奈米生物醫學新策略開發

New strategy development for nanomedicine

鍾采樺(Tsai-Hua Chung)

指導教授 ： 郭彥彬

共同指導教授 ： 黃東明

國立臺灣大學/牙醫專業學院/臨床牙醫學研究所/博士(2012年)

https://doi.org/10.6342/NTU.2012.01391

摘要

奈米技術的快速發展是個備受重視的研究領域，尤其在醫藥生技（奈米醫學）的應用潛力更是無限。本論文有五個部份將分別陳述，主要是結合奈米醫學、幹細胞學與藥物生理學三大領域，並且發展以利用奈米粒子或結合幹細胞的模式應用於癌症治療，另瞭解抗癌藥物對幹細胞生理的影響，試圖發展奈米生物醫學應用之新策略。
（1）氧化鐵奈米粒子促進人類幹細胞表面生長因子受體大量表現以作為癌症治療模式之應用：氧化鐵奈米粒子在幹細胞治療應用中，具有可標定幹細胞且可透過MRI偵測及追蹤。然而，氧化鐵奈米粒子對於幹細胞的影響仍不明確，並且是個不容忽視的議題。在本論文，我們將氧化鐵奈米粒子標定幹細胞後，發現會促進幹細胞表面生長因子受體EGFR大量表現。而標定後的幹細胞趨向於腫瘤的能力以及抗腫瘤生長的情形均比未標定的幹細胞優。而這些現象起因於幹細胞大量表現EGFR而抑制了腫瘤因自泌作用產生的EGF所造成的腫瘤生長、血管新生以及因腫瘤的VEGF表現所造成的腫瘤癌化與發育。我們的結果顯示，氧化鐵奈米粒子對於幹細胞的影響將可提供一個全新的幹細胞治療之應用。
（2）探討基因轉殖於人類幹細胞以結合氧化鐵奈米粒子於癌症治療應用：我們藉由氧化鐵奈米粒子促進幹細胞移動的能力，試圖合成Dextran和CM-Dextran不同材質的氧化鐵奈米粒子，利用幹細胞來攜帶具有抑制腫瘤生長的基因，加強毒殺腫瘤的能力。根據初步結果顯示，Dextran和CM-Dextran氧化鐵奈米粒子會促進人類幹細胞EGFR的大量表現，亦提昇幹細胞本身具有趨向腫瘤的能力。我們預計，幹細胞的基因轉殖技術克服後，將進一步在動物模式中進行腫瘤毒殺測試。
（3）探討人類幹細胞傳遞磁性氧化鐵奈米粒子結合抗癌藥物釋放於腫瘤治療之應用：根據上述，利用幹細胞具有趨向腫瘤且具有毒殺腫瘤能力的應用，亦可利用幹細胞將具有抑制腫瘤生長能力的基因或藥物，攜帶到腫瘤的位置，提昇毒殺腫瘤的能力。本部份，試圖合成具有可控制藥物釋放的奈米粒子，再透過幹細胞具有移動的能力，攜帶藥物到腫瘤的位置。由於，具有抗癌藥物釋放能力的奈米粒子，在合成上具有挑戰性及困難度，因此，突破在合成上的瓶頸後，接著在後續的腫瘤毒殺作用便將可順利進行。
（4）可見光誘導之二氧化鈦奈米粒子於腫瘤毒殺作用：透過浸漬法（impregnation method）的方式改變二氧化鈦吸收光的光譜，從原本UV光增加到可見光（藍光）可吸收的範圍。改質後的二氧化鈦奈米粒子容易被黑色素瘤細胞吞入，且隨著培養的時間增加，吞入的二氧化鈦奈米粒子愈多。吞入二氧化鈦奈米粒子後的黑色素瘤細胞於照光1小時後會產生活性氧，進而攻擊細胞造成細胞凋亡的現象，因此可作為黑色素瘤細胞之癌症治療應用。
（5）YC-1可減緩惡質病現象藉由脂肪裂與脂肪生成之調控：脂肪組織的減少，主要是由於脂肪裂解速度增加以及脂肪生成能力降低所造成的現象，而這也是癌症惡質病造成體重減輕的主要特徴。有太多相關致病的訊息調控影響了脂肪組織的減少，因此有必要發展有效抑制惡質病造成脂肪減少的治療藥物。根據我們的結果證明，由於YC-1具有抗腫瘤生長等多功能的生理作用，因此，YC-1影
響了3T3-L1 preadipocytes分化時的脂肪生成、也參與了TNF-α與腫瘤細胞造成的脂肪裂解調控以及腸癌細胞CT26-WT動物模式造成的惡質病的現象。YC-1會活化Akt和ERK的調控機制，促進了3T3-L1 preadipocytes 提早分化為脂肪細胞的過程，其中也活化了PPARγ、IRα、IRS3和GLUT-4等與脂肪生成相關的蛋白質；在活體外的脂肪裂解模式中，YC-1具有抑制因TNF-α或腫瘤細胞所促進而活化ERK的訊息傳遞，使得下游PLIN的蛋白質表現佭低所造成脂肪裂解的情形。在腫瘤腸癌細胞動物模式中，YC-1能抑制因腫瘤造成血液中胰島素減少，使得能延緩體重減輕的能力。綜合上述，YC-1是個新的癌症治療藥物，不僅具有抗腫瘤生長，亦具有抗腫瘤惡質病的能力。

關鍵字

氧化鐵 ； 奈米粒子 ； 幹細胞 ； 惡質病 ； YC-1 ； 3T3-L1

並列摘要

With the recent progress of nanotechnology in biomedical applications, nanomedicine is a rapidly evolving area of intensive research and has received considerable attention. In this thesis, the combination of nanotechnology or nanomedicine, stem cell biology and pharmacology is used to develop novel stem cell-based tumor therapy and new strategy for nanomedicial applications. There are five topics: (1) Iron oxide nanoparticles-induced epidermal growth factor receptor expression in human stem cells for tumor therapy. (2) The enhancement of stem cell-based gene delivery by iron oxide nanoparticles for tumor therapy. (3) Stem cell-targeting delivery of controlled drug released-nanoparticles for tumor therapy. (4) Toxic effects of titanium dioxide nanoparticles in melanoma. (5) YC-1 rescues cancer cachexia by affecting lipolysis and adipogenesis.
(1) Iron oxide nanoparticles-induced epidermal growth factor receptor expression in human stem cells for tumor therapy.
Superparamagnetic iron oxide (SPIO) nanoparticles show promise as labels for cellular magnetic resonance imaging (MRI) in the application of stem cell-based therapy. However, the unaddressed concerns about the impact of SPIO nanoparticles on stem cell attributes make the feasibility of SPIO labeling uncertain. Here, we show that the labeling of human mesenchymal stem cells (hMSCs) with ferucarbotran can induce epidermal growth factor receptor (EGFR) overexpression. Labeled hMSCs with their overexpressed EGFR were attracted by tumorous EGF and more effectively migrated toward tumor than unlabeled cells, resulting in more potent intrinsic antitumor activity. Moreover, the captured binding of tumorous EGF by overexpressed EGFR of labeled hMSCs blocked EGF/EGFR signaling-derived tumor growth, tumorous angiogenesis, and tumorous VEGF expression also responsible for tumor progression and development. Our results show that the impact of SPIO nanoparticles on stem cell. attributes is not necessarily harmful but can be cleverly used to be beneficial to stem cell-based therapy.
(2) The enhancement of stem cell-based gene delivery by iron oxide nanoparticles for tumor therapy.
With the promotion stem cell migration toward tumor by iron oxide nanoparticles, we have been trying to synthesized dextran- or Carboxymethyl-dextran (CM-dextran) -coated SPIO nanoparticles to enhance stem cell-based gene delivery. We found that as-synthesized dextran-and CM-dextran-coated SPIO nanoparticles could induce the expression of EGFR and hence stimulate the tumor tropism of stem cells as demonstrated by in vitro migration assay. A stable transfection of stem cells with TNF-a gene is proceeding. We suggest that with the establishment of transgenic stem cells SPIO-stimulated tumor tropism would be benificical to stem cell-based gene therapy for tumor.
(3) Stem cell-targeting delivery of controlled drug released-nanoparticles for tumor therapy.
According to the above, SPIO-induced stem cells tropism toward tumor is also used to deliver drug-loading nanoparticles stem cells used for specific tumor targeting. Therefore, we would like to integrate the targeted delivery ability of stem cells with the carrying efficiency for anticancer drugs of nanoparticles to develop a novel drug delivery system for cancer therapy. Anticancer drugs will be encapsulated into a soft-material core (polyvinyl alcohol, PVA) and then coated with a biocompatible silica shell that can protectively construct the nanoparticles and can enhance the internalization of nanoparticles into stem cells. Moreover, a controlled release mechanism (high-frequency magnetic field, HFMF) will be installed in the nanoparticle core. After the targeted-migration of stem cells with intracellular internalized nanoparticles toward tumors, HFMF could induce the release of anticancer drugs from the nanoparticle core to kill the tumor cells.
(4) Visible-light inducible carbon-modified titanium dioxide nanoparticles for tumor therapy.
Visible-light inducible of carbon-modified titanium dioxide were synthesized by impregnation method, and therefore these particles are called TiO2-200 nanoparticles. The photocatalytic activity that under the irradiation of visible-light was ROS produced and was measured by reduction methyl orange assay. TiO2-200 nanoparticles are easily uptaken by melanoma cells in a time-dependent manner. By irradiation with visible-light (400-500 nm) the decrease of cell viability was measured by trypan blue and induced apoptosis was measured by TUNEL assay. Intracellular ROS were generated dependent on uptake of TiO2-200 nanoparticles in treated melanoma cells. Taken together, we suggest that the carbon-modified titanium dioxide would be ideality beneficial to therapy for tumor.
(5) YC-1 rescues cancer cachexia by affecting lipolysis and adipogenesis.
Loss of adipose tissue, primarily due to increased lipolysis but also to an impairment of adipogenesis, is a key feature of weight loss in cancer cachexia. Because of the myriad pathogenic signaling pathways essential for atrophy of adipose tissue, effective therapeutic agents for cachectic adipose loss are lacking and urgently needed. The authors evaluated the effects of YC-1 on adipogenesis of 3T3-L1 preadipocytes, TNF-α- and tumor-cell-induced lipolysis in 3T3-L1 adipocytes, and cachectic weight loss in colon-26 adenocarcinoma-bearing mice because YC-1 has been shown to possess versatile pharmacological actions, including anticancer activity. It was found that YC-1 promotes the differentiation of 3T3-L1 preadipocytes into adipocytes through activation of Akt and extracellular signal-regulated kinase (ERK) signaling pathways as well as activation of several adipogenic mediators, such as peroxisome proliferator-activated receptor γ(PPARγ), insulin receptor α (IRα), insulin receptor substrate-3 (IRS-3) and glucose transporter-4 (GLUT-4). In the in vitro lipolysis models, YC-1 attenuates TNF-α induced lipolysis of adipocytes by antagonizing TNF-a-mediated activation of ERK and downregulation of perilipin (PLIN). It was also found that YC-1 inhibits colon-26 adenocarcinoma cell-induced lipolysis of 3T3-L1 adipocytes. Moreover, YC-1 effectively rescues cachectic weight loss in colon-26 adenocarcinoma-bearing mice by blocking lipolysis, involving insulin. Taken together the results show that YC-1 with its anticancer and anticachexia talents is highly worth developing as a novel agent for cancer therapy.

並列關鍵字

iron oxide ； nanoparticles ； stem cell ； cachexia ； YC-1 ； 3T3-L1

參考文獻

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