the development of an in vitro assay to screen lipid based nanoparticles for sirna delivery
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Journal of Controlled Release 174 (2014) 714
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Journal of Controlled Release
j ourna l homepage: www.e lsev ie r .com/ locate / jconre lThe development of an in vitro assay to screen lipid based nanoparticlesfor siRNA deliveryYe Zhang , Leticia Arrington, David Boardman, Jared Davis, Yan Xu, Katie DiFelice, Steve Stirdivant,Weimin Wang, Brian Budzik, Jack Bawiec, James Deng, Greg Beutner, Darla Seifried, Matthew Stanton,Marian Gindy, Anthony LeoneDepartment of RNAi Therapeutics, Merck Research Laboratories, 770 Sumneytown Pike, West Point, 19486, USA Corresponding author. Tel.: +1 215 652 2401; fax: +E-mail address: firstname.lastname@example.org (Y. Zhang).
0168-3659/$ see front matter 2013 Elsevier B.V. All rihttp://dx.doi.org/10.1016/j.jconrel.2013.11.006a b s t r a c ta r t i c l e i n f oArticle history:Received 28 August 2013Accepted 4 November 2013Available online 12 November 2013
Keywords:siRNA deliveryLipid nanoparticlesSerum stabilityEndosome escapesiRNA releaseIn order to rapidly screen and select lead candidates for in vivo evaluation of lipid nanoparticles (LNPs) for sys-temic small interfering RNA (siRNA) delivery, an in vitro assay amenable to high-throughput screening (HTS)is developed. The strategy is tomimic the in vivo experience of LNPs after systemic administration, such as inter-actions with serum components, exposure to endosomal pH environments, and interactions with endosomalmembrane lipids. It is postulated that the amount of siRNA released from LNPs after going through these treat-ments can be used as a screening tool to rank order the effectiveness of siRNA delivery by lipid nanoparticlesin vivo. LNPs were incubated with 50% serum from different species (i.e. mouse, rat, or rhesus) at 37 C. Theresulting samples were then reacted with anionic, endosomal-mimicking lipids at different pHs. The amount ofsiRNA released from LNPs was determined using spectrophotometry employing the fluorescent indicator SYBRGold. Our results indicated that the amount of siRNA liberated was highly dependent upon the species ofserum used and the pH to which it was exposed. LNPs treated with mouse serum showed higher levels ofsiRNA release, as did those subjected to endosomal pH (6.0), compared to physiological pH. Most interestingly,a good correlation between the amount of siRNA released and the in vivo efficacy was observed. In conclusion,an in vitro siRNA release assay was developed to screen and rank order LNPs for in vivo evaluation.
2013 Elsevier B.V. All rights reserved.1. Introduction
Since the discovery of RNA interference (RNAi) by Fire and Mello in1998  and the demonstration of synthetic small interfering RNA(siRNA) mediated RNAi by Tuschl and colleagues in 2001 , therapeu-tic development of siRNA drugs to silence disease genes has been pur-sued extensively by the pharmaceutical industry and academicresearchers. Similar to gene therapy, efficient delivery of siRNA to targetcells and tissues with minimal toxicity is the major challenge for thesuccessful development of siRNA based therapeutics. Various deliveryapproaches, including lipid or polymer based nanoparticles, and siRNAconjugates have been investigated . For systemic delivery ofsiRNA, one of themost advanced delivery technologies is lipid nanopar-ticles (LNPs).
Typically, LNPs consist of 4 to 5 components: cationic lipid, neutralhelper lipid (s), polyethylene glycol (PEG) conjugated lipid, and siRNA[4,6,7]. Cationic lipids such as 3-dimethylamino-2-(Cholest-5-en-3oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA) and 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA)  are1 215 652 7310.
ghts reserved.designed to have an apparent pKa of 6.58.0 and to produce neutralparticles in blood circulation at physiological pH. LNPs are expected tobe protonated at endosomal pH 6.0 to facilitate interactions with thenegatively charged endosomal lipids, and the subsequent release ofsiRNA into the cytoplasm . Strong positive charge in general circula-tion increases the potential of LNP opsonization, reticuloendothelialsystem (RES) clearance, immune response in the form of cytokine in-duction and complement activation, as well as other toxicities [6,9,10].Therefore, a particle with pKa ~ 7 might be ideal. Neutral helper lipidssuch as cholesterol and phosphatidylcholine (PC) confer rigidity andstability to the bilayer . The PEG lipid provides stealthness andstability to LNPs; however, it may also interfere with interactions be-tween particle and target cells, hindering endosome escape andinhibiting transfection [14,15]. Therefore, it is designed to diffuse tosome extent when interacting with serum components in circulation,to maintain the balance between stealth, stability, and cell transfection.
After intravenous administration, LNPs are exposed to various con-stituents found in whole blood, namely serum proteins, lipoproteins,and blood cells (Fig. 1).
Interactions between LNPs and these elements have been shown tochange the particle size and/or surface properties of LNPs [9,2025]. Fol-lowing circulation, LNPs accumulate in the liver cells, either passively or
Fig. 1. Illustration of the barriers and potential LNP transformations after intravenous ad-ministration.Adapted and reproduced with permission from Journal of Medicinal Chemistry. 2010, 22,7888. Copyright 2010 American Chemical Society.
8 Y. Zhang et al. / Journal of Controlled Release 174 (2014) 714through receptormediated endocytosis. Serum component Apolipopro-tein E (ApoE) has been reported as an endogenous targeting ligand forhepatocyte delivery of ionizable LNP . The pHwithin the endosomesdecreases from 7.4 to 5 or 6within 1020 min. The ability of LNP encap-sulated siRNA to escape early endosomes is critical to efficacy, failureresults in residence in late stage endosomes or lysosomes, where degra-dation occurs.
Considering the cost and labor associated with testing delivery vehi-cles in animals, many in vitro assays have been proposed and used byresearchers to screen lipid based delivery vectors for oligonucleotides.These include cell based transfection, serum stability, and/or endosomeescape propensity [8,20,24,2731]. Most recently, a multiparametricapproach was also proposed to evaluate LNP for siRNA delivery .However, a discrepancy was found between in vitro and in vivo efficacydata [32,33] and none of these assays have proven good predictors forin vivo efficacy. This is mainly because the in vitro environment fails toadequately approximate the in vivo situation. For example, in culture,cells were treated with delivery vehicles either in the presence or ab-sence of 10% fetal bovine serum (FBS). This value is approximately onefifth of the physiological serum concentration (50% v/v). Similarly,although the serum stability assay has been conducted at a higher,more relevant concentration of 50% serum, it only evaluates LNP stabil-ity in serum. It cannot provide information on endosome escape andsiRNA release [27,34]. In the present study, we report a new in vitroassay for LNP screening which better emulates the barriers for systemicdelivery. By mimicking the in vivo environment LNPs experience aftersystemic administration, such as interactions with serum componentsand exposure to endosomal pHand lipids, greater predictive capabilitiesare garnered.
2. Materials and methods
1,2-Dioleoyl-sn-glycero-3-(phosphor-L-serine) (DOPS), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (NBD-PE) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissaminerhodamine B sulfonyl) (N-Rh-PE) were purchased from AvantiPolar Lipids (Alabaster, AL). Cholesterol, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)and1,2-distearoyl-sn-glycero-3-phosphocholine(DSPC) were purchased from Northern Lipids Inc. (Burnaby, BC, Canada).Poly(ethylene glycol)2000dimyristoylglycerol (PEGDMG)wasmanufac-tured by NOF Corporation (Kanagawa, Japan). All cationic lipids used inthe study including CLinDMA and DLinDMA were synthesized at Merck(West Point, PA). Mouse, rat, and rhesus serum were obtained fromBioreclamation LLC (East Meadow, NY). SYBR Gold was acquired fromInvitrogen (Carlsbad, CA). Chemically modified ApoB siRNA, targetingthe mRNA transcript for the ApoB gene (accession # NM_019287), wassynthesized at Merck (Rahway, NJ). The primary sequence (presentedin the 5 to 3 direction) and chemical modification pattern of the ApoBsiRNA strands are as follows:
Sense strand: iB; omeC; omeU; omeU; omeU;fluA;fluA; omeC;fluA;fluA; omeU; omeU; omeC; omeC; omeU; fluG; fluA; fluA; fluA;omeU; dTsdT-iB.Antisense strand: rA; srU; srU; someU; omeC; fluA; fluG; fluG; fluA;fluA; omeU; omeU; fluG; fluU; omeU; fluA; fluA; fluA; fluG; omeU;
Nucleotide modifications are represented as deoxy (d), 2-fluoro(flu), 2-o-methyl (ome), and ribo (r), with abbreviations immediatelypreceding the altered base. Phosphorothioate linkages replacing phos-phate bonds are represented by a subscript (s) between the two nucle-otides linked. Additionally, the sense strand is blocked with an invertedabasic nucleotide on the 5 and 3 ends (iB). All chemicals were ofreagent grade or higher quality.
2.2. Preparation of siRNA encapsulated LNPs and anionic liposomes
siRNA encapsulated LNPs were prepared as described previously byAbrams et al. . LNP A contains CLinDMA, cholesterol, and PEGDMGat a molar ratio of 68:30:2 and a nitrogen/phosphate (N/P) ratio of2.8. LNP B an