pharmaceutics Article
Enhanced In Vitro Antiviral Activity of Ivermectin-Loaded Nanostructured Lipid Carriers against Porcine Epidemic Diarrhea Virus via Improved Intracellular Delivery Xiaolin Xu 1 , Shasha Gao 1 , Qindan Zuo 1 , Jiahao Gong 1 , Xinhao Song 1 , Yongshi Liu 1 , Jing Xiao 1 , Xiaofeng Zhai 1,2 , Haifeng Sun 1 , Mingzhi Zhang 3 , Xiuge Gao 1 and Dawei Guo 1, * 1
2 3 *
Citation: Xu, X.; Gao, S.; Zuo, Q.; Gong, J.; Song, X.; Liu, Y.; Xiao, J.; Zhai, X.; Sun, H.; Zhang, M.; et al. Enhanced In Vitro Antiviral Activity of Ivermectin-Loaded Nanostructured Lipid Carriers against Porcine Epidemic Diarrhea Virus via Improved Intracellular Delivery. Pharmaceutics 2024, 16, 601. https://doi.org/10.3390/ pharmaceutics16050601
Engineering Center of Innovative Veterinary Drugs, Center for Veterinary Drug Research and Evaluation, MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, 1 Weigang, Nanjing 210095, China Academy for Advanced Interdisciplinary Studies, Nanjing Agricultural University, Nanjing 210095, China Jiangsu Key Laboratory of Pesticide Science, College of Sciences, Nanjing Agricultural University, 1 Weigang, Nanjing 210095, China Correspondence: gdawei0123@njau.edu.cn; Tel.: +86-25-8439-6215; Fax: +86-25-8439-8669
Abstract: Porcine epidemic diarrhea virus (PEDV) is an acute enteric coronavirus, inducing watery diarrhea and high mortality in piglets, leading to huge economic losses in global pig industry. Ivermectin (IVM), an FDA-approved antiparasitic agent, is characterized by high efficacy and wide applicability. However, the poor bioavailability limits its application. Since the virus is parasitized inside the host cells, increasing the intracellular drug uptake can improve antiviral efficacy. Hence, we aimed to develop nanostructured lipid carriers (NLCs) to enhance the antiviral efficacy of IVM. The findings first revealed the capacity of IVM to inhibit the infectivity of PEDV by reducing viral replication with a certain direct inactivation effect. The as-prepared IVM-NLCs possessed hydrodynamic diameter of 153.5 nm with a zeta potential of −31.5 mV and high encapsulation efficiency (95.72%) and drug loading (11.17%). IVM interacted with lipids and was enveloped in lipid carriers with an amorphous state. Furthermore, its encapsulation in NLCs could enhance drug internalization. Meanwhile, IVM-NLCs inhibited PEDV proliferation by up to three orders of magnitude in terms of viral RNA copies, impeding the accumulation of reactive oxygen species and mitigating the mitochondrial dysfunction caused by PEDV infection. Moreover, IVM-NLCs markedly decreased the apoptosis rate of PEDV-induced Vero cells. Hence, IVM-NLCs showed superior inhibitory effect against PEDV compared to free IVM. Together, these results implied that NLCs is an efficient delivery system for IVM to improve its antiviral efficacy against PEDV via enhanced intracellular uptake. Keywords: ivermectin; antivirus; nanostructured lipid carriers; porcine epidemic diarrhea virus; intracellular delivery
Academic Editor: Sofia Lima Received: 28 March 2024 Revised: 18 April 2024 Accepted: 25 April 2024 Published: 29 April 2024
Copyright: © 2024 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).
1. Introduction Swine diarrhea is one of the main infectious diseases affecting the pig industry, among which viral diarrhea has the highest incidence and causes the most serious damage [1]. Coronaviruses are important pathogens causing diarrhea in piglets and mainly include porcine epidemic diarrhea virus (PEDV), porcine infectious gastroenteritis virus (TGEV), porcine delta coronavirus (PDCoV), and porcine acute diarrhea syndrome coronavirus (SADS-CoV). Currently, PEDV and TGEV are the most widely distributed viral pathogens of porcine diarrhea [2]. PEDV is a member of the genus α-coronavirus, a single-stranded, positive-stranded RNA virus with a capsid, which can cause acute diarrhea, vomiting, and dehydration in piglets, with a lethality rate of up to 90%, inducing significant economic losses to the pig industry worldwide [3–5]. Available PEDV vaccines include inactivated and attenuated vaccines, but these diseases are still frequent in the pig breeding industry, Pharmaceutics 2024, 16, 601. https://doi.org/10.3390/pharmaceutics16050601
partly due to the emergence of new mutant strains [6]. With the risk of emerging or re-emerging diseases, there is an urgent need to develop new antiviral compounds [4,7,8]. Recently, drug repurposing has become an attractive option for treating emerging diseases, as developing new effective drugs against a particular pathogen is time-consuming and expensive [9]. In contrast, approved substances are easily available, and their potential side effects are well characterized [10]. Drug repurposing is a strategy for converting an FDA-approved or investigational drug from its original use to a new use [11]. The greatest benefit of repurposed drugs is the omission of critical and time-consuming drug development phases, which significantly reduces the time required to produce effective antiviral drugs [12]. Ivermectin (IVM) is an FDA-approved, broad-spectrum, high-efficiency, low-toxicity antiparasitic drug [13,14]. It is a kind of hexadecameric ring macrolide drug, and its biological activity is broad [15]. Studies have shown that it has potential therapeutic effects against viruses [16]. In vitro investigations have demonstrated that IVM could effectively restrict infection caused by a variety of RNA and DNA viruses, including HIV-1, dengue virus (DENV), related flaviviruses, influenza A, and Venezuelan equine encephalitis virus (VEEV) [17–21]. Recent studies have indicated that it is a potent inhibitor of SARS-CoV2 [22]. However, the low solubility of IVM in water, only 6-9 micrograms per milliliter, and the poor bioavailability limit its clinical applications [23,24]. Nanostructured lipid carriers are a new generation of lipid nanoparticles consisting of a mixture of solid and liquid lipids [25]. It is a cutting-edge nano-delivery system that enhances the solubility, stability, and bioavailability of the encapsulated bioactive compounds by protecting them from adverse environmental conditions and regulates their release by enabling them to exert their active effects at the right time and site [26–28]. In this study, we determined that IVM could inhibit the infectivity of PEDV, and then, formulated nanostructured lipid carriers could be used strategically to tackle the low solubility and the poor bioavailability of IVM. The results showed that NLCs could enhance the antiviral activity of IVM by improving intracellular delivery. This study aimed to verify the potential of IVM-NLC as an alternative promising therapeutic drug for PEDV. 2. Materials and Methods 2.1. Chemical, Cells, and Viruses Ivermectin (IVM, 91%) was purchased from the China Institute of Veterinary Drug Control (Beijing, China). African green monkey epithelial cells (Vero) and PEDV strain CV777 (GenBank Accession No. KT323979) were maintained in the laboratory. 2.2. Preparation of IVM-NLCs The high-shear-ultrasound and high-pressure homogenization methods were used to prepare IVM-NLCs [29,30]. Briefly, a certain amount of oleic acid (OA) (Aladdin, Shanghai, China), palmitic acid (PA, Aladdin, Shanghai, China), Tween 20 (Aladdin, Shanghai, China), and IVM was mixed as the oil phase and heated up to 70 ◦ C until complete melting. In parallel, poloxamer 188 (Yunhong Chemical Preparations and Accessories Technology Corporation, Shanghai, China) was dissolved in water as the aqueous phase and heated at the same temperature. Then, the aqueous phase was poured into the oil phase under magnetic stirring at 700 rpm. Subsequently, the mixture was homogenized at 11,000 rpm for 5 min with a High Shear Dispersion Emulsifying Machine (FM200, IKA, Staufen, Germany), and then treated by probe sonication at 300 W for 20 min with an Ultrasonic Cell Disruptor (JY96-II, Scientz, Ningbo, China). The obtained oil-in-water (O/W) emulsion was rapidly transferred into cold water under high-shear conditions for 1 min and cooled to form the NLCs. The hot O/W system was cycled four times in a high-pressure homogenizer (AH-BASIC, ATS, Suzhou, China) at 700 bar to obtain IVM-NLCs in bulk.
2.3. Characterization of IVM-NLCs 2.3.1. Hydrodynamic Diameter (HD), Polydispersity Index (PDI), and Zeta Potential (ZP) Prior to measurement, all the samples were diluted appropriately with deionized water. The HD, PDI, and ZP of IVM-NLCs were characterized using dynamic light scattering (DLS) with the Zetasizer (Malvern Instruments Ltd., Malvern, Worcestershire, UK). 2.3.2. Transmission Electron Microscopy (TEM) The morphology of the IVM-NLCs was observed by TEM (Tecnai 12, Philips, Amsterdam, The Netherlands). Briefly, the diluted IVM-NLCs were dropped onto 300-mesh copper grids. After drying, the samples were negatively stained for 2 min using a 2% (w/v) phosphotungstic acid solution. The dried samples were then subjected to TEM analysis. 2.3.3. X-ray Diffraction (XRD) and Fourier Transform Infrared Spectroscopy (FT-IR) PA, IVM, physical mixtures of PA and IVM, lyophilized NLCs, and IVM-NLC powder were studied using XRD (D8 Advance, Bruker AXS, Karlsruhe, Germany) to analyze the changes in the crystal structure of IVM during NLC formation. In addition, the above samples were placed in a crucible and compacted onto slides. Subsequently, XRD with Cu/Kα radiation source were scanned at 4◦ /min in the range of 5◦ ~50◦ under the conditions of 40 kV/40 mA. After that, PA, IVM, physical mixture of IVM and PA, lyophilized NLCs, and IVM-NLC powder IRs were recorded using an FT-IR spectrometer (IS5&N380, Nicolet, Waltham, MA, USA). Prior to FT-IR spectroscopy, the samples were mixed with potassium bromide (KBr) in a ratio of 1:150 (w/w) and pressed into thin slices using a high-pressure hydraulic press. The prepared flake samples were then placed on an FT-IR spectrometer and scanned under a wave number range from 4000 cm−1 to 500 cm−1 . 2.3.4. Entrapment Efficiency (EE) and Drug Loading (DL) The EE and DL of IVM-NLCs were determined by ultrafiltration centrifugation combined with high-performance liquid chromatography (HPLC). The diluted IVM-NLCs were added to methanol, vortexed, and sonicated to break the emulsion. The supernatant was centrifuged, and the weight of IVM (Wtotal ) in the emulsion was determined by HPLC. In addition, the dilution of IVM-NLCs was placed in ultra-filtration centrifuge tube (MWCO: 100 kDa, Millipore, Bilrika, MA, USA) at high speed, and the weight of free IVM (Wfree ) in the emulsion was determined by HPLC. The DL and EE of IVM-NLCs were calculated using the following formulas: EE (%) = (Wtotal − Wfree )/Wtotal DL (%) = (Wtotal − Wfree )/WNLCs where Wtotal is the total content of applied IVM in NLCs, Wfree is the amount of free IVM in the supernatant phase, and WNLCs is the content of the lipid used in the preparation of the IVM-NLCs. 2.4. Cell Culture Vero cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, Waltham, MA, USA) with 1% penicillin–streptomycin (HyClone, Logan, UT, USA) and 5% Fetal Bovine Serum (FBS) (Gibco, CA, USA). Cells were placed in a humidified cell culture incubator with 5% CO2 at 37 ◦ C. 2.5. Cell Viability Evaluation The cytotoxicity of IVM and IVM-NLCs was tested in Vero cells using the Cell Counting kit-8 (CCK-8, KeyGen Biotech Co., Ltd., Nanjing, China) method. Vero cells were seeded in a 96-well plate overnight at a density of 10,000 cells per well in 100 µL fresh medium. After plating, cells were treated with IVM and IVM-NLCs for 24 h and 48 h. Subsequently, CCK-8 (10 µL/well) was added to each well and incubated at 37 ◦ C for 1 h, and the OD
values at 450 nm were measured. Cell viability was calculated using the following formula: cell viability (%) = (ODtreated − ODblank )/(ODuntreated − ODblank ) × 100%. 2.6. In Vitro Cellular Uptake Qualitative and quantitative assessment of in vitro cellular uptake of coumarin-6 (C6) in Vero cells using fluorescence microscopy (Thermo Fisher, Waltham, MA, USA) and flow cytometry (BD Biosciences, New York, NY, USA), respectively. Briefly, Vero cells were seeded into a 6-well culture plate at the density of 2 × 105 cells per well and incubated overnight. The cells were then treated with free C6 or C6-NLCs, and cells without any treatment were used as a control. After 4 h, cells were washed with cold PBS, fixed in 4% paraformaldehyde for 10–15 min, labeled with DAPI, and visualized by fluorescence microscopy. In addition, to further investigate the cellular uptake process of C6 and C6NLCs, after the termination of uptake, treated cells were separated with 0.25% trypsin, centrifuged at 1000 rpm for 5 min, and suspended in PBS, and fluorescent signals in cells were analyzed by flow cytometry. 2.7. TCID50 Assay To determine the 50% tissue culture infectious dose (TCID50 ), PEDV samples were diluted 10-fold with maintenance solution and used to inoculate Vero cells. Briefly, Vero cells were seeded in a 96-well plate overnight. Then, the medium was discarded, washed twice with maintenance solution, and 100 µL diluted virus solution per well and maintenance solution was added to the negative control wells. The cell culture plates were placed in an incubator at 37 ◦ C and incubated for 1 h. After that, the old maintenance solution was discarded, and 100 µL of maintenance solution was added to the incubator for continued cultivation and monitoring of CPE. TCID50 /mL was calculated according to the method of Reed–Muench. 2.8. Antiviral Assay Cells were infected with PEDV at multiplicity of infection (MOI) of 0.05. One hour later, the inoculum was removed, and the cells were washed with serum-free DMEM. Subsequently, cells were incubated separately with DMEM, NLCs, IVM, and IVM-NLCs. The antiviral effect of NLCs, IVM, and IVM-NLCs on PEDV infection was then evaluated by quantitative real-time polymerase chain reaction (RT-qPCR), Western blot, and indirect immunofluorescence assay (IFA). There exists a possible target for inhibition at each stage of viral infection [31,32]. To determine at which stage IVM blocked infection, IVM as well as PEDV were added to cells with different treatments: Inactivation assay: The mixture of IVM with PEDV was placed at 37 ◦ C for 3 h. Vero cells were infected with the pretreated PEDV for 1 h. After incubation, the supernatant was discarded and replaced with a maintenance solution without any drugs to continue the culture. Total RNA was extracted and quantified by RT-qPCR. Attachment assay: Vero cells were pre-cooled at 4 ◦ C, followed by incubation with 5 µM IVM for 1 h, and then infection with PEDV for 1 h at 4 ◦ C. After discarding the supernatant, the cells were washed twice with pre-cooled serum-free DMEM. Total RNA was extracted and quantified by RT-qPCR. Adsorption assay: After pre-cooling at 4 ◦ C, Vero cells were infected with PEDV at ◦ 4 C for 1 h. After discarding the supernatant and washing with pre-cooled serum-free DMEM, the cells were treated with 5 µM IVM in DMEM containing 2% FBS for 1 h at 37 ◦ C. Total RNA was extracted and quantified by RT-qPCR. Replication assay: Vero cells were infected with PEDV for 1 h at 37 ◦ C. To remove non-adsorbed virus particles, the cells were washed with pre-cooled serum-free DMEM, incubated with DMEM for 4 h, and then treated with 5 µM IVM for 12 h. Total RNA was extracted and quantified by RT-qPCR.
Release assay: Vero cells were infected with PEDV for 10 h at 37 ◦ C. The supernatant was discarded, and after washing with serum-free DMEM, the cells were incubated separately with IVM in DMEM containing 2% FBS. Total RNA was extracted and quantified by RT-qPCR. 2.9. One-Step Growth Curve Vero cells at 80–90% confluence were infected with 0.05 MOI of PEDV. Subsequently, the control DMEM, NLCs, IVM, and IVM-NLCs were added and subjected to further incubation at 37 ◦ C. The TCID50 was recorded at 1, 4, 8, 12, 24, 36, 48, 60, and 72 hpi according to the Reed–Muench method. PEDV titers were calculated, and viral growth curves were plotted by determining TCID50 at different time points. 2.10. RT-qPCR RT-qPCR was based on SYBR Green method. The PEDV total RNA was extracted from the cells in a 6-well plate using RNAiso Plus (Takara, Tokyo, Japan) according to the manufacturer’s protocol. The concentration and purity of total RNA were assessed by Nanodrop (Thermo Fisher Scientific, Waltham, MA, USA). The total RNA was reverse transcribed into cDNA using cDNA synthesis kit (Vazyme, Nanjing, China) and stored at −20 ◦ C. The target genes were evaluated in triplicate using SYBR qPCR Master Mix (Vazyme, Nanjing, China). Absolute fluorescence was quantitatively referenced as described in [33]. 2.11. Western Blot Analysis Vero cells were cultured to approximately 80–90% confluence in 6-well plates, and infected with 0.05 MOI of PEDV. After 1 h of infection, the cell monolayers were incubated with control DMEM, NLCs, IVM, and IVM-NLCs (5 µM) for 24 h, and then treated with lysis buffer (100 µL/well) to extract total protein, followed by the quantification of protein concentration using bicinchoninic acid (BCA) protein assay kit according to the manufacturer’s instructions (Solarbio, Beijing, China). Sodium dodecyl sulfate (SDS) loading buffer was added to the collected cell extracts and boiled for 10 min. Equivalent amounts of proteins were loaded and electrophoresed on 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to nitrocellulose filter (NC) membranes followed by blocking with 5% skim milk for 2 h. Subsequently, the expression of the PEDV N protein was determined. The expression of GAPDH was investigated to represent the same amounts of protein sample loading. 2.12. IFA The inhibitory effect of NLCs, IVM, and IVM-NLCs against PEDV in Vero cells was further evaluated by immunofluorescence. Vero cells in 24-well plates were infected with PEDV at 0.05 MOI. After 1 h of incubation, free viruses were removed by extensive rinsing. Then, the cells were incubated with control DMEM (containing 2% FBS) or NLCs, IVM, and IVM-NLCs, respectively. Twenty-four hours later, cells were fixed with 4% formaldehyde for 15 min. After permeabilization with 0.1% Triton X-100, the cells were incubated with the mouse monoclonal antibody against the PEDV N protein (1:200 dilution) at 4 ◦ C overnight and washed three times with phosphate-buffered solution with Tween 20 (PBST) for 10 min each. Then, the cells were incubated with FITC-conjugated goat anti-mouse antibody (1:200 dilution) for 1 h, and counterstained with DAPI at room temperature for 10 min. After washing three times, the photographs were obtained by fluorescence microscopy. 2.13. Determination of Reactive Oxygen Species (ROS) Generation ROS level was assessed using a 2′ ,7′ -dichlorofluorescein diacetate (DCFH-DA)-based ROS assay kit (Nanjing Kaiji Biotechnology Co., Ltd., Nanjing, China). Briefly, wellgrown Vero cells were inoculated in 12-well plates, and an inoculum of 0.05 MOI of PEDV (500 µL/well) was added to Vero cells grown to 80–90% fusion. After 1 h of infection, the cell monolayer was rinsed and then covered with NLCs, IVM, and IVM-NLCs, respectively.
After that, the cells were incubated with DCFH-DA for 20 min at 37 ◦ C in the dark. The fluorescence changes in cells in each group were observed under a fluorescence microscope (Ex = 488 nm, Em = 507 nm) (Thermo Fisher, MA, USA). 2.14. Mitochondrial Membrane Potential (MMP) Analysis JC-1 assay kit (KeyGen Biotech Co., Ltd., Nanjing, China) was used to detect MMP changes. Well-grown Vero cells were inoculated in 12-well plates, and an inoculum of 0.05 MOI of PEDV (500 µL/well) was added to Vero cells grown to 80–90% fusion. After 1 h of infection, the cell monolayer was rinsed and then covered with NLCs, IVM, and IVM-NLCs. Subsequently, the cells were washed with PBS. JC-1 (10 µg/mL) was added to each sample and incubated at 37 ◦ C in the dark for 15 min. The fluorescence changes in cells in each group were observed under a fluorescence microscope (green fluorescence, Ex = 488 nm, Em = 507 nm; red fluorescence, Ex = 525 nm, Em = 590 nm) (Thermo Fisher, MA, USA). 2.15. Apoptosis Assay Apoptosis rate of PEDV-infected Vero cells was assessed with Annexin V-FITC/PI double staining kit (KeyGen Biotech Co., Ltd., Nanjing, China) according to the manufacturer’s instructions. The cells were seeded into 6-well plates and then stained with Annexin V-FITC and PI in dark. Cells were analyzed by flow cytometry (Ex = 488 nm, Em = 525 nm). Cell-Quest software (Becton Dickinson, San Jose, CA, USA) was employed to analyze the data. 2.16. Statistical Analysis All data are presented as mean ± standard deviation (SD), and experiments were conducted in triplicate. GraphPad Prism 8.0 software was used for statistical analysis. Differences between the two groups were assessed using t-test (mean ± SD). When comparing several groups, one-way analysis of variance (ANOVA) was employed. Statistics were applied to differences with * p < 0.05 and ** p < 0.01. 3. Results and Discussion 3.1. IVM Inhibited the Infectivity of PEDV Numerous studies have recently reported the antiviral properties of IVM, which could impede viruses by hindering nuclear input that was reliant on specific IMPα/β1dependent viral proteins [2]. This study investigated the antiviral activity of IVM against PEDV in vitro. Prior to assessing its inhibitory potency, cytotoxicity assays were conducted on Vero cells. The results indicated that cell viability remained above 80% at concentrations ranging from 0 to 10 µM (Figure 1A). After excluding cytotoxicity, the inhibitory effect of IVM on PEDV-infected Vero cells was evaluated. Specifically, Vero cells were infected with 0.05 MOI of PEDV and subsequently treated with varying concentrations of IVM. Furthermore, a discernible difference was observed among the virus control group after 2.5 or 5 µM IVM treatment, and cell viability could reach more than 80% after 10 µM IVM treatment, as illustrated in Figure 1B. We calculated the concentration for 50% of maximal effect (EC50 ) value, which reflected the concentration of IVM required to abolish infectious virus particle production by 50%. The EC50 value for IVM following infection with PEDV in Vero cells was 4.63 µM (Figure 1C). Furthermore, the administration of IVM at concentrations of 1.25, 2.5, 5, and 10 µM resulted in a reduction of PEDV RNA N copies at 12 h post-infection from 6.1 to 5.8, 5.5, 4.8, and 3.8 lg copies, respectively, in comparison to the untreated cohort (Figure 1D). Notably, the PEDV nucleocapsid (N) protein, an RNA-binding protein crucial for the virus life cycle, could serve as a precise and early diagnostic target for PEDV infection [34,35]. The inhibitory effect of IVM on PEDV proliferation was verified by detecting the expression level of PEDV N protein. Specifically, the infected cells were incubated with different concentrations of IVM. As shown in Figure 1E, at 24 hpi, a strong fluorescent signal was observed within the PEDVinfected Vero cells treated with DMSO by indirect immunofluorescence. However, we
to the untreated cohort (Figure 1D). Notably, the PEDV nucleocapsid (N) protein, an RNA-binding protein crucial for the virus life cycle, could serve as a precise and early diagnostic target for PEDV infection [34,35]. The inhibitory effect of IVM on PEDV proliferation was verified by detecting the expression level of PEDV N protein. Specifically, the 7 of 15 infected cells were incubated with different concentrations of IVM. As shown in Figure 1E, at 24 hpi, a strong fluorescent signal was observed within the PEDV-infected Vero cells treated with DMSO by indirect immunofluorescence. However, we observed a significant observed a significant and concentration-dependent infected and concentration-dependent difference between difference untreatedbetween infecteduntreated cells and IVMcells and IVM-treated infected cells, which was in accordance with the growth curves of treated infected cells, which was in accordance with the growth curves of the virus. the virus.
Figure 1. 1. IVM inhibited the infectivity of PEDV. (A) Cytotoxicity of Vero Vero cells treated treated with with different different Figure concentrations of IVM at the appointed time via CCK-8 assay. Medium containing 0.05% DMSO concentrations of IVM at the appointed time via CCK-8 assay. Medium containing 0.05% DMSO 50 of IVM (v/v) served as control. (B) Antiviral activity of IVM was measured by CCK-8 assay. (C) EC (v/v) served as control. (B) Antiviral activity of IVM was measured by CCK-8 assay. (C) EC50 of was calculated with GraphPad Prism 8.0. (D) PEDV N RNA copies of Vero cells infected with PEDV IVM was calculated with GraphPad Prism 8.0. (D) PEDV N RNA copies of Vero cells infected with after different treatments of IVM by RT-qPCR. (E) Indirect immunofluorescence assay of PEDVPEDV after different treatments of IVM by RT-qPCR. (E) Indirect immunofluorescence assay of PEDVinfected Vero cells after treatment with different concentrations of IVM or without treatment. Blue, infected Vero cells after treatment with different concentrations IVM orµm. without Blue, DAPI; green, FITC-conjugated goat anti-mouse antibody. Scaleofbar = 50 Errortreatment. bars represent DAPI; green, deviation FITC-conjugated goat anti-mouse antibody. Scale barvalue = 50 was µm.calculated Error barsby represent the standard from three repeated experiments. The mean the onethe from three repeated mean wascompared calculatedwith by the waystandard analysis deviation of variance (ANOVA) (mean ± experiments. SD, n = 3). * pThe < 0.05, ** value p < 0.01, the one-way analysis of variance (ANOVA) (mean ± SD, n = 3). * p < 0.05, ** p < 0.01, compared with the PEDV group. PEDV group.
3.2. Effect of IVM in Diverse Stages of PEDV Life Cycle 3.2. Effect of IVM in Diverse Stages of PEDV Life Cycle The present study investigated the underlying mechanism of the antiviral properties Thebypresent study investigated mechanism of the its antiviral properties of IVM analyzing their impact on the the underlying proliferation of PEDV during replication cycle. of IVM by analyzing their impact on the proliferation of PEDV during its replication cycle. The schematic diagram is shown in Figure 2A. The direct inactivation potential of IVM on The schematic diagram is shown in Figure 2A. The direct inactivation potential of IVM PEDV was initially evaluated, and the results demonstrated a 10-fold reduction in the on PEDV was initially evaluated, and the results demonstrated a 10-fold reduction in number of PEDV via RT-qPCR depicted in Figure 2B. In the adsorption process of PEDV, the number of PEDV via RT-qPCR depicted in Figure 2B. In the adsorption process of there was no significant difference between the experimental group and the control group PEDV, there was no significant difference between the experimental group and the control group in terms of their suppression effect on PEDV adsorption (Figure 2C). In the invasion process of PEDV, the results revealed that IVM treatment decreased the infectious virus titer by about 10-fold relative to the control group (Figure 2D), implying IVM had slight influence on PEDV invasion. In addition, as shown in Figure 2E, IVM reduced the number of PEDV N RNA copies by nearly 102 -fold, implying that IVM may suppress PEDV mainly via inhibiting PEDV replication. In addition, the effect of IVM on the release of PEDV progeny is shown in Figure 2F. There was no noticeable difference in the virus titers of PEDV compared to the control group, suggesting that IVM had no inhibitory effect on the release of PEDV progeny. To sum up, IVM exerted its suppressive effect on PEDV primarily by inhibiting virus invasion and replication with a certain direct inactivation effect in vitro.
via inhibiting PEDV replication. In addition, the effect of IVM on the release of PEDV progeny is shown in Figure 2F. There was no noticeable difference in the virus titers of PEDV compared to the control group, suggesting that IVM had no inhibitory effect on the release of PEDV progeny. To sum up, IVM exerted its suppressive effect on PEDV primarily by inhibiting virus invasion and replication with a certain direct inactivation effect in 8 of 15 vitro.
Figure2.2.IVM IVMtreatment treatmentatat multiple stages of inhibition of PEDV proliferation. (A) Schematic diaFigure multiple stages of inhibition of PEDV proliferation. (A) Schematic diagram gram of the effect of IVM on the replication cycle of PEDV. (B) Effect of IVM on direct inactivation of the effect of IVM on the replication cycle of PEDV. (B) Effect of IVM on direct inactivation of PEDV. of PEDV. Effect of IVM on the (C) adsorption, (D) invasion, (E) replication, and (F) release processes Effect of IVM on the (C) adsorption, (D) invasion, (E) replication, and (F) release processes of infected of infected cells. The mean value was calculated by the t-test (mean ± SD, n = 3). * p < 0.05, ** p < 0.01, cells. The mean value was calculated by the t-test (mean ± SD, n = 3). * p < 0.05, ** p < 0.01, compared compared with the PEDV group. with the PEDV group.
3.3. Characterization Characterization of of IVM-Loaded IVM-Loaded Nanostructured Nanostructured Lipid Lipid Carriers Carriers 3.3. Theprepared preparedIVM-NLCs IVM-NLCsthrough throughthe the high-pressure homogenization technique exThe high-pressure homogenization technique exhibhibited characteristics a homogeneous, opaque, milky white liquid fluited characteristics of a of homogeneous, opaque, and and milky white liquid withwith highhigh fluidity. idity. The hydrodynamic diameter (HD) zeta potential (ZP)IVM-NLCs, of the IVM-NLCs, as ilThe hydrodynamic diameter (HD) and zetaand potential (ZP) of the as illustrated lustrated in Figure 3A,B, demonstrated a narrow normal distribution. The as-prepared in Figure 3A,B, demonstrated a narrow normal distribution. The as-prepared IVM-NLCs IVM-NLCsanpossessed an HD of 153.5 ± 0.80anm with a polydispersity index (PDI)±of0.007, 0.153 possessed HD of 153.5 ± 0.80 nm with polydispersity index (PDI) of 0.153 ± 0.007, indicating a high particle size distribution homogeneity indicating a high degree ofdegree particleofsize distribution homogeneity (Table S1). (Table ZP wasS1). alsoZP a was so a critical factor to stability of colloidal dispersion. In general, stable critical factor to evaluate theevaluate stabilitythe of colloidal dispersion. In general, stable dispersion dispersion of a nanoparticle system was when achieved the absolute of ZP exceeded of a nanoparticle system was achieved thewhen absolute value of value ZP exceeded 30 mV due to electrical repulsion [36]. The of ZP IVM-NLCs was − 31.5−31.5 ± 0.569 mV,mV, indicat30 mV due to electrical repulsion [36].ZP The of IVM-NLCs was ± 0.569 indiing favorable stability. The morphology of IVM-NLCs was examined using transmission electron microscopy (TEM), revealing spherical or ellipsoidal particles with uniform size distribution and no observed agglomeration (Figure 3C). The mean distribution size of IVM-NLCs was determined to be 39.54 ± 9.17 nm (Figure 3D). Notably, the particle size of IVM-NLCs was significantly smaller than the HD. The reason for this disparity is that dynamic light scattering (DLS) provides an indirect measurement of particle size by detecting the fluctuation in scattered light intensity due to Brownian motion in a hydrated state, whereas TEM requires the sample to be in a dry state during testing [37,38]. DL and EE were important parameters for evaluating the preparation of NLCs. Increasing EE could enhance drug efficacy and reduce adverse drug reactions. Increasing DL could lead to a more stable formulation, while reducing the use of excipients and thus their potential toxicity. The EE and DL of the prepared IVM-NLCs measured by HPLC were 95.72 ± 0.30% and 11.17 ± 0.75%, respectively (Table S1). The prepared IVM-NLCs was based on the
tecting the fluctuation in scattered light intensity due to Brownian motion in a hydrated state, whereas TEM requires the sample to be in a dry state during testing [37,38]. DL and EE were important parameters for evaluating the preparation of NLCs. Increasing EE could enhance drug efficacy and reduce adverse drug reactions. Increasing DL could lead to a more stable formulation, while reducing the use of excipients and thus their potential 9 of 15 toxicity. The EE and DL of the prepared IVM-NLCs measured by HPLC were 95.72 ± 0.30% and 11.17 ± 0.75%, respectively (Table S1). The prepared IVM-NLCs was based on laboratory preparation ofof IVM-SLNs IVM-NLCshad had the laboratory preparation IVM-SLNs[30]. [30].Compared Compared with with IVM-SLNs, IVM-NLCs smallerHD HDand andmore moreuniform uniformdistribution, distribution,which whichimproved improvedEE EEand andDL. DL. smaller
Figure of of thethe optimized IVM-NLCs. (A) (A) Hydrodynamic diameter (HD)(HD) and and (B) Figure3.3.Characterization Characterization optimized IVM-NLCs. Hydrodynamic diameter zeta potential (ZP) of the IVM-NLCs were determined by DLS. (C) The morphology of the IVM(B) zeta potential (ZP) of the IVM-NLCs were determined by DLS. (C) The morphology of the IVMNLCs NLCswas wasobserved observedby byTEM, TEM,and and(D) (D)the thesize sizedistribution distributionwas wasobtained obtainedvia viaanalysis analysisofofthe theparticles particles from several TEM images. Scale bar = 100 nm. (E) X-ray diffraction (XRD) spectra and (F) Fourier from several TEM images. Scale bar = 100 nm. (E) X-ray diffraction (XRD) spectra and (F) Fourier transform infrared (FT-IR) spectra for IVM-NLCs, NLCs, physical mixture, IVM, and PA were transform infrared (FT-IR) spectra for IVM-NLCs, NLCs, physical mixture, IVM, and PA were shown. shown.
The conversion of drugs from a crystalline to an amorphous state had been found to The conversion of drugs from a crystalline to an amorphous state had been found to enhance drug loading and improve the stability of nanodrug delivery systems [39]. The enhance drug loading and improve the stability of nanodrug delivery systems [39]. The X-ray diffraction pattern was used for crystallographic analysis [40]. As shown in Figure 3E, X-ray diffraction pattern was usedbefor crystallographic Asthe shown in Figure apparent diffraction peaks could observed near 10◦ , analysis 15◦ , and [40]. 20◦ in pattern of IVM, 3E, apparent diffraction peaks could be observed near 10°, 15°, and 20° in the pattern of indicating hat IVM was a crystalline structure. The diffraction peaks observed in the IVM, indicating that IVM was a crystalline structure. The diffraction peaks observed in XRD patterns of IVM were still observed in the physical mixture of PA and IVM. The the XRD patterns of were still observedsignificantly in the physical of PA IVM. The sharp diffraction of IVM IVM-NLCs disappears nearmixture 10◦ , while theand characteristic sharp diffraction of IVM-NLCs disappears significantly near 10°, while the characteristic ◦ ◦ diffraction peaks persist near 15 and 20 but unlike IVM, which suggest that the IVM has diffraction persist near 15° and 20° but unlike forces, IVM, which suggest that theofIVM has reacted in peaks the NLCs, weakening its intermolecular and the crystallinity the IVM reacted in the NLCs, weakening its intermolecular forces, and the crystallinity of the IVM has also weakened. In addition, the diffraction peaks observed in NLCs were consistent has also weakened. In addition, diffraction peaksinobserved were consistent with IVM-NLCs, indicating thatthe IVM was dispersed NLCs inin anNLCs amorphous form. The FT-IR spectra of PA, IVM, physical mixture of IVM and PA, freeze-dried IVM-NLC powder, and NLC powder are displayed in Figure 3F. The characteristic absorption peak of C=C at 1680 cm−1 in IVM-NLCs disappeared and the characteristic peak of C-O-C stretching vibration of IVM group at 1050–1200 cm−1 was significantly reduced; the sharp peak at 3650 cm−1 for IVM is a stretching vibration of the alcohol hydroxyl group, which also occurs in IVM-PA mixtures and IVM-NLCs; IVM had the O-H stretching vibration peak of hydroxyl group at 3468 cm−1 , and so did the physical mixture of IVM and PA; while the O-H stretching vibration peak of IVM-NLCs was blue-shifted, suggesting that the binding between drug and carrier occurred, marking the successful preparation of nanostructured lipid carriers; the waveforms of IVM and IVM-NLCs were basically similar, indicating that the lipid carriers did not change the skeletal structure of IVM, and IVM was wrapped in the lipid carriers in non-crystalline form. The XRD and FT-IR results were in agreement, Pharmaceutics 2024, 16, 601
occurs in IVM-PA mixtures and IVM-NLCs; IVM had the O-H stretching vibration peak of hydroxyl group at 3468 cm−1, and so did the physical mixture of IVM and PA; while the O-H stretching vibration peak of IVM-NLCs was blue-shifted, suggesting that the binding between drug and carrier occurred, marking the successful preparation of nanostructured lipid carriers; the waveforms of IVM and IVM-NLCs were basically similar, indicating 10 of 15 that the lipid carriers did not change the skeletal structure of IVM, and IVM was wrapped in the lipid carriers in non-crystalline form. The XRD and FT-IR results were in agreement, and IVM was transformed from crystals to amorphous in IVM-NLCs, encapsulated into and IVM was transformed from crystals to amorphous in IVM-NLCs, encapsulated into the nanostructured lipid matrix in an amorphous state. the nanostructured lipid matrix in an amorphous state. 3.4. NLCs NLCs Improved Improved Cellular Cellular Uptake Uptake of of IVM IVM 3.4. Sincethe thevirus virusisisparasitized parasitizedwithin within host cells, increasing intracellular drug upSince thethe host cells, increasing intracellular drug uptake takeimprove can improve antiviral efficacy [41,42]. In order to evaluate in vitro biocompatibilcan antiviral efficacy [41,42]. In order to evaluate the inthe vitro biocompatibility of ity of IVM-NLCs and IVM, cytotoxicity assays were conducted by incubating Vero cells IVM-NLCs and IVM, cytotoxicity assays were conducted by incubating Vero cells with with varying concentrations of IVM or IVM-NLCs and respectively.The Theresults results varying concentrations of IVM or IVM-NLCs for for 24 24 and 4848 h,h,respectively. for Vero cells are presented in Figure S1. The relative survival of cells after exposure to for Vero cells are presented in Figure S1. The relative survival of cells after exposure to IVM-NLCs (0–10 µM) for 24 and 48 h was greater than 80%, indicating that IVM-NLCs IVM-NLCs (0–10 µM) for 24 and 48 h was greater than 80%, indicating that IVM-NLCs have good good biocompatibility. biocompatibility. The The experimental experimental findings findings demonstrated demonstrated aa significant significant enenhave hancement in in the the biocompatibility biocompatibility of of IVM-NLCs IVM-NLCs in in comparison comparison to to IVM. IVM. Additionally, Additionally,the the hancement cytotoxicityofofIVM-NLCs IVM-NLCsononVero Vero cells exhibited a dosetime-dependent relationcytotoxicity cells exhibited a doseandand time-dependent relationship. ship.fluorescence The fluorescence microscopy results depicted in Figure 4A indicated that C6-NLCs The microscopy results depicted in Figure 4A indicated that C6-NLCs disdisplayed more pronounced fluorescence signals than free C6. Moreover, the fluorescence played more pronounced fluorescence signals than free C6. Moreover, the fluorescence intensity exhibited exhibited by by C6-NLCs C6-NLCs was was significantly significantly higher higher than than that that of of free free C6, C6, as as depicted depicted intensity in Figure Figure4B. 4B. Furthermore, Furthermore, the the mean mean fluorescence fluorescence intensity intensity of of C6-NLCs C6-NLCs in in Vero Verocells cellswas was in 3.7-fold greater greater than than that that of of free free C6, C6, as as determined determined through through flow flow cytometry cytometry analysis analysis in in 3.7-fold Figure Figure 4C. 4C. These These findings findings collectively collectively suggested suggested that that NLCs NLCs hold hold potential potential as as aa delivery delivery carrier carrierfor foraugmenting augmentingthe thecellular cellularuptake uptakeof ofIVM. IVM.
Figure 4. Effect of NLCs on the cellular uptake of coumarin-6 (C6) in Vero cells. (A) Representative fluorescence microscope images of various preparations uptake in Vero cells. (B) Cell uptake was determined by flow cytometry of C6 in Vero cells after treatment with free C6 and C6-NLCs and (C) mean intracellular fluorescence intensity. Scale bar = 50 µm. The mean value was calculated by the t-test (mean ± SD, n = 3). ** p < 0.01, for C6-NLCs vs. free C6.
3.5. NLCs Enhanced the Antiviral Activity of IVM against PEDV The present study initially assessed the cell viability of Vero cells infected with PEDV using IVM-NLCs through the CCK-8 assay (Figure S1). The inhibitory effect of IVM-NLCs on PEDV-infected Vero cells is shown in Figure S2. The EC50 value for IVM-NLCs following infection with PEDV in Vero cells was 3.57 µM (Figure 5A). The EC50 value for IVM-NLCs was lower than IVM and confirmed that IVM-NLCs exhibited higher antiviral activity than IVM. The results illustrated in Figure 5B indicated that NLCs improved the viability of Vero cells at 48 h post-treatment in comparison to free IVM. To assess the impact of NLCs, IVM, and IVM-NLCs on PEDV replication, the one-step growth curve was generated through
using IVM-NLCs through the CCK-8 assay (Figure S1). The inhibitory effect of IVM-NLCs on PEDV-infected Vero cells is shown in Figure S2. The EC50 value for IVM-NLCs following infection with PEDV in Vero cells was 3.57 µM (Figure 5A). The EC50 value for IVMNLCs was lower than IVM and confirmed that IVM-NLCs exhibited higher antiviral activity than IVM. The results illustrated in Figure 5B indicated that NLCs improved 11 ofthe 15 viability of Vero cells at 48 h post-treatment in comparison to free IVM. To assess the impact of NLCs, IVM, and IVM-NLCs on PEDV replication, the one-step growth curve was generated the through the PEDV titerwith following treatment withIn5Figure µM of5C, each measuring PEDVmeasuring titer following treatment 5 µM of each agent. at agent. In Figure 5C,toatproliferate, 12 hpi, PEDV to proliferate, with a period 12 hpi, PEDV began withbegan a period of rapid proliferation fromof24rapid hpi toprolifer48 hpi 6.5 TCID50/0.1 mL at 60 hpi. After 60 ationa from hpititer to 48ofhpi and a peak viral titer of 10 and peak 24 viral 106.5 TCID mL at 60 hpi. After 60 h, PEDV proliferation 50 /0.1 h, PEDV proliferation decreased due to cell collapse. Compared to the negative control decreased due to cell collapse. Compared to the negative control group, significant viral group, significant viral titerininhibition was observed in cells treatedthe with IVM-NLCs. titer inhibition was observed cells treated with IVM-NLCs. Therefore, changes in the titer of PEDV that IVM-NLCs indeedverified possessed antiviral activity against Therefore, theverified changes in the titer of PEDV thatsuperior IVM-NLCs indeed possessed suviral replication. perior antiviral activity against viral replication.
Figure 5.
5. Anti-PEDV
(A)(A)
ECEC
50 of IVM-NLCs
waswas
calculated
with
Figure
Anti-PEDV activity
activityof
ofIVM-NLCs
IVM-NLCson
onVero
Verocells.
cells.
calculated
50 of IVM-NLCs
GraphPad
Prism
8.0.
(B)
Antiviral
activity
of
NLCs,
IVM,
and
IVM-NLCs
(5.0
µM)
measured
by
with GraphPad Prism 8.0. (B) Antiviral activity of NLCs, IVM, and IVM-NLCs (5.0 µM) measured by
CCK-8
assay.
(C)
One-step
growth
curve
of
virus
after
treatment
or
without
treatment
with
NLCs,
CCK-8 assay. (C) One-step growth curve of virus after treatment or without treatment with NLCs,
IVM, and IVM-NLCs. (D) PEDV N RNA copies of Vero cells infected with PEDV after the treatment
IVM, and IVM-NLCs. (D) PEDV N RNA copies of Vero cells infected with PEDV after the treatment
with NLCs, IVM, and IVM-NLCs by RT-qPCR. (E) Western blot analysis of the expression level of
with NLCs, IVM, and IVM-NLCs by RT-qPCR. (E) Western blot analysis of the expression level of
PEDV N protein under the treatment of NLCs, IVM, and IVM-NLCs. (F) Indirect immunofluoresPEDV
protein
under thecells.
treatment
NLCs,
IVM, FITC-conjugated
and IVM-NLCs. (F)
Indirect
immunofluorescence
cence N
assay
of infected
Blue, of
DAPI;
green,
goat
anti-mouse
antibody. (G)
assay
of infected
Blue,
DAPI;(ROS)
green,level
FITC-conjugated
goatpost
anti-mouse
(G)(H)
Cellular
Cellular
reactivecells.
oxygen
species
in infected cells
differentantibody.
treatments.
Mitoreactive
oxygen
species
(ROS) level
in infected
cells
post
different
treatments.
Mitochondrial
chondrial
membrane
potential
(MMP)
infected
cells
post
different
treatments.(H)
Scale
bar = 50 µm.
membrane
in infected
cells post
treatments.
Scale barThe
= 50mean
µm. Error
Error bars potential
represent(MMP)
the standard
deviation
fromdifferent
three repeated
experiments.
valuebars
was
calculatedthe
bystandard
the one-way
analysis
variance
(ANOVA)
(mean ±The
SD,mean
n = 3).value
* p Additionally, RT-qPCR analysis revealed that IVM-NLCs exhibited a greater reduction
in PEDV N RNA copies, as expected in Figure 5D. To validate the inhibitory impact
of IVM-NLCs on the proliferation of PEDV, we assessed the expression level of PEDV
N protein. Specifically, the Western blot assay demonstrated a significant reduction in
the expression level of PEDV N protein upon treatment with IVM-NLCs (Figure 5E).
Although the downregulation of PEDV N protein expression was observed in both IVM
and IVM-NLCs treatment groups, a more pronounced effect was observed in the IVM-NLCtreated group. Additionally, we incubated infected cells with NLCs, IVM, and IVM-NLCs.
At 24 hpi, a strong fluorescent signal was observed in PEDV-infected Vero cells by IFA.
However, a marked difference was observed in the number of infected cells in the IVMNLCs treated group versus the untreated PEDV-infected group, as indicated by green
fluorescence (Figure 5F). These findings collectively indicated that NLCs may enhance the
antiviral activity of IVM against PEDV. Reactive oxygen species (ROS) were toxic byproducts of cellular metabolism, primarily
generated by mitochondria in mammalian cells, and were involved in regulating multiple
physiological functions of cells [43]. We investigated the effect of IVM-NLCs on ROS
production during PEDV infection. Our findings, as depicted in Figure 5G, revealed a
substantial increase in DCF fluorescence intensity in infected Vero cells. Conversely, cells
treated with IVM-NLCs exhibited a significant reduction in ROS generation compared to
those treated with IVM alone. The results indicated that ROS was involved in the antiviral
effect of IVM-NLCs. ROS caused mitochondrial membrane damage, resulting in MMP
disorder [44]. In normal cells, JC-1 emitted red fluorescence. In contrast, in PEDV-infected
Vero cells, JC-1 exhibited green fluorescence, which indicated that PEDV had disrupted
the mitochondrial membrane potential of Vero cells, resulting in its decline. Following
treatment with IVM-NLCs, the mitochondrial membrane potential was notably restored
(Figure 5H). In summary, compared with IVM, IVM-NLCs could improve the inhibition of
MMP damage and impede intracellular ROS accumulation in infected Vero cells.
3.6. Effect of IVM-NLCs on the Apoptosis Rate in PEDV-Infected Vero Cells
To investigate the mechanism of PEDV inhibition by ivermectin, an AnnexinV-FITC/PI
kit was used to detect the apoptosis of the cells by flow cytometry. The results showed
that PEDV could induce apoptosis rate of 20.9 ± 1.89% (Figure 6A), and the apoptosis
rates of IVM- and IVM-NLC-treated PEDV-infected groups were significantly reduced
to 16.4 ± 1.17 and 13.9 ± 1.59 (Figure 6B), which indicated that they play an important
Pharmaceutics 2024, 16, x FOR PEER biological
REVIEW function in PEDV-induced apoptosis. These findings suggested that IVM-NLCs
13 of 16
reduced ROS accumulation in PEDV-infected Vero cells by improving the inhibition of
MMP damage, thereby reducing apoptosis in infected cells. Figure
of of
PEDV-infected
VeroVero
cellscells
afterafter
treatment
with IVM-NLCs.
(A) Apoptosis
assay
Figure6.6.Apoptosis
Apoptosis
PEDV-infected
treatment
with IVM-NLCs.
(A) Apoptosis
was
performed
in PEDV-infected
Vero cells
treated
NLCs,
(5 µM),
IVM-NLCs
(5 µM).
assay
was performed
in PEDV-infected
Vero
cells with
treated
withIVM
NLCs,
IVM and
(5 µM),
and IVM-NLCs
(5 The
µM).graph
(B) The
graph represents
the percentage
of apoptosis
in Vero
cells
after treatment.
Thevalue
mean
(B)
represents
the percentage
of apoptosis
in Vero cells
after
treatment.
The mean
value
was calculated
by the one-way
of variance
(ANOVA)
(mean
** p < 0.01.
was
calculated
by the one-way
analysisanalysis
of variance
(ANOVA)
(mean ±
SD, n±=SD,
3). n**=p3).
< 0.01. 4.4.Conclusions
Conclusions
Herein,
Herein,the
theinhibitory
inhibitoryeffect
effectofofIVM
IVMon
onPEDV
PEDVininvitro
vitrowas
wasfirst
firstdemonstrated.
demonstrated.IVM
IVM
could
inhibit
PEDV
by
the
direct
inactivation
of
viral
particles
and
the
could inhibit PEDV by the direct inactivation of viral particles and theinhibition
inhibitionofofthe
the
replication
replicationphase.
phase.Subsequently,
Subsequently,IVM-NLCs
IVM-NLCswere
weresuccessfully
successfullydeveloped
developedwith
withexcellent
excellent
physicochemical
andand
improved
solubility,
it could
a promising
nanocarphysicochemicalproperties
properties
improved
solubility,
it serve
couldasserve
as a promising
rier
for IVM with
an increased
enhanced
efficacy. According
nanocarrier
for IVM
with an solubility
increasedand
solubility
andpharmacological
enhanced pharmacological
efficacy.
toAccording
biological to
tests,
IVM-NLCs
exhibited
stronger
antiviral
activity
against
than
free
biological tests, IVM-NLCs exhibited stronger antiviral PEDV
activity
against PEDV than free IVM and reduced PEDV-induced mitochondrial dysfunction, which prevented ROS generation and improved viability of infected Vero cell. Moreover, IVMNLCs also reduced PEDV-induced cell apoptosis rate. In view of the in vitro results, it
would be necessary to carry out in vivo tests as soon as possible, to explore its potential
in the clinical treatment of PEDV. Consequently, IVM-NLCs were demonstrated to be a Pharmaceutics 2024, 16, 601 13 of 15 IVM and reduced PEDV-induced mitochondrial dysfunction, which prevented ROS generation and improved viability of infected Vero cell. Moreover, IVM-NLCs also reduced
PEDV-induced cell apoptosis rate. In view of the in vitro results, it would be necessary to
carry out in vivo tests as soon as possible, to explore its potential in the clinical treatment of
PEDV. Consequently, IVM-NLCs were demonstrated to be a potential drug against PEDV,
which might provide a basis for the development of novel drugs to antagonize PEDV.