Poly(lactic- co-glycolic acid) Microspheres Encapsulating a Viral-Binding Protein, PmRab7, for Preventing White Spot Syndrome Virus in Shrimp

Abstract

White spot syndrome virus (WSSV) is one of the most devastating pathogens affecting shrimp. Within a short time, it leads to a hundred percent mortality rate, which causes substantial economic losses. PmRab7 has been reported to bind to the envelope protein of WSSV, VP28, resulting in a reduction of viral replication. In order to apply PmRab7 in shrimp feed, the development of delivery systems is crucial. Poly(lactic-co-glycolic acid) (PLGA) is a biodegradable polymer extensively studied for drug delivery in the form of nanoparticles or microspheres (MSs). Despite its potential, PLGA has not been previously reported for antiviral use in shrimp. This study is the first to demonstrate the potential use of PLGA and chitosan-coated PLGA (PLGA/CS) MSs for the delivery of PmRab7 in shrimp. Both PLGA and PLGA/CS were optimized and characterized to allow for a sustained release of encapsulated PmRab7. Initial in vitro and in vivo evaluations demonstrated that both MSs are safe for use in shrimp, can sustain the release of PmRab7, and enhance its antiviral activity as shown by a decrease in the mortality rate in shrimp. The development of these MSs has the potential to significantly enhance disease control in shrimp aquaculture, leading to more effective and sustainable practices that will ultimately bolster the industry's growth and long-term stability.

Keywords: PLGA; PmRab7; chitosan; microspheres; white spot syndrome virus.

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Scheme of microsphere preparation (created by biorender.com).

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Characterization of PLGA and PLGA/CS microspheres prepared from different organic solvents: ethyl acetate and dichloromethane. (A) Size. (B) Zeta potential. Values represent mean ± standard deviation (n = 3 independent batches). Error bars reflect variation among three separately prepared microsphere batches. Two-way ANOVA with post hoc analysis using the uncorrected Fisher’s least significant difference (LSD) test was used for statistical analysis. Statistical significance is denoted as follows: ****p ≤ 0.0001, ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05; ns, not significant (p > 0.05). Scanning electron microscopy (SEM) images: (C) PLGA was prepared from EA; (D) PLGA/CS was prepared from EA; (E) PLGA was prepared from DCM; and (F) PLGA/CS was prepared from DCM. (G) FTIR spectra of PLGA and PLGA/CS microspheres. Scale bar = 1 μm.

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Fluorescence images of (a) PLGA, (b) (PmRab7-FITC)-PLGA, (c) PLGA/CS, and (d) (PmRab7-FITC)-PLGA/CS. Scale bar = 50 μm.

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Characterization of PmRab7-encapsulated PLGA microspheres (PmRab7-PLGA) and PmRab7-encapsulated PLGA/CS microspheres (PmRab7-PLGA/CS). (A) Size. (B) Zeta potential. Data are shown as mean ± standard deviation from three independently prepared batches (n = 3). Measurements were performed using a Zetasizer Nano ZS90. The unpaired t-test was used for statistical analysis. Statistical significance is denoted as follows: ****p ≤ 0.0001, ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05; ns, not significant (p > 0.05). (C) SEM image of (PmRab7)-PLGA. (D) SEM image of (PmRab7)-PLGA/CS. Teal bar: (PmRab7)-PLGA, orange bar: (PmRab7)-PLGA/CS, and scale bar = 1 μm.

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Release profile of PmRab7 in different environmental factors. (A) (PmRab7)-PLGA in different salinities of artificial seawater. (B) (PmRab7)-PLGA/CS in different salinities of artificial seawater. (C) (PmRab7)-PLGA in different pH conditions. (D) (PmRab7)-PLGA/CS in different pH conditions. (E) (PmRab7)-PLGA at different temperatures. (F) (PmRab7)-PLGA/CS at different temperatures.

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Percentage of shrimp survival (n = 11) after oral injection and observed it for 10 days. Control: noninjection and PBS injection. Shrimps were fed with PLGA (red), PLGA/CS (brown), (PmRab7)-PLGA (orange), and (PmRab7)-PLGA/CS (yellow) at 0.72, 7.2, and 72 μg/g shrimp.

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Neutralization of WSSV with PmRab7 and microspheres. On day 0 (A) shrimp were orally intubated with either PBS, PmRab7, PLGA, or (PmRab7)-PLGA. (B) Shrimp were orally intubated with PBS, PLGA/CS, or (PmRab7)-PLGA/CS before immersion with WSSV (10⁶ copies/ml water). The survival rate of shrimp after WSSV challenge following oral administration of different microsphere formulations. Each treatment group consisted of 2 replicates of 15 shrimp each, with individual shrimp serving as biological replicates (n = 30). (C). Survival rate of shrimp in all groups at day 8. One-way ANOVA followed by Dunnett’s multiple comparison test was used. Error bars indicate standard deviations. **p ≤ 0.01, *p ≤ 0.05. The survival rate of shrimp after WSSV challenge following oral administration of different microsphere formulations.

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Histopathological examination in shrimp post microparticle administration and virus challenge. The pictures represent stomach epithelial cells infected by WSSV and hypertrophic nuclei were observed (indicated by red arrows), especially in moribund. Less number of hypertrophic cells was mainly observed in surviving shrimp when administered with (PmRab7)-PLGA or (PmRab7)-PLGA/CS. Scale bar = 20 μm.