## 5 Conclusions  
In summary, we offered a report on temperature-sensitive star-shaped 4 sPLGA-mPEG block copolymer hydrogels with loading multi-contraceptive for vaginal application. In vitro controlled release profiles of three contraceptives were investigated via the HPLC method. The HPLC condition was first determined as follows: the maximum absorption peaks of IMC and EE were \(\sim\)210 nm, and the one of GSD was \(\sim\)240 nm; volume ratio of optimal mobile phase was 53:47; and the retention time of IMC, GSD, and EE were 11.18, 13.11, and 15.57 min. In vitro release study showed that cumulative release amount of IMC, GSD, and EE in copolymer hydrogels increased with decreasing LA/GA mol ratio from 9 to 1, suggested that drugs release was not only controlled by drugs diffusion but also mainly by degradability of copolymer hydrogels. In addition, in vitro release profiles of IMC, GSD, and EE were well fitted with the Higuchi model. Lastly, in vivo evaluation of copolymer hydrogels with loading drugs, including acute toxicity and antifertility experiment, was subsequently investigated. For high dosage group, slight hydroncus of the uterus was observed on day 1. Swelling of the uterus became extremely severe and intestinal adhesion was also found at day 3. Uterus and intestinesulcerated at day 5. Regarding the middle dosage group, no festering of tissues was observed and blood coagulum existed in the uterus at a different defined time. For low dosage group, no significant tissue necrosis was observed at the different indicated time. No embryo was observed in the low dosage drug-loaded hydrogel group, indicated that prepared copolymer hydrogel system with packing multi-drug was feasible as a novel contraception method. The following are available online at [[http://www.mdpi.com/2076-3417/9/8/1638/s1](http://www.mdpi.com/2076-3417/9/8/1638/s1)]([http://www.mdpi.com/2076-3417/9/8/1638/s1](http://www.mdpi.com/2076-3417/9/8/1638/s1)), Table S1: Preparation of simulated vaginal fluid solution by using different chemicals, Table S2: Fitting parameters of various drug release by using Higuchi kinetic model, Figure S1: GPC traces of prepared CP-1, CP-2, and CP-3 block copolymers, Figure S2: Standard curves of IMC, GSD, and EE for measuring release of drugs, Figure S3: The release amounts of IMC, GSD, and EE from the prepared 4 sPLGA-mPEG block copolymer hydrogels, Figure S4: Fitting curve of cumulative release of GSD using Higuchi kinetic model, Figure S5: HE staining of SD rat hearts at day 1, 3, and 5 after injecting 4 sPLGA-mPEG block copolymer hydrogels with different dosages into vagina, Figure 56: HE staining of SD rat lungs at day 1, 3, and 5 after injecting 4 sPLGA-mPEG block copolymer hydrogels with different dosages into vagina, Figure S7: HE staining of SD rat kidneys at day 1, 3, and 5 after injecting 4 sPLGA-mPEG block copolymer hydrogels with different dosages into vagina, Figure S8: TEM of CP-3 block copolymer solution at different concentrations. All authors discussed and agreed on the contents of the manuscript. Conceptualization, L.N., P.Z., and J.S. ; methodology, L.N., H.Y., P.Z., and J.S. ; formal analysis, L.N., P.Z., J.D., M.S., and J.S. ; investigation, L.N., P.Z., P.D., C.J., and J.S. ; resources, [PERSON], P.Z., Y.H., and J.S. ; data curation, [PERSON], P.Z., Q.Z., and J.S. ; writing--original draft preparation, [PERSON], P.Z., and J.S. ; writing--review and editing, [PERSON], J.D., M.S., C.J., H.Y., Y.H., and J.S. ; visualization, [PERSON], Q.Z., and J.S. ; supervision, L.N. and J.S. ; project administration, L.N. and J.S. ; funding acquisition, L.N. and J.S. This research was funded by the National Natural Science Foundation of China No. 31700840, and the Key Scientific Research Project of Henan Province (18B430013, 18A150049), Research Fund from Health Commission of Hubei Province (WJ2019M038), and Wuhan Municipal Health and Family Planning Research Fund (WX18C23). This research was supported by the Nanhu Scholars Program for Young Scholars of XYNU.