2.1 .1 Materials and reagents
Sal B, TSN and GA used as liposome raw materials were purchased from Baoji GuoKang Biotechnology Co., Ltd. (Shanxi, China) with the purity >98%. The same reagents used as quantitative analytes and chloromycetin were obtained from National Institutes for Food and Drug Control (Beijing, China). Cholesterol was purchased from Amresco, Inc. (OH, USA). Soybean phospholipid was purchased from Lipoid (Germany). DiR iodide [1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide] was purchased from Fan Bo Biochemical Co., Ltd. (Beijing, China). Fetal bovine serum was purchased from Gibco Co., Ltd. (USA), DEME liquid medium and PBS buffer were purchased from Corning Co., Ltd. (USA), Trypsin and pancreatin termination solution were gained from Sciencell Co., Ltd. (USA), MTT assay kit was purchased from Tianenze Gene Technology Ltd.Human HSC cell line were provided by the Beijing Wangjing Hospital Pathology department.Urethane was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Acetonitrile and methanol were HPLC grade and all other reagents were of analytical grade.
2.1 .2 Instruments
Incubator Model BB16UV/BB5060UVCO2 was purchased from Heraeus, The low-speed medical centrifuge Model 40C was from Baiyang centrifuge factory (Anxin, China ),The triple purpose thermostatic water tank Model SHH.W21.600 was purchased from Shuli Instrument Co.,Ltd.(Shanghai, China) , The clean bench Model HF safe 1500 was obtained from Lishen Scientific Instrument Co.,Ltd.(Shanghai, China), The low-speed shock meter Model DTY-2000 was purchased from Detianyou Technology Development Co., Ltd.(Beijing, China), The microplate reader Model 680 was from BIO RAD.The electron microscope Model Eclipse TE2000-5 was purchased from Nikon Corporation(Japan),The flow cytometry Model FACSC antoⅡwas from BD Corporation(USA).
2.2 18-GA-Suc synthesis process
The first step: weigh 2.7g of stearyl alcohol in 20mL of anhydrous dichloromethane, add 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC-I). After stirring at 80 ° C with constant temperature, 4.7 g of GA was added and stirring was continued. The reaction solution was concentrated, and separated by silica gel column chromatography, eluting with a solvent gradient of methanol-dichloromethane, and recrystallized to get a white solid, which was confirmed to be intermediate 18-GA by NMR and carbon structure.
Second step: 695 mg of 18-GA was weighed and dissolved in 20 mL of anhydrous dimethylformamide (DMF) solution, 150 mg of succinic anhydride and an appropriate amount of EDC-I were added, and the mixture was stirred under vacuum at 100 °C. The residue was diluted and stirred at room temperature and extracted with dichloromethane. The combined organic layer was washed with brine, dried over sodium sulfate, and then evaporated. The spectrum and carbon spectrum structure were confirmed to be 18-GA-Suc.
2.3 Preparation and characterization of GTS-lip and Suc-GTS-lip
GTS-lip was produced as described previously (Lin et al., 2014) and Suc-GTS-lip was made, with some modification, on the basis of this previous method. Two hydrophobic ingredients and the ligand, namely, GA, TSN and 18-GA-Suc, were embedded in phospholipid bilayers by the thin film-ultrasound method to form Suc-GT-lip. The ratio between soy lecithin (SPC) and cholesterol (CH) was 6:1 (w/w), SPC and TSN 30:1 (w/w), and SPC and GA 24:1 (w/w). The hydrophilic ingredient Sal B was dissolved in glycine-HCl buffer with pH 3.32. This solution was then added dropwise into Suc-GT-lip and altogether placed into a water bath at 30 oC for 30 min to obtain Suc-GTS-lip. The liposome was characterized for particle size, size distribution and entrapment efficiency.
2.4 In vitro release experiments
Three copies of Suc-GTS-lip mixed with GA and TSN were absorbed in parallel. Each 3 mL was placed in 100 mL 0.5% SDS release effluent. 3 mL of release medium was added to the pretreated dialysis bags, and the two ends were tightly immersed in the release of the external liquid. The stirring paddle speed was 100 r·min-1, and the temperature was controlled at 37 °C. One dialysis bag was taken out at 1h, 2h, 4h, 8h, 12h, 18h, 24h, 36h, and an equal amount of isothermal release medium was added. The TSN and GA contents were determined by HPLC, and the cumulative release percentage of the drug was calculated and fitted to the model.
2.5 In vivo pharmacokinetics and tissue distribution study
2.5.1 Animals
5-week-old, 20 ± 2 g male Kunming mice [SCXK (Jing) 2011-0004] were all purchased from SPF Biotechnology Co., Ltd. (Beijing, China). All animal procedures were performed according to animal care protocols approved by the Ethics Committee of Beijing University of Chinese Medicine in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals.
2.5.2 Drug administration and sampling
For the pharmacokinetics and tissue distribution study, one hundred mice were randomly distributed into two groups, each containing fifty animals. One group was administered GTS-lip at a dose of 48.25 mg/kg body weight for Sal B, 4.88 mg/kg body weight for TSN and 26.12 mg/kg body weight for GA, while the other group received Suc-GTS-lip by tail intravenous injection at equivalent doses as the GTS-lip group. Mice eyeballs were extracted and blood samples were collected into heparinized Eppendorf tubes and the heart, lungs, liver, spleen, and kidneys were collected at 0.083, 0.17, 0.33, 0.5, 0.75, 1, 1.5, 2, 4, and 6h. Plasma was collected from the blood samples by centrifugation at 15000 rpm for 10 min and stored at -20 oC. All of the tissue samples were flushed with saline and stored at -20 oC until further analysis.
2.5.3 Plasma sample treatment
50 μL of formic acid:water (v:v = 1:3) solution and 10 μL chloromycetin internal standard (0.2 mg/mL) were added to 150 μL plasma. Acetonitrile (1 mL) was then added, and the samples were vortexed for 3 min and centrifuged at 15000 rpm for 10 min to precipitate the proteins. The supernatants were transferred to another Eppendorf tube and to the remaining precipitations was added another 1 mL acetonitrile. The supernatants of this double extraction were combined to dry up. The residues were redissolved in 100 μL of methanol, vortexed for 1 min and centrifuged for 10 min at 15000 rpm. Samples were then analyzed by UPLC.
2.5.4 Tissue sample treatment
Tissue samples were thawed, cut into pieces and added into saline for further homogenization. Formic acid:water (v:v = 1:3) solution (70 μL for liver and kidney, 50 μL for spleen, lung and heart) and 10 μL of chloromycetin were added into a certain amount of tissue homogenates (500 μL of liver and kidney, 150 μL of heart and spleen, 250 μL of lung). The extraction and other process were same as above.
2.5.5 UPLC analysis
UPLC analyses of mouse plasma and tissue samples were performed using a Waters ACQUITY UPLC series with binary solvent management system, on-line degasser, automatic sampler and photo-diode array (Waters Co., USA). Chromatography was recorded on an ACQUITY UPLC® BEH C18 reverse-phase column (2.1 mm×50 mm, particle size 1.7 μm) (Waters Co., USA). The injection volume was 1 μL. The mobile phase consisted of a mixture of acetonitrile (solvent A) and 0.5% formic acid in water (solvent B) with gradient elution and was pumped at a flow rate of 0.4 mL/min with 30 °C column oven temperature, and column eluents were monitored by dual-wavelength spectrophotometry (289 nm for Sal B, 265 nm for TSN, 254 nm for GA). The linear gradient elution program was as follows: (1) from 20% A to 25% A in 0-1.5min, (2) from 25% A to 64% A in 1.5-3 min, (3) held at 64% A in 3-5.1 min, (4) from 64% A to 90% A in 5.1-5.8min, (5) from 90% A to 20% A in 5.8-6.0 min.
2.5.6 In vivo imaging and ex vivo imaging of mouse organs
Mice were anesthetized by urethane, and intravenously injected with 0.2 mL Suc-GTS-lip, which was labeled with a lipophilic near-infrared fluorescent dye (DiR-iodide, 10 µg/kg). A small animal in vivo imaging device was used obtain fluorescent images at 10, 30, 60, 120, 240, and 360 min after administration, and images were taken at the same exposure intensity and exposure time.
Experimental animals were sacrificed after intravenous administration of GTS-lip and Suc-GTS-lip at 10, 30, 60, 120, 240, and 360 min (n = 3 for each time point), and the heart, liver, spleen, lungs and kidneys were collected.
After removing the blood, the organs were imaged using the small animal fluorescence imager and their exposure intensity and time were normalized.
2.5.7 Pharmacokinetic and tissue distribution data analysis
Pharmacokinetic parameters and tissue distribution of Sal B, TSN and GA were calculated using non-compartmental methods (Winnonlin 5.2, Pharsight, Certara USA, Inc.).
SPSS statistical software version 17.0 (SPSS Inc., USA) was used for statistical data analysis. Student’s t-test was used for comparative analysis between two groups of pharmacokinetic parameters. One-way ANOVA was employed to compare data between multiple groups.
2.6 Effect of Suc-GTS-lip on proliferation inhibition and apoptosis of hepatic stellate cells
2.6.1 Cell Culture
Both cell lines were cultured with Dulbecco’s Modified Eagle’s Media (DMEM) supplemented with 10% fetal bovine serum in a humidified atmosphere of 5% CO2 at 37℃. The medium was replaced with fresh medium every other day until the cells grew to about approximately 80%.Then Suc-GTS-lip were diluted at different concentrations with DMEM.
2.6.2 Grouping and drug administration:
HSC cells were divided into two groups. One group was used to study the effect of Suc-GTS-lip on the proliferation inhibition of hepatic stellate cells in different doses, and the other was used to study the effect of Suc-GTS-lip on apoptosis of hepatic stellate cells in different doses.
In the study of the effect of Suc-GTS-lip on the proliferation inhibition of hepatic stellate cells, the groups and the drug loading concentrations of liposomal Suc-GTS-lip were shown in table 1 below. 200μL single cell suspension(1.0 × 105/mL) were plated in 96-well plates and incubated at 37°C and 5% CO2 until the culture plate was covered around 80%,different concentrations of drugs were administered to 7 groups mentioned below. Every group was provided with 6 sane holes and cultured under the same condition.
Table 1 Grouping and dosing of Suc-GTS-lip
Groups
|
SalB(μM)
|
TSN(μM)
|
GA(μM)
|
18-GA-Suc(μM)
|
Suc-GTS-lip1
|
600
|
80
|
300
|
300
|
Suc-GTS-lip2
|
300
|
40
|
150
|
150
|
Suc-GTS-lip3
|
150
|
20
|
75
|
75
|
Suc-GTS-lip4
|
75
|
10
|
37.5
|
37.5
|
Suc-GTS-lip5
|
37.5
|
5
|
18.75
|
18.75
|
Blank-lip
|
——
|
——
|
——
|
——
|
Healthy Cells(Control Group))
|
——
|
——
|
——
|
——
|
In the study of the effect of Suc-GTS-lip on apoptosis of hepatic stellate cells, the groups and the drug loading concentrations of liposomal Suc-GTS-lip were shown in table 2 below. 2mL single cell suspension(1.0 × 105/mL) were plated in 6-well plates and incubated at 37°C and 5% CO2 until the culture plate was covered around 80%,different concentrations of drugs were administered to 4 groups mentioned below. Every group was provided with 3 same holes and cultured under the same condition.
Table 2 Grouping and dosing of Suc-GTS-lip
Groups
|
SalB(μM)
|
TSN(μM)
|
GA(μM)
|
18-GA-Suc(μM)
|
Suc-GTS-lip(High-dose)
|
300
|
40
|
150
|
150
|
Suc-GTS-lip(Medium-dose)
|
150
|
20
|
75
|
75
|
Suc-GTS-lip(Low-dose)
|
75
|
10
|
37.5
|
37.5
|
Healthy Cells(Control Group)
|
——
|
——
|
——
|
——
|
2.6.3 MTT Assay
In the study of the effect of Suc-GTS-lip on the proliferation inhibition of hepatic stellate cells, after administration ,the cells were incubated at 37°C and 5% CO2 for 24 and 48 hours. After removing the supernatant we added 100 μL fresh medium and 10 μL solution A (containing MTT component) were added into 96-well plates. After incubation for 4 hours, original medium was removed and 110 μL solution B was added into each well and shaked 10 min by low volatility. Absorbance was detected at 490 nm with the enzyme-linked immunosorbent detector (EIA). The inhibition rate of cell proliferation was calculated according to the following formula, and the A was absorbance.
inhibition rate of cell proliferation=(A0-Ai)/A0×100%
Ai indicated the absorbance value which was in the experimental group, and A0 indicated the absorbance value which was in the control group.
2.6.4 Determination of HSC apoptosis rate by Annexin V-FITC/PI method
In the study of the effect of Suc-GTS-lip on apoptosis of hepatic stellate cells, after administration, the cells were incubated at 37°C and 5% CO2 for 24 hours. After removing the supernatant, the HSC cells were monitored the morphology of in each group.
The corresponding loading liposomes (Suc-GTS-lip) were added into the incubator according to the grouping requirements. After 24 hours, the primary culture medium was discarded and the cells were collected by 0.25% trypsin digestion without Ethylene Diamine Tetraacetic Acid (EDTA). 1 mL cells mentioned above were centrifuged for 10 min in 1000 rpm and the supernatant was discarded.Then1 mL pre-chilled PBS was added, the cells were suspended with shaking and centrifuged in 1000 rpm for 10 min and the supernatant was discarded. Repeat the step above two times. And then the cells were resuspended in 200 μL Binding Buffer. Next,10 µL Annexin V-FITC and 10 µL PI were added and mixed and put them in dark room for 15 min.Last, Detect apoptosis rate by flow cytometry within 1 h.
2.6.5 Primer design and RNA electrophoresis separation
Primer design was shown in Table 3.
Table 3 Primer design
Primer name
|
Primer sequence (5'to3')
|
Amplification product size (BP)
|
MMP-1 F
|
CCTCTGGCTTTCTGGAAGGG
|
267
|
MMP-1 R
|
CCACATCAGGCACTCCACAT
|
TIMP-1 F
|
CTCGTCATCAGGGCCAAGTT
|
312
|
TIMP-1R
|
GTAGGTCTTGGTGAAGCCCC
|
TIMP-2 F
|
TGCACATCACCCTCTGTGAC
|
TIMP-2R
|
TGGACCAGTCGAAACCCTTG
|
362
|
Collagen –I F
|
AGAGGTCGCCCTGGAGC
|
425
|
Collagen –I R
|
GGAGAGCCATCAGCACCTTT
|
Collagen –III F
|
GAGCTGGCTACTTCTCGCTC
|
285
|
Collagen –III R
|
CCTTGACCATTAGGAGGGCG
|
Beta-Actin F
|
TGACGTGGACATCCGCAAAG
|
185
|
Beta-Actin R
|
CTGGAAGGTGGACAGCGAGG
|
8 μL RNA was electrophoresis analyzed with 1% agarose gel.
2.6.6 Reverse Transcription
HiFi-MMLV cDNA(Cat#CW0744)was used to reverse transcription. Experimental operation follows the product instructions:
(1) RNA template, primers, 5xRT mix and RNase-free Water were dissolved and placed on ice to spare.
(2) Add and mix 20ul the first part of the reaction system to the tube as follows.
Reagents
|
20μL Reaction System
|
Final concentration
|
Primer mix
|
2 μL
|
50 uM
|
RNA Template
|
2 μL
|
10 ng
|
RNase-Free water
|
To 15 μL
|
|
(3) Incubate for 5 min at 65℃, then put it into the ice bath immediately for 2 min and centrifuge briefly, last collect the solution.
(4) Continue add the following reagents:
Reagents
|
20μL Reaction System
|
Final concentration
|
RT Mix,5x
|
4μl
|
1×
|
HiFi-MMLV Enzyme Mix
|
1μl
|
|
(5) Mix and incubate for 40 min at 37℃. After the reaction, keep warm at 70 ℃for 10 minutes.
2.6.7 Reverse Transcription PCR
(1) RT-PCR reaction system:
Reagents
|
20μL Reaction System
|
Pcr Mixture (2 ×)
|
10 μL
|
The upstream primer (10μM)
|
1.5 μL
|
Downstream primer (10μM)
|
1.2 μL
|
Template
|
2 μL
|
Sterile distilled water
|
To 20 μL
|
(2) The reaction system was used to amplified, the experimental operation followed by PCR mixture product instructions. The amplification program was: 95℃ for 10 min, 95℃ for 15 s, 59℃ for 60 s × 30 cycles.
(3) All the amplified products were electrophoretic analyzed by 5ul.
2.6.8 The Statistical Treatment
The relative quantitative analysis of the data was carried out by gray scale analysis with BIOER PCR instrument.
The quantitative gray value is expressed by X + s, and the data are processed by SPSS17.0 statistical software. Two samples were compared by T test, and One-way ANOVA was used among the groups.