3.1 BAPL Preparation and Encapsulation Efficiency Calculation
3.1.1 Preparation of BAPL by the Film Dispersion Ultrasonic Method
Step 1: Lecithin and cholesterol were dissolved in a mixture of 30 mL of petroleum benzine and 10 mL of absolute ethanol, and then the mixed liquid was placed in an eggplant-shaped flask to obtain the suspension. Step 2: The suspension obtained in Step 1 was rotated and evaporated to form a light-yellow translucent film at 80 r/min in a constant temperature water bath at 50 °C. Step 3: Twenty milliliters of phosphate buffer with AP was added to the eggplant-shaped flask to elute and disperse the film. Then, 5 mg of borneol dissolved in 0.5 mL of absolute ethanol was added and shaken for 10 min. Step 4: The suspension in Step 3 was placed in a 300 W probe ultrasonic water bath at 40 °C for 15 min. Step 5: The suspension in Step 4 was filtered three times with a 0.45-μm microporous membrane to prepare the BAPL suspension and was refrigerated at 4 °C until further use. The flowchart of BAPL preparation is as follows:
3.1.2 Calculation of the Liposome Encapsulation Efficiency
(1) Determination of the detection wavelength: 4 mL of 4 mg/mL AP solution was drawn accurately, and 5 mL of concentrated sulfuric acid and 1 mL of 5% phenol solution were added and mixed well. The mixed solution was placed in a boiling water bath for 15 min and cooled with running water. With pure water as the blank control, a Multiscan Spectrum was used to detect the wavelength range of 200 nm to 800 nm, and the absorption value was the maximum at 490 nm; therefore, the detection wavelength was determined to be 490 nm.
(2) Preparation of the standard curve: The standard curve was prepared with glucose standard substance. Ten milligrams of glucose standard was weighed precisely and dissolved in pure water. Then, a volume of 100 mL was made constant to obtain a 1 mg/mL glucose solution. Then, 10 mL of glucose solution was drawn from it and diluted to 100 mL with water to obtain the 0.1 mg/mL glucose standard solution. Glucose standard solutions of 0, 0.2, 0.4, 0.6, 0.8, 1.0 and 1.2 mL were drawn into centrifuge tubes, and pure water was added until 2 mL was reached. Then, 5 mL of concentrated sulfuric acid and 1 mL of 5% phenol solution were added to each tube and mixed well. All mixed tubes were placed in a boiling water bath for 15 min and cooled with running water. After cooling, the absorbance was detected at 490 nm with a Multiscan Spectrum. The glucose concentration (X) was used to regress the absorbance (Y) to obtain the regression equation: Y=0.041X+0.12, R2=0.998. The results showed that glucose had a good linear relationship in the range of 2.5～15 μg/mL.
- Determination of encapsulation efficiency: Four milliliters of prepared BAPL suspension was absorbed and centrifuged in a chilled ultracentrifuge (4 °C, 10,000 r/min, 30 min). The supernatant was extracted in a 50-mL volumetric flask, diluted with pure water to 50 mL and then mixed well. Four milliliters was accurately drawn into a 10-ml centrifuge tube. Five milliliters of concentrated sulfuric acid and 1 mL of 5% phenol solution were added and mixed. The solution was then heated in boiling water for 15 min and cooled with running water. After cooling, 100 µL was dropped into 96-well plates, and 100 µL of pure water was added to dilute each plate. The absorbance was detected with a Multiscan Spectrum, and the results were matched to the standard curve to calculate the concentration of free AP and obtain the mass of free AP from the concentration and ultimately to calculate the encapsulation efficiency. The formula for the encapsulation efficiency was encapsulation efficiency=(1-Afree/Atotal)×100%, where Afree was the mass of free AP and Atotal was the mass of added AP when preparing BAPL.
- The flow chart of BAPL encapsulation efficiency determination is shown below.
3.1.3 Determination of Borneol
According to the existing liposome preparation process, 3～10 mg of borneol was added as an adjuvant drug. Combined with the previous experiments in this study, it was found that the encapsulation efficiency of borneol was the highest when the mass of borneol was 5 mg; therefore, the mass of borneol used in this study was 5 mg.
3.1.4 Measurement of BAPL Particle Size and Surface Potential
(1) Measurement of liposome particle size: A 1-mL sample of BAPL suspension was slowly added into the sample tank with a 1-mL syringe and inserted into the groove. The DTS software was opened, and the number of measurements was set to 3. Then, the test was initiated, and the results were automatically saved. (2) Zeta potential test on the liposome surface: BAPL suspension was added to the side of the sample tank with a syringe until the sample was filled in a U-tube and inserted into the groove. The DTS software was opened, and the number of measurements was set to 3. Then, the test was initiated, and the results were automatically saved.
3.1.5 Electron Microscope Observation of Liposomes
A total of 10 µL of sample suspension was dropped on a copper mesh carrier with film. The sample was negatively stained with 2% phosphotungstic acid and dried in a static state. The microscopic morphology of BAPL was observed by transmission electron microscopy.
3.1.6 Statistical Analysis
The experimental results are expressed as`c ± s and were analyzed with SPSS 20.0 software. After quadratic regression analysis, the fitting equation of the relationship between encapsulation efficiency Y and influencing factor X was obtained. P < 0.05 indicated that the difference was statistically significant.
3.2 Study of the Anti-Cerebral Ischemia-Reperfusion Inflammatory Reaction of BAPL
In this study, a cerebral ischemia-reperfusion rat model was designed as the research object. TTC staining and Western blot analysis were used to explore the effect and mechanism of BAPL in the anti-cerebral ischemia-reperfusion inflammatory reaction.
The SD rats were fed for one week, weighed and then divided into 5 groups with 5 rats in each group: the BAPL group (cerebral ischemia-reperfusion model with BAPL suspension administration); the APL group (cerebral ischemia reperfusion model with APL suspension administration); the AP group (cerebral ischemia reperfusion model with AP administration); the Model group (cerebral ischemia reperfusion model with saline administration); and the Sham group, which was given saline.
3.2.2 Drug Administration and Model Preparation
The SD rats were fed adaptively for 7 days followed by tail vein injection once a day for 3 days (BAPL suspension, APL suspension and AP solution were injected at a single dose of 30 mg/kg AP). The rats were modeled 2 hours after the last administration, and the rats were fasted 12 hours before the operation. The cerebral ischemia-reperfusion rat model was established by the suture method. The Sham group underwent the same operation without inserting a suture through the common carotid artery. After cerebral ischemia in rats was established, penicillin was used to avoid infection, and then the skin was sutured. After 2 hours of ischemia, the rats were anesthetized again, and the sutures were pulled out to restore the blood supply of the left common carotid artery and left internal carotid artery; thus, the rat model of cerebral ischemia was established for 2 hours, followed by reperfusion for 24 hours. During reperfusion, the drug administration schedule was the same as before. During the operation, the rats were continuously heated with a heating blanket and incandescent lamp to maintain their rectal temperature at 37.5±0.3 °C. The dry bedding materials were paved at 3 to 5 cm height. After the operation, the rats were placed in a thermostat until they awakened. Then, the rats were moved to a feeding box paved with 3 to 5 cm dry bedding materials.
3.2.3 Scores of Neurological Function
The neurological deficits of rats were scored by Zea-Longa’s score. The scores ranged from 0 to 4 points. The higher the score, the more severe the neurological deficits were. The standards of Zea-Longa’s scores are shown in Table 1.
Table 1 Five-grade Standard Scores by Zea-Longa
Behavioral and Psychological Symptoms Points
Normal movement; when lifting the tail, the two fore claws can
straighten to the ground without neurological deficit. 0
When lifting the tail, the right fore claw cannot straighten
completely, and the rats circle around to the right when crawling. 1
When lifting the tail, the right fore claw is incurvate, and
the rats circle around to the right when crawling. 2
Stand unsteadily and fall to hemiplegia side when crawling. 3
Conscious disorder and unable to walk spontaneously. 4
3.2.4 Observation of Cerebral Infarction Volume by TTC Staining
After ischemia for 2 hours and reperfusion for 24 hours, pentobarbital was injected intraperitoneally into the rats, and brain tissue was collected after death. TTC staining was used to observe cerebral infarction: 3 rats were randomly selected in each group. Their brain tissue was removed by decapitation and placed in a refrigerator at -20 °C for several minutes, and then the olfactory bulb, cerebellum and low brainstem were removed. Five coronary slices were cut from the frontal pole to the occipital pole, immersed in 2% TTC phosphate buffer, stained in the dark at 37 °C for 30 min, fixed with 4% formaldehyde solution for 30 min and photographed. The white part was the infarct area, while the red part was the normal brain tissue. Image-Pro Plus 6.0 software was used to analyze the cerebral infarction area of rats in each group. The calculation formula was V=A(B1+B2 … …+ Bn) for the calculation of cerebral infarction volume in rats, and its percentage to the whole rat brain was further calculated, where A was the slice thickness and B was the infarct area.
3.2.5 Western-Blot Analysis of Protein Expression
The rat brain tissues from the infarction area were removed and repeatedly ground at low temperature to obtain brain tissue homogenates. After adding the appropriate RIPA lysate, the supernatant was collected by centrifugation at 4 °C. The total protein concentration was measured by the BCA method. The sample was loaded with 20 μg of total protein, subjected to constant voltage electrophoresis and then transferred to a PVDF membrane. After sealing and rinsing, the IL-1β (1:1000), IL-6 (1:1000), IL-8 (1:1000), IL-10 (1:1000), NF-κBp65 (1:2000), TLR-4 (1:750), ZO-1 (1:5000), ZO-2 (1:5000), and β-a primary antibodies (1:5000) were added and incubated overnight and were then washed 3 times with TBST for 15 min each time. Then, goat anti-rabbit secondary antibody (1:6000) or goat anti-mouse secondary antibody (1:5000) was added, incubated at 37 °C for 1 hour and washed 3 times with TBST for 10 min each time. ECL chemiluminescence agent was added to the gel image analysis system, and the exposed films were scanned. Quantity One software was used for professional gray analysis, and the relative values of the target proteins were obtained from the ratio of the gray levels between the IL-1β, IL-6, IL-8, IL-10, NF-κBp65, TLR-4, ZO-1, ZO-2 and β-actin protein bands.