2.1 Materials
Maleimide poly(ethylene glycol) amine (Mal-PEG-NH2, 5000 Da) was obtained from Xi’an Ruixi Biological Technology Co., Ltd. Hyaluronic acid (HA, 40–100 kDa), cysteamine hydrochloride and ethyl oxalyl chloride were purchased from Shanghai McLean Biochemical Technology Co., Ltd. L-Tyrosine N-carboxyanhydride (Tyr-NCA) was brought from Shanghai Bide Pharmaceutical Technology Co., Ltd. Lipopolysaccharide (LPS) was brought from Sigma-Aldrich. Cy5 NHS ester was purchased from Shanghai Yuanye Biotechnology Co., Ltd. 2’, 7’-Dichlorofluorescin diacetate (DCFH-DA) was obtained from Beyotime Institute of Biotechnology. Anhydrous N, N-dimethylformamide (DMF) and dichloromethane (DCM) were purchased from Energy Chemical. Other solvents, including dimethylsulfoxide (DMSO), tetrahydrofuran (THF) and triethylamine (TEA) were brought from Greagent and used as received.
2.2 Synthesis of Mal-PEG-Ptyr
Mal-PEG-Ptyr was synthesized according to the report [23, 24]. Briefly, Mal-PEG-NH2 (1.0 g, 0.2 mmol) and Tyr-NCA (1.0 g, 4.8 mmol) were dissolved in anhydrous 15 mL DMF. The reaction was performed at 35 oC under N2 for 72 h. The reaction mixture was concentrated under vacuum and poured into excess cold diethyl ether for five times. The precipitation was collected and dried under vacuum.
2.3 Synthesis of Mal-PEG-Ptyr-EO
Mal-PEG-Ptyr (0.5 g) was dissolved in anhydrous DMF. TEA (0.6 mL) was added into DMF solution. Ethyl oxalyl chloride (0.3 g, 2.2 mmol) in DCM was added dropwise in ice bath. The reaction was stirred for 24 h. After that, the reaction mixture was filtrated and washed by saturated NaCl solution for three times. The organic phase was collected and poured into excess cold diethyl ether for three times. The precipitation was collected and dried under vacuum.
2.4 Synthesis of amino-PEG-Ptyr-EO
Mal-PEG-Ptyr-EO (0.3 g) was dissolved in 10 mL DMF with cysteamine hydrochloride (0.1 g) added. The reaction was stirred at room temperature for 24 h. After that, the mixture was diluted with DCM, washed with saturated NaCl solution for three times and dried with anhydrous Na2SO4. After concentration, the mixture was poured into cold diethyl ether. Amino-PEG-Ptyr-EO was obtained by filtration and drying under vacuum. To label amino-PEG-Ptyr-EO with fluorescence dye, 10 mg of Cy5 NHS ester was reacted with 50 mg of amino-PEG-Ptyr-EO to generate Cy5-PEG-Ptyr-EO.
2.5 ROS degradation
Mal-PEG-Ptyr-EO was dissolved completely in DMSO-d6 with addition of 50 mM H2O2 in a nuclear magnetic tube. The tube was incubated at 37 oC for 30 min, 60 min and 120 min. The chemical structure was characterized by 1H NMR.
2.6 Preparation of micelles
Before preparing micelles, the molecular weights and their corresponding distribution of polymers were measured by the gel permeation chromatography system (GPC, Waters). THF was used as the eluent at a low flow rate of 1.0 mL/min. The standard curve was constructed by monodispersed polystyrene standard. The critical micelle concentrations (CMC) for the block copolymers were determined using pyrene as the probe [25]. Briefly, a constant concentration of pyrene (5.0 × 10− 7 mol/L) was incubated with varying concentrations of lyophilized block copolymer (0.05–51.2 µg/mL) in phosphate buffer solution (PBS) at 37 oC for 36 h. All samples were run with a total sample volume of 1.2 mL in a quartz cuvette. The spectrophotometer (Hengping F9600) was used to record the excitation spectra for each polymer concentration at 338 and 333 nm. A plot of the I338/I333 ratio versus log of the block copolymer concentration was drawn to calculate the CMC.
Micelles based on amino-PEG-Ptyr-EO were prepared by dialysis method. Briefly, 10 mg amino-PEG-Ptyr-EO dissolved in 1 mL DMSO completely was placed into a dialysis tube (Mw 3500) and dialyzed against pure water for 24 h. Amino-PEG-Ptyr-EO micelles (PEMs) were obtained by lyophilization. HA-coated PEMs (HPEMs) were prepared by electrostatic attraction. Briefly, PEMs suspension was slowly added to a HA solution under vigorous stirring at room temperature. The mass ration of HA to PEMs and the concentration of HA were further investigated to obtain the optimum formulation. The method to prepare Cy5-labeled micelles was as mentioned above, except that amino-PEG-Ptyr-EO was replaced with amino-PEG-Ptyr-EO/Cy5-PEG-Ptyr-EO (95/5, w/w).
2.7 Characterization of micelles
The size distribution and Zeta potential of micelles were recorded by dynamic light scattering (DLS, Malvern Zetasizer). The morphologies of micelles were investigated by transmission electron microscopy (TEM, JEM-2100). In brief, a drop of micelle suspension was placed onto the surface of the copper grid. After stained for 5 min, the suspension on the copper grid was absorbed by filter paper. The TEM photos were taken after air-drying.
2.8 Drug encapsulation and release
SIM-loaded PEMs (SPEMs) were prepared by dialysis method. Briefly, 100 mg amino-PEG-Ptyr-EO and 10 mg SIM were dissolved in 2 mL DMSO completely. Then, the mixture was placed into a dialysis tube (Mw 3500) and dialyzed against pure water for 48 h to remove DMSO and unloaded SIM. After that, the suspension was filtrated through 0.45 µm syringe filter. Afterwards, SIM-loaded HPEMs (SHPEMs) were obtained by HA coating with 0.5 mg/mL HA solution with the mass ratio of 3/1 (HA to SPEMs). Besides, SIM-loaded Mal-PEG-Ptyr micelles (SPMs) were prepared by the same method and used in drug release experiments.
To determine the drug loading efficiency (DLE) and drug loading content (DLC), a known amount of SPEMs was dissolved in 1 mL of THF. This solution was then analyzed by an ultraviolet (UV) spectrophotometer at 238 nm with PEMs suspension as the control [26]. The drug concentration in the solution was calculated based on the standard curve. DLE and DLC were obtained in accordance with the following formulae:
The drug release was conducted based on SPEMs and SPMs. Briefly, 1 mL of SPEMs or SPMs suspension with the same SIM amount were placed into the dialysis bag (Mw 3500) suspended in 20 mL PBS (pH 7.4) containing 0.2% SDS with or without 10 mM H2O2. The sealed vials were shaken at 37 oC for 72 h. At predetermined time intervals, the release medium was withdrawn to measure the amount of SIM by UV method and 20 mL fresh medium was added. The experiment was performed in triplicates.
2.9 Cell culture
RAW264.7 cells were employed to evaluate the cellular uptake and cytotoxicity against SIM and SIM-loaded micelles. RAW264.7 cells, highly expressed CD44 receptors, were cultured in Dulbecco’s modified eagle’s medium (DMEM) containing 10% FBS, 100 mg/mL of penicillin and 100 mg/mL of streptomycin at 37 oC in an incubator with 5% CO2 humid atmosphere. To induce inflammatory reaction, RAW264.7 cells were simulated with 100 ng/mL LPS for 24 h.
2.10 Cellular uptake
LPS-simulated RAW264.7 cells were reported to highly express CD44 receptors that can specifically bind HA ligands. Therefore, RAW264.7 cells were seeded into a 6-well plate with the density of 3.0 × 105 cells/well. After incubation for 12 h, the culture medium was replaced with fresh medium containing Cy5-labeled PEMs and HPEMs with or without HA pretreatment. After incubation for another 2 h, cells were washed with PBS for two times and fixed with 4% paraformaldehyde for 15 min. After stained with DAPI for 10 min, fluorescence images of cells were obtained using a fluorescence microscopy (CKX53, Olympus).
2.11 Cytotoxicity
The cytotoxicity of SIM and SIM-loaded micelles against RAW264.7 cells was measured by MTT assay. Briefly, LPS-simulated RAW264.7 cells were seeded at a density of 4 × 103 cells/well in 96-well plates. When the cells reached 80% confluence, blank micelles, free SIM, SPEMs or SHPEMs at different SIM concentrations (0.25, 0.5, 1.0, 2.0, 4.0, and 8.0 µg/mL) or different polymer concentrations (50, 100, 200, 300, 400 and 500 µg/mL) in DMEM were added and cultured for 24 h. To evaluate the time-dependent cytotoxicity, cells were incubated with free SIM and SHPEMs (SIM: 4.0 µg/mL) for 24 h, 48 h and 72 h. To survey the influence of HA coating on the cytotoxicity, cells were incubated with free SIM, SPEMs and SHPEMs at the same SIM concentration (8.0 µg/mL) for 4 h. Then, the culture medium was replaced with fresh medium and cells were incubated for another 20 h. 10 µL of 5 mg/mL MTT solution was added to each well and the cells were incubated for another 4 h. Subsequently, the medium was carefully removed and 100 µL of DMSO was added to each well. The absorbance of each well was recorded on a microplate reader at the wavelength of 562 nm. The experiment was performed in triplicates.
where ODsample means the OD value from wells treated with drugs, ODcontrol from wells treated with DMEM medium and ODblank from wells without cells but culture medium.
2.12 ROS generation
DCFH-DA was used to evaluate the generation of intracellular ROS by fluorescence imaging and flow cytometry. In brief, RAW264.7 and LPS-induced RAW264.7 cells were seeded in a 6-well plate and cultured for 12 h at 37 °C. Next, cells were incubated with DCFH-DA (10 µM) for 30 min. Then the cells were washed with PBS and incubated with different samples respectively (PBS, free SIM, HPEMs and SHPEMs). After incubation for another 12 h, the cells were washed with PBS to remove free samples, following by observation using a fluorescence microscopy and analysis by flow cytometry.
2.13 Aminals treatment
Apolipoprotein E-deficient (ApoE−/−) mice (six-week old) were obtained from the Nanjing University in Nanjing, China. All the animal care and experimental protocols were carried out with review and approval from the Laboratory Animal Welfare and Ethics Committee of the Nanjing University. ApoE−/− mice were fed with a high-fat diet for 8 weeks and treated once a week with saline, free SIM, SPEMs and SHPEMs at a dose of 30 mg/kg for SIM.
2.14 ORO and H&E staining
After treatment for 4 weeks, the mice were sacrificed, and the aortas and major organs were harvested. Aortas, from the heart to the iliac bifurcation, were fixed by perfusion with paraformaldyhyde (4% in PBS). After removing the periadventitial tissue, aortas were dissected longitudinally and stained with Oil Red O (ORO) to quantify the lesion areas of the plaques by NIH ImageJ software. Besides, sections of the major organs including heart, liver, spleen, lung, and kidney were also analyzed by hematoxylin-eosin (H&E) staining.