CN111228520B

CN111228520B - Cell membrane coated ultra-small ferroferric oxide nanocluster and preparation and application thereof

Info

Publication number: CN111228520B
Application number: CN202010059064.4A
Authority: CN
Inventors: 沈明武; 贾亮; 史向阳
Original assignee: Donghua University
Current assignee: Donghua University
Priority date: 2020-01-19
Filing date: 2020-01-19
Publication date: 2021-04-02
Anticipated expiration: 2040-01-19
Also published as: CN111228520A

Abstract

The invention relates to a cell membrane coated ultra-small ferroferric oxide nanocluster and a preparation method and application thereof. The method comprises the following steps: preparing ultra-small ferroferric oxide nanoparticles, preparing surface aminated ultra-small ferroferric oxide nanoparticles, preparing a ferroferric oxide nanocluster, preparing an adriamycin-loaded ferroferric oxide nanocluster, preparing a cell suspension, and preparing the membrane-coated ultra-small ferroferric oxide nanocluster. After the nanoclusters enter a mouse body through tail vein injection, T can be achieved₁/T₂The double-mode MR imaging conversion can also play a role in resisting tumors, and the chemotherapy effect can be further enhanced by using a UTMD auxiliary means, so that the method has potential clinical application value.

Description

Cell membrane coated ultra-small ferroferric oxide nanocluster and preparation and application thereof

Technical Field

The invention belongs to the field of nano medical diagnosis and treatment materials and preparation and application thereof, and particularly relates to a cell membrane coated ultra-small ferroferric oxide nanocluster and a preparation method and application thereof.

Background

Today, cancer has become one of the major health-threatening factors. At present, although many chemotherapy drugs used clinically can inhibit the growth of tumors to a certain extent, the chemotherapy drugs have the defects of large toxic and side effects, low drug utilization rate, large cumulative dosage and the like. However, the nano platform constructed by the nano material loaded with the small molecule anticancer drug has the advantages of targeted delivery of the drug, high drug release amount at a focus part and the like, so that normal tissue damage can be reduced, and the treatment effect can be improved. Ultra-small ferroferric oxide nanoparticles (USIONP) compared to numerous nanocarriers_S) Has the characteristics of good biocompatibility, no tissue damage and the like, and achieves targetingAfter the part, the MR imaging of the target part can be realized, and the loaded chemotherapeutic drug can be controlled and released. Therefore, the method is considered to be an excellent carrier for constructing the diagnosis and treatment integrated nano platform.

Research shows that ferroferric oxide (Fe)₃O₄) The imaging properties of nanoparticles are closely related to their size, when the particle size is<At 5nm, T can be used as MR₁The imaging contrast agent is weighted. When having T₁The aggregation of the nanoparticles with imaging performance to form larger-sized nanoparticles can be used as T₂The imaged contrast agent is weighted. Because the microenvironment of the tumor part is weakly acidic, and the pH around the normal tissue is about 7.4, the difference of the pH can be used for realizing the T of the MR₁/T₂Bimodal imaging and drug delivery, enabling precise imaging and targeted therapy. For example, Li and the like prepare ultra-small ferroferric oxide nanoclusters with pH response performance to be used as T of tumor sites₁/T₂Bimodal MR imaging contrast agents (Fangyuan Li, et al nano lett, 2019,19, 4213-. Liu et al prepared a drug delivery nano-platform with pH-responsive properties, enabling precise dosing at tumor sites (Junjie Liu, et al biomaterials,2016,83, 51-65).

Driven by the continuous development of nanotechnology and biotechnology, the drug delivery, diagnostic imaging and therapeutic modalities in the field of nano-biomedicine have revolutionized. However, current delivery systems for use in vivo still suffer from immune clearance, bioadhesion, and difficulty in targeting to specific sites. Therefore, it is necessary to improve the existing nano-synthesis system, achieve immune evasion of the nano-material and prolong the blood circulation time thereof. Nanomaterials coated by cancer cell membranes have been designed as novel carriers for in vivo drug delivery. Because the surface of the cancer cell membrane has immune evasion protein and homologous targeting protein, immune clearance can be evaded, and accurate imaging and targeted therapy of tumor parts are realized. For example, Xie et al prepared mesoporous silica nanoparticles coated with cell membrane for drug delivery to achieve targeted therapy of tumor sites (Wei Xie, et al ACS Nano,2019,13, 2849-2857).

Ultrasonic Targeted Microbubble Destruction (UTMD) is a technology that promotes the enrichment of drug-loaded nanomaterials around Targeted tissues. Under the action of ultrasonic alternating sound pressure, the microbubbles or the microcapsules injected in advance can generate instant cavitation effect, so that the permeability of cell membranes is increased, non-lethal sound holes which can be opened and closed reversibly are generated, and the nano material enters cells through the sound holes to play a role. The literature reports before the subject group (Lin et al Theransosics, 2018.8(7): p.1923-1939) promote the uptake of tumor cells to the dendrimer nano-carrier loading genes and chemotherapeutic drugs by means of the UTMD technology, thereby improving the tumor treatment effect.

The research on domestic and foreign documents and patents does not find the preparation of cell membrane coated ultra-small ferroferric oxide nanoclusters with pH response performance, the loading of anticancer drug adriamycin (DOX) in the cavity of the ultra-small ferroferric oxide nanoclusters and the combination of ultrasonic targeted microbubble destruction technology (UTMD) for the T in vivo₁/T₂Bimodal MR imaging and chemotherapy.

Disclosure of Invention

The invention aims to solve the technical problem of providing a cell membrane coated ultra-small ferroferric oxide nanocluster and a preparation method and application thereof so as to fill the blank in the prior art.

The invention provides a cell membrane coated ultra-small ferroferric oxide nanocluster, which is obtained by amination of activated ultra-small ferroferric oxide nanoparticles, addition of a coupling agent for reaction, addition of the obtained ferroferric oxide nanocluster into an adriamycin hydrochloride aqueous solution for stirring, mixing of the obtained adriamycin-loaded ferroferric oxide nanocluster with B16 cell membrane suspension or erythrocyte membrane suspension and extrusion.

The invention also provides a preparation method of the cell membrane coated ultra-small ferroferric oxide nanocluster, which comprises the following steps:

(1) dissolving ferric trichloride in solvent, adding sodium citrate, stirring, adding anhydrous sodium acetate, continuously stirring, carrying out solvothermal reaction, cooling, centrifuging, and drying to obtain ultra-small ferroferric oxide nanoparticles USIONP_SWherein ferric trichloride, a solvent, sodium citrate, anhydrous sodium acetateThe ratio is 0.62-0.66 g: 38-42 mL: 0.46-0.51 g: 1.3-1.4 g;

(2) ultrasonically dispersing the ultra-small ferroferric oxide nanoparticles obtained in the step (1) in ultrapure water, activating by EDC and NHS, adding into EDA solution of ethylenediamine (electronic design automation) for reaction, dialyzing, and concentrating to obtain the surface aminated ultra-small ferroferric oxide nanoparticles USIONP_S-NH₂The solution is prepared from 50-60 mg of ultra-small ferroferric oxide nanoparticles, EDC, NHS and ethylenediamine: 145-155 mg: 90-100 mg: 200-220 μ L;

(3) the USIONP in the step (2)_S-NH₂Mixing the solution with precooled DMSO solution, adding a coupling agent, stirring for reaction, dialyzing, and freeze-drying to obtain the ferroferric oxide nanocluster IONC_SWherein USIONP_S-NH₂The proportion of the solution, the coupling agent and the DMSO is 2-4 mL: 8-10 mg: 10-20 mL;

(4) dissolving doxorubicin hydrochloride in ultrapure water, adding the ferroferric oxide nanoclusters obtained in the step (3), stirring, centrifuging, suspending the obtained precipitate in ultrapure water, and freeze-drying to obtain doxorubicin-loaded ferroferric oxide nanocluster IONC_SThe mass ratio of the ferroferric oxide nanoclusters to the doxorubicin hydrochloride is 0.98: 1-1: 1;

(5) centrifuging B16 cells (melanoma cells) or mouse blood cells, adding hypotonic cell lysate into the obtained B16 cell sediment or mouse blood cell sediment, repeatedly freezing and thawing and crushing, extracting B16 cell membranes or erythrocyte membranes by gradient centrifugation, and suspending the obtained sediment in PBS solution to obtain B16 cell membrane suspension or erythrocyte membrane suspension, wherein the ratio of the cells in the B16 cell sediment or mouse blood cell sediment to the hypotonic cell lysate is 10⁷The method comprises the following steps: 2-4 mL;

(6) dissolving the adriamycin-loaded ferroferric oxide nanoclusters in the step (4) into the B16 cell membrane suspension or the erythrocyte membrane suspension in the step (5), then extruding and centrifuging to obtain the cell membrane-coated ultra-small ferroferric oxide nanoclusters, wherein the ratio of the adriamycin-loaded ferroferric oxide nanoclusters to the B16 cell membrane suspension or the erythrocyte membrane suspension is 200 mug: 0.5-1 mL.

The solvent in the step (1) is diethylene glycol.

And (2) stirring for 10min in the step (1) until the sodium citrate is completely dissolved.

And (2) cooling to 50 ℃ before adding anhydrous sodium acetate in the step (1).

The continuous stirring in the step (1) comprises the following steps: stirring for 2h at 70-75 ℃ in air atmosphere until anhydrous sodium acetate is completely dissolved.

In the step (1), the solvothermal reaction temperature is 190-220 ℃, and the solvothermal reaction time is 3-5 hours.

The drying in the step (1) comprises the following steps: vacuum drying at 50-60 deg.C for 6-8 hr.

The activation in step (2) by EDC and NHS is as follows: EDC is added firstly, stirred and reacted for 0.5-1h, NHS is added, stirred and reacted for 1-4h, and carboxyl is activated.

In the step (2), the reaction temperature is room temperature, and the reaction time is 3-5 days.

The precooling of the DMSO solution in the step (3) is as follows: the DMSO solution was pre-cooled to 10-15 ℃.

Before adding the coupling agent in the step (3), carrying out ice bath for 1-3 min; the coupling agent is terephthalaldehyde.

The stirring reaction in the step (3) is as follows: firstly stirring and reacting for 24-48h at 40-60 ℃, and then stirring and reacting for 24-48h at normal temperature.

The dialysis in the step (3) is as follows: selecting a fiber dialysis bag with the molecular weight cut-off of 8000-14000, wherein the dialysis solution is 0.01M NaHCO₃The salt solution has pH of 8-9 and volume of 2L, and water is changed for 6-8 times within 24 h.

The ferroferric oxide nanocluster IONC in the step (3)_SHas pH response performance.

And (4) stirring at room temperature for 4-6 h.

And (4) centrifuging for 3-8 min at 8000 rpm.

The volume fraction of the phenylmethylsulfonyl fluoride PMSF in the hypotonic cell lysate in the step (5) is 10%.

The technological parameters of repeated freeze-thaw crushing in the step (5) are as follows: quick freezing at-20 deg.C, quick thawing at 37 deg.C, and repeating for 3-5 times.

The gradient centrifugation in the step (5) is as follows: the centrifugation temperature is 4 ℃, the centrifugation is firstly carried out for 10-20min by the centrifugal force of 700-850g, the supernatant is left, and then the centrifugation is carried out for 30-40min by the centrifugal force of 14000-16000g, the supernatant is removed, and the precipitate is left.

The concentration of the B16 cell membrane suspension or the erythrocyte membrane suspension in the step (5) is 10⁷ 2X 10 per mL⁷one/mL.

The extrusion in the step (6) is as follows: the solution is extruded 10-14 times using an Avanti micro extruder.

The centrifugation rotating speed in the step (6) is 8000-14000rpm, and the centrifugation time is 30-40 min.

The invention also provides a cell membrane coated ultra-small ferroferric oxide nanocluster prepared by the method₁/T₂Application in bimodal nuclear magnetic resonance imaging contrast and diagnosis and treatment agents for inhibiting tumor cell proliferation.

The invention synthesizes ultra-small ferroferric oxide nano particles (USIONP) with stable surface citric acid by a solvothermal method_S) And modifying the mixture with Ethylenediamine (EDA) to synthesize the surface aminated ultra-small ferroferric oxide nano-particles (USIONP)_S-NH₂). Reacting terephthalaldehyde with the terephthalaldehyde to generate Schiff base sensitive to pH, and synthesizing ferroferric oxide nanocluster (IONC)_S). Finally through electrostatic adsorption at the IONC_SThe cavity and the surface are loaded with chemotherapeutic drug adriamycin (DOX), and the cell membrane is coated to enhance the biocompatibility and the homologous targeting of the nano material, so the nano material is used for T of a tumor model₁/T₂The bimodal MR imaging and the targeted therapy realize the diagnosis and treatment integration of the tumor model.

The invention uses terephthalaldehyde to couple ultra-small ferroferric oxide nano particles into a cluster structure. By controlling coupling agent and ultra-small ferroferric oxide nanoparticles (USIONP)_S) The feeding proportion of the material can prepare the ferroferric oxide nanocluster (IONC) with stable structure and controllable size_S). The antitumor drug adriamycin can be loaded on the cavities and the surfaces of the cluster structures by means of electrostatic adsorption, and the surfaces of the materials are coated by a physical extrusion modeA cell membrane. Successfully prepared ferroferric oxide nanocluster (IONC)_S/DOX @ CCM) shows good pH response performance in vitro, and can realize T under the weak acidic tumor microenvironment condition₁/T₂Bimodal MR imaging; the loaded adriamycin and the surface-coated cell membrane can also realize the targeted treatment of tumors.

The physical and chemical properties of the prepared ferroferric oxide nanocluster are represented by means of Zeta potential and dynamic light scattering analysis (DLS), infrared spectroscopy (FT-IR), ultraviolet-visible spectroscopy (UV-vis), thermogravimetric analysis (TGA), inductively coupled plasma atomic emission spectroscopy (ICP-OES), SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and the like. And the T of the ferroferric oxide nanocluster is determined by an MR imager₁/T₂Bimodal imaging performance, in vitro imaging performance of the material is measured by measuring the change of relaxation performance of the material in solutions with different pH values. And then, evaluating the cytotoxicity of the ferroferric oxide nanocluster by using a CCK-8 method, and detecting the phagocytosis of the material by the cells by using flow cytometry. And finally, establishing a white mouse subcutaneous tumor model for MR imaging and anti-tumor experiments. The specific test results are as follows:

zeta potential and hydrodynamic diameter test results

Taking synthetic USIONP_S、IONC_S、IONC_S/DOX、IONC_SEach 1mg of/DOX @ CMM was diluted to 50. mu.g/mL with ultrapure water, and 50. mu.L of USIONP was sampled_S-NH₂Dissolved in 950. mu.L of ultrapure water for the determination of surface potential and hydrodynamic diameter. As shown in Table 1, USIONP_SHas a potential of-40.3 mV, USIONP_S-NH₂The potential of (a) was-17.2 mV, and the potential was elevated, demonstrating the successful modification of Ethylenediamine (EDA). Coupling USIONP with terephthalaldehyde_S-NH₂The potential is changed to-14.6 mV, the hydrated particle size is increased to 372.6nm, and the ferroferric oxide nanocluster (IONC) is proved_S) The successful synthesis of the compound. When the IONC is_SAfter loading the chemotherapeutic drug DOX, the potential is changed from negative to positive, and the hydrated particle size is increased to 746.2nm, thus proving the successful loading of DOX. By physical squeezing of the IONC_SAfter the surface of the/DOX is coated with cancer cell membrane, the potential is changed10.7nm, hydrated particle size 231.4nm, demonstrating IONC_SSuccessful coating of cell membranes on the surface of DOX. IONC_SThe hydrodynamic diameter of/DOX @ CMM in water, PBS solution and 1640 medium can be constant over a longer period of time (FIG. 2), demonstrating that the IONC_Sthe/DOX @ CMM has good colloidal stability.

2. Infrared (FT-IR) test:

the USIONP prepared by the FT-IR test_S、USIONP_S-NH₂、IONC_SThe characterization was performed, as shown in FIG. 3, curve a is 3400-3500cm^-1The vicinity is a stretching vibration absorption peak of free hydroxyl (-OH), 2882-2987cm^-1The nearby characteristic absorption peak belongs to the stretching vibration absorption peak of methylene in sodium citrate, 1488-1718cm^-1The characteristic absorption peak in the vicinity is attributed to the stretching vibration absorption peak of carbonyl (C ═ O). 1546cm in curve b^-1Near is the stretching vibration absorption peak of carbon-nitrogen bond (C-N), and 1598cm^-1Partial overlapping of absorption peaks of stretching vibration of carbonyl (C ═ O) proves that Ethylenediamine (EDA) is in ultra-small ferroferric oxide nanoparticles (USIONP)_S) Successful modification of the surface. Curve c 1573-^-1Near is a characteristic absorption peak of imine bond (C ═ N), and a fingerprint area is 800cm^-1The vicinity is a characteristic absorption peak of benzene ring para-substituted, 1900cm^-1The frequency doubling peak absorbed by the benzene ring is nearby, and the successful modification of the coupling agent terephthalaldehyde is proved.

3. Thermogravimetric analysis (TGA) test:

the invention is tested by TGA to USIONP_S、USIONP_S-NH₂、IONC_S、IONC_S/DOX、IONC_Sthe/DOX @ CCM was characterized as shown in FIG. 4, ultra-small ferroferric oxide nanoparticles (USIONP)_S) The heat loss of (1) is 22.33%, USIONP_S-NH₂The heat loss of (A) was 25.50%, thereby quantifying the amount of Ethylenediamine (EDA) in USIONP_SThe mass ratio of the above modification was 3.17%. Ferroferric oxide nanoclusters (IONC)_S) The heat loss of the reaction solution was 31.09%, from which terephthalaldehyde was quantitatively determined in the IONC_SThe mass ratio of the components is 5.59 percent. IONC_S/DOX、IONC_Sof/DOX @ CCMThe heat loss was 60.56% and 66.43%, respectively, from which the IONC was quantified_SThe mass proportion of Doxorubicin (DOX) loaded on is 29.47%, compared with that in the IONC_SThe mass ratio of the cell membrane coated on the DOX surface is 5.87%.

SDS-polyacrylamide gel electrophoresis (SDS-PAGE) test:

cell membrane suspension and IONC using BCA protein quantification kit_SThe protein content in/DOX @ CCM was determined and adjusted to 1mg/mL with PBS. Adding 5 μ L of protein Marker into the first protein lane, and respectively collecting 20 μ L of cell membrane suspension and IONC_S(200. mu.g in 1mL PBS) and IONC_Sthe/DOX @ CCM was added to the lane, setting the running current at 100A for 30 min. As shown in FIG. 5, the IONC_Sthe/DOX group did not run out lanes with protein streaks, whereas the IONC_SSimilar protein stripe lanes were run in the/DOX @ CCM group and the cell membrane (CCM) group, demonstrating that the cell membranes are in the IONC_SSuccessful coating of the/DOX surface.

5. Material T₁And T₂And (3) testing relaxation performance:

IONC determination by ICP-OES test method_SThe content of Fe element in the/DOX @ CCM. In order to test the pH response performance of the ferroferric oxide nanocluster, the IONC is adopted_SThe concentration gradient solutions of pH 5.5 and 7.4 were prepared as 2mL each (Fe element concentration 0.1, 0.2, 0.4, 0.8, 1.6mM) in/DOX @ CCM, respectively. Separately determining the IONC_ST of/DOX @ CCM at different pH conditions (pH 5.5, 7.4)₁And T₂The slope of the relaxation time is the relaxation rate (shown in FIGS. 6 and 7) (T)₁Relaxation rate of imaging by r₁Denotes, T₂Relaxation rate of imaging by r₂Representation). Generally, r₂/r₁Less than 5 indicates that the material has a good T₁Imaging performance; if r is₂/r₁If greater than 5, this indicates that the material has a good T₂And (4) imaging performance. At pH 5.5, IONC_SR of/DOX @ CCM₁Is 1.044mM^-1S^-1And r is₂1.854mM^-1S^-1，r₂/r₁Less than 5, and is characterized in that,indicating that under this pH condition, the IONC_Sthe/DOX @ CCM has good T₁Imaging performance; at pH 7.4, IONC_SR of/DOX @ CCM₁0.6404mM^-1S^-1And r is₂4.666mM^-1S^-1，r₂/r₁Greater than 5, indicating that under this pH condition, the IONC is_Sthe/DOX @ CCM has good T₂And (4) imaging performance. The above results are combined to further illustrate that pH is the response mechanism, IONC_Sthe/DOX @ CCM has good T₁/T₂The bimodal MR imaging performance can be used as good T in MRI molecular imaging diagnosis₁/T₂A bimodal contrast agent.

6. In vitro drug release testing:

preparing buffer solutions with pH 7.4 and pH 5.5 respectively, and mixing the prepared IONC_S(DOX and IONC)_SThe solution of/DOX @ CCM was dissolved in 1mL of the above two buffer solutions with different pH to prepare a 1mg/mL solution, which was then placed in a dialysis bag, which was then placed in a container containing 9mL of the above corresponding pH buffer solution and shaken in a constant temperature shaker at 37 ℃. At different time points, 1mL of the dialysate outside the bag was aspirated, the container was replenished with the corresponding pH buffer solution, and the absorbance of the aspirated fluid at 480nm was measured. After the slow release is finished, drawing the IONC_S(DOX and IONC)_SThe drug release profile of/DOX @ CCM at different pH conditions is shown in FIG. 8, IONC_SThe drug release rate of/DOX is 38.85% at pH 5.5 and 19.58% at pH 7.4, the former being higher than the latter, indicating IONC_Sthe/DOX has pH response performance and is more beneficial to the release of the drug under the acidic condition. IONC_S(DOX @ CCM) and IONC_SThe drug release behavior of the/DOX is similar, and the fact that the ferroferric oxide nanocluster (IONC) is not influenced by coating the cell membrane is proved_S) The drug release properties of (1).

7. Cytotoxicity experiments:

evaluation of Free DOX and IONC with B16 cells as cell model_S/DOX、IONC_S/DOX@CCM、IONC_SCytotoxicity of/DOX @ CCM-UTMD. B16 cells were packed at 1X 10⁴Individual cellThe density per well was seeded on 4 96-well plates in 5% CO₂Incubation was carried out at 37 ℃ for 12 hours. Then the medium was changed to contain Free DOX, IONC_S/DOX、IONC_SCulture medium and cells in/DOX @ CCM (DOX concentrations were set to 0. mu.g/mL, 1.6. mu.g/mL, 3.2. mu.g/mL, 6.4. mu.g/mL, 12.8. mu.g/mL, 25.6. mu.g/mL) at 5% CO₂Co-cultivation at 37 ℃ for 24h, for IONC_Sthe/DOX @ CCM-UTMD group, added with 20% (v/v) SonoVue and with IONC_SThe total volume of the fresh medium of/DOX @ CCM was 1mL (the concentrations of DOX in the mixture were 0, 1.6, 3.2, 6.4, 12.8 and 25.6. mu.g/mL), and the concentration was 0.4W/cm²And (3) carrying out ultrasonic treatment for 30s under the condition of 1KHz PRF, and then placing the treated product in an incubator for incubation for 24 h. Subsequently, a solution of 1640 medium (100. mu.L/well) containing 10% (v/v) CCK-8 (10. mu.L) was added and the culture was continued in the incubator for 2-4 hours. And finally, testing the absorbance of each hole at the position with the wavelength of 450nm by using a multifunctional microplate reader, taking the PBS-treated cells as a blank control, and marking the cell activity as 100%. The results are shown in fig. 9, where the cytotoxicity of each group of materials increased gradually with increasing DOX concentration over the range of experimental concentrations. Because of the targeting ability of cell membranes (CCM), the uptake of material by cells can be increased, thus IONC is the same concentration of DOX_S(DOX @ CCM ratio IONC)_Sthe/DOX showed stronger cytotoxicity. At the same time, at the same DOX concentration, the IONC_SCytotoxicity ratio IONC of/DOX @ CCM-UTMD_Sthe/DOX @ CCM is high, and proves that the UTMD technology can further enhance the material uptake of cells, thereby enhancing the IONC_SThe effect of/DOX @ CCM on inhibiting cancer cell proliferation. As can be seen from Table 2, DOX and IONC are free_S/DOX、IONC_S(DOX @ CCM) and IONC_SDOX half-Inhibitory Concentration (IC) after 24h of co-incubation of/DOX @ CCM-UTMD with B16 cells₅₀) 0.376. mu.g/mL, 19.4. mu.g/mL, 7.9. mu.g/mL, 6.7. mu.g/mL, respectively, demonstrate that the IONC is free of DOX under the same experimental operating conditions_Sthe/DOX @ CCM-UTMD showed the strongest killing power on B16 cells.

8. Immune evasion and homology targeting ability test:

evaluation of IONC Using RAW264.7 cells as cell model_SImmune evasion ability of/DOX @ CCM, with B16 cells as cell model to evaluate IONC_SThe homologous targeting ability of/DOX @ CCM. RAW264.7 cells and B16 cells were mixed at 15X 10⁴The density of each cell per well was seeded on 12-well plates in 5% CO₂Incubation was carried out at 37 ℃ for 12 h. Subsequently, the medium was changed to contain IONC_S/DOX,IONC_S/DOX@RBCM,IONC_S/DOX@CCM([Fe]50. mu.g/mL) of medium with cells at 5% CO₂Co-incubation at 37 ℃ and collection of cells in the well plate at different time points (0,1,2,4,8,12h) and digestion with 1mL of aqua regia for 4 h. And finally, measuring the content of the Fe element in the cells by an inductively coupled plasma emission spectrometer (ICP-OES), and taking the cells treated by the PBS as a blank control (marking the content of the Fe element to be 0 pg/cell). As can be seen from FIG. 10(a), at the same time point, RAW264.7 cells are paired with IONC_S(DOX @ CCM) and IONC_SThe amount of phagocytosis of/DOX @ RBCM was comparable but significantly less than for IONC_SPhagocytosis of/DOX, indicating IONC_S(DOX @ CCM) and IONC_Sthe/DOX @ RBCM can escape from phagocytosis of RAW264.7 cells and has immune evasion capability. As can be seen from FIG. 10(B), at the same time point, B16 cells were paired with IONC_SThe phagocytic amount of/DOX @ CCM is obviously higher than that of IONC_S/DOX，IONC_Sthe/DOX @ RBCM shows that the cancer cell membrane (B16 cell membrane) can increase the IONC pair of B16 cells_SPhagocytosis of/DOX @ CCM, conferring its cognate targeting ability.

9. Cell phagocytosis assay:

evaluation of cell pairs Free DOX and IONC with B16 cells as cell model_S/DOX、IONC_S/DOX@CCM、IONC_S(ii) phagocytic potency of/DOX @ CCM-UTMD. B16 cells were packed at 15X 10⁴The density of each cell per well was seeded on 12-well plates in 5% CO₂Incubation was carried out at 37 ℃ for 12 h. Then the medium was changed to contain Free DOX, IONC_S/DOX、IONC_SCulture medium and cells in/DOX @ CCM (concentrations of DOX are set to 0.5. mu.g/mL, 1.0. mu.g/mL, 2.0. mu.g/mL) at 5% CO₂Co-cultivation at 37 ℃ for 6h, for IONC_Sthe/DOX @ CCM-UTMD group, added with 20% (v/v) SonoVue and with IONC_S1mL (DO) of fresh culture medium of/DOX @ CCMThe concentrations of X in the mixed solution were 0.5, 1.0, 2.0. mu.g/mL), respectively, at 0.4W/cm²Ultrasonic treatment is carried out for 30s under the condition of 1KHz PRF, and the treated product is placed in an incubator for 6 h. Cells from all plates were then digested, centrifuged, collected and the fluorescence intensity of the cell samples was measured by flow cytometry. As can be seen in FIG. 11, the fluorescence intensity of each group of cells increased with increasing concentration of DOX. By contrast of IONC_S(DOX and IONC)_SThe cell fluorescence intensity of the/DOX @ CCM group can be seen, under the same conditions, the IONC_Sthe/DOX @ CCM panel showed higher fluorescence intensity, indicating that the targeting ability of the cell membrane (CCM) can increase phagocytosis of the material by cells; under the same conditions, the IONC_S/DOX @ CCM-UTMD group ratio IONC_Sthe/DOX @ CCM panel shows a stronger fluorescence signal intensity, which may be caused by the sonoporation effect caused by UTMD.

10. In vivo tumor MR imaging results:

the prepared USIONP_S、IONC_S/DOX、IONC_S/DOX@CCM、IONC_Sthe/DOX @ CCM-UTMD was prepared as a PBS solution with a Fe concentration of 500. mu.g/mL. 2 x 10 to⁶B16 cells were inoculated into the medial thigh of a white mouse until the tumor volume reached 200mm³On the left and right, groups of materials were injected (200. mu.L) via the tail vein of mice. The tumor sites of the tumor-bearing mice at different time points (0, 15, 25, 45 and 75min) after the injection of the material are scanned by a nuclear magnetic resonance imager for MR imaging, and the T is evaluated₁/T₂Bimodal MR contrast effect (see fig. 12). After 15min of injection, IONC_STumor sites of the/DOX @ CCM group were darkened, T₂The MR signal reaches a minimum; after 25min of injection, the tumor site becomes bright and T₁The MR signal reaches a peak value, which indicates that the slightly acidic (pH about 5.5) environment of the tumor site can cause cluster-structured ferroferric oxide nanoparticles (IONC)_S) Degradation occurs, thereby realizing dynamic T of the tumor part₁/T₂Bimodal accurate MR imaging. For IONC_Sthe/DOX @ CCM-UTMD group in the injection IONC_Safter/DOX @ CCM, SonoVue (1.18mg/mL,0.2mL of physiological saline) was injected via tail vein and sonicated at the tumor site for 2min (1MHz, 0.4W/cm)²20% microvesicles) observed, upon injectionIONC_S15min after/DOX @ CCM, T at tumor site₂The MR signal value became the lowest, and at 25min, the tumor site T₁The MR signal value reaches the peak value, which shows that UTMD can enhance IONC_SMR imaging performance of/DOX @ CCM.

11. Tissue distribution:

to study the distribution and metabolism of nanomaterials in various organs and tissues of the body, the group of nanomaterials (USIONP) prepared in example 1 was used_S、IONC_S/DOX、IONC_S/DOX@CCM、IONC_S/DOX @ CCM-UTMD) was prepared as a PBS solution with a Fe concentration of 500. mu.g/mL. 2 x 10 to⁶B16 cells were inoculated into the medial thigh of a white mouse until the tumor volume reached 200mm³On the left and right, each group of materials was injected (200. mu.L) via the tail vein of mice. Tumor bearing mice were sacrificed and dissected at different time points (1,12,24h), and using tumor bearing mice injected with PBS (200. mu.L) into tail vein as blank control, the heart, liver, spleen, lung, and kidney were removed, weighed, cut into 2X 2mm fragments, and digested with aqua regia for 24 h. Meanwhile, tumor-bearing mice were sacrificed at different time points (15min,25min,45min,12h,24h) and dissected, tumor-bearing mice injected with PBS (200 μ L) into tail vein were used as blank control, tumors were taken out and weighed, cut into 2 × 2mm pieces, and then digested with aqua regia for 24 h. Finally, the content of iron element in each sample was measured by ICP-OES, and the content of iron element in each organ and tumor was calculated (fig. 13). As can be seen from FIGS. 13(a-d), the iron content of each organ (heart, liver, spleen, lung, kidney) was restored to the pre-injection level 24 hours after the tail vein injection of each group of materials, and these results demonstrate that each group of materials (USIONP) prepared in example 1_S、IONC_S/DOX、IONC_S/DOX@CCM、IONC_S/DOX @ CCM-UTMD) can be normally eliminated in the body of the mouse. As can be seen from FIG. 13(e), each group of materials (USIONP) was injected in the tail vein_S、IONC_S/DOX、IONC_S/DOX@CCM、IONC_SThe Fe content of a tumor part is obviously increased after 15min of/DOX @ CCM-UTMD), the Fe content reaches a peak value after about 25min, and material retention caused by an EPR effect is possible; after 45min, the Fe content decreased, probably due to the cluster structure of the IONC_SDegradation into individual USIONPs_SAnd is transported to other organs and tissues of the mouse through blood circulation.

12. Tumor treatment results:

2 x 10 to⁶B16 cells were inoculated into the medial thigh of a white mouse until the tumor volume reached 200mm³On the left and right, mice were randomly divided into 5 groups (5 mice per group) as follows: control group (PBS,100 μ L); free DOX ([ DOX ]]＝5mg/kg,100μL)；IONC_S/DOX([DOX]＝5mg/kg,100μL)；IONC_S/DOX@CCM([DOX]5mg/kg,100 μ L) and then the respective materials were injected into mice through the tail vein. For IONC_SThe group of/DOX @ CCM-UTMD was injected into tail vein with IONC_Safter/DOX @ CCM, SonoVue (1.18mg/mL,0.2mL of physiological saline) was injected via tail vein and sonicated at the tumor site for 2min (1MHz, 0.4W/cm)²20% microbubbles). The day of treatment initiation was recorded as day 0, once every 4 days, and mouse body weight and tumor size were recorded every 2 days. As can be seen from FIG. 14(a), free DOX, IONC are relative to the PBS group_S/DOX、IONC_S/DOX@CCM、IONC_Sthe/DOX @ CCM-UTMD shows a certain anti-tumor effect. Under the same conditions, IONC compared to free DOX group_SThe better antitumor effect of/DOX is probably due to IONC_Sthe/DOX has better EPR effect and can effectively reach the interior of the tumor, which indicates that the IONC is_SCan be used as a good drug carrier. Under the same conditions, the IONC_SThe anti-tumor effect of the/DOX @ CCM is superior to that of the IONC_Sthe/DOX, probably due to the presence of immune evasion proteins and cognate targeting proteins on the cell membrane surface, can prolong the blood circulation time of the material and enrich it at the tumor site. Under the same conditions, the IONC_SThe treatment effect of the/DOX @ CCM-UTMD is better than that of the IONC_Sthe/DOX @ CCM shows that UTMD can play a role in assisting in enhancing the effect of chemotherapy. As can be seen from fig. 14(b), the body weight of the mice in the free DOX group was significantly reduced, and the body weight of the mice in the other groups was not significantly changed, indicating that free DOX may cause toxic and side effects to the body of the mice.

Advantageous effects

(1) The ferroferric oxide nanocluster (IONC) prepared by the invention_S) In thatAnd the pH response performance is good in vitro. Ferroferric oxide (IONC) with cluster structure in tumor microenvironment (weak acidity, pH about 5.5)_S) Will disperse into single ferroferric oxide (USIONP)_S) Nanoparticles of a material consisting of T₂Conversion of imaging to T₁Imaging, effecting T of material₁/T₂Bimodal MR imaging.

(2) The IONC prepared by the invention_SAfter the/DOX @ CCM enters a mouse body through tail vein injection, T can be realized₁/T₂The double-mode MR imaging conversion can also play a role in resisting tumors, and the chemotherapy effect can be further enhanced by using a UTMD auxiliary means, so that the method has potential clinical application value.

Drawings

FIG. 1 shows the nano-material IONC of the present invention_SA synthetic scheme of/DOX @ CCM;

FIG. 2 is an IONC prepared in accordance with the present invention_SGraph of hydrodynamic diameter over time in aqueous solutions at pH 7.4(a) and pH 5.5(b), in PBS and in 1640 medium,;

FIG. 3 shows USIONP prepared by the present invention_S、USIONP_S-NH₂、IONC_SAn infrared spectrum of (1);

FIG. 4 shows USIONP prepared by the present invention_S、USIONP_S-NH₂、IONC_S、IONC_S(DOX and IONC)_SThermogravimetric analysis (TGA) profile of/DOX @ CCM;

FIG. 5 shows a cell membrane suspension, IONC, prepared according to the present invention_S(DOX and IONC)_SSDS-polyacrylamide gel electrophoresis (SDS-PAGE) of/DOX @ CCM;

FIG. 6 is an IONC made in accordance with the present invention_ST of/DOX @ CCM at pH 5.5 and pH 7.4₁Relaxation Rate (r)₁)；

FIG. 7 is an IONC made in accordance with the present invention_ST of/DOX @ CCM at pH 5.5 and pH 7.4₂Relaxation Rate (r)₂)；

FIG. 8 is an IONC made in accordance with the present invention_S(DOX and IONC)_S(pH 5) under different conditionsThe drug release kinetics profile of 5 or 7.4);

FIG. 9 is a DOX, IONC free_S/DOX、IONC_S(DOX @ CCM) and IONC_SCell viability map after 24h of co-incubation of/DOX @ CCM-UTMD with B16 cells;

FIG. 10 is an IONC made in accordance with the present invention_S/DOX、IONC_S(DOX @ RBCM and IONC)_SFe elemental measurement analysis plot after incubation of/DOX @ CCM with RAW264.7 cells (a) and B16 cells (B), respectively, for different time periods;

FIG. 11 is an IONC made in accordance with the present invention_S/DOX、IONC_S(DOX @ CCM) and IONC_SFlow cytometric analysis plot after 6h of co-incubation of/DOX @ CCM-UTMD with B16 cells;

FIG. 12 is an IONC made in accordance with the present invention_S(DOX @ CCM) and IONC_ST of mouse tumor at different time points before and after tail vein injection of/DOX @ CCM-UTMD₁/T₂A bimodal MR imaging map (a), a map (b) of the MR signal-to-noise ratio variation of the corresponding tumor site;

FIG. 13 shows USIONP injected via tail vein in accordance with the present invention_S、IONC_S/DOX、IONC_S(DOX @ CCM) and IONC_SDistribution of nanoparticles in organs (a-d) and tumors (e) in mice after different time points of/DOX @ CCM-UTMD (combined with UTMD assistance);

FIG. 14 shows the present invention injected via tail vein with PBS, free DOX, and IONC_S/DOX、IONC_S(DOX @ CCM) and IONC_SThe tumor was treated with/DOX @ CCM-UTMD (in combination with UTMD adjuvant), and the tumor volume change (a) and body weight change (b) were recorded over 12 days.

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.

Unless otherwise specified, all chemical reagents were commercially available and used without further purification. Iron (III) chloride was purchased from Adamas reagents ltd. Sodium citrate, anhydrous sodium acetate, ethylene diamine and other reagents were purchased from national drug-regulated chemical reagents, ltd (shanghai, china). 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC. HCl) and N-hydroxysuccinimide (NHS) were purchased from GL Biochem (Shanghai, China). Terephthalaldehyde was purchased from alatin industries, inc (shanghai, china). Doxorubicin (DOX) was purchased from beijing huafeng pharmaceutical limited (beijing, china). Cell lysis buffers for bicinchoninic acid (BCA) assay kit, phenylmethylsulfonyl fluoride (PMSF), Western and IP were purchased from Beyotime (shanghai, china). SDS sample buffer and SDS-polyacrylamide gels were purchased from Tanon Science & Technology co., Ltd. (shanghai, china). Avanti extruders are available from Avanti Polar Lipids, Inc. B16 cells (murine melanoma cell line) and raw264.7 cells (mouse macrophage line) were from the institute of biochemistry and cell biology, chinese academy of sciences. RPMI-1640 medium (1640 medium, GIBCO, Invitrogen, Carlsbad, CA), fetal bovine serum (FBS, GIBCO), penicillin-streptomycin (HyClone, Thermo Scientific, Logan, UT) and trypsin 0.25% solution (HyClone) were purchased from Gino biomedical technologies, Inc., Hangzhou (Hangzhou, China). Cell Counting Kit-8(CCK-8) was from 7Sea Biotech Co., Ltd. (Shanghai, China). Mice about 4-6 weeks old were purchased from the Shanghai Slac center for laboratory animals (Shanghai, China). The water used in all experiments with a resistivity higher than 18.2 M.OMEGA.cm was purified by a laboratory water purification system (Cascada I, PALL, Beijing, China). Regenerated cellulose dialysis membranes with molecular weight cut-offs (MWCO) of 8,000 and 10,000 were purchased from Fisher (pittsburgh, pa).

Example 1

(1) 0.6488g of ferric chloride was dissolved in 40mL of diethylene glycol (also known as diethylene glycol, DEG), and 0.47g of sodium citrate (Na)₃Cit) is dissolved in the solution, and is stirred for 1h at 80 ℃ under the air atmosphere, after sodium citrate is completely dissolved, 1.312g of anhydrous sodium acetate is added into the solution, the stirring is continued until sodium acetate powder is completely dissolved, and then the solution is transferred into a 50mL high-pressure reaction kettle and is reacted for 4h at 200 ℃; after the reaction is finished, naturally cooling to room temperature,transferring the product into a 50mL centrifuge tube, centrifuging at the rotating speed of 8500rpm for 15min, discarding the supernatant, redissolving the precipitate with ultrapure water, centrifuging at the rotating speed of 8500rpm for 15min, repeating the operation for 3 times, and drying the precipitate in a vacuum drying oven at the temperature of 60 ℃ to obtain the ultra-small Fe with stable surface sodium citrate₃O₄Nanoparticles (USIONP)_S)。

(2) Taking 56mg USIONP_SDissolving in 16mL ultrapure water, stirring, adding EDC (150mg,1mL ultrapure water) dropwise, 30min later, adding NHS (95mg,1mL ultrapure water) dropwise to activate USIONP_SCarboxyl group on 3 h. Then, the solution is rapidly added into an aqueous solution (2mL) containing ethylenediamine (217 μ L), reacted for 3 days, and dialyzed in ultrapure water for 3 days by using a fiber membrane dialysis bag with molecular weight cutoff of 10000, thus obtaining USIONP_S-NH₂And (3) solution.

(3) Precooling 15mL of DMSO to 10-15 ℃, and taking 3mL of USIONP_S-NH₂Dissolving the solution in water, ice-bathing for 3min, adding 9mg of terephthalaldehyde, stirring at 40 deg.C for 24 hr, and stirring at room temperature for 24 hr to allow full reaction. After the reaction was complete, the reaction was quenched with 2L of 0.01M NaHCO₃(pH 8-9) dialyzing in salt solution (molecular weight cut-off of 8000-_S)。

(4) 5mg of doxorubicin hydrochloride was dissolved in 10mL of ultrapure water, and 5mg of IONC was added to the above solution_SStirring for 4h at room temperature, centrifuging (8000rpm,5min), collecting precipitate, suspending the precipitate in 2mL of ultrapure water, and freeze-drying to obtain doxorubicin-loaded ferroferric oxide nanocluster (IONC)_S(DOX). Meanwhile, the absorbance of the supernatant at 480nm was measured by ultraviolet to calculate the drug loading rate to be 48.23% and the encapsulation rate to be 93.16%.

(5) Taking B16 cells 10 in logarithmic growth phase⁷Centrifuging (1000rpm,5min) to obtain cell precipitate, washing with sterile PBS solution, adding 3mL of hypotonic cell lysis buffer solution (containing 10% volume fraction) PMSF into the precipitate, ice-bathing for 15min, and repeatedly freezing and thawing by freeze-thaw method (quick freezing at-20 deg.C and quick freezing at-37 deg.C)Snap) 3 times. Setting the centrifugal temperature at 4 ℃, centrifuging for 15min at a centrifugal force of 850g, and removing precipitates; and centrifuging the supernatant for 30min by using a centrifugal force of 16000g, reserving the precipitate, and suspending the precipitate in 1mL of PBS (phosphate buffer solution) to obtain a suspension of B16 cell membranes.

(6) 200 ug of IONC_SThe IONC is prepared by dissolving/DOX in 1mL cell membrane suspension, extruding the solution 11 times with Avanti micro extruder, centrifuging (10000rpm,30min), removing excessive cell membrane vesicles_S/DOX@CCM。

Example 2

Take USIONP synthesized in example 1_S、IONC_S、IONC_S/DOX、IONC_SEach 1mg of/DOX @ CMM was diluted to 50. mu.g/mL with ultrapure water, and 50. mu.L of USIONP was sampled_S-NH₂Dissolved in 950. mu.L of ultrapure water for the determination of surface potential and hydrodynamic diameter. As shown in Table 1, USIONP_SHas a potential of-40.3 mV, USIONP_S-NH₂The potential of (a) was-17.2 mV, and the potential was elevated, demonstrating the successful modification of Ethylenediamine (EDA). Coupling USIONP with terephthalaldehyde_S-NH₂The potential is changed to-14.6 mV, the hydrated particle size is increased to 372.6nm, and the ferroferric oxide nanocluster (IONC) is proved_S) The successful synthesis of the compound. When the IONC is_SAfter loading the chemotherapeutic drug DOX, the potential is changed from negative to positive, and the hydrated particle size is increased to 746.2nm, thus proving the successful loading of DOX. By physical squeezing of the IONC_SAfter the surface of the/DOX is coated with cancer cell membranes, the potential is changed to-10.7 nm, the hydrated particle size is changed to 231.4nm, and the IONC is proved_SSuccessful coating of cell membranes on the surface of DOX.

IONC_SThe hydrodynamic diameter of/DOX @ CMM in water, PBS solution and 1640 medium can be constant over a longer period of time (FIG. 2), demonstrating that the IONC_Sthe/DOX @ CMM has good colloidal stability.

TABLE 1

Sample (I)	Surface potential (mV)	Hydrodynamic diameter (nm)	Polydispersity index (PDI)
				USIONP_S	-40.3±0.929	60.7±1.421	0.374±0.03
USIONP_S-NH₂	-17.2±1.276	80.23±1.42	0.25±0.044
				IONC_S	-14.6±0.448	372.6±7.53	0.233±0.027
IONC_S/DOX	3.82±0.446	746.2±3.59	0.222±0.092
				IONC_S/DOX@CCM	-10.7±0.493	231.4±3.579	0.448±0.037

Example 3

The USIONP prepared in example 1 was taken_S、USIONP_S-NH₂、IONC_SFT-IR characterization was performed, as shown in FIG. 3, curve a at 3400-^-1The vicinity is a stretching vibration absorption peak of free hydroxyl (-OH), 2882-2987cm^-1The nearby characteristic absorption peak belongs to the stretching vibration absorption peak of methylene in sodium citrate, 1488-1718cm^-1The characteristic absorption peak in the vicinity is attributed to the stretching vibration absorption peak of carbonyl (C ═ O). 1546cm in curve b^-1Near is the stretching vibration absorption peak of carbon-nitrogen bond (C-N), and 1598cm^-1Partial overlapping of absorption peaks of stretching vibration of carbonyl (C ═ O) proves that Ethylenediamine (EDA) is in ultra-small ferroferric oxide nanoparticles (USIONP)_S) Successful modification of the surface. Curve c 1573-^-1Near is a characteristic absorption peak of imine bond (C ═ N), and a fingerprint area is 800cm^-1The vicinity is a characteristic absorption peak of benzene ring para-substituted, 1900cm^-1The frequency doubling peak absorbed by the benzene ring is nearby, and the successful modification of the coupling agent terephthalaldehyde is proved.

Example 4

The USIONP prepared in example 1 was taken_S、USIONP_S-NH₂、IONC_S、IONC_S/DOX、IONC_STGA characterization was performed on/DOX @ CCM, shown in FIG. 4, for ultra-small ferroferric oxide nanoparticles (USIONP)_S) The heat loss of (1) is 22.33%, USIONP_S-NH₂The heat loss of (A) was 25.50%, thereby quantifying the amount of Ethylenediamine (EDA) in USIONP_SThe mass ratio of the above modification was 3.17%. Ferroferric oxide nanoclusters (IONC)_S) The heat loss of the reaction solution was 31.09%, from which terephthalaldehyde was quantitatively determined in the IONC_SThe mass ratio of the components is 5.59 percent. IONC_S/DOX、IONC_SThe heat loss of the/DOX @ CCM was 60.56% and 66.43%, respectively, from which the IONC was quantified_SThe mass proportion of Doxorubicin (DOX) loaded on is 29.47%, compared with that in the IONC_SThe mass ratio of the cell membrane coated on the DOX surface is 5.87%.

Example 5

Cell membrane suspension and IONC from example 1 were quantitated using BCA protein quantification kit_SThe protein content of/DOX @ CCM was determined and adjusted to 1mg/mL with PBS. Adding 5 μ L of protein Marker into the first protein lane, and respectively collecting 20 μ L of cell membrane suspension and IONC_S(200. mu.g in 1mL PBS) and IONC_Sthe/DOX @ CCM (200. mu.g in 1mL PBS) was added to the lane and the gel run current was set at 100A for 30 min. As shown in FIG. 5, the IONC_Sthe/DOX group did not run out lanes with protein streaks, whereas the IONC_SSimilar protein stripe lanes were run in the/DOX @ CCM group and the cell membrane (CCM) group, demonstrating that the cell membranes are in the IONC_SSuccessful coating of the/DOX surface.

Example 6

IONC determination by ICP-OES test method_SThe content of Fe element in the/DOX @ CCM. In order to test the pH response performance of the ferroferric oxide nanocluster, the IONC is adopted_SThe concentration gradient solutions of pH 5.5 and 7.4 were prepared as 2mL each (Fe element concentration 0.1, 0.2, 0.4, 0.8, 1.6mM) in/DOX @ CCM, respectively. Separately determining the IONC_ST of/DOX @ CCM at different pH conditions (pH 5.5, 7.4)₁And T₂The slope of the relaxation time is the relaxation rate (shown in FIGS. 6 and 7) (T)₁Relaxation rate of imaging by r₁Denotes, T₂Relaxation rate of imaging by r₂Representation). Generally, r₂/r₁Less than 5 indicates that the material has a good T₁Imaging performance; if r is₂/r₁If greater than 5, this indicates that the material has a good T₂And (4) imaging performance. At pH 5.5, IONC_SR of/DOX @ CCM₁Is 1.044mM^-1S^-1And r is₂1.854mM^-1S^-1，r₂/r₁Less than 5, indicating that under this pH condition, the IONC is_Sthe/DOX @ CCM has good T₁Imaging performance; at pH 7.4, IONC_SR of/DOX @ CCM₁0.6404mM^-1S^-1And r is₂4.666mM^-1S^-1，r₂/r₁Greater than 5, indicating that under this pH condition, the IONC is_Sthe/DOX @ CCM has good T₂And (4) imaging performance. The above results are combined to further illustrate that pH is the response mechanism, IONC_Sthe/DOX @ CCM has good T₁/T₂The bimodal MR imaging performance can be used as good T in MRI molecular imaging diagnosis₁/T₂A bimodal contrast agent.

Example 7

Buffer solutions (PBS, i.e., phosphate buffer solutions) at pH 7.4 and pH 5.5 were prepared, respectively, and the prepared iocc was used_S(DOX and IONC)_SThe solution of/DOX @ CCM is dissolved in 1mL of buffer solution with different pH respectively to prepare 1mg/mL solution, and then the solution is placed in a dialysis bag, and then the dialysis bag is placed in a container containing 9mL of buffer solution with different pH and placed in a constant temperature shaking table at 37 ℃ for shaking. At different time points, 1mL of the external fluid of the dialysis bag was aspirated, the corresponding pH buffer solution was replenished into the container, and the absorbance at 480nm of the withdrawn fluid was measured. After the slow release is finished, drawing the IONC_S(DOX and IONC)_SThe drug release profiles of/DOX @ CCM at different pH conditions are shown in FIG. 8. IONC_SThe drug release rate of/DOX is 38.85% at pH 5.5 and 19.58% at pH 7.4, the former being higher than the latter, indicating IONC_Sthe/DOX has pH response performance and is more beneficial to the release of the drug under the acidic condition.

IONC_S(DOX @ CCM) and IONC_SThe drug release behavior of the/DOX is similar, and the fact that the coating does not influence the ferroferric oxide nanocluster (IONC) is proved_S) The drug release properties of (1).

Example 8

Evaluation of free DOX and IONC with B16 cells as a cell model_S/DOX、IONC_S/DOX@CCM、IONC_SCytotoxicity of/DOX @ CCM-UTMD. B16 cells were packed at 1X 10⁴The density of each cell per well was seeded on a cell culture plate of 4 96-well plates and placed in 5% CO₂Incubation was carried out at 37 ℃ for 12 hours. Then the medium is changed to contain free DOX, IONC_S/DOX、IONC_S(DOX @ CCM) (concentrations of DOX are set to 0. mu.g/mL and 1.6. mu.g/mL respectivelyg/mL, 3.2. mu.g/mL, 6.4. mu.g/mL, 12.8. mu.g/mL, 25.6. mu.g/mL) of medium and cells in 5% CO₂Co-cultivation at 37 ℃ for 24h, for IONC_Sthe/DOX @ CCM-UTMD group, added with 20% (v/v) SonoVue and with IONC_SThe total volume of the fresh medium of/DOX @ CCM was 1mL (the concentrations of DOX in the mixture were 0, 1.6, 3.2, 6.4, 12.8 and 25.6. mu.g/mL), and the concentration was 0.4W/cm²And (3) carrying out ultrasonic treatment for 30s under the condition of 1KHz PRF, and then placing the treated product in an incubator for incubation for 24 h. Subsequently, a solution of 1640 medium (100. mu.L/well) containing 10% (v/v) CCK-8 (10. mu.L) was added, and the culture was continued in the incubator for 4 hours. And finally, testing the absorbance of each hole at the position with the wavelength of 450nm by using a multifunctional microplate reader, taking the PBS-treated cells as a blank control, and marking the cell activity as 100%. The results are shown in fig. 9, where the cytotoxicity of each group of materials increased gradually with increasing DOX concentration over the range of experimental concentrations. Because of the targeting ability of cell membranes (CCM), the uptake of material by cells can be increased, thus IONC is the same concentration of DOX_S(DOX @ CCM ratio IONC)_Sthe/DOX showed stronger cytotoxicity. At the same time, at the same DOX concentration, the IONC_SCytotoxicity ratio IONC of/DOX @ CCM-UTMD_Sthe/DOX @ CCM is high, and proves that the UTMD technology has enhanced IONC_SThe effect of/DOX @ CCM on inhibiting cancer cell proliferation. As can be seen from Table 2, DOX and IONC are free_S/DOX、IONC_S(DOX @ CCM) and IONC_SDOX half-Inhibitory Concentration (IC) after 24h of co-incubation of/DOX @ CCM-UTMD with B16 cells₅₀) 0.376. mu.g/mL, 19.4. mu.g/mL, 7.9. mu.g/mL, 6.7. mu.g/mL, respectively, demonstrate that the IONC is free of DOX under the same experimental operating conditions_Sthe/DOX @ CCM-UTMD showed the strongest killing power on B16 cells.

TABLE 2

Sample (I)	IC₅₀(μg/mL)
		Free DOX	0.376
IONC_S/DOX	19.4
		IONC_S/DOX@CCM	7.9
IONC_S/DOX@CCM-UTMD	6.7

Example 9

Evaluation of IONC Using RAW264.7 cells as cell model_SImmune evasion ability of/DOX @ CCM, and IONC was evaluated using B16 cells as a cell model_SThe homologous targeting ability of/DOX @ CCM. RAW264.7 cells and B16 cells were mixed at 15X 10⁴The density of each cell per well was seeded on 12-well plates in 5% CO₂Incubation was carried out at 37 ℃ for 12 h. Subsequently, the medium was changed to contain IONC_S/DOX,IONC_S/DOX@RBCM,IONC_S/DOX@CCM([Fe]50. mu.g/mL) of medium with cells at 5% CO₂Co-incubation at 37 ℃ and collection of cells in the well plate at different time points (0,1,2,4,8,12h) and digestion with 1mL of aqua regia for 4 h. And finally, measuring the content of the Fe element in the cells by an inductively coupled plasma emission spectrometer (ICP-OES), and taking the cells treated by the PBS as a blank control (marking the content of the Fe element to be 0 pg/cell). As can be seen from FIG. 10(a), at the same time point, RAW264.7 cells are paired with IONC_S(DOX @ CCM) and IONC_SThe amount of phagocytosis of/DOX @ RBCM was comparable but significantly less than for IONC_SPhagocytosis of/DOX, indicating IONC_S(DOX @ CCM) and IONC_Sthe/DOX @ RBCM can escape from phagocytosis of RAW264.7 cells and has immune evasion capability. As can be seen from FIG. 10(B), at the same time point, B16 cells were paired with IONC_SThe phagocytic amount of/DOX @ CCM is obviously higher than that of IONC_S/DOX，IONC_Sthe/DOX @ RBCM shows that the cancer cell membrane (B16 cell membrane) can increase the IONC pair of B16 cells_SPhagocytosis of/DOX @ CCM, conferring its cognate targeting ability.

Example 10

Evaluation of cell pairs Free DOX and IONC with B16 cells as cell model_S/DOX、IONC_S/DOX@CCM、IONC_S(ii) phagocytic potency of/DOX @ CCM-UTMD. B16 cells were packed at 15X 10⁴The density of each cell per well was seeded on 12-well plates in 5% CO₂Incubation was carried out at 37 ℃ for 12 h. Then the medium is changed to contain free DOX, IONC_S/DOX、IONC_SCulture medium and cells in/DOX @ CCM (concentrations of DOX are set to 0.5. mu.g/mL, 1.0. mu.g/mL, 2.0. mu.g/mL) at 5% CO₂Co-cultivation at 37 ℃ for 6h, for IONC_Sthe/DOX @ CCM-UTMD group, added with 20% (v/v) SonoVue and with IONC_SThe total volume of the fresh medium of/DOX @ CCM was 1mL (the concentrations of DOX in the mixture were 0.5, 1.0, and 2.0. mu.g/mL), and the concentration was 0.4W/cm²Ultrasonic treatment is carried out for 30s under the condition of 1KHz PRF, and the treated product is placed in an incubator for 6 h. Cells from all plates were then digested, centrifuged, collected and the fluorescence intensity of the cell samples was measured by flow cytometry. As can be seen in FIG. 11, the fluorescence intensity of each group of cells increased with increasing concentration of DOX. By contrast of IONC_S(DOX and IONC)_SThe cell fluorescence intensity of the/DOX @ CCM group can be seen, under the same conditions, the IONC_Sthe/DOX @ CCM panel showed higher fluorescence intensity, indicating that the targeting ability of the cell membrane (CCM) can increase phagocytosis of the material by cells; under the same conditions, the IONC_S/DOX @ CCM-UTMD group ratio IONC_Sthe/DOX @ CCM panel shows a stronger fluorescence signal intensity, which may be caused by the sonoporation effect caused by UTMD.

Example 11

USIONP prepared in example 1_S、IONC_S/DOX、IONC_S/DOX@CCM、IONC_Sthe/DOX @ CCM-UTMD was prepared as a PBS solution with a Fe concentration of 500. mu.g/mL. 2 x 10 to⁶B16 cells were inoculated into the medial thigh of white miceThe tumor volume reaches 200mm³On the left and right, groups of materials were injected (200. mu.L) via the tail vein of mice. The T of the tumor-bearing mice is evaluated by scanning the tumor sites of the tumor-bearing mice at different time points (0, 15, 25, 45 and 75min) after the injection of the materials through an MRI (magnetic resonance imaging) instrument₁/T₂Bimodal MR contrast effect (see fig. 12). After 15min of injection, IONC_ST of/DOX @ CCM set₂The MR signal becomes dark at the tumor part, and the signal reaches the minimum value; after 25min of injection, the tumor site becomes bright and T₁The MR signal reaches a peak value, which indicates that the slightly acidic (pH about 5.5) environment of the tumor site can cause cluster-structured ferroferric oxide nanoparticles (IONC)_S) Degradation occurs to realize T of tumor₁/T₂Bimodal accurate MR imaging. For IONC_Sthe/DOX @ CCM-UTMD group in the injection IONC_Safter/DOX @ CCM, SonoVue (1.18mg/mL,0.2mL of physiological saline) was injected via tail vein and sonicated at the tumor site for 2min (1MHz, 0.4W/cm)²20% microbubbles). It can be observed that IONC is injected_S15min after/DOX @ CCM, T at tumor site₂The MR signal value was lowest, and at 25min, the tumor site T₁The MR signal value reaches the peak value, which shows that UTMD can enhance IONC_SMR imaging performance of/DOX @ CCM.

Example 12

To study the distribution and metabolism of nanomaterials in various organs and tissues of the body, the group of nanomaterials (USIONP) prepared in example 1 was used_S、IONC_S/DOX、IONC_S/DOX@CCM、IONC_S/DOX @ CCM-UTMD) was prepared as a PBS solution with a Fe concentration of 500. mu.g/mL. 2 x 10 to⁶B16 cells were inoculated into the medial thigh of a white mouse until the tumor volume reached 200mm³On the left and right, each group of materials was injected (200. mu.L) via the tail vein of mice. Tumor bearing mice were sacrificed and dissected at different time points (1,12,24h), and using tumor bearing mice injected with PBS (200. mu.L) into tail vein as blank control, the heart, liver, spleen, lung, and kidney were removed, weighed, cut into 2X 2mm fragments, and digested with aqua regia for 24 h. At the same time, tumor-bearing mice were sacrificed at time points (15min,25min,45min,12h,24h) and dissected, with tumor-bearing mice injected with PBS (200. mu.L) into tail vein as blank control, tumors were removed and weighed, and cut into 2X 2mm piecesThe pieces were then digested with aqua regia for 24 h. Finally, the content of iron element in each sample was measured by ICP-OES, and the content of iron element in each organ and tumor was calculated (fig. 13). As can be seen from FIGS. 13(a-d), the iron content of each organ (heart, liver, spleen, lung, kidney) was restored to the pre-injection level 24 hours after the tail vein injection of each group of materials, and these results demonstrate that each group of materials (USIONP) prepared in example 1_S、IONC_S/DOX、IONC_S/DOX@CCM、IONC_S/DOX @ CCM-UTMD) can be normally eliminated in the body of the mouse. As can be seen from FIG. 13(e), each group of materials (USIONP) was injected in the tail vein_S、IONC_S/DOX、IONC_S/DOX@CCM、IONC_SThe Fe content of a tumor part is obviously increased after 15min of/DOX @ CCM-UTMD), the Fe content reaches a peak value after about 25min, and material retention caused by an EPR effect is possible; after 45min, the Fe content decreased, probably due to the cluster structure of the IONC_SDegradation into individual USIONPs_SAnd is transported to other organs and tissues of the mouse through blood circulation.

Example 13

The B16 white mouse tumor model constructed in example 10 was used to study the therapeutic effect of each group of materials in example 1. Mice were randomly divided into 5 groups (5 per group) as follows: control group (PBS,100 μ L); free DOX ([ DOX ]]＝5mg/kg,100μL)；IONC_S/DOX([DOX]＝5mg/kg,100μL)；IONC_S/DOX@CCM([DOX]5mg/kg,100 μ L) and then the respective materials were injected into mice through the tail vein. For IONC_SThe group of/DOX @ CCM-UTMD was injected into tail vein with IONC_Safter/DOX @ CCM, SonoVue (1.18mg/mL,0.2mL of physiological saline) was injected via tail vein and sonicated at the tumor site for 2min (1MHz, 0.4W/cm)²20% microbubbles). The day of treatment initiation was recorded as day 0, once every 4 days, and mouse body weight and tumor size were recorded every 2 days. As can be seen from FIG. 14(a), free DOX, IONC are relative to the PBS group_S/DOX、IONC_S/DOX@CCM、IONC_Sthe/DOX @ CCM-UTMD shows a certain anti-tumor effect. Under the same conditions, IONC compared to free DOX group_SThe better antitumor effect of/DOX is probably due to IONC_S/DOXHas better EPR effect, is easy to reach the inside of the tumor, and shows that the IONC is_SCan be used as a good drug carrier. Under the same conditions, the IONC_SThe anti-tumor effect of the/DOX @ CCM is superior to that of the IONC_Sthe/DOX, probably due to the presence of immune evasion proteins and homologous targeting proteins on the cell membrane surface, can prolong the blood circulation time of the material and target enrichment at the tumor site. Under the same conditions, the IONC_SThe treatment effect of the/DOX @ CCM-UTMD is better than that of the IONC_Sthe/DOX @ CCM shows that UTMD can play a role in assisting in enhancing the effect of chemotherapy. As can be seen from fig. 14(b), the body weight of the mice in the free DOX group was significantly reduced, and the body weight of the mice in the other groups was not significantly changed, indicating that free DOX may cause toxic and side effects to the body of the mice.

Claims

1. A preparation method of cell membrane coated ultra-small ferroferric oxide nanoclusters comprises the following steps:

(1) dissolving ferric trichloride in solvent, adding sodium citrate, stirring, adding anhydrous sodium acetate, continuously stirring, carrying out solvothermal reaction, cooling, centrifuging, and drying to obtain ultra-small ferroferric oxide nanoparticles USIONP_SWherein the proportion of ferric trichloride, solvent, sodium citrate and anhydrous sodium acetate is 0.62-0.66 g: 38-42 mL: 0.46-0.51 g: 1.3-1.4 g;

(2) ultrasonically dispersing the ultra-small ferroferric oxide nano particles in the step (1) in ultrapure water, activating by EDC and NHS, then adding the ultra-small ferroferric oxide nano particles into an ethylenediamine solution for reaction, dialyzing and concentrating to obtain the surface aminated ultra-small ferroferric oxide nano particles USIONP_S-NH₂The solution is prepared from 50-60 mg of ultra-small ferroferric oxide nanoparticles, EDC, NHS and ethylenediamine: 145-155 mg: 90-100 mg: 200-220 μ L;

(3) the USIONP in the step (2)_S-NH₂Mixing the solution with precooled DMSO solution, adding a coupling agent, stirring for reaction, dialyzing, and freeze-drying to obtain the ferroferric oxide nanocluster IONC_SWherein USIONP_S-NH₂The proportion of the solution, the coupling agent and the DMSO solution is 2-4 mL: 8-10 mg: 10-20 mL;

(5) centrifuging B16 cells or mouse blood cells, adding hypotonic cell lysis solution into the obtained B16 cell sediment or mouse blood cell sediment, repeatedly freezing and thawing and crushing, extracting B16 cell membranes or erythrocyte membranes by gradient centrifugation, and suspending in PBS solution to obtain B16 cell membrane suspension or erythrocyte membrane suspension, wherein the ratio of the B16 cells or mouse blood cells to the hypotonic cell lysis solution is 10⁷2-4 mL;

2. The method according to claim 1, wherein the solvent in step (1) is diethylene glycol; the stirring is continued as follows: stirring for 2 hours at 70-75 ℃ in air atmosphere; the solvothermal reaction temperature is 190-220 ℃, and the solvothermal reaction time is 3-5 h.

3. The method of claim 1, wherein the EDC and NHS activation in step (2) is: firstly adding EDC, stirring and reacting for 0.5-1h, then adding NHS, stirring and reacting for 1-4 h; the reaction temperature is room temperature, and the reaction time is 3-5 days.

4. The method according to claim 1, wherein the pre-cooling of the DMSO solution in step (3) is: pre-cooling the DMSO solution to 10-15 ℃; adding coupling agent, and ice-cooling for 1-3 min; the coupling agent is terephthalaldehyde.

5. The method according to claim 1, wherein the stirring reaction in the step (3) is: firstly stirring and reacting for 24-48h at 40-60 ℃, and then stirring and reacting for 24-48h at normal temperature.

6. The method according to claim 1, wherein in the step (4), the stirring temperature is room temperature, and the stirring time is 4-6 h; the centrifugation time is 3-8 min.

7. The method according to claim 1, wherein the process parameters of the repeated freeze-thaw disruption in the step (5) are as follows: quick freezing at-20 deg.C, quick thawing at 37 deg.C, and repeating for 3-5 times; gradient centrifugation is as follows: the centrifugation temperature is 4 ℃, the centrifugation is firstly carried out for 10-20min by the centrifugal force of 700-850g, the supernatant is left, and then the centrifugation is carried out for 30-40min by the centrifugal force of 14000-16000g, the supernatant is removed, and the precipitate is left.

8. The method of claim 1, wherein the extruding in step (6) is: extruding the solution for 10-14 times by using an Avanti micro extruder; the centrifugal speed is 8000-14000rpm, and the centrifugal time is 30-40 min.

9. A cell membrane-coated ultra-small ferroferric oxide nanocluster prepared according to the method of claim 1.

10. Preparation of cell membrane-coated ultra-small ferroferric oxide nanoclusters according to claim 9 with T₁/T₂Application in bimodal nuclear magnetic resonance imaging contrast and diagnosis and treatment agents for inhibiting tumor cell proliferation.