感溫性水膠對蛋白質吸收和釋放行為之探討

The Study of protein Absorption and Release Behaviors of Thermally Sensitive Hydrogel

聚氮異丙基丙烯醯胺（poly（N-isopropyl acrylamide）, PNIPAAm） 是已知具感溫性的高分子材料。當溫度高於其較低臨界溶解溫度（Lower Critical Solution Temperature, LCST） 時， 聚合物是厭水性而且不溶於水；反之，溫度低於其LCST 時， 聚合物會在水中呈現水合膨潤狀態。本研究利用添加不同比例的第二單體丙烯酸（acrylic acid, AAc） 與NIPAAm 形成共聚物， 來調整水膠的LCST 更接近體溫。同時製造並純化紅色螢蛋白質DsRed 重組蛋白質當追蹤劑，來偵測水膠對DsRed 蛋白質的吸收與釋放行為。為確保DsRed 蛋白質之螢光活性，不使用化學性固定劑而選擇物理性的包覆法，利用水膠低於其LCST 時對蛋白質進行膨潤吸收，而將蛋白質分子包覆於水膠中， 高於其LCST 時水膠收縮而將蛋白質分子排出。實驗結果顯示，在磷酸緩衝溶液（PBS, pH7.4） 中， 水膠的丙烯酸含量0% 增至0.27% 時， 其LCST 由28 ℃提昇到40℃， 同時對蛋白質吸附量也隨之升高， 並且LCST 接近於體表溫度之丙烯酸含量為0.19% 之水膠仍保有良好溫度感應性。以含0.11% 與0.19% 丙烯酸之水膠進行蛋白質吸收和釋放實驗，將膠體浸泡在25℃ 之紅色螢光蛋白質DsRed （0.075mg/ml） 溶液24 小時後，轉置於35℃ 之緩衝液，在不同時間後照像分析膠體內殘留紅螢光蛋白質之影像強度。結果發現兩種膠體在30 分鐘時即可釋放約為80 至90% 之蛋白質量，顯示這些水膠在不喪失對溫度的敏感性的情況下， 仍具良好包載和釋放蛋白質的能力。

Poly N-isopropylacrylamide (PNIPAAm) is a well-known temperature-sensitive polymer. When the temperature is higher than the lower critical solution temperature (LCST), the polymer becomes hydrophobic and insoluble. In contrast, as the temperature decreases below the LCST, the polymer is hydrated in water. In this study, we co-polymerized NIPAAm with weakly acidic acrylic monomers (acrylic acid, AAc) to modify the LCST of polymer towards the body temperature. As a tracer, the recombinant red fluorescent protein DsRed (Discosoma Red) was produced and purified to detect the release and absorbing behaviors of hydrogel. For maintaining DsRed chromophore, the hydrogels absorb DsRed protein when these polymers swell under the LCST, and release DsRed protein when these polymers shrink above the LCST without any chemical fixation procedure. The results demonstrated that the percentage of AAc in hydrogels were increased from 0 % to 0.27%, the LCSTs were increased from about 28 to 40 in the phosphate buffer saline (PBS, pH7.4) and the absorbed ℃ amount of DsRed protein were also increased. Meanwhile the hydrogels containing 0.19% AAc, those LCST were near the human body temperature, still showed good thermo-sensitivity. After the hydrogels with 0.11 and 0.19% AAc were soaked in 0.075 mg/ml DsRed protein solution at 25℃ for 24 hours, those gels were put in the buffer at 35℃ for different time, and then were measured the DsRed protein remained in the gel by photographic analysis of the signal intensity, we found those hydrogels could release about 80 to 90% of DsRed protein within 30 minutes. These results show those hydrogel posse good protein-loading capacity without losing their thermosensitve properties.