CN108324962B

CN108324962B - Preparation method of ferroferric oxide nanoparticles with cluster structure

Info

Publication number: CN108324962B
Application number: CN201810074527.7A
Authority: CN
Inventors: 史向阳; 马丹; 彭琛; 张九龙; 史梦晗; 陆诗怡
Original assignee: Suzhou Biowisetech Co ltd; Donghua University
Current assignee: Suzhou Biowisetech Co ltd; Donghua University
Priority date: 2018-01-25
Filing date: 2018-01-25
Publication date: 2020-06-23
Anticipated expiration: 2038-01-25
Also published as: CN108324962A

Abstract

The invention relates to a preparation method of ferroferric oxide nanoparticles with cluster structures, which comprises the following steps: preparing ultra-small ferroferric oxide nano particles, preparing activated ultra-small ferroferric oxide nano particle solution, and preparing the ferroferric oxide nano particles with cluster structures. The invention is simple, and the prepared Fe₃O₄the/Cystamine nano-particles have good stability and biocompatibility, are sensitive to reducing conditions, and can realize T of tumor parts in animal bodies₁‑T₂The bi-modal MR imaging effect can be effectively used as an MR imaging contrast agent, and has industrial and commercial application prospect.

Description

Preparation method of ferroferric oxide nanoparticles with cluster structure

Technical Field

The invention belongs to the field of preparation of Magnetic Resonance Imaging (MRI) contrast agents, and particularly relates to a preparation method of ferroferric oxide nanoparticles with cluster structures.

Background

Malignant tumors are the first killers harmful to human life all the time, and have the characteristics of high mortality rate, difficult treatment, rapid deterioration and the like. Therefore, early diagnosis and specific treatment of tumors are important. At present, the main means for detecting tumors are: ultrasound imaging, CT imaging, nuclear medicine (PET or SPECT) imaging, and Magnetic Resonance Imaging (MRI). With the development of the magnetic resonance technology, the scanning time is gradually shortened, the resolution is gradually improved, and the detection of small lesions is more accurate, so that the magnetic resonance imaging technology becomes a novel disease detection means developed in recent years. In order to improve the sensitivity and specificity of MRI imaging diagnostics it is necessary to select a suitable MRI contrast agent. Conventional MRI contrast agents fall into two main categories: one is T₁Weighted MRI contrast agents, one type being T₂Weighted MRI contrast agents. T is₂Weighted MRI contrast agents are the first method of detection of soft tissue damage and a large number of reports have been made in the literature to date using magnetic iron oxide nanoparticles as MRI negative contrast agents for cancer diagnosis. However, in human blood, calcium ion-rich regions, metal ion deposition, and human tissue damage sites are at T₂The negative contrast images obtained by signal attenuation during imaging often interfere with clinical diagnosis, which requires a higher resolution T₁A weighted MRI contrast agent. In summary, single modality detection methods often fail to give highly accurate and comprehensive diagnostic results. With the development of science and technology, the study of MRI contrast agents tends to be multifunctional and multimode, and the treatment scheme can be guided and formulated better by integrating different diagnostic information. Wherein, T₁-T₂Bimodal mri contrast agents have become an emerging research direction that has attracted a great deal of attention.

Construction of T₁-T₂The following 3 methods are common for bimodal contrast agents: (1) a certain T₂Passage of contrast agent through T₁The contrast agent is combined to make the obtained composite nano material have T₁-T₂Bimodal MR imaging performance; (2) by aligning magnetic nanoparticles (e.g. Fe)₃O₄Nano particles) is regulated and controlled, and the magnetic nano material with proper size is synthesized. Research shows that Fe₃O₄The magnetic properties of the nanoparticles are strongly correlated with size, the particle size<5nm of Fe₃O₄Nanoparticles (ultra small Fe)₃O₄Nanoparticles) a substantial reduction in magnetic moment, T₂The effect is suppressed and can therefore be used as T₁Weighting or T₁-T₂Contrast agents for bimodal MR imaging; (3) cluster type nanoparticles. Having a T₁The weak magnetic nanoparticles of the effect obtain T due to cluster formation, size enlargement and magnetic enhancement₂And (4) imaging effect. The study shows that the compound has T per se₁Ultra-small Fe for weighting MR imaging effects₃O₄The magnetic nanoparticles become larger after forming clusters, so that the magnetism of the magnetic nanoparticles is enhanced, and the magnetic moment is increased. Therefore, cluster type ultra small Fe₃O₄T of nanoparticles₁The effect is suppressed and then converted into T₂Imaging the contrast agent. Construction of cluster-type ultra-small Fe by a suitable method₃O₄Nanoparticles, can also realize T₁-T₂A bimodal imaging effect. For example, Mao et al prepared oligosaccharide-encapsulated Fe with a particle size of 3.5nm₃O₄And (3) nanoparticles. Such monodisperse nanoparticles exhibit good T₁Effect, but Fe that can form cluster structure under acidic tumor microenvironment₃O₄Self-assembling and converting into T₂Effect, achieving T at the tumor site₁-T₂Bimodal MR imaging contrast (Mao et al, ACS nano.2017,11, 4582-.

The method is used for searching domestic and foreign documents, and no research on the construction of cluster type ultra-small ferroferric oxide nano particles and the application of the cluster type ultra-small ferroferric oxide nano particles in T in organisms is found₁-T₂Report on bimodal MRI diagnosis.

Disclosure of Invention

The invention aims to solve the technical problem of providing a preparation method of ferroferric oxide nano particles with cluster structures, which has the advantages of simplicity, mild reaction conditions, easiness in operation, lower cost and capability of preparing Fe with cluster structures₃O₄the/Cystamine nano-particle has good stability and biocompatibility, is easy to be endocytosed by cells, and can realize T in organisms₁-T₂Transformed bimodal MR imaging.

The invention relates to a preparation method of ferroferric oxide nano particles with cluster structures, which comprises the following specific steps:

(1) dissolving a trivalent ferric salt in a solvent, adding a stabilizer, stirring, adding a reaction auxiliary agent, carrying out thermal reaction on the solvent, cooling, centrifuging and drying to obtain ultra-small ferroferric oxide nanoparticles, wherein the ratio of the trivalent ferric salt to the solvent to the reaction auxiliary agent is 0.62-0.68 g, 38-40 mL, 0.47-0.50 g and 1.312-1.33 g;

(2) dispersing the ultra-small ferroferric oxide nanoparticles in the step (1) in a solvent, performing ultrasonic treatment, and activating by EDC and NHS to obtain an activated ultra-small ferroferric oxide nanoparticle solution, wherein the mass ratio of the ultra-small ferroferric oxide nanoparticles to EDC to NHS is 13-16: 28-36: 17-20, and the ratio of the ultra-small ferroferric oxide nanoparticles to the solvent is 28-32 mg: 2-4 mL;

(3) dispersing Cystamine dihydrochloride dihydrate in a solvent, performing ultrasonic treatment, adding the activated ultra-small ferroferric oxide nano-particle solution obtained in the step (2) to react, dialyzing, and performing freeze drying to obtain Fe with a cluster structure₃O₄the/Cystamine nano-particles comprise activated ultra-small ferroferric oxide nano-particles and Cystamine dihydrochloride, wherein the molar ratio of the activated ultra-small ferroferric oxide nano-particles to the Cystamine dihydrochloride is 3-5: 1-2, and the ratio of the Cystamine dihydrochloride to a solvent is 15-19 mg: 2-4 mL.

The ferric salt in the step (1) is anhydrous ferric chloride; the solvent is diethylene glycol DEG; the stabilizer is sodium citrate; the reaction auxiliary agent is anhydrous sodium acetate; the conditions of adding the stabilizer and stirring are as follows: stirring for 1-2 h at 80 ℃ in an air atmosphere.

In the step (1), the solvothermal reaction temperature is 190-200 ℃, and the solvothermal reaction time is 3-4 h.

The centrifugation in the step (1) comprises the following specific steps: centrifuging at 8500-9000 rpm for 10-15 min, discarding the supernatant, redissolving with absolute ethanol, centrifuging at 8500-9000 rpm for 10-15 min, and repeating the operation for 2-3 times.

The solvent thermal reaction in the step (1) is carried out in a 50mL high-pressure reaction kettle; the drying is carried out at the temperature of 60-65 ℃.

And (3) the solvents in the steps (2) and (3) are ultrapure water.

The activation time in the step (2) is 2.5-3 h.

And (3) adding the activated ultra-small ferroferric oxide nano particle solution in the step (2) to react at room temperature for 60-72 hours.

The specific steps of dialysis in step (3) are as follows: dialyzing with a dialysis bag with the cut-off molecular weight of 8000-14000 for 2-3 days (1.5-2L of distilled water is used in each dialysis, and 6-9 times of water is changed in total).

The invention uses Cystamine dihydrochloride (Cystamine dihydrate) to convert ultra-small Fe₃O₄The nanoparticles are cross-linked into a cluster structure. By controlling the cross-linking agent and Fe₃O₄The cluster type Fe with stable property and controllable size can be prepared according to the proportion₃O₄the/Cystamine nano-particles endow the particles with higher T₂Relaxation Rate (26.4 mM)^-1s^-1). Cluster structure of Fe₃O₄the/Cystamine nano-particles show the characteristic of responding to a reducing agent GSH in vitro, and S-S in Cystamine dihydrochloride can be broken under reducing conditions, so that cluster type Fe is generated₃O₄dispersing/Cystamine nano particles into Fe₃O₄Nanoparticles, higher T₁Relaxation Rate (4.3 mM)^-1s^-1) Effecting T of the material₁-T₂A dual modality MR imaging functionality. With ultra-small Fe alone₃O₄In contrast, Fe of cluster structure₃O₄the/Cystamine nanoparticles are larger in hydrodynamic diameter (134.4nm), more easily endocytosed by cells, and sensitive to the reducing environment inside 4T1 cells, and can realize MR imaging at a cellular level. In addition to this, cluster-structured Fe₃O₄the/Cystamine can be applied to animal bodies through tail vein injection or intratumoral injection, and Fe is injected to tumor parts in tumor-bearing mice₃O₄T can be clearly observed after/Cystamine₁-T₂Transformation procedure for bimodal MR imaging by injection of Fe via tail vein₃O₄after/Cystamine, an increase in MR imaging signal at the tumor site can be observed. Therefore, the cluster-structured Fe prepared by the invention₃O₄the/Cystamine nano-particles have potential MR diagnosis application value.

The physical and chemical properties of the prepared magnetic nanoparticles are characterized by methods such as infrared (FTIR), thermogravimetric analysis (TGA), inductively coupled plasma atomic emission spectrometry (ICP-OES), Zeta potential and hydrated particle size (DLS). And determining T of the nanoparticles by MRI imager₁-T₂Bimodal imaging performance, in-vitro reduction responsiveness is tested by testing relaxation performance change of the material after mixing with a GSH solution. Then, the hemocompatibility and cytotoxicity of the nanoparticles are evaluated by a hemolysis experiment and a CCK-8 method, and the phagocytosis capacity of the 4T1 cells on the nanoparticles is verified by a Prussian blue staining method and an ICP-OES method. By injecting nanoparticles into the tumor or tail vein of the tumor-bearing mice, T of the material is observed₁-T₂Bimodal MR imaging contrast effects.

Advantageous effects

(1) The invention is simple, and adopts ultra-small Fe with high relaxation rate₃O₄Nanoparticles, crosslinking of ultra-small Fe by cystamine dihydrochloride₃O₄Nanoparticle formation of stable cluster type Fe₃O₄/Cystamine nano-particles, and cluster type Fe prepared by using the same₃O₄the/Cystamine nano-particles have good stability and biocompatibility, T₂The relaxation rate is higher (26.4 mM)^-1s^-1)；

(2) Cluster type Fe prepared₃O₄the/Cystamine nano-particles show the characteristic of responding to a reducing agent GSH in vitro, and S-S in Cystamine dihydrochloride can be broken under reducing conditions, so that cluster type Fe is generated₃O₄dispersing/Cystamine nano particles into Fe₃O₄Nanoparticles, higher T₁Relaxation Rate (4.3 mM)^-1s^-1) Effecting T of the material₁-T₂The dual-mode MR imaging function has the prospect of commercialization implementation;

(3) preparing the obtained Fe with cluster structure₃O₄The Cystamine can be applied to animal bodies through intratumoral injection, and Fe is injected to tumor parts in tumor-bearing mice₃O₄T can be clearly observed after/Cystamine₁-T₂Transformation procedure of bimodal MR imaging. After injection through the tail vein, Fe₃O₄the/Cystamine nano-particles have good in vivo T₁Weighted MR imaging capability enabling T in vivo₁Weighted MRI diagnosis; these advantages lead to the preparation of Fe according to the invention₃O₄the/Cystamine nanoparticles can be effectively used as an MR imaging contrast agent.

Drawings

FIG. 1 is Fe in example 3₃O₄(a) And Fe₃O₄The infrared spectrum of/cystamine (b);

FIG. 2 is Fe in example 3₃O₄(a) And Fe₃O₄Thermogravimetric analysis of/cystamine (b);

FIG. 3 is Fe in example 4₃O₄A stability test chart of the/Cystamine nano particles;

FIG. 4 is Fe in example 5₃O₄(a)、Fe₃O₄/Cystamine (b) and Fe after glutathione GSH treatment₃O₄TEM image of/Cystamine (c and d) nanoparticles;

FIG. 5 is Fe in example 6₃O₄，Fe₃O₄Fe treated with glutathione GSH and Cystamine₃O₄(a) T of/Cystamine nanoparticles₁Relaxation rate and (b) T₂A relaxation rate;

FIG. 6 is Fe in example 6₃O₄，Fe₃O₄Fe treated with glutathione GSH and Cystamine₃O₄(a) T of/Cystamine nanoparticles₁Weighted MR imaging and (b) T₂Weighting the MR imaging images;

FIG. 7 shows the results of CCK-8 assay in example 7 in which 4T1 cells were treated with PBS buffer (control), Fe₃O₄And Fe₃O₄Cell viability test results of Cystamine nanoparticles after 24 hours of treatment under the condition of iron concentration of 5-100 mug/mL;

FIG. 8 is Fe in example 8₃O₄The hemolysis experiment result of the/Cystamine nano-particles under the condition that the iron concentration is 10-200 mug/mL;

FIG. 9 shows the 4T1 cells of example 9 after being treated with PBS buffer (controls a and e), Fe₃O₄PBS solution of nanoparticles (b-d) and Fe₃O₄Results of Prussian blue staining after 4 hours of treatment with PBS/Cystamine (f-h) with an iron concentration of 25. mu.g/mL, (c, g) an iron concentration of 50. mu.g/mL, and (d, h) an iron concentration of 100. mu.g/mL;

FIG. 10 shows the 4T1 cells in example 9 after being treated with PBS buffer and Fe₃O₄PBS solution of nanoparticles and Fe₃O₄Treating the cells for 4 hours by using PBS (the concentration of Fe is within the range of 5-100 mu g/mL) of/Cystamine nano particles, and determining by using ICP-OES after the cells are digested by aqua regia to obtain the iron content result endocytosed by each cell;

FIG. 11 shows the intratumoral injection of Fe in mice of example 10₃O₄T at tumor sites at various time points after PBS solution of/Cystamine (Fe concentration 3.2mM, 20. mu.L)₁-T₂A bimodal MR imaging picture test result;

FIG. 12 shows the respective injections of Fe into the tail vein of mouse in example 11₃O₄PBS solution of nanoparticles (a) and Fe₃O₄T of tumor site at different time points (0-120 min) after PBS solution (b) of/Cystamine nanoparticles (Fe concentration 0.1M, 150. mu.L)₁Weighting the MR imaging pictures;

FIG. 13 shows the respective injections of Fe into the tail vein of mouse in example 11₃O₄PBS solution of nanoparticles and Fe₃O₄T of nude mouse tumor site at different time points after PBS solution of/Cystamine nanoparticles (Fe concentration 0.1M, 150. mu.L)₁Weighting changes in signal-to-noise ratio values of the MRI;

FIG. 14 shows the injection of PBS and Fe into the tail vein of mouse in example 12₃O₄PBS solution of nanoparticles and Fe₃O₄After 12 hours, the nanoparticles were distributed in each organ of nude mice in PBS (Fe concentration 0.1M, 150. mu.L).

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.

Example 1

(1) 0.65g of anhydrous ferric chloride was dissolved in 40mL of diethylene glycol (also known as diethylene glycol, DEG), and 0.47g of sodium citrate (Na) was added₃Cit), stirring for 1h at 80 ℃ in an air atmosphere, adding 1.312g of anhydrous sodium acetate powder after the sodium citrate is completely dissolved, continuously stirring until the sodium acetate powder is completely dissolved, then transferring the solution to a 50mL high-pressure reaction kettle, and reacting for 4 hours at 200 ℃; after the reaction is finished, naturally cooling to room temperature, transferring the product into a 50mL centrifuge tube to centrifuge at 8500rpm for 15 minutes, discarding the supernatant, redissolving with absolute ethyl alcohol, centrifuging at 8500rpm for 15 minutes, repeating the operation for 3 times, and drying the precipitate at 60 ℃ to obtain the ultra-small Fe₃O₄Nanoparticles, i.e., ultra-small iron oxide nanoparticles with surface sodium citrate stabilization.

(2) Ultra-small Fe in the step (1)₃O₄Dispersing nanoparticles (30mg) in 3mL of ultrapure water, dissolving with ultrasound for 10min, dissolving EDC (59mg) and NHS (35mg) in 1mL of ultrapure water respectively, dissolving completely, and adding into the above-mentioned ultra-small Fe₃O₄Reacting in the nano-particle solution for 3 hours to activate Fe₃O₄And (4) obtaining activated ultra-small ferroferric oxide nano-particle solution through carboxyl on the surface of the nano-particles.

(3) Dispersing cystamine dihydrochloride (17mg) in 3mL of ultrapure water, performing ultrasonic treatment for 10min to dissolve the cystamine dihydrochloride, then dropwise adding the activated ultra-small ferroferric oxide nanoparticle solution in the step (2), reacting for 72h at room temperature, dialyzing for 3 days by using a dialysis bag with the molecular weight cutoff of 8000-14000 (2L of distilled water used in each dialysis, and 9 times of water replacement), and performing vacuum freeze drying to obtain Fe of clusters₃O₄a/Cystamine nanoparticle.

Example 2

Respectively taking the ultra-small Fe in the example 1₃O₄Nanoparticles and clustersCluster of Fe₃O₄2mg of/Cystamine nano particles are dissolved in 2mL of ultrapure water to obtain a nano particle suspension, the nano particle suspension is subjected to uniform ultrasonic treatment, the surface potential and the hydrated particle size are measured, and the test results are shown in Table 1. The test result shows that: the prepared ultra-small Fe₃O₄Fe of nanoparticles and clusters₃O₄The surface potential of the/Cystamine nano-particles is-33.2 mV and-21.4 mV respectively; the hydrated particle sizes were 25.6 and 134.4nm, respectively. From the experimental results, it was found that the surface potential of the monodisperse ultra small iron oxide nanoparticles rises after cross-linking into clusters and the hydrodynamic diameter increases significantly. Changes in surface potential and hydrated particle size indicate Fe of the cluster₃O₄the/Cystamine nanoparticles have been formed.

TABLE 1

Sample (I)	Electric potential (mV)	Hydrodynamic diameter (nm)	Polydispersity index
				Fe₃O₄	-33.2±1.7	25.6±1.6	0.53
Fe₃O₄/Cystamine	-21.4±2.6	134.4±2.6	0.18

Example 3

Respectively taking the ultra-small Fe in the example 1₃O₄Fe of nanoparticles and clusters₃O₄the/Cystamine nanoparticles were measured at 5mg for infrared spectroscopy (as shown in FIG. 1) and thermogravimetric analysis (as shown in FIG. 2). 466-601cm by analyzing infrared spectrum (as shown in figure 1)^-1The characteristic absorption peak appears as Fe₃O₄Stretching vibration of upper Fe-O, 3451cm^-1The nearby peak is the stretching vibration peak of OH on water molecule, 2931cm^-1And 2800cm^-1The nearby characteristic absorption peak is attributed to the stretching vibration of the methylene group in sodium citrate. At the same time at 1396-^-1Stretching vibration of C ═ O. While the strong absorption peak of 1736 in curve b belongs to the amino group and Fe of cystamine dihydrochloride₃O₄The surface carboxyl groups are combined to generate amido bonds. 551cm^-1The characteristic peak at (A) is clearly enhanced and should be attributed to the S-S bond in cystamine dihydrochloride. The infrared spectrogram result shows that Fe₃O₄The nanoparticles were successfully linked to cystamine dihydrochloride. In addition, the TGA test results (as shown in FIG. 2) indicate that Fe₃O₄Weight loss of 35.3%, Fe₃O₄Weight loss of/Cystamine was 37.7%, whereby the coupling of Cystamine dihydrochloride to Fe was quantified₃O₄The mass ratio on the nanoparticles was 2.4%.

Example 4

Fe prepared in example 1 was weighed₃O₄the/Cystamine nanoparticles 2mg, dissolved in 2mL water, PBS, DMEM and FBS respectively, were monitored for hydrodynamic diameter after 24 hours of nanoparticle dispersion (as shown in figure 3). The experimental result shows that Fe₃O₄The solution of the/Cystamine dissolved in different media shows a clear state, no precipitation or aggregation occurs after 24 hours of standing, and the hydrodynamic diameter of the nanoparticles dispersed in different media does not change significantly in value. Description of Fe₃O₄the/Cystamine has good colloidal stability.

Example 5

Separately, Fe prepared in example 1 of the present invention₃O₄/Cystamine and control Material (Fe)₃O₄Nanoparticles) were dissolved in 100 μ L of ultrapure water to prepare a nanoparticle suspension. Each 5. mu.L of Fe₃O₄Cystamine and Fe₃O₄The nanoparticle suspension was dropped onto the copper mesh surface and dried in air before being used for TEM testing (as shown in figure 4). To observe cluster type Fe₃O₄Changing the shape of Cystamine nano particles under reducing condition, dissolving glutathione GSH in ultrapure water to prepare reducing solution with the concentration of 10mM, and adding the reducing solution into Fe₃O₄Taking 5 mu L of glutathione-treated Fe in water solution of/Cystamine nano particles₃O₄the/Cystamine nanoparticles were used for TEM testing after air drying (as shown in figure 4). The TEM result shows that: ultra-small Fe₃O₄Exhibits a monodisperse state and is relatively uniform in size, Fe after crosslinking by cystamine dihydrochloride₃O₄Agglomerated into Fe₃O₄the/Cystamine, forms a cluster structure and becomes larger in size. Under the action of a reducing agent GSH, S-S in cystamine dihydrochloride is broken, and cluster Fe₃O₄the/Cystamine nano particles are decomposed into monodisperse ultra-small Fe₃O₄. Description of Fe₃O₄the/Cystamine nano-particles have a cluster type structure, are sensitive to oxidation-reduction conditions, and can be converted from an aggregation state to a monodispersion state in a reduction environment.

Control Material (Fe)₃O₄Nanoparticles) is prepared by the following specific method:

0.65g of anhydrous ferric chloride was dissolved in 40mL of diethylene glycol (also known as diethylene glycol, DEG), and 0.47g of sodium citrate (Na) was added₃Cit), stirring for 1h at 80 ℃ in an air atmosphere, adding 1.312g of anhydrous sodium acetate powder after the sodium citrate is completely dissolved, continuously stirring until the sodium acetate powder is completely dissolved, then transferring the solution to a 50mL high-pressure reaction kettle, and reacting for 4 hours at 200 ℃; after the reaction is finished, naturally cooling to room temperature, transferring the product into a 50mL centrifuge tube, centrifuging at 8500rpm for 15 minutes, discarding the supernatant, redissolving with absolute ethyl alcohol, centrifuging at 8500rpm for 15 minutes, repeating the operation for 3 times, and then precipitating at 60 DEG CDrying to obtain ultra-small Fe₃O₄And (3) nanoparticles.

Example 6

The relaxation rate reflects the efficiency of the nanoparticles as MRI contrast agents and can be calculated by inverse fitting of the relaxation times at different concentrations. Fe in example 1 was measured by ICP-OES test method₃O₄Cystamine and Fe in example 5₃O₄The Fe content in the nanoparticles was 192. mu.g/mg and 234. mu.g/mg, respectively. Then, 2mL of each of aqueous solutions having Fe concentrations of 0.1, 0.2, 0.4, 0.8 and 1.6mM in this order was prepared with ultrapure water. To test cluster type Fe₃O₄Reduction responsiveness of/Cystamine nanoparticles, glutathione GSH is dissolved in ultrapure water to prepare a reducing solution with the concentration of 10mM, and then added into Fe₃O₄Fe added with glutathione in water solution of/Cystamine nano-particles₃O₄the/Cystamine nano-particles are prepared into 2mL of aqueous solutions with Fe concentrations of 0.1, 0.2, 0.4, 0.8 and 1.6mM in sequence. Three materials (Fe) were measured separately₃O₄/Cystamine，Fe₃O₄And glutathione-added Fe₃O₄/Cystamine nanoparticles) T of nanoparticles at different Fe concentrations₁And T₂Relaxation time, linear fitting of the reciprocal relaxation time to the Fe concentration (as shown in FIG. 5) and testing of T₁And T₂Weighted imaging plots (as shown in fig. 6). The relaxation rate test result shows that the T of the three materials is within the range of 0.1-1.6mM of Fe concentration₁And T₂The relaxation time and the Fe concentration have good linear relation. Wherein, Fe₃O₄T of₁High relaxation rate, r₁Is 4.3mM^-1s^-1. Cluster-forming Fe₃O₄after/Cystamine nanoparticles, T of the Material₁Decrease in relaxation rate (r)₁Is 1.4mM^-1s^-1) And under GSH conditions, Fe₃O₄Re-dissolution of/Cystamine into Fe₃O₄T of nanoparticles₁The relaxation rate is also obviously increased to reach the state of monodisperse Fe₃O₄Approximate value of relaxation rate. On the other hand, Fe₃O₄T of₂Lower relaxation rate (r)₂Is 7.3mM^-1s^-1) Cluster-forming Fe₃O₄After the/Cystamine nanoparticles, their T₂The relaxation rate is remarkably increased to 26.4mM^-1s^-1Under GSH conditions, Fe₃O₄Re-dissolution of/Cystamine into Fe₃O₄T of nanoparticles₂The relaxation rate is also restored to monodisperse Fe₃O₄Approximate value of relaxation rate. This result indicates that Fe₃O₄the/Cystamine not only has higher T₂Relaxation rate convertible into monodisperse Fe under reducing conditions₃O₄To obtain higher T₁And (4) imaging performance. Fe₃O₄the/Cystamine has good T₁-T₂Bimodal MR imaging performance. As can be seen from FIG. 6, with the change of Fe concentration (0.1-1.6mM), the MRI signal shows good gradient change trend, and the test result shows that the material has good MRI imaging capability and can be used as excellent T in MRI molecular imaging diagnosis₁-T₂A bimodal contrast agent.

Example 7

Fe prepared by evaluating 4T1 cells as model cells₃O₄And Fe₃O₄Effects of/Cystamine nanoparticles on cell survival. Fe with the iron content of 1mg in example 5 was weighed₃O₄And Fe in example 1₃O₄the/Cystamine nano-particles are dispersed in sterile PBS to prepare PBS solution with the iron concentration of 1mg/mL, and are sterilized by ultraviolet irradiation overnight. Then, Fe was formulated in sterile PBS at iron concentrations of 5, 10, 25, 50 and 100. mu.g/mL in a clean bench₃O₄And Fe₃O₄a/Cystamine nanoparticle suspension. 4T1 cells were seeded in 96-well plates and then separately mixed with Fe₃O₄And Fe₃O₄the/Cystamine nanoparticles (

iron concentrations

5, 10, 25, 50 and 100. mu.g/mL) were co-incubated at 37 ℃ for 24 hours. Then, 20. mu.L of CCK-8 was added to the well of the plate, after further incubation at 37 ℃ for 4 hours, the culture solution was discarded, and 100. mu.L of DMSO was added, and after shaking for 20 minutes, the absorbance was measured at 450nm, and the buffer PBS group was usedThe absorbance of 4T1 cells was calculated by comparing the absorbance of the treated material with the absorbance of the treated material (see fig. 7). Fe compared to control PBS-treated cells₃O₄And Fe₃O₄The activity of the/Cystamine nano-particles to 4T1 cells is not significantly different within the range of experimental concentration of 5-100 mu g/mL, and the cell activity is over 85 percent. This fully illustrates the Fe prepared in example 5₃O₄And Fe prepared in example 1₃O₄the/Cystamine nano-particles have good cell compatibility and can be applied to MRI imaging detection in organisms.

Example 8

When contrast medium is injected into the body, it is necessary to directly contact with blood, and the intervention of contrast medium without hemolysis or other adverse symptoms becomes one of the important factors that researchers have to consider. To ensure that the nanoparticles prepared according to the present invention can be safely used for in vivo bioimaging diagnosis, the prepared Fe was evaluated₃O₄The blood compatibility of the/Cystamine nanoparticles. Fe in example 1 was weighed₃O₄the/Cystamine nano-particles are dispersed in PBS to prepare a solution with the iron concentration of 1mg/mL as a mother solution, and then PBS is used for preparing nano-particle suspensions with the iron concentrations of 10 mug/mL, 20 mug/mL, 50 mug/mL, 100 mug/mL and 200 mug/mL in sequence. An appropriate amount of fresh human blood was taken, first centrifuged (2000rpm, 5 minutes) to remove the supernatant, then the red blood cells were washed 5 times with PBS, and healthy red blood cells were collected and diluted 10-fold with PBS. Then Fe with different iron concentrations₃O₄the/Cystamine nanoparticle suspensions were mixed with red blood cells, and after standing for 1 hour, centrifuged at 10000rpm for 1 minute, photographed and the supernatant was measured for UV absorbance (as shown in FIG. 8). The process used ultrapure water as a positive control and PBS as a negative control. Fe is shown in FIG. 8₃O₄Results of hemolytic test of/Cystamine nanoparticles at a given iron concentration. And quantitatively evaluating the hemolysis of the nano material by measuring the absorbance value of the supernatant. As can be seen from the test results, Fe reached 200. mu.g/mL of iron concentration₃O₄The hemolysis rate of the/Cystamine nanoparticles is still less than 5%, which illustrates the preparation of example 1Fe (b) of₃O₄the/Cystamine has good blood compatibility, so that the/Cystamine can be safely used for in vivo MR imaging.

Example 9

Detection of Fe at different concentrations by Prussian blue staining₃O₄And Fe₃O₄Endocytosis effect of/Cystamine nanoparticles after 4 hours of coculture with 4T1 cells (see FIG. 9). 4T1 cells in 2 × 10⁵One cell/well was planted in 12-well plates and cultured overnight before Fe was added to each of the cells in example 1₃O₄Cystamine and Fe in example 5₃O₄Nanoparticle solutions in PBS (

Fe concentrations

25, 50 and 100. mu.g/mL) were co-incubated at 37 ℃ for 4 hours, and PBS-treated cells were used as controls. And (3) discarding the culture medium after co-culture, washing the cells for three times by using PBS, photographing and recording by using a phase contrast microscope after the cells are dyed by Prussian blue, and qualitatively analyzing the amount of the nanoparticles phagocytized by the cells according to the blue dyeing depth of the cells. In fig. 9, the blue color of the cells co-cultured with the nanoparticles gradually deepens after staining with increasing Fe concentration, indicating that the phagocytosis of both nanoparticles by the 4T1 cells is concentration-dependent. At the same Fe concentration, Fe₃O₄The blue color degree of cells treated by Cystamine is obviously deeper than that of Fe₃O₄The cells after nanoparticle treatment showed 4T1 cells vs Fe₃O₄The phagocytic efficiency of the/Cystamine nano-particles is higher. In addition, the average Fe phagocytosis per cell was also quantitatively analyzed using ICP-OES technique₃O₄And Fe₃O₄Amount of/Cystamine nanoparticles. When various concentrations of nanoparticles (Fe concentration of 5, 10, 20, 50 and 100 μ g/mL) in PBS solution were co-cultured with 4T1 cells for 4 hours, the cells were washed 3 times with PBS, then trypsinized, counted, and finally the cells were digested with aqua regia, the total amount of phagocytic iron elements per well was measured with ICP-OES, the phagocytic iron content per cell was calculated by dividing the number of cells, and PBS-treated cells were used as a control group. As shown in FIG. 10, the PBS-treated cells had almost no Fe element, while Fe₃O₄The iron content of the/Cystamine treated cells increased significantly with increasing concentration. When the iron concentration is 100. mu.g/mL, the single 4T1 is fineIntracellular Fe₃O₄The amount of iron element in the/Cystamine nano-particle reaches 6.2 pg/cell; and Fe₃O₄The phagocytosis amount of the treated cells is less increased along with the increase of the iron concentration, and when the iron concentration is 100 mu g/mL, a single 4T1 cell phagocytoses Fe₃O₄The nano-particle iron element is only 4.6 pg/cell. And the phagocytosis amount of the two groups of materials under the same iron concentration can be seen in comparison, the Fe of the cluster₃O₄The phagocytosis of the/Cystamine nano-particles by 4T1 cells is obviously higher than that of ultra-small Fe₃O₄And (3) nanoparticles. The results of this experiment demonstrate that Fe₃O₄the/Cystamine can be more effectively endocytosed by 4T1 cells, thereby providing reliable basis for the material to be effectively applied to in vivo MR imaging.

Example 10

Fe prepared in example 1₃O₄Percystamine was prepared as a 20. mu.L PBS dispersion with a Fe concentration of 3.2mM 2 × 10⁶Inoculating 4T1 cells into nude mice, and injecting Fe into mice tumor after tumor diameter reaches 0.6-1cm₃O₄PBS solution of/Cystamine nanoparticles, MR imaging of tumor sites at different time points (0, 5, 20 min) after material injection in mice bearing tumors using a NMR imager, and evaluation of T₁-T₂Bimodal MRI contrast effect (see fig. 11). The results show that Fe is injected₃O₄MR T of tumor sites in tumor-bearing mice after Cystamine₂The image was clearly darkened and the optimal T was reached 5min after injection₂And (4) imaging effect. Then due to cluster type Fe₃O₄the/Cystamine nano-particles are gradually dispersed into monodisperse Fe₃O₄，T₂Gradual transformation of effect into T₁And (4) effect. From T₁As can be seen in the image, T of the tumor site of the mouse₁The weighted MR imaging effect gradually increased after injection and tumor T₁The image reached the brightest at 20min after injection. This result also confirms the above conclusion, indicating that cluster type Fe₃O₄the/Cystamine nano-particles can be dispersed into Fe in a tumor microenvironment in a living body₃O₄Is realized by MR T₂Imaging to T₁And (5) converting the imaging effect. These results indicate Fe₃O₄the/Cystamine has excellent and switchable T₁-T₂The bimodal MRI tumor diagnosis effect and can be successfully applied to in-vivo MRI tumor diagnosis.

Example 11

Fe prepared in example 1₃O₄Cystamine and Fe prepared in example 5₃O₄150 μ L of PBS dispersion having an Fe concentration of 0.1M was prepared in accordance with the iron concentration measured by ICP-OES 2 × 10⁶Inoculating 4T1 cells into nude mice, and respectively injecting Fe through tail vein when tumor diameter reaches 0.6-1cm after two weeks₃O₄Cystamine and Fe₃O₄Nanoparticle PBS solution was used to evaluate the MR imaging effect at the tumor site (see figure 12). Fe was injected within 0 to 120 minutes after injection₃O₄The light and shade change of the tumor part of the nude mouse is not obvious, and Fe is injected₃O₄Nude mice tumors with/Cystamine nanoparticles brighten significantly, reaching brightest 30 minutes after injection. Exhibit Fe₃O₄the/Cystamine nano-particles have excellent T in vivo₁Weighted MR imaging effect with better imaging effect than Fe₃O₄Nanoparticles, probably due to Fe₃O₄the/Cystamine nano-particles can be more effectively caused by tumor enrichment through enhanced tumor penetration retention effect. FIG. 13 is T at tumor site at different time points after injection₁Weighted MR imaging SNR variation, Fe injected within 0 to 120 minutes after injection₃O₄The change of the tumor MRI signal value of the nude mice is not obvious, and Fe is injected₃O₄Tumor MRI signal values were significantly enhanced in nude mice for/Cystamine and the signal to noise ratio reached a maximum at 30 min. This is consistent with the results of fig. 12. These results illustrate the Fe prepared in example 1₃O₄the/Cystamine nano-particles have good in vivo T₁Weighted MR imaging capability enabling T in animals by intravenous injection₁Weighted MRI diagnosis.

Injecting cluster-structured Fe into vein of mouse₃O₄/Cystamine nano-particlesAnd the material Fe₃O₄Nanoparticles, observation of T thereof₂In the case of weighted MR imaging, T was found to be exhibited by neither material₂Weighted MR imaging effect. Possibly Fe₃O₄the/Cystamine nano-particles are very sensitive to the redox condition of organisms, and can rapidly disperse Fe in the reducing microenvironment in vivo₃O₄Nanoparticles, thereby losing T₂The effect is caused.

Example 12

To investigate the biological tissue distribution of nanoparticles, Fe prepared in example 1 was used₃O₄Cystamine and Fe prepared in example 5₃O₄(control group) 150. mu.L of PBS dispersion containing 0.1M Fe concentration 2 × 10⁶Inoculating 4T1 cells into nude mice, and respectively injecting Fe through tail vein when tumor diameter reaches 0.6-1cm after three weeks₃O₄And Fe₃O₄After 12 hours of PBS solution of/Cystamine nanoparticles, nude mice were sacrificed and dissected, and nude mice injected with PBS at tail vein were used as a blank group, the heart, liver, spleen, lung, kidney, and tumor were taken out, weighed, cut into 2 × 2mm fragments, digested with aqua regia for 24 hours, and then measured for iron content of each sample by ICP-OES, and finally calculated for iron content in each vital organ (FIG. 14). Fe prepared in example 1 was injected intravenously can be seen from the graph₃O₄/Cystamine nanoparticles or control material Fe₃O₄After 12 hours of nanoparticle administration (Fe concentration 0.1M, 150 μ L), the iron content in the liver and spleen of mice increased significantly compared to those before injection, while in other organs, such as: heart, lung, kidney and tumor, with low iron content. These results demonstrate that the nanoparticles prepared in example 1 are able to be cleared metabolically normally in mice.

Claims

1. A preparation method of ferroferric oxide nanoparticles with cluster structures comprises the following specific steps:

(1) dissolving ferric salt in a solvent, adding a stabilizer, stirring, adding a reaction auxiliary agent, carrying out solvothermal reaction for 4 hours at 200 ℃, cooling, centrifuging and drying to obtain the ultra-small ferroferric oxide nano-particles, wherein the ratio of the ferric salt to the solvent to the stabilizer to the reaction auxiliary agent is 0.65g to 40mL to 0.47g to 1.312g, the ferric salt is anhydrous ferric chloride, and the solvent is diethylene glycol DEG; the stabilizer is sodium citrate; the reaction auxiliary agent is anhydrous sodium acetate;

(2) dispersing the ultra-small ferroferric oxide nanoparticles obtained in the step (1) in ultrapure water, performing ultrasonic treatment, and activating for 3 hours by EDC and NHS to obtain an activated ultra-small ferroferric oxide nanoparticle solution, wherein the mass ratio of the ultra-small ferroferric oxide nanoparticles to EDC to NHS is 15:29.5:17.5, and the ratio of the ultra-small ferroferric oxide nanoparticles to the ultrapure water is 30mg:3 mL;

(3) dispersing Cystamine dihydrochloride hydrate in ultrapure water, performing ultrasonic treatment, adding the activated ultra-small ferroferric oxide nano particle solution in the step (2), reacting for 72 hours at room temperature, dialyzing, and freeze-drying to obtain Fe with a cluster structure₃O₄the/Cystamine nano-particles comprise activated ultra-small ferroferric oxide nano-particles and Cystamine dihydrochloride, wherein the molar ratio of the activated ultra-small ferroferric oxide nano-particles to the Cystamine dihydrochloride is 3-5: 1-2, and the ratio of the Cystamine dihydrochloride to ultrapure water is 17mg:3 mL.

2. The method for preparing cluster-structured ferroferric oxide nanoparticles according to claim 1, wherein the stabilizer is added in the step (1) and stirred under the conditions that: stirring for 1-2 h at 80 ℃ in an air atmosphere.

3. The preparation method of the cluster-structured ferroferric oxide nanoparticles according to claim 1, wherein the centrifugation in the step (1) comprises the following specific steps: centrifuging at 8500-9000 rpm for 10-15 min, discarding the supernatant, redissolving with absolute ethanol, centrifuging at 8500-9000 rpm for 10-15 min, and repeating the operation for 2-3 times.

4. The preparation method of the cluster-structured ferroferric oxide nanoparticles according to claim 1, wherein the specific dialysis step in the step (3) is as follows: dialyzing for 2-3 days by using a dialysis bag with the molecular weight cutoff of 8000-14000.