CN111388668B - Magnetic composite nano material and preparation method and application thereof - Google Patents
Magnetic composite nano material and preparation method and application thereof Download PDFInfo
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- CN111388668B CN111388668B CN202010160598.6A CN202010160598A CN111388668B CN 111388668 B CN111388668 B CN 111388668B CN 202010160598 A CN202010160598 A CN 202010160598A CN 111388668 B CN111388668 B CN 111388668B
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
- A61K41/0052—Thermotherapy; Hyperthermia; Magnetic induction; Induction heating therapy
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- 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
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- 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
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
- A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
- A61K49/10—Organic compounds
- A61K49/12—Macromolecular compounds
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- A—HUMAN NECESSITIES
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Abstract
The application discloses magnetic composite nano material and preparation method and application thereof, including modified magnetic nanoparticle and being located the modified macromolecule layer of modified magnetic nanoparticle surface, modified macromolecule layer include the macromolecule and through pH response group with the polymer that the macromolecule is connected, pH response group's response pH value < 6.8. The magnetic composite nano material exists in a cluster form before reaching a tumor part, and is difficult to be rapidly metabolized and removed by a kidney due to large particle size; after reaching the tumor part, the assembly (magnetic composite nano material) gradually disperses under the slightly acidic condition of the tumor, and exists at the tumor part in the form of small-particle-size magnetic nano particles, so that the penetration depth of the tumor tissue is increased, the shedding of the surface modified polymer increases the surface positive charge, and the nano particles are more easily taken up by cells. Therefore, the pH response type magnetic composite nano material cluster provided by the application not only enhances the MRI contrast effect, but also improves the tissue penetrability.
Description
Technical Field
The application relates to a magnetic composite nano material and a preparation method and application thereof, belonging to the field of nano materials and biomedicine.
Background
Cancer is one of the major factors threatening human life, and cancer research institutes have pointed out that lack of effective screening and early diagnosis is an important factor in the high number of cancer cases. Early diagnosis is an important factor affecting the prognosis of malignant tumors and is also a prerequisite for early treatment.
Magnetic Resonance Imaging (MRI) is a clinically common noninvasive early tumor diagnosis means, has the advantages of high resolution, high tissue penetration, no wound, no radiation, low examination cost and the like, and often needs a contrast agent to improve the diagnosis capability.
Superparamagnetic iron oxide NPs are popular in the biomedical field because they contain degradable iron elements, which can be recycled by cells utilizing biochemical pathway for iron metabolism, and they are also approved by FDA as MRI contrast agents, have stronger T2 effect, and therefore have higher sensitivity on MRI than gadolinium complexes. Theoretically, the relaxation characteristics of iron oxide NPs are closely related to particle size. Studies have shown that the saturation magnetization increases with increasing particle size and thus the relaxation rate. However, the increase in relaxation rate tends to be gradual as the particle size increases, and another method of enhancing magnetic properties to obtain a high MRI contrast effect is to manufacture a magnetic nano-assembly composed of small-particle-size magnetic nanoparticles, thereby increasing the effective magnetic size.
The small-particle-size magnetic nanoparticles have good tissue penetrability, but are easy to be cleared by rapid renal metabolism through intravenous injection. Polyethylene glycol as a surface modification of nanoparticles is known to prolong the in vivo circulation time of nanoparticles, but also to affect cellular uptake of nanoparticles.
Disclosure of Invention
According to a first aspect of the application, there is provided a magnetic composite nanomaterial comprising a modified magnetic nanoparticle and a modified macromolecular layer located on the outer surface of the modified magnetic nanoparticle, the modified macromolecular layer comprising macromolecules and a polymer linked to the macromolecules via pH-responsive groups, the pH-responsive groups having a responsive pH < 6.8. The pH-responsive group has pH responsiveness under the condition that the pH is less than 6.8, the magnetic composite nano material exists in a cluster form before reaching a tumor part, and is difficult to be rapidly removed by kidney metabolism due to large particle size; after reaching the tumor part, the assembly (magnetic composite nano material) gradually disperses under the slightly acidic condition of the tumor, and exists at the tumor part in the form of small-particle-size magnetic nano particles, so that the penetration depth of the tumor tissue is increased, the shedding of the surface modified polymer increases the surface positive charge, and the nano particles are more easily taken up by cells. Therefore, the pH response type magnetic composite nano material cluster provided by the application not only enhances the MRI contrast effect, but also improves the tissue penetrability.
Herein, the pH-responsive group is linked between the modified magnetic nanoparticle and the modified macromolecule layer, and the response pH <6.8 means that the acid-sensitive bond in the pH-responsive group is cleaved, for example, C ═ N cleavage in a phenylimide bond, when the pH is < 6.8.
Optionally, the pH-responsive group is a benzoylimine group.
Optionally, the modified magnetic nanoparticles are carboxyl-modified magnetic nanoparticles, preferably carboxyl-modified monodisperse magnetic nanoparticles;
the magnetic nano particles are oxides of magnetic metals;
the magnetic metal is selected from at least one of Fe, Mn, Zn, Co and Ni, and Fe is preferred;
the particle size of the magnetic nanoparticles is 4-400 nm. Optionally, the polymer is a polymer with aldehyde phenyl at the terminal;
the aldehyde phenyl is phenyl in which at least one hydrogen atom on a benzene ring is substituted by aldehyde.
The polymer with aldehyde phenyl at the tail end is selected from at least one of methoxy polyethylene glycol-p-aldehyde benzoic acid;
the macromolecule is at least one of a macromolecular polymer containing amino in a repeating unit and a modified macromolecular polymer containing amino in a repeating unit;
the high molecular polymer containing amino in the repeating unit is at least one of polyether high molecular polymer containing amino in the repeating unit, polyester high molecular polymer containing amino in the repeating unit, protein, polypeptide, oligopeptide and polysaccharide containing amino in the repeating unit, preferably polylysine;
the modified high molecular polymer containing amino in the repeating unit is a high molecular polymer containing amino in the repeating unit modified by polyethylene glycol and/or polyacrylic acid;
optionally, the macromolecule has a molecular weight of 15,000 to 30,000. Suitable macromolecules may be selected according to particular needs.
According to a second aspect of the present application, there is provided a method of preparing a magnetic composite nanomaterial described in any of the above, comprising at least the steps of:
and (3) carrying out reaction I on a mixed solution I containing an activating agent I, modified magnetic nanoparticles and modified macromolecules to obtain the magnetic composite nanomaterial.
Alternatively, the modified macromolecule is prepared by:
and (3) carrying out aldehyde-amine reaction on the polymer with aldehyde phenyl at the tail end and the high molecular polymer containing amino in the repeating unit to obtain the modified macromolecule.
Alternatively, the polymer having an aldehyde phenyl group at the terminal is prepared by the following method:
and (3) reacting the mixed solution II containing the polymer to be modified, an activating agent II and aldehyde phenyl acid to obtain the polymer with aldehyde phenyl at the tail end.
Optionally, the polymer to be modified is methoxypolyethylene glycol;
optionally, the mass concentration of the methoxypolyethylene glycol in the mixed solution II is 30-60mg/mL, preferably 30-59 mg/mL;
optionally, the aldehyde phenyl acid is p-aldehyde benzoic acid, and the molar concentration ratio of the methoxy polyethylene glycol to the p-aldehyde benzoic acid is 0.1-0.4: 1;
optionally, the mixed solution II further comprises an organic solvent II, wherein the organic solvent II is selected from dichloromethane; preferably, the mixed solution II further contains a cosolvent, wherein the cosolvent is selected from at least one of N, N dimethylformamide and dimethyl sulfoxide; the volume ratio of the solvent II to the cosolvent is 8-16.
Optionally, the activating agent II is selected from at least one of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, dicyclohexylcarbodiimide and 4-dimethylaminopyridine;
preferably, the activating agent II is prepared by mixing dicyclohexylcarbodiimide and 4-dimethylaminopyridine according to a molar ratio of 2-4: 1, preparing a composition;
optionally, the high molecular polymer containing amino groups in the repeating units is polylysine, and the conditions of the aldehyde-amine reaction specifically include:
dissolving the polymer with aldehyde phenyl at the tail end and polylysine in an organic solvent to form a reaction solution;
optionally, the organic solvent is dimethyl sulfoxide and/or N, N dimethylformamide;
optionally, the concentration of the polymer with the aldehyde phenyl group at the end in the reaction solution is 3-8 mg/L, preferably 4-8 mg/mL;
optionally, the concentration of the polylysine is 0.4-1 mg/L, preferably 0.5-1 mg/mL;
optionally, the mass ratio of the polylysine to the polymer with the aldehyde phenyl group at the terminal is 0.05-0.13: 1;
optionally, the reaction is carried out under a stirring condition, the reaction temperature is 20-50 ℃, and the stirring time is 16-36 hours.
Optionally, after the aldehyde-amine reaction is performed on the polymer with the aldehyde phenyl at the terminal and the high molecular polymer containing amino in the repeating unit, the method further comprises:
adding polyacrylic acid and/or polyethylene glycol into the reaction system after the reaction for reaction.
Optionally, the modified magnetic nanoparticles are prepared by:
and (3) carrying out a reaction III on the reaction liquid III containing the magnetic metal source and the alkali source, and then adding a modifier to carry out a reaction IV to obtain the modified magnetic nanoparticles.
Optionally, the magnetic metal source comprises at least one of a hydrochloride of a magnetic metal, a nitrate of a magnetic metal, a sulfate of a magnetic metal, an oxalate of a magnetic metal;
optionally, the magnetic metal source is an iron salt; preferably, the ferric salt is prepared from a ferric salt and a ferrous salt according to a mol ratio of 1.5-2: 1 in proportion;
optionally, the modifier is selected from at least one of citric acid, polyacrylic acid and sodium citrate;
optionally, the mass ratio of the modifier to the iron salt is 0.4-8.5: 1, preferably 0.4 to 8.4: 1;
optionally, the alkali source is selected from at least one of ammonia water, sodium hydroxide, sodium carbonate and sodium acetate;
optionally, the molar ratio of the alkali source to the iron salt is 5-10: 1;
optionally, the reaction temperature of the reaction III is 60-180 ℃, and the reaction time is 30-90 min;
optionally, the reaction temperature of the reaction IV is 60-180 ℃, and the reaction time is 60-90 min;
optionally, the reaction III and the reaction IV are carried out under the condition of non-activity and stirring, and the rotating speed is 400-600 rpm. The inactive condition may be by passing N2And inert gas, and can be sealed.
Optionally, the activating agent I is a mixture of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide or a mixture of dicyclohexylcarbodiimide and 4-dimethylaminopyridine.
Optionally, the mixed solution I further contains an organic solvent I, wherein the organic solvent I is at least one selected from dimethyl sulfoxide and N, N-dimethylformamide;
optionally, the concentration of the modified magnetic nanoparticles in the mixed solution I is 0.4-1.7 μmol/L, preferably 0.8-1.7 μmol/L;
optionally, the mass ratio of the modified macromolecules to the modified magnetic nanoparticles is 3-15: 1.
optionally, the activating agent I is prepared from 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in a molar ratio of 1.5-2: 1 in proportion;
optionally, the mass concentration of the activator I is 2.4-6 mg/mL based on the total mass of the activator I.
In an alternative embodiment, a method of making a magnetic composite nanomaterial includes:
(S1) stirring a mixture containing an activator II, methoxy polyethylene glycol and p-aldehyde benzoic acid at room temperature to react to obtain modified polyethylene glycol; dispersing the product to obtain a dispersion of polyethylene glycol;
(S2) heating the mixture of the polyethylene glycol dispersion liquid and the macromolecular material in the step (S1) for reaction to obtain a polyethylene glycol modified macromolecular material;
(S3) adding an activating agent I and the modified magnetic nanoparticles into the polyethylene glycol modified macromolecule dispersion liquid obtained in the step (S1), and stirring for reaction to obtain the nanomaterial.
In another alternative embodiment, a method of making a magnetic composite nanomaterial, comprising:
(S1) stirring a mixture containing an activator II, methoxy polyethylene glycol and p-aldehyde benzoic acid at room temperature to react to obtain modified polyethylene glycol; dispersing the product to obtain a dispersion of polyethylene glycol;
(S2) heating the mixture of the polyethylene glycol dispersion liquid and the macromolecular material in the step (S1) for reaction to obtain a mixed liquid containing the polyethylene glycol modified macromolecular material;
(S3) adding polyacrylic acid into the mixed solution of the polyethylene glycol modified macromolecular material in the step (S2), and stirring for reaction to obtain a multi-modified macromolecular material;
(S4) adding an activating agent I and the nano particles into the multi-modified macromolecule dispersion liquid obtained in the step (S3), and stirring for reaction to obtain the nano material.
In a specific embodiment, 1) modifying the surface of the prepared magnetic nanoparticles to obtain carboxyl-modified monodisperse magnetic nanoparticles;
2) carrying out esterification reaction on methoxy polyethylene glycol (mPEG) and p-aldehyde benzoic acid (FBA) to form mPEG-FBA with aldehyde group at the tail end;
3) performing an aldehyde-amine condensation reaction on the mPEG-FBA and Polylysine (PLL) to obtain polylysine PLL-mPEG modified by polyethylene glycol;
4) and performing amide reaction on the carboxyl modified magnetic nanoparticles and PLL-mPEG to obtain the pH response type magnetic nanoparticle assembly.
The particle size of the magnetic nanoparticles in the step 1) is 4-20 nm, and specifically, ferric salt and ferrous salt are dissolved in deionized water, nitrogen is introduced into the deionized water to raise the temperature to a certain temperature, ammonia water is added to react for a period of time, citric acid is added to continue to react for a period of time, and dialysis is performed after the reaction is finished. Wherein the molar ratio of the added ferric salt to the ferrous salt is 1.5-2; the molar ratio of the ammonia water to the ferric salt is 5-10; the total mass ratio of the citric acid to the ferric salt is 0.4-1.7; the heating temperature is 60-100 ℃, the first stage reaction time is 30-90 min, and the second stage reaction time is 60-90 min; the rotating speed range is 400-600 rpm
The step 2) of the scheme is specifically that methoxy polyethylene glycol, p-aldehyde benzoic acid, dicyclohexyl carbodiimide and 4-dimethylamino pyridine are sequentially dissolved in dichloromethane and N, N-dimethylformamide for dissolution, wherein the molar concentration ratio of the methoxy polyethylene glycol to the p-aldehyde benzoic acid is 0.1-0.4, and the mass concentration of the methoxy polyethylene glycol is 30-60 mg/mL; the molar ratio of dicyclohexylcarbodiimide to 4-dimethylaminopyridine is 2-4: 1; the molar concentration ratio of dicyclohexylcarbodiimide to p-aldehyde benzoic acid is 1-4; the volume ratio of the dichloromethane to the N, N-dimethylformamide is 8-16.
The method comprises the following steps of 3) specifically dissolving mPEG-FBA in an organic solvent, adding polylysine, and stirring at 20-50 ℃ to obtain PLL-mPEG, wherein the organic solvent comprises dimethyl sulfoxide and N, N-dimethylformamide; wherein the concentration of mPEG-FBA is 3-8 mg/mL, and the concentration of PLL is 0.4-1 mg/mL; the mass ratio of PLL to mPEG-FBA is 0.05-0.13: 1; stirring for 16-36 h at constant temperature.
The step 4) of the scheme is specifically that carboxyl modified magnetic iron oxide and PLL-mPEG are dissolved in an organic solvent, then 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC. HCl) and N-hydroxysuccinimide (NHS) are added and stirred together to obtain PLL @ mPEG @ SPION; wherein the volume ratio of the PLL-mPEG solution to the organic solvent is 0.3-0.5; the molar weight of the ferric oxide is 25-50 mu mol; the molar ratio of EDC & HCl to NHS is 1.5-2; the stirring time is 12-24 h.
In a third aspect of the present application, there is provided a use of at least one of the magnetic composite nanomaterial described in any one of the above and the magnetic composite nanomaterial prepared by the method for preparing the magnetic composite nanomaterial described in any one of the above in a contrast agent or a therapeutic agent for tumor diagnosis and treatment.
In a fourth aspect of the present application, there is provided a contrast agent comprising at least one of the magnetic composite nanomaterial described in any one of the above, and a magnetic composite nanomaterial produced by the method for producing a magnetic composite nanomaterial described in any one of the above.
Optionally, the contrast agent is a magnetic resonance imaging contrast agent.
Optionally, the contrast agent is a fluorescence imaging contrast agent.
In a fifth aspect of the present application, there is provided a therapeutic agent comprising at least one of the magnetic composite nanomaterial described in any one of the above, and a magnetic composite nanomaterial produced by the method for producing a magnetic composite nanomaterial described in any one of the above.
The pH response type iron oxide nanoparticle assembly (magnetic composite nanomaterial) formed by chemical crosslinking overcomes the limitation of the particle size on the imaging effect of enhanced nuclear magnetic resonance T2, and the small particle size improves the saturation magnetization of the nanoparticles by forming the assembly, so that the nuclear magnetic resonance T2 imaging signal is improved; the problem of rapid renal clearance of small-particle-size iron oxide nanoparticles in vivo is solved by forming an assembly, and the in-vivo circulation time of the nanoparticles is prolonged by connecting PEG on the surface of the assembly; secondly, the surface PEG falls off under the slightly acidic condition of the tumor, more positive charge groups are exposed, the interaction of the nanoparticles and the cell membrane is promoted, and the endocytosis efficiency of the cell is improved; in addition, the self-assembly agglomeration state of the iron oxide nanoparticles is removed, so that the iron oxide nanoparticles are relatively dispersed, and the tissue penetration capability is relatively good. Simultaneously, because of increase material accumulation and the tissue penetrability at the tumour position for the magnetic heat treatment in-process, tumour regional temperature is more even, and the treatment is better.
The preparation method of the nanoparticle assembly is also suitable for preparing magnetic nanoparticles modified by carboxyl by various methods, and has universality within the particle size range.
Optionally, the therapeutic agent is a magnetocaloric therapeutic agent.
The beneficial effects that this application can produce include:
the magnetic composite nano material exists in a cluster form before reaching a tumor part, and is difficult to be rapidly metabolized and removed by a kidney due to large particle size; after reaching the tumor site, the benzoyl imine bond has pH responsiveness under the condition that the pH is less than 6.8 under the slightly acidic condition of the tumor, the assembly is gradually dispersed and exists at the tumor site in the form of small-particle-size magnetic nanoparticles, so that the penetration depth of the tumor tissue is increased, the surface positive charge is increased due to the falling of the surface modification material, and the nanoparticles are more easily taken up by cells. Therefore, the pH response type magnetic composite nano material cluster provided by the application not only enhances the MRI contrast effect, but also improves the tissue penetrability.
The combination of MRI diagnostic imaging and therapy at the same time can be used to further facilitate cancer therapy. Magnetic hyperthermia is a kind of hyperthermia, which is characterized in that the heating area and temperature are more controllable due to the properties of the magnetic material, the alternating magnetic field is used as the heating source, the penetrating power is strong, and the magnetic hyperthermia is hardly influenced by the organism tissue. The nano ferric oxide is used for magnetic heat treatment, so that the temperature of a tumor area is more uniform in the treatment process, and the treatment effect is better.
Drawings
FIG. 1 is a TEM image of superparamagnetic ferroferric oxide (SPION) prepared in example 1;
FIG. 2 is a TEM image of PLL @ mPEG @ SPION prepared in example 1;
FIG. 3 is a DLS particle size distribution plot of superparamagnetic ferroferric oxide (SPION) prepared in example 1;
FIG. 4 is a DLS particle size distribution plot of PLL @ mPEG @ SPION prepared in example 1;
FIG. 5 is an infrared absorption plot of PLL @ mPEG @ SPION prepared in example 1;
FIG. 6 is a hysteresis curve diagram of superparamagnetic ferroferric oxide (SPION) prepared in example 1;
FIG. 7 is a hysteresis graph of PLL @ mPEG @ SPION prepared in example 1;
FIG. 8 is an MRI image of superparamagnetic ferroferric oxide (SPION), PLL @ mPEG @ SPION in example 1;
FIG. 9 is a graph of the relaxation rates of superparamagnetic ferroferric oxide (SPION), PLL @ mPEG @ SPION in example 1, wherein FIG. 9a is a graph of the relaxation rate of superparamagnetic ferroferric oxide, and FIG. 9b is a graph of the relaxation rate of PLL @ mPEG @ SPION;
FIG. 10 is a TEM image of PLL @ mPEG @ SPION after response at pH 6.0 in example 1;
FIG. 11 is an MRI image plot of PLL @ mPEG @ SPION after response at pH 6.0 in example 1;
FIG. 12 is a graph of the relaxation rate of PLL @ mPEG @ SPION after response at pH 6.0 in example 1.
FIG. 13 is an in vivo MRI imaging plot of PLL @ mPEG @ SPION in example 1.
Detailed Description
The present application will be described in detail with reference to examples, but the present application is not limited to these examples.
The raw materials in the examples of the present application were all purchased commercially, unless otherwise specified.
Wherein mPEG is purchased from Aladdin and has a molecular weight of 2000;
Poly-L-lysine hydrobromide (polylysine (PLL) for short) having a molecular weight of 15000-30000, purchased from Sigma
PAA is purchased from Huijai Biotech and has a molecular weight of 2000.
The general test method comprises the following steps:
particle size distribution test
Testing an instrument: zetasizer Nano ZS type dynamic light scattering particle size analyzer, test conditions: the scatter angle is 173 °.
TEM
Testing an instrument: transmission electron microscope model Tecnai F20; and (3) testing conditions are as follows: 200Kv, 101. mu.A; and the nano particles to be tested are dispersed in water for testing. (test after drying by dropping on a copper mesh)
T1 weighted imaging of MRI
Testing an instrument: a MesoMR23-060H-I nmr analysis and imaging system; test conditions were T1：TR＝300ms，TE＝18.2ms。
T2 weighted imaging of MRI
Testing an instrument: a MesoMR23-060H-I nmr analysis and imaging system; test conditions were T2：TR＝2500ms，TE＝40ms。
MRI relaxation rate measurement
Testing an instrument: a MesoMR23-060H-I nmr analysis and imaging system; the test condition is r1Test TW＝16500ms；r2Test TW＝5500ms。
Infrared absorption spectrum test
Testing an instrument: NICOLET 6700 intelligent fourier infrared spectrometer (FTIR); the test conditions were resolution: 0.09cm-1(ii) a And testing the PLL @ mPEG @ SPION mixed KBr tablet to be tested.
Example 1
A preparation method of a pH response type magnetic composite nanoparticle diagnosis and treatment reagent comprises the following steps:
step 1: 8g of methoxy PEG (mPEG) and 1.8g of p-aldehyde benzoic acid (FBA) were dissolved in 160mL of dichloromethane, 20mL of Dimethylformamide (DMF) was added, followed by 2.48g of Dicyclohexylcarbodiimide (DCC) and 732mg of 4-Dimethylaminopyridine (DMAP), and stirred for 24h.
Step 2: and (3) carrying out vacuum filtration on the system obtained in the step (1) for 3 times to remove precipitates, introducing nitrogen into the obtained clear system to remove the solvent, dispersing the clear system into 50mL of deionized water, and carrying out freeze drying to obtain mPEG-FBA (polymer with aldehyde phenyl at the tail end) powder.
And step 3: synthesizing superparamagnetic ferroferric oxide nanoparticles (SPION): 0.81g of ferric chloride hexahydrate (FeCl) was taken3·6H2O), 0.556g of ferrous sulfate heptahydrate (FeSO)4·7H2O), dissolving in 15mL of deionized water, introducing nitrogen into the reaction system for 50min, heating to 75 ℃, and rapidly adding 3mL of ammonia water (NH with the mass concentration of 35%) (NH)3·H2O), mixing and stirring for 30min to obtain a mixed solution containing SPION (magnetic nanoparticles).
And 4, step 4: and (3) adding 0.6g of citric acid into the mixed solution obtained in the step (3), continuously mixing and stirring at 75 ℃ for 1h, and cooling to room temperature under the protection of nitrogen to obtain the SPION mixed solution containing the surface modified carboxyl.
And 5: and (3) dialyzing and purifying the mixed solution obtained in the step (4) for 72 hours by using a 14kDa dialysis bag, and concentrating by using a 10kDa ultrafiltration tube to obtain a concentrated solution containing the carboxyl modified SPION (modified magnetic nanoparticles).
Step 6: the mPEG-FBA powder obtained in the step 2 is dissolved in dimethyl sulfoxide (DMSO), and the concentration of mPEG-FBA is 0.2 g/mL.
And 7: and (3) dissolving 5mg of Polylysine (PLL) in 10mL of dimethyl sulfoxide (DMSO), adding 200 mu L of the solution obtained in the step 6, carrying out ultrasonic treatment on the obtained mixture for 5min, mixing and stirring the reaction system in an oil bath at 40 ℃ for 16h, and forming a phenylformimino bond with pH responsiveness through an aldehyde-amine condensation reaction to obtain the PLL-mPEG micelle (modified macromolecule).
And step 8: 60mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC. HCl) and 27mg of N-hydroxysuccinimide (NHS) were dissolved in 6mL of dimethyl sulfoxide (DMSO) as a catalyst (activator I).
And step 9: and (3) adding 5mL of the mixed solution obtained in the step (8) into the concentrated solution containing the carboxyl modified SPION in the step (5) (containing 25 mu mol of SPION), performing ultrasonic treatment for 1min to form a dispersed mixed solution, adding the dispersed mixed solution into the system obtained in the step (7), performing ultrasonic dispersion for 5min, adding dimethyl sulfoxide (DMSO) to fix the volume of the system to 30mL, mechanically stirring for 12h, and connecting the carboxyl modified superparamagnetic ferroferric oxide nanoparticles (SPION) with the micelles obtained in the step (7) through amidation reaction to obtain a mixed system containing PLL @ mPEG @ SPION (magnetic composite nanomaterial) with pH responsiveness.
Step 10: and (3) gradually adding deionized water into the mixed system obtained in the step (9) to enable the organic phase/water phase to be 5% -10%, centrifuging the obtained system, setting the parameters to be 11,000rpm for 10min, carrying out centrifugal washing for 3 times by using the deionized water with the same parameters, and dispersing the obtained product in the deionized water to obtain a pH-responsive ferroferric oxide nanoparticle assembly PLL @ mPEG @ SPION, wherein the sample is marked as sample 1.
The reaction is not particularly limited, and the ambient temperature of the reaction is about 20 ℃.
Example 2
Step 1: 8g of methoxy PEG (mPEG) and 1.8g of p-aldehyde benzoic acid (FBA) were dissolved in 160mL of dichloromethane, 20mL of Dimethylformamide (DMF) was added, followed by 2.48g of Dicyclohexylcarbodiimide (DCC) and 732mg of 4-Dimethylaminopyridine (DMAP), and stirred for 24h.
Step 2: and (3) carrying out vacuum filtration on the system obtained in the step (1) for 3 times to remove precipitates, introducing nitrogen into the obtained clear system to remove the solvent, dispersing the clear system into 50mL of deionized water, and carrying out freeze drying to obtain mPEG-FBA powder.
And step 3: synthesizing superparamagnetic ferroferric oxide nanoparticles (SPION): 0.81g of ferric chloride hexahydrate (FeCl) was taken3·6H2O), 0.556g of ferrous sulfate heptahydrate (FeSO)4·7H2O), dissolving in 15mL of deionized water, introducing nitrogen into the reaction system for 50min, heating to 75 ℃, and quickly adding 3mL of ammonia water (NH with the mass concentration of 35%) (NH)3·H2O), mixing and stirring for 30min to obtain a mixed solution containing SPION.
And 4, step 4: and (3) adding 0.6g of citric acid into the mixed solution obtained in the step (3), mixing and stirring at 75 ℃ for 1h, and cooling to room temperature under the protection of nitrogen to obtain the SPION mixed solution containing the surface modified carboxyl.
And 5: and (3) dialyzing and purifying the mixed solution obtained in the step (4) for 72 hours by using a 14kDa dialysis bag, and concentrating by using a 10kDa ultrafiltration tube to obtain a concentrated solution containing the carboxyl modified SPION.
Step 6: the mPEG-FBA powder obtained in the step 2 is dissolved in dimethyl sulfoxide (DMSO), and the concentration of mPEG-FBA is 0.2 g/mL.
And 7: and (3) dissolving 5mg of Polylysine (PLL) in 10mL of dimethyl sulfoxide (DMSO), adding 400 mu L of the solution obtained in the step 6, carrying out ultrasonic treatment on the obtained mixture for 5min, mixing and stirring the reaction system in an oil bath at 40 ℃ for 16h, and forming a phenylformimino bond with pH responsiveness through an aldehyde-amine condensation reaction to obtain the PLL-mPEG micelle.
And 8: 60mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC. HCl) and 27mg of N-hydroxysuccinimide (NHS) were dissolved in 6mL of dimethyl sulfoxide (DMSO) as a catalyst.
And step 9: and (3) adding 5mL of the mixed solution obtained in the step (8) into the concentrated solution containing the carboxyl modified SPION in the step (5) (containing 25 mu mol of SPION), performing ultrasonic treatment for 1min to form a dispersed mixed solution, adding the dispersed mixed solution into the system obtained in the step (7), performing ultrasonic dispersion for 5min, adding dimethyl sulfoxide (DMSO) to fix the volume of the system to 30mL, mechanically stirring for 12h, and connecting superparamagnetic ferroferric oxide nanoparticles (SPION) with the micelles obtained in the step (7) through amidation reaction to obtain a mixed system containing PLL @ mPEG @ SPION with pH responsiveness.
Step 10: and (3) gradually adding deionized water into the mixed system obtained in the step (9) to enable the organic phase/water phase to be 5% -10%, centrifuging the obtained system, setting the parameters to be 11,000rpm for 10min, carrying out centrifugal washing for 3 times by using the deionized water with the same parameters, and dispersing the obtained product in the deionized water to obtain a pH-responsive ferroferric oxide nanoparticle assembly PLL @ mPEG @ SPION, wherein the sample is marked as sample 2.
The reaction is not particularly limited, and the ambient temperature of the reaction is about 20 ℃.
Example 3
Step 1: 8g of methoxy PEG (mPEG) and 1.8g of p-aldehyde benzoic acid (FBA) were dissolved in 160mL of dichloromethane, 20mL of Dimethylformamide (DMF) was added, followed by 2.48g of Dicyclohexylcarbodiimide (DCC) and 732mg of 4-Dimethylaminopyridine (DMAP), and stirred for 24h.
Step 2: and (3) carrying out vacuum filtration on the system obtained in the step (1) for 3 times to remove precipitates, introducing nitrogen into the obtained clear system to remove the solvent, dispersing the clear system into 50mL of deionized water, and carrying out freeze drying to obtain mPEG-FBA powder.
And step 3: synthesizing superparamagnetic ferroferric oxide nanoparticles (SPION): 0.81g of ferric chloride hexahydrate (FeCl) was taken3·6H2O), 0.556g of ferrous sulfate heptahydrate (FeSO)4·7H2O), dissolving in 15mL of deionized water, introducing nitrogen into the reaction system for 50min, heating to 75 ℃, and rapidly adding 3mL of ammonia water (NH with the mass concentration of 35%) (NH)3·H2O), mixing and stirring for 30min to obtain a mixed solution containing SPION.
And 4, step 4: and (3) adding 0.6g of citric acid into the mixed solution obtained in the step (3), mixing and stirring at 75 ℃ for 1h, and cooling to room temperature under the protection of nitrogen to obtain the SPION mixed solution containing the surface modified carboxyl.
And 5: and (3) dialyzing and purifying the mixed solution obtained in the step (4) for 72 hours by using a 14kDa dialysis bag, and concentrating by using a 10kDa ultrafiltration tube to obtain a concentrated solution containing the carboxyl modified SPION.
Step 6: the mPEG-FBA powder obtained in the step 2 is dissolved in dimethyl sulfoxide (DMSO), and the concentration of mPEG-FBA is 0.2 g/mL.
And 7: and (3) dissolving 5mg of Polylysine (PLL) in 10mL of dimethyl sulfoxide (DMSO), adding 400 mu L of the solution obtained in the step 6, carrying out ultrasonic treatment on the obtained mixture for 5min, mixing and stirring the reaction system in an oil bath at 40 ℃ for 16h, and forming a phenylformimino bond with pH responsiveness through an aldehyde-amine condensation reaction to obtain the PLL-mPEG micelle.
And 8: 60mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC. HCl) and 27mg of N-hydroxysuccinimide (NHS) were dissolved in 6mL of dimethyl sulfoxide (DMSO) as a catalyst.
And step 9: and (3) adding 5mL of the mixed solution obtained in the step (8) into the concentrated solution containing the carboxyl modified SPION in the step (5) (containing 50 mu mol of SPION), performing ultrasonic treatment for 1min to form a dispersed mixed solution, adding the dispersed mixed solution into the system obtained in the step (7), performing ultrasonic dispersion for 5min, adding dimethyl sulfoxide (DMSO) to fix the volume of the system to 30mL, mechanically stirring for 12h, and connecting superparamagnetic ferroferric oxide nanoparticles (SPION) with the micelles obtained in the step (7) through amidation reaction to obtain a mixed system containing PLL @ mPEG @ SPION with pH responsiveness.
Step 10: and (3) gradually adding deionized water into the mixed system obtained in the step (9) to enable the organic phase/water phase to be 5% -10%, centrifuging the obtained system, setting the parameters to be 11,000rpm for 10min, carrying out centrifugal washing for 3 times by using the deionized water with the same parameters, and dispersing the obtained product in the deionized water to obtain a pH-responsive ferroferric oxide nanoparticle assembly PLL @ mPEG @ SPION, wherein the sample is marked as sample 3.
The reaction is not particularly limited, and the ambient temperature of the reaction is about 20 ℃.
Example 4
Step 1: 8g of methoxy PEG (mPEG) and 1.8g of p-aldehyde benzoic acid (FBA) were dissolved in 160mL of dichloromethane, 20mL of Dimethylformamide (DMF) was added, followed by 2.48g of Dicyclohexylcarbodiimide (DCC) and 732mg of 4-Dimethylaminopyridine (DMAP), and stirred for 24h.
Step 2: and (3) carrying out vacuum filtration on the system obtained in the step (1) for 3 times to remove precipitates, introducing nitrogen into the obtained clear system to remove the solvent, dispersing the clear system into 50mL of deionized water, and carrying out freeze drying to obtain mPEG-FBA powder.
And step 3: synthesizing superparamagnetic ferroferric oxide nanoparticles (SPION): 1.6g of ferric chloride hexahydrate (FeCl) was taken3·6H2O), 1.2g ferrous sulfate heptahydrate (FeSO)4·7H2O), dissolving in 15mL of deionized water, introducing nitrogen into the reaction system for 50min, heating to 80 ℃, and rapidly adding 3mL of ammonia water (NH with the mass concentration of 35%) (ammonia water)3·H2O), mixing and stirring for 60min to obtain a mixed solution containing SPION.
And 4, step 4: and (3) adding 1.2g of citric acid into the mixed solution obtained in the step (3), mixing and stirring at 80 ℃ for 1h, and cooling to room temperature under the protection of nitrogen to obtain the SPION mixed solution containing the surface modified carboxyl.
And 5: and (3) dialyzing and purifying the mixed solution obtained in the step (4) for 72 hours by using a 14kDa dialysis bag, and concentrating by using a 10kDa ultrafiltration tube to obtain concentrated solution containing the carboxyl modified SPION.
Step 6: the mPEG-FBA powder obtained in the step 2 is dissolved in dimethyl sulfoxide (DMSO), and the concentration of mPEG-FBA is 0.2 g/mL.
And 7: dissolving 5mg of Polylysine (PLL) in 10mL of dimethyl sulfoxide (DMSO), adding 200 mu L of the solution obtained in the step 6, carrying out ultrasonic treatment on the obtained mixture for 5min, mixing and stirring the reaction system in an oil bath at 40 ℃ for 16h, and forming a pH-responsive benzoylimine bond through an aldehyde-amine condensation reaction to obtain the PLL-mPEG micelle.
And 8: 60mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC. HCl) and 27mg of N-hydroxysuccinimide (NHS) were dissolved in 6mL of dimethyl sulfoxide (DMSO) as a catalyst.
And step 9: and (3) adding 5mL of the mixed solution obtained in the step (8) into the concentrated solution containing the carboxyl modified SPION in the step (5) (containing 25 mu mol of SPION), performing ultrasonic treatment for 1min to form a dispersed mixed solution, adding the dispersed mixed solution into the system obtained in the step (7), performing ultrasonic dispersion for 5min, adding dimethyl sulfoxide (DMSO) to fix the volume of the system to 30mL, mechanically stirring for 12h, and connecting superparamagnetic ferroferric oxide nanoparticles (SPION) with the micelles obtained in the step (7) through amidation reaction to obtain a mixed system containing PLL @ mPEG @ SPION with pH responsiveness.
Step 10: and (3) gradually adding deionized water into the mixed system obtained in the step (9) to enable the organic phase/water phase to be 5% -10%, centrifuging the obtained system, setting the parameters to be 11,000rpm for 10min, carrying out centrifugal washing for 3 times by using the deionized water with the same parameters, and dispersing the obtained product in the deionized water to obtain a pH-responsive ferroferric oxide nanoparticle assembly PLL @ mPEG @ SPION, wherein the sample is marked as sample 4.
The reaction is not particularly limited, and the ambient temperature of the reaction is about 20 ℃.
Example 5
Step 1: 8g of methoxy PEG (mPEG) and 1.8g of p-aldehyde benzoic acid (FBA) were dissolved in 160mL of dichloromethane, 20mL of Dimethylformamide (DMF) was added, followed by 2.48g of Dicyclohexylcarbodiimide (DCC) and 732mg of 4-Dimethylaminopyridine (DMAP), and stirred for 24h.
Step 2: and (3) carrying out vacuum filtration on the system obtained in the step (1) for 3 times to remove precipitates, introducing nitrogen into the obtained clear system to remove the solvent, dispersing the clear system into 50mL of deionized water, and carrying out freeze drying to obtain mPEG-FBA powder.
And step 3: synthesizing superparamagnetic ferroferric oxide nanoparticles (SPION): 1.08g of ferric chloride hexahydrate (FeCl) was taken3·6H2O), 0.4g of ferrous chloride tetrahydrate (FeCl)2·4H2O), dissolving in 50mL of diethylene glycol, introducing nitrogen into the reaction system for 30min, heating to 80 ℃, and rapidly adding 3.4mL of ammonia water (with the mass concentration of 35% NH)3·H2O), heating to 180 ℃, mixing and stirring for 60min to obtain a mixed solution containing SPION.
And 4, step 4: and (3) adding 12.5g of citric acid into the mixed solution obtained in the step (3), mixing and stirring at 180 ℃ for 90min, and cooling to room temperature under the protection of nitrogen to obtain the SPION mixed solution containing the surface modified carboxyl.
And 5: and (4) adding acetone into the system obtained in the step (4) for precipitation, centrifuging at the rotating speed of 12,000rpm for 15min, and carrying out centrifugal cleaning on ethanol for 2 times and water for 2 times under the same centrifugation condition to obtain the carboxyl modified SPION.
Step 6: the mPEG-FBA powder obtained in the step 2 is dissolved in dimethyl sulfoxide (DMSO), and the concentration of mPEG-FBA is 0.2 g/mL.
And 7: and (3) dissolving 5mg of Polylysine (PLL) in 10mL of dimethyl sulfoxide (DMSO), adding 400 mu L of the solution obtained in the step 6, carrying out ultrasonic treatment on the obtained mixture for 5min, mixing and stirring the reaction system in an oil bath at 40 ℃ for 16h, and forming a phenylformimino bond with pH responsiveness through an aldehyde-amine condensation reaction to obtain the PLL-mPEG micelle.
And 8: 60mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC. HCl) and 27mg of N-hydroxysuccinimide (NHS) were dissolved in 6mL of dimethyl sulfoxide (DMSO) as a catalyst.
And step 9: and (3) adding 50 mu mol of carboxyl modified SPION (calculated by the molar amount of the SPION) into 5mL of the mixed solution obtained in the step (8), performing ultrasonic treatment for 1min to form a dispersed mixed solution, adding the dispersed mixed solution into the system obtained in the step (7), performing ultrasonic dispersion for 5min, adding dimethyl sulfoxide (DMSO) to fix the volume of the system to 30mL, mechanically stirring for 12h, and connecting superparamagnetic ferroferric oxide nanoparticles (SPION) with the micelles obtained in the step (7) through amidation reaction to obtain a mixed system containing pH-responsive PLL @ mPEG @ SPION.
Step 10: and (3) gradually adding deionized water into the mixed system obtained in the step (9) to enable the organic phase/water phase to be 5% -10%, centrifuging the obtained system, setting the parameters to be 11,000rpm for 10min, carrying out centrifugal washing for 3 times by using the deionized water with the same parameters, and dispersing the obtained product in the deionized water to obtain a pH-responsive ferroferric oxide nanoparticle assembly PLL @ mPEG @ SPION, wherein the sample is marked as sample 5.
The reaction is not particularly limited, and the ambient temperature of the reaction is about 20 ℃.
Example 6
Step 1: 8g of methoxy PEG (mPEG) and 1.8g of p-aldehyde benzoic acid (FBA) were dissolved in 160mL of dichloromethane, 20mL of Dimethylformamide (DMF) was added, followed by 2.48g of Dicyclohexylcarbodiimide (DCC) and 732mg of 4-Dimethylaminopyridine (DMAP), and stirred for 24h.
Step 2: and (3) carrying out vacuum filtration on the system obtained in the step (1) for 3 times to remove precipitates, introducing nitrogen into the obtained clear system to remove the solvent, dispersing the clear system into 50mL of deionized water, and carrying out freeze drying to obtain mPEG-FBA powder.
And step 3: synthesizing superparamagnetic ferroferric oxide nanoparticles (SPION): 0.81g of ferric chloride hexahydrate (FeCl) was taken3·6H2O), 0.556g of ferrous sulfate heptahydrate (FeSO)4·7H2O), dissolved in15mL of deionized water, introducing nitrogen into the reaction system for 50min, heating to 75 ℃, and rapidly adding 3mL of ammonia water (NH with the mass concentration of 35%) (ammonia water)3·H2O), mixing and stirring for 30min to obtain a mixed solution containing SPION.
And 4, step 4: and (3) adding 0.6g of citric acid into the mixed solution obtained in the step (3), mixing and stirring at 75 ℃ for 1h, and cooling to room temperature under the protection of nitrogen to obtain the SPION mixed solution containing the surface modified carboxyl.
And 5: and (3) dialyzing and purifying the mixed solution obtained in the step (4) for 72 hours by using a 14kDa dialysis bag, and concentrating by using a 10kDa ultrafiltration tube to obtain a concentrated solution containing the carboxyl modified SPION.
Step 6: the mPEG-FBA powder obtained in the step 2 is dissolved in dimethyl sulfoxide (DMSO), and the concentration of mPEG-FBA is 0.2 g/mL.
And 7: and (3) dissolving 5mg of Polylysine (PLL) in 10mL of dimethyl sulfoxide (DMSO), adding 200 mu L of the solution obtained in the step 6, carrying out ultrasonic treatment on the obtained mixture for 5min, mixing and stirring the reaction system in an oil bath at 40 ℃ for 16h, and forming a phenylformimino bond with pH responsiveness through an aldehyde-amine condensation reaction to obtain the PLL-mPEG micelle.
And 8: 60mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC. HCl) and 27mg of N-hydroxysuccinimide (NHS) were dissolved in 6mL of dimethyl sulfoxide (DMSO) as a catalyst.
And step 9: and (3) adding 2mL of polyacrylic acid (PAA) and DMSO as a solvent into the system obtained in the step (7) at a concentration of 10mg/mL, adding 3mL of the mixed solution obtained in the step (8), and stirring for 12h to obtain PLL @ mPEG @ PAA.
Step 10: adding 25 mu mol of carboxyl modified SPION (counted by the mol amount of the SPION) into 5mL of the solution obtained in the step 8, performing ultrasonic treatment for 1min to form a dispersed mixed solution, adding the dispersed mixed solution into the system obtained in the step 9, performing ultrasonic dispersion for 5min, adding dimethyl sulfoxide (DMSO) to fix the volume of the system to 30mL, mechanically stirring for 12h, and connecting superparamagnetic ferroferric oxide nanoparticles (SPION) with the micelles obtained in the step 9 through amidation reaction to obtain PLL @ mPEG @ PAA @ SPION with pH responsiveness.
Step 10: gradually adding deionized water into the system obtained in the step 9 to enable the organic phase/water phase to be 5% -10%, centrifuging the obtained system, setting the parameters to be 11,000rpm for 10min, carrying out centrifugal washing for 3 times by using the deionized water with the same parameters, and dispersing the obtained product in the deionized water to obtain a pH-responsive ferroferric oxide nanoparticle assembly PLL @ mPEG @ PAA @ SPION, which is marked as sample 6.
The reaction is not particularly limited, and the ambient temperature of the reaction is about 20 ℃.
Example 7 topographical characterization of the samples and/or intermediates provided in examples 1-6
The magnetic nanoparticles (SPION) obtained in examples 1 to 6 all have a particle size of 5 to 10nm, exhibit monodispersity, and samples 1 to 6 all have a particle size of 100 to 130 nm. Taking the example 1 as a typical representative, as can be seen from fig. 1-2, the particle size of SPION provided in the example 1 is 10nm, the particle size of the sample 1 is 120nm, the particle size of SPION hydrate shown in fig. 3 is 40nm, and the particle size of the sample 1 hydrate shown in fig. 4 is 148 nm.
Taking sample 1 provided in example 1 as a representative, see FIG. 5, an infrared absorption spectrum containing an absorption peak of a phenylimidine bond and Fe3O4Characteristic absorption peaks, indicating that PLL and mPEG-FBA are connected through a benzoyl imide bond, and magnetic nanoparticles are also successfully connected to PLL-mPEG
Example 8 Performance testing of samples and/or intermediates provided in examples 1-6
The magnetic nanoparticles (SPION) obtained in examples 1 to 6 have superparamagnetism. Taking example 1 as a representative example, as can be seen from fig. 6, SPION provided in example 1 is superparamagnetic, saturation magnetization is 45.35emu/g, and sample 1 also has superparamagnetic property, as shown in fig. 7, PLL @ mPEG @ SPION saturation magnetization is 35.45 emu/g.
The SPION and PLL @ mPEG @ SPION obtained in examples 1-6 are configured into different concentration aqueous solutions for MR in vitro T2 imaging and relaxation test, wherein as typified by example 1, as can be seen from FIG. 8, the SPION provided in example 1 has T2 imaging effect, the T2 effect of sample 1 is enhanced, and the longitudinal relaxation rate r of the SPION provided in example 1 is shown in FIG. 9116.06 indicates mM-1s-1Transverse relaxation rate r2＝203.64mM-1s-1，r2/r112.68. Example 1 providesSample 1 (5.78 mM) longitudinal relaxation rate r1-1s-1Transverse relaxation rate r2＝220.12mM-1s-1，r2/r1When it is 38.08, it is clear that not only r but also r are formed after cluster formation2Increase in value of r2/r1The value also increased significantly. Illustrating that the formation of SPION clusters can enhance Fe3O4Transverse relaxation property of the nano-particles.
The samples obtained in examples 1 to 6 were subjected to in vitro T2 imaging and relaxation testing by preparing aqueous solutions of different concentrations in response to pH 6.0. Taking example 1 as a representative example, it can be seen from fig. 11 that T2 imaging effect of sample 1 provided in example 1 is reduced after pH response. It can be seen from FIG. 12 that the longitudinal relaxation rate r of sample 1 provided in example 1 after pH response is1＝2.22mM-1s-1Transverse relaxation rate of r only2＝22.62，r2/r110.19, which decreased significantly relative to before the pH response.
The experimental results of examples 2 to 6 are substantially the same as those of example 1.
Example 9 animal Experimental testing of samples provided in examples 1-6
The test method comprises the following steps:
respectively preparing the samples 1-6 into injection samples with the concentration of 5mg/mL, and tail vein injecting the injection samples into the tumor parts of the tumor model mice according to the injection amount of 10mg/kg for MR imaging exploration.
The MR imaging method specifically comprises: tumor models were established in the right leg of nude mice with 4T1 cells and mice were imaged with T2 using 1.5T medical MR nmr.
Typically, in example 1, as shown in FIG. 13, the tumor site image was darker than that before injection 6h after injection of 10mg/kg sample into tail vein, and the same phenomenon was observed in other examples.
Although the present application has been described with reference to a few embodiments, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the application as defined by the appended claims.
Claims (19)
1. A method for preparing a magnetic composite nano material is characterized by at least comprising the following steps:
carrying out reaction I on a mixed solution I containing an activating agent I, modified magnetic nanoparticles and modified macromolecules to obtain the magnetic composite nanomaterial;
the magnetic composite nano material comprises modified magnetic nano particles and a modified macromolecule layer positioned on the outer surfaces of the modified magnetic nano particles, wherein the modified macromolecule layer comprises macromolecules and a polymer connected with the macromolecules through pH response groups, and the response pH value of the pH response groups is less than 6.8.
2. The method of claim 1, wherein the pH-responsive group is a benzoylimine group.
3. The preparation method according to claim 1, wherein the modified magnetic nanoparticles are carboxyl-modified magnetic nanoparticles;
the magnetic nano particles are oxides of magnetic metals;
the magnetic metal is selected from at least one of Fe, Mn, Zn, Co and Ni;
the particle size of the magnetic nanoparticles is 4-400 nm.
4. The method according to claim 1, wherein the polymer is a polymer having an aldehyde phenyl group at a terminal thereof;
the polymer with aldehyde phenyl at the tail end is selected from methoxy polyethylene glycol-p-aldehyde benzoic acid;
the macromolecule is at least one of a macromolecular polymer containing amino in a repeating unit and a modified macromolecular polymer containing amino in a repeating unit;
the high molecular polymer containing amino in the repeating unit is at least one of polyether high molecular polymer containing amino in the repeating unit, polyester high molecular polymer containing amino in the repeating unit, protein, polypeptide, oligopeptide and polysaccharide containing amino in the repeating unit;
the modified high molecular polymer containing amino in the repeating unit is a high molecular polymer containing amino in the repeating unit modified by polyethylene glycol and/or polyacrylic acid.
5. The method of claim 1, wherein the modified macromolecule is prepared by:
and (3) carrying out aldehyde-amine reaction on the polymer with aldehyde phenyl at the tail end and the high molecular polymer containing amino in the repeating unit to obtain the modified macromolecule.
6. The method of claim 5, wherein the polymer having an aldehyde phenyl group at the terminal is prepared by:
and (3) reacting the mixed solution II containing the polymer to be modified, an activating agent II and aldehyde phenyl acid to obtain the polymer with aldehyde phenyl at the tail end.
7. The method according to claim 6, wherein the polymer to be modified is methoxypolyethylene glycol, and the aldehyde phenyl acid is p-aldehyde benzoic acid;
the mixed solution II also comprises an organic solvent II, and the organic solvent II is selected from dichloromethane;
the activating agent II is dicyclohexylcarbodiimide and 4-dimethylaminopyridine.
8. The method according to claim 5, wherein the step of subjecting the polymer having an aldehyde phenyl group at the terminal thereof and the high molecular polymer having an amino group in the repeating unit thereof to an aldehyde-amine reaction further comprises:
adding polyacrylic acid and/or polyethylene glycol into the reaction system after the reaction for reaction.
9. The method of claim 1, wherein the modified magnetic nanoparticles are prepared by:
and (3) carrying out a reaction III on the reaction liquid III containing the magnetic metal source and the alkali source, and then adding a modifier to carry out a reaction IV to obtain the modified magnetic nanoparticles.
10. The method of claim 9, wherein the magnetic metal source comprises at least one of a hydrochloride of a magnetic metal, a nitrate of a magnetic metal, a sulfate of a magnetic metal, an oxalate of a magnetic metal;
the modifier is selected from at least one of citric acid, polyacrylic acid and sodium citrate;
the alkali source is at least one selected from ammonia water, sodium hydroxide, sodium carbonate and sodium acetate.
11. The process according to claim 1, wherein the activating agent I is a mixture of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide or a mixture of dicyclohexylcarbodiimide and 4-dimethylaminopyridine.
12. The method according to claim 1, wherein the mixed solution I further contains an organic solvent I selected from at least one of dimethyl sulfoxide and N, N-dimethylformamide;
the concentration of the modified magnetic nanoparticles in the mixed solution I is 0.4-1.7 mu mol/mL in terms of the mole number of the magnetic nanoparticles;
the mass ratio of the modified macromolecules to the modified magnetic nanoparticles is 3-15: 1.
13. the preparation method of claim 1, wherein the activating agent I is prepared from 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in a molar ratio of 1.5-2: 1 in proportion;
and the mass concentration of the activator I is 2-6 mg/mL based on the total mass of the activator I.
14. Use of the magnetic composite nanomaterial prepared by the method for preparing the magnetic composite nanomaterial of any one of claims 1 to 13 in preparation of contrast agents and therapeutic agents for tumor diagnosis and treatment.
15. A contrast agent comprising at least one of the magnetic composite nanomaterials produced by the method for producing a magnetic composite nanomaterial described in any one of claims 1 to 13.
16. The contrast agent according to claim 15, wherein the contrast agent is a magnetic resonance imaging contrast agent.
17. The contrast agent according to claim 15, wherein the contrast agent is a fluorescence imaging contrast agent.
18. A therapeutic agent comprising at least one of the magnetic composite nanomaterials produced by the method for producing a magnetic composite nanomaterial claimed in any one of claims 1 to 13.
19. The therapeutic agent of claim 18, wherein the therapeutic agent is a magnetocaloric therapeutic agent.
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