CN111956811A - Preparation method of ASGPR (advanced red-fluorescence tomography-based red fluorescent protein) multi-modal image display contrast agent - Google Patents
Preparation method of ASGPR (advanced red-fluorescence tomography-based red fluorescent protein) multi-modal image display contrast agent Download PDFInfo
- Publication number
- CN111956811A CN111956811A CN202010819064.XA CN202010819064A CN111956811A CN 111956811 A CN111956811 A CN 111956811A CN 202010819064 A CN202010819064 A CN 202010819064A CN 111956811 A CN111956811 A CN 111956811A
- Authority
- CN
- China
- Prior art keywords
- mpeg
- contrast agent
- preparation
- asgpr
- display contrast
- 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
- 239000002872 contrast media Substances 0.000 title claims abstract description 42
- 238000002360 preparation method Methods 0.000 title claims abstract description 40
- 101150075175 Asgr1 gene Proteins 0.000 title claims abstract 11
- 238000003325 tomography Methods 0.000 title abstract description 6
- 108010054624 red fluorescent protein Proteins 0.000 title description 2
- 238000003384 imaging method Methods 0.000 claims abstract description 19
- 238000000034 method Methods 0.000 claims abstract description 13
- 230000002902 bimodal effect Effects 0.000 claims abstract description 3
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 72
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 claims description 71
- 238000003756 stirring Methods 0.000 claims description 42
- 239000000412 dendrimer Substances 0.000 claims description 36
- 229920000736 dendritic polymer Polymers 0.000 claims description 36
- 229920000962 poly(amidoamine) Polymers 0.000 claims description 34
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 30
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 29
- 239000000243 solution Substances 0.000 claims description 24
- 238000006243 chemical reaction Methods 0.000 claims description 22
- 229910004042 HAuCl4 Inorganic materials 0.000 claims description 21
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 17
- 238000004108 freeze drying Methods 0.000 claims description 15
- NWZSZGALRFJKBT-KNIFDHDWSA-N (2s)-2,6-diaminohexanoic acid;(2s)-2-hydroxybutanedioic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O.NCCCC[C@H](N)C(O)=O NWZSZGALRFJKBT-KNIFDHDWSA-N 0.000 claims description 14
- IKDUDTNKRLTJSI-UHFFFAOYSA-N hydrazine monohydrate Substances O.NN IKDUDTNKRLTJSI-UHFFFAOYSA-N 0.000 claims description 14
- 125000003277 amino group Chemical group 0.000 claims description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 13
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 claims description 12
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 claims description 12
- 239000012279 sodium borohydride Substances 0.000 claims description 11
- 229910000033 sodium borohydride Inorganic materials 0.000 claims description 11
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims description 9
- 239000008101 lactose Substances 0.000 claims description 9
- SZVJSHCCFOBDDC-UHFFFAOYSA-N iron(II,III) oxide Inorganic materials O=[Fe]O[Fe]O[Fe]=O SZVJSHCCFOBDDC-UHFFFAOYSA-N 0.000 claims description 8
- 108010088751 Albumins Proteins 0.000 claims description 7
- 102000009027 Albumins Human genes 0.000 claims description 7
- 239000002105 nanoparticle Substances 0.000 claims description 7
- 238000001338 self-assembly Methods 0.000 claims description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- 241000399119 Spio Species 0.000 claims description 6
- 230000010355 oscillation Effects 0.000 claims description 6
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 5
- 230000004913 activation Effects 0.000 claims description 5
- 229940098773 bovine serum albumin Drugs 0.000 claims description 5
- 238000002156 mixing Methods 0.000 claims description 5
- UQSXHKLRYXJYBZ-UHFFFAOYSA-N Iron oxide Chemical compound [Fe]=O UQSXHKLRYXJYBZ-UHFFFAOYSA-N 0.000 claims description 4
- BEOOHQFXGBMRKU-UHFFFAOYSA-N sodium cyanoborohydride Chemical compound [Na+].[B-]C#N BEOOHQFXGBMRKU-UHFFFAOYSA-N 0.000 claims description 4
- 229920005654 Sephadex Polymers 0.000 claims description 3
- 239000012507 Sephadex™ Substances 0.000 claims description 3
- 238000004440 column chromatography Methods 0.000 claims description 3
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Substances [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 claims description 3
- 239000008055 phosphate buffer solution Substances 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 3
- 239000006228 supernatant Substances 0.000 claims description 3
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 claims description 2
- -1 lactosyl Chemical group 0.000 claims description 2
- 230000008569 process Effects 0.000 claims description 2
- 239000002904 solvent Substances 0.000 claims description 2
- 238000012800 visualization Methods 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 7
- 238000013170 computed tomography imaging Methods 0.000 abstract description 6
- 238000003745 diagnosis Methods 0.000 abstract description 6
- 206010073071 hepatocellular carcinoma Diseases 0.000 abstract description 5
- 230000008685 targeting Effects 0.000 abstract description 5
- 206010028980 Neoplasm Diseases 0.000 abstract description 3
- 230000015572 biosynthetic process Effects 0.000 abstract description 3
- 201000011510 cancer Diseases 0.000 abstract description 3
- 238000003748 differential diagnosis Methods 0.000 abstract description 3
- 125000002519 galactosyl group Chemical group C1([C@H](O)[C@@H](O)[C@@H](O)[C@H](O1)CO)* 0.000 abstract description 3
- 238000003786 synthesis reaction Methods 0.000 abstract description 3
- 239000007864 aqueous solution Substances 0.000 abstract description 2
- 230000009286 beneficial effect Effects 0.000 abstract description 2
- 238000000338 in vitro Methods 0.000 abstract description 2
- 210000004881 tumor cell Anatomy 0.000 abstract description 2
- 102000005427 Asialoglycoprotein Receptor Human genes 0.000 description 14
- 108010006523 asialoglycoprotein receptor Proteins 0.000 description 14
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 4
- 231100000844 hepatocellular carcinoma Toxicity 0.000 description 4
- 239000011630 iodine Substances 0.000 description 4
- 229910052740 iodine Inorganic materials 0.000 description 4
- 210000004185 liver Anatomy 0.000 description 4
- 238000011161 development Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 210000005229 liver cell Anatomy 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 210000004738 parenchymal cell Anatomy 0.000 description 3
- WQZGKKKJIJFFOK-SVZMEOIVSA-N (+)-Galactose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-SVZMEOIVSA-N 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000002595 magnetic resonance imaging Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 206010016654 Fibrosis Diseases 0.000 description 1
- 229910052688 Gadolinium Inorganic materials 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- OVRNDRQMDRJTHS-CBQIKETKSA-N N-Acetyl-D-Galactosamine Chemical compound CC(=O)N[C@H]1[C@@H](O)O[C@H](CO)[C@H](O)[C@@H]1O OVRNDRQMDRJTHS-CBQIKETKSA-N 0.000 description 1
- MBLBDJOUHNCFQT-UHFFFAOYSA-N N-acetyl-D-galactosamine Natural products CC(=O)NC(C=O)C(O)C(O)C(O)CO MBLBDJOUHNCFQT-UHFFFAOYSA-N 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- YVPYQUNUQOZFHG-UHFFFAOYSA-N amidotrizoic acid Chemical compound CC(=O)NC1=C(I)C(NC(C)=O)=C(I)C(C(O)=O)=C1I YVPYQUNUQOZFHG-UHFFFAOYSA-N 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 230000031018 biological processes and functions Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000007882 cirrhosis Effects 0.000 description 1
- 208000019425 cirrhosis of liver Diseases 0.000 description 1
- 238000002059 diagnostic imaging Methods 0.000 description 1
- 229960005223 diatrizoic acid Drugs 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 230000012202 endocytosis Effects 0.000 description 1
- UIWYJDYFSGRHKR-UHFFFAOYSA-N gadolinium atom Chemical compound [Gd] UIWYJDYFSGRHKR-UHFFFAOYSA-N 0.000 description 1
- 108010042430 galactose receptor Proteins 0.000 description 1
- 125000003147 glycosyl group Chemical group 0.000 description 1
- 230000002440 hepatic effect Effects 0.000 description 1
- 208000006454 hepatitis Diseases 0.000 description 1
- 231100000283 hepatitis Toxicity 0.000 description 1
- PNDPGZBMCMUPRI-UHFFFAOYSA-N iodine Chemical compound II PNDPGZBMCMUPRI-UHFFFAOYSA-N 0.000 description 1
- NTHXOOBQLCIOLC-UHFFFAOYSA-N iohexol Chemical compound OCC(O)CN(C(=O)C)C1=C(I)C(C(=O)NCC(O)CO)=C(I)C(C(=O)NCC(O)CO)=C1I NTHXOOBQLCIOLC-UHFFFAOYSA-N 0.000 description 1
- 229960001025 iohexol Drugs 0.000 description 1
- 208000019423 liver disease Diseases 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000001613 neoplastic effect Effects 0.000 description 1
- 238000009206 nuclear medicine Methods 0.000 description 1
- 238000012634 optical imaging Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000036285 pathological change Effects 0.000 description 1
- 231100000915 pathological change Toxicity 0.000 description 1
- 238000012805 post-processing Methods 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- MNWBNISUBARLIT-UHFFFAOYSA-N sodium cyanide Chemical compound [Na+].N#[C-] MNWBNISUBARLIT-UHFFFAOYSA-N 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
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
- A61K49/00—Preparations for testing in vivo
- A61K49/04—X-ray contrast preparations
-
- 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
- A61K49/14—Peptides, e.g. proteins
- A61K49/143—Peptides, e.g. proteins the protein being an albumin, e.g. HSA, BSA, ovalbumin
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Epidemiology (AREA)
- Molecular Biology (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Radiology & Medical Imaging (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention discloses a preparation method of an ASGPR (advanced red-fluorescence tomography-based red-fluorescence) multimodal image display contrast agent. Belongs to the technical field of biomedicine. The method comprises the following steps: preparation of Gal-BSA-SPIO { (Au)0)25‑G5.NH2‑mPEG20Preparation of target display contrast agent. Compared with the prior art, the invention has the beneficial effects that: the CT imaging device has longer CT imaging time and better CT imaging effect; the prepared target display contrast agent has good stability, aqueous solution dispersibility and biocompatibility, shows good in-vitro good and malignant tumor cell targeting effect, and has specific targeting tumor CT/MR bimodal image display imaging effect; fe modified by synthesis of ASGPR based galactosyl albumin3O4The multi-modal (CT + MR) targeted contrast agent realizes the imaging diagnosis and differential diagnosis of HCC.
Description
Technical Field
The invention relates to the technical field of biomedicine, in particular to a preparation method of an ASGPR (advanced resolved fluorescence tomography imaging) based multi-modal image display contrast agent.
Background
Molecular imaging is a product combining molecular biology technology and modern medical imaging, and the biological process of living tissues on the molecular and cellular level is displayed by detecting the physiological and pathological change processes of organisms by a precise imaging technology and then by a series of image post-processing technologies. Molecular imaging reveals gene level abnormalities in the early development of the disease, which can detect the disease condition when the molecules of diseased cells are changed, thereby realizing early and accurate diagnosis and treatment of cancer. The main imaging techniques of molecular imaging mainly include nuclear medicine imaging, ultrasonic imaging, magnetic resonance imaging (MRI for short), optical imaging, tomography imaging (CT for short) and other methods. Among them, the CT imaging technique has the advantages of high spatial resolution, short image acquisition time, and capability of integral imaging, and meanwhile, the cost is low, and plays a very important role in medical clinical examination.
Currently, contrast agents for CT diagnosis widely used in clinical practice mainly include iodine-containing contrast agents and gadolinium contrast agents, wherein the iodine-containing contrast agents mainly include small molecular compounds containing iodine, including organic iodine and inorganic iodine small molecular compounds, such as diatrizoic acid and iohexol. However, the above-mentioned contrast agents for image diagnosis have disadvantages of short development time and difficulty in surface modification, and also lack specificity.
In summary, it is an urgent need to solve the above-mentioned problems by those skilled in the art to provide an image display contrast agent with long development time, easy surface modification, and strong specificity.
Disclosure of Invention
In view of the above, the present invention provides a method for preparing an ASGPR-based contrast agent for multimodal image display.
In order to achieve the purpose, the invention adopts the following technical scheme:
the preparation method of the contrast agent based on ASGPR multi-modal image display comprises the following steps:
s1, preparation of Gal-BSA-SPIO:
(11) allowing the nanometer iron oxide SPIO to pass through a chromatographic column, separating the small-particle-size SPIO, dialyzing, and concentrating until the Fe content is 6-8 mg/ml;
(12) then adjusting the pH value to 6.5-7.0, adding a lactose-based albumin solution with the same volume and mass concentration of 1%, and carrying out ice bath ultrasonic oscillation for 1-3 h;
(13) after ultrasonic oscillation, adding 1M NaCl solution to remove unbound lactoalbumin, and adjusting the pH value of the solution to 7.3-7.5 to obtain Gal-BSA-SPIO solution;
S2、{(Au0)25-G5.NH2-mPEG20preparation of }:
(21) dissolving mPEG-COOH, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to perform activation reaction for 1-2.5 h to obtain activated mPEG-COOH;
(22) reacting activated mPEG-COOH with fifth generation polyamidoamine dendrimer G5.NH2Mixing the DMSO solution, adding HAuCl4Reacting for 30-40 min, and then adding NaBH4Continuously stirring and reacting for 1-2 h, then adding triethylamine, stirring for 10-30 min, then adding acetic anhydride, continuously stirring and reacting for 18-25 h, dialyzing, and freeze-drying to obtain { (Au)0)25-G5.NH2-mPEG20};
S3, preparation of a target display contrast agent:
(31) dissolving Gal-BSA-SPIO obtained in step S1 in water, and adding { (Au) obtained in step S20)25-G5.NH2-mPEG20Will { (Au) by self-assembly0)25-G5.NH2-mPEG20Assembled at Fe3O4A nanoparticle surface;
(32) and then adding hydrazine hydrate, stirring for 1-2 h, adding triethylamine, stirring for 10-30 min, adding acetic anhydride, stirring for reacting for 10-15 h, dialyzing, and freeze-drying to obtain the target display contrast agent.
An asialoglycoprotein receptor (ASGPR), also known as the galactose receptor, is specifically present in mammalian liver parenchymal cells with specific recognition. ASGPR is mainly expressed on the surface of liver parenchymal cell of sinusoid of liver of mammal, normal liver cell membraneThe surface of the liver cell is rich in ASGPG, one cell has up to 500000 receptors on average, the liver cell can recognize and specifically combine with a sugar chain of which the terminal glycosyl is D-galactose or N-acetyl-D-galactosamine, and the efficient target liver effect of a ligand-carried substance is realized through endocytosis. When liver diseases such as primary Hepatocellular carcinoma (HCC), hepatitis, cirrhosis and the like occur, the expression quantity and the function of ASGPR are reduced. For non-neoplastic hepatic parenchymal cells, low expression of ASGPR is a marker of high proliferative potential. To this end, the present invention provides for the synthesis of ASGPR based galactosyl albumin modified Fe3O4The CT/MR bimodal targeting contrast agent realizes the imaging diagnosis and differential diagnosis of HCC.
Further, in the step (21), the solvent DMSO is used to dissolve mPEG-COOH.
In the step (21), the molar ratio of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide to mPEG-COOH is (8-10) 10 (1-2), and the concentrations of mPEG-COOH, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide are 6-10 mg/mL, 5-8 mg/mL and 3-6 mg/mL, respectively.
Further, mPEG-COOH and fifth generation polyamidoamine dendrimer G5.NH are carried out in the step (22)2The molar ratio of (15-18) to (1), the fifth generation of polyamide-amine dendrimer G5.NH2The concentration of (b) is 5-15 mg/mL.
Further, in step S2, HAuCl4And { (Au)0)25-G5.NH2-mPEG20The molar ratio of (165-180): 1; NaBH4And HAuCl4In a molar ratio of 2: 1; triethylamine is fifth generation polyamidoamine dendrimer G5.NH2The mole number of the terminal amino is 3-8 times that of the terminal amino; acetic anhydride is fifth generation polyamidoamine dendrimer G5.NH2The number of moles of the terminal amino groups is 2 to 6 times.
Further, in step S3, hydrazine hydrate and HAuCl4In a molar ratio of 2: 1; triethylamine is fifth generation polyamidoamine dendrimer G5.NH2The molar number of the terminal amino groups is 3-6 times that of the terminal amino groups; acetic anhydride is fifth generation polyamidoamine dendrimer G5.NH2The number of moles of the terminal amino groups is 2 to 5 times.
Further, the preparation of the lactoalbumin comprises the following specific steps: dissolving bovine serum albumin, lactose and sodium cyanoborohydride in a phosphate buffer solution, stirring in a water bath for 24-80 h for reaction, dialyzing, centrifuging to obtain a supernatant, separating and purifying by using sephadex column chromatography, and freeze-drying to obtain the lactosyl albumin, wherein the mass ratio of the bovine serum albumin, the lactose and the sodium cyanoborohydride is 1 (0.002-0.005) to 3.2-3.8).
The invention also aims to provide application of the ASGPR (advanced selective absorption tomography) based multi-modal image display contrast agent prepared by the method to CT/MR dual-modal image imaging.
According to the technical scheme, compared with the prior art, the invention has the following beneficial effects: (1) the CT imaging time is longer and the CT imaging effect is better; (2) the prepared ASGPR-based multi-modal image display contrast agent has good stability, aqueous solution dispersibility and biocompatibility, shows good in-vitro good and malignant tumor cell targeting effect, has a specific targeting tumor CT/MR dual-modal image display imaging effect, and has a potential application prospect; (3) fe modified by synthesis of ASGPR based galactosyl albumin3O4The multi-modal (CT + MR) targeted contrast agent realizes the imaging diagnosis and differential diagnosis of HCC.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The required medicament is a conventional experimental medicament purchased from a market channel; the unrecited experimental method is a conventional experimental method, and is not described in detail herein.
Example 1
The preparation method of the contrast agent based on ASGPR multi-modal image display comprises the following steps:
s1, preparation of Gal-BSA-SPIO:
(11) taking a proper amount of SPIO to pass through a SepHarose 4B (with the height of 100cm and the width of 1cm) chromatographic column, separating the SPIO with small particle size, dialyzing the solution at low temperature (4 ℃) for 24h by using a standard buffer solution with the pH value of 7.4, and concentrating the solution until the Fe content is 8 mg/ml;
(12) adjusting pH to 6.5, adding lactose-based albumin solution (dissolving appropriate amount of product in pH 6.5 standard buffer solution) with equal volume and mass concentration of 1%, and performing ultrasonic oscillation in ice bath;
(13) after ultrasonic oscillation, adding 1M NaCl solution to remove unbound lactose-based albumin, and adjusting the pH value of the solution to 7.4 to obtain Gal-BSA-SPIO solution;
S2、{(Au0)25-G5.NH2-mPEG20preparation of }:
(21) dissolving mPEG-COOH by using DMSO, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to perform activation reaction for 2h to obtain activated mPEG-COOH;
(22) reacting activated mPEG-COOH with fifth generation polyamidoamine dendrimer G5.NH2Mixing the DMSO solution, adding HAuCl4Stirring to react for 35min, and adding NaBH4Continuously stirring for reaction for 2h, then adding triethylamine, stirring for reaction for 20min, finally adding acetic anhydride, continuously stirring for reaction for 22h, then dialyzing, and freeze-drying to obtain { (Au)0)25-G5.NH2-mPEG20};
Wherein the molar ratio of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and mPEG-COOH is 9:10: 1; mPEG-COOH and fifth generation polyamidoamine dendrimers G5.NH2In a molar ratio of 16:1, HAuCl4And { (Au)0)25-G5.NH2-mPEG20The molar ratio of (1) } is 170: 1; NaBH4And HAuCl4In a molar ratio of 2: 1; triethylamine is fifth generation polyamidoamine dendrimer G5.NH25 times of the mol number of the terminal amino; acetic anhydride is fifth generation polyamidoamine dendrimer G5.NH2Terminal amino group molo3 times the number of moles;
s3, preparation of a target display contrast agent:
(31) dissolving Gal-BSA-SPIO obtained in step S1 in water, and adding { (Au) obtained in step S20)25-G5.NH2-mPEG20Will { (Au) by self-assembly0)25-G5.NH2-mPEG20Assembled at Fe3O4A nanoparticle surface;
(32) then adding hydrazine hydrate, stirring for 1.5 times, adding triethylamine, continuing to stir for 20min, then adding acetic anhydride, continuing to stir for reaction for 12h, dialyzing, and freeze-drying to obtain the target display contrast agent.
Among them, hydrazine hydrate and HAuCl4In a molar ratio of 2: 1; triethylamine is fifth generation polyamidoamine dendrimer G5.NH24 times of the mol number of the terminal amino; acetic anhydride is fifth generation polyamidoamine dendrimer G5.NH23 times of the mol number of the terminal amino;
the preparation of the lactoalbumin comprises the following specific steps: weighing 240mg of bovine serum albumin, 1.2mg of lactose and 816mg of cyano sodium borohydride by adopting a reducing amine method, dissolving in 30ml of 0.2M phosphate buffer solution with the pH value of 8.0, and stirring in a water bath at 37 ℃ for reacting for 35 hours; dialyzing the obtained reaction solution against distilled water at low temperature for 3d, and alternately changing dialyzed external solution; centrifuging the dialyzed solution at low temperature, collecting supernatant, separating with Sephadex G-25 column chromatography, and freeze drying the purified solution to obtain white solid powder.
Example 2
S1, preparation of Gal-BSA-SPIO: the preparation is the same as example 1;
S2、{(Au0)25-G5.NH2-mPEG20preparation of }:
(21) dissolving mPEG-COOH in DMSO, and then adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to perform activation reaction for 2h to obtain activated mPEG-COOH;
(22) reacting activated mPEG-COOH with fifth generation polyamidoamine dendrimer G5.NH2Mixing with the DMSO solution, adding HAuCl4Stirring to react for 30min, and adding NaBH4Continuously stirring for reaction for 1h, then adding triethylamine, stirring for reaction for 10min, finally adding acetic anhydride, continuously stirring for reaction for 20h, then dialyzing, and freeze-drying to obtain { (Au)0)25-G5.NH2-mPEG20};
Wherein the molar ratio of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and mPEG-COOH is 9:10: 1; mPEG-COOH and fifth generation polyamidoamine dendrimers G5.NH2In a molar ratio of 18:1, HAuCl4And { (Au)0)25-G5.NH2-mPEG20The molar ratio of 165: 1; NaBH4And HAuCl4In a molar ratio of 2: 1; triethylamine is fifth generation polyamidoamine dendrimer G5.NH23 times of the mol number of the terminal amino; acetic anhydride is fifth generation polyamidoamine dendrimer G5.NH22 times of the mol number of the terminal amino;
s3, preparation of a target display contrast agent:
(31) dissolving Gal-BSA-SPIO obtained in step S1 in water, and adding { (Au) prepared in step (2)0)25-G5.NH2-mPEG20Will { (Au) by self-assembly0)25-G5.NH2-mPEG20Assembled at Fe3O4A nanoparticle surface;
(32) then adding hydrazine hydrate, stirring for 1.5 times, adding triethylamine, continuing to stir for 20min, then adding acetic anhydride, continuing to stir for reaction for 12h, dialyzing, and freeze-drying to obtain the target display contrast agent.
Among them, hydrazine hydrate and HAuCl4In a molar ratio of 2: 1; triethylamine is fifth generation polyamidoamine dendrimer G5.NH24 times of the mol number of the terminal amino; acetic anhydride is fifth generation polyamidoamine dendrimer G5.NH2The number of moles of the terminal amino group is 3 times.
Example 3
S1, preparation of Gal-BSA-SPIO: the preparation is the same as example 1;
S2、{(Au0)25-G5.NH2-mPEG20preparation of }:
(21) dissolving mPEG-COOH by using DMSO, and then adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to perform activation reaction for 2h to obtain activated mPEG-COOH;
(22) reacting activated mPEG-COOH with fifth generation polyamidoamine dendrimer G5.NH2Mixing with the DMSO solution, adding HAuCl4Stirring to react for 35min, and adding NaBH4Continuously stirring for reaction for 2h, then adding triethylamine, stirring for reaction for 30min, finally adding acetic anhydride, continuously stirring for reaction for 25h, then dialyzing, and freeze-drying to obtain { (Au)0)25-G5.NH2-mPEG20};
Wherein the molar ratio of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and mPEG-COOH is 9:10: 1; mPEG-COOH and fifth generation polyamidoamine dendrimers G5.NH2In a molar ratio of 15:1, HAuCl4And { (Au)0)25-G5.NH2-mPEG20The molar ratio of (1)/(180); NaBH4And HAuCl4In a molar ratio of 2: 1; triethylamine is fifth generation polyamidoamine dendrimer G5.NH23 times of the mol number of the terminal amino; acetic anhydride is fifth generation polyamidoamine dendrimer G5.NH2The mol number of the terminal amino is 6 times that of the terminal amino;
s3, preparation of a target display contrast agent:
(31) dissolving Gal-BSA-SPIO obtained in step S1 in water, and adding { (Au) obtained in step S20)25-G5.NH2-mPEG20Will { (Au) by self-assembly0)25-G5.NH2-mPEG20Assembled at Fe3O4A nanoparticle surface;
(32) then adding hydrazine hydrate, stirring for 1.5 times, adding triethylamine, continuing to stir for 20min, then adding acetic anhydride, continuing to stir for reaction for 12h, dialyzing, and freeze-drying to obtain the target display contrast agent.
Among them, hydrazine hydrate and HAuCl4In a molar ratio of 2: 1; triethylamine is fifth generation polyamidoamine dendrimer G5.NH25 times of the mol number of the terminal amino; acetic acidAnhydride is fifth generation polyamidoamine dendrimer G5.NH2The number of moles of the terminal amino group is 2 times.
Example 4
S1, preparation of Gal-BSA-SPIO: the preparation is the same as example 1;
S2、{(Au0)25-G5.NH2-mPEG20preparation of }: the preparation is the same as example 1;
s3, preparation of a target display contrast agent:
(31) dissolving Gal-BSA-SPIO obtained in step S1 in water, and adding { (Au) prepared in step S20)25-G5.NH2-mPEG20Will { (Au) by self-assembly0)25-G5.NH2-mPEG20Assembled at Fe3O4A nanoparticle surface;
(32) and then adding hydrazine hydrate, stirring for 2h, adding triethylamine, continuing to stir for 10min, adding acetic anhydride, continuing to stir for reaction for 15h, dialyzing, and freeze-drying to obtain the target display contrast agent.
Among them, hydrazine hydrate and HAuCl4In a molar ratio of 2: 1; triethylamine is fifth generation polyamidoamine dendrimer G5.NH2The mol number of the terminal amino is 6 times that of the terminal amino; acetic anhydride is fifth generation polyamidoamine dendrimer G5.NH2The number of moles of the terminal amino group is 4 times.
Example 5
S1, preparation of Gal-BSA-SPIO: the preparation is the same as example 1;
S2、{(Au0)25-G5.NH2-mPEG20preparation of }: the preparation is the same as example 1;
s3, preparation of a target display contrast agent:
(31) dissolving Gal-BSA-SPIO obtained in step S1 in water, and adding { (Au) prepared in step (2)0)25-G5.NH2-mPEG20Will { (Au) by self-assembly0)25-G5.NH2-mPEG20Assembled at Fe3O4A nanoparticle surface;
(32) then adding hydrazine hydrate, stirring for 1.5 times, adding triethylamine, continuing to stir for 20min, then adding acetic anhydride, continuing to stir for reaction for 12h, dialyzing, and freeze-drying to obtain the target display contrast agent.
Among them, hydrazine hydrate and HAuCl4In a molar ratio of 2: 1; triethylamine is fifth generation polyamidoamine dendrimer G5.NH23 times of the mol number of the terminal amino; acetic anhydride is fifth generation polyamidoamine dendrimer G5.NH2The number of moles of the terminal amino group is 5 times.
The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (8)
1. The preparation method of the contrast agent based on ASGPR multi-modal image display is characterized by comprising the following steps:
s1, preparation of Gal-BSA-SPIO:
(11) allowing the nanometer iron oxide SPIO to pass through a chromatographic column, separating the small-particle-size SPIO, dialyzing, and concentrating until the Fe content is 6-8 mg/ml;
(12) then adjusting the pH value to 6.5-7.0, adding a lactose-based albumin solution with the same volume and mass concentration of 1%, and carrying out ice bath ultrasonic oscillation for 1-3 h;
(13) after ultrasonic oscillation, adding 1M NaCl solution to remove unbound lactoalbumin, and adjusting the pH value of the solution to 7.3-7.5 to obtain Gal-BSA-SPIO solution;
S2、{(Au0)25-G5.NH2-mPEG20preparation of }:
(21) dissolving mPEG-COOH, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to perform activation reaction for 1-2.5 h to obtain activated mPEG-COOH;
(22) reacting activated mPEG-COOH with fifth generation polyamidoamine dendrimer G5.NH2Mixing the DMSO solution, adding HAuCl4Reacting for 30-40 min, and then adding NaBH4Continuously stirring and reacting for 1-2 h, then adding triethylamine, stirring for 10-30 min, then adding acetic anhydride, continuously stirring and reacting for 18-25 h, dialyzing, and freeze-drying to obtain { (Au)0)25-G5.NH2-mPEG20};
S3, preparation of a target display contrast agent:
(31) dissolving Gal-BSA-SPIO obtained in step S1 in water, and adding { (Au) obtained in step S20)25-G5.NH2-mPEG20Will { (Au) by self-assembly0)25-G5.NH2-mPEG20Assembled at Fe3O4A nanoparticle surface;
(32) and then adding hydrazine hydrate, stirring for 1-2 h, adding triethylamine, stirring for 10-30 min, adding acetic anhydride, stirring for reacting for 10-15 h, dialyzing, and freeze-drying to obtain the target display contrast agent.
2. The method for preparing an ASGPR based multimodal imaging display contrast agent according to claim 1, wherein mPEG-COOH is dissolved in the solvent DMSO in step (21).
3. The method for preparing an ASGPR multi-modal image display contrast agent according to claim 1, wherein in the step (21), the molar ratio of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and mPEG-COOH is (8-10): 10 (1-2), and the concentrations of mPEG-COOH, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide are 6-10 mg/mL, 5-8 mg/mL and 3-6 mg/mL, respectively.
4. According to claim 1The preparation method of the ASGPR-based multi-modal image display contrast agent is characterized in that mPEG-COOH and fifth generation polyamidoamine dendrimer G5.NH are adopted in the step (22)2The molar ratio of (15-18) to (1), the fifth generation of polyamide-amine dendrimer G5.NH2The concentration of (b) is 5-15 mg/mL.
5. The method of claim 1, wherein in step S2, HAuCl is added to the ASGPR-based multi-modal image display contrast medium4And { (Au)0)25-G5.NH2-mPEG20The molar ratio of (165-180): 1; NaBH4And HAuCl4In a molar ratio of 2: 1; triethylamine is fifth generation polyamidoamine dendrimer G5.NH2The mole number of the terminal amino is 3-8 times that of the terminal amino; acetic anhydride is fifth generation polyamidoamine dendrimer G5.NH2The number of moles of the terminal amino groups is 2 to 6 times.
6. The method for preparing contrast agent based on ASGPR multi-modal image display, according to the claim, wherein in step S3, hydrazine hydrate and HAuCl4In a molar ratio of 2: 1; triethylamine is fifth generation polyamidoamine dendrimer G5.NH2The molar number of the terminal amino groups is 3-6 times that of the terminal amino groups; acetic anhydride is fifth generation polyamidoamine dendrimer G5.NH2The number of moles of the terminal amino groups is 2 to 5 times.
7. The method for preparing an ASGPR-based multimodal imaging display contrast agent according to claim 1, wherein the preparation of lactoalbumin comprises the steps of: dissolving bovine serum albumin, lactose and sodium cyanoborohydride in a phosphate buffer solution, stirring in a water bath for 24-80 h for reaction, dialyzing, centrifuging to obtain a supernatant, separating and purifying by using sephadex column chromatography, and freeze-drying to obtain the lactosyl albumin, wherein the mass ratio of the bovine serum albumin, the lactose and the sodium cyanoborohydride is 1 (0.002-0.005) to 3.2-3.8).
8. Use of an ASGPR based multimodal image visualisation contrast agent prepared according to the process of any one of claims 1 to 7 for CT/MR bimodal image imaging.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202010819064.XA CN111956811A (en) | 2020-08-14 | 2020-08-14 | Preparation method of ASGPR (advanced red-fluorescence tomography-based red fluorescent protein) multi-modal image display contrast agent |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202010819064.XA CN111956811A (en) | 2020-08-14 | 2020-08-14 | Preparation method of ASGPR (advanced red-fluorescence tomography-based red fluorescent protein) multi-modal image display contrast agent |
Publications (1)
Publication Number | Publication Date |
---|---|
CN111956811A true CN111956811A (en) | 2020-11-20 |
Family
ID=73366104
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202010819064.XA Pending CN111956811A (en) | 2020-08-14 | 2020-08-14 | Preparation method of ASGPR (advanced red-fluorescence tomography-based red fluorescent protein) multi-modal image display contrast agent |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN111956811A (en) |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102671217A (en) * | 2012-06-05 | 2012-09-19 | 东华大学 | Preparation of CT/MR bimodal imaging nano contrast medium with folate targeting function |
CN103212091A (en) * | 2013-04-28 | 2013-07-24 | 东华大学 | Preparation method of hepatocarcinoma target CT (Computed Tomography) contrast medium based on Au DENPs-LA (Dendrimer-entrapped Gold Nanoparticles-Lactobionic acid) |
CN103223178A (en) * | 2013-05-09 | 2013-07-31 | 东华大学 | Preparation method of folic acid modified multifunctional targeted contrast agent magnetic iron oxide/gold nanoparticles |
CN108079319A (en) * | 2016-11-21 | 2018-05-29 | 任秀敏 | A kind of nanogold particle composite material and preparation method thereof of polyethyleneglycol modified the 5th generation dendrimer package and application |
CN108079320A (en) * | 2016-11-21 | 2018-05-29 | 任秀敏 | The nanogold particle composite material and preparation method and application of a kind of the 5th generation dendrimer package of modified with folic acid |
-
2020
- 2020-08-14 CN CN202010819064.XA patent/CN111956811A/en active Pending
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102671217A (en) * | 2012-06-05 | 2012-09-19 | 东华大学 | Preparation of CT/MR bimodal imaging nano contrast medium with folate targeting function |
CN103212091A (en) * | 2013-04-28 | 2013-07-24 | 东华大学 | Preparation method of hepatocarcinoma target CT (Computed Tomography) contrast medium based on Au DENPs-LA (Dendrimer-entrapped Gold Nanoparticles-Lactobionic acid) |
CN103223178A (en) * | 2013-05-09 | 2013-07-31 | 东华大学 | Preparation method of folic acid modified multifunctional targeted contrast agent magnetic iron oxide/gold nanoparticles |
CN108079319A (en) * | 2016-11-21 | 2018-05-29 | 任秀敏 | A kind of nanogold particle composite material and preparation method thereof of polyethyleneglycol modified the 5th generation dendrimer package and application |
CN108079320A (en) * | 2016-11-21 | 2018-05-29 | 任秀敏 | The nanogold particle composite material and preparation method and application of a kind of the 5th generation dendrimer package of modified with folic acid |
Non-Patent Citations (1)
Title |
---|
张晓东: "肝细胞去唾液酸糖蛋白受体介导的半乳糖基白蛋白-SPIO纳米微粒的合成及其肝脏MR成像研究", 《中国优秀博硕士学位论文全文数据库(博士) 医药卫生科技辑》 * |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Zhao et al. | Chlorotoxin peptide-functionalized polyethylenimine-entrapped gold nanoparticles for glioma SPECT/CT imaging and radionuclide therapy | |
Shu et al. | Facile preparation of a new gadofullerene-based magnetic resonance imaging contrast agent with high 1H relaxivity | |
Huang et al. | Biodegradable polydisulfide dendrimer nanoclusters as MRI contrast agents | |
Wei et al. | pH-responsive pHLIP (pH low insertion peptide) nanoclusters of superparamagnetic iron oxide nanoparticles as a tumor-selective MRI contrast agent | |
Tan et al. | Peptide-targeted nanoglobular Gd-DOTA monoamide conjugates for magnetic resonance cancer molecular imaging | |
Li et al. | A new interleukin-13 amino-coated gadolinium metallofullerene nanoparticle for targeted MRI detection of glioblastoma tumor cells | |
AU2004244811B9 (en) | Magnetic nanoparticles linked to a ligand | |
Chen et al. | Triblock copolymer coated iron oxide nanoparticle conjugate for tumor integrin targeting | |
Fan et al. | Design and Biomedical Applications of Poly (amidoamine)‐Dendrimer‐Based Hybrid Nanoarchitectures | |
Li et al. | Peptide-enhanced tumor accumulation of upconversion nanoparticles for sensitive upconversion luminescence/magnetic resonance dual-mode bioimaging of colorectal tumors | |
JPH08501097A (en) | Biocompatible polymers including clinical diagnostic and therapeutic moieties | |
Sun et al. | Radiolabeled ultra-small Fe3O4 nanoprobes for tumor-targeted multimodal imaging | |
Zhang et al. | Paramagnetic albumin decorated CuInS 2/ZnS QDs for CD133+ glioma bimodal MR/fluorescence targeted imaging | |
KR20050121673A (en) | Conjugates for medical imaging comprising carrier, targeting moiety and a contrast agent | |
Li et al. | Arg-Gly-Asp-D-Phe-Lys peptide-modified PEGylated dendrimer-entrapped gold nanoparticles for targeted computed tomography imaging of breast carcinoma | |
US20180117188A1 (en) | Radiolabeled active targeting pharmaceutical composition and the use thereof | |
Tian et al. | Glypican-3 (GPC3) targeted Fe 3 O 4 core/Au shell nanocomplex for fluorescence/MRI/photoacoustic imaging-guided tumor photothermal therapy | |
CN105079826A (en) | Preparation method and application of RGD@BBN double-targeted MR (magnetic resonance)/optical dual-mode molecular probe | |
Li et al. | Melanin-gelatin nanoparticles with both EPR effect and renal clearance for PA/MRI dual-modal imaging of tumors | |
Qiu et al. | Liver injury long-term monitoring and fluorescent image-guided tumor surgery using self-assembly amphiphilic donor-acceptor NIR-II dyes | |
Wang et al. | Engineering of 177Lu-labeled gold encapsulated into dendrimeric nanomaterials for the treatment of lung cancer | |
Xin et al. | Stearic acid-grafted chitooligosaccharide nanomicelle system with biocleavable gadolinium chelates as a multifunctional agent for tumor imaging and drug delivery | |
Zheng et al. | Recent progress of molecular imaging probes based on gadofullerenes | |
Wang et al. | Multifunctional nanoprobe for multi-mode imaging and diagnosis of metastatic prostate cancer | |
Gong et al. | Ultrasound-enhanced theranostics of orthotopic breast cancer through a multifunctional core–shell tecto dendrimer-based nanomedicine platform |
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: 20201120 |
|
RJ01 | Rejection of invention patent application after publication |