Materials
Inonotus obliquus was purchased from the Northeast Natural Products Trading Company (Harbin, China). Ultrapure water was obtained from Millipore DQ-8 Ultrapure water system at SPST Public Platform and was used throughout all experiments. Tween 80 and cholesterol (Chol) are bought from Shanghai Aladin Chemical (Shanghai, China). 1,2-dialmitoyl-sn-glycero-3-phosphocholine (DPPC) is bought from Corden Pharma (Switzerland). 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(poly(ethylene glycol))-2000] (DSPE-PEG2000) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[folate (poly(ethylene glycol))-2000] (FA-DSPE-PEG2000) are purchased from Shanghai Yare Biotech (Shanghai, China). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Dimethyl sulfoxide (DMSO, ≥99%) and phosphate buffered saline (PBS) are all purchased from Solarbio Science & Technology Co., Ltd (Beijing, China).
L02 (normal human hepatic cells) were obtained from Chinese Academy of Sciences Committee on Type Culture Collection Cell Bank (Shanghai, China). HepG2 (human liver cancer cells), HeLa (human cervical cancer cells) and MCF-7 (human breast cancer cells) were previously purchased from Procell Life Science & Technology Co., Ltd. (Shanghai, China) and were frozen in liquid nitrogen tanks in laboratory. Fetal bovine serum (FBS), penicillin–streptomycin solution and Dulbecco’s modified Eagle’s medium (DMEM, high glucose) were purchased from Gibco Life Technology (Gibco, USA). 96-well plate, pipet and cell culture bottle were bought from Corning (NY, USA). MTT, DMSO are cell culture grade, other reagents were at least of analytical grade and were used without further purification.
The chemical structure of DPPC (A), cholesterol (B), DSPE-PEG2000 (C) and inotodiol (D) as Fig. 1.
Simulated gastric fluid (SGF) was prepared as Dr. Gao’ s method [31]: 23.4 mL 38% concentrated hydrochloric acid and added 100 mL distilled water to prepare diluted hydrochloric acid, 1.64 mL dilute hydrochloric acid and 1 g of pepsin was added to 80 mL distilled water, then diluted to 100 mL with distilled water after they were mixed well.
Isolation of inotodiol
According to Lishuai Ma’ s extraction method [32]: 2.0 kg sclerotia of Inonotus obliquus was ground to a fine power, followed by extraction with 60 L 80% ethanol for 2 h at 78 °C for thrice and the ethanol extracts were then mixed, evaporation solvent using rotavapor, the resulting fractions were dried in vacuum, then extracted with petroleum ether (PE), followed by ethyl acetate (EA) extracted. The yield of petroleum ether fraction (PEF) is 38 g.
For faster separation, the gradient was optimized on the basis of Ma’ s[32]: 25 g PEF (5 g) was subjected to normal phase silica gel column chromatography and gradient elution [750 g, P.E.–EtOAc (80:20 → 70:30 → 65:35 → 60:40 →55:45 →50:40 → 40:60)] to give 9 fractions (Fr.1 ∼ Fr.9). The fraction 4 was eluted by P.E.–EtOAc 60:40, after recrystallization using MeOH to give white acicular crystal compound, it was later identified by comparison of their spectral with reported values of TLC and 1H-NMR to be inotodiol and the purity is over 98%.[δ 0.75, 0.82, 0.88, 0.99,1.01, 1.67, 1.77 (3H, s, H3‐18, 29, 30, 19, 28, 26, 27), δ 0.95 (3H, d, J=6.6 Hz, H3‐21),δ 3.24 (1H, dd, J=11.3, 4.6 Hz, H‐5),δ 3.69 (1H, m, J=6.3, 3.3 Hz, H‐22), δ 5.19 (1H, t, J=7.2 Hz, H‐24)]
Preparation of IOP liposomes (IOP-Lps) and FA-IOP-Lps
IOP-Lps was prepared on the basis of the previously reported traditional methods[33], the preparation steps are shown in Fig. 2.
Briefly, IOP (1 mg), DPPC (15.98 mg), cholesterol (3.75 mg) and DSPE-PEG2000 (0.9 mg) were dissolved in methanol[34], after sonication, the supernatant were transferred into a 100 mL round bottom flask by pipet, then 15 ml chloroform was added, dealt with ultrasonic for 5 min, evaporate all solvent by rotary evaporation under high vacuum for 12 h, a thick blue film, that is the IOP liposomes[35]. Finally, 100 mL ultrapure water was added into the flask and sonicated for 5 min to obtain the aqueous dispersion and was kept in 4 °C refrigerator for further use.
Formulations of different materials is list as Table 1.
Table 1 Orthogonal test design: L9(34)of inotodiol liposomes
Factor
|
Level
|
1
|
2
|
3
|
A Chol(mg)
|
2.5
|
3.75
|
5
|
B DPPC(mg)
|
10.65
|
15.98
|
21.3
|
C DSPE-PEG2000 (mg)
|
0.6
|
0.9
|
1.2
|
D IOP(mg)
|
0.5
|
1
|
1.5
|
FA-IOP-Lps was later prepared at the optimal formulation of IOP-Lps by adding of extra 1 mg FA- DSPE-PEG2000.
Determination of entrapment efficiency (EE) and drug loading capacity (DL) of IOP-Lps
The EE and DL of liposome samples was determined by ultra centrifugation methods[36] and analyzed by HPLC.
The newly prepared liposome was quickly hydrated by 10.00 mL 0.01X PBS then centrifuged at 12,000 g for 30 min. Then the supernatant was analyzed by RP-HPLC (Agilent 1220, USA), the absorbance at 244 nm UV were recorded and peak area were calculated. The amount of free drug was determined by standard calibration curve of peak area. The standard calibration curve was obtained using different concentration of pure inotodiol[37].
Entrapment efficiency (EE) and drug loading capacity (DL) were calculated using the following formula[38]:
EE (%) = Mentrapped /Mtotal × 100% (1)
DL(%) = Mentrapped /MLps × 100% (2)
where Mentrapped is the IOP amount entrapped in liposomes, Mtotal is the total IOP amount in the resulted preparation and MLps is the amount of total IOP in the resulted preparation.
In vitro release of of IOP -Lps
2 mL of IOP-Lps was suspended in a MW 3500 dialysis bag[39,40] (activated by boiling water for 15 minutes) and then immersed into beaker with either 50 mL simulated gastric fluid (SGF), pH 5.5 phosphorous buffer and pH 7.4 PBS, each with 0.5% (v/v) Tween-80, and stirring at 37 °C under 200 rpm[47], beakers were wrapped with tin foil to protect them from light.
Characterization of of blank liposome, IOP-Lps and FA-IOP-Lps
IR spectra of blank liposome, IOP-Lps and FA-IOP-Lps
The infrared (IR) spectra of inotodiol, blank liposome, IOP-Lps and FA-IOP-Lps were characterized by potassium bromide (KBr) tableting method[41] using Bruker infra-red spectrophotometer (TENSOR 27, Germany). A tiny drop of hydrated IOP-Lps and was drop onto the surface of potassium bromide tablet, the transmittance within the wave number range of 4000-400cm-1 was measure after drying by infrared drying oven.
Morphology of IOP-Lps
One drop of IOP-Lps was taken and dropped on the copper net by the method of phosphotungstic acid negative staining[42]. After natural drying, it was negative stained with 1% phosphotungstic acid for 2min. After drying, the morphology of samples was characterized with transmission electron microscopy (TEM, JEM-2100F, JEOL, Japan).
Particle size and zeta potential of IOP-Lps and FA-IOP-Lps
The average particle size and zeta potential of samples was detected by dynamic light scattering (DLS) method by Malvern Zetasizer (Nano ZS 90, Malvern Instruments, UK) at room temperature[35]. Samples were suspensions with concentration below 1 mg/ml.
All procedures were carried out in triplicate.
Culture of HepG2, L02, MCF-7 and HeLa cells
The frozen HepG2, L02, MCF-7 and HeLa cells were taken out from the liquid nitrogen tank and resuscitated rapidly, they were cultured with the medium consist of 90% DMEM, 10% (v/v) FBS plus an extra 1% (v/v) penicillin/streptomycin under standard incubation condition (37 °C, 5% CO2). The cells were carried out to subculture for 3 times until the growth condition is stable.
In Vitro Cytotoxicity of inotodiol, IOP-Lps and FA-IOP-Lps
The cytotoxicity of cancer and normal cells were assessed using MTT assay. HepG2, L02, MCF-7 and HeLa cells (1 × 104 cells per well) were seeded in a 96-well plate and incubated overnight under standard incubation condition (37 °C, 5% CO2). Various concentrations of samples were added into each well to replace the original medium, followed by incubating for 12 h, 24h, 36h under standard incubation condition. Afterward, MTT solution (5 mg/mL, 20 μL) was added into each well and further incubated for 4 h. Finally, after the removal of old media and the generated formazan were dissolved with DMSO under mild shaking, the OD value at 490 nm (570 nm as reference) of each well were immediately measured by an enzyme-linked immunosorbent assay reader[43] to calculate the viability and IC50.
Statistical analysis
In this experiment, datum were tested three times unless otherwise stated. ANOVA (analysis of variance) were used for statistically significance. IC50 value were obtain from SPSS software. Data are expressed as mean ± standard deviation (SD).