Supramolecular self-assembled nanoparticles mediate oral delivery of therapeutic TNF-α siRNA against systemic inflammation.

Intervention of the inflammation cascade with tumor necrosis factor-a (TNF-a) monoclonal antibodies or receptors represents a major approach in clinical immunotherapy against inflammatory diseases, which however, often suffers from high cost, autoimmunity to antibodies, and various side effects. siRNA-mediated RNA interference (RNAi) has recently emerged as a potent modality in regulating gene expression by suppressing mRNA translation; its high efficiency and specificity has made it a promising treatment paradigm for TNF-a-mediated inflammatory disorders. The therapeutic potential of siRNAwas recently exemplified by a report of attenuating systemic inflammation by targeting orally delivered Map4k4 siRNA to gut-associated macrophages (GAMs). Owing to the infiltration of GAMs to systemic reticuloendothelial tissues, Map4k4 siRNA-mediated TNF-a knockdown in GAMs extended to other tissues and thus induced systemic anti-inflammatory effects. Despite its biological potency, the clinical potential of orally delivered siRNA has been hampered by the lack of efficient delivery technologies. siRNA is anionic, hydrophilic, and easily degraded by nucleases in the body. As such, it cannot survive the harsh conditions of the gastrointestinal (GI) tract or effectively penetrate the intestinal epithelia or membranes of target cells. Hence, an effective carrier is needed not only to protect siRNA from degradation in the GI tract but also to improve the intestinal absorption as well as transfection in macrophages, thereby maximizing the in vivo RNAi efficiency and anti-inflammatory effect of orally delivered siRNA. To address the dearth of techniques for oral siRNA delivery, here we report the design of supramolecular selfassembled nanoparticles (SSNPs) that are able to overcome the absorption and transfection barriers posed by intestinal macrophages and exhibit remarkable in vivo oral RNAi efficiency. SSNPs were constructed through the electrostatic and hydrophobic self-assembly of several rationally designed or selected building blocks, including oleyl trimethyl chitosan (OTMC), poly(g-(4-(((2-(piperidin-1-yl)ethyl)amino)methyl)benzyl-l-glutamate) (PVBLG-8), oleyl-PEGmannose (OPM), oleyl-PEG-cysteamine (OPC), sodium tripolyphosphate (TPP), and TNF-a siRNA (Figure 1A). Trimethyl chitosan (TMC) is an effective intestinal absorption enhancer as well as transfection reagent. As a more hydrophobic derivative of TMC, OTMCwith the oleyl conjugation ratio of 20.3% was synthesized and expected to display further enhanced permeation-enhancing and gene transfection capabilities. PVBLG-8 is a cationic a-helical polypeptide we recently developed which exhibits potent membrane activities and gene delivery efficiencies. We incorporated PVBLG-8 (degree of polymerization (DP)= 195) into the SSNPs in attempt to promote the cellular internalization and endosomal escape. PVBLG-8 adopts a superstable a-helical structure between pH 2 and 9 (Figure S2 in the Supporting Information), making it an ideal material for oral delivery applications as it is able to maintain helical secondary structure after passing through both stomach (acidic) and intestinal (weakly basic) environments. Incorporating OPM is expected to target SSNPs to enterocytes and macrophages that express mannose receptors, thus improving the intestinal absorption and macrophage uptake. OPC will improve the mucoadhesion of SSNPs by forming disulfide bonds with mucin glycoproteins enriched in the intestinal mucosa and on cell surfaces. OPM and OPC were synthesized by conjugation methods and their structures were confirmed by MALDI-TOF mass spectrometry (Figures S3 and S4 in the Supporting Information). The 2’-Omethyl-modified siRNA duplex against TNF-a was reported previously, wherein the 2’-O-methyl modification on the antisense strand eliminates off-target effects, minimizes nonspecific immune responses, and improves siRNA stability. With these building blocks, SSNPs are expected to promote the intestinal absorption of TNF-a siRNA, facilitate RNAi in macrophages, and thus mediate systemic TNF-a knockdown against lipopolysaccharide (LPS)-induced hepatic injury. For the construction of SSNPs, TNF-a siRNA was condensed with cationic OTMC and PVBLG-8 through [*] Dr. L. Yin, Z. Song, K. H. Kim, N. Zheng, C. Yao, I. Chaudhury, Dr. H. Tang, Prof. Dr. J. Cheng Department of Materials Science and Engineering University of Illinois, Urbana-Champaign 1304 West Green Street, Urbana, IL 61801 (USA) E-mail: [email protected] Homepage: http://cheng.matse.illinois.edu/