Berberine-loaded lipid nanoparticle inhibits H2O2-induced chondrocyte injury by promoting mitochondria autophagy in C28/I2 cells 

Berberine-loaded lipid nanoparticle inhibits H2O2-induced chondrocyte injury by promoting mitochondria autophagy in C28/I2 cells | 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 Berberine-loaded lipid nanoparticle inhibits H2O2-induced chondrocyte injury by promoting mitochondria autophagy in C28/I2 cells Guoliang Yi, Peng Xie, Qingqi Meng, Siming Li This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2688076/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Although berberine (BBR) is considered to be of therapeutic value in treating various disorders; however, its low bioavailability hinders its further development on clinical application. To evaluate the effects of BBR against chondrocyte damage, we synthesized liposomal nanoparticles loaded with BBR (BBR@LNPs) with an average particle size of 137 nm using microfluidic technology. The CCK8 assay was used to determine the cytotoxicity of the BBR@LNPs in C28/I2. Additionally, flow cytometry and western blotting were used to investigate the mechanism of its protect chondrocytes from H2O2-induced oxidative damage in C28/I2. Our findings showed that BBR@LNPs had stable nanoscale structure and low cytotoxicity. Importantly, administration of BBR@LNPs significantly inhibited ROS release and cell apoptosis in H 2 O 2 -induced C28/I2, BBR@LNPs enhanced mitochondrial autophagy by up-regulating PINK1 and Parkin expression. Our findings may provide some guidance BBR-loaded lipid nanoparticle delivery system in cartilage injury. berberine lipid nanoparticle mitochondrial autophagy apoptosis chondrocyte damage Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Cartilage injury has a high prevalence and disability rate worldwide [ 1 ] . Chronic degenerative joint diseases such as osteoarthritis and rheumatoid arthritis [ 2 ] , can be caused by ageing, obesity, strain, trauma, congenital anomalies of the joints, and joint deformities, resulting in cartilage degeneration in the large joints and later severe limitation of joint movement and inconvenience [ 3 ] . Acute and chronic pathological aspects of cartilage are associated with a succession of damaging pathological events such as oxidative stress, apoptosis, necrosis of chondrocytes, and inflammatory response [ 4 , 5 ] . Cartilage injury not only causes serious physical and psychological damage, but also imposes a significant financial burden on the family and society. Although the available clinical treatment options can improve the prognosis of some patients. The complex pathology of the injury has limited the development of relevant clinical interventions. It is consequently necessary and critical to focused on developing targeted and effective interventions or targeted drugs to systematically treat or repair damaged cartilages [ 6 ] . Mitochondrial autophagy, also known as mitophagy, is a highly conserved and selective autophagic process that has been shown to be the major physiological mechanism governing the quality and quantity of mitochondria [ 7 ] . Mitochondria are the site of intracellular ATP production and play an important role in maintaining cellular homeostasis, physiological functions, and cell survival [ 8 ] . Most illnesses are caused by dysfunctional mitochondrial autophagy such as AD, PD, cerebral ischemia-reperfusion injury, spinal cord injury, and cartilage injury [ 9 – 11 ] . Oscar Alvarez-Garcia discovered that REDD1, a gene that governs development and DNA damage response, was faulty in injured chondrocytes, preventing chondrocyte autophagy and mitochondrial biogenesis and thereby exacerbating cartilage destruction [ 12 ] . This may suggest that mitochondrial function is defective in the course of cartilage injury and that the lack of timely and effective clearance of functionally defective mitochondria may have an essential role in the inhibition of cartilage injury or in the functional recovery of damaged cartilages. The utilization of mitochondrial autophagy as a therapeutic target could be beneficial in the treatment cartilage injury. Berberine (BBR), also known as hydrochloride, is a class of flavonoid small molecule compounds [ 13 ] derived from the genus Berberis, which has received much attention in recent years due to its potent antioxidant and anti-inflammatory properties [ 14 ] . BBR suppresses oxidative stress through the ER and TrkA pathways in a model of hydrogen peroxide-induced oxidative stress injury in PC12 cells, therefore inhibiting oxidative damage in neuronal cells [ 15 ] . The above studies suggest that BBR may have an inhibitory role in oxidative stress and impaired autophagy induced cell damage, such as cartilage injury, spinal cord injury, and ischemic stroke. However, the poor cellular uptake of BBR hinders its clinical use. Therefore, the development of delivery technologies to improve BBR bioavailability might significantly contribute to its increased therapeutic value. Lipid nanoparticles (LNPs) have attracted tremendous attention in the biomedical field due to their simple preparation process, high drug-carrying capacity, and excellent bio-solubility. The use of LNPs to carry mRNA, siRNA, and vaccines for the treatment of diseases has attracted increasing attention from scientists [ 16 , 17 ] . BBR combined with liposomal nanocarriers offer the potential for enhanced intracellular drug delivery. Numerous studies have revealed that BBR exhibits an anti-inflammatory effect. However, its effectiveness in cartilage injury is unknown, and no studies on cartilage injury have been reported. Therefore, the synthesis of lipid nanoparticle systems loaded with berberine BBR and the exploration of their use for the treatment or intervention of cartilage injury is of substantial interest. Using liposomal BBR-encapsulated nanoparticles (BBR@LNPs) prepared by microfluidic technology, we propose to investigate whether liposomal nanoparticles loaded with BBR can inhibit cartilage damage or promote the repair of sue-injured cartilage in a rat model of cartilage knee joint cartilage injury. We further investigated whether they inhibit cartilage damage and promote cartilage repair by inhibiting oxidative stress and promoting mitochondrial autophagy, and investigated putative molecular mechanisms or regulatory signaling pathways. It is expected to enrich the pathological mechanisms of cartilage damage, and identify drug interventions and delivery systems with clinical therapeutic implications. Materials And Methods 2.1. Materials 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and cholesterol were purchased from Xi’an Ruixi Biological Technology Co., Ltd., China. Isopropyl alcohol (IPA) was obtained from Shanghai Macklin Biochemical Co., Ltd, China. Phosphate buffered saline (PBS) (10 mM, pH 7.4) was obtained from Codow Chemical Co., Ltd, China. 2.2. Synthesis And Preparation Of Bbr@lnps The synthesis method refered to the published paper from our partner [ 18 ] . A modified method briefly introduced as follow. DPPC (30 mM) and cholesterol with a molar ratio of 3:1 were dissolved in isopropanol and sonicated to aid dissolution; berberine hydrochloride was sonicated to aid in dissolution and dissolved in PBS at a concentration of 5 mM, and the solution was injected into a flow-focusing microfluidic chip at an aqueous phase: organic phase volumetric flow rate ratio of 4 and a total flow rate of 30 ml/h. The microfluidic chip was fabricated by a 3D printer (microArch™ S140; Boston Micro Fabrication, China), and the microchannels have a rectangular cross section with 200 µm depth and 400 µm width. The prepared liposomes were dialyzed using a 7 KD dialysis bag for 48 h, during which the dialysis medium was changed with fresh PBS three times; the final purified products were LNPs loaded with berberine hydrochloride (BBR@LNPs). 2.3. Characteristic Of Bbr@lnps The BBR@LNPs were characterized with size, polydispersity index (PDI), morphology, zeta potential, and efficiency (EE%). The morphology was studied using a transmission electron microscope (TEM). The particle size and PDI of BBR@LNPs were measured by dynamic light scattering (DLS) using a Zeta sizer Nano ZS90, whereas the zeta potential was measured using the Zeta sizer Nano ZS90 at 25 ℃ and a scattering angle of 173 o by measuring the electrophoretic mobility with laser Doppler velocimetry. For characterization study, various parameters of optimal formulation were then measured three times. For the determination of the EE%, the drug content was analyzed by reversed phase HPLC. The un-encapsulated BBR was separated from the LNPs by an ultrafiltration method at the bottom of the Amicon UltraVR tube and subjected to HPLC analysis to determine the content of BBR. The total drug content in BBR@LNPs was determined after lysis of lipid core with methanol. EE% could be calculated by the following equation: Encapsulation efficiency(EE%)= (Wt-Wu)/Wt ×100% Where Wu is the drug untrapped in the LNPs and Wt is the total drug in the BBR@LNPs. 2.4. In Vitro Release Study Of Bbr@lnps A modified dialysis method was used to evaluate the in vitro release of BBR loaded in LNPs. Briefly, 1 ml of BBR@LNPs with a concentration of 10 mg/ml was placed in a regenerated cellulose dialysis bag against 200 ml of 0.5% Triton X-100 aqueous solution, which was maintained at 37 ℃ with continuous magnetic stirring at 100 rpm. At predetermined time points (0, 10, 20, 30, 40, and 50 h), 1 ml of solution in the receiving phase was taken for HPLC analysis of the released BBR. Each 1 ml sample was collected for various time points and immediately replaced with the same volume of fresh media to maintain the total volume at 200 ml. The free BBR was solubilized by the presence of 50% of PEG in solution. All experiments were carried out in sextuplicate. 2.5. Cytotoxicity Analysis C28/I2 cells are owned by our laboratory [ 19 ] . C28/I2 was cultured in DMEM/F12 medium containing 10% fetal bovine serum. After the cell density reached about 80%. The cells were digested by trypsin, prepared into a cell suspension, inoculated into 96-well plates at 1 × 105 cells/well and incubated for 24 h. After 24 h, the cells were incubated with medium containing different concentrations of BBR@LNPs medium for 24 h. The old medium was aspirated and discarded, and CCK8 working solution was prepared at a ratio of 10:1 medium: CCK8, added at 100 ml per well, and incubated at 37°C in a CO 2 incubator for 1.5 h. The OD value was detected at 450 nm using an Universal microplate reader. 2.6. Flow Cytometry Analysis After C28/I2 cells were inoculated in 6-well culture at appropriate density and cells were cultured overnight, cells were divided into control, H 2 O 2 , and H 2 O 2 + LNPs@BBR groups. BBR@LNPs was added to C28/I2 cell medium 2 h before the addition of H 2 O 2 , and the culture was continued for 22 h after subsequent addition of the indicated concentrations of H 2 O 2 . The cells were washed twice with pre-chilled 1× PBS after aspiration of the old medium, digested with EDTA-free trypsin and the cell precipitate was collected. The cell precipitate was centrifuged twice in PBS and incubated for 15 min at room temperature with FITC and PI binding buffer at working concentrations before flow-on assay. FlowJo-v10.8.1 software was used to analysis the data. 2.7. Western Blot Analysis The treated groups of cells were collected and lysed on ice for 30 min by adding appropriate amount of lysis solution containing PMSF, and then centrifuged at 12000 r/min for 15 min.The protein supernatant was collected, followed by quantitative analysis of protein concentration of each sample by BCA protein, adjustment of protein concentration, dilution of protein supernatant with 5×SDS-PAGE loading buffer at a ratio of protein supernatant: 5×SDS-PAGE loading buffer of 4:1. After denaturing for 5 min at 95°C, the samples were spotted on SDS-PAGE gel at 20 µg/well, the proteins were separated by electrophoresis and transferred to PVDF membrane, 5% skimmed milk powder was incubated at room temperature for 1.5 h. The primary antibody was incubated overnight at 4°C in the refrigerator, 1×TBST washed the membrane three times for 10 min each time, and the corresponding secondary antibody coupled with HRP was incubated for 1.5 h at room temperature. The bands were saved after ECL luminescence development, and the protein bands were analyzed by ImageJ software using β-actin as the sample content control. 2.8. Data Analysis Statistics analysis was carried out by the unpaired student’s t-test using GraphPad Prism (8.01), with a statistical significance level of p < 0.01. All data were expressed as the means ± SEM. Resullts 3.1 Physical characterization As shown in Fig. 1 A, the effectively synthesized BBR@LNPs solution was a faint yellow liquid. According to the transmission electron microscopy data shown in Fig. 1 C, the produced BBR@LNPs were uniformly distributed in shape and size and had a regular morphology. In addition, the particle size distribution results as shown in Fig. 1 D showed that the average particle size value of BBR@LNPs was 137 nm; the PDI value was 0.18 and the zeta potential value was − 22.4 mV; furthermore, the drug loading of the nanoliposomes to be 7.7% and the encapsulation rate of the BBR@LNPs drug delivery system with BBR-loaded nanoliposomes synthesized by our microfluidic technology is around 80%. 3.2. In-vitro Release The results of the in vitro release experiments of BBR@LNPs were shown in Fig. 1 E. The release rate showed a sharp increase within 10 h of release, followed by a gentle increase, and after 30 h, the release reaches a maximum peak of about 70%, and the release rate no longer increases, reaching a plateau in release. This suggests that a duration of in vitro administration of up to 30 h is appropriate for our subsequent experiments. In addition, this will also serve as a reference for in vivo administration of release in subsequent animal experiments. 3.3. Cytotoxicity Analysis By using CCK-8 method and protein immunoblotting experiments to assess the effects of H 2 O 2 and BBR@LNPs on the activity of chondrocytes C28/I2 and HEK293, we found that with different concentrations of H 2 O 2 stimulation of C28/12 cells, cell activity decreased significantly with increased H 2 O 2 concentration, as shown in Fig. 2 A. Interestingly, along with BBR@ LNPs treatment, the viability of C28/I2 and HEK293 cells did not show a sharp decrease in activity at higher concentrations of BBR@LNPs stimulation, as shown in Fig. 2 B. Analysis determined the IC50 value of BBR@LNPs in C28/I2 cells to be around 500 µg/ml. Furthermore, as shown in Figs. 2 C,E, the results of protein immunoblotting showed that 50, 100 and 200 µM H 2 O 2 significantly decreased the expression of the antioxidant enzyme GPX4 and significantly increased the expression of cleaved caspase-3, suggesting that H 2 O 2 significantly induced oxidative damage in C28/I2 cells; Accordingly, in subsequent experiments, we chose 100 µM H 2 O 2 to establish the concentration of oxidative damage in C28/I2 cells. 3.4. Inhibition Of Ho-induced Apoptosis By Bbr@lnps To analyze whether BBR@LNPs had an inhibitory effect on H 2 O 2 -stimulated C28/I2 chondrocyte injury, we used flow cytometry to detect and analyze the apoptosis levels and the antioxidant proteins GPX4 and Cleaved cas-3 in the control H2O2 (100 µM) and H2O2 + BBR@LNPs (1×103 µg/ml) groups expression changes. Interestingly, when the cells were pretreated with 1 µg/ml of BBR@LNPs for 2 h and then exposed to H2O2 for 24 h, the H 2 O 2 -induced increase in apoptosis was significantly reduced. In addition, we also analyzed the changes in the expression of antioxidant molecule GPX4 and apoptotic molecule Cleaved cas-3 under the same treatment conditions, and the results were shown in Fig. 3 B and 3 C: BBR@LNPs significantly reduced the H 2 O 2 -induced increase in the expression of Cleaved cas-3 and increased the expression of antioxidant molecule GPX4. It suggests that BBR@LNPs can inhibit H 2 O 2 -induced apoptosis and increase the antioxidant properties of C28/I2 cells. 3.5. Bbr@lnps Reduce H2o2-induced Ros Release Increased ROS release is an important driver of oxidative damage in chondrocytes; therefore, we employed fluorescence microscopy and flow cytometry to investigate the quantities of ROS released from H 2 O 2 -stimulated C28/I2 cells alone and C28/I2 cells co-cultured with BBR@LNPs and H 2 O 2 . The results are shown in Fig. 4 A and 4 B. The BBR@LNPs were able to significantly reduce the H 2 O 2 -stimulated ROS release, and the same results were obtained for both flow cytometry and fluorescence microscopy assays. These findings suggest that our synthesized BBR@LNPs can inhibit the increase in H2O2-stimulated ROS release, which may be an important mechanism for the BBR@LNPs to play a role in inhibiting oxidative damage in chondrocytes. 3.6. Bbr@lnps Enhance Mitophagy Level It has been reported that berberine activates the mitochondrial autophagy level of cells and functions as an inhibitor of oxidative damage. Therefore, we examined the expression of mitochondrial autophagy marker molecules PINK1 and Parkin as well as apoptosis analysis Caspase-3 and antioxidant molecule GPX4 by protein immunoblotting assay in C28/I2 cells co-cultured with BBR@LNPs and autophagy inhibitor 3-MA with H 2 O 2 stimulation; the results was shown in Fig. 5 , BBR@LNP increased the expression of PINK1, Parkin, and GPX4; however, treatment with the autophagy inhibitor 3-MA intensified the expression of the apoptotic molecule Caspase-3 and decreased the expression of PINK1 and Parkin. The above results may suggest that the inhibition of H 2 O 2 -induced increase in apoptosis by BBR@LNPs may be achieved through the activation of mitochondrial autophagy; suggesting that BBR@LNPs may be an agonist of mitochondrial autophagy and play a function in activating mitochondrial autophagy. Discussion We used a microfluidic method to effectively manufacture lipid nanoparticles loaded with choline hydrochloride with particle sizes of around 137 nm and assessed their zeta potential values of -10.82 mV and PDI values of 0.18. In addition, we also assessed their in vitro drug release rate after 48 h. With longer release times, the release rate of BBR@LNPs peaked at around 30 h. With increasing release duration, the release rate of BBR@LNPs peaked at 70% at around 30 h. after which it did not continue to rise. The IC50 values were interestingly found to be within a high concentration range, indicating that the toxic effects were low. The mechanism by which BBR@LNPs inhibit H 2 O 2 -induced cartilage damage is achieved by enhancing mitochondrial autophagy. The primary purpose of mitochondrial autophagy, which is substantially conserved in eukaryotes, and its main function is to clear damaged or dysfunctional mitochondria and tightly control mitochondrial mass [ 7 ] . Mitochondrial autophagy has an important role in regulation of apoptosis, inflammatory response, and cell differentiation [ 20 , 21 ] . Sun et al. reported that mitochondrial autophagy has a regulatory role in the development of degenerative joint diseases [ 22 ] . Here, we found that exposure of human chondrocytes C28/I2 to the strong oxidant H 2 O 2 resulted in increased ROS release, leading to decreased mitochondrial autophagy and the production of ROS disrupts the dynamic homeostasis of mitochondria and activates mitochondrial autophagy to remove damaged mitochondria [ 23 ] . However, dysfunctional mitochondrial autophagy in turn leads to the emergence of processes, for example, apoptosis, which exacerbates oxidative cellular damage. It is suggested that pharmacological interventions or target-based therapies can increase or repair mitochondrial autophagy and thereby maintain cellular homeostasis and promote recovery of cellular function. In this study, we discovered that BBR-loaded liposomal nanoparticles dramatically increased mitochondrial autophagy and prevented H 2 O 2 -induced oxidative damage in chondrocytes. In our previous study, BBR was found to inhibit the process of excessive oxidative stress activated by oxygen glucose deprivation-reoxygenation [ 24 ] . Interestingly, BBR reduced the generation of ROS by inhibiting the activation of inflammatory vesicles and promoting higher amounts of mitochondrial autophagy in influenza virus-infected cells [ 25 ] . Our results are partially corroborated by our study. However, less research has been reported on chondrocyte damage by BBR. BBR is derived from the flavonoids of Rhizoma coptidis , which has antioxidant, anti-apoptotic, anticancer and neuroprotective effects [ 26 ] . However, its poor water solubility and its tendency to precipitate crystals in solution hinder its potential clinical application [ 27 ] . The establishment of an effective drug delivery system is of great significance for the drug utilization of chalcone hydrochloride. Numerous clinical therapies have demonstrated the effectiveness of nanocarrier-based medicines [ 28 , 29 ] . The first liposome-based nanomedicine, Doxorubicin, was developed in 1995 and has been used in tumor therapy [ 30 ] . Until today, scientists have developed around 50 nanoparticle-based drugs. The new use of old drugs has been attracting a lot of attention, because it saves costs in drug development, reduces the drug development cycle, and has a clear pharmacological mechanism of action. Precious metal-based drug delivery technologies, such as colloidal gold, are pricy, difficult to create, and unsuited for large-scale drug manufacturing [ 31 ] . Nanoliposomes were the first carriers developed for nucleic acid transfection technology for basic science research and have gained attention in recent years for their value in drug delivery, with the novel nucleic acid vaccine for coronavirus being the best classical use of nanoliposomes for drug delivery [ 32 – 34 ] . Using cholesterol and low-cost DPPC as liposomal cofactors, we created liposomal nanoparticles loaded with small cleavage hydrochloride, we then examined their characteristics using a variety of techniques, including transmission electron microscopy and zeta sizer, and assayed their drug release rates in vitro, and further applied them in a model of H 2 O 2 -induced oxidative damage in C28/12 cells The pharmacological effects of the nano-liposomes loaded with chondroitin hydrochloride were demonstrated by flow cytometry, CCK-8, immunofluorescence analysis, and protein immunoblotting. The mechanism of its pharmacology may be achieved by inhibiting the release of ROS and increasing the physiological function of mitochondrial autophagy. In conclusion, our study has successfully produced nanoliposomes loaded with chondroitin hydrochloride using microfluidic technology, and explored the mechanism of their pharmacological effects by reducing the release of ROS and increasing the level of mitochondrial autophagy in a model of oxidative damage in chondrocytes. The potential mechanism of action of BBR@LNPs may be as Graphical Abstract showed. Collectively, our results may be useful in the development of chondroitin hydrochloride drugs for cartilage injury, as well as in providing some insight into the pathological process of cartilage injury. Declarations Data availability No new data were generated or analysed in support of this research. Author contributions Gl Y and P X conducted the experiments and the data analysis. Qq M and Sm L conceived the project, designed the study, arranged the results and revised the manuscript. All authors approved the final version of the manuscript. Sm L accepted full responsibility for the finished work, had access to the data and controlled the decision to publish. Funding This work was supported by the Clinical Medical Technology Innovation guidance Program of Human Province(2021SK51821) and the Natural Science Foundation of Guangdong Province (2019A1515011085). Acknowledgements We thank doctoral candidate Han shan at Department of Dermatology, Xiangya Hospital, Central South University for guidance of the synthesis method of BBR@LNPs and rewired the manuscript. Competing interests All authors declared that there are no interests between them and the research was conducted under absence of any commercial or financial relationships. References RIM Y A, NAM Y, JU J H. The Role of Chondrocyte Hypertrophy and Senescence in Osteoarthritis Initiation and Progression [J]. 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COVID-19 vaccines: The status and perspectives in delivery points of view [J]. Advanced drug delivery reviews, 2021, 170: 1-25. LAMB Y N. BNT162b2 mRNA COVID-19 Vaccine: First Approval [J]. Drugs, 2021, 81(4): 495-501. Additional Declarations No competing interests reported. Supplementary Files GA.png Graphical Abstract BBR@LNPs suppresses the ROS release, and inhibits chondrocytes apoptosis through enhancing the expression of Parkin and PINK1 mediated mitophagy. Cite Share Download PDF Status: Posted Version 1 posted 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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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-2688076","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":183400184,"identity":"0607f3bd-4d7d-4d33-a15b-26b4e1d2a886","order_by":0,"name":"Guoliang Yi","email":"","orcid":"","institution":"Guizhou medical university","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Guoliang","middleName":"","lastName":"Yi","suffix":""},{"id":183400185,"identity":"36aa2eee-8cf4-4e37-914b-c16ccd935857","order_by":1,"name":"Peng Xie","email":"","orcid":"","institution":"Guizhou medical university","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Peng","middleName":"","lastName":"Xie","suffix":""},{"id":183400186,"identity":"d2d696bb-66d9-4bf7-99db-1baea2c2abf9","order_by":2,"name":"Qingqi Meng","email":"","orcid":"","institution":"Jinan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Qingqi","middleName":"","lastName":"Meng","suffix":""},{"id":183400187,"identity":"08400d8f-f329-43e1-86ff-7afd6ab2d83f","order_by":3,"name":"Siming Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAu0lEQVRIiWNgGAWjYDCCA0D8wMAmAcxJKCBWS4JBWgIDG5hBtBaGwxAtDMRo4bt9/OmGhILzefzy3YkfHhgwyPOLHcCvRfJcjtmNBIPbxZJtvJslgA4znDk7Ab8WgzM8bCAtiRuO8W4AaQGyCWphfwbUcg6kZfMPIrUwgBx2AKRlG3G2SJ7hAWlJTpzZlrvNIsFAgrBf+EAO+/DHLrGf+ezmmz8qbOT5pQloQQcSpCkfBaNgFIyCUYAdAACbN0dhKlq6tgAAAABJRU5ErkJggg==","orcid":"","institution":"Guizhou medical university","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Siming","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2023-03-13 15:44:43","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2688076/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2688076/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":34443527,"identity":"965b28ea-2a1d-4c40-ab66-cc36e8555faf","added_by":"auto","created_at":"2023-03-17 21:48:02","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":782131,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterization of BBR@LNPs.\u003c/strong\u003e A. the chemical formula of BBR, B. the schematic diagram of BBR@LNPs. C. transmission electron microscopic image. D. size distribution. E. zeta potential. F. In vitro release profiles of BBR@LNPs suspensions in PBS at 37℃.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-2688076/v1/8a5105d48d040a2bb16f7c90.png"},{"id":34441389,"identity":"f389ff57-1b5c-47bc-9c88-cd569c080f02","added_by":"auto","created_at":"2023-03-17 21:32:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":202252,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCytotoxicity analysis of H2O2 and BBR@LNPs in C28/I2.\u003c/strong\u003e A. C28/I2 cells were exposed to the indicated concentrations of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 24 h, and B. C28/I2 and HEK293 cells were treated with different concentrations of BBR@LNPs for 24 h. The cell viability was determined by CCK-8 method. C. C28/I2 cells were exposed to 50, 100, and 200 μM of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 24 h. The protein level of GPX4 and Cleaved cas-3 were detected by Western blot. D. and E. The quantitative analysis of GPX4 and Cleaved cas-3 expression. (**\u003cem\u003eP\u003c/em\u003e＜0.01 vs control group).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-2688076/v1/4ad6f8cc01ab6d391c816f8c.png"},{"id":34441392,"identity":"fb301d0b-eccf-4f5c-a476-102c263eab51","added_by":"auto","created_at":"2023-03-17 21:32:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":563774,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBBR@LNPs suppress apoptosis induced by H\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e.\u003c/strong\u003e A. The apoptotic C28/12 cells were detected by flow cytometry (FITC-Annexin V apoptotic detection assay). B. The quantitative assay of Apoptotic cells. C. The protein expression of GPX4 and Cleaved cas-3 were detected with Western blot. C. The quantitative assay of GPX4 and Cleaved cas-3. (**\u003cem\u003eP\u003c/em\u003e＜0.01, vs control group. \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e＜0.01, vs H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e group).\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-2688076/v1/b94702cc56226777aaebcfae.png"},{"id":34441391,"identity":"0cdc75ab-6d14-41c6-8c9c-fe8a84216602","added_by":"auto","created_at":"2023-03-17 21:32:02","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":362208,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of BBR@LNPs on the ROS release in C28/I2 cells induced by H\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e.\u003c/strong\u003e A. Intracellular ROS was measured under fluorescence microscope (scar bar =100 μM). B. The intracellular ROS level was measured by flow cytometry.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-2688076/v1/f3fcb63ffdfb6498fcd9f272.png"},{"id":34442536,"identity":"dc2ec42e-c544-41c2-81de-3f3f332006c5","added_by":"auto","created_at":"2023-03-17 21:40:02","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":762139,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of BBR@LNPs on mitophagy in H2O2 stimulated C28/I2 cells. \u003c/strong\u003eA. Western blot band of related protein. The quantitative assay of PINK1 (B), Parkin (C), Cleaved cas-3 (D), GPX4 (E), and LC3-Ⅱ/LC3-Ⅰ (F). (\u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e＜0.01 vs control group. \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e＜0.01，vs H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e group. \u003csup\u003e\u0026amp;\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e＜0.01,vs BBR@LNPs group).\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-2688076/v1/6f451b240c75936e0cdad342.png"},{"id":34443549,"identity":"802fa7e8-cef4-4d5c-9334-9c8e9985e6a7","added_by":"auto","created_at":"2023-03-17 21:48:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1981420,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2688076/v1/51d8dfd0-73ee-4ef5-aed2-42d3dadc8734.pdf"},{"id":34442534,"identity":"ca41631e-a291-45e6-a937-2e575cb43e0a","added_by":"auto","created_at":"2023-03-17 21:40:02","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":329805,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical Abstract\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBBR@LNPs suppresses the ROS release, and inhibits chondrocytes apoptosis through enhancing the expression of Parkin and PINK1 mediated mitophagy.\u003c/p\u003e","description":"","filename":"GA.png","url":"https://assets-eu.researchsquare.com/files/rs-2688076/v1/beb313560930350951a047aa.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Berberine-loaded lipid nanoparticle inhibits H2O2-induced chondrocyte injury by promoting mitochondria autophagy in C28/I2 cells ","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCartilage injury has a high prevalence and disability rate worldwide\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. Chronic degenerative joint diseases such as osteoarthritis and rheumatoid arthritis\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e, can be caused by ageing, obesity, strain, trauma, congenital anomalies of the joints, and joint deformities, resulting in cartilage degeneration in the large joints and later severe limitation of joint movement and inconvenience\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. Acute and chronic pathological aspects of cartilage are associated with a succession of damaging pathological events such as oxidative stress, apoptosis, necrosis of chondrocytes, and inflammatory response\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. Cartilage injury not only causes serious physical and psychological damage, but also imposes a significant financial burden on the family and society. Although the available clinical treatment options can improve the prognosis of some patients. The complex pathology of the injury has limited the development of relevant clinical interventions. It is consequently necessary and critical to focused on developing targeted and effective interventions or targeted drugs to systematically treat or repair damaged cartilages\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMitochondrial autophagy, also known as mitophagy, is a highly conserved and selective autophagic process that has been shown to be the major physiological mechanism governing the quality and quantity of mitochondria\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. Mitochondria are the site of intracellular ATP production and play an important role in maintaining cellular homeostasis, physiological functions, and cell survival\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. Most illnesses are caused by dysfunctional mitochondrial autophagy such as AD, PD, cerebral ischemia-reperfusion injury, spinal cord injury, and cartilage injury\u003csup\u003e[\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. Oscar Alvarez-Garcia discovered that REDD1, a gene that governs development and DNA damage response, was faulty in injured chondrocytes, preventing chondrocyte autophagy and mitochondrial biogenesis and thereby exacerbating cartilage destruction\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. This may suggest that mitochondrial function is defective in the course of cartilage injury and that the lack of timely and effective clearance of functionally defective mitochondria may have an essential role in the inhibition of cartilage injury or in the functional recovery of damaged cartilages. The utilization of mitochondrial autophagy as a therapeutic target could be beneficial in the treatment cartilage injury.\u003c/p\u003e \u003cp\u003eBerberine (BBR), also known as hydrochloride, is a class of flavonoid small molecule compounds\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e derived from the genus Berberis, which has received much attention in recent years due to its potent antioxidant and anti-inflammatory properties\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. BBR suppresses oxidative stress through the ER and TrkA pathways in a model of hydrogen peroxide-induced oxidative stress injury in PC12 cells, therefore inhibiting oxidative damage in neuronal cells\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. The above studies suggest that BBR may have an inhibitory role in oxidative stress and impaired autophagy induced cell damage, such as cartilage injury, spinal cord injury, and ischemic stroke. However, the poor cellular uptake of BBR hinders its clinical use. Therefore, the development of delivery technologies to improve BBR bioavailability might significantly contribute to its increased therapeutic value. Lipid nanoparticles (LNPs) have attracted tremendous attention in the biomedical field due to their simple preparation process, high drug-carrying capacity, and excellent bio-solubility. The use of LNPs to carry mRNA, siRNA, and vaccines for the treatment of diseases has attracted increasing attention from scientists\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. BBR combined with liposomal nanocarriers offer the potential for enhanced intracellular drug delivery. Numerous studies have revealed that BBR exhibits an anti-inflammatory effect. However, its effectiveness in cartilage injury is unknown, and no studies on cartilage injury have been reported. Therefore, the synthesis of lipid nanoparticle systems loaded with berberine BBR and the exploration of their use for the treatment or intervention of cartilage injury is of substantial interest.\u003c/p\u003e \u003cp\u003eUsing liposomal BBR-encapsulated nanoparticles (BBR@LNPs) prepared by microfluidic technology, we propose to investigate whether liposomal nanoparticles loaded with BBR can inhibit cartilage damage or promote the repair of sue-injured cartilage in a rat model of cartilage knee joint cartilage injury. We further investigated whether they inhibit cartilage damage and promote cartilage repair by inhibiting oxidative stress and promoting mitochondrial autophagy, and investigated putative molecular mechanisms or regulatory signaling pathways. It is expected to enrich the pathological mechanisms of cartilage damage, and identify drug interventions and delivery systems with clinical therapeutic implications.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Materials\u003c/h2\u003e \u003cp\u003e1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and cholesterol were purchased from Xi\u0026rsquo;an Ruixi Biological Technology Co., Ltd., China. Isopropyl alcohol (IPA) was obtained from Shanghai Macklin Biochemical Co., Ltd, China. Phosphate buffered saline (PBS) (10 mM, pH 7.4) was obtained from Codow Chemical Co., Ltd, China.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003e2.2. Synthesis And Preparation Of Bbr@lnps\u003c/h3\u003e\n\u003cp\u003eThe synthesis method refered to the published paper from our partner\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. A modified method briefly introduced as follow. DPPC (30 mM) and cholesterol with a molar ratio of 3:1 were dissolved in isopropanol and sonicated to aid dissolution; berberine hydrochloride was sonicated to aid in dissolution and dissolved in PBS at a concentration of 5 mM, and the solution was injected into a flow-focusing microfluidic chip at an aqueous phase: organic phase volumetric flow rate ratio of 4 and a total flow rate of 30 ml/h. The microfluidic chip was fabricated by a 3D printer (microArch\u0026trade; S140; Boston Micro Fabrication, China), and the microchannels have a rectangular cross section with 200 \u0026micro;m depth and 400 \u0026micro;m width. The prepared liposomes were dialyzed using a 7 KD dialysis bag for 48 h, during which the dialysis medium was changed with fresh PBS three times; the final purified products were LNPs loaded with berberine hydrochloride (BBR@LNPs).\u003c/p\u003e\n\u003ch3\u003e2.3. Characteristic Of Bbr@lnps\u003c/h3\u003e\n\u003cp\u003eThe BBR@LNPs were characterized with size, polydispersity index (PDI), morphology, zeta potential, and efficiency (EE%). The morphology was studied using a transmission electron microscope (TEM). The particle size and PDI of BBR@LNPs were measured by dynamic light scattering (DLS) using a Zeta sizer Nano ZS90, whereas the zeta potential was measured using the Zeta sizer Nano ZS90 at 25 ℃ and a scattering angle of 173\u003csup\u003eo\u003c/sup\u003e by measuring the electrophoretic mobility with laser Doppler velocimetry. For characterization study, various parameters of optimal formulation were then measured three times.\u003c/p\u003e \u003cp\u003eFor the determination of the EE%, the drug content was analyzed by reversed phase HPLC. The un-encapsulated BBR was separated from the LNPs by an ultrafiltration method at the bottom of the Amicon UltraVR tube and subjected to HPLC analysis to determine the content of BBR. The total drug content in BBR@LNPs was determined after lysis of lipid core with methanol. EE% could be calculated by the following equation:\u003c/p\u003e \u003cp\u003eEncapsulation efficiency(EE%)= (Wt-Wu)/Wt \u0026times;100%\u003c/p\u003e \u003cp\u003eWhere Wu is the drug untrapped in the LNPs and Wt is the total drug in the BBR@LNPs.\u003c/p\u003e\n\u003ch3\u003e2.4. In Vitro Release Study Of Bbr@lnps\u003c/h3\u003e\n\u003cp\u003eA modified dialysis method was used to evaluate the in vitro release of BBR loaded in LNPs. Briefly, 1 ml of BBR@LNPs with a concentration of 10 mg/ml was placed in a regenerated cellulose dialysis bag against 200 ml of 0.5% Triton X-100 aqueous solution, which was maintained at 37 ℃ with continuous magnetic stirring at 100 rpm. At predetermined time points (0, 10, 20, 30, 40, and 50 h), 1 ml of solution in the receiving phase was taken for HPLC analysis of the released BBR. Each 1 ml sample was collected for various time points and immediately replaced with the same volume of fresh media to maintain the total volume at 200 ml. The free BBR was solubilized by the presence of 50% of PEG in solution. All experiments were carried out in sextuplicate.\u003c/p\u003e\n\u003ch3\u003e2.5. Cytotoxicity Analysis\u003c/h3\u003e\n\u003cp\u003eC28/I2 cells are owned by our laboratory\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. C28/I2 was cultured in DMEM/F12 medium containing 10% fetal bovine serum. After the cell density reached about 80%. The cells were digested by trypsin, prepared into a cell suspension, inoculated into 96-well plates at 1 \u0026times; 105 cells/well and incubated for 24 h. After 24 h, the cells were incubated with medium containing different concentrations of BBR@LNPs medium for 24 h. The old medium was aspirated and discarded, and CCK8 working solution was prepared at a ratio of 10:1 medium: CCK8, added at 100 ml per well, and incubated at 37\u0026deg;C in a CO\u003csub\u003e2\u003c/sub\u003e incubator for 1.5 h. The OD value was detected at 450 nm using an Universal microplate reader.\u003c/p\u003e\n\u003ch3\u003e2.6. Flow Cytometry Analysis\u003c/h3\u003e\n\u003cp\u003eAfter C28/I2 cells were inoculated in 6-well culture at appropriate density and cells were cultured overnight, cells were divided into control, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;LNPs@BBR groups. BBR@LNPs was added to C28/I2 cell medium 2 h before the addition of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, and the culture was continued for 22 h after subsequent addition of the indicated concentrations of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. The cells were washed twice with pre-chilled 1\u0026times; PBS after aspiration of the old medium, digested with EDTA-free trypsin and the cell precipitate was collected. The cell precipitate was centrifuged twice in PBS and incubated for 15 min at room temperature with FITC and PI binding buffer at working concentrations before flow-on assay. FlowJo-v10.8.1 software was used to analysis the data.\u003c/p\u003e\n\u003ch3\u003e2.7. Western Blot Analysis\u003c/h3\u003e\n\u003cp\u003eThe treated groups of cells were collected and lysed on ice for 30 min by adding appropriate amount of lysis solution containing PMSF, and then centrifuged at 12000 r/min for 15 min.The protein supernatant was collected, followed by quantitative analysis of protein concentration of each sample by BCA protein, adjustment of protein concentration, dilution of protein supernatant with 5\u0026times;SDS-PAGE loading buffer at a ratio of protein supernatant: 5\u0026times;SDS-PAGE loading buffer of 4:1. After denaturing for 5 min at 95\u0026deg;C, the samples were spotted on SDS-PAGE gel at 20 \u0026micro;g/well, the proteins were separated by electrophoresis and transferred to PVDF membrane, 5% skimmed milk powder was incubated at room temperature for 1.5 h. The primary antibody was incubated overnight at 4\u0026deg;C in the refrigerator, 1\u0026times;TBST washed the membrane three times for 10 min each time, and the corresponding secondary antibody coupled with HRP was incubated for 1.5 h at room temperature. The bands were saved after ECL luminescence development, and the protein bands were analyzed by ImageJ software using β-actin as the sample content control.\u003c/p\u003e\n\u003ch3\u003e2.8. Data Analysis\u003c/h3\u003e\n\u003cp\u003eStatistics analysis was carried out by the unpaired student\u0026rsquo;s t-test using GraphPad Prism (8.01), with a statistical significance level of p\u0026thinsp;\u0026lt;\u0026thinsp;0.01. All data were expressed as the means\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM.\u003c/p\u003e"},{"header":"Resullts","content":"\u003cdiv class=\"Section2\" id=\"Sec12\"\u003e\n \u003ch2\u003e3.1 Physical characterization\u003c/h2\u003e\n \u003cp\u003eAs shown in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA, the effectively synthesized BBR@LNPs solution was a faint yellow liquid. According to the transmission electron microscopy data shown in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC, the produced BBR@LNPs were uniformly distributed in shape and size and had a regular morphology. In addition, the particle size distribution results as shown in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD showed that the average particle size value of BBR@LNPs was 137 nm; the PDI value was 0.18 and the zeta potential value was \u0026minus;\u0026thinsp;22.4 mV; furthermore, the drug loading of the nanoliposomes to be 7.7% and the encapsulation rate of the BBR@LNPs drug delivery system with BBR-loaded nanoliposomes synthesized by our microfluidic technology is around 80%.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003e3.2. In-vitro Release\u003c/h3\u003e\n\u003cp\u003eThe results of the in vitro release experiments of BBR@LNPs were shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eE. The release rate showed a sharp increase within 10 h of release, followed by a gentle increase, and after 30 h, the release reaches a maximum peak of about 70%, and the release rate no longer increases, reaching a plateau in release. This suggests that a duration of in vitro administration of up to 30 h is appropriate for our subsequent experiments. In addition, this will also serve as a reference for in vivo administration of release in subsequent animal experiments.\u003c/p\u003e\n\u003ch3\u003e3.3. Cytotoxicity Analysis\u003c/h3\u003e\n\u003cp\u003eBy using CCK-8 method and protein immunoblotting experiments to assess the effects of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and BBR@LNPs on the activity of chondrocytes C28/I2 and HEK293, we found that with different concentrations of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e stimulation of C28/12 cells, cell activity decreased significantly with increased H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e concentration, as shown in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA. Interestingly, along with BBR@ LNPs treatment, the viability of C28/I2 and HEK293 cells did not show a sharp decrease in activity at higher concentrations of BBR@LNPs stimulation, as shown in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB. Analysis determined the IC50 value of BBR@LNPs in C28/I2 cells to be around 500 \u0026micro;g/ml. Furthermore, as shown in Figs. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC,E, the results of protein immunoblotting showed that 50, 100 and 200 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e significantly decreased the expression of the antioxidant enzyme GPX4 and significantly increased the expression of cleaved caspase-3, suggesting that H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e significantly induced oxidative damage in C28/I2 cells; Accordingly, in subsequent experiments, we chose 100 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e to establish the concentration of oxidative damage in C28/I2 cells.\u003c/p\u003e\n\u003ch3\u003e3.4. Inhibition Of Ho-induced Apoptosis By Bbr@lnps\u003c/h3\u003e\n\u003cp\u003eTo analyze whether BBR@LNPs had an inhibitory effect on H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-stimulated C28/I2 chondrocyte injury, we used flow cytometry to detect and analyze the apoptosis levels and the antioxidant proteins GPX4 and Cleaved cas-3 in the control H2O2 (100 \u0026micro;M) and H2O2\u0026thinsp;+\u0026thinsp;BBR@LNPs (1\u0026times;103 \u0026micro;g/ml) groups expression changes. Interestingly, when the cells were pretreated with 1 \u0026micro;g/ml of BBR@LNPs for 2 h and then exposed to H2O2 for 24 h, the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced increase in apoptosis was significantly reduced. In addition, we also analyzed the changes in the expression of antioxidant molecule GPX4 and apoptotic molecule Cleaved cas-3 under the same treatment conditions, and the results were shown in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB and \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC: BBR@LNPs significantly reduced the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced increase in the expression of Cleaved cas-3 and increased the expression of antioxidant molecule GPX4. It suggests that BBR@LNPs can inhibit H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced apoptosis and increase the antioxidant properties of C28/I2 cells.\u003c/p\u003e\n\u003ch3\u003e3.5. Bbr@lnps Reduce H2o2-induced Ros Release\u003c/h3\u003e\n\u003cp\u003eIncreased ROS release is an important driver of oxidative damage in chondrocytes; therefore, we employed fluorescence microscopy and flow cytometry to investigate the quantities of ROS released from H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-stimulated C28/I2 cells alone and C28/I2 cells co-cultured with BBR@LNPs and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. The results are shown in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA and \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB. The BBR@LNPs were able to significantly reduce the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-stimulated ROS release, and the same results were obtained for both flow cytometry and fluorescence microscopy assays. These findings suggest that our synthesized BBR@LNPs can inhibit the increase in H2O2-stimulated ROS release, which may be an important mechanism for the BBR@LNPs to play a role in inhibiting oxidative damage in chondrocytes.\u003c/p\u003e\n\u003ch3\u003e3.6. Bbr@lnps Enhance Mitophagy Level\u003c/h3\u003e\n\u003cp\u003eIt has been reported that berberine activates the mitochondrial autophagy level of cells and functions as an inhibitor of oxidative damage. Therefore, we examined the expression of mitochondrial autophagy marker molecules PINK1 and Parkin as well as apoptosis analysis Caspase-3 and antioxidant molecule GPX4 by protein immunoblotting assay in C28/I2 cells co-cultured with BBR@LNPs and autophagy inhibitor 3-MA with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e stimulation; the results was shown in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e, BBR@LNP increased the expression of PINK1, Parkin, and GPX4; however, treatment with the autophagy inhibitor 3-MA intensified the expression of the apoptotic molecule Caspase-3 and decreased the expression of PINK1 and Parkin. The above results may suggest that the inhibition of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e -induced increase in apoptosis by BBR@LNPs may be achieved through the activation of mitochondrial autophagy; suggesting that BBR@LNPs may be an agonist of mitochondrial autophagy and play a function in activating mitochondrial autophagy.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe used a microfluidic method to effectively manufacture lipid nanoparticles loaded with choline hydrochloride with particle sizes of around 137 nm and assessed their zeta potential values of -10.82 mV and PDI values of 0.18. In addition, we also assessed their in vitro drug release rate after 48 h. With longer release times, the release rate of BBR@LNPs peaked at around 30 h. With increasing release duration, the release rate of BBR@LNPs peaked at 70% at around 30 h. after which it did not continue to rise. The IC50 values were interestingly found to be within a high concentration range, indicating that the toxic effects were low. The mechanism by which BBR@LNPs inhibit H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced cartilage damage is achieved by enhancing mitochondrial autophagy.\u003c/p\u003e \u003cp\u003eThe primary purpose of mitochondrial autophagy, which is substantially conserved in eukaryotes, and its main function is to clear damaged or dysfunctional mitochondria and tightly control mitochondrial mass\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. Mitochondrial autophagy has an important role in regulation of apoptosis, inflammatory response, and cell differentiation\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. Sun et al. reported that mitochondrial autophagy has a regulatory role in the development of degenerative joint diseases\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. Here, we found that exposure of human chondrocytes C28/I2 to the strong oxidant H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e resulted in increased ROS release, leading to decreased mitochondrial autophagy and the production of ROS disrupts the dynamic homeostasis of mitochondria and activates mitochondrial autophagy to remove damaged mitochondria\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. However, dysfunctional mitochondrial autophagy in turn leads to the emergence of processes, for example, apoptosis, which exacerbates oxidative cellular damage. It is suggested that pharmacological interventions or target-based therapies can increase or repair mitochondrial autophagy and thereby maintain cellular homeostasis and promote recovery of cellular function. In this study, we discovered that BBR-loaded liposomal nanoparticles dramatically increased mitochondrial autophagy and prevented H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced oxidative damage in chondrocytes. In our previous study, BBR was found to inhibit the process of excessive oxidative stress activated by oxygen glucose deprivation-reoxygenation\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. Interestingly, BBR reduced the generation of ROS by inhibiting the activation of inflammatory vesicles and promoting higher amounts of mitochondrial autophagy in influenza virus-infected cells\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. Our results are partially corroborated by our study. However, less research has been reported on chondrocyte damage by BBR. BBR is derived from the flavonoids of \u003cem\u003eRhizoma coptidis\u003c/em\u003e, which has antioxidant, anti-apoptotic, anticancer and neuroprotective effects\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. However, its poor water solubility and its tendency to precipitate crystals in solution hinder its potential clinical application\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. The establishment of an effective drug delivery system is of great significance for the drug utilization of chalcone hydrochloride. Numerous clinical therapies have demonstrated the effectiveness of nanocarrier-based medicines\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. The first liposome-based nanomedicine, Doxorubicin, was developed in 1995 and has been used in tumor therapy\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. Until today, scientists have developed around 50 nanoparticle-based drugs. The new use of old drugs has been attracting a lot of attention, because it saves costs in drug development, reduces the drug development cycle, and has a clear pharmacological mechanism of action. Precious metal-based drug delivery technologies, such as colloidal gold, are pricy, difficult to create, and unsuited for large-scale drug manufacturing\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. Nanoliposomes were the first carriers developed for nucleic acid transfection technology for basic science research and have gained attention in recent years for their value in drug delivery, with the novel nucleic acid vaccine for coronavirus being the best classical use of nanoliposomes for drug delivery\u003csup\u003e[\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e. Using cholesterol and low-cost DPPC as liposomal cofactors, we created liposomal nanoparticles loaded with small cleavage hydrochloride, we then examined their characteristics using a variety of techniques, including transmission electron microscopy and zeta sizer, and assayed their drug release rates in vitro, and further applied them in a model of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced oxidative damage in C28/12 cells The pharmacological effects of the nano-liposomes loaded with chondroitin hydrochloride were demonstrated by flow cytometry, CCK-8, immunofluorescence analysis, and protein immunoblotting. The mechanism of its pharmacology may be achieved by inhibiting the release of ROS and increasing the physiological function of mitochondrial autophagy.\u003c/p\u003e \u003cp\u003eIn conclusion, our study has successfully produced nanoliposomes loaded with chondroitin hydrochloride using microfluidic technology, and explored the mechanism of their pharmacological effects by reducing the release of ROS and increasing the level of mitochondrial autophagy in a model of oxidative damage in chondrocytes. The potential mechanism of action of BBR@LNPs may be as \u003cb\u003eGraphical Abstract\u003c/b\u003e showed. Collectively, our results may be useful in the development of chondroitin hydrochloride drugs for cartilage injury, as well as in providing some insight into the pathological process of cartilage injury.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo new data were generated or analysed in support of this research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGl Y and P X conducted the experiments and the data analysis. Qq M and Sm L conceived the project, designed the study, arranged the results and revised the manuscript. All authors approved the final version of the manuscript. Sm L accepted full responsibility for the finished work, had access to the data and controlled the decision to publish.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Clinical Medical Technology Innovation guidance Program of Human Province(2021SK51821) and the Natural Science Foundation of Guangdong Province (2019A1515011085).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank doctoral candidate Han shan at Department of Dermatology, Xiangya Hospital, Central South University for guidance of the synthesis method of BBR@LNPs and rewired the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors declared that there are no interests between them and the research was conducted under absence of any commercial or financial relationships.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eRIM Y A, NAM Y, JU J H. 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Materials (Basel, Switzerland), 2021, 14(19).\u003c/li\u003e\n\u003cli\u003eZHAO M, VAN STRATEN D, BROEKMAN M L D, et al. Nanocarrier-based drug combination therapy for glioblastoma [J]. Theranostics, 2020, 10(3): 1355-72.\u003c/li\u003e\n\u003cli\u003ePETERSEN A L, HANSEN A E, GABIZON A, et al. Liposome imaging agents in personalized medicine [J]. Advanced drug delivery reviews, 2012, 64(13): 1417-35.\u003c/li\u003e\n\u003cli\u003eSIEGAL T, HOROWITZ A, GABIZON A. Doxorubicin encapsulated in sterically stabilized liposomes for the treatment of a brain tumor model: biodistribution and therapeutic efficacy [J]. Journal of neurosurgery, 1995, 83(6): 1029-37.\u003c/li\u003e\n\u003cli\u003eAUSTIN L A, MACKEY M A, DREADEN E C, et al. The optical, photothermal, and facile surface chemical properties of gold and silver nanoparticles in biodiagnostics, therapy, and drug delivery [J]. Archives of toxicology, 2014, 88(7): 1391-417.\u003c/li\u003e\n\u003cli\u003eANDRIES O, MC CAFFERTY S, DE SMEDT S C, et al. N(1)-methylpseudouridine-incorporated mRNA outperforms pseudouridine-incorporated mRNA by providing enhanced protein expression and reduced immunogenicity in mammalian cell lines and mice [J]. Journal of controlled release : official journal of the Controlled Release Society, 2015, 217: 337-44.\u003c/li\u003e\n\u003cli\u003eCHUNG J Y, THONE M N, KWON Y J. COVID-19 vaccines: The status and perspectives in delivery points of view [J]. Advanced drug delivery reviews, 2021, 170: 1-25.\u003c/li\u003e\n\u003cli\u003eLAMB Y N. BNT162b2 mRNA COVID-19 Vaccine: First Approval [J]. Drugs, 2021, 81(4): 495-501.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"berberine, lipid nanoparticle, mitochondrial autophagy, apoptosis, chondrocyte damage","lastPublishedDoi":"10.21203/rs.3.rs-2688076/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2688076/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAlthough berberine (BBR) is considered to be of therapeutic value in treating various disorders; however, its low bioavailability hinders its further development on clinical application. To evaluate the effects of BBR against chondrocyte damage, we synthesized liposomal nanoparticles loaded with BBR (BBR@LNPs) with an average particle size of 137 nm using microfluidic technology. The CCK8 assay was used to determine the cytotoxicity of the BBR@LNPs in C28/I2. Additionally, flow cytometry and western blotting were used to investigate the mechanism of its protect chondrocytes from H2O2-induced oxidative damage in C28/I2. Our findings showed that BBR@LNPs had stable nanoscale structure and low cytotoxicity. Importantly, administration of BBR@LNPs significantly inhibited ROS release and cell apoptosis in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced C28/I2, BBR@LNPs enhanced mitochondrial autophagy by up-regulating PINK1 and Parkin expression. Our findings may provide some guidance BBR-loaded lipid nanoparticle delivery system in cartilage injury.\u003c/p\u003e","manuscriptTitle":"Berberine-loaded lipid nanoparticle inhibits H2O2-induced chondrocyte injury by promoting mitochondria autophagy in C28/I2 cells ","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-03-17 21:31:57","doi":"10.21203/rs.3.rs-2688076/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"db6fa8b3-a309-4e22-9c1f-a88243e4543b","owner":[],"postedDate":"March 17th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-03-17T21:31:59+00:00","versionOfRecord":[],"versionCreatedAt":"2023-03-17 21:31:57","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2688076","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2688076","identity":"rs-2688076","version":["v1"]},"buildId":"cBFmMYwuxLRRLfASyISRj","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}