Photodynamic therapy (PDT) is a minimally invasive treatment modality for some human diseases, including cancer. To destroy the targeted cells or tissues, PDT relies on the reactive oxygen species (ROS) generated from a series of photochemical reactions by the light-activated photosensitizers administered to the patients. Many dyes could be modified to become photosensitizers. The BODIPY-based fluorophores could be converted into potent photosensitizers with highly efficient singlet oxygen generation as well as considerable brightness of fluorescence. Some of them exhibit potent in vitro PDT effects. However, carriers are often required for an effective delivery of the BODIPY-based dyes in biological system. / Silica nanoparticles are ceramic-based materials prepared by condensation of silanes along the surfactant-based templating agents. Mesoporous silica nanoparticles (MSNs) and organically modified silica nanoparticles (OMSNs) represent two major types of silica nanocarriers used for loading of photosensitizers in PDT. Typical MSNs have a diameter of 100-500 nm and a highly ordered hexagonal porous structure for loading of guest molecules. The OMSNs are smaller in size (diameter ~20 nm). Both MSNs and OMSNs are known to be chemically inert and biocompatible. Therefore, they were selected as the carriers for BODIPY-based photosensitizers in the present study. / A BODIPY-based photosensitizer with an absorption maximum at the red region (~660 nm) was modified to carry a carboxyl group at the meso-position. This photosensitizer was conjugated to an amine-functionalized MSN of diameter 80-120 nm by a post-synthesis grafting approach. This strategy allowed the entrapment of delicate dyes by the MSN under mild reaction conditions. The resulting composites with different photosensitizer loading were all spherical and with diameter from 80-120 nm. Dispersing the composites in H₂O, the fluorescence emission was moderately quenched due to dye aggregation. However, compared with the surfactant solubilized free dye, the MSN conjugated BODIPY produced singlet oxygen more efficiently. The dye loading per the unit mass of MSN did not impose any significant effect on the photophysical properties of the composites. The in vitro PDT effects of the BODIPY-based dye entrapped in the MSN were evaluated by using the human adenocarcinoma cells HT-29. The dyes loaded in the MSNs were more cytotoxic than the free dye, but slightly less cytotoxic when compared with the surfactant-formulated dye. The rate of intracellular ROS production of the MSN entrapped dye was much higher than that of the free dye with or without surfactant, which was consistent with the results obtained in the studies of the photophysical properties. The dye in MSN was more efficient as an inducer of apoptosis than the dye in surfactant, as shown by the annexin V/PI staining. Subcellular localization studies using confocal microscopy revealed that the dye in MSN was mainly found in endoplasmic reticulum and lysosome of the cells. / This amine-functionalized MSN platform was further modified on the surface. On a batch of amine-bearing MSN (~200 nm in diameter) loaded with the BODIPY-based photosensitizer, a layer of polyethylene glycol (PEG) was grafted. The PEG layer was comprised of short PEG chains (~15 repeating units) with a methyl end and another long PEG chains (~44 repeating units) with an azide end. The alkyne-modified tLyP-1 peptide targets the cancer cell and blood vessel surface marker, neuropilin, was conjugated to the MSN via azide-alkyne Huisgen cycloaddition (click reaction). Unlike many other reported designs of photosensitizer-MSN composites utilizing only amine functionalization, this approach prevented the competition for conjugation site between the photosensitizer and the targeting ligand. The use of click reaction allowed a greater feasibility in targeting ligand design. In H₂O, the surface decorated composites could still generate singlet oxygen as effectively as the bare composite. The in vitro PDT effects toward human prostate adenocarcinoma cells PC-3 with a high level of neuropilin expression of the composites were evaluated. The peptide-bearing MSN had a faster initial uptake in cells, which could be moderately suppressed by the addition of free peptide. The peptide-linked MSN had a higher photocytotoxicity toward the PC-3 cells when compared with the MSN without the peptide due to the enhanced cellular uptake. Both composites were confined to the lysosome of the cells, which might be the consequence of lacking surface positive charge to help endosomal escape. Therefore, further optimization of the composite by adjusting the PEG loading, chain length and the amount of targeting ligand loading should be made. / OMSNs are homogenous spherical particles with a smaller size (~20 nm). The capability of OMSNs to be multi-functional dye nanocarriers was also explored. Besides a BODIPY-based photosensitizer, a phthalocyanine-based photosensitizer and an aza-BODIPY-based imaging dye were also chosen for OMSN entrapment. It was found that only those surfactant-soluble dyes could be successfully entrapped in the OMSNs. Dyes in OMSNs remained non-aggregated, thus emitting a bright fluorescence. The photosensitizers generated singlet oxygen with the same efficiency as the surfactant-formulated free dyes. The potency of the OMSN-entrapped photosensitizers toward the HeLa derived KB cells was similar to that of the surfactant-solubilized free dyes. OMSNs are hence alternative carrier to the surfactant-based emulsifiers for in vitro photosensitizer delivery. To decorate the surface of OMSN, folate was conjugated to the composite by one-pot approach. However, the resulting composite failed to exhibit any tumor targeting effect toward KB cells having a high level of folate receptor expression. Besides, in order to prevent premature dye leakage in culture media as a result of the interaction with serum proteins, an attempt was also made to prepare OMSNs with a covalently linked BODIPY-based dye. However, the conjugated dye was aggregated, which diminished the singlet oxygen generation and quenched the fluorescence of the composites, although the surface of this composite was successfully decorated with PEG or the azide-bearing silane. / In conclusion, the BODIPY-based photosensitizers could be entrapped in MSNs and OMSNs could be successfully delivered into cancer cells in vitro. The PDT effects induced by these composites were often comparable to those caused by the the surfactant-solubilized dyes. Although surface decorations could be made for the particles, further fine adjustment on the surface properties of the composites is needed to improve the specificity and potency of the composites in the future. / 光動力治療(PDT)可用作治療一些人類疾病如癌症。這種低創傷度治療模式的主要原理是先把光敏劑注入病人體內，然後以光照射患處，激活當中的光敏劑。經過一系列的光化學反應後，病患組織附近的氧分子會被光敏劑轉化成為活性氧物種，從而破壞病變的組織或細胞。在眾多可改造成光敏劑的染色劑中，氟硼二吡咯(BODIPY)熒光團可以轉成兼具高活性氧轉化率及強熒光的光敏劑。研究顯示，部份BODIPY衍生的光敏劑在細胞實驗中有強大的PDT效果。可是，這些光敏劑往往需要由載體輔助才可以送達目標組織或細胞。 / 二氧化矽納米粒子是由硅烷在介面活性劑模板上聚合而成的陶瓷基類納米粒子，其中介孔二氧化矽納米粒子(MSN)和有機改造二氧化矽納米粒子(OMSN)為兩種最常用的光敏劑載體。MSN的直徑通常介乎100至500納米，並有高度整齊排列的六角介孔以供承載客分子。OMSN則比較細小，直徑約20納米。MSN和OMSN皆為化學惰性以及生物相容的物料，所以在本研究中它們被選為BODIPY類光敏劑的載體。 / 在一個BODIPY光敏劑（它的吸收峰位於紅光，波長約660納米範圍內）上，羧基被加到中央位置上。這個帶羧基的光敏劑可以嫁接到己成形的帶胺功能團MSN上。由於後合成嫁接法可以於溫和的條件下進行，相信其亦可應用其他脆弱的染色劑及帶胺MSN的連結上。即使有不同光敏劑載入量，同一系列的合成物的直徑皆是80至120納米。當這些合成物分散在水中，MSN內光敏劑會因聚合作用，其熒光強度會明顯減弱。可是，比起沒有載體或以介面活性劑配方的光敏劑，包裹於MSN內的光敏劑能更有效的產生活性氧。在光物理的測試中，光敏劑的承載量對MSN化合物的光物理性質俱沒有明顯的影響。 / 對人工培植的人類大腸腺癌細胞HT-29而言，承載於MSN內的光敏劑的比沒有載體的具光毒性。可是對比起以介面活性劑配方的BODIPY，承載於MSN內的光敏劑還是光毒性稍弱。另外，承載於MSN內的光敏劑在細胞內產生活性氧的效率，遠高於介面活性劑配方內、或沒有載體的光敏劑，這項發現跟光物理的檢測結果類近。另外，annexin v/PI染色實驗結果顯示，MSN承載的光敏劑能更有效的引發細胞淍亡。共焦顯微鏡進行的細胞內定位法顯示，MSN承載的光敏劑主要分佈在內質網和溶酶體之中。 / 這類帶胺官能團的MSN的粒子表面可作進一步修飾。本研究嘗試用聚乙二醇層修飾一種直徑約200納米、以及載有BODIPY光敏劑的MSN。該聚乙二醇層由帶甲基端的短鏈(約15個重複單位)和帶疊氮端的長鏈(約44個重複單位)PEG組成。然後，以疊氮──炔烴Huisgen環加成法（點擊反應）將帶炔烴的tLyP-1短肽連接到PEG上。該tLYP-1短肽對在一些癌細胞和癌組織血管上常見的neuropilin有高度結合親和力，故可作靶向分子使用。另外，本研究裏採用的嫁接方法跟現時常用於只有胺官能團的MSN處理法不同：光敏劑和靶向份子是分別連接於MSN的胺和疊氮官能團上，故能避免光敏劑與靶向分子競爭。同時因為使用簡易的點擊化學反應，靶向分子的設計可以有更多選擇。 / 在水中，有和沒有表面修飾的MSN同樣有效地產生活性氧。對人工培植、並在細胞表面高度表達neuropilin的人類前列腺癌細胞PC-3而言，帶tLyP-1的MSN合成物有更高光毒性，其中的原因包括帶tLyP-1的MSN有更快的攝入初速。另外，自由的tLyP-1短肽可稍為抑制這種MSN合成物的細胞吸收，亦可見tLyP-1足以影響MSN的細胞攝入。可是，不論有沒有帶短肽，兩種以PEG修飾的MSN皆是因為沒有表面電荷而無法逃離細胞的溶酶體，以致其PDT效果改善幅度有限。所以在往後的研究，PEG的覆蓋量、長度和靶向分子的接連率需作進一步探討。 / OMSN是直徑約20納米均勻的圓形粒子。本研究亦會探討如何可以用OMSN作為多功能的染色劑載體。除了BODIPY光敏劑之外，另一個鋅酞菁和一個氮雜氟硼二吡咯的熒光顯影劑亦包裹在OMSN中。結果顯示，只有可以用介面活性劑溶解的染色劑才可以成功包進OMSN之內。OMSN內的染色劑基本上沒有聚合現象，故能發出強熒光，而包覆在光敏劑之內的光敏劑轉化氧作活性氧的效率跟介面活性劑配方的光敏劑一樣。 / 不論是鋅酞菁或BODIPY，包裹在OMSN內的光敏劑對在人工培植、及源自HeLa的KB癌細胞的PDT效果跟介面活性劑配方的光敏劑相若。因此，OMSN可替代活性介面劑作為光敏劑的載體。本研究亦嘗試在OMSN的表面上以葉酸作修飾。可是，即使用了表面上有大量的葉酸受體的KB細胞，葉酸修飾過的OMSN並沒有靶向性。另外，為了解決OMSN內吸附的染色劑會因粒子與血清蛋白的互動而漏出粒子外的問題，本研究亦嘗試將OMSN與BODIPY衍生的光敏劑以共價鍵連結。同時其表面亦以帶疊氮聚乙二醇層作修飾。可是，BODIPY光敏劑在以這些OMSN合成物內有嚴重的聚合現象，以致於活性氧產生和熒光亮度大幅度減弱。 / 總括來說，本研究內設計的BODIPY衍生的光敏劑皆可以用MSN或OMSN來承載，並成功送達至人工培植的癌細胞內。這些化合物的PDT效果往往媲美那些以介面活性劑配方的同類光敏劑。雖然證明了這些粒子表面可以予以修飾，其表面修飾層仍需要進一步的改善，以增強它們的靶向專一度和PDT功效。 / Yeung, Sin Lui. / Thesis Ph.D. Chinese University of Hong Kong 2015. / Includes bibliographical references (leaves 148-159). / Abstracts also in Chinese. / Title from PDF title page (viewed on 24, October, 2016). / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only.
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_1291550 |
| Date | January 2015 |
| Contributors | Yeung, Sin Lui (author.), Ng, Dennis K. P. (thesis advisor.), Chinese University of Hong Kong Graduate School. Division of Chemistry. (degree granting institution.) |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography, text |
| Format | electronic resource, electronic resource, remote, 1 online resource (6 unnumbered leaves, xiii, 173 leaves) : illustrations (some color), computer, online resource |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons "Attribution-NonCommercial-NoDerivatives 4.0 International" License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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