Gold Nanoparticle-Mediated Delivery of siRNA: A Promising Strategy in the Treatment of Mosquito-Borne Viral Diseases?

KEYWORDS:

• gold nanoparticles
• mosquito-borne viruses
• siRNA delivery

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 mosquito-borne 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 ambi-sensed, linear, ssRNA viruses. The main cellular entry pathway for most mosquito-borne 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 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 flaviviruses. 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]. Among other surface modifications, such as N-(2-hydroxypropyl) methacrylamide (poly-HPMA) 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 lipid-based 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 reagent-siRNAs 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; 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/m² 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 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.

Additional information

Funding

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.