CN113842473A - 一种用于小梁类细胞标记的双模态纳米探针及其制备方法和应用 - Google Patents
一种用于小梁类细胞标记的双模态纳米探针及其制备方法和应用 Download PDFInfo
- Publication number
- CN113842473A CN113842473A CN202111193841.5A CN202111193841A CN113842473A CN 113842473 A CN113842473 A CN 113842473A CN 202111193841 A CN202111193841 A CN 202111193841A CN 113842473 A CN113842473 A CN 113842473A
- Authority
- CN
- China
- Prior art keywords
- plga
- nanoprobe
- solution
- spio
- bimodal
- 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.)
- Granted
Links
- 230000002902 bimodal effect Effects 0.000 title claims abstract description 30
- 238000002372 labelling Methods 0.000 title claims abstract description 8
- 238000002360 preparation method Methods 0.000 title abstract description 9
- 239000003814 drug Substances 0.000 claims abstract description 22
- 239000000523 sample Substances 0.000 claims abstract description 17
- 229940079593 drug Drugs 0.000 claims abstract description 15
- 239000003590 rho kinase inhibitor Substances 0.000 claims abstract description 9
- 238000012377 drug delivery Methods 0.000 claims abstract description 4
- 229920001606 poly(lactic acid-co-glycolic acid) Polymers 0.000 claims description 18
- 239000002105 nanoparticle Substances 0.000 claims description 17
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 16
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 14
- 241000399119 Spio Species 0.000 claims description 13
- 238000000034 method Methods 0.000 claims description 11
- 239000004372 Polyvinyl alcohol Substances 0.000 claims description 10
- 239000002245 particle Substances 0.000 claims description 10
- 229920002451 polyvinyl alcohol Polymers 0.000 claims description 10
- GRAVJJAQKJDGPM-UHFFFAOYSA-N 3-[2-[7-[3-(2-carboxyethyl)-1,1-dimethylbenzo[e]indol-3-ium-2-yl]hepta-2,4,6-trienylidene]-1,1-dimethylbenzo[e]indol-3-yl]propanoic acid;bromide Chemical compound [Br-].OC(=O)CCN1C2=CC=C3C=CC=CC3=C2C(C)(C)\C1=C/C=C/C=C/C=C/C1=[N+](CCC(O)=O)C2=CC=C(C=CC=C3)C3=C2C1(C)C GRAVJJAQKJDGPM-UHFFFAOYSA-N 0.000 claims description 9
- 230000001804 emulsifying effect Effects 0.000 claims description 8
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 6
- 239000002244 precipitate Substances 0.000 claims description 4
- 238000004945 emulsification Methods 0.000 claims description 3
- WTFXARWRTYJXII-UHFFFAOYSA-N iron(2+);iron(3+);oxygen(2-) Chemical compound [O-2].[O-2].[O-2].[O-2].[Fe+2].[Fe+3].[Fe+3] WTFXARWRTYJXII-UHFFFAOYSA-N 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims description 2
- 238000003756 stirring Methods 0.000 claims description 2
- 210000004027 cell Anatomy 0.000 abstract description 46
- 238000002595 magnetic resonance imaging Methods 0.000 abstract description 14
- 210000001585 trabecular meshwork Anatomy 0.000 abstract description 8
- 210000001742 aqueous humor Anatomy 0.000 abstract description 7
- 238000000799 fluorescence microscopy Methods 0.000 abstract description 7
- 238000003333 near-infrared imaging Methods 0.000 abstract description 5
- 230000037361 pathway Effects 0.000 abstract description 3
- 238000005481 NMR spectroscopy Methods 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 31
- 238000003384 imaging method Methods 0.000 description 9
- 239000000463 material Substances 0.000 description 8
- 230000000694 effects Effects 0.000 description 7
- 238000010586 diagram Methods 0.000 description 6
- 230000005540 biological transmission Effects 0.000 description 5
- 238000000338 in vitro Methods 0.000 description 5
- 238000001514 detection method Methods 0.000 description 4
- NGOGFTYYXHNFQH-UHFFFAOYSA-N fasudil Chemical compound C=1C=CC2=CN=CC=C2C=1S(=O)(=O)N1CCCNCC1 NGOGFTYYXHNFQH-UHFFFAOYSA-N 0.000 description 4
- 229960002435 fasudil Drugs 0.000 description 4
- 239000007850 fluorescent dye Substances 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- 238000010186 staining Methods 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- 208000010412 Glaucoma Diseases 0.000 description 3
- 206010057249 Phagocytosis Diseases 0.000 description 3
- OURRXQUGYQRVML-AREMUKBSSA-N [4-[(2s)-3-amino-1-(isoquinolin-6-ylamino)-1-oxopropan-2-yl]phenyl]methyl 2,4-dimethylbenzoate Chemical compound CC1=CC(C)=CC=C1C(=O)OCC1=CC=C([C@@H](CN)C(=O)NC=2C=C3C=CN=CC3=CC=2)C=C1 OURRXQUGYQRVML-AREMUKBSSA-N 0.000 description 3
- UREBDLICKHMUKA-CXSFZGCWSA-N dexamethasone Chemical compound C1CC2=CC(=O)C=C[C@]2(C)[C@]2(F)[C@@H]1[C@@H]1C[C@@H](C)[C@@](C(=O)CO)(O)[C@@]1(C)C[C@@H]2O UREBDLICKHMUKA-CXSFZGCWSA-N 0.000 description 3
- 229960003957 dexamethasone Drugs 0.000 description 3
- 238000009826 distribution Methods 0.000 description 3
- 230000005284 excitation Effects 0.000 description 3
- 229910001385 heavy metal Inorganic materials 0.000 description 3
- DCYOBGZUOMKFPA-UHFFFAOYSA-N iron(2+);iron(3+);octadecacyanide Chemical compound [Fe+2].[Fe+2].[Fe+2].[Fe+3].[Fe+3].[Fe+3].[Fe+3].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-] DCYOBGZUOMKFPA-UHFFFAOYSA-N 0.000 description 3
- 230000005415 magnetization Effects 0.000 description 3
- 230000008782 phagocytosis Effects 0.000 description 3
- 229960003351 prussian blue Drugs 0.000 description 3
- 239000013225 prussian blue Substances 0.000 description 3
- 230000008685 targeting Effects 0.000 description 3
- CMDJNMACGABCKQ-XVSRHIFFSA-N 4-fluoro-5-[[(2s)-2-methyl-1,4-diazepan-1-yl]sulfonyl]isoquinoline;dihydrate;hydrochloride Chemical compound O.O.Cl.C[C@H]1CNCCCN1S(=O)(=O)C1=CC=CC2=CN=CC(F)=C12 CMDJNMACGABCKQ-XVSRHIFFSA-N 0.000 description 2
- 101000979572 Homo sapiens NLR family CARD domain-containing protein 4 Proteins 0.000 description 2
- 101710196550 Myocilin Proteins 0.000 description 2
- 102100029839 Myocilin Human genes 0.000 description 2
- 102100023435 NLR family CARD domain-containing protein 4 Human genes 0.000 description 2
- KPKZJLCSROULON-QKGLWVMZSA-N Phalloidin Chemical compound N1C(=O)[C@@H]([C@@H](O)C)NC(=O)[C@H](C)NC(=O)[C@H](C[C@@](C)(O)CO)NC(=O)[C@H](C2)NC(=O)[C@H](C)NC(=O)[C@@H]3C[C@H](O)CN3C(=O)[C@@H]1CSC1=C2C2=CC=CC=C2N1 KPKZJLCSROULON-QKGLWVMZSA-N 0.000 description 2
- IYOZTVGMEWJPKR-IJLUTSLNSA-N Y-27632 Chemical compound C1C[C@@H]([C@H](N)C)CC[C@@H]1C(=O)NC1=CC=NC=C1 IYOZTVGMEWJPKR-IJLUTSLNSA-N 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 230000003915 cell function Effects 0.000 description 2
- 210000003855 cell nucleus Anatomy 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- 210000000805 cytoplasm Anatomy 0.000 description 2
- 210000004292 cytoskeleton Anatomy 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 230000004064 dysfunction Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000000684 flow cytometry Methods 0.000 description 2
- 239000012216 imaging agent Substances 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 230000001965 increasing effect Effects 0.000 description 2
- 229910010272 inorganic material Inorganic materials 0.000 description 2
- 239000011147 inorganic material Substances 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000000242 pagocytic effect Effects 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 229910052761 rare earth metal Inorganic materials 0.000 description 2
- 150000002910 rare earth metals Chemical class 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 201000004569 Blindness Diseases 0.000 description 1
- 102400000888 Cholecystokinin-8 Human genes 0.000 description 1
- 101800005151 Cholecystokinin-8 Proteins 0.000 description 1
- 238000000116 DAPI staining Methods 0.000 description 1
- 229910052688 Gadolinium Inorganic materials 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 206010030043 Ocular hypertension Diseases 0.000 description 1
- 206010030348 Open-Angle Glaucoma Diseases 0.000 description 1
- 108010009711 Phalloidine Proteins 0.000 description 1
- 238000003917 TEM image Methods 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000000862 absorption spectrum Methods 0.000 description 1
- 239000000695 adrenergic alpha-agonist Substances 0.000 description 1
- 229940006133 antiglaucoma drug and miotics carbonic anhydrase inhibitors Drugs 0.000 description 1
- 230000001640 apoptogenic effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000002876 beta blocker Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229910052793 cadmium Inorganic materials 0.000 description 1
- 239000003489 carbonate dehydratase inhibitor Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000000064 cholinergic agonist Substances 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 239000002872 contrast media Substances 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 238000002651 drug therapy Methods 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 230000004406 elevated intraocular pressure Effects 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- UIWYJDYFSGRHKR-UHFFFAOYSA-N gadolinium atom Chemical compound [Gd] UIWYJDYFSGRHKR-UHFFFAOYSA-N 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000004410 intraocular pressure Effects 0.000 description 1
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Substances [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 1
- UQSXHKLRYXJYBZ-UHFFFAOYSA-N iron oxide Inorganic materials [Fe]=O UQSXHKLRYXJYBZ-UHFFFAOYSA-N 0.000 description 1
- 238000010859 live-cell imaging Methods 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 239000002086 nanomaterial Substances 0.000 description 1
- 229950009210 netarsudil Drugs 0.000 description 1
- 231100001083 no cytotoxicity Toxicity 0.000 description 1
- 238000013421 nuclear magnetic resonance imaging Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- NDLPOXTZKUMGOV-UHFFFAOYSA-N oxo(oxoferriooxy)iron hydrate Chemical compound O.O=[Fe]O[Fe]=O NDLPOXTZKUMGOV-UHFFFAOYSA-N 0.000 description 1
- 230000000149 penetrating effect Effects 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 201000006366 primary open angle glaucoma Diseases 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 150000003180 prostaglandins Chemical class 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 230000000171 quenching effect Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 108700002895 trabecular meshwork-induced glucocorticoid response Proteins 0.000 description 1
- 238000001392 ultraviolet--visible--near infrared spectroscopy Methods 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/0002—General or multifunctional contrast agents, e.g. chelated agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
- A61K31/4409—Non condensed pyridines; Hydrogenated derivatives thereof only substituted in position 4, e.g. isoniazid, iproniazid
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/47—Quinolines; Isoquinolines
- A61K31/472—Non-condensed isoquinolines, e.g. papaverine
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
- A61K31/551—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having two nitrogen atoms, e.g. dilazep
-
- 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
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/32—Macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. carbomers, poly(meth)acrylates, or polyvinyl pyrrolidone
-
- 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
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
-
- 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
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
-
- 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
- A61K49/0054—Macromolecular compounds, i.e. oligomers, polymers, dendrimers
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
- A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
- A61K49/10—Organic compounds
- A61K49/12—Macromolecular compounds
- A61K49/126—Linear polymers, e.g. dextran, inulin, PEG
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
- A61P27/06—Antiglaucoma agents or miotics
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Epidemiology (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Engineering & Computer Science (AREA)
- Inorganic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Nanotechnology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Biomedical Technology (AREA)
- Ophthalmology & Optometry (AREA)
- Organic Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Radiology & Medical Imaging (AREA)
- Biophysics (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Medical Informatics (AREA)
- Molecular Biology (AREA)
- Crystallography & Structural Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
本发明提供了一种用于小梁类细胞标记的双模态纳米探针及其制备方法和应用，所述双模态纳米探针以PLGA为载体，包被功能性近红外染料cypate和超顺磁性氧化铁纳米粒子SPIO，从而形成PLGA‑Cypate‑SPIO多功能纳米探针。本发明所述的双模态纳米探针作为近红外成像的显影剂，可用于共聚焦荧光显微镜对小梁类细胞进行近红外荧光成像；作为磁共振成像的显影剂，可用于核磁共振成像仪对小梁类细胞进行磁共振成像；同时该磁性纳米探针可作为药物递送载体，可载送包括Rho激酶抑制剂在内的多种药物，实现Rho激酶抑制剂的靶向递送，有效提高小梁网通路房水外排效率。
Description
技术领域
本发明属于纳米材料技术领域，特别涉及一种用于小梁类细胞标记的双模态纳米探针及其制备方法和应用。
背景技术
青光眼是全球第二大致盲原因，主要与眼压升高有关。而小梁网（trabecularmeshwork，TM）是常规房水（aqueous humor，AH）外流通路的重要组成部分，在调节AH流出发挥重要作用。TM功能失调是导致高眼压症和原发性开角型青光眼的主要原因。
目前，药物治疗是降低青光眼患者眼压的主要手段。主要包括用于减少AH产生的经典药物（包括β-肾上腺素能拮抗剂、α-肾上腺素能激动剂和碳酸酐酶抑制剂）、增加AH外流的常见药物（包括胆碱能激动剂和前列腺素类似物）。但由于这些药物尚存在一定的副作用、单药治疗存在一定的局限性，多药治疗患者的依从性较差等问题，这些药物的临床应用始终面临着很大的挑战。
具体来说，上述药物不具靶向性，应用于青光眼患者后，其未靶向作用于小梁网组织结构，可能影响治疗作用的发挥，还有可能破坏其他组织的功能障碍，产生一定副作用。
因此，如何开发一种能够靶向作用于小梁网的药物，是一个需要解决的问题。
发明内容
针对现有技术中存在的问题，本发明的目的在于提供一种用于小梁类细胞标记的双模态纳米探针及其制备方法和应用，所述纳米探针在不影响细胞生物学特性的前提下，可用于共聚焦荧光显微镜对小梁类细胞进行NIR近红外荧光成像，MRI核磁共振成像示踪细胞，实时监测小梁类细胞的状态；同时该探针可载送药物，使其靶向小梁网，可有效提高药物的治疗效果。
本发明的目的是通过以下技术方案实现的：
一种用于小梁类细胞标记的双模态纳米探针，所述双模态纳米探针以PLGA为载体，包被功能性近红外染料cypate和超顺磁性氧化铁纳米粒子SPIO，从而形成PLGA-Cypate-SPIO多功能纳米探针。
进一步的，所述双模态纳米探针的粒径为200 nm~300 nm。
通过乳化法制备所述双模态纳米探针，其制备方法包括以下步骤：
S1，将PLGA-PEG-COOH充分溶解在二氯甲烷（CH2Cl2）中形成PLGA溶液，然后将SPIO和Cypate一起添加到PLGA溶液中，通过使用超声波探头对溶液进行乳化，从而得到第一乳化溶液；
S2，将S1获得的第一乳化溶液缓慢倒入聚乙烯醇（PVA）溶液中，并通过超声波探头进行乳化，从而得到第二乳化溶液；
S3，向S2获得的第二乳化溶液中异丙醇溶液，过夜搅拌从而挥发CH2Cl2，再经离心处理后，得到的沉淀即为PLGA-Cypate-SPIO多功能纳米探针，于4 ℃下保存。
进一步的，S1中所述PLGA-PEG-COOH、SPIO和Cypate的重量比为（12.5~50）：（0.4~1.2）：（1~4）。
进一步的，S2中所述聚乙烯醇溶液的质量浓度为3%~5%。
进一步的，S3中所述异丙醇溶液的体积浓度为1%~3%。
进一步的，S3中所述离心处理的条件为10000~12000 rpm离心6~11分钟。
本发明的另一方面：
上述双模态纳米探针作为药物递送载体的用途，其中，所述药物包括Rho激酶抑制剂；包括但不限于小分子抑制剂Y-27632（MedChemExpress 美国MCE生物科技公司）、法苏地尔Fasudil（HA-1077；Asahi Kasei Corporation日本旭化成公司）、利帕苏地尔Ripasudil（K-115，Kowa兴和制药）和内塔苏地尔Netarsudil（AR-13324，艾瑞制药有限公司AeriePharmaceuticals Inc.）等商业药物，以及处于临床试验阶段的AMA0076（Amakem）和PG324（Aerie Pharmaceuticals Inc.）。
本发明相比现有技术的有益效果为：
1、在生物安全方面，现有的显影剂多为金属等无机材料，生物相容性较差；在成像方面，现有成像探针多为单一成像，如光学荧光成像，材料多为Cd, Te等重金属组成的无机材料、稀土材料、有机荧光分子等。一方面这些重金属，稀土材料在体内安全性存在争议；另一方面部分材料存在代谢快，荧光淬灭性快，深层组织成像受限，自身荧光影响较大等缺点，不适合做长期标记细胞。磁共振成像MRI显影剂多为重金属，基于氧化铁或钆鳌合物之类的磁性造影剂，存在体内残留，无特异性分布，对人体产生毒性副作用等问题；而本发明所述PLGA-Cypate-SPIO多功能纳米探针，所采用的材料均为FDA批准或改良的，生物相容性较好，安全性高；
2、本发明所述的PLGA-Cypate-SPIO多功能纳米探针，本发明采用荧光/MRI双模态成像，弥补了单一成像的不足。选择合适的具有较高灵敏度和荧光性能的荧光成像材料，选择合适的具有高弛豫性能和安全性能的磁共振成像材料。即选用生物相容性高，组织穿透力强，波长范围内自身荧光弱，灵敏度高的近红外荧光染料cypate；易代谢，成本低，灵敏度高，生物安全性高（Fe是红细胞的重要成分）的超顺磁性氧化铁纳米粒子SPIO；通过PLGA聚合物包被可有效保护荧光染料，减缓有效成分的分解代谢，达到缓释，长时间标记的效果，同时可以增强细胞的标记，提高纳米探针的生物相容性；
3、本发明所述的双模态纳米探针可以作为药物递送载体，可载送包括Rho激酶抑制剂在内的多种药物，实现Rho激酶抑制剂的准确靶向，有效提高小梁网通路房水外排效率。
附图说明
下面结合附图和实施例对本发明作进一步说明：
图1为实施例所述双模态纳米探针的制备示意图；
图2为实施例所述双模态纳米探针PLGA-Cypate-SPIO的粒径分布图及电镜图；其中，A为马尔文激光粒度仪测得的粒径分布图；B为未包裹任何物质的PLGA纳米粒子透射电镜图；C, D为双模态纳米探针PLGA-Cypate-SPIO的透射电镜图，D为纳米粒子的局部放大图；
图3为各粒子的可见近红外吸收光谱图、荧光激发/发射图及磁滞回线；其中，A为PLGA, Cypate , SPIO, PLGA-SPIO, PLGA-Cypate, PLGA-Cypate-SPIO 双模态纳米探针的可见近红外吸收光谱图；B为纳米粒子的荧光激发/发射图；C为纳米粒子的磁滞回线；
图4为实施例所述双模态纳米探针PLGA-Cypate-SPIO对小梁类细胞的细胞毒性结果图；
图5为细胞的吞噬情况图；其中，A为细胞吞噬与未吞噬的流式检测图；B, C为细胞生物透射电镜图；D为细胞普鲁士蓝染色；
图6为对标记后细胞标记物的检测结果图；
图7为免疫荧光鬼笔环肽染色结果图；
图8为地塞米松处理后，Western blot蛋白（myocilin）检测结果图；
图9为为体外细胞近红外荧光成像结果图；其中，A为标记后三种细胞的共聚焦荧光图；B为小动物活体成像拍摄细胞团块图；
图10为体外细胞MRI成像结果图；其中，A为不同浓度下的多功能纳米粒子NPs溶液的MRI成像图及标记的细胞的成像图；B为三种细胞的MRI成像图。
具体实施方式
实施例1——纳米探针的制备及表征
本实施例提供了一种用于小梁类细胞标记的双模态纳米探针，如图1所示，通过乳化法制备所述纳米探针，其制备方法包括以下步骤：
S1，将25 mg PLGA-PEG-COOH充分溶解在2 mL二氯甲烷（CH2Cl2）中形成PLGA溶液，然后将800 μg SPIO（10 nm，水分散型）和2 mg Cypate一起添加到PLGA溶液中，通过使用超声波探头对溶液进行乳化，从而得到第一乳化溶液；
S2，将S1获得的第一乳化溶液缓慢倒入5 mL聚乙烯醇（PVA）溶液（4% w/v）中，并通过超声波探头进行乳化，从而得到第二乳化溶液；
S3，向S2获得的第二乳化溶液中50 mL异丙醇溶液（2% v/v），过夜搅拌从而挥发CH2Cl2，再经11000 rpm离心7分钟后，得到的沉淀即为PLGA-Cypate-SPIO多功能纳米探针，于4 ℃下保存。
其中，PLGA是一种递送载体，生物降解性好、延长循环时间和易于表面修饰等优点，在显像剂和药物递送中发挥着重要作用。SPIO具有超顺磁性，不仅能够磁靶向而且具有优异的生物相容性和低毒性，还能作为显像剂。Cypate是FDA批准的有机近红外荧光染料ICG衍生物，生物相容性好，灵敏度高、空间分辨率高。
为了验证是否成功制备纳米粒子，双模态纳米探针是否具有近红外成像NIR，磁共振成像MRI及磁靶向的能力。需要对制备得到的纳米粒子进行相应的表征测试。本实施例通过马尔文激光粒度仪测试其粒径大小、Zeta电位值。TEM透射电镜观察NPs形态，SPIO是否已经被PLGA包被。紫外分光光度计测试NPs吸光度，荧光分光光度计测试NPs荧光性。振动样品磁强计检测NPs磁性参数。
如图2的马尔文激光粒度仪测试显示，本实施例所述PLGA-Cypate-SPIO多功能纳米探针的粒径在243 nm左右，Zeta电位-21.7 mv。PLGA NPs和PLGA-Cypate-SPIO NPs的TEM图片显示合成的纳米粒子呈球型且边缘清晰，SPIO被包裹在PLGA骨架材料中。
如图3中UV-VIS-NIR显示在780 nm处，PLGA -Cypate-SPIO有较大吸收，荧光分光光度计显示NPs有明显的激发（781 nm）/发射峰（815 nm）。通过振动样品磁强计得到的S型磁滞曲线可以看出，随着外磁场增加的磁化过程中，磁化强度由负到正，在接近零磁场时，磁化曲线呈线性趋势，并且经过原点，证明PLGA-Cypate-SPIO NPs具有超顺磁性特征。
实施例2——双模态纳米探针PLGA-Cypate-SPIO性质测定
本实施例测定了不同浓度下实施例1所述PLGA-Cypate-SPIO多功能纳米探针，与小梁类细胞（miPSC-TM，hpTM，HTM三种细胞）孵育24h，利用CCK8法检测PLGA-Cypate-SPIONPs对细胞的增殖活性影响。
如图4所示，纳米探针PLGA-Cypate-SPIO与小梁类细胞孵育24h后，并未对细胞造成凋亡，说明PLGA-Cypate-SPIO多功能纳米探针不具有细胞毒性。
本实施例测定了不同浓度下实施例1所述PLGA-Cypate-SPIO多功能纳米探针的细胞吞噬能力，选用不同浓度的PLGA-Cypate-SPIO多功能纳米探针与细胞进行孵育，通过流式细胞仪，生物电镜，普鲁士蓝染色及荧光成像，观察细胞对纳米粒子的吞噬情况。
根据测图5可以看到流式细胞仪检测可以看出吞噬了纳米粒的细胞信号明显强于对照组的，细胞透射电镜表明纳米粒子主要分布在细胞质中已被吞噬，普鲁士蓝染色表明纳米粒子被细胞吞噬主要分布在细胞核周围（箭头指向的蓝色）。
为验证PLGA-Cypate-SPIO多功能纳米探针对细胞的影响，是否影响其细胞功能及特性。我们对标记的细胞（标记浓度35μg/mL）的细胞标记物及小梁特性（地塞米松诱导细胞骨架形成交叉连接蛋白网CLANs，增强Myocilin蛋白的表达）进行检测。由图6-8可以看出，多功能纳米粒子标记的细胞与未标记的细胞相比，细胞标记物未发生改变，都能检测到。在地塞米松的诱导下，能够形成CLANs细胞骨架，MYOC蛋白表达增加。由上可见PLGA-Cypate-SPIO多功能纳米探针没有影响细胞的正常细胞功能及特性。
本实施例还进行了体外细胞示踪定位实验，具体如下：
体外细胞近红外成像实验：
由图9可以看出，miPSC-TM，hpTM，HTM三种细胞都能在近红外荧光下成像。参照图10 A中DAPI染色，可以看出PLGA-Cypate-SPIO多功能纳米探针主要存在于细胞质内，细胞核的周围，进一步表明PLGA-Cypate-SPIO多功能纳米探针被细胞吞噬。
体外细胞MRI成像：
由图10可见，实施例1所制备的PLGA-Cypate-SPIO多功能纳米粒子具有MRI成像能力，可以标记细胞，可用于MRI成像。
实施例3——双模态纳米探针PLGA-Cypate-SPIO载药
本实施例提供了一种利用实施例1所述PLGA-Cypate-SPIO多功能纳米探针载送Rho激酶抑制剂的方法，包括以下步骤：
S1，将25 mg PLGA-PEG-COOH充分溶解在2 mL二氯甲烷（CH2Cl2）中形成PLGA溶液；
S2，混溶800 μg SPIO（10 nm，水分散型）和1 mg的Rho激酶抑制剂，得到混合液；
所述Rho激酶抑制剂可选用包括Y-27632，HA-1077，K-115，AR-13324等在内的水溶性药物；
S3，将S2所得混合液与2 mg 荧光染料Cypate一起添加到S1中PLGA溶液中，通过使用超声波探头对溶液进行乳化，从而得到第一乳化溶液；
S4，将S3获得的第一乳化溶液缓慢倒入5 mL聚乙烯醇（PVA）溶液（4% w/v）中，并通过超声波探头进行乳化，从而得到第二乳化溶液；
S5，向S4获得的第二乳化溶液中50 mL异丙醇溶液（2% v/v），过夜搅拌从而挥发CH2Cl2，再经11000 rpm离心7分钟后，得到的沉淀即为Rho@PLGA-Cypate-SPIO多功能纳米药物，于4 ℃下保存。
最后应说明的是，以上仅用以说明本发明的技术方案而非限制，尽管参照较佳布置方案对本发明进行了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的精神和范围。
Claims (8)
1.一种用于小梁类细胞标记的双模态纳米探针，其特征在于，所述双模态纳米探针以PLGA为载体，包被功能性近红外染料cypate和超顺磁性氧化铁纳米粒子SPIO，从而形成PLGA-Cypate-SPIO多功能纳米探针。
2. 根据权利要求1所述的双模态纳米探针，其特征在于，所述纳米探针的粒径为200nm~300 nm。
3.一种如权利要求1或2所述双模态纳米探针的制备方法，其特征在于，通过乳化法制备所述双模态纳米探针，包括以下步骤：
S1，将PLGA-PEG-COOH充分溶解在二氯甲烷（CH2Cl2）中形成PLGA溶液，然后将SPIO和Cypate一起添加到PLGA溶液中，通过使用超声波探头对溶液进行乳化，从而得到第一乳化溶液；
S2，将S1获得的第一乳化溶液缓慢倒入聚乙烯醇（PVA）溶液中，并通过超声波探头进行乳化，从而得到第二乳化溶液；
S3，向S2获得的第二乳化溶液中异丙醇溶液，过夜搅拌从而挥发CH2Cl2，再经离心处理后，得到的沉淀即为PLGA-Cypate-SPIO多功能纳米探针，于4 ℃下保存。
4.根据权利要求3所述双模态纳米探针的制备方法，其特征在于，S1中所述PLGA-PEG-COOH、SPIO和Cypate的重量比为（12.5~50）：（0.4~1.2）：（1~4）。
5.根据权利要求3所述双模态纳米探针的制备方法，其特征在于，S2中所述聚乙烯醇溶液的质量浓度为3%~5%。
6.根据权利要求3所述双模态纳米探针的制备方法，其特征在于，S3中所述异丙醇溶液的体积浓度为1%~3%。
7. 根据权利要求3所述双模态纳米探针的制备方法，其特征在于，S3中所述离心处理的条件为10000~12000 rpm离心6~11分钟。
8.一种如权利要求1或2所述双模态纳米探针作为药物递送载体的用途，其特征在于，所述药物包括Rho激酶抑制剂。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111193841.5A CN113842473B (zh) | 2021-10-13 | 2021-10-13 | 一种用于小梁类细胞标记的双模态纳米探针及其制备方法和应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111193841.5A CN113842473B (zh) | 2021-10-13 | 2021-10-13 | 一种用于小梁类细胞标记的双模态纳米探针及其制备方法和应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113842473A true CN113842473A (zh) | 2021-12-28 |
| CN113842473B CN113842473B (zh) | 2024-01-30 |
Family
ID=78978355
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111193841.5A Active CN113842473B (zh) | 2021-10-13 | 2021-10-13 | 一种用于小梁类细胞标记的双模态纳米探针及其制备方法和应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113842473B (zh) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110212032A1 (en) * | 2007-08-29 | 2011-09-01 | Korea Research Institute Of Bioscience And Biotechnology | Polymer particles for nir/mr bimodal molecular imaging and method for preparing the same |
| CN102743764A (zh) * | 2012-04-11 | 2012-10-24 | 中山大学中山眼科中心 | RhoA的RNA干扰靶点序列在制备治疗青光眼药物中的应用 |
| US20140178308A1 (en) * | 2011-07-29 | 2014-06-26 | Institut National De La Sante Et De La Recherche Medicale (Inserm) | Method for increasing the intraocular pressure in an animal |
| CN105056253A (zh) * | 2015-07-21 | 2015-11-18 | 福州市传染病医院 | 用于脂肪干细胞标记、活体示踪和治疗的多功能纳米探针 |
| WO2021126989A1 (en) * | 2019-12-17 | 2021-06-24 | The Regents Of The University Of California | Two-way magnetic resonance tuning nanoprobe enhanced subtraction imaging |
-
2021
- 2021-10-13 CN CN202111193841.5A patent/CN113842473B/zh active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110212032A1 (en) * | 2007-08-29 | 2011-09-01 | Korea Research Institute Of Bioscience And Biotechnology | Polymer particles for nir/mr bimodal molecular imaging and method for preparing the same |
| US20140178308A1 (en) * | 2011-07-29 | 2014-06-26 | Institut National De La Sante Et De La Recherche Medicale (Inserm) | Method for increasing the intraocular pressure in an animal |
| CN102743764A (zh) * | 2012-04-11 | 2012-10-24 | 中山大学中山眼科中心 | RhoA的RNA干扰靶点序列在制备治疗青光眼药物中的应用 |
| CN105056253A (zh) * | 2015-07-21 | 2015-11-18 | 福州市传染病医院 | 用于脂肪干细胞标记、活体示踪和治疗的多功能纳米探针 |
| WO2021126989A1 (en) * | 2019-12-17 | 2021-06-24 | The Regents Of The University Of California | Two-way magnetic resonance tuning nanoprobe enhanced subtraction imaging |
Non-Patent Citations (8)
Also Published As
| Publication number | Publication date |
|---|---|
| CN113842473B (zh) | 2024-01-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Guller et al. | Cytotoxicity and non-specific cellular uptake of bare and surface-modified upconversion nanoparticles in human skin cells | |
| Yang et al. | Hyaluronic acid conjugated magnetic prussian blue@ quantum dot nanoparticles for cancer theranostics | |
| Lu et al. | Hybrid drug nanocrystals | |
| Gilad et al. | MR tracking of transplanted cells with “positive contrast” using manganese oxide nanoparticles | |
| Ling et al. | Temozolomide loaded PLGA-based superparamagnetic nanoparticles for magnetic resonance imaging and treatment of malignant glioma | |
| Zhou et al. | Ultrabright NIR‐II emissive polymer dots for metastatic ovarian cancer detection | |
| US9987378B2 (en) | Prussian blue-inspired constructs for multimodal imaging and therapy | |
| Chen et al. | Dispersion stability and biocompatibility of four ligand-exchanged NaYF4: Yb, Er upconversion nanoparticles | |
| Wadajkar et al. | Dual‐imaging enabled cancer‐targeting nanoparticles | |
| Sulek et al. | Peptide functionalized superparamagnetic iron oxide nanoparticles as MRI contrast agents | |
| Wu et al. | Negatively charged magnetite nanoparticle clusters as efficient MRI probes for dendritic cell labeling and in vivo tracking | |
| Orringer et al. | In vitro characterization of a targeted, dye-loaded nanodevice for intraoperative tumor delineation | |
| US20120045399A1 (en) | Positively-charged superparamagnetic iron oxide nanoparticle, contrast agent using the same and method of preparing the same | |
| Wilk et al. | Photo-oxidative action in MCF-7 cancer cells induced by hydrophobic cyanines loaded in biodegradable microemulsion-templated nanocapsules | |
| US20150265728A1 (en) | Magnetic and fluorescent reverse nanoassemblies | |
| Xu et al. | Monodisperse magnetite nanoparticles coupled with nuclear localization signal peptide for cell‐nucleus targeting | |
| Xu et al. | Fabrication of folic acid functionalized pH-responsive and thermosensitive magnetic chitosan microcapsules via a simple sonochemical method | |
| JP6618680B2 (ja) | 光力学的治療用自己組立型薬学の組成物 | |
| Zhao et al. | Multifunctional magnetic nanoparticles for simultaneous cancer near-infrared imaging and targeting photodynamic therapy | |
| Iliasov et al. | Non-magnetic shell coating of magnetic nanoparticles as key factor of toxicity for cancer cells in a low frequency alternating magnetic field | |
| JP6425486B2 (ja) | 癌細胞の造影用ｍｒｉ造影剤組成物 | |
| Linot et al. | PEGylated anionic magnetofluorescent nanoassemblies: impact of their interface structure on magnetic resonance imaging contrast and cellular uptake | |
| Zhang et al. | Holmium (III)-doped multifunctional nanotheranostic agent for ultra-high-field magnetic resonance imaging-guided chemo-photothermal tumor therapy | |
| CN113842473B (zh) | 一种用于小梁类细胞标记的双模态纳米探针及其制备方法和应用 | |
| KR101717010B1 (ko) | 암의 조기 진단 또는 치료용 폴리아미노산 기반의 생체환경 감응형 나노입자 |
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 |