Chemical and biological kinetics of click chemistry reactions for surface modification of Feraheme nanoparticles

Objectives: Click reactions are widely used in organic chemistry due to fast reaction rate, solvent independency, aqueous media compatibility, modularity, and bioorthogonality in reactions with small molecules. These properties have not been established in the context of nanoparticle (NP) modifications. We have previously developed derivatives of Feraheme (FH) nanoparticles using heat-induced radiolabeling (HIR) to dope the iron oxide core with radiometals and using click chemistry for surface modifications. The present study advances our understanding of Strain Promoted Alkyne and Azide Cycloaddition (SPAAC) click chemistry between dibenzocyclooctyne (DBCO) derivatives and Azide-FH, including: (1) quantitative verification of heat stability of azide groups at various surface densities; (2) study of click reaction kinetics dependence on number of azide groups, different DBCO-ligands, and reaction media; (3) the impact of PEG sizes on the in vitro and in vivo kinetics of PEGylated FH NPs.

Methods: FH NPs were subjected to surface modification by the attachment of azide groups through the reactions between the NP surface carboxylic groups and amino-PEG-azide linker (linker) under the coupling of EDC/HOBT. The kinetic studies were carried out in the correspondent media by the reactions of Azide-FH NPs with designated DBCO-ligands for each study. Quantitative firstorder exponential correlations, such as the azide density (N₃/NP) vs. amount of 1M linker (uL) or click yield (%) vs. time (hr), were determined by non-linear regression. The in vitro stability of click PEGylated FH NPs was evaluated by the variations of particle sizes (diameter: nm) and relaxivity (T₁and T₂) over time. Monocyte uptake was performed by the incubation of PEG-FH-Cy5.5 fluorescent NPs with monkey blood under 37 ^oC for 1 or 3 hrs and labeled monocyte populations were analyzed by flow cytometry. For the extension of blood circulation time, 30k Da PEGylated ⁸⁹Zr-FH NPs were synthesized by the combination of HIR radiochemistry and click surface PEGylation, followed by the injection of 147.5±5.4 μCi of ⁸⁹Zr-FH-30kPEG into healthy female C57B6 mice (N=4, 18.25±1.56 g). Dynamic PET imaging was performed for 1 hr and static scans acquired at 2, 8, 24, and 96 hrs.

Results: Our results indicated that the number of azide groups could be finely controlled by adjusting the ratio of FH NPs/azide-PEG linkers indicated by a first order exponential non-linear regression between N₃/NP and amount of linker. 48-70% of azide groups survived the heating challenge of HIR (120 ^oC, 2 h). The small molecular ligands, such as widely used fluorescent dyes (Cy5.5 and Alexa 647) and small peptide (KUE, a PSMA inhibitor), affected the reaction kinetics in the first 5 hrs. When the click reactions of DBCO-Cy5.5 were carried out in 10 mM buffers for 2 h, the click yields showed a sharp difference as >90% (in DPBS), 50% (in MES and Tris), and <40% (in HEPES and pure water). When the buffer concentrations were increased, Tris and MES increased the click kinetics. All the kinetics (click yield, %) fitted the equation of first order exponential non-linear regression. The surface PEGylation showed a clear effect on their monocyte uptake indicated by the decrease of fluorescent intensity of monocytes in cytometry when larger PEGs were used. The surface click attached 30 kDa PEGs extended the blood half-life of FH NPs from 1 h to 4.75 h in mice, likely due to reduced phagocytosis as evidenced by the aforementioned monocyte uptake assay.

Conclusions: Click reactions for surface modification of Feraheme nanoparticles did not have the same kinetics as have been widely found in small molecule chemistry. This principle should be investigated with other nanoparticle formulations to determine the generality of the present findings and evaluate the impact on click chemistry’s utility as a tool for surface modification of nanomaterials. Acknowledgements: NIH R01MH100350, R01EB017699, P41EB022544

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