Abstract:

Many pH- and temperature-responsive polymers have been designed for preparing hydrogel. In the present study, in order to decrease the pH sensitivity of reported poly(amidoamine)-poly(ethyleneglycol)-poly(amidoamine) (PAA₁₅₈₀-PEG₄₆₀₀-PAA₁₅₈₀), we designed and synthesized poly(amidoamine)-poly(ethyleneglycol)-poly(amidoamine) (PAA-PEG-PAA) with shorter length of PAA chain by decreasing reaction temperature for preparing PAA-PEG-PAA hydrogel solution containing doxorubicin (DOX). The PAA-PEG-PAA was synthesized via the Michael-addition polymerization. The characteristic of PAA-PEG-PAA was evaluated.The PAA-PEG-PAA hydrogel solution was prepared and investigated. DOX-loaded PAA-PEG-PAA hydrogel solution was prepared, and its in vitro DOX release and in vitro anti-tumor activity were evaluated. Our results indicated that the viscosity of PAA-PEG-PAA hydrogel solution was concentration- and temperature-dependent. The sol-gel transition temperature of PAA-PEG-PAA hydrogel solution (12 %, w/w) ranged from 35 to 29 ºC, and its pH ranged from 6.0 to 7.4. The released DOX from DOX-loaded PAA-PEG-PAA hydrogel showed sustained release characteristics. The in vitro anti-tumor activity of DOX-loaded PAA-PEG-PAA hydrogel was confirmed in B16F10 cell line. Considering the acidic tumor microenvironment, this DOX-loaded PAA-PEG-PAA hydrogel solution would be easy in situ administration for intra-tumor injection or para-tumor injection forming hydrogel at body temperature. We suggested that this DOX-loaded PAA-PEG-PAAhydrogel solution, if containing photothermal conversion agents, would have a potential further use for photothermal therapy.      

Key words: Poly(amidoamine)-poly(ethyleneglycol)-poly(amidoamine), pH- and temperature-responsive polymer, Hydrogel, Doxorubicin, B16F10 cells  

CLC Number: 

• R944

Supporting:

Methods

Drug release

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.

 

 N_D: number of death cells;

 N_L: 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.

 

Result

Drug release

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.