A Scalable Method for Squalenoylation and Assembly of Multifunctional 64Cu-Labeled Squalenoylated Gemcitabine Nanoparticles


Authors: S.T. Tucci, J.W. Seo, H. Kakwere, A. Kheirolomoom, E.S. Ingham, L.M. Mahakian, S. Tam, S. Tumbale, M. Baikoghli, H. Cheng and K.W. Ferrara

Journal: Nanotheranostics

DOI: 10.7150/ntno.26969

Publication - Abstract

September 05, 2018

Abstract

Squalenoylation of gemcitabine, a front-line therapy for pancreatic cancer, allows for improved cellular-level and system-wide drug delivery. The established methods to conjugate squalene to gemcitabine and to form nanoparticles (NPs) with the squalenoylated gemcitabine (SqGem) conjugate are cumbersome, time-consuming and can be difficult to reliably replicate. Further, the creation of multi-functional SqGem-based NP theranostics would facilitate characterization of in vivo pharmacokinetics and efficacy.

Methods:

Squalenoylation conjugation chemistry was enhanced to improve reliability and scalability using tert-butyldimethylsilyl (TBDMS) protecting groups. We then optimized a scalable microfluidic mixing platform to produce SqGem-based NPs and evaluated the stability and morphology of select NP formulations using dynamic light scattering (DLS) and transmission electron microscopy (TEM). Cytotoxicity was evaluated in both PANC-1 and KPC (KrasLSL-G12D/+; Trp53LSL-R172H/+; Pdx-Cre) pancreatic cancer cell lines. A 64Cu chelator (2-S-(4-aminobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid, NOTA) was squalenoylated and used with positron emission tomography (PET) imaging to monitor the in vivo fate of SqGem-based NPs.

Results:


Squalenoylation yields of gemcitabine increased from 15% to 63%. Cholesterol-PEG-2k
inclusion was required to form SqGem-based NPs using our technique, and additional cholesterol inclusion increased particle stability at room temperature; after 1 week the PDI of SqGem NPs with cholesterol was ~ 0.2 while the PDI of SqGem NPs lacking cholesterol was ~ 0.5. Similar or superior cytotoxicity was achieved for SqGem-based NPs compared to gemcitabine or Abraxane® when evaluated at a concentration of 10 µM. Squalenoylation of NOTA enabled in vivo monitoring of SqGem-based NP pharmacokinetics and biodistribution.

Conclusion:

We present a scalable technique for fabricating efficacious squalenoylatedgemcitabine nanoparticles and confirm their pharmacokinetic profile using a novel multifunctional
64Cu-SqNOTA-SqGem NP.

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