Folate receptor-targeted delivery of small interfering RNA to cancer cells

Yilin Zhang


The vitamin folic acid (folate, FA) has been extensively explored as a targeting ligand to deliver a variety of diagnostic/prognostic/therapeutic agents into various tumors through the assistance of its receptor - the folate receptor (FR). FR is over-expressed in many types of human cancer and can mediate internalization of FA-conjugates through an endocytic pathway. The discovery of small interfering RNA (siRNA), which is cable of inducing potent gene silencing in a sequence-specific manner, provides an excellent molecular tool to suppress aberrant gene expression in malignancies, and therefore siRNA has the potential to revolutionize cancer therapeutics. Towards the goal of developing an efficient and cancer-specific siRNA strategy, three types of FA-conjugated molecules have been synthesized to investigate targeted siRNA delivery to cancer cells in vitro. In the first section, FA-linked siRNA was synthesized through our one-step in vitro transcription using FA-HAD-AMP as a transcriptional initiator. FR-dependent cellular uptake and moderate specific gene down-regulation (50%) were observed in a stable cell line (Gluc-KB), which was established in this work by integrating Gaussia luciferase (Gluc) gene to the genome of KB cells (human nasopharyngeal carcinoma). Gluc-KB provides a platform to better evaluate gene expression changes upon siRNA treatment. In the second section, a FA-functionalized, multivalent copolymer (FAPol13) was introduced to: (i) complex with siRNA to form a FAPol13/siRNA complex, (ii) protect siRNAs from enzymatic degradation, (iii) enhance cellular uptake by conjugating several FA molecules to copolymer, and (iv) increase RNAi efficacy eventually. A typical FAPol13 compound has three functional groups: cationic, hydrophilic, and FA moieties for providing siRNA packaging site, water solubility, and cell-selectivity, respectively. This nontoxic polymer successfully delivered siRNAs against Gluc, survivin (Sur), Caspase 8 associated protein 2 (Casp8ap2) genes in KB cells and efficiently reduced their expression (i.e., 62% and 68% downregulation from siSv and siGLuc treatments, respectively). Strikingly, treatment of KB cells with FAPol13/siCasp8ap2 significantly repressed cell growth and induced cells into apoptosis (24.5%, p = 0.01). Furthermore, a real-time imaging system, employing fluorescence approaches (time-lapse, z-stacking, and colocalization analysis), has been developed to evaluate siRNA binding, cellular uptake, and escape from compartments, respectively. In particular, Pearson's correlation coefficient (PCC) was employed to quantify siRNA endosomal escape, one of the crucial steps of siRNA intracellular trafficking. This method was further applied to the analysis of siRNA delivery in HeLa (human cervical carcinoma) and SKOV3 (human ovary carcinoma). In the third section, a gold nanoparticle (AuNP) capable of packaging and protecting siRNAs was utilized to transport siRNA to cancer cells. Cytotoxicity assay, cellular uptake, and gene down-regulation studies of the AuNP-siRNA system indicate that AuNP can be an efficient siRNA platform. In the fourth section, a correlation between FR expression level and the delivering effectiveness of FA-conjugates was established by comparing cellular absorption, cellular uptake, and RNAi efficiency among KB, HeLa, SKOV3 and A549 (human lung carcinoma) cells. Collectively, we have demonstrated effective FR-mediated and cancer cell-specific siRNA delivery by several approaches. Further studies may lead to the development of therapeutic siRNA delivery systems.