RNAi-Based PLGA Nanoparticles for Targeted Silencing of NNV Capsid Protein

RNAi-Based PLGA Nanoparticles for Targeted Silencing of NNV Capsid Protein | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article RNAi-Based PLGA Nanoparticles for Targeted Silencing of NNV Capsid Protein Mingguang Mao, Yaru Wang, Shiyu Zhu, Jielan Jiang, Xiaoming Geng, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7394032/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract Nervous necrosis virus (NNV) can be transmitted vertically from parents to offspring, causing significant mortality in young fish. Therefore, effective decontamination of fertilized eggs is essential for sustainable aquaculture production. This study devel-oped siRNA-loaded PLGA nanoparticles (NPs) for targeted silencing of NNV capsid protein (CP) and evaluated their therapeutic potential in vitro. Using a dou-ble-emulsion method, siRNA-PLGA NPs were synthesized with optimized physico-chemical properties: the PLGA-PEI/trichloromethane/PEG (W/O/W) system yielded monodisperse, spherical nanoparticles (~ 110 nm post-sonication) with a high zeta po-tential (+ 30.91 mV), ensuring colloidal stability and efficient cellular delivery. Over-expression of NNV CP in EPC cells induced rapid cytotoxicity, including nuclear ab-normalities and cell death within 48 hours, which was fully rescued by co-administering three rationally designed CP-targeting siRNAs. Strikingly, nanopar-ticles resuspended in phosphate-buffered saline (PBS) exhibited a higher siRNA en-capsulation efficiency, whereas those suspended in Diethyl Pyrocarbonate water showed negligible loading. These findings underscore the importance of formulation parameters in nanoparticle design and highlight the promise of RNAi-based PLGA nanotherapeutics for combating aquatic viral infections. PLGA Nanoparticle Nervous Necrosis Virus Coat Protein Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Nervous Necrosis Virus (NNV) represents a formidable challenge to aquaculture, particularly marine fish populations, where it inflicts substantial damage by compromising the nervous system and precipitating high mortality rates (Mao et al. 2015b ). The pathogenicity of NNV predominantly affects larval and juvenile stages, sparing adults (Munday and Nakai,1997; Iwamoto 2004; Doan et al. 2017 ). Current prophylactic and management strategies for NNV encompass diverse approaches targeting both the viral pathogen and the aquaculture environment. Among these, vaccines are extensively studied but remain ineffective in larval stages due to the immaturity of their immune systems (Thiery et al. 2006 ; Pakingking et al. 2010 ; Mao et al. 2013 ; Mao et al. 2015a ; Bandín and Souto, 2020 ). Alternatively, advancements in gene-editing technologies have demonstrated potential for enhancing host resistance through targeted modification of immune-related genetic loci. Effective water quality management further complements these strategies, albeit as an indirect control measure. NNV transmission occurs via horizontal and vertical pathways, with the latter posing significant concern due to the potential for perpetuation of infection across generations. Vertical transmission facilitates viral passage from broodstock to progeny through reproductive processes or within eggs and larvae, creating reservoirs of infection that persist independently of external exposure (Breuil et al. 1991 ; Munday and Nakai,1997; Munday et al. 2002 ; Toffan et al. 2017 ). Our study seeks to address this challenge by employing small interfering RNAs (siRNAs) to suppress or eliminate NNV in fertilized eggs, thereby interrupting vertical transmission. However, siRNAs are inherently unstable in marine environments (Suksai et al. 2025 ), and their intracellular delivery to target sites within fertilized eggs necessitates an efficacious carrier system. Poly (lactic-co-glycolic acid) (PLGA) emerges as a prominent candidate for siRNA delivery. As a biodegradable and biocompatible polymer, PLGA offers superior drug delivery properties, including enhanced stability, tunable degradation rates, and minimal cytotoxicity (Anderson and Shive, 1997 ; Jain 2000 ; Makadia and Siegel, 2011 ; Danhier et al. 2012 ). PLGA nanoparticles (NPs) have garnered considerable attention as delivery vehicles for siRNA, as their encapsulation capabilities mitigate enzymatic degradation and facilitate targeted delivery. Optimizations in molecular weight and the lactic-to-glycolic acid ratio enable precise control over release kinetics. Hybrid systems incorporating materials such as polyethyleneimine (PEI) and lipids further augment the stability, cellular uptake efficiency, and immunocompatibility of PLGA-based NPs (Khalil et al. 2006 ; Dobrovolskaia and McNeil, 2007 ; Danhier et al. 2012 ; Sun et al. 2018 ; Kumar et al. 2020 ). Preclinical investigations have validated the efficacy of PLGA-loaded siRNA NPs across diverse biomedical applications, including oncology, viral infections, and gene therapy (Khalil et al. 2006 ; Wang et al. 2010 ; Zhao et al. 2017 ; Kumar et al. 2020 ; Sun et al. 2018 ). For instance, murine tumor models demonstrated substantial gene silencing and therapeutic outcomes with PLGA-siRNA formulations. Despite these advancements, there remains a paucity of studies exploring PLGA NPs in aquaculture. This study seeks to bridge this gap by employing a double-emulsion (w/o/w) technique to construct siRNA-PLGA nanoparticles targeting NNV, with the ultimate goal of establishing a novel intervention framework for aquaculture. 2. Materials and Methods 2.1 Materials PLGA was synthesized by copolymerizing lactic acid and glycolic acid at a ratio of 75:25, with a molecular weight of 10 kDa. Ultrafiltration spin columns (catalog number RT-VS2052-5) were procured for purification processes. Additional reagents included Tween-80, trichloromethane, N,N-dimethylformamide (DMF), polyethylene glycol (PEG), and polyethyleneimine (PEI, molecular weight 25,000), the latter sourced from Shanghai manufacturers. EPC (Epithelioma Papulosum Cyprini) cell lines were utilized as provided by the laboratory, while fertilized grouper eggs, 18 hours post-fertilization, were acquired for experimental applications. 2.2 Synthesis and Characterization of siRNA-PLGA Nanoparticles (NPs) siRNA-PLGA nanoparticles were synthesized via a double-emulsion (w/o/w) method coupled with ultrafiltration. Briefly, 5 mg of PLGA was dissolved in 1 mL of an organic solvent (trichloromethane or DMF) to constitute the oil phase. The primary emulsion was generated by ultrasonication of the oil phase with a water solution containing siRNA, PEI or Tween-80 for 6 minutes in an ice bath (amplitude: 40%, pulse on: 5 s, pulse off: 2 s, for 5min). This emulsion was subsequently dispersed into 30 mL of an aqueous solution containing PEG or Tween-80, forming the secondary emulsion, which was also generated by ultrasonication (amplitude: 40%, pulse on: 5 s, pulse off: 2 s, for 5min). The nanoparticle suspension was centrifuged at 3000 rpm for 30 minutes, collected using Ultrafiltration spin columns, washed and resuspended with 1 mL of PBS or distilled water. The size and surface charge of the nanoparticles were characterized using dynamic light scattering (DLS) and laser Doppler velocimetry (LDV) with a Nano ZS Zetasizer (Malvern Instruments, UK). Nanoparticle morphology was visualized via transmission electron microscopy (TEM). 2.3 Expression of Nervous Necrosis Virus Capsid Protein (CP) in EPC Cells The capsid protein (CP) gene of the NNV isolate RG-NNV, previously cloned and preserved in the laboratory, was inserted into the pEGFP-N1 plasmid vector to generate the recombinant plasmid pEGFP-CP. The plasmid was amplified in Escherichia coli and purified using an endotoxin-free plasmid extraction kit, with sequence integrity confirmed by sequencing. Transfection of EPC cells with pEGFP-CP was performed using Lipofectamine 3000 (Invitrogen, L3000) (Table 1 ). CP expression was assessed 48 hours post-transfection by fluorescence microscopy. To evaluate cell viability and nuclear morphology, transfected cells were stained with Hoechst 33342 at 24 hours post-transfection, prior to the onset of cell death and detachment. Imaging was conducted using confocal microscopy. Table 1 Targets for siRNA Silencing Name Sequence (5’-3’) Length (bp) Target siRNA18F GCAAAGGGAAUAAGAAAUUTT 21 Position 18 siRNA18R AAUUUCUUAUUCCCUUUGCTT 21 siRNA144F GCCUCGACUAUCACGGGAUTT 21 Position 144 siRNA144R AUCCCGUGAUAGUCGAGGCTT 21 siRNA720F GCAGCCACUGAUUUCAAAUTT 21 Position 720 siRNA720R AUUUGAAAUCAGUGGCUGCTT 21 siRNA-Cy3-F UUCUCCGAACGUGUCACGUTT 21 Control siRNA-Cy3-R ACGUGACACGUUCGGAGAATT 21 2.4 siRNA Entrapment Efficiency in PLGA Nanoparticles PLGA nanoparticles encapsulating siRNA targeting the CP gene of NNV were synthesized as described. Encapsulation efficiency was quantified as the ratio of siRNA content retained within nanoparticles relative to the total amount used in synthesis. This was determined using Nano-Flow Cytometry (NanoFCM, www.nanofcm.com ). 2.5 Effect of siRNA-PLGA Nanoparticles on Fish Egg Survival Rate The siRNA (siRNA144)-PLGA NPs was diluted into 1000× and 10× concentrations respectively and added to the fish egg culture water. The fertilized fish eggs (12 hours post-fertilization at the start of the experiment) were randomly allocated into 9 culture dishes (15 cm in diameter, 3 cm in height) that had been thoroughly disinfected with KMnO4, with an initial quantity of 50 fish eggs per dish. The experiment was divided into 3 groups with different concentrations of the siRNA-PLGA nanoparticles, and each group had 3 replicates. Mortality rate and hatching rate of fish eggs were rec-orded at different time points during the experiment. Constant temperature, light, and aeration were maintained throughout the process. 3. Results 3.1 Characterization of siRNA-PLGA Nanoparticles (NPs) The NPs synthesized using the double-emulsion method exhibited the desired particle size distribution and surface properties essential for effective gene delivery. When chloroform was used as the primary solvent and PEG was added to the external aqueous phase, the resulting nanoparticle size was 156.6 nm (Fig. 1 A); when Tween-80 was added to the external aqueous phase instead of PEG, the nanoparticle size was 146 nm (Fig. 1 B). Using DMF as the primary solvent and PEG in the external aqueous phase resulted in nanoparticles with a size of 189.2 nm (Fig. 1 C); when Tween-80 was added to the external aqueous phase in the DMF system, two peaks were observed, indicating a heterogeneous size distribution (Fig. 1 D). The average particle size, determined by dynamic light scattering (DLS), was approximately 164 nm. Based on these results, we selected the PLGA-PEI/Trichloromethane/PEG (W/O/W) system for the next experiment. The nanoemulsions synthesized using the PLGA-PEI/Trichloromethane/PEG (W/O/W) system were subjected to a second round of sonication, followed by filtration using an ultrafiltration membrane. The nanoparticles on the membrane were then resuspended in DEPC water. After measurement, the nanoparticle size was found to be approximately 110 nm, with a unimodal light intensity distribution (Fig. 2 A), which was smaller than that of the nanoparticles synthesized after the first round of sonication. The zeta potential of the nanoparticles was measured at approximately 30.91 ± 2 mV(Fig. 2 B), indicating good colloidal stability and minimal aggregation under physiological conditions( https://www.malvernpanalytical.com/en/learn/knowledge-center/application-notes/an101130suspensionstability ). The NPs on the filter membrane were resuspended in PBS, dried on a copper grid, and observed under electron microscopy. The nanoparticle size was consistent with the results from the nanoparticle size analyzer, and spherical particles with a size of approximately 110 nm were clearly observed, as shown in Fig. 3 . Transmission electron microscopy (TEM) images confirmed the spherical morphology of the NPs, with a consistent and well-defined nanoparticle structure (Fig. 3 ). No significant distortion or aggregation was observed, supporting the robustness of the nanoparticle formulation. 2.2. NNV model establishment The recombinant plasmid pEGFP-CP, harboring the NNV capsid protein gene, was transfected into EPC cells to establish a model for investigating viral protein expression dynamics. Fluorescence microscopy analysis demonstrated robust expression of the GFP-CP fusion protein, confirming successful transfection and intracellular protein synthesis. In control groups (transfected with empty vector), GFP fluorescence exhibited homogeneous cytoplasmic distribution at 48 hours post-transfection (Fig. 4 A-B). In contrast, cells transfected with pEGFP-CP displayed extensive cell death, evidenced by a significant population of detached, non-viable cells in the culture medium. Fluorescent imaging revealed fragmented GFP signals (Fig. 4 C), suggesting potential cytotoxic effects associated with CP overexpression. To further characterize CP-induced cellular pathology, confocal microscopy was performed on adherent cells at 24 hours post-transfection, prior to observable cell detachment. Nuclear staining followed by high-resolution imaging revealed marked abnormalities in nuclear morphology (Fig. 4 D), including chromatin condensation and irregular nuclear envelope contours, indicative of CP-triggered nuclear stress. These morphological aberrations correlate temporally with the onset of cytotoxicity, implying a potential mechanistic link between CP accumulation, nuclear destabilization, and subsequent cell death. 3.3 Screening of Effective siRNAs Three CP-targeting siRNA duplexes were rationally designed to silence NNV capsid protein expression. Transfection of EPC cells with pEGFP-CP for 48h induced significant cytotoxicity, characterized by extensive cell detachment and reduced viability (Fig. 5 A, Control). Co-administration of all three siRNA candidates markedly rescued cell viability (Fig. 5 B). Notably, all siRNAs demonstrated superior silencing potency and they were prioritized for subsequent nanoparticle encapsulation studies. 3.4 siRNA Encapsulation Efficiency The encapsulation efficiency of FAM-labeled siRNA within PLGA nanoparticles was systematically quantified using nano-flow cytometry (nFCM) to evaluate the impact of suspension media on siRNA entrapment. Strikingly, nanoparticles resuspended in phosphate-buffered saline (PBS) exhibited a higher siRNA encapsulation efficiency (Fig. 6 A), whereas those suspended in DEPC-treated water showed negligible loading (Fig. 6 B). Consistent with the physicochemical characterization in Section 3.1 , nanoparticle tracking analysis (NTA) confirmed a monodisperse population centered at 160 ± 5.8 nm (Fig. 6 A-B), aligning with TEM result (Fig. 3 ) 2.5. The effect of the siRNA-PLGA NPs on the survival of fish eggs To investigate the effects of the siRNA-PLGA NPs on fish eggs, this study diluted the NPs to 1000× and 10× concentrations and added them to the seawater for fish egg hatching. The results showed a survival rate of 86% in the control group, 86% after 1000× dilution and a 4% decrease in survival after 10× dilution. It showed a slight decrease in the survival rate of fish eggs as the number of times of drug dilution decreased (increase in drug concentration) (Fig. 7 ) 4. Discussion 4.1. Optimized Nanoparticle Design for siRNA Delivery The siRNA-PLGA NPs synthesized using the PLGA-PEI/trichloromethane/PEG (W/O/W) system demonstrated superior physicochemical properties, including a reduced particle size (~ 110 nm post-sonication/filtration), monodisperse distribution (Fig. 2 A, 6 A-B), and a high zeta potential (~ 30.91 mV). These attributes are critical for efficient cellular uptake and endosomal escape, as smaller nanoparticles (< 200 nm) are preferentially internalized via clathrin-mediated endocytosis, while a positive surface charge enhances interactions with negatively charged cell membranes (Gratton et al. 2008 ; Choi et al. 2010 ; He et al. 2010 ; Iversen et al. 2011 ). The spherical morphology observed via TEM (Fig. 3 ) further corroborates the structural integrity of the NPs, minimizing aggregation risks in physiological environments (Chithrani et al. 2006 ; Gratto et al. 2008; Albanese et al. 2012; Zhang et al. 2016). Notably, the choice of solvent and surfactant significantly influenced nanoparticle homogeneity. The heterogeneity observed in the DMF/Tween-80 system (Fig. 1 D) likely stems from incomplete emulsification, underscoring the importance of solvent-surfactant compatibility in NP synthesis. The transition to PEG in the external aqueous phase improved colloidal stability, aligning with PEG’s well-established role in steric stabilization and biocompatibility (Harris and Zalipsky, 1997 ; Suk et al. 2016 ). 4.2. NNV Capsid Protein (CP)-Induced Cytotoxicity and siRNA Rescue The overexpression of NNV CP in EPC cells triggered rapid cytotoxicity, characterized by cell detachment, fragmented GFP signals (Fig. 4 B), and nuclear abnormalities such as chromatin condensation (Fig. 4 C). These observations suggest that CP accumulation induces apoptosis or necroptosis, potentially through nuclear stress pathways (Vandenabeel et al. 2010; Green and Llambi, 2015 ; Galluzz et al. 2018). The temporal correlation between nuclear destabilization (24 h) and overt cell death (48 h) implies a cascade of intracellular damage, positioning CP as a critical virulence factor. The complete rescue of cell viability by all three CP-targeting siRNAs (Fig. 5 B) validates their silencing efficacy and highlights the therapeutic potential of RNAi in mitigating NNV-induced pathology. The lack of significant differences in siRNA potency suggests redundant target accessibility or uniform siRNA design efficiency, warranting further mechanistic studies to identify optimal silencing regions within the CP transcript. 4.3. siRNA Encapsulation Efficiency: Role of Suspension Medium A striking finding was the stark contrast in siRNA encapsulation efficiency between nanoparticles resuspended in PBS (> 10%) versus DEPC-treated water (< 3%) (Fig. 6 C-D). While DEPC water is traditionally employed for RNA-related applications due to its RNase-free properties (Farrell 2010 ; Rio et al. 2010), its poor performance here may arise from ionic incompatibility with the PLGA matrix (Gilman 1993 ; Makadia and Siegel, 2011 ). PBS, with its physiological ion concentration and buffering capacity, likely stabilizes electrostatic interactions between siRNA and the cationic PLGA-PEI polymer, enhancing entrapment (Panyam and Labhasetwar 2003 ; Kumar et al. 2004 ; Jeon et al. 2007; Whitehead et al. 2009 ). This underscores the necessity of tailoring suspension media to the physicochemical properties of both the nanoparticle and payload. PLGA degradation primarily occurs in endosomes/lysosomes (pH 4.5–5.5). The acidic environment accelerates hydrolysis, promoting siRNA escape into the cytoplasm to exert its function. (1) Diffusion Release: Initial siRNA partially diffuses out through pores formed by PLGA swelling. (2) Degradation-Controlled Release: Later-stage PLGA backbone cleavage leads to massive siRNA release (burst effect). Ester bonds (-COO-) in PLGA chains are hydrolyzed by water molecules, generating lactic acid (LA) and glycolic acid (GA) monomers. LA is naturally produced in the body. While GA is not endogenous in fish, it exhibits low toxicity at controlled concentrations. In sum, this study demonstrates the successful design of siRNA-loaded PLGA nanoparticles with optimized size, stability, and encapsulation efficiency, capable of rescuing CP-induced cytotoxicity in EPC cells. The critical role of formulation parameters (solvent, surfactant, suspension medium) in NP performance highlights the need for meticulous optimization in nanomedicine development. Future work should focus on scaling up NP production, validating efficacy in vivo, and expanding the siRNA library to target additional NNV virulence factors (Whitehea et al. 2009; Petros and DeSimone 2010 ; Chen et al. 2015). These findings pave the way for RNAi-based therapies against aquatic viral pathogens, with broader implications for antiviral nanotechnology. Declarations Author contribution M. Mao and J. Jiang designed the experiments and obtained the funding. Y. Wang and S. Zhu carried out the experiment. Y. Wang, S. Zhu, X. Geng and X. Tang carried out the data analysis. M. Mao and J. Jiang wrote the manuscript with support from Y. Wang and S. Zhu, and all authors agree to be accountable for all aspects of the work. Funding This research was funded by the National key research and development program of China (2024YFD2401803), the Hainan Provincial Natural Science Foundation of China (322RC716), the National Natural Science Foundation of China (Grant No. 32360923), the Hainan Provincial Natural Science Foundation of China (422MS083), the Scientific Research Foundation of Hainan Tropical Ocean University (RHDRC202206). Any field work in this study complied with the current laws of China, where it was performed. Data Availability No datasets were generated or analysed during the current study. Competing interest The authors declare no competing interests. Consent for publication All authors agreed to publish the current study through Aquaculture International. References Anderson JM, Shive MS (1997) Biodegradation and biocompatibility of PLA and PLGA microspheres. Adv Drug Deliv Rev 28:5–24. https://doi.org/10.1016/S0169-409X(97)00048-3 Bandín I, Souto S (2020) Betanodavirus and VER disease: A 30-year research review. Pathogens 9:106. https://doi.org/10.3390/pathogens9020106 Breuil G, Bonami JR, Pepin JF, Pichot Y (1991) Viral infection (picorna-like virus) associated with mass mortalities in hatchery-reared sea bass ( Dicentrarchus labrax ) larvae and juveniles. Aquaculture 97:109–116. https://doi.org/10.1016/0044-8486(91)90258-9 Chithrani BD, Ghazani AA, Chan WCW (2006) Determining the size and shape dependence of gold nanoparticle uptake into mammalian cells. Nano Lett 6:662–668. https://doi.org/10.1021/nl052396o Choi CH, Alabi CA, Webster P, Davis ME (2010) Mechanism of active targeting in solid tumors with transferrin-containing gold nanoparticles. Proc Natl Acad Sci 107:1235–1240. https://doi.org/10.1073/pnas.0914140107 Danhier F, Ansorena E, Silva JM et al (2012) PLGA-based nanoparticles: An overview of biomedical applications. J Control Release 161:505–522. https://doi.org/10.1016/j.jconrel.2012.01.043 Dobrovolskaia MA, McNeil SE (2007) Immunological properties of engineered nanomaterials. Nat Nanotechnol 2:469–478. https://doi.org/10.1038/nnano.2007.223 Doan QK, Vandeputte M, Chatain B, Morin T, Allal F (2017) Viral encephalopathy and retinopathy in aquaculture: a review. J Fish Dis 40:717–742. https://doi.org/10.1111/jfd.12550 Farrell RE (2010) RNA Methodologies: A Laboratory Guide for Isolation and Characterization (4th ed.). Academic Press. Galluzzi L, Vitale I, Aaronson S et al (2018) Molecular mechanisms of cell death: Recommendations of the Nomenclature Committee on Cell Death 2018. Cell Death Differ 25:486–541. https://doi.org/10.1038/s41418-017-0012-4 Gilman M (1993) Ribonuclease inhibition by diethyl pyrocarbonate. Methods in Molecular Biology 16:21–23. Gratton SE, Ropp PA, Pohlhaus PD et al (2008) The effect of particle design on cellular internalization pathways. Proc Natl Acad Sci 105:11613–11618. https://doi.org/10.1073/pnas.0801763105 Green DR, Llambi F (2015) Cell death signaling. Csh Perspect Biol 7: a006080. https://doi.org/10.1101/cshperspect.a006080 Harris JM, Zalipsky S (1997) Poly (ethylene glycol): Chemistry and Biological Applications . ACS Symposium Series. He C, Hu Y, Yin L, Tang C, Yin C (2010) Effects of particle size and surface charge on cellular uptake and biodistribution of polymeric nanoparticles. Biomaterials 31:3657–3666. https://doi.org/10.1016/j.biomaterials.2010.01.065 Iversen TG, Skotland T, Sandvig K (2011) Endocytosis and intracellular transport of nanoparticles: Present knowledge and need for future studies. Nano Today 6:176–185. https://doi.org/10.1016/j.nantod.2011.02.003 Iwamoto T, Okinaka Y, Mise K et al (2004) Identification of host-specificity determinants in betanodaviruses by using reassortants between striped jack nervous necrosis virus and sevenband grouper nervous necrosis virus. J Virol 78:1256–1262. https://doi.org/10.1128/JVI.78.3.1256-1262.2004 Jain RA (2000) The manufacturing techniques of various drug loaded biodegradable poly(lactide-co-glycolide) (PLGA) devices. Biomaterials 21:2475–2490. https://doi.org/10.1016/S0142-9612(00)00115-0 Jeong JH, Mok H, Oh YK et al (2007) siRNA conjugate delivery systems. Bioconjugate Chem 18:5–14. https://doi.org/10.1021/bc800278e Khalil IA, Kogure K, Akita H, Harashima H (2006) Uptake pathways and subsequent intracellular trafficking in nonviral gene delivery. Pharmacol Rev 58:32–45. https://doi.org/10.1124/pr.58.1.8 Kumar MNVR et al (2004) Cationic poly(lactide-co-glycolide) nanoparticles as efficient in vivo gene transfection agents. J Nanosci Nanotechno 4:990–994. https://doi.org/10.1166/jnn.2004.141 Kumar R, Santa Chalarca CF, Bockman MR et al (2020) Polymeric delivery of therapeutic nucleic acids. Chem Rev 120:10716–10776. https://doi.org/10.1021/acs.chemrev.0c00697 Makadia HK, Siegel SJ (2011) Poly lactic-co-glycolic acid (PLGA) as biodegradable controlled drug delivery carrier. Polymers 3:1377–1397. https://doi.org/10.3390/polym3031377 Mao MG, Jiang JL, Perálvarez-Marín A, Wang KJ, Lei JL (2013) Characterization of the Mx and hepcidin genes in Epinephelus akaara asymptomatic carriers of the nervous necrosis virus. Aquaculture 408-409:175–183. https://doi.org/10.1016/j.aquaculture.2013.05.038 Mao MG, Li X, Peralvarez-Marin A et al (2015a). Transcriptomic analysis and biomarkers (Rag1 and Igmu) for probing the immune system development in Pacific cod, Gadus macrocephalus . Fish Shellfish Immun 44:622–632. https://doi.org/10.1016/j.fsi.2015.03.035 Mao MG, Wen SH, Peralvarez-Marin, A et al (2015b) Evidence for and characterization of nervous necrosis virus infection in Pacific cod ( Gadus macrocephalus ). Arch Virol 160:2237–2248. https://doi.org/10.1007/s00705-015-2498-8 Munday BL, Kwang J, Moody N (2002) Betanodavirus infections of teleost fish: a review. J Fish Dis 25:127–142. https://doi.org/10.1046/j.1365-2761.2002.00350.x. Munday BL, Nakai T (1997) Nodaviruses as pathogens in larval and juvenile marine finfish. World J Microb Biot 13:375–381. https://doi.org/10.1023/A:1018575210357 Pakingking R, Bautista NB, de Jesus-Ayson EG, Reyes O (2010) Protection of grouper ( Epinephelus coioides ) against nervous necrosis virus by recombinant capsid protein vaccine. Fish Shellfish Immun 28:140–147. https://doi.org/10.1016/j.fsi.2009.10.007 Panyam J, Labhasetwar V (2003) Biodegradable nanoparticles for drug and gene delivery to cells and tissue. Adv Drug Deliver Rev 55:329–347. https://doi.org/10.1016/S0169-409X(02)00228-4 Petros RA, DeSimone JM (2010) Strategies in the design of nanoparticles for therapeutic applications. Nat Rev Drug Discov 9:615–627. https://doi.org/10.1038/nrd2591 Rio DC (2010) RNA: A Laboratory Manual . Cold Spring Harbor Laboratory Press. Suk JS, Xu Q, Kim N, Hanes J, Ensign LM (2016) PEGylation as a strategy for improving nanoparticle-based drug and gene delivery. Adv Drug Deliver Rev 99:28–51. https://doi.org/10.1016/j.addr.2015.09.012 Suksai S, Attasart P, Angsujinda K et al (2025) Delivery of virus-specific dsRNA using a composite nanomaterial improves the protection of shrimp ( Litopenaeus vannamei ) against yellow head virus challenge. Aquaculture 595:123456. https://doi.org/10.1016/j.aquaculture.2024.741570 Sun X, Zhang N, Yin C (2018) PLGA-based nanoparticles for siRNA delivery: Strategies and applications. J Mater Chem B 6:6615–6631. https://doi.org/10.1039/C8TB01662F Thiery R, Cozien J, Cabon J et al (2006) Induction of a protective immune response against viral nervous necrosis in the European sea bass Dicentrarchus labrax by using betanodavirus virus-like particles. J Virol 80:10201–10207. https://doi.org/10.1128/JVI.01098-06 Toffan A, Pascoli F, Pretto T et al (2017). Viral nervous necrosis in gilthead sea bream ( Sparus aurata ) caused by reassortant betanodavirus RGNNV/SJNNV: an emerging threat for Mediterranean aquaculture. Scientific Reports 7:46755. https://doi.org/10.1038/srep46755 Vandenabeele P, Galluzzi L, Vanden Berghe T et al, (2010) Molecular mechanisms of necroptosis: An ordered cellular explosion. Nat Rev Mol Cell Bio 11:700–714. https://doi.org/10.1038/nrm2970 Wang J, Lu Z, Wientjes MG, Au JL (2010) Delivery of siRNA therapeutics: Barriers and carriers. The AAPS Journal 12:492–503. https://doi.org/10.1208/s12248-010-9210-4 Whitehead KA, Langer R, Anderson D (2009) Knocking down barriers: advances in siRNA delivery. Nat Rev Drug Discov 8:129–138. https://doi.org/10.1038/nrd2742 Zhao Y, Huang L, Zhang C (2017) PLGA-based nanoparticles for siRNA delivery: Preclinical and clinical advances. Biomaterials 120:1–14. https://doi.org/10.1016/j.biomaterials.2017.01.016 Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7394032","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":505140878,"identity":"587d8a6f-3862-48c9-867f-9419e74ac9dd","order_by":0,"name":"Mingguang Mao","email":"","orcid":"","institution":"Yazhou Bay Innovation Institute, Hainan Tropical Ocean University","correspondingAuthor":false,"prefix":"","firstName":"Mingguang","middleName":"","lastName":"Mao","suffix":""},{"id":505140879,"identity":"b1432af7-b9e8-43e1-aa2b-8f91a424b907","order_by":1,"name":"Yaru Wang","email":"","orcid":"","institution":"Yazhou Bay Innovation Institute, Hainan Tropical Ocean University","correspondingAuthor":false,"prefix":"","firstName":"Yaru","middleName":"","lastName":"Wang","suffix":""},{"id":505140880,"identity":"35e4e96c-28fd-477e-831a-eeb2c46a96b7","order_by":2,"name":"Shiyu Zhu","email":"","orcid":"","institution":"Yazhou Bay Innovation Institute, Hainan Tropical Ocean University","correspondingAuthor":false,"prefix":"","firstName":"Shiyu","middleName":"","lastName":"Zhu","suffix":""},{"id":505140881,"identity":"6fb3e611-9776-4d5f-ae1c-5a4a77904f04","order_by":3,"name":"Jielan Jiang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4klEQVRIiWNgGAWjYBACxmYGxgMMDBJy/MwHQHwJGWK0MADV2hhLtiWAtfAQZRNQS1qiwTGwFgbCWpjbmQ8c+LjjcILxMeZjUjdqLHgY2A8f3YDfYWwJB2eeOZxndowt2TjnGNBhPGlpN/Br4TE4zNt2uNjsfo/h4xw2oBYJHjMCWvg/HP7bdjhxcxtQb84/orTwMBxmbEtL3MDGY/g4t40oLWwGB3vbbIwlQH7J7ZPgYSPkF8P+ww8f/GwDRmUb8zHpnG91cvzsh4/h19KALsKGTzkIyBNSMApGwSgYBaOAAQAkzUcjoegePQAAAABJRU5ErkJggg==","orcid":"","institution":"Yazhou Bay Innovation Institute, Hainan Tropical Ocean University","correspondingAuthor":true,"prefix":"","firstName":"Jielan","middleName":"","lastName":"Jiang","suffix":""},{"id":505140883,"identity":"eaabd3a1-3729-4b85-99f0-71bd83052156","order_by":4,"name":"Xiaoming Geng","email":"","orcid":"","institution":"Yazhou Bay Innovation Institute, Hainan Tropical Ocean University","correspondingAuthor":false,"prefix":"","firstName":"Xiaoming","middleName":"","lastName":"Geng","suffix":""},{"id":505140885,"identity":"32b8ab4f-73d0-4a20-95c6-46d72c795473","order_by":5,"name":"Xiangcheng Tang","email":"","orcid":"","institution":"Yazhou Bay Innovation Institute, Hainan Tropical Ocean University","correspondingAuthor":false,"prefix":"","firstName":"Xiangcheng","middleName":"","lastName":"Tang","suffix":""}],"badges":[],"createdAt":"2025-08-17 18:53:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7394032/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7394032/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":90003755,"identity":"14e7bab3-a9c5-441b-afa0-b742e956288e","added_by":"auto","created_at":"2025-08-27 09:14:38","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":62180,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of different reagents on nanoparticle formation\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7394032/v1/5596b339dc34cdef632a417f.png"},{"id":90003758,"identity":"e2022b4d-5251-4b43-84ac-22cd86f1c5a9","added_by":"auto","created_at":"2025-08-27 09:14:38","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":60460,"visible":true,"origin":"","legend":"\u003cp\u003eSize Distribution and Zeta Potential of Nanoparticles after Secondary Ultrasonication and Ultrafiltration\u003c/p\u003e\n\u003cp\u003e(A) Size distribution of nanoparticles based on light intensity. (B) Zeta potential measured by voltage-current analysis.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7394032/v1/5aeef1c607df6b253208002d.png"},{"id":90003763,"identity":"82fb36c5-97f5-46e3-b2d7-96834aa6137a","added_by":"auto","created_at":"2025-08-27 09:14:39","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":476181,"visible":true,"origin":"","legend":"\u003cp\u003eTransmission Electron Microscopy (TEM) Images of Nanoparticles\u003cbr\u003e\n(A) Image captured at a scale bar of 400 nm. (B) Image of a single nanoparticle at a scale bar of 100 nm.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7394032/v1/74e8fda8c3489290a84eb4d5.png"},{"id":90004187,"identity":"630f7b67-6788-42a1-a8d8-f23c2c03f930","added_by":"auto","created_at":"2025-08-27 09:22:38","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":225547,"visible":true,"origin":"","legend":"\u003cp\u003eNNV CP with GFP Expression in EPC\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA-B\u003c/strong\u003e) Observation under fluorescence microscope 48h post-transfection with pEGFP-N1 (\u003cstrong\u003eC\u003c/strong\u003e) Observation under fluorescence microscope 48h post-transfection with pEGFP-CP (\u003cstrong\u003eD\u003c/strong\u003e) Observation of adherent cells under confocal microscope 24h post-transfection with pEGFP-CP.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7394032/v1/d96fdec8868b64f7fa91ef5e.png"},{"id":90003756,"identity":"b2fda672-82a0-4fff-bbc2-65ce306a7509","added_by":"auto","created_at":"2025-08-27 09:14:38","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":116336,"visible":true,"origin":"","legend":"\u003cp\u003eCell Viability of EPCs Treated with Various Reagents\u003cbr\u003e\n(\u003cstrong\u003eA\u003c/strong\u003e) EPCs were transfected with pEGFP, siRNA-Cys, or pEGFP-CP, with untreated EPCs serving as the control; (\u003cstrong\u003eB\u003c/strong\u003e) Cell viability of EPCs co-transfected with siRNA and pEGFP-CP. \u003cstrong\u003eNote:\u003c/strong\u003e Asterisks (**) indicate a highly significant difference (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01) compared to the control group.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7394032/v1/9c5ce59cc77b21502beb2543.png"},{"id":90003767,"identity":"d7d262a2-b299-4bbe-a075-de72422f500a","added_by":"auto","created_at":"2025-08-27 09:14:39","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":68551,"visible":true,"origin":"","legend":"\u003cp\u003esiRNA Encapsulation in NPs\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Fluorescence-based size distribution profile of NPs resuspended in PBS after ultrafiltration membrane recovery; (\u003cstrong\u003eB)\u003c/strong\u003e Corresponding siRNA encapsulation efficiency for PBS-resuspended NPs.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7394032/v1/1ad8ff63e69e474bbc313fa1.png"},{"id":90003762,"identity":"b4e37794-ebcf-4277-83e2-cb7df11fe437","added_by":"auto","created_at":"2025-08-27 09:14:39","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":15242,"visible":true,"origin":"","legend":"\u003cp\u003eSurvival of fish eggs treated with the siRNA-PLGA NPs\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7394032/v1/809f171275891be55ccfdf8d.png"},{"id":90006260,"identity":"549dd5d2-658a-4f66-bf5d-bc4c8d19f22b","added_by":"auto","created_at":"2025-08-27 09:38:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1578239,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7394032/v1/f6ec40f5-4d88-4be1-b566-20b8ecaacbc8.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"RNAi-Based PLGA Nanoparticles for Targeted Silencing of NNV Capsid Protein","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eNervous Necrosis Virus (NNV) represents a formidable challenge to aquaculture, particularly marine fish populations, where it inflicts substantial damage by compromising the nervous system and precipitating high mortality rates (Mao et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2015b\u003c/span\u003e). The pathogenicity of NNV predominantly affects larval and juvenile stages, sparing adults (Munday and Nakai,1997; Iwamoto 2004; Doan et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Current prophylactic and management strategies for NNV encompass diverse approaches targeting both the viral pathogen and the aquaculture environment. Among these, vaccines are extensively studied but remain ineffective in larval stages due to the immaturity of their immune systems (Thiery et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Pakingking et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Mao et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Mao et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2015a\u003c/span\u003e; Band\u0026iacute;n and Souto, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Alternatively, advancements in gene-editing technologies have demonstrated potential for enhancing host resistance through targeted modification of immune-related genetic loci. Effective water quality management further complements these strategies, albeit as an indirect control measure.\u003c/p\u003e\u003cp\u003eNNV transmission occurs via horizontal and vertical pathways, with the latter posing significant concern due to the potential for perpetuation of infection across generations. Vertical transmission facilitates viral passage from broodstock to progeny through reproductive processes or within eggs and larvae, creating reservoirs of infection that persist independently of external exposure (Breuil et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1991\u003c/span\u003e; Munday and Nakai,1997; Munday et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Toffan et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Our study seeks to address this challenge by employing small interfering RNAs (siRNAs) to suppress or eliminate NNV in fertilized eggs, thereby interrupting vertical transmission. However, siRNAs are inherently unstable in marine environments (Suksai et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), and their intracellular delivery to target sites within fertilized eggs necessitates an efficacious carrier system.\u003c/p\u003e\u003cp\u003ePoly (lactic-co-glycolic acid) (PLGA) emerges as a prominent candidate for siRNA delivery. As a biodegradable and biocompatible polymer, PLGA offers superior drug delivery properties, including enhanced stability, tunable degradation rates, and minimal cytotoxicity (Anderson and Shive, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Jain \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Makadia and Siegel, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Danhier et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). PLGA nanoparticles (NPs) have garnered considerable attention as delivery vehicles for siRNA, as their encapsulation capabilities mitigate enzymatic degradation and facilitate targeted delivery. Optimizations in molecular weight and the lactic-to-glycolic acid ratio enable precise control over release kinetics. Hybrid systems incorporating materials such as polyethyleneimine (PEI) and lipids further augment the stability, cellular uptake efficiency, and immunocompatibility of PLGA-based NPs (Khalil et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Dobrovolskaia and McNeil, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Danhier et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Sun et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Kumar et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Preclinical investigations have validated the efficacy of PLGA-loaded siRNA NPs across diverse biomedical applications, including oncology, viral infections, and gene therapy (Khalil et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Zhao et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Kumar et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Sun et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). For instance, murine tumor models demonstrated substantial gene silencing and therapeutic outcomes with PLGA-siRNA formulations. Despite these advancements, there remains a paucity of studies exploring PLGA NPs in aquaculture. This study seeks to bridge this gap by employing a double-emulsion (w/o/w) technique to construct siRNA-PLGA nanoparticles targeting NNV, with the ultimate goal of establishing a novel intervention framework for aquaculture.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Materials\u003c/h2\u003e\u003cp\u003ePLGA was synthesized by copolymerizing lactic acid and glycolic acid at a ratio of 75:25, with a molecular weight of 10 kDa. Ultrafiltration spin columns (catalog number RT-VS2052-5) were procured for purification processes. Additional reagents included Tween-80, trichloromethane, N,N-dimethylformamide (DMF), polyethylene glycol (PEG), and polyethyleneimine (PEI, molecular weight 25,000), the latter sourced from Shanghai manufacturers. EPC (Epithelioma Papulosum Cyprini) cell lines were utilized as provided by the laboratory, while fertilized grouper eggs, 18 hours post-fertilization, were acquired for experimental applications.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Synthesis and Characterization of siRNA-PLGA Nanoparticles (NPs)\u003c/h2\u003e\u003cp\u003esiRNA-PLGA nanoparticles were synthesized via a double-emulsion (w/o/w) method coupled with ultrafiltration. Briefly, 5 mg of PLGA was dissolved in 1 mL of an organic solvent (trichloromethane or DMF) to constitute the oil phase. The primary emulsion was generated by ultrasonication of the oil phase with a water solution containing siRNA, PEI or Tween-80 for 6 minutes in an ice bath (amplitude: 40%, pulse on: 5 s, pulse off: 2 s, for 5min). This emulsion was subsequently dispersed into 30 mL of an aqueous solution containing PEG or Tween-80, forming the secondary emulsion, which was also generated by ultrasonication (amplitude: 40%, pulse on: 5 s, pulse off: 2 s, for 5min). The nanoparticle suspension was centrifuged at 3000 rpm for 30 minutes, collected using Ultrafiltration spin columns, washed and resuspended with 1 mL of PBS or distilled water. The size and surface charge of the nanoparticles were characterized using dynamic light scattering (DLS) and laser Doppler velocimetry (LDV) with a Nano ZS Zetasizer (Malvern Instruments, UK). Nanoparticle morphology was visualized via transmission electron microscopy (TEM).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Expression of Nervous Necrosis Virus Capsid Protein (CP) in EPC Cells\u003c/h2\u003e\u003cp\u003eThe capsid protein (CP) gene of the NNV isolate RG-NNV, previously cloned and preserved in the laboratory, was inserted into the pEGFP-N1 plasmid vector to generate the recombinant plasmid pEGFP-CP. The plasmid was amplified in \u003cem\u003eEscherichia coli\u003c/em\u003e and purified using an endotoxin-free plasmid extraction kit, with sequence integrity confirmed by sequencing. Transfection of EPC cells with pEGFP-CP was performed using Lipofectamine 3000 (Invitrogen, L3000) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). CP expression was assessed 48 hours post-transfection by fluorescence microscopy. To evaluate cell viability and nuclear morphology, transfected cells were stained with Hoechst 33342 at 24 hours post-transfection, prior to the onset of cell death and detachment. Imaging was conducted using confocal microscopy.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eTargets for siRNA Silencing\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eName\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSequence (5\u0026rsquo;-3\u0026rsquo;)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eLength (bp)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eTarget\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003esiRNA18F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGCAAAGGGAAUAAGAAAUUTT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003ePosition 18\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003esiRNA18R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAAUUUCUUAUUCCCUUUGCTT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e21\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003esiRNA144F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGCCUCGACUAUCACGGGAUTT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003ePosition 144\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003esiRNA144R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAUCCCGUGAUAGUCGAGGCTT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e21\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003esiRNA720F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGCAGCCACUGAUUUCAAAUTT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003ePosition 720\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003esiRNA720R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAUUUGAAAUCAGUGGCUGCTT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e21\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003esiRNA-Cy3-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eUUCUCCGAACGUGUCACGUTT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003esiRNA-Cy3-R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eACGUGACACGUUCGGAGAATT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e21\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 siRNA Entrapment Efficiency in PLGA Nanoparticles\u003c/h2\u003e\u003cp\u003ePLGA nanoparticles encapsulating siRNA targeting the CP gene of NNV were synthesized as described. Encapsulation efficiency was quantified as the ratio of siRNA content retained within nanoparticles relative to the total amount used in synthesis. This was determined using Nano-Flow Cytometry (NanoFCM, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.nanofcm.com\u003c/span\u003e\u003cspan address=\"http://www.nanofcm.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Effect of siRNA-PLGA Nanoparticles on Fish Egg Survival Rate\u003c/h2\u003e\u003cp\u003eThe siRNA (siRNA144)-PLGA NPs was diluted into 1000\u0026times; and 10\u0026times; concentrations respectively and added to the fish egg culture water. The fertilized fish eggs (12 hours post-fertilization at the start of the experiment) were randomly allocated into 9 culture dishes (15 cm in diameter, 3 cm in height) that had been thoroughly disinfected with KMnO4, with an initial quantity of 50 fish eggs per dish. The experiment was divided into 3 groups with different concentrations of the siRNA-PLGA nanoparticles, and each group had 3 replicates. Mortality rate and hatching rate of fish eggs were rec-orded at different time points during the experiment. Constant temperature, light, and aeration were maintained throughout the process.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1 Characterization of siRNA-PLGA Nanoparticles (NPs)\u003c/h2\u003e\n \u003cp\u003eThe NPs synthesized using the double-emulsion method exhibited the desired particle size distribution and surface properties essential for effective gene delivery. When chloroform was used as the primary solvent and PEG was added to the external aqueous phase, the resulting nanoparticle size was 156.6 nm (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA); when Tween-80 was added to the external aqueous phase instead of PEG, the nanoparticle size was 146 nm (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB). Using DMF as the primary solvent and PEG in the external aqueous phase resulted in nanoparticles with a size of 189.2 nm (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC); when Tween-80 was added to the external aqueous phase in the DMF system, two peaks were observed, indicating a heterogeneous size distribution (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD). The average particle size, determined by dynamic light scattering (DLS), was approximately 164 nm. Based on these results, we selected the PLGA-PEI/Trichloromethane/PEG (W/O/W) system for the next experiment.\u003c/p\u003e\n \u003cp\u003eThe nanoemulsions synthesized using the PLGA-PEI/Trichloromethane/PEG (W/O/W) system were subjected to a second round of sonication, followed by filtration using an ultrafiltration membrane. The nanoparticles on the membrane were then resuspended in DEPC water. After measurement, the nanoparticle size was found to be approximately 110 nm, with a unimodal light intensity distribution (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA), which was smaller than that of the nanoparticles synthesized after the first round of sonication. The zeta potential of the nanoparticles was measured at approximately 30.91\u0026thinsp;\u0026plusmn;\u0026thinsp;2 mV(Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB), indicating good colloidal stability and minimal aggregation under physiological conditions(\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.malvernpanalytical.com/en/learn/knowledge-center/application-notes/an101130suspensionstability\u003c/span\u003e\u003c/span\u003e ). The NPs on the filter membrane were resuspended in PBS, dried on a copper grid, and observed under electron microscopy. The nanoparticle size was consistent with the results from the nanoparticle size analyzer, and spherical particles with a size of approximately 110 nm were clearly observed, as shown in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e\n \u003cp\u003eTransmission electron microscopy (TEM) images confirmed the spherical morphology of the NPs, with a consistent and well-defined nanoparticle structure (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). No significant distortion or aggregation was observed, supporting the robustness of the nanoparticle formulation.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003e2.2. NNV model establishment\u003c/h2\u003e\n \u003cp\u003eThe recombinant plasmid pEGFP-CP, harboring the NNV capsid protein gene, was transfected into EPC cells to establish a model for investigating viral protein expression dynamics. Fluorescence microscopy analysis demonstrated robust expression of the GFP-CP fusion protein, confirming successful transfection and intracellular protein synthesis. In control groups (transfected with empty vector), GFP fluorescence exhibited homogeneous cytoplasmic distribution at 48 hours post-transfection (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA-B). In contrast, cells transfected with pEGFP-CP displayed extensive cell death, evidenced by a significant population of detached, non-viable cells in the culture medium. Fluorescent imaging revealed fragmented GFP signals (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC), suggesting potential cytotoxic effects associated with CP overexpression.\u003c/p\u003e\n \u003cp\u003eTo further characterize CP-induced cellular pathology, confocal microscopy was performed on adherent cells at 24 hours post-transfection, prior to observable cell detachment. Nuclear staining followed by high-resolution imaging revealed marked abnormalities in nuclear morphology (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD), including chromatin condensation and irregular nuclear envelope contours, indicative of CP-triggered nuclear stress. These morphological aberrations correlate temporally with the onset of cytotoxicity, implying a potential mechanistic link between CP accumulation, nuclear destabilization, and subsequent cell death.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3 Screening of Effective siRNAs\u003c/h2\u003e\n \u003cp\u003eThree CP-targeting siRNA duplexes were rationally designed to silence NNV capsid protein expression. Transfection of EPC cells with pEGFP-CP for 48h induced significant cytotoxicity, characterized by extensive cell detachment and reduced viability (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA, Control). Co-administration of all three siRNA candidates markedly rescued cell viability (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB). Notably, all siRNAs demonstrated superior silencing potency and they were prioritized for subsequent nanoparticle encapsulation studies.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003e3.4 siRNA Encapsulation Efficiency\u003c/h2\u003e\n \u003cp\u003eThe encapsulation efficiency of FAM-labeled siRNA within PLGA nanoparticles was systematically quantified using nano-flow cytometry (nFCM) to evaluate the impact of suspension media on siRNA entrapment. Strikingly, nanoparticles resuspended in phosphate-buffered saline (PBS) exhibited a higher siRNA encapsulation efficiency (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA), whereas those suspended in DEPC-treated water showed negligible loading (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eB). Consistent with the physicochemical characterization in Section \u003cspan class=\"InternalRef\"\u003e3.1\u003c/span\u003e, nanoparticle tracking analysis (NTA) confirmed a monodisperse population centered at 160\u0026thinsp;\u0026plusmn;\u0026thinsp;5.8 nm (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA-B), aligning with TEM result (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e)\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003e2.5. The effect of the siRNA-PLGA NPs on the survival of fish eggs\u003c/h2\u003e\n \u003cp\u003eTo investigate the effects of the siRNA-PLGA NPs on fish eggs, this study diluted the NPs to 1000\u0026times; and 10\u0026times; concentrations and added them to the seawater for fish egg hatching. The results showed a survival rate of 86% in the control group, 86% after 1000\u0026times; dilution and a 4% decrease in survival after 10\u0026times; dilution. It showed a slight decrease in the survival rate of fish eggs as the number of times of drug dilution decreased (increase in drug concentration) (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e)\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e4.1. Optimized Nanoparticle Design for siRNA Delivery\u003c/h2\u003e\u003cp\u003eThe siRNA-PLGA NPs synthesized using the PLGA-PEI/trichloromethane/PEG (W/O/W) system demonstrated superior physicochemical properties, including a reduced particle size (~\u0026thinsp;110 nm post-sonication/filtration), monodisperse distribution (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-B), and a high zeta potential (~\u0026thinsp;30.91 mV). These attributes are critical for efficient cellular uptake and endosomal escape, as smaller nanoparticles (\u0026lt;\u0026thinsp;200 nm) are preferentially internalized via clathrin-mediated endocytosis, while a positive surface charge enhances interactions with negatively charged cell membranes (Gratton et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Choi et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; He et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Iversen et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The spherical morphology observed via TEM (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) further corroborates the structural integrity of the NPs, minimizing aggregation risks in physiological environments (Chithrani et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Gratto et al. 2008; Albanese et al. 2012; Zhang et al. 2016).\u003c/p\u003e\u003cp\u003eNotably, the choice of solvent and surfactant significantly influenced nanoparticle homogeneity. The heterogeneity observed in the DMF/Tween-80 system (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD) likely stems from incomplete emulsification, underscoring the importance of solvent-surfactant compatibility in NP synthesis. The transition to PEG in the external aqueous phase improved colloidal stability, aligning with PEG\u0026rsquo;s well-established role in steric stabilization and biocompatibility (Harris and Zalipsky, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Suk et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e4.2. NNV Capsid Protein (CP)-Induced Cytotoxicity and siRNA Rescue\u003c/h2\u003e\u003cp\u003eThe overexpression of NNV CP in EPC cells triggered rapid cytotoxicity, characterized by cell detachment, fragmented GFP signals (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), and nuclear abnormalities such as chromatin condensation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). These observations suggest that CP accumulation induces apoptosis or necroptosis, potentially through nuclear stress pathways (Vandenabeel et al. 2010; Green and Llambi, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Galluzz et al. 2018). The temporal correlation between nuclear destabilization (24 h) and overt cell death (48 h) implies a cascade of intracellular damage, positioning CP as a critical virulence factor. The complete rescue of cell viability by all three CP-targeting siRNAs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB) validates their silencing efficacy and highlights the therapeutic potential of RNAi in mitigating NNV-induced pathology. The lack of significant differences in siRNA potency suggests redundant target accessibility or uniform siRNA design efficiency, warranting further mechanistic studies to identify optimal silencing regions within the CP transcript.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e4.3. siRNA Encapsulation Efficiency: Role of Suspension Medium\u003c/h2\u003e\u003cp\u003eA striking finding was the stark contrast in siRNA encapsulation efficiency between nanoparticles resuspended in PBS (\u0026gt;\u0026thinsp;10%) versus DEPC-treated water (\u0026lt;\u0026thinsp;3%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC-D). While DEPC water is traditionally employed for RNA-related applications due to its RNase-free properties (Farrell \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Rio et al. 2010), its poor performance here may arise from ionic incompatibility with the PLGA matrix (Gilman \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Makadia and Siegel, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). PBS, with its physiological ion concentration and buffering capacity, likely stabilizes electrostatic interactions between siRNA and the cationic PLGA-PEI polymer, enhancing entrapment (Panyam and Labhasetwar \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Kumar et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Jeon et al. 2007; Whitehead et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). This underscores the necessity of tailoring suspension media to the physicochemical properties of both the nanoparticle and payload.\u003c/p\u003e\u003cp\u003ePLGA degradation primarily occurs in endosomes/lysosomes (pH 4.5\u0026ndash;5.5). The acidic environment accelerates hydrolysis, promoting siRNA escape into the cytoplasm to exert its function. (1) Diffusion Release: Initial siRNA partially diffuses out through pores formed by PLGA swelling. (2) Degradation-Controlled Release: Later-stage PLGA backbone cleavage leads to massive siRNA release (burst effect). Ester bonds (-COO-) in PLGA chains are hydrolyzed by water molecules, generating lactic acid (LA) and glycolic acid (GA) monomers. LA is naturally produced in the body. While GA is not endogenous in fish, it exhibits low toxicity at controlled concentrations.\u003c/p\u003e\u003cp\u003eIn sum, this study demonstrates the successful design of siRNA-loaded PLGA nanoparticles with optimized size, stability, and encapsulation efficiency, capable of rescuing CP-induced cytotoxicity in EPC cells. The critical role of formulation parameters (solvent, surfactant, suspension medium) in NP performance highlights the need for meticulous optimization in nanomedicine development. Future work should focus on scaling up NP production, validating efficacy in vivo, and expanding the siRNA library to target additional NNV virulence factors (Whitehea et al. 2009; Petros and DeSimone \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Chen et al. 2015). These findings pave the way for RNAi-based therapies against aquatic viral pathogens, with broader implications for antiviral nanotechnology.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contribution\u003c/strong\u003e M. Mao and J. Jiang designed the experiments and obtained the funding. Y. Wang and S. Zhu carried out the experiment. Y. Wang, S. Zhu, X. Geng and X. Tang carried out the data analysis. M. Mao and J. Jiang wrote the manuscript with support from Y. Wang and S. Zhu, and all authors agree to be accountable for all aspects of the work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e This research was funded by the National key research and development program of China (2024YFD2401803), the Hainan Provincial Natural Science Foundation of China (322RC716), the National Natural Science Foundation of China (Grant No. 32360923), the Hainan Provincial Natural Science Foundation of China (422MS083), the Scientific Research Foundation of Hainan Tropical Ocean University (RHDRC202206). Any field work in this study complied with the current laws of China, where it was performed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e No datasets were generated or analysed during the current study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interest\u003c/strong\u003e The authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e All authors agreed to publish the current study through Aquaculture International.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAnderson JM, Shive MS (1997) Biodegradation and biocompatibility of PLA and PLGA microspheres. Adv Drug Deliv Rev 28:5\u0026ndash;24. https://doi.org/10.1016/S0169-409X(97)00048-3\u003c/li\u003e\n\u003cli\u003eBand\u0026iacute;n I, Souto S (2020) Betanodavirus and VER disease: A 30-year research review. Pathogens 9:106. https://doi.org/10.3390/pathogens9020106\u003c/li\u003e\n\u003cli\u003eBreuil G, Bonami JR, Pepin JF, Pichot Y (1991) Viral infection (picorna-like virus) associated with mass mortalities in hatchery-reared sea bass (\u003cem\u003eDicentrarchus labrax\u003c/em\u003e) larvae and juveniles. Aquaculture 97:109\u0026ndash;116. https://doi.org/10.1016/0044-8486(91)90258-9\u003c/li\u003e\n\u003cli\u003eChithrani BD, Ghazani AA, Chan WCW (2006) Determining the size and shape dependence of gold nanoparticle uptake into mammalian cells. Nano Lett 6:662\u0026ndash;668. https://doi.org/10.1021/nl052396o\u003c/li\u003e\n\u003cli\u003eChoi CH, Alabi CA, Webster P, Davis ME (2010) Mechanism of active targeting in solid tumors with transferrin-containing gold nanoparticles. Proc Natl Acad Sci 107:1235\u0026ndash;1240. https://doi.org/10.1073/pnas.0914140107\u003c/li\u003e\n\u003cli\u003eDanhier F, Ansorena E, Silva JM et al (2012) PLGA-based nanoparticles: An overview of biomedical applications. J Control Release 161:505\u0026ndash;522. https://doi.org/10.1016/j.jconrel.2012.01.043\u003c/li\u003e\n\u003cli\u003eDobrovolskaia MA, McNeil SE (2007) Immunological properties of engineered nanomaterials. Nat Nanotechnol 2:469\u0026ndash;478. https://doi.org/10.1038/nnano.2007.223\u003c/li\u003e\n\u003cli\u003eDoan QK, Vandeputte M, Chatain B, Morin T, Allal F (2017) Viral encephalopathy and retinopathy in aquaculture: a review. J Fish Dis 40:717\u0026ndash;742. https://doi.org/10.1111/jfd.12550\u003c/li\u003e\n\u003cli\u003eFarrell RE (2010) \u003cem\u003eRNA Methodologies: A Laboratory Guide for Isolation and Characterization\u003c/em\u003e (4th ed.). Academic Press.\u003c/li\u003e\n\u003cli\u003eGalluzzi L, Vitale I, Aaronson S et al (2018) Molecular mechanisms of cell death: Recommendations of the Nomenclature Committee on Cell Death 2018. Cell Death Differ 25:486\u0026ndash;541. https://doi.org/10.1038/s41418-017-0012-4\u003c/li\u003e\n\u003cli\u003eGilman M (1993) Ribonuclease inhibition by diethyl pyrocarbonate. Methods in Molecular Biology 16:21\u0026ndash;23.\u003c/li\u003e\n\u003cli\u003eGratton SE, Ropp PA, Pohlhaus PD et al (2008) The effect of particle design on cellular internalization pathways. Proc Natl Acad Sci 105:11613\u0026ndash;11618. https://doi.org/10.1073/pnas.0801763105\u003c/li\u003e\n\u003cli\u003eGreen DR, Llambi F (2015) Cell death signaling. Csh Perspect Biol 7: a006080. https://doi.org/10.1101/cshperspect.a006080\u003c/li\u003e\n\u003cli\u003eHarris JM, Zalipsky S (1997) \u003cem\u003ePoly (ethylene glycol): Chemistry and Biological Applications\u003c/em\u003e. ACS Symposium Series.\u003c/li\u003e\n\u003cli\u003eHe C, Hu Y, Yin L, Tang C, Yin C (2010) Effects of particle size and surface charge on cellular uptake and biodistribution of polymeric nanoparticles. Biomaterials 31:3657\u0026ndash;3666. https://doi.org/10.1016/j.biomaterials.2010.01.065\u003c/li\u003e\n\u003cli\u003eIversen TG, Skotland T, Sandvig K (2011) Endocytosis and intracellular transport of nanoparticles: Present knowledge and need for future studies. Nano Today 6:176\u0026ndash;185. https://doi.org/10.1016/j.nantod.2011.02.003\u003c/li\u003e\n\u003cli\u003eIwamoto T, Okinaka Y, Mise K et al (2004) Identification of host-specificity determinants in betanodaviruses by using reassortants between striped jack nervous necrosis virus and sevenband grouper nervous necrosis virus. J Virol 78:1256\u0026ndash;1262. https://doi.org/10.1128/JVI.78.3.1256-1262.2004\u003c/li\u003e\n\u003cli\u003eJain RA (2000) The manufacturing techniques of various drug loaded biodegradable poly(lactide-co-glycolide) (PLGA) devices. Biomaterials 21:2475\u0026ndash;2490. https://doi.org/10.1016/S0142-9612(00)00115-0\u003c/li\u003e\n\u003cli\u003eJeong JH, Mok H, Oh YK et al (2007) siRNA conjugate delivery systems. Bioconjugate Chem 18:5\u0026ndash;14. https://doi.org/10.1021/bc800278e\u003c/li\u003e\n\u003cli\u003eKhalil IA, Kogure K, Akita H, Harashima H (2006) Uptake pathways and subsequent intracellular trafficking in nonviral gene delivery. Pharmacol Rev\u003cem\u003e \u003c/em\u003e58:32\u0026ndash;45. https://doi.org/10.1124/pr.58.1.8\u003c/li\u003e\n\u003cli\u003eKumar MNVR et al (2004) Cationic poly(lactide-co-glycolide) nanoparticles as efficient in vivo gene transfection agents. J Nanosci Nanotechno 4:990\u0026ndash;994. https://doi.org/10.1166/jnn.2004.141\u003c/li\u003e\n\u003cli\u003eKumar R, Santa Chalarca CF, Bockman MR et al (2020) Polymeric delivery of therapeutic nucleic acids. Chem Rev 120:10716\u0026ndash;10776. https://doi.org/10.1021/acs.chemrev.0c00697\u003c/li\u003e\n\u003cli\u003eMakadia HK, Siegel SJ (2011) Poly lactic-co-glycolic acid (PLGA) as biodegradable controlled drug delivery carrier. Polymers 3:1377\u0026ndash;1397. https://doi.org/10.3390/polym3031377\u003c/li\u003e\n\u003cli\u003eMao MG, Jiang JL, Per\u0026aacute;lvarez-Mar\u0026iacute;n A, Wang KJ, Lei JL (2013) Characterization of the Mx and hepcidin genes in \u003cem\u003eEpinephelus akaara\u003c/em\u003e asymptomatic carriers of the nervous necrosis virus. Aquaculture 408-409:175\u0026ndash;183. https://doi.org/10.1016/j.aquaculture.2013.05.038\u003c/li\u003e\n\u003cli\u003eMao MG, Li X, Peralvarez-Marin A et al (2015a). Transcriptomic analysis and biomarkers (Rag1 and Igmu) for probing the immune system development in Pacific cod, \u003cem\u003eGadus macrocephalus\u003c/em\u003e. Fish Shellfish Immun 44:622\u0026ndash;632. https://doi.org/10.1016/j.fsi.2015.03.035\u003c/li\u003e\n\u003cli\u003eMao MG, Wen SH, Peralvarez-Marin, A et al (2015b) Evidence for and characterization of nervous necrosis virus infection in Pacific cod (\u003cem\u003eGadus macrocephalus\u003c/em\u003e). Arch Virol 160:2237\u0026ndash;2248. https://doi.org/10.1007/s00705-015-2498-8\u003c/li\u003e\n\u003cli\u003eMunday BL, Kwang J, Moody N (2002) Betanodavirus infections of teleost fish: a review. J Fish Dis 25:127\u0026ndash;142. https://doi.org/10.1046/j.1365-2761.2002.00350.x.\u003c/li\u003e\n\u003cli\u003eMunday BL, Nakai T (1997) Nodaviruses as pathogens in larval and juvenile marine finfish. World J Microb Biot 13:375\u0026ndash;381. https://doi.org/10.1023/A:1018575210357\u003c/li\u003e\n\u003cli\u003ePakingking R, Bautista NB, de Jesus-Ayson EG, Reyes O (2010) Protection of grouper (\u003cem\u003eEpinephelus coioides\u003c/em\u003e) against nervous necrosis virus by recombinant capsid protein vaccine. Fish Shellfish Immun 28:140\u0026ndash;147. https://doi.org/10.1016/j.fsi.2009.10.007\u003c/li\u003e\n\u003cli\u003ePanyam J, Labhasetwar V (2003) Biodegradable nanoparticles for drug and gene delivery to cells and tissue. Adv Drug Deliver Rev 55:329\u0026ndash;347. https://doi.org/10.1016/S0169-409X(02)00228-4\u003c/li\u003e\n\u003cli\u003ePetros RA, DeSimone JM (2010) Strategies in the design of nanoparticles for therapeutic applications. Nat Rev Drug Discov 9:615\u0026ndash;627. https://doi.org/10.1038/nrd2591\u003c/li\u003e\n\u003cli\u003eRio DC (2010) \u003cem\u003eRNA: A Laboratory Manual\u003c/em\u003e. Cold Spring Harbor Laboratory Press.\u003c/li\u003e\n\u003cli\u003eSuk JS, Xu Q, Kim N, Hanes J, Ensign LM (2016) PEGylation as a strategy for improving nanoparticle-based drug and gene delivery. Adv Drug Deliver Rev 99:28\u0026ndash;51. https://doi.org/10.1016/j.addr.2015.09.012\u003c/li\u003e\n\u003cli\u003eSuksai S, Attasart P, Angsujinda K et al (2025) Delivery of virus-specific dsRNA using a composite nanomaterial improves the protection of shrimp (\u003cem\u003eLitopenaeus vannamei\u003c/em\u003e) against yellow head virus challenge. Aquaculture 595:123456. https://doi.org/10.1016/j.aquaculture.2024.741570\u003c/li\u003e\n\u003cli\u003eSun X, Zhang N, Yin C (2018) PLGA-based nanoparticles for siRNA delivery: Strategies and applications. J Mater Chem B 6:6615\u0026ndash;6631. https://doi.org/10.1039/C8TB01662F\u003c/li\u003e\n\u003cli\u003eThiery R, Cozien J, Cabon J et al (2006) Induction of a protective immune response against viral nervous necrosis in the European sea bass \u003cem\u003eDicentrarchus labrax \u003c/em\u003eby using betanodavirus virus-like particles. J Virol 80:10201\u0026ndash;10207. https://doi.org/10.1128/JVI.01098-06\u003c/li\u003e\n\u003cli\u003eToffan A, Pascoli F, Pretto T et al (2017). Viral nervous necrosis in gilthead sea bream (\u003cem\u003eSparus aurata\u003c/em\u003e) caused by reassortant betanodavirus RGNNV/SJNNV: an emerging threat for Mediterranean aquaculture. Scientific Reports 7:46755. https://doi.org/10.1038/srep46755\u003c/li\u003e\n\u003cli\u003eVandenabeele P, Galluzzi L, Vanden Berghe T et al, (2010) Molecular mechanisms of necroptosis: An ordered cellular explosion. Nat Rev Mol Cell Bio\u003cem\u003e \u003c/em\u003e11:700\u0026ndash;714. https://doi.org/10.1038/nrm2970\u003c/li\u003e\n\u003cli\u003eWang J, Lu Z, Wientjes MG, Au JL (2010) Delivery of siRNA therapeutics: Barriers and carriers. The AAPS Journal 12:492\u0026ndash;503. https://doi.org/10.1208/s12248-010-9210-4\u003c/li\u003e\n\u003cli\u003eWhitehead KA, Langer R, Anderson D (2009) Knocking down barriers: advances in siRNA delivery. Nat Rev Drug Discov\u003cem\u003e \u003c/em\u003e8:129\u0026ndash;138. https://doi.org/10.1038/nrd2742\u003c/li\u003e\n\u003cli\u003eZhao Y, Huang L, Zhang C (2017) PLGA-based nanoparticles for siRNA delivery: Preclinical and clinical advances. Biomaterials 120:1\u0026ndash;14. https://doi.org/10.1016/j.biomaterials.2017.01.016\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"","identity":"aquaculture-international","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"10499","submissionUrl":"https://submission.nature.com/new-submission/10499/3","title":"Aquaculture International","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"","reportingPortfolio":"VoR Journals","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"PLGA, Nanoparticle, Nervous Necrosis Virus, Coat Protein","lastPublishedDoi":"10.21203/rs.3.rs-7394032/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7394032/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNervous necrosis virus (NNV) can be transmitted vertically from parents to offspring, causing significant mortality in young fish. Therefore, effective decontamination of fertilized eggs is essential for sustainable aquaculture production. This study devel-oped siRNA-loaded PLGA nanoparticles (NPs) for targeted silencing of NNV capsid protein (CP) and evaluated their therapeutic potential in vitro. Using a dou-ble-emulsion method, siRNA-PLGA NPs were synthesized with optimized physico-chemical properties: the PLGA-PEI/trichloromethane/PEG (W/O/W) system yielded monodisperse, spherical nanoparticles (~\u0026thinsp;110 nm post-sonication) with a high zeta po-tential (+\u0026thinsp;30.91 mV), ensuring colloidal stability and efficient cellular delivery. Over-expression of NNV CP in EPC cells induced rapid cytotoxicity, including nuclear ab-normalities and cell death within 48 hours, which was fully rescued by co-administering three rationally designed CP-targeting siRNAs. Strikingly, nanopar-ticles resuspended in phosphate-buffered saline (PBS) exhibited a higher siRNA en-capsulation efficiency, whereas those suspended in Diethyl Pyrocarbonate water showed negligible loading. These findings underscore the importance of formulation parameters in nanoparticle design and highlight the promise of RNAi-based PLGA nanotherapeutics for combating aquatic viral infections.\u003c/p\u003e","manuscriptTitle":"RNAi-Based PLGA Nanoparticles for Targeted Silencing of NNV Capsid Protein","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-27 09:14:34","doi":"10.21203/rs.3.rs-7394032/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-22T19:03:24+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-22T19:02:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"37000702803564863493914328969939834464","date":"2025-09-22T19:01:11+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-21T21:04:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"44798854347942041900027323808614179372","date":"2025-09-08T15:43:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"249402621676642582881741838293531520480","date":"2025-08-21T16:04:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"99419031702307897095287869257985039817","date":"2025-08-21T11:58:39+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-19T09:09:34+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-19T09:08:14+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-19T04:54:54+00:00","index":"","fulltext":""},{"type":"submitted","content":"Aquaculture International","date":"2025-08-17T18:48:29+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"","identity":"aquaculture-international","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"10499","submissionUrl":"https://submission.nature.com/new-submission/10499/3","title":"Aquaculture International","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"","reportingPortfolio":"VoR Journals","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"f4d10cf5-f40d-4e36-bc10-15bede1dcb84","owner":[],"postedDate":"August 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-10-23T12:08:51+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-27 09:14:34","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7394032","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7394032","identity":"rs-7394032","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}