Target‐Cell‐Specific Delivery, Imaging, and Detection of Intracellular MicroRNA with a Multifunctional SnO2 Nanoprobe

Abstract

MicroRNAs (miRNAs) are a class of short, endogenous, noncoding regulatory RNAs (approximately 18–25 nucleotides), encoded in the genomes of plants, animals, and viruses. They partially complement the 3’ untranslated region of target mRNAs, causing mRNA cleavage or inhibiting protein synthesis within the Dicer/Argonaute complex. MiRNAs play key regulatory roles in a diverse range of biological processes, including cell development, differentiation, metabolism, and apoptosis. In particular, distinct miRNA expression patterns are associated with various cancer phenotypes. Therefore, miRNAs are also an emerging class of useful diagnostic and prognostic markers. However, miRNA analysis is challenging owing to the unique characteristics of miRNAs, such as their small size, sequence similarity among family members, low abundance, susceptibility to degradation, and the technical impediments in delivering miRNAs across the plasma membrane of cells. An improved delivery strategy is thus needed to successfully monitor intracellular miRNA levels in situ. Generally, transmission of oligonucleotides for gene therapy falls into two broad categories, viral vectors or nonviral carriers. Viral vectors exhibit high transfection efficiency but also some fundamental problems, such as immunogenicity and toxicity. These problems limit their broad application. Currently, the nonviral vectors, such as liposomes, cationic polymers, dendrimers, polypeptides, and nanomaterials have attracted significant interest, owing to their good biocompatibility and potential for large-scale production, in contrast to viral vectors. However, many of these carrier systems do not inhibit genes in a cell-specific manner and cannot be used to monitor their intracellular delivery or therapeutic response. An excellent nonviral vector should efficiently facilitate cell-specific gene-probe (a single-stranded oligonucleotide designed to recognize a single target-nucleotide sequence) uptake and gene-probe endosomal escape for intracellular delivery. Fluorescent nanoparticles are highly attractive materials for gene-probe delivery because of their unique properties, including uniform size, superior imaging characteristics, and facile surface modification. The rational functionalization of these nanomaterials with target-specific moieties, such as antibodies, aptamers, and other molecules, to recognize receptors on the cell surface has led to versatile theragnostic nanosystems. These multifunctional nanosystems not only allow efficient delivery of gene probes to target cells with fewer side effects, but also allow the simultaneous monitoring of delivery and the therapeutic response of the targeted genes, using the advanced optical properties of the nanosystem. However, these nanosystems have not been used for in situ detection of intracellular miRNAs yet, because the detection strategy has not been optimized. In this study we design a novel multifunctional SnO2 nanoprobe (mf-SnO2), which contains a cell-targeting moity, as well as a conjugated gene probe to specifically recognize the target sequence, thus providing a detection strategy or inhibitor. Also, visualization of the delivery and intracellular response is possible through fluorescence of the SnO2. As shown in Scheme 1, cell-specific delivery is achieved by functionalizing SnO2 nanoparticles (SnO2NPs) with folic acid (FA), which targets cancer cells; a gene probe, in this case a molecular beacon (MB) to detect target miRNAs, is conjugated by a disulfide linkage, which is sensitive to pH values. Cleavage of the disulfide linkage between the gene probe and the nanoparticle enhances the efficiency of intracellular delivery. Using miRNA-21 in HeLa cells as a model, a method for in situ detection of intracellular miRNA by the multifunctional nanoprobe is reported. To verify the practicality of this approach, another nanoprobe was also designed, by substituting the MB with an anti-miR of miRNA-21, to down-regulate the expression of a target miRNA. The proposed method, with a MB as the recognition probe, can be used subsequently to monitor the change in miRNA levels from negligible cytotoxicity, and to monitor the ability of the multifunctional nanoprobe to [*] Dr. H. Dong, Prof. J. Lei, Prof. H. Ju, Z. Zhu State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University Nanjing 210093 (P.R. China) E-mail: hxju@nju.edu.cn