Skip to main content
eScholarship
Open Access Publications from the University of California

Targeted delivery of siRNA to human cancer and human embryonic stem cells with cell level resolution

  • Author(s): Huang, Xiao
  • Advisor(s): Reich, Norbert O.
  • et al.
Abstract

RNA interference (RNAi), a process that can silence specific genes, has been recognized for its potential in applications from basic research, cancer therapy to tissue engineering. However, the routine use of RNAi for disease treatment or prevention still calls for novel and efficient methods of delivery with targeting control. We recently developed an approach using hollow gold nanoshells (HGNs) and near-infrared (NIR) light: siRNAs loaded on HGNs via gold-thiol bonds are internalized in cells through targeting peptide-mediated pathways; laser illumination at a wavelength resonant with the nanoparticle plasmon (~800nm) triggers the release of siRNAs from gold surface to cytosol without any cell damage. A cancer-therapeutic siRNA (under phase I/II clinical trial) targeting Polo-like kinase (plk1) gene is delivered via this strategy to specifically target human prostate cancer cells. We demonstrated that this method has no off-target toxicity to non-cancerous prostate cells, and requires 10-fold less material than standard transfection methods.

Human embryonic stem cells (hESC) hold immense promise in tissue engineering due to their ability to differentiation into all types of cells, and RNAi can serve as a powerful tool to attain this. We developed our HGN-based construct for effective cell penetration and optimized the protocol to accommodate delivery into hESCs. The effectiveness and biocompatibility of this light-activated RNAi approach were demonstrated by targeting GFP and Oct4 genes in hESCs, and no adverse effects to differentiation were detected.

The in vitro generation of tissues and organs for transplantation therapy and disease models for drug screening demand proficient control over the three-dimensional (3D) patterning of gene regulation. The exquisite activation of siRNA activity by tissue penetrable NIR light irradiation in our technique is well suited for this purpose. We used a two-photon microscope to discriminate targeted cell(s) from neighboring cell(s) by simply focusing NIR light at selected x, y and z position to activate the siRNA. The ability to induce light-mediated gene knockdown persists for at least two days, offering a time window for temporal control.

Furthermore, we developed a universal surface module for the maximized delivery of short functional nucleic acids in versatile mammalian cell lines. A modular biotin-modified thiol-RNA is designed as the scaffold for streptavidin and biotin-TAT attaching, sharing the surface with thiolated functional RNA. We found interesting surface chemistries with direct impact on cellular delivery outcomes. This should provide an important basis for various cell engineering applications.

Main Content
Current View