奈米樹枝狀高分子對於細胞胞飲體及溶小體pH值的影響

從1980年代被研發以來，樹枝狀高分子已經被廣泛應用在生物醫學領域上。因為樹枝狀高分子具有高度分支且帶大量正電官能基的結構特殊性，因此能夠因應不同的需求，藉由對其表面官能基的改質，接上不同的物質，而進一步加以應用。我們實驗室之前已將第四代的樹枝狀高分子與螢光物質-FITC鍵結後餵予細胞，再利用不同的細胞胞器標定染劑，以共軛焦螢光顯微鏡和流氏細胞儀，觀察樹枝狀高分子在細胞內的傳輸與分布；也發現在我們追蹤的12小時時間裡，螢光強度沒有減弱的趨勢，帶螢光的樹枝狀高分子也沒有被分解；但理論上隨著胞飲作用的進行，當樹枝狀高分子到達較酸性環境的後期胞飲體或溶小體時，與其鍵結的FITC螢光應該會有減弱的現象。因此，我們設計利用FITC在不同酸鹼環境下其發散光強度的變化來間接測定螢光物質所在的胞器的pH值，並結合溶小體的免疫螢光染色，發現可能因為樹枝狀高分子本身結構的特性，帶有大量正電荷的表面氨基官能基，進而改變所在胞器的pH值，甚至因而延遲被溶小體分解的時間。本研究的結果解開了樹枝狀高分子在胞飲作用過程中對胞飲體及溶小體pH值的影響，對於利用樹枝狀高分子作為藥物或基因傳輸的載體的設計上，有相當大的幫助；延長的胞飲作用過程，增加了與其他方法如光化學內化法和光動力療法等等的合併使用時機選擇，亦或能配合藥物的作用機制或基因的特性，發展出新型更具有潛力的藥物傳輸與基因傳輸載體。

關鍵字

胞飲作用；樹枝狀高分子； pH值；溶小體

並列摘要

PAMAM dendrimers have been implemented in various biomedical applications in recent years. Because of the unique highly branched structure, multivalent functional groups, PAMAM dendrimers could be easily conjugated with tumor-targeting ligands, chemotherapeutical drugs, or plasmids for further usage. In our previous studies, we have used fluorescein isothiocyanate (FITC) – conjugated G = 4 PAMAM dendrimers (G4-FITC) to elucidate its intracellular trafficking and subcellular distribution. We found the majority of the dendrimers accumulated in the lysosomes 24-48 hour after incubation without being degraded. Increased fluorescence intensity of G4-FITC after uptake by cells could also be observed. However, FITC is a pH sensitive probe that should be inactive in organelles with acidic luminal pH, i.e. the lysosomes. These findings prompted us to study the effects of G4-FITC on the luminal pH of the endo-lysosomal compartments after uptake by the cells. In this study, we established the Dextran-FITC and G4-FITC pH standard curves by using flow cytometry. The luminal pH of the endosomes/lysosomes after uptake of Dextran-FITC and G4-FITC was also identified. Immunofluorescence microscopy was used to further elucidate the trafficking of G4-FITC to the late endosomes and lysosomes. Our results showed the luminal pH of endosomes/lysosomes was buffered by the PAMAM dendrimers, which may result in the decreased lysosomal enzyme activity and hence the maintenance of fluorescence intensity in the cells. The results from this study bring us further insight into the biological effects of PAMAM dendrimers. Combination with other approaches, such as photochemical internalization (PCI), may help us to design better strategies for efficient intracellular drug or gene delivery in the future.

並列關鍵字

lysosome ； pH value ； dendrimer ； endocytosis

參考文獻

Al-Awqati, Q. (1986) Proton-translocating ATPase. Annual Review of Cell Biology 2, 179-199

Bielinska, A.U., Chen, C., Johnson, J. and Baker, J.R. (1999) DNA complexing with polyamidoamine dendrimers: implications for transfection. Bioconjugate Chemistry 10, 843-850

Cain, C.C., Sipe, D.M. and Murphy, R.F. (1989) Regulation of endocytic pH by the Na+,K+-ATPase in living cells. Proceedings of the National Academy of Sciences of the United States of America 86, 544-548

Fuchs, R., Schmid, S. and Mellman, I. (1989) A possible role for the Na+ ,K+-ATPase in regulating ATP-dependent endosome acidification. Proceedings of the National Academy of Sciences of the United States of America 86, 539-543

Gruenberg, J., Griæths, G. and Howell, K.E. (1989) Characterisation of the early endosome and putative endocytic carrier vesicles in vivo and with an assay of vesicle fusion in vitro. Journal of Cell Biology 108, 1301-1316

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奈米樹枝狀高分子對於細胞胞飲體及溶小體pH值的影響

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