A novel approach to alleviate acetaminophen-induced hepatotoxicity with hybrid balloon flower root-derived exosome-like nanoparticles (BDEs) with silymarin via inhibition of hepatocyte MAPK pathway and apoptosis

A novel approach to alleviate acetaminophen-induced hepatotoxicity with hybrid balloon flower root-derived exosome-like nanoparticles (BDEs) with silymarin via inhibition of hepatocyte MAPK pathway and apoptosis | 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 A novel approach to alleviate acetaminophen-induced hepatotoxicity with hybrid balloon flower root-derived exosome-like nanoparticles (BDEs) with silymarin via inhibition of hepatocyte MAPK pathway and apoptosis Jisu Kim, Chao Gao, Pengcheng Guo, Jianyong Sheng, Jianxin Wang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4035317/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 18 Jun, 2024 Read the published version in Cell Communication and Signaling → Version 1 posted 11 You are reading this latest preprint version Abstract Introduction: Balloon flower root-derived exosome-like nanoparticles (BDEs) have recently been proposed as physiologically active molecules with no cytotoxicity. However, the therapeutic effects of drug-induced hepatotoxicity of BDEs have not been elucidated. BDEs contain a large amount of platycodin D, which is widely known to be effective in regulating inflammation and ameliorating systemic toxicity. Thus, the main therapeutic activity of BDEs is attributed to inhibiting the inflammatory response and alleviating toxicity. Objective: Considering the potential therapeutic effects of BDEs, and the potential to achieve synergistic effects to improve therapeutic outcomes, we constructed hybrid BDEs with a soy lecithin-based liposome loaded with silymarin (SM). Since liposomes can provide higher thermal stability and have greater structural integrity, these might be more resistant to clearance and enzymatic degradation of drug molecules. Methods: Hybrid BDEs with liposome-loaded SM (BDEs@lipo-SM) were fabricated by thin-film hydration and extrusion. BDEs@lipo-SM were characterized using dynamic light scattering and high-performance liquid chromatography. After confirmation of the physical properties of BDEs@lipo-SM, various therapeutic properties were evaluated. Results: BDEs@lipo-SM were internalized by hepatocytes and immune cells and significantly decreased mRNA expression of apoptosis and inflammation-relevant cytokines by inhibiting the hepatocyte MAPK pathway. BDEs@lipo-SM significantly induced an increase in glutathione levels and inhibited APAP-induced hepatotoxicity. Conclusion: BDEs@lipo-SM have therapeutic benefits for acute liver injury and can alleviate cell death or toxicity. BDEs@lipo-SM can accelerate liver recovery in the APAP-induced acute liver injury model, which highlights thathybrid BDEs represent an attractive delivery vehicle for therapeutic applications. plant-derived exosome-like nanoparticles balloon flower root-derived exosome-like nanoparticles hybrid exosomes N-acetyl-ρ-aminophen-induced liver injury drug delivery systems Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Acetaminophen (N-acetyl-ρ-aminophen, APAP) is the most popular analgesic and antipyretic drug and is widely used for mild to moderate pain and fever worldwide. APAP is considered safe to use at the recommended doses, but it can induce severe liver toxicity, even death, following intentional or non-intentional overdose. In the United States, drug-induced liver injury (DILI), including APAP-induced hepatotoxicity, is the main cause of acute liver failure, which suddenly affects healthy individuals and requires the urgent emergency liver transplantation. 1, 2 Optimal management of DILI resulting from APAP toxicity remains to be elucidated and is associated with adverse patient outcomes with increased morbidity. Thus, the development of a novel approach to the treatment of DILI is urgently needed. In 1963, the US Food and Drug Administration (FDA) approved N-acetylcysteine (NAC) as the main treatment agent for APAP overdose. 3, 4 APAP hepatotoxicity occurs through the formation of the toxic N-acetyl-ρ-benzoquinone imine (NAPQI) metabolite, resulting in glutathione (GSH) depletion and the formation of APAP protein adducts. 5 Adduct formation on mitochondrial proteins modulates respiratory chain function, producing elevated levels of free radicals such as superoxide, which promotes extensive cell death. NAC, a GSH precursor, is used as an antidote to APAP overdose by providing cysteine for glutathione synthesis and can act to detoxify oxidizing radicals of APAP. Unfortunately, therapy with NAC comes with limitations such as a need of high doses and long treatment times due to its poor bioavailability. Furthermore, cases of nausea, vomiting, diarrhea, and anaphylactoid reactions have been reported. Thus, a novel treatment approach to the treatment of DILI is required without such limitations and severe side effects. 6, 7 In Chinese medicine, balloon flower root has been traditionally used for the treatment of bronchitis, asthma, hypertension, allergies, and other inflammatory diseases due to its beneficial bioactive properties. 8-11 Balloon flower root contains saponin, specifically platycodin D, as the main active component that can potentially inhibit inflammatory pathways, promote AMPK expression and protect from oxidative stress damage. Recently, the protective effects of balloon flower root and its bioactive molecules on acute liver toxicity have been reported. 12 According to a previous report, platycodin D may partially inhibit hepatocyte apoptosis by inactivating the MAPK signaling pathway, while reducing mitochondrial oxidative stress in vivo . 10, 13, 14 However, it is not warranted to investigate therapeutic doses of the original raw form balloon flower root for its clinical benefits and bioavailability of phytochemical and bioactive molecules in the circulation 15 Herein, to improve therapeutic outcomes through enhanced bioavailability and to achieve a better therapeutic effect on drug-induced liver injury, we successfully isolated BDEs at high yield using a tangential flow filtration (TFF) method and demonstrated the development of a BDE-based hybrid strategy and discussed the therapeutic potential of the formulation in treating APAP-induced hepatotoxicity. 16-18 In addition, hybrid BDEs were prepared with improved stability, showing significantly enhanced accumulation in the liver and prolonged circulation compared to the free BDE liposome. In conclusion, BDEs can be a promising candidate as a drug delivery system for liver injury therapy and the combination of BDEs with SPC-based liposome-loaded silymarin (BDEs@lipo-SM) can be exploited to deliver therapeutic cargoes to cells and to provide improved drug protection during delivery without hepatotoxicity. Method and material Materials Lecithin (Soya L-a-Phosphatidylcholine, SPC), Silibinin, acetaminophen (APAP), and Bortezomib were obtained from Meilun Biotechnology (Dalian, China) and used as received. Cholestrol was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). 1,1’-dioctadecyl-3,3,3’,3’–tetramethylind-odicarbocyanine, 4-chlorobenzenesulfonate salt (DiD) was purchased from Fanbo Biochemical Co., Ltd. (Beijing, China). Dimethyl sulfoxide (DMSO) was purchased from Sigma-Aldrich (St. Louis, USA). Fetal bovine serum (FBS), Dulbecco's modified Eagle's medium (DMEM), phosphate buffer saline (PBS, pH 7.4), and trypsin were purchased from Yuanpei Biological Technology Co., Ltd (Shanghai, China). Mouse monoclonal antibodies were purchased from BioLegend (London, UK). Isolation of BDEs 100 g of Platycodon grandiflorum , harvested from Chifeng, Inner Mongolia, was purchased from a local market. For isolation of exosome-like nanoparticles, Platycodon grandiflorum was washed with a running water. Then, Platycodon grandiflorum was thoroughly ground in 300 mL 1X PBS with a blender for 5 min. The juice was squeezed on a sieve to remove rough debris and centrifuged at 2000 g for 20 min and the supernatant was further centrifuged at 10,000 g for 60 min. After centrifugation, the supernatant was collected and filtered using a 0.22 μm microporous filter membrane (Collins, USA). To isolate the BDEs, we used a tangential flow filtration method (TFF) using a Minimate EVO Tangential flow ultrafiltration system (Pall Biotech, Dreieich, Germany) to achieve high yield of the exosome-like nanoparticles without sucrose. 16 200 mL of the multivesicular exosomal fluid was transferred into a feed tank and separated through the tangential filter, which is an Omega™ 500 kDa TFF Membrane (Pall Biotech, Dreieich, Germany), at the 20 mL/min of the feed flow rate and 2 bar of the transmembrane pressure. Particles larger than 100 nm concentrated were retained in the main vessel and particles smaller than 100 nm were in the filtrate vessel. While passing through the tangential filter, the final exosome-like nanoparticles were successfully diafiltered and concentrated about 6 times to reach a final volume of 30 mL of BDEs. Preparation of liposome loaded SM The liposome loaded SM (lipo-SM) was prepared by thin-film hydration method. 25.5 mg SPC and 1.5 mg Cholesterol were dissolved in organic solvent (Chloroform : Methanol, 9:1, v/v). The solution was mixed with 1 mg SM dissolved in DMSO. Then, the organic solvent was removed using a rotary vacuum evaporation (ZX-98 rotary evaporator, LOOYE, China) for 10 min in a water bath at 48 ℃ to form a SM-containing lipid membrane. 500 μL of 1X PBS was added to the SM-containing lipid membrane and the lipid solution was stirred to get a preliminary nanoliposomal solution. The nanoliposomal solution was vortexed and sonicated using an ultrasonic cell pulverizer (20% amplitude, 2 sec pulse on/off) for 2 min to turn the multilamellar lipid vesicles into smaller vesicles and the final nanoliposome was obtained by filtering through 0.22 μm filters. Fabrication of BDEs@lipo-SM To promote hybrid BDEs with liposome loaded SM, the BDEs@lipo-SM was synthesized by simple thin film hydration followed by a membrane extrusion method. Functional siRNA Delivery by Extracellular Vesicle–Liposome Hybrid Nanoparticles The fresh BDEs were added in the dry lipid layer to hydrate (1:1, v/v) in the final volume of 500 μL. Then, the mixture was then vortexed and sonicated at 20% amplitude for 2 sec pulse on/off for 2 min. The formed multilamellar BDEs@lipo-SM was extruded through 800, 400, 200 and 100 nm size polycarbonate membranes, respectively, to achieve nano-sized unilamellar BDEs@lipo-SM. The final BDEs@lipo-SM was obtained by filtering through 0.22 μm filters. 19, 20 Nanoparticle tracking analysis Nanoparticle tracking analysis (NTA) was performed by NTA (NanoSight LM10, Malvern Instrument, UK). Size distribution and concentration of BDEs in liquid suspension were measured. BDEs were diluted to a final dilution of 1 : 10,000. 1 mL of BDEs was injected in the chamber with disposable sterile syringes (BD Discardit II, New Jersey, USA) until the solution came out at the end of the nozzle in a way. The sample was detected for 60 second duration using NanoSight automatic analysis settings at room temperature. The average and SD values were analysed using the NTA 3.0 software (Malvern Instruments) Measurement of of protein concentration of BDEs To quantify BDEs, the protein concentration was measured by Bradford protein assay (Yeasen, China). The fresh BDEs were diluted for 10 times with 1X PBS and 5 mL of the diluted BDEs was used for measurement of the protein concentration. Bradford assay was performed as described in the previous report. 21, 22 DLS measurement The BDEs and fabricated BDEs@lipo-SM were diluted 100 times using diethylpyrocarbonate (DEPC) and 1 mL BDEs and fabricated BDEs@lipo-SM were transferred into a cuvette (12 mm square polystyrene cuvette, DTS0012, Malvern, UK) and a disposable cuvette (disposable capillary cell, DTS1070, Malvern, UK) for the size and zeta potential measurement, respectively. The cuvette was placed into the dynamic light scattering (DLS) device to start measuring and the size and surface charge were measured using a Zetasizer Nano ZS (Malvern Instrument, UK). The measurement was conducted at 25 ℃, and all experiments were run in triplicate. Encapsulation efficiency analysis by HPLC The encapsulation efficiency of SM was determined by high-performance liquid chromatography (HPLC) (Agilent, USA). Drug loading capacity was calculated as SM encapsulated/liposome material×100%. To remove free SM in liposomes, the Lipo-SM was centrifuged at 64000 g for 60 min at 4℃ with 10 mL 1X PBS. Then, the supernatant was removed and pellet was dissolved in 500 μL of an organic solvent consisting 10% Triton X and methanol (1:9, v/v). The sample was prepared by filtering through 0.22 μm filters for HPLC analysis. When performing HPLC analysis, SM was eluted through the column of Agilent ZORBAX SB-Aq 5 μm, 4.6 Х 250 mm (CA, USA) at a flow rate of 1 ml/min and separated using a mobile-phase gradient. (Mobile phase A; phosphate buffer (pH 5.0; 10 mM), mobile phase B; acetonitrile (ACN)) The analysis time was set for 22 min and the gradient was subsequently changed in mobile phase B to 29% at 0 min, 41% at 10 min, 29% at 20 min, and ended at 22 min. SM was analyzed using an Agilent 1260 Infinity II instrument (CA, USA). The drug encapsulation efficiency was calculated according to the following formula: The drug encapsulation efficiency = (total drug / free drug)/the initial added total drug × 100%. Cell culture Huh7 liver cells and RAW 264.7 were purchased from Meilun Biotechnology (Dalian, China). All cells were cultured in DMEM with High glucose containing 10% FBS, 100 U/mL penicillin at 37 °C in a humidified atmosphere with 5% CO 2 . For subculturing, the culture media was removed and washed with PBS, followed by adding 0.25% Trypsin-EDTA. The cells were collected and centrifuged for 5 min at 1500 g. Cells were seeded on a 12 well plate at the density of 1×10 5 for Huh7 and RAW 264.7 cell lines, respectively. Analysis of cellular uptake of BDEs@lipo-SM by flow cytometry To determine cellular uptake of BDEs@lipo-SM, Huh7 and RAW 264.7 cells were seeded on a 12 well plate at the density of 1×10 5 . The cells were incubated for 24 h and subsequently 20 μL of DiD-labeled BDEs@lipo-SM was conducted in the cells. The cells were incubated at 37℃ for 1, 3, and 6 h. Then, the cells were collected for detection of the internalized fluorescence of BDEs@lipo-SM by flow cytometry. Immunostaining and flow cytometry To analyze the M2 macrophage proliferation by the macrophage markers in RAW 264.7 cells, flow cytometry was performed. RAW 264.7 cell line was seeded in a 12 well plate at 1×10 5 cells/well. After incubation for 24 h, BDEs and BDEs@lipo-SM were added in the cells and the plate was incubated for 48 h. Non-modified BDEs or BDEs@lipo-SM were considered as a control. Then, the media was removed and cells were collected after washing with 1X PBS. The cells were blocked with 3% BSA on ice for 30 min and centrifuged at 1500 g for 5 min at 4 ℃. Then, the supernatant was removed and the cells were resuspended with 1X PBS and incubated with an eFluore 450-labeled anti-CD45 mAb, PE-labeled anti-CD86 mAb, and APC-labeled anti-CD206 mAb (dilution, 1:100) for 30 min at room temperature avoiding light. Subsequently, the cells were washed 3 times with 1X PBS and detected on The Cytomics™ FC 500 Flow Cytometry (Beckman Coulter, Brea, CA, USA). The result was organized by FlowJo software (BD biosciences, USA). Flow cytometry assay using annexin V/PI staining To evaluate cell apoptosis of BDEs@lipo-SM, apoptosis was detected using an FITC-Annexin V apoptosis detection kit (BD Pharmingen, USA). RAW 264.7 cells were seeded at the density of 1 × 10 5 cell/well in a 12-well plate and incubated for 24 h before treatment. After 48 h of treatment of BDEs@lipo-SM at concentrations of from 0 to 200 µg/ml, both treated and untreated cells were trypsinized and collected. Then, the cells were suspended in 1X binding buffer and incubated with 2.5 µL of fluorescein isothiocyanate (FITC)-conjugated Annexin V and 5 µl of phycoerythrin (PE)-conjugated PI for 15 min in the dark at room temperature followed by flow cytometry. RT-PCR Total RNA was isolated from Huh7 and RAW 264.7 cells treated with a serial dilution of BDEs@lipo-SM ranging from 1 μg/mL to 50 μg/mL for 48 h. cDNA was prepared from the total RNA and consistently RT-PCR was performed using Hifair III One Step RTqPCR SYBR Green Kit (Yeasen, China) according to the manufacturer’s instructions. Primer sets are described in Table 1. To determine apoptosis of immune cells, apoptosis and inflammatory-related genes were examined including BAX, IL-6, IL-1b, TNF-α and the reference gene, GAPDH. To evaluate the gene expression of the targets, the solution containing DNA polymerase, primer pair and cDNA was pre-heated for 5 min at 95˚C to activate and denature non-specific DNA binding. Immediately, the reaction was performed by 40 cycles of 95˚C for 20 s, 55˚C for 30 s and 68˚C for 20 s using QuantStudio 3. Relative gene expression was calculated as the ΔΔCT method and normalized to the expression of GAPDH as a standard for gene expression quantification. All qRT-PCRs were performed in triplicate, and the data are presented as means±standard errors of the means (S.E.M). Western blot analysis The cells were washed with 1X PBS on ice. Then, the ice-cold RIPA buffer including protease and phosphatase inhibitor was added in the cells and the cells were scraped off and the cell suspension was transferred into a 1.5mL microcentrifuge tube. The tube was centrifuged at 14,000g at 4°C for 10 min. The supernatant was transferred to a fresh tube. Cell lysates were analyzed for total protein concentration using the BCA protein assay kit (Beyotime, Shanghai, China). 25 μg of total protein were loaded on 10% acrylamide gel. Relative enzyme levels were quantified using antibodies; mouse mAb p-ERK 1/2; ERK 1/2; p-JNK; JNK; p-p38; p38. Measurement of AST/ALT level To evaluate the liver damage, after treatment of BDEs@lipo-SM, the blood was collected and placed at room temperature for 2 h to separate the serum sample. The blood was then centrifuged at 1000 g for 15 min and the supernatant was taken followed by stored at -80 ℃. serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were assessed in blood plasma by Wuhan Servicebio Technology Co., Ltd. (Wuhan, China) using Chemray 240 automatic biochemical analyzer (Rayto, Shenzhen, China). Animal studies and ethical approval of animal experiment 18-20 g of male C57BL/6J mice at the age of 6-7 weeks were purchased from Sino-British SIPPR/BK Lab. Animal Co., Ltd. (Shanghai, China) and housed under pathogen-free conditions. The animal experiments were conducted at School of Pharmacy in Fudan University in accordance with the Guiding Principles for the Care and Use of Experimental Animals (Shanghai, China). To induce hepatotoxicity, the mice were injected intraperitoneally with 500 mg/kg of APAP and right after treated with 200 μL of 1 mg/mL of BDEs, 150 mg/kg NAC, and 200 μL of 1 mg/mL of BDEs@lipo-SM (based on the BDEs concentration) once. Before being treated, mice were fasted for 24 h. After 24 h of administration, the mice were anesthetized and humanly sacrificed by cervical dislocation and the liver was collected for subsequent analysis. Statistical analysis Statistical significance was determined using the t-test, and a p-value < 0.05 was considered statistically significant, and non-significant result was recorded as N.S. The t-test was conducted through GraphPad Prism 7.0 software (CA, USA). Results Isolation and characterization of BDEs To isolate BDEs with high purity grade and yield, BDEs were obtained using the TFF method. After differential centrifugation of a homogeneous balloon flower root juice, the multivesicular exosomal fluid was collected and exposed to a Tangential flow ultrafiltration system. The fluid was separated through the tangential filter and the filtrate containing particles <100 nm was collected in a separate vessel. The final filtrate of exome-like nanoparticles had a 6-fold higher concentration of BDEs. The morphology of BDEs was observed by TEM, as shown in Figure. 1a , confirming the isolation of 100-nm spherical nanovesicles enclosed by a bilayer lipid membrane without aggregation or degradation. To characterize BDEs, we evaluated the size distribution and particle number using NTA. (Figure 1b) The homogeneous vesicles revealed a concentration of 5.61 ´10 11 particles/mL. DLS measurements based on number were performed to determine the most abundant size population. Similar to the TEM results, the average diameter of BDEs was approximately 80.75±22.95 nm with a low polydispersity index (PDI) of 0.285. The surface charge of the BDEs indicated that the BDEs were covered by a negative charge of –18 mV. (Figure. 1c) The yield of BDE was quantified by the total protein concentration through Bradford protein quantification analysis. The protein concentration of BDEs was approximately 2.08±0.2 mg/mL. Because the purity of BDEs is considered a key indicator of biological activity and availability of exosomes, the purity of BDEs was calculated by the ratio of particle counts to protein concentration, which resulted in approximately 2.69±0.11×10 11 particles/mg protein. (Figure. 1d) To clearly define the content of the bioactive component of BDEs, we performed an HPLC analysis. After extraction, the evaluation of the overall chemical compositions of BDE revealed an enrichment of platycodin D, which is believed to contribute to the efficacy of BDEs in inflammation and related conditions. (Figure. 1e) Preparation and characterization of BDEs@lipo-SM To develop an effective therapy for APAP-induced hepatotoxicity, we designed hybrid BDEs with SPC liposome-loaded SM (BDEs@lipo-SM). To fabricate BDEs@lipo-SM, SPC-based liposomes were prepared for the encapsulation of SM using a thin-film hydration method. After fabrication of the SPC-based liposomes loaded with SM, BDEs were hybridized with the liposomes by a membrane extrusion method. The BDEs@lipo-SM were then examined using DLS. The average particle size of BDEs@lipo-SM obtained from the DLS was 108.09±14.26 nm. The zeta potential was also determined to be -10.16±0.58 mV. (Figure 1f) After hybridization, the average particle size increased slightly compared to BDE, and it appeared that the SPC-based liposome was larger than the BDE. Similar to the change in size distribution, the negative zeta potential of BDEs@lipo-SM was slightly increased. It was assumed that this increase in size also contributed to the increase in surface charge of the SPC-based liposome. (Figure 1g) Evaluation of encapsulation efficiency To demonstrate the efficacy of the SPC-based liposomes loaded with SM, we first optimized the formulation conditions. 20 Under our experimental conditions, the optimized formulation with SPC and cholesterol content showed the best encapsulation and integrity with good storage stability (Data not shown). Then, the encapsulation efficiency of lipo-SM was further determined by HPLC, using the ratio of encapsulated SM to the initial added SM. The encapsulation efficiency was estimated to be approximately 39.82%. (Figure. 1g) Cellular uptake of BDEs and BDEs@lipo-SM in vitro A key factor in the development of drug delivery technologies with lipid-based systems is their capacity for internalization by target cells. Thus, DiD-labeled BDEs and BDEs@lipo-SM were evaluated for cellular uptake via flow cytometry. The cellular uptake of DiD-labeled BDEs increased for up to 6 h in Huh7 cells, achieving 13.2%±2.12, nonetheless, it was not considered very efficient. (Figure 2a) In Huh7 cells exposed to DiD-labeled BDEs@lipo-SM, the cellular uptake of DiD-labeled BDEs@lipo-SM increased to 97.6%±0.75 at 6 h. This increase indicates that the internalization efficiency of the preparation was approximately 7.3-fold higher that achieved by cells in the presence of DiD-labeled BDEs. (Figure 2c) Regulation of apoptosis and inflammation To evaluate whether BDEs and BDEs@lipo-SM can regulate apoptosis and inhibit the inflammatory response, the level of expression genes related to apoptosis and to inflammatory inflammation was analyzed by RT-qPCR. Huh7 cells were exposed to 1 and 10 μg/mL BDEs and BDE@lipo-SM treatment for 48 h and the apoptosis-related gene, BAX, and the inflammatory markers iNOS, IL6, IL1b, and TNF-α were quantified. As shown in Figure 2b and d, BAX expression (a pro-apoptotic factor) decreased significantly following treatment with both BDE and BDE@lipo-SM. Thus, BDEs and BDEs@lipo-SM can induce an antiapoptotic effect on Huh7 cells. A similar trend of down-regulation of inflammatory-related genes was observed following treatment with BDE and BDEs@lipo-SM. However, treatment with BDEs@lipo-SM exhibited a stronger anti-inflammatory effect than treatment with BDEs, as evidenced by the greatly reduced level of gene expression of inflammatory markers. (Figure 2b and d) Because well-fabricated BDEs@lipo-SM intrinsically contain natural bioactive molecules, such as PD that can successfully encapsulate SM and be delivered in cells, inducing significant anti-apoptosis and inflammatory effects. Thus, BDEs@lipo-SM could be utilized as drug delivery agents to enhance therapeutic efficacy. Proliferation of M2 macrophages To evaluate whether BDEs@lipo-SM can induce anti-inflammatory reactions in the macrophage, we treated RAW 264.7 cells with BDEs@lipo-SM, and the effects of BDEs@lipo-SM on macrophage polarization cells was analyzed by flow cytometry. As shown in Figure 3 , the M2 macrophage marker, CD206, markedly increased in the presence of BDEs@lipo-SM, while the M1 macrophage marker, CD86, and levels of pro-inflammatory cytokines, did not increase and even decreased in a concentration-dependent manner. (Figure 3a and b) Interestingly, the M2 macrophage marker CD206 was markedly up-regulated after treatment with 100 μg/mL BDEs@lipo-SM to 91±1.52%, while the M1 macrophage marker CD86 decreased to 0.23±0.17%. Furthermore, macrophage morphological changes were observed in M2 macrophages at different concentrations of BDE@lipo-SM under an inverted phase contrast microscope. The cells treated with free BDEs@lipo-SM showed mainly homogeneous rounded-shaped morphology, whereas cells treated with BDEs@lipo-SM appeared as a heterogeneous cell population with variable pleomorphic morphology. The elongated cell bodies with cytoplasmic extensions were slightly larger than those of the untreated cells. (Figure 3c) In vivo biodistribution of BDEs@lipo-SM and blood pharmacokinetics To validate the biodistribution of BDEs@lipo-SM in vivo , the main organs including the brain, heart, liver, spleen, and kidneys were collected from C57BL/6j mice and analyzed by the IVIS imaging system after 24 h of intravenous administration of DiD-BDEs, DiD-lipo-SM, and DiD-BDEs@lipo-SM. As shown in Figure 4a, DiD-BDEs@lipo-SM were mainly found in the liver and redistributed to the lung and spleen. The detailed quantitative fluorescence values of the IVIS images showed that the accumulation of DiD-BDEs@lipo-SM in the liver was significantly increased by approximately 2-fold compared to DiD-lipo-SM, showing a more prominent hepatic accumulation than DiD-BDEs and DiD-lipo-SM. (Figure 4b) DiD-BDEs@lipo-SM displayed effective delivery to the liver, indicating that BDEs@lipo-SM could be an attractive vehicle for a drug delivery system targeting drugs to the liver. After confirming the biodistribution of BDEs@lipo-SM, we performed serum pharmacokinetic studies to evaluate the retention time of SPC-based liposomes and BDEs@lipo-SM in vivo in the blood circulation. Blood samples were collected at different time intervals (0.125, 0.25, 0.5, 1, 2, 3, 4, 8, and 12 h) after a single intravenous injection of DiD-PBS, DiD-lipo-SM, and DiD-BDEs@lipo-SM, respectively; DiD-PBS was used as a control. The average fluorescence emission was determined and quantified using the IVIS imaging system. For the hematic pharmacokinetic studies, we first confirmed the same fluorescence intensity of DiD-lipo-SM and DiD-BDEs@lipo-SM to ensure equal analysis conditions (Data not shown). As shown in Figure 4c, DiD-BDEs@lipo-SM achieved a prolonged circulation time in the blood. Furthermore, an evident bright fluorescence signal for DiD-BDEs@lipo-SM could still be observed in the blood sample 12 h after injection, indicating that hybrid BDEs could effectively escape drug clearance by the reticuloendothelial system (RES) and thus obtain enhanced blood retention in vivo compared to liposomes. Pharmacokinetic studies showed BDEs@lipo-SM exhibited significantly longer half-life and slower clearance compared to liposomes. In summary, BDEs@lipo-SM would enable enhanced targetability in the liver with no toxicity and possess a prolonged half-life. These results suggest that treatment with BDEs@lipo-SM is safe for APAP-induced hepatotoxicity and provides opportunities for recovery from liver damage after a single treatment. Effects on alleviation of APAP-induced hepatotoxicity in vivo To evaluate the improvement of APAP-induced hepatotoxicity, blood biochemistry tests were performed to measure serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and GSH levels. After intraperitoneal injection of 500 mg/mL of APAP, BDEs, NAC, lipo-SM, and BDEs@lipo-SM were injected intravenously and serum and liver markers were evaluated for each mouse after 24 h of injection. (Figure 4d) As shown in Figure 4e, after BDE treatment @lipo-SM, ALT, and AST levels decreased significantly compared to the APAP-treated group. Treatment with NAC, a glutathione precursor, an antidote for APAP overdose, produced significantly decreased serum conditions of ALT and AST 24 h after treatment. In the course of APAP hepatotoxicity, GSH depletion plays a pivotal role in inducing oxidative stress. 23-25 Thus, it is important to restore GSH levels to overcome long-term damage to liver tissue. GSH levels showed a similar trend as that observed for ALT and AST levels. GSH levels were significantly increased in the treatment of BDEs@lipo-SM compared to the APAP-treated group. Overall, these results suggest that BDEs@lipo-SM allowed to restore GSH levels, which can counteract oxidative stress conditions; thus, attenuating liver injury and inducing liver regeneration. 26-28 Histopathological findings of APAP-induced hepatotoxicity Consistently, based on the results of the blood chemistry evaluations, we further performed the histopathological evaluation of liver tissues from BDEs@lipo-SM-treated mice after 24 h and compared tissues of control mice without exposure to APAP, and exposed to APAP, BDEs, NAC, and lipo-SM to verify the degree of severity of APAP-induced hepatotoxicity. As shown in Figure 4f , after a 24 h treatment, liver tissue in BDEs@lipo-SM treated mice showed comparable intact liver structure with round liver cells and less inflammatory infiltration or cell necrosis surrounding the central veins of the liver, whereas hepatocyte disruption was observed in areas of severe sinusoidal congestion accompanied by extensive centrilobular necrosis, loss of hepatocyte nuclei, and hemorrhaging in the APAP treated group and in the other experimental groups. The results of these pathological changes indicated that treatment with BDEs@lipo-SM can alleviate inflammation and protect the liver, as revealed by decreases in hemorrhage and inflammation infiltration and recovery in hepatocytes. Mechanism of antihepatotoxicity of BDEs@lipo-SM in the MAPK/ERK 1/2 pathway To further evaluate the mechanism of antihepatotoxic activity of BDEs@lipo-SM on APAP-induced hepatotoxicity, the protein expression of components of the mitogen-activated protein kinase (MAPK)/ERK 1/2 pathway was examined by western blotting. In a previous report, it was shown that the MAPK signaling pathway is involved in APAP-induced hepatotoxicity, but the regulatory mechanism of BDEs@lipo-SM on APAP-induced hepatotoxicity remains unknown, thus we analyzed protein expression of ERK1/2, JNK, and p38, which belong to the MAPK family. As shown in Figure 5a , high levels of phosphorylation of ERK1/2, JNK, and p38 were observed in Huh7 cells. However, the level of protein expression of ERK1/2, JNK, and p38 was markedly suppressed following treatment with BDEs@lipo-SM. The relative band densities of ERK1/2, JNK, and p38 were also analyzed. (Figure 5b) It implies that the hybrid BDE strategy can be used to improve antihepatotoxicitic effects through the inhibition of the MAPK pathway and be a good candidate to prevent APAP-induced hepatotoxicity. Discussion In recent years, PENs-based nanotechnologies have gained much attention because they exhibit a variety of functions ranging from cell-to-cell interaction and therapeutic applications. PENs are considered natural nanoparticles, with cargos of phospholipids, proteins, and genetic material that are not harmful. Unlike synthetic nanoparticles, PENs are safe for use as a topical and systemic treatment and may be suitable for certain therapeutic applications. PENs can be used as alternative drug delivery agents in a variety of medicinal applications because of their high stability, inherent biocompatibility, and specificity to target cells. Previous studies investigating PENs have reported the therapeutic potential of PENs and their cell signaling mechanism in various diseases. Balloon flower root contains enriched bioactive compounds and isolated BDEs from their preparation include the various platycosides, specifically PD. A recent study showed that BDEs have great wound healing properties with anti-inflammatory and antioxidant effects in chronic skin wounds. 29 The enriched phytochemicals and bioactive molecules of BDEs can induce antioxidant activity against oxidative stress and can reduce the expression of pro-inflammatory cytokines. As shown in Figure 1e, HPLC analysis indicates that BDEs contain different molecules, including PD. Therefore, BDEs can be expected to contribute to counteract inflammatory disease and exert anti-apoptotic, and cell survival signaling. Consequently, to achieve a higher thermal stability benefit and greater structural integrity, we fabricated hybrid BDEs. Firstly, SPC-based liposomes loaded with SM were prepared and incorporated in BDEs. Next, BDEs@lipo-SM were produced by extrusion. To validate the stability of the fabricated BDEs@lipo-SM, size and zeta potential analyses were performed, with values of 108.09±14.26 nm and -10.16±0.58 mV, respectively, indicating that the engineered BDEs possess great stability in aqueous suspension. To evaluate the entrapment of SM in BDEs, the encapsulation efficiency was assessed to be approximately 39.82%, which is quite low because of the hydrophobicity of the chemical drug and non-ionizable chemical structure. BDEs@lipo-SM exhibit an advantage over original BDEs as they are rapidly captured by cells within 1 h and induce an increase in inhibitory activity against APAP-induced hepatotoxicity due to the synergistic effect of BDEs with SM. BDEs@lipo-SM distinctly evoke an anti-inflammatory immune response in immune cells due to their intrinsic bioactive components in BDEs, while reducing pro-inflammatory signals, albeit with no toxicity. Synergistically, BDEs@lipo-SM significantly increased the levels of GSH following liver injury, indicating that SM is successfully delivered in the liver through hybrid BDEs as a drug carrier and enhances cysteine availability, leading to an increase in GSH levels. 30 Synergistic BDEs@lipo-SM activity can more effectively inhibit the MAPK pathway and lead to liver cell recovery. As described above, BDEs are obtained in high yield with high-grade purity and are expected to become reliable therapeutic carriers for the treatment of drug-induced hepatotoxicity. In the fabrication of the hybrid BDEs@lipo-SM, the BDEs remain structurally stable and retain their biological function, which means that BDEs@lipo-SM can act as a biocompatible systematic delivery system. To exploit the opportunities and potential applications for using BDEs as a drug delivery vehicle, more comprehensive applications based on the peculiar properties of BDEs and hybrid BDEs are necessary. Additionally, to expand the potential applications of BDEs, basic studies of hybrid PENs need to include a comprehensive discussion of the potential of current technology to develop efficient cargo loading, gene loading, and membrane engineering techniques to enhance specific delivery and avoid the systemic adverse effects induced by hybrid PENs over prolonged treatment. Conclusion In this study, the incorporation of BDEs and liposome-loaded SM was significantly enhanced in vitro and in vivo, and BDEs demonstrated therapeutic potential for hepatoprotection and as an anti-inflammatory treatment, supported by evidence of reduced liver injury on histopathological examination compared to treatment with either non-modified BDEs and NAC alone. By inhibiting the MAPK pathway, BDEs@lipo-SM effectively inhibited APAP-induced hepatotoxicity by reducing the protein expression of ERK1/2, JNK, and p38. The results indicate that BDEs@lipo-SM could be successfully delivered in the liver and induce the required therapeutic effect, such as anti-inflammatory and promotion of immunity, due to the stability of BDEs@lipo-SM in vivo , as a carrier of intrinsic bioactive molecules and chemical agents. These findings provide the basis for further development and the fabrication of hybrid BDEs (or PENs) and applications as drug delivery agents to inhibit APAP-induced hepatotoxicity and liver injury. However, thus far, we have a limited understanding of the potential of PENS, and there are still several unanswered questions that need to be addressed, such as processes for highly optimized fabrication of hybrid BDEs and their systemic effects as vehicles of therapeutic agents in clinical trials for various diseases. Further research is required to improve the applicability of the hybrid BDEs as a drug delivery system. Declarations Conflict of interest There authors declare there are no conflicts of interest. Acknowledgment This work was supported by the 72nd round of the China Postdoctoral Science Foundation (No. KLF301807) and the National Natural Science Foundation of China (No. 82074277 and 81773911). References Chang CY, Schiano TD. Review article: drug hepatotoxicity. Aliment Pharmacol Ther . 2007;25(10):1135-1151. 10.1111/j.1365-2036.2007.03307.x Russo MW, Galanko JA, Shrestha R, Fried MW, Watkins P. Liver transplantation for acute liver failure from drug induced liver injury in the United States. Liver Transpl . 2004;10(8):1018-1023. 10.1002/lt.20204 Khayyat A, Tobwala S, Hart M, Ercal N. N-acetylcysteine amide, a promising antidote for acetaminophen toxicity. Toxicol Lett . 2016;241(133-142. 10.1016/j.toxlet.2015.11.008 Chughlay MF, Kramer N, Spearman CW, Werfalli M, Cohen K. 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Oncol Lett . 2018;15(2):2266-2272. 10.3892/ol.2017.7594 Santacroce G, Gentile A, Soriano S, Novelli A, Lenti MV, Di Sabatino A. Glutathione: Pharmacological aspects and implications for clinical use in non-alcoholic fatty liver disease. Front Med (Lausanne) . 2023;10(1124275. 10.3389/fmed.2023.1124275 Vairetti M, Di Pasqua LG, Cagna M, Richelmi P, Ferrigno A, Berardo C. Changes in Glutathione Content in Liver Diseases: An Update. Antioxidants (Basel) . 2021;10(3):10.3390/antiox10030364 Dentico P, Volpe A, Buongiorno R, Grattagliano I, Altomare E, Tantimonaco G, Scotto G, Sacco R, Schiraldi O. [Glutathione in the treatment of chronic fatty liver diseases]. Recenti Prog Med . 1995;86(7-8):290-293. Huang ZZ, Li H, Cai J, Kuhlenkamp J, Kaplowitz N, Lu SC. Changes in glutathione homeostasis during liver regeneration in the rat. Hepatology . 1998;27(1):147-153. 10.1002/hep.510270123 Sacco R, Eggenhoffner R, Giacomelli L. Glutathione in the treatment of liver diseases: insights from clinical practice. Minerva Gastroenterol Dietol . 2016;62(4):316-324. Kim M, Jang H, Park JH. Balloon Flower Root-Derived Extracellular Vesicles: In Vitro Assessment of Anti-Inflammatory, Proliferative, and Antioxidant Effects for Chronic Wound Healing. Antioxidants (Basel) . 2023;12(6):10.3390/antiox12061146 Song Z, Deaciuc I, Song M, Lee DY, Liu Y, Ji X, McClain C. Silymarin protects against acute ethanol-induced hepatotoxicity in mice. Alcohol Clin Exp Res . 2006;30(3):407-413. 10.1111/j.1530-0277.2006.00063.x Scheme 1 Scheme 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Graphicalabstract.png Schematicillustrationv2.0.png Schematic illustration. Molecular mechanism underlying ameliorative effects of BDEs@lipo-SM against APAP-induced cytotoxicity. (a) BDEs@lipo-SM was fabricated by thin film hydration and extrusion method. (b) Therapeutic benefits of BDE@lipo-SM in APAP-induced hepatotoxicity. (c) Acetaminophen (APAP) metabolic pathway and molecular mechanism ofBDEs@lipo-SM in APAP-induced hepatotoxicity. APAP is bioactivated by cytochromes P450 to form a toxic intermediate, N-acetyl-p-benzoquinone imine (NAPQI). NAPQI is conjugated with gluthatione (GSH) resulting in non-toxic metabolites. During APAP overdose, a large amount of APAP is metabolized, which increases NAPQI generation depleting GSH. NAPQI-protein adducts in hepatocytes induce oxidative stress, mitochondrial dysfunction and cell death. BDEs@lipo-SM can increase GSH level and induce hepato-preventive and anti-apoptotic effect through MARK pathway. Supportinginformation.PrimersusedforPCRamplification.xlsx Cite Share Download PDF Status: Published Journal Publication published 18 Jun, 2024 Read the published version in Cell Communication and Signaling → Version 1 posted Editorial decision: Revision requested 24 Apr, 2024 Reviews received at journal 23 Apr, 2024 Reviews received at journal 22 Apr, 2024 Reviewers agreed at journal 14 Apr, 2024 Reviewers agreed at journal 14 Apr, 2024 Reviews received at journal 02 Apr, 2024 Reviewers agreed at journal 24 Mar, 2024 Reviewers invited by journal 23 Mar, 2024 Submission checks completed at journal 17 Mar, 2024 Editor assigned by journal 17 Mar, 2024 First submitted to journal 07 Mar, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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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-4035317","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":280641227,"identity":"d81c8fd3-5da2-4212-8185-e4e20eecfa5a","order_by":0,"name":"Jisu Kim","email":"","orcid":"","institution":"Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Jisu","middleName":"","lastName":"Kim","suffix":""},{"id":280641229,"identity":"7f9af6ad-96cf-4f41-a229-3d855b19a581","order_by":1,"name":"Chao Gao","email":"","orcid":"","institution":"Guangzhou University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Chao","middleName":"","lastName":"Gao","suffix":""},{"id":280641231,"identity":"eab91829-0bb7-4cea-a08e-c6225531f158","order_by":2,"name":"Pengcheng Guo","email":"","orcid":"","institution":"Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Pengcheng","middleName":"","lastName":"Guo","suffix":""},{"id":280641234,"identity":"d057bb31-c20a-48fc-935a-8e2bb8d8d5c3","order_by":3,"name":"Jianyong Sheng","email":"","orcid":"","institution":"Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Jianyong","middleName":"","lastName":"Sheng","suffix":""},{"id":280641235,"identity":"8de816e9-7a09-440c-95ff-aac2718a3e01","order_by":4,"name":"Jianxin Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3klEQVRIiWNgGAWjYBACxgYwZcPPwJAAYjATrSVNsoFoLVBwmAQtzDNyDD8X/DovYXA8+dkDhgrrxAb2swfwO6znjLH0zL7bEgZnnpkbMJxJT2zgyUvAr6W9d4M0b8/tOoMbCWYSjG2HExskeAzwa2nm3fybt+echMGN9G8SjP+I0dLeu02a58cBoJYcoC0NxGjpOf/NmrchWULyzJsyiYRj6cZtPDn4tRjOSEu+zfPHToLvePo2iQ811rL97GcIaGkAWdUG5SUAMRte9UAgDyb/EFI2CkbBKBgFIxoAANAvRorc4Z3lAAAAAElFTkSuQmCC","orcid":"","institution":"Fudan University, Ministry of Education","correspondingAuthor":true,"prefix":"","firstName":"Jianxin","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2024-03-08 03:19:38","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4035317/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4035317/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12964-024-01700-z","type":"published","date":"2024-06-18T15:11:24+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":53098545,"identity":"c03930ab-a538-49aa-bd6e-87b35111f610","added_by":"auto","created_at":"2024-03-20 14:29:37","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":465627,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterization of BDEs and BDEs@lipo-SM\u003c/strong\u003e. (a) The round-shaped morphology of BDEs was observed by transmission electron microscopy (TEM). (b) The concentration of 5.61 × 10\u003csup\u003e11\u003c/sup\u003e particles/mL was measured by nanoparticle tracking analysis (NTA) and the round-shaped morphology of BDEs was recorded (inset). (c) Size distribution and zeta potential were determined with a serial dilution of BDEs by dynamic light scattering (DLS). (d) The total protein concentration and the purity of BDEs were calculated in the graph. (e) Platycodin D in BDEs was evaluated by HPLC analysis. (f) Size distribution and zeta potential of BDEs@lipo-SM were measured by DLS. (g) Tabular form of the size, PDI, and zeta potential of BDEs, liposomes-loaded SM, and BDEs@lipo-SM, respectively and an encapsulation efficiency (EE) of SM in liposomes was determined.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-4035317/v1/2c190ca91873260ddc5075f0.png"},{"id":53098551,"identity":"faf7f02c-430f-4f9e-ba45-20fa49fae529","added_by":"auto","created_at":"2024-03-20 14:29:38","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":170720,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCellular uptake of BDEs and BDEs@lipo-SM in hepatocyte and gene expression of apoptosis and inflammatory related genes.\u003c/strong\u003e(a) The representative histograms and cellular uptake of DiD-labeled BDEs in Huh7 cells was determined at the different time points using flow cytometry analysis. (b) Gene expression of pro-apoptotic related genes including iNOS, BAX, IL6, IL1b, and TNF-α in Huh7 cells in the presence of 1 and 10 μg/mL BDEs. (c) The representative histograms and cellular uptake of DiD-labeled BDEs@lipo-SM in Huh7 cells was determined at the different time points using flow cytometry analysis. (d) Gene expression of pro-apoptotic related genes including iNOS, BAX, IL6, IL1b, and TNF-α in Huh7 cells in the presence of 1 and 10 μg/mL BDEs@lipo-SM. (*P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001)\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-4035317/v1/46b7e63880bb28dbf28a6c79.png"},{"id":53098547,"identity":"b10f3e49-e32d-470d-a438-9ce3f70308e8","added_by":"auto","created_at":"2024-03-20 14:29:37","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":288425,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePromotion of M2 macrophage of BDEs@lipo-SM in RAW 264.7 cells. \u003c/strong\u003e(a) RAW 264.7 cells were proliferated into M2 macrophages with treatment of BDEs@lipo-SM. (b) Evaluation of cell population of M1 (CD45+, CD86+) and M2 (CD45+, CD206+) macrophages. (c) The morphological differences of macrophages in M2 macrophage with treatment of BDEs@lipo-SM.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-4035317/v1/f573709f05c337003c149398.png"},{"id":53098550,"identity":"01000bb9-7f89-49c1-86f9-9b31d38da44c","added_by":"auto","created_at":"2024-03-20 14:29:38","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1487034,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAlleviate effect of BDEs@lipo-SM in APAP-induced liver toxicity at doses of 500 mg/kg in vivo assay and pharmacokinetics study of BDEs@lipo-SM. \u003c/strong\u003e(a) Biodistribution of DiD-labeled BDEs, DiD-labeled lipo-SM, and DiD-labeled BDEs@lipo-SM. In vivo imaging system (IVIS) was used to image brain, heart, liver, spleen and kidneys. (b) Increased liver accumulation of DiD-labeled BDEs@lipo-SM by approximately 2 folds compared to lipo-SM. (c) Average radiant efficiency of blood in mice after treatment of DiD-PBS, DiD-labeled lipo-SM, DiD-labeled BDEs@lipo-SM at the indicated time points (0.125, 0.25, 0.5, 1, 2, 3, 4, 8 and 12 h) post injection. (d) Schematic illustration of APAP-induced hepatotoxicity in vivo assay. (e) Blood chemistry test of AST, ALT and GSH to indicate liver toxicity. (f) H\u0026amp;E staining of liver tissue obtained from mice with non-treated APAP, APAP, APAP+BDEs, APAP+NAC, APAP+lipo-SM and APAP+BDEs@lipo-SM.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-4035317/v1/e946edda0a5cff4f17d0477f.png"},{"id":53098553,"identity":"d8ad20da-fc04-410b-a507-6d37a7c16036","added_by":"auto","created_at":"2024-03-20 14:29:38","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":246979,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of BDEs@lipo-SM on the phosphorylation of ERK1/2, JNK, and p38. \u003c/strong\u003eWestern blot analyses showed activation of the MAPK/ERK 1/2 pathway by BDEs@lipo-SM in Huh7 cells. (1: NC; 2: BDEs; 3:NAC; 4: BDEs@lipo-SM)\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-4035317/v1/ac12c792fccfd89ab28ad41a.png"},{"id":58822821,"identity":"512d655d-aa56-4378-b6d6-71c7aeaccfb2","added_by":"auto","created_at":"2024-06-21 16:48:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3628594,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4035317/v1/f0de8a92-c9e9-4d6c-bde6-564936850224.pdf"},{"id":53098546,"identity":"2257ef88-8c37-4ed6-9a5d-a2057c822623","added_by":"auto","created_at":"2024-03-20 14:29:37","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":265517,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Graphicalabstract.png","url":"https://assets-eu.researchsquare.com/files/rs-4035317/v1/1425bb8432c58f8523a16d88.png"},{"id":53098552,"identity":"3ebe24ba-bd89-4f48-9495-463df4fb317a","added_by":"auto","created_at":"2024-03-20 14:29:38","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":359996,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic illustration. Molecular mechanism underlying ameliorative effects of BDEs@lipo-SM against APAP-induced cytotoxicity. \u003c/strong\u003e(a) BDEs@lipo-SM was fabricated by thin film hydration and extrusion method. (b) Therapeutic benefits of BDE@lipo-SM in APAP-induced hepatotoxicity. (c) Acetaminophen (APAP) metabolic pathway and molecular mechanism ofBDEs@lipo-SM in APAP-induced hepatotoxicity. APAP is bioactivated by cytochromes P450 to form a toxic intermediate, N-acetyl-p-benzoquinone imine (NAPQI). NAPQI is conjugated with gluthatione (GSH) resulting in non-toxic metabolites. During APAP overdose, a large amount of APAP is metabolized, which increases NAPQI generation depleting GSH. NAPQI-protein adducts in hepatocytes induce oxidative stress, mitochondrial dysfunction and cell death. BDEs@lipo-SM can increase GSH level and induce hepato-preventive and anti-apoptotic effect through MARK pathway.\u003c/p\u003e","description":"","filename":"Schematicillustrationv2.0.png","url":"https://assets-eu.researchsquare.com/files/rs-4035317/v1/fdac5a1f776d09eb3ee7245c.png"},{"id":53098548,"identity":"2322dcb1-a186-4a24-a07e-611e068eee63","added_by":"auto","created_at":"2024-03-20 14:29:37","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":9275,"visible":true,"origin":"","legend":"","description":"","filename":"Supportinginformation.PrimersusedforPCRamplification.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4035317/v1/8eb0ee916ff907e6cd6f23ed.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"A novel approach to alleviate acetaminophen-induced hepatotoxicity with hybrid balloon flower root-derived exosome-like nanoparticles (BDEs) with silymarin via inhibition of hepatocyte MAPK pathway and apoptosis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAcetaminophen (N-acetyl-\u0026rho;-aminophen, APAP) is the most popular analgesic and antipyretic\u0026nbsp;drug\u0026nbsp;and is widely used for mild to moderate pain and fever worldwide. APAP is\u0026nbsp;considered\u0026nbsp;safe to use at the recommended doses,\u0026nbsp;but\u0026nbsp;it\u0026nbsp;can induce severe liver\u0026nbsp;toxicity,\u0026nbsp;even\u0026nbsp;death,\u0026nbsp;following intentional or non-intentional overdose.\u0026nbsp;In\u0026nbsp;the\u0026nbsp;United States, drug-induced liver injury (DILI), including APAP-induced hepatotoxicity, is the main cause of acute liver failure, which suddenly affects healthy individuals and\u0026nbsp;requires\u0026nbsp;the\u0026nbsp;urgent emergency liver transplantation.\u003csup\u003e1, 2\u003c/sup\u003e\u0026nbsp; Optimal management of DILI resulting from APAP toxicity remains to be elucidated and is associated with adverse patient outcomes with increased morbidity. Thus, the development of a novel approach to the treatment of DILI is urgently needed.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn\u0026nbsp;1963,\u0026nbsp;the US Food and Drug Administration\u0026nbsp;(FDA)\u0026nbsp;approved N-acetylcysteine (NAC)\u0026nbsp;as the main treatment agent for APAP overdose.\u003csup\u003e3, 4\u003c/sup\u003e APAP hepatotoxicity occurs\u0026nbsp;through\u0026nbsp;the\u0026nbsp;formation of the toxic N-acetyl-\u0026rho;-benzoquinone imine (NAPQI) metabolite, resulting in\u0026nbsp;glutathione (GSH)\u0026nbsp;depletion\u0026nbsp;and\u0026nbsp;the\u0026nbsp;formation of APAP protein adducts.\u003csup\u003e5\u003c/sup\u003e Adduct formation on mitochondrial proteins modulates respiratory chain function, producing elevated levels of free radicals such as superoxide, which promotes extensive cell death. NAC, a GSH precursor, is used as an antidote to APAP overdose by providing cysteine for glutathione synthesis and can act to detoxify oxidizing radicals of APAP. Unfortunately, therapy with NAC comes with limitations such as a need of high doses and long treatment times due to its poor bioavailability. Furthermore, cases of nausea, vomiting, diarrhea, and anaphylactoid reactions have been reported. Thus, a novel treatment approach to the treatment of DILI is required without such limitations and severe side effects.\u003csup\u003e6, 7\u003c/sup\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn Chinese medicine, balloon flower root has been traditionally used for the treatment of bronchitis, asthma, hypertension, allergies, and other inflammatory diseases due to its beneficial bioactive properties.\u003csup\u003e8-11\u003c/sup\u003e Balloon flower root contains saponin, specifically platycodin D,\u0026nbsp;as\u0026nbsp;the\u0026nbsp;main active component that can potentially inhibit inflammatory pathways, promote AMPK\u0026nbsp;expression\u0026nbsp;and protect from oxidative stress damage. Recently, the protective effects of balloon flower root and its bioactive molecules on acute liver toxicity have been reported.\u003csup\u003e12\u003c/sup\u003e According to a previous report, platycodin D may partially inhibit hepatocyte apoptosis by inactivating the MAPK signaling pathway, while reducing mitochondrial oxidative stress \u003cem\u003ein vivo\u003c/em\u003e.\u003csup\u003e10, 13, 14\u003c/sup\u003e However, it is not warranted to investigate therapeutic doses of the original raw form balloon flower root for its clinical benefits and bioavailability of phytochemical and bioactive molecules in the circulation \u003csup\u003e15\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eHerein, to improve therapeutic outcomes through enhanced bioavailability and to achieve a better therapeutic effect\u0026nbsp;on\u0026nbsp;drug-induced liver injury, we successfully isolated BDEs at high yield using a tangential flow filtration (TFF) method and demonstrated the development\u0026nbsp;of\u0026nbsp;a BDE-based\u0026nbsp;hybrid strategy and discussed the therapeutic potential of the formulation in treating APAP-induced hepatotoxicity.\u0026nbsp;\u003csup\u003e16-18\u003c/sup\u003e In addition, hybrid BDEs were prepared with improved stability, showing significantly enhanced accumulation in the liver and prolonged circulation compared\u0026nbsp;to\u0026nbsp;the\u0026nbsp;free\u0026nbsp;BDE\u0026nbsp;liposome. In conclusion, BDEs can be a promising candidate as a drug delivery system for liver injury therapy\u0026nbsp;and\u0026nbsp;the combination of BDEs with SPC-based\u0026nbsp;liposome-loaded\u0026nbsp;silymarin (BDEs@lipo-SM) can be exploited to deliver therapeutic cargoes to cells and to provide improved drug protection during delivery\u0026nbsp;without\u0026nbsp;hepatotoxicity.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"Method and material","content":"\u003cp\u003e\u003cstrong\u003eMaterials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLecithin (Soya L-a-Phosphatidylcholine, SPC), Silibinin, acetaminophen (APAP), and Bortezomib were obtained from Meilun Biotechnology (Dalian, China) and used as received. Cholestrol was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). 1,1\u0026rsquo;-dioctadecyl-3,3,3\u0026rsquo;,3\u0026rsquo;\u0026ndash;tetramethylind-odicarbocyanine, 4-chlorobenzenesulfonate salt (DiD) was purchased from Fanbo Biochemical Co., Ltd. (Beijing, China). Dimethyl sulfoxide (DMSO) was purchased from Sigma-Aldrich (St. Louis, USA). Fetal bovine serum (FBS), Dulbecco\u0026apos;s modified Eagle\u0026apos;s medium (DMEM), phosphate buffer saline (PBS, pH 7.4), and trypsin were purchased from Yuanpei Biological Technology Co., Ltd (Shanghai, China). Mouse monoclonal antibodies were purchased from BioLegend (London, UK).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIsolation of BDEs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e100 g of \u003cem\u003ePlatycodon grandiflorum\u003c/em\u003e, harvested from Chifeng, Inner Mongolia, was purchased from a local market. For isolation of exosome-like nanoparticles, \u003cem\u003ePlatycodon grandiflorum\u003c/em\u003e was washed with a running water. Then, \u003cem\u003ePlatycodon grandiflorum\u003c/em\u003e was thoroughly ground in\u0026nbsp;300\u0026nbsp;mL 1X PBS with a blender for 5 min. The juice was squeezed on a sieve to remove rough debris and centrifuged at 2000 g for 20 min and the supernatant was further centrifuged at 10,000 g for 60 min. After centrifugation, the supernatant was collected and filtered using\u0026nbsp;a 0.22 \u0026mu;m microporous filter membrane (Collins, USA). To isolate the BDEs, we used a tangential flow filtration method\u0026nbsp;(TFF)\u0026nbsp;using a Minimate EVO Tangential flow ultrafiltration system (Pall Biotech, Dreieich, Germany)\u0026nbsp;to achieve high yield of the exosome-like nanoparticles without sucrose.\u0026nbsp;\u003csup\u003e16\u003c/sup\u003e 200 mL of the multivesicular exosomal fluid was transferred into a feed tank and separated through the tangential filter, which is an Omega\u0026trade; 500 kDa TFF Membrane (Pall Biotech, Dreieich, Germany), at the 20 mL/min of the feed flow rate and 2 bar of the transmembrane pressure.\u0026nbsp;Particles larger than 100 nm concentrated were retained in the main vessel and particles smaller than 100 nm were in the filtrate vessel.\u0026nbsp;While passing through the tangential filter, the final exosome-like nanoparticles were successfully diafiltered and concentrated about 6 times to reach a final volume of 30 mL of BDEs.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of liposome loaded SM\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe liposome loaded SM (lipo-SM) was prepared by thin-film hydration method. 25.5 mg SPC and 1.5 mg Cholesterol were dissolved in organic solvent (Chloroform : Methanol, 9:1, v/v). The solution was mixed with 1 mg SM dissolved in DMSO. Then, the organic solvent was removed using a rotary vacuum evaporation (ZX-98 rotary evaporator, LOOYE, China) for 10 min in a water bath at 48\u0026nbsp;℃ to form a SM-containing lipid membrane. 500\u0026nbsp;\u0026mu;L of 1X PBS was added to the SM-containing lipid membrane and the lipid solution was stirred to get a preliminary nanoliposomal solution. The nanoliposomal solution was vortexed and sonicated using an ultrasonic cell pulverizer (20% amplitude, 2 sec pulse on/off) for 2 min to turn the multilamellar lipid vesicles into smaller vesicles and the final nanoliposome was obtained by filtering through 0.22 \u0026mu;m filters.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFabrication of BDEs@lipo-SM\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo promote hybrid BDEs with liposome loaded SM, the BDEs@lipo-SM was synthesized by simple thin film hydration followed by a membrane extrusion method. Functional siRNA Delivery by Extracellular Vesicle\u0026ndash;Liposome Hybrid Nanoparticles The fresh BDEs were added in the dry lipid layer to hydrate (1:1, v/v) in the final volume of 500 \u0026mu;L. Then, the mixture was then vortexed and sonicated at 20% amplitude for 2 sec pulse on/off for 2 min. The formed multilamellar BDEs@lipo-SM was extruded through 800, 400, 200 and 100 nm size polycarbonate membranes, respectively, to achieve nano-sized unilamellar BDEs@lipo-SM. The final BDEs@lipo-SM was obtained by filtering through 0.22 \u0026mu;m filters.\u0026nbsp;\u003csup\u003e19, 20\u003c/sup\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNanoparticle tracking analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNanoparticle tracking analysis (NTA) was performed by NTA (NanoSight LM10, Malvern Instrument, UK). Size distribution and concentration of BDEs in liquid suspension were measured. BDEs were diluted to a final dilution of 1 : 10,000. 1 mL of BDEs was injected in the chamber with disposable sterile syringes (BD Discardit II, New Jersey, USA) until the solution came out at the end of the nozzle in a way. The sample was detected for 60 second duration using NanoSight automatic analysis settings at room temperature. The average and SD values were analysed using the NTA 3.0 software (Malvern Instruments)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMeasurement of of protein concentration of BDEs\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo quantify BDEs, the protein concentration was measured by Bradford protein assay (Yeasen, China). The fresh BDEs were diluted for 10 times with 1X PBS and 5 mL of the diluted BDEs was used for measurement of the protein concentration. Bradford assay was performed as described in the previous report.\u0026nbsp;\u003csup\u003e21, 22\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDLS measurement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe BDEs and fabricated BDEs@lipo-SM were diluted 100 times using diethylpyrocarbonate (DEPC) and 1 mL BDEs and fabricated BDEs@lipo-SM were transferred into a cuvette (12 mm square polystyrene cuvette, DTS0012, Malvern, UK) and a disposable cuvette (disposable capillary cell, DTS1070, Malvern, UK) for the size and zeta potential measurement, respectively. The cuvette was placed into the dynamic light scattering (DLS) device to start measuring and the size and surface charge were measured using a Zetasizer Nano ZS (Malvern Instrument, UK). The measurement was conducted at 25\u0026nbsp;℃, and all experiments were run in triplicate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEncapsulation efficiency analysis by HPLC\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe encapsulation efficiency of SM was determined by high-performance liquid chromatography (HPLC) (Agilent, USA). Drug loading capacity was calculated as SM encapsulated/liposome material\u0026times;100%. To remove free SM in liposomes, the Lipo-SM was centrifuged at 64000 g for 60 min at 4℃\u0026nbsp;with 10 mL 1X PBS. Then, the supernatant was removed and pellet was dissolved in 500 \u0026mu;L of an organic solvent consisting 10% Triton X and methanol (1:9, v/v). The sample was prepared by filtering through 0.22 \u0026mu;m filters for HPLC analysis. When performing HPLC analysis, SM was eluted through the column of Agilent ZORBAX SB-Aq 5 \u0026mu;m, 4.6 Х 250 mm (CA, USA) at a flow rate of 1 ml/min and separated using a mobile-phase gradient. (Mobile phase A; phosphate buffer (pH 5.0; 10 mM), mobile phase B; acetonitrile (ACN)) The analysis time was set for 22 min and the gradient was subsequently changed in mobile phase B to 29% at 0 min, 41% at 10 min, 29% at 20 min, and ended at 22 min. SM was analyzed using an Agilent 1260 Infinity II instrument (CA, USA). The drug encapsulation efficiency was calculated according to the following formula: The drug encapsulation efficiency\u0026thinsp;=\u0026thinsp;(total drug / free drug)/the initial added total drug \u0026times; 100%.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuh7\u0026nbsp;liver cells and RAW 264.7 were purchased from Meilun Biotechnology (Dalian, China). All cells were cultured in DMEM with High glucose containing 10% FBS, 100 U/mL penicillin at 37 \u0026deg;C in a humidified atmosphere with 5% CO\u003csub\u003e2\u003c/sub\u003e. For subculturing, the culture media was removed and washed with PBS, followed by adding 0.25% Trypsin-EDTA. The cells were collected and centrifuged for 5 min at 1500 g. Cells were seeded on a 12 well plate at the density of 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e for Huh7 and RAW 264.7 cell lines, respectively.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of cellular uptake of BDEs@lipo-SM by flow cytometry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine cellular uptake of BDEs@lipo-SM, Huh7 and RAW 264.7 cells were seeded on a 12 well plate at the density of 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e. The cells were incubated for 24 h and subsequently 20 \u0026mu;L of DiD-labeled BDEs@lipo-SM was conducted in the cells. The cells were incubated at 37℃\u0026nbsp;for 1, 3, and 6 h. Then, the cells were collected for detection of the internalized fluorescence of BDEs@lipo-SM by flow cytometry.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunostaining and flow cytometry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo analyze the M2 macrophage proliferation by the macrophage markers in RAW 264.7 cells, flow cytometry was performed. RAW 264.7 cell line was seeded in a 12 well plate at 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/well. After incubation for 24 h, BDEs and BDEs@lipo-SM were added in the cells and the plate was incubated for 48 h. Non-modified BDEs or BDEs@lipo-SM were considered as a control. Then, the media was removed and cells were collected after washing with 1X PBS. The cells were blocked with 3% BSA on ice for 30 min and centrifuged at 1500 g for 5 min at 4\u0026nbsp;℃. Then, the supernatant was removed and the cells were resuspended with 1X PBS and incubated with an eFluore 450-labeled anti-CD45 mAb, PE-labeled anti-CD86 mAb, and APC-labeled anti-CD206 mAb (dilution, 1:100) for 30 min at room temperature avoiding light. Subsequently, the cells were washed 3 times with 1X PBS and detected on The Cytomics\u0026trade; FC 500 Flow Cytometry (Beckman Coulter, Brea, CA, USA). The result was organized by FlowJo software (BD biosciences, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFlow cytometry assay using annexin V/PI staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate cell apoptosis of BDEs@lipo-SM, apoptosis was detected using an FITC-Annexin V apoptosis detection kit (BD Pharmingen, USA). RAW 264.7 cells were seeded at the density of 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cell/well in a 12-well plate and incubated for 24 h before treatment. After 48 h of treatment of BDEs@lipo-SM at concentrations of from 0 to 200 \u0026micro;g/ml, both treated and untreated cells were trypsinized and collected. Then, the cells were suspended in 1X binding buffer and incubated with 2.5 \u0026micro;L of fluorescein isothiocyanate (FITC)-conjugated Annexin V and 5 \u0026micro;l of phycoerythrin (PE)-conjugated PI for 15 min in the dark at room temperature followed by flow cytometry.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRT-PCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was\u0026nbsp;isolated\u0026nbsp;from Huh7 and RAW 264.7 cells treated with a serial dilution of BDEs@lipo-SM ranging from 1 \u0026mu;g/mL to 50 \u0026mu;g/mL for 48 h. cDNA was prepared from the total RNA and consistently RT-PCR was performed using Hifair III One Step RTqPCR SYBR Green Kit (Yeasen, China) according to the manufacturer\u0026rsquo;s instructions. Primer sets are described in Table 1. To determine apoptosis of immune cells, apoptosis and inflammatory-related genes were examined including BAX, IL-6, IL-1b, TNF-\u0026alpha; and the reference gene, GAPDH. To evaluate the gene expression of the targets, the solution containing DNA polymerase, primer pair and cDNA was pre-heated for 5 min at 95˚C to activate and denature non-specific DNA binding. Immediately, the reaction was performed by 40 cycles of 95˚C for 20 s, 55˚C for 30 s and 68˚C for 20 s using QuantStudio 3. Relative gene expression was calculated as the \u0026Delta;\u0026Delta;CT method and normalized to the expression of GAPDH as a standard for gene expression quantification. All qRT-PCRs were performed in triplicate, and the data are presented as means\u0026plusmn;standard errors of the means (S.E.M).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blot analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe cells were washed with 1X PBS on ice. Then, the ice-cold RIPA buffer including protease and phosphatase inhibitor was added in the cells and the cells were scraped off and the cell suspension was transferred into a 1.5mL microcentrifuge tube. The tube was centrifuged at 14,000g at 4\u0026deg;C for 10 min. The supernatant was transferred to a fresh tube. Cell lysates were analyzed for total protein concentration using the BCA protein assay kit (Beyotime, Shanghai, China). 25\u0026nbsp;\u0026mu;g\u0026nbsp;of total protein were loaded on 10% acrylamide gel. Relative enzyme levels were quantified using antibodies; mouse mAb p-ERK 1/2; ERK 1/2; p-JNK; JNK; p-p38; p38.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMeasurement of AST/ALT level\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate the liver damage, after treatment of BDEs@lipo-SM, the blood was collected and placed at room temperature for 2 h to separate the serum sample. The blood was then centrifuged at 1000 g for 15 min and the supernatant was taken followed by stored at -80\u0026nbsp;℃.\u0026nbsp;serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were assessed in blood plasma by Wuhan Servicebio Technology Co., Ltd. (Wuhan, China) using Chemray 240 automatic biochemical analyzer (Rayto, Shenzhen, China).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimal studies and ethical approval of animal experiment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e18-20 g of male C57BL/6J mice at the age of 6-7 weeks were purchased from Sino-British SIPPR/BK Lab. Animal Co., Ltd. (Shanghai, China) and housed under pathogen-free conditions. The animal experiments were conducted at School of Pharmacy in Fudan University in accordance with the Guiding Principles for the Care and Use of Experimental Animals (Shanghai, China). To induce hepatotoxicity, the mice were injected intraperitoneally with 500 mg/kg of APAP and right after treated with 200 \u0026mu;L of 1 mg/mL of BDEs, 150 mg/kg NAC, and 200 \u0026mu;L of 1 mg/mL of BDEs@lipo-SM (based on the BDEs concentration) once. Before being treated, mice were fasted for 24 h. After 24 h of administration, the mice were anesthetized and humanly sacrificed by cervical dislocation and the liver was collected for subsequent analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical significance was determined using the t-test, and a p-value \u0026lt; 0.05 was considered statistically significant, and non-significant result was recorded as N.S. The t-test was conducted through GraphPad Prism 7.0 software (CA, USA).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIsolation and characterization of BDEs\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo isolate BDEs\u0026nbsp;with\u0026nbsp;high purity grade\u0026nbsp;and\u0026nbsp;yield, BDEs were obtained using the TFF method. After differential centrifugation of a homogeneous balloon flower root juice, the multivesicular exosomal fluid was collected and exposed to a Tangential flow ultrafiltration system. The fluid was separated through the tangential filter and the filtrate containing particles \u0026lt;100 nm was collected in a separate vessel. The final filtrate of exome-like nanoparticles had a 6-fold higher concentration of BDEs. The morphology of BDEs was observed by TEM, as shown in \u003cstrong\u003eFigure. 1a\u003c/strong\u003e, confirming the isolation of\u0026nbsp;100-nm\u0026nbsp;spherical nanovesicles enclosed by a bilayer lipid membrane without aggregation or degradation. To characterize\u0026nbsp;BDEs,\u0026nbsp;we evaluated\u0026nbsp;the size distribution and particle\u0026nbsp;number\u0026nbsp;using NTA. \u003cstrong\u003e(Figure 1b)\u0026nbsp;\u003c/strong\u003eThe\u0026nbsp;homogeneous\u0026nbsp;vesicles revealed a concentration of 5.61\u0026nbsp;\u0026acute;10\u003csup\u003e11\u003c/sup\u003e particles/mL. DLS measurements based on number were performed to determine the most abundant size population.\u0026nbsp;Similar\u0026nbsp;to the TEM results, the average diameter of BDEs was approximately 80.75\u0026plusmn;22.95 nm with a low polydispersity index (PDI) of 0.285. The surface charge\u0026nbsp;of\u0026nbsp;the\u0026nbsp;BDEs indicated\u0026nbsp;that\u0026nbsp;the\u0026nbsp;BDEs\u0026nbsp;were\u0026nbsp;covered by a negative charge of \u0026ndash;18 mV. \u003cstrong\u003e(Figure. 1c)\u0026nbsp;\u003c/strong\u003eThe yield of\u0026nbsp;BDE\u0026nbsp;was quantified by the total protein concentration through Bradford protein quantification analysis. The protein concentration of BDEs was approximately 2.08\u0026plusmn;0.2 mg/mL. Because the purity of BDEs is\u0026nbsp;considered\u0026nbsp;a key indicator\u0026nbsp;of\u0026nbsp;biological activity and availability of exosomes, the purity of BDEs was calculated by the ratio of particle counts to protein concentration, which resulted in approximately 2.69\u0026plusmn;0.11\u0026times;10\u003csup\u003e11\u003c/sup\u003e particles/mg\u0026nbsp;protein. \u003cstrong\u003e(Figure. 1d)\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo\u0026nbsp;clearly define the content\u0026nbsp;of\u0026nbsp;the\u0026nbsp;bioactive component of BDEs, we performed an HPLC analysis. After extraction, the evaluation of the overall chemical compositions of\u0026nbsp;BDE revealed an\u0026nbsp;enrichment of platycodin D,\u0026nbsp;which is believed to contribute to the efficacy of BDEs in inflammation and related conditions. \u003cstrong\u003e(Figure. 1e)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePreparation and characterization of BDEs@lipo-SM\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo\u0026nbsp;develop\u0026nbsp;an\u0026nbsp;effective therapy for APAP-induced hepatotoxicity, we\u0026nbsp;designed\u0026nbsp;hybrid BDEs with\u0026nbsp;SPC\u0026nbsp;liposome-loaded\u0026nbsp;SM (BDEs@lipo-SM). To fabricate BDEs@lipo-SM, SPC-based\u0026nbsp;liposomes\u0026nbsp;were prepared\u0026nbsp;for the encapsulation of\u0026nbsp;SM\u0026nbsp;using a thin-film hydration method. After fabrication of the SPC-based\u0026nbsp;liposomes\u0026nbsp;loaded with SM, BDEs were hybridized with the liposomes by a membrane extrusion method. The BDEs@lipo-SM were then examined using DLS. The average particle size of BDEs@lipo-SM obtained from the DLS was 108.09\u0026plusmn;14.26 nm. The zeta potential was also determined to be -10.16\u0026plusmn;0.58 mV. \u003cstrong\u003e(Figure 1f)\u0026nbsp;\u003c/strong\u003eAfter\u0026nbsp;hybridization, the average particle size\u0026nbsp;increased\u0026nbsp;slightly compared to BDE,\u0026nbsp;and it appeared that the SPC-based liposome was larger than the BDE.\u0026nbsp;Similar\u0026nbsp;to the change in size distribution, the negative zeta potential of BDEs@lipo-SM was slightly increased.\u0026nbsp;It\u0026nbsp;was\u0026nbsp;assumed that\u0026nbsp;this increase in size also\u0026nbsp;contributed to the increase in surface charge of the SPC-based liposome. \u003cstrong\u003e(Figure 1g)\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEvaluation of encapsulation efficiency\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo demonstrate the efficacy of the SPC-based liposomes loaded with SM, we first optimized the formulation conditions.\u003csup\u003e20\u003c/sup\u003e Under our experimental conditions, the optimized formulation with SPC and cholesterol\u0026nbsp;content\u0026nbsp;showed the best encapsulation and integrity with good storage stability (Data not shown). Then, the encapsulation efficiency of lipo-SM was further determined by HPLC, using the ratio of encapsulated SM to the initial added SM. The encapsulation efficiency was estimated to be approximately 39.82%. \u003cstrong\u003e(Figure. 1g)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCellular uptake of BDEs and BDEs@lipo-SM in vitro\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA key factor in the development of drug delivery technologies with lipid-based systems is their capacity for internalization by target cells. Thus, DiD-labeled BDEs and BDEs@lipo-SM were evaluated for cellular uptake via flow cytometry. The cellular uptake of DiD-labeled\u0026nbsp;BDEs\u0026nbsp;increased for up to 6 h in Huh7 cells, achieving 13.2%\u0026plusmn;2.12, nonetheless, it\u0026nbsp;was\u0026nbsp;not considered very efficient. \u003cstrong\u003e(Figure 2a)\u0026nbsp;\u003c/strong\u003eIn\u0026nbsp;Huh7 cells exposed to DiD-labeled BDEs@lipo-SM, the cellular uptake of DiD-labeled\u0026nbsp;BDEs@lipo-SM\u0026nbsp;increased to 97.6%\u0026plusmn;0.75 at 6 h. This increase indicates that the internalization efficiency of the preparation was approximately 7.3-fold\u0026nbsp;higher that achieved by cells in the presence of DiD-labeled BDEs. \u003cstrong\u003e(Figure 2c)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eRegulation of apoptosis and inflammation\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate whether BDEs and BDEs@lipo-SM can regulate apoptosis and\u0026nbsp;inhibit\u0026nbsp;the\u0026nbsp;inflammatory response,\u0026nbsp;the\u0026nbsp;level of\u0026nbsp;expression\u0026nbsp;genes related to apoptosis\u0026nbsp;and\u0026nbsp;to\u0026nbsp;inflammatory\u0026nbsp;inflammation\u0026nbsp;was analyzed by RT-qPCR. Huh7 cells were exposed to 1 and 10 \u0026mu;g/mL BDEs and\u0026nbsp;BDE@lipo-SM\u0026nbsp;treatment for 48 h and the\u0026nbsp;apoptosis-related\u0026nbsp;gene, BAX, and the inflammatory markers iNOS, IL6, IL1b, and TNF-\u0026alpha; were quantified. As shown in\u0026nbsp;\u003cstrong\u003eFigure\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;2b and d,\u0026nbsp;\u003c/strong\u003eBAX expression (a pro-apoptotic factor) decreased\u0026nbsp;significantly\u0026nbsp;following treatment with both BDE\u0026nbsp;and\u0026nbsp;BDE@lipo-SM.\u0026nbsp;Thus, BDEs and BDEs@lipo-SM can induce an antiapoptotic effect on Huh7 cells. A similar\u0026nbsp;trend\u0026nbsp;of down-regulation of inflammatory-related\u0026nbsp;genes was observed\u0026nbsp;following treatment with\u0026nbsp;BDE\u0026nbsp;and\u0026nbsp;BDEs@lipo-SM.\u0026nbsp;However, treatment with BDEs@lipo-SM exhibited a stronger anti-inflammatory effect than treatment with\u0026nbsp;BDEs,\u0026nbsp;as evidenced by the greatly\u0026nbsp;reduced\u0026nbsp;level of\u0026nbsp;gene\u0026nbsp;expression\u0026nbsp;of inflammatory markers.\u003cstrong\u003e\u0026nbsp;(Figure 2b and d)\u003c/strong\u003e Because well-fabricated BDEs@lipo-SM intrinsically contain natural bioactive molecules, such as PD that can successfully encapsulate SM and be delivered\u0026nbsp;in\u0026nbsp;cells, inducing significant anti-apoptosis and inflammatory effects. Thus, BDEs@lipo-SM could be utilized as drug delivery agents to\u0026nbsp;enhance\u0026nbsp;therapeutic efficacy.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eProliferation of M2 macrophages\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate whether BDEs@lipo-SM can induce anti-inflammatory reactions in the macrophage, we treated RAW 264.7 cells with BDEs@lipo-SM, and the effects of BDEs@lipo-SM\u0026nbsp;on\u0026nbsp;macrophage polarization cells was analyzed by flow cytometry. As shown in \u003cstrong\u003eFigure 3\u003c/strong\u003e,\u0026nbsp;the\u0026nbsp;M2\u0026nbsp;macrophage\u0026nbsp;marker,\u0026nbsp;CD206,\u0026nbsp;markedly\u0026nbsp;increased\u0026nbsp;in the presence of BDEs@lipo-SM, while\u0026nbsp;the\u0026nbsp;M1\u0026nbsp;macrophage\u0026nbsp;marker,\u0026nbsp;CD86, and levels of\u0026nbsp;pro-inflammatory\u0026nbsp;cytokines, did\u0026nbsp;not\u0026nbsp;increase\u0026nbsp;and even decreased in a concentration-dependent manner. \u003cstrong\u003e(Figure 3a and b)\u0026nbsp;\u003c/strong\u003eInterestingly, the M2 macrophage marker CD206 was markedly up-regulated after treatment with 100 \u0026mu;g/mL BDEs@lipo-SM to 91\u0026plusmn;1.52%,\u0026nbsp;while\u0026nbsp;the M1 macrophage marker CD86 decreased to 0.23\u0026plusmn;0.17%. Furthermore, macrophage morphological changes were observed in M2\u0026nbsp;macrophages\u0026nbsp;at\u0026nbsp;different concentrations of\u0026nbsp;BDE@lipo-SM\u0026nbsp;under an inverted phase contrast microscope. The cells treated with free BDEs@lipo-SM showed mainly\u0026nbsp;homogeneous\u0026nbsp;rounded-shaped morphology, whereas cells treated with BDEs@lipo-SM appeared as a heterogeneous cell population with variable pleomorphic morphology. The elongated cell bodies with cytoplasmic extensions were slightly larger\u0026nbsp;than\u0026nbsp;those of\u0026nbsp;the untreated cells. \u003cstrong\u003e(Figure 3c)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn vivo biodistribution of BDEs@lipo-SM and blood pharmacokinetics\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo validate the biodistribution of BDEs@lipo-SM \u003cem\u003ein vivo\u003c/em\u003e, the main organs\u0026nbsp;including\u0026nbsp;the\u0026nbsp;brain, heart, liver,\u0026nbsp;spleen,\u0026nbsp;and kidneys were collected from C57BL/6j mice and analyzed\u0026nbsp;by\u0026nbsp;the\u0026nbsp;IVIS imaging system after 24 h of intravenous administration of DiD-BDEs, DiD-lipo-SM, and DiD-BDEs@lipo-SM. As shown in \u003cstrong\u003eFigure 4a,\u0026nbsp;\u003c/strong\u003eDiD-BDEs@lipo-SM were mainly found in the liver and redistributed to the lung and spleen. The detailed quantitative fluorescence values of the IVIS images showed\u0026nbsp;that\u0026nbsp;the\u0026nbsp;accumulation of DiD-BDEs@lipo-SM in the liver was significantly increased by approximately 2-fold compared to DiD-lipo-SM,\u0026nbsp;showing\u0026nbsp;a\u0026nbsp;more prominent hepatic accumulation than DiD-BDEs and DiD-lipo-SM. \u003cstrong\u003e(Figure 4b)\u0026nbsp;\u003c/strong\u003eDiD-BDEs@lipo-SM\u0026nbsp;displayed effective delivery to the liver, indicating that BDEs@lipo-SM could be an attractive vehicle\u0026nbsp;for\u0026nbsp;a\u0026nbsp;drug delivery system targeting drugs to the liver.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAfter confirming the biodistribution of BDEs@lipo-SM, we performed serum\u0026nbsp;pharmacokinetic\u0026nbsp;studies to evaluate the retention time of SPC-based liposomes and BDEs@lipo-SM\u0026nbsp;\u003cem\u003ein vivo\u003c/em\u003e in the blood circulation. Blood samples were collected at different time intervals (0.125, 0.25, 0.5, 1, 2, 3, 4, 8, and 12 h) after a single intravenous injection of DiD-PBS, DiD-lipo-SM, and DiD-BDEs@lipo-SM, respectively; DiD-PBS was used as a control. The average fluorescence emission was determined and quantified\u0026nbsp;using\u0026nbsp;the\u0026nbsp;IVIS imaging system. For the hematic\u0026nbsp;pharmacokinetic\u0026nbsp;studies, we first confirmed the same fluorescence intensity of DiD-lipo-SM and DiD-BDEs@lipo-SM to ensure equal analysis conditions (Data not shown). As shown in \u003cstrong\u003eFigure 4c,\u0026nbsp;\u003c/strong\u003eDiD-BDEs@lipo-SM\u0026nbsp;achieved\u0026nbsp;a\u0026nbsp;prolonged circulation time in the blood. Furthermore, an\u0026nbsp;evident\u0026nbsp;bright fluorescence signal for DiD-BDEs@lipo-SM could still be observed\u0026nbsp;in\u0026nbsp;the\u0026nbsp;blood sample 12 h\u0026nbsp;after injection,\u0026nbsp;indicating\u0026nbsp;that\u0026nbsp;hybrid BDEs could effectively\u0026nbsp;escape\u0026nbsp;drug clearance by the\u0026nbsp;reticuloendothelial system\u0026nbsp;(RES)\u0026nbsp;and thus obtain enhanced blood retention \u003cem\u003ein vivo\u003c/em\u003e compared to liposomes. Pharmacokinetic studies showed BDEs@lipo-SM exhibited significantly longer half-life and slower clearance compared to liposomes. In summary, BDEs@lipo-SM would enable enhanced targetability in the liver with no toxicity and\u0026nbsp;possess\u0026nbsp;a\u0026nbsp;prolonged half-life. These results suggest that treatment with BDEs@lipo-SM is safe for APAP-induced hepatotoxicity and\u0026nbsp;provides\u0026nbsp;opportunities for recovery from liver damage after a single\u0026nbsp;treatment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEffects on alleviation of APAP-induced hepatotoxicity in vivo\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate the improvement of APAP-induced hepatotoxicity, blood biochemistry\u0026nbsp;tests\u0026nbsp;were performed\u0026nbsp;to\u0026nbsp;measure\u0026nbsp;serum alanine aminotransferase (ALT), aspartate aminotransferase\u0026nbsp;(AST),\u0026nbsp;and\u0026nbsp;GSH levels.\u0026nbsp;After\u0026nbsp;intraperitoneal\u0026nbsp;injection of 500\u0026nbsp;mg/mL\u0026nbsp;of\u0026nbsp;APAP, BDEs, NAC, lipo-SM, and BDEs@lipo-SM\u0026nbsp;were\u0026nbsp;injected\u0026nbsp;intravenously\u0026nbsp;and\u0026nbsp;serum and liver markers were evaluated for each mouse after 24 h of injection. \u003cstrong\u003e(Figure 4d)\u0026nbsp;\u003c/strong\u003eAs\u0026nbsp;shown in\u003cstrong\u003e\u0026nbsp;Figure 4e,\u0026nbsp;\u003c/strong\u003eafter\u0026nbsp;BDE\u0026nbsp;treatment\u0026nbsp;@lipo-SM,\u0026nbsp;ALT, and AST levels decreased\u0026nbsp;significantly\u0026nbsp;compared to the APAP-treated group. Treatment with NAC, a glutathione precursor, an antidote for APAP overdose, produced significantly decreased serum conditions of ALT and\u0026nbsp;AST\u0026nbsp;24 h after\u0026nbsp;treatment.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn the course of APAP hepatotoxicity, GSH depletion plays a pivotal role in inducing oxidative stress.\u003csup\u003e23-25\u003c/sup\u003e Thus, it is important to restore GSH levels to overcome long-term damage to liver tissue. GSH levels showed a similar\u0026nbsp;trend\u0026nbsp;as that observed for ALT and AST levels. GSH levels were significantly increased in the treatment of BDEs@lipo-SM compared to the APAP-treated group. Overall, these results suggest that BDEs@lipo-SM allowed to\u0026nbsp;restore\u0026nbsp;GSH levels, which can counteract oxidative stress conditions; thus,\u0026nbsp;attenuating\u0026nbsp;liver\u0026nbsp;injury\u0026nbsp;and inducing liver regeneration.\u0026nbsp;\u003csup\u003e26-28\u003c/sup\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eHistopathological findings of APAP-induced hepatotoxicity\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConsistently, based on the results\u0026nbsp;of\u0026nbsp;the\u0026nbsp;blood chemistry evaluations, we further performed the histopathological evaluation of liver tissues from BDEs@lipo-SM-treated mice after 24\u0026thinsp;h and compared tissues of control mice without exposure to\u0026nbsp;APAP,\u0026nbsp;and exposed to APAP, BDEs, NAC, and\u0026nbsp;lipo-SM\u0026nbsp;to verify the degree of severity of APAP-induced hepatotoxicity. As shown in \u003cstrong\u003eFigure 4f\u003c/strong\u003e, after a 24\u0026thinsp;h\u0026nbsp;treatment,\u0026nbsp;liver tissue in\u0026nbsp;BDEs@lipo-SM\u0026nbsp;treated\u0026nbsp;mice showed comparable intact liver structure with round liver cells and less inflammatory infiltration or cell necrosis surrounding the central veins of the liver, whereas hepatocyte disruption was observed in areas of severe sinusoidal congestion accompanied by extensive centrilobular necrosis, loss of hepatocyte nuclei, and hemorrhaging in the\u0026nbsp;APAP\u0026nbsp;treated group and in the other experimental groups. The results of these pathological changes indicated that treatment with BDEs@lipo-SM can alleviate inflammation and protect the liver, as revealed by decreases in hemorrhage and inflammation infiltration and recovery in hepatocytes.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMechanism of antihepatotoxicity of\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eBDEs@lipo-SM\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003ein the\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e\u0026nbsp;MAPK/ERK\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e\u0026nbsp;1/2 pathway\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further evaluate the mechanism of antihepatotoxic activity of\u0026nbsp;BDEs@lipo-SM\u0026nbsp;on\u0026nbsp;APAP-induced hepatotoxicity, the protein expression of components of the\u0026nbsp;mitogen-activated protein kinase (MAPK)/ERK\u0026nbsp;1/2 pathway was examined by western\u0026nbsp;blotting. In a previous report, it was shown\u0026nbsp;that\u0026nbsp;the\u0026nbsp;MAPK\u0026nbsp;signaling pathway is involved in APAP-induced hepatotoxicity, but the regulatory mechanism of BDEs@lipo-SM on APAP-induced hepatotoxicity remains unknown, thus we\u0026nbsp;analyzed\u0026nbsp;protein expression of ERK1/2, JNK, and p38, which belong to the\u0026nbsp;MAPK\u0026nbsp;family. As shown in \u003cstrong\u003eFigure 5a\u003c/strong\u003e, high levels of phosphorylation of\u0026nbsp;ERK1/2, JNK,\u0026nbsp;and p38 were observed in Huh7 cells. However,\u0026nbsp;the\u0026nbsp;level of\u0026nbsp;protein\u0026nbsp;expression\u0026nbsp;of\u0026nbsp;ERK1/2, JNK,\u0026nbsp;and p38 was markedly suppressed following treatment with BDEs@lipo-SM. The relative band densities of ERK1/2, JNK, and p38 were also analyzed. \u003cstrong\u003e(Figure 5b)\u003c/strong\u003e It implies that the hybrid BDE strategy can be used to improve antihepatotoxicitic effects through the inhibition of the MAPK pathway and be a good candidate to prevent APAP-induced hepatotoxicity.\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn recent years, PENs-based nanotechnologies have gained much attention because they exhibit a variety of functions ranging from\u0026nbsp;cell-to-cell\u0026nbsp;interaction and therapeutic applications. PENs are considered natural nanoparticles, with cargos of phospholipids, proteins, and genetic\u0026nbsp;material\u0026nbsp;that\u0026nbsp;are not harmful. Unlike synthetic nanoparticles, PENs are safe for use as a topical and systemic treatment and may be suitable for certain therapeutic applications. PENs can be used as alternative drug delivery agents in a variety of medicinal applications because of their high stability, inherent\u0026nbsp;biocompatibility,\u0026nbsp;and specificity to target cells. Previous studies investigating PENs have reported the therapeutic\u0026nbsp;potential\u0026nbsp;of PENs\u0026nbsp;and their cell signaling mechanism\u0026nbsp;in\u0026nbsp;various diseases.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBalloon flower root contains enriched bioactive\u0026nbsp;compounds\u0026nbsp;and isolated BDEs from their preparation include the various platycosides, specifically PD. A recent study showed that BDEs have great wound healing properties with anti-inflammatory and antioxidant effects in chronic skin wounds.\u003csup\u003e29\u003c/sup\u003e The enriched phytochemicals and bioactive molecules of BDEs can\u0026nbsp;induce\u0026nbsp;antioxidant activity against oxidative stress and can reduce the expression of pro-inflammatory cytokines. As shown in \u003cstrong\u003eFigure\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e1e,\u003c/strong\u003e HPLC analysis indicates that BDEs contain different molecules, including PD. Therefore, BDEs can be expected to contribute to counteract inflammatory disease and exert anti-apoptotic, and cell\u0026nbsp;survival\u0026nbsp;signaling. Consequently, to\u0026nbsp;achieve\u0026nbsp;a\u0026nbsp;higher thermal stability benefit and greater structural\u0026nbsp;integrity,\u0026nbsp;we fabricated hybrid BDEs. Firstly, SPC-based liposomes loaded with SM were prepared and incorporated in BDEs. Next, BDEs@lipo-SM were produced by extrusion. To validate the stability of the fabricated\u0026nbsp;BDEs@lipo-SM,\u0026nbsp;size and zeta potential analyses were performed, with values of 108.09\u0026plusmn;14.26 nm and -10.16\u0026plusmn;0.58 mV, respectively, indicating that the engineered BDEs possess great stability in aqueous suspension. To evaluate the entrapment of SM in BDEs, the encapsulation efficiency was assessed to be approximately 39.82%, which is quite low because of the hydrophobicity of the chemical drug and non-ionizable chemical structure.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBDEs@lipo-SM exhibit\u0026nbsp;an advantage\u0026nbsp;over\u0026nbsp;original BDEs as they are rapidly captured\u0026nbsp;by\u0026nbsp;cells within 1 h and induce an increase in inhibitory activity against APAP-induced hepatotoxicity due to the synergistic effect of BDEs with SM. BDEs@lipo-SM distinctly\u0026nbsp;evoke\u0026nbsp;an\u0026nbsp;anti-inflammatory immune response in immune cells due to their intrinsic bioactive components in BDEs, while\u0026nbsp;reducing\u0026nbsp;pro-inflammatory signals, albeit with no toxicity. Synergistically,\u0026nbsp;BDEs@lipo-SM\u0026nbsp;significantly\u0026nbsp;increased\u0026nbsp;the\u0026nbsp;levels\u0026nbsp;of\u0026nbsp;GSH following liver injury, indicating that SM is successfully delivered in the liver\u0026nbsp;through\u0026nbsp;hybrid BDEs as a drug carrier and enhances cysteine availability, leading to an increase in GSH levels.\u003csup\u003e30\u003c/sup\u003e Synergistic\u0026nbsp;BDEs@lipo-SM\u0026nbsp;activity can more effectively inhibit the MAPK pathway and lead to liver cell recovery.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAs described above, BDEs are obtained in high yield with high-grade purity and are expected to become reliable therapeutic carriers for the treatment of drug-induced hepatotoxicity. In the fabrication of the hybrid BDEs@lipo-SM, the BDEs remain structurally stable and retain their biological function, which means that BDEs@lipo-SM can act as a biocompatible systematic delivery system. To exploit the opportunities and potential applications for using BDEs as a drug delivery vehicle, more comprehensive applications based on the peculiar properties of BDEs and hybrid BDEs are necessary. Additionally, to expand the potential applications of BDEs, basic studies of hybrid PENs need to include a comprehensive discussion of the potential of current technology to develop efficient cargo loading, gene loading, and membrane engineering techniques to enhance specific delivery and avoid the systemic adverse effects induced by hybrid PENs over prolonged treatment.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, the incorporation of\u0026nbsp;BDEs\u0026nbsp;and\u0026nbsp;liposome-loaded\u0026nbsp;SM was significantly enhanced \u003cem\u003ein vitro\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;in vivo,\u0026nbsp;\u003c/em\u003eand\u0026nbsp;BDEs\u0026nbsp;demonstrated therapeutic potential for hepatoprotection and as an\u0026nbsp;anti-inflammatory treatment, supported by evidence of\u0026nbsp;reduced liver injury on histopathological examination compared to treatment with either non-modified BDEs and NAC alone.\u0026nbsp;By inhibiting the\u0026nbsp;MAPK pathway, BDEs@lipo-SM effectively inhibited APAP-induced hepatotoxicity by reducing the protein expression of\u0026nbsp;ERK1/2, JNK,\u0026nbsp;and p38. The results indicate that BDEs@lipo-SM could be successfully delivered in the liver and induce the required therapeutic\u0026nbsp;effect,\u0026nbsp;such as anti-inflammatory and promotion of\u0026nbsp;immunity,\u0026nbsp;due to the stability of BDEs@lipo-SM\u003cem\u003e\u0026nbsp;in vivo\u003c/em\u003e, as a carrier of intrinsic bioactive molecules and chemical agents. These findings provide the basis for further development and the fabrication of hybrid BDEs (or PENs) and applications as drug delivery agents to inhibit APAP-induced hepatotoxicity and liver injury. However, thus far, we have a limited understanding of the potential of PENS, and there are still several unanswered questions that need to be addressed, such as processes for highly optimized fabrication of hybrid BDEs and their systemic effects as vehicles of therapeutic agents in clinical trials for various diseases. Further research is required to improve the applicability of the hybrid BDEs as a drug delivery system.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere authors declare there are no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the 72nd round of the China Postdoctoral Science Foundation (No. KLF301807) and the National Natural Science Foundation of China (No. 82074277 and 81773911).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eChang CY, Schiano TD. Review article: drug hepatotoxicity. \u003cem\u003eAliment Pharmacol Ther\u003c/em\u003e. 2007;25(10):1135-1151. 10.1111/j.1365-2036.2007.03307.x\u003c/li\u003e\n\u003cli\u003eRusso MW, Galanko JA, Shrestha R, Fried MW, Watkins P. 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Silymarin protects against acute ethanol-induced hepatotoxicity in mice. \u003cem\u003eAlcohol Clin Exp Res\u003c/em\u003e. 2006;30(3):407-413. 10.1111/j.1530-0277.2006.00063.x\u003c/li\u003e\n\u003c/ol\u003e\n"},{"header":"Scheme 1","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\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":"[email protected]","identity":"cell-communication-and-signaling","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ccas","sideBox":"Learn more about [Cell Communication and Signaling](http://biosignaling.biomedcentral.com/)","snPcode":"12964","submissionUrl":"https://submission.nature.com/new-submission/12964/3","title":"Cell Communication and Signaling","twitterHandle":"@bmc","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"plant-derived exosome-like nanoparticles; balloon flower root-derived exosome-like nanoparticles; hybrid exosomes; N-acetyl-ρ-aminophen-induced liver injury; drug delivery systems","lastPublishedDoi":"10.21203/rs.3.rs-4035317/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4035317/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eIntroduction: \u003c/strong\u003eBalloon flower root-derived exosome-like nanoparticles (BDEs) have recently been proposed as physiologically active molecules with no cytotoxicity. However, the therapeutic effects of drug-induced hepatotoxicity of BDEs have not been elucidated. BDEs contain a large amount of platycodin D, which is widely known to be effective in regulating inflammation and ameliorating systemic toxicity. Thus, the main therapeutic activity of BDEs is attributed to inhibiting the inflammatory response and alleviating toxicity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eObjective: \u003c/strong\u003eConsidering the potential therapeutic effects of BDEs, and the potential to achieve synergistic effects to improve therapeutic outcomes, we constructed hybrid BDEs with a soy lecithin-based liposome loaded with silymarin (SM). Since liposomes can provide higher thermal stability and have greater structural integrity, these might be more resistant to clearance and enzymatic degradation of drug molecules.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eHybrid BDEs with liposome-loaded SM (BDEs@lipo-SM) were fabricated by thin-film hydration and extrusion. BDEs@lipo-SM were characterized using dynamic light scattering and high-performance liquid chromatography. After confirmation of the physical properties of BDEs@lipo-SM, various therapeutic properties were evaluated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eBDEs@lipo-SM were internalized by hepatocytes and immune cells and significantly decreased mRNA expression of apoptosis and inflammation-relevant cytokines by inhibiting the hepatocyte MAPK pathway. BDEs@lipo-SM significantly induced an increase in glutathione levels and inhibited APAP-induced hepatotoxicity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eBDEs@lipo-SM have therapeutic benefits for acute liver injury and can alleviate cell death or toxicity. BDEs@lipo-SM can accelerate liver recovery in the APAP-induced acute liver injury model, which highlights thathybrid BDEs represent an attractive delivery vehicle for therapeutic applications.\u003c/p\u003e","manuscriptTitle":"A novel approach to alleviate acetaminophen-induced hepatotoxicity with hybrid balloon flower root-derived exosome-like nanoparticles (BDEs) with silymarin via inhibition of hepatocyte MAPK pathway and apoptosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-20 14:29:32","doi":"10.21203/rs.3.rs-4035317/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-04-24T12:17:22+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-04-23T06:47:05+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-04-23T03:37:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"6951c12c-284e-44fe-8d44-65814d5bad2b","date":"2024-04-15T00:28:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"ebd1a214-8d07-487c-82f0-ab03b8fbf641","date":"2024-04-14T16:36:26+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-04-02T09:01:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"0f577850-2501-47ab-a396-deafc616ef33","date":"2024-03-24T05:16:51+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-03-23T17:32:48+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-03-18T01:27:51+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-03-18T01:27:51+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell Communication and Signaling","date":"2024-03-08T02:56:29+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-communication-and-signaling","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ccas","sideBox":"Learn more about [Cell Communication and Signaling](http://biosignaling.biomedcentral.com/)","snPcode":"12964","submissionUrl":"https://submission.nature.com/new-submission/12964/3","title":"Cell Communication and Signaling","twitterHandle":"@bmc","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a1e42274-741a-4e42-a3fd-3846c08e6658","owner":[],"postedDate":"March 20th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-06-21T15:11:25+00:00","versionOfRecord":{"articleIdentity":"rs-4035317","link":"https://doi.org/10.1186/s12964-024-01700-z","journal":{"identity":"cell-communication-and-signaling","isVorOnly":false,"title":"Cell Communication and Signaling"},"publishedOn":"2024-06-18 15:11:24","publishedOnDateReadable":"June 18th, 2024"},"versionCreatedAt":"2024-03-20 14:29:32","video":"","vorDoi":"10.1186/s12964-024-01700-z","vorDoiUrl":"https://doi.org/10.1186/s12964-024-01700-z","workflowStages":[]},"version":"v1","identity":"rs-4035317","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4035317","identity":"rs-4035317","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}