Alkane-modified short polyethyleneimine for siRNA delivery

https://doi.org/10.1016/j.jconrel.2011.11.030Get rights and content

Abstract

RNA interference (RNAi) is a highly specific gene-silencing mechanism triggered by small interfering RNA (siRNA). Effective intracellular delivery requires the development of potent siRNA carriers. Here, we describe the synthesis and screening of a series of siRNA delivery materials. Short polyethyleneimine (PEI, Mw 600) was selected as a cationic backbone to which lipid tails were conjugated at various levels of saturation. In solution these polymer–lipid hybrids self-assemble to form nanoparticles capable of complexing siRNA. The complexes silence genes specifically and with low cytotoxicity. The efficiency of gene knockdown increased as the number of lipid tails conjugated to the PEI backbone increased. This is explained by reducing the binding affinity between the siRNA strands to the complex, thereby enabling siRNA release after cellular internalization. These results highlight the importance of complexation strength when designing siRNA delivery materials.

Introduction

Since its discovery by Fire and Mello [1], RNA interference (RNAi) has been studied pre-clinically for its potential to silence genes that are associated with cancer [2], [3], infection and inflammation [4], respiratory and neurological disorders [5], [6], and autoimmune diseases [7]. Clinical investigations of the therapeutic potential of RNAi are currently under evaluation [8], [9]. However, additional advances are needed to make broad application of siRNA. This is explained by a series of hurdles that make delivery difficult. Carriers must bind sufficient amounts of siRNA, protect it from degradative enzymes, cross the anionic cell membrane and release the payload into the cytoplasm [3]. Moreover, the carrier must perform these tasks without damaging the cell. To overcome these hurdles, diverse delivery systems have been developed. Some of the most effective have been synthesized from cationic polymers, lipids, and lipid-like materials [10], [11], [12], [13].

Amine-based cationic materials have been effective in binding and delivering nucleic acids. Of these, one of the most rigorously studied is polyethyleneimine (PEI). PEI was first used as a delivery vehicle for long nucleic strands due to its capacity to form condensed and stable structures with DNA and RNA [14]. These polyplexes are taken up by cells via endocytotic pathways [15], [16] before being released to the cytosol [16], [17]. However, toxicity and efficacy have both been reported to increase as the molecular weight of PEI increases [18], [19], [20]. More specifically, 25 kDa molecular weight PEI, which has been used in many studies, is effective, but is toxic to multiple cell lines [19]. Several approaches have been employed to reduce the toxicity associated with high molecular weight PEI [21], [22]. For example, biodegradable bonds were inserted into the polymer backbone [23], [24], [25]. It was proposed that the long polymer would deliver nucleic payloads effectively and then degrade into well-tolerated short PEI fragments [26]. Another approach is to conjugate chemical groups, such as lipids, to high Mw PEI, thereby reducing toxicity [27], [28], [29], [30].

Here, we sought to study the ability of low molecular-weight PEI (0.6 kDa) modified with lipid tails to act as siRNA delivery materials. Our hypothesis was that these short lipid-polymer hybrids would be well tolerated. Furthermore, we tested the effect of the carriers' molecular structure on their capacity to induce RNAi. For this, lipid tails were conjugated to the amine backbone at increasing levels of saturation, and the expression of a targeted gene was measured. We found that increasing the degree of saturation, up to a point at which the complex was not stable in solution, was most effective for siRNA delivery.

Section snippets

Lipid-modified PEI

Polyethyleneimine, ethylendiamine end capped, Mw 600 (Aldrich, Natick, MA) was conjugated to 1,2-Epoxyoctadecane, MW 268.49 (Alfa Aesar, Ward Hill, MA) via an epoxide ring-opening reaction as shown schematically in Fig. 1A and described previously in detail in [11]. In brief, epoxide-terminated C18 alkanes were dissolved in 200-proof ethanol and reacted with PEI at 90 °C for 48 h. The epoxide:PEI molar ratios were varied from 0:1 up to 3:1; above this ratio the product formed unstable complexes

Results

This study examines the potential of alkane-modified low molecular weight PEI as siRNA delivery materials.

Sixteen-carbon-long lipid tails were conjugated to PEI at increasing levels of saturation. When immersed in aqueous solutions these compounds self-assembled to form nanoparticles. As the number of tails conjugated to the PEI backbone increased, the size of the self-assembled particles increased too (Fig. 2A). When having, on average, between one and two conjugated lipid tails per PEI

Discussion

RNAi has broad clinical applications. Realizing the full potential of RNAi will require tailoring new delivery materials. Here, we tested the ability of short lipid-modified PEI to act as siRNA delivery materials.

Sixteen-carbon-long lipid tails were conjugated to a 0.6 kDa PEI backbone at various levels of saturation. While the compounds were well tolerated, capable of complexing siRNA and facilitated cellular uptake, we found a structure–function relationship to occur. As the number of lipid

Acknowledgements

AS was supported by the Misrock Foundation. This work was supported by MIT-Harvard Center for Cancer Nanotechnology Excellence Grant U54 CA151884 from the National Cancer Institute and by NIH grant # EB000244. JD thanks the NDSEG, NSF and MIT Presidential Fellowships. We thank Natalia Schiller from the Biopolymers and Proteomics Core Facility at the David H. Koch Institute for Integrative Cancer Research at MIT for helping with mass spectrometry. The authors thank Alnylam Pharmaceuticals for

References (39)

  • A. Fire et al.

    Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans

    Nature

    (1998)
  • F. Takeshita et al.

    Therapeutic potential of RNA interference against cancer

    Cancer Sci.

    (2006)
  • K.A. Whitehead et al.

    Knocking down barriers: advances in siRNA delivery

    Nat. Rev. Drug Discov.

    (2009)
  • B.C. Ponnappa

    siRNA for inflammatory diseases

    Curr. Opin. Investig. Drugs

    (2009)
  • O.B. Garbuzenko et al.

    Intratracheal versus intravenous liposomal delivery of siRNA, antisense oligonucleotides and anticancer drug

    Pharm. Res.

    (2009)
  • G.J. Prud'homme

    Gene Therapy of Autoimmune Diseases

    (2005)
  • M.E. Davis et al.

    Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles

    Nature

    (2010)
  • A. Cervantes et al.

    Phase I dose-escalation study of ALN-VSP02, a novel RNAi therapeutic for solid tumors with liver involvement

    J. Clin. Oncol.

    (2011)
  • A. Schroeder et al.

    Lipid-based nanotherapeutics for siRNA delivery

    J. Intern. Med.

    (2010)
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