Materials
1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-mPEG2000) and DSPE-mPEG2000-COOH were purchased from Xi'an Ruixi Biological Technology Co., Ltd. (Xi’an, China). Cholesterol was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). SHP1i were achieved from Sigma-Aldrich (USA). CD36 Polyclonal Antibody and CD130 Polyclonal Antibody was purchased from Beijing Biodragon Immunotechnologies Co., Ltd. (Beijing, China). TLR4 Polyclonal Antibody, IL-6R Polyclonal Antibody, TNFR1 Polyclonal Antibody, IFNGR1 Polyclonal Antibody, SRA Polyclonal Antibody, CD80 Polyclonal Antibody and CD206 Polyclonal Antibody were purchased from Proteintech Group, Inc (Wuhan; China). Oxidized Low Density Lipoprotein (oxLDL) was obtained from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Lipopolysaccharide (LPS) was supplied by Sigma-Aldrich (USA). oxLDL ELISA kit was received from Jiangsu Mei Biao Biological Technology Co., Ltd. (Yancheng, China). Limulus amebocyte lysate (LAL) assay was purchased from Thermo Fisher Scientific (USA). Coumarin-6 and Oil Red O solution were purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). Total cholesterol (TC) determination kit was received from Shanghai Cablebridge Biotechnology Co., Ltd. (Shanghai, China). Mouse Tumor Necrosis Factor Alpha ELISA kit was obtained from ABclonal Biotechnology Co., Ltd. (Wuhan; China). Mouse IL-6 ELISA kit and Mouse IFN-γ ELISA kit were purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). 2′,7′- Dichlorodihydrofluorescein diacetate (DCFH-DA) was purchased from Dalian Meilun Biotechnology Co., Ltd. (Dalian, China). Annexin V-FITC Apoptosis Detection Kit was purchased from Jiangsu KeyGEN BioTECH Co., Ltd. (Nanjing, China). Cell-Tracker Red CMTPX and Cell-Tracker Green CMTPX were purchased from Shanghai Maokang Biotechnology Co., Ltd. (Shanghai, China). CD68 Polyclonal Antibody, CD14 Polyclonal Antibody, MMP-9 Polyclonal Antibody, α-SMA Polyclonal Antibody, CD31 Polyclonal Antibody, KI67 Polyclonal Antibody and Caspase-3 Polyclonal Antibody were purchased from Wuhan Servicebio Technology Co., Ltd. (Wuhan, China). Anti-Macrophage Inflammatory protein 3 alpha antibody was obtained from Abcam Plc (Shanghai, China). Rabbit Anti-phospho-PTPN6 antibody was purchased from Beijing Biosynthesis Biotechnology Co., Ltd. (Beijing, China).
Isolation and verification of macrophage membrane
Macrophage membranes were acquired with a previously reported method, with a minor modification [19] [20]. Specifically, RAW264.7 macrophage cells (cell number ≈ 1 × 108) were isolated from culture dish with 0.25% Trypsin–EDTA and washed with PBS for 3 times (500 × g for 10 min each time), and dispersed in PBS. Next, a hypotonic lysing buffer containing 1 mmol L−1 NaHCO3, 0.2 mmol L−1 EDTA and 1 mmol L−1 PMSF, was added to disperse cells and incubated in 4 °C overnight. Then, the cell suspension was put into a non-contact automatic ultrasonic disruptor and was destroyed after 60 cycles. The obtained suspension was centrifuged at 3200 × g at 4 °C for 5 min to remove large debris. The collected supernatant was further centrifuged at 20,000 × g for 25 min to discard the pellet. Finally, the supernatant was centrifuged at 100,000 × g for 35 min to collect the cell membranes in the bottom, which were dispersed in PBS (pH = 7.4). To obtain macrophage membrane vesicles, the extracted macrophage membranes were extruded 15–20 times through a 400 nm and 200 nm polycarbonate porous membrane using an Avestin Mini-extruder. The harvested macrophage membrane vesicles were stored in PBS at 4 °C for later use.
In order to verify the successful isolation of macrophage membrane, the cultured macrophages were double-stained with DiI and DAPI to distinguish the isolated membrane components from the nuclear components. Briefly, RAW264.7 cells were fixed with 4% paraformaldehyde for 15 min and washed with PBS for 3 times. Then, the cells were stained with DAPI for 3–5 min and washed with PBS for 3 times. Next, the cells were stained with 10 µM DiI for 15–20 min and washed with PBS for 3 times. The macrophage membrane was then extracted according to the above steps. The extract in each step was observed under the fluorescence microscope and photographed.
Synthesis of blank liposomes and lips-SHPIi nanoparticles
Blank liposomes were prepared by lipid film hydration and extrusion method, according to the article we previously published with minor modification [21]. Briefly, 0.0198 g DSPC, 0.0048 g cholesterol, 0.001875 g DSPE-mPEG2000 and 0.001875 g DSPE-mPEG2000-COOH were dissolved in a mixture of chloroform (4 mL) and methanol (1 mL). The above solution was sonicated with an ultrasonic cell disruptor for 30 min. Then, the organic solvent was evaporated to dryness using a vacuum rotary evaporator, to obtain a lipid film attached to the bottle wall. Then it was placed in a vacuum drying oven to dry for 6 h. The obtained thin film was hydrated by vortexing in a 5 mL PBS solution and stirred to form blank liposomes suspension. Finally, the blank liposomes suspension can be extruded repeatedly 10 times through a 400 nm and 200 nm polycarbonate porous membrane in a liposome extruder to get well size-distributed blank liposomes for further use.
For SHP1i loading, the SHP1i solution was added to blank liposomes overnight to form Lips-SHP1i nanoparticles. After 24 h of stirring, Lips-SHP1i was centrifuged to remove unloaded SHP1i molecules. The concentration of the loaded SHP1i was measured using a NanoDrop (Nanodrop2000; Thermo Scientific) according to its absorption of 320 nm [18]. The SHP1i loading efficiency (LE) and encapsulation efficiency (EE) was determined by the following equations:
$$\mathrm{Loading\,Efficiency }\,(\mathrm{LE})=\frac{{\mathrm{m}}_{\mathrm{SHP}1\mathrm{i},\mathrm{total}}-{\mathrm{m}}_{\mathrm{SHP}1\mathrm{i},\mathrm{free}}}{{\mathrm{m}}_{\mathrm{blank liposomes}}}\times 100\mathrm{\%}$$
$$\mathrm{Encapsulation\,Efficiency }\,(\mathrm{EE})=\frac{{\mathrm{m}}_{\mathrm{SHP}1\mathrm{i},\mathrm{total}}-{\mathrm{m}}_{\mathrm{SHP}1\mathrm{i},\mathrm{free}}}{{\mathrm{m}}_{\mathrm{SHP}1\mathrm{i},\mathrm{total}}}\times 100\mathrm{\%}$$
Preparation and characterization of MM@Lips and MM@Lips-SHP1i
A mechanical co-extrusion method was used to fuse the macrophage membrane and the blank liposomes or Lips-SHP1i nanoparticles to obtain the macrophage membrane biomimetic nanoparticles. Firstly, the collected macrophage membrane vesicles were mixed with nanoparticle cores with a membrane protein-to-polymer weight ratio of 1:1, and the mixture was sonicated with a bath sonicator at a frequency of 42 kHz and a power of 100 W for 2 min. Then, the mixture was extruded 15–20 times through a mini-extruder to obtain MM@Lips and MM@Lips-SHP1i nanoparticles. Finally, the resulting solution was centrifuged at 10,000 × g for 30 min to remove the uncoated membrane. The prepared macrophage membrane biomimetic nanoparticles are stored at 4 °C for further use.
The size/polydispersity coefficient (PDI) and zeta potential of MM vesicles, Lips, Lips-SHP1i, MM@Lips and MM@Lips-SHP1i were measured by dynamic light scattering detector (DLS) (Nano ZS90; Malvern, UK). All measurements were done in triplicate at room temperature. The morphology of MM vesicles, Lips and MM@Lips-SHP1i were visually observed using a transmission electron microscope (TEM) (JEM-2100F; JEOL, Japan). To verify the successful synthesis of Lips-SHP1i, the ultraviolet–visible absorption spectrum of SHP1i, Lips, Lips-SHP1i, MM vesicles, MM@Lips and MM@Lips-SHP1i were measured by UV–vis spectrophotometer. The particle sizes of MM@Lips-SHP1i nanoparticles dispersed in PBS and 10% mouse serum were determined over a span of 7 days, to evaluate the stability of biomimetic macrophage membrane nanoparticles.
Membrane protein characterization
The surface membrane proteins were characterized by polyacrylamide gel electrophoresis (SDS-PAGE). Briefly, the Lips-SHP1i, macrophages, macrophage membrane vesicles and MM@Lips-SHP1i were prepared in a RIPA buffer supplemented with protease inhibitor and quantified by the BCA Protein Assay (Beyotime; China). Then, the samples were mixed with 5 × loading buffer before heating at 100 °C for 5 min. Approximately, 30 μg proteins for each sample were loaded into each well in 12% SDS-PAGE and run at 100 V for 2 h. After that, the SDS-PAGE gel was stained by Coomassie Blue Staining Kit (Beyotime; China) according to the provided protocol until imaged with investigator prolmage.
The specific surface markers on macrophage, macrophage membrane and MM@Lips-SHP1i were determined by Western blotting. Specifically, the proteins were transferred from the gel to the poly (vinylidene diflfluoride) membranes followed by blocking for 2 h with 5% skimmed milk powder in tris-buffered saline after the electrophoresis. Then, membranes were incubated overnight at 4 °C with primary antibodies, including CD36, SRA, TLR4, IL-6R, CD130, TNFR1, IFNGR1. Followed by incubation of a secondary HRP-conjugated affinipure goat anti-rabbit IgG (H + L) antibody (Proteintech; China) at room temperature for 1 h, specific bands were visualized using an enhanced chemiluminescent detection kit (NCM Biotech, China) under a chemiluminescence/fluorescence image analysis system (Tanon 5200, China).
The macrophage phenotype was also confirmed by evaluating the expression of CD80 (M1 macrophage phenotype marker) and CD206 (M2 macrophage phenotype marker) by Confocal Laser Scanning Microscope (CLSM). In brief, RAW264.7 cells were seeded in a 6-well plates containing cell slide at a density of 1 × 104 cells per well. After overnight incubation, M1/M2 macrophages were polarized in vitro. After polarization, cells were fixed with 4% paraformaldehyde and subsequently incubated with 0.3% TritonX-100 for 30 min. After washing with PBS, the cells were blocked with blocking solution (10% NGS, 0.3% TritonX-100) for 2 h at room temperature. Then, cells were incubated overnight at 4 °C with primary antibodies CD80 or CD206. Followed by incubation of a fluorescence secondary antibody for 2 h, the nuclei were counterstained with DAPI staining solution. Finally, the slides were sealed with anti-fluorescence quenching agent, and the cells were observed and photographed under CLSM.
oxLDL and LPS binding studies
To quantify oxLDL clearance rate by MM@Lips, MM@Lips (0.5 mg) were mixed with oxLDL of varying amount (10, 20, 30, 60 μg), respectively, in 1 × PBS containing 10% FBS [19]. In a parallel experiment, the removal amount was studied by fixing oxLDL amount of 60 μg but varying the amount of MM@Lips at 0.1, 0.2, 0.3, 0.4 and 0.5 mg, respectively. In both cases, the mixtures were incubated for 30 min and then centrifuged at 20,000 × g for 15 min to pellet the nanoparticles. The free oxLDL content in the supernatant was measured by using enzyme linked immunosorbent assay (ELISA) kit according to manufacturer’s instructions. All experiments were performed in triplicate.
Similarly, to quantify LPS clearance rate by MM@Lips, MM@Lips (0.5 mg) were mixed with different amout of LPS (5, 10, 25, and 50 ng), respectively. In parallel, 50 ng of LPS was mixed with different concentrations of MM@Lips (0.1, 0.2, 0.3, 0.4 and 0.5 mg). After incubated for 30 min, the free LPS content in the supernatant was quantified using limulus amebocyte lysate (LAL) assay according to the manufacturer’s instructions. All experiments were performed in triplicate.
Cell culture
The RAW 264.7 macrophage cells and Human Umbilical Vein Endothelial Cells (HUVECs) were purchased from Cell Bank/Stem Cell Bank, Chinese Academy of Sciences. The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (Gibco, USA), penicillin (100 U/mL) and streptomycin (0.1 mg/mL). Cells were kept in an incubation chamber at 37 °C and 5% CO2 with a humidified atmosphere.
Cytotoxicity evaluation
In vitro cytotoxicity of Lips, Lips-SHP1i, MM@Lips, MM@Lips-SHP1i were evaluated with RAW264.7 and HUVECs cells by MTT assays, respectively. Mainly, the cells were seeded in 96-well plates (1 × 104 cells per well). After incubation for 24 h, fresh DMEM medium containing varying concentrations of Lips, Lips-SHP1i, MM@Lips, MM@Lips-SHP1i (0.1, 0.2, 0.3, 0.4, 0.5, 0.6 mg/mL) were incubated with cells for another 24 h. After discarding the medium, 100 μL of 1 mg/mL MTT solution was added into each well and incubated for 4 h in dark at 37 °C. Afterward, 100 μL of dimethylsulfoxide (DMSO) was introduced to dissolve the formed formazan crystals in each well. The absorbance at 490 nm was recorded by microplate reader (Thermo Scientific, USA). Each experiment was conducted 3 times in parallel. The cell viability was calculated by GraphPad Prism 9 according to their absorbances at 490 nm in each well.
MM@Lips-SHP1i NPs escape clearance from immune system
To verify the effect of macrophage membrane coating in evading clearance from the immune system, fluorescently labeled nanoparticles were incubated with RAW264.7 macrophages to observe the phagocytosis reduction by macrophages. To prepare the fluorescently labeled nanoparticles, 1 wt % Coumarin-6 (excitation/emission wavelength = 466/504 nm) was loaded into blank liposomes. Briefly, RAW264.7 cells were seeded in 6-well plates at a density of 1 × 104 cells per well and cultured overnight. Then, the media were replaced with fresh media containing 100 μg Lips-C6 NPs and MM@Lips-C6 NPs. After incubation for different times (0.25, 0.5, 1, and 2 h), the cells were washed with PBS for 3 times and fixed by paraformaldehyde for 15 min at room temperature. Subsequently, the cells were washed with PBS for three more times and the nuclei were stained with DAPI. After washing with PBS for 3 times again, the cells were observed via inverted fluorescence microscope.
Inhibition of macrophage foaming in vitro
RAW264.7 macrophages were seeded in a 24-well plate (1 × 104 cells per well). After incubation for 12 h, the cells were co-treated with oxLDL (60 μg/mL) and Lips, Lips-SHP1i, MM@Lips, MM@Lips-SHP1i NPs for 24 h, respectively. The degree of macrophage foaming was evaluated by oil red O (ORO) staining and the determination of intracellular total cholesterol (TC) concentration.
The ORO staining in macrophages was carried out according to the instructions. Specifically, the saturated oil red O stock solution was added to distilled water in a ratio of 3:2 (oil red O: distilled water) and filtered twice with filter paper before use. After fixed with 4% paraformaldehyde at room temperature for 15 min and washed with PBS, the macrophages were stained by freshly prepared ORO working solution for 15 min, and rinsed with 60% isopropanol. After washing with PBS, the cells were observed under microscope. The obtained images were further analyzed with Image J software.
Inhibition of proinflammatory cytokines, ROS and iNO by LPS neutralization in vitro
RAW 264.7 macrophages were seeded in a 24-well plate at a density of 1 × 105 cells/well and cultured overnight. Then the cells were co-treated with LPS (20 ng/mL) and Lips, Lips-SHP1i, MM@Lips, MM@Lips-SHP1i NPs for another 24 h. After incubation, the concentrations of proinflammatory cytokines including TNF-α, IL-6, IFN-γ in the supernatant were quantified using ELISA kits.
Furthermore, the ROS levels were analyzed to confirm the ROS production. Briefly, RAW264.7 macrophages were cultured in 6-well plates at a density of 1 × 104 cells/well for 12 h. After co-treatment of cells by LPS (20 ng/mL) and different groups (Lips, SHP1i, Lips-SHP1i, MM@Lips and MM@Lips-SHP1i NPs) for 4 h, cells were rinsed and treated with 10 µM 2′,7′-dichlorofluorescin diacetate (DCFH-DA) in PBS for 30 min. Afterwards, the cells were washed with PBS for 3 times and the intracellular ROS generation was observed by Inverted Fluorescence Microscope. Results were analyzed using Image J software (version 7.6.1).
Production of intracellular nitric oxide (iNO) was also used to evaluate the influence of LPS neutralization with MM@Lips-SHP1i. Briefly, 2 × 104 RAW264.7 macrophages were seeded in each well of a 96-well plate and incubated overnight. Then, 180 μL of medium containing 20 ng/mL of LPS was added to each well, followed with the addition of 20 μL of SHP1i, Lips-SHP1i, MM@Lips or MM@Lips-SHP1i, respectively. As control, 20 μL of PBS was added into control wells. Cells without any treatment served as the background. The plate was incubated at 37 °C for 24 h and the iNO productions were measured by a nitric oxide detection kit. The above-mentioned cell supernatant was collected, and 50 μL of the supernatant, 50 μL of Griess Reagent I and 50 μL of Griess Reagent II were added to each well, respectively. Finally, the absorbance value of each well at 540 nm was read with a microplate reader, and the concentration of nitric oxide in each sample was calculated according to the standard curve.
Detection of scavenger receptor expression on macrophages
RAW264.7 macrophages were seeded in a 6-well plate at a density of 1 × 105 cells/well and cultured overnight. After co-treated with oxLDL and SHP1i, Lips-SHP1i, MM@Lips, MM@Lips-SHP1i NPs for 24 h, respectively, the total cell protein of each group was extracted according to the previous steps. Western Blot was used to evaluate the expression of scavenger receptors (mainly CD36 and SRA) on the surface of macrophages in each group.
Activated macrophage apoptosis assay
Briefly, RAW264.7 macrophages were cultured in 6-well plates at a density of 1 × 104 cells/well for 12 h. After treated by oxLDL and SHP1i, Lips-SHP1i, MM@Lips or MM@Lips-SHP1i NPs for 24 h, the cells were collected by trypsinization without EDTA. Then the cells were washed twice with PBS and suspended in 500 μL of binding buffer. Subsequently, after adding 5 μL of Annexin V-fluorescein isothiocyanate (V-FITC) and 5 μL of propidium iodide (PI) and reacting for 15 min at room temperature, cells were analyzed by flow cytometer to determine the apoptosis rates. Results were analyzed using FlowJo software (version 7.6.1).
In vitro efferocytosis assay
In vitro efferocytosis assays were performed by cell tracker. Briefly, RAW264.7 macrophages were labelled with CellTracker Red and pretreated with SHP1i, Lips-SHP1i, MM@Lips or MM@Lips-SHP1i for 30 min. To produce labelled apoptotic cells, RAW264.7 cells labelled with CellTracker Green were incubated with tumor necrosis factor-α (TNF-α) for 24 h. Then, apoptotic cells were plated in 6-well dishes at a density of 1.5 × 105 cells per well and RAW264.7 cells were added at a density of 3 × 105 cells per well. They were co-incubated for 2 h in serum-free media. Efferocytic activity was observed by fluorescence microscopy and evaluated by Image J software (version 7.6.1).
Animals
Female C57BL/6 mice were obtained from the Animal Center of Xuzhou Medical University. Female apolipoprotein E knockout (ApoE-/-) mice aged 8 weeks were purchased from GemPharmatech Co., Ltd. (Nanjing, China). All animals were maintained under standard housing conditions and all animals were acclimatized for at least 3 days before the experiments started. All animal protocols were approved by the Ethics Committee of Xuzhou Medical University.
Blood half-life determination
Six C57BL/6 mice aged 8 weeks, were randomly divided into two groups. In each group, 200 μL of Lips-C6 or MM@Lips-C6 nanoparticles (5 mg/mL) were injected intravenously. 10 μL of blood was quickly collected from the tail of the mouse at different time points post-injection (5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h, and 48 h). The collected blood was immediately diluted with 10 μL of 2 mM EDTA-2 K PBS solution. The fluorescence intensities of the samples were measured with a fluorescence microplate reader. Fitting half-life curve and half-life (T1/2) was calculated according to the obtained fluorescence intensities with the time passing by.
Construction of AS in ApoE-/- mice
Eight-week-old female ApoE-/- mice were fed a normal diet for 12 weeks in the negative control group while a high-fat diet (HFD) containing 20% fat and 1.25% cholesterol was given to another group to induce atherosclerosis.
In vivo targeting of atherosclerotic plaque
Eight-week-old female ApoE-/- mice fed with high fat diet for 3 months were administered with Lips-C6 and MM@Lips-C6 via the tail vein, respectively. After 2 h, the mice were euthanized and subsequently perfused with PBS to remove the blood and unbound dyes. The aortas were isolated for imaging and fluorescence quantitative analysis using an chemiluminescence/fluorescence image analysis system.
In vivo treatment of AS in ApoE-/- mice
ApoE-/- mice were randomized into 6 groups (7 mice per group) and injected with saline, SHP1i, Lips-SHP1i, MM@Lips or MM@Lips-SHP1i NPs every 4 days for 4 weeks. Mice in negative control group were given normal diet and those in other groups were fed with high fat diet. At the end of treatment, the ApoE-/- mice were euthanized and the degree of pathological evolution were evaluated by measuring the lesion area of the aorta from the heart to the iliac bifurcation. To determine the extent of atherosclerosis at the aortic root, aortic arch, and brachiocephalic artery, Oil Red O (ORO) staining was performed to confirm the formation of atherosclerotic plaque in mice.
Furthermore, quantitative analysis of atherosclerotic plaques was also determined by ORO staining with sequential 10 cryosections at 100 μm intervals from the aorta tissues. Subsequently, for histological analysis, aortic root, aortic arch and brachiocephalic artery from mice with various treatments were collected for hematoxylin–eosin (H&E) staining, Masson’s trichrome staining and toluidine blue staining. For immunohistochemistry analysis, sections of aortic root, aortic arch and brachiocephalic artery were incubated with antibodies, including CD68, CD14, MMP-9, and α-SMA, CD31 and KI67, respectively.
In vivo efferocytosis assay
To confirm the interruption of MM@Lips-SHP1i NPs to CD47-SIRPα signaling in vivo, sections were co-stained with phospho-SHP1 and Mac-3, respectively, to assess the lesional SHP-1 activity. The phospho-SHP1 area was quantified and normalized to the Mac-3 area. To assess apoptotic cells in lesions, sections were stained for cleaved caspase-3. The percentage of cleaved caspase-3 + area was calculated and divided by the total atherosclerotic plaque area. To study the efferocytosis of apoptotic cells by macrophages, sections were co-stained with cleaved caspase-3 and Mac-3. And then the in vivo phagocytic index was calculated by manually counting the number of free apoptotic cells versus phagocytosed (macrophage-associated) apoptotic cells.
Safety evaluation
During the above treatment, the changes of body weight of mice in all groups were recorded. At the endpoint of experiment, all mice were sacrificed, and the heart, livers, spleen, lungs, and kidneys were processed for histological study. In addition, the blood was collected to quantitate the immune-associated cells, such as red blood cells (RBCs), white blood cells (WBCs), platelets (PLTs), and hemoglobin (HGB). The serum was also collected for blood chemistry analysis, including the liver function biomarkers (ALT and AST), kidney function biomarkers (UREA and CREA).
In addition, the biocompatibility of MM@Lips-SHP1i was also evaluated by hemolysis assay [22]. Briefly, fresh mouse blood samples were collected and centrifuged to separate RBCs. After washed several times and diluted with saline, 200 μL of RBCs suspension was mixed with 800 μL of different nanoparticles. Red blood cells treated with the same volume of deionized water and saline were set as positive and negative controls. After incubation at 37 °C for 2 h, the mixture was centrifuged at 5000 rpm for 5 min, and the absorbance of the supernatant at 541 nm was measured by UV–vis spectrophotometer. The hemolysis rate of each sample was calculated by the following formula:
$$\mathrm{Hemolysis}(\mathrm{\%})=\frac{{\mathrm{A}}_{\mathrm{sample}}-{\mathrm{A}}_{\mathrm{negative\,control}}}{{\mathrm{A}}_{\mathrm{positive\,control}}-{\mathrm{A}}_{\mathrm{negative\,control}}}\times 100\mathrm{\%}$$
Statistical analysis
The data obtained by at least three independent repeated experiments were presented as the mean ± standard deviation in this study. SPSS 22.0 software was used for the statistical analysis. One-way analysis of variance (ANOVA) was utilized for revealing differences among the groups, and when the overall difference was statistically significant, pairwise comparison was performed with least significant difference (LSD) method inspection. Values of *P < 0.05, **P < 0.01 and ***P < 0.001 were applied to annotate statistical significance.
