NanomedicineVol. 5, No. 10 EditorialFree AccessTargeted delivery of RNAi therapeutics for cancer therapyJun Li & Leaf HuangJun LiDivision of Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USASearch for more papers by this author & Leaf Huang† Author for correspondenceDivision of Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. Search for more papers by this authorEmail the corresponding author at Leafh@unc.eduPublished Online:14 Dec 2010https://doi.org/10.2217/nnm.10.124AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinkedInRedditEmail Keywords: cancer therapynanoparticlesiRNASince the discovery of RNA interference (RNAi) [1], siRNA) has become the focus of attention for pharmaceutical development. siRNA is more potent than antisense oligonucleotides owing to the endogenous mechanism of action catalyzed by the RNA-induced silencing complex [2]. Many oncogenic targets involved in survival, specifically antiapoptosis, angiogenesis and drug resistance, have been tested for siRNA-mediated cancer therapy. However, siRNA can only become an anticancer agent when it is specifically and effectively delivered to the target cells in vivo[3]. An ideal siRNA delivery vehicle for cancer therapy must be able to evade the reticuloendothelial system to be effectively taken up by the tumor cells and to escape from the endosome after endocytosis. The existence of a leaky vasculature in most of the solid tumors supports the enhanced permeability and retention effect for nanoparticle (NP)-mediated delivery of anticancer agents, for example the successful Doxil® formulation of doxorubicin. A well-designed NP of less than 200 nm in diameter is suitable for siRNA delivery to the tumor. NPs composed of different materials have been developed for siRNA delivery. Two major classes of biomaterials have been employed for siRNA delivery. They will be discussed separately in this article.Lipid base NPs for targeted siRNA deliveryCationic lipids have been widely used for siRNA delivery in vitro. Many commercially available transfection reagents are made of cationic lipids but these reagents usually do not support in vivo delivery owing to the presence of excess cationic charges in the lipoplex. Ideally, siRNA should be entrapped in a large quantity inside the unilamellar liposomes that are protected with a polyethylene glycol (PEG) brush layer on the surface. The PEG brush effectively reduces blood protein adsorption to the NP and reduces its uptake by the reticuloendothelial system [4]. A targeting ligand is further tethered at the distal end of the PEG chain for specific binding and internalization mediated by tumor-specific receptors. Lipids with an ionizable headgroup having a pKa of approximately 6 are particularly attractive. siRNA can associate with the lipid at low pH because the lipid is protonated and positively charged. Being entrapped at low pH, substantial amounts of siRNA are still liposome-associated after the pH returns to neutral. The lipid will again be cationic in the acidic endosome. It is well known that cationic lipid binds with anionic lipid to form inverted micelles, or the HII phase, which is a nonbilayer structure [5]. Lipids with bulky hydrocarbon chains are prone to assume such structure [6]. Several lipids containing a tertiary amine headgroup (pKa ∼6) and two hydrocarbon chains with two cis double bonds (bulky) have been designed and used to deliver siRNA [7]. The activity of the ionizable lipid NPs for siRNA delivery to the liver hepatocytes depends on the circulating ApoE, which facilitates an avid uptake of the NP via the low-density lipoprotein receptor [8]. The 50% effective dose (ED50) of siRNA for silencing factor VII, which is exclusively produced in the liver, was as low as 0.01 mg/kg in rodents and 0.1 mg/kg in nonhuman primates. No noticeable toxicity at these low doses was reported. N-acetyl galactosamine can also be used to target the iLNPs to the liver via the galactose receptor. This is a remarkable achievement, although the formulation has not yet been used for cancer therapy.The stable nucleic acid lipid particle is a similar ionizable lipid base formulation developed by the same group for systemic siRNA administration [9]. The original study focused on hepatic cells, which demonstrated the downregulation of ApoB in the liver with a dose of 2.5 mg/kg in nonhuman primates [10]. High therapeutic doses, however, usually cause some type of immune response. Recently, a clinical trial was terminated owing to the immunotoxicity revealed in the trial [101].In an alternative approach, a combinatorial library of lipid-like materials was developed and described with the term ‘lipidoids’ [11]. The one-step synthetic scheme provided some choice of cationic lipid for efficient transfection of siRNA [12]. The selected cationic LNPs was able to achieve siRNA-mediated gene silencing effect for hepatic cell at a dose of 0.01 mg/kg in mice and 0.03 mg/kg in nonhuman primates [13]. This formulated NP is again a remarkable achievement, although the toxicity of these lipidoids has not been thoroughly studied.Instead of application in hepatic cells, a preclinical study of 2´-O-methyl-modified siRNA formulated in LNPs was reported to show potent antitumor efficacy in both hepatic and subcutaneous tumor models [14]. The siRNA is targeted to the essential cell-cycle proteins polo-like kinase 1 (PLK1) and kinesin spindle protein (KSP) in mice. A very mild immune response was observed in mice with intravenous twice-weekly administration at a dose of 2 mg/kg. A Phase I clinical trial was launched to determine the safety, tolerability, pharmacokinetics and pharmacodynamics of intravenous LNPs in patients with advanced solid tumors involving the liver [102].Another lipid-base NP for cancer therapy is known as lipid/protamine/DNA (LPD) formulation, which was originally developed for plasmid DNA delivery [15]. LPD was engineered by combining cationic liposomes and polycation to condense plasmid DNA. When mixed, the components spontaneously rearranged to form a virus-like structure with the condensed DNA core located inside the lipid membranes [16]. Recently, siRNA was entrapped in the core of LPD and the surface of the particle was wrapped with cationic lipid and PEG. Anisamide was linked to the terminal of PEG as a targeting ligand of s receptor, which is overexpressed in many human tumor cells [17]. The most important feature of the modified LPD is its ability to evade the reticuloendothelial system, which is due to the fact that a high amount (∼10 mol%) of PEG chains could be grafted on the surface to form a brush protective layer on the NPs [18]. The xenograft model demonstrated that LPD NPs could deliver 60–80% of intravenous injected siRNA per gram of tissue weight to the lung cancer xenograft and effectively silence the expression of EGF receptor in the entire tumor. Two consecutive intravenous administrations significantly reduced the lung metastasis of melanoma (70–80%) at a relatively low dose (0.45 mg/kg) [19]. To reduce the potential toxicity of calf thymus DNA in the formulation, liposome–polycation–hyaluronic acid was developed to deliver siRNA systemically into the tumor, which resulted in very little immunotoxicity in a wide dose range (0.15–11.2 mg/kg) [20].To improve the efficiency of LPD formulation, an acid-sensitive material, calcium phosphate, was investigated to replace the stable protamine/DNA complex. The formulation contained a PEGylated membrane similar to that of LPD, but the core that encapsulated siRNA is replaced with calcium phosphate amorphous nanoprecipitate [21]. The proposed mechanism of action is described as follows. After entering the endosome, calcium phosphate dissolves rapidly in acidic pH, increasing the osmotic pressure, and resulting in swelling and rupture of the endosome to release siRNA into the cytoplasm. The new formulation is known as lipid–calcium–phosphate and the ED50 is similar to LPD formulation with very mild immune toxicity.There are several reports using neutral phospholipids to formulate siRNA (neutral nanoliposome) [22]. The formulation has a potent activity in silencing Epha2, an oncogene involved in ovarian cancer progression. Impressive antitumor activity was shown by intravenous administration of the siRNA formulation. The study has already been advanced to a Phase I clinical trial, but it is not clear how siRNA could be entrapped with high efficiency in the neutral liposomes.Polymer-based NPs for targeted siRNA deliveryIn addition to cationic lipid, positively charged polymer is another type of material for the delivery of nucleic acid. The complex is termed polyplex. Polyethyleneimine, a highly cationically charged polymer, is a potent in vitro transfection reagent and has been used to deliver VEGF siRNA for cancer therapy [23]. With grafted PEG, the complex spontaneously forms micelles, which have a polyethyleneimine–siRNA core wrapped with PEG. Tail vein injection of 1.5 nmol (1.1 mg/kg) siRNA in the micelles significantly suppressed tumor growth in a xenograft tumor model. In addition, arg–gly–asp peptide-labeled chitosan NP was employed as a vehicle for siRNA delivery, targeting to integrins overexpressed in the tumor cells and the tumor endothelial cells. The NP significantly increased siRNA delivery in orthotopic animal models of ovarian cancer and the injected dose of siRNA was 0.15 mg/kg [24].Other cationic polymer-based NPs for siRNA delivery consist of a cyclodextrin-containing polymer for binding with siRNA, a PEG as steric stabilization agent, and transferrin as a targeting ligand for cancer cells [25]. The cationic polymer with siRNA stays inside of the NP and the cyclodextrin serves as an adapter, where different PEG and the target ligand conjugated to adamantane can be ‘plugged’ into the complex. The targeted NPs that systematically deliver siRNA against the EWS-FLI1 gene could inhibit tumor growth in a murine model of metastatic Ewing’s sarcoma. The most exciting characteristic is that the resulting siRNA NPs (2.5 mg/kg) did not induce immune response. When administered to cynomolgus monkeys at doses of 3 and 9 mg siRNA/kg, the NPs were well tolerated. However, elevated levels of blood urea nitrogen and creatinine were observed, indicating kidney toxicity when the dose was 27 mg siRNA/kg. This targeted NP is currently in a clinical trial with patients with solid tumors [26]. Tumor biopsies from melanoma patients in this trial showed the presence of intracellularly localized NPs. Moreover, expected mRNA degradation fragments by the RNA-induced silencing complex were observed in the biopsies.Recently, several multifunctional polyconjugates have been developed to avoid the off-target delivery and facilitate the escape of siRNA from the endosome. For example, a pH-sensitive polyconjugate was attached to siRNA through a disulfide linkage [27]. The complex was shielded by PEG and modified with a hepatocyte-targeting ligand. After entering the endosome, the polymer shed both the targeting ligand and the PEG to expose its multiple amino groups to destabilize the endosome membrane. siRNA was released into the cytoplasm due to its reductive environment. Over 80% of the ApoB expression was downregulated in vivo by this method with a siRNA dose of 2.5 mg/kg. Another reducible polyconjugate was also prepared by modifying the siRNA with phosphothioethanol portion via a disulfide bond [28]. Although no tumor model was tested to date, the multifunctional polyconjugate is probably safer than other polymers, but its activity appears to be significantly lower than that of ionizable or cationic LNPs.In addition to the synthetic polymers, biodegradable and biocompatible proteins have been employed as biopolymers for siRNA delivery. For example, a protamine–antibody fusion protein was designed to deliver siRNA to HIV-infected or envelope-transfected cells [29]. Intratumoral or intravenous injection of the antibody–siRNA complex into mice showed a targeted delivery to melanoma cells. Inhibition of subcutaneous B16F10 tumor growth was also observed. Atelocollagen, a chemically modified collagen, was used to deliver siRNA to bone metastasis of prostate cancer [30]. It is interesting to note that the siRNA–atelocollagen complex could be detected in the tumor 24 h after injection and the target silencing lasted for at least 3 days.ConclusionNonviral vectors for delivering nucleic acid have improved dramatically in the last decade. Their activity is comparable with that of viral vectors in many cases. Their clinical development has also been accelerated recently. Despite these encouraging advances, toxicity of nonviral vectors has not been adequately evaluated, especially in clinical trials. Cationic lipids and polymers induce a rapid increase in cellular reactive oxygen species [31]. This is a primary signal in macrophages and other innate immune cells to secrete proinflammatory cytokines. In addition, siRNA is not without its own immunostimulating toxicity, which often leads to off-target effects [32]. Fortunately, various chemical modifications of siRNA tend to limit this type of unwanted toxicity [33]. Still, the best strategy is to develop potent delivery formulation such that only a small dose is needed to silence the target gene for activity to be therapeutic. Toxicity would not be an issue at low doses. In addition, it will significantly decrease the cost of the drug because both siRNA and the formulation of lipid or polymer can be very expensive. Nevertheless, the field of siRNA delivery for cancer therapy has already come a long way. It is not hard to see an even brighter future on the horizon.Financial & competing interests disclosureThe authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.No writing assistance was utilized in the production of this manuscript.Bibliography1 Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC: Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.Nature391,806–811 (1998).Crossref, Medline, CAS, Google Scholar2 Zamore PD, Tuschl T, Sharp PA, Bartel DP: RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell101,25–339 (2000).Crossref, Medline, CAS, Google Scholar3 Whitehead KA, Langer R, Anderson DG: Knocking down barriers: advances in siRNA delivery. Nat. Rev. Drug Discov.8,129–138 (2009).Crossref, Medline, CAS, Google Scholar4 Harris JM, Chess RB: Effect of pegylation on pharmaceuticals. Nat. Rev. Drug Discov.2,214–221 (2003).Crossref, Medline, CAS, Google Scholar5 Xu Y, Szoka FC Jr: Mechanism of DNA release from cationic liposome/DNA complexes used in cell transfection. Biochemistry35,5616–5623 (1996).Crossref, Medline, CAS, Google Scholar6 Hafez IM, Maurer N, Cullis PR: On the mechanism whereby cationic lipids promote intracellular delivery of polynucleic acids. Gene Ther.8,1188–1196 (2001).Crossref, Medline, CAS, Google Scholar7 Semple SC, Akinc A, Chen J et al.: Rational design of cationic lipids for siRNA delivery. Nat. Biotechnol.28,172–176 (2010).Crossref, Medline, CAS, Google Scholar8 Akinc A, Querbes W, De S et al.: Targeted delivery of RNAi therapeutics with endogenous and exogenous ligand-based mechanisms. Mol. Ther.18,1357–1364 (2010).Crossref, Medline, CAS, Google Scholar9 Jeffs LB, Palmer LR, Ambegia EG, Giesbrecht C, Ewanick S, MacLachlan I: A scalable, extrusion-free method for efficient liposomal encapsulation of plasmid DNA. Pharm. Res.22,362–372 (2005).Crossref, Medline, CAS, Google Scholar10 Zimmermann TS, Lee AC, Akinc A et al.: RNAi-mediated gene silencing in non-human primates. Nature441,111–114 (2006).Crossref, Medline, CAS, Google Scholar11 Akinc A, Zumbuehl A, Goldberg M et al.: A combinatorial library of lipid-like materials for delivery of RNAi therapeutics. Nat. Biotechnol.26,561–569 (2008).Crossref, Medline, CAS, Google Scholar12 Akinc A, Goldberg M, Qin J et al.: Development of lipidoid-siRNA formulations for systemic delivery to the liver. Mol. Ther17,872–879 (2009).Crossref, Medline, CAS, Google Scholar13 Love KT, Mahon KP, Levins CG et al.: Lipid-like materials for low-dose, in vivo gene silencing. Proc. Natl Acad. Sci. USA 107,1864–1869 (2010).Crossref, Medline, CAS, Google Scholar14 Judge AD, Robbins M, Tavakoli I et al.: Confirming the RNAi-mediated mechanism of action of siRNA-based cancer therapeutics in mice. J. Clin. Invest.119,661–673 (2009).Crossref, Medline, CAS, Google Scholar15 Li S, Huang L. In vivo gene transfer via intravenous administration of cationic lipid–protamine–DNA (LPD) complexes. Gene Ther.4,891–900 (1997).Crossref, Medline, CAS, Google Scholar16 Li S, Rizzo MA, Bhattacharya S, Huang L: Characterization of cationic lipid–protamine–DNA (LPD) complexes for intravenous gene delivery. Gene Ther.5,930–937 (1998).Crossref, Medline, CAS, Google Scholar17 Li SD, Chen YC, Hackett MJ, Huang L: Tumor-targeted delivery of siRNA by self-assembled nanoparticles. Mol. Ther.16,163–169 (2008).Crossref, Medline, CAS, Google Scholar18 Li SD, Huang L: Nanoparticles evading the reticuloendothelial system: role of the supported bilayer. Biochim. Biophys. Acta1788,2259–2266 (2009).Crossref, Medline, CAS, Google Scholar19 Li SD, Chono S, Huang L: Efficient oncogene silencing and metastasis inhibition via systemic delivery of siRNA. Mol. Ther.16,942–946 (2008).Crossref, Medline, CAS, Google Scholar20 Chono S, Li SD, Conwell CC, Huang L: An efficient and low immunostimulatory nanoparticle formulation for systemic siRNA delivery to the tumor. J. Control. Release131,64–69 (2008).Crossref, Medline, CAS, Google Scholar21 Li J, Chen YC, Tseng YC, Mozumdar S, Huang L: Biodegradable calcium phosphate nanoparticle with lipid coating for systemic siRNA delivery. J. Control. Release142,416–421 (2010).Crossref, Medline, CAS, Google Scholar22 Landen CN, Jr, Chavez-Reyes A, Bucana C et al.: Therapeutic EphA2 gene targeting in vivo using neutral liposomal small interfering RNA delivery. Cancer Res.65,6910–6918 (2005).Crossref, Medline, CAS, Google Scholar23 Kim SH, Jeong JH, Lee SH, Kim SW, Park TG: PEG conjugated VEGF siRNA for anti-angiogenic gene therapy. J. Control. Release116,123–129 (2006).Crossref, Medline, CAS, Google Scholar24 Han HD, Mangala LS, Lee JW et al.: Targeted gene silencing using RGD-labeled chitosan nanoparticles. Clin. Cancer Res.16,3910–3922 (2010).Crossref, Medline, CAS, Google Scholar25 Hu-Lieskovan S, Heidel JD, Bartlett DW, Davis ME, Triche TJ: Sequence-specific knockdown of EWS-FLI1 by targeted, nonviral delivery of small interfering RNA inhibits tumor growth in a murine model of metastatic Ewing’s sarcoma. Cancer Res.65,8984–8992 (2005).Crossref, Medline, CAS, Google Scholar26 Davis ME, Zuckerman JE, Choi CH et al.: Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles. Nature464,1067–1070 (2010).Crossref, Medline, CAS, Google Scholar27 Rozema DB, Lewis DL, Wakefield DH et al.: Dynamic polyconjugates for targeted in vivo delivery of siRNA to hepatocytes. Proc. Natl Acad. Sci. USA 104,12982–12987 (2007).Crossref, Medline, CAS, Google Scholar28 Musacchio T, Vaze O, D’Souza G, Torchilin VP: Effective stabilization and delivery of siRNA: reversible siRNA–phospholipid conjugate in nanosized mixed polymeric micelles. Bioconjug. Chem.21,1530–1536 (2010).Crossref, Medline, CAS, Google Scholar29 Song E, Zhu P, Lee SK et al.: Antibody mediated in vivo delivery of small interfering RNAs via cell-surface receptors. Nat. Biotechnol.23,709–717 (2005).Crossref, Medline, CAS, Google Scholar30 Takeshita F, Minakuchi Y, Nagahara S et al.: Efficient delivery of small interfering RNA to bone-metastatic tumors by using atelocollagen in vivo. Proc. Natl Acad. Sci. USA 102,12177–12182 (2005).Crossref, Medline, CAS, Google Scholar31 Yan W, Chen W, Huang L: Reactive oxygen species play a central role in the activity of cationic liposome based cancer vaccine. J. Control. Release130,22–28 (2008).Crossref, Medline, CAS, Google Scholar32 Fedorov Y, Anderson EM, Birmingham A et al.: Off-target effects by siRNA can induce toxic phenotype. RNA12,1188–1196 (2006).Crossref, Medline, CAS, Google Scholar33 Jackson AL, Burchard J, Leake D et al.: Position-specific chemical modification of siRNAs reduces “off-target” transcript silencing. RNA12,1197–1205 (2006).Crossref, Medline, CAS, Google Scholar101 Clinicaltrials.gov: Study to evaluate the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of liposomal siRNA in Subjects with high cholesterol (2010) http://clinicaltrials.gov/ct2/show/NCT00927459?term=siRNA&rank=10Google Scholar102 Clinicaltrials.gov: Dose escalation trial to evaluate the safety, tolerability, pharmacokinetics and pharmacodynamics of intravenous ALN-VSP02 in patients with advanced solid tumors with liver involvement (2010) http://clinicaltrials.gov/ct2/show/NCT00882180?term=Alnylam&rank=3Google ScholarFiguresReferencesRelatedDetailsCited ByPre-Clinical and Clinical Applications of Small Interfering RNAs (siRNA) and Co-Delivery Systems for Pancreatic Cancer Therapy29 November 2021 | Cells, Vol. 10, No. 12Nanoparticle‐Mediated siRNA Delivery and Multifunctional Modification Strategies for Effective Cancer Therapy14 May 2021 | Advanced Materials Technologies, Vol. 6, No. 10Efficient and tumor-specific knockdown of MTDH gene attenuates paclitaxel resistance of breast cancer cells both in vivo and in vitro18 September 2018 | Breast Cancer Research, Vol. 20, No. 1Nanomaterials as Protein, Peptide and Gene Delivery AgentsThe Open Biotechnology Journal, Vol. 12, No. 1Optimization of Formulations Consisting of Layered Double Hydroxide Nanoparticles and Small Interfering RNA for Efficient Knockdown of the Target Gene2 May 2018 | ACS Omega, Vol. 3, No. 5Knockdown of RRS1 by lentiviral-mediated RNAi promotes apoptosis and suppresses proliferation of human hepatocellular carcinoma cells14 August 2017 | Oncology Reports, Vol. 38, No. 4Bioconjugates for targeted delivery of therapeutic oligonucleotidesAdvanced Drug Delivery Reviews, Vol. 87Co-delivery of HIF1α siRNA and gemcitabine via biocompatible lipid-polymer hybrid nanoparticles for effective treatment of pancreatic cancerBiomaterials, Vol. 46High-throughput screening identifies small molecules that enhance the pharmacological effects of oligonucleotides6 February 2015 | Nucleic Acids Research, Vol. 43, No. 4RNA Interference: a Promising Therapy for Gastric CancerAsian Pacific Journal of Cancer Prevention, Vol. 15, No. 14General Synthesis and Physicochemical Characterisation of a Series of Peptide-Mimic Lysine-Based Amino-Functionalised Lipids9 August 2013 | Chemistry - A European Journal, Vol. 19, No. 38Cdk2 Silencing via a DNA/PCL Electrospun Scaffold Suppresses Proliferation and Increases Death of Breast Cancer Cells20 December 2012 | PLoS ONE, Vol. 7, No. 12Targeted Expression of BikDD Eliminates Breast Cancer with Virtually No Toxicity in Noninvasive Imaging Models6 September 2012 | Molecular Cancer Therapeutics, Vol. 11, No. 9Synthesis of 2′-O-guanidinopropyl-modified nucleoside phosphoramidites and their incorporation into siRNAs targeting hepatitis B virusBioorganic & Medicinal Chemistry, Vol. 20, No. 4Delivery and biodistribution of siRNA for cancer therapy: challenges and future prospectsTherapeutic Delivery, Vol. 3, No. 2Superparamagnetic Nanoparticles and RNAi-Mediated Gene Silencing: Evolving Class of Cancer Diagnostics and TherapeuticsJournal of Nanomaterials, Vol. 2012Targeted siRNA delivery to diseased microvascular endothelial cells-Cellular and molecular concepts15 July 2011 | IUBMB Life, Vol. 63, No. 8To Be Targeted: Is the Magic Bullet Concept a Viable Option for Synthetic Nucleic Acid Therapeutics?Human Gene Therapy, Vol. 22, No. 7 Vol. 5, No. 10 Follow us on social media for the latest updates Metrics History Published online 14 December 2010 Published in print December 2010 Information© Future Medicine LtdKeywordscancer therapynanoparticlesiRNAFinancial & competing interests disclosureThe authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.No writing assistance was utilized in the production of this manuscript.PDF download