1. Introduction
Porcine reproductive and respiratory syndrome (PRRS) is a viral disease that affects domestic pigs of all ages, causing significant economic losses in the swine industry due to its devastating effect on production [1] [2] . The causative agent Betaarterivirus suid (PRRSV) shows great genetic and antigenic variability with a marked increase in virulence, so vaccines should be useful in controlling the problems caused [3] [4] [5] .
The study of new strategies to combat this disease is a promising field of research highlighted by the development of technologies that combine the advantages of modern nanomedicine with compounds of natural origin that have been used in traditional medicine since ancient times. Indeed, applications of nanotechnology in the health sciences have led to the formulation of nanometric-scale systems called nanoparticle drug carriers (NPDCs) that consist of at least two components: the active principle, or biologically active molecule, and a delivery system. Compounds of this kind can perform special functions related to diagnosing, treating, and preventing certain diseases [6] . The specific properties that determine their biological impact include chemical composition, primary particle size, morphology, and solubility. These systems offer advantages like the ability to cross biological barriers to reach target organs, tissues, cell groups, or intracellular compartments, while also controlling the release of the active molecule once it reaches its action or absorption site. In these ways they contribute to resolving the challenges associated with the solubility, bioavailability, immunocompatibility and cytotoxicity of many drugs [7] . These systems can also function at the molecular level in a range of 10 - 1000 nm in at least one dimension [8] .
Nanosystems made with polymers (PNPs) are solid vectors with high stability that, depending on the type of polymer used, provide such properties as controlled release, vectorization, and transport of hydrophilic and lipophilic substances that cannot otherwise cross cell membranes in living organisms [9] .
Nanomedicine, in combination with traditional medicine, has allowed the development of new products, including NPDCs as vehicles for active ingredients of natural origin, such as glycyrrhizic acid (GA) [10] , an antiviral compound extracted from the sweet root legume (glycyrrhiza glabra) that grows in subtropical regions [11] [12] . GA has therapeutic applications in various diseases of interest in both human and veterinary medicine. In this study, the in vitro effect of this antiviral content in poly-ε-caprolactone nanoparticles containing glycyrrhizic acid (PNPsGA) was evaluated as a new therapeutic strategy for treating PRRSV infections.
2. Materials and Methods
2.1. Preparation of the PNPsGA
The PNPs were prepared by the modified emulsification-diffusion method [13] . Solvent saturation was performed by mixing equal parts of water and ethyl acetate for 20 min. After separation, the two phases were recovered independently and 2 g of Mowiol 4 - 8 were dissolved in 40 ml of the aqueous phase, and 400 mg of poly-ε-caprolactone in 20 ml of the organic phase. Both phases were emulsified at 11,000 rpm for 10 min in Ultraturax® equipment (Cole-Parmer, USA) to form an oil-in-water emulsion (o/w). Next, 100 ml of water were added (diffusion stage) and subsequently evaporated from the solvent in a rotary evaporator for 1 h at 40˚C and 60 rpm. The PNPs were left to stabilize for 24 h at 4˚C, then centrifuged at 30,000 rpm for 30 min to resuspend them in 2 ml of H2O. Finally, they were added by slow flow to a 20-ml solution of acetate buffer that contained 37 mg of glycyrrhizic acid. This formulation was maintained under magnetic stirring at 500 rpm for 1 h to complete drug absorption. All reagents used were of reagent grade, obtained from Sigma-Aldrich (USA), and used with no additional purification. The water was of Milli-Q quality (Millipore®, Bedford, MD, USA).
2.2. Characterization
The characterization of the size, shape and stability of systems determines the properties and behavior in biological environments. Particle size by Dynamic light-scattering (DLS) was performed with a submicron laser particle counter at a scattering angle of 90˚ (Nanosizer® N4 Plus) and a wavelength of 680 nm for 3 min at 25˚C. Samples were diluted in distilled water to within the device’s sensitivity range. The scattered light data were examined using a digital correlator in the unimodal analysis mode. Only the formulations with a polydispersity index (PI) < 1 were selected. The samples were analyzed at six intervals: 1, 2, 3, 4, 15, and 30 days post-processing. After measuring them, were recovered to assess stability (zeta potential) by Doppler laser microelectrophoresis using a Zetasizer Nano HS (ZEN3600) (Malvern® Instruments Limited, UK) equipped with an He/Ne laser with a wavelength of 633 nm and 4 mW of output power. An average of 100 readings was performed. Determinations were made in triplicate for all batches prepared.
For the morphology analysis by Scanning electron microscopy (SEM) of the PNPsGA, the samples were placed in a 0.22-µm pore filtration membrane, dried at 25˚C, and placed on a grid covered with a thin gold film (~20 nm thick) for 5 min at 7 amps in a vacuum (Denton Vacuum, USA) for subsequent visualization at 25,000X using a Jeol® JSM-6010LA (USA) low vacuum, scanning electron microscope.
2.3. Adsorption Efficiency (EA) and Loading Capacity (LC)
The EA and LC of the GA in the PNPsGA were determined by ultracentrifugation at 20,000 rpm and 10˚C for 30 min. The resulting supernatant containing glycyrrhizic acid not associated with the nanoparticles was measured by high-performance liquid chromatography (HPLC). The supernatant was then brought to a volume of 250 ml, and a 4-ml aliquot was taken and brought to a final volume of 25 ml. In the next step, 20 microliters of that solution were injected into a reverse-phase C18 column (5 μm, 4.6 mm × 150 mm). The mobile phase was a mixture of methanol/acidified water (68.5:31.5) at 1% with glacial acetic acid. The flow rate was 1 ml/min. The adsorption efficiency (AE%, Equation (1)) and loading capacity of the GA (LC%, Equation (2)) of the nanoparticles were calculated with the following equations:
(1)
(2)
2.4. MARC-145 Cell Line
The MARC-145 cell line (clone of the African green monkey kidney cell line MA-104) was used in the infection challenges with PRRSV and exposure to the PNPsGA. The cells were cultured in Roswell Park Memorial Institute 1640 medium (RPMI), supplemented with 10% and 2% of fetal bovine serum (FBS) for growth and maintenance, respectively, and antibiotics (5000 IU/ml penicillin and 5 µg/ml streptomycin). The cultures were maintained in a humidified incubator with a mixture of 95:5 air/CO2 at 37˚C until they reached a confluence of 80% - 90%, which was considered adequate for use in the ensuing experiments.
2.5. Viral Spread
The VR-2332 strain of PRRSV was obtained from the commercial live virus vaccine Ingelvac® PRRS MLV at the Bôehringer Ingelheim laboratory. For propagation, MARC-145 cultures were covered with the viral solution and incubated for 2 h, then the viral solution was removed and 1 ml of RPMI medium supplemented with 2% FBS (2% RPMI) was added. The cultures were monitored every 12 hours post-infection (hpi). Upon observing cytopathic effects, they were subjected to three freezing/thawing steps to lyse the cells and release the viral load into the medium. The resulting medium was centrifuged at 5000 rpm for 20 min. The supernatant was recovered, filtered through a 0.22-μm membrane, and aliquoted under sterile conditions for later use. After propagation and recovery, the viral titers were calculated by the Reed-Muench method.
2.6. Viral Infection
MARC-145 cells were cultured in 24-well plates to 70% confluence. Five systems were tested:
System 1 (to determine a possible preventive effect): 50 µl/ml of PNPsGA were added to each well. After 4 h, the cultures were washed with sterile 3% RPMI and infected for 1 h with 200 µl of PRRSV, then the virus was removed and 3% RPMI medium was added to monitor the course of the infection.
System 2 (to determine a possible inhibitory effect): cell cultures with 70% confluence were infected for 1 h with 200 μl of PRRSV. When the viral solution was added, 50 μl/well of PNPsGA were administered, then the virus and PNP solution were removed and 3% RPMI medium was added to monitor the course of the infection.
System 3 (to determine a possible therapeutic effect): after viral infection and the addition of RPMI 3% medium, 50 μl/well of the PNPsGA were added to monitor the course of the infection.
System 4 (control of viral infection): cultures were infected for 1 h with 200 µl of PRRSV, then the virus was removed and 3% RPMI medium was added to monitor the course of the infection.
System 5 (uninfected culture): once confluence was reached, the 3% RPMI medium was changed to continue with the culture.
All systems were maintained for 96 hpi and evaluated at 4 intervals (24, 48, 72, 96 hpi).
2.7. Hematoxylin and Eosin Staining
After exposure to the different systems, cells seeded on circular slides were fixed with a 4% paraformaldehyde solution for 10 min, washed with phosphate buffered saline (PBS), and covered with a 1% hematoxylin solution for 20 min. At that point, washes were carried out with PBS and the cells were covered for 10 min with eosin. Finally, they were washed with PBS and mounted on slides with resin.
2.8. Cell Viability
For the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay [14] , the culture medium was added to a 0.5 mg/ml MTT solution and incubated for 3 h in a humidified environment with 5% CO2 at 37˚C. Subsequently, 330 μl of dimethyl sulfoxide (DMSO) were added and product intensity was analyzed by spectrophotometry at 570 nm in a plate reader (enzyme-linked immunosorbent assay, ELISA).
2.9. Cell Viability by Exclusion of Trypan Blue
These samples were incubated at a 1:1 ratio of 0.4% (w/v) trypan blue solution for 10 min using automated equipment (TC20TM Automated Cell Counter, Bio-Rad, USA) in a detection concentration range of 5 × 104 - 1 × 107 cells/ml. Next, 10 μl of the cell suspension were placed in duplicate on a slide with a double counting chamber (C20TM counting slides, Bio-Rad, USA), and introduced immediately into the equipment for counting.
2.10. RT-qPCR
For the real-time quantitative polymerase chain reaction (RT-qPCR) technique, the POCKIT TM PRRSV-NA reagent set (GeneReach Biotech, Taichung, Taiwan) was used to detect two of the best conserved regions of the ORF6 and ORF7 genes in the PRRSV genome. Samples were processed using the TRIzol® method (Reagent Total RNA Isolation Reagent, Life Technologies). The primers used to identify ORF5-7 were forward 5'-TTT GGC AAT GTG TCA GGC ATC GTG-3' and reverse 5'-CCA TTC ACC ACA CAT TCT TCC-3'.
2.11. Statistical Analysis
The data obtained were analyzed independently using a completely randomized design. The separation of means was performed using a Tukey’s test. The data were subjected to an analysis of variance (ANOVA) using the GraphPad Prism 7 package with a significance level of α = 0.05 to detect between-group differences. ImageJ software was used for image analysis.
3. Results
The PNPsGA were elaborated following the reference in Jardon et al., (2018) [15] . Subsequent measurement by the DLS technique determined that the PNPsGA had a nanometric size with a maximum intensity peak at 217.43 ± 11.44 nm, and a polydispersity index of 0.17 ± 0.04, which is indicative of a homogeneous size distribution. During the evaluation period, the partial particle size of each system was measured weekly to evaluate the physical stability of the formulations, which remained on a nanometric scale for up to 6 months at room temperature without light protection. To validate the data obtained from the partial particle size study using the DLS technique, the samples were observed under scanning electron microscopy. Measurements of the nanometric size obtained by DLS were consistent with those of the micrographs, which not only showed structures <1 micrometer but also allowed us to determine that each system had a spherical morphology that tended to agglomerate during drying and then form films (Figure 1).
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Figure 1. Scanning electron microscopy of PNPs. Images were taken with SEM at 15,000X (A, B) and 20,000X (C). Micrographs show that the nanotransporters have a size < 1 micrometer and spherical shapes. Bar = 1 μm. PNP intensity peak graph (D).
After obtaining and characterizing the systems, the PNPsGA were sterilized with UV light to assess their antiviral effect on MARC-145 cultures infected with PRRSV. The size of the PNPsGA was determined before and after exposure to UV light to verify that nanometric size was maintained. During preparation of the lots of PNPsGA, the load capacity of the samples for GA was evaluated, determining a capacity of 58.81 ± 0.14.
All tests of the cell cultures were performed using MARC-145 cells, as this infection model is useful in research into the action mechanisms of this virus and the effectiveness of new antiviral systems [15] .
The cytopathic effect produced by infection with PRRSV was visible when the cultures were evaluated by bright field microscopy. This effect was characterized by the acquisition of a rounded shape, loss of the nucleus-cytoplasm relation, vacuolization, formation of syncytia, and cell lysis (Figure 2). Results showed that PRRSV had a severe cytopathic effect after 48 hpi, as it destroyed 76.8% of the monolayer. To evaluate the effects on morphology during acute infection, the cultures were stained with hematoxylin and eosin (H&E), which showed changes after 24 hpi (Figure 3) that included lysis, vacuolization, and the formation of syncytia (Figure 3B).
The most pronounced cytopathic effect occurred in the range of 48-72 hpi, where the damage to the integrity of the monolayer was >70% (Figure 2). The adhered cells presented an isolated distribution with numerous vacuoles at the cytoplasmic level that produced a granular aspect, loss of the nucleus-cytoplasm relation, and morphological changes ranging from rounded forms (Figure 3D) to a fusiform shape with pyknotic nuclei (Figure 3E) that ended with the detachment of the monolayer due to cell lysis (Figure 3F).
Based on the cytopathic effects induced by in vitro infection with PRRSV, 48 and 72 hpi were chosen to evaluate cell viability by the MTT assay (Figure 4) and exclusion with trypan blue (Figure 5). Pretreatment with 50 μl of PNPs for 4 h decreased the cytotoxicity determined by the MTT assay by 38% compared to untreated infected cells, while the behavior of cell viability measured by the trypan blue test was 22%.
The effect of the PNPsGA on viral replication was evaluated by reverse transcription, followed by real-time quantitative PCR (RT-qPCR) analysis of the PRRSV RNA level. As Figure 6 shows, treatment with the PNPsGA reduced the number of RNA copies of PRRSV in 3 cycles.
4. Discussion
This study analyzed the antiviral potential of a model of nanoparticles with glycyrrhizic acid (Figure 1), a component of natural origin that has been shown to have an antiviral effect [11] [16] [17] . On viruses such as influenza A [18] , (Harada 2005), herpes simplex virus type 1 (HSV-1), human papillomavirus [19] and Newcastle virus [16] [20] [21] [22] . It also decreases the replication of viruses like severe acute respiratory syndrome (SARS) [23] . Glycyrrhizinic acid contained in carbon dots has been reported to suppress the spread of pseudorabies virus (PRV) and porcine epidemic diarrhea virus (PEDV), suggesting the broad antiviral activity in pigs [24] [25] . However, GA has certain drawbacks; namely, poor solubility in water, and some degree of cytotoxicity [26] . For this reason, it is necessary to use nanotransporters; that is, systems designed to improve the pharmacokinetics of antivirals because their nanometric size confers important characteristics, including the ability to cross biological barriers and avoid the premature degradation of active agents, among others [6] . The nature of the materials used to make them gives unique properties. Polymeric NPs have a limited loading capacity, but the nature of their components means they are considered biodegradable with low toxicity and good tolerance in biological systems [10] [15] [27] [28] [29] .
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Figure 2. Effect of PNP pretreatment on MARC-145 cell cultures. PRRSV-induced cytopathic changes in MARC-145 cells. Foci of damage were observed during the period from 24 - 72 hpi. Top row: MARC-145 control cultures. Middle row: MARC-145 cell culture infected with PRRSV. Bottom row: MARC-145 culture cells treated with PNPS 4 h prior to PRRSV infection. Cytopathic damage in the cultures decreased during PRRSV infection with PNP pretreatment 4 hours prior to MARC-145 infection. Clear field microscopy at 20X and 40X. Images obtained from 3 independent experiments.
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Figure 3. Morphological evaluation of MARC-145 cells infected in vitro with PRRSV. (A) control, (B) 24 hpi, (C) 48 hpi, (D, E) 72 hpi, (F) 96 hpi with H&E staining. Black arrows indicate the presence of pycnotic nuclei. Yellow arrows indicate the presence of r-type basophilic material at the cytoplasmic level. Red arrows indicate vacuolization zones.
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Figure 4. Assessment of viability in cultures of MARC-145 cells treated with GA (A). Data from PNP treatments at 48 hpi showed no significant difference between cultures infected with PRRSV and those infected with the virus incubated 12 h prior to infection with PNPsGA (PRRSV Tx) and those treated with PNPs 4 h pre-infection (PNPs 4 h-PRRSV). However, at 72 hpi, a protective effect was observed with a significant difference (B). ANOVA, ***P < 0.001, **P < 0.01, and *P < 0.05. Data obtained from 3 independent experiments by MTT.
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Figure 5. Trypan blue exclusion. The effect of PNPsGA treatment on cell viability in PRRSV-infected cultures at 72 hpi was also demonstrated by trypan blue staining. (A) Refringent cells that correspond to viable cells in the control culture; and (B) cell stained with dye in an PRRSV-infected culture, revealed as a dead cell. Culture cells treated with PNPsGA 4 h pre-infection with PRRSV, viable (refringent) and dead (blue) cells at 48 hpi (C) and 72 hpi (D). Figure (D) shows an increase in cell number and viability.
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Figure 6. RT-qPCR vs. PRRSV curves. Quantification data and amplification curves for PRRSV-infected crops (red) and cultures treated with PNPs (olive green). Treatment with PNPs decreased the amplification and detection of PRRSV sequences by RT-PCR by 2 - 3 cycles (bridged green line vs. dotted red line (PRRSv).
Viral agents represent a significant challenge for human and animal health. One of the diseases recognized as having the greatest negative economic impact on the global swine industry is porcine respiratory and reproductive syndrome [1] [30] [31] [32] . In their studies of this disease, Kim (1993) [33] and Dea (1995) [34] reported that the MARC-145 cell line is highly permissive for replicating PRRSV. Based on those reports, MARC-145 cultures have emerged as the primary choice for replicating this pathogen. In the present study, 4 intervals (24 - 96 hpi) were tested to evaluate the cytopathic changes induced by PRRSV and the effects of PNPsGA pre- and post-infection and at coinfection [35] [36] . Some authors have reported that the cytopathic effects in porcine alveolar macrophages occur at 16 hpi and end at 24 hpi with total cell destruction. This effect occurs later in MARC-145 cultures, where cytopathic effects are reported at 48 h, a time similar to the changes observed in this study, followed by a predominant period of damage from 48 - 72 hpi, with the maximum peak of affectation occurring at 72 h [37] [38] , (Zhang 2011; Howerth 2002), concluding with the detachment of the monolayer at 96 hpi [39] .
In the viral infection assays, coadministration (PNPsGA + PRRSV) and 12 hpi treatment (PNPsGA and PRRSV administered at 12 hpi) showed no significant differences in the infected cultures (PRRSV), thus ruling out a possible inhibitory and/or therapeutic effect. The course of infection was similar to that in the positive control (PRRSV) in that it ended with the destruction of the monolayer, a result similar to that obtained by Duan et al., (2015) [40] , who reported no evidence of effects on viral penetration and absorption capacity in MARC-145 cells when the virus was treated with GA. Their results may be related to reports that GA mainly inhibits the viral penetration but has little effect on the absorption or release phases of PRRSV infection during its life-cycle [40] [41] .
The presence of direct inhibitory action of PNPsGA on the PRRSV viral particles was thus excluded. In addition, incubation of PRRSV with PNPsGA for 24 h to evaluate a possible inhibitory effect on the infective capacity of the particles led to this being ruled out, as well, because effects similar to those on the infected cells were observed with PRRSV. These findings highlight the fact that GA inhibits the proliferation of PRRSV mainly during the internalization stage of the virus, but has no apparent effect on the direct inactivation of PRRSV particles. In 2015, Duan [40] similarly reported that coincubation of PRRSV with GA at concentrations of 20, 40, and 80 μM had no effect on the ability of PRRSV to infect MARC-145 cells, thus demonstrating that GA does not directly inactivate PRRSV particles.
The effects reported in earlier assays are attributable to active infection by PRRSV, so the RT-qPCR test showed the presence of the virus in cultures infected with PRRSV, without a positive signal in the control cultures (Figure 6). This led to the determination that the cytopathic changes observed are attributable to the process of viral infection. The greatest antiviral effect of PNPsGA was observed in MARC-145 cells pretreated for 4 h. These results can be explained by the recognized capacity of nanotransporters to act as structures that improve the cell penetration capacity of certain drugs. This effect is observed even with the concentration of the active principle in PNPsGA. In cultures treated 4 h pre-infection with PRRSV (Figure 6, olive green line), the number of cycles necessary for the threshold cycle (TC) was slightly higher than in the ones infected with PNPsGA without pretreatment (Figure 6, red line). This suggests the need for assays carried out at distinct concentrations of PNPsGA and pre-infection times.
An active ingredient in herbal medicine, glycyrrhizic acid has played an important role as an antioxidant, anti-inflammatory, antiviral immunoregulator, and liver protector [22] [42] ; as recent research shows [41] . However, it presents problems of solubility, cytotoxicity, degradation, and biocompatibility, so the nontoxic metabolism of polymer nanomaterials is an alternative for transporting this type of drug. The antiviral mechanism of PNPsGA was demonstrated by the inhibition of invasion and in vitro replication of PRRSV, which showed that this formulation has good antiviral activity against PRRSV (≈3 orders of magnitude) and increases cell viability by 38% for up to 72 h.
5. Conclusion
In this study, the antiviral effect of a PNPsGA was evaluated, determining that this a stable system has glycyrrhizic acid loading capacity, which inhibits the invasion and replication of PRRSV as its the most important mechanism, was developed and evaluated. It reduced cellular damage induced by PRRSV infection by 38% in MARC cells, with a decrease of 3 amplification cycles and detection of PRRSV sequences by RT-qPCR when administered in cell cultures 4 h preinfection. The development of this technology could suggest a preventive therapeutic effect. However, additional studies of the effect of the viral penetration process are needed, the formulation needs to be optimized to increase its capacity to transport GA and evaluate the therapeutic potential associated with the concentration of the active ingredient. Finally, our cellular model of infection is a useful tool for conducting research on the action mechanisms of this virus, and evaluating the effectiveness of new nanoparticle systems for the transport of antiviral substances that provide a more effective way to deal with PRRSV.
Ethics Statement
The authors confirm that the ethical policies of the journal, as noted on the author guidelines page, have been adhered to. No ethical approval was required as the work conducted is original research on cell cultures.