Zhantao Li1,2, Ai Liao2, Man Liu1,2, Shuang Zhang1,2, Zhenhan Feng1, Guangxue Wang2, Jingru Wang1,2, Meiqi Xu1,2, Zhuoyue Li1,2, Xiaochuan Duan1,2, Yanli Hao1,2, Xiuchai Zheng2, Hui Li1, Qin Na2, Hua Zhang3, Bilin Liu4, Xuan Zhang1,2*
As shown in Scheme 2, DOX loaded PAA-PEG-PAA hydrogel solution (3 mg/g) was loaded onto the upper insert of a transwell insert (the pore size of the transwell insert membrane was 8 μm). The transwell insert was incubated 30 min at 37 ºC for DOX loaded PAA-PEG-PAA hydrogel solution completed gelation. After that, the transwell was incubated in 1 mL of PBS at 42 ºC. At determined time, a volume of 200 μL release media were taken out and replaced with an equal volume of pre-warmed fresh PBS. The samples were diluted 10 times for determining the concentration of DOX with Spectrofluorophotometer (SHIMADZU, RF-5301PC).
The Cytotoxicity of DOX loaded PAA-PEG-PAA hydrogel
As shown in Scheme 3a, B16F10 cells were dispersed in FBS free DMEM and were added 200 μL onto the upper insert of the transwell inserts (the pore size of the transwell insert membrane was 8 μm), then the transwell inserts were placed in 48-well plates that was loaded with 1 mL of DMEM that contains FBS previously. Under the driven effect of FBS, cells migrant from the upper insert to the lower insert through the pores of the Polyester membrane of the transwell insert and adhere to the lower layer of the Polyester membrane after incubating at 37 °C for 48 h.
As shown in Scheme 3b, after cells are adhered on the lower insert membrane, a volume of 200 μL of DOX loaded PAA-PEG-PAA hydrogel solution was added onto the upper transwell insert. The transwell inserts were incubated 15 min at 42 ºC. After incubation, DOX loaded PAA-PEG-PAA hydrogel was removed and each insert was washed with PBS three times. After that, B16F10 cells were treated with Live-Dead Cell Staining Kit for 30 min. After removing the dyeing agent, transwell inserts were observed with Inverted Fluorescence Microscope (OLYMPUS, IX83). Living cells were observed with exciter filter BP530-550, barrier filter BA575-625. Dead cells were observed with exciter filter BP460-495, barrier filter BA510-550. GrapHical calculating software Image J was used to calculate the number of dead and living cells in the photos. Death cell ratio was calculated.
ND: number of death cells;
NL: number of living cells.
Negative control group was incubated with cell culture medium. Positive control group was incubated with saponin solution. Both negative and positive group was incubated at 37 ºC.
As shown in Figure S1, the released DOX from DOX loaded PAA-PEG-PAA hydrogel at 42 ºC was about 10.0 % at 144 h time point, slightly higher than that of at 37 ºC (7.8 %), also showing the sustained release characteristics.
The Cytotoxicity of DOX loaded PAA-PEG-PAA hydrogel
The calculated dead cell ratios were shown in Figure S21 The dead cell ratio in 0.5 mg/g, 1 mg/g and 3 mg/g DOX loaded PAA-PEG-PAA hydrogel groups at 42 ºC was about 64 %, 87 % and 86 %, respectively, significantly higher than that of control and blank gel groups, 1% and 3% (P<0.01). Also, the dead cell ratio in 0.5 mg/g, 1 mg/g and 3 mg/g DOX loaded PAA-PEG-PAA hydrogel groups at 42 ºC was higher than that of at 37 ºC.
Figure S1. The released DOX from DOX-loaded PAA-PEG-PAA hydrogel at 42 ºC.
Figure S2. The dead cell ratio of DOX-loaded PAA-PEG-PAA hydrogel at 42 ºC.
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