具表面修飾之磁性奈米粒子其交流磁場效應對乳癌細胞生長影響

Abstract

熱療法已被廣泛應用於腫瘤及癌症的臨床治療。在熱療法中，磁性奈米顆粒（簡稱MNP）被成功地運用；這種方法的基本原理是將MNP注射到患部，然後利用交流磁場來引發熱炙效應。當MNP被注射到患處後，癌細胞會吞噬這些顆粒。在施加交流磁場的情況下，MNP在癌細胞內部受到磁場影響，產生轉動及熱效應，造成細胞結構的損傷及凋亡。 胞外體(Exosome)在癌症研究中扮演著關鍵角色。它們會在癌細胞之前先轉移到目的地，並釋放功能性蛋白來調整微環境。當環境適宜時，癌細胞接著轉移。利用這一特性，將MNP接上胞外體的表面，使其成為可被交流磁場處理的目標。本研究旨在探討MNP及其在交流磁場作用下對三陰性乳腺癌細胞系MDA-MB-4175的影響。此外，我們也研究了使用牛血清白蛋白(BSA)修飾MNP的過程，以優化其與胞外體的結合。 實驗主要分為乳癌細胞的生存狀態與表面修飾兩大部分。在乳癌細胞的部分，分別評估了癌細胞與作為正常細胞參照的NIH 3T3細胞，在多種MNP濃度和不同交流磁場持續時間下的存活情況。此外，透過特定的實驗設計，進一步探索了環境和細胞內MNP的具體影響。在表面修飾的部分，將MNP與BSA混合，並於不同的反應時間點測量BSA的濃度，以了解其在MNP上的結合比率。最後，利用Rabbit igG作為接合橋樑，並在其上連接螢光抗體，再以顯微鏡進行觀察。 實驗結果表明，當細胞與MNP共同培養後，細胞會吞噬MNP。隨著MNP濃度的增加，3T3細胞和MDA-MB-4175細胞的存活率都會逐步下降，但在交流磁場作用下的0至60分鐘內並無明顯變化。然而，當被細胞吞噬的MNP受到交流磁場的影響時，其在細胞內的旋轉和熱效應會進一步降低存活率，大約下降了20%。當MNP被PEG-400包裹後，其對細胞的毒性得以降低，使存活率提高約13%。至於MNP的表面修飾，研究發現在37℃、0.1M的EDC條件下，反應時間為50分鐘時，MNP能成功地與BSA結合，其比例是每毫克MNP能結合0.3毫克的BSA。免疫螢光影像也證實了這一成功的結合過程。

Thermal therapy has been widely applied to clinical treatments for tumors and cancers. In hyperthermia, Magnetic Nanoparticles (MNPs) have been successfully utilized; the fundamental principle of this method is the injection of MNPs into the afflicted area, followed by the application of an alternating magnetic field to induce a hyperthermic effect. After MNPs are injected into the affected area, cancer cells engulf these particles. When subjected to an alternating magnetic field, MNPs within the cancer cells respond to the magnetic influence, generating rotational and thermal effects, leading to cellular structural damage and apoptosis. Exosomes play a pivotal role in cancer research. They migrate to target locations before cancer cells and release functional proteins to modify the microenvironment. Once the environment is conducive, cancer cells follow suit. Exploiting this characteristic, MNPs are attached to the surface of exosomes, turning them into targets for alternating magnetic field treatment. This research aims to investigate the impact of MNPs and their effects on the triple-negative breast cancer cell line MDA-MB-4175 under an alternating magnetic field. Additionally, we studied the process of modifying MNPs using Bovine Serum Albumin (BSA) to optimize their binding with exosomes. The experiments are divided into the survival state of breast cancer cells and surface modification. In the breast cancer cell segment, we evaluated the survival of cancer cells and NIH 3T3 cells, which served as a model for normal cells, under various MNP concentrations and alternating magnetic field durations. Furthermore, through specific experimental designs, we delved deeper into the precise impacts of the environment and intracellular MNPs. In the surface modification segment, we mixed MNPs with BSA and measured the concentration of BSA at different reaction time points to understand its binding ratio on MNPs. Finally, we used Rabbit IgG as a binding bridge and attached fluorescent antibodies to it, followed by microscopic observation. The results indicate that cells engulf MNPs after co-culturing. As the concentration of MNPs increases, the survival rates of both 3T3 cells and MDA-MB-4175 cells progressively decline, but no noticeable changes are observed within 0 to 60 minutes under the alternating magnetic field. However, when the MNPs ingested by cells are influenced by the alternating magnetic field, their intracellular rotation and thermal effects further reduce the survival rate, decreasing by approximately 20%. When MNPs are encapsulated with PEG-400, their cytotoxicity is diminished, leading to an increase in survival rate by about 13%. Regarding MNP surface modification, we discovered that at 37°C with 0.1M EDC conditions, with a reaction time of 50 minutes, MNPs successfully bind with BSA at a ratio of 0.3 mg BSA per mg of MNP. Immunofluorescence images also confirmed this successful binding process.