Discoidal porous silicon particles (iNPG) with dimensions 2.6 μm by 700 nm were fabricated as described previously (16). Particles were then partially oxidized and modified by 2% (v/v) 3-aminopropyltriethoxysilane (APTES) in isopropanol for 48 hours at 55°C to confer primary amines on the particle surface. Polymeric doxorubicin (pDox) was separately synthesized by conjugating the drug Dox to side chains of poly(l-glutamic acid) via a pH-sensitive hydrazone linker following previous protocols (8).

For radiolabeling, iNPG was first conjugated to NOTA (Macrocyclics, Dallas, TX) (17). Briefly, 3 billion APTES-modified (-NH2-bearing) iNPG particles were dissolved in 0.5 ml of N,N′-dimethylformamide, followed by addition of 0.8 mg of p-SCN-Bn-NOTA in 0.5 ml of dimethyl sulfoxide (DMSO). The mixture was sonicated until a homogeneous suspension was observed, followed by the addition of 20 μl of diisopropylethylamine. The reaction was allowed to proceed at 25°C for 4 hours under vigorous shaking at 700 rpm. The final conjugate was washed by centrifugation successively, twice with DMSO, followed by isopropanol and water. As-synthesized NOTA-iNPG was suspended in water and freeze-dried for further usage. For radioactive labeling, NOTA-iNPG was suspended in 0.1 M Na2CO3 solution, and the pH was adjusted to 5.5. Copper-64 [64CuCl2; 37 megabecquerel (MBq); Washington University, St. Louis, MO] was added, and the mixture was incubated at 37°C for 2 hours. Excess non-chelated 64Cu was removed by centrifugation at 4000 rpm. To obtain the final constructs, polymeric pDox monomers (3 mg) were loaded into [64Cu]NOTA-iNPG constructs, in DMSO solution (70 mg ml−1), following vigorous vortexing for 4 hours. Unloaded pDox was removed by centrifugation. Radio–thin-layer chromatography (radio-TLC; Bioscan AR-2000 Radio-TLC Imaging Scanner, Eckert & Ziegler, Valencia, CA) using 50 mM EDTA as the mobile phase was used to calculate radiochemical yields and assess time-dependent radiostability in 1× mouse serum.

A similar procedure was adopted to develop fluorescent iNPG by replacing chelator NOTA with NIR dye, AlexaFluor AF647 (excitation: 640 nm; emission: 680 nm, Sigma, St. Louis, MO) to form AF647-iNPG. AF647-iNPG microparticles were filled with pDox monomers as described above to produce AF647-iNPG-pDox for multiplexed OI.

To perform 89Zr radiolabeling of pDox NPs, pDox was first conjugated to p-SCN-Bn-Deferoxamine (DFO; Macrocyclics, Dallas, TX) via amine-isothiocyanate conjugation chemistry as described above (26). Final conjugates were obtained after filtration through PD-10 desalting columns (GE Healthcare, USA). As-synthesized pDox-DFO NPs were suspended in 0.1 M HEPES buffer, and the pH was adjusted to 7 to 7.5. Zirconium-89 (89Zr oxalate; 37 MBq; Washington University, St. Louis, MO) was added, and the mixture was incubated at 37°C for 2 hours. Excess non-chelated 89Zr was removed by PD-10 columns to obtain the final radiolabeled product [89Zr]DFO-pDox.

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