Preparation method and application of superparamagnetic bionic nano diagnosis and treatment agent

Abstract

The invention belongs to the technical field of biomedicine, and particularly relates to a preparation method and application of a superparamagnetic bionic nanometer diagnosis and treatment agent.The preparation method of the superparamagnetic bionic nanometer diagnosis and treatment agent.The preparation method of the superparamagnetic bionic nanometer diagnosis and treatment agent.The preparation method comprises the following steps that 1, MnFe2O4 nanoparticles are synthesized; step 2, loading DHA (docosahexaenoic acid) on the MnFe2O4 nano particles, so as to prepare a MnFe2O4-DHA nano drug; 3, the outer side of the MnFe2O4-DHA nano drug is wrapped with a GL261 cell membrane, and MnFe2O4-DHA (at) CM is prepared. The nano-drug can effectively penetrate through BBB and is actively targeted to a tumor site, so that the MnFe2O4-DHA nano-drug is accurately released in cells, tumor marker targeted therapy is realized, H2O2 is efficiently catalyzed by the synthesized bionic nano-drug into toxic dual ROS-hydroxyl free radicals and singlet oxygen, tumor cells are killed, and the tumor marker targeted therapy is realized. An in-vivo anti-oxidation system is jointly inhibited inside and outside, a cascade amplification ferroptosis treatment effect is generated, and a relatively good killing effect on tumors can be achieved.

Description

Technical Field

[0001] The invention belongs to the field of biomedical technology, and specifically relates to a preparation method and application of a superparamagnetic bionic nano diagnostic and therapeutic agent. Background Art

[0002] Gliomas are primary malignant brain tumors of the central nervous system (CNS) with high mortality and recurrence rates, among which glioblastomas are characterized by grade IV astrocytomas, which are the most invasive. Standard treatments in the fields of surgery, chemotherapy, radiotherapy, and novel tumor therapies already exist, but the median survival of glioma patients remains almost unchanged (approximately 8-15 months for glioblastoma patients). Given the highly invasive nature of gliomas and the anatomical structure of the brain, complete resection is almost impossible to achieve with surgery, resulting in a high recurrence rate. Protected by the blood-brain barrier (BBB) ​​composed of dense capillary endothelial cells (ECs), intact basement membranes, pericytes, and astrocytes, it is usually difficult for drugs to cross the BBB, resulting in poor response of glioma cells to conventional chemotherapeutic drugs. Therefore, developing alternative strategies for the effective treatment of gliomas remains a pressing challenge. Summary of the invention

[0003] The purpose of the present invention is to provide a preparation method and application of a superparamagnetic bionic nano-diagnostic and therapeutic agent, which can effectively pass the nano-drug through the BBB and actively target the tumor site, so that the MnFe2O4-DHA nano-drug is accurately released in the cell, achieving targeted therapy of tumor markers, and jointly inhibiting the antioxidant system in the body internally and externally, producing a cascade amplification of ferroptosis treatment effect, and can achieve a better killing effect on the tumor.

[0004] The technical solution adopted by the present invention is as follows:

[0005] A method for preparing a superparamagnetic bionic nano-diagnostic agent comprises the following steps:

[0006] Step 1: Synthesis of MnFe2O4 nanoparticles;

[0007] Step 2: Load DHA on MnFe2O4 nanoparticles to prepare MnFe2O4-DHA nanomedicine;

[0008] Step 3: Wrap the GL261 cell membrane on the outside of the MnFe2O4-DHA nanodrug to prepare MnFe2O4-DHA@CM.

[0009] Furthermore, the step 1 comprises the following steps:

[0010] Step 101: add 0.6 g of Fecl3·6H2O, 0.45 g of MnCL4.4H2O, 30 mL of ultrapure water, 30 mL of anhydrous ethanol and 50 mL of n-hexane into a three-necked flask, put in a magnet, place on a magnetic stirrer for stirring, introduce argon gas, evacuate to remove water, then heat to 70°C and reflux for 4 h, then turn off the temperature to cool, pour into a beaker, and stir;

[0011] Step 102: then use ultrapure water and cyclohexane to wash and remove impurities, take the upper organic layer, put it into a magnetic stirrer for 8-12 hours, and then place it in a vacuum oven for drying to obtain an oleic acid iron-manganese complex;

[0012] Step 103: 66.67 g of octadecene, 1.9 g of oleic acid and 10 g of oleic acid iron-manganese complex were added to a single-necked bottle, heated to 110° C. under argon, vacuumized, and dehydrated after 15 min;

[0013] Step 104: reflux under argon and heat to 313° C., react for 30 min, then cool to 60° C., add 50 mL of n-hexane for precipitation;

[0014] Step 105: After the sedimentation is completed, the upper organic layer is taken, and anhydrous ethanol is added to wash repeatedly, centrifuged at 10000 rpm for 20 min, the supernatant is removed, cyclohexane is added to precipitate, anhydrous ethanol is added to wash again, centrifuged, and placed in a 60°C oven for drying to finally obtain MnFe2O4 nanoparticles;

[0015] The step 2 comprises the following steps:

[0016] Step 201: dispersing 10 mg of MnFe2O4 and 20 mg of dihydroartemisinin in 10 mL of chloroform solution, and stirring at 37° C. for 24 h to obtain an organic solvent;

[0017] Step 202: adding the obtained organic solvent dropwise to a suspension of sodium dodecylbenzenesulfonate dispersed in dichloromethane, and then continuing to stir for 24 hours;

[0018] Step 203: stirring the solvent at a constant speed, and adding ultrapure water dropwise while stirring at a constant speed;

[0019] Step 204: washing the product obtained in step 203 with ethanol and ultrapure water respectively, centrifuging at 10000 rpm for 10 min, and collecting the supernatant;

[0020] Step 205: drying the precipitate in a vacuum oven at 60° C. for 8-12 hours;

[0021] Step 206: using the supernatant collected in step 204 to measure the drug loading of DHA;

[0022] The step 3 comprises the following steps:

[0023] Step 301: collect and culture GL261 tumor cells, and wash them three times with PBS;

[0024] Step 302: disrupting the cells using an ultrasonic disruptor to form a cell disruption solution, and centrifuging the cell disruption solution at a relative centrifugal force of 900 g for 10 minutes at 4° C. to collect the supernatant;

[0025] Step 303: centrifuging the supernatant collected in step 302 at 4° C. and a relative centrifugal force of 14,000 g for 30 minutes, collecting the precipitate as the cell membrane, and storing it in a refrigerator at 4° C.;

[0026] Step 304: Re-disperse the cell membrane in PBS by ultrasonication, and then repeatedly squeeze the filter head 10 times;

[0027] Step 305: The cell membrane in step 304 is dispersed in PBS, placed in an ultrasonic cleaner, and the MD nanodrug solution dispersed in PBS is added dropwise to the PBS, and the mixture is treated with ultrasound for 2 hours, and pushed, and centrifuged at a relative centrifugal force of 900g for 10 minutes at a temperature of 4°C to remove excess cell membrane to obtain a nanodrug suspension of MDM, wherein the nanodrug suspension is MnFe2O4-DHA@CM.

[0028] A superparamagnetic bionic nano-diagnostic and therapeutic agent is used to prepare drugs for treating tumors.

[0029] Furthermore, the tumor is a glioma.

[0030] The technical effects achieved by the present invention are:

[0031] (1) The preparation method and application of a superparamagnetic bionic nano-diagnostic agent of the present invention utilizes the biological homotypic recognition surface receptor function and magnetic properties of the tumor cell membrane of bionic nano-materials, applies a magnetic field effect outside the tumor to dual-target guide the directional movement and positioning concentration of nano-drugs in the body, effectively passes the BBB and actively targets the nano-drugs to the tumor site. In addition, the homologous recognition function of the bionic nanoparticles accelerates the internalization of tumor cells, and the magnetism of the nano-drug also plays a role in fueling the process. Due to the characteristics of the weakly acidic microenvironment of the tumor and the heat generated by applying an alternating magnetic field outside the tumor, the drug and MnFe2O4 can be accurately released inside the cell to achieve therapeutic effects.

[0032] (2) The preparation method and application of a superparamagnetic bionic nano-diagnostic agent of the present invention is to synthesize bionic nano-medicine to efficiently catalyze H2O2 into toxic dual ROS-hydroxyl radical (Hydroxyl Radical, ·OH) and singlet oxygen (Singlet oxygen, 1 O2), causing the killing of tumor cells. It not only consumes GSH from the endogenous pathway by using its enzymatic activity, but also blocks the Xc-system from the exogenous pathway by inducing autophagy. It inhibits the antioxidant system in the body from both inside and outside, produces a cascade amplification of ferroptosis therapeutic effect, and can achieve a better killing effect on tumors and realize the treatment of tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 It is the TEM image and XRD spectrum of the MnFe2O4 nanomaterial of the present invention;

[0034] Figure 2 It is the FTIR spectrum of MD and the XRD spectrum of MD nanomedicine of the present invention;

[0035] Figure 3 It is the TEM image, DLS spectrum and ZETA spectrum of the MDM nanomedicine of the present invention;

[0036] Figure 4 is the ESR image of the nanomedicine of the present invention;

[0037] Figure 5 It is the hysteresis regression of MDM with different concentrations and the magnetic thermal image of temperature rise under the alternating magnetic field of the present invention;

[0038] Figure 6 are T1-T2 weighted images and signal intensities of different concentrations of MDM of the present invention;

[0039] Figure 7 is a graph of the POD-like activity of the MDM of the present invention;

[0040] Figure 8 is a graph of GSH-Ox activity of the MDM of the present invention;

[0041] Fig. 9 is the CAT-like activity graph of the MDM of the present invention;

[0042] Fig.10 It is the cytotoxicity graph of different groups and the cell killing graph of different groups of the present invention;

[0043] Fig.11 It is a schematic diagram of the in vitro BBB model constructed by the Transwell system of the present invention, a TEER detection diagram, and a fluorescence diagram of cell phagocytosis of nanomedicine;

[0044] Fig.12It is the ROS produced by different groups of the present invention;

[0045] Fig.13 It is a graph of GSH consumption of stimulated cells in different groups of the present invention;

[0046] Fig.14 is a graph of the Western blot evaluation of ferroptosis markers of the present invention;

[0047] Fig.15 It is a biological transmission electron microscopic image of autophagosome and mitochondrial ferroptosis of the present invention;

[0048] Fig.16 It is a tumor growth diagram of MRI imaging of mouse glioma treated with different treatment methods of the present invention;

[0049] Fig.17 It is a schematic diagram of the present invention. DETAILED DESCRIPTION

[0050] In order to make the purpose and advantages of the present invention more clearly understood, the present invention is specifically described below in conjunction with embodiments. It should be understood that the following text is only used to describe one or several specific embodiments of the present invention, and does not strictly limit the scope of protection of the specific claims of the present invention.

[0051] This scheme designs an innovative bionic nano-platform, which consists of GL261 cell membrane wrapped with MnFe2O4 loaded with DHA (MnFe2O4-DHA@CM, abbreviated as MDM). Ferroptosis is a very promising non-invasive treatment technology and is increasingly used in the treatment of systemic tumors. However, the hypoxic environment, high content of hydrogen peroxide (H2O2), redox homeostasis, insufficient iron and the presence of BBB in gliomas limit their effects. The MDM bionic nano-platform of the present invention achieves effective penetration of the BBB by dual-targeting of the carrier by utilizing biological homotypic recognition surface receptors and physical magnetic targeting of the external magnetic field of the tumor site, and actively targets the glioma site to release nano-drugs. Specifically, the magnetic nano-material of MnFe2O4 is first prepared, then DHA is loaded, and then the extracted tumor cell membrane is wrapped. The interaction between MnFe2O4 and DHA released from the tumor site can generate oxygen using the higher H2O2 in the tumor microenvironment, producing dual ROS. At the same time, it can also efficiently consume GSH. The magnetic heat generated under the alternating magnetic field promotes the release of DHA to induce multiple autophagy pathways, block endogenous and exogenous pathways, and enhance the amplification of ferroptosis treatment. In addition, due to Mn 2+Manganese-doped ferrite has a higher spin number (carrying five unpaired electrons), a longer electron relaxation time, and unstable water exchange. This enhances the T1-weighted MRI signal, produces bright images, and also improves the T2-weighted signal. Therefore, the superparamagnetic effect of manganese ferrite further realizes real-time in vivo imaging of tumor MRI. This system can effectively penetrate the BBB, autonomously target and locate, improve the hypoxic acidic microenvironment of the tumor, enhance autophagy and amplify ferroptosis treatment through magnetic hyperthermia, and enhance the real-time monitoring effect of T1 and T2 magnetic resonance in vivo. The materials used have strong tissue compatibility, which is conducive to rapid clinical application and promotion, and can provide a new idea for the treatment of gliomas.

[0052] Embodiment 1:

[0053] like Figure 1-17 As shown, a method for preparing a superparamagnetic bionic nano-diagnostic agent comprises the following steps:

[0054] Step 1: Synthesis of MnFe2O4 nanoparticles;

[0055] In order to prepare nanoparticles of smaller size, the synthesis of MnFe2O4 nanoparticles in step 1 adopts a thermal decomposition method. Specifically, step 1 includes the following steps:

[0056] Step 101: add 0.6 g of Fecl3·6H2O, 0.45 g of MnCL4.4H2O, 30 mL of ultrapure water, 30 mL of anhydrous ethanol and 50 mL of n-hexane into a three-necked flask, put in a magnet, place on a magnetic stirrer for stirring, introduce argon gas, evacuate to remove water, then heat to 70°C and reflux for 4 h, then turn off the temperature to cool, pour into a beaker and stir;

[0057] Step 102: then use ultrapure water and cyclohexane to wash and remove impurities, take the upper organic layer, put it into a magnetic stirrer overnight (stirring for 8-12 hours), and then place it in a vacuum oven for drying to obtain an oleic acid iron-manganese complex;

[0058] Step 103: 66.67 g of octadecene, 1.9 g of oleic acid and 10 g of oleic acid iron-manganese complex were added to a single-necked bottle, heated to 110° C. under argon, vacuumized, and dehydrated after 15 min;

[0059] Step 104: reflux under argon and heat to 313° C., react for 30 min, then cool to 60° C., add 50 mL of n-hexane for precipitation;

[0060] Step 105: After the sedimentation is completed, take the upper organic layer, add anhydrous ethanol to wash repeatedly, centrifuge (10000rpm, 20min), remove the supernatant, add cyclohexane to precipitate, add anhydrous ethanol to wash again, centrifuge, put into a 60°C oven to dry, and finally obtain MnFe2O4 nanoparticles.

[0061] like Figure 1 As shown in a-1b, Figure 1 a is the TEM image of MnFe2O4 nanomaterials. Figure 1 b is the XRD spectrum of MnFe2O4 nanomaterials;

[0062] After the MnFe2O4 nanoparticles were prepared, the morphology of the MnFe2O4 nanoparticles was observed using a transmission electron microscope (TEM). Figure 1 As shown in a, it can be seen that MnFe2O4 is in a spherical structure. These small nanoparticles are evenly distributed in size, with an average particle diameter of about 12nm. The crystal structure of the powder sample was analyzed using X-ray diffraction (XRD) to obtain XRD pattern 1b. For MnFe2O4, all observed peaks can correspond to the standard card (JCPDS73-1964), indicating that MnFe2O4 was successfully prepared.

[0063] Step 2: Load DHA onto MnFe2O4 nanoparticles to prepare MnFe2O4-DHA (abbreviation: MD) nanodrug core;

[0064] Specifically, step 2 includes the following steps:

[0065] Step 201: dispersing 10 mg of MnFe2O4 and 20 mg of dihydroartemisinin DHA in 10 mL of chloroform solution, and stirring at 37° C. for 24 h to obtain an organic solvent;

[0066] Step 202: Then slowly drop the obtained organic solvent into a suspension of sodium dodecylbenzenesulfonate dispersed in dichloromethane (30 mg / mL, 10 mL), and then continue stirring for 24 hours until most of the solvent is evaporated.

[0067] Step 203: Stir the solvent at a constant speed continuously, and then slowly add ultrapure water dropwise while stirring at a constant speed continuously to ensure that the organic solvent is completely volatilized.

[0068] Step 204: The product obtained in step 203 is then washed with ethanol and ultrapure water, respectively, centrifuged (10000 rpm, 10 min), and the supernatant is collected.

[0069] Step 205: Place the precipitate in a vacuum oven at 60°C and dry overnight (dry for 8-12 hours).

[0070] Step 206: The supernatant collected in step 204 is used to measure the drug loading of DHA.

[0071] Specifically, the drug loading of DHA measured in step 206 is that the DHA in the supernatant is mixed with a 0.2% NaOH aqueous solution and heated at 55±1° C. for 30 minutes to convert the DHA in the supernatant into a compound that absorbs ultraviolet light, and the absorption value at 238 nm is detected by a UV-vis spectrometer.

[0072] like Figure 2 As shown in a-2b, Figure 2 a is the FTIR spectrum of MD, Figure 2 b is the XRD pattern of MD nanomedicine;

[0073] The powder was tested using a Fourier transform infrared spectrometer (FTIR). Figure 2 a shows that the DHA-loaded MnFe2O4 nanoparticles have a wavelength of 800–1200 cm -1 The absorption peaks in the range of 1227 cm -1 ), COC(1025cm -1 ) mode. In addition, the strong methyl group (-CH3) present in DHA can be seen at 2925cm -1 The presence of OH can also be determined by the peak at 3376 cm -1 The peak at 679 cm-1 is also confirmed by the peak of MnFe2O4. -1 The characteristic peak of FeO at 240 nm was found, which confirmed that DHA was successfully loaded into MnFe2O4. The XRD pattern of the composite nanomedicine shown in 2b showed that the crystal structure peak was consistent with the crystal structure of the single MnFe2O4 nanoparticles synthesized previously, and the study confirmed the successful synthesis of MD.

[0074] Step 3: Wrap the GL261 cell membrane on the outside of the MnFe2O4-DHA (abbreviation: MD) nanodrug to establish the MnFe2O4-DHA@CM (abbreviation: MDM) system, and prepare MnFe2O4-DHA@CM, that is, GL261 cell membrane wrapped with DHA-loaded MnFe2O4.

[0075] Specifically, step 3 includes the following steps:

[0076] Step 301: first collect and culture GL261 tumor cells by centrifugation (1500 rpm, 4° C., 4 min), and wash with PBS three times;

[0077] Step 302: using an ultrasonic disruptor to disrupt the cells (75W, 3s / 5s, 3min), then centrifuging the cell disruption solution at a low speed (900g, 4°C, 10min), and carefully collecting the supernatant;

[0078] In step 302, the collected GL261 tumor cells are added to a buffer to form a cell suspension, and then the GL261 tumor cells are disrupted using an ultrasonic disruptor so that the substances in the cells are mixed with the buffer to form a cell disruption solution.

[0079] Step 303: centrifuging the supernatant collected in step 302 at high speed (14000 g, 4° C., 30 min), collecting the precipitate as the cell membrane, and storing it in a refrigerator at 4° C.;

[0080] Step 304: Re-disperse the cell membrane in PBS (pH 7.4) by ultrasonication, and then repeatedly push the filter head 10 times;

[0081] Step 305: Disperse the cell membrane prepared in step 304 in PBS, place it in an ultrasonic cleaner, add the MD nanodrug solution dispersed in PBS (cell membrane: MD nanodrug 1: 2.5 w / w) dropwise into the PBS, treat with ultrasound for 2 hours, and push, and finally centrifuge at 900g, 10min, 4°C to remove excess cell membrane to obtain a nanodrug suspension of MDM, i.e., GL261 cell membrane-encapsulated MnFe2O4 loaded with DHA, and then store the nanodrug suspension in a refrigerator at 4°C.

[0082] Here, the MD nano drug solution dispersed in PBS in step 305 refers to a solution formed by dispersing MD nano drug in PBS.

[0083] like Figure 3 As shown in a-3c, Figure 3 a is the TEM image of MDM nanomedicine, Figure 3 b is the DLS spectrum of MDM nanomedicine, Figure 3 c is the ZETA diagram of MDM nanomedicine;

[0084] Among them, the TEM image of MDM is as follows Figure 3 As shown in a, the observed shape is spherical. These biomimetic nanomedicines are not aggregated, and the average particle diameter is about 68nm. The particle size of MnFe2O4, MD and MDM nanoparticles is detected by nanoparticle size and Zeta potential analyzer (DLS). Figure 3 As shown in b, their average particle sizes are 11.97±0.30nm, 25.67±1.79nm, and 83.57±10.94nm, respectively; the corresponding ZETA potentials of MnFe2O4, DHA, MD, and MDM are detected as follows Figure 3 As shown in c, they are 19.30±0.38 mV, -17.70±0.79 mV, -7.15±0.10 mV, and -35.90±6.29 mV, respectively, indicating the successful synthesis of MDM.

[0085] Embodiment 2:

[0086] This embodiment further discloses the MDM function and performance characteristics, which are as follows:

[0087] ROS detection of MDM:

[0088] like Figure 4 As shown in a-4b, the ability of MDM to generate ROS was evaluated by electron spin resonance (ESR) spectroscopy, using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as a spin trap for ·OH and 2,2,6,6-tetramethyl-4-piperidone hydrochloride (TEMP) as 1 The representative ESR signal of OH was observed in the presence of H2O2. Compared with H2O, DHA, and MnFe2O4, MDM showed a higher signal intensity ( Figure 4 a), In addition, MDM produces 1 O2 is also more capable than H2O, DHA, and MnFe2O4 ( Figure 4 b), indicating that manganese and iron ions can promote the rupture of DHA peroxide bridges and produce more ROS (including ·OH, 1 O2), and as the temperature increased to 42 °C, more ROS were produced, confirming that the increase in temperature increased the ·OH and 1 The efficiency of O2 production indicated that MDM is an ideal agent capable of producing both ROS.

[0089] Magnetic, magnetothermal and MRI imaging performance testing of MDM:

[0090] Before evaluating the magnetocaloric properties of MDM, its magnetic properties were first tested. Hysteresis regression was used to determine the magnetic properties of MnFe2O4( Figure 5 a) and MDM( Figure 5 b) The magnetic properties of MnFe2O4 and MDM nanoparticles were tested. At 300K, the saturation magnetization (MS) of MnFe2O4 and MDM nanoparticles were 19.13 and 8.68emu / g, respectively, and the coercive force and residual magnetization were both 0, indicating that they were superparamagnetic. In addition, a 0.04T magnet ( Figure 5 b), after 50s, the MDM nanodrug was completely adsorbed to the side with ferromagnetic addition, indicating that MDM has good superparamagnetic response performance.

[0091] The magnetothermal properties of MDM nanoparticles were studied under AMF. A magnetic field strength of (1.2 mT, 496 kHz) was applied for 600 s. MDM aqueous solutions with different concentrations in EP tubes were placed in the magnetic field, and the temperature increase and magnetothermal images were recorded by an infrared (IR) camera. Figure 5c shows that the temperature increases with different concentrations of MDM. When the NP concentration is 2 mg / mL, the temperature increases by 22 °C, which is high for killing cancer cells. In magnetic hyperthermia, when tissue is exposed to high temperature (about 42 °C), malignant tissue is destroyed or becomes more sensitive to the effects of anticancer drugs. It is more ideal to control the temperature at around 42 °C.

[0092] In order to prove that MDM has T1 and T2 NMR weighted enhancement imaging effects, MDM was dispersed in 2% agarose gel with Fe-Mn content ranging from 0 to 0.2 mM. A GE 3.0T NMR instrument was used to scan and obtain the following images: Figure 6 (a, b), the results show that the signal intensity on T1-weighted images increases with the increase of NP concentration, and a higher longitudinal relaxation rate of about (r1 = 6.84 mM -1 S -1 ), and the signal intensity on the T2-weighted image gradually decreased with the increase of NP concentration, and the corresponding transverse relaxation rate was (r2=130.90mM -1 S -1 ), and the linear correlation coefficients were high, all greater than 0.99, indicating that MDM is a promising T1-T2 dual-modality MRI contrast agent.

[0093] Enzyme activity detection of MDM:

[0094] The peroxidase-like activity of the prepared MDM was determined by the colorimetric reaction of 3,3,5,5'-tetramethylbenzidine (TMB). That is, TMB was oxidized by ·OH to obtain the blue product oxTMB, and its absorbance at A = 652nm was detected. First, different concentrations of nanomaterials were added to 0.1M acetic acid-sodium acetate buffer solutions of different pH values, in which the final concentration of H2O2 was 5mM and the final concentration of TMB was 20mg / mL. After mixing, the mixture was incubated for 30min, and the supernatant was taken by centrifugation to measure the absorbance at 652nm. The results are shown in Figure 2. Figure 7 As shown in a, with the increase of MDM concentration, the amount of oxidized oxTMB produced increased, indicating that the POD-like enzyme activity was higher, which was 2-3 times that of MnFe2O4@CM (abbreviation: MM) without DHA. In addition, in order to detect the effect of simulated magnetic thermal heating on the POD-like enzyme activity of bionic nanomedicine, Figure 7 b It was observed that the absorbance value at elevated temperature was nearly twice that of normal body temperature, indicating that heat can enhance the catalytic activity of the enzyme.

[0095] In order to prove that the prepared MDM also has GSH-Ox enzyme activity, 5,5'-dithiobis(DTNB) was used to detect the GSH consumption characteristics of biomimetic nanomedicines, such as Figure 8As shown in the figure, as the concentration of MDM increases, the consumption of GSH is faster, indicating that the activity of GSH-Ox is better, and there is no significant difference with MM without DHA. In addition, Figure 8 As shown in b, the bionic nanomedicine consumes slightly more GSH than normal body temperature when simulating magnetic thermal heating, indicating that heat can slightly enhance the catalytic activity of GSH-Ox enzyme.

[0096] In order to prove that the prepared MDM also has CAT-like activity, a dissolved oxygen meter was used to detect the oxygen level generated by H2O2 catalyzed by the biomimetic nanomedicine dispersed in water. Fig. 9 As shown in a, as the concentration of MDM increases, more oxygen is produced, indicating a higher CAT-like activity. In addition, Fig. 9 As shown in b, the oxygen produced by bionic nanomedicine under simulated magnetic thermal heating is twice that at normal body temperature, indicating that heat can enhance the catalytic activity of CAT-like enzymes, effectively produce O2 to alleviate the hypoxic microenvironment at the tumor site, thereby increasing the lethality to the tumor.

[0097] Embodiment 3:

[0098] This example introduces the anti-tumor effect of the composite nanomaterial at the cellular level, which is as follows:

[0099] In order to study the cytotoxicity of nanomaterials, MnFe2O4, DHA and MDM were co-incubated with normal mouse fibroblast L929 cells for 24 h, and the cell viability was determined by MTT method. Fig.10 As shown in a, Fig.10 a is the cytotoxicity of different groups. With the increase of NP concentration, cell viability gradually decreased. When the MDM concentration reached 300ug / mL, the survival rate of normal cells was more than 80%, while the survival rate of cells treated with 100ug / mL DHA and 200ug / mL MnFe2O4 was less than 80%, indicating that MDM has higher biosafety to normal cells than DHA / MnFe2O4. Subsequently, in the presence of H2O2 and / or AMF, 8 different groups of PBS, DHA, MnFe2O4, MDM, MM+H2O2+AMF, MDM+H2O2, MDM+AMF, and MDM+H2O2+AMF with the same concentrations were set up and incubated with mouse glioblastoma GL261 cells for 24 hours. Fig.10 As shown in b, Fig.10b is the cell killing of different groups. The bionic nanodrug MDM has a higher killing effect on tumor cells than DHA / MnFe2O4, reaching 48.59±3.46%. When H2O2 and AMF stimulation conditions exist at the same time, MDM exerts a stronger killing effect on tumor cells, and the cell survival rate is only 9.98±2.28%, indicating that MDM produces more dual ROS under magnetic heat, and jointly blocks the endogenous and exogenous antioxidant systems, amplifies the ferroptosis effect, and greatly improves the killing of tumor cells.

[0100] like Fig.11 a- Fig.11 As shown in c, Fig.11 a is a schematic diagram of constructing an in vitro BBB model using the transwell system. Fig.11 b is the TEER detection diagram, Fig.11 c is the fluorescence image of cells engulfing nanomedicine.

[0101] In order to study whether MDM has the ability to cross the blood-brain barrier, this technical solution uses transwell chambers to construct an in vitro BBB model, such as Fig.11 As shown in a, the system consists of an upper chamber containing brain microvascular endothelial (bEnd.3) cells and a bottom chamber containing glioma (GL261) cells. Transendothelial electrical resistance (TEER) is widely used to assess the tightness of cell barriers. As shown in 11b, when the TEER value was ≥200Ω·cm on day 11, 2 When the in vitro blood-brain barrier model was successfully constructed with tight junctions, it can be used for subsequent experiments. Rhodamine B (Rh B) was used to label MDM and MD and added to the top chamber. After incubation for 2, 4, and 6 hours, we observed the fluorescence signals of GL261 cells in the bottom wells by confocal laser scanning microscopy (CLSM). Fig.11 c As shown in the results, compared with MD, MDM showed a stronger red fluorescence signal. At the same time, when a magnet was applied to the bottom, MDM showed an even stronger red fluorescence signal, indicating that tumor cell membrane wrapping combined with nanomagnetic force can effectively cross the BBB and accelerate phagocytosis by homologous tumor cells.

[0102] like Fig.12 As shown, Fig.12 Among them, (1) is PBS, (2) is H2O2+AMF, (3) is DHA, (4) is MnFe2O4, (5) is MDM, (6) is MDM+H2O2, (7) is MDM+AMF, and (8) is MDM+H2O2+AMF;

[0103] To demonstrate the ROS-generating ability of biomimetic nanomaterials in tumor cells, the classic ROS probe 2′,7′-dichlorofluorescein diacetate (DCFH-DA) was used to assess the ROS levels in living cells, e.g. Fig.12 The green fluorescence observed in the image represents the oxidation of DCFH by ROS, and the fluorescence intensity reflects the amount of intracellular ROS generated. Almost no fluorescence was observed in the blank control group, and weak fluorescence was observed in the DHA-treated group. On the contrary, the MDM-treated group showed appropriate green fluorescence, indicating that manganese and iron ions can destroy the DHA peroxide bridge to produce more free radicals. It is worth noting that the strongest fluorescence was observed in the GL261 cell group treated with both H2O2 and AMF, indicating that a large amount of ROS was generated, which was due to the complementary effects of manganese, iron ions and DHA.

[0104] In order to verify the ability of biomimetic nanomedicine to consume glutathione, the level of GSH in GL261 cells after different groups were treated was detected using a kit. Fig.13 The experimental results showed that compared with the untreated group, the single DHA and MnFe2O4 groups had the ability to consume GSH, the MDM treatment group enhanced the consumption of GSH, and the catalytic activity of GSH-Ox enzyme activity was further enhanced when stimulation conditions (H2O2 or AMF) were applied. Similarly, MDM treatment under simultaneous conditions (H2O2 and AMF) significantly reduced the GSH level in the cells, indicating that MDM has excellent GSH-Ox activity in the cells.

[0105] It is well known that GPX4 plays a crucial role in the cellular lipid repair system and can be inactivated by GSH depletion. Based on the high GSH depletion capacity of MDM, the intracellular GPX4 level was further monitored using Western blotting. Single DHA, MnFe2O4, and MDM could all reduce GPX4 levels, while MDM under the applied conditions (H2O2 or / and AMF) showed significantly higher inhibition efficiency, as shown in Figure 2. Fig.14 In addition, compared with the blank control group, MDM can activate the Beclin1 autophagy pathway to block the ferroptosis extrinsic pathway Xc - These results demonstrate that under the conditions of H2O2 and / or AMF, MDM can induce tumor ferroptosis by internal and external linkage, that is, nanomedicine activates the Beclin1 autophagy pathway and binds to SLC7A11 to block the Xc-system, prevent the synthesis of GSH, and enhance ferroptosis from the exogenous pathway.

[0106] In addition, the underlying microstructures were determined to evaluate the biomimetic nanomedicine-induced autophagy and ferroptosis. Morphologically, ferroptotic cells showed shrunken mitochondria with thickened membranes, and autophagic cells showed many autophagic vesicles, including autophagosomes and autolysosomes. Compared with the normal oblong mitochondria ( Fig.15 a), GL261 cells treated with MDM under the conditions of H2O2 and AMF ( Fig.15b), TEM images showed that GL261 cells had many shrunken mitochondria, concentrated membrane density, reduced mitochondrial cristae, and ruptured mitochondrial outer membrane (yellow arrows). In addition, autophagosomes and autophagic lysosomes (blue arrows) also appeared, proving that MDM has the ability to induce autophagy and efficiently enhance ferroptosis.

[0107] Embodiment 4:

[0108] This example introduces the anti-tumor effect of composite nanomaterials at the animal level, which is as follows:

[0109] like Fig.16 As shown, Fig.16 MRI images of gliomas in mice treated with different treatments show tumor growth (blue arrows point to tumor areas);

[0110] A mouse orthotopic glioma model was constructed. When treatment started one week later, the mice were randomly divided into 6 groups (n=6), namely PBS group, MDM group, MDM+magnetic targeting group, MM+magnetic targeting+AMF group, MDM+AMF group, and MDM+magnetic targeting+AMF group. The bionic nanomedicine prepared above was prepared into a concentration of 5 mg / mL. After one week, it was injected into the tumor-bearing mice through the tail vein every two days, with or without the application of AMF or magnet. The results are shown in Fig.16 As shown, compared with the control group, the tumors of the experimental mice in the MDM group and the MDM+magnetic targeting group had a certain degree of inhibitory effect. Compared with the MM+magnetic targeting+AMF group without DHA, the tumor inhibition effect of the experimental mice in the MDM+magnetic targeting+AMF group was stronger. Compared with the MDM+AMF group without magnetic targeting, the tumors of the mice in the MDM+magnetic targeting+AMF group showed a significant shrinking trend.

[0111] This technical solution uses magnetothermal enhancement of Mn and Fe ions to induce DHA to produce two types of ROS (·OH, 1 O2), compared with the normal body temperature POD-like, CAT-like and GSH-Ox enzyme activities, hyperthermia can increase their catalytic activity, the developed nanomedicine activates the Beclin1 autophagy pathway and combines with SLC7A11 to block the Xc-system, prevents the synthesis of GSH, and enhances ferroptosis from the exogenous pathway. Compared with blocking the endogenous pathway of conventional GPX4 to induce ferroptosis, MDM can simultaneously block the endogenous GPX4 and exogenous SLC7A11 pathways to enhance ferroptosis. Compared with passively targeted nanocarriers, MDM uses homologous targeting biomembranes and nanomagnetism to achieve dual targeting and actively reach the target through the BBB.

[0112] In summary, based on the above effects, it can be seen that the technical solution utilizes the biological homotypic recognition surface receptor function and magnetic properties of the tumor cell membrane of bionic nanomaterials, applies a magnetic field effect outside the tumor to dual-target guide the directional movement and positioning of nanomedicines in the body, and effectively passes the nanomedicine through the BBB and actively targets the tumor site. In addition, the homologous recognition function of bionic nanoparticles accelerates the internalization of tumor cells, and the magnetism of nanomedicines also plays a role in fueling the process. Due to the characteristics of the weakly acidic microenvironment of the tumor and the heat generated by applying an alternating magnetic field outside the tumor, the drug and MnFe2O4 can be precisely released in the cell to achieve therapeutic effects. The synthesized bionic nanomedicine efficiently catalyzes H2O2 into toxic dual ROS—hydroxyl radicals (Hydroxyl Radical, ·OH) and singlet oxygen (Singlet oxygen, 1 O2), causing the killing of tumor cells. It not only consumes GSH from the endogenous pathway by utilizing its enzymatic activity, but also blocks the Xc-system from the exogenous pathway by inducing autophagy. It inhibits the antioxidant system in the body from both inside and outside, produces a cascade amplification of ferroptosis therapeutic effect, and can achieve a better killing effect on tumors.

[0113] The above is only a preferred embodiment of the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the principles of the present invention, and these improvements and modifications should also be considered as the protection scope of the present invention. The structures, devices and operating methods not specifically described and explained in the present invention shall be implemented according to the conventional means in the art unless otherwise specified and limited.

Claims

1. A method for preparing a superparamagnetic bionic nano-diagnostic agent, characterized in that: The following steps are involved: Step 1: Synthesis of MnFe2O4 nanoparticles; Step 2: Load DHA on MnFe2O4 nanoparticles to prepare MnFe2O4-DHA nanomedicine; Step 3: Wrap the GL261 cell membrane on the outside of the MnFe2O4-DHA nanodrug to prepare MnFe2O4-DHA@CM.

2. The method for preparing a superparamagnetic bionic nano-diagnostic agent according to claim 1, characterized in that: In step 1, the synthesis of MnFe2O4 nanoparticles is carried out by thermal decomposition method.

3. The method for preparing a superparamagnetic bionic nano-diagnostic agent according to claim 1, characterized in that: The step 1 comprises the following steps: Step 101: add 0.6 g of Fecl3·6H2O, 0.45 g of MnCL4.4H2O, 30 mL of ultrapure water, 30 mL of anhydrous ethanol and 50 mL of n-hexane into a three-necked flask, put in a magnet, place on a magnetic stirrer for stirring, introduce argon gas, evacuate to remove water, then heat to 70°C and reflux for 4 h, then turn off the temperature to cool, pour into a beaker, and stir; Step 102: then use ultrapure water and cyclohexane to wash and remove impurities, take the upper organic layer, put it into a magnetic stirrer for 8-12 hours, and then place it in a vacuum oven for drying to obtain an oleic acid iron-manganese complex; Step 103: 66.67 g of octadecene, 1.9 g of oleic acid and 10 g of oleic acid iron-manganese complex were added to a single-necked bottle, heated to 110° C. under argon, vacuumized, and dehydrated after 15 min; Step 104: reflux under argon and heat to 313° C., react for 30 min, then cool to 60° C., add 50 mL of n-hexane for precipitation; Step 105: After the sedimentation is completed, take the upper organic layer, add anhydrous ethanol to wash repeatedly, centrifuge at 10000rpm for 20min, remove the supernatant, add cyclohexane to precipitate, add anhydrous ethanol to wash, centrifuge, put into a 60℃ oven to dry, and finally obtain MnFe2O4 nanoparticles.

4. The method for preparing a superparamagnetic bionic nano-diagnostic agent according to claim 3, characterized in that: The step 2 comprises the following steps: Step 201: dispersing 10 mg of MnFe2O4 and 20 mg of dihydroartemisinin in 10 mL of chloroform solution, and stirring at 37° C. for 24 h to obtain an organic solvent; Step 202: adding the obtained organic solvent dropwise to a suspension of sodium dodecylbenzenesulfonate dispersed in dichloromethane, and then continuing to stir for 24 hours; Step 203: stirring the solvent at a constant speed, and adding ultrapure water dropwise while stirring at a constant speed; Step 204: washing the product obtained in step 203 with ethanol and ultrapure water respectively, centrifuging at 10000 rpm for 10 min, and collecting the supernatant; Step 205: drying the precipitate in a vacuum oven at 60° C. for 8-12 hours; Step 206: The supernatant collected in step 204 is used to measure the drug loading of DHA.

5. The method for preparing a superparamagnetic bionic nano-diagnostic agent according to claim 4, characterized in that: The drug loading amount of DHA measured in step 206 is obtained by mixing DHA in the supernatant with a 0.2% NaOH aqueous solution and heating them at 55±1° C. for 30 minutes.

6. The method for preparing a superparamagnetic bionic nano-diagnostic agent according to claim 4, characterized in that: The step 3 comprises the following steps: Step 301: collect and culture GL261 tumor cells, and wash them three times with PBS; Step 302: disrupting the cells using an ultrasonic disruptor to form a cell disruption solution, and centrifuging the cell disruption solution at a relative centrifugal force of 900 g for 10 minutes at 4° C. to collect the supernatant; Step 303: centrifuging the supernatant collected in step 302 at 4° C. and a relative centrifugal force of 14,000 g for 30 minutes, collecting the precipitate as the cell membrane, and storing it in a refrigerator at 4° C.; Step 304: Re-disperse the cell membrane in PBS by ultrasonication, and then repeatedly squeeze the filter head 10 times; Step 305: The cell membrane in step 304 is dispersed in PBS, placed in an ultrasonic cleaner, and the MD nanodrug solution dispersed in PBS is added dropwise to the PBS, and the mixture is treated with ultrasound for 2 hours, and pushed, and centrifuged at a relative centrifugal force of 900g for 10 minutes at a temperature of 4°C to remove excess cell membrane to obtain a nanodrug suspension of MDM, wherein the nanodrug suspension is MnFe2O4-DHA@CM.

7. The method for preparing a superparamagnetic bionic nano-diagnostic agent according to claim 6, characterized in that: The GL261 tumor cells in step 301 are collected and cultured by centrifugation at 1500 rpm at 4° C. for 4 minutes.

8. The method for preparing a superparamagnetic bionic nano-diagnostic agent according to claim 6, characterized in that: In step 305, the mass ratio of the cell membrane to the MD nanomedicine is 1:2.

5.

9. Application of a superparamagnetic bionic nano-diagnostic agent, prepared by the preparation method according to any one of claims 1 to 8, characterized in that: Superparamagnetic bionic nano-diagnostic and therapeutic agents are used to prepare drugs for treating tumors.

10. The use of a superparamagnetic bionic nano-diagnostic agent according to claim 9, characterized in that: The tumor is a glioma.

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