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Shell crosslinked nanoparticles (SCKs) are amphiphilic core-shell nanoparticles inspired from biological constructs (1-2). They are prepared by the self-assembly and subsequent crosslinking of block copolymer micelles and can be optimized for guest packaging. The easily controlled structural and chemical features play a critical synergistic role in their resulting binding, sequestration, toxicity, and immunogenic properties. To explore the biomedical application of these SCKs, we have designed biocompatible nanoscale scaffolds that will provide multi-/poly-valent presentation of functionalities for detection, diagnosis and intervention of disease states by coupling various target molecules to SCKs, in which radionuclides are included for imaging and/or radiotherapy. Herein, we present an approach to complex 64 Cu to the SCK via conjugation of a TETA derivative, and preliminary in vivo evaluation of the 64 Cu-labeled nanoparticles.
Methods. The SCKs and nanocages (NCs) (resulting from ozone degradation of the SCK core) were prepared as previously described (3-5).
The conjugation of TETA-CONH-(C 2 H 4 O) 2 C 2 H 4 NH 2 (TETA-NH 2 ) to the SCK or NC was carried out by modifying our DOTA-mAb conjugation procedure. 64 Cu-labeling was accomplished by incubation of 64 Cu(OAc) 2 with TETA-NH-SCK (or NC) for 4 h at 70 C, followed by a DTPA challenge and Centricon separation. The radiochemical purity was monitored by FPLC. The biodistribution studies used mature Sprague-Dawley rats (n = 4, ca.12 mCi/100 mL), and the PET imaging was performed on the first commercially available microPET (Concorde Microsystems, Knoxville, TN) using Balb/c mice (n = 2, 150 mCi/100 mL).
Results and Discussion. Because self condensation between the carboxyl groups and the amino groups of TETA-NH 2 molecules occurred when direct conjugation to the nanostructures was attempted, a step was introduced to produce and separate the activated SCK-NHS ester. Through the modified procedure, both SCKs and NCs showed up to 70% radiolabeling yields, and over 95% radiochemical purity after a DTPA challenge and centricon separation, as indicated by FPLC. The preliminary biodistribution data of 64 Cu-TETA-NH-NC (57-nm, Mw 1,510,900) and 64 Cu-TETA-NH-SCK (13.7-45 nm, Mw 523 -4,004 KDa) showed rapid 64 Cu uptake in liver (NC: 69.23 -1.31 %ID/liver at 10 min p.i.; SCK with MW 4,004 KDa: 46.88 -3.27%ID/liver at 10 min p.i.), and slow clearance via kidneys and intestine, which was further confirmed by the microPET imaging studies. To lower the liver burden, a PEG-derivatized SCK (MW 4,004 KDa plus added PEG) was prepared (PEG: poly(ethylene glycol)). The microPET images clearly showed both kidneys, which indicates that an increased renal clearance resulted from the PEG derivatization. Future studies include: determination of SCK and NC size effects on the in vivo behavior; further modification of the SCK and NC surface chemistries; and coupling of ligands for specific mo lecular targeting.
Acknowledgment.
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