## 4 Discussion
The drug released from hydrogel might be mainly controlled by drugs' diffusion and the degradability of copolymer hydrogel during the first week, after then, the degradability of copolymer hydrogel became the main factor to regulate drug release profiles. The biodegradation rate of star-shaped 4 sPLGA-mPEG copolymer hydrogel could be regulated by LA/GA mol ratio. In addition, the acid environment of the vagina could urge the degradation of prepared block copolymer hydrogels, mainly due to the break of ester bonds in block copolymers. The degradable mechanism of the star-shaped 4 sPLGA-mPEG copolymer was shown in Scheme 2. In brief, the degradation process of the copolymer was divided into the following three steps. In the first step, the ester bond linked with the mPEG blocks preferentially hydrolyzed and the composition of PLGA segments were invariant. In the second step,the GA units in the PLGA blocks and the ester bonds linked with the mPEG blocks mainly hydrolyzed. In the final step, the LA units in the PLGA blocks mainly hydrolyzed [46]. In addition, the in vitro release profiles of indomethacin (IMC), gestodene (GSD), and ethinyl estradiol (EE) were fitted well with the [PERSON] model as shown in Table S2. The Higuchi equation was written as Equation (1). Professor [PERSON] published the derivation of an equation to evaluate the quantification of drug release from thin ointment films into the skin, then to learn more about the drug release mechanism [59; 60]. Although in vitro release profile of drugs was indeed agreed with the Higuchi model, in vivo release profiles still need to be further analyzed. Here, the prepared 4 sPLGA-mPEG copolymer was used as a drug release matrix, the transport from this copolymer hydrogel in vaginal site might often lead to release under conditions that were inconsistent with Higuchi's behavior, especially in the vaginal environment, such as that the hydrogel has adhered to mucus layer, so edge effects need to be considered (edge effects are negligible as one of Higuchi conditions) [61]. Furthermore, the anatomy of the vagina and vascular supply makes this organ a special route of drug administration, and the contraceptive drugs are particularly suitable delivering through the vaginal epithelium, which allows quick absorption and unaffected by gastrointestinal disturbance [18; 62]. It was known that the vaginal epithelium with a large area of high vascularization, was highly permeable to small molecules [63]. The multi-drug released from 4 sPLGA-mPEG block copolymer hydrogel by drug diffusion and the biodegradability rate of the copolymer has a significant influence on the drug release profile. In the vaginal environment, some of the drugs were released to the vagina, some were absorbed by vaginal epithelial cells, as shown in Scheme 3. In vivo release profiles of drugs in simulated vagina closer to real vaginal environment need to be explored in the future. \[M_{\mathrm{t}}/M_{\infty}=kt^{1/2}+b \tag{1}\]  
where \(M_{\mathrm{t}}\) is defined as the amount of drug released at time \(t\), and \(M_{\infty}\) is the amount of drug released as time approaches infinity. Next, the 4 sPLGA-mPEG copolymer hydrogels loaded with multi-drug with different dosages were injected into the vagina of SD rats to investigate the acute toxicity. For the high dosage group, the morphological changes of uterus, vagina, and intestines, such as hydroncus and adhesion, could be easily estimated by photographic anatomical observation, as shown in Figure 4. At high dosage, the drugs were released from the hydrogel in the vagina, because of a high toxicity on surrounded tissues, some drugs were excluded through the autoimmune responses of SD rats [64]. The histocompatibility and toxicity of multi-drug loaded hydrogel on the whole body need to be further analyzed by organ pathology. Hearts, livers, vagina, uterus, spleen, lungs, and kidneys were collected and sectioned for hematoxylin and eosin staining.