CN113244417A - CaO2/MnFe2O4Nanocomposite material, preparation and application thereof - Google Patents

Abstract

The invention discloses CaO₂/MnFe₂O₄Nanocomposite and preparation thereofAnd the application, the preparation of the nano composite material: with CaCl₂Adopts a one-step method to prepare PEG modified CaO₂Nanoparticles; with Fe (acac)₃And Mn (acac)₂Preparation of MnFe as raw material₂O₄Nanoparticles; taking CaO₂Ethanol dispersion of nanoparticles, dropwise adding MnFe₂O₄The CaO is obtained by ultrasonically stirring ethanol dispersion of the nano particles₂/MnFe₂O₄A nanocomposite material. The composite material of the invention not only can be used for treating cancers by combining ion interference method and chemical kinetics therapy, but also can be used as a contrast agent of MRI to realize diagnosis and treatment. Meanwhile, the composite material has the characteristics of pH response degradation and magnetic targeting, can be metabolized out of a body, and has wide clinical application prospect in the field of biomedicine.

Description

CaO2/MnFe2O4Nanocomposite material, preparation and application thereof

Technical Field

The invention belongs to the technical field of nano composite particle preparation, and relates to CaO₂/MnFe₂O₄Nanocomposite materials, their preparation and use.

Background

Malignant tumors are one of the major diseases that seriously endanger human health and are also the leading cause of death worldwide. Chemotherapy, as the most traditional treatment modality, has toxic and side effects due to low bioavailability. Under the influence of nontoxic substances released in tumors, Fenton reaction can be converted into toxic anticancer substances in situ in the tumor microenvironment, so that the cancer treatment with remarkably enhanced treatment effect and negligible toxic and side effects is realized. MnFe₂O₄The nano particle can convert H under neutral condition₂O₂Decompose to non-toxic O₂And H₂And O. More importantly, they can efficiently remove H under acidic conditions₂O₂Decompose into highly toxic hydroxyl radicals (. OH). Therefore, magnetic nanoparticles are considered as a promising tumor-targeting nanocatalysis enzyme, because hydroxyl radicals are generated in an acidic tumor microenvironment, resulting in apoptosis or necrosis of tumor cells. However, intratumoral H₂O₂Too low to generate significant hydroxyl radicals to achieve satisfactory catalytic therapeutic effects.

In order to overcome the defect of poor therapeutic effect of the magnetic nanoparticles, many attempts are made by those skilled in the art. For example, patent CN 202010973050.3 discloses a preparation method and application of copper sulfide/hyperbranched macromolecular nano-bionic enzyme, which uses hyperbranched macromolecular solution and CuCl₂·2H₂O and Na₂S·9H₂And preparing CuS @ G5 nano-particles by taking O as a reactant, and then sequentially preparing the GOD-loaded copper sulfide/hyperbranched macromolecular nano-bionic enzyme. According to the preparation method, a copper sulfide nanoparticle core is used as a photo-thermal agent and a Fenton-like catalyst, a hyperbranched macromolecular polymer is used as a template to wrap copper sulfide and graft glucose oxidase, and researches show that the nano bionic enzyme system has excellent chemical kinetics and photo-thermal treatment performance under the micro-acid environment of tumors. However, the product prepared in this patent is not easily degraded and the remaining material may cause damage to vital organs of the organism, resulting in organ failure and death of the organism.

Therefore, the development of the magnetic nanoparticles with good tumor treatment effect, easy degradation and good human safety has practical significance.

Disclosure of Invention

The invention aims to overcome the defect that the magnetic nanoparticles in the prior art cannot realize good consideration of tumor treatment effect and human safety, and provides the magnetic nanoparticles which have good tumor treatment effect, are easy to degrade and have good human safety.

In order to achieve the purpose, the invention provides the following technical scheme:

CaO₂/MnFe₂O₄nanocomposite material comprising CaO₂Nanoparticles and coating CaO₂MnFe of the outer surface of the nanoparticles₂O₄Nanoparticles.

Compared with the prior art, the magnetic nano particle-MnFe₂O₄Nano particles, the invention introduces exogenous CaO₂Thereby increasing the amount of H in the tumor₂O₂Due to MnFe₂O₄And CaO₂All of which are sensitive to pH and are degraded in the tumor environment (under the acidic environment) to release a large amount of ions in the tumor environment, thereby generating hydrogen peroxide and hydroxyl radicals, and CaO₂The rapid release of degraded calcium ions in the tumor microenvironment can effectively induce calcium overload and calcification, both of which are detrimental to tumor survival. The strategy of combining chemokinetic therapy and ion interference therapy (calcium ion interference method) highlights the availability of the compound in tumor treatment (obviously improves the treatment effect on tumor cells), and opens a new door for further clinical cancer treatment.

As a preferred technical scheme:

CaO as described above₂/MnFe₂O₄Nanocomposite of said CaO₂/MnFe₂O₄The average particle size of the nano composite material is 80-200 nm.

The invention also provides CaO as described above₂/MnFe₂O₄The preparation method of the nano composite material comprises the following steps:

(1) with CaCl₂Adopts a one-step method to prepare PEG modified CaO₂Nanoparticles; CaO modified by PEG₂The nano particles are stable nano particles, so that the nano particles have better dispersity, are easy to collect, are not easy to degrade and are positively charged;

specifically, CaCl is added by one-step method₂Raw materials, hydrogen peroxide as an oxidant and ammonia water as an auxiliary agent are used for gradually synthesizing CaO₂Nano particles, which are prepared for the next experiment;

(2) with Fe (acac)₃Namely iron triacetylacetonate and Mn (acac)₂Namely, manganese acetylacetonate is used as a raw material to prepare MnFe by a hydrothermal method₂O₄The method is mainly used for synthesizing the negatively charged nanoparticles (namely the MnFe) with the particle diameter of less than 10nm₂O₄Nano particles) capable of coating CaO on the surface better and more fully through electrostatic adsorption₂；

(3) Taking CaO₂Ethanol dispersion of nanoparticles, dropwise adding MnFe₂O₄Ethanol dispersion of nanoparticles, ultrasonic agitation (using ultrasonic-assisted mechanical agitation, synthesized MnFe₂O₄The nano-particles have magnetic characteristics, and the synthesized material can be more uniform and has good dispersibility only through mechanical stirring) to obtain the CaO₂/MnFe₂O₄Nanocomposites, i.e. by electrostatic adsorption of MnFe₂O₄The nano particles coat CaO₂The surface of the nano-particle makes the whole nano-composite material more sensitive to pH, thereby achieving the response and release of the tumor area.

The invention firstly synthesizes PEG modified CaO by a one-step method₂Nanoparticles, then coated with MnFe₂O₄Nano particles, synthesis of CaO with good dispersity₂@MnFe₂O₄The nano composite material has wide clinical application prospect in the aspects of chemokinetic therapy/ion interference method. Through the biological imaging guide and the magnetic target of the material, the multiple treatment modes are mutually cooperated, and the tumor can be completely eliminated. After treatment, the degradability of the material can reduce the toxic and side effects of the material on organisms.

As a preferred technical scheme:

the preparation method comprises the step (1) of preparing CaO by a one-step method₂The process of the nano particles is specifically as follows:

taking CaCl₂Dropping the mixture and PEG solution into anhydrous methanol, and adding H₂O₂Stirring the mixture and ammonia water (the concentration is 25 to 28 weight percent) at normal temperature, centrifuging the mixture by using ethanol, and washing the mixture to obtain CaO₂And dispersing the nano particles in ethanol for later use.

Preparation method as described above, the CaCl₂、PEG、H₂O₂And ammonia water in a ratio of (0.2 to 0.3) g: (0.5-1) mg: (1-3) mL: (0.2-0.3) mL. The protection scope of the present invention is not limited thereto, and those skilled in the art can adjust the ratio of the added amount within a certain range, but the adjustment range is not too large, for example, excessive addition of PEG will cause CaO₂Nanoparticle agglomeration (i.e. CaO)₂The particle size of the nano particles is increased), and further the particle size of the final material is influenced, the addition of PEG is too little, and CaO cannot be ensured₂Dispersibility of the nanoparticles.

The production method as described above, in step (2), MnFe is produced₂O₄The process of the nano particles is specifically as follows:

taking Fe (acac)₃、Mn(acac)₂The oleylamine, the oleic acid and the benzyl alcohol are put into a hydrothermal reaction kettle for continuous reaction for 10 hours, and the MnFe is obtained after centrifugation and washing₂O₄Nanoparticles.

Preparation method as described above, Fe (acac)₃、Mn(acac)₂And the addition amount ratio of oleylamine, oleic acid and benzyl alcohol is (0.7-0.8) g: (0.2-0.3) g: (3-4) mL: (3-4) mL: (10-20) mL; the protection scope of the present invention is not limited thereto, and those skilled in the art can adjust the ratio of the addition amount within a certain range, but the adjustment range is not too large, such as Fe (acac)₃Or Mn (acac)₂If the addition amount of (A) is too large, the particle size of the final product is influenced too large; too little oleic acid will affect incomplete reaction, uneven particle size and poor dispersibility.

The temperature of the water bath heating is 180-190 ℃, and the reaction time is 8-10 h. The protection scope of the present invention is not limited thereto, and those skilled in the art can adjust the temperature and reaction time of the water bath heating within a certain range, but the adjustment range is not easy to be too large, and the excessive reaction temperature and the excessive reaction time can cause MnFe₂O₄The particle size of the nano particles is too large, so that agglomeration and growth are caused; the reaction temperature is too low, the reaction time is too short, the reagent can not react completely, and MnFe is influenced₂O₄Yield of nanoparticles.

The preparation method as described above, in the step (3), CaO₂Nanoparticles and MnFe₂O₄The mass ratio of the nanoparticles is 10-15: 3 to 5. The scope of the present invention is not limited thereto, and those skilled in the art can apply CaO to a certain extent₂Nanoparticles and MnFe₂O₄The mass ratio of the nano particles is adjusted, but the adjustment range is not easy to be overlarge, such as MnFe₂O₄If the amount of the nanoparticles added is too small, CaO will be formed₂/MnFe₂O₄The catalytic performance and nuclear magnetism effect of the nano composite material are greatly reduced; MnFe₂O₄Excessive addition of nanoparticles can result in MnFe₂O₄The nanoparticles are free and lost during later processes.

In addition, the invention also provides CaO as described above₂/MnFe₂O₄The application of the nano composite material in preparing tumor diagnosis and treatment reagents, chemical kinetic treatment reagents, biological imaging reagents or pH sensitive degradation reagents.

Has the advantages that:

(1) CaO of the present invention₂/MnFe₂O₄The preparation method of the nano composite material is simple, and the synthesized nano composite material has good dispersibility and smaller particle size;

(2) CaO of the present invention₂/MnFe₂O₄The nano composite material can realize the synergistic effect of the magnetic targeting combined chemical kinetics and the ion interference method;

(3) CaO of the present invention₂/MnFe₂O₄The nano composite material can realize biological imaging, can degrade the pH dependently, reduces the toxic and side effects on organisms, has good tumor treatment effect and high human body safety, and has great application prospect.

Drawings

FIG. 1 shows CaO obtained in example 1₂/MnFe₂O₄Transmission electron microscopy of the nanocomposite;

FIG. 2 shows CaO obtained in example 1₂/MnFe₂O₄NanocompositeAn elemental map of the material;

FIG. 3 shows CaO obtained in example 1₂/MnFe₂O₄The particle size distribution profile of the nanocomposite;

FIG. 4 shows CaO obtained in example 1₂/MnFe₂O₄ROS plots generated by the nanocomposite at different pH;

FIG. 5 shows CaO obtained in example 1₂/MnFe₂O₄MRI imaging of the nanocomposite material;

FIG. 6 shows CaO obtained in example 1₂/MnFe₂O₄A plot of hemolysis experiments for the nanocomposite;

FIG. 7 shows CaO obtained in example 1₂/MnFe₂O₄CCK-8 diagram of the nanocomposite.

Detailed Description

The invention is described in detail below with reference to the figures and specific embodiments. The present embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation manner and a specific operation process are given, but the scope of the present invention is not limited to the following embodiments.

In the following examples, unless otherwise specified, all the materials or treatment techniques are conventional and commercially available materials or conventional treatment techniques in the art, and NH used in the following examples₃H₂O is 25 to 28 wt%.

Example 1

CaO₂/MnFe₂O₄The preparation method of the nano composite material comprises the following steps:

(1) generally, CaO₂Synthesized at room temperature. 1mL of CaCl was added under vigorous stirring₂(2mol/L) and 1mL of PEG (0.5mg/mL) were added to 60mL of anhydrous methanol. Thereafter, 2mL of H₂O₂Mixed into the above mixture and then added dropwise with 0.2mL NH three seconds later₃H₂And O. When the reaction lasted 2 hours, CaO was obtained by centrifugation₂And washed with ethanol.

(2)MnFe₂O₄The nanoparticles were prepared according to a hydrothermal method.0.7285g Fe (acac)₃And 0.2532g Mn (acac)₂Dissolved in a mixture containing 3.0mL OA (oleic acid), 3.0mL OM (oleylamine) and 10mL benzyl alcohol. After 1 minute of sonication, the solution was reacted in an autoclave at 180 ℃ for 10 hours. The product was collected by magnetic separation and washed several times with ethanol.

(3) First, under mechanical agitation and ultrasonic treatment, 15mgCaO was added₂Dissolved in 10mL of ethanol. Then 5mg MnFe₂O₄Dispersed in 10mL of ethanol, and the dispersion was slowly dropped into the above solution. After 6h of treatment, CaO was separated by centrifugation₂/MnFe₂O₄The nanocomposite was washed 3 times with ethanol.

Example 2

CaO₂/MnFe₂O₄Detecting the generated ROS of the composite nano material: the DPBF solution (130. mu.L, 1mg/mL) was added to the cuvette first, followed by 600. mu.g.ml^-1CaO of (2)₂/MnFe₂O₄The nanomaterial was compounded with PBS buffer (pH 6.5 or 7.4) to make the total reaction volume 3 ml. The absorbance of DPBF at 410nm was measured at different times.

Example 3

MRI performance testing: in vivo magnetic resonance imaging, HeLa tumor-bearing mice were injected with 200 μ L CaO₂/MnFe₂O₄A nanocomposite material. Mice were scanned before and 3 hours after injection. A T2-weighted MRI cross-sectional scan image of the mouse was obtained.

Example 4

Hemolysis experiment: the red blood cells are washed and diluted phosphate buffer solution is mixed with CaO₂/MnFe₂O₄The nanocomposites were kept at room temperature for 4h at different concentrations (6.25-100. mu.g/mL) and with deionized water and PBS as positive and negative controls. Then, the supernatant is obtained by centrifugation, and the hemolysis rate is determined and calculated.

Example 5

Evaluation of cytotoxicity: cells were plated at 1X 10 per well⁴Inoculating into 96-well plate at a density of 37 deg.C and 5% CO₂Incubate for 24h under conditions. Then, using a solution containingWith different concentrations (0, 6.25, 12.5, 25, 50, 100, 200. mu.g/mL, 400. mu.g/mL) of CaO₂/MnFe₂O₄Replacing the original culture medium with the fresh culture medium, continuously culturing for 24h, replacing the culture medium with a culture medium solution containing 10% CCK-8, continuously culturing for 1h, and detecting the OD value (the detection wavelength is 450nm) of each well on an enzyme-labeling instrument.

The final products synthesized in the above examples were characterized as shown in fig. 1 to 7:

as can be seen from the transmission electron microscope of FIG. 1 and the mapping of FIG. 2, CaO was successfully synthesized₂/MnFe₂O₄Nano material and small grain size.

As is clear from the particle size diagram in FIG. 3, CaO₂/MnFe₂O₄The particle size of (A) is uniform and the hydrated particle size is about 150 nm.

As can be seen from FIG. 4, CaO₂/MnFe₂O₄Compared with pH7.4, the nanocomposite generates more ROS in PBS buffer solution with pH6.5, which indicates that the nanocomposite degrades under acidic condition and can effectively generate a large amount of ROS to kill tumor cells.

As can be seen from FIG. 5, the nanocomposite CaO₂/MnFe₂O₄The nuclear magnetic signal in the tumor area is obvious, which indicates that the nano composite material is suitable to be used as an MRI imaging contrast agent for MRI imaging.

As can be seen from the hemolysis experiment of FIG. 6, the material CaO₂/MnFe₂O₄The hemolysis rate of (2) is low, which shows that the material has excellent biocompatibility and water solubility and can be used for in vivo experiments.

As can be seen from the CCK-8 experiment of FIG. 7a, normal cells and high CaO concentration₂/MnFe₂O₄The survival rate is still about 80% when the materials are cultivated, and the low toxicity of the materials is proved. As can be seen from FIG. 7b, the survival rate of tumor cells at pH6.5 was significantly lower than that at pH7.4 when the material was incubated with tumors of different pH, demonstrating that the material had a good effect of killing tumor cells in an acidic response.

Example 6

CaO₂/MnFe₂O₄The preparation method of the nano composite material comprises the following steps:

(2) generally, CaO₂Synthesized at room temperature. Under vigorous stirring, 1mL of CaCl₂(2mol/L) and 1mL of PEG (0.5mg/mL) were added to 60mL of anhydrous methanol. Thereafter, 1mL of H₂O₂Mixed into the above mixture and then added dropwise with 0.2mL NH three seconds later₃H₂And O. When the reaction lasted 2 hours, CaO was obtained by centrifugation₂And washed with ethanol.

(2)MnFe₂O₄The nanoparticles were prepared according to a hydrothermal method. 0.73g Fe (acac)₃And 0.26gMn (acac)₂Dissolved in a mixture containing 3.0mL OA (oleic acid), 3.0mL OM (oleylamine) and 10mL benzyl alcohol. After 1 minute of sonication, the solution was reacted in an autoclave at 180 ℃ for 10 hours. The product was collected by magnetic separation and washed several times with ethanol.

(3) First, 10mgCaO was added under mechanical agitation and ultrasonic treatment₂Dissolved in 10mL of ethanol. Then 3mgMnFe₂O₄Dispersed in 10mL of ethanol, and the dispersion was slowly dropped into the above solution. After 6h of treatment, CaO was separated by centrifugation₂/MnFe₂O₄The nanocomposite was washed 3 times with ethanol.

Example 7

CaO₂/MnFe₂O₄The preparation method of the nano composite material comprises the following steps:

(1) generally, CaO₂Synthesized at room temperature. Under vigorous stirring, 1mL of CaCl₂(2.1mol/L) and 1mL of PEG (0.5mg/mL) were added to 60mL of anhydrous methanol. Thereafter, 2mL of H₂O₂Mixed into the above mixture and then added dropwise with 0.3mL of NH three seconds later₃H₂And O. When the reaction lasted 2 hours, CaO was obtained by centrifugation₂And washed with ethanol.

(2)MnFe₂O₄The nanoparticles were prepared according to a hydrothermal method. Mixing 0.75g Fe (acac)₃And 0.26gMn (acac)₂Dissolved in a solution containing 3.0mLOA (oleic acid), 3.0mL OM (oleylamine) and 10mL benzyl alcohol. After 1 minute of sonication, the solution was reacted in an autoclave at 180 ℃ for 10 hours. The product was collected by magnetic separation and washed several times with ethanol.

(3) Firstly, under mechanical stirring and ultrasonic treatment, 15mg CaO is added₂Dissolved in 10mL of ethanol. Then 5mg MnFe₂O₄Dispersed in 10mL of ethanol, and the dispersion was slowly dropped into the above solution. After 6h of treatment, CaO was separated by centrifugation₂/MnFe₂O₄The nanocomposite was washed 3 times with ethanol.

Example 8

CaO₂/MnFe₂O₄The preparation method of the nano composite material comprises the following steps:

(1) generally, CaO₂Synthesized at room temperature. Under vigorous stirring, 1mL of CaCl₂(2mol/L) and 2mL of PEG (0.5mg/mL) were added to 60mL of anhydrous methanol. After that, 3mL of H₂O₂Mixed into the above mixture and then added dropwise with 0.3mL of NH three seconds later₃H₂And O. When the reaction lasted 2 hours, CaO was obtained by centrifugation₂And washed with ethanol.

(2)MnFe₂O₄The nanoparticles were prepared according to a hydrothermal method. 0.73g Fe (acac)₃And 0.25gMn (acac)₂Dissolved in a mixture containing 3.0mL OA (oleic acid), 3.0mL LOM (oleylamine) and 10mL benzyl alcohol. After 1 minute of sonication, the solution was reacted in an autoclave at 180 ℃ for 10 hours. The product was collected by magnetic separation and washed several times with ethanol.

(3) First, under mechanical agitation and ultrasonic treatment, 15mgCaO was added₂Dissolved in 10mL of ethanol. Then 5mg MnFe₂O₄Dispersed in 10mL of ethanol, and the dispersion was slowly dropped into the above solution. After 6h of treatment, CaO was separated by centrifugation₂/MnFe₂O₄The nanocomposite was washed 3 times with ethanol.

The embodiments described above are described to facilitate an understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.

Claims (10)

1.CaO₂/MnFe₂O₄Nanocomposite material, characterized in that it comprises CaO₂Nanoparticles and coating CaO₂MnFe of the outer surface of the nanoparticles₂O₄Nanoparticles.

2. CaO according to claim 1₂/MnFe₂O₄Nanocomposite material, characterized in that said CaO₂/MnFe₂O₄The average particle size of the nano composite material is 80-200 nm.

3. CaO according to claim 1 or 2₂/MnFe₂O₄The preparation method of the nano composite material is characterized by comprising the following steps:

(1) with CaCl₂Adopts a one-step method to prepare PEG modified CaO₂Nanoparticles;

(2) with Fe (acac)₃And Mn (acac)₂Preparation of MnFe as raw material₂O₄Nanoparticles;

(3) taking CaO₂Ethanol dispersion of nanoparticles, dropwise adding MnFe₂O₄The CaO is obtained by ultrasonically stirring ethanol dispersion of the nano particles₂/MnFe₂O₄A nanocomposite material.

4. The method according to claim 3, wherein CaO is prepared in one step in step (1)₂The process of the nano particles is specifically as follows:

taking CaCl₂Dropping with PEG solutionAdding H into anhydrous methanol₂O₂Mixing with ammonia water, stirring at normal temperature, centrifuging with ethanol, and washing to obtain CaO₂And dispersing the nano particles in ethanol for later use.

5. The method of claim 4, wherein the CaCl is₂、PEG、H₂O₂And ammonia water in a ratio of (0.2 to 0.3) g: (0.5-1) mg: (1-3) mL: (0.2-0.3) mL.

6. The method according to claim 3, wherein in the step (2), MnFe is prepared₂O₄The process of the nano particles is specifically as follows:

taking Fe (acac)₃、Mn(acac)₂The oleylamine, the oleic acid and the benzyl alcohol are put into a hydrothermal reaction kettle for continuous reaction for 10 hours, and the MnFe is obtained after centrifugation and washing₂O₄Nanoparticles.

7. The method according to claim 6, wherein the Fe (acac)₃、Mn(acac)₂And the addition amount ratio of oleylamine, oleic acid and benzyl alcohol is (0.7-0.8) g: (0.2-0.3) g: (3-4) mL: (3-4) mL: (10-20) mL;

the temperature of the water bath heating is 180-190 ℃, and the reaction time is 8-10 h.

8. The method according to claim 3, wherein in the step (3), CaO is added₂Nanoparticles and MnFe₂O₄The mass ratio of the nanoparticles is 10-15: 3 to 5.

9. CaO according to claim 1 or 2₂/MnFe₂O₄Use of a nanocomposite material for the preparation of a chemokinetic therapeutic agent, a bioimaging agent or a pH sensitive degradation agent.

10. CaO according to claim 1 or 2₂/MnFe₂O₄The application of the nano composite material in preparing a tumor diagnosis and treatment reagent.

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