Lipophilic siRNA targets albumin in situ and promotes bioavailability, tumor penetration, and carrier-free gene silencing

Abstract

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.

Recent Advances in the Design of Self‐Delivery Amphiphilic Drugs and Vaccines

“…Beyond the linker length and composition, the optimization of a particular amphiphilic drug also includes the amphiphilic modification that tunes the overall hydrophilic/lipophilic balance. For example, lipid composition and hydrophobicity have been shown to control the protein binding and biodistribution of lipid‐conjugate siRNA . Taken together, the above examples demonstrated the structures and physicochemical properties of amphiphilic drugs are highly tunable and can be tailored for targeted drug delivery.…”

“…Since lipid molecules exhibit strong affinity toward albumin and lipoproteins, lipid‐drug conjugates are widely used to hijack the trafficking pathways of serum proteins. The affinity and specificity of protein binding are primarily determined by the structures and hydrophobicity of the lipid …”

“…While cholesterol‐siRNA binds to lipoprotein and achieves organ/tissue targeting via lipoprotein receptors, diacyl lipid modified siRNA binds albumin in situ and hijacks the extracellular and intracellular albumin trafficking pathways . Compared to unmodified siRNA, lipophilic siRNA exhibited significantly longer circulation time in blood and increased tumor penetration and accumulation.…”

“…Compared to unmodified siRNA, lipophilic siRNA exhibited significantly longer circulation time in blood and increased tumor penetration and accumulation. The improved pharmacokinetic characteristics resulted in significant tumor gene silencing in a xenograft model in mice . This albumin‐hitchhiking approach is simple and offers tremendous promise for the development of siRNA‐based drug in cancer.…”

Hydrophobicity drives the systemic distribution of lipid-conjugated siRNAs via lipid transport pathways

“…The second major class of molecular siRNA conjugates are lipids, which have been shown to enhance circulation time and promote local and systemic delivery and efficacy. Cholesterol-modified siRNA, one of the first reported lipid conjugates, silences liver apolipoprotein B (ApoB) expression at high doses (50 mg kg -1 ), while α-tocopherol and fatty acid conjugates have also shown potential for liver delivery and gene silencing [5][6][7] . Productive internalization of cholesterol-conjugated siRNAs hinges on its interactions with circulating lipoproteins and tissue receptors 6 .…”