1342787 九、發明說明： 【發明所屬之技術領域】 ^ · •.奈米科技將來能被顧於各種日常生活產品當中，也包括縣雜未來之 應用。相氣祕標紐術，使岐綠子作⑽彡像對比劑最主要的 優點是能夠避免総白，而且奈絲子是很好的醫_帶物，因此將其表 面做適當的修飾，奈練子即可_來標定特定的生物分子或紐，因此 Φ 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㈣光分子包括螢光有機分子、勞光蛋 白等’有機分子的好處是小，容祕飾’且在長崎展後，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料研究之觀察。近來來則發現許多 奈米粒子也具有·的絲雜，如量子點，可膽撕纽大小不同產 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-代螢光標的物質的籠心金或銀奈錄子則可利用 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TI遲緩與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 1342787 以產生明顯的造影。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。 但奈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求化的 絲’常常使騎料_性紐觀變，原料活化的金齡魏相當活 潑，以至於和活體内的生物分子交互_，造成相當大的毒性反性。因此 經過脂質表祕飾可崎低非特異性的結合和不必要的毒性。 Φ 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、螢光或共概焦顯微鏡的存在已 經是生物實驗上不可獲缺馳取具，然而，縣標的卻錢個致命的缺 點，一者，螢光物質一旦受到激發，釋出能量的同時，便可能對樣品本身 產生傷害，一者，受到強光照射致使分子的共耗長度改變，導致光漂白現 象，更是讓研究人員不勝其擾。然而，倍頻技術卻不牽涉螢光，且能量不 ® 累積，樣品不受傷害，故成為近幾年來全世界備受注目的新穎光學技術， 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，故可產生三件頻气號， 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， 1342787 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’訊號不強一般來說，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， 需要相當靈敏的紀錄器才能擷取正確的訊號.相對的，脂質是一種排列規 • 則的生物聚合物，經雷射光源激發後，本身也能產生三倍頻的訊號，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。本發明所揭示的脂質奈米粒子，所包覆的奈米顆粒材料， •不管是金屬或非金屬，皆能產生大於上述個別單體訊號十至一百倍的訊號 強度(如本發明之圖十、計_)，其影像之清晰程度，使脂f奈米粒子變 成即時影像的顯影對比劑。 •【發明内容】 本發明所揭示的奈米生物技術，主要是利用直徑小於兩百奈米之金屬 顆粒或非金屬料雜量子財，經職f包额触成轉定且無生物 母性的脂質奈米粒子师如如叩牆㈣’再藉由表面適當的修飾它能將 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 時作為多重功能影像(multimodaUty imaging)之影像對比劑，來同步觀察藥物 即時作用的機制’或了解疾病成因的分子基礎。 1342787 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，結果發現在50奈米以上之奈米球，即可產生明顯可辨識之三倍頻訊號， 1〇〇奈米之奈米粒子訊號更強，100奈米以上者，強度雖未明顯增強但卻 ;·可明確觀_雛大小的變化，並在細胞峨察分子間的祕反應，故5 確認此種奈米球可作為三倍頻技術之體外(invitr〇)或活體(inviv〇)之顯影劑 (contrast agent) 〇 /丁、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， 籲《基因遞送。但是奈米粒子過大(>彻奈米)也會讓生物體認出，造成生體 内的網狀内皮緖清除後’短納會堆積在肝臟、腎臟等處，造成非特異 之毒性，或產生背景雜訊會造成影像觸上_擾。由於生物體内本來就 存在脂肪，故毒性小’生物適性高，用於奈米粒子的包袠，可減少奈米粒 子的毒性。湘正價脂質和聚乙二醇(PEG)修飾之脂f自組裝㈣f_assemb丨ed) 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 經超音波震盤，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、圓九）’也可制專_性遞送β在遞送生醫藥物的雜巾，可利用倍 頻技術檢職分析。或者在標定蛋白，長_觀測蛋自㈣分子作用 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 技術不會職本構成傷害’可_此技術進行幹細胞分化難之制分析。 1342787 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 不含氧化鐵 (Fe3〇4) 包裹氧化雄 (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. 1,2-一-(油醯基氧基)-3-(三甲基錢)丙炫（1 2-bis(di〇|eoyloxy)-3-(tri-methylarnonio) propane) PEG-DSPE:聚乙醇-雙硬脂酸峨脂醯乙醇胺 (PEG2000-1,2-distearoyl-sn-glycero-3^phosphoethanolamine)1342787 Nine, invention description: [Technical field to which the invention belongs] ^ · • Nano technology can be taken into account in various daily life products, including the application of the future of the county. The most important advantage of the 气 秘 秘 , ( ( ( 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 最 最 最 最 最 最 最 最 最 最 最 最 最 最 最The practice can be used to calibrate specific biomolecules or nucleus, so Φ will have potential for future applications in molecular medicine. [Prior Art] The application of nanoparticle in imaging is used in the optical application part. Traditionally, (4) optical molecules including fluorescent organic molecules, laurel proteins, etc. 'The benefits of organic molecules are small, and the secrets are decorated' and after the exhibition in Nagasaki 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 studies. Recently, many nanoparticles have been found to have filaments, such as quantum dots, which can produce different wavelengths of fluorescence. The molecular absorption coefficient is high, and the peak width of the radiation is narrow, so that multicolor dyeing can be achieved. And the phenomenon of photobleaching is not much more obvious than the traditional materials. It can be used for long-term observation, and it can also be used as a new-generational cursor. The cage gold or silver na[iota] can be detected by surface plasma resonance. A few reports are using its fluorescent light. In the nuclear magnetic resonance imaging technique, the nanoparticles are different according to their TI retardation and T2 retardation, and the 'iron oxide Τ2 is slow and short, so the Τ2 slow measurement can be 1342787 to produce obvious Contrast. In addition, the biolipid particles of the microlipids have excellent applications in ultrasonic imaging. However, the toxicity problem of nanoparticle in vivo is also the focus of current research. Because the silk of the present is often used to change the material, the golden age of the activated material is quite active, so that it can be biomolecules in vivo. Interaction _, causing considerable toxicity. Therefore, lipid-based secrets can be used to reduce non-specific binding and unnecessary toxicity. The development of Φ frequency doubling technology uses the technology of the fluorescent cursor to understand the interaction of the microscopic or macroscopic world of the organism has been widely used in various biological fields. Therefore, the existence of optical, fluorescent or total focus microscopy is already In biological experiments, it is impossible to obtain a lack of equipment. However, the county standard has a fatal flaw. Once the fluorescent substance is excited, the energy may be released, and the sample itself may be harmed. Light irradiation causes the length of the co-consumption of molecules to change, leading to photobleaching, which is even more disturbing for researchers. However, the frequency doubling technology does not involve fluorescence, and the energy does not accumulate and the sample is not damaged. Therefore, it has become a novel optical technology in the world in recent years. Among them, the double frequency signal comes from the neatly arranged nanometer. The crystal structure is graded, while the triple frequency is derived 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 three frequency codes can be generated, but the optical signal is affected by the particle size. Only fat balls 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 for less than 4 〇〇 nanometer, the 1342787 signal is weakly. Therefore, this method can only be applied to fat cells or tissues that produce a large amount of fat. , • Signals of metal under frequency doubling technology, · Several metals or alloys are known to produce triple frequency signals, such as gold, silver, iron alloys, etc. However, 'signals are not strong, generally, gold, silver, etc. The metal itself has the effect of surface resonance, which can be excited by the laser to generate a double or triple frequency signal, but the signal strength is not high, and a fairly sensitive recorder is needed to capture the correct signal. In contrast, lipid is a kind of The biopolymer, which is arranged by the laser source, can also generate a triple-frequency signal itself, but similarly, its signal is quite weak, and the signal-to-noise ratio is not good, even with a good recorder record. The image is also quite blurry. The lipid nanoparticle disclosed in the present invention, the coated nanoparticle material, whether metal or non-metal, can generate a signal intensity greater than ten to one hundred times of the individual monomer signals (as shown in the figure of the present invention) Ten, _), the clarity of the image, so that the fat f nanoparticle becomes a development contrast agent for instant images. • [Summary of the Invention] The nano biotechnology disclosed in the present invention mainly utilizes metal particles or non-metal materials with a diameter of less than two hundred nanometers, and is a nutrient-rich nutrient. The rice particler can use the appropriate surface modification to efficiently deliver the drug molecules carried to the surface or body of the living organism cell, and can simultaneously serve as an image of multimodaUty imaging. Contrast agents, to simultaneously observe the mechanism of immediate action of drugs' or to understand the molecular basis of the cause of the disease. 1342787 This technology combines the benefits of both lipids and metals to construct metal or non-metallic lipid nanoparticles. It has been found that nanospheres above 50 nm can produce clearly identifiable triple-frequency signals. The rice particle signal is stronger, and the intensity of 100 nm or more is not obviously enhanced, but it can clearly determine the change of the size of the chick, and observe the secret reaction between the molecules in the cell, so 5 confirm this kind of nai Rice ball can be used as a three-frequency technology in vitro (invitr〇) or in vivo (inviv〇) developer (contrast agent) 〇 / butyl, rice particles have been used in the application of organisms for many years, can be used for drug carrying , called "gene delivery. However, if the nanoparticle is too large (>Chenna), the organism will recognize it, and after the reticuloendothelial resection in the living body, the short-term will accumulate in the liver, kidney, etc., causing non-specific toxicity, or Producing background noise can cause the image to be stunned. Since there is inherently fat in the living body, the toxicity is small, and the biocompatibility is high, and it is used for the packing of the nanoparticles to reduce the toxicity of the nanoparticles. Xiangzheng valence lipid and polyethylene glycol (PEG) modified lipid f self-assembly (four) f_assemb丨ed) principle, adjust the ratio of the two (such as Table 1, Table 2), or add neutral lipids (such as Table 3 And through the ultrasonic shock disk, it can effectively control the size of the nanoparticles, increase the efficiency of the particles themselves, reduce the non-specific toxicity, and achieve the purpose of gene delivery. Through simple surface modification (Figure 8, circle IX) 'Can also be used to deliver _ sexually delivered β in the delivery of raw materials, can be analyzed by frequency doubling technology. Or in the calibration of protein, long _ observation of egg from (four) molecular action and movement. After cell calibration can be Perform high-resolution biopsies analysis, and because of the frequency-doubled technique, it will not cause damage. 'This technology is difficult to analyze stem cell differentiation. 1342787 Table 1: Lipid nanoparticles with different formula synthesis Diameter (nm) '· * DOTAP PEG-DSPE Contains no iron oxide (Fe3〇4) Encapsulated oxidized male (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-I-(oleryloxy)-3-(trimethyl hydroxy)propanol (1 2-bis(di〇|eoyloxy) -3-(tri-methylarnonio) propane) PEG-DSPE: Polyethanol-bisstearate PEG2000-1,2-distearoyl-sn-glycero-3^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 10 1342787 GEC-Chol: (2-狐基乙基)-胺甲基胳固酵（3-beta-[N-(2-guanidinoethyl)carbamoyn-cholesterol) PEG-DSPE:聚乙酵-雙硬脂酸磷脂醯乙醇胺 (PEG2000-1,2-distearoyl-sn-glycero-3-phosphoethanolamine) 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 GEC-Chol Choi PEG- DSPE 不含氧化嫌 包裹氧化嫌 粒徑（nm> 三倍頻 訊號 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: (2-胍基乙基)-胺甲基膀固醇（3-beta-[N-(2-guanidinoethyl)carbamoyl]-cholestero|) Choi:膽固醇（Cholesterol) PEG-DSPE:聚乙醇-雙硬脂酸磷脂醯乙酵胺 (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三維(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。 11 ΙΓ ·'! 1342787 【實施方式】 磁鐵對氧化鐵脂質奈米粒子(MION-micelles)運送至人類子宮頸癌細胞 .-HeLa細胞的影響》 ,•細胞以5萬/孔的密度種植於24孔之培養盤内，15小時後以1奈莫爾數/孔 (nmole/well)濃度的氧化鐵脂質奈米粒子(100% PEG-DSPE與微量之 Rhodamine-DOPE ; a，b)或氧化鐵正價脂質奈米粒子(25%PEG-DSPE、 750/〇DOTAP 與微量之 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氧化鐵脂質奈米粒 子之細胞載入的結果(放大倍率為1〇〇倍）。（圖一） 氧化鐵脂質奈米粒子(MION-miceHes)停留於人類子宮頸癌細胞HeLa細胞 内之時效與其對細胞生長之影響》 細胞以5萬/扎的密度種植於24孔之培養盤内，15小時後以1奈莫爾數/孔 濃度的氧化鐵脂質奈米粒子(25% PEG-DSPE、75%DOTAP與微量之 Rhodamine-DOPE)處理細胞12小時。經磷酸食鹽水緩衝液(PBS)清洗除去未 載入之脂質奈米粒子後，將細胞培養於含有1〇%胎牛血清的細胞培養液 内。於不同時間點以胰蛋白酶切離貼附之細胞，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度，證 明氧化鐵奈米粒子存留的情況。（圖三） 12 1342787 以量子點正價脂質奈米粒子遞送短鍵反義核苷酸。 人類子宮頸癌細胞HeLa細胞以1萬/孔的密度種植於包含玻璃片之 r · 12孔培養盤内，15小時後，將1微克(Ug)經螢光標定之短鏈反義核苷酸 * (antisense oligodeoxynucleotide，antisense ODN)與 2.5 微克之量子點正價脂質 奈米粒子混合物加入，處理細胞24小時，經磷酸食鹽水緩衝液清洗兩次’ 以4%甲酸·(formaldehyde)固定細胞15分鐘，再以填酸食鹽水緩衝液清洗二 次後，用螢光染色劑Hoechst33342作核染色，以共軛焦螢光顯微鏡觀察。 φ (圖四） 以氧化鐵正償脂質奈米粒子遞送短键反義核苷酸》 人類乳腺癌細胞AU565、大鼠骨髓幹細胞rBMSC及人類子宮頸癌細胞HeLa 細胞以1萬/孔的密度種植於包含玻璃片之12孔培養盤内’ 15小時 後，將1微克經螢光標定之短鏈反義核苷酸與2.5微克之量子點正價脂質奈 米粒子混合物加入，處理細胞24小時，經磷酸食鹽水緩衝液清洗兩次，以 4°/〇曱醛固定細胞15分鐘，再以磷酸食鹽水緩衝液清洗三次後，用螢光染色 劑Hoechst33342作核染色，以共輥焦螢光顯微鏡觀察。（圓五） 利用量子點正價脂質奈米粒子標定大鼠之骨髓幹細胞(rBMSC)，並顯示其 細胞標定量與處理劑量之關係。 大鼠骨髓幹細胞rBMSC細胞以1萬/孔的密度種植於12孔之培養盤 内’丨5小時後以125, 2.5及5微克/孔(pg / well)之量子點含量處理細胞24 小時，去除舊有培養液，經磷酸食鹽水緩衝液清洗兩次，加入新培養液後， 13 1342787 以螢光顯微鏡觀察。（圈六) 利用量子點脂質奈米粒子標定人類之骨髄幹細胞(hBMSC)，並利用TAT胜 ， * 肽修飾提高遞送效率。 ' (A)人類骨髓幹細胞hBMSC細胞以1萬/孔的密度種植於12孔之培養 盤内，15小時後分別以量子點中性脂質奈米粒子、經鍵徽親合素(streptavidin) 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微克/孔之量子點含量處理細胞24小時，去除舊有培 養液，經鱗酸食鹽水緩衝液清洗兩次，加入新培養液後，以螢光顯微鏡觀 察。（圈七） 利用抗體修飾量子點脂質奈米粒子，專一性標定氨基呔酶CD13過度表現 之人類肺腺癌細胞(CD13+CL1-0細胞)〇氨基呔酶過度表現之人類肺腺癌細 胞CD13+ CL1-0細胞以10萬/孔的密度種植於12孔之培養盤内，15 小時後以量子點、量子點中性脂質奈米粒子，以及經抗氨基呔酶CD13之抗 • 體表面修飾之量子點中性脂質奈米粒子1.25微克/孔之量子點含量處理細胞 16小時’去除舊有培養液，經磷酸食鹽水緩衝液清洗兩次，加入新培養液 後，以螢光顯微鏡觀察。（圓八） 利用抗艟修飾量子點脂質奈米粒子，專一性標定人類乳腺癌細胞AU565細 胞。（A-Η)人類乳腺癌細胞AU565細胞以1萬/孔的密度種植於12孔 之培養盤内’ 15小時後以量子點、量子點中性脂質奈米粒子 '經人類免疫 球蛋白G(IgG)表面修飾之量子點中性脂質奈米粒子，以及經抗人類上皮生Different Formulated Lipid Nanoparticle Size Lipid Component Particle 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 10 1342787 GEC-Chol: (2-fox 3-beta-[N-(2-guanidinoethyl)carbamoyn-cholesterol) PEG-DSPE: Polyethylenic acid-bis-stearate phospholipid ethanolamine (PEG2000-1,2- Distearoyl-sn-glycero-3-phosphoethanolamine) Table 3 The size of the lipid nanoparticle synthesized by different formulations and its relative triple frequency signal lipid component GEC-Chol Choi PEG- DSPE does not contain the oxidized oxidized susceptibility particle size (nm> Triple frequency signal 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: (2-mercaptoethyl)-amine A 3-beta-[N-(2-guanidinoethyl)carbamoyl]-cholestero|) Choi: Cholesterol PEG-DSPE: Polyethanol-bisstearic acid Phospholipid Ethylamine (PEG2000-1,2-distearoyl-sn-glycero-3-phosphoethanolamine) Because of the frequency-doubled technique, non-invasive, non-energy-generating microscopy, and irrelevant photobleaching can be used to observe living organisms, Three-dimensional (3D) images of living cells or other types of biological samples, but without the developer, this technique can only be used to observe changes in tissue or cell type. The figure shows that we use lipids containing metals or non-metals. Nanoparticles are used as image contrast agents for triple frequency microscopy to study examples of markers for cancer and stem cells. Therefore, the use of multiple image developers in conjunction with the present invention can extend this technique to more microscopic and precise. The application of molecular detection to image detection. 11 ΙΓ ·'! 1342787 [Embodiment] The effect of magnets on the transport of iron oxide lipid nanoparticles (MION-micelles) to human cervical cancer cells.-HeLa cells, • Cells are planted at a density of 50,000/well. Iron oxide lipid nanoparticles (100% PEG-DSPE and trace amounts of Rhodamine-DOPE; a, b) or iron oxide at a concentration of 1 nmole/well after 15 hours in the well plate Positive-valency lipid nanoparticles (25% PEG-DSPE, 750/〇DOTAP and trace amounts of Rhodamine-DOPE; c, d) treat cells in the presence of (b, d) or without (a, c) magnets. Hour' and the results of cell loading of iron oxide lipid nanoparticles under different conditions were recorded by fluorescence image (magnification: 1〇〇). (Fig. 1) Time-dependent effect of iron oxide lipid nanoparticles (MION-miceHes) in human cervical cancer cells HeLa cells and its effect on cell growth. Cells were seeded in 24-well culture plates at a density of 50,000/zab. After 15 hours, the cells were treated with iron oxide lipid nanoparticles (25% PEG-DSPE, 75% DOTAP and trace amounts of Rhodamine-DOPE) at a Naimor number/well concentration for 12 hours. After the unloaded lipid nanoparticles were washed with phosphate buffered saline (PBS), the cells were cultured in a cell culture medium containing 1% fetal calf serum. The attached cells were trypsinized at different time points, and the number of cells was counted by a cell counter. (Fig. 2) The magnet adsorption degree of the cells was tested with a magnet holder. After the cells were labeled with iron oxide nanoparticles, the magnets were used to test the degree of magnet adsorption of the cells, and the presence of iron oxide nanoparticles was confirmed. (Fig. 3) 12 1342787 Short-chain antisense nucleotides are delivered by quantum dot positive-valency lipid nanoparticles. Human cervical cancer HeLa cells were seeded at a density of 10,000/well in a r·12-well culture plate containing glass slides. After 15 hours, 1 μg (Ug) of short-chain antisense nucleotides were fluoresced. * (antisense oligodeoxynucleotide, antisense ODN) was added to a mixture of 2.5 micrograms of quantum dot normal lipid nanoparticle. The cells were treated for 24 hours and washed twice with phosphate buffer. 'Fixed cells with 4% formic acid for 15 minutes. After washing twice with saline solution buffer, the fluorescent stain Hoechst33342 was used for nuclear staining and observed by a conjugated fluorescence microscope. φ (Fig. 4) Delivery of short-bond antisense nucleotides with iron oxide-compensated lipid nanoparticles. Human breast cancer cells AU565, rat bone marrow stem cells rBMSC and human cervical cancer cells HeLa cells were planted at a density of 10,000/well. After 15 hours in a 12-well culture dish containing glass slides, 1 μg of a mixture of short-chain antisense nucleotides fluorescein and 2.5 μg of quantum dot-valency lipid nanoparticles were added and the cells were treated for 24 hours. After washing twice with phosphate buffer solution, the cells were fixed with 4 ° / furfural for 15 minutes, washed three times with phosphate buffer solution, and stained with fluorescent staining agent Hoechst 33342, and observed by a co-roller fluorescence microscope. (Ruan 5) Rat bone marrow stem cells (rBMSC) were calibrated using quantum dot normal-valency lipid nanoparticles, and the relationship between the cell standard quantitation and the treatment dose was shown. Rat bone marrow stem cell rBMSC cells were seeded in a 12-well culture dish at a density of 10,000/well. After 5 hours, cells were treated with quantum dots at 125, 2.5 and 5 μg/well (pg / well) for 24 hours. The old culture medium was washed twice with a phosphate buffer solution, and after adding a new culture solution, it was observed by a fluorescence microscope at 13 1342787. (Circle 6) The use of quantum dot lipid nanoparticles to calibrate human osteophyte stem cells (hBMSC) and utilize TAT wins, * peptide modification to improve delivery efficiency. ' (A) Human bone marrow stem cells hBMSC cells were seeded in a 12-well culture dish at a density of 10,000/well. After 15 hours, they were surface-modified with quantum dot neutral lipid nanoparticles and streptavidin. The quantum dot neutral lipid nanoparticle, and the quantum dot content of the quantum dot neutral lipid nanoparticle modified by TAT peptide on the surface of the nanoparticle of 2.5 micrograms/hole, the cells were treated for 24 hours, and the old culture solution was removed, and the scalar brine was removed. The buffer was washed twice, and after adding a new culture solution, it was observed with a fluorescence microscope. (Circle 7) Using human antibodies to modify quantum dot lipid nanoparticles, specifically calibrating human lung adenocarcinoma cells (CD13+CL1-0 cells) with over-expression of aminoguanidine CD13, human lung adenocarcinoma CD13+ CL1-0 cells were seeded in a 12-well culture dish at a density of 100,000/well. After 15 hours, the cells were modified with quantum dots, quantum dot neutral lipid nanoparticles, and anti-aminopurine CD13. Quantum Dots Neutral Lipid Nanoparticles 1.25 μg/well Quantum Dot Content Treatment of cells for 16 hours 'Removal of the old culture medium, washing twice with phosphate buffer solution, adding a new culture solution, and observing with a fluorescence microscope. (Yuan 8) Using the anti-mite modified quantum dot lipid nanoparticles, the human breast cancer cell AU565 cells were specifically calibrated. (A-Η) Human breast cancer cells AU565 cells were seeded in a 12-well culture dish at a density of 10,000/well '15 hours later with quantum dots, quantum dot neutral lipid nanoparticles' via human immunoglobulin G ( IgG) surface-modified quantum dot neutral lipid nanoparticles, and anti-human epithelial
S 14 1342787 長因子接受器第2蛋白(human epidermal growth factor recept〇r 2,出⑺之抗 體賀平癌(Herceptin)表面修飾之量子點中性脂質奈米粒子丨25微克/孔之量 ，’子點含量處理細胞16小時，去除舊有培養液，經磷酸食鹽水緩衝液清洗兩 :.次，加入新培養液後，以螢光顯微鏡觀察。（I，J)人類乳腺癌細胞MCF7細 胞以1萬/孔的密度種植於12孔之培養盤内，μ小時後以經抗人類上 皮生長因子接受器第2蛋白之抗體賀平癌表面修飾之量子點中性脂質奈米 粒子1.25萬/孔之量子點含量處理細胞16小時，去除舊有培養液，經碟酸 # 食鹽水緩衝液清洗兩次，加入新培養液後，以螢光顯微鏡觀察。（圖九） 測量董子點脂質奈米粒子之訊號。 以鉻鎂撖欖石1230奈米(nm)之雷射，偵測410奈米之三倍頻訊號。（a)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。（b) 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 0 頻訊號及三光子螢光訊號分析。（圓十） 人類子宮頸癌細胞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9〇〇伏特(v) ^ (匕) 經處理量子點脂質奈米粒子，偵測光電倍增管電壓5〇〇伏特。（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 1342787 其三倍頻訊號。（圓十二) 顯示氧化鐵脂質奈米粒子之細胞吞適量與處理剤量之關係。 (A)人類子宮頸癌細胞HeLa細胞以1萬/孔的密度種植種植於12孔 . · 之培養盤内，15小時後分別以不同量之鐵含量處理細胞4小時，經磷酸食 鹽水緩衝液清洗後，以4%甲醛固定細胞15分鐘，再以磷酸食鹽水緩衝液 清洗三次後’觀察其三倍頻訊號。（B)分別為〇, 0.25, 0.5, 1.25, 2.5, 5, 7.5, 15, ^ 25微克/孔之鐵含量處理細胞4小時後之三倍頻訊號量化結果。（圖十三） 顯示氧化鐵脂質奈米粒子之細胞呑適量與處理時間之關係。 (A)人類子宮頸癌細胞HeLa細胞以1萬/孔的密度種植種植於12孔 之培養盤内’ 15小時後以7.5微克/孔之鐵含量處理細胞，分別在不同時間 點’將細胞經碟酸食鹽水緩衝液清洗後’以4〇/〇曱醛固定細胞15分鐘，再 以磷酸食鹽水緩衝液清洗三次後，觀察其三倍頻訊號。（B)以7 5微克/孔之 鐵含量處理細胞，分別在處理〇, 0.25, 0.5, 1，2, 4, 8, 16, 24小時後將細胞固 • 定’測量三倍頻訊號後之量化結果。（圓十四） 16 1342787 【圖式簡單說明】 圓一、外加磁場能加速氧化鐵奈米粒子被貼覆培養之人類子宮頸癌細胞 -‘ HeLa細胞呑堪》 « - 囷二、氧化鐵奈米粒子停留於人類子宮頭癌細胞HeLa細胞内之時效與其 對人類子宮頸癌細胞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 φ 度’證明氧化鐵奈米粒子存留的情況。 圊四、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、利用量子點正價脂質奈米粒子標定大鼠之骨髄幹細胞(rBMSC)，並 顯示其細胞標定量舆處理劑量之關係，顯示其生物相容性明顯提昇。 籲 圖七、利用量子點脂質奈米粒子標定人類之骨效幹細胞(hBMSC)，並利用 TAT胜肽修飾提高遞送效率》 圓八、利用抗艘修飾量子點脂質奈米粒子，專一性標定氨基呔酶CD13過 度表現之人類肺旅癌細胞(CD13+CL1-0細胞） 圖九、利用抗艟修飾董子點脂質奈米粒子，專一性標定人類乳腺癌細胞 AU565細胞。 ; £Γ ) 17 1342787 圖十、測量量子點脂質奈米粒子之倍頻訊號β 圖·^"一、人類子宮頸癌細胞HeLa細胞吞噬奈米粒子後顯示其水平切面之 三倍頻訊號。 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適量舆處理劑量之關係，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吞適量與處理時間之關係。 【主要元件符號說明】 奈米粒子：nanoparticles 量子點：quantum dotsS 14 1342787 Human epidermal growth factor recept〇r 2, the antibody (Herceptin) surface-modified quantum dot neutral lipid nanoparticle 丨25 μg/well, ' The cells were treated for 16 hours at the sub-point content, and the old culture solution was removed and washed with phosphate buffer solution for two times. After adding the new culture solution, it was observed by a fluorescence microscope. (I, J) Human breast cancer cells MCF7 cells were The density of 10,000/well was planted in a 12-well culture plate. After μ hours, the surface of the anti-human epithelial growth factor receptor 2 protein was modified with a quantum dot neutral lipid nanoparticle of 125,000/well. The cells were treated with quantum dots for 16 hours, and the old culture solution was removed, washed twice with a dish of acid acid saline, and then added with a new culture solution, and observed under a fluorescent microscope (Fig. 9). Signal of the particle. Detecting a triple-frequency signal of 410 nm with a laser of 1230 nm (nm) of chrome-magnesium sapphire. (a) Direct measurement of suspended quantum dot lipid nanoparticles in aqueous solution Triple frequency signal. (b) take less The quantum dot lipid nanoparticles are dried on a glass slide and measured to measure the triple frequency signal after drying. (c) Direct measurement of tripled 0-frequency signals and three-photon fluorescence generated by suspended quantum dot lipid nanoparticles in aqueous solution. Signal analysis. (Circle 10) Triple-frequency signal of a horizontal slice of HeLa cells in human cervical cancer cells (a) Untreated quantum dot lipid nanoparticles, detecting photomultiplier voltage 9 volts (v) ^ (匕) After processing the quantum dot lipid nanoparticles, the photomultiplier tube voltage is detected at 5 volts (Fig. 11). The triple frequency signal generated by different sizes of metal lipid nanoparticles is described. (A) Direct measurement The triple frequency signal generated by the iron oxide lipid nanoparticles suspended in the aqueous solution (B) was taken on a glass slide to dry a small amount of iron oxide lipid nanoparticles, and after drying, the triple frequency signal of 1342787 was measured. (Circle 12) The relationship between the amount of cell phagocytosis of iron oxide lipid nanoparticles and the amount of sputum was shown. (A) Human cervical cancer cells HeLa cells were planted in 12 wells at a density of 10,000/well. Different amounts The cells were treated with iron content for 4 hours. After washing with phosphate buffered saline, the cells were fixed with 4% formaldehyde for 15 minutes, and then washed three times with phosphate buffered saline to observe the triple frequency signal. (B) 0.25, 0.5, 1.25, 2.5, 5, 7.5, 15, ^ 25 μg/well of iron content Quantitative results of three-fold frequency signal after treatment of cells for 4 hours. (Fig. 13) Cells showing iron oxide lipid nanoparticles The relationship between the right amount and the treatment time. (A) Human cervical cancer cells HeLa cells were planted in a 12-well culture dish at a density of 10,000/well. After 15 hours, the cells were treated with 7.5 μg/well of iron, respectively. At different time points, 'the cells were washed with a dish of acid saline buffer', the cells were fixed with 4 〇/furfural for 15 minutes, and then washed three times with phosphate buffered saline to observe the triple frequency signal. (B) The cells were treated with iron content of 75 μg/well, and the cells were fixed after measuring 〇, 0.25, 0.5, 1, 2, 4, 8, 16 24 hours, respectively. Quantify the results. (Circle 14) 16 1342787 [Simple description of the diagram] Round one, external magnetic field can accelerate the oxidation of iron oxide nanoparticles to human cervical cancer cells--"HeLa cells" « - 囷二, iron oxide The aging of rice particles in HeLa cells of human uterine cancer cells and its effect on the growth of human cervical cancer cells HeLa cells. In Fig. 3, after the cells were labeled with iron oxide nanoparticles, the magnet adsorption phase φ degree of the cells was measured by a magnet holder to prove that the iron oxide nanoparticles remained. Fourth, the short-chain antisense nucleotides are efficiently delivered by quantum dot positive-valency lipid nanoparticles. Figure 5. Efficient delivery of short-chain antisense nucleotides with iron oxide positive-valency lipid nanoparticles. Figure 6. Calibration of rat osteophyte stem cells (rBMSC) using quantum dot-valence lipid nanoparticles and showing the relationship between the cell dose and the dose of guanidine, indicating a significant increase in biocompatibility. Figure 7. Identification of human bone-derived stem cells (hBMSC) using quantum dot lipid nanoparticles, and using TAT peptide modification to improve delivery efficiency. Round eight, using anti-canada modified quantum dot lipid nanoparticles, specificity calibration of aminoguanidine Human lung cancer cells (CD13+CL1-0 cells) with over-expressed enzyme CD13 Figure 9. The human breast cancer cell AU565 cells were specifically labeled with anti-tuberculosis modified Dongzi point lipid nanoparticles. ; £Γ ) 17 1342787 Figure 10. Measuring the frequency-doubled signal of the quantum dot lipid nanoparticle β. Figure 1. The human cervical cancer cell HeLa cells show the triple-frequency signal of the horizontal section after phagocytizing the nanoparticle. Round 12, indicating the triple frequency signal generated by different sizes of metal lipid nanoparticles. The relationship between the amount of 呑 and the amount of 舆 treatment of the iron oxide lipid nanoparticles showed that the lipid-modified nanoparticles had no significant biological toxicity. Round XIV shows the relationship between the amount of cell phagocytosis of iron oxide lipid nanoparticles and the treatment time. [Main component symbol description] Nanoparticles: nanoparticles quantum dots: 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: (2-胍基乙基)-胺曱基膽固醇 (3-beta-[N-(2-guanidinoethyl)carbamoyl]-cholesterol) 1342787 PEG-DSPE:聚乙醇-雙硬脂酸碟脂醯乙醇胺 (PEG2000 1,2 ^^stear〇yl-sn-gIycero-3-phosphoethanolamine) DOTAP: 1，2-二-(油醯基氧基)_3_(三甲基敍)丙烷 (1,2-bis (dioleoyloxy)-3-(tri-methylamonio) propane)Iron oxide nanoparticles: iron oxide nanoparticles positively charged lipids: cationic lipids positively charged cholesterol: cationic cholesterol positively charged double lipid chain lipids: cationic diacyllipid frequency doubled microscopy: harmonic generation microscopy GEC-Chol: (2-mercaptoethyl -3-beta-[N-(2-guanidinoethyl)carbamoyl]-cholesterol) 1342787 PEG-DSPE: Polyethanol-bis-stearic acid saponin ethanolamine (PEG2000 1,2 ^^stear〇yl -sn-gIycero-3-phosphoethanolamine) DOTAP: 1,2-bis-(oleoyloxy)_3_(trimethyls)propane (1,2-bis (dioleoyloxy)-3-(tri-methylamonio) propane )
Rhodamine-DOPE:羅丹明-雙油酸磷脂醯乙醇胺 (1，2-Dioleoyl-j«-Glycero-3-Phosphoethanolamine-N-(Lissamine Rhodamine B Sulfonyl))Rhodamine-DOPE: Rhodamine-D-Lilyoyl-j«-Glycero-3-Phosphoethanolamine-N-(Lissamine Rhodamine B Sulfonyl)
1919