CN108559487B - 一种靶向线粒体的荧光纳米材料及其制备方法与应用 - Google Patents
一种靶向线粒体的荧光纳米材料及其制备方法与应用 Download PDFInfo
- Publication number
- CN108559487B CN108559487B CN201810410907.3A CN201810410907A CN108559487B CN 108559487 B CN108559487 B CN 108559487B CN 201810410907 A CN201810410907 A CN 201810410907A CN 108559487 B CN108559487 B CN 108559487B
- Authority
- CN
- China
- Prior art keywords
- fluorescent
- nano material
- mitochondrion
- fluorescent nano
- gold
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 239000002086 nanomaterial Substances 0.000 title claims abstract description 49
- 238000002360 preparation method Methods 0.000 title abstract description 10
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims abstract description 33
- 239000010931 gold Substances 0.000 claims abstract description 29
- 229910052737 gold Inorganic materials 0.000 claims abstract description 29
- RIOQSEWOXXDEQQ-UHFFFAOYSA-N triphenylphosphine Chemical compound C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1 RIOQSEWOXXDEQQ-UHFFFAOYSA-N 0.000 claims abstract description 16
- 239000003550 marker Substances 0.000 claims abstract description 10
- 239000000126 substance Substances 0.000 claims abstract description 4
- 230000008878 coupling Effects 0.000 claims abstract description 3
- 238000010168 coupling process Methods 0.000 claims abstract description 3
- 238000005859 coupling reaction Methods 0.000 claims abstract description 3
- 230000002438 mitochondrial effect Effects 0.000 claims description 15
- 239000003446 ligand Substances 0.000 claims description 9
- IZFHEQBZOYJLPK-SSDOTTSWSA-N (R)-dihydrolipoic acid Chemical compound OC(=O)CCCC[C@@H](S)CCS IZFHEQBZOYJLPK-SSDOTTSWSA-N 0.000 claims description 5
- 239000000463 material Substances 0.000 claims description 4
- 239000002245 particle Substances 0.000 claims description 2
- 210000003470 mitochondria Anatomy 0.000 abstract description 18
- 210000004027 cell Anatomy 0.000 abstract description 12
- 230000008685 targeting Effects 0.000 abstract description 11
- 238000006243 chemical reaction Methods 0.000 abstract description 10
- 238000000034 method Methods 0.000 abstract description 6
- 230000000694 effects Effects 0.000 abstract description 4
- 230000008569 process Effects 0.000 abstract description 4
- 239000002994 raw material Substances 0.000 abstract description 4
- 230000008901 benefit Effects 0.000 abstract description 3
- 239000000090 biomarker Substances 0.000 abstract description 3
- 239000000243 solution Substances 0.000 description 34
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Substances CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 description 10
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 8
- RUVJFMSQTCEAAB-UHFFFAOYSA-M 2-[3-[5,6-dichloro-1,3-bis[[4-(chloromethyl)phenyl]methyl]benzimidazol-2-ylidene]prop-1-enyl]-3-methyl-1,3-benzoxazol-3-ium;chloride Chemical compound [Cl-].O1C2=CC=CC=C2[N+](C)=C1C=CC=C(N(C1=CC(Cl)=C(Cl)C=C11)CC=2C=CC(CCl)=CC=2)N1CC1=CC=C(CCl)C=C1 RUVJFMSQTCEAAB-UHFFFAOYSA-M 0.000 description 6
- 239000002609 medium Substances 0.000 description 6
- 238000000108 ultra-filtration Methods 0.000 description 6
- 238000003384 imaging method Methods 0.000 description 5
- AYEKOFBPNLCAJY-UHFFFAOYSA-O thiamine pyrophosphate Chemical compound CC1=C(CCOP(O)(=O)OP(O)(O)=O)SC=[N+]1CC1=CN=C(C)N=C1N AYEKOFBPNLCAJY-UHFFFAOYSA-O 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 4
- 230000004913 activation Effects 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- 239000000975 dye Substances 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- DYAOREPNYXXCOA-UHFFFAOYSA-N 2-sulfanylundecanoic acid Chemical compound CCCCCCCCCC(S)C(O)=O DYAOREPNYXXCOA-UHFFFAOYSA-N 0.000 description 3
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 description 3
- 206010028980 Neoplasm Diseases 0.000 description 3
- 239000002246 antineoplastic agent Substances 0.000 description 3
- 229940041181 antineoplastic drug Drugs 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 230000008045 co-localization Effects 0.000 description 3
- 239000007850 fluorescent dye Substances 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 125000000524 functional group Chemical group 0.000 description 3
- 230000006872 improvement Effects 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 2
- 102000004142 Trypsin Human genes 0.000 description 2
- 108090000631 Trypsin Proteins 0.000 description 2
- 238000000862 absorption spectrum Methods 0.000 description 2
- 230000006907 apoptotic process Effects 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000005540 biological transmission Effects 0.000 description 2
- 238000006555 catalytic reaction Methods 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- IZFHEQBZOYJLPK-UHFFFAOYSA-N dihydrolipoic acid Chemical compound OC(=O)CCCCC(S)CCS IZFHEQBZOYJLPK-UHFFFAOYSA-N 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- IWBOPFCKHIJFMS-UHFFFAOYSA-N ethylene glycol bis(2-aminoethyl) ether Chemical compound NCCOCCOCCN IWBOPFCKHIJFMS-UHFFFAOYSA-N 0.000 description 2
- 238000000695 excitation spectrum Methods 0.000 description 2
- 238000005286 illumination Methods 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 238000004020 luminiscence type Methods 0.000 description 2
- 238000001000 micrograph Methods 0.000 description 2
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 239000012588 trypsin Substances 0.000 description 2
- -1 1-ethyl- (3-dimethylaminopropyl) -carbodiimides hydrochloride Chemical class 0.000 description 1
- BVKRDNIULHRLCO-UHFFFAOYSA-N 2-carboxyethyl(triphenyl)phosphanium;bromide Chemical compound [Br-].C=1C=CC=CC=1[P+](C=1C=CC=CC=1)(CCC(=O)O)C1=CC=CC=C1 BVKRDNIULHRLCO-UHFFFAOYSA-N 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 229920001661 Chitosan Polymers 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 125000000304 alkynyl group Chemical group 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000000692 anti-sense effect Effects 0.000 description 1
- 230000005775 apoptotic pathway Effects 0.000 description 1
- 125000000852 azido group Chemical group *N=[N+]=[N-] 0.000 description 1
- 230000003851 biochemical process Effects 0.000 description 1
- 238000004061 bleaching Methods 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 238000009510 drug design Methods 0.000 description 1
- 238000000295 emission spectrum Methods 0.000 description 1
- 125000003700 epoxy group Chemical group 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 238000002284 excitation--emission spectrum Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000004129 fatty acid metabolism Effects 0.000 description 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
- 238000001215 fluorescent labelling Methods 0.000 description 1
- 230000007674 genetic toxicity Effects 0.000 description 1
- 231100000025 genetic toxicology Toxicity 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 150000002634 lipophilic molecules Chemical class 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 210000003463 organelle Anatomy 0.000 description 1
- 230000010627 oxidative phosphorylation Effects 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 229910052697 platinum Inorganic materials 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 238000006862 quantum yield reaction Methods 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 230000004102 tricarboxylic acid cycle Effects 0.000 description 1
- CMSYDJVRTHCWFP-UHFFFAOYSA-N triphenylphosphane;hydrobromide Chemical compound Br.C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1 CMSYDJVRTHCWFP-UHFFFAOYSA-N 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/08—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials
- C09K11/58—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials containing copper, silver or gold
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y20/00—Nanooptics, e.g. quantum optics or photonic crystals
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/02—Use of particular materials as binders, particle coatings or suspension media therefor
- C09K11/025—Use of particular materials as binders, particle coatings or suspension media therefor non-luminescent particle coatings or suspension media
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
Landscapes
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Nanotechnology (AREA)
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Materials Engineering (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Optics & Photonics (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Crystallography & Structural Chemistry (AREA)
- Analytical Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Inorganic Chemistry (AREA)
- Pathology (AREA)
- Composite Materials (AREA)
- Condensed Matter Physics & Semiconductors (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
本发明属于纳米材料及生物标记领域，具体公开了一种靶向线粒体的荧光纳米材料及其制备方法与应用。本发明所述的荧光纳米材料由金纳米簇与三苯基膦或其衍生物通过化学共价偶联而成，所述金纳米簇优选为配体保护的金纳米簇。本发明所提供的荧光纳米材料中修饰有线粒体靶向分子，可以对细胞中的线粒体产生较好的靶向效果。且不需要外加荧光分子，本发明所述荧光纳米材料本身就具有优异发光性能，可作为生物荧光标记物应用。不仅如此，所述荧光纳米材料的原材料易得、且价格低廉，工艺简单、反应温和。
Description
技术领域
本发明属于纳米材料及生物标记领域，具体地说，涉及一种靶向线粒体的荧光纳米材料。
背景技术
线粒体是普遍存在于真核细胞中的一种重要细胞器，参与细胞内三羧酸循环、脂肪酸代谢、氧化磷酸化等多项重要的生理和生化过程，是细胞生存的关键。
近年来的研究表明，线粒体与细胞凋亡密切相关，因此线粒体可以作为抗肿瘤药物的靶点；通过诱导细胞凋亡的途径杀死肿瘤癌细胞，还可以克服铂类药物的耐药性和遗传毒性。基于线粒体的抗肿瘤药物研究，已经成为肿瘤药物设计的热点。
目前已报道的以线粒体凋亡途径为靶点的抗肿瘤药物大都是小分子化合物和反义核酸，但是这些小分子化合物存在水溶性低等缺陷。
荧光标记技术是开展细胞生物学研究的重要手段之一，通过对目标物的标记并结合荧光显微成像技术可以实现细胞及亚细胞生命过程可视化的分析及监测。金纳米簇由几到几十个金原子组成，其荧光性质随组成纳米簇的原子个数的增加而发生变化。2009年，Xie等(J.Am.Chem.Soc.，2009，131(3)，888-889)报道了利用牛血清白蛋白介导合成红色荧光金纳米簇，合成过程温和无毒，且产物量子产率较高。2009年，Lin等(ACS NANO，3(2)，395-401)报道了表面包被二氢硫辛酸DHLA的金纳米簇(AuNCs@DHLA)，并研究了AuNC@DHLA与细胞的非特异性吸附。2014年，Zhuang等(Biosensors and Bioelectronics 55，2014，76-82)报道了蓝色发光的金纳米簇，表面包裹壳聚糖后，与2-羧乙基三苯基溴化磷偶联，制备了可以靶向线粒体的金纳米探针。
然而，上述技术方案仍然存在以下不足：(1)光稳定性差，容易光漂泊，不适于长时间观察，大大限制了其应用范围；(2)双光子发光亮度低，不适于双光子成像。(3)生物相容性差。因此，需要开发一种光稳定性更高、双光子发光亮度高并靶向线粒体的荧光纳米材料。
发明内容
为了解决现有技术中存在的问题，本发明的目的是提供一种靶向线粒体的荧光纳米材料及其制备方法与应用。
为了实现本发明目的，本发明的技术方案如下：
第一方面，本发明提供了一种靶向线粒体的荧光纳米材料，所述荧光纳米材料由金纳米簇(Gold nanocluster，AuNCs)与三苯基膦或其衍生物通过化学共价偶联而成。
所述金纳米簇与三苯基膦或其衍生物之间通过1-乙基-(3-二甲基氨基丙基)-碳酰二亚胺盐酸盐和N-羟基琥珀酰亚胺反应或者点击化学(Click chemistry)反应形成肽键。
进一步地，所述金纳米簇优选为配体保护的金纳米簇。配体保护后的金纳米簇可更好地与三苯基膦或其衍生物通过化学共价实现偶联，并提高所述荧光纳米材料的水分散性。
在金纳米簇上修饰配体的技术手段，可参考《金纳米簇的制备及其在生物医学中的应用》(化学通报，2015年，第78卷)一文。本发明对现有技术的改进不在于此，且本领域技术人员依据现有技术可知晓具体的操作手段，故本发明对此不另做限定。
进一步地，所述配体保护的金纳米簇具有以下一种或几种表面官能团：-NH2、-COOH、-PO3、-SH、-S-S-、环氧基、叠氮基、炔基。
在本发明的具体实施方式中，以二氢硫辛酸和巯基十一烷酸作为配体为例进行示例性说明，二者作为配体保护金纳米簇后，其表面官能团为-COOH。
本领域技术人员应当理解，虽然本发明在具体实施方式中以上述两种配体为例进行示例性说明，但并不代表对配体的选择仅限于此。且由配体所带来的表面官能团也并不仅限于-COOH。
进一步地，所述荧光纳米材料的粒子直径优选为0.1-10nm。在该直径下，所述荧光纳米材料可在生物标记、纳米药物递送等方面发挥更佳作用。
进一步地，所述荧光纳米材料优选含有1-200个金原子，更有选为5-30个金原子，如此，更有利于发光效率保持及高效的生物标记。
第二方面，本发明提供了所述荧光纳米材料的制备方法，包括如下步骤：
(1)将配体保护的金纳米簇与三苯基膦或其衍生物配成溶液并混合，加入1-乙基-(3-二甲基氨基丙基)-碳酰二亚胺盐酸盐反应后，再加入N-羟基琥珀酰亚胺反应；
或：将配体保护的金纳米簇与三苯基膦或其衍生物配成溶液并混合，在Cu(I)催化下进行点击化学反应；
(2)将所得溶液用超滤管超滤或者离心，收集上清，即得。
作为一种示例性说明，所述荧光纳米材料的制备方法步骤包括：
a)将金纳米簇用水配成1-2mg/mL的溶液；
b)取三苯基溴化磷TPP，用水配成1-2mg/mL的溶液；
c)将步骤a)和步骤b)所配溶液混合，所得混合液中加入1-乙基-(3-二甲基氨基丙基)-碳酰二亚胺盐酸盐，搅拌条件下室温反应30-40min，再加入N-羟基琥珀酰亚胺反应30-40min；
或者：
将步骤a)和步骤b)所配溶液混合，Cu(I)催所下进行点击化学反应。
d)步骤c)所得溶液，用超滤管超滤或者离心3次，收集上清，即得。
第三方面，本发明所述的荧光纳米材料具有多种功能，既可用作生物荧光标记物，又可用作细胞中线粒体的靶向材料。
因此，本发明进一步提供了所述荧光纳米材料作为生物荧光标记物的应用，以及其作为靶向线粒体材料的应用。
本发明所提供的荧光纳米材料的优势在于，可以实现单光子成像/双光子成像。
且本发明所述荧光纳米材料与商业的线粒体Marker相比，双光子性质更优、光稳定性更好。
本发明所述荧光纳米材料在应用时，需在300～700nm的波长下进行单光子照射，或在760～1300nm的波长下进行双光子照射。
本发明涉及到的原料或试剂均为普通市售产品，涉及到的操作如无特殊说明均为本领域常规操作。
在符合本领域常识的基础上，上述各优选条件，可以相互组合，得到具体实施方式。
本发明的有益效果在于：
本发明提供了一种靶向线粒体的荧光纳米材料，其核心为金纳米簇AuNCs，具备激发发光的功能，且其表面暴露的氨基或羧基，可增加其水溶性和生物相容性，其进一步偶联的三苯基膦或其衍生物作为亲脂性分子，具备大量正电荷，可作为线粒体靶向分子。
进一步，本发明所提供的荧光纳米材料具有很好的双光子发光性质(105GM)，可以特异性靶向线粒体，实现单光子成像/双光子成像。与商业的线粒体Marker想比，本发明所提供的荧光纳米材料的双光子性质更优(商业marker双光子性质一般小于102GM)、光稳定性更好、灵敏度更高。
与现有技术相比，本发明所提供的荧光纳米材料中修饰有线粒体靶向分子，可以对细胞中的线粒体产生较好的靶向效果。且不需要外加荧光分子，本发明所述荧光纳米材料本身就具有优异发光性能，可作为生物荧光标记物应用。不仅如此，所述荧光纳米材料的原材料易得、且价格低廉，工艺简单、反应温和。
附图说明
图1为实施例1制备的靶向线粒体的荧光纳米探针AuNCs@DHLA-TPP的吸收谱、激发谱和发射谱图。
图2为实施例1制备的靶向线粒体的荧光纳米探针AuNCs@DHLA-TPP的透射电镜图。
图3为实施例1制备的靶向线粒体的荧光纳米探针AuNCs@DHLA-TPP对线粒体靶向效果的共聚焦图片。
图4为488nm光照条件下，实施例1制备的靶向线粒体的荧光纳米探针AuNCs@DHLA-TPP与商用线粒体染料MitoTracker荧光稳定性对比情况。
图5为800nm光照条件下，实施例1制备的靶向线粒体的荧光纳米探针AuNCs@MUA-TPP与商用线粒体染料MitoTracker荧光稳定性对比情况。
具体实施方式
下面将结合实施例对本发明的优选实施方式进行详细说明。需要理解的是以下实施例的给出仅是为了起到说明的目的，并不是用于对本发明的范围进行限制。本领域的技术人员在不背离本发明的宗旨和精神的情况下，可以对本发明进行各种修改和替换。
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1
本实施例以AuNCs@DHLA为核，偶联TPP制备靶向线粒体的荧光纳米探针AuNCs@DHLA-TPP。
1、二氢硫辛酸DHLA包裹的金纳米材料(AuNCs@DHLA)制备。
参照：Lin等，Synthesis,Characterization,and Bioconjugation ofFluorescent Gold Nanoclusters toward Biological Labeling Applications，ACSNANO，3(2)，395-401。
2、线粒体靶向金纳米材料AuNCs@DHLA-TPP的制备方法步骤：
(1)取AuNCs@DHLA溶液1mL(0.5mg/mL)，加PBS(pH7.4)4mL，配成0.1mg/mL的AuNCs@DHLA溶液a。
(2)向溶液a中加入0.4mg EDC(1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐)和0.3mg NHS(N-羟基琥珀酰亚胺)，活化15min后获得溶液b。
(3)向溶液b中加入2mg H2N-PEG-NH2(Mw：2000)，反应2h后超滤离心获得溶液c。
(4)取1mg TPP加入5mL PBS(pH 7.4)获得溶液d。
(5)向溶液d中加入0.4mg EDC和0.3mg NHS，活化15min后获得溶液e。
(6)将溶液c加入溶液e中反应5h后超滤离心获得AuNCs@DHLA-TPP溶液。
对获得的荧光纳米材料AuNCs@DHLA-TPP进行检测，其吸收谱、激发谱和发射谱图如图1所示，透射电镜图如图2所示。
实施例2
本实施例以AuNCs@MUA为核，偶联TPP制备靶向线粒体的荧光纳米探针AuNCs@MUA-TPP。
1、巯基十一烷酸MUA包裹的金纳米簇(AuNCs@MUA)制备。
参照：Lin等，Synthesis,Characterization,and Bioconjugation ofFluorescent Gold Nanoclusters toward Biological Labeling Applications，ACSNANO，3(2)，395-401。
2、线粒体靶向金纳米材料AuNCs@MUA-TPP的制备方法步骤：
(1)取AuNCs@MUA溶液1mL(0.5mg/mL)，加PBS(pH7.4)4mL，配成0.1mg/mL的AuNCs@DHLA溶液a。
(2)向溶液a中加入0.4mg EDC和0.3mg NHS，活化15min后获得溶液b。
(3)向溶液b中加入2mg H2N-PEG-NH2(Mw：2000)，反应2h后超滤离心获得溶液c。
(4)取1mg TPP加入5mL PBS(pH 7.4)获得溶液d。
(5)向溶液d中加入0.4mg EDC和0.3mg NHS，活化15min后获得溶液e。
(6)将溶液c加入溶液e中反应5h后超滤离心获得AuNCs@MUA-TPP溶液。
实施例3
本实施例用于说明实施例1和实施例2做制备的荧光纳米探针AuNCs@DHLA-TPP和AuNCs@MUA-TPP在线粒体靶向标记中的应用。
取对数生长期的HepG2细胞，用0.25％的胰蛋白酶消化后，以大约5×104个/cm2的密度接种于共聚焦培养皿中。加入0.2ml DMEM培养基，在含有5％CO2的培养箱中于37℃培养24h，取出后用10mM PBS冲洗除去培养基，将AuNCs@DHLA-TPP用DMEM培养基稀释200倍加入培养皿，在含有5％CO2的培养箱中于37℃培养8h。然后用商用线粒体染料MitoTrackergreen荧光染料(购自Thermo Fisher Scientific)标记线粒体，在荧光共聚焦显微镜(Zeiss，德国)下观察MitoTracker green通道与AuNCs@DHLA-TPP通道荧光共定位情况。在488nm激光照射下，相同条件下观察两种荧光物质的光稳定性。
荧光纳米材料AuNCs@DHLA-TPP对线粒体靶向效果的共聚焦图片如图3所示，由图可以看出，AuNCs@DHLA-TPP发出的红色信号与商用线粒体marker发出的绿色信号具有很好的共定位，该结果表明本发明所述的AuNCs@DHLA-TPP具有很好特异性靶向线粒体功能，可以作为线粒体Marker。
结果见图4，由图可以看出，AuNCs@DHLA-TPP比商用MitoTracker green具有更好的光稳定性，这有利于其在长时间动态观察等方面应用。
取对数生长期的HepG2细胞，用0.25％的胰蛋白酶消化后，以大约5×104个/cm2的密度接种于共聚焦培养皿中。加入0.2ml DMEM培养基，在含有5％CO2的培养箱中于37℃培养24h，取出后用10mM PBS冲洗除去培养基，将AuNCs@MUA-TPP用DMEM培养基稀释200倍加入培养皿，在含有5％CO2的培养箱中于37℃培养8h。然后用商用线粒体染料MitoTrackergreen荧光染料(购自ThermoFisher Scientific)标记线粒体，在荧光共聚焦显微镜(Zeiss，德国)下观察MitoTracker green通道与AuNCs@MUA-TPP通道荧光共定位情况。在800nm飞秒激光照射下，相同条件下观察两种荧光物质的光稳定性。
结果见图5，由图可以看出，AuNCs@DHLA-TPP比商用MitoTracker green具有更好的光稳定性。
应当理解的是，对上述实施例所用试剂或原料的用量进行等比例扩大或者缩小后的技术方案，与上述实施例的实质相同。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
Claims (7)
1.一种靶向线粒体的荧光纳米材料，其特征在于，所述荧光纳米材料由金纳米簇与三苯基膦或其衍生物通过化学共价偶联而成；所述金纳米簇为以二氢硫辛酸为配体保护的金纳米簇。
2.根据权利要求1所述的荧光纳米材料，其特征在于，所述荧光纳米材料的粒子直径为0.1-10nm。
3.根据权利要求2所述的荧光纳米材料，其特征在于，所述荧光纳米材料含有1-200个金原子。
4.权利要求1或2所述的荧光纳米材料作为生物荧光标记物的应用。
5.根据权利要求4所述的应用，其特征在于，在300～700nm的波长下进行单光子照射。
6.根据权利要求4所述的应用，其特征在于，在760～1300nm的波长下进行双光子照射。
7.权利要求1-3任一项所述的荧光纳米材料作为靶向线粒体材料的应用。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810410907.3A CN108559487B (zh) | 2018-05-02 | 2018-05-02 | 一种靶向线粒体的荧光纳米材料及其制备方法与应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810410907.3A CN108559487B (zh) | 2018-05-02 | 2018-05-02 | 一种靶向线粒体的荧光纳米材料及其制备方法与应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108559487A CN108559487A (zh) | 2018-09-21 |
| CN108559487B true CN108559487B (zh) | 2020-11-13 |
Family
ID=63537717
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810410907.3A Active CN108559487B (zh) | 2018-05-02 | 2018-05-02 | 一种靶向线粒体的荧光纳米材料及其制备方法与应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108559487B (zh) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110455430B (zh) * | 2019-08-16 | 2021-04-02 | 中国科学院理化技术研究所 | 一种靶向线粒体的荧光寿命温度传感器及其制备方法及应用 |
| CN114644836B (zh) * | 2021-03-09 | 2023-05-12 | 浙江大学 | 一种以芴环为母体的羧酸盐双光子染料、合成方法及应用 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103215032A (zh) * | 2013-04-24 | 2013-07-24 | 安徽大学 | 一种生物相容性强荧光金银混合团簇及其制备方法 |
| CN105499599A (zh) * | 2015-12-15 | 2016-04-20 | 安徽大学 | 一类小尺寸金纳米簇的制备方法及金纳米簇 |
-
2018
- 2018-05-02 CN CN201810410907.3A patent/CN108559487B/zh active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103215032A (zh) * | 2013-04-24 | 2013-07-24 | 安徽大学 | 一种生物相容性强荧光金银混合团簇及其制备方法 |
| CN105499599A (zh) * | 2015-12-15 | 2016-04-20 | 安徽大学 | 一类小尺寸金纳米簇的制备方法及金纳米簇 |
Non-Patent Citations (2)
| Title |
|---|
| Gold nanoclusters with bright near-infrared photoluminescence;Goutam Pramanik等;《Nanoscale》;20180108;第10卷;第3792-3798页 * |
| Targeted surface-functionalized gold nanoclusters for mitochondriali maging;Qianfen Zhuang等;《Biosensors and Bioelectronics》;20131210;第55卷;第76-82页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108559487A (zh) | 2018-09-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Iravani et al. | Green synthesis, biomedical and biotechnological applications of carbon and graphene quantum dots. A review | |
| Ali et al. | Red fluorescent carbon nanoparticle-based cell imaging probe | |
| Gandra et al. | Bacteriophage bionanowire as a carrier for both cancer-targeting peptides and photosensitizers and its use in selective cancer cell killing by photodynamic therapy | |
| Zhao et al. | Designing luminescent ruthenium prodrug for precise cancer therapy and rapid clinical diagnosis | |
| Weng et al. | Luminescent quantum dots: a very attractive and promising tool in biomedicine | |
| Hada et al. | Folic acid functionalized gold nanoclusters for enabling targeted fluorescence imaging of human ovarian cancer cells | |
| Wang et al. | Peptide‐engineered fluorescent nanomaterials: Structure design, function tailoring, and biomedical applications | |
| Zhuang et al. | Immunoglobulin G-encapsulated gold nanoclusters as fluorescent tags for dot-blot immunoassays | |
| Li et al. | Europium-complex-grafted polymer dots for amplified quenching and cellular imaging applications | |
| Dhamodharan et al. | Carbon nanodots: Synthesis, mechanisms for bio-electrical applications | |
| CN108559487B (zh) | 一种靶向线粒体的荧光纳米材料及其制备方法与应用 | |
| Zalmi et al. | Recent advances in aggregation-induced emission active materials for sensing of biologically important molecules and drug delivery system | |
| Kritchenkov et al. | Functionalized Pt (II) and Ir (III) NIR emitters and their covalent conjugates with polymer-based nanocarriers | |
| Guo et al. | A new approach to developing diagnostics and therapeutics: Aggregation‐induced emission‐based fluorescence turn‐on | |
| Yu et al. | Highly excretable gold supraclusters for translatable in vivo raman imaging of tumors | |
| Nasrin et al. | Conjugated ternary doped carbon dots from vitamin B derivative: Multispectral nanoprobes for targeted melanoma bioimaging and photosensitization | |
| Chen et al. | Far-red/near-infrared fluorescence light-up probes for specific in vitro and in vivo imaging of a tumour-related protein | |
| Ramya et al. | New insight of squaraine-based biocompatible surface-enhanced Raman scattering nanotag for cancer-cell imaging | |
| Srivastava et al. | Rational design of surface-state controlled multicolor cross-linked carbon dots with distinct photoluminescence and cellular uptake properties | |
| Chen et al. | Fluorogenic reactions in chemical biology: seeing chemistry in cells | |
| US20130216588A1 (en) | Insulin-gold nanocluster, pharmaceutical composition for reducing blood glucose comprising the same, and method for detecting adipose cells in tissue by using the same | |
| Mutas et al. | Specific binding and internalization: an investigation of fluorescent aptamer-gold nanoclusters and cells with fluorescence lifetime imaging microscopy | |
| Chen et al. | ATP-responsive near-infrared fluorescence MOF nanoprobe for the controlled release of anticancer drug | |
| Zhang et al. | A Lysosome-Targetable Fluorescence Probe Based on L-Cysteine-Polyamine-Morpholine-Modified Quantum Dots for Imaging in Living Cells | |
| Asgher et al. | Multifunctional materials conjugated with near-infrared fluorescent organic molecules and their targeted cancer bioimaging potentialities |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |