The following reagents, drugs and chemicals were used in this study: paclitaxel (PTX, purchased from Luzhou Renkang Biotechnology Co., Ltd.), curcumin derivatives (CUD, homemade in laboratory), dipalmitoyl phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000, Lipoid GmbH, Germany), Poloxamer F68 (Shanghai Aladdin Biochemical Technology Co., Ltd.), trichloromethane (purchased from Luzhou Renkang Biotechnology Co., Ltd.) and thiazole blue (MTT, Beijing Soleboro Technology Co., Ltd.). All reagents were analytical or high-performance liquid chromatography grade.
Human breast cancer MCF-7 cells (purchased from the Cell Bank Center of Chinese Academy of Sciences) were cultured in DMEM high glucose medium containing 10% fetal bovine serum and 1% penicillin/streptomycin. The cells were cultured in a 37°C cell incubator containing 5% carbon dioxide humidified air.
Female BALB/c mice, aged 7 weeks old and weighing 20 ± 3 g, were purchased from Chengdu Dashuo Laboratory Animal Co., Ltd. (Laboratory Animal Use License: SYXK (Sichuan) 2018-065). Female Sprague Dawley (SD) mice, weighing 220 ± 20g, were purchased from the Experimental Animal Center of Southwest Medical University (Laboratory Animal License: SYXK (Sichuan) 2018 − 181). All mice were housed indoors with alternating light and dark cycles for 12 h, and had free access to water and food during the adaptation period. Four-week-old female BALB/c nude mice (Laboratory Animal Production License No. SCXK (Beijing) 2016-0002) were purchased from Sparford (Beijing) Biotechnology Co., Ltd. and raised in SPF animal room of Chengbei Animal Center of Southwest Medical University.
Synthesis of CUD
After 110 mg curcumin (CU) was dissolved in anhydrous dichloromethane, 96 µL triethylamine (TEA) was added. Subsequently, 135 mg of cholesteryl chloroformate dissolved in anhydrous dichloromethane was slowly dropped to react under ice bath for 1 h. The solvent was removed by distillation under reduced pressure after completion of the reaction, and the desired product was obtained by an extraction method. The precipitate was retained after five ultrasonic washing with methanol, and the pellet was washed with isopropanol followed by centrifugation to collect the supernatant. CUD was obtained by rotary evaporation of the supernatant to remove the solvent.
Preparation of Nanoparticles
The defined amounts of PTX, CUD, F68, DSPE-PEG2000 were accurately weighed and dissolved via appropriate amounts of trichloromethane. Lipid membranes were formed by stirring with a magnetic stirrer and evaporating the solvent at 300 r/min and 40°C. To ensure no residual trichloromethane, the solvent was further removed with nitrogen and then cured in an oven at 40 ℃ for about 30 min. The particles are uniformly dispersed by ultrasonic probe after adding deionized water for hydration. The clarified CUD-PTX-LN nano-solution was obtained by ultrasonic fragmentation at the intensity of 185 W for 10 min . In order to long-term storage, a certain amount of sucrose (w/v) was added as a lyophilization protectant . The yellow loose powder was obtained after freeze-drying for about 28 h.
Characterization of Nanoparticles
Encapsulation Efficiency (EE) and Drug Loading (DL)
Encapsulation efficiency (EE) and drug loading (DL) were measured by centrifuging freshly prepared CUD-PTX-LN nano-solution at 3000 r/min for 10 min. The free PTX was precipitated while the supernatant contained homogeneous PTX Nanoparticles. To break the nanoparticles, 4 mL methanol was added to 100 µL of the supernatant of CUD-PTX-LN, followed by sonication for 5 min. In addition, 100 µL of uncentrifuged CUD-PTX-LN was processed as described above. The content of PTX was detected by HPLC to determine the drug content entrapped in the nano-liquid.
The encapsulation efficiency and drug loading formula are as follows:
EE (%) = Actual loading of PTX in nanoparticles /actual amount of PTX used for nanoparticles preparation ×100%
DL (%) = Amount of PTX in nanoparticles /Total amount of nanoparticles×100%
The chromatographic conditions are described below. Liquid chromatographic protection column: Phenomenex C18 (4.0 mm x 3.0 mm), liquid chromatographic column: Luna 5 µm C18 (2) 100Å (4.6 mm × 250 mm), elution time: 10 min, mobile phase: acetonitrile-water phosphate (PH 4.0) = 58:42 (v/v), wavelength: 227 nm, injection volume: 20 µL, column temperature: 30 ℃, flow rate: 1 mL/min.
Particle Size, Zeta Potential (ZP) and Polydispersity Index (PDI)
The CUD-PTX-LN solution was diluted to an appropriate concentration and placed into the sample pool (1 mL). Particle size, polydispersity index (representing the uniformity of particle size distribution) and zeta potential were measured using a Malvern particle size analyzer at 25℃.
In vitro Release Study
The release profile of CUD-PTX-LN was investigated with a total volume of 200 mL (containing 0.2% Tween-80) of PBS phosphate buffer solution as the release medium . After 1 mL of the same concentration of CUD-PTX-LN and free PTX were separately placed into a dialysis bag (8000–14000 Da), the release trial was performed using a dissolution apparatus at 37°C under 120 r/min. Equal amounts of release medium (3 mL) were withdrawn at 0.25, 0.5, 1, 2, 4, 8, 12, 24, 48, 72, 96, 120, 144 and 168 h, as well as centrifuged at 8000 r/min for 10 min. The supernatant was subjected to HPLC detection for analysis of cumulative release.
Nanoparticles were stored at room temperature, and the appropriate amounts were collected at 24 h. After determination of particle size, PDI, entrapment efficiency, and drug loading, the four indexes were compared with the original. The coefficient of variation (CV) was calculated to investigate the sample use stability. To guarantee the long-term preservation of the liquid formulation of the nanoparticles, the powdered solid formulation was prepared by freeze-drying technique. The CUD-PTX-LN lyophilized samples were kept in brown ampoules for 6 months, accompanied by a temperature of 25 ± 2°C and a relative humidity of 40 ± 5%. The same procedure described above was performed after dissolving the samples with deionized water at 1, 2, 3 and 6 months, respectively.
The toxicity of nanoparticles on MCF-7 cells was determined by thiazole blue colorimetry (MTT colorimetry). In the logarithmic growth phase, MCF-7 cells were trypsinized to prepare a cell suspension. The cells were counted and seeded into a 96-well plate at a density of 5×103 cells/well. After the cells were cultured at 5% CO2 and 37°C for 24 h, five different concentrations (5, 10, 20, 40 and 80 ug/mL) of free PTX, CUD-PTX-LN and blank nanoparticles were added to the cultured cells, respectively. Each well was supplemented with 20 µL MTT solution (5 mg/mL) after 24, 48, 72, and 96 h of continuous culture. The cultures were terminated after 3–4 h, then the medium was aspirated and 150 µL dimethyl sulfoxide was added. In order to dissolve the crystals sufficiently, the mixing was shaken for 10 min. The absorbance value (OD) of each well was measured with a microplate reader at 490 nm wavelength. The growth inhibition rate of drugs on tumor cells was calculated according to the formula, and then the dose-effect curve was plotted with drug concentration (ug/mL) as abscissa.
Cell growth inhibition rate (%) = [1-(OD experiment-A blank) / (A control-A blank)] *100
In vitro Hemolysis
An appropriate amount of SD rat blood was continuously stirred clockwise through a glass rod for 5 min to destroy fibrinogen. After adding 0.9% sodium chloride solution, centrifugation (1500 r/min, 10 min) was performed and the supernatant was decanted. To obtain erythrocytes, the addition of 0.9% sodium chloride solution was continued and the process of centrifugation described above was repeated until the supernatant exhibited colorless. A erythrocyte suspension with a concentration of 2% was configured via supplementation with 0.9% sodium chloride solution. Different concentration groups of CUD-PTX-LN (1–7), negative control (8) and positive blank control (9) were placed in the 37 ℃ incubator. All groups were assessed for hemolysis after centrifugation (1500 r/min, 10 min) at 1, 3, and 5 h, respectively. The supernatant of each sample after 5 h was supplemented to a 96 well plate. The microplate reader determined the absorbance (A) at the wavelength of 540 nm, and the hemolysis rate (%) was calculated by the following formula .
Hemolysis (%) = (A sample - A negative)/ (A positive - A negative) × 100%
Acute and Cumulative Toxicity In vivo
Sixty mice were randomly divided into six groups (10 per group) and received the following administration forms: (A) PTX at 40 mg/kg, (B) CUD-PTX-LN at 40 mg/kg, (C) normal saline with the same volume as the administration group, (D) PTX at 10 mg/kg, (E) CUD-PTX-LN at 10 mg/kg and (F) normal saline with the same volume as the administration group. The first three groups constituted the acute toxicity trial, and each mouse was dosed once through the abdominal cavity after being weighed. In addition, panels C, D, and E were utilized for the evaluation of cumulative toxicity. All mice were injected intraperitoneally after weighing, and the drug was administered every 3 days for 60 days.
Throughout the experiment, the status of the mice as well as the number of deaths were observed and recorded daily. On day 60th after drug administration, plasma and tissue (heart, liver, kidney) samples were collected from three randomly selected mice in each group for analysis and detection. The blood biochemical identification of liver and kidney function is mainly manifested in plasma samples (the following parameters were measured: alanine transaminase (ALT), aspartate transaminase (AST), lactate dehydrogenase (LDH), urea (urea), creatinine (crea) and uric acid (UA)). Tissue (heart, liver, and kidney) samples were stained with hematoxylin eosin (HE) before pathological section analysis. All operating procedures were performed in accordance with the requirements of ethical regulations.
Pharmacokinetics of CUD-PTX-LN
Ten SD rats were randomly divided into two groups (five in each group), which were treated sequentially with the following dosage: 10 mg/kg of PTX and 10 mg/kg of CUD-PTX-LN. For each rat after drug administration, cardiac blood was sampled at 0.08, 0.25, 0.5, 1, 2, 4, 8, 12, 24, 48, 72, 96 and 120 h. The removed raw blood (0.3 mL) was centrifuged for 3 min (5000 rpm/min) in a sodium heparin containing centrifuge tube, and 100 µL of the supernatant plasma was collected for detection. The plasma was vortexed for 3 min in 0.5 mL of extractant (methanol: ethyl acetate = 10:90, v/v), and the supernatant was obtained after centrifugation at 8000 rpm/min for 3 min. In order to ensure adequate extraction, the remaining precipitate was again added with 0.5 mL of extractant to repeat the extraction. The two supernatants were combined and blown dry with nitrogen at 35°C. 200 µL of the complex solvent [phosphoric acid: acetonitrile = 75:25 (pH 3.5)] was added and vortexed for 4 min until complete dissolution. After sonication for 4 min and centrifugation at 10000 rpm/min for 10 min, the supernatant was collected and detected by HPLC.
The chromatographic conditions are described below. Liquid chromatographic protection column: Phenomenex C18 (4.0 mm x 3.0 mm), liquid chromatographic column: Luna 5 µm C18 (2) 100Å (4.6 mm × 250 mm), elution time: 10 min, mobile phase: acetonitrile-water phosphate (PH 4.0) = 65: 35 (v/v), wavelength: 227 nm, injection volume: 20 µL, column temperature: 30 ℃, flow rate: 0.8 mL/min.
In vivo Antitumor Assessment
The in vivo antitumor effect of PTX nanoparticles was investigated with tumor-bearing nude mice. 0.1 mL tumor cell suspension (3 × 107 cells/mL) was inoculated into the left armpit of female BALB / tumor bearing nude mice. Thirty nude mice were randomly divided into three groups: (1) control group (normal saline), (2) 10 mg/kg PTX group, (3) 10 mg/kg CUD-PTX-LN group. All groups were administrated intraperitoneally every 3 days for 20 days.
After drug intervention on tumor-bearing nude mice, the body weight and tumor volume of all tumor-bearing nude mice were measured every 3 days. In addition, the cumulative number of deaths of tumor-bearing nude mice in each group was tallied every 5 days. The formula V = π / 6 (AB2) was chosen to calculate the volume (V) of the tumor, where A and B represent the largest and shortest vertical axes, respectively. The average body weight and tumor volume change curves of each group were plotted. After administration for 20 days, the nude mice were sacrificed and the tumor mass was removed for weighing, and the tumor inhibition rate (IR) was calculated by the following formula: IR = (1-tumor weightdrug/tumor weightcontrol×100%).
Tumor tissues were dehydrated with absolute ethanol after being fixed in 4% paraformaldehyde for 24 h, and then embedded by paraffin to prepare tumor sections. Antigen retrieval of tissue sections was performed at a high temperature environment in 0.01 M citric acid buffer (pH 6.0) for 5 min. Sections were incubated in primary antibody for KI67 overnight at 4°C, followed by dropwise biotinylated secondary antibody incubation for 30 min at 37°C. Ultimately, the sections were subjected to image acquisition by microscopic camera system after color development with 3,30-diaminobenzidine (DAB) and hematoxylin staining. All experimental procedures were performed in a blinded manner.
All data were expressed as mean ± SEM deviation by SPSS 19.0 software. One-way ANOVA and Tukey's test were used for comparison between groups. P ≤ 0.05 was considered statistically significant.