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
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- UIWYJDYFSGRHKR-UHFFFAOYSA-N gadolinium atom Chemical compound [Gd] UIWYJDYFSGRHKR-UHFFFAOYSA-N 0.000 description 1
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- 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
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- 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
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
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- 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.
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