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

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.

Anionic Polymer and Quantum Dot Excipients to Facilitate siRNA Release and Self-Reporting of Disassembly in Stimuli-Responsive Nanocarrier Formulations.

The incorporation of anionic excipients into polyplexes is a promising strategy for modulating siRNA binding versus release and integrating diagnostic capabilities; however, specific design criteria and structure-function relationships are needed to facilitate the development of nanocarrier-based theranostics. Herein, we incorporated poly(acrylic acid) (PAA) and quantum dot (QD) excipients into photolabile siRNA polyplexes to increase gene silencing efficiencies by up to 100% and enable self-reporting of nanocarrier disassembly. Our systematic approach identified the functional relationships between gene silencing and key parameters such as excipient loading fractions and molecular weights that facilitated the establishment of design rules for optimization of nanocarrier efficacy. For example, we found that PAA molecular weights ∼10-20× greater than that of the coencapsulated siRNA exhibited the most efficient release and silencing. Furthermore, siRNA release assays and RNAi modeling allowed us to generate a PAA "heat map" that predicted gene silencing a priori as a function of PAA molecular weight and loading fraction. QDs further promoted selective siRNA release and provided visual as well as Förster resonance energy transfer (FRET)-based monitoring of the dynamic changes in nanostructure in situ. Moreover, even with the addition of anionic components, our formulations exhibited substantially improved stability and shelf life relative to typical formulations, with complete stability after a week of storage and full activity in the presence of serum. Taken together, this study enabled synergistic improvements in siRNA release and diagnostic capabilities, along with the development of mechanistic insights that are critical for advancing the translation of nucleic acid theranostics into the clinic.

Efficient tuning of siRNA dose response by combining mixed polymer nanocarriers with simple kinetic modeling.

Two of the most prominent challenges that limit the clinical success of siRNA therapies are a lack of control over cargo release from the delivery vehicle and an incomplete understanding of the link between gene silencing dynamics and siRNA dosing. Herein, we address these challenges through the formulation of siRNA polyplexes containing light-responsive polymer mixtures, whose varied compositions and triggered release behavior provide enhanced gene silencing and controlled dose responses that can be predicted by simple kinetic models. Through the straightforward mixing of two block copolymers, the level of gene knockdown was easily optimized to achieve the maximum level of GAPDH protein silencing in NIH/3T3 cells (~70%) using a single siRNA dose. The kinetic model was used to describe the dynamic changes in mRNA and protein concentrations in response to siRNA treatment. These predictions enabled the application of a second dose of siRNA to maximally suppress gene expression over multiple days, leading to a further 50% reduction in protein levels relative to those measured following a single dose. Furthermore, polyplexes remained dormant in cells until exposed to the photo-stimulus, demonstrating the complete control over siRNA activity as well as the stability of the nanocarriers. Thus, this work demonstrates that pairing advances in biomaterials design with simple kinetic modeling provides new insight into gene silencing dynamics and presents a powerful strategy to control gene expression through siRNA delivery. STATEMENT OF SIGNIFICANCE: Our manuscript describes two noteworthy impacts: (1) we designed mixed polymer formulations to enhance gene silencing, and (2) we simultaneously developed a simple kinetic model for determining optimal siRNA dose responses to maintain silencing over several days. These advances address critical challenges in siRNA delivery and provide new opportunities in therapeutics development. The structure-function relationships prevalent in these formulations were established to enable tuning and forecasting of nanocarrier efficiency a priori, leading to siRNA dosing regimens able to maximally suppress gene expression. Our advances are significant because the mixed polymer formulations provide a straightforward and scalable approach to tailor siRNA delivery regimens. Moreover, the implementation of accurate dosing frameworks addresses a major knowledge gap that has hindered clinical implementation of siRNA.

Attenuation of Maladaptive Responses in Aortic Adventitial Fibroblasts through Stimuli-Triggered siRNA Release from Lipid-Polymer Nanocomplexes.

Lipid-siRNA assemblies are modified with photo-responsive polymers to enable spatiotemporally-controlled silencing of interleukin 1 beta (IL1ß) and cadherin 11 (CDH11), two genes that are essential drivers of maladaptive responses in human aortic adventitial fibroblasts (AoAFs). These hybrid nanocomplexes address the critical challenge of locally mitigating fibrotic actions that lead to the high rates of vascular graft failures. In particular, the lipid-polymer formulations provide potent silencing of IL1ß and CDH11 that is precisely modulated by a photo-release stimulus. Moreover, a dynamic modeling framework is used to design a multi-dose siRNA regimen that sustains knockdown of both genes over clinically-relevant timescales. Multi-dose suppression illuminates a cooperative role for IL1ß and CDH11 in pathogenic adventitial remodeling and is directly linked to desirable functional outcomes. Specifically, myofibroblast differentiation and cellular proliferation, two of the primary hallmarks of fibrosis, are significantly attenuated by IL1ß silencing. Meanwhile, the effects of CDH11 siRNA treatment on differentiation become more pronounced at higher cell densities characteristic of constrictive adventitial remodeling in vivo. Thus, this work offers a unique formulation design for photo-responsive gene suppression in human primary cells and establishes a new dosing method to satisfy the critical need for local attenuation of fibrotic responses in the adventitium surrounding vascular grafts.

Mechanistic Design of Polymer Nanocarriers to Spatiotemporally Control Gene Silencing.

Current siRNA delivery systems lack the ability to precisely tune siRNA release and maximize gene silencing in a spatiotemporal manner. Herein, we investigate photoresponsive block copolymer solution assemblies, for which stimuli-triggered changes in polymer structure altered nanocarrier stability and defined siRNA activity. Uniquely, our biomaterials design enabled the development and validation of a simple kinetic model that accurately predicted the extent of intracellular nanocarrier disassembly and silencing. Moreover, our constructs showed that maximal gene silencing could be achieved using concentrations of siRNA 5-fold lower than typical formulations due to the ability to rapidly release sufficient amounts of siRNA to saturate the cellular RISC machinery. The ability of our nanocarriers to remain dormant prior to phototriggered siRNA release allowed for the generation of cell patterns in gene expression with spatial control on cellular length scales and no detectable off-target effects. Furthermore, precisely tuned changes in nanocarrier structure enabled the modulation of protein and mRNA knockdown levels in murine fibroblasts and terminally differentiated human primary cells. These advances lead to increased precision, potency, and utility relative to other recent spatiotemporally controlled nucleic acid delivery vehicles reported in the literature. Moreover, the combination of experimental examination and kinetic modeling described herein should be applicable to a host of systems for which temporal control over nucleic acid delivery is a critical parameter in influencing cellular responses.

Light-mediated activation of siRNA Release in diblock copolymer assemblies for controlled gene silencing.

Controllable release is particularly important for the delivery of small interfering RNA (siRNA), as siRNAs have a high susceptibility to enzymatic degradation if release is premature, yet lack silencing activity if they remain inaccessible within the cytoplasm. To overcome these hurdles, novel and tailorable mPEG-b-poly(5-(3-(amino)propoxy)-2-nitrobenzyl methacrylate) (mPEG-b-P(APNBMA)) diblock copolymers containing light-sensitive o-nitrobenzyl moieties and pendant amines are employed to provide both efficient siRNA binding, via electrostatic and hydrophobic interactions, as well as triggered charge reversal and nucleic acid release. In particular, siRNA/mPEG-b-P(APNBMA)23.6 polyplexes show minimal aggregation in physiological salt and serum, and enhanced resistance to polyanion-induced unpackaging compared to polyethylenimine preparations. Cellular delivery of siRNA/mPEG-b-P(APNBMA)23.6 polyplexes reveals greater than 80% cellular transfection, as well as rapid and widespread cytoplasmic distribution. Additionally, UV irradiation indicates ≈70% reduction in targeted gene expression following siRNA/mPEG-b-P(APNBMA)23.6 polyplex treatment, as compared to 0% reduction in polyplex-treated cells without UV irradiation, and only ≈30% reduction for Lipofectamine-treated cells. The results here highlight the potential of these light-sensitive copolymers with a well-defined on/off switch for applications including cellular patterning for guided cell growth and extension, and cellular microarrays for exploring protein and drug interactions that require enhanced spatiotemporal control of gene activation.

Catch and Release: Photocleavable Cationic Diblock Copolymers as a Potential Platform for Nucleic Acid Delivery.

Binding interactions between DNA and cationic carriers must be sufficiently strong to prevent nuclease-mediated degradation, yet weak enough to permit transcription. We demonstrate cationic diblock copolymers containing PEG and o-nitrobenzyl moieties that facilitated tailorable DNA complexation and light-activated release. This design unlocks a new approach to advance non-viral gene packaging.