臺灣博碩士論文加值系統

本研究主要著重於對癌症的治療及診斷的藥物開發，實驗中我們合成一些簡單的螢光有機分子，並藉由其聚集誘導螢光(Aggregation-Induced Emission，AIE)的獨特性質而增強其在細胞內的表現。首先，我們合成了一個不對稱1,6-雙取代萘分子(NV-12P)，其可以在光源照射下產生特定的活性氧物質，以進行I型(type I)的光動力治療，並在癌細胞中經由標定粒線體而表現出選擇性的暗毒性，以及藉由細胞內的螢光亮點而有效的產生光毒性，結果顯示此化合物可成為一個具有潛力的雙重毒性功效分子。另外，我們基於光致電子轉移(photo-induced electron transfer，PET)及分子內電荷轉移(intramolecular charge transfer，ICT)的原理而成功設計並合成出一個水溶性高的有機分子(ADA)，其可藉由螢光顏色的轉換而辨識該環境的酸鹼值。而此pH值的辨識範圍，可使ADA應用在標定細胞內隨著pH值改變的微環境。最後，我們除了利用ADA來辨識不同細胞株之間的細胞內pH梯度，並藉由ADA在正常細胞中所表現獨特的螢光強度而將ADA發展成為標記正常細胞的螢光分子，使其有效的應用在做癌症檢測時的對照組。

In this study, we focused on developing the newly organic molecules on the therapy and diagnosis of cancers. In the experiments, we synthesized some simple fluorescent organic molecules and enhanced their performances in the cells by the unique properties of Aggregation-Induced Emission (AIE). First, we synthesized an asymmetric 1, 6-disubstituted naphthalene (NV-12P), which can generate particular reactive oxygen species to undergo type I photodynamic therapy under irradiation. Furthermore, this compound can specifically localize in mitochondria in cancer cells to exhibit selective dark cytotoxicity, and exhibit efficient photodamage in cancer cells due to intracellular bright spots. Results suggested that this compound can be a potential dual-toxic efficacy molecule. Additionally, a water-soluble pH sensor ADA was designed and synthesized based on the molecular design of photo-induced electron transfer (PET) and intramolecular charge transfer (ICT). The fluorescent emission response against a pH value located in the range of 3~6, which is suitable to label intracellular pH dependent microenvironments. Finally, we not only used ADA to identify intracellular pH gradients between different cell lines, but also developed ADA as a fluorescent molecule that labeled normal cells by the unique fluorescence intensity of ADA in normal cells, making it effective as the control group at the time of cancer diagnosis.

第一章 序論 1
1.1 分子內電荷轉移 (Intramolecular Charge Transfer, ICT) 1
1.2 扭轉的分子內電荷轉移 (Twisted Intramolecular Charge Transfer, TICT) 3
1.3 光誘導電子轉移 (Photo-induced Electron Transfer, PET) 5
1.4 聚集誘導螢光(Aggregation Induced Emission, AIE)的螢光有機奈米粒(Fluorescence Organic Nanoparticles, FONs) 6
1.5 光動力治療 (Photodynamic Therapy, PDT) 7
1.6 研究大綱 9
1.7 實驗簡介 10
(一) 一個雙重抗癌療效分子：一個選擇性暗毒性光敏物質 10
(二) 一個用來偵測活細胞中酸性胞器的螢光pH探針 11
第二章 實驗方法與合成 14
2.1 實驗用藥品 14
2.2 儀器設備 14
2.2.1 核磁共振光譜儀（Nuclear Magnet Resonance Spectrometer , NMR） 14
2.2.2 紫外光-可見光吸收光譜儀（UV-Vis Spectrophotometer，UV-vis） 14
2.2.3 螢光光譜儀（Fluorescence Spectrometer，PL） 15
2.2.4 酸鹼度計（pH-meter） 15
2.2.5 氙光燈源(Xenon light source) 15
2.2.6 螢光顯微鏡 (Fluorescence microscope) 15
2.3 細胞培養條件及細胞影像 16
2.3.1 細胞株 16
2.3.2 細胞培養液 16
2.3.3 細胞培養 16
a. 細胞解凍程序 16
b. 繼代培養 17
c. 冷凍保存細胞 17
2.3.4 細胞影像 17
2.4 細胞內特定胞器追蹤 (Cellular tracker assay) 17
2.5 細胞暗毒性及光毒性實驗 18
2.5.1 細胞暗毒性 18
2.5.2 細胞光毒性 18
2.6 細胞內pH值校正 (Intracellular pH calibration) 19
2.7 化學合成 19
第三章 實驗結果與討論 25
(I) A dual anticancer efficacy molecule : a selective dark cytotoxicity photosensitizer 25
一個雙重抗癌療效分子：一個選擇性暗毒性光敏物質 25
3.1.1 分子設計 25
3.1.2 特定光學性質 (基於AIEE特性的螢光有機奈米粒，FON) 26
3.1.3 細胞滲透性與新型ROS 28
3.1.4 針對癌細胞的選擇性光毒性及暗毒性 33
3.1.5 NV-12P在細胞內的堆積及位置分佈 37
3.1.6 細胞光毒性及暗毒性機制 43
3.1.7 結合暗毒性及光毒性之雙重療效 45
(II) A fluorescent pH probe for acidic organelles in living cells 50
一個用來偵測活細胞中酸性胞器的螢光pH探針 50
3.2.1 分子設計與合成 50
3.2.2 基本光譜探討 55
3.2.2.1 溶劑效應 55
3.2.2.2 ICT / TICT以及trans / cis form的決定 65
3.2.3 酸滴定(Acidic titration)相關實驗 68
3.2.3.1 決定ANB、ANB-1及ADA的酸化路徑 68
3.2.3.2 水中的酸滴定實驗及pKa數值的決定 75
3.2.3.3 逆向(reverse)反應實驗 79
3.2.3.4 ANB及ADA的滴定實驗之NMR光譜分析 84
3.2.4 分子的AIE (aggregation-induced emission)特性 89
3.2.5 化合物的活細胞影像及ADA的細胞內pH值校正(intracellular pH calibration) 95
3.2.6 分子的免疫染色及Time dependent、Concentration dependent實驗 98
3.2.7 細胞的固定影像 (Cellular fixation image) 106
3.2.8 結合ADA及BMVC2分子辨識正常細胞及癌細胞 108
第四章 結論 113
參考文獻 114

1.Salerni A. A chemical and photophysical analysis of a push-pull compound. A major qualifying project report: submitted to the faculty of the Woreceter Polytechnic Institute. 2014, May, 1st.
2.Loving G. S.; Sainlos M.; Imperiali B. Monitoring protein interactions and dynamics with solvatochromic fluorophores. Trends Biotechnol. 2010, 28, 73-83.
3.Haidekker M. A.; Theodorakis E. A. Environment-sensitive behavior of fluorescent molecular rotors. Journal of Biological Engineering, 2010, 4:11.
4.Grabowski Z. R.; Rotkiewicz K.; Rettig W. Structural changes accompanying intramolecular electron transfer: focus on twisted intramolecular charge-transfer states and structures. Chem. Rev. 2003, 103, 3899-4031.
5.de Silva A. P.; Gunaratne H. Q. N.; Gunnlaugsson T.; Huxley A. J. M.; McCoy C. P.; Rademacher J. T.; Rice T. E. Signaling recognition events with fluorescent sensors and switches. Chem. Rev. 1997, 97, 1515-1566.
6.Horn D.; Rieger J. Organic nanoparticles in the aqueous phase-theory, experiment, and use. Angew. Chem. Int. Ed. Engl. 2001, 40, 4330-4361.
7.Kasai H.; Kamatani H.; Okada S.; Oikawa H.; Matsuda1 H.; Nakanishi H. Size-dependent colors and luminescences of organic microcrystals. Jpn. J. Appl. Phys. 1996, 35, 221-223.
8.An B.-K.; Kwon S.-K.; Jung S.-D.; Park S. Y. Enhanced emission and its switching in fluorescent organic nanoparticles. J. Am. Chem. Soc. 2002, 124, 14410-14415.
9.Deing K. C.; Mayerhöffer U.; Würthner F.; Meerholz K. Aggregation-dependent photovoltaic properties of squaraine/PC61BM bulk heterojunctions. Phys. Chem. Chem. Phys. 2012, 14, 8328-8334.
10.Lipson R. L.; Baldes E. J. The photodynamic properties of a particular hematoporphyrin derivative. Arch. Dermatol. 1960, 82, 508-516.
11.Dai T.; Fuchs B. B.; Coleman J. J.; Prates R. A.; Astrakas C.; St Denis T. G.; Ribeiro M. S.; Mylonakis E.; Hamblin M. R.; Tegos G. P. Concepts and principles of photodynamic therapy as an alternative antifungal discovery platform. Front Microbiol. 2012, 3, Article 120.
12.Moan J.; Berg K. The photodegradation of porphyrins in cells can be used to estimate the lifetime of singlet oxygen. Photochem. Photobiol. 1991, 53, 549-553.
13.Castano, A. P.; Mroz, P.; Hamblin, M. R. Photodynamic therapy and anti-tumour immunity. Nat. Rev. Cancer 2006, 6, 535-545.
14.Pass, H. I. Photodynamic therapy in oncology: mechanisms and clinical use. J. Natl. Cancer Inst. 1993, 85, 443-456.
15.DeRosa, M. C.; Crutchley, R. J. Photosensitized singlet oxygen and its applications. Coord. Chem. Rev. 2002, 233-234, 351-371.
16.Ding, H.; Sumer, B. D.; Kessinger, C. W.; Dong, Y.; Huang, G.; Boothman, D. A.; Gao, J. Nanoscopic micelle delivery improves the photophysical properties and efficacy of photodynamic therapy of protoporphyrin IX. J. Controlled Release 2011, 151, 271-277.
17.Lai, Y. C.; Su, S. Y.; Chang, C. C. Special reactive oxygen species generation by a highly photostable BODIPY-based photosensitizer for selective photodynamic therapy. ACS Appl. Mater. Interfaces 2013, 5, 12935-12943.
18.Hsieh, T. S.; Wu, J. Y.; Chang, C. C. Synthesis of a photostable near-infrared-absorbing photosensitizer for selective photodamage to cancer cells. Chem.- Eur. J. 2014, 20, 9709-9715.
19.Chou, Y. S.; Chang, C. C.; Chang, T. C.; Yang, T. L.; Young, T. H.; Lou, P. J. Photo-induced antitumor effect of 3,6-bis(1-methyl-4-vinylpyridinium) carbazole diiodide. BioMed Res. Int. 2013, 2013, 1.
20.Dias, N.; Bailly, C. Drugs targeting mitochondrial functions to control tumor cell Growth. Biochem. Pharmacol. 2005, 70, 1-12.
21.Rodriguez, M. E.; Azizuddin, K.; Zhang, P.; Chiu, S. M.; Lam, M.; Kenney, M. E.; Burda, C.; Oleinick, N. L. Targeting of mitochondria by 10-N-alkyl acridine orange analogues: role of alkyl chain length in determining cellular uptake and localization. Mitochondrion 2008, 8, 237-246.
22.Sakhrani, N. M.; Padh, H. Organelle targeting: third level of drug targeting. Drug Des. Dev. Ther. 2013, 7, 585-599.
23.Xu, J.; Zeng, F.; Wu, H.; Hu, C.; Wu, S. Enhanced photodynamic efficiency achieved via a dual-targeted strategy based on photosensitizer/micelle structure. Biomacromolecules 2014, 15, 4249-4259.
24.Wang, Y.; Wang, K.; Zhao, J.; Liu, X.; Bu, J.; Yan, X.; Huang, R. Multifunctional mesoporous silica-coated graphene nanosheet used for chemo-photothermal synergistic targeted therapy of glioma. J. Am. Chem. Soc. 2013, 135, 4799-4804.
25.Postiglione, I.; Chiaviello, A.; Palumbo, G. Enhancing photodynamyc therapy efficacy by combination therapy: dated, current and oncoming strategies. Cancers 2011, 3, 2597-2629.
26.Li, H.; Li, Z.; Liu, L.; Lu, T.; Wang, Y. An efficient gold nanocarrier for combined chemo-photodynamic therapy on tumour cells. RSC Adv. 2015, 5, 34831-34838.
27.He, C.; Liu, D.; Lin, W. Self-assembled core-shell nanoparticles for combined chemotherapy and photodynamic therapy of resistant head and neck cancers. ACS Nano 2015, 9, 991-1003.
28.Castano, A. P.; Mroz, P.; Wu, M. X.; Hamblin, M. R. Photodynamic therapy plus low-dose cyclophosphamide generates antitumor immunity in a mouse model. Proc. Natl. Acad. Sci. USA. 2008, 105, 5495-5500.
29.Gao, L.; Zhang, C.; Gao, D.; Liu, H.; Yu, X.; Lai, J.; Wang, F.; Lin, J.; Liu, Z. Enhanced anti-tumor efficacy through a combination of integrin AVB6-targeted photodynamic therapy and immune checkpoint inhibition. Theranostics 2016, 6, 627-637.
30.Casey J. R.; Grinstein S.; Orlowski J. Sensors and regulators of intracellular pH. Nat. Rev. Mol. Cell Biol. 2010, 11, 50-61.
31.Gallagher F. A.; Kettunen M. I.; Day S. E.; Hu D. E.; Ardenkjær-Larsen J. H.; Zandt R. i.; Jensen P. R.; Karlsson M.; Golman K.; Lerche M. H.; Brindle K. M. Magnetic resonance imaging of pH in vivo using hyperpolarized 13C labelled bicarbonate. Nature 2008, 453, 940-943.
32.Kato Y.; Ozawa S.; Miyamoto C.; Maehata Y.; Suzuki A.; Maeda T.; Baba Y. Acidic extracellular microenvironment and cancer. Cancer Cell Int. 2013, 13, 89-96.
33.Webb B. A.; Chimenti M.; Jacobson M. P.; Barber D. L. Dysregulated pH : a perfect storm for cancer progression. Nat. Rev. Cancer 2011, 9, 671-677.
34.Swietach P.; Hulikova A.; Patiar S.; Vaughan-Jones R. D.; Harris A. L. Importance of intracellular pH in determining the uptake and efficacy of the weakly basic chemotherapeutic drug, doxorubicin. PLoS One 2012, 7, e35949.
35.Canton I.; Battaglia G. Endocytosis at the nanoscale. Chem. Soc. Rev. 2012, 41, 2718-2739.
36.Han J.; Burgess K. Fluorescent indicators for intracellular pH. Chem. Rev. 2010, 110, 2709-2728.
37.Luo J.; Xie Z.; Lam J. W.; Cheng L.; Chen H.; Qiu C.; Kwok H. S.; Zhan X.; Liu Y.; Zhu D.; Tang B. Z. Aggregation-induced emission of 1-methyl-1,2,3,4,5-pentaphenylsilole. Chem. Commun. 2001, 18, 1740-1741.
38.Joseph K.-H. W.; Matthew H. T.; Peter J. R. Recent advances in macrocyclic fluorescent probes for ion sensing. Molecules 2017, 22, 200.
39.Yang W.; Li C.; Zhang M.; Zhou W.; Xue R.; Liu H.; Li Y. Aggregation-induced emission and intermolecular charge transfer effect in triphenylamine fluorophores containing diphenylhydrazone structures. Phys. Chem. Chem. Phys. 2016, 18, 28052-28060.
40.Kwok R. T.; Leung C. W.; Lam J. W.; Tang B. Z. Biosensing by luminogens with aggregation-induced emission characteristics. Chem. Soc. Rev. 2015, 44, 4228-4238.
41.Liang J.; Tang B. Z.; Liu B., Specific light-up bioprobes based on AIEgen conjugates. Chem. Soc. Rev. 2015, 44, 2798-2811.
42.Feng Q.; Li Y.; Wang L.; Li C.; Wang J.; Liu Y.; Li K.; Hou H. Multiple-color aggregation-induced emission (AIE) molecules as chemodosimeters for pH sensing. Chem. Commun. 2016, 52, 3123-3126.
43.Yang Z.; Qin W.; Lam J. W. Y.; Chen S.; Sung H. H. Y.; Williams I. D.; Tang B. Z. Fluorescent pH sensor constructed from a heteroatom-containing luminogen with tunable AIE and ICT characteristics. Chem. Sci. 2013, 4, 3725-3730.
44.Wu D.; Shao L.; Li Y.; Hu Q.; Huang F.; Yu G.; Tang G. A boron difluoride dye showing the aggregation-induced emission feature and high sensitivity to intra- and extra-cellular pH changes. Chem. Commun. 2016, 52, 541-544.
45.Zhou Z.; Gu F.; Peng L.; Hu Y.; Wang Q. Spectroscopic analysis and in vitro imaging applications of a pH responsive AIE sensor with a two-input inhibit function. Chem. Commun. 2015, 51, 12060-12063.
46.Smith, R. A.; Hartley, R. C.; Murphy, M. P. Mitochondria-targeted small molecule therapeutics and probes. Antioxid. Redox Signaling 2011, 15, 3021-3038.
47.Kang, C. C.; Kouh, C. W.; Wang, Z. F.; Cho, C. C.; Chang, C. C.; Wang, C. L.; Chang, T. C.; Seemann, J.; Huang, L. J. S. Chemical principles for the design of a novel fluorescent probe with high cancer-targeting selectivity and sensitivity. Integr. Biol. 2013, 5, 1217-1228.
48.Huang, W. C.; Tseng, T. Y.; Chen, Y. T.; Chang, C. C.; Wang, Z. F.; Wang, C. L.; Hsu, T. N.; Li, P. T.; Chen, C. T.; Lin, J. J.; Lou, P. J.; Chang, T. C. Direct evidence of mitochondrial G-quadruplex DNA by using fluorescent anti-cancer agents. Nucleic Acids Res. 2015, 43, 10102-10113.
49.Lin, H. H.; Su, S. Y.; Chang, C. C. Fluorescent organic nanoparticle formation in lysosomes for cancer cell recognition. Org. Biomol. Chem. 2009, 7, 2036-2039.
50.Su, S. Y.; Lin, H. H.; Chang, C. C. Dual optical responses of phenothiazine derivatives: near-IR chromophore and water-soluble fluorescent organic nanoparticles. J. Mater. Chem. 2010, 20, 8653-8658.
51.Lin, H. H.; Chan, Y. C.; Chen, J. W.; Chang, C. C. Aggregation-induced emission enhancement characteristics of naphthalimide derivatives and their applications in cell imaging. J. Mater. Chem. 2011, 21, 3170-3177.
52.Hsieh, M. C.; Chien, C. H.; Chang, C. C.; Chang, T. C. Aggregation induced photodynamic therapy enhancement based on linear and nonlinear excited FRET of fluorescent organic nanoparticles. J. Mater. Chem. B 2013, 1, 2350-2357.
53.Chang, C. C.; Hsieh, M. C.; Lin, J. C.; Chang, T. C. Selective photodynamic therapy based on aggregation-induced emission enhancement of fluorescent organic nanoparticles. Biomaterials 2012, 33, 897-906.
54.Mei, J.; Leung, N. L. C.; Kwok, R. T. K.; Lam, J. W. Y.; Tang, B. Z. Aggregation-induced emission: together we shine, united we soar! Chem. Rev. 2015, 115, 11718-11940.
55.Charras, G.; Yarrow, J.; Horton, M.; Mahadevan, L.; Mitchison, T. Non-equilibration of hydrostatic pressure in blebbing cells. Nature 2005, 435, 365-369.
56.Gollnick, K.; Griesbeck, A. Singlet oxygen photooxygenation of furans: isolation and reactions of (4 + 2)-cycloaddition products (unsaturated sec.-ozonides). Tetrahedron 1985, 41, 2057-2068.
57.Bulina, M. E.; Chudakov, D. M.; Britanova, O. V.; Yanushevich, Y. G.; Staroverov, D. B.; Chepurnykh, T. V.; Merzlyak, E. M.; Shkrob, M. A.; Lukyanov, S.; Lukyanov, K. A. A genetically encoded photosensitizer. Nat. Biotechnol. 2006, 24, 95-99.
58.Zhao, E.; Deng, H.; Chen, S.; Hong, Y.; Leung, C. W. T.; Lam, J. W. Y.; Tang, B. Z. A dual functional AEE fluorogen as a mitochondrial-specific bioprobe and an effective photosensitizer for photodynamic therapy. Chem. Commun. 2014, 50, 14451-14454.
59.Chan, D. C. Fusion and fission: interlinked processes critical for mitochondrial health. Annu. Rev. Genet. 2012, 46, 265-287.
60.Ly, J. D.; Grubb, D. R.; Lawen, A. The mitochondrial membrane potential (deltapsi(M)) in apoptosis; an update. Apoptosis 2003, 8, 115-128.
61.Karbowski, M.; Youle, R. J. Dynamics of mitochondrial morphology in healthy cells and during apoptosis. Cell Death Differ. 2003, 10, 870-880.
62.Suen, D. F.; Norris, K. L.; Youle, R. J. Mitochondrial dynamics and apoptosis. Genes Dev. 2008, 22, 1577-1590.
63.Kwok, R. T. K.; Leung, C. W. T.; Lam, J. W. Y.; Tang, B. Z. Biosensing by luminogens with aggregation-induced emission characteristics. Chem. Soc. Rev. 2015, 44, 4228-4238.
64.Yuan, Y.; Zhang, C. J.; Gao, M.; Zhang, R.; Tang, B. Z.; Liu, B. Specific light-up bioprobe with aggregation-induced emission and activatable photoactivity for the targeted and image-guided photodynamic ablation of cancer cells. Angew. Chem., Int. Ed. 2015, 54, 1780-1786.
65.Verma, S.; Watt, G. M.; Mai, Z.; Hasan, T. Strategies for enhanced photodynamic therapy effects. Photochem. Photobiol. 2007, 83, 996-1005.
66.Belloc, F.; Dumain, P.; Boisseau, M. R.; Jalloustre, C.; Reiffers, J.; Bernard, P.; Lacombe, F. A flow cytometric method using hoechst 33342 and propidium iodide for simultaneous cell cycle analysis and apoptosis determination in unfixed cells. Cytometry 1994, 17, 59-65.
67.Niu C. G.; Zeng G. M.; Chen L. X.; Shena G. L.; Yu R. Q. Proton "off-on" behaviour of methylpiperazinyl derivative of naphthalimide: a pH sensor based on fluorescence enhancement. Analyst 2004, 129, 20-24.
68.Cui D.; Qian X.; Liu F.; Zhang R. Novel fluorescent pH sensors based on intramolecular hydrogen bonding ability of naphthalimide. Org. Lett 2004, 16, 2757-2760.
69.Trupp S.; Hoffmann P.; Henkel T.; Mohr G. J. Novel pH indicator dyes for array preparation via NHS ester activation or solid-phase organic synthesis. Org. Biomol. Chem. 2008, 6, 4319-4322.
70.Kim H. M.; An M. J.; Hong J. H.; Jeong B. H.; Kwon O.; Hyon J. Y.; Hong S. C.; Lee K. J.; Cho B. R. Two-photon fluorescent probes for acidic vesicles in live cells and tissue. Angew. Chem. Int. Ed. 2008, 47, 2231–2234.
71.Jin X.; Zhao J.; Zhang L.; Huang Y.; Zhao S. An enhanced fluorescence polarization strategy based on multiple protein–DNA–protein structures for sensitive detection of PDGF-BB. RSC Adv. 2014, 4, 6850-6853.
72.Doz. P.; Rettig W. Charge separation in excited states of decoupled systems—TICT compounds and implications regarding the development of new laser dyes and the primary process of vision and photosynthesis. Angew. Chem. Int. Ed. Engl. 1986, 25, 971–988.
73.Ashis N.; Kankan B. Role of twisted intramolecular charge transfer in the fluorescence sensitivity of biological probes: Diethylaminocoumarin laser dyes. Chem. Phys. Lett. 1990,169, 12.
74.Mao M.; Ren M. G.; Song Q. H. Thermodynamics and conformations in the formation of excited states and their interconversions for twisted donor-substituted tridurylboranes. Chemistry 2012, 18, 15512-15522.
75.Auweter H.; Haberkorn H.; Heckmann W.; Horn D.; Lüddecke E.; Rieger J.; Weiss H. Supramolecular structure of precipitated nanosize beta-carotene particles, Angew. Chem. Int. Ed. 1999, 38, 2188-2191.
76.Lee M. H.; Han J. H.; Lee J. H.; Park N.; Kumar R.; Kang C.; Kim J. S. Two-color probe to monitor a wide range of pH values in cells, Angew. Chem. Int. Ed. 2013, 52, 6206-6209.
77.Tafani M.; Cohn J. A.; Karpinich N. O.; Rothman R. J.; Russo M. A.; Farber J. L. Regulation of intracellular pH mediates Bax activation in HeLa cells treated with staurosporine or tumor necrosis factor-alpha, J. Biol. Chem. 2002, 277, 49569-49576.
78.Willem J.; ter Beest M.; Scherphof G.; Hoekstra D. A non-exchangeable fluorescent phospholipid analog as a membrane traffic marker of the endocytic pathway, Eur. J. Cell Biol. 1990, 53, 173-184.
79.Somapuram S. R.; Vani K.; Messana E.; Bogen S. A. A molecular mechanism of formalin fixation and antigen retrival. Am. J. Clin. Pathol. 2004, 121, 190-199.
80.Kiernan J. A. Formaldehyde, formalin, paraformaldehyde and glutaraldehyde: What they are and what they do. Microscopy Today 2000, 1, 8-12.
81.Thavarajah R.; Mudimbaimannar V. K.; Elizabeth J.; Rao U. K.; Ranganathan K. Chemical and physical basics of routine formaldehyde fixation. J. Oral Maxillofac. Pathol. 2012, 16, 400-405.
82.Bruke F. V. The pH of formalin-A factor in fixation: Adjustment and stabilization of the hydrogen Ion concentration of formalin solutions. Am. J. Pathol. 1933, 9, 915-920.
83.Swietach P.; Vaughan-Jones R. D.; Harris A. L.; Hulikova A. The chemistry, physiology and pathology of pH in cancer, Philos. Trans. R. Soc. Lond. B. Biol. Sci. 2014, 369, 1638-1646.
84.Stüwe L.; Müller M.; Fabian A.; Waning J.; Mally S.; Noël J.; Schwab A.; Stock C. pH dependence of melanoma cell migration: protons extruded by NHE1 dominate protons of the bulk solution, J. Physiol. 2007, 585, 351-360.