(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was obtained from Amresco (Solon, OH, USA); potassium bis(trimethylsilyl)amide (KHMDS), dimethyl maleic anhydride, ethylene oxide and lactide were obtained from Xi'an Ruixi Biological Technology Co., Ltd.; D-tocopherol polyethylene glycol 1000 succinate (TPGS) was purchased from Sigma; chloroform was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China); and FeCl3-6H2O, ethylene glycol, dimethyl sulfoxide (DMSO), urea, ethylene Diamine Tetraacetic Acid (EDTA), doxorubicin (DOX) and anhydrous ethanol were purchased from Hao Sai Biotechnology Co., Ltd. (Jinan, China). 1640 medium, penicillin-streptomycin, fetal bovine serum (FBS) and 0.25% trypsin-ethylene diaminetetraacetic acid solution were purchased from Invitrogen-Gibco (Waltham, MA, USA). Reactive Oxygen Species Assay Kit was purchased from Hao Sai Biotechnology Co., Ltd. (Jinan, China).
Cell culture
Human cervical carcinoma (HeLa) cells were purchased from the Shanghai Institute for Biological Sciences (Shanghai, People’s Republic of China) and grown in 1640 medium containing 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin-streptomycin (100 mg/mL penicillin G and 100 mg/mL streptomycin) at 37°C in a 5% CO2 atmosphere at 95% relative humidity.
Animals
Female Sprague-Dawley (6-8 weeks old, 150 g) and female nude mice (6-8 weeks old, 20 g) were purchased from Weitong Lihua Biotechnology Co., Ltd. (Beijing, China). All animal procedures were performed in accordance with the protocol approved by the Animal Care and Use Committee of Shandong University, Qilu Hospital (DWLL-2019-005).
Preparation of DOX-Fe3O4-PEG-PLA-NPs
Synthesis of oily Fe3O4
The synthesis of MNPS-EDTA by the solvent thermal method was prepared by the hydrothermal method using FeCl3-6H2O as the single Fe ion source. FeCl3-6H2O (0.5 g) was dissolved in ethylene glycol (30 mL) to form a clear solution. Then, urea (1.8 g) and EDTA (0.35 g) were added to the mixed solution and vigorously stirred for 30 minutes. The mixture was put into a Teflon-lined stainless steel autoclave (50 mL); thereafter, the autoclave was heated to 198°C for 6 h in a furnace and allowed to cool to room temperature. The reaction products were collected with magnets and cleaned and reused in anhydrous ethanol and deionized water. Finally, the nanoparticles were dried in a vacuum oven at 50°C for 24 h. On this basis, Fe3O4 magnetic nanoparticles (MNPs) were prepared without EDTA.
Synthesis of H2N-PEG-b-PLA nanoparticle micelles
With potassium bis(trimethylsilyl)amide (KHMDS) as the initiator, ethylene oxide and lactide were successively added to carry out anionic ring-opening polymerization, and the protecting group was removed by acid hydrolysis to produce the H2N-PEG-b-PLA block copolymer. Then, a certain proportion of dimethyl maleic anhydride was added to obtain HOOC-PEG-PLA.
Synthesis of DOX-PEG-PLA micelles
First, 500 µL of PEG5k-PLA5k (100 mg/mL), 1 mL of DOX (2.5 mg/mL) and 2.2 mL of chloroform were uniformly mixed and added into a mixture of 12 mL of 1% PVA and TPGS (external aqueous phase, PVA:TPGS 1:5). An oil-water emulsion was formed by using probe ultrasound for 5 minutes (80 W) in an ice bath. Finally, the mixture was added to 60 mL of 0.3% PVA (dispersed phase) and stirred overnight to volatilize the chloroform and solidify the PLA ball surface. A 100 kD ultrafiltration tube was used for ultrafiltration concentration cleaning. Finally, the sample was collected and stored at 4°C after a constant volume of pure water was added for a final volume of 10 mL.
Preparation of DOX-Fe3O4-PEG-PLA micelles
First, 500 µL of PEG5k-PLA5k (100 mg/mL), 1 mL of DOX (2.5 mg/mL) and 2.2 mL of chloroform were uniformly mixed and added into a mixture of 12 mL of 1% PVA and TPGS (external aqueous phase, PVA:TPGS 1:5). An oil-water emulsion was formed by using probe ultrasound for 5 minutes (80 W) in an ice bath. Finally, the mixture was added to 60 mL of 0.3% PVA (dispersed phase) and stirred overnight to volatilize the chloroform and solidify the PLA ball surface. A 100 kD ultrafiltration tube was used for ultrafiltration concentration cleaning. Finally, the sample was collected and stored at 4°C after a constant volume of pure water was added for a final volume of 10 mL.
Characterization of the nanoparticles
TEM images were taken with a JEM-2100 HR transmission electron microscope (JEOL, Tokyo, Japan) with a tungsten filament at an accelerating voltage of 200 kV. The UV-vis absorption values of the different nanoparticle samples were measured with a UV-vis-NIR spectrophotometer (UV-1780, Shimadzu, Japan). The hydrodynamic size and surface potential of the nanoparticles were determined in the aqueous phase by using a Malvern Zetasizer Nano-ZS (Malvern Instruments, Worcestershire, UK). Fluorescence spectra of the drug-loaded PLA-PEG-FITC nanoparticles were measured with a Tecan multifunctional enzyme standard instrument (excitation wavelength was 488 nm, emission wavelength was 500 nm-750 nm). Infrared spectra of the nanoparticles were obtained with an FT-IR spectrometer (Nexus 870 FT-IR, Thermo Nicolet, USA). The magneto-thermal conversion efficiency of materials was measured by infrared thermal camera in vitro.
Drug loading and encapsulation rate
One hundred microliters of DOX-Fe3O4-PEG-PLA nanoparticles (a), DOX-PEG-PLA nanoparticles (b), and PEG-PLA nanoparticles (c) were dried in a drying centrifuge tube, and the concentration of nanoparticles was obtained by the weight difference method. Then, 1 mL of methanol solution was added with ultrasound to dissolve the drug. The content of DOX loaded into the PEG-PLA-FITC micelles was determined by a UV-vis method. PEG-PLA-FITC micella were used as the control group because free FITC could interfere with the measurement. The absorption of DOX at 530 nm was measured. Equations (1) and (2) were used to calculate the encapsulation rate and drug loading efficiency (DL%) of the nanoparticles.
Encapsulation rate = WA/WB×100% (1)
Drug load = WA/WC ×100% (2)
WA: DOX (mg) loaded into the PEG-PLA-FITC nanoparticles; WB: total dose of DOX (mg); and WC: total mass of the drug-loaded nanoparticles (mg).
Release study of nanocarriers in vitro
The release characteristics of DOX from nanocarriers were studied by the dialysis method. First, freeze-dried DOX-NPs and free DOX solutions (1 mg) were dissolved in DMSO (1 mL), transferred to dialysis bags (Snakeskin, Pierce Biotechnology, Rockford, IL, USA) (3500 kDa) and immersed in 50 mL of neutral saline (pH=7.4) or acidic saline (pH=6.5). The vials were shaken horizontally in a shaking water bath (100 rpm) at 37°C for 160 h. At predetermined time intervals, 5 mL from each saline sample was collected and replaced with an equal amount of fresh saline. Then, the DOX concentration in the physiological saline samples was analyzed by ultraviolet spectrophotometry to determine the cumulative DOX content released.
Cell culture and cytotoxicity measurements
The MTT method was used to determine the toxicity of the blank nanocarriers and the different DOX-NP groups to HeLa cells. To detect the effects of the blank nanocarriers on HeLa cell cytotoxicity, different concentrations of the nanocarriers (5, 20, 50, 100 µg/mL) were incubated with the cells for different lengths of time (8, 24, 48 and 72 h). Different drug-loaded nanocarriers with or without the alternating magnetic field conditions used this same method to explore the impacts of different drug concentrations (0.5, 1, 2, 4 µg/mL in doxorubicin concentration) and different times of action on the tumor cell survival rate. The specific experimental procedures were as follows: HeLa cells were seeded in a 96-well plate at a density of 1×104 cells per well. The cells were incubated in an incubator overnight until the cells were 80% confluent, and then the medium was removed. After the groups with different concentrations of nanoparticles and tumor cells were cocultured, 20 µL of MTT solution (in phosphate-buffered saline, 5 mg/mL) was added to each well followed by culturing for another 4 h. Then, 150 µL of dimethyl sulfoxide (DMSO) was added to dissolve the blue formazan crystals produced by the proliferating cells, the plates were shaken at 37°C for 10 minutes, and a victor 3 microplate reader (PerkinElmer, Boston, MA, USA) was used to measure the absorbance at 490 nm.
In vitro functional tests
Transwell assay
Cell invasion was analyzed in a transwell chamber (8 µm). In brief, 300 µL of complete medium was added to the bottom chamber, and then HeLa cells (1 × 105 cells/mL) were seeded into the upper chambers of 24-well Transwell plates. Then, HeLa cells were incubated with DOX, DOX-PEG-PLA or DOX-Fe3O4-PEG-PLA-NP at a DOX concentration equivalent to 2 µg/mL for 24 h. The concentration of free PEG-PLA-NPs was 8 µg/mL. After 24 h, the filter was removed from the plate, and the cells remaining on the upper filter were gently wiped. The cells that migrated to the bottom chamber were fixed with methanol and stained with crystal violet for 2 minutes. The average number of migrating cells was determined from six independent views photographed using a microscope.
Scratch experiment
First, two horizontal lines were drawn on the back of a 6-well plate. After the cells were digested, a cell suspension was made and plated, and when the confluence was above 90%, the cells were scratched with a 10 µL pipette tip on the bottom horizontal line, followed by gently rinsing with PBS 2-3 times, and replacing the medium with serum-free dosing medium for cell stimulation. Cultivation was continued in a 37°C, 5% CO2 incubator. After 24 h, pictures were taken under a microscope, and the scratch area was calculated.
Flow cytometry
Flow cytometry was also utilized to examine the effects of the PEG-PLA-NPs on HeLa cell apoptosis. Briefly, HeLa cells were exposed to DOX, DOX-PEG-PLA or DOX-Fe3O4-PEG-PLA-NPs at an equivalent DOX concentration of 2 µg/mL for 24 h, while the concentration of free the PEG-PLA-NPs was 50 µg/mL. The cells were then washed twice with PBS. Next, the cells were collected and stained with annexin V-FITC/PI for 30 minutes in the dark. Finally, the stained cells were examined by flow cytometry, and the data were analyzed with Flow Jo software (Treestar, Ashland, OR, USA).
Cellular uptake of the nanoparticles
In order to prove the killing effects of the nanoparticles on tumors in vitro, the uptake capacity of DOX and the DOX-Fe3O4-PEG-PLA-NP and DOX-PEG-PLA-NP formulations were evaluated in HeLa cells. We used DAPI to normalize the cell densities of the different groups. HeLa cells were cultured for 24 h, and different culture media sample solutions with the same DOX concentration were added. The cells were incubated with tumor cells for different lengths of time (1, 2, 4, 8 h), washed with cold PBS 3 times to terminate cellular uptake, fixed with 4% formaldehyde for 30 minutes and finally washed with PBS 3 times for 10 minutes each time. PBS (5% Triton-X-100) was permeated for 15 minutes, and then the cells were rinsed with PBS 3 times for 10 minutes each time followed by blocking with PBS (10% NGS) for 1 h. Cells were then incubated with the specific primary antibody at 37°C for 30 minutes. The cells were rinsed with PBS (0.1% Tween-20) three times for 10 minutes each. Sections were coated with aluminum foil, incubated with secondary antibodies for 30 minutes, and rinsed with PBS 3 times to remove the secondary antibodies. DAPI was added for 15 minutes of incubation followed by rinsing with PBS (0.1% Tween-20) three times for 10 minutes each time. FITC coupled with PEG-PLA-NP was used for green light excitation imaging in the cellular uptake experiment. The cellular uptake characteristics of the different groups were analyzed by fluorescence microscopy (Olympus, Tokyo, Japan).
Trajectory detection
HeLa cells were seeded in petri dishes at a density of 1×104 cells/well. The culture medium was removed after cell adherence, then add DOX-Fe3O4-PEG-PLA-NP solution (10 µg/mL). Attracted by a magnet on one side, the movement of the nanoparticles by microscope and plots the mean square displacement and plane trajectory.
Pharmacokinetic data analysis in rats
Female Sprague-Dawley rats (n=6/group) were injected with DOX or DOX-NPs (5 mg/kg DOX concentration) into the tail vein. Blood samples were collected at predetermined time points (0.17, 0.34, 0.51, 0.68, 1, 2, 4, 6, 12, 24, 36, 48 and 60 h). Mobile phase (200 µL of methanol and 2 mL of ethyl ether) was mixed with the blood samples collected at different time points (200 µL), followed by vortexing for 5 minutes, centrifuging at 10,000 rpm for 10 minutes, and then drying the samples under nitrogen. Finally, the DOX concentration in each sample was analyzed using high-performance liquid (HPLC).
Tissue-distribution study in tumor-bearing mice
HeLa cells (Luc-transfected cells) (8× 106/100 µL) were subcutaneously inoculated into the right upper limbs of female BALB/c nude mice to establish a subcutaneous tumor mouse model to construct a standard tissue curve and to study drug tissue distribution. To construct the standard curve of DOX, blood and tissue samples (liver, spleen, kidney, heart, lung, uterus, small intestine and tumor) from the tumor-bearing mice were homogenized with 5 mL of PBS. Subsequently, 20 µL of different quantitative standard DOX solutions (1.00, 5.00, 10.00, 25.00, 50.00, 75.00, 100.00, 150.00 and 200 µg/mL) were added to the homogenates (200 µL), followed by HPLC analysis. A linear regression equation was then determined based on the DOX concentration (X) and AUC value (Y) from the HPLC experiment. Subcutaneous tumor-bearing mice were randomly divided into 4 groups (20 mice/group). Each mouse was intravenously injected with DOX or DOX nanocarrier solution (5 mg/kg in DOX concentration). At predetermined time points (1, 2, 4, 12, and 24 h), 28 mice in each group were euthanized, their plasma and tissue samples were rapidly collected for HPLC analysis and then the DOX concentrations in the samples were calculated based on the linear regression equation mentioned above.
Anti-tumor efficiency
Intratumoral injection of DOX-Fe3O4-PEG-PLA-NP (2µg/mL) under different AMF treatment time, the magnetothermal conversion efficiency of tumor-bearing mice was detected by near-infrared thermal imaging camera and the tumor temperature curve was drawn. The antitumor efficiencies of the different nanocarriers in HeLa subcutaneous tumor-bearing mice were studied. Subcutaneous tumor-bearing mice (n = 25) were randomly divided into 5 groups. When the tumor volume reached 100 mm3, different drugs were injected. The experimental groups were DOX, DOX-Fe3O4-PEG-PLA-NPs, DOX-Fe3O4-PEG-PLA-NPs (AMF), DOX-PEG-PLA-NPs (AMF) (DOX concentration of 10 mg/kg), and the control group was injected with normal saline. The drug was injected every three days for eight treatment cycles. Mouse weights and tumor volumes were measured every 3 days during treatment (on days 14, 16, 18, 20, 22, 24 and 26). One week after the last injection, the mice were imaged to observe the tumor killing effects of the different treatments. The tumor-bearing mice were euthanized, and specimen tissues (heart, liver, spleen, lung, kidney and tumor) were taken for histological staining and sequencing. Its tumor volume was measured with calipers and calculated according to the formula:
Tumor volume (cm3) = 1/2 (tumor length × tumor width2) (3)
Abdominal cavity tumor-bearing mouse model were randomly divided into five groups (n =5 each group): (I) saline, (II) DOX, (III) DOX-PEG-PLA-NPs(AMF), (IV) DOX-Fe3O4-PEG-PLA-NPs and (V) DOX-Fe3O4-PEG-PLA-NPs (AMF). Each mouse in the different groups was given a different treatment as designed, its survival was tracked separately throughout the experiment, and then a survival curve was drawn.
Histological examination
Different groups of tumor-bearing mice were sacrificed by inhalation anesthesia with excessive ether. The tumor, heart, liver, spleen, lung, and kidney were immediately removed, weighed, fixed with 10% formalin, and then paraffin-embedded. Histological changes and cell apoptosis were observed by hematoxylin and eosin (H&E) staining, TUNEL staining and Ki67 immunohistochemical staining. The distribution of ferric oxide in different tissues was observed by Prussian blue staining. These images were obtained at 400× magnification.
Sequencing
Tumor-bearing mice in different groups were sacrificed, and the tumor tissues were harvested for second-generation sequencing and proteomic detection.
T2-weighted magnetic resonance imaging
The T2-corrected MRI formaldehyde molecular probe DOX-Fe3O4-PEG-PLA-NP relaxation rate was determined with different concentrations (0.1, 0.5, 2.0, 10.0, 50.0 and 100.0 µg/mL iron oxide concentration) of DOX-Fe3O4-PEG-PLA-NPs, USPIO standard product and 2 mL of glucosamine solution. Using a CE3.0T MR scanner (Shanghai Niumag Company, Shanghai, China), the above three different sets of solutions were placed on the 8-channel head coil, and the T2 relaxation rate data collected by the spin echo sequence (SE): TR 3000 msec; TE 10, 20, 40, 60, 80 and 100 msec was used. T2 Map was used to measure the T2 values corresponding to different solution concentrations and the concentration-relaxation rate curve was fit according to 1/T2 and the solution concentration. The slope of the curve is the relaxation rate r2. A subcutaneous tumor-bearing mouse model was then established. When the mouse tumor diameter was greater than 1 cm for MRI scanning, an 8-channel mouse special MR coil was used to collect aberration parameters at position T2. The instrument parameters were set as follows: TE 13.4 mm, slice thickness 3 mm, TR 660 msec, FOV 10, matrix: 512×512 µm, NEX1. The mice were anesthetized by intraperitoneal injection of chloral hydrate (3%), and MRI of the neck and chest was performed. First, a plain MRI scan was performed, and then DOX-Fe3O4-PEG-PLA-NP solutions of different concentrations (10.0, 15.0, 20.0, 25.0, 30.0 µg/mL) were injected through the tail veins of the mice, with diatrizoate injection as a negative control. MRI scans were performed at different time points (5 minutes, 30 minutes, 1 h, 2 h, 4 h, 12 h and 24 h) with the same sequence parameters as before. After scanning, the data before and after enhancement will be measured and analyzed.
Statistical analysis
Statistical Package for the Social Sciences version 11.0 software (SPSS Inc., Chicago, IL, USA) was used for statistical analyses. The data are presented as the mean ± standard deviation and were analyzed by one-way analysis of variance followed by Student’s t-test. Statistical significance was set at alpha values of p <0.001 (very significant), p <0.01 (highly significant), and p <0.05 (significant).
