200843801 九、發明說明： 【發明所屬之技術領域】 奈米科技將來能被應用於各種日常生活產品當中，也包括醫華景<像未來之 應用。相較於傳統染色標定技術，使用奈米粒子作為影像對比劑最主要的 優點是能夠避免光漂白，而且奈米粒子是很好的醫藥攜帶物，因此將其表 面做適當的修飾，奈米粒子即可以用來標定特定的生物分子或病灶，因此 將來在分子醫藥上的應用頗具潛力。 【先前技術】 奈米粒子在影像上的應用 在光學應用部分，傳統上使用的螢光分子包括螢光有機分子、螢光蛋 白等，有機分子的好處是小，容易修飾，且在長期發展後，已產生許多光 度強且放光穩定之分子，大多是應用於抗體上；螢光蛋白則需大量的基因 工程操作，但可以利於蛋白質交互作用等研究之觀察。近來來則發現許多 奈米粒子也具有特殊的光學特性，如量子點，可以依據材質及大小不同產 生不同波段之螢光，分子吸收係數高，放射光峰寬窄，故可以達到多色染 色的目的，且光漂白(photobleaching)現象較傳統物質不明顯許多，可用於長 時間觀察’也成為新一代螢光標的物質的寵兒。金或銀奈米粒子則可利用 表面電漿共振做檢測，也有少數報告是利用其螢光。 在核磁共震造影技術中，奈米粒子依據其T1遲缓與T2遲緩的不同， 而有不同的顧，其中，氧化鐵的Τ2遲緩短，故在Τ2遲緩的測量上，可 6 200843801 '產生;Μ的！^。另外’微脂體類之生物性奈米粒子則在超音波影像上 有極佳的應用。 C不米粒子在生物體内的毒性問題也是目前研究的重點，因為奈米化的 結果’常常使得材料的物性雜都改變，縣不活化的金屬會變得相當活 潑以至於和活體内的生物分子交互作用，造成相當大的毒性反性。因此 經過脂質表面修飾可崎低非特異性的結合和不必要的毒性。 倍頻技術的發展 利用螢光標的技術來了解生物體微觀或巨觀世界的交互作用已被廣泛 應用在各個生物學的領域上，因此，光學、螢光或共補、顯微鏡的存在已 經是生物實驗上不可躲峨測王具，然而，#光標的卻有幾個致命的缺 點’一者，螢光物質-旦受到激發，釋出能量的同時，便可能對樣品本身 產生傷害，二者，受到強光照射致使分子的共輛長度改變，導致光漂白現 象，更是讓研究人員不勝其擾。然而，倍頻技術卻不牵涉螢光，且能量不 累積，樣品衫傷害，故成為近辭來全世界備受注目_穎光學技術， 其中，二倍頻的訊號來自整齊排列的奈米級晶體結構，而三倍頻則是來自 不連續介面。 脂質在倍頻技術偵測得到的訊號 脂肪球在懸浮於水溶液時便可產生油水介面，故可產生三倍頻气號， 但光學訊號受顆粒大小影響，唯有大於400奈米之脂肪球可以產生可辨之 訊號，大於1微米之脂肪球的訊號強度才利於分析，而小於4〇〇奈米者， 200843801 訊號微弱，故此法只能應用於脂肪細胞或產生大量脂肪之組織檢測。 金屬在倍頻技術下的訊號 目前已知有數種金屬或合金可以產生三倍頻訊號，例如金、銀、鐵金 合金等’然而’峨不強。_般來說，金、銀等金屬本身具有表面共振的 效應，可以被雷射激發後產生二倍頻或三倍頻的訊號，但是訊號強度不高， 需要相當靈敏❸紀錄器才能擷取正確的訊號。相制，脂質是一種排列規 則的生物聚合物，經雷射絲激發後，树也能產生三倍頻軌號，但同 樣的，它的訊號相當弱，且訊噪比不佳，即使利用優良的紀錄器紀錄，其 影像也相當模糊。本發明所揭示的脂f奈米粒子，所包覆的奈米顆粒材料， 不官是金屬或非金屬，皆能產生大於上述個別單體訊號十至一百倍的訊號 強㈣如本發明之針、針―），其影像之清_度，使崎奈米粒讀 成即時影像的顯影對比劑。 【發明内容】 本發明所揭補奈米生物技術，主要是糊直徑小麵百奈米之金屬 =粒或非金屬辭導體量子點等，經職質包覆後所喊的穩定且無生物 母性的脂質奈米粒子(lipidnan〇particles)，再藉由表面適當的修飾，編 所攜帶的醫藥分子有效率的遞送到生物活體細胞表面或體内，並且可以同 树為夕重功麟像(multim〇dality i腿忌㈣之影像對比劑，來同步觀察 藥物即時_的機制，或了解疾病成因的分子基礎。 200843801 本技術結合脂質與金屬兩者之好處’構築金屬或非金屬脂質奈米粒 子、果S現在5G奈米以上之奈米球’即可產生_可辨識之三倍頻訊號， 100奈米之奈米粒子訊號更強，奈米以上者，強度雖未明顯增強但卻 可明雜麵難大小_化，並在_峨察分子騎祕反應，故可 確認此種奈树可作為三倍雜狀體外(in叫或涵(in V⑽之顯影劑 (contrast agent)。 不米粒子在生物體的應用上已有多年歷史，可同時應用於藥物攜帶， 或基因遞运。但是奈米粒子過大(>彻nm)也會讓㈣體認出，造成生體内 的網狀内皮系統清除後，短期内會堆積在肝臟、腎臟等處，造成非特異之 母I4生’或產生背景雜訊會造成影像判讀上的珊。由於生物體内本來就存 在脂肪，故毒性小，生物適性高，用於奈米粒子的包裹，可減少奈米粒子 的毒性。利用正價脂質和PEG修飾之脂質自組裝(self_assembled)的原理， 調整二者之比例(如表一、表二），或再加上中性脂質(如表三），並經超音波 震盪’可有效控制奈米粒子的大小，增加粒子本身的遞送效率，減少非特 異性的毒性，同時達到基因遞送的目的。經由簡易的表面修飾(如圖八、圖 九）也可達到專-性遞送。在遞送生醫藥物的過程中，可利用倍頻技術檢 測並分析。或者在標定蛋白，長時間觀測蛋白f的分子義及運動情 形。細胞標定後可以進行高解度的生物體切片分析，並由於倍頻技術不會 對樣本構成傷害’可糊此技術進行幹細胞分化過程之應用分析。 200843801 表一： 不同配方合成之脂質奈米粒子大小 脂質成分 粒徑（nm) DOTAP PEG-DSPE w/o Fe3〇4 with Fe3〇4 75 25 28·5 ± 1·6 30_2 ± 0·8 50 50 27_0 ± 1_1 26.1 ± 4.6 25 75 24.9 ± 0.9 32.4 ± 0.6 10 90 23·6 ± 1_8 37.7 ± 5.9 0 100 18_9 ± 5_7 30.7 ± 5.6 DOTAP : 1,2-bis (dioleoyloxy)-3-(tri-methylamonio) propane PEG-DSPE : PEG2000-1，2-distearoyl-sn-glycero3-phosphoethanolamine 不同配方合成之脂質奈米粒子大小 脂質成分 粒徑（nm) GEC-Chol PEG-DSPE 100 0 95.4 90 10 81.0 75 25 70.0 50 50 30.4 25 75 21.1 10 90 17.0 GEC-Chol: 3-beta-[N-(2-guanidinoethyl)carbamoyl]-cholesterol PEG-DSPE : PEG2000-1,2-distearoyl-sn-glycero-3-phosphoethanolamine 10 200843801 表三 不同配方合成之脂質奈米粒子大小及其相對之三倍頻訊號 脂質成分 ：C-Chol Choi PEG- DSPE w/o Fe3〇4 with Fe3〇4 三訊倍號頻 粒徑（nm} 三倍頻訊 號 50 50 0 221.0 ±54.6 - 387.0 ± 80.7 +++++ 50 50 0 104.0 ±28.2 - 101.0 ±38.1 ++++ 50 50 0 81.6 ±23.2 - 79.0 ± 25.8 ++ 37.5 37.5 25 49.2 ± 3.7 55.3 ±17.6 + 25 25 50 28.5 ± 6.4 - 26.3 ± 5.8 _ 12.5 12.5 75 10± 1_6 - 27.6 ± 7.9 GEC-Chol: 3-beta-[N-(2-guanidinoethyl)carbamoyl]-cholesterol Choi: Cholesterol PEG-DSPE : PEG2000-1,2-distearoyl-sn-glycero-3-phosphoethanolamine 由於倍頻技術為非侵入式，無能量累積之顯微技術，且無關光漂白作用， 可用以觀測活體、活細胞或其他種類生物樣本之3D影像，但若無顯影劑， 此技術也只能單純用以觀測組織或細胞型態的改變，附圖即是我們利用含 有金屬或非金屬的脂質奈米粒子作為三倍頻顯微術的影像對比劑，來研究 癌症和幹細胞的標記之實例。因此，配合本發明之多重影像顯影劑的使用 則可將此技術推廣到更微觀、更精密的分子作用之影像檢測應用。 200843801 【實施方式】 磁鐵對氧化鐵腊質奈米粒子(MION-micelles)運送至HeLa細胞的影響。 細胞以5萬/well的密度種植於24 well之培養盤内，15小時後以Inmole/well 濃度的 MION-micelles(l〇〇% PEG-DSPE 與微量之 Rh〇damine-DOPE ; a，b) 或 MION_cationic micelles(25% PEG-DSPE、75%DOTAP 與微量之200843801 IX. Invention: [Technical field of invention] Nano technology can be applied to various daily life products, including medical Huajing < future applications. Compared with the traditional dyeing calibration technology, the main advantage of using nanoparticle as the contrast agent is to avoid photobleaching, and the nanoparticle is a good drug carrier, so the surface is properly modified, nanoparticle It can be used to calibrate specific biomolecules or lesions, so there is potential for future applications in molecular medicine. [Prior Art] The application of nano particles in imaging is in the optical application part. Traditionally used fluorescent molecules include fluorescent organic molecules, fluorescent proteins, etc. The benefits of organic molecules are small, easy to modify, and after long-term development. Many molecules with strong luminosity and stable light emission have been produced, mostly applied to antibodies; fluorescent proteins require a large number of genetic engineering operations, but can be beneficial for the observation of protein interaction and other research. Recently, many nano particles have been found to have special optical properties, such as quantum dots, which can produce different wavelengths of fluorescence depending on the material and size. The molecular absorption coefficient is high and the emission peak width is narrow, so that multi-color dyeing can be achieved. And the phenomenon of photobleaching is not much more obvious than traditional substances, and can be used for long-term observations, and it has become the darling of the material of the new generation of cursors. Gold or silver nanoparticles can be detected by surface plasmon resonance, and a few reports use their fluorescence. In the nuclear magnetic resonance imaging technique, the nanoparticles are different according to the retardation of T1 and the retardation of T2, and the Τ2 of iron oxide is slow and short, so in the measurement of 迟2 delay, it can be generated. Hey! ^. In addition, the biolipid particles of the microlipids have excellent applications in ultrasonic imaging. The toxicity problem of C-grain particles in living organisms is also the focus of current research, because the result of nanocrystallization often changes the physical properties of materials, and the inactive metal in the county becomes quite active and in vivo. Molecular interactions cause considerable toxic counteraction. Therefore, lipid surface modification can reduce non-specific binding and unnecessary toxicity. The development of frequency doubling technology uses the technology of the fluorescent cursor to understand the interaction of the microscopic or giant world of the organism has been widely used in various biological fields. Therefore, the existence of optics, fluorescence or co-compensation, the microscope is already a biological In the experiment, you can't hide the testware. However, the #cursor has several fatal flaws. One, the fluorescent material is excited, and when the energy is released, it may cause damage to the sample itself. The exposure of strong light causes the length of the molecules to change, resulting in photobleaching, which is even more difficult for researchers. However, the frequency doubling technology does not involve fluorescence, and the energy does not accumulate, the sample shirt hurts, so it has become a close-knit whistle in the world. Among them, the double frequency signal comes from neatly arranged nano-crystals. Structure, and triple frequency is from the discontinuous interface. The signal fat ball detected by the frequency doubling technique can produce an oil-water interface when suspended in an aqueous solution, so that a triple frequency gas number can be generated, but the optical signal is affected by the particle size, and only the fat ball larger than 400 nm can be used. Producing a identifiable signal, the signal intensity of a fat globule larger than 1 micron is good for analysis, while the less than 4 〇〇 nanometer, 200843801 signal is weak, so this method can only be applied to fat cells or tissues that produce a lot of fat. Signals for Metals in Frequency Doubled Techniques Several metals or alloys are known to produce triple frequency signals, such as gold, silver, iron alloys, etc. 'However' _ In general, gold, silver and other metals have the effect of surface resonance, which can be excited by laser to generate double or triple frequency signals, but the signal strength is not high, and it needs to be quite sensitive to the recorder to get the correct Signal. According to the phase, lipid is a regularly arranged biopolymer. After being excited by a laser, the tree can also generate a triple frequency track number. However, its signal is quite weak, and the signal-to-noise ratio is not good, even if it is excellent. The record of the recorder is also quite vague. The lipid f nanoparticle disclosed in the present invention, the coated nanoparticle material, which is not metal or non-metal, can generate a signal intensity greater than ten to one hundred times of the individual monomer signals (IV). Needle, needle --), the image clear _ degree, so that the Nai Na rice grain as a real-time image development contrast agent. SUMMARY OF THE INVENTION The nano biotechnology disclosed in the present invention is mainly a metal of a paste diameter of a small surface, a grain of a grain or a non-metal terminus of a quantum dot, etc., which is stable and has no biological motherhood after being coated with a job. Lipid nanoparticles (lipid nano particles), and then modified by the appropriate surface, the drug molecules carried can be efficiently delivered to the surface or body of living cells, and can be used as the same tree (multim〇) Dality i leg avoidance (four) image contrast agent, to simultaneously observe the mechanism of drug immediate, or to understand the molecular basis of the cause of the disease. 200843801 This technology combines the benefits of both lipid and metal 'construct metal or non-metallic lipid nanoparticles, fruit S now the nanosphere ball above 5G nanometer can generate _ identifiable triple frequency signal, 100 nanometer nano particle signal is stronger, above the nano, the intensity is not obviously enhanced but it can be clear It is difficult to size _, and in the _ observation molecule riding reaction, it can be confirmed that this nai tree can be used as a triple heterogeneous body (in called or culvert (in V (10) developer (contrast agent). Body application It has been used for many years and can be used for drug carrying, or gene delivery. However, if the nanoparticles are too large (> ng), the body will be recognized, resulting in the removal of the reticuloendothelial system in the body. Will accumulate in the liver, kidneys, etc., causing non-specific mother I4' or background noise will cause image interpretation. Because there is inherent fat in the organism, it is less toxic, bio-adapted, used in Nai The wrapping of rice particles can reduce the toxicity of nano particles. Using the principle of self-assembled lipids modified by regular valency lipids and PEG, adjust the ratio of the two (such as Table 1 and Table 2), or add neutral Lipids (as shown in Table 3) and ultrasonically oscillated' can effectively control the size of the nanoparticles, increase the efficiency of the particles themselves, reduce non-specific toxicity, and achieve the purpose of gene delivery. Through simple surface modification (such as Figure VIII and Figure IX) can also achieve specialized delivery. In the process of delivering raw medicinal materials, it can be detected and analyzed by frequency doubling technique. Or in the calibration of proteins, the molecular meaning and function of protein f can be observed for a long time. In the case of cell calibration, a high-resolution biopsies analysis can be performed, and because the frequency doubling technique does not cause damage to the sample, the technique can be applied to analyze the application of the stem cell differentiation process. 200843801 Table 1: Different formulations of lipids Rice particle size lipid particle size (nm) DOTAP PEG-DSPE w/o Fe3〇4 with Fe3〇4 75 25 28·5 ± 1·6 30_2 ± 0·8 50 50 27_0 ± 1_1 26.1 ± 4.6 25 75 24.9 ± 0.9 32.4 ± 0.6 10 90 23·6 ± 1_8 37.7 ± 5.9 0 100 18_9 ± 5_7 30.7 ± 5.6 DOTAP : 1,2-bis (dioleoyloxy)-3-(tri-methylamonio) propane PEG-DSPE : PEG2000-1,2 -distearoyl-sn-glycero3-phosphoethanolamine Lipid Nanoparticle size synthesized by different formulations Lipid size (nm) GEC-Chol PEG-DSPE 100 0 95.4 90 10 81.0 75 25 70.0 50 50 30.4 25 75 21.1 10 90 17.0 GEC- Chol: 3-beta-[N-(2-guanidinoethyl)carbamoyl]-cholesterol PEG-DSPE : PEG2000-1,2-distearoyl-sn-glycero-3-phosphoethanolamine 10 200843801 Table 3 Lipid Nanoparticle Sizes Synthesized by Different Formulations And its relative triple frequency signal Ingredients: C-Chol Choi PEG- DSPE w/o Fe3〇4 with Fe3〇4 Three-way frequency particle size (nm} Triple frequency signal 50 50 0 221.0 ±54.6 - 387.0 ± 80.7 +++++ 50 50 0 104.0 ±28.2 - 101.0 ±38.1 ++++ 50 50 0 81.6 ±23.2 - 79.0 ± 25.8 ++ 37.5 37.5 25 49.2 ± 3.7 55.3 ±17.6 + 25 25 50 28.5 ± 6.4 - 26.3 ± 5.8 _ 12.5 12.5 75 10 ± 1_6 - 27.6 ± 7.9 GEC-Chol: 3-beta-[N-(2-guanidinoethyl)carbamoyl]-cholesterol Choi: Cholesterol PEG-DSPE : PEG2000-1,2-distearoyl-sn-glycero-3-phosphoethanolamine Frequency technology is a non-invasive, energy-free microscopy technology, and has no photobleaching effect. It can be used to observe 3D images of living, living cells or other kinds of biological samples. However, if there is no developer, this technology can only be used. To observe changes in tissue or cell type, the figure is an example of a marker for cancer and stem cells using a lipid-nanoparticle containing metal or non-metal as an image contrast agent for triple-frequency microscopy. Thus, the use of the multi-image developer in accordance with the present invention extends this technique to more microscopic, more sophisticated molecular imaging applications. 200843801 [Embodiment] The effect of magnets on the transport of iron oxide waxy nanoparticles (MION-micelles) to HeLa cells. The cells were seeded in a 24 well culture dish at a density of 50,000/well, and MION-micelles (10% PEG-DSPE and trace amounts of Rh〇damine-DOPE; a, b) at an Inmole/well concentration after 15 hours. Or MION_cationic micelles (25% PEG-DSPE, 75% DOTAP and trace amounts)
Rhodamine-DOPE ; c，d)在有(b，d)或沒有(a，c)磁鐵的存在下處理細胞12小 時’並以螢光影像紀錄不同條件下micelle細胞載入的結果(放大倍率為1〇〇 倍）。（围一）Rhodamine-DOPE ; c, d) Treat cells for 12 hours in the presence of (b, d) or without (a, c) magnets and record the results of micelle cell loading under different conditions with fluorescent images (magnification) 1〇〇). (circle 1)
氧化鐵脂質奈米粒子(MION-micelles)停留於HeLa細胞内之時效與其對 HeLa細胞生長之影響Q 細胞以5萬/well的密度種植於24 well之培養盤内，15小時後以1 n mole/well 濃度的 MION-cationicmicelles(25%PEG-DSPE、75%DOTAP 與微量之 Rhodamine-DOPE)處理細胞12小時。經PBS清洗去除為載入之mieelles|， 將細胞培養於含有10%胎牛血清的DMEM培養液内。於不同時間點以 trypsin切離貼附之細胞，以細胞計數器計算細胞數量。（圖二） 以磁鐵座測試細胞之磁鐵吸附程度。 細胞經過標記氧化鐵奈米粒子後，以磁鐵座測試細胞之磁鐵吸附程户，《 明氧化鐵奈米粒子存留的情況。（圓三） 以量子點正價脂質奈米粒子遞送短鍵反義核苷酸。The aging of iron oxide lipid nanoparticles (MION-micelles) in HeLa cells and its effect on the growth of HeLa cells. Q cells were seeded in 24 well plates at a density of 50,000/well, and 1 n mole after 15 hours. Cells were treated with /well concentrations of MION-cationicmicelles (25% PEG-DSPE, 75% DOTAP and trace amounts of Rhodamine-DOPE) for 12 hours. The mieelles| was loaded by washing with PBS, and the cells were cultured in DMEM containing 10% fetal calf serum. The attached cells were trypsin at different time points and the number of cells was counted in a cell counter. (Fig. 2) The magnet adsorption degree of the cells was tested with a magnet holder. After the cells are labeled with iron oxide nanoparticles, the magnets of the cells are tested by magnet holders, and the "iron oxide nanoparticles" remain. (Round 3) Short-chain antisense nucleotides are delivered by quantum dot positive-valency lipid nanoparticles.
HeLa細胞以1萬/孔的密度種植於包含玻璃片之l2Wei丨培養般内 12 200843801 15小時後，將1 Ug DNA與2.5 ug之量子點正價脂質奈米粒子混合物加入， 處理細胞24小時，經PBS清洗兩次，以4% formaldehyde固定細胞15分 鐘，再以PBS清洗三次後，用Hoechst33342作核染色，以共軛焦螢光顯微 鏡觀察。（圓四） 以氧化鐵正價脂質奈米粒子遞送短鏈反義核苷酸。 AU565、rBMSC及HeLa細胞以1萬/孔的密度種植於包含玻璃片之 12 well培養盤内，15小時後，將1 ugDNA與2·5 ug之量子點正價脂質奈 米粒子混合物加入，處理細胞24小時，經PBS清洗兩次，以4% formaldehyde 固定細胞15分鐘，再以PBS清洗三次後，用Hoechst33342作核染色，以 共輛焦螢光顯微鏡觀察。（圖五） 利用量子點正價脂質奈米粒子標定大鼠之骨越幹細胞，並顯示其細胞標定 量與處理劑量之關係。 rBMSC細胞以1萬/孔的密度種植於I2 well之培養盤内，15小時後以 1.25, 2.5及5 ug/well之量子點含量處理細胞24小時，去除舊有培養液， 經PBS清洗兩次，加入新培養液後，以螢光顯微鏡觀察。（圓六） 利用量子點脂質奈米粒子標定人類之骨趙幹細胞，並利用TAT胜肽修飾提 高遞送效率。 (A)hBMSC細胞以1萬/孔的密度種植於12 well之培養盤内，15小時 後分別以量子點中性脂質奈米粒子、經stepavidin表面修飾之量子點中性脂 質奈米粒子，以及經TAT胜肽表面修飾之量子點中性脂質奈米粒子2 5/wdl 13 200843801 之量子點含量處理細胞24小時，去除舊有培養液，經pBS清洗兩次，加入 新培養液後，以螢光顯微鏡觀察。（圖七） 利用抗體修飾量子點脂質奈米粒子，專一性標定CD13+ CL1-0細胞。 CD13+ CL1-0細胞以1 0萬/孔的密度種植於12 weii之培養盤内，15 小時後以量子點、量子點中性脂質奈米粒子，以及經抗CD13之抗體表面修 . 飾之量子點中性脂質奈米粒子1·25 / well之量子點含量處理細胞16小時， . 去除舊有培養液，經PBS清洗兩次，加入新培養液後，以螢光顯微鏡觀察。 (圓八） 利用抗體修飾量子點脂質奈米粒子，專一性標定AU565細胞。（A-H)AU565 細胞以1萬/孔的密度種植於12 well之培養盤内，15小時後以量子點、 量子點中性脂質奈米粒子、經人類IgG表面修飾之量子點中性脂質奈米粒 子，以及經抗Her2之抗體Herceptin表面修飾之量子點中性脂質奈米粒子 1·25/well之量子點含量處理細胞16小時，去除舊有培養液，經PBS清洗 兩次，加入新培養液後，以螢光顯微鏡觀察。（I，J)MCF7細胞以1萬/孔 的密度種植於12 well之培養盤内，15小時後以經抗Her2之抗體 Herceptin表面修飾之量子點中性脂質奈米粒子1·25 / well之量子點含量處 理細胞16小時，去除舊有培養液，經PBS清洗兩次，加入新培養液後，以 螢光顯微鏡觀察。（圖九） 測量量子點脂質奈米粒子之訊號。 以鉻鎂撖欖石1230 nm之雷射，偵測410 nm之三倍頻訊號。⑻於水溶液 200843801 中直接測量餅之量子蹄質奈米粒子缝生之三倍頻峨。⑼取少 量量子點脂質奈米粒子置於玻片上乾燥，乾燥後測量其三倍頻訊號。（C) 於水溶液中直接測量懸浮之量子點脂質奈米粒子所產生之三倍頻訊號 及三光子螢光訊號分析。（圖十）HeLa cells were planted at a density of 10,000/well in a l2Wei丨 culture containing glass slides for 12 hours. After 12 hours, a mixture of 1 Ug of DNA and 2.5 ug of quantum dot-valency lipid nanoparticles was added and the cells were treated for 24 hours. After washing twice with PBS, the cells were fixed with 4% formaldehyde for 15 minutes, washed three times with PBS, and stained with Hoechst 33342 as a nucleus, and observed under a conjugated fluorescence microscope. (Round 4) Short-chain antisense nucleotides are delivered as iron oxide positive-valency lipid nanoparticles. AU565, rBMSC and HeLa cells were planted in a 12 well plate containing glass slides at a density of 10,000/well. After 15 hours, 1 ug of DNA and 2·5 ug of quantum dot-valency lipid nanoparticle mixture were added. The cells were washed twice with PBS for 24 hours, fixed in 4% formaldehyde for 15 minutes, washed three times with PBS, stained with Hoechst 33342, and observed under a co-fluorescence microscope. (Fig. 5) The bone-derived stem cells of the rat were calibrated using quantum dot-valence lipid nanoparticles and the relationship between the cell calibrated amount and the treatment dose was shown. rBMSC cells were seeded in I2 well plates at a density of 10,000/well. After 15 hours, the cells were treated with quantum dots at 1.25, 2.5 and 5 ug/well for 24 hours. The old culture medium was removed and washed twice with PBS. After adding a new culture solution, it was observed with a fluorescence microscope. (Circle 6) The human bone Zhao stem cells were calibrated using quantum dot lipid nanoparticles, and TAT peptide modification was used to improve delivery efficiency. (A) hBMSC cells were seeded in a 12 well plate at a density of 10,000/well, and after 90 hours, quantum dot neutral lipid nanoparticles, stepavidin surface-modified quantum dot neutral lipid nanoparticles, and The TUN peptide surface-modified quantum dot neutral lipid nanoparticle 2 5/wdl 13 200843801 The quantum dot content was treated for 24 hours, the old culture solution was removed, washed twice with pBS, and the new culture solution was added to the firefly. Light microscopy. (Fig. 7) The antibody was used to modify the quantum dot lipid nanoparticle, and the CD13+ CL1-0 cells were specifically calibrated. CD13+ CL1-0 cells were seeded in a 12 usii culture dish at a density of 100,000/well. After 15 hours, quantum dots, quantum dot neutral lipid nanoparticles, and anti-CD13 antibody surface were repaired. The cells were treated with a quantum dot content of neutral lipid particles of 1·25 / well for 16 hours. The old culture solution was removed, washed twice with PBS, and a new culture solution was added, followed by observation with a fluorescence microscope. (Circle 8) The AU565 cells were specifically calibrated by modifying the quantum dot lipid nanoparticles with antibodies. (AH) AU565 cells were seeded in a 12 well plate at a density of 10,000/well. After 15 hours, quantum dots, quantum dot neutral lipid nanoparticles, and human dot IgG surface-modified quantum dot neutral lipid nanoparticles were used. The particles, and the quantum dot content of the quantum dot neutral lipid nanoparticles of the Herceptin surface modified by Herceptin, were treated for 16 hours, the old culture solution was removed, washed twice with PBS, and a new culture solution was added. After that, observe with a fluorescent microscope. (I, J) MCF7 cells were seeded in a 12 well plate at a density of 10,000/well. After 15 hours, the quantum dot neutral lipid nanoparticles modified with the anti-Her2 antibody Herceptin surface was used. The cells were treated with quantum dot content for 16 hours, the old culture solution was removed, washed twice with PBS, and a new culture solution was added, followed by observation with a fluorescence microscope. (Fig. 9) Signals for measuring quantum dot lipid nanoparticles. A 4030 nm laser is used to detect a triple frequency signal at 410 nm. (8) The triple-frequency enthalpy of the quantum hoof nanoparticle of the cake was directly measured in the aqueous solution 200843801. (9) Take a small amount of quantum dot lipid nanoparticles and dry them on a glass slide. After drying, measure the triple frequency signal. (C) Direct measurement of triple-frequency signals and three-photon fluorescence signal analysis of suspended quantum dot lipid nanoparticles in aqueous solution. (Figure 10)
HeLa細胞一水平切面之三倍頻訊號。 (a)未處理量子點脂質奈米粒子，偵測光電倍增管電壓9〇〇v。作）經處 理ϊ子點脂質奈米粒子，彳貞測光電倍增管電壓5〇〇v。（圖十一） 說明不同大小之金屬脂質奈米粒子所產生之三倍頻訊號。 (A)直接量測於水溶液中懸浮之氧化鐵脂質奈米粒子所產生之三倍 頻訊號（B)取少量氧化鐵脂質奈米粒子置於玻片上乾燥，乾燥後測量 其三倍頻訊號。（圖十二） 顯示氧化鐵脂質奈米粒子之細胞呑嗤量與處理劑量之關係。 (A) HeLa細胞以1萬/孔的密度種植種植於12 weU之培養盤内，15 小時後刀別以不同量之鐵含量處理細胞4小時，經pbs清洗後，以4% formaldehyde固定細胞15分鐘，再以pBS清洗三次後，觀察其三倍頻訊號。 (B) 分別為 0, 0.25, 0.5, U5, 2.5, 5, 7·5, 15, 25 ug / well 之鐵含量處理細胞 4 小時後之三倍頻訊號量化結果。（圈十三） 顯不氧化鐵脂質奈米粒子之細胞呑噬量與處理時間之關係。 (A) HeLa細胞以1萬/孔的密度種植種植於12weU之培養盤内，15 15 200843801 小時後以7·5 ug / well之鐵含量處理細胞，分別在不同時間點，將細胞經pBS 清洗後，以4% formaldehyde固定細胞15分鐘，再以PBS清洗三次後，觀 察其三倍頻訊號。(B)以7·5 ug / well之鐵含量處理細胞，分別在處理〇, 〇 25, 0·5, 1，2, 4, 8, I6, 24小時後將細胞固定，測量三倍頻訊號後之量化結果。（圖 十四） 200843801 【圖式簡單說明】 圖一、外加磁場能加速氧化鐵奈米粒子被貼覆培養之HeLa細胞呑噬。 圖二、氧化鐵奈米粒子停留於HeLa細胞内之時效與其對HeLa細胞生長 之影響。 圖三、細胞經過標記氧化鐵奈米粒子後，以磁鐵座測試細胞之磁鐵吸附程 度’證明氧化鐵奈米粒子存留的情況。 圖四、以量子點正價脂質奈米粒子有效遞送短鏈反義核苷酸。 圖五、以氧化鐵正價脂質奈米粒子有效遞送短鏈反義核苷酸。 圖六、利用量子點正償脂質奈米粒子標定大鼠之骨趙幹細胞，並顯示其細 胞標定量與處理劑量之關係，顯示其生物相容性明顯提昇。 圖七、利用量子點脂質奈米粒子標定人類之骨趙幹細胞，並利用TAT胜肽 j 修飾提高遞送效率。 圖八、利用抗體修飾量子點脂質奈米粒子，專一性標定CD13+ CL1_0細胞。 圖九、利用抗體修飾量子點脂質奈米粒子，專一性標定AU565細胞。 圖十、測量量子點脂質奈米粒子之倍頻訊號。 圖十一、HeLa細胞呑噬奈米粒子後顯示其水平切面之三倍頻訊號。 17 200843801 圓十二、說明不同大小之金屬脂質奈米粒子所產生之三倍頻訊號。 圓十三、顯示氧化鐵脂質奈米粒子之細胞呑噬量與處理劑量之關 係’證明脂質修飾後之奈米粒子沒有顯著的生物毒性。 圓十四、顯示氧化鐵脂質奈米粒子之細胞呑噬量與處理時間之關 係0 【主要元件符號說明】 奈米粒子:nanoparticles 量子點：quantum dots 氧化鐵奈米顆粒: iron oxide nanoparticles 正電荷脂質：cationic lipid 正電荷膽固醇：cationic cholesterol 正電荷雙脂鏈脂質：cationic diacyllipid 倍頻顯微術:harmonic generation microscopy GEC-Chol: 3-beta-[N-(2-guanidinoethyl)carbamoyl]-cholesterol PEG-DSPE :PEG2000-1,2-distearoyl-sn-glycero-3-phosphoethanolamine 18The triple frequency signal of a horizontal slice of HeLa cells. (a) Untreated quantum dot lipid nanoparticles, detecting photomultiplier tube voltage 9 〇〇 v. The membrane was treated with hazelnut lipid nanoparticles and the photomultiplier tube voltage was measured at 5 〇〇 v. (Fig. 11) Describe the triple frequency signal generated by different sizes of metalloid nanoparticles. (A) Direct measurement of the triple-frequency signal generated by the iron oxide lipid nanoparticles suspended in an aqueous solution (B) A small amount of iron oxide lipid nanoparticles were placed on a glass slide, dried, and the triple-frequency signal was measured after drying. (Fig. 12) shows the relationship between the amount of cell iron oxide particles and the treatment dose. (A) HeLa cells were planted in a 12 usU culture dish at a density of 10,000/well. After 15 hours, the cells were treated with different amounts of iron for 4 hours. After washing with pbs, the cells were fixed with 4% formaldehyde. Minutes, after washing three times with pBS, observe the triple frequency signal. (B) Quantification results of the three-fold frequency signal after 4 hours of treatment of the iron content of 0, 0.25, 0.5, U5, 2.5, 5, 7·5, 15, 25 ug / well, respectively. (Circle 13) The relationship between the amount of cell phagocytosis of the non-oxidized iron lipid nanoparticles and the treatment time. (A) HeLa cells were planted in a 12weU culture dish at a density of 10,000/well. After 15 15 200843801 hours, the cells were treated with iron content of 7.5 ug / well, and the cells were washed with pBS at different time points. Thereafter, the cells were fixed in 4% formaldehyde for 15 minutes, and then washed three times with PBS, and the triple frequency signal was observed. (B) The cells were treated with iron content of 7.5 ug / well, and the cells were fixed after treatment of 〇, 〇25, 0·5, 1, 2, 4, 8, I6, 24 hours later, and the triple frequency signal was measured. Quantitative results afterwards. (Fig. 14) 200843801 [Simple description of the diagram] Figure 1. The external magnetic field can accelerate the helium phagocytosis of the iron oxide nanoparticles coated with HeLa cells. Figure 2. The aging of iron oxide nanoparticles in HeLa cells and their effects on HeLa cell growth. Fig. 3. After the cells were labeled with iron oxide nanoparticles, the magnet adsorption degree of the cells was measured by a magnet holder to confirm the retention of the iron oxide nanoparticles. Figure 4. Effective delivery of short-chain antisense nucleotides by quantum dot positive-valency lipid nanoparticles. Figure 5. Efficient delivery of short-chain antisense nucleotides with iron oxide positive-valency lipid nanoparticles. Fig. 6. Using the quantum dot positively compensated lipid nanoparticles to calibrate the bone stem cells of the rats, and showed the relationship between the cell standard quantitation and the treatment dose, indicating that the biocompatibility was significantly improved. Figure 7. Calibration of human bone stem cells using quantum dot lipid nanoparticles and modification of TAT peptide j to improve delivery efficiency. Figure 8. Modification of CD13+ CL1_0 cells by antibody-modified quantum dot lipid nanoparticles. Figure 9. Quantification of AU565 cells by antibody-modified quantum dot lipid nanoparticles. Figure 10. Measurement of the frequency-doubled signal of quantum dot lipid nanoparticles. Figure 11. HeLa cells show three-fold frequency signals of their horizontal sections after phagocytizing nanoparticles. 17 200843801 Round 12, illustrating the triple frequency signal produced by different sizes of metalloid nanoparticles. Round Thirteen shows that the relationship between the amount of cellular phagocytosis of iron oxide lipid nanoparticles and the treatment dose has demonstrated that the lipid-modified nanoparticle has no significant biological toxicity. Round XIV, showing the relationship between the amount of cellular phlegm and the treatment time of iron oxide lipid nanoparticles. [Key element symbol description] Nanoparticles: Nanoparticles Quantum dots: quantum dots Iron oxide nanoparticles: iron oxide nanoparticles Positively charged lipids :cationic lipid positively charged cholesterol:cationic cholesterol positively charged double lipid chain lipid:cationic diacyllipid frequency doubled microscopy:harmonic generation microscopy GEC-Chol: 3-beta-[N-(2-guanidinoethyl)carbamoyl]-cholesterol PEG-DSPE :PEG2000-1,2-distearoyl-sn-glycero-3-phosphoethanolamine 18