藉由胜肽輸送進入活細胞內溶小體之具有酸鹼值偵測能力的中孔洞二氧化矽奈米粒子

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國立臺灣大學理學院化學研究所碩士論文

Department of Chemistry College of Science

National Taiwan University Master Thesis

藉由胜肽輸送進入活細胞內溶小體之具有酸鹼值偵測能力的中孔洞二氧化矽奈米粒子

Peptide-mediated delivery of pH-sensing Mesoporous Silica Nanoparticles into Lysosome in living cells

張榮麟 Rong-Lin Zhang

指導教授：牟中原博士 Advisor: Chung-Yuan Mou, Ph.D.

中華民國 105 年 7 月

July, 2016

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誌謝

時間飛逝，轉眼間就快完成兩年的碩班研究，在這段期間，感謝足智多謀的牟老師給予我很多寶貴的知識和意見以及豐富的研究資源，讓我有充分學習的機會，

且站在科學巨人的肩膀上開啟我的研究旅程，也讓我獲益良多。而在這一路學習的過程中，首先要感謝思翰學長，給予我實驗的方向，也教導我實驗的規劃並且耐心地與我討論實驗的結果，讓一開始懵懂無知的我逐漸有了研究的概念，也讓我慢慢了解這個奈米材料的世界。而面對完全陌生的光學系統，很慶幸有劫叡學長的幫助，

對於我無數的問題，都能耐心教導我，讓我實驗能夠順利的進行，另外對於程式一竅不通的我，很感謝有中央大學簡汎清教授的協助，才能讓數據能夠更順利的分析，

對於光學儀器的架設，感謝呂杰翰博士耐心地給予指導，也給了我很多光學相關知識，讓我更熟系這套光學系統，在每次遇到的光學瓶頸，還好有您們從深淵中把我拉起，是您們重新建立起我的信心，讓實驗可以持續的進行。在生物實驗方面，要非常感謝陳奕平學長的協助，細心的教導我實驗的步驟和技巧，也不辭辛勞的提供實驗上載送的服務，讓我能更快速取得完美漂亮的數據，另外在研究上也給予我很多的關心讓我感到非常欣慰。在這兩年的時光裡，最懷念還是實驗室的夥伴們，在忙碌的碩班生活中，有你們的陪伴是支持我的動力，謝謝明瀚學長給予我很多豐富的經驗與人生道理，感謝乃元給予我實驗和課業上的幫助，你是我學習的好榜樣，

謝謝怡婷高亢美妙的歌聲撫慰了我的心靈，感謝每天都很歡樂的梓安學長時常找我吃飯和送便當，讓我即使在 12 樓也不會感到孤獨，謝謝祖源讓我吃到我生平都沒吃過的大餐，謝謝你們的陪伴，才能更豐富我的碩班生活。

最後，感謝愛我的家人，謝謝你們給予我生活上的幫助，有你們背後默默的付出和支持，才有今天的我，僅以此論文獻給我最摯愛的家人。

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中文摘要

具中孔洞的二氧化矽奈米粒子作為平台可有效地運輸酵素抵達受損的組織，

因酵素裝載在此多功能的中孔洞二氧化矽奈米粒子裡，能夠增加在血液中的循環時間、有更精準的標靶能力以及保護酵素免於免疫反應的發生。在這個研究裡，此中孔洞的二氧化矽奈米粒子被修飾上兩個酸鹼值靈敏的螢光染劑，分別是 fluorescein isothiocyanate (FITC, pKa = 6.7)和 oregon green succinimidyl ester (OG, pKa = 4.8)，以及一個參考染劑為 rhodamine B isothiocyanate (RITC)，使其成為一個 pH 值感測器並具有一個寬廣的酸鹼值偵測範圍 (pH = 4.2 ─ 7.4)，因此我們可以利用單一粒子追蹤的顯微技術來做酸鹼值的即時偵測，藉此提供一個新穎的方式來辨識此中孔洞二氧化矽奈米粒子在活細胞中的位置。在另一方面，此具有酸鹼值感測能力的中孔洞二氧化矽奈米粒子被修飾上具有溶小體標靶能力的胜肽鏈 (YXXØ )可作為酵素取代治療的生醫應用，我們展示了酸鹼值感測的中孔洞二氧化矽奈米粒子對於細胞環境 pH 值的即時傳訊是一個有用的技術來評估其在活細胞中的移動途徑，而藉著 YXXØ 胜肽的功能，此連接著 YXXØ 的中孔洞二氧化矽奈米粒子確實能夠到達溶小體，相較於裸露的中孔洞二氧化矽奈米粒子幾乎只累積在細胞質的環境。酵素取代治療已經在臨床上被使用來治療溶小體囤積症，然而目前為止，仍然有許多的問題尚待解決，其中像是造成各種副作用的免疫反應以及不佳的治療效果等等，因此具有溶小體標靶能力的中孔洞二氧化矽奈米粒子將會是一個有潛力的奈米載體作為改善酵素取代治療對於溶小體囤積症的效果。

關鍵字:中孔洞二氧化矽奈米粒子、酸鹼值感測器、三維空間單一粒子追蹤、溶小體標靶、溶小體囤積症

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Abstract

Mesoporous silica nanoparticles (MSNs) as a platform can efficiently transport

enzymes to the impaired tissues because enzymes loaded in multiple functional MSNs

can increase the circulation time in blood, have more precisely targeting ability and

protect them from some immune reaction. In this study, MSNs were made as a pH

nanosensor which has a broad pH-sensing range from 4.2 to 7.4 by conjugating two pH-

sensitive fluorescent dyes, fluorescein isothiocyanate (FITC, pKa=6.7) and oregon green

succinimidyl ester (OG, pKa=4.8), and a referent dye, rhodamine B isothiocyanate (RITC)

onto MSNs. So we can provide a novel method to identify the localization of the MSNs

in living cells with real-time pH detection by single particle tracking microscope

technique. In the other hand, the pH-sensing MSNs were modified with lysosomal sorting

peptides (YXXØ ) which can target lysosomes for potential enzyme replacement therapy.

We showed that the real-time signaling of the environment pH values from the pH-sensing

MSNs is a useful technique for evaluating the pathway of them in living cells, and the

specific peptide-conjugated MSNs were indeed delivered into lysosomes by the function

of the YXXØ sequences. Enzyme replacement therapy has been used clinically for

treating lysosomal storage diseases. However there are still many problems such as

immune reaction causing much side-effect need to be solved. So the lysosome-targeting

MSNs will be a promising nanocarrier for improving the enzyme replacement therapy to

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lysosomal storage disease.

Keywords: mesoporous silica nanoparticles、pH sensor、3D single particle tracking、

lysosome targeting 、lysosomal storage disease

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List of Figures

Figure 1.1. Schematic presentation of F-UiO synthesis. (b) Correlation between FITC

absorbance and fluorescence at various FITC loadings. (c-e) pH-calibration curves of free

FITC (c) and F-UiO acquired by fluorimetry (d) and by confocal laser scanning

microscopy (e). 488/435 in the Y-axis represents I488/520/I435/520. (F) CLSM images

showing the overlay of green (488 nm excitation) and red (435 nm excitation) colors of

F-UiO particles in HBSS buffers with different pHvalues.¹⁴ ... 3

Figure 1.2. QD-FP FRET-based pH sensor. (a) Schematic demonstration of the pH-

dependent energy transfer between the quantum dot and fluorescent protein. In an acidic

environment, energy transfer to the FP FRET acceptor is minimal, yielding a high QD

signal; at neutral or basic pH, energy transfer is more efficient, producing an enhanced

FRET signal. (b) Absorbance spectroscopy indicates multiple proteins bound to each QD,

as depicted in the inset. (c and d) Titration of QD-FP probes containg the FP acceptors

mOrange and mOrange M163K, respectively, showing increased energy transfer at

alkaline pHs with clear isosbestic points. Representative spectra of one of three

independent titrations are shown. (e) The ratio of acceptor emission to donor emission

increases with increasing pH for both probes. Data points are means ± standard deviations

for three independent titration. (f) The changes in the nanoprobe acceptor to donor ratios

are compared to the ratiometric signal change for the pH-sensitive fluorophore BCECF.

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One representative titration is shown.¹⁰ ... 4

Figure 1.3. Schematic of the cross-linked poly acrylamide nanoparticle. (b) In vitro

calibration of the triple-labeled sensor with both OG and FS, and two dual-labeled sensors

with either OG or FS. (c) Uptake of the triple-labeled sensor by a HepG2 cell after 24h

and washing and imaged with confocal microscopy. Scale bar = 10µm. OG = Oregon

Green; FS = fluorescein.⁷ ... 7

Figure 1.4. Schematic of the confocal 3-D tracking microscope optical configuration.

The fiber optic pairs are configured to form a 3-D tetrahedron-like detection volume in

sample space.^22c ... 9

Figure 1.5. The scheme of 3D STORM. (A) Three-dimensional localization of individual

fluorophores. The simplified optical diagram illustrates the principle of determining the

z coordinate of a fluorescent object from the ellipticity of its image by introducing a

cylindrical lens into the imaging path. The right panel shows images of a fluorophore at

various z positions. EMCCD, electron-multiplying charge-coupled device. (B)

Calibration curve of image widths Wx and Wy as a function of z obtained from single

Alexa 647 molecules. Each data point represents the average value obtained from six

molecules. The data were fit to a defocusing function (red curve).^23d ... 10

Figure 1.6. Structures of mesoporous M41S materials: (a) MCM-41 (2D hexagonal,

space group p6mm), (b) MCM-48 (cubic, space group Ia3d), and (c) MCM-50 (lamellar,

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space group p2).³⁰ ... 12

Figure 1.7. Formation of mesoporous materials by structure-directing agent: (a) true

liquid-crystal template mechanism, (b) cooperative liquid-crystal template mechanism.³⁰

... 13

Figure 1.8. Schematic illustration for the synthesis and selective functionalization of

MSNs.³³ ... 14

... 14

Figure 1.9. TEM images of mesoporous silica with different average sizes: (a) 280 nm;

inset: FFT analysis of the TEM image; (b) 170, (c) 110, (d) 50, (e) 30 nm. (f) High-

resolution TEM image of a single particle in (c).³⁴ ... 14

Figure 1.10. Effect of pH value on the silica condensation rate, charge properties and

charge density on the surface of the silica species.³⁴ ... 15

Figure 1.11. Functionalization of MSNs by co-condensation (top) or the post-synthetic

method (bottom). A trialkoxysilane molecule bearing a functional group (green) is shown

as an example of a silica precursor. The structure-directing agent is represented by

micelles (red).³⁷ ... 16

Figure 1.12. Pathways of mesoporous silica nanoparticles for entry into cells. Large

particles are internalized by phagocytosis, whereas fluid uptake occurs by

micropinocytosis. In the case of MSN materials, most internalization is via endocytic

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pathways. These pathways differ with regard to the nature of the surface functionalization

and structural properties of MSNs.⁵⁰ ... 26

Figure 1.13. Intracellular trafficking of mesoporous silica nanoparticles. (a) MSNs are

wrapped through specific (ligand-receptor) and nonspecific (hydrophobic, Coulombic)

binding interaction. (b) Once the MSNs are internalized, depending on the endocytic

pathway, it can be delivered to intermediate compartments (e.g., caveosomes). (c) Later

these compartments are transported to early endosomes and then to sorting endosomes.

From sorting endosomes, a fraction of the MSNs are sorted back to the cell exterior

through recycling endosomes (not shown in the scheme). (d) The remaining fraction is

transported to secondary endosomes, (e) which then fuse with lysosomes. (f) The MSNs

escape the endolysosomes and enter the cytosolic compartment.⁵⁰ ... 27

Figure 1.14. Confocal fluorescence images of HeLa cells stained with FM 4-64 and 40

µg/mL suspensions of (a) FITC-MSN and (b) FAP-MSN after 6 hours of introduction.

The fluorescent images (left) show the MSNs (green) and FM 4-64-labeled endosomes

(red) are shown on the right.⁵² ... 28

Figure 1.15. Ratiometric imaging of pH in various intracellular compartments using

confocal microscopy. HeLa cells were incubated at 37 ℃ with MSN-PP and MSN-TA

for 4 hours, respectively. The images (overlaid on bright field) of pH sensors in HeLa

cells showing (a) MSN-PP, and (b) MSN-TA.⁴⁰ ... 29

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Figure 1.16. Confocal microscopy analysis of (a) MSN-PP and (b) MSN-TA in HeLa

cells. The living unfixed cells were co-treated with endosome-specific marker FM 4-64

(5 µg/mL) and analyzed by confocal microscopy for an endosomal co-localization image.

The fluorescent images show the MSNs (green, FITC and red, RITC) and FM 4-64-

labeled endosomes (blue).⁴⁰ ... 29

Figure 1.17. Confocal images of 10 nm mesoporous silica nanoparticles with lysosomes.

Cells adherent on coverslips were preincubated for 10 minutes with Lysotracker Green or

Red, then washed and incubated with nanoparticles, and imaged at 1, 5, and 30 minutes.⁵⁷

... 30

Figure 1.18. Comparison of uptake and intracellular localization of 50 nm mesoporous

silica nanoparticles functionalized or not with either COOH or NH2 groups in SKOV3

and NIH-OVCAR cells after incubation times of one and 24 hours with 20 µg of

nanoparticles.⁵⁷ ... 30

Figure 1.19. Intracellular distributions of R6G-FITC-MSNs as compared to LysoTracker

Blue DND-22. Bar, 10 µm.⁵⁸ ... 31

Figure 1.20. The intracellular localization of spherical MS nano- and microparticles with

different sizes within lysosomes of MDA-MB-468 cells. (a) E-MS-1 (190 nm), (b) E-MS-

2 (420 nm), (c) E-MS-3 (1220 nm), (d) C-MS-1 (190 nm), (e) C-MS-2 (420 nm), and (f)

C-MS-3 (1220 nm).⁵⁹ ... 31

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Figure 1.21. Schematic representation of adaptor protein (AP) complexes. The four

heterotetrameric adaptor protein (AP) complexes (i.e., AP-1, AP-2, AP-3 and AP-4) are

composed of homologous subunits, some of which occur as multiple isoforms. The

subunits assemble into complexes organized into core, hinge and ear domains. AP

complexes participate in protein sorting to lysosomes at different stages of biosynthetic

and endocytic pathways.⁶⁰ ... 35

Figure 1.22. Proposed role for AP-3 in the trafficking of lysosomal membrane proteins

(E, early endosome; LE, late endosome; LYS, lysosome; PM, plasma membrane).⁶⁵ ... 35

Figure 3.1. Characterization of FRMSNs with different synthesis conditions by TEM (left)

and DLS (right). (a) 0.768 M NH3 (aq). (b) 0.896 M NH3 (aq). (c) 1.024 M NH3 (aq). (d)

1.024 M NH3 (aq) + triethylamine (TEA). (e) 1.024 M NH3 (aq) + ethylene glycol (EG).

... 49

Figure 3.2. Scheme of 3D single particle tracking apparatus. ... 51 Figure 3.3. TEM images (Top) and pH calibration (Bottom) of double-labeled MSN with

different mole ratio and addition time of FITC and RITC. (a) FITC : RITC = 28.3 : 1/3 ;

FITC addition with RITC. (b) FITC : RITC = 28.3 : 1 ; FITC addition with RITC. (c)

FITC : RITC = 28.3 : 3 ; FITC addition with RITC. (d) FITC : RITC = 28.3 : 1/3 ; RITC

addition after FITC in 30min. . (e) FITC : RITC = 28.3 : 1 ; RITC addition after FITC in

30min. . (f) FITC : RITC = 28.3 : 3 ; RITC addition after FITC in 30min. ... 52

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Figure 3.4. Characterization of double-labeled MSNs by TEM (left) and SPT

fluorescence microscopy (right). (a) double-labeled MSN without PEI (b) double-labeled

MSN with PEI. ... 54

Figure 3.5. Fluorescence emission spectra of FRMSN-PEI (80 µg/mL) in 10 mM

phosphate buffer at various pH values. (b) The ratio of the fluorescence intensity of FITC

to that of RITC (I520 / I593) versus pH measured by Fluorimetry in 10 mM phosphate buffer.

(c) The ratio of the fluorescence intensity of FITC to that of RITC (I520 / I593) versus pH

measured by Fluorescence Microscopy in 10 mM phosphate buffer. ... 57

Figure 3.6. pH reversibility study of double-labeled MSNs between pH 4 and 8. ... 57 Figure 3.7. Fluorescence intensity ratio for the double-labeled MSNs (80 µg/mL) in

phosphate buffer solution (10 mM, pH 7.4) upon the addition of of various metal ions.

The concentrations of all the cations are 50 µM. ... 58

Figure 3.8. Fluorescence intensity ratio for the double-labeled MSNs (80 µg/mL) in

phosphate buffer solution (10 mM, pH 7.4) containing KCl with various ionic strengths

(0, 0.01, 0.05, 0.15, 0.20Ｍ). ... 58

Figure 3.9. Design and characterizations of triple-labeled MSN. (a) Schematic of triple-

labeled MSN and FRET effect when being excited by single wavelength laser. (b)

Characterization of triple-labeled MSNs by TEM image (Top) and DLS (Bottom). (c)

Fluorescence spectrum of triple-labeled MSNs with pH variation from pH 3.19 to pH 8.84

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in 10 mM phosphate buffer. (d) The ratio of the fluorescence intensity of FITC to that of

RITC (I520 / I593) versus pH measured by Fluorimetry in 10 mM phosphate buffer. ... 60

Figure 3.10. Nitrogen adsorption-desorption isotherm and (b) corresponding pore size

distribution plot of triple-labeled MSNs. The pore size distribution plot is obtained from

the analysis of the adsorption or desorption isotherms using BJH (Barrett-Joyner-Halenda)

method. ... 61

Figure 3.11. The ratio ( I520 / I593 ) of the fluorescence intensity of Triple-labeled MSNs

versus pH measured in different environment by Fluorescence Microscopy. (a) In 10 mM

phosphate buffer solution. (b) In Medium ( DMEM+FBS+P/S). (c) Inside HeLa cells ( in

situ ). ... 63

Figure 3.12. The in-situ pH calibration curves of triple-labeled MSNs before and after

being stored in 99.5% EtOH for 3 months. ... 63

Figure 3.13. Scheme of 3D single particle tracking apparatus combined with pH reporter.

... 67

Figure 3.14. The z calibration curve of the widths from the fluorescence spots in x and y

direction as a function of z locations obtained from five triple-labeled MSNs. The

fluorescence spots of the images were fit to an elliptical Gaussian function carried out

using MatLab software. ... 67

Figure 3.15. Three dimensional localization distribution of triple-labeled MSNs.

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Histograms of the distribution in x, y and z are fit to a Guassian function, yielding

standard deviations of 24 nm in x, 10 nm in y, and 18 nm in z. ... 68

Figure 3.16. X, Y, and Z drift study. Triple-labeled MSNs were immobilized on a

coverslip for 30 min tracking (1 frame per 15 s). (b) The corresponding root mean square

speed variations over time from (a). ... 68

Figure 3.17. The variations of fluorescence intensity ratio when triple-labeled MSNs

were excited with different laser power. The 10 nanoparticles in one image were

employed for calculation. (b) The relationship between FITC/RITC ratios and

fluorescence intensity of RITC. The 13 triple-labeled MSNs immobilized on coverslip

are tracked for a long time to examine the influence of weak RITC intensity. ... 69

Figure 3.18. Photo-stability of Triple-labeled MSNs is measured for 30 min by

Fluorescence Microscopy (1 frames per 15s). (a) The intensity variation of both I520 (FITC

and OG) and I593 (RITC) over time. (b) The corresponding intensity ratios (I520 : I593) over

time, which remains the same value in this duration. ... 69

Figure 3.19. TEM images (left) and DLS plot (right) of (a) triple-labeled MSN, (b) triple-

labeled MSN@PM PEI, and (c) triple-labeled MSN@PM PEI@L. ... 72

Figure 3.20. Zeta potential plot of triple-labeled MSN (blue line) and triple-labeled

MSN@PM PEI (red line) in water solution with different pH values. ... 73

Figure 3.21. The in-situ calibration curves of (a) triple-labeled MSN and (b) triple-

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labeled MSN@PM PEI@L after 30 minutes (red) and 12 hours (blue) of incubation with

HeLa cells. ... 73

Figure 3.22. The percent of uptake and (b) the fluorescence intensity of the nanoparticles

by flow cytometry analysis of cell-uptake. HeLa cells were incubated at 37 ℃ with

triple-labeled MSN or triple-labeled MSN@PM PEI@L for 4 h. ... 75

Figure 3.23. Co-localization images of triple-labeled MSN@PM PEI @L with (a) early

endosome marker, EEA-1, and (b) lysosomal marker, LAMP-1 antibody, at 4, 8 and 12

hours in HeLa cells. The percentages on the images, representing the overlapping ratio of

the nanoparticle with the markers, were analyzed by Image J software. ... 77

Figure 3.24. Co-localization images of early endosome marker, EEA-1, with (a) triple-

labeled MSN and (b) triple-labeled MSN@PM PEI@L at 4 and 12 hours in HeLa cells.

The percentages on the images, representing the overlapping ratio of the nanoparticle with

the two markers, were analyzed by Image J software. ... 77

Figure 3.25. Co-localization images of lysosomal marker, LAMP-1 antibody, with (a)

triple-labeled MSN and (b) triple-labeled MSN@PM PEI@L at 4 and 12 hours in HeLa

cells. The percentages on the images, representing the overlapping ratio of the

nanoparticle with the two markers, were analyzed by Image J software. ... 78

Figure 3.26. A representative case of triple-labeled MSN for staying at cytoplasm. The

trajectory of a triple-labeled MSN after 12 hours incubation with HeLa cells as a function

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of (a) time and (b) pH value. (c) The corresponding RMS speed variation over time. (d)

The corresponding pH variation over time. The images were taken at 1 frame per 5

seconds. ... 82

Figure 3.27. A representative case of triple-labeled MSN@PM PEI@L for entering late-

endosome from cytosol. The trajectory of a triple-labeled MSN@PM PEI@L after 12

hours incubation with HeLa cells as a function of (a) time and (b) pH value. (c) The

corresponding RMS speed variation over time. (d) The corresponding pH variation over

time. The images were taken at 1 frame per 5 seconds. ... 83

Figure 3.28. A representative case of triple-labeled MSN@PM PEI@L for maturing to

the lysosome. The trajectory of a triple-labeled MSN@PM PEI@L after 12 hours

incubation with HeLa cells as a function of (a) time and (b) pH value. (c) The

corresponding RMS speed variation over time. (d) The corresponding pH variation over

time. The images were taken at 1 frame per 5 seconds. ... 84

Figure 3.29. A representative case of triple-labeled MSN@PM PEI@L for staying at

lysosome. The trajectory of a triple-labeled MSN@PM PEI@L after 12 hours incubation

with HeLa cells as a function of (a) time and (b) pH value. (c) The corresponding RMS

speed variation over time. (d) The corresponding pH variation over time. The images

were taken at 1 frame per 5 seconds. ... 85

Figure 3.30. Statistic of average pH values of (a) triple-labeled MSN and (b) triple-

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labeled MSN@PM PEI@L incubated with HeLa cells for 12 hours. In the right chart, the

colors of red, green and blue separately represent the environment of lysosome (pH<5.2),

late-endosome (pH 5.2 to 6), and others (pH>6) in HeLa cells. ... 86

Figure 3.31. The percentages of the three kinds of the pH variation types of (a) triple-

labeled MSN and (b) triple-labeled MSN@PM PEI@L. The colors of red, green and

purple separately represent acidification, basification and stabilization. ... 86

Figure 3.32. Histogram of average pH values of triple-labeled MSN@PM PEI@L

classified by three kinds of pH variation types, (a) acidification, (b) basification and (c)

stabilization. ... 87

Figure 3.33. Mechanism of the lysosome targeting of the YXXФ-mediated MSNs. .... 87

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List of Tables

Table 1.1. Lysosomal Storage Diseases⁴⁶ ... 21

Table 1.2. Endocytic pathways for the internalization of MSNs.⁵⁰ ... 26

Table 1.3. Zeta-potentials and ED50 for cellular uptake of the MSNs.⁵² ... 28

Table 1.4. Examples of dileucine-based and tyrosine-based sorting signals.⁶⁰ ... 34

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Chapter 1 General Introduction

Intracellular pH homeostasis plays a pivotal role in various cellular processes such

as endocytosis¹, drug resistance², cellular metabolism, cellular signaling, and cell

proliferation and apoptosis.³ In eukaryotic cells, the subcellular organelles are highly

compartmentalized for maintaining the specific pH values or function; for example,

lysosomes responsible for digestion processes have low pH values of 4─5.5 for activating

enzymes or denature proteins.⁴ Abnormal pH values are often associated with cell

dysfunction and can be observed in some diseases including cancer⁵, Alzheimer’s

disease⁶, and others. On the other hand, the real-time pH-sensing of nanoparticles in

endosome-lysosome pathway is also highly significant to design pH-sensitive drug

delivery systems.⁷ Thus, quantification of pH values and their fluctuation measured by a

nanosensor in living cells is very potential for understanding physiological and

pathological process, and even the special carrier design.

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1.1 Measuring Intracellular pH by Fluorescence

It has been known that measurement of intracellular pH has variety of techniques

including nuclear magnetic resonance (NMR), absorbance spectroscopy, proton-

permeable microelectrodes, and fluorescence imaging.⁸ Compared to other methods,

fluorescence-based techniques provide a more powerful tool for pH-sensing in intact cells

and subcellular regions, owing to their high sensitivity, outstanding spatial and temporal

resolution, and noninvasive feature.⁴ In particular, ratiometric measurement by detecting

fluorescence signals from two (or more) excitation or emission wavelengths of a pH

sensor can overcome the influence of fluctuation in the local probe concentration,

allowing reliable and accurate pH sensing.⁹

To detect the intracellular pH, a great amount of ratiometric fluorescence-based pH

nanosensors including quantum dot¹⁰, polymetric nanoparticle¹¹, gold nanoparticle¹²,

nanogel¹³, Metal-organic framework¹⁴ and MSN¹⁵ have been reported in recent year.

Wenbin Lin et al.¹⁴ (Figure 1.1) developed a nanoscale metal-organic frameworks

(NMOFs) for ratiometric pH-sensing in living cells by covalently conjugating fluorescein

isothiocyanate (FITC) onto UiO NMOF. The pH-response of F-UiO exhibits the sensitive

dynamic range at pH = 5.0 ─ 7.0 depending on the pKa of the FITC. Gang Bao et al.¹⁰

(Figure 1.2) developed the pH nanosensor comprising a bright and photostable

semiconductor quantum dot (QD) and GEP-like FPs exhibits a Förster resonance energy

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transfer (FRET) effect and enables ratiometric measurement. The FRET pairs give

heightened sensitivity by eliciting opposing changes in fluorescence emission at two

wavelengths between pH 6 and 8.

Figure 1.1. Schematic presentation of F-UiO synthesis. (b) Correlation between FITC

absorbance and fluorescence at various FITC loadings. (c-e) pH-calibration curves of free

FITC (c) and F-UiO acquired by fluorimetry (d) and by confocal laser scanning

microscopy (e). 488/435 in the Y-axis represents I488/520/I435/520. (F) CLSM images

a)

b) c)

d) e)

f)

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showing the overlay of green (488 nm excitation) and red (435 nm excitation) colors of

F-UiO particles in HBSS buffers with different pHvalues.¹⁴

Figure 1.2. QD-FP FRET-based pH sensor. (a) Schematic demonstration of the pH-

dependent energy transfer between the quantum dot and fluorescent protein. In an acidic

environment, energy transfer to the FP FRET acceptor is minimal, yielding a high QD

signal; at neutral or basic pH, energy transfer is more efficient, producing an enhanced

FRET signal. (b) Absorbance spectroscopy indicates multiple proteins bound to each QD,

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as depicted in the inset. (c and d) Titration of QD-FP probes containg the FP acceptors

mOrange and mOrange M163K, respectively, showing increased energy transfer at

alkaline pHs with clear isosbestic points. Representative spectra of one of three

independent titrations are shown. (e) The ratio of acceptor emission to donor emission

increases with increasing pH for both probes. Data points are means ± standard deviations

for three independent titration. (f) The changes in the nanoprobe acceptor to donor ratios

are compared to the ratiometric signal change for the pH-sensitive fluorophore BCECF.

One representative titration is shown.¹⁰

However, a precise pH measurement in the endosome-lysosome region by using

ratiometric fluorescence-based pH nanosensors is often not well accomplished because

the nanosensors usually only have a narrow dynamic range.⁷ The limitation of the pH

sensing range results from the acid dissociation constant (Ka) of the pH-sensitive

fluorescence molecules, which exhibit a sigmoidal calibration curve in a pH range of the

pKa±1, as a rule of thumb, with a nonlinear relationship between fluorescence ratio and

pH. In several studies about endosome-lysosome system,¹⁶ the nanosensors with a

maximum range of two pH units often detect the pH out of the range of the sensors since

the pH variation has more than 2 pH units between early endosomes and lysosomes. The

result probably give misleading information and misinterpretation to the localization of

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the sensors in the living cell. Thus, developing a broad dynamic range of pH sensing is

very important for the research of the endosome-lysosome system. Here we focus on

developing the necessary features of the nanosensor such as good suspension, high

brightness and a broad pH-sensitive range for biomedical application with a 3D-single

particle tracing (SPT) microscopy technique.

1.2 Broadening the Sensing Range of Nano-Sized pH sensor

The pH-sensing ability of nanosensors reported in mostly former literatures is

usually limited since the pH value in some systems of living cells may falls outside the

limitation of the dynamic range and the read out nearing the limit is very reliable. For

improving the capacity of pH probe, novel designs of the nanosensors have been reported.

To break through the limitation of the dynamic measurement range, the triple-labeled

cross-linked polyacrylamide nanoparticles have been designed⁷. The triple-labeled

nanosensors were conjugated with two pH-sensitive dyes, fluorescein and Oregon Green,

and the pH-insensitive rhodamine B. The pKa values of Oregon Green (OG) and

fluorescein (FS) are 4.1 and 6.0, respectively. The mixing of the two dyes with different

pKa gives a doubling of the measurement pH range about 3.2-7.0 in comparison to

nanosensors with only one pH-sensitive fluorophore. The results are showed in Figure

1.3.⁷

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Figure 1.3. Schematic of the cross-linked poly acrylamide nanoparticle. (b) In vitro

calibration of the triple-labeled sensor with both OG and FS, and two dual-labeled sensors

with either OG or FS. (c) Uptake of the triple-labeled sensor by a HepG2 cell after 24h

and washing and imaged with confocal microscopy. Scale bar = 10µm. OG = Oregon

Green; FS = fluorescein.⁷

1.3 Fluorescence Microscopy with 3D-Single Particle Tracking Technique

Fluorescence microscopy is a powerful and common tool to collect cellular

information, such as the intracellular pH values, which is difficult to be detected using

conventional approaches. For continuous intracellular pH monitoring, three dimensional

(3D) single-particle tracking (SPT) spectroscopy is needed. 3D-SPT spectroscopy can

record the motion of individual particles in a complex system.¹⁷ It has been applied for

visualization of membrane dynamics,¹⁸ following protein traffic in living cells,¹⁹

evaluating the local viscosity conditions within the cells,²⁰ and early stage of cellular

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uptake of nanoparticles.²¹ Two common 3D-SPT methods are categorized into active

(confocal feedback-based methods)^{19-20, 22} or passive (wide field imaging based

techniques)²³ tracking techniques. The active method is a sophisticated technique

equipped with active feedback in x-y-z directions, and it requires higher cost and can only

focus on a single target at one time (Figure 1.4).^22c In contrast, the passive technique

allows multiple single probes be detected simultaneously unless the emitters diffuse out

of the stationary detection region, which is about 1 µm in the z-axis around the focal plane

(Figure 1.5).^23d Considering the potential of widespread applications, the passive 3D-SPT

technique is chosen to perform the 3D tracking of a nanoparticle, and the pH sensing.

In this work, the nanoparticles equipped with ratiometric pH sensing and imaging

ability is prepared by co-conjugating with the pH sensitive dyes, fluorescein

isothiocyanate (FITC) and Oregon Green® 488 (OG), and the reference dye, rhodamine

B isothiocyanate (RITC), into the MSNs. The 3D-SPT microscope combined with the

pH-sensing MSNs would be a powerful technique to the biomedical application. Here we

report the simultaneous real-time detection of the 3D trajectory of the pH sensors and the

local pH values in HeLa cells is a novel method for the evaluation of the traffic pathways

of the nanoparticles with the special surface modification. The transportation of the pH

sensors between endocytosis and cytoplasm has the significant pH variation, which is a

crucial information for the design of the particular drug delivery system.

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Figure 1.4. Schematic of the confocal 3-D tracking microscope optical configuration.

The fiber optic pairs are configured to form a 3-D tetrahedron-like detection volume in

sample space.^22c

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Figure 1.5. The scheme of 3D STORM. (A) Three-dimensional localization of individual

fluorophores. The simplified optical diagram illustrates the principle of determining the

z coordinate of a fluorescent object from the ellipticity of its image by introducing a

cylindrical lens into the imaging path. The right panel shows images of a fluorophore at

various z positions. EMCCD, electron-multiplying charge-coupled device. (B)

Calibration curve of image widths Wx and Wy as a function of z obtained from single

Alexa 647 molecules. Each data point represents the average value obtained from six

molecules. The data were fit to a defocusing function (red curve).^23d

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1.4 Introduction to Mesoporous Silica Nanoparticles (MSNs)

Based on the combination of sol-gel chemistry and liquid-crystalline templating

technique, mesoporous silica was discovered and named as MCM-41 (Mobil Crystalline

Materials number 41) by researchers at Mobil Oil Corporation in the early 1990s.²⁴ Since

then, a massive study has been devoted to the synthesis of mesoporous silica nanoparticles

with multiple features and functions.²⁵ The multi-functional mesoporous silica materials

with controlled morphologies,²⁶ particle size,²⁷ and excellent thermal stability²⁸ have

received tremendous attention because of their unique physicochemical properties such

as large surface area, uniform pore size, available for further modification of diverse

functional groups, and good biocompatibility.²⁹ In the past few decades, the more

mesoporous silica materials (M41S family) were fabricated based on the concept of the

formation of MCM-41, and the discovery of MCM-41 is believed to the starting point of

ordered-mesoporous-material research. The members of M41S include MCM-41

(hexagonal phase), MCM-48 (cubic phase), and MCM-50 (lamellar phase)³⁰. The

structures are shown in Figure1.6.

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Figure 1.6. Structures of mesoporous M41S materials: (a) MCM-41 (2D hexagonal,

space group p6mm), (b) MCM-48 (cubic, space group Ia3d), and (c) MCM-50 (lamellar,

space group p2).³⁰

1.4.1 Synthesis Mechanism of MSNs

In order to explain the construction of MCM-41, the‘liquid-crystal templating’

(LCT) mechanism based on the similarity between the mesostructure of lyotropic liquid

crystals from surfactant assemblies and the structures of M41S materials was suggested

by Beck^{24, 31}. There are two main general pathways in this mechanism, in which either (i)

the liquid-crystal phase pre-exists before the silicate species are added or (ii) the addition

of the silicate anions promotes the long-range ordering of the surfactant to form the

hexagonal arrangement (Figure 1.7).³⁰

The synthesis of mono-disperse MSNs is based on the method which was developed

by Mou and co-workers with the separation of nuclei formation and particle growth into

two steps in a dilute alkaline solution.³² The method can be realized in a LaMer diagram

of concentration variation during nucleation and growth. In the first step (step (A), Figure

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1.8), the full amount of surfactant (CTAB) and a small amount of TEOS are mixed to

create a clear solution of micelle/silicate clusters containing nuclei. Then, a larger amount

of TEOS is added to start the growth process without further nucleation (step (B), Figure

1.8). Eventually, with the accelerating of the growth process, the materials are exhausted

and a uniform finite size is formed.³³

In addition, the size of the MSNs can be modulated by controlling the pH (10.86 ─

11.52) of the reaction solution (Figure 1.9).³⁴ A lower pH provided a smaller size of MSN

due to the increased condensation rate and thus more nuclei (Figure 2.0).³⁴

Figure 1.7. Formation of mesoporous materials by structure-directing agent: (a) true

liquid-crystal template mechanism, (b) cooperative liquid-crystal template mechanism.³⁰

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Figure 1.8. Schematic illustration for the synthesis and selective functionalization of

MSNs.³³

Figure 1.9. TEM images of mesoporous silica with different average sizes: (a) 280 nm;

inset: FFT analysis of the TEM image; (b) 170, (c) 110, (d) 50, (e) 30 nm. (f) High-

resolution TEM image of a single particle in (c).³⁴

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Figure 1.10. Effect of pH value on the silica condensation rate, charge properties and

charge density on the surface of the silica species.³⁴

1.4.2 Applications of MSNs

The MSNs have been utilized to many application like drug/enzyme delivery, ^{25b, 35}

imaging^{32, 36} and pH sensing.¹⁵ Generally, chemical functionalized MSNs were

synthesized by one-step or two-step method: (i) co-condensation reaction and (ii) post-

synthetic grafting process, both of which are schemed in Figure 1.11.³⁷ In co-

condensation reaction, the functional silane is mixed with silicate precursor and added to

the surfactant-contained basic aqueous solution. As the result the functional silane will be

either confined in the structure or grafted on the surface of MSNs. On the other hand in

post-synthetic grafting method, the functional silane is added after the surfactant of the

as-synthesized MSN is removed. Due to the densely distributed silanol groups on the

surface of the MSNs, the functional silane or other chemicals can be easily modified on

the wall of channels and/or the external surface.

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Figure 1.11. Functionalization of MSNs by co-condensation (top) or the post-synthetic

method (bottom). A trialkoxysilane molecule bearing a functional group (green) is shown

as an example of a silica precursor. The structure-directing agent is represented by

micelles (red).³⁷

The ability to trap and release molecules from MSNs gives the promise in sensor

and drug-delivery applications. Hence various stimuli-responsive controlled release

systems which are able to respond to environmental changes, such as pH value, redox

potential, temperature, photo-irradiation, and biomolecules have been achieved.³⁸ For

example, Feng et al. has introduced a responsive polymer-coated mesoporous silica as a

pH-sensitive nanocarrier, in which a pH-responsive polymer poly(4-vinyl pyridine) is

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coated on mesoporous silica through the facile post-synthetic grafting method. The

grafted polymer nanoshell can serve as a pH-sensitive valve to control the release of

trapped molecules from mesoporous silica.³⁹ The nanoscale MSNs are suitable for the

cellular uptake and thus the MSNs conjugated with fluorescent molecules can be utilized

as the cell markers.³² Moreover, a fluorescent dye-loaded MSNs can also be used to

measure the local pH value in living cells.⁴⁰

1.5 Ratiometric pH Sensor based on Mesoporous Silica Nanoparticles

It has been known the ratiometric pH sensing based on the fluorescent molecules is

a powerful tool in recent years. Moreover, The sensors with the manner of Förster

resonance energy transfer (FRET) are more potential because they have following unique

advantages: (i) they can render dual or multiple emissions under a single-wavelength

excitation, providing more convenient and effective ratiometric detection superior to

those that require two excitation wavelengths and (ii) they give great flexibility for sensor

design by changing the FRET components.¹⁵ However, the construction of ratiometric pH

sensors with the application of FRET is still infancy despite the pH monitoring has a great

demand in many fields like medicine, biology and the environment. In the past study,

there are some pH sensors which use FRET between quantum dots (QDs) and analyte

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responsive dye have been reported.^{10, 41} However, the cytotoxicity of ODs and difficulties

in acquiring modified ODs limit their applications.¹⁵

MSNs hold great promise as a platform for the design of a FRET-based ratiometric

pH sensors due to their high stability, large surface areas, tunable pore sizes, abundant

surface functionalization sites and biocompatibility.⁴² In this study, the MSNs conjugated

with two pH-sensitive dyes, FITC and OG, and one referent dye, RITC, were prepared

by the co-condensation reaction at the ammonia-catalyzed condition. The FITC and OG

on pH sensors can be excited by a single-wavelength (473 nm), and RITC can receive the

energy by the FRET effect. Thus, simultaneously detecting the emissions from the three

fluorescent molecules can be achieved. In addition, the dynamic range of the pH sensing

almost covers the entire physiological environment from pH 4.3 to pH 7.4 by utilizing the

different pKa of FITC and OG. On the other hand, the MSNs modified with PEG have

excellent suspension in medium (DMEM+FBS+P/S) for appropriate biomedical

applications. Therefore, the dyes-conjugated MSNs would be a potential sensors for pH

sensing in living cells.

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1.6 Lysosome-Targeting Nanocarriers for Lysosomal Storage Disorder Therapy

1.6.1 Introduction to LSD

Lysosomal storage diseases (LSD) comprise about 50 diseases in which the

deficiency of a single lysosomal enzyme results in the progressive accumulation of

undegraded substrates in the lysosomes in cells of most tissues.⁴³ Lysosomal dysfunction

has also been indicated as a significant risk factor for a variety of other disease,⁴⁴

especially neurodegenerative diseases.⁴⁵ The pathology related to substrate accumulation

can vary remarkably depending on the specific storage material, where the materials

accumulate, as well as the degree to which the activity of enzymes is compromised.⁴⁶ The

enzyme activity losing completely or near completely would result in the most severe

pathology, often at the early stage and death in the first few years of life. On the other

hand, the residual activity of some lysosomal enzymes would lead to pathology that is

often not diagnosed until adolescence or later. Although each LSD is individually

somewhat rare, as a group they have an incidence of about 1 per 7,000 to 8,000 live births,

with regional and genetic population variation.⁴⁷ Some of the most common LSDs, the

affected enzymes, and the accumulated storage products are listed in Table 1.1.⁴⁶

In the United States, the term “enzyme replacement therapy” (ERT) refer to a

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group of nine commercially available glycoprotein products, each intended to enhance or

replace the activity of a specific endogenous catabolic enzyme within cellular

lysosomes.⁴⁷ This strategy works is that lysosomal enzymes contain oligosaccharide

residue such as mannose 6-phosphate (M6P) which can bind to cellular receptors like

M6P receptors (M6PR), resulting in internalization of the enzymes by clathrin-mediated

endocytosis and subsequently trafficking to lysosomes.⁴⁸ However, poor access of

recombinant enzymes to these tissues, the altered M6PR expression in LSDs, and immune

reaction against the injected enzyme reduce the therapy effect.⁴⁹ Thus, these factor

emphasizes the need for alternative therapeutic options.

A strategy that enhances delivery of lysosomal enzymes is that of loading the

enzymes to nanocarriers which have ability to efficiently target the specific site and

prevent from the attack of the immune system. In order to achieve the purpose, a lysosome

targeting nanoccarier is essential. The MSN is a suitable material to be utilized for

designing the lysosome targeting nanoccariers because of their large surface areas,

mesoporous properties, and simply being modified.

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Table 1.1. Lysosomal storage diseases⁴⁶

1.6.2 The Trafficking of MSNs in Living Cells

The cell membranes are usually the most important barrier for intracellular drug

delivery. Clear understanding of the pathways for the cellular internalization of MSNs is

a significant for many of its applications in biomedicine and biotechnology. There are

various pathways for internalization of external materials in mammalian cells. In general,

these mechanisms can be divided in two categories: pinocytosis and phagocytosis (Figure

1.12).⁵⁰ The cells can utilize any of these internalization process depending on the size of the particles. The cellular uptake of small particles (< 200 ─ 300nm) such as MSNs is

usually involved in endocytosis for the majority of cases.⁵¹ The mechanisms of

endocytosis that transport the MSNs into the cells include clathrin-dependent, caveolin-

dependent, receptor-mediated, and clathrin- and caveolin-independent processes (Figure

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1.12).⁵⁰ So far there is no specific endocytic pathways that either MSNs or functionalized

MSNs materials will follow when they are internalized by cells. Hence thorough

understanding and controlling the internalization pathway is of extreme importance in the

development of drug delivery platforms. The endocytic pathways of MSNs with different

functional groups have been studied (Table 1.2).⁵⁰ In addition to the different

functionalization, the MSNs with various surface charge, shape or size also have effect

on the cellular uptake.⁵⁰

After the MSNs overcome the cell membrane barrier and enter cells by endocytic

pathways, the series of events can be divided into the following sequence: the MSNs are

first transported to primary endosomes followed by transport to sorting endosomes. And

then, a fraction of MSNs are directed back to the cell exterior through recycling

endosomes, while the remaining fraction is transported to secondary endosomes that fuse

with lysosomes. And then the MSNs escape from the endolysosomes into the cytosolic

compartment (Figure 1.13).⁵⁰ On the other hand, MSNs with different surface properties

could quickly escape the early-endosomes before they reach the lysosomes. For example,

the effect of the surface charge of MSNs were studied by S.-Y. Lin et al..⁵² The FITC-

MSNs with the diameter of 150 nm separately modified with different functional groups

(AP-, GP-, GEGP-, and FAP-) have the different surface charge and 50% effective dose

(ED50) as shown in Table 1.3.⁵² The results exhibit the more negatively charged FITC-

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and AP-MSNs appear to be able to escape from endosomes within 6 hours, while those

with more positive surface charge, such as GP-, GEGP- and FAP-MSNs, remained

trapped within endosomes (Figure 1.14)⁵². This behavior demonstrate the more negatively

charged materials have the better Proton Sponge effect or buffering capacity which is

important for the endosome escape.⁵³ The proton sponge effect implies that the weak acid

or basic compounds such as polyethyleneimine (PEI) buffers the protons being pumped

into the lysosomal compartment by the v-ATPase (proton pump). This results in

heightened pump activity, leading to the accumulation of a Cl^─ and a water molecule for

each proton that is retained; ultimately, this leads to osmotic rupture of the endosome.⁵⁴

The negatively charged MSNs by surface attachment of phosphonate groups (MSN-PP)

and positively charged MSNs by conjugating quaternary ammonium groups (MSN-TA)

to FITC&RITC@MSN were also researched by C.-Y. Mou et al.⁴⁰ The results suggest the

presence of positively charged MSN-TA in the cytoplasm, but negatively charged MSN-

PP is probably trapped in the endosome/lysosome analyzed by the pH detection (Figure

1.15) and confocal images (Figure 1.16).⁴⁰ The MSNs coated with polyethyleneimine

(PEI) for the delivery of siRNA and DNA construct is also reported by Andre E. Nel et

al.⁵⁴ PEI (10k)-coated MSN (100-130 nm in diameter) is a versatile delivery system that

can facilitate cellular uptake to increase drug delivery payload and also be utilized to

improve nucleic acids delivery into cytoplasm with the proton sponge effect. David

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Oupicky et al. reported the polycation- (PDMAEMA or PDEAEMA) and PEG-coated

mesoporous silica nanoparticles were able to successfully deliver plasmid DNA and

siRNA in cell culture.⁵⁵ Shi Zhang Qiao et al. also reported that the poly-L-lysine-

functionalized large-pore MSNs with cubic mesostructured have a true potential for

delivery of nucleic acids into HeLa cells for gene therapy applications.⁵⁶ The effect of

different sizes and surface charge of the naked MSNs on the cellular trafficking were also

studied by Ciro Isidoro et al.⁵⁷ They found the 10 nm naked MSNs can quickly

accumulate in lysosome in 5 minutes with the almost 98% co-localization, while more

90% of MSNs escaped the lysosomes at 30 minutes (Figure 1.17)⁵⁷. Moreover, the

increasing size from 10 nm to 50 nm reduces the uptake efficiency of MSNs. The

negatively charged groups (-COOH) impair endocytosis of MSNs regardless of the

presence or absence of caveolin-1 on the plasma membrane, and the 50 nm MSNs

functionalized or not with either COOH or NH2 groups would all enter and reside

permanently in lysosomes (Figure 1.18).⁵⁷ Shoufa Han et al. reported the internalized

R6G-FITC-MSNs with post modification of PEG (110 nm in diameter, surface charge : -

6.76 mV) are site-specifically delivered into lysosomes (Figure 1.19).⁵⁸ The effect of

spherical mesoporous silica (MS) nano- and microparticles with the treatment of

extraction (E-MS) or calcination (C-MS) on the intracellular localization was also

investigated by Yaping Li et al.⁵⁹ The results qualitatively indicate the intracellular

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distribution of RhB-labeled MS-1, 2, and 3 particles (190 nm, 420 nm, and 1220 nm) in

lysosomes, but there is a very limited number of the E-MS-3 can enter the MDA-MB-468

cells and locate in lysosomes, compared to the E-MS-1 and E-MS-2 with a much larger

number in lysosomes. However, the behavior of the C-MS is largely different from that

of the E-MS. Only a limited number of C-MS particles can go into cells and accumulate

in lysosomes, even at the particle size of 190 nm (Figure 1.20).⁵⁹ From the above research,

we found the MSNs reaching the lysosomes involve the various factors such as size, shape,

surface charge, surface functionalization, and even the method of surfactant removing.

Thus, designing a lysosome-targeting MSNs by adjusting these elements is very

ineffective. In this study, we would modify the MSNs with a lysosome targeting peptide.

If the peptide could successfully deliver MSNs into the lysosomes in living cells by a

biological mechanism, that would be a more convenient and effective manner for

targeting lysosomes of nanoparticles.

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Figure 1.12. Pathways of mesoporous silica nanoparticles for entry into cells. Large

particles are internalized by phagocytosis, whereas fluid uptake occurs by

micropinocytosis. In the case of MSN materials, most internalization is via endocytic

pathways. These pathways differ with regard to the nature of the surface functionalization

and structural properties of MSNs.⁵⁰

Table 1.2. Endocytic pathways for the internalization of MSNs.⁵⁰

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