Dual bioluminescence and near-infrared fluorescence monitoring to evaluate spherical nucleic acid nanoconjugate activity in vivo

Significance Small interfering (si) and micro (mi)RNA-carrying nanomaterials emerged as a new class of anticancer therapeutics. To enable quantification of intratumoral gene expression in response to RNAi nanoconjugate treatment in vivo, we developed a dual reporter glioblastoma xenograft model using cell lines that stably coexpress optical reporters for luciferase and a near-infrared fluorescent protein (iRFP670). We generated orthotopic glioblastoma multiforme tumors expressing an iRFP670-O6-methylguanine-DNA-methyltransferase (MGMT) fusion protein for real-time assessment of MGMT-targeting spherical nucleic acids (i.e., gold-based nanoconjugates functionalized with siRNA oligonucleotides targeted to MGMT). We demonstrate that dual noninvasive bioluminescence and fluorescence imaging can determine intratumoral protein expression in response to systemic spherical nucleic acid treatment and represents an in vivo testing platform to facilitate preclinical investigations of nanoscale gene silencing therapeutics. RNA interference (RNAi)-based gene regulation platforms have shown promise as a novel class of therapeutics for the precision treatment of cancer. Techniques in preclinical evaluation of RNAi-based nanoconjugates have yet to allow for optimization of their gene regulatory activity. We have developed spherical nucleic acids (SNAs) as a blood–brain barrier-/blood–tumor barrier-penetrating nanoconjugate to deliver small interfering (si) and micro (mi)RNAs to intracranial glioblastoma (GBM) tumor sites. To identify high-activity SNA conjugates and to determine optimal SNA treatment regimens, we developed a reporter xenograft model to evaluate SNA efficacy in vivo. Engrafted tumors stably coexpress optical reporters for luciferase and a near-infrared (NIR) fluorescent protein (iRFP670), with the latter fused to the DNA repair protein O6-methylguanine-DNA-methyltransferase (MGMT). Using noninvasive imaging of animal subjects bearing reporter-modified intracranial xenografts, we quantitatively assessed MGMT knockdown by SNAs composed of MGMT-targeting siRNA duplexes (siMGMT-SNAs). We show that systemic administration of siMGMT-SNAs via single tail vein injection is capable of robust intratumoral MGMT protein knockdown in vivo, with persistent and SNA dose-dependent MGMT silencing confirmed by Western blotting of tumor tissue ex vivo. Analyses of SNA biodistribution and pharmacokinetics revealed rapid intratumoral uptake and significant intratumoral retention that increased the antitumor activity of coadministered temozolomide (TMZ). Our study demonstrates that dual noninvasive bioluminescence and NIR fluorescence imaging of cancer xenograft models represents a powerful in vivo strategy to identify RNAi-based nanotherapeutics with potent gene silencing activity and will inform additional preclinical and clinical investigations of these constructs.