*2.1. Materials*

L-lactide was purchased from Purac Co., Ltd. (Shanghai, China). Dichloromethane (DCM), ethanol, tert-butyl methyl ether, and isopropanol were purchased from Tianjin Damao Chemical Reagent Co., Ltd. (Tianjin, China) and were all analytical grade. ASAs were purchased from Kunming Institute of Botany, Chinese Academy of Sciences, batch number 20180501 (Kunming, China). ASAs is a mixture of brown solid powder. Two of main active ingredients are Vallesamine and Picrinine, which were detected by HPLC with peak times of 43.133 min and 72.190 min. NaH2PO4, KH2PO4, and NaCl were purchased from Chengdu Chron Chemicals Co., Ltd. (Chengdu, China). Stannous octoate (95%, analytical grade) and polyvinyl alcohol (PVA; Mw = 75,000 Da and 88% alcoholysis degree) were purchased from Shanghai Jingchun Chemicals Co., Ltd. (Shanghai, China). mPEG (Mw 5000) was purchased from Shanghai Seebio Biotechnology Co., Ltd. (Shanghai, China). HL-7702 cells (normal human hepatocytes) were purchased from Procell Life Sciences and Technology Co., Ltd. (Wuhan, China). High glucose and 1640 medium were provided by HyClone Company. The 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) was purchased from APExBIO (Houston, TX, USA). High-quality fetal bovine serum (FBS) was purchased from Shanghai ExCell Bio Co., Ltd. (Shanghai, China). Ethylenediaminetetraacetic acid (EDTA)-trypsin solution, cyan-streptomycin, and cisplatin were provided by Solarbio (Shanghai, China). Kunming mice, Sprague-Dawley (SD) rats, and rabbits were purchased from Kunming Medical University (Kunming, China). Dengtaiye Pian (DP) was purchased from Yunnan Datang Hanfang Pharmaceutical Co., Ltd. (Kunming, China). Aspirin was purchased from Bayer HealthCare Manufacturing Co., Ltd. (Beijing, China).

#### *2.2. Preparation of mPEG-PLA*

mPEG-PLA was prepared by ring-opening polymerization. L-LA (18 g) and mPEG (2 g) were putted in reaction flask as raw materials. Protected by nitrogen, catalyzed by stannous octoate (0.02 g), the mPEG-PLA was polymerized at 130–160 ◦C for 24 h. Using recrystallization, the product was precipitated by methylene chloride and ethanol. Product was filtered and washed with hot water to remove the mPEG bulk polymer. The obtained product was dried with P2O5 for 48 h to obtain a white copolymer. Figure 1 shows the synthesis process.

**Figure 1.** The synthesis process of mPEG-PLA.

#### *2.3. Determination of Molecular Mass*

3 mg copolymer were dissolved in 1 mL chromatography grade THF and was filtered by using a 0.45 μm nylon 66 filter membrane. Then using gel permeation chromatography (GPC) which was purchased from Waters Inc. (Milford, MA, USA) to define the molecular weight and distribution of the compounds, and THF was used as the eluent. The system is equipped with a column (7.8 × 300 mm, Waters Styragel, Waters Inc., Milford, MA, USA), a Waters 515 pump and a Waters 2414 refractive index detector. When the column temperature of GPC is 40 ◦C, the flow rate is 1 mL/min, and the baseline is smooth. The filtrate was pulled into an injection needle, and the sample was slowly and uniformly injected into the sampler when the air was removed. All data were obtained under the same standard curve.

#### *2.4. Preparing the Blank and ASAs-Loaded mPEG-PLA Microspheres*

The ASAs-loaded mPEG-PLA microspheres were prepared using the W/O/W double-emulsion technique. First, 100 mg of ASAs were dissolved in 1 mL methanol solution, which was the internal water phase (W1). The 500-mg mPEG-PLA copolymer material was weighed and completely dissolved in 10 mL of methylene chloride as the oil phase (O). The external water phase (W2) was a 2% PVA solution. Next, the ASAs solution was injected into the oil phase in an ice bath at a constant rate. The mixture was emulsified at high speed (21,000 rpm) for 2 min to form the first emulsion. Similarly, the first emulsion was continuously dropped into 20 mL of 2% PVA (W2) in an ice bath and emulsified at high speed (21,000 rpm) for 2 min to form a double emulsion. The double emulsion was poured into 400 mL of 5% isopropanol solution and stirred at low speed at room temperature for 6 h. After the organic solvent had completely evaporated, the ASAs-loaded microsphere solution was obtained. Finally, the ASAs-loaded microsphere solution was centrifuged (6500 rpm, 10 min), and the supernatant was discarded. The microspheres were collected (white solid), washed with pure water 3 times, and lyophilized at −50 ◦C for 24 h. The obtained microspheres were dried at −20 ◦C. Blank microspheres were prepared by the same method.

#### *2.5. Morphology, Particle size and Particle Size Distribution of the Microspheres*

The morphological characteristics of the ASAs-loaded mPEG-PLA microspheres were observed using SEM (NOVA NANOSEM-450, FEI, Hillsboro, OR, USA). Conductive adhesive tape was applied to the surface of the loading platform. A few lyophilized microspheres (white powder) were evenly spread on a conductive adhesive, and the surface was sprayed with gold to observe. The particle size distribution of the microspheres was measured using a laser particle-size analyzer (Mastersizer 3500, Microtrac, Malvern, UK). A small portion of the microsphere suspension was diluted in a 15 mL centrifuge tube until the solution became nearly transparent. The particle size distribution of the microspheres was determined using a particle-size analyzer.

#### *2.6. Measuring the EE of the Microspheres*

Twenty milligrams of lyophilized microspheres were weighed, and 500 μL of methylene chloride was added to completely dissolve them. Next, 2 mL of methanol solution was added. After centrifugation, the supernatant was filtered through a 0.45-μm microfiltration membrane. Next, 20 μL of filtered solution was injected via HPLC, and the area normalization method was used to calculate the EE% and LE% using the following equations.

$$\text{LE\%} = \frac{\text{weight of ASAs in microsphere}}{\text{total weight of microsphere}} \times 100\%$$

$$\text{EE\%} = \frac{\text{weight of encapsulated drug}}{\text{weight of initial drug loading}} \times 100\%$$

#### *2.7. In Vitro Microsphere Release*

Fifty milligrams of lyophilized microspheres were weighed and placed in a 15 mL centrifuge tube, then 5 mL of phosphate buffer saline (PBS) was added and shaken in a thermostatic oscillator at 37 ◦C. The tube was removed at set time intervals and centrifuged at 5000 rpm for 6 min. Next, 1 mL of the release solution was removed from the supernatant, an equal amount of fresh PBS was added to the tube, and the tube was placed back in the thermostatic oscillator and shaken at 37 ◦C. The ASAs content in the release solution was determined by HPLC, and the cumulative drug release amount was calculated using the following formula [20]. The amount (%) of the released ASAs-loaded microspheres was plotted against the release time (t, in days) to obtain the in vitro release curve of the microspheres.

$$\mathbf{Q} = \mathbf{C}\_{\mathbf{n}} \times \mathbf{V}\_1 + \mathbf{V}\_2 \sum \mathbf{C}\_{\mathbf{n}-1}$$

Q: cumulative drug release (μg);

Cn: concentration of the release solution (μg/mL) removed at time t;

V1: volume of the released medium (mL);

V2: volume of the medium to be withdrawn each time (mL).

#### *2.8. In Vitro Microsphere Degradation*

Fifty milligrams of lyophilized microspheres were weighed and placed in a 15 mL centrifuge tube, and then, 5 mL of PBS was added and shaken at 37 ◦C. The tube was removed at set time intervals and centrifuged at 5000 rpm. The supernatant was collected to determine the pH changes in the microspheres. Buffer salts on the microsphere surface were washed with pure water and frozen at −50 ◦C for 24 h. The microspheres were weighed using an analytical balance, and the dry weight loss of the degraded microspheres was calculated. The molecular weight of 3 mg of the lyophilized microspheres was measured via GPC. The pH value, the microsphere weight and the relative molecular weight (Mn) were separately plotted against degradation time to obtain the in vitro degradation curve of the microspheres.

#### *2.9. Blood Compatibility Testing of The Microspheres*

All the trials were approved by the laboratory animals Ethics Committee of Yunnan Minzu University and were registered on the Kunming Science and Technology Bureau (SYXK (Yunnan) K2017-0001, 16 January 2017).

## 2.9.1. Hemolysis Experiment

The samples were dissolved in deionized water (DI water), and the solutions were prepared at concentrations of 10.6 μg/mL, 1.06 mg/mL, and 0.106 mg/mL. Eight milliliters of fresh anticoagulated rabbit blood (blood mixed with sodium citrate at a volume ratio of 9:1) was diluted with 10 mL of 0.9% sodium chloride solution. Two hundred microliters of the sample was placed in a test tube, and 5 mL of 0.9% sodium chloride solution was added. The tube was then kept in a 37 ◦C water bath for 30 min. Next, 100 μL of diluted blood was added, and the mixture was gently mixed and incubated for 60 min. The positive control was treated with 5 mL DI water and 100 μL blood (D = 0.8 ± 0.3). The negative

control was treated with 5 mL 0.9% NaCl solution and 100 μL blood. After centrifugation at 800 r/min for 5 min, the supernatant was transferred into a cuvette, and an ultraviolet (UV) spectrophotometer was used to measure the absorbance at 540 nm. Hemolysis of the sample was calculated per the formula below. The final sample concentrations were 40 μg/mL, 4 μg/mL, and 0.4 μg/mL. The material was considered hemolyzed when the hemolysis rate was above 5% [21–27].

$$\text{Hemolys is rate} (\%) = \frac{\text{Abs}\_{\text{sample}} - \text{Abs}\_{\text{negative control}}}{\text{Abs}\_{\text{positive control}} - \text{Abs}\_{\text{negative control}}} \times 100\%$$

## 2.9.2. Coagulation Experiment

The samples were dissolved in distilled water to prepare solutions with concentrations of 245.2 μg/mL, 24.52 μg/mL, and 2.452 mg/mL. The final concentrations were 40 μg/mL, 4 μg/mL, and 0.4 μg/mL. Next, 200 μL of the solution was placed in a 15-mL centrifuge tube and kept at 37 ◦C for 5 min. Fifty microliters of fresh anticoagulated rabbit blood was added to the samples, which were and kept at a constant temperature for 5 min. Ten microliters of aqueous calcium chloride solution (0.2 mL/L) was added to the blood sample, and the centrifuge tube was shaken to evenly mix the calcium chloride and blood and kept at a constant temperature for 5 min. The centrifuge tube was then removed, 12 mL of DI water was added to the solution, and the supernatant was collected. The blood was measured at 540 nm using a UV spectrophotometer. The optical absorbance (i.e., the optical density [OD] of the free hemoglobin remaining in the beaker containing 50 μL of whole blood treated with 12 mL of DI water) was used as a reference. The average of 5 measurements was taken. The sample's anticoagulant activity was expressed as the relative absorbance [28,29]:

$$\text{BCI} = \frac{\text{I}\_{\text{o}}}{\text{I}\_{\text{w}}} \times 100\% \text{ o}$$

Io: relative absorbance of the mixture of blood and calcium chloride after contact with the sample for a set period of time;

Iw: relative absorbance of blood mixed with a certain amount of DI water.

#### *2.10. Cytotoxicity of The Microspheres*

#### 2.10.1. Cell Culture

Normal human hepatocytes (HL-7702) were inoculated into culture flasks, and RPMI-1640 medium (containing 10% FBS, 100 U/mL penicillin, and 100 U/mL streptomycin) was added. Cells were cultured in a 5% CO2 incubator at 37 ◦C. Cells grew as monolayers adherent to glass and were passaged once every 3–5 days using 0.25% trypsin.

#### 2.10.2. MTT Assay of Sample Inhibition on Tumor Cell Proliferation

HL-7702 cells (180 μL; 5 × 10<sup>4</sup> cells/mL) in the logarithmic growth phase were inoculated in a 96-well plate, and 20 μL of the sample was added to each well after incubating overnight. Three concentration gradients were set with 3 wells per concentration. After a 48-h incubation, 20 μL of 5 mg/mL MTT was added to each well, and the cells were continuously cultured for 4 h. The culture medium was aspirated and discarded, and 150 μL dimethylsulfoxide (DMSO) was added to terminate the reaction. The plate was shaken for 15 min in a shaker. The OD value at 490 nm was measured in a microplate reader [30,31], and the inhibition rate was calculated [32]:

$$\text{Cell viability} = \frac{\text{ODsample}}{\text{ODnegative control}} \times 100\% \text{ \%}$$

#### *2.11. Anti-Inflammatory Activity Testing of The Microspheres*

To determine the influence of xylene-induced auricle swelling on the mice [33], 35 Kunming mice (19–22 g) were randomly divided into 7 groups by body weight and sex, with 5 mice per group. Except for the aspirin group, only one intragastric administration on the day of the experiment, the other groups were pre-administered once daily for 3 consecutive days. The control group was administered the same volume of 1% CMC-Na (carboxymethyl cellulose-sodium). The gavage volume was 20 mL/kg per group. Thirty minutes after the last gastric gavage, 0.05 mL of xylene was evenly applied on both sides of each mouse's right ear, while the left ear was used as the control. The mice were sacrificed via cervical dislocation 1 h after inflammation. The same area of both ears was cut o ff using a 10-mm-diameter puncher, and the weight di fference between the two ears was used as the swelling degree.

To determine the influence of the egg-white-induced pedal swelling in the rats, 35 male SD rats (170–220 g) were randomly divided into 7 groups by body weight, with 5 rats per group. All groups were intragastrically administered the microspheres once daily for 3 consecutive days, except the aspirin group, which received the microspheres via intragastric administration on the day of the experiment. The control group received the same volume of 1% CMC-Na (carboxymethyl cellulose-sodium). The gavage volume was 10 mL/kg per group. Thirty minutes after the last gastric gavage, 0.1 mL of fresh egg white was injected subcutaneously into the pedal of the right rear foot of each rat to induce inflammation. The foot areas before inflammation and at 0.5, 1, 2, 3, 4, and 5 h after inflammation were measured. The di fference in the foot area before and after inflammation was determined as the degree of swelling, and the swelling rate was calculated.

$$\text{Swell rate} = \frac{\mathbf{A}\_{\text{t1}} - \mathbf{A}\_{\text{t0}}}{\mathbf{A}\_{\text{t0}}} \times 100\% \text{ }$$

At0: foot area before administration At1: foot area after administration

#### **3. Results and Discussion**

#### *3.1. Characterization of Copolymer mPEG-PLA*

Figure 2 shows the nuclear magnetic resonance (NMR) spectra of the mPEG-PLA copolymer. The 1H NMR image shows the characteristic peaks of hydrogen in the mPEG-PLA. The peaks at 5.19 ppm and 1.5 ppm correspond to the PLA's methine and methyl peaks, respectively. The methylene peak at 3.6 ppm is the repeating unit in the PEG. The peak at 3.39 ppm corresponds to the hydrogen of the CH3O- group of the mPEG at the end of the copolymer. NMR spectroscopy was performed at 25 ◦C (Bruker 400 MHz, Karlsruhe, Germany).

Figure 3 shows the mPEG-PLA gel permeation chromatography (GPC) curve with the di fferent molecular weights of mPEG. Data were processed using di fferent molecular weights of polystyrene as the reference material. Table 1 shows the weight-average molecular weight (Mw), number-average molecular weight (Mn) and polydispersity index of the molecular weight distribution (PDI) of the copolymer. The obtained copolymers have relatively small PDIs and relatively uniform molecular weights. Four copolymers with di fferent molecular weights were prepared by copolymerization of four mPEG with the same amount of L-LA. The GPC curves show that the di fferent molecular weights of the four mPEG-PLA, they all have a single peak, indicating that no other copolymers were formed. The copolymer synthesized from mPEG of di fferent molecular weights, wherein the larger the molecular weight of mPEG, the higher the molecular weight of the obtained copolymer and the shorter the peak time of the copolymer, which is consistent with the results obtained by the GPC test.

**Figure 2.** 1H nuclear magnetic resonance (NMR) spectrum of mPEG-PLA.

**Figure 3.** Gel permeation chromatography (GPC) of mPEG-PLA.

**Table 1.** The Mn, Mw and PDI of different molecular weight of mPEG-PLA.


1 Determined by GPC via a universal calibration curve and appropriate Mark−Houwink parameters for THF. 2 Calculated by 1H NMR. Mn: The number-average molecular weight; Mw: The weight-average molecular weight; PDI: polydispersity index of the molecular weight distribution.

#### *3.2. Morphology and Size Distribution of the Microspheres*

Figure 4 shows the scanning electron microscopy (SEM) images of the mPEG-PLA microspheres loaded with ASAs. Figure 4A shows mPEG550-PLA microspheres, which have a small particle diameter, a round smooth spherical shape, and no obvious adhesion. Figure 4B shows mPEG2000-PLA microspheres with small pores on the surface and varied particle sizes. Figure 4C shows mPEG5000-PLA microspheres with a few small pores on the surface and with di fferent pore sizes and numbers. Figure 4D shows mPEG10000-PLA microspheres. Compared with the mPEG550-PLA, mPEG2000-PLA, and mPEG5000-PLA microspheres, the mPEG10000-PLA's particle size is larger, its surface has few small pores and its shape is smooth. The adhesion is due to the relatively large molecular weight of the mPEG-PLA copolymer. The pores on the surface are caused by water molecule di ffusion during lyophilization. Figure 5 shows the particle size distribution of the mPEG-PLA microspheres loaded with ASAs using a laser particle-size analyzer (Mastersizer 3500, Microtrac, Malvern, UK). The average diameter of the mPEG550-PLA microspheres was 1.803 ± 0.21 μm, and the PDI was 0.15. The average diameter of the mPEG2000-PLA microspheres was 2.083 ± 0.17 μm, and the PDI was 0.14. The average diameter of the mPEG5000-PLA microspheres was 2.631 ± 0.2 μm, and the PDI was 0.20. The average diameter of the mPEG10000-PLA microspheres was 3.84 ± 0.30 μm, and the PDI was 0.18(Table 2). These results are nearly the same as those shown in the SEM images. The experimental results showed that the diameters of the 4 microspheres were smaller than 5 μm, indicating that these microspheres are suitable for oral of drug delivery, which is consistent with the study by Wei et al. [34].

**Figure 4.** Scanning electron microscopy (SEM) images of mPEG-PLA microsphere. ( **A**) mPEG 550-PLA, (**B**) mPEG 2000-PLA, ( **C**) mPEG5000-PLA, ( **D**) mPEG10000-PLA. The magnification of SEM image was 2000.

**Figure 5.** Size distribution of mPEG-PLA microsphere.

## *3.3. Drug Loads*

mPEG-PLA microspheres loaded with ASAs were prepared using the W/O/W double-emulsion technique, and the EE and LE were measured by high-performance liquid chromatography (HPLC; Table 2). With the increased molecular weight, the microsphere's EE and LE increased, with trends similar to those of the mPEG-PLA microspheres loaded with recombinant human growth hormone (rhGH) prepared by Yi et al. [35]. While preparing the microspheres, a portion of the drug diffused into the external water phase due to solvent evaporation, resulting in drug loss. A copolymer with a high molecular weight has a large molecular gap, enabling the drug to diffuse outward, thus increasing drug loss.

**Table 2.** The average diameter, PDI, EE, LE of mPEG-PLA microspheres loaded ASAs.


1PDI: polydispersity index of the particle size distribution. EE: encapsulation efficiency. LE: loading efficiency.

#### *3.4. Analysis of In Vitro Microsphere Release*

Figure 6 shows the release curve of mPEG-PLA microspheres loaded with ASAs in phosphate-buffered saline (PBS) (pH = 7.40). The cumulative amount of mPEG550-PLA microspheres released on the first day was 24.32 ± 0.37%, and then, the release rate slowed, reaching 64.38 ± 1.21% after a two-week stable release. The cumulative amount of mPEG2000-PLA microspheres released on the first day was 20.74 ± 0.29%, and then, the release rate slowed, reaching 57.09 ± 1.28% after a two-week stable release. The cumulative amount of mPEG5000-PLA microspheres released on the first day was 19.54 ± 0.22%, and then, the release rate slowed, reaching 52.45 ± 1.28% after a two-week

stable release. The cumulative amount of the mPEG10000-PLA microspheres released on the first day was 15.03 ± 0.51%, and then, the release rate slowed, reaching 45.12 ± 1.04% after a two-week stable release. The relatively large amount of the drug released on the first day occurred because a small amount of the drug adhered to the microsphere surface when the microspheres encapsulated the drug. With the microsphere's increased molecular weight, in vitro release of the drug from the microspheres was relatively slow. The release time of the mPEG-PLA microspheres in this study was longer than that of the microspheres prepared by Zheng et al. [36] and Xiong et al. [37], indicating that the mPEG-PLA was well encapsulated on the ASAs.

**Figure 6.** Release curve of mPEG-PLA microspheres loaded ASAs in PBS

#### *3.5. Analysis of In Vitro Microsphere Degradation*

Based on the morphology, particle size, EE, LE, and in vitro microsphere release, we selected mPEG10000-PLA as the optimal microspheres for the study. Figure 7 shows the degradation process of the ASAs-loaded mPEG10000-PLA microsphere. The figure shows the changes in the microsphere's dry weight, system pH and molecular weight over time. In PBS (pH = 7.40, 10 mM), the three curves all decreased with time. Within 60 days, the microspheric pH decreased from 7.40 to 5.89, the microspheres lost 47.16% of their dry weight, and their molecular weight dropped from 10307 Da to 8258 Da. As its degradation progressed, the mPEG-PLA was hydrolyzed to produce CO2, which was dissolved in water to decrease the pH. mPEG-PLA is an amphiphilic material that absorbs water in PBS and breaks the hydrophilic segment, thus decreasing the molecular weight. As the hydrophilic segmen<sup>t</sup> breaks, the molecular weight of the copolymer decreases, resulting in a loss of copolymer quality. Li et al. [38] studied the degradation of mPEG-PLA nanoparticles and found that the mPEG-PLA degraded very slowly, and the Mn decreased by 27.6% within 30 days. Simone [39] et al. studied the polymer degradation conditions at pH and found that the pH decreased correspondingly over time, which is similar to the mPEG-PLA degradation trend in this study.

**Figure 7.** The degradation process of the ASAs-loaded mPEG10000-PLA microspheres in the phosphate buffer saline (PBS) (pH = 7.40): the pH (**A**) Mn (**B**) and mass (**C**) changed with time.

#### *3.6. Analysis of In Vitro Hemolytic Properties of Microspheres*

Figure 8 shows the hemolysis and anticoagulation rates of the ASAs-loaded mPEG10000-PLA microsphere, the mPEG10000-PLA, and the ASAs. The hemolysis rates of the ASAs-loaded mPEG10000-PLA microsphere, the mPEG10000-PLA, and the ASAs increased as the concentration increased but did not exceed 5% at 40 μg/mL. Studies have shown that hemolysis rates below 5% indicate blood compatibility, which is consistent with the International Organization for Standardization (ISO) hemolysis standard, indicating applicability for intravenous injection. The anticoagulant chart shows that the blood clotting index (BCI) increases as the concentration increases, indicating that its anticoagulant activity increases as the sample concentration increases.

**Figure 8.** Hemolytic activity (**A**) and blood clotting index (**B**) of the ASAs-loaded mPEG10000-PLA microsphere. \* *P* < 0.05 and \*\* *P* < 0.01, compared with control, and the whole blood sample with the ASAs was used as a control.

#### *3.7. In Vitro Cytotoxicity Analysis of The Microspheres*

Figure 9 shows the cytotoxicity of the ASAs-loaded mPEG10000-PLA microspheres, the mPEG10000-PLA, and the ASAs to the HL-7702 (normal human hepatocyte) cell line. To study the cytotoxicity of the ASAs-loaded mPEG10000-PLA microspheres, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to detect HL-7702 cell viability under different concentrations of ASAs-loaded mPEG10000-PLA microspheres, mPEG10000-PLA, and ASAs. Figure 8 shows the toxicity at concentrations from 0.4 μg/mL to 40 μg/mL. The results showed that mPEG10000-PLA microspheres could be used for drug loading. The microspheres prepared by Song et al. [40] showed that the HL-7702 cells were dose- and time-dependent, and as the concentration increased, the HL-7702 cell survival rate decreased, which is similar to the HL-7702 cell viability under the ASAs in this study.

**Figure 9.** The cytotoxicity of the ASAs-loaded mPEG10000-PLA microspheres, the mPEG10000-PLA, and the ASAs to the HL-7702 cells. \* *P* < 0.05 and \*\* *P* < 0.01, compared with control, and the whole blood sample with the ASAs was used as a control.

#### *3.8. Analysis of the Microspheric Anti-Inflammatory Activity*

Table 3 shows the effect of the ASAs on xylene-induced auricle swelling in mice. The ASAs-loaded microspheres significantly inhibited the xylene-induced auricle swelling in the mice. The effect of the ASAs-loaded microspheres at 10 mg/kg was comparable to that of the 0.3 g/kg DP, and the efficacies at 20 and 40 mg/kg were stronger than that of 10 mg/kg aspirin.

**Group Doses (**/**kg) Auricular Swelling Degree (x** ± **s, mg) Inhibition Rate (%)** Control — 50.85 ± 3.61 — Aspirin 10 mg 34.65 ± 2.90 31.86% DP 0.3 g 37.65 ± 3.04 25.96% ASAs 0.1 g 39.65 ± 0.78 22.03% 10 mg 39.00 ± 2.69 23.30% Loaded 20 mg 31.35 ± 6.86 38.35% microspheres 40 mg 27.65 ± 7.57 45.62%

**Table 3.** Effect of the ASAs on xylene-induced auricle swelling in mice.

Table 4 shows the effect of the ASAs on egg-white-induced pedal swelling in rats. The ASAs-loaded microspheres significantly inhibited the egg-white-induced pedal swelling in the rats, and the action time of the ASAs-loaded microspheres at 20 mg/kg was longer than that of the DP, and the action time of ASAs-loaded microspheres at 80 mg/kg was similar to that of aspirin.


**Table 4.** Effect of the ASAs on egg-white-induced pedal swelling in rats.
