Supplies
Tetraethyl orthosilicate (TEOS), polyvinylpyrrolidone (PVP, Mw = 10000), polyallylamine hydrochloride (PAH, Mw ≈ 15000), polyacrylic acid (PAA, Mw ≈ 1800), resorcinol (A.R., 99%), formaldehyde answer, sodium carbonate (NaOH), potassium permanganate (KMnO4), ammonia answer, and hexadecyltrimethoxysilane (HDTMS, G.C.≥85%) had been bought from Sino-pharm Chemical Reagent Co., Ltd. Colchicine was bought from Chengdu Should Biotechnology Co., Ltd. (Chengdu, China). RAW 264.7 cells had been bought from the Chinese language Academy of Science (Shanghai, China). Fetal bovine serum (FBS) was bought from Inside Mongolia Opcel Biotechnology Co., Ltd. (Inside-Mongolia, China). Calcein-AM, propidium iodide (PI), DiI, DiR, DHE, 4,6-diamidino-2-phenylindole (DAPI), paraformaldehyde, DCFH-DA, DAF-FM DA and Actin-Tracker Pink-Rhodamine had been bought from Beyotime Biotechnology (Shanghai, China). Ldl cholesterol was obtained from Avanti Polar Lipids, Inc. (USA). A Cell Counting Equipment-8 (CCK-8) was bought from Beyotime Institute of Biotechnology (Shanghai, China). CD86, CD206, HIF-1α and ABCG1 antibodies had been bought from Beyotime Biotechnology (Shanghai, China). Integrin α, Integrin β, CD47 and CCR2 antibodies had been obtained from AiFang Organic (Hunan, China). ELISA kits for IL-6, IL-1β, TNF-α, IL-18, and CRP had been obtained from Jiangsu Sumeike Organic Expertise Co., Ltd. (Jiangsu, China).
Synthesis of mesoporous manganese dioxide and the profitable coating of macrophage membrane
Preparation of mesoporous manganese dioxide
First, we have to synthesize the silica spheres (SiO2) within the middle. The synthesis process was derived from an earlier methodology with a number of alterations [45]. 98 mL of ethanol and 10 mL of water had been combined collectively, after which 1.25 mL of ammonia (28%) and 5 mL of TEOS had been added. After stirring at room temperature (25 °C) for 20 h, the SiO2 was collected by centrifugation and rinsed a number of instances with ethanol and water.
Within the second step, we synthesized SiO2@MnO2. 50 mg of SiO2 and 0.5 g of PVP had been stirred at room temperature in a single day, after which the combination was washed with 8,000 r/min of centrifugal water thrice to acquire the SiO2@PVP product. Then, 25.5 mL of water, 20 mg of metiodephenol, and 28 µL of formaldehyde had been added to the obtained product. After thorough mixing, 100 µL of ammonia water (2.8%) was added, the temperature was elevated to 60 °C, the combination was stirred for two h, the temperature was elevated to 100 °C, the combination was stirred for two h, and the response was terminated by cooling and washing with centrifuged water thrice to acquire the SiO2@RF product. 5 mL water was added to 10 mg of SiO2@RF, combined completely, after which 3 mL of KMnO4 (0.2 M) was added. The combination was stirred at room temperature for 4 h, adopted by three washes with centrifuged water to acquire the specified SiO2@MnO2 product.
Lastly, we efficiently ready the hole mesoporous manganese dioxide (HMnO2). To realize a hole construction, a mix of 20 mL of 1 M NaOH and 50 mg of SiO2@MnO2 was stirred in a single day at room temperature. The combination was then centrifuged and washed thrice with water to acquire HMnO2. The obtained HMnO2 was dispersed in 5 mL of H2O, 5 mL of PAH answer (5 mg/mL) was added below ultrasonic stirring, the combination was stirred for 3 h, and the answer was collected. The pattern was rinsed with deionized water to yield a colorless and clear supernatant. The synthesized HMnO2-PAH was slowly added to five mL of PAA answer (5 mg/mL) after which subjected to ultrasonic stirring for 3 h, adopted by washing and assortment for future use.
The profitable coating of macrophage membrane
We ready the macrophage membranes (MM). RAW264.7 cells had been inoculated right into a tradition dish and incubated for twenty-four h. Subsequent, the tradition medium was collected, and the cells had been centrifuged to separate the cells and particles. The supernatant was concentrated in a rotating column (Millipore, USA) with an IOkDa molecular weight at 2500 × g and 4 °C for 8 min. The collected liquid was subsequently centrifuged at 800 × g and 4 °C for 40 min, after which the ensuing precipitate was resuspended in sterile phosphate-buffered saline (PBS) and saved at -80 °C for subsequent experiments.
Lastly, we efficiently wrapped the macrophage membrane to acquire the Col@HMnO2-MM. Owing to the modification of PAA/PAH, HMnO2 was stirred with Col at room temperature for twenty-four h to acquire Col@HMnO2. To organize the MM coating Col@HMnO2-MM, the combination of Col@HMnO2 and the MM was extruded by a polycarbonate membrane with 400 nm pores utilizing a liposome extruder, which was repeated 18 instances, adopted by centrifugation at 13,000 rpm for 10 min to acquire the Col@HMnO2-MM nanomedicine.
Characterization of Col@HMnO2-MM
The morphologies of SiO2, SiO2@RF, SiO2@MnO2, HMnO2, and Col@HMnO2-MM had been noticed through transmission electron microscopy (TEM) (FEI Tecnai G2 F30, USA). The basic mapping and EDX spectrum of Col@HMnO2-MM had been additionally examined by TEM. The construction of HMnO2 was investigated by XRD (BRUKER D8 ADVANCE, Germany), XPS (Thermo Scientific Ok-Alpha), and FTIR (Thermo Scientific Nicolet iS5). The particular floor space and corresponding pore measurement distribution of HMnO2 had been measured by a BET floor space adsorption analyzer (ASAP 2460, USA). The unloaded Col within the supernatant of Col@HMnO2 was measured utilizing a BCA protein assay package, and the drug loading and encapsulation effectivity had been calculated as follows: encapsulation effectivity = (WComplete added Col -WUnloaded Col) / WComplete added Col × 100%; loading effectivity = (WComplete added Col -WUnloaded Col) / (WLoaded Col + WComplete added HMnO2) × 100%. A dynamic mild scattering (DLS) laser particle measurement analyzer (Zetasizer Nano ZS90, UK) was used to research the scale and zeta potential of MM, Col@HMnO2, and Col@HMnO2-MM, and to watch the scale stability of Col@HMnO2-MM at 1, 3, 5 and seven d. The protein profiles of HMnO2, MM and Col@HMnO2-MM had been validated through western blot evaluation.
Launch property of col
To evaluate the drug launch sample of Col, 1 mL of both Col@HMnO2-MM (1 mg/mL) or Col@HMnO2 (2 mg/mL) was positioned into dialysis luggage with a molecular weight cutoff of 3500 Da. These dialysis luggage had been immersed in 50 mL of PBS at both pH 7.4 or 5.5 and maintained at 37 °C. At particular time intervals, samples of the discharge medium (500 µL) had been obtained. The Col focus was measured utilizing UV − vis spectroscopy.
ROS scavenging properties of Col@HMnO2-MM in vitro
•O2
− scavenging property
The superoxide anion (•O2−) was ready by reacting xanthine oxidase with xanthine. Then, Col@HMnO2-MM was added and allowed to react for five min and 10 min, and the residual •O2− was measured by electron paramagnetic resonance (EPR).
•OH scavenging property
Hydroxyl radicals (•OH) had been ready by a TiO2/UV system below 340 nm ultraviolet mild. Then, 30 µg/mL Col@HMnO2-MM was added and allowed to react for five min and 10 min, and the residual •OH was measured by EPR.
H2O2 scavenging property
H2O2 (10 mM) was used because the management, the Col@HMnO2-MM pattern was added to the response combination for five min and 10 min, and hydrogen peroxide clearance was noticed by EPR.
O2 focus monitoring
Col@HMnO2-MM (30 µg/mL) was added to 10 mL of H2O2 (10 mM) answer. Col@HMnO2-MM alone and H2O2 answer alone had been used because the management teams. Dissolved oxygen was utilized to measure the O2 ranges within the answer for 10 min.
DPPH scavenging property
DPPH answer (0.04 mg/mL) was ready with anhydrous ethanol. Then, Col@HMnO2-MM was added and allowed to react for five min and 10 min, and the residual DPPH was measured by EPR.
Elimination of alkyl and alkoxy radicals
Alkyl radicals and alkyl radicals had been ready through a PAA/UV system. Then, Col@HMnO2-MM was added and allowed to react for five min and 10 min, and the residual alkyl radicals and alkoxy radicals had been measured by EPR.
LPO inhibition assay of Col@HMnO2-MM
400 µL completely different Col@HMnO2-MM options (10, 20, 30, 40, and 50 µg/mL) had been combined with the as-prepared MDA working answer. Subsequent, the samples had been heated in boiling water at 95 °C for 50 min. After cooling, the examined answer was centrifuged at 15,000 rpm for 20 min, and the absorbance of the supernatant was measured at 535 nm through a UV − vis spectrophotometer.
Biosafety of Col@HMnO2-MM
We first accomplished the hemolysis experiment of nanomedicine in vitro. Pink blood cells had been obtained from contemporary blood samples from SD rats by centrifugation (3000 r/min) and washing thrice. The erythrocytes had been incubated with HMnO2-MM, Col, or Col@HMnO2-MM for 4 h after which centrifuged at 13,500 rpm for five min. The absorbance of the supernatant at 540 nm was assessed through a microplate reader. Pink blood cells had been positioned in water because the constructive management and in PBS because the adverse management. The hemolysis charge was calculated as follows: hemolysis charge% = (experimental group absorbance − adverse group absorbance) / (constructive group absorbance – adverse group absorbance) × 100%.
We investigated the cytotoxicity of nanomedicine. First, we explored the optimum focus of colchicine [46, 47]. RAW264.7 cells had been inoculated in 96-well plates, completely different concentrations of colchicine (0.5, 1, 2, 4, 8, 16, 32, or 64 µg/mL) had been added to the cell cultures for twenty-four h, after which the CCK8 assay was used to detect cytotoxicity. Equally, we explored the security of various doses of Col@HMnO2-MM (10, 20, 30, 40, and 50 µg/mL) on Raw264.7 cells. Varied concentrations of nanomedicine had been added to the cells for twenty-four h, and CCK-8 assays had been carried out to judge their cytotoxicity.
Lastly, we verified the in vivo biosafety of nanomedicine. HMnO2-MM, Col, and Col@HMnO2-MM had been injected into wholesome C57BL/6J mice. Three days later, we performed weight testing on the mice within the numerous teams. Recent blood was collected for biochemical and routine blood exams, after which organs and tissues had been collected for H&E staining.
Mobile uptake
We examined the phagocytosis of nanomedicine in vitro with activated and un-activated macrophages. We first explored the nanomedicine uptake by un-activated macrophages. RAW 264.7 cells had been obtained from the Chongqing Key Laboratory of Ultrasound Molecular Imaging. RAW 264.7 cells (5 × 104) had been seeded in confocal dishes and co-incubated for twenty-four h. DiI-labeled Col@HMnO2 and Col@HMnO2-MM (30 µg/mL) had been added, and the cells had been cultured for 4 h. The cell nuclei had been stained with DAPI for 15 min after fixation with paraformaldehyde. Lastly, the cells had been noticed through CLSM.
Subsequently, we explored the nanomedicine uptake by activated macrophages. RAW264.7 cells (5 × 104) had been plated in confocal dishes for twenty-four h after which handled with LPS (1 µg/mL). DiI-labeled Col@HMnO2 and Col@HMnO2-MM had been added, and the cells had been cultured for 4 h. The cells had been washed with PBS, and the nuclei had been stained with DAPI for 15 min after fixation with paraformaldehyde. The cells had been subsequently visualized through CLSM. As well as, organic TEM was used to watch Col@HMnO2-MM within the activated macrophages after 0.5 h and 4 h.
In vitro antioxidative protecting results
We used the CCK8 assay to judge cell survival. RAW 264.7 macrophages had been plated in 96-well plates and handled with HMnO2-MM, free Col or Col@HMnO2-MM for two h. For the management teams, the cells had been cultured in medium with none therapy. The cells had been handled with 400 µM H2O2 for twenty-four h. For the management, the cells had been handled with contemporary medium for comparability. CCK-8 assays had been carried out to judge the cell survival charge on the second day.
The mitochondrial membrane potential adjustments had been detected by JC-1. RAW264.7 cells (5 × 104) had been seeded in confocal dishes and incubated with HMnO2-MM, free Col or Col@HMnO2-MM for two h. Subsequent, the cells had been uncovered to H2O2 (400 µM) for a interval of 24 h. A JC-1 detection package was used to establish alterations within the mitochondrial membrane potential.
We additionally evaluated the apoptosis detection by circulation cytometry. RAW 264.7 cells had been plated at a density of 1 × 106 cells per effectively in six-well plates and incubated in a single day for adhesion. HMnO2-MM, Col, and Col@HMnO2-MM had been added to the wells for a 2 h pretreatment. The cells had been subsequently stimulated with H2O2 (at a remaining focus of 400 µM) for twenty-four h. Move cytometry was used to detect macrophage apoptosis.
Intracellular ROS scavenging exercise
RAW 264.7 macrophages had been cultured in confocal dishes and handled with HMnO2-MM, free Col or Col@HMnO2-MM for two h. For the management teams, the cells had been cultured in medium with none therapy. Then, the cells had been handled with 1 µg/mL LPS for twenty-four h, whereas the adverse management was handled with contemporary medium once more. After washing with PBS, the cells had been handled with 10 µM DCFH-DA in serum-free medium for 30 min of incubation. The cells had been visualized through CLSM.
In vitro anti‑inflammatory results
After receiving the identical therapy described above, the cells had been stimulated with LPS, an ELISA package was used to detect numerous inflammatory components within the tradition supernatant, and a BCA package was used to detect complete protein ranges.
M1 to M2 polarization of macrophages
For the RT-PCR experiments, RAW 264.7 macrophages had been seeded right into a 6-well plate and activated with LPS (1 µg/mL) to induce M1 polarization. Afterward, HMnO2-MM, free Col or Col@HMnO2-MM was added, and the combination was incubated for twenty-four h. After the cells had been collected, complete RNA was extracted with TRIzol reagent. The mRNA ranges of the macrophages had been measured through RT‒PCR. Then, we noticed the expression of CD86 and CD206 through CLSM. The cells had been subjected to equivalent remedies and stuck with 4% paraformaldehyde for immunofluorescence staining. The membranes had been uncovered to the first antibody and incubated in a single day at 4 °C. Secondary antibodies labeled with fluorescent tags had been added and incubated for 1 h. The nuclei had been costained with DAPI, and CLSM was used for commentary and imaging. LPS-stimulated RAW 264.7 cells had been cocultured with completely different nanomedicine therapy teams. We used a FITC-labeled anti-mouse CD86 antibody and an APC- labeled anti-mouse CD206 antibody. The expression of CD86 and CD206 floor proteins was detected through circulation cytometry.
Mobile uptake of ox-LDL
Confocal dishes had been used to detect the inhibitory impact of Col@HMnO2-MM on the uptake of oxidized low-density lipoprotein (ox-LDL). RAW 264.7 cells had been initially uncovered to 1 µg/mL LPS for twenty-four h, adopted by a 2-hour incubation with Col@HMnO2-MM. Second, the above cells had been cultured with DiI-oxLDL at a focus of 40 µg/mL for 4 h. Remark of ox-LDL uptake by the cells was carried out through CLSM after staining with Hoechst 33,342 for 30 min.
Regulation of HIF-1α, ABCA1 and ABCG1 protein expression
To induce the upregulation of HIF-1α protein expression, the cells had been incubated with CoCl2 (100 µM) in serum-free medium for 4 h. Later, the cells had been subjected to numerous remedies for twenty-four h, and protein expression was detected through CLSM, RT-PCR and Western blotting. To detect the expression of ABCA1, the cells had been subjected to completely different remedies. Following the extraction of complete RNA through TRIzol reagent, the relative expression of ABCA1 mRNA was decided through RT-PCR. To detect the expression of ABCG1, the cells had been subjected to completely different remedies. The outcomes had been visualized utilizing CLSM. After the qPCR and immunofluorescence outcomes had been obtained, we used the identical therapy strategies to confirm the expression of the ABCA1 and ABCG1 proteins through Western blotting.
Ldl cholesterol efflux assay
We utilized fluorescent NBD-cholesterol to analyze ldl cholesterol efflux from macrophages. After the cells had been handled in numerous methods for 12 h, 1 µg/mL NBD-cholesterol was added to the cell tradition dishes. Then, the cells had been washed with PBS and cultured for a further 6 h. The supernatant of the medium was collected and centrifuged (14000 rpm, 6 min) to take away particles, whereas the cells had been lysed with sodium hydroxide (1 mL, 0.05 M). The focus of NBD-cholesterol was detected by a fluorescence enzyme spectrometer at Ex = 500 nm and Em = 535 nm, and the share of ldl cholesterol efflux was calculated.
Oil pink O staining for the formation of froth cells
After the cells had been handled with 30 µg/mL ox-LDL, the ready nanomedicine was added to the completely different teams and incubated for twenty-four h. The cells had been subsequently mounted with 4% paraformaldehyde for 25 min, adopted by staining with oil pink O for 15 min. Then, the cells had been washed with isopropyl alcohol as soon as and washed with water twice earlier than they had been stained with hematoxylin for 1 min. After being washed with water, lipid droplets within the cells had been noticed below a lightweight microscope.
Institution of the APOE−/− mouse AS mannequin
All animal procedures and protocols had been accredited by the Experimental Animal Ethics Committee of the Second Affiliated Hospital of Chongqing Medical College. The experiments had been carried out based on the Nationwide Rules on the Use of Experimental Animals. Male C57BL/6 mice (6–8 weeks) and male apolipoprotein E-deficient (ApoE−/−) mice (roughly 6–8 weeks previous) had been supplied by Ensville Biotechnology Co., Ltd (Chengdu, China). APOE−/− mice had been fed a high-fat food regimen for 13 weeks to ascertain the atherosclerosis mannequin.
Distribution and pharmacokinetic research in vivo
Preparation of DiR-labeled nanomedicine
6 µL DiR (10 mg/mL) was added to 0.6 mg/mL Col@HMnO2-MM options (20 mL), which had been stirred utterly at room temperature for two h, centrifuged (10000 r *8 min), washed twice, and at last dispersed in PBS [48, 49].
Organic concentrating on of DiR-labeled nanomedicine in vivo
DiR-labeled Col@HMnO2-MM had been injected into the tail veins of AS mannequin mice (3 mg/kg), and after completely different cycle instances (2, 4, and 6 h), the aortas had been eliminated for fluorescence imaging in vivo to judge the concentrating on of the plaque websites within the atherosclerotic mice. Afterward, 6 h was chosen because the time level to match the concentrating on efficiency of the nanomedicine in vivo.
Metabolism of DiR-labeled nanomedicine in vivo
Fluorescence imaging was performed on the guts, liver, spleen, lungs and kidneys at 6 h and 48 h postadministration of the nanomedicine to judge their metabolism within the organs. Within the pharmacokinetic research, the ocular blood of the mice was injected with DiR-labeled Col@HMnO2-MM by the tail vein and picked up at particular time factors (2, 4, 6, 8, 10, 12, 24 and 48 h). Blood samples (100 µL) had been obtained by static storage and centrifugation (3000 r*10 min) for fluorescence imaging to judge the half-life of the nanomedicine Col@HMnO2-MM.
In vivo analysis of therapeutic results and research of therapeutic mechanisms
In vivo animal experiments, APOE−/− mice had been fed a Western food regimen to ascertain an AS mannequin, whereas management mice had been fed a traditional food regimen. On the finish of the fifth week, the mice within the above mannequin teams had been randomly divided into 4 teams with completely different therapy strategies (the conventional saline group, the HMnO2-MM group, the Col group and the Col@HMnO2-MM group), and nanomedicine was injected by the tail vein twice per week. After 8 weeks of therapy, the aorta was excised, and the mice had been killed. The aorta was washed with 10% paraformaldehyde for 30 min for follow-up analysis. The complete aorta was stained with oil pink O, and the plaque space was subsequently analyzed with Picture-Professional Plus 6.0 software program. Aorta sections had been mounted in 10% paraformaldehyde after which embedded in 3.5 μm thick paraffin for H&E staining previous to histological examination. The next paraffin sections had been evaluated by Masson’s trichrome staining and with CD68, MMP-9 and α-SMA antibodies. Aortic sections had been frozen and DHE fluorescent dye was utilized to evaluate ROS manufacturing on the plaque location. To check the polarization of macrophages, aortic sections had been incubated with anti-Arg-1 and anti-iNOS antibodies. Serum samples had been collected, and the degrees of TNF-α, IL-6, CRP, IL-1β and IL-18 had been measured by ELISA kits to judge the extent of serum irritation. We collected the attention blood of the mice and measured their serum levels of cholesterol. In the course of the therapy interval, main organs had been collected and stained with H&E for biosafety evaluation.
Statistical evaluation
The information used within the experiments are expressed because the means ± commonplace deviations. The variances had been in contrast through Scholar’s t-test and one-way ANOVA. (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001).
