Principal devices and reagents
Bovine serum albumin (BSA) and MES answer have been bought from Sigma (St. Louis, MO, USA); sodium hydroxide, hydrochloric acid, Sodium dodecyl sulfate (SDS) and DL-Dithiothreitol (DTT) have been bought from Aladdin (Shanghai, China); liraglutide was bought from MCE Firm (St. Louis, MO, USA); Liraglutide (LIR) detection package (enzyme-linked immunosorbent assay) was bought from Wuhan Cloud Clone Expertise Co., Ltd.(Wuhan, China); cell and tissue lysate have been bought from Shanghai Beyotime Biotechnology Co., Ltd. (Shanghai, China); CO2 incubator (Thermo Firm, St. Louis, MO, USA); inverted microscope (Olympus Firm, Japan); confocal laser microscope (Zeiss, Germany); transmission electron microscope (FEI, USA); particle measurement analyzer (Malvern Firm) and UV spectrophotometer (Shimadzu Firm).
Preparation of platelet membrane fragments
Platelets have been manually concentrated from contemporary mouse complete blood and saved at − 80 °C. The next steps have been taken to arrange platelet membrane fragments (PMFs). Briefly, the platelets have been thawed thrice till they reached room temperature. Then, a centrifugation course of was carried out for 3 min at 4000 g to separate the platelet membranes. The pellets have been thereafter suspended in a PBS buffer containing 2 mM of protease inhibitor. Lastly, a sonication course of at 40 kHz and an influence of 100 W was carried out for five min, leading to a suspension of PMFs.
Preparation of nanoparticles
To arrange BSA nanoparticles, 120 mg BSA, 60 mg sodium dodecyl sulfate (SDS) and 4.4 mg dithiothreitol (DTT) have been weighed and added to a 5 mL pattern bottle. Subsequent, 3 mL of ultrapure water was added and the answer was positioned in a 90 °C oil bathtub at 180 r/min for two h. Subsequent, 0.97 g PMF was dissolved in 50 mL ultrapure water, and the pH worth was adjusted to 4.25–4.8 with 0.1 mol/L NaOH. Then, 25 μL of activated BSA answer (40 mg/mL) was taken and added right into a 24-well plate, adopted by including 975 μL PMF. The combination was positioned at 37 °C with shaking at 800 r/min for 4 h. The synthesized nanoparticles have been washed 2 to three occasions with a 100 kd ultrafiltration tube at 4 000 r/min for 10 min and at last saved at 4 °C for later use. For Getting ready BSA@LIR nanoparticles, 25 μL of activated BSA answer (40 mg/mL) was added to a 24-well plate, then 950 μL PMF was added, adopted by mixing underneath shaking situations. Subsequent, 25 μL of LIR answer was added, adopted by synthesis at 37 °C underneath shaking at 800 r/min for 4 h. The nanoparticles that have been synthesized underwent washing two to 3 occasions utilizing a 100 kd ultrafiltration tube at 4,000 r/min for 10 min. Afterward, the BSA protein focus was measured using a BCA protein package, and it was saved at a temperature of 4 °C. For the preparation of BSA@LIR-PMF nanoparticles, 1 mg/mL BSA@LIR answer and 1 mg/mL PMF answer have been ready, and blended based on the amount ratio of 1:1. Subsequent, the above-mixed answer was handed by 0.22 μm polycarbonate. The membrane was extruded forwards and backwards 20 occasions, and the sediment was resuspended for later use.
Characterization of the nanoparticles
The micromorphology of BSA, BSA@LIR, and BSA@LIR-PMF was examined utilizing transmission electron microscopy (TEM). Dynamic Gentle Scattering (DLS) was used to check the hydration kinetic diameter and Zeta potential of the nanoparticles earlier than and after drug loading and coating. The LIR detection package (enzyme-linked immunosorbent assay) was utilized to detect the encapsulation charge and loading charge of LIR, and the discharge of LIR in BSA@LIR-PMF.
Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE)
To show that the modified BSA@LIR-PMF has intact membrane proteins, RIPA lysate was used to cleave PMF and BSA@LIR-PMF adopted by centrifugation (14 000 r/min, 5 min) to acquire the extracted membrane proteins, after which the protein focus was decided utilizing a BCA protein focus assay package. The pattern was blended with loading buffer and boiled for five min. PMF and BSA@LIR-PMF have been loaded right into a 15% SDS-PAGE gel (35 μg per effectively) run at 120 V for 1.7 h and subsequently stained with Coomassie Good Blue for 60 min.
ATr5 tradition and remedy
The ATr5 cell traces have been bought from the Cell Financial institution of the Chinese language Academy of Sciences in Shanghai. These cell traces have been cultivated in Dulbecco’s modified Eagle’s medium (DMEM) from Gibco, Life Applied sciences, Grand Island, NY, USA. The medium was supplemented with 10% fetal bovine serum (FBS) from GIBCO Industries, USA. The cells have been then incubated in a humidified chamber at 37 °C with 5% CO2. Afterward, the cells have been handled with ox-LDL (10 μg/ml) and excessive glucose situation (30 mmol/L) for twenty-four h for pattern assortment and subsequent testing.
Cytotoxicity of nanoparticles
The cells have been seeded in a 96-well plate at a density of 5000 cells per effectively with a closing quantity of 200 μL. They have been then cultured at 37 °C, 5% CO2, and saturated humidity. As soon as the cells have been utterly hooked up, 100 nM BSA and BSA@PMF have been added in sequence (Subsequent in vitro experiments have been carried out utilizing the identical concentrations). The cells have been then incubated for varied durations (4 h, 8 h, 12 h). Following this, 20 μL of CCK-8 answer (Beyotime, China) was added to every effectively, and the cells have been incubated for a further 2 h in a 37 °C, 5% CO2 cell tradition incubator. The microplate reader was preheated prematurely, and the absorbance worth was measured at OD450 and saved for additional evaluation.
Cell phagocytosis
The experiment concerned seeding cells in a 6-well plate with a density of 4 × 105 cells per effectively and a closing quantity of two mL. The cells have been cultured at 37 °C, 5% CO2, and saturated humidity till they have been utterly hooked up. Then, isothiocyanate was used to label BSA@LIR-PMF with fluorescein (FITC) (Aladdin, China). The labeled nanoparticles have been incubated with cells for 4 and eight h, stained with DAPI (1:1000) (Thermo Fisher Scientific, USA) for 15 min, and washed with CLSM (ZEISS, Germany) to watch the mobile uptake of nanoparticles. Lastly, the fluorescence depth was measured utilizing Picture J (Nationwide Institutes of Well being, Bethesda, MD), and the outcomes have been expressed as imply FITC fluorescence depth (MFI).
Cell proliferation
Cells have been plated right into a 96-well plate at an preliminary density of 5000 cells per effectively and a closing quantity of 200 μl. The cells have been cultured for 12 h at 37 °C, 5% CO2, and saturated air humidity. Afterward, they have been uncovered to ox-LDL and excessive glucose situation (30 mmol/L) for twenty-four h.[21] Then separate units of cultures have been used that included BSA@PMF in a preheated microplate reader and, the OD450 studying was carried out.
Cell migration
There have been about 4 × 105 cells seeded in every of the 6 wells. 2 mL of the amount was used. The cells have been then cultured for 12 h at 37 °C, 5% CO2, and saturated humidity. Thereafter, the cells underwent the remedy of ox-LDL and excessive glucose (30 mmol/L) for twenty-four h. The tradition medium was subsequently eliminated after which BSA@PMF, LIR, BSA@LIR, and BSA@LIR-PMF have been added to the wells. It remained for 12 h. Then, we rigorously eliminated the chamber with tweezers, and the higher chamber liquid was dried and glued at room temperature with methanol for 30 min. Afterwards, the mounted liquid within the higher chamber was absorbed and crystal violet answer was used for staining at room temperature for 30 min. Subsequent, after rinsing and soaking in PBS a number of occasions, the liquid from the higher chamber was eliminated, adopted by switch to a glass slide for sealing with impartial resin. Lastly, 5 random fields have been chosen to rely underneath a microscope.
Vitality metabolism evaluation
PBS was used because the homogenization medium to interrupt the cells, after which the supernatant was taken by centrifugation. The intracellular lactate degree was detected utilizing the lactate check package (Nanjing Jiancheng, China), and the colorimetric measurement wavelength was 530 nm. Equally, the intracellular ATP degree was measured utilizing the ATP content material assay package (Nanjing Jiancheng, China), and the colorimetric measurement wavelength was 636 nm.
Detection of reactive oxygen species (ROS) ranges
The diploma of mobile oxidative harm was evaluated by measuring the content material of ROS utilizing 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA). The cells have been seeded in a 48-well plate at a density of 5 × 104 cells per effectively and cultured for 12 h at 37 °C, 5% CO2, and saturated humidity. After 24 h of publicity to ox-LDL, the tradition medium was eliminated, and the cells have been handled with BSA@PMF, LIR, and BSA@LIR-PMF for 12 h. Then, the cells have been incubated with 20 μM DCFH-DA answer (Thermo Fisher Scientific, USA) for 30 min at 37 °C, adopted by inexperienced fluorescence imaging utilizing a fluorescence microscope (infinite M200, TECAN, Switzerland). The imply fluorescence depth (MFI) of dichlorofluorescein (DCF) was analyzed utilizing Picture J. Untreated cells have been used as controls.
Detection of extracellular acidification charge (ECAR) and oxygen consumption charge (OCR)
The speed of dissolved O2 round cultured cells was measured utilizing the Seahorse Bioscience X96 mannequin from Agilent Applied sciences, which is an extracellular flux analyzer. The XF96 V3 cell tradition microplate developed by Seahorse Bioscience was used for culturing A7r5 cells in DMEM supplemented with 0.5% FBS at a density of 0.8 × 104 cells per effectively. The oxygen consumption charges (OCR) have been measured utilizing the mitochondria stress check package, which concerned injecting varied compounds into the microplate. The glycolytic exercise was assessed utilizing the identical XF-96 analyzer, which offered extracellular acidification charge (ECAR) related to glycolysis, most glycolytic capability, and non-glycolytic ECAR after injecting D-glucose (2 g/L), oligomycin (1 μM), and 2-Deoxyglucose (100 mM) onto the microplate.
Institution of diabetic atherosclerosis mouse mannequin
The Animal Care and Use Committee of Huashan Hospital, College of Medication, Fudan College, Shanghai accredited our research, which utilized C57BL/6 mice and APOE-/- weighing 20 ± 2 g and aged 6 weeks. These mice have been procured from Charles River Laboratories Animal Co., Ltd. (Beijing, China). They have been supplied with meals and water advert libitum in a managed surroundings for every week, with a 12 h gentle–darkish cycle at 25 ± 1 °C and 50 ± 10% relative humidity.
Male APOE-/- mice aged 6 weeks have been bought and fed a high-fat and high-cholesterol weight loss plan for 8–12 weeks. After that, the mice have been injected with streptozotocin (STZ) (50 mg/kg, Sigma) answer for 5 consecutive days for induction of diabetes. After 4 weeks of remedy with BSA@PMF, LIR, BSA@LIR, and BSA@LIR-PMF by tail-vain injection at a nanoparticle focus of 332 µg/ml (6 µg per animal), the mice have been sacrificed and their aortas have been collected for subsequent testing.
Oil pink staining of the aorta
To arrange the Oil Pink O working answer, we weighed 0.5 g of Oil Pink O powder and added 100 ml of 98% isopropyl alcohol. The combination was left in a 90 °C water bathtub for 1 h and filtered to acquire the Oil Pink O saturated liquid. The saturated liquid was then blended with double-distilled water at a ratio of three:2 and left to face for 10 min. After 10 min, the combination was filtered to acquire the Oil Pink O working answer. Each the saturated answer and dealing answer have been saved in a light-protected container. To stain the blood vessels, the glass dish was full of the Oil Pink O working answer till the blood vessel was utterly submerged. The dish was then positioned in a 37 °C water bathtub for 30 min. The staining answer was discarded, and 75% ethanol was added for differentiation twice, every for five min. The ethanol was then washed away with physiological saline or PBS. Lastly, the blood vessels have been immersed in physiological saline or PBS within the dish and footage have been taken.
Animal imaging
After administering isoflurane anesthesia, the mice have been positioned in a darkish field for recording. The imaging system used was a small animal multispectral in vivo imaging system. Fluorescent dyes have been used to label nanoparticles (BSA@LIR, BSA@LIR-PMF), which have been then injected into the mouse physique by the tail vein at a quantity of 100 μL/mouse. The imaging system captured the fluorescence emitted by the animal at a hard and fast time, permitting researchers to investigate the distribution of fluorescent medicine.
Histological evaluation
All aorta tissue specimens of mice have been analyzed by HE staining. Briefly, paraffin Sects. (5 μm) have been ready and, after deparaffinization with xylene for 10 min and rehydration in an ethanol gradient, hematoxylin (1%) was used to stain the sections for five min. Subsequent, the slides have been washed, adopted by differentiation with 1% hydrochloric acid alcohol for five s. Subsequent, the slides have been stained with 1% eosin alcohol for 1 min. After staining, the specimens have been dehydrated in an alcohol gradient and counterstained in xylene. Lastly, the specimens have been mounted and noticed underneath a lightweight microscope to investigate histological options.
For Masson staining, after being deparaffinized, the sections have been refixed in Bouin’s answer at 56–60 °C for 1 h. Subsequent, the sections have been stained utilizing hematoxylin, washed completely, and handled with Masson Ponceau acidic fuchsin answer, aniline blue, and glacial acetic acid aqueous answer. Lastly, the sections have been handled with alcohol, mounted with a coverslip, and examined underneath a microscope.
Oil pink staining of aorta sections
To arrange the frozen sections for microscopic examination, the sections have been positioned right into a dye vat full of Oil Pink O for 10 to fifteen min after which washed with distilled water at a temperature of about 37 °C for five to twenty s. They have been then stained with gentle hematoxylin counterstaining answer for 3 to five min and washed with water for about 30 to 60 s. The reagent three-aqueous mounting agent was added dropwise to the floor of the glass slide earlier than the floor water was utterly dry and the slide was sealed (the aqueous mounting agent was heated to a liquid state in 60 °C heat water earlier than sealing the slide). Lastly, a microscopic examination of the ready sections was carried out.
Immunofluorescence assay
The aortic tissue sections have been ready, dewaxed, hydrated, and soaked in 3% H2O2 methanol for 10 min to take away endogenous catalase. After high-pressure antigen retrieval, serum blocking was carried out earlier than incubation with Anti-Cleaved Caspase-3 (Abcam, ab32042) at a dilution ratio of 1:100, Anti-alpha clean muscle Actin (Abcam, ab7817) at a closing focus of 1 µg/ml, and Anti-CD31 (Abcam, ab222783) in a single day. After washing with PBS, Biotinylated Goat Anti-Rabbit IgG (H + L) (Abcam, ab64256) was added at a dilution ratio of 1:200 and incubated at room temperature for two h. DAPI counterstaining was carried out for 15 min, after which the slides have been mounted. The samples have been noticed and footage have been taken underneath a fluorescence microscope (infinite M200, TECAN, Switzerland). ImageJ software program (Nationwide Institutes of Well being, Bethesda, MD) was used for quantitative evaluation to acquire the cumulative optical density IOD.
Biochemical indicators
Serum was separated from arterial blood, and plasma whole ldl cholesterol (TC), triglyceride (TG), high-density lipoprotein (HDL) ldl cholesterol, and low-density lipoprotein (LDL) ldl cholesterol concentrations have been measured utilizing a lipid meter (PTS, USA).
Biosafety evaluation
First, we collected the samples and ready paraffin sections utilizing a sequence of steps, together with assortment and fixation, washing, dehydration, transparency, waxing, embedding, slicing, unfolding, fishing, patching, and baking. Subsequent, we dewaxed the sections utilizing two rounds of xylene remedy, adopted by a hydration course of involving a sequence of alcohol washes. After hydration, we dyed the sections utilizing hematoxylin and eosin staining, with a short differentiation step utilizing hydrochloric acid alcohol. Lastly, we dehydrated the sections once more utilizing a sequence of alcohol washes and made them clear utilizing xylene, earlier than sealing the slides.
Quantitative real-time PCR
Complete RNA was extracted from cells and tissue samples utilizing the Trizol (Thermo Fisher Scientific, USA) methodology, and cDNA was obtained by a reverse transcription package (Takara Bio Inc, Japan). RT-qPCR was carried out utilizing SYBR Inexperienced qPCR Grasp Combine (Takara Bio Inc, Japan) as follows: an preliminary denaturation was carried out at 95 °C for 30 s, adopted by 40 cycles of 95 °C for five s and 60 °C for 30–60 s. Relative expression of mRNA (normalized to actin) was calculated by the 2-ΔΔCq methodology.
Western blot
Protein was extracted from each cells and tissue samples utilizing RIPA lysis buffer from Beyotime, China. Subsequently, protein quantification was carried out utilizing the BCA protein focus assay package, additionally from Beyotime, China. The pattern was then loaded along with PageRuler™ pre-stained protein ladder from Thermo Fisher Scientific, USA, and underwent SDS gel electrophoresis, switch, and blocking. The pattern was then mixed with CHOP (D46F1) Rabbit mAb (1:1000) (#5554, Cell Signaling Expertise, USA), eIF2α Antibody (1:1000) (#9722, Cell Signaling Expertise, USA), GRP78 (BiP) Antibody (1:1000) (#3183, Cell Signaling Expertise, USA), PGC-1a Antibody (1:1000) (ab191838, Abcam, USA) and CPT1 Antibody (1:1000) (ab189182, Abcam, USA) and incubated in a single day at room temperature. Goat Anti-Rabbit IgG H&L (HRP) (ab205719, Abcam, UK) or Goat Anti-Mouse IgG H&L (HRP) (ab205719, Abcam, UK) was incubated for two h and developed by a chemiluminescence imaging system from Shanghai Ouxiang Scientific Instrument Co., Ltd., China. Lastly, the grey worth of the strips was measured by Picture J from the Nationwide Institutes of Well being in Bethesda, MD.
Statistics
Knowledge was expressed as imply ± SD utilizing GraphPad Prism. The variations among the many teams have been carried out utilizing one-ANOVA adopted by Turkeys’ posttest. The importance cutoff was 0.05.