Although siRNA-based nanomedicines hold promise for cancer treatment, conventional siRNA–polymer complex (polyplex) nanocarrier systems have poor pharmacokinetics following intravenous delivery, hindering tumor accumulation. Here, we determined the impact of surface chemistry on the in vivo pharmacokinetics and tumor delivery of siRNA polyplexes. A library of diblock polymers was synthesized, all containing the same pH-responsive, endosomolytic polyplex core-forming block but different corona blocks: 5 kDa (benchmark) and 20 kDa linear polyethylene glycol (PEG), 10 kDa and 20 kDa brush-like poly(oligo ethylene glycol), and 10 kDa and 20 kDa zwitterionic phosphorylcholine-based polymers (PMPC). In vitro, it was found that 20 kDa PEG and 20 kDa PMPC had the highest stability in the presence of salt or heparin and were the most effective at blocking protein adsorption. Following intravenous delivery, 20 kDa PEG and PMPC coronas both extended circulation half-lives 5-fold compared to 5 kDa PEG. However, in mouse orthotopic xenograft tumors, zwitterionic PMPC-based polyplexes showed highest in vivo luciferase silencing (>75% knockdown for 10 days with single IV 1 mg/kg dose) and 3-fold higher average tumor cell uptake than 5 kDa PEG polyplexes (20 kDa PEG polyplexes were only 2-fold higher than 5 kDa PEG). These results show that high molecular weight zwitterionic polyplex coronas significantly enhance siRNA polyplex pharmacokinetics without sacrificing polyplex uptake and bioactivity within tumors when compared to traditional PEG architectures.

Clinical translation of therapies based on small interfering RNA (siRNA) is hampered by siRNA's comprehensively poor pharmacokinetic properties, which necessitate molecule modifications and complex delivery strategies. We sought an alternative approach to commonly used nanoparticle carriers by leveraging the long-lived endogenous serum protein albumin as an siRNA carrier. We synthesized siRNA conjugated to a diacyl lipid moiety (siRNA-L2), which rapidly binds albumin in situ. siRNA-L2, in comparison with unmodified siRNA, exhibited a 5.7-fold increase in circulation half-life, an 8.6-fold increase in bioavailability, and reduced renal accumulation. Benchmarked against leading commercial siRNA nanocarrier in vivo jetPEI, siRNA-L2 achieved 19-fold greater tumor accumulation and 46-fold increase in per-tumor-cell uptake in a mouse orthotopic model of human triple-negative breast cancer. siRNA-L2 penetrated tumor tissue rapidly and homogeneously; 30 min after i.v. injection, siRNA-L2 achieved uptake in 99% of tumor cells, compared with 60% for jetPEI. Remarkably, siRNA-L2 achieved a tumor:liver accumulation ratio >40:1 vs. <3:1 for jetPEI. The improved pharmacokinetic properties of siRNA-L2 facilitated significant tumor gene silencing for 7 d after two i.v. doses. Proof-of-concept was extended to a patient-derived xenograft model, in which jetPEI tumor accumulation was reduced fourfold relative to the same formulation in the orthotopic model. The siRNA-L2 tumor accumulation diminished only twofold, suggesting that the superior tumor distribution of the conjugate over nanoparticles will be accentuated in clinical situations. These data reveal the immense promise of in situ albumin targeting for development of translational, carrier-free RNAi-based cancer therapies.

Formation of stable, long-circulating siRNA polyplexes is a significant challenge in translation of intravenously-delivered, polymeric RNAi cancer therapies. Here, we report that siRNA hydrophobization through conjugation to palmitic acid (siPA) improves stability, in vivo pharmacokinetics, and tumor gene silencing of PEGylated nanopolyplexes (siPA-NPs) with balanced cationic and hydrophobic content in the core (relative to the analogous polyplexes formed with unmodified siRNA, si-NPs). Hydrophobized siPA loaded into the NPs at a lower charge ratio (N+:P−) relative to unmodified siRNA, and the siPA-NPs had superior resistance to siRNA cargo unpackaging in comparison to si-NPs upon exposure to the competing polyanion heparin and serum. In vitro, siPA-NPs increased uptake in MDA-MB-231 breast cancer cells (100% positive cells vs. 60% positive cells) but exhibited equivalent silencing of the model gene luciferase relative to si-NPs. In vivo in a murine model, the circulation half-life of intravenously-injected siPA-NPs was double that of si-NPs, resulting in a >2-fold increase in siRNA biodistribution to orthotopic MDA-MB-231 mammary tumors. The increased circulation half-life of siPA-NPs was dependent upon the hydrophobic interactions of the siRNA and the NP core component and not just siRNA hydrophobization, as siPA did not contribute to improved circulation time relative to unmodified siRNA when delivered using polyplexes with a fully cationic core. Intravenous delivery of siPA-NPs also achieved significant silencing of the model gene luciferase in vivo (~40% at 24 hours after one treatment and ~60% at 48 hours after two treatments) in the murine MDA-MB-231 tumor model, while si-NPs only produced a significant silencing effect after two treatments. These data suggest that stabilization of PEGylated siRNA polyplexes through a combination of hydrophobic and electrostatic interactions between siRNA cargo and the polymeric carrier improves in vivo pharmacokinetics and tumor gene silencing relative to conventional formulations comprising only electrostatic interactions.

siRNA delivered from ROS-degradable tissue engineering scaffolds promotes diabetic wound healing in rats. Porous poly(thioketal-urethane) (PTK-UR) scaffolds implanted in diabetic wounds locally deliver siRNA that inhibits the expression of prolyl hydroxylase domain protein 2 (PHD2), thereby increasing the expression of pro-growth genes and increasing vasculature, proliferating cells, and tissue development in diabetic wounds.

The discovery of RNAi in the late 1990s unlocked a new realm of therapeutic possibilities by enabling potent and specific silencing of theoretically any desired genetic target. Better elucidation of the mechanism of action, the impact of chemical modifications that stabilize and reduce nonspecific effects of siRNA molecules, and the key design considerations for effective delivery systems has spurred progress toward developing clinically-successful siRNA therapies. A logical aim for initial siRNA translation is local therapies, as delivering siRNA directly to its site of action helps to ensure that a sufficient dose reaches the target tissue, lessens the potential for off-target side effects, and circumvents the substantial systemic delivery barriers. While topical siRNA delivery has progressed into numerous clinical trials, an enormous opportunity also exists to develop sustained-release, local delivery systems that enable both spatial and temporal control of gene silencing. This review focuses on material platforms that establish both localized and controlled gene silencing, with emphasis on the systems that show most promise for clinical translation.