結合碳酸鈣與溫感性水膠作為卡鉑控制釋放及小鼠神經膠質瘤治療之藥物載體

Abstract

惡性膠質瘤為成人最常見的惡性腦瘤，約佔所有顱內腫瘤的30%至40%與所有顱內惡性腫瘤的80%。即便以手術加上術後同步放射化學治療，仍預後不佳。為使腫瘤切除後的傷口能夠順利恢復，通常放射治療必須等待術後兩週才能進行。此等待期間，腫瘤仍繼續生長。此空窗期可以藥物來抑制腫瘤生長，其中一種策略是藉由腫瘤原位注射可控制釋放藥物的載體來達成，此類局部藥物傳輸的方式可突破血腦屏障的限制，提高局部抗癌藥物的濃度並降低全身副作用。卡鉑透過鉗合DNA干擾癌細胞複製，具有放射性增強抗癌效果。卡鉑依附碳酸鈣顆粒，在中性環境下維持穩定不釋放，而在內體-溶酶體複合物或其他酸性環境中，經碳酸鈣被降解後釋放，藉此來達到藥物控制釋放的效果。水膠作為藥物載體能攜載大量藥物、提供適當的穿透深度並且具有與腦組織較為接近的力學性質，原位成膠以及良好的粘附特性可使水膠以較大的接觸面積來釋放藥物，單劑小體積注射可以降低腦水腫及癲癇的風險，適合用於惡性膠質瘤的治療。本研究使用溫感性的氧化透明質酸/己二酸二醯胼水膠來攜載卡鉑並且摻雜載藥的碳酸鈣顆粒來達到初期的抑制效果，而載藥的碳酸鈣會隨著水膠的降解逐漸被釋放出來，進一步延長卡鉑的釋放，透過兩週的卡鉑緩釋使得卡鉑的化學治療能與放射治療合併治療，來達到加成的治療效果。載藥碳酸鈣係透過化學沉澱法來製備，透明質酸的改值係先將透明質酸以過碘酸鈉溶液進行開環氧化，再以己二酸二醯胼來進行交聯。從SEM影像中可以證實本研究所製備出來的載藥碳酸鈣微粒呈現球型外貌，並且大小落在677奈米左右，粒徑大小也經由DLS進一步地被驗證。本研究使用ICP-MS來測定碳酸鈣的載藥效率以及藥物釋放曲線，載藥碳酸鈣的載藥效率為18.7 ± 2 %，而在pH 7的中性環境下，卡鉑在載藥碳酸鈣的釋放非常緩慢，而以pH 4來模擬內體-溶酶體複合物的酸性環境時，卡鉑能快速地從載藥碳酸鈣中被釋放出來，摻雜載藥碳酸鈣的載藥水膠則是在中性環境下可以快速地釋放水膠中的卡鉑來達到初期的抑制效果。摻雜載藥碳酸鈣的載藥水膠的降解時間約為兩週，因此載藥的碳酸鈣能在適當的時間內被釋放出來，而摻雜載藥碳酸鈣的載藥水膠在37℃的成膠時間為7.6分鐘，因此可以在人體的溫度下快速成膠。而從WST-1、LDH及Live/Dead染色當中證實了所選用的材料都具有良好的生物相容性。WST-1、Live/Dead染色、西方點墨法及gamma H2A.X的測定結果中也證實了載藥碳酸鈣及摻雜載藥碳酸鈣的載藥水膠具有良好的抑制腫瘤效果。這些結果證實了攜載卡鉑之碳酸鈣及攜載卡鉑之氧化透明質酸/己二酸二醯胼水膠所形成之複合載體用作神經膠質瘤之局部注射治療的潛力。

Malignant gliomas are the most common adult brain tumors, which account for 30% to 40% of all intracranial tumors and 80% of all malignant intracranial tumors. Radiotherapy usually starts 2 weeks after surgery for patients’ recovery. Therefore, adequate intratumoral drug injection with sustained release was proposed to control the tumor growth after surgery. The site-specific property of local drug delivery increases the local drug concentration, reduces the systemic toxicity, and circumvents the restriction of blood–brain-barrier (BBB). Carboplatin is a platinum-based antineoplastic agent, which is usually combined with radiotherapy for cancer treatment. CaCO3 can incorporate carboplatin and remain stable until being degraded in the endosome/lysosome complex or acidic environment and thus achieves the controlled-release. In-situ forming potency, high drug encapsulation capability, biocompatibility, biodegradability, adhesive properties, moderate mechanical properties, and moderate penetration depth make hydrogel a great candidate for local drug delivery and gliomas treatment. In this study, we adopted thermogelling oxidized hyaluronic acid/adipic acid dihydrazide (oxi-HA/ADH) hydrogel to load carboplatin and combined with carboplatin-loaded CaCO3 (CPCC) to provide an initial suppressive effect and enable the co-treatment of the chemical therapy and the radiotherapy via 2-week controlled-release. CPCC particles were synthesized by chemical precipitation method. HA was oxidized by sodium periodate and then crosslinked with ADH. The sphere-shaped CPCC particles with averaged size of 677 nm were characterized by SEM, and the size was further confirmed by DLS. ICP-MS was used to characterize the encapsulation efficiency of CPCC and the drug release profile. The encapsulation efficiency of CPCC was 18.7 ± 2 %, and the release of carboplatin from CPCC was slow in neutral environment (pH 7.4) while the release in acidic environment (pH 4) was relative fast. The release of CPCC-incorporated carboplatin-loaded oxi-HA/ADH hydrogel (CPH) was fast in neutral environment, indicating the potency of initial suppressive effect. The degradation took 2 weeks and the gelation time was 7.6 min at °C, indicating the release of CPCC particles with moderate time and the fast gelation of CPCC-incorporated CPH at human body temperature. WST-1, LDH, and Live/Dead staining results of L929 cells and ALTS1C1-Luc cells confirmed the biocompatibility of chosen materials. Antineoplastic effect of CPCC and CPCC-incorporated CPH were further proved by WST-1, Live/Dead staining, western blot, and gamma-H2A.X analysis. From these results, the potency of CPCC-incorporated CPH for gliomas treatment was demonstrated.