以電化學方法促進順鉑-幾丁聚醣奈米顆粒貼片的口腔腫瘤治療效果-動物試驗

Abstract

口腔癌目前在台灣癌症發生率排名第五。口腔癌的治療基本是手術合併放射線治療以及化療。化療常以順鉑(cisplatin)作為全身性化療藥物，但cisplatin經過全身循環到達腫瘤的藥量會大幅減少，若增加劑量則會因為非專一性而傷害健康組織。因此已有研究顯示包覆cisplatin的奈米粒子可局部導入腫瘤，降低全身循環的副作用，並增加抗腫瘤的療效。為了更有效將奈米粒子導入腫瘤，我們設計包覆cisplatin的奈米載體為幾丁聚醣(chitosan)帶正電，再加上電化學離子電滲療法(iontophoresis)促進奈米粒子導入腫瘤中，本研究的目的是評估不同電化學離子導入法導入cisplatin的奈米粒子的抗腫瘤療效。 本研究分為體外(in vitro)試驗及動物(in vivo)試驗，體外試驗將鼠皮架設至橫式玻璃槽內，並貼上含有cisplatin奈米粒子的貼片，測試不同參數對cisplatin奈米粒子的滲透量，參數設定初步分為計時電流組(Chronopotentiometry, CP)、變動電流組(CP (on/off ratio: 1:1 (sec)))以及微分脈衝伏安組(Differential Pulse Voltammetry, DPV)，找出各組最佳參數後則進行體內試驗。動物實驗使用野生型C57BL/6小鼠，在口腔黏膜上施打MOC2口腔癌細胞，當腫瘤約長到10~20 mm³時進行治療。分為控制組、被動滲透組(Passive)、CP組、變動電流組以及DPV組，共五個組別。將含有cisplatin奈米粒子貼片貼在有腫瘤口腔內側，搭配恆電位儀(Potentiostat)依照各組參數進行治療，頻率為每隻小鼠一次30分鐘每週三次直至實驗結束，治療期間會使用電子尺與超音波影像(ultrasonography)進行腫瘤大小的測量，實驗結束會犧牲小鼠取出腫瘤分別進行組織切片、H&E和IHC染色(Ki-67、pan CK (AE1/AE3))，並利用ICP-MS分析腫瘤內Pt(鉑)含量。 結果顯示在體外測試中DPV 16 cycle (0~0.06V) 組作用30分鐘具有最大的cisplatin釋放量；在動物實驗方面，變動電流組與DPV組的腫瘤體積皆小於其他組別並有顯著差異；而以腫瘤體積增長量的結果來看，變動電流組與DPV組在初期(前兩次治療)較能有效壓制腫瘤生長，並有較少的Ki-67 marker表現；在ICP-MS的分析中， DPV組測得較高的Pt(鉑)含量，並略高於變動電流組但兩組間無顯著差異，因此推論變動電流組與DPV組優於其他治療組。 結論，在本研究的動物模型中，DPV組與變動電流組有較佳的cisplatin奈米粒子滲透率，並能有效抑制腫瘤生長。因此，以離子電滲療法促進貼片釋放cisplatin奈米粒子在未來可用於臨床治療口腔癌的選擇。

Currently, oral cancer ranks fifth in the incidence of cancers in Taiwan. The treatment for oral cancer generally involves a combination of surgery, radiation therapy, and chemotherapy. Cisplatin is commonly used as a systemic chemotherapy drug, but the amount of cisplatin reaching the tumor significantly decreases after systemic circulation. Increasing the dosage can harm healthy tissues due to nonspecific effects. Therefore, studies have shown that cisplatin-encapsulated chitosan nanoparticles can locally target tumors, reduce systemic side effects, and enhance the anti-tumor efficacy. To enhance the delivery of nanoparticles to tumors, we designed a cisplatin-encapsulated chitosan nanoparticle, which is positively charged. Additionally, we employed the technique of iontophoresis to facilitate the entry of nanoparticles into tumors. The aim of this study was to evaluate the tumor suppression efficacy of cisplatin-encapsulated chitosan nanoparticles using different iontophoresis methods.

The study comprised in vitro and in animal experiments. In the in vitro experiments, mouse skin was mounted in a horizontal glass chamber, and patches containing cisplatin nanoparticles were applied to test the permeation under different electric parameters (Chronopotentiometry (CP), CP (on/off ratio 1:1 (sec)) and Differential Pulse Voltammetry (DPV)). After identifying the optimal parameters for each group, in animal experiments was conducted. Wild-type C57BL/6 mice were used, and MOC2 oral cancer cells were implanted onto the oral mucosa. Treatment was initiated when tumors reached approximately 10~20 mm³. Patches containing cisplatin-encapsulated chitosan nanoparticles were applied to oral tumor, and treatment frequency was 30 minutes per session, three times a week. The animal experiment included a control group, a passive diffusion group, CP group, CP (on/off ratio 1:1 (sec)) group, and DPV group, totaling five groups. Tumor measurements were taken during treatment using ultrasonography. At the end of the experiment, mice were sacrificed, tumors were extracted for histological sections, and H&E and IHC staining (Ki-67, pan CK (AE1/AE3)) were performed. Furthermore, ICP-MS analysis was conducted to examine Pt (platinum) content within the tumors, evaluating the efficacy of iontophoresis of cisplatin nanoparticles.

In results, DPV 16 cycle (0~0.06V) group showed best cisplatin delivery in vitro mouse skin study. In the animal models, the tumor volumes in both the CP (on/off ratio 1:1 (sec)) group and the DPV group were significantly smaller than those in the other groups. In terms of tumor volume growth, the CP (on/off ratio 1:1 (sec)) group and the DPV group were more effective in suppressing tumor growth at the initial stage (the first two treatments). The expression of the Ki-67 marker in the CP (on/off ratio 1:1 (sec)) group and the DPV group were the lowest among the treatment groups. In ICP-MS analysis, the DPV group showed a higher Pt (platinum) content, slightly surpassing the CP (on/off ratio 1:1 (sec)) group, although the difference between the two groups was not statistically significant. Therefore, both the CP (on/off ratio 1:1 (sec)) and DPV showed best tumor suppression efficacy.

Overall, the tumor suppression efficacy of the DPV group and the CP (on/off ratio 1:1 (sec)) group showed better diffusion of cisplatin-encapsulated chitosan nanoparticles. The iontophoresis facility for delivering cisplatin-encapsulated chitosan nanoparticles can be utilized in future clinical treatment.