## 3 Results
### In Vivo Acute Toxicity of Copolymer Hydrogels with Loading Multi-drug  
Figure 4 showed photographs of SD rats at indicated days after injecting hydrogels with different mixed drug dosages into the vagina. For the control group (injected hydrogels without drugs), no significant tissue necrosis was found on dates specified throughout our observation. This result was consistent with our previously reported paper [40; 44], which used a subcutaneous injection into the neck of SD rats instead of injecting into the vagina and, confirmed a good biocompatibility of star-shaped PLGA-mPEG copolymer hydrogel as drug carrier. For the high dosage group, slight hydroncus of the uterus was observed on day 1. The swelling of uterus became extremely serious and intestinal adhesion was also found at day 3. Uterus and intestines ulcerated at day 5. With regarding the middle dosage group, no tester of tissues was observed, and blood coagulum existed in the uterus at a different defined time. For the low dosage group, no significant tissue necrosis was found from day 1. Figure 4: Photographs of Sprague-Dawley (SD) rats at various indicated days after injecting hydrogels loading with different drug dosages into the vagina (high dosage, middle dosage, and low dosage). The CP-3 block copolymer solution at the concentration of 30 wt% was used. The volume of drug-loaded copolymer solution injected into the vagina is 0.2 mL. Copolymer solutions without loading drug were injected into the vagina of SD rats as a control group. In the control group, liver sections showed normal hepatic cells with well-preserved cytoplasm, prominent nucleus and nucleolus, and central vein, as shown in Figure 5. No abnormalities were observed in liver organs in the low dosage and middle dosage group. However, on day 3 and day 5 hepatocytes necrosis exhibiting pale cytoplasm color with an unclear interface and lymphocytic infiltration in liver were observed in high dosage group, suggested that copolymer hydrogels packed higher dose was toxic to the hepatic organ [47]. Figure 6 showed the HE staining of uterus sections at various indicated days after injecting hydrogels with different mixed drug dosages into the vagina. In the control and low dosage group, no lesion was found in the uterus. Endometrium was composed of a cylindrical epithelium with basally located nuclei which lined both the uterine lumen and glands and lamina propria is densely packed [48]. As to the middle dosage group, epithelial hyperplasia was presented in almost all animal samples, and uterus congestion was found at day 3. In the high dosage group, extremely severe swelling of the cylindrical epithelium of uterus was observed at day 1, 3, and 5. Figure 7 showed the HE staining of vagina sections at the various indicated time after injecting hydrogels with different mixed drug dosages into the vagina. In the vagina, no histological changes were observed in the control and low dosage group. Squamous cell hyperplasia was prominent in the middle dosage group [49]. However, in the high dosage group, vaginal bleeding was found at all indicated times. The histological photomicrographs of the spleen sections were shown in Figure 8. No severe damages of spleen tissue were reflected in the control and the low dosage group, while lymph nodule proliferation was observed in the middle and high dosage group [50]. In addition, there were no abnormal pathology changes in the heart, lung and kidney tissues, as shown in Figures S5-S7. Figure 5: Hematoxylin-eosin (HE) staining of Sprague-Dawley (SD) rat livers at various indicated days after injecting hydrogels with different drug dosages into the vagina (high dosage, middle dosage, and low dosage). The CP-3 block copolymer solution at the concentration of 30 wt% was used. The volume of drug-loaded copolymer solution injected into the vagina is 0.2 mL. Copolymer solutions without loading drug were injected into the vagina of SD rats as a control group, scale bar: 5 μm.