The Study of Porous Hydroxyapatite Particles as Anti-Glioma Drug Carrier

高度神經膠質瘤為最常見且侵略性高的原發性腦腫瘤，現今治療腦瘤方式以外科手術為主，然而其因形狀不規則且邊緣癌細胞普遍有向外浸潤情形，造成術後極高的復發風險。因此術後多會以輔助性化療抑制腦中殘留的腫瘤。然而藥物經由口服或注射進入人體有諸多不足，首先腦中血腦屏障 (BBB) 結構將大幅限制藥物由血液循環滲透到腦組織，且循環代謝過程難以維持有效藥物濃度，循環過程亦對人體其他正常組織產生毒性，因此發展有別於傳統的給藥方式有其必要。近年來，利用生物可分解的生醫材料攜帶藥物，直接於手術後進行顱內給藥是重要的研究方向，如臨床上的 GLIADELR Wafer，該藥物載體將直接在腦中進行藥物控制釋放，使目標區域周邊能維持有效藥物濃度並長期存在，增加藥物的治療效果。 本研究將以氫氧基磷灰石 (hydroxyapatite, HAp) 作為基材，利用其良好生物相容性及可降解性作為吸附和厚朴酚 (honokiol) 藥物的載體 (HAp-honokiol) ，探討其以疏水性吸附攜帶抗癌藥物、利用 pH 敏感特性作為藥物釋放機制之可行性及效果，試圖克服臨床產品面臨的問題以提供治療神經膠質瘤的選擇方案。此外亦分析 honokiol 抑制膠質瘤細胞的凋亡途徑，並建立本實驗室 C57BL/6 小鼠神經膠質瘤的動物模式。 本研究以共沈澱方式合成 HAp 微粒，並在合成過程添加蛋白以幫助合成後的孔隙形成。接著為了使疏水性的和厚朴酚較易吸附在微粒表面，以硬脂酸做氫氧基磷灰石微粒的疏水修飾。樣品檢驗首先以X光散射圖譜 (X-Ray Diffraction Spectrum, XRD) 觀察 HAp 微粒之結晶相，以傅立葉轉換紅外線光譜儀 (FT-IR) 確認疏水性硬脂酸的修飾，以熱重分析儀 (thermogravimetric analysis, TGA) 得知藥物含量。再進行藥物釋放特性實驗，以紫外/可見光譜儀 (UV/Vis spectrum) 定量樣品分別在酸性及中性水相環境下藥物釋放情形。本實驗選用小鼠膠質瘤ALTS1C1 作細胞活性、毒性實驗。並以免疫染色及蛋白質表現量檢驗分析該藥物對細胞造成的細胞凋亡機制。最後以立體定位儀顱內注射 ALTS1C1 細胞於 C57BL/6 小鼠腦部以建立腦小鼠膠質瘤動物模式。 實驗顯示，合成之樣品具備相當高的藥物吸附量，以 TGA 測得每 100 μg的 HAp-honokiol 含有 15 μg 的藥物。經由藥物釋放特性實驗，在酸性環境下本實驗樣品將於 48 小時內快速釋放出所有的藥物，而中性環境下僅會緩慢釋放出 65 % 的藥物而停止釋放。而經細胞活性毒性測試，觀察到 HAp-honokiol 能在體外環境延長釋放時間。相較於藥物本身，該樣品能長期抑制膠質瘤細胞生長。本研究亦探討了和厚朴酚對於ALTS1C1神經膠質瘤細胞的抑制效果與途徑，Annexin V-PI雙染 及 西方點墨法 (Western Blot) 確認藥物將會抑制 Akt 進而誘導細胞產生一系列步入凋亡的機制。High-grade gliomas are the most common and aggressive types of primary brain tumor. Surgery remains the first line therapy of gliomas nowadays because of its effectiveness. However, high-grade gliomas have feature of diffuse infiltration that may cause the difficulty and failure of surgical resection, that is, the relapse rate after surgery remains high. Therefore, it is necessary to apply adjuvant chemotherapy to inhibit the cancer recurrence. However, when drugs are given via systemic circulation, Blood-Brain Barrier (BBB) restricts the entry of chemotherapeutic agents from blood side to brain side, and consequently limits the concentration and the effect of drugs. The drugs would also cause damage of normal tissues or organs when circulating. As a result, different route of administration is needed in the treatment of gliomas. Using biodegradable biomaterials as drug carriers for local delivery in brain is a promising therapy lately. For instance, the only drug carriers product approved by FDA in clinic, GLIADELR Wafer, can achieve long-term drug release and maintain the effective drug concentration in the brain tumor site. This study is willing to develop hydroxyapatite particles as drug carriers carrying honokiol (HAp-honokiol) and provides an alternative option when dealing with gliomas in clinical. The effectivity of the drug adsorption, drug-releasing profile, and tumor cell inhibition effect and its mechanism would be analyzed. And the animal model of C57BL/6 mice glioma would be established in this study. In this research, the synthesis of hydroxyapatite (HAp) particles is conducted by modified co-precipitation method. The addition of egg white helps the formation of porous structures afterwards. XRD is for observing the crystal phase of the HAp particles and FTIR is used to analyze the modification of stearic acid on the HAp particles. The drug loading capacity is then evaluated by TGA. And the drug releasing profiles in neutral and acid environment are conducted by UV-Vis. ALTS1C1 is the glioma cell model in this experiment. Cell viability tests, toxicity tests, and immunostaining are used to analyzed the inhibition effect and apoptosis pathway by HAp-honokiol. Finally, the intercerebral injection of ALTS1C1 to establish the mice glioma model is conducted by the stereotactic apparatus. The results show promisingly high adsorption rate of HAp-honokiol, that 100 μg sample contains 15 μg of honokiol. Through the drug releasing experiments, honokiol would be rapidly released within 48 hours in acid environment, while the accumulative concentration of releasing honokiol reach the plateau of 65 % and stop rising. And the in vitro studies show well inhibiting effect of tumor growth and induction of cell apoptosis of HAp-honokio. The apoptosis pathway of ALTS1C1 by honokiol is also confirmed through Annexin V-PI double staining and western blot