A macrophage-targeted theranostic nanoparticle for biomedical applications.

Sequelae of atherosclerotic vascular disease (for example, acute myocardial infarction or stroke) are leading causes of death worldwide. Given that atherogenesis is an insidious process occurring over decades, there exists a need to first diagnose and then treat subclinical but high-risk (so-called vulnerable) atherosclerotic plaques prior to clinical manifestations. In particular, the macrophage has emerged as a key biological, imaging, and therapeutic target for atherosclerosis. Thus, antimacrophage therapeutic strategies are becoming more prevalent. These therapies include systemic modulation of macrophage activity and localized photodynamic therapy (PDT), a clinical approach that utilizes injectable photosensitizers and subsequent photoangioplasty to debulk atherosclerotic lesions. Commonly used photosensitizers are usually of porphyrinic origin and, once excited by light of the appropriate wavelength, are capable of producing cytotoxic singlet oxygen through interactions between the excited porphyrinic molecule and molecular oxygen. One of the main advantages of PDT is its ability to spare healthy tissues surrounding the site of interest, due to the fact that treatment only takes place upon illumination, which can be localized. PDT has recently found clinical utility in the treatment of cardiovascular diseases, such as atherosclerosis and in-stent restenosis. 8] A number of photosensitizers have shown preferential uptake in atherosclerotic plaques, probably due to the interactions between the relatively hydrophobic porphyrin derivatives and the lipid-rich core of the atheroma. The most studied of these, motexafin lutetium (Antrin) is currently in clinical trials for the treatment of coronary artery disease. Unfortunately, systemic delivery of most photosensitizing agents also leads to significant side effects, such as prolonged photosensitivity due to localization of the drug within the skin. Numerous methodologies have been developed to increase the efficacy of PDT, such as conjugation of photosensitizers to peptides and antibodies, for the active targeting of cell-surface receptors or other biomolecules. PDT agents have also been encapsulated within polymeric nanoparticles, in order to yield higher local concentrations at therapeutic sites. We hypothesized that the efficacy of PDT in killing local macrophages could be augmented by conjugation of photosensitizers to macrophage-avid nanoparticles. Using a mouse model of atherosclerosis ACHTUNGTRENNUNG(apoE / ), we have previously shown that a dextrancoated magnetofluorescent nanoparticle (MFNP) had enhanced uptake in atherosclerotic plaques in vivo, as well as specificity for macrophages. Importantly, these nanoparticles do not localize in the epidermis in significant amounts, a fact suggesting that a MFNP-based PDT approach could lessen the risk of skin toxicity as compared to that with available PDT agents. Based on the previous results and biodistribution studies of magnetic nanoparticles (MNPs) in clinical use, we reasoned that an atherosclerotic-plaque/macrophage-targeted delivery vehicle could be based upon dextran coatings. Indeed, macrophages contain a previously cloned dextran receptor, SIGNR1. Other design criteria included the need for the nanoparticles to be detectable by magnetic resonance imaging (MRI; magnetic core) as well as by catheter-based fluorescence imaging. Since dextrans and other carbohydrates dissociate from iron oxide crystal surfaces, we crosslinked the coating through epichlorohydrin and functionalized the coating with primary amines. The monocrystalline nanoparticles had an overall diameter (volume weighted) in aqueous solution of 33 nm (polydispersity= 0.21), a superparamagnetic core of 5 nm containing 8000 Fe atoms per particle and magnetic relaxivities, R1 and R2, of 21 and 62 mm s 1 (37 8C, 0.5T), respectively. Alexa Fluor 750 (AF750, Invitrogen) was conjugated to the particle in a ratio of 3 fluorophores per particle, through reaction of the amine groups with the corresponding succinimidyl ester of AF750. In order to impart upon the nanoparticle the ability to generate singlet oxygen, a potent photosensitizer, 5-(4-carboxyphenyl)-10,15,20-triphenyl-2,3-dihydroxychlorin (TPC), was covalently conjugated to the primary amines of the MFNP. TPC has a singlet-oxygen quantum yield of 0.65 when excited at 646 nm. It has also proven more efficacious than the commonly used chlorin e6. [34] This sensitizer is convenient to synthesize and utilize as it is generated directly from the corresponding 5,10,15,20-tetraphenylporphyrin derivative in relatively high yields and purity. The presence of one carboxylic acid moiety, which is easily activated, also simplifies conjugation to peptides or other amine-containing molecules. TPC (2) was synthesized from the corresponding mesotetraphenylporphyrin analogue as shown in Scheme 1. OsO4-mediated dihydroxylation of porphyrin 1 yields the chlorin 2, while activation of 2 with DSC in DMF gives 3. In order to conjugate the activated chlorin to the AF750– MFNP, the phosphate-buffered saline (PBS) in which the particles were originally synthesized and suspended was exchanged for dimethylsulfoxide (DMSO) by filtration through Sephadex G-25 with DMSO as the eluent. Conjuga[*] Dr. J. R. McCarthy, Dr. F. A. Jaffer, Prof. R. Weissleder Center for Molecular Imaging Research Massachusetts General Hospital and Harvard Medical School Building 149, 13th Street, Room 5406 Charlestown, MA 02129 (USA) Fax: (+1)617-726-5708 E-mail: jason_mccarthy@hms.harvard.edu weissleder@helix.mgh. harvard.edu