Gold nanoparticle-mediated delivery of siRNA - Future Medicine

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Gold nanoparticle-mediated delivery of siRNA: a promising strategy in the treatment of mosquito-borne viral diseases? “Recent progress on using gold nanoparticles to deliver siRNAs in vitro and in vivo indicates a novel, potential strategy against mosquito-borne viral infections.” Amber M Paul1, Faqing Huang2 & Fengwei Bai*,1 Since vaccine and antiviral therapeutics for mosquito-borne viruses are limited, an alternative approach to control infection is necessary. Nanoparticles (NPs) have been used as promising vehicles to deliver small molecules, genes or proteins as therapeutics to treat viral infection and disease [1,2] . Specifically, gold nanoparticles (AuNPs) have been recently utilized to deliver siRNA that can target host gene [3] and viral gene expression [1] . AuNPs display several features that make them well suited for biomedical applications, including ease of preparation, potential for incorporation of secondary selective tags and properties of biocompatibility. In this editorial, we will briefly discuss the perspectives of using AuNPs to deliver siRNAs to control mosquito-borne viral infections. Human mosquito-borne viral diseases The viral families that cause mosquitoborne diseases in humans include flaviviruses of the family Flaviviridae, alphaviruses of the family Togaviridae and several

genera in the family Bunyaviridae. These viruses are positive-, negative- or ambisensed, linear, ssRNA viruses. The main cellular entry pathway for most mosquitoborne viruses is by a clathrin-dependent process, whereby virus binds to host cellular receptors triggering endocytosis. Within the clathrin-coated endosomes, the viruses are uncoated and release their viral RNA genome into the cellular cytoplasm where the viral genome replicates. Currently there are some human vaccines available to control mosquito-borne viral infections for Yellow Fever [4] , Rift valley fever virus [5] and Japanese Encephalitis virus [4] . In addition, there are horse vaccines against West Nile virus, Venezuelan equine encephalitis virus, Eastern equine encephalitis virus and Western equine encephalitis virus [6] . However, there is no licensed vaccine or specific therapeutic available to most mosquito-transmitted viral infections in humans. Some mosquito-transmitted alphaviruses, including Venezuelan equine encephalitis virus, Eastern equine encephalitis

KEYWORDS

• gold nanoparticles • mosquito-borne viruses • siRNA

delivery

“...direct inhibition of viral genome

replication may be a promising alternative strategy. siRNAs can cleave viral RNA sequences thereby repressing viral RNA translation and progeny formation.”

Department of Biological Sciences, University of Southern Mississippi, Hattiesburg, MS 39406, USA Department of Chemistry & Biochemistry, University of Southern Mississippi, Hattiesburg, MS 39406, USA *Author for correspondence: Tel.: +1 601 266 4748; Fax: +1 601 266 5797; [email protected] 1 2

10.2217/FVL.14.64 © 2014 Future Medicine Ltd

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“The configurations of gold nanoparticle-siRNAs, including gold nanoparticle size, charge, surface modifications and additional modifications to polyethylenimine can be optimized to increase transfection efficiency and render these complexes biocompatible in vivo.”

virus, Western equine encephalitis virus, Sindbis virus, Ross River virus, Chikungunya virus and Semliki Forest virus, can cause human infections with symptoms from arthritis to life-threatening encephalitis [6] . Recent studies have introduced promising alphavirus vaccine candidates that have not yet reached successful clinical trials [7] . The problems associated with the development of an alphavirus vaccine are partially due to the plasticity of the RNA genome. RNA viruses utilize RNA-dependent RNA polymerase for RNA synthesis, which lacks proofreading capability. Therefore, integration of mutational errors that can alter the phenotype of the virus may allow the virus to escape neutralization by a defined vaccine. Similarly, no human vaccine is available for most mosquito-transmitted f laviviruses. In addition to viral RNA plasticity in flaviviruses, dengue virus (DENV) in particular, can induce antibody-dependent enhancement reaction, whereby infection against one serotype can provide lifelong immunity against that serotype, but can enhance DENV infectivity of other cocirculating serotypes [4] . Antibody-dependent enhancement reaction hinders the development of an antibody-based vaccine against DENV infection. Therefore, direct inhibition of viral genome replication may be a promising alternative strategy. siRNAs can cleave viral RNA sequences thereby repressing viral RNA translation and progeny formation. However, efficient delivery of siRNAs remains a great challenge for clinical applications because siRNAs are vulnerable to degradation by serum nucleases and to rapid renal excretion due to their small size and anionic character. Recently, we have reported that AuNPs can be used to construct well-defined AuNP-siRNA complexes whose size, charge density, and siRNA loading can be controlled. These AuNP-siRNA complexes have been shown to deliver antiviral siRNAs to control DENV infection in vitro. In addition, we found that AuNPs can significantly increase siRNA stability by protecting siRNA from RNase degradation, suggesting this approach may be further developed as a novel therapeutic against DENV and other mosquito-borne viral or nonviral diseases [1] . Optimization of AuNPs for siRNA delivery Surface modifications of AuNPs to contain a net positive charge are necessary for efficient siRNA delivery into cells and to maintain stability [8] .

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Among other surface modifications, such as N-(2-hydroxypropyl) methacrylamide (polyHPMA) and polyethylene glycol (PEG) [9] , the gold-standard surface coating for AuNP gene delivery is with the cationic chemical, polyethylenimine (PEI) [1,8] . Although PEI is useful to conjugate and stabilize siRNA onto AuNPs, this unmodified chemical may induce apoptosis [10] and be toxic at high concentrations [11] . Therefore, modifying PEI, such as conjugating PEI to thiol groups [1,12] , have been reported to maintain biocompatibility of NPs in different applications. It has been noted that AuNPs are not as efficient as well-established, lipid-based transfection reagents such as Lipofectamine® 2000 (Invitrogen, CA, USA) and Invivofectamine® 2.0 (Invitrogen, CA, USA), for siRNA delivery in vitro [1] and in vivo [13] . However, there are some caveats associated with using lipidbased transfection reagents. For instance, siRNA-lipoplexes have been demonstrated to selectively target liver cells in vivo, which limits the distribution of siRNA into other tissues [14] . Moreover, to ensure siRNAs silence a targeted gene, lipoplexes are usually required at high dosages and with multiple administrations, which can lead to tissue damage [15] and cellular stress responses or tissue death [16] . Furthermore, lipid-based transfection reagentsiRNAs complexes are not stable and need to be used immediately, which limits the use of these reagents as optimal therapeutics in clinical settings [8] . There is great potential for AuNPs to be utilized as alternative siRNA delivery vehicles in vivo. The configurations of AuNP-siRNAs, including AuNP size, charge, surface modifications and additional modifications to PEI can be optimized to increase transfection efficiency and render these complexes biocompatible in vivo. In addition, several characteristics of AuNPs make them suitable to deliver siRNA in vivo, which includes stability under enzymatic degradation (in vivo serum nuclease degradation and intracellular lysosome degradation), high siRNA loading capacity (∼300 siRNAs/AuNP) and biocompatibility [1] . AuNPs deliver siRNAs & proteins in vivo AuNPs have been implicated as biocompatible carriers for siRNAs and proteins in mice models. A recent report utilized AuNPs to deliver siRNAs against a lung cancer model in mice;

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Gold nanoparticle-mediated delivery of siRNA whereby, introduction of AuNP-siRNA complexes resulted in a significant reduction of the targeted gene. In that study, the authors also analyzed the proportion of inflammatory cells within the bronchoalveolar lavage collection following AuNP-siRNA treatment. The results showed that the proportion of macrophages, lymphocytes and neutrophils were not significantly altered between days 1 and 14 post treatment, suggesting AuNPs are biocompatible and not toxic to immune cells [17] . Another report showed that AuNP-siRNAs (1.5 μM siRNA, 50 nM AuNP) were able to almost completely abolish EGFR gene expression with no clinical or histological evidence of toxicity. In addition, these complexes were undetected in the internal organs 3 weeks after topical administration [3] . Two other reports have utilized AuNPs conjugated to recombinant human TNF-α (CYT-6091) to inhibit tumor growth in a mouse tumor progression model. The results showed that the treatment successfully inhibited tumor development [18] with minimal systemic cytotoxicity [19] . Importantly, data collected from a Phase I human clinical trial showed that treatment with CYT-6091 displayed promising results in late-phase cancer patients and the AuNP-TNF-α complexes could be administered at 600 μg/m 2 without encountering dose-limiting toxicities or immunogenicity [20] . These animal studies and clinical results suggest that AuNPs may be feasible vectors to deliver siRNAs and proteins in vivo. The clinical use of AuNPs, however, still pose certain challenges in terms of their potential pharmacokinetic, bioavailability, bioaccumulation, clearance and toxic effects. Biodistribution of AuNPs have been investigated in vivo, whereby bioaccumulation of AuNPs have been shown to limit within phagocytic-like cells (macrophages) and reticuloendothelial cells in lymph nodes, bone marrow, spleen, adrenals, liver and kidneys [21] . The location of AuNP accumulation seems to be dependent on AuNP size: 5–10 nm (liver), 30 nm (spleen), 5–60 nm (blood and bone marrow) [22] , while AuNPs less than 2.5 nm have been reported to be renally excreted [23] . To evaluate the bioaccumulation and toxic effects of different doses of AuNPs, Lasagna-Reeves et al. administrated 12.5 nm AuNPs (40, 200 and 400 μg/kg/day) intraperitoneally in mice daily for 8 days. They found that the tissue accumulation pattern of AuNPs depended on the


Editorial

dose administered and the accumulation of the AuNPs did not produce subacute physiological damage [24] . AuNP clearance from the body is another concern regarding AuNP administration in vivo. To investigate this, Sadauskas et al. reported that 40 nm AuNPs can be detected in the liver 6 months after intravenous injections, suggesting the clearance rate of AuNPs might be slow [25] . The cores of AuNPs are inert, but the potential extent of toxicity depends on their size, surface coating charge and chemical complexity [26] . For example, when 10 and 60 nm PEG-coated AuNPs were introduced in vivo, aminase and aspartate transaminase levels increased, suggesting some liver damage. However, this was not observed with 5- and 30-nm PEG-AuNPs delivery [22] . In addition, in vivo exposure to PEG coated AuNPs (13 nm) induced acute liver inflammation and apoptosis [27] . Furthermore, AuNPs coated with various surface modifiers, such as cysteine and glucose, displayed some toxicity [28] . Therefore, toxicity of AuNPs may also be due to variable surface modifications. In brief, there are still many questions that need to be addressed before using AuNPs as delivery vehicles for small molecules, siRNAs or proteins in clinical trials. Conclusion Recent progress on using AuNPs to deliver siRNAs in vitro and in vivo indicates a novel, potential strategy against mosquito-borne viral infections. However, there are some challenges to face before clinical trials, such as the optimization of AuNP size and the necessary AuNP-siRNA configurations that can increase biocompatible siRNA delivery efficiency without yielding unwanted AuNP accumulation in tissues. These questions require intensive future investigations. Nevertheless, delivery of siRNAs with AuNPs may provide an advantageous alternative option to treat mosquito-borne viral diseases in the future. Financial & competing interests disclosure This work was supported by F Bai’s University of Southern Mississippi new faculty start-up funding. The authors have no other relevant affiliations or involvement with any organization or entity with a financial interest or financial conflict with the subject matter or materials discussed in the manuscript. No writing assistance was utilized in the production of this manuscript.

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Paul AM, Shi Y, Acharya D et al. Delivery of anti-viral siRNA with gold-nanoparticles inhibits dengue virus infection in vitro. J. Gen. Virol. 8, 1712–1722 (2014). Davis ME, Chen ZG, Shin DM. Nanoparticle therapeutics: an emerging treatment modality for cancer. Nat. Rev. Drug Discov. 7(9), 771–782 (2008). Zheng D, Giljohann DA, Chen DL et al. Topical delivery of siRNA-based spherical nucleic acid nanoparticle conjugates for gene regulation. Proc. Natl Acad. Sci. USA 109(30), 11975–11980 (2012). Heinz FX, Stiasny K. Flaviviruses and flavivirus vaccines. Vaccine 30(29), 4301–4306 (2012). Ikegami T, Makino S. Rift valley fever vaccines. Vaccine 27(Suppl. 4), D69–D72 (2009). Go YY, Balasuriya UB, Lee CK. Zoonotic encephalitides caused by arboviruses: transmission and epidemiology of alphaviruses and flaviviruses. Clin. Exp. Vaccine Res. 3(1), 58–77 (2014). Thiboutot MM, Kannan S, Kawalekar OU et al. Chikungunya: a potentially emerging epidemic? PLoS Negl. Trop. Dis. 4(4), e623 (2010).

8

Gao K, Huang L. Nonviral methods for siRNA delivery. Mol. Pharm. 6(3), 651–658 (2009).

9

Gilstrap K, Hu X, Lu X, He X. Nano technology for energy-based cancer therapies. Am. J Cancer Res. 1(4), 508–520 (2011).

10 Hunter AC, Moghimi SM. Cationic carriers

of genetic material and cell death: a mitochondrial tale. Biochim. Biophys. Acta 1797(6–7), 1203–1209 (2010). 11 Zintchenko A, Philipp A, Dehshahri A,

Wagner E. Simple modifications of branched

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PEI lead to highly efficient siRNA carriers with low toxicity. Bioconjug. Chem. 19(7), 1448–1455 (2008). 12 Huang F, Shi Y. Synthesis of symmetrical

thiol-adenosine conjugate and 5’ thiol-RNA preparation by efficient one-step transcription. Bioorg. Med. Chem. Lett. 20(21), 6254–6257 (2010). 13 Stein DA, Perry ST, Buck MD et al.

Inhibition of dengue virus infections in cell cultures and in AG129 mice by a small interfering RNA targeting a highly conserved sequence. J. Virol. 85(19), 10154–10166 (2011). 14 Wang X, Yu B, Ren W et al. Enhanced

hepatic delivery of siRNA and microRNA using oleic acid based lipid nanoparticle formulations. J. Control. Release 172(3), 690–698 (2013). 15 Zimmermann TS, Lee AC, Akinc A et al.

RNAi-mediated gene silencing in non-human primates. Nature 441(7089), 111–114 (2006). 16 Zhong YQ, Wei J, Fu YR et al. [Toxicity of

cationic liposome Lipofectamine 2000 in human pancreatic cancer Capan-2 cells]. Nan Fang Yi Ke Da Xue Xue Bao 28(11), 1981–1984 (2008). 17 Conde J, Tian F, Hernandez Y et al. In vivo

tumor targeting via nanoparticle-mediated therapeutic siRNA coupled to inflammatory response in lung cancer mouse models. Biomaterials 34(31), 7744–7753 (2013). 18 Goel R, Swanlund D, Coad J, Paciotti GF,

Bischof JC. TNF-alpha-based accentuation in cryoinjury – dose, delivery, and response. Mol. Cancer Ther. 6(7), 2039–2047 (2007). 19 Goel R, Shah N, Visaria R, Paciotti GF,

Bischof JC. Biodistribution of TNF-alphacoated gold nanoparticles in an in vivo model system. Nanomedicine (Lond.) 4(4), 401–410 (2009).

Future Virol. (2014) 9(11)

20 Libutti SK, Paciotti GF, Byrnes AA et al.

Phase I and pharmacokinetic studies of CYT-6091, a novel PEGylated colloidal gold-rhTNF nanomedicine. Clin. Cancer Res. 16(24), 6139–6149 (2010). 21 Dobrovolskaia MA, Aggarwal P, Hall JB,

Mcneil SE. Preclinical studies to understand nanoparticle interaction with the immune system and its potential effects on nanoparticle biodistribution. Mol. Pharm. 5(4), 487–495 (2008). 22 Zhang XD, Wu D, Shen X et al. Size-

dependent in vivo toxicity of PEG-coated gold nanoparticles. Int. J. Nanomed. 6, 2071–2081 (2011). 23 Liu J, Yu M, Zhou C, Yang S, Ning X,

Zheng J. Passive tumor targeting of renal-clearable luminescent gold nano particles: long tumor retention and fast normal tissue clearance. J. Am. Chem. Soc. 135(13), 4978–4981 (2013). 24 Lasagna-Reeves C, Gonzalez-Romero D,

Barria MA et al. Bioaccumulation and toxicity of gold nanoparticles after repeated administration in mice. Biochem. Biophys. Res. Commun. 393(4), 649–655 (2010). 25 Sadauskas E, Danscher G, Stoltenberg M,

Vogel U, Larsen A, Wallin H. Protracted elimination of gold nanoparticles from mouse liver. Nanomedicine 5(2), 162–169 (2009). 26 Lewinski N, Colvin V, Drezek R.

Cytotoxicity of nanoparticles. Small 4(1), 26–49 (2008). 27 Cho WS, Cho M, Jeong J et al. Acute toxicity

and pharmacokinetics of 13 nm-sized PEG-coated gold nanoparticles. Toxicol. Appl. Pharmacol. 236(1), 16–24 (2009). 28 Connor EE, Mwamuka J, Gole A, Murphy CJ,

Wyatt MD. Gold nanoparticles are taken up by human cells but do not cause acute cytotoxicity. Small 1(3), 325–327 (2005).
