Graphical Abstract

Small interfering RNA (siRNA) is a powerful and highly effective method to regulate gene expression in vitro and in vivo. However, the susceptibility to serum nuclease-catalyzed degradation is a major challenge and it remains unclear whether the strategies developed to improve the stability of siRNA free in serum solution are ideal for siRNA conjugated to nanoparticle surfaces. Herein, we use spherical nucleic acid nanoparticle conjugates, consisting of gold nanoparticles (AuNPs) with siRNA chemisorbed to the surface, as a platform to study how a model siRNA targeting androgen receptor degrades in serum (SNA-siRNA AR ). In solutions of 10% (vol/vol) FBS, we find rapid endonuclease hydrolysis at specific sites near the AuNP-facing terminus of siRNA AR , which were different from those of siRNA AR free in solution. These data indicate that the chemical environment of siRNA on a nanoparticle surface can alter the recognition of siRNA by serum nucleases and change the inherent stability of the nucleic acid. Finally, we demonstrate that incorporation of 2′-O-methyl RNA nucleotides at sites of nuclease hydrolysis on SNA-siRNA AR results in a 10-fold increase in siRNA lifetime. These data suggest that strategies for enhancing the serum stability of siRNA immobilized to nanoparticles must be developed from a dedicated analysis of the siRNAnanoparticle conjugate, rather than a reliance on strategies developed for siRNA free in solution. We believe these findings are important for fundamentally understanding interactions between biological media and oligonucleotides conjugated to nanoparticles for the development of gene regulatory and therapeutic agents in a variety of disease models.biological recognition | nanotechnology | OliGreen | polyacrylamide gel electrophoresis

Learning how to assemble inorganic nanoparticles into ordered lattices may prove to be important for applications, such as, electronics, photonics, and catalysis. [1] Indeed, theoretical studies have shown that certain types of crystalline arrays of nanoparticles could potentially be used to generate photonic band-gap materials, negative index materials, and metamaterials at visible and infrared length scales. [2,3] The vast majority of work in this area has focused on the assembly of spherical particles. However, anisotropic nanoparticles, which display rich assembly behavior owing to their reduced symmetry, and have unique physical properties that can be engineered by controlling interparticle spacing and orientation, may provide access to even more interesting materials. [4][5][6][7][8] Moreover, they require design rules for predicting the way such nanoparticles can assemble and the types of structures that may be realized. The rapidly expanding library of available anisotropic nanoparticle building blocks provides exciting new opportunities to study colloidal assembly as a function of particle shape. [9][10][11] Herein, we introduce a directional entropic force approach (DEFA) for controlling the assembly of anisotropic nanoparticles into crystalline lattices. The method relies on surfactant micelle-induced depletion interactions [12] to assemble anisotropic gold nanoparticles into reconfigurable, nonclose-packed (open) superlattices in solution. The anisotropic nanoparticles align along their flat facets to maximize entropy, and therefore minimize the free energy of the system, leading to assemblies with long-range order. [13,14] Importantly, our experimental work complements recent theoretical work that proposes directional entropic forces between nanoparticle facets as a viable means for thermodynamically assembling nanoparticle superlattices. [14][15][16][17] The experimental work herein uses depletants to create strong attractive forces that can drive assembly of reversible superlattices with tunable spacing in solution. These directional entropic forces are analogous to the directional bonding between atoms in molecules. [14,15,18] The resulting crystalline superlattices are therefore shape-dependent. We show that the electrostatic and depletion interactions combine to determine the lattice spacing, and can be tuned independently with surfactant concentration and ionic strength to reconfigure the lattice constant.The DEFA presented herein complements several assembly strategies that have been developed to create superlattices of anisotropic nanostructures including evaporationinduced [19,20] and sedimentation-based methods, [8] as well as ones based upon the manipulation of electrostatic, [21,22] entropic, [13,[23][24][25][26][27][28] block copolymer, [29,30] and biological recognition interactions. [31] However, unlike the majority of these methods, our approach yields reversible crystals with tunable lattice constants in situ.Depletion interactions are purely entropic in nature and arise when non-ads...

Ribonucleic acids (RNAs) are key components in many cellular processes such as cell division, differentiation, growth, aging, and death. RNA spherical nucleic acids (RNA-SNAs), which consist of dense shells of double-stranded RNA on nanoparticle surfaces, are powerful and promising therapeutic modalities because they confer advantages over linear RNA such as high cellular uptake and enhanced stability. Due to their three-dimensional shell of oligonucleotides, SNAs, in comparison to linear nucleic acids, interact with the biological environment in unique ways. Herein, the modularity of the RNA-SNA is used to systematically study structure–function relationships in order to understand how the oligonucleotide shell affects interactions with a specific type of biological environment, namely, one that contains serum nucleases. We use a combination of experiment and theory to determine the key architectural properties (i.e., sequence, density, spacer moiety, and backfill molecule) that affect how RNA-SNAs interact with serum nucleases. These data establish a set of design parameters for SNA architectures that are optimized in terms of stability.

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.