clotted plasma proteins in tumor vessels. Previous results show that CREKA-coated superparamagnetic iron oxide particles can cause additional clotting in tumor vessels, which creates more binding sites for the peptide. We have used this self-amplifying homing system to develop theranostic nanoparticles that simultaneously serve as an imaging agent and inhibit tumor growth by obstructing tumor circulation through blood clotting. The CREKA nanoparticles were combined with nanoparticles coated with another tumor-homing peptide, CRKDKC, and nanoparticles with an elongated shape (nanoworms) were used for improved binding efficacy. The efficacy of the CREKA peptide was then increased by replacing some residues with nonproteinogenic counterparts, which increased the stability of the peptide in the circula-

We report the controlled radical copolymerization of N-(2-hydroxypropyl)methacrylamide (HPMA) with a monomer containing an active ester, N-methacryloyloxysuccinimide (NMS), by reversible addition fragmentation chain transfer (RAFT). The large difference in the reactivity ratios of HPMA and NMS resulted in significant variations in copolymer composition with increasing conversion during batch copolymerization. The use of a semi-batch copolymerization method, involving the gradual addition of the more reactive NMS, allowed uniformity of copolymer composition to be maintained during the polymerization. We synthesized polymers in a wide range of molecular weights (M(n) = 3000-50,000 Da) with low polydispersities (1.1-1.3). The effect of the ratio of monomer to chain transfer agent (CTA) on the molecular weight of the polymer was investigated. Given the numerous applications of poly(HPMA)-based conjugates in designing polymeric therapeutics, these controlled molecular weight activated polymers represent attractive scaffolds for biofunctionalization. As a demonstration, we attached a peptide to the activated polymer backbone to synthesize a potent controlled molecular weight polyvalent inhibitor of anthrax toxin.