CN110478493B - Construction method of nano graphene composite material carrying CCL2 antibody - Google Patents

Construction method of nano graphene composite material carrying CCL2 antibody Download PDF

Info

Publication number
CN110478493B
CN110478493B CN201910802748.6A CN201910802748A CN110478493B CN 110478493 B CN110478493 B CN 110478493B CN 201910802748 A CN201910802748 A CN 201910802748A CN 110478493 B CN110478493 B CN 110478493B
Authority
CN
China
Prior art keywords
nano
peg
rgo
ionp
composite
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN201910802748.6A
Other languages
Chinese (zh)
Other versions
CN110478493A (en
Inventor
战跃福
吴烨华
王毅翔
鲁宏
郭子义
陈建强
关莹
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Maternal And Child Health Hospital Of Hainan Province
Original Assignee
Maternal And Child Health Hospital Of Hainan Province
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Maternal And Child Health Hospital Of Hainan Province filed Critical Maternal And Child Health Hospital Of Hainan Province
Priority to CN201910802748.6A priority Critical patent/CN110478493B/en
Publication of CN110478493A publication Critical patent/CN110478493A/en
Application granted granted Critical
Publication of CN110478493B publication Critical patent/CN110478493B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
    • 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
    • A61K47/02—Inorganic compounds
    • 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
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • 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
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/52—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an inorganic compound, e.g. an inorganic ion that is complexed with the active ingredient
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Epidemiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Inorganic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Organic Chemistry (AREA)
  • Biophysics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Biomedical Technology (AREA)
  • Oncology (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Carbon And Carbon Compounds (AREA)
  • Medicinal Preparation (AREA)

Abstract

The invention provides a construction method of a nano graphene composite material carrying a CCL2 antibody, which comprises the following steps of (1) synthesizing C18-PMH-PEG-NH with an amino functional group on the surface2A nanocomposite; (2) using RGO-IONP and C18-PMH-PEG-NH2Carrying out ultrasonic mixing on the nano composite for 80-90 min to obtain RGO-IONP-PEG-NH containing amino functional groups on the surface2A nanocomposite; (3) contacting the CCL2 antibody with RGO-IONP-PEG-NH2Adding the nano-composite into a microwave tube, carrying out dark ultrasonic mixing for 20-25 min, putting the nano-metal oxide and the bis (2-chlorophenyl) boric acid combined as a catalyst under microwave radiation for catalytic reaction, wherein the microwave temperature is less than or equal to 50 ℃, and the microwave time is 15-20 min, so as to promote the CCL2 antibody and the RGO-IONP-PEG-NH2And (3) performing reaction connection on the nano-composite, centrifuging, collecting supernatant, and removing the CCL2 antibody which is not connected by using an ultrafiltration tube to obtain the RGO-IONP-PEG-CCL2 nano-functional composite. The yield of the RGO-IONP-PEG-CCL2 nano functional composite obtained by the construction method is improved, the reaction temperature is low, the reaction condition is mild, and the high loading rate of the nano graphene composite material on the CCL2 antibody is realized.

Description

Construction method of nano graphene composite material carrying CCL2 antibody
Technical Field
The invention relates to the technical field of biomedical composite materials, in particular to a construction method of a nano graphene composite material carrying a CCL2 antibody.
Background
Nano graphene is a two-dimensional planar material formed of a single layer of carbon atoms, and in recent years, functional complexes based on nano graphene have attracted much attention for application in the biomedical field, and can be used as various biomolecule carriers for biological detection, drug delivery, and gene delivery. At present, different nano graphene functional composite materials are obtained by continuously researching a new construction method of carrier graphene, and are functionally modified to form excellent carriers for in vivo delivery of drugs, antibodies, genes and the like, so that the nano graphene functional composite materials are applied to targeted diagnosis and treatment in ischemic diseases and tumors. However, different construction methods of the graphene carrier often have obvious differences in the load rates of small molecule drugs, antibodies, genes and the like, and further influence the final in vivo drug treatment effect.
At present, in the process of researching how to break through the immune barrier mainly based on tumor-related neutrophils, inhibit the invasion/metastasis cascade mechanism induced by the tumor-related neutrophils and fully exert the anti-tumor curative effect of salmonella, graphene is often required to be functionally modified so as to efficiently carry a CCL2 antibody, so that the CCL2 antibody is used for interfering with the tumor microenvironment neutrophil chemokine CCL2 and regulating the immune barrier mainly based on neutrophils. However, in the process of constructing the nano graphene composite material carrying the CCL2 antibody, the existing construction method is often insufficient in reaction, and the finally obtained nano graphene composite material has a low loading rate on the CCL2 antibody, so that the aim of carrying the CCL2 antibody efficiently cannot be fulfilled.
Disclosure of Invention
In view of the above, the invention provides a construction method of a nano graphene composite material carrying a CCL2 antibody, which has a high loading rate and mild reaction conditions.
The technical scheme of the invention is realized as follows:
the invention provides a construction method of a nano graphene composite material carrying a CCL2 antibody, which comprises the following steps:
(1) synthesis of C18-PMH-PEG-NH2
Using C18PMH, mPEG-NH2And NH2PEG-BOC, C18-PMH-PEG-NH with amino functional group on the surface2A nanocomposite; c18PMH is polymaleic anhydride-C18 alkene;
(2) synthesis of RGO-IONP-PEG-NH2
The RGO-IONP is adopted to react with the C18-PMH-PEG-NH obtained in the step (1)2The nano composite is prepared from the following components in a mass ratio of 1: (10-15) carrying out ultrasonic mixing for 80-90 min to remove redundant C18-PMH-PEG-NH2Nano-composite to obtain RGO-IONP-PEG-NH containing amino functional group on surface2A nanocomposite; RGO is reduced graphene, IONP is ferroferric oxide;
(3) synthesis of RGO-IONP-PEG-CCL2 nanometer functional complexes
The CCL2 antibody is first mixed with RGO-IONP-PEG-NH obtained in step (2)2The nano-composite is prepared from the following components in percentage by mass of 1: (8-9) adding the mixture into a microwave tube, carrying out dark ultrasonic mixing for 20-25 min, then using the combination of the nano metal oxide and the 2-chlorphenyl boric acid as a catalyst, and placing the catalyst under microwave radiation for catalytic reaction, wherein the microwave temperature is less than or equal to 50 ℃, and the microwave time is 15-20 min, so that the CCL2 antibody and RGO-IONP-PEG-NH are promoted2And connecting the nano-composite through amidation reaction, finally centrifuging the reaction solution at 6000-8000 rpm for 2-3 min, removing unstable particles or agglomerates, collecting supernatant, and removing the unconnected CCL2 antibody by adopting a 100-110 KD ultrafiltration tube to obtain the RGO-IONP-PEG-CCL2 nano-functional composite. The invention provides a construction method of a nano graphene composite material carrying a CCL2 antibody, which aims to efficiently connect the CCL2 antibody to a composite (RGO-IONP-PEG) of reduced graphene, ferroferric oxide and polyethylene glycol, and firstly synthesizes C18-PMH-PEG-NH with amino functional group protection on the surface2Nano-composite, then, reducing graphene and ferroferric oxide composite (RGO-IONP) and C18-PMH-PEG-NH are utilized2Ultrasonic mixing the nano-composite to obtain RGO-IONP-PEG-NH containing amino functional groups on the surface2A nanocomposite; finally, the CCL2 antibody was passed through RGO-IONP-PEG-NH2The nano composite is subjected to ultrasonic mixing according to a certain proportion, simultaneously, nano metal oxide and 2-chlorophenyl boronic acid are combined to serve as a catalyst, and under the condition of microwave radiation, a nano graphene composite (RGO-IONP-PEG-CCL2 nano functional composite) carrying a CCL2 antibody is catalytically synthesized, the yield of the RGO-IONP-PEG-CCL2 nano functional composite obtained through amidation reaction is obviously improved, the reaction temperature is low, the reaction condition is mild, and therefore the high loading rate of the nano graphene composite on the CCL2 antibody is effectively achieved.
Further, in the step (3), the mass ratio of the nano metal oxide to the 2-chlorophenyl boronic acid is 1 (1-2). Promoting the mass ratio of the CCL2 antibody to the RGO-IONP-PEG-NH by controlling the mass ratio of the nano metal oxide to the 2-chlorphenyl boronic acid in the catalyst2And the amidation reaction of the nano-composite improves the yield.
Further, in the step (3), the mass ratio of the catalyst to the reactant is 1 (8-10). The mass ratio of the catalyst to the reactant is strictly controlled, so that the reactant can realize effective amidation reaction under the condition of microwave radiation at a lower temperature.
Further, in the step (3), the nano metal oxide is nano magnesium oxide or nano aluminum oxide. The combination of nano-magnesia or nano-alumina and 2-chlorphenyl boric acid as the catalyst has better catalytic effect, and the highest yield can reach 96 percent.
Further, in the step (3), the temperature of the microwave is 40-50 ℃, and the microwave time is 18 min. The temperature and the microwave radiation time of the microwave are adjusted, the heat conduction time is shortened, and the reaction rate of reactants is improved.
Further, in step (3), the CCL2 antibody was conjugated with RGO-IONP-PEG-NH2The nano-composite is prepared from the following components in percentage by mass of 1:8 into a microwave tube.
Further explaining, in the step (3), after the microwave radiation reaction is finished, standing in a constant-temperature water bath at 30-40 ℃ for 2-3 min, and then centrifuging the reaction solution at the rotating speed of 7500rpm for 3min to remove unstable particles or agglomerates.
Further illustrates that, in the step (1), C18-PMH-PEG-NH2The synthesis of the nano-composite comprises the following steps:
(1) mixing C18PMH and mPEG-NH2、NH2-Mixing and adding PEG-BOC into a round-bottom flask according to the mass ratio of 1:10:5, then adding dichloromethane, dissolving, sequentially adding EDC, triethylamine and 2-chlorophenyl boric acid to serve as a catalyst, and magnetically stirring for 24 hours under the protection of nitrogen at normal temperature to obtain a reaction initial solution;
(2) adding trifluoroacetic acid into the reaction initial solution, magnetically stirring for 3 hours, removing BOC protecting group, blowing dry with nitrogen to remove excessive trifluoroacetic acid, dissolving the residue in ultrapure water, transferring into dialysis bag with molecular weight of 14kDa, dialyzing for 48 hours, removing unreacted mPEG-NH2And other unreacted substances are subjected to freeze drying to obtain white solid C18PMH-PEG-NH2The nanocomposite, and stored at-20 ℃ for later use. At C18-PMH-PEG-NH2In the synthesis process of the nano-composite, EDC, triethylamine and 2-chlorphenyl boric acid are used as a catalyst in a combined manner to improve C18PMH-PEG-NH2Yield of the nanocomposite.
Further, in step (2), RGO-IONP was reacted with C18-PMH-PEG-NH obtained in step (1)2The nano composite is prepared from the following components in a mass ratio of 1: 12 ultrasonic mixing is carried out for 85 min.
Compared with the prior art, the invention has the beneficial effects that: the invention firstly synthesizes C18-PMH-PEG-NH with amino functional group protection on the surface2The nano-composite is prepared by reducing graphene and ferroferric oxide composite and C18-PMH-PEG-NH2Ultrasonic mixing the nano-composite to obtain RGO-IONP-PEG-NH2A nanocomposite; finally, the CCL2 antibody was mixed with RGO-IONP-PEG-NH2The nano-composite is prepared according to the following steps of 1: (8-9) on the basis of ultrasonic mixing in mass ratio, nano metal oxide and 2-chlorophenyl boronic acid are combined to serve as a catalyst, and a nano graphene composite material carrying a CCL2 antibody is catalytically synthesized under the condition of microwave radiation, so that the yield of the RGO-IONP-PEG-CCL2 nano functional composite finally obtained through amidation reaction is obviously improved, the reaction temperature is low, and the reaction conditions are lowThe method is mild, so that the high loading rate of the nano graphene composite material on the CCL2 antibody is effectively realized.
Detailed Description
In order to better understand the technical content of the invention, specific examples are provided below to further illustrate the invention.
The experimental methods used in the examples of the present invention are all conventional methods unless otherwise specified.
The materials, reagents and the like used in the examples of the present invention can be obtained commercially without specific description.
Example 1-a method of constructing a nanographene composite carrying CCL2 antibody, comprising the steps of:
(1) synthesis of C18-PMH-PEG-NH2
Using C18PMH, mPEG-NH2And NH2PEG-BOC, C18-PMH-PEG-NH with amino functional group on the surface2A nanocomposite;
(2) synthesis of RGO-IONP-PEG-NH2
The RGO-IONP is adopted to react with the C18-PMH-PEG-NH obtained in the step (1)2The nano composite is prepared from the following components in a mass ratio of 1: 15 ultrasonic mixing for 90min to remove the excessive C18-PMH-PEG-NH2Nano-composite to obtain RGO-IONP-PEG-NH containing amino functional group on surface2A nanocomposite;
(3) synthesis of RGO-IONP-PEG-CCL2 nanometer functional complexes
The CCL2 antibody is first mixed with RGO-IONP-PEG-NH obtained in step (2)2The nano-composite is prepared from the following components in percentage by mass of 1:9, adding the mixture into a microwave tube, carrying out dark ultrasonic mixing for 20-25 min, and then using the combination of the nano metal oxide and 2-chlorphenyl boric acid as a catalyst, wherein the mass ratio of the nano metal oxide to the 2-chlorphenyl boric acid is 1: 1; the mass ratio of the catalyst to the reactants is 1: 8; placing the sample in a microwave radiation environment for catalytic reaction, wherein the microwave temperature is 50 ℃ and the microwave time is 15min, and promoting the CCL2 antibody and the RGO-IONP-PEG-NH2Connecting the nano-composite through amidation reaction, finally centrifuging the reaction solution at 6000rpm for 3min, removing unstable particles or agglomerates, collecting supernatant, and collectingRemoving the unconnected CCL2 antibody by using an ultrafiltration tube with 100-110 KD to obtain the RGO-IONP-PEG-CCL2 nano functional compound.
Example 2-a method of constructing a nanographene composite carrying CCL2 antibody, comprising the steps of:
(1) synthesis of C18-PMH-PEG-NH2
Using C18PMH, mPEG-NH2And NH2PEG-BOC, C18-PMH-PEG-NH with amino functional group on the surface2A nanocomposite comprising the steps of:
a. mixing C18PMH and mPEG-NH2、NH2-Mixing and adding PEG-BOC into a round-bottom flask according to the mass ratio of 1:10:5, then adding dichloromethane, dissolving, sequentially adding EDC, triethylamine and 2-chlorophenyl boric acid to serve as a catalyst, and magnetically stirring for 24 hours under the protection of nitrogen at normal temperature to obtain a reaction initial solution;
b. adding trifluoroacetic acid into the reaction initial solution, magnetically stirring for 3 hours, removing BOC protecting group, blowing dry with nitrogen to remove excessive trifluoroacetic acid, dissolving the residue in ultrapure water, transferring into dialysis bag with molecular weight of 14kDa, dialyzing for 48 hours, removing unreacted mPEG-NH2And other unreacted substances are subjected to freeze drying to obtain white solid C18PMH-PEG-NH2The nano-composite is stored at-20 ℃ for later use;
(2) synthesis of RGO-IONP-PEG-NH2
The RGO-IONP is adopted to react with the C18-PMH-PEG-NH obtained in the step (1)2The nano composite is prepared from the following components in a mass ratio of 1:10 ultrasonic mixing for 80min to remove excessive C18-PMH-PEG-NH2Nano-composite to obtain RGO-IONP-PEG-NH containing amino functional group on surface2A nanocomposite;
(3) synthesis of RGO-IONP-PEG-CCL2 nanometer functional complexes
The CCL2 antibody is first mixed with RGO-IONP-PEG-NH obtained in step (2)2The nano-composite is prepared from the following components in percentage by mass of 1:9, adding the mixture into a microwave tube, carrying out dark ultrasonic mixing for 20-25 min, and then using the combination of nano magnesium oxide and 2-chlorphenyl boric acid as a catalyst, wherein the nano magnesium oxide and the 2-chlorphenyl boric acid are used as the catalystThe mass ratio of (A) to (B) is 1: 1; the mass ratio of the catalyst to the reactants is 1: 10; placing in microwave radiation at 40 deg.C for 20min for catalytic reaction, standing in 30 deg.C constant temperature water bath for 3min to promote CCL2 antibody and RGO-IONP-PEG-NH2And connecting the nano-composite through amidation reaction, finally centrifuging the reaction solution at 8000rpm for 2min, removing unstable particles or agglomerates, collecting supernatant, and removing the unconnected CCL2 antibody by adopting an ultrafiltration tube with 100-110 KD to obtain the RGO-IONP-PEG-CCL2 nano-functional composite.
Example 3-a method of constructing a nanographene composite carrying CCL2 antibody, comprising the steps of:
(1) synthesis of C18-PMH-PEG-NH2
Using C18PMH, mPEG-NH2And NH2PEG-BOC, C18-PMH-PEG-NH with amino functional group on the surface2A nanocomposite comprising the steps of:
a. 20mg of C18PMH, 200mg of mPEG-NH2100mg of NH2-Mixing PEG-BOC, adding the mixture into a 30mL round-bottom flask, adding 5mL dichloromethane, dissolving, sequentially adding 11mg EDC, 13uL triethylamine and 2mg 2-chlorophenylboronic acid as a catalyst, and magnetically stirring for 24 hours under the protection of nitrogen at normal temperature to obtain a reaction initial solution;
b. adding 2mL of trifluoroacetic acid into the initial reaction solution, magnetically stirring for 3 hours, removing BOC protecting group, blowing and drying excess trifluoroacetic acid by using nitrogen, dissolving the residue in ultrapure water, transferring the residue into a dialysis bag, dialyzing for 48 hours, removing unreacted mPEG-NH2And other unreacted substances, and then freeze-drying to obtain white solid C18PMH-PEG-NH2A nanocomposite, stored at-20 ℃ for later use;
(2) synthesis of RGO-IONP-PEG-NH2
The RGO-IONP is adopted to react with the C18-PMH-PEG-NH obtained in the step (1)2The nano composite is prepared from the following components in a mass ratio of 1: 12 ultrasonic mixing for 85min to remove excessive C18-PMH-PEG-NH2Nanocomposite to obtain a composition containing ammonia on the surfaceFunctional group-based RGO-IONP-PEG-NH2A nanocomposite;
(3) synthesis of RGO-IONP-PEG-CCL2 nanometer functional complexes
The CCL2 antibody is first mixed with RGO-IONP-PEG-NH obtained in step (2)2The nano-composite is prepared from the following components in percentage by mass of 1:8, adding the mixture into a microwave tube, carrying out dark ultrasonic mixing for 20-25 min, and then using the combination of nano alumina and 2-chlorophenylboronic acid as a catalyst, wherein the mass ratio of the nano alumina to the 2-chlorophenylboronic acid is 1: 2; the mass ratio of the catalyst to the reactants is 1: 9; placing in microwave radiation at 45 deg.C for 18min for catalytic reaction, standing in 40 deg.C constant temperature water bath for 2min to promote CCL2 antibody and RGO-IONP-PEG-NH2And connecting the nano-composite through amidation reaction, finally centrifuging the reaction solution at 7500rpm for 3min, removing unstable particles or agglomerates, collecting supernatant, and removing the unconnected CCL2 antibody by adopting an ultrafiltration tube with 100-110 KD to obtain the RGO-IONP-PEG-CCL2 nano-functional composite.
Comparative example 1-according to the method for constructing a nano graphene composite material carrying CCL2 antibody in example 3, only the mass ratio of the nano metal oxide to 2-chlorophenylboronic acid in step (3) is changed to 1:3, and the rest steps are the same as example 3.
Comparative example 2-according to the method for constructing the nano graphene composite material carrying the CCL2 antibody in example 3, only the mass ratio of the nano metal oxide to the 2-chlorophenylboronic acid in step (3) is changed to 2:1, and the rest steps are the same as in example 3.
Comparative example 3-according to the construction method of the nano graphene composite carrying the CCL2 antibody in example 3, only the step (3) is changed, the nano metal oxide is nano iron oxide, and is combined with EDC as a catalyst, the mass ratio of the nano iron oxide to DEC is 1:2, and the rest steps are the same as example 3.
Comparative example 4-according to the method for constructing the nano graphene composite carrying the CCL2 antibody in example 3, only the mass ratio of the catalyst to the reactant in step (3) is changed to 1:11, and the rest steps are the same as in example 3.
Comparative example 5-according to the method for constructing the nano graphene composite carrying the CCL2 antibody in example 3, only the microwave temperature is 35 ℃ and the microwave time is 25min in step (3) are changed, and the rest steps are the same as in example 3.
Comparative example 6-construction of a nanographene composite carrying CCL2 antibody according to one of the methods of example 3, changing only the CCL2 antibody and RGO-iopp-PEG-NH in step (3)2The nano-composite is prepared from the following components in percentage by mass of 1: 7 was added to the microwave tube and the remaining steps were the same as in example 3.
Comparative example 7-according to the method for constructing the nano graphene composite material carrying the CCL2 antibody in example 3, only changing the step (1), adding EDC and triethylamine as a catalyst in combination, and magnetically stirring for 24 hours under the protection of nitrogen at normal temperature to obtain a reaction initial solution; the remaining steps were the same as in example 3.
According to the methods for constructing the nano graphene composite materials carrying the CCL2 antibody in examples 1 to 3 and comparative examples 1 to 7, 20 groups of test groups are respectively set, and the average yield of the RGO-iopp-PEG-CCL 2 nano functional composites obtained in examples 1 to 3 and comparative examples 1 to 7 is respectively counted, and the results are as follows:
Figure GDA0002844344760000071
Figure GDA0002844344760000081
according to the above table results, it can be seen that the construction methods of the nano graphene composite materials carrying the CCL2 antibody according to embodiments 1 to 3 of the present invention can increase the yield of the RGO-iopp-PEG-CCL 2 nano functional complex to 95% or more, and can link the CCL2 antibody to the RGO-iopp-PEG-NH under the condition of microwave irradiation at a lower temperature2On the nano composite, the reaction temperature is low, and the nano graphene composite material can be used for preparing the CCL2 antibodyHigh load factor. In addition, in example 3, after the microwave radiation reaction is completed, the reaction mixture is left standing in a constant temperature water bath at 30-40 ℃ for 2-3 min, so that the CCL2 antibody and the RGO-IONP-PEG-NH can be further promoted2The nano-composite is connected through amidation reaction, so that the yield of the nano-graphene composite material carrying the CCL2 antibody is improved. Comparing the experimental results of example 3 with those of comparative examples 1 to 3, it is shown that under the same reaction conditions, the change of the ratio and components of the nano metal oxide of the catalyst and the 2-chlorophenylboronic acid has a significant effect on the yield of the RGO-iopp-PEG-CCL 2 nano functional composite, and the yield is significantly reduced. According to the comparative example 4, it can be seen that changing the mass ratio of the catalyst and the reactant also has a certain influence on the yield of the final product, and controlling the reasonable mass ratio range thereof enables the reactant to effectively perform stable amidation reaction under the condition of microwave radiation at a lower temperature. According to comparative example 5, it can be seen that when the temperature of microwave irradiation is too low, the yield of the final RGO-IONP-PEG-CCL2 nano-functional complex is reduced, and therefore, the optimal reaction conditions for controlling the microwave irradiation are that the microwave temperature is 40 to 50 ℃ and the microwave time is 18min, based on the use of a specific catalyst. As can be seen from comparative example 6, by adjusting the CCL2 antibody to RGO-IONP-PEG-NH2The nano-composite is in a reasonable proportion, so that the yield of the nano-graphene composite material carrying the CCL2 antibody is ensured. As can be seen from comparative example 7, the use of EDC, triethylamine and 2-chlorophenylboronic acid in combination as catalysts can increase the C18PMH-PEG-NH content over the existing combination of EDC and triethylamine2The yield of the nano-complex is further beneficial to the subsequent synthesis of the RGO-IONP-PEG-CCL2 nano-functional complex.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims (7)

1. A construction method of a nano graphene composite material carrying a CCL2 antibody is characterized by comprising the following steps: the method comprises the following steps:
(1) synthesis of C18-PMH-PEG-NH2
Using C18PMH, mPEG-NH2And NH2PEG-BOC, C18-PMH-PEG-NH with amino functional group on the surface2A nanocomposite; c18PMH is polymaleic anhydride-C18 alkene;
(2) synthesis of RGO-IONP-PEG-NH2
The RGO-IONP is adopted to react with the C18-PMH-PEG-NH obtained in the step (1)2The nano composite is prepared from the following components in a mass ratio of 1: (10-15) carrying out ultrasonic mixing for 80-90 min to remove redundant C18-PMH-PEG-NH2Nano-composite to obtain RGO-IONP-PEG-NH containing amino functional group on surface2A nanocomposite; RGO is reduced graphene, IONP is ferroferric oxide;
(3) synthesis of RGO-IONP-PEG-CCL2 nanometer functional complexes
The CCL2 antibody is first mixed with RGO-IONP-PEG-NH obtained in step (2)2The nano-composite is prepared from the following components in percentage by mass of 1: (8-9) adding the mixture into a microwave tube, carrying out dark ultrasonic mixing for 20-25 min, then using the combination of the nano metal oxide and the 2-chlorphenyl boric acid as a catalyst, and placing the catalyst under microwave radiation for catalytic reaction, wherein the microwave temperature is less than or equal to 50 ℃, and the microwave time is 15-20 min, so that the CCL2 antibody and RGO-IONP-PEG-NH are promoted2Connecting the nano-composite through amidation reaction, finally centrifuging the reaction solution at 6000-8000 rpm for 2-3 min, removing unstable particles or agglomerates, collecting supernatant, and removing the unconnected CCL2 antibody by adopting a 100-110 KD ultrafiltration tube to obtain an RGO-IONP-PEG-CCL2 nano-functional composite; the mass ratio of the nano metal oxide to the 2-chlorphenyl boric acid is 1 (1-2); the nano metal oxide is nano magnesium oxide or nano aluminum oxide.
2. The method for constructing a nano graphene composite material carrying a CCL2 antibody according to claim 1, wherein the method comprises the following steps: in the step (3), the mass ratio of the catalyst to the reactant is 1 (8-10).
3. The method for constructing a nano graphene composite material carrying a CCL2 antibody according to claim 1, wherein the method comprises the following steps: in the step (3), the temperature of the microwave is 40-50 ℃, and the microwave time is 18 min.
4. The method for constructing a nano graphene composite material carrying a CCL2 antibody according to claim 1, wherein the method comprises the following steps: in step (3), the CCL2 antibody is conjugated with RGO-IONP-PEG-NH2The nano-composite is prepared from the following components in percentage by mass of 1:8 into a microwave tube.
5. The method for constructing a nano graphene composite material carrying a CCL2 antibody according to claim 1, wherein the method comprises the following steps: and (3) standing in a constant-temperature water bath at 30-40 ℃ for 2-3 min after the microwave radiation reaction is finished, and centrifuging the reaction solution at the rotating speed of 7500rpm for 3min to remove unstable particles or agglomerates.
6. The method for constructing a nano graphene composite material carrying a CCL2 antibody according to claim 1, wherein the method comprises the following steps: in the step (1), C18-PMH-PEG-NH2The synthesis of the nano-composite comprises the following steps:
(1) mixing C18PMH and mPEG-NH2、NH2-Mixing and adding PEG-BOC into a round-bottom flask according to the mass ratio of 1:10:5, then adding dichloromethane, dissolving, sequentially adding EDC, triethylamine and 2-chlorophenyl boric acid to serve as a catalyst, and magnetically stirring for 24 hours under the protection of nitrogen at normal temperature to obtain a reaction initial solution;
(2) adding trifluoroacetic acid into the reaction initial solution, magnetically stirring for 3 hours, removing BOC protecting group, blowing dry with nitrogen to remove excessive trifluoroacetic acid, dissolving the residue in ultrapure water, transferring into dialysis bag with molecular weight of 14kDa, dialyzing for 48 hours, removing unreacted mPEG-NH2And other unreacted substances are subjected to freeze drying to obtain white solid C18PMH-PEG-NH2The nanocomposite, and stored at-20 ℃ for later use.
7. The nanographene complex carrying CCL2 antibody according to claim 1The method for constructing the composite material is characterized by comprising the following steps: in step (2), RGO-IONP is reacted with C18-PMH-PEG-NH obtained in step (1)2The nano composite is prepared from the following components in a mass ratio of 1: 12 ultrasonic mixing is carried out for 85 min.
CN201910802748.6A 2019-08-28 2019-08-28 Construction method of nano graphene composite material carrying CCL2 antibody Active CN110478493B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201910802748.6A CN110478493B (en) 2019-08-28 2019-08-28 Construction method of nano graphene composite material carrying CCL2 antibody

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201910802748.6A CN110478493B (en) 2019-08-28 2019-08-28 Construction method of nano graphene composite material carrying CCL2 antibody

Publications (2)

Publication Number Publication Date
CN110478493A CN110478493A (en) 2019-11-22
CN110478493B true CN110478493B (en) 2021-04-06

Family

ID=68554843

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201910802748.6A Active CN110478493B (en) 2019-08-28 2019-08-28 Construction method of nano graphene composite material carrying CCL2 antibody

Country Status (1)

Country Link
CN (1) CN110478493B (en)

Also Published As

Publication number Publication date
CN110478493A (en) 2019-11-22

Similar Documents

Publication Publication Date Title
Wang et al. Metal-catalyzed azide-alkyne “click” reactions: Mechanistic overview and recent trends
Xu et al. Hyper-cross-linked polymer supported rhodium: an effective catalyst for hydrogen evolution from ammonia borane
Majedi et al. Theoretical view on interaction between boron nitride nanostructures and some drugs
JP5991510B2 (en) Method for producing copper nanowires
TWI306907B (en) Non-electrolytic pre-plating treating agent, non-electrolytic plating method using the agent and non-electrolytic plating product
JP6316011B2 (en) Nanoparticles carrying anticancer agents
WO2019181018A1 (en) Nano-silica particles containing boron isotope and serving as boron neutron capture agent
Srinivasan et al. Functionalization of magnetic nanoparticles with organic molecules: Loading level determination and evaluation of linker length effect on immobilization
Smitha et al. Highly functionalized heterogeneous dendrigraft catalysts with peripheral copper moieties for the facile synthesis of 2-substituted benzimidazoles and 2, 2-disubstituted benzimidazoles
CN110478493B (en) Construction method of nano graphene composite material carrying CCL2 antibody
Leland et al. Sustainable preparation of aminosilane monomers, oligomers, and polymers through Si–N dehydrocoupling catalysis
Hollingsworth et al. Preparation of metalloporphyrin-bound superparamagnetic silica particles via “Click” reaction
CN113368077B (en) Polydopamine-modified nano covalent organic framework and preparation method and application thereof
Mudgal et al. Exploring the Catalytic Efficiency of Copper-Doped Magnetic Carbon Aerogel for the Coupling Reaction of Isatin Oxime with Phenylboronic Acid Derivatives
CN1208852C (en) Method for coating nucleic acid with metal by means of non-field produced metal nanoparticles
Alvarez-Paino et al. Surface modification of magnetite hybrid particles with carbohydrates and gold nanoparticles via “click” chemistry
Liu et al. A nanoscale MOF-based heterogeneous catalytic system for the polymerization of N-carboxyanhydrides enables direct routes toward both polypeptides and related hybrid materials
Ciacchi et al. Nucleation of platinum clusters on biopolymers: a first principles study of the molecular mechanisms
Chakrabarti et al. Nanotechnology-driven chemistry of boron materials
Zainal et al. Characterization of the modified nickel-zinc ferrite nanoparticles coated with APTES by salinization reaction
CN113166072A (en) Carbene compound, carbene-metal nanoparticle complex and preparation method thereof
JP6760655B2 (en) Method for producing Group 10 element cluster carrier with precise control of atomic number in sub-nano region, platinum cluster carrier and catalyst
Kurnoskin Epoxy polymer modification with metals
Neves et al. PdNP@ Cyclodextrin on Cu/Al LDH-containing nanocomposites: Cage effect, crystallite size tuning and composite topology towards cross-couplings
Aghabozorg et al. Different complexation behavior of a proton transfer compound obtained from 1, 10‐phenanthroline and pyridine‐2, 6‐dicarboxylic acid with Sn (IV), Sb (III) and Tl (I)

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