CN113512415A - 一种细胞核靶向上转换荧光探针及其制备方法和应用 - Google Patents
一种细胞核靶向上转换荧光探针及其制备方法和应用 Download PDFInfo
- Publication number
- CN113512415A CN113512415A CN202110788639.0A CN202110788639A CN113512415A CN 113512415 A CN113512415 A CN 113512415A CN 202110788639 A CN202110788639 A CN 202110788639A CN 113512415 A CN113512415 A CN 113512415A
- Authority
- CN
- China
- Prior art keywords
- fluorescent probe
- conversion
- conversion fluorescent
- fluorescent
- cell nucleus
- 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.)
- Pending
Links
- 238000006243 chemical reaction Methods 0.000 title claims abstract description 50
- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 43
- 238000002360 preparation method Methods 0.000 title abstract description 11
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 54
- 239000002105 nanoparticle Substances 0.000 claims abstract description 36
- 210000003855 cell nucleus Anatomy 0.000 claims abstract description 28
- 230000008685 targeting Effects 0.000 claims abstract description 22
- 239000010410 layer Substances 0.000 claims abstract description 21
- 239000000377 silicon dioxide Substances 0.000 claims abstract description 20
- 239000003446 ligand Substances 0.000 claims abstract description 14
- 229910052814 silicon oxide Inorganic materials 0.000 claims abstract description 14
- 239000011247 coating layer Substances 0.000 claims abstract description 12
- 229910052761 rare earth metal Inorganic materials 0.000 claims abstract description 12
- 229940044683 chemotherapy drug Drugs 0.000 claims abstract description 10
- -1 rare earth ion Chemical class 0.000 claims abstract description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 21
- 239000008367 deionised water Substances 0.000 claims description 14
- 229910021641 deionized water Inorganic materials 0.000 claims description 14
- 238000000034 method Methods 0.000 claims description 13
- 230000004048 modification Effects 0.000 claims description 12
- 238000012986 modification Methods 0.000 claims description 12
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 9
- 230000008569 process Effects 0.000 claims description 9
- WOWHHFRSBJGXCM-UHFFFAOYSA-M cetyltrimethylammonium chloride Chemical compound [Cl-].CCCCCCCCCCCCCCCC[N+](C)(C)C WOWHHFRSBJGXCM-UHFFFAOYSA-M 0.000 claims description 7
- 239000002245 particle Substances 0.000 claims description 5
- 229920001184 polypeptide Polymers 0.000 claims description 4
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 4
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 4
- FZHAPNGMFPVSLP-UHFFFAOYSA-N silanamine Chemical compound [SiH3]N FZHAPNGMFPVSLP-UHFFFAOYSA-N 0.000 claims description 4
- 229910052710 silicon Inorganic materials 0.000 claims description 4
- 239000010703 silicon Substances 0.000 claims description 4
- BOTDANWDWHJENH-UHFFFAOYSA-N Tetraethyl orthosilicate Chemical compound CCO[Si](OCC)(OCC)OCC BOTDANWDWHJENH-UHFFFAOYSA-N 0.000 claims description 3
- 239000003795 chemical substances by application Substances 0.000 claims description 3
- 239000000523 sample Substances 0.000 claims description 3
- 239000004094 surface-active agent Substances 0.000 claims description 3
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 claims description 2
- 239000003054 catalyst Substances 0.000 claims description 2
- 239000011248 coating agent Substances 0.000 claims 1
- 238000000576 coating method Methods 0.000 claims 1
- 230000004807 localization Effects 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 claims 1
- 230000025308 nuclear transport Effects 0.000 claims 1
- 239000002246 antineoplastic agent Substances 0.000 abstract description 6
- 238000000799 fluorescence microscopy Methods 0.000 abstract description 5
- 230000035515 penetration Effects 0.000 abstract description 5
- 210000001519 tissue Anatomy 0.000 abstract description 5
- 150000002910 rare earth metals Chemical group 0.000 abstract description 4
- 230000035945 sensitivity Effects 0.000 abstract description 4
- 239000011148 porous material Substances 0.000 abstract description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 18
- 229940079593 drug Drugs 0.000 description 9
- 239000003814 drug Substances 0.000 description 9
- 239000000047 product Substances 0.000 description 9
- 210000004940 nucleus Anatomy 0.000 description 8
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 description 6
- 230000015572 biosynthetic process Effects 0.000 description 6
- 210000004027 cell Anatomy 0.000 description 6
- 230000004083 survival effect Effects 0.000 description 6
- 206010028980 Neoplasm Diseases 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 238000003786 synthesis reaction Methods 0.000 description 5
- XDTMQSROBMDMFD-UHFFFAOYSA-N Cyclohexane Chemical compound C1CCCCC1 XDTMQSROBMDMFD-UHFFFAOYSA-N 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 239000003937 drug carrier Substances 0.000 description 4
- 238000002073 fluorescence micrograph Methods 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 229910052786 argon Inorganic materials 0.000 description 3
- 230000001413 cellular effect Effects 0.000 description 3
- 238000010438 heat treatment Methods 0.000 description 3
- 238000003384 imaging method Methods 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 241000699670 Mus sp. Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000004140 cleaning Methods 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 238000002595 magnetic resonance imaging Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 230000032258 transport Effects 0.000 description 2
- 210000003462 vein Anatomy 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- WYTZZXDRDKSJID-UHFFFAOYSA-N (3-aminopropyl)triethoxysilane Chemical compound CCO[Si](OCC)(OCC)CCCN WYTZZXDRDKSJID-UHFFFAOYSA-N 0.000 description 1
- WRIDQFICGBMAFQ-UHFFFAOYSA-N (E)-8-Octadecenoic acid Natural products CCCCCCCCCC=CCCCCCCC(O)=O WRIDQFICGBMAFQ-UHFFFAOYSA-N 0.000 description 1
- LQJBNNIYVWPHFW-UHFFFAOYSA-N 20:1omega9c fatty acid Natural products CCCCCCCCCCC=CCCCCCCCC(O)=O LQJBNNIYVWPHFW-UHFFFAOYSA-N 0.000 description 1
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- QSBYPNXLFMSGKH-UHFFFAOYSA-N 9-Heptadecensaeure Natural products CCCCCCCC=CCCCCCCCC(O)=O QSBYPNXLFMSGKH-UHFFFAOYSA-N 0.000 description 1
- ZQPPMHVWECSIRJ-UHFFFAOYSA-N Oleic acid Natural products CCCCCCCCC=CCCCCCCCC(O)=O ZQPPMHVWECSIRJ-UHFFFAOYSA-N 0.000 description 1
- 239000005642 Oleic acid Substances 0.000 description 1
- 206010057249 Phagocytosis Diseases 0.000 description 1
- LDDQLRUQCUTJBB-UHFFFAOYSA-N ammonium fluoride Chemical compound [NH4+].[F-] LDDQLRUQCUTJBB-UHFFFAOYSA-N 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000003501 co-culture Methods 0.000 description 1
- 238000002591 computed tomography Methods 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 238000012632 fluorescent imaging Methods 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- QXJSBBXBKPUZAA-UHFFFAOYSA-N isooleic acid Natural products CCCCCCCC=CCCCCCCCCC(O)=O QXJSBBXBKPUZAA-UHFFFAOYSA-N 0.000 description 1
- 102000035118 modified proteins Human genes 0.000 description 1
- 108091005573 modified proteins Proteins 0.000 description 1
- CCCMONHAUSKTEQ-UHFFFAOYSA-N octadecene Natural products CCCCCCCCCCCCCCCCC=C CCCMONHAUSKTEQ-UHFFFAOYSA-N 0.000 description 1
- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(O)=O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 description 1
- 238000012831 peritoneal equilibrium test Methods 0.000 description 1
- 230000008782 phagocytosis Effects 0.000 description 1
- 238000012636 positron electron tomography Methods 0.000 description 1
- 238000012877 positron emission topography Methods 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- LIVNPJMFVYWSIS-UHFFFAOYSA-N silicon monoxide Chemical group [Si-]#[O+] LIVNPJMFVYWSIS-UHFFFAOYSA-N 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 238000005979 thermal decomposition reaction Methods 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 238000005303 weighing Methods 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
- C09K11/02—Use of particular materials as binders, particle coatings or suspension media therefor
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
- A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
- A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0063—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres
- A61K49/0069—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form
- A61K49/0089—Particulate, powder, adsorbate, bead, sphere
- A61K49/0091—Microparticle, microcapsule, microbubble, microsphere, microbead, i.e. having a size or diameter higher or equal to 1 micrometer
- A61K49/0093—Nanoparticle, nanocapsule, nanobubble, nanosphere, nanobead, i.e. having a size or diameter smaller than 1 micrometer, e.g. polymeric nanoparticle
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
- A61K9/5115—Inorganic compounds
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- 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
- C09K11/77—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials containing rare earth metals
- C09K11/7766—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials containing rare earth metals containing two or more rare earth metals
- C09K11/7772—Halogenides
- C09K11/7773—Halogenides with alkali or alkaline earth metal
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Epidemiology (AREA)
- Pharmacology & Pharmacy (AREA)
- Medicinal Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biomedical Technology (AREA)
- Organic Chemistry (AREA)
- Nanotechnology (AREA)
- Materials Engineering (AREA)
- Inorganic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Physics & Mathematics (AREA)
- Optics & Photonics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Immunology (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
本发明公开了一种细胞核靶上转换荧光探针及其制备方法和应用，所述荧光探针为壳核结构，其中，所述荧光探针的内核为稀土离子掺杂的上转换荧光纳米颗粒，所述荧光探针的外壳为介孔氧化硅包裹层；所述介孔氧化硅包裹层的介孔中装载有化疗药物，所述介孔氧化硅包裹层外表面嫁接细胞核靶向配体。本发明提出的荧光探针不仅能够通过介孔氧化硅层的介孔孔道装载化疗药物并靶向输送至细胞核，还能够通过稀土离子掺杂的上转换荧光纳米颗粒UCNPs实现NIR‑vis、NIR‑NIR上转换荧光成像，利用稀土离子掺杂的上转换荧光纳米颗粒具有较高灵敏度和皮肤组织穿透深度的特点，实现细胞核靶向输运和上转换荧光示踪定位。
Description
技术领域
本发明属于生物医学影像材料技术，具体涉及一种细胞核靶向上转换荧光探针及其制备方法和应用。
背景技术
众所周知，只有药物进入细胞核并且破坏核内的DNA，才能真正杀死癌细胞，目前相关领域内已经提出多种具有细胞核靶向功能的药物载体，用于将化疗药物直接运输至细胞核内。此外，高效的细胞核靶向输运离不开精确的影像示踪观察，目前，通常借助影像技术(荧光、PET、CT扫描、磁共振成像等)实时跟踪观察药物载体的细胞核靶向输运。但是，CT和MRI成像虽然具有很高的空间分辨率，但灵敏度较低；荧光影像虽然具有很高的灵敏度，但空间分辨率较低、组织穿透深度较浅。
因此，亟待开发一种具有细胞核靶向功能且组织穿透力强的荧光探针，便于全方位观察药物载体靶向进入细胞核。
发明内容
发明目的：本发明旨在提供一种细胞核靶向上转换荧光探针及其制备方法和应用，实现针对恶性肿瘤的细胞核靶向输运和上转换荧光影像精确定位。
发明内容：为实现上述目的，本发明提出以下技术方案：
一方面，本发明提出一种细胞核靶向上转换荧光探针，所述荧光探针为粒径小于50nm的壳核结构，其中，所述荧光探针的内核为稀土离子掺杂的上转换荧光纳米颗粒，所述荧光探针的外壳为介孔氧化硅包裹层；所述介孔氧化硅包裹层的介孔中装载有化疗药物，所述介孔氧化硅包裹层外表面嫁接TAT多肽作为细胞核靶向配体。
所述荧光探针以稀土离子掺杂的上转换荧光纳米颗粒UCNPs作为内核，可以用于NIR-vis、NIR-NIR上转换荧光成像，具有较高的灵敏度和皮肤组织穿透深度；所述荧光探针中介孔氧化硅层的介孔孔道用于装载化疗药物，从而实现诊疗一体化；介孔氧化硅层外表面嫁接的细胞核靶向配体赋予了荧光探针靶向细胞核输运功能，从而实现细胞核靶向输运和上转换荧光示踪定位。
另一方面，本发明还提出一种制备所述细胞核靶向上转换荧光探针的方法，包括步骤：
S1、制备稀土离子掺杂的上转换荧光纳米颗粒；
S2、用盐酸对所述上转换荧光纳米颗粒进行亲水改性；
S3、对进行亲水改性后的上转换荧光纳米颗粒进行介孔氧化硅层包裹；
S4、在所述介孔氧化硅层外表面嫁接细胞核靶向配体：先利用氨基羧基结合反应，对细胞核靶向配体进行氨基硅烷修饰；然后基于硅羟基键结合的原理，将进行氨基硅烷修饰后的配体通过共价键结合的形式嫁接于介孔氧化硅层的外表面，得到所述细胞核靶向上转换荧光探针。
对于所述制备方法，以下还提供了若干可选方式，但并不作为对上述总体方案的额外限定，仅仅是进一步的增补或优选，在没有技术或逻辑矛盾的前提下，各可选方式可单独针对上述总体方案进行组合，还可以是多个可选方式之间进行组合。
可选的，所述步骤S3中，对进行亲水改性后的上转换荧光纳米颗粒进行介孔氧化硅层包裹的具体步骤为：将进行亲水改性后的上转换荧光纳米颗粒溶于水，并加入表面活性剂CTAC作为造孔剂，加入TEA作为催化剂，再加入硅源TEOS，整个过程在80度水浴中进行，反应1～2h后，离心收集样品，最后分散在去离子水中，得到介孔氧化硅层包裹的上转换荧光纳米颗粒。
再一方面，本发明还提出所述细胞核靶向上转换荧光探针在细胞核靶向输运过程中进行荧光示踪定位的应用。
有益效果：与现有的具有细胞核靶向功能的药物载体相比，本发明至少具有以下优势：
1、本发明提出的荧光探针不仅能够通过介孔氧化硅层的介孔孔道装载化疗药物并靶向输送至细胞核，还能够通过稀土离子掺杂的上转换荧光纳米颗粒UCNPs实现NIR-vis、NIR-NIR上转换荧光成像，利用稀土离子掺杂的上转换荧光纳米颗粒具有较高灵敏度和皮肤组织穿透深度的特点，实现细胞核靶向输运和上转换荧光示踪定位。
2、本发明合成工艺简单易行、制备成本低、效率高，所制得的材料具有非常好的分散性、稳定性以及生物相容性，在生物医学影像领域具有广阔的应用前景，尤其适用于细胞核靶向药物输运和荧光影像示踪观察。
附图说明
图1为实施例1涉及的细胞核靶向上转换荧光探针在合成过程中各个阶段产物的结构示意图；
图2为实施例2涉及的细胞核靶向上转换荧光探针的合成流程图；
图3为实施例1涉及的细胞核靶向上转换荧光探针合成过程中每步产物的TEM照片：图3(a)为上转换荧光纳米颗粒UCNPs，图3(b)为进行亲水改性后的上转换荧光纳米颗粒H+-UCNPs，图3(c)为包裹了介孔氧化硅层的上转换荧光纳米颗粒UCMSNs；
图4为实施例1涉及的细胞核靶向上转换荧光探针UCMSNs-TAT在细胞水平的生物安全性评价结果对比图；图4(a)为培养了24h的存活率，图4(b)为培养了48h的存活率；
图5为实施例1涉及的细胞核靶向上转换荧光探针UCMSNs-TAT通过尾静脉注射入荷MCF-7/ADR耐药肿瘤小鼠后的荧光成像图：图5(a)为明场像，图5(b)为上转换荧光成像，图5(c)为图5(a)和图5(b)的叠加图。
具体实施方式
下面将结合附图和具体实施例对本发明作更进一步的说明。但应当理解的是，本发明可以以各种形式实施，以下在附图中出示并且在下文中描述的一些示例性和非限制性实施例，并不意图将本发明限制于所说明的具体实施例。
实施例1：
本实施例旨在提供一种细胞核靶向上转换荧光探针，所述荧光探针为粒径小于50nm的壳核结构，其中，所述荧光探针的内核为稀土离子掺杂的上转换荧光纳米颗粒，所述荧光探针的外壳为介孔氧化硅包裹层；所述介孔氧化硅包裹层的介孔中装载有化疗药物，所述介孔氧化硅包裹层外表面嫁接TAT多肽作为细胞核靶向配体。
本实施例中所述荧光探针的结构如图1所示，本实施例优选采用钆掺杂的上转换荧光纳米颗粒，化学式为NaYF4:Yb/Er/Tm/Gd。本实施例将钆掺杂上转换荧光纳米颗粒UCNPs与介孔氧化硅MSNs复合于同一结构体系中，得到包裹了介孔氧化硅层的上转换荧光纳米颗粒UCMSNs，保证了良好的分散性和稳定性。通过嫁接细胞核核靶向配体TAT，得到具有细胞核靶向功能的上转换荧光探针UCMSNs-TAT，用于细胞核靶向药物输运和荧光影像示踪观察。
实施例2：
本实施例针对实施例1中所述的细胞核靶向上转换荧光探针，提出其制备方法，具体流程如图2所示，包括以下步骤：
S1、采用高温分解法制备钆掺杂的上转换荧光纳米颗粒
1.1、稀土氯化物的准备。分别称取1.28mmol(388.3008mg)YCl3-6H2O，0.36mmol(139.4964mg)YbCl3-6H2O,0.04mmol(15.2684mg)ErCl3-6H2O,0.02mmol(5.5087mg)TmCl3，0.3mmol(79.083mg)GdCl3-6H2O。这些粉末放置于同一个样品瓶中，然后用4mL去离子水溶解，转入100mL三口烧瓶中。
1.2、向三口瓶中加入15mL油酸，30mL十八烯，室温下搅拌1h。然后开始通5min氩气，除去瓶中的空气，体系开始进行缓慢的除水过程。先升温到80度，保持1h或者更长时间(将自由水除尽)；随后升到120度，保持1h；然后升到156度左右，保持1h，获得黄色澄清液。停止加热，让体系自然降温至室温。
1.3、NaOH(上海凌锋)-200mg，NH4F(Sigma)-296.3mg，用10mL甲醇溶解，超声加速分散。然后小心加入体系中。室温下搅拌2h，促进离子之间的交换和前驱体的形成。期间，将氩气撤去，塞住三口瓶。
1.4、2h后，体系开始进入除甲醇环节。同样是缓慢进行。先通5min氩气，然后升温到50度，1h；80～100度，1h。直到体系中看不见白色气泡为止，表明甲醇已除完。也可以升到120度，保持1h；确保甲醇基本去除干净。
1.5、甲醇除完之后。将冷凝管接好，然后开始升温，将最后的温度稳定在280度左右，并保持1.5h。自然降至室温。
1.6、清洗过程：首先，向体系中加入20mL无水乙醇，搅拌30min。然后分装到两个50mL离心管中，用11000r/min，离心10min；收集到略带黄色的产物(第一次)。随后，各加入5～10mL环己烷，小心摇晃，并超声，可以发现产物迅速溶解，得到浑浊溶液，然后加入15mL无水乙醇，超声约5min，离心收集(第二次)。重复清洗3～5次。最后产物用20mL环己烷分散，获得100mM内核。
步骤S1所制备的钆掺杂上转换荧光纳米颗粒的粒径尺寸约为19nm，其化学组成为NaYF4:Yb/Er/Tm/Gd。具有钆掺杂上转换荧光纳米颗粒在980nm近红外光的激发下，不仅可以发出可见光，还可以发出紫外光和800nm近红外光。
S2、用盐酸对所述上转换荧光纳米颗粒进行亲水改性
将2.5mL 100mM UCNPs的环己烷溶液加入到5mL去离子水中，随后滴加20μL的浓盐酸，密封搅拌2h。UCNPs便从上层的环己烷转移到去离子水中，离心收集。整个过程用去离子水清洗并且超声分散，重复三次，最后改性后的H+-UCNPs分散在10mL去离子水中。
S3、对进行亲水改性后的上转换荧光纳米颗粒进行介孔氧化硅层包裹
将2g CTAC和0.02g TEA溶于20mL去离子水中，室温下搅拌1.5h。随后加入H+-UCNPs的去离子水溶液，继续搅拌1.5h。然后将整个反应体系转移到80度水浴中，逐滴加入200μL TEOS，反应1h后立即取出来。之后先用乙醇反复清洗3遍，再将其溶于质量分数为1wt％的NaCl的甲醇溶液中，室温下搅拌3h，整个过程持续3遍，直至CTAC完全除去为止。最后，将产物UCMSNs分散至10mL去离子水中。
S4、在所述介孔氧化硅层外表面嫁接细胞核靶向配体
将38mg EDC，57mg NHS和200μg TAT溶解于8mL去离子水中，搅拌5分钟后，加入45μL APTES，继续反应24h。然后逐滴加入2mL UCMSNs的去离子水溶液，继续反应24h后，用去离子水清洗三遍，最后，将产物UCMSNs-TAT分散在5mL去离子水中。
图3显示了UCMSNs的合成过程中每步产物的TEM照片。其中，图3(a)为上转换荧光纳米颗粒UCNPs；图3(b)为进行亲水改性后的上转换荧光纳米颗粒H+-UCNPs；图3(c)为包裹了介孔氧化硅层的上转换荧光纳米颗粒UCMSNs。从图中可以看出，每步合成的产物均呈现规则均一的球形，且具有非常好的分散性和稳定性，便于后续的生物学效应研究。
为验证上述细胞核靶向上转换荧光探针UCMSNs-TAT在细胞水平的荧光成像效果，我们进行了以下实验：首先，将不同浓度的UCMSNs-TAT与MCF-7/ADR耐药细胞共培养24h和48h后，利用MTT试剂检测UCMSNs-TAT的细胞相容性。其次，将UCMSNs-TAT与MCF-7/ADR细胞共培养24h后，利用DAPI染色细胞核，然后在共聚焦显微镜下观察UCMSNs-TAT靶向进入MCF-7/ADR细胞核内的荧光影像。
图4显示了MCF-7/ADR耐药细胞与不同浓度的UCMSNs-TAT共培养24h和48h后存活率，其表明了细胞核靶向上转换荧光探针UCMSNs-TAT在细胞水平的生物安全性评价，其中，图4(a)为培养了24h的存活率，图4(b)为培养了48h的存活率。从图中可以看出，MCF-7/ADR耐药细胞与不同浓度的UCMSNs-TAT共培养24h和48h后，仍然保持接近100％左右的存活率，表明UCMSNs-TAT具有非常好的生物相容性。
图5为细胞核靶向上转换荧光探针UCMSNs-TAT通过尾静脉注射入荷MCF-7/ADR耐药肿瘤小鼠后的荧光成像图，其中，图5(a)为明场像，图5(b)为上转换荧光成像，图5(c)为图5(a)和图5(b)的叠加图。从图中可以看出，MCF-7/ADR耐药肿瘤区出现了明显的荧光信号，说明UCMSNs-TAT可以靶向富集到肿瘤部位。
综上所述，本实施例首先采用高温热分解法，制备粒径尺寸约为19nm的钆掺杂上转换荧光纳米颗粒UCNPs(NaYF4:Yb/Er/Tm/Gd)；然后，直接用微量浓盐酸对其进行亲水改性(记作H+-UCNPs)；接着，以表面活性剂CTAC作为造孔剂，在H+-UCNPs表面均匀包裹一层厚度可控的介孔氧化硅，得到尺寸在50nm以下的介孔氧化硅包裹的UCNPs(记作UCMSNs)。最后，在UCMSNs的外表面修饰细胞核靶向配体多肽TAT，从而得到具有细胞核靶向功能的上转换荧光探针(记作UCMSNs-TAT)。其中，内核UCNPs具有上转换荧光成像功能；介孔氧化硅结构可以用于后续载药，TAT可以高效靶向细胞核。本发明的合成工艺简单易行、制备成本低、效率高。所制得的材料具有非常好的分散性、稳定性以及生物相容性，并且整体尺寸可以控制在50nm以下，可望减少免疫系统(RES)的吞噬，在生物医学影像领域具有广阔的应用前景，尤其适用于细胞核靶向药物输运和荧光影像示踪观察。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。
Claims (4)
1.一种细胞核靶向上转换荧光探针，其特征在于，所述荧光探针为粒径小于50nm的壳核结构，其中，所述荧光探针的内核为稀土离子掺杂的上转换荧光纳米颗粒，所述荧光探针的外壳为介孔氧化硅包裹层；所述介孔氧化硅包裹层的介孔中装载有化疗药物，所述介孔氧化硅包裹层外表面嫁接TAT多肽作为细胞核靶向配体。
2.一种制备如权利要求1所述细胞核靶向上转换荧光探针的方法，其特征在于，包括步骤：
S1、制备稀土离子掺杂的上转换荧光纳米颗粒；
S2、用盐酸对所述上转换荧光纳米颗粒进行亲水改性；
S3、对进行亲水改性后的上转换荧光纳米颗粒进行介孔氧化硅层包裹；
S4、在所述介孔氧化硅层外表面嫁接细胞核靶向配体：先利用氨基羧基结合反应，对细胞核靶向配体进行氨基硅烷修饰；然后基于硅羟基键结合的原理，将进行氨基硅烷修饰后的配体通过共价键结合的形式嫁接于介孔氧化硅层的外表面，得到所述细胞核靶向上转换荧光探针。
3.根据权利要求2所述的制备细胞核靶向上转换荧光探针的方法，其特征在于，所述步骤S3中，对进行亲水改性后的上转换荧光纳米颗粒进行介孔氧化硅层包裹的具体步骤为：将进行亲水改性后的上转换荧光纳米颗粒溶于水，并加入表面活性剂CTAC作为造孔剂，加入TEA作为催化剂，再加入硅源TEOS，整个过程在80度水浴中进行，反应1～2h后，离心收集样品，最后分散在去离子水中，得到介孔氧化硅层包裹的上转换荧光纳米颗粒。
4.如权利要求1所述细胞核靶向上转换荧光探针在细胞核靶向输运过程中进行荧光示踪定位的应用。
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110788639.0A CN113512415A (zh) | 2021-07-13 | 2021-07-13 | 一种细胞核靶向上转换荧光探针及其制备方法和应用 |
| PCT/CN2021/122096 WO2023284134A1 (zh) | 2021-07-13 | 2021-09-30 | 一种细胞核靶向上转换荧光探针及其制备方法和应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110788639.0A CN113512415A (zh) | 2021-07-13 | 2021-07-13 | 一种细胞核靶向上转换荧光探针及其制备方法和应用 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113512415A true CN113512415A (zh) | 2021-10-19 |
Family
ID=78067641
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110788639.0A Pending CN113512415A (zh) | 2021-07-13 | 2021-07-13 | 一种细胞核靶向上转换荧光探针及其制备方法和应用 |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN113512415A (zh) |
| WO (1) | WO2023284134A1 (zh) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023284134A1 (zh) * | 2021-07-13 | 2023-01-19 | 南京诺源医疗器械有限公司 | 一种细胞核靶向上转换荧光探针及其制备方法和应用 |
| CN116823836A (zh) * | 2023-08-30 | 2023-09-29 | 南京诺源医疗器械有限公司 | 适用于细胞核靶向上转换荧光探针的数据处理方法 |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103285409A (zh) * | 2013-06-25 | 2013-09-11 | 中国科学院上海硅酸盐研究所 | 具有化/放疗协同作用的磁共振/上转换荧光双模式成像造影剂及其制备方法 |
| CN103432597A (zh) * | 2013-08-08 | 2013-12-11 | 中国科学院上海硅酸盐研究所 | 一种基于肿瘤多模式协同治疗的新型多功能纳米诊疗剂及其制备方法 |
| WO2014200441A1 (en) * | 2013-06-14 | 2014-12-18 | National University Of Singapore | Core-shell fluorescent upconversion nanoparticles for photoactivation of multiple biomolecules |
| CN104288790A (zh) * | 2014-10-28 | 2015-01-21 | 中国科学院上海硅酸盐研究所 | 一种细胞核靶向放疗增敏剂及其制备方法 |
| CN104383539A (zh) * | 2014-10-28 | 2015-03-04 | 中国科学院上海硅酸盐研究所 | 一种细胞核靶向生物光子诊疗剂及其制备方法 |
| CN110947006A (zh) * | 2019-08-26 | 2020-04-03 | 上海大学 | 一种稀土上转换-铋诊疗一体化纳米杂化体系、制备方法及应用 |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104174027A (zh) * | 2014-09-15 | 2014-12-03 | 中国科学院上海硅酸盐研究所 | 肿瘤血管-肿瘤细胞膜-细胞核连续靶向药物输运体系及其制备方法和应用 |
| CN104804741B (zh) * | 2015-03-31 | 2017-04-12 | 复旦大学 | 一种单发射上转换纳米荧光探针及其合成方法 |
| CN113512415A (zh) * | 2021-07-13 | 2021-10-19 | 南京诺源医疗器械有限公司 | 一种细胞核靶向上转换荧光探针及其制备方法和应用 |
-
2021
- 2021-07-13 CN CN202110788639.0A patent/CN113512415A/zh active Pending
- 2021-09-30 WO PCT/CN2021/122096 patent/WO2023284134A1/zh unknown
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014200441A1 (en) * | 2013-06-14 | 2014-12-18 | National University Of Singapore | Core-shell fluorescent upconversion nanoparticles for photoactivation of multiple biomolecules |
| CN103285409A (zh) * | 2013-06-25 | 2013-09-11 | 中国科学院上海硅酸盐研究所 | 具有化/放疗协同作用的磁共振/上转换荧光双模式成像造影剂及其制备方法 |
| CN103432597A (zh) * | 2013-08-08 | 2013-12-11 | 中国科学院上海硅酸盐研究所 | 一种基于肿瘤多模式协同治疗的新型多功能纳米诊疗剂及其制备方法 |
| CN104288790A (zh) * | 2014-10-28 | 2015-01-21 | 中国科学院上海硅酸盐研究所 | 一种细胞核靶向放疗增敏剂及其制备方法 |
| CN104383539A (zh) * | 2014-10-28 | 2015-03-04 | 中国科学院上海硅酸盐研究所 | 一种细胞核靶向生物光子诊疗剂及其制备方法 |
| CN110947006A (zh) * | 2019-08-26 | 2020-04-03 | 上海大学 | 一种稀土上转换-铋诊疗一体化纳米杂化体系、制备方法及应用 |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023284134A1 (zh) * | 2021-07-13 | 2023-01-19 | 南京诺源医疗器械有限公司 | 一种细胞核靶向上转换荧光探针及其制备方法和应用 |
| CN116823836A (zh) * | 2023-08-30 | 2023-09-29 | 南京诺源医疗器械有限公司 | 适用于细胞核靶向上转换荧光探针的数据处理方法 |
| CN116823836B (zh) * | 2023-08-30 | 2023-11-07 | 南京诺源医疗器械有限公司 | 适用于细胞核靶向上转换荧光探针的数据处理方法 |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2023284134A1 (zh) | 2023-01-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Liu et al. | Controlled synthesis of uniform and monodisperse upconversion core/mesoporous silica shell nanocomposites for bimodal imaging | |
| US10987435B2 (en) | Ultrafine nanoparticles comprising a functionalized polyorganosiloxane matrix and including metal complexes; method for obtaining same and uses thereof in medical imaging and/or therapy | |
| CN113512415A (zh) | 一种细胞核靶向上转换荧光探针及其制备方法和应用 | |
| CN102782573A (zh) | 在治疗和诊断中应用的稀土掺杂上转换纳米颗粒 | |
| WO2022116672A1 (zh) | 一种肿瘤微环境响应型off-on上转换荧光探针及其制备方法和应用 | |
| CN108130069B (zh) | 稀土上转换纳米诊疗剂及其制备方法 | |
| CN108904800B (zh) | 基于纳米钯异质生长的上转换纳米杂化体系、制备方法及应用 | |
| Liu et al. | A smart tumor microenvironment responsive nanoplatform based on upconversion nanoparticles for efficient multimodal imaging guided therapy | |
| Yin et al. | Folic acid-conjugated organically modified silica nanoparticles for enhanced targeted delivery in cancer cells and tumor in vivo | |
| CN107812200B (zh) | Bsa-钆离子络合物包载的空心金纳米壳及制备方法 | |
| CN113927027B (zh) | 一种类病毒空心氧化锰负载近红外二b区激发的稀土纳米晶及其制备方法和应用 | |
| CN109172587A (zh) | 一种pH响应双药物释放的金属有机框架-上转换纳米体系的制备方法与应用 | |
| CN109078196B (zh) | 一种骨髓间充质干细胞介导的纳米水凝胶及其制备和应用 | |
| CN106635021A (zh) | 一种稀土掺杂纳米颗粒的制备方法 | |
| US20230109863A1 (en) | Compositions and methods for targeted delivery of therapeutic and/or diagnostic species | |
| CN109674764A (zh) | 一种抗肿瘤磁性载药杂化纳米胶囊及其制备方法 | |
| CN104383539B (zh) | 一种细胞核靶向生物光子诊疗剂及其制备方法 | |
| Rodponthukwaji et al. | Facile synthesis of magnetic silica-mannan nanocomposites for enhancement in internalization and immune response of dendritic cells | |
| CN113769115B (zh) | 一种上转换纳米诊疗一体化平台探针及其制备方法 | |
| CN109913201A (zh) | 基于黑磷的近红外二区荧光纳米探针及其制备和应用 | |
| Hu et al. | Dendritic cells reprogrammed by CEA messenger RNA loaded multi-functional silica nanospheres for imaging-guided cancer immunotherapy | |
| CN115607669A (zh) | 一种诊疗一体化稀土纳米颗粒及其制备方法 | |
| CN112870387B (zh) | 一种磁性纳米药物载体及其制备方法和应用 | |
| CN108619514B (zh) | 一种复合Au纳米粒子及其制备方法与应用 | |
| CN103007295A (zh) | 一种医用成像材料及其制备方法和应用 |
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 | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20211019 |
|
| RJ01 | Rejection of invention patent application after publication |