Mechanistic Design of Polymer Nanocarriers to Spatiotemporally Control Gene Silencing

Abstract

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.

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

“…Moreover, the on/off control over siRNA activity afforded by the hybrid nanocomplexes can be easily extended to spatially regulate gene expression at cellular length scales using previously described procedures. [19, 23] …”

“…[19] Each siRNA dose was modeled by increasing the normalized siRNA concentration by 100 units following time points corresponding to light-triggered siRNA release. The siRNA turnover rate was estimated by determining the cell doubling time of AoAFs cultured in standard growth medium, which was measured to be ~38 h (Figure S7).…”

“…[18] Moreover, our previous work established other beneficial properties of our mPEG- b -poly(5-(3-(amino)propoxy)-2-nitrobenzyl methacrylate) [mPEG- b -P(APNBMA)] block copolymer system for vascular applications, including high stability and the capacity to locally regulate the extent of protein silencing on cellular length scales. [19] Such features make our nanocomplexes ideal for incorporation into biomaterials, such as abluminally-applied hydrogels, to enable regulation of adventitial cell behavior through a combination of mechanical stimulation and spatial regulation of gene expression in anastomoses. Additionally, the precisely controlled nature of the system allows for accurate predictions of siRNA dosing regimens that facilitate gene knockdown over clinically-relevant timescales associated with adventitial remodeling (one week).…”

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

“…As shown in Figure 2, the 0/100 polyplexes [containing only mPEG- b -P(APNBMA) 23.6 ] exhibited ~40% protein knockdown, consistent with the literature. [48] Interestingly, the 100/0 formulation also was able to achieve a silencing efficiency of ~40%; however, all polyplexes comprised of a mixture of the two polymers exhibited enhanced knockdown levels, demonstrating that the degree of silencing was tunable on the basis of polyplex composition. In particular, the 50/50 formulation was able to mediate gene silencing most efficiently, with ~70% protein knockdown; this silencing effect is near the highest degree achievable with a single dose of siRNA given the half-lives of the targeted mRNA and protein.…”

“…In particular, the 50/50 formulation was able to mediate gene silencing most efficiently, with ~70% protein knockdown; this silencing effect is near the highest degree achievable with a single dose of siRNA given the half-lives of the targeted mRNA and protein. [48] This simple, yet powerful, strategy of mixing BCPs produced polyplexes with greater silencing efficiencies than nanocarriers comprised of a single polymer species, implying that the polymers were functioning in a synergistic manner.…”