Supplies
PLGA-PEG-COOH (PLGA (lactide:glycolide = 50:50, MW: 10000 Da); PEG (MW: 2000 Da)) was bought from Xi’an Ruixi Organic Know-how Co. (Xi’an, China). Dulbecco’s modified Eagle’s medium (DMEM) was bought from HACAKA (Shanghai, China). Colchicine, polyvinyl alcohol (PVA; MW: 30,000–70,000 Da), the fluorescent dyes 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI), 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide (DiR), 4′,6-diamidino-2-phenylindole (DAPI), N-(3-dimethylaminopropyl)-N’-ethylcarbodii-mide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) have been supplied by Sigma‒Aldrich Company (St Louis, MO, USA). VHPK (VHPKQHRGGSKGC) and FITC-VHPK peptides have been bought from Qiangyao Organic Know-how Co. (Suzhou, China). The Cell Counting Package-8 (CCK-8) assay equipment was bought from Dojindo Laboratories (Mashiki-machi, Tabaru, Japan). Recombinant human tumor necrosis issue α (TNF-α) was bought from NoVo Protein Scientific, Inc. (Canada). The anti-mouse CD68 antibody, anti-mouse VCAM-1 antibody, anti-mouse MMP-9 antibody, anti-mouse NLRP3 antibody, anti-mouse IL-1β antibody, anti-mouse IL-18 antibody, anti-mouse p65 antibody, and anti-mouse caspase-1 antibody have been bought from Cell Signaling Know-how (MA, USA). Enzyme-linked immunosorbent assay (ELISA) kits have been bought from Boster Organic Know-how Co. Ltd. (Wuhan, China). Oil crimson O (ORO) and hematoxylin and eosin (H&E) have been bought from Servicebio (Wuhan, China). Human umbilical vein endothelial cells (HUVECs) have been bought from Science Cell (Carlsbad, CA).
Preparation of VHPK-PLGA@COL and affirmation of VHPK peptide binding
Preparation of VHPK-PLGA@COL
PLGA@COL was ready utilizing a modified double-emulsion technique (W/O/W technique) [28, 29]. In keeping with this technique, 50 mg of PLGA-PEG-COOH and 25 mg of colchicine have been absolutely dissolved in 2 mL of chloroform (CHCl3) because the oil part. Then, 200 μL of double-distilled water was added to function the internal aqueous part, and the combination was emulsified with a sonicator (Sonics & Supplies Inc., Newtown, Connecticut, USA) to acquire a major emulsion. Subsequently, 5 mL of 4% PVA resolution was added for the second sonication cycle to kind a W/O/W double emulsion. Ten milliliters of two% isopropanol resolution was then added, and the answer was magnetically stirred for 4 h till the natural solvents had evaporated utterly and the surfaces of the NPs solidified. Lastly, PLGA@COL was purified by centrifugation (10,000 rpm, 7 min).
VHPK-PLGA@COL was fabricated by the carbodiimide technique [30]. Briefly, extra EDC and NHS at a molar ratio of two:1 have been added to 0.1 M MES buffer resolution (pH = 5.2) to activate the carboxyl group of PLGA@COL, and the combination was allowed to react on a shaker for two h. The unreacted EDC and NHS have been eliminated by centrifugal washing. Then, the activated PLGA@COL and 5 mg of VHPK peptide have been dispersed in 0.1 M MES buffer resolution (pH = 8) and reacted in a shaker for 12 h. Lastly, VHPK-PLGA@COL was rinsed with double-distilled water thrice to take away the unreacted supplies.
DiI- or DiR-labeled PLGA@COL or VHPK-PLGA@COL was added to an applicable quantity of DiI or DiR when dissolving PLGA-PEG-COOH. We fabricated FITC-labeled NPs utilizing an analogous technique besides we changed the VHPK peptide with FITC-VHPK peptide. VHPK-PLGA was ready utilizing the identical procedures described above with out the addition of colchicine in step one as a clean management.
Affirmation of VHPK peptide binding
To verify the conjugation of the VHPK peptide to PLGA@COL, VHPK-PLGA@COL was visualized by observing the colocalization of DiI-labeled PLGA@COL (crimson) and FITC-labeled VHPK peptide (inexperienced) utilizing confocal laser scanning microscopy (CLSM; A1R, Nikon, Tokyo, Japan). The service charge of the VHPK peptide was analyzed by stream cytometry (FCM; FACS Vantage SE, Becton Dickinson, San Jose, CA, USA). To additional consider the density of VHPK peptide on VHPK-PLGA@COL, the focus of VHPK peptide within the supernatant was decided utilizing high-performance liquid chromatography (HPLC, Kromasil 100-5C18: 5 µm, 4.6 mm × 250 mm; Temperature: 25 ℃; Cell part: 0.1% Trifluoroacetic Acid in Acetonitrile and 0.1% Trifluoroacetic Acid in water; Circulate Charge: 1.0 ml/min; Run Time: 20 min; Wavelength: 220 nm). The VHPK peptide binding capability (BC) and binding effectivity (BE) have been calculated as follows:
$$BC,(% ), = ,frac{mass,of,VHPK,peptide,binding,on,nanoparticles}{{mass,of,nanoparticles}}, instances ,100%$$
$$BE,(% ), = ,frac{mass,of,VHPK,peptide,binding,on,nanoparticles}{{mass,of,VHPK,peptide,used}}, instances ,100%$$
The mass of VHPK peptide on nanoparticles = mass of VHPK peptide used – mass of VHPK peptide within the supernatant.
Characterization of VHPK-PLGA@COL
The morphology and construction of VHPK-PLGA@COL have been noticed utilizing scanning electron microscopy (SEM; Hitachi S-3400N, Hitachi, Ltd., Tokyo, Japan) and transmission electron microscopy (TEM; Hitachi H-7600, Hitachi, Ltd., Tokyo, Japan). The dimensions, polydispersity indexes (PDIs), and zeta potentials of VHPK-PLGA and VHPK-PLGA@COL have been measured by a dynamic mild scattering detector (DLS, Malvern Devices, Malvern, UK). To check the steadiness of VHPK-PLGA@COL, the dimensions and PDI have been monitored utilizing DLS in phosphate-buffered saline (PBS) containing 10% fetal bovine serum (FBS) for 7 d.
The usual curve of colchicine dissolved in double-distilled water was established by measuring the absorbance of options with completely different concentrations with a UV‒vis–NIR spectrophotometer at a wavelength of 353 nm. The focus of colchicine was calculated based mostly on the corresponding absorbance of the UV spectrum at 353 nm. The drug loading capability (LC) and encapsulation effectivity (EE) have been calculated as follows:
$$LC,(% ), = ,frac{mass,of,COL,encapsulated,on,nanoparticles}{{mass,of,nanoparticles}}, instances ,100%$$
$$EE,(% ), = ,frac{mass,of,COL,encapsulated,on,nanoparticles}{{mass,of,COL,used}}, instances ,100%$$
The mass of COL encapsulated in nanoparticles = mass of COL used—mass of COL within the supernatant.
Drug launch evaluation
In vitro drug launch evaluation
The cumulative launch of colchicine from VHPK-PLGA@COL was investigated by including the nanoparticles right into a dialysis bag (MWCO 3500 Da) and immersing it in 50 mL of PBS (pH 7.4) at 37 °C or 4 °C with shaking at 100 rpm. At particular time intervals (0, 0.5, 1, 1.5, 2, 2.5, 3, 6, 10, 16, 20, 24, 30, 39, and 48 h), 1 mL of pattern was withdrawn from the buffer resolution and changed with an equal quantity of contemporary PBS. As a management, an equal quantity of free colchicine was added to a dialysis bag (MWCO 3500 Da) at 37 °C with shaking at 100 rpm. The cumulative launch of colchicine was calculated in keeping with the usual curve.
In vivo drug launch evaluation
Male SD rats have been bought from the Animal Heart of Chongqing Medical College. Rats have been randomly divided into two teams (n = 3 for every group). Colchicine and VHPK-PLGA@COL have been intravenously administered through the tail vein at an equal dose of 0.1 mg/kg colchicine. Blood samples (200 µL) have been drawn from the carotid vein on the preset time factors, e.g., 0.25, 0.5, 0.75, 1, 2, 4, 8, 12, 24, and 48 h, after intravenous administration, and centrifuged instantly at 3000 rpm for five min to acquire plasma. The plasma was positioned in a 3 KDa centrifugal filter and centrifuged for 10 min at 10,000 rpm to separate free colchicine from plasma. The ultrafiltrate (50 µL) was positioned in a glass tube with 50 µL of the interior normal (20 ng/ml tegafur), and a pair of ml of n-hexane:dichloromethane:isopropanol (300:150:15, v/v/v) was added. The combination was vortexed for 3 min and centrifuged at 3500 rpm for 10 min. Then, the higher natural layer was decanted into one other tube and evaporated to dryness at 40 ℃ below a delicate stream of nitrogen. The residue was reconstituted in 50 µL of cellular part after which was injected right into a liquid chromatography–tandem mass spectrometer (LC–MS/MS) to measure the colchicine focus. The LC–MS/MS system consisted of an HPLC (Agilent Applied sciences, Palo Alto, CA, USA) and a mass spectrometer (MS, Utilized Biosystems Sciex, Ontario, Canada) utilizing electrospray ionization (ESI). Chromatography was carried out on a Zorbax Prolong C18 column (5 µm, 150 mm × 4.6 mm i.d. Agilent Applied sciences) maintained at 40 ℃ with a cellular part of formic acid:10 mM ammonium acetate:methanol (1:49:75, v/v/v) at a stream charge of 1.1 ml/min. For quantitative evaluation, the MS was run in a number of response monitoring (MRM) mode. The MRM transitions for colchicine have been 400.1 → 358.3 (m/z) and for tegafur have been 200.5 → 130.9 (m/z).
Cell tradition and institution of the inflammatory cell mannequin
HUVECs have been cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin and incubated at 37 °C in a 5% CO2 ambiance. Cultured cells within the logarithmic development part have been used for cell experiments. Inflammatory endothelial cells have been induced by incubation with 20 ng/mL TNF-α [4] at 37 °C for twenty-four h.
Institution of the mouse mannequin of atherosclerosis
Six-week-old homozygous male apolipoprotein E knockout C57BL/6 mice (ApoE − / − mice) have been bought from Beijing Huafukang Biotechnology Co., Ltd. (License SCXK 2019e0008), quarantined, and acclimatized for one week earlier than the experiments. All mice have been subjected to a 12 h mild/darkish cycle below particular pathogen-free situations at 27 °C and correctly dealt with in accordance with the rules of the Institutional Animal Care and Use Committee (IACUC) of Chongqing Medical College. All animal experiments have been accredited by the Animal Ethics Committee of Chongqing Medical College. ApoE − / − mice have been fed a high-cholesterol weight loss plan (HCD; 40% fats, 40% carbohydrate, and 20% protein) with a excessive degree of ldl cholesterol (D12108C-high-fat rodent weight loss plan with 1.25% ldl cholesterol, FBSH, Shanghai, China) for 10 weeks [4, 31] to induce atherosclerotic plaque formation within the mouse aortic area and set up an atherosclerotic mouse mannequin. As the conventional group, six-week-old ApoE − / − mice have been fed a standard chow weight loss plan (NCD; 10% fats, 70% carbohydrate, and 20% protein).
In vitro cytotoxicity and blood compatibility checks of VHPK-PLGA@COL
Cytotoxicity check
The toxicity of colchicine and VHPK-PLGA@COL was detected by CCK-8 assays, reside/lifeless cell staining, and FCM. First, HUVECs have been seeded in a 96-well plate at a density of 1 × 104 cells per effectively and incubated for twenty-four h. The cells have been handled with colchicine or VHPK-PLGA@COL at completely different concentrations (0.1, 0.2, 0.4, 0.8, 1, 30 and 50 μg/mL) for twenty-four h and 48 h, respectively. Cell viability was measured utilizing a CCK-8 assay. As well as, HUVECs (5 × 105) have been seeded in laser confocal cell tradition dishes and cultured for twenty-four h, adopted by remedy with 0.8 µg/mL colchicine and VHPK- PLGA@COL for twenty-four h. Then, the cells have been stained with M5 HiPer Calcein AM/PI and noticed by CLSM (Beijing, China). In the meantime, the cell survival charge following drug remedy for twenty-four h was decided by FCM.
Blood compatibility check
For the hemolysis analysis, contemporary blood was collected from anesthetized C57BL/6 mice with anticoagulant containing ethylenediaminetetraacetic acid (EDTA), and the entire blood was diluted with saline to acquire diluted complete blood. Twenty microliters of the diluted complete blood was added to 1 mL of VHPK-PLGA@COL resolution at numerous concentrations (0.4, 0.8, 2, 3, 4, and 5 µg/mL) and incubated at 37 °C for 1 h. Crimson blood cells in saline have been used as unfavorable controls, and people in double-distilled water have been considered constructive controls. Subsequently, the supernatant of every pattern was collected by centrifugation (3000 rpm, 5 min), and the absorbance at 540 nm was measured utilizing a microplate reader.
Evaluation of the concentrating on potential of VHPK-PLGA@COL
Focusing on potential evaluation in vitro
The flexibility of VHPK-PLGA@COL to focus on HUVECs was evaluated utilizing each CLSM and FCM. The inactivated and activated cells (induced by 20 ng/mL TNF-α) have been seeded into laser confocal cell tradition dishes and cultured for twenty-four h. Then, the unique medium was changed with contemporary medium containing 0.4 µg/mL DiI-labeled VHPK-PLGA@COL or PLGA@COL and incubated for an additional 2 h. After washing thrice with PBS, the cells have been fastened with 4% paraformaldehyde for 15 min and stained with DAPI resolution for an additional 10 min, adopted by washing twice with PBS. Cell photographs have been captured by CLSM. For the blocking experiment, activated HUVECs have been cultured with medium containing free VHPK peptide resolution in a single day, adopted by incubation with medium containing 0.4 µg/mL DiI-labeled VHPK-PLGA@COL for two h. Cell photographs have been captured by CLSM. For dynamic uptake evaluation, activated HUVECs have been incubated with 0.4 µg/mL DiI-labeled VHPK-PLGA@COL or PLGA@COL for numerous durations (0, 0.5, 2, and 4 h), and the uptake of VHPK-PLGA@COL or PLGA@COL by HUVECs was quantitatively decided and analyzed by FCM.
In vivo concentrating on evaluation
To find out the biodistribution and concentrating on functionality of VHPK-PLGA@COL in vivo, equal volumes of DiR-labeled VHPK-PLGA@COL and PLGA@COL or saline because the management group have been intravenously administered to ApoE − / − atherosclerotic mouse fashions (3 mice/group). After 24 h, the aortas from the aortic root to the bifurcation of the iliac artery, hearts, livers, spleens, lungs, and kidneys have been dissected from completely different teams of mice and imaged by an in vivo imaging system (IVIS, Perkin Elmer, U.Okay.). As well as, sections of the remoted aortic sinuses have been immediately immunofluorescence staining of VCAM-1 (inexperienced) adopted by staining with DAPI resolution to find out the distribution of VHPK-PLGA@COL and PLGA@COL by CLSM.
Animal experiments
Animal experimental protocol
As illustrated within the scheme (Fig. 1c), six-week-old male ApoE − / − mice have been randomly and investigator-blindly divided into 5 teams (G1-G5: regular, management, VHPK-PLGA, colchicine, VHPK-PLGA@COL; 5 mice/group). Mice in G1, the conventional group, have been fed a standard chow weight loss plan (NCD) all through the experimental interval. All mice from G2 to G5 have been first fed a high-cholesterol weight loss plan (HCD) for 10 weeks to ascertain the atherosclerotic mannequin adopted by remedy with saline because the management (G2), VHPK-PLGA (G3), colchicine (G4), and VHPK-PLGA@COL (G5), respectively.
Schematic illustration of the preparation of VHPK-PLGA@COL and the therapeutic paradigm and potential mechanisms. a After encapsulating COL within the nanoparticles, the VHPK peptide was subsequently added to focus on AS, forming VHPK-PLGA@COL. b These nanoparticles may accumulate in infected endothelial cells that overexpressed VCAM-1 and limit the development of AS by inhibiting NF-κB/NLRP3 pathways and lowering the secretion of proinflammatory cytokines reminiscent of IL-1β and IL-18. c In our examine, six-week-old male ApoE − / − mice have been divided into 5 teams (G1-G5). In G1, the conventional group, mice have been fed a standard chow weight loss plan (NCD). In G2 to G5, mice have been fed a high-cholesterol weight loss plan (HCD) for 10 weeks adopted by remedy with saline, VHPK-PLGA, COL, or VHPK-PLGA@COL, respectively, for 8 weeks
The indicated formulations have been administered intravenously through the tail vein each two days for 8 weeks at a dosage of 0.1 mg/kg colchicine and an equal quantity of saline. The colchicine dosage used was based mostly on beforehand printed research [32, 33]. Upon termination of the examine, the physique weights of mice have been recorded, and the animals have been sacrificed below anesthesia. Blood was collected in EDTA spray-coated tubes and centrifuged at 3000 rpm for five min to gather plasma. The aortas from the center to the iliac bifurcation and organs have been fastidiously dissected and glued with 4% paraformaldehyde resolution.
In vivo biosafety analysis of VHPK-PLGA@COL
Hematological parameters, together with crimson blood cells (RBCs), platelets (PLTs), white blood cells (WBCs), and hemoglobin (HGB) content material, in blood samples collected from completely different teams of mice have been analyzed, respectively. In the meantime, biochemical parameters within the corresponding plasma samples, together with alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CRE), blood urea nitrogen (BUN), whole ldl cholesterol (TC), triglyceride (TG), low-density lipoprotein ldl cholesterol (LDL-C), and high-density lipoprotein ldl cholesterol (HDL-C), have been additionally evaluated accordingly. As well as, the key organs of mice, together with coronary heart, liver, spleen, lung, and kidney, have been harvested and glued with 4% paraformaldehyde for H&E staining.
Analysis of the in vivo anti-atherosclerotic impact of VHPK-PLGA@COL
Quantitative evaluation of the atherosclerotic plaques
Digital photographs of the aortic arch have been obtained after the aortas have been dissected from the mice. The plaque form, measurement, and distribution all through your entire aorta have been noticed intuitively with gross pathological specimens. The entire aorta was longitudinally opened and stained with ORO, and photographs of the stained aortas have been quantitatively analyzed by ImageJ to judge the therapeutic efficacy of the completely different formulations.
Evaluation of the steadiness of atherosclerotic plaques
To evaluate stability of the atherosclerotic plaques, sections of the aortic sinus have been stained with H&E and ORO and incubated with antibodies, together with anti-CD68 and anti-matrix metalloproteinase-9 (MMP-9), respectively, for immunofluorescence evaluation. Then, the sections have been visualized utilizing CLSM. Moreover, quantitative evaluation of atherosclerotic plaques or the constructive space was decided by ImageJ.
Quantification of inflammatory cytokines in plasma
The degrees of TNF-α, IL-1β, IL-18, and CRP within the plasma of mice have been analyzed and quantified utilizing business ELISA kits in keeping with the producer’s directions.
Western blot
Whole proteins have been extracted from total aortas dissected from completely different teams of mice. The protein focus was decided utilizing BCA assay. Equal quantities of denatured protein samples have been separated by 12% SDS‒PAGE, transferred to PVDF membranes (Bio-Rad Laboratories, Hercules, CA, USA), and blocked with 5% bovine serum albumin (BSA) for 1 h at room temperature. The membranes have been then incubated in a single day at 4 °C with major antibodies towards NF-κB p65, NLRP3, caspase-1, IL-1β, IL-18, α-Tubulin, and GAPDH, adopted by incubation with HRP-conjugated secondary antibodies for 1 h at room temperature. The blot indicators have been visualized by enhanced chemiluminescent (ECL) reagents and detected by way of a Bio-Rad imaging system (Bio-Rad, USA). The expression degree of the goal proteins was semi-quantified by measuring the relative grey worth of every goal protein band with the corresponding GAPDH or α-Tubulin as inner management for the normalization of information.
Statistical evaluation
All statistical evaluation have been carried out utilizing GraphPad Prism (model 8.02). One-way evaluation of variance (ANOVA) was carried out for a number of comparisons. All information are displayed because the imply ± SD and P values < 0.05 have been thought-about statistically vital.
