Predicting Gene Silencing Through the Spatiotemporal Control of siRNA Release from Photo-responsive Polymeric Nanocarriers

Summary

We present a novel method that uses photo-responsive block copolymers for more efficient spatiotemporal control of gene silencing with no detectable off-target effects. Additionally, changes in gene expression can be predicted using straightforward siRNA release assays and simple kinetic modeling.

Abstract

New materials and methods are needed to better control the binding vs. release of nucleic acids for a wide range of applications that require the precise regulation of gene activity. In particular, novel stimuli-responsive materials with improved spatiotemporal control over gene expression would unlock translatable platforms in drug discovery and regenerative medicine technologies. Furthermore, an enhanced ability to control nucleic acid release from materials would enable the development of streamlined methods to predict nanocarrier efficacy a priori, leading to expedited screening of delivery vehicles. Herein, we present a protocol for predicting gene silencing efficiencies and achieving spatiotemporal control over gene expression through a modular photo-responsive nanocarrier system. Small interfering RNA (siRNA) is complexed with mPEG-b-poly(5-(3-(amino)propoxy)-2-nitrobenzyl methacrylate) (mPEG-b-P(APNBMA)) polymers to form stable nanocarriers that can be controlled with light to facilitate tunable, on/off siRNA release. We outline two complementary assays employing fluorescence correlation spectroscopy and gel electrophoresis for the accurate quantification of siRNA release from solutions mimicking intracellular environments. Information gained from these assays was incorporated into a simple RNA interference (RNAi) kinetic model to predict the dynamic silencing responses to various photo-stimulus conditions. In turn, these optimized irradiation conditions allowed refinement of a new protocol for spatiotemporally controlling gene silencing. This method can generate cellular patterns in gene expression with cell-to-cell resolution and no detectable off-target effects. Taken together, our approach offers an easy-to-use method for predicting dynamic changes in gene expression and precisely controlling siRNA activity in space and time. This set of assays can be readily adapted to test a wide variety of other stimuli-responsive systems in order to address key challenges pertinent to a multitude of applications in biomedical research and medicine.

Introduction

Small interfering RNAs (siRNAs) mediate post-transcriptional gene silencing through a catalytic RNAi pathway that is highly specific, potent, and tailorable to virtually any target gene¹. These promising characteristics have enabled siRNA therapeutics to advance in human clinical trials for the treatment of numerous diseases, including metastatic melanoma and hemophilia^2,3. However, significant delivery issues persist that have hindered translation⁴. In particular, delivery vehicles must remain stable and protect siRNAs from extracellular degradation, yet also re....

Protocol

1. Formulation of siRNA Nanocarriers

1. Prepare separate solutions of siRNA and mPEG-b-P(APNBMA) with equal volumes diluted in 20 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) buffer at pH 6.0.
   1. Add siRNA at a concentration of 32 µg/mL to 20 mM HEPES solution.
      NOTE: The siRNA was a non-targeted, universal negative control sequence; however, the siRNA can be designed to target any gene of interest.
   2. Dissolve mPEG-b-P(APNBMA) polymers into a 20 mM HEPES solution. Add an appropriate amount of mPEG-b-P(APNBMA) to make a 220 µg/mL solution so that the N/P ratio (N, amine groups ....

Results

Following the formulation of the nanocarriers, siRNA release assays were conducted to inform the irradiation conditions to be used in the in vitro transfections. Various dosages of light were applied to determine the percent of siRNA that was released. The first assay used gel electrophoresis to separate the free siRNA molecules from the siRNA molecules still complexed/associated with the polymer. Nanocarriers that were not treated with light remained stable and did not release a.......

Discussion

There are a few steps in the method that are particularly critical. When formulating the nanocarriers, the order of component addition and mixing speed are two important parameters influencing efficacy³⁹. This protocol requires that the cationic component, mPEG-b-P(APNBMA), is added to the anionic component, siRNA, in a dropwise fashion while vortexing. Depending on the total formulation volume, this mixing process takes 3-6 s. To test if the nanocarriers were formed properly, measure the.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
siRNA	Sigma-Aldrich	SIC001	non-targeted, universal negative control
mPEG-b-P(APNBMA)	synthesized in our lab	N/A	photo-responsive polymer
HEPES	Fisher Scientific	BP310-100
sodium dodecyl sulfate	Sigma-Aldrich	436143
rubber gasket	McMaster-Carr	3788T21	0.5 mL thick
UV laser	Excelitas Technologies	Omnicure S2000	collimating lens and 365 nm filter used
agarose	Fisher Scientific	BP160-100
ethidium bromide	Fisher Scientific	BP1302-10
siRNA labelled with Dy547	GE Healthcare Dharmacon, Inc.	custom order	fluorophore conjugated to 5’ end of sense strand
microscope slide	Fisher Scientific	12-550-A3	pre-cleaned glass
Secure-Seal Spacer	Life Technologies	S24735	double-sided adhesive
LSM 780	Carl Zeiss	N/A	confocal microscope
ZEN 2010	Carl Zeiss	N/A	FCS analysis software
MATLAB	MathWorks	N/A	programming language
NIH/3T3 cells	ATCC	ATCC CRL-1658
DMEM	Mediatech	10-013-CV	growth media
fetal bovine serum	Mediatech	35-011-CV	heat-inactivated
penicillin-streptomycin	Mediatech	30-002-CI
6-well plates	Fisher Scientific	08-772-1B
Opti-MEM	Life Technologies	11058021	transfection media