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

Abstract

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 RNAibased 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 nearinfrared (NIR) fluorescent protein (iRFP670), with the latter fused to the DNA repair protein O 6 -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.

Nanomedical Devices and Cancer Theranostics

“…Nano-encapsulation of multiplexed RNAi in lipopolymeric NPs is a therapeutic targeting strategy to meet the challenges of therapeutic resistance and tumor heterogeneity [112]. Spherical nucleic acids (SNAs) nanoconjugate-based RNAi constitute an in vivo nanotherapeutic strategy, they are capable of performing noninvasive imaging and inactivating in vivo intratumoral proteins [113].…”

Near-Infrared Fluorescent Proteins and Their Applications

“…iRFP670 was used for evaluating the efficiency of the new method for delivery of small interfering RNAs (siRNAs) into intracranial glioblastoma cells in mice [99]. For this purpose, glioblastoma cells were generated that stably expressed a chimeric protein consisting of iRFP670 and protein target of the siRNA.…”

Spherical Nucleic Acid Nanoparticles: Therapeutic Potential

“…Small-molecule drugs and antibodies that are currently used in the clinic to attack glioblastoma are extremely ineffective due to their limited ability to cross blood–brain barrier or blood–tumor barrier, which leads to the need for high doses that cause systemic toxicity [45–47]. Because SNAs could transfect cells in vitro to regulate gene expression, Mirkin and colleagues investigated whether SNAs could deliver siRNA and miRNA to brain tumors for therapeutic gene regulation [22, 41, 48]. They designed 13-nm gold core SNAs to deliver siRNA targeting Bcl2L12 [41], an anti-apoptotic gene that is overexpressed in glioblastoma, or miR-182 [22], a miRNA that is downregulated in glioblastoma and that functions to suppress Bcl2L12 .…”

“…Additionally, these SNAs exhibited no apparent toxicity, as evidenced by histopathology of various tissues and analysis of blood chemistry [22, 41]. SNAs have also been developed to suppress the DNA repair protein O6-methylguanine-DNA-methyltransferase ( MGMT ), which is associated with drug resistance in glioblastoma, and these siMGMT-SNAs potentiated the effects of co-administered temozolomide in murine tumor models [48]. Together, these findings demonstrate the immense potential of SNAs as a new treatment for glioblastoma, either alone or in combination with other modalities.…”