Photomagnetic in-situ self-assembled photothermal gas cascade bionic nanoreactor, preparation method and application

Abstract

The invention belongs to the field of application of inorganic biomimetic materials to tumor targeted drugs, and particularly discloses a photomagnetic in-situ self-assembled photothermal gas cascade bionic nanoreactor, a preparation method and application. The nanoreactor is composed of spherical carbonyl iron powder in the innermost layer, sea urchin mastoid Fe3O4 attached to the surface of the spherical carbonyl iron powder and a platelet membrane wrapping the outermost layer. The preparation method comprises the following steps: 1) preparation of carbonyl iron powder (CIP): converting sponge iron elementary substance into multi-carbonyl iron under the conditions of high temperature and high pressure, and introducing CO for annealing to obtain the carbonyl iron powder; (2) preparing CIP-Fe3O4: depositing ferroferric oxide on the surface of carbonyl iron powder by using the protection of NH3 on carbonyl in an in-situ coprecipitation manner; and 3) preparation of CIP-coated Fe3O4-coated PM: coating the surface of CIP-coated Fe3O4 with the extracted platelet membrane through ultrasonic and extrusion methods. The CIP (at) Fe3O4 (at) PM nano-reactor disclosed by the invention has the characteristics of optomagnetic in-situ self-assembly capability and photothermal gas cascade, and is used for enhancing the tumor diagnosis and treatment effect.

Description

Technical Field

[0001] The invention belongs to the field of using inorganic bionic materials for tumor-targeted drugs, and specifically discloses a photothermal gas cascade bionic nanoreactor that can be used for anti-cancer photomagnetic in-situ self-assembly. Background Art

[0002] Nano-diagnosis and treatment system is one of the important research directions in precision tumor treatment. It uses nano-systems to achieve visualized drug delivery and treatment monitoring, and improves the diagnosis and treatment effects of tumors through collaboration between diagnosis and treatment.

[0003] Among them, the effective aggregation of nano-diagnostic and therapeutic systems at the tumor site is the key to the diagnosis and treatment of tumors. However, the complex physiological and pathological barriers of tumors cause insufficient penetration and short retention time of nano-diagnostic and therapeutic systems in tumors, making it difficult for them to accumulate at the tumor site, seriously affecting and restricting the effectiveness of tumor diagnosis and treatment.

[0004] In recent years, although traditional magnetic nanoparticles can be guided to the tumor area through an external magnetic field, they are difficult to overcome the high interstitial pressure (IFP) and dense extracellular matrix of the tumor due to the lack of a mechanism to actively destroy the physical barrier of the tumor, resulting in insufficient penetration depth (<200μm) and can only act on the edge area of ​​the tumor. In addition, other studies have shown that although existing photothermal therapy nanosystems (such as gold nanorods and black phosphorus quantum dots) can ablate tumor cells through photothermal effects, their single function cannot achieve integrated diagnosis and treatment, and they lack long-term retention capacity and are easily washed away by dynamic fluids in the tumor microenvironment, and the retention time is usually less than 24 hours. This further reveals the limitations of existing nanocarriers in terms of biocompatibility. Some synthetic materials (such as cationic polymers and inorganic nanocrystals) may induce immunogenic reactions or long-term metabolic toxicity, which seriously restricts their clinical transformation potential.

[0005] In view of the delivery difficulties of nano-diagnostic and therapeutic systems in tumor penetration and retention, breaking through the tumor barrier to effectively penetrate into the tumor and overcoming the elution effect of the high IFP of the tumor on nanoparticles so that they can be fully retained in the tumor are the keys to improving the efficacy of nano-systems in tumor diagnosis and treatment, and are also the key scientific issues that need to be urgently solved in the current tumor nano-diagnostic and therapeutic research. Summary of the invention

[0006] In view of this, the present invention discloses a photothermal-gas cascade bionic nanoreactor with photomagnetic in-situ self-assembly, a preparation method and an application thereof.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] The first technical object of the present invention is to provide a photo-thermal-gas cascade biomimetic nanoreactor with photo-magnetic in-situ self-assembly, and the nanoreactor is composed of spherical carbonyl iron powder at the innermost layer, sea urchin papilla-shaped Fe3O4 attached to the surface of the spherical carbonyl iron powder, and platelet membrane wrapped at the outermost layer.

[0009] It should be noted that the designed CIP@Fe3O4@PM sea urchin-shaped nanoreactor of the present invention has the ability of photo-magnetic in-situ self-assembly and the characteristics of photo-thermal-gas cascade for enhancing the tumor diagnosis and treatment effect.

[0010] The present invention constructs a photo-thermal-gas cascade nanoreactor through nano-carbonyl iron powder, and due to the inheritance of the physical properties of iron powder by nano-carbonyl iron powder, it has photo-thermal characteristics and can realize photo-thermal imaging, can realize ultrasonic imaging due to gas production when heated, and realizes nuclear magnetic resonance imaging with different weightings due to its composite magnetism, thereby enhancing the tumor diagnosis and treatment effect.

[0011] The second technical object of the present invention is to provide a preparation method of the above-mentioned photo-thermal-gas cascade biomimetic nanoreactor with photo-magnetic in-situ self-assembly, including the following steps:

[0012] 1) Preparation of nano-carbonyl iron powder (CIP): Under high temperature and high pressure conditions, sponge iron is converted into polycarbonyl iron, and then CO is introduced for annealing to obtain carbonyl iron powder;

[0013] 2) Preparation of CIP@Fe3O4 nanoreactor: By using the in-situ co-precipitation method and the protection of NH3 for carbonyl groups, magnetite is deposited on the surface of the carbonyl iron powder prepared in step 1) to prepare the CIP@Fe3O4 nanoreactor;

[0014] 3) Preparation of CIP@Fe3O4@PM: The extracted platelet membrane is wrapped on the surface of the CIP@Fe3O4 nanoreactor prepared in step 2) by ultrasonic and extrusion methods to prepare CIP@Fe3O4@PM, that is, the above-mentioned photo-thermal-gas cascade biomimetic nanoreactor with photo-magnetic in-situ self-assembly.

[0015] It should be noted that the present invention innovatively prepares nano-carbonyl iron powder (CIP) by imitating the blast furnace iron-making method; deposits magnetite (Fe3O4) on the surface of nano-carbonyl iron powder (CIP) by the in-situ co-precipitation method to prepare the CIP@Fe3O4 nanoreactor; and prepares the platelet membrane-coated CIP@Fe3O4 nanoreactor (CIP@Fe3O4@PM) by extracting the platelet membrane and applying the biomimetic cell membrane engineering technology.

[0016] Furthermore, in step 1), the preparation of the carbonyl iron powder CIP includes the following steps:

[0017] S1 Use scaly sponge iron as a synthetic raw material, grind it into a powder of about 80 mesh by a ball mill, then carry out reduction at 740 - 760 °C under a hydrogen atmosphere to obtain highly active iron powder, and load it into a high-pressure synthesis reactor while isolating oxygen.

[0018] S2 Pressurize carbon monoxide gas to 290 - 310 MPa with a high-pressure compressor and introduce it into the reactor. At the same time, heat the reactor body to 190 - 210 °C to carry out a high-pressure synthesis reaction to generate iron carbonyl (mainly pentacarbonyl iron), and gradually reduce the pressure and cool it to a liquid state.

[0019] S3 After multiple temperings (150 - 160 °C), pressure reduction (9 - 11 MPa) annealing, and calcination passivation under a nitrogen protection atmosphere, nano iron carbonyl powder is prepared.

[0020] It should be noted that using the prodrug design idea of protecting the central metal iron element with carbonyl coordination, a donor of the NIR light-stimulated responsive gas therapeutic molecule carbon monoxide (CO), nano iron carbonyl powder (CIP), is innovatively prepared by metal smelting technology. It not only inherits the excellent photothermal conversion and ferromagnetic properties of iron powder but also improves the instability of iron powder. At the same time, Fe3O4 is plated on the surface of nano iron carbonyl powder (CIP@Fe3O4) by in-situ self-assembly, endowing the system with superparamagnetism and further protecting the stability of nano iron carbonyl powder.

[0021] Furthermore, in step 2), the preparation of CIP@Fe3O4 includes the following steps:

[0022] Under the conditions of 80 - 90 °C and pH = 10, deposit and grow Fe3O4 on the surface of nano iron carbonyl powder by in-situ coprecipitation using ammonia water, ferrous salt, and ferric salt solutions to prepare a CIP@Fe3O4 core-shell structure nano-reactor with nano iron carbonyl powder as the core and Fe3O4 as the shell layer.

[0023] Furthermore, in step 3), the preparation of CIP@Fe3O4@PM includes the following steps:

[0024] Collect whole blood using ACD vacuum anticoagulant tubes and centrifuge at a centrifugal force of 100 - 200 g for 20 min at room temperature; carefully aspirate the upper white platelet-rich plasma (PRP) and transfer it to another EP tube, and add PGE1 diluent with a final concentration of 1 μM (to avoid platelet activation); centrifuge at a centrifugal force of 800 - 1000 g for 20 min to precipitate platelets from PRP, discard the supernatant plasma, carefully add 0.5 mL of PBS containing 1 - 3 mM PMSF (as a protease inhibitor) along the wall of the EP tube, resuspend the purified platelets, and place them in an -80°C refrigerator for 3 cycles of repeated freezing and thawing, then wash and centrifuge with PBS, and the obtained platelet membrane is resuspended in PBS and stored in an -80°C refrigerator;

[0025] Extract the platelet membrane, apply biomimetic cell membrane engineering technology, and use the methods of ultrasonic treatment and extrusion through a polycarbonate membrane to prepare a platelet membrane-coated CIP@Fe3O4 nanoreactor (CIP@Fe3O4@PM).

[0026] It should be noted that in the present invention, a platelet membrane with a photothermal recruitment effect is coated through cell membrane biomimetic technology to construct a biomimetic platelet membrane nano-carbonyl iron powder@iron tetroxide (CIP@Fe3O4@PM) nanoreactor. The components of this system have a simple composition, a clear structure, and a controllable preparation process. It is worth noting that nano-carbonyl iron powder and Fe3O4 have been separately approved by the FDA for use as iron supplements for clinical injection and contrast agents, and can be metabolized in the body to synthesize hemoglobin, ferritin, etc., and have good biosafety.

[0027] The third technical objective of the present invention is to provide an application of the CIP@Fe3O4@PM nanoreactor as described above in the preparation of anti-tumor drugs.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] The photomagnetic nanoreactor (CIP@Fe3O4@PM) developed in the present invention significantly overcomes the core defects of traditional nanodiagnosis and treatment technologies through multi-dimensional innovative design, and demonstrates breakthrough advantages in tumor targeting penetration, synergistic diagnosis and treatment functions, and clinical application value.

[0030] Aiming at the problems of low-efficiency penetration and short-term retention caused by the tumor barrier in traditional nanodrugs, the present invention proposes a magnetic-optical synergistic driving strategy: after the magnetic field guides the nanoparticles to accurately accumulate in the tumor area, the photothermal effect synergistically destroys the physical barrier, greatly improving the penetration depth; at the same time, in-situ self-assembly is triggered through magnetic field-induced aggregation and biomimetic membrane adhesion, forming stable aggregates, effectively resisting the erosion of the tumor microenvironment, and significantly prolonging the drug retention time, providing a guarantee for long-term treatment.

[0031] In terms of the integration of diagnosis and treatment functions, this system innovatively integrates multimodal imaging and multi-mechanism treatment in depth. Through multi-modal complementarity, the imaging function significantly improves the accuracy of tumor contour recognition and the ability of dynamic monitoring. The treatment function, through the synergistic effects of photothermal, gas, and ferroptosis, can achieve low-temperature and highly efficient ablation of tumor cells while disrupting the tumor metabolic defense mechanism, significantly improving the tumor suppression effect. In particular, its biomimetic platelet membrane coating design not only greatly extends the blood circulation time but also reduces the clearance rate of the body through its immune escape function, significantly enhancing the targeting efficiency.

[0032] In terms of safety and application value, this technology uses biocompatible components to effectively avoid the toxicity risks of traditional nanomaterials, and its biomimetic design significantly reduces immunogenicity, ensuring the safety of long-term use. Its "diagnosis-treatment-monitoring" integrated closed-loop mode not only greatly reduces medical costs but also optimizes clinical decisions through precise visualization of treatment, especially providing an efficient and inclusive new option for patients with deep tumors and advanced patients lacking targeted treatment options.

[0033] Overall, this technology provides the possibility for the clinical transformation of the nano-diagnosis and treatment system by breaking through the penetration and retention bottleneck, realizing the synergy of diagnosis and treatment functions, and modular expansion potential. Brief Description of the Drawings

[0034] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on the provided drawings.

[0035] Figure 1 Transmission electron microscope (TEM), scanning electron microscope (SEM), high-angle annular dark-field scanning transmission electron microscope (HADDF-STEM), and energy-dispersive X-ray spectroscopy mapping (EDS-Mapping) images of nano-carbonyl iron powder (Nano-CIP); scale bar = 50 nm.

[0036] Figure 2 Transmission electron microscope (TEM), scanning electron microscope (SEM), high-angle annular dark-field scanning transmission electron microscope (HADDF-STEM), and energy-dispersive X-ray spectroscopy mapping (EDS-Mapping) images of CIP@Fe3O4 nano-reactor; scale bar = 50 nm.

[0037] Figure 3 Particle size and Zeta potential of CIP@Fe3O4 and CIP@Fe3O4@PM (mean ± standard deviation, n = 3).

[0038] Figure 4 Infrared spectrum of the CIP@Fe3O4 nanoreactor.

[0039] Figure 5 X-ray diffraction pattern of the CIP@Fe3O4 nanoreactor.

[0040] Figure 6 Mössbauer spectrum of the CIP@Fe3O4 nanoreactor.

[0041] Figure 7 Transmission electron microscope image of platelet membrane vesicles.

[0042] Figure 8 Transmission electron microscope (TEM), scanning electron microscope (SEM), high-angle annular dark-field scanning transmission electron microscope (HADDF-STEM), and energy-dispersive X-ray spectroscopy mapping (EDS-Mapping) images of CIP@Fe3O4@PM, scale bar = 30 nm.

[0043] Figure 9 Photothermal imaging of different concentrations of CIP@Fe3O4@PM (A) and heating curve (B).

[0044] Figure 10 In vitro ultrasound B-mode and contrast-enhanced mode images of CIP@Fe3O4@PM.

[0045] Figure 11 In (A), hysteresis loop; in (B), atomic force microscope (AFM, upper figure) and magnetic force microscope (MFM, lower figure) images of CIP@Fe3O4; in (C), magnetic needle deflection response of nanoparticles under MFM scanning; in (D, E), T1-weighted MRI images and relaxation rate analysis of CIP@Fe3O4 and CIP@Fe3O4@PM at specified concentrations; in (F, G), T2-weighted MRI images and relaxation rate analysis. Detailed implementation manners

[0046] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0047] The special term "embodiment" here, any embodiment described as "exemplary" does not have to be construed as superior to or better than other embodiments. For the performance index tests in the embodiments of this application, unless otherwise specified, conventional test methods in the art are used. It should be understood that the terms described in this application are only for describing specific embodiments and are not used to limit the content disclosed in this application.

[0048] Unless otherwise specified, the technical and scientific terms used in this document have the same meanings as those commonly understood by those of ordinary skill in the technical field to which this application belongs; the test methods and technical means not specifically noted in this application refer to the experimental methods and technical means commonly adopted by those of ordinary skill in the art.

[0049] In the description of the present invention, it should be understood that the orientation or positional relationships indicated by the terms "middle", "upper", "lower", "rise", "fall", "vertical", "surface", "top", "bottom", "inner", "outer", etc. are based on the orientation or positional relationships shown in the drawings. These are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and thus should not be construed as a limitation to the present invention.

[0050] To better illustrate the content of this application, numerous specific details are given in the following specific embodiments. Those skilled in the art should understand that the present application can still be implemented without certain specific details. In the embodiments, some methods, means, instruments, devices, etc. well-known to those skilled in the art are not described in detail in order to highlight the gist of this application.

[0051] On the premise of no conflict, the technical features disclosed in the embodiments of this application can be combined arbitrarily, and the obtained technical solutions belong to the content disclosed in the embodiments of this application; and for the reagents or instruments used in the embodiments of the present invention that are not marked with the manufacturer, they are all conventional reagent products that can be obtained through commercial purchase.

[0052] The present invention discloses a photo-thermal gas cascade biomimetic nanoreactor for anti-cancer and its preparation method by photo-magnetic in-situ self-assembly.

[0053] To better understand the present invention, the following embodiments are used to further specifically elaborate on the present invention, but it should not be construed as a limitation to the present invention. For those non-essential improvements and adjustments made by those skilled in the art based on the above-mentioned inventive content, they are also considered to fall within the protection scope of the present invention.

[0054] Example 1

[0055] (1) Preparation of nano-carbonyl iron powder (CIP):

[0056] First, take scaly sponge iron as the synthetic raw material, grind it into a powder of about 80 mesh by a ball mill, then reduce it at 750 °C under a hydrogen atmosphere to obtain highly active iron powder, which is filled into a high-pressure synthesis reactor while isolating oxygen. Press carbon monoxide gas to 300 MPa with a high-pressure compressor and introduce it into the reactor. At the same time, heat the reactor body to 200 °C to carry out a high-pressure synthesis reaction to generate iron carbonyl (mainly pentacarbonyl iron), which is gradually depressurized and cooled to a liquid state. After multiple temperings (155 °C), pressure reduction (10 MPa) annealing, and calcination passivation under a nitrogen protection atmosphere, solid-state nano iron carbonyl powder is prepared.

[0057] (2) Preparation of CIP@Fe3O4 nano-reactor:

[0058] First, add 10 mL of an alcohol-water (5:1) system to a three-necked flask as the bottom liquid and stir magnetically at 25 °C for 20 min. Weigh 75 mg of Fe2(SO4)3·6H2O and 55 mg of FeSO4·7H2O and dissolve them in 10 mL of deionized water, then ultrasonicate for 10 min to obtain a mixed iron salt solution. Dilute 0.25 mL of ammonia water (w = 25%) to 10 mL with deionized water. Then, drop the iron salt solution and ammonia water into the three-necked flask at a rate of one drop every 3 seconds (1 mL syringe). After the dropping is complete, stir for 20 min, raise the water bath temperature to 80 °C, and add 1 mL of an aqueous solution containing 37.5 mg of PEG-2000. Continue to stir for 30 min, then cool the temperature to room temperature. Stir and age for one day, wash several times with deionized water and ethanol, and then dry in a vacuum oven at 70 °C for 12 hours. In-situ co-precipitation method is used to deposit and grow Fe3O4 on the surface of nano iron carbonyl powder to prepare a CIP@Fe3O4 core-shell structure nano-reactor with nano iron carbonyl powder as the core and Fe3O4 as the shell layer.

[0059] (3) Preparation of CIP@Fe3O4@PM nano-reactor:

[0060] Collect whole blood using an ACD vacuum anticoagulation tube and centrifuge at a centrifugal force of 150 g for 20 min at room temperature; carefully aspirate the upper white platelet-rich plasma (PRP) and transfer it to another EP tube, and add a PGE1 dilution solution with a final concentration of 1 μM; centrifuge at a centrifugal force of 900 g for 20 min to precipitate platelets from PRP, discard the supernatant plasma, carefully add 0.5 mL of PBS containing 2 mM PMSF along the wall of the EP tube, resuspend the purified platelets, and place them in a -80 °C refrigerator for 3 repeated freeze-thaw cycles, then wash and centrifuge with PBS. The obtained platelet membrane is resuspended in PBS and stored in a -80 °C refrigerator.

[0061] The uniformly sized platelet membranes prepared were ultrasonically treated with CIP@Fe3O4 sea urchin-like core-shell nanoparticles at a power of 90 - 110 W and a frequency of 40 kHz for 10 min, and then repeatedly extruded using a liposome extruder through polycarbonate (PC) membranes with different pore sizes, with 10 - 15 repeated extrusions under each PC membrane with a different pore size, to obtain CIP@Fe3O4@PM nano-reactors.

[0062] The chemical structures and elemental compositions of the prepared nano-carbonyl iron powder (CIP), CIP@Fe3O4 nano-reactors, and CIP@Fe3O4@PM nano-reactors were detected as follows:

[0063] The apparent state was analyzed by SEM. The nanoparticles were spherical with a smooth surface. Combining with TEM characterization, the average particle size was 40 nm ( Figure 1 ). Elemental analysis by EDS-Mapping showed that Fe, C, and O were uniformly distributed, and the signal values of the elemental contents of C and O were close to 1:1, which also confirmed the successful preparation of nano-carbonyl iron powder. In addition, the results of selected area electron diffraction (SAED) showed that there was a polycrystalline structure in the nano-carbonyl iron powder.

[0064] Based on the bottom-up theory, Fe3O4 was deposited on the surface of nano-carbonyl iron powder (nano-CIP) by in-situ co-precipitation method ( Figure 2 ), to form CIP@Fe3O4 nano-reactors with a core-shell structure, whose morphology was similar to that of a sea urchin. The elemental composition and chemical state of CIP@Fe3O4 were analyzed by using high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) combined with energy spectrum mapping (EDS-mapping) technology. The results showed that the characteristic elements Fe, C, and O were uniformly distributed in the CIP@Fe3O4 nano-reactors, with an average particle size of 63.7 nm and a Zeta potential of 5.6 mV ( Figure 3 ). The Fourier transform infrared spectrum (FTIR) of the CIP@Fe3O4 nano-reactors showed ( Figure 4 ) that there were metal coordination carbonyl vibration peaks and Fe-O bond vibration peaks. Among them, the absorption peaks at 638 cm - -1 and 617 cm - -1 corresponded to Fe-O vibration, and the reflection peak at 793 cm - -1 corresponded to Fe atoms. In addition, a radiation peak consistent with Fe atoms was observed at 1034 cm - -1, and the absorption peak at 1609 cm - -1 could be attributed to the C=O bond. The X-ray diffraction (XRD) pattern showed the characteristic diffraction peaks of Fe-O bonds, indicating that Fe3O4 was deposited on the surface of nano-CIP in a crystalline form. Figure 5)。To further verify the valence state of iron, Mössbauer spectroscopy analysis was carried out, as Figure 6 shown. The Mössbauer spectrum of CIP@Fe3O4 presented sextuplet fine-splitting peaks, indicating the simultaneous presence of Fe, Fe2 + and Fe3 + in CIP@Fe3O4.

[0065] After multiple centrifugations to remove impurities, the protein impurities in the lipid were significantly reduced. Under a fluorescent light source, a transparent light yellow lipid precipitate could be observed at the bottom of the EP tube after centrifugation, indicating that the platelet membrane had been preliminarily purified. Subsequently, by ultrasonic method and polycarbonate membrane extrusion method, the platelet membrane was successfully prepared into a uniform platelet vesicle solution (the same method as in Example 1), and its appearance showed a uniform and transparent state. As Figure 7 shown, the transmission electron microscope (TEM) image showed that the platelet vesicles were of uniform size, further proving the reliability and good reproducibility of this method.

[0066] Platelet membrane vesicles (PM vesicles) were prepared by differential centrifugation of ultrasonically disrupted platelets; subsequently, the PM vesicles were coated on the surface of CIP@Fe3O4 nanoreactors by ultrasonic method and polycarbonate membrane extrusion method (the same method as in Example 1). As Figure 8 , high-resolution transmission electron microscope (HR-TEM) characterization showed that there was a membrane structure on the protruding surface of the CIP@Fe3O4 nanoreactor, and the size was uniform and well-dispersed. In addition, the results of high-angle annular dark-field scanning transmission electron microscope (HAADF-STEM) showed that the PM vesicles were successfully attached to the surface of the sea urchin-like nanostructure. The relatively high biomimetic membrane coverage rate on the surface of CIP@Fe3O4@PM might enhance its photothermal recruitment and aggregation ability. Compared with the EDS energy spectrum mapping analysis results (Fe, C, and O elements) of CIP@Fe3O4, the EDS energy spectrum mapping of CIP@Fe3O4@PM showed that its outer layer contained N, P, and S elements, while the core region was still Fe, C, and O elements. In addition, due to the coating of negatively charged PM vesicles, the average particle size of CIP@Fe3O4@PM increased to 85.6 nm, and the Zeta potential reversed to -11.3 mV ( Figure 3 ).

[0067] Test Examples

[0068] It should be noted that the CIP@Fe3O4@PM nanoreactors used in the following test examples were prepared by the method described in Example 1.

[0069] 1) Experiment on exploring the photothermal conversion ability

[0070] The photothermal performance of CIP@Fe3O4@PM nanoreactors under 808 nm laser irradiation was evaluated using an infrared camera system. Aqueous solutions of CIP@Fe3O4@PM nanoreactors with concentration ranges of 25, 50, 100, 200 μg / mL were irradiated with an 808 nm laser at a power of 0.5 W / cm 2 for 5 minutes, and the temperature changes were recorded every 30 seconds using an infrared camera, with water as a control for comparison. The photothermal conversion efficiency of CIP@Fe3O4@PM was calculated using the following formula.

[0071]

[0072] t = -τ s lnθ

[0073]

[0074] By monitoring the temperature changes of the nanoreactor aqueous dispersion under 808 nm laser (power of 0.5 W / cm 2 ) irradiation, the ability of CIP@Fe3O4@PM nanoreactors to capture near-infrared light and convert it into heat was evaluated. Compared with nano-CIP, the CIP@Fe3O4@PM nanoreactors showed a higher photothermal conversion efficiency (46.1%) under 808 nm near-infrared light (0.5 W / cm 2 ) irradiation, and the highest temperature of the 200 μg / mL solution rose to 64.3 °C after 5 minutes of irradiation ( Figure 9 ). The stronger photothermal effect of CIP@Fe3O4@PM can be attributed to the deposition of Fe3O4.

[0075] 2) Gas generation evaluation and in vitro ultrasonic imaging ability investigation experiment

[0076] To prepare an agarose hydrogel of CIP@Fe3O4@PM nanoreactors at 200 μg / mL, first, 2 g of agarose powder was precisely weighed, added to ultrapure water and made up to 100 mL in a mold, boiled, and when cooled to about 37 °C, 20 mg of precisely weighed CIP@Fe3O4@PM nanoreactors was quickly added and stirred evenly. It was cooled overnight at 4 °C, and the next day, it was checked with a strong flashlight to ensure there were no bubbles and it was clear and transparent. Then it was compared with a blank agarose hydrogel without CIP@Fe3O4@PM nanoreactors for standby. An ultrasonic imaging system (GE Logiq E9) was used to record the in vitro energy Doppler and B-mode ultrasonic imaging of CIP@Fe3O4@PM nanoreactors at a concentration of 0.1 mg / mL in aqueous solution. The experiment was carried out at a power of 0.5 W / cm 2It was carried out 5 minutes after irradiation with 808 nm laser. For ultrasonic imaging, a 15L8-w broadband high-frequency linear probe with a frequency of 10 MHz and a transmission power of 18 dB was used. The CIP@Fe3O4@PM nanoreactor without laser irradiation was used as a control group for comparison.

[0077] The CO gas-responsive release behavior triggered by the cleavage of Fe-CO coordination bonds under near-infrared light (808 nm, 0.5 W / cm 2 ) irradiation of CIP@Fe3O4@PM was verified by in vitro ultrasonic (US) imaging experiments. As shown in Figure 18, after laser irradiation, a large number of CO microbubbles were generated by CIP@Fe3O4@PM, showing high-intensity signals in the ultrasonic B-mode and obvious cloud-like echo signals could be observed in the contrast-enhanced mode ( Figure 10 ). It should be noted that an explosive CO microbubble release phenomenon was observed at the boundary of the irradiation area of CO release: the microbubbles showed an edge diffusion effect and formed an olive-shaped contour.

[0078] 3) Gas generation evaluation and in vitro ultrasonic imaging ability investigation experiment

[0079] Measurement of the hysteresis curve: The completely freeze-dried CIP@Fe3O4 sea urchin-like core-shell nanoparticles were fixed on the sample rod to ensure that the sample position was centered and firm. The instrument was calibrated to eliminate zero drift and background signals. The magnetic field strength was gradually changed, and the magnetization intensity of the sample at different magnetic fields (M-H curve) was recorded, and the key magnetic parameters, coercivity (Hc), remanence (Mr), and saturation magnetization intensity (Ms) were calculated.

[0080] Investigation of magnetic domains by magnetic microscopy: The completely freeze-dried CIP@Fe3O4 sea urchin-like core-shell nanoparticles were sampled by the "tapping mode", and preliminary atomic force microscopy mode scanning was carried out. After obtaining the preliminary nanoparticle profile, the nanoparticles were lightly scanned in the "lift mode" to obtain an intuitive observation and deflection angle of the magnetic domain region.

[0081] Investigation of transverse relaxation rate and longitudinal relaxation rate: CIP@Fe3O4 and CIP@Fe3O4@PM nanoreactors with concentration gradients from 0.001 to 1.0 mg / mL were analyzed by T1 (longitudinal) and T2 (transverse) weighted magnetic resonance imaging (MRI) using a nuclear magnetic resonance imaging instrument. T1 weighted MRI measurement used the T1 sequence with the following parameters: repetition time (TR) = 1000 ms, echo time (TE) = 8.5 ms, inversion time decreased from 5500 ms to 327.103 ms, and the matrix size was 256×256. T2 weighted MRI measurement used the T2 sequence with the following parameters: TR = 2500 ms, TE = 33 ms, inversion time from 11 ms to 165 ms, and the matrix size was 256×256.

[0082] The response characteristics of CIP@Fe3O4@PM to an external magnetic field were evaluated using a vibrating sample magnetometer (VSM) and a magnetic force microscope (MFM). VSM tests showed that the hysteresis loop of CIP@Fe3O4 had low coercivity and high remanence characteristics, and the saturation magnetization was as high as 204.8 emu / g( Figure 11 A), indicating its fast magnetic response ability. The morphology and ferromagnetic properties of CIP@Fe3O4 were characterized by the tapping mode of atomic force microscopy (AFM) and the lift mode of MFM( Figure 11 B-C). As Figure 3 shown in C, a 1.3° deflection occurred when the magnetic needle swept over the nanoparticles, confirming its ferromagnetic properties.

[0083] Based on the formula Emag = -μHmI / I, Mössbauer spectroscopy analysis showed that although the asymmetric electric field would cause the splitting of nuclear magnetic energy levels, the magnetic quantum numbers (±mI) still belonged to the same energy level. Under the action of an external magnetic field, the interaction between the nuclear magnetic moment and the magnetic field caused further splitting of the nuclear magnetic energy levels, forming 2I + 1 magnetic energy levels. The γ-ray diffraction analysis of CIP@Fe3O4 showed that the incident γ photons excited the Fe nuclei to the I = 1 / 2 and 3 / 2 energy levels, which split into 6 energy levels under the action of the intrinsic magnetic field H( Figure 6 ). The splitting of the nuclear magnetic energy levels led to an increase in the number of Mössbauer spectral lines, manifested as magnetic hyperfine splitting (i.e., the Zeeman effect).

[0084] In view of the presence of superparamagnetic Fe3O4 in the nanoreactor, the potential of CIP@Fe3O4@PM as a T1-MRI contrast agent was further evaluated. Experiments showed that the CIP@Fe3O4 solution showed a significant signal enhancement effect in the T1-weighted mode, while it showed a signal attenuation in the T2-weighted mode, and the signal intensity was positively correlated with the concentration( Figure 11 D-E). After calculation, the longitudinal relaxation rate (r1) of CIP@Fe3O4 was 88.1 mM - 1·s - 1( Figure 11 F), and the transverse relaxation rate (r2) was 601.5 mM - 1·s - 1( Figure 11 G). After the biomimetic coating of platelet membrane (PM), the signal enhancement effect of T1-weighted and the signal attenuation effect of T2-weighted decreased slightly. The r1 and r2 of CIP@Fe3O4@PM decreased to 59.07 mM - 1·s - 1 and 543.5 mM - 1·s - 1, respectively. In summary, CIP@Fe3O4@PM can be used as a high-performance T1-T2 dual-weighted MRI contrast enhancer, providing a new strategy for accurate tumor imaging.

[0085] The foregoing description of the disclosed embodiments enables those skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A photo-thermal gas cascade biomimetic nanoreactor with in-situ photo-magnetic self-assembly, characterized in that The described nano-reactor consists of spherical carbonyl iron powder in the innermost layer, sea urchin papilla-shaped Fe3O4 attached to the surface of the spherical carbonyl iron powder, and a platelet membrane wrapped around the outermost layer.

2. A preparation method of the photo-thermal-gas cascade biomimetic nanoreactor with photo-magnetic in-situ self-assembly as described in claim 1, characterized in that, It includes the following steps: 1) Preparation of nano-carbonyl iron powder CIP: Under high temperature and high pressure conditions, sponge iron is converted into polycarbonyl iron, and then annealed with CO to obtain carbonyl iron powder; 2) Preparation of CIP@Fe3O4 nano-reactor: By means of in-situ co-precipitation, using NH3 to protect the carbonyl group, Fe3O4 is deposited on the surface of the carbonyl iron powder prepared in step 1) to prepare the CIP@Fe3O4 nano-reactor; 3) Preparation of CIP@Fe3O4@PM: The extracted platelet membrane is wrapped around the surface of the CIP@Fe3O4 nano-reactor prepared in step 2) by ultrasonic and extrusion methods to prepare CIP@Fe3O4@PM, that is, the described photo-magnetic in-situ self-assembled photo-thermal gas cascade biomimetic nano-reactor.

3. The preparation method of the photo-thermal-gas cascade biomimetic nano-reactor with photo-magnetic in-situ self-assembly according to claim 2, wherein, In step 1), the preparation of the carbonyl iron powder CIP includes the following steps: S1. Use scaly sponge iron as the synthesis raw material, grind it into a powder of 70-90 mesh by a ball mill, and then reduce it at 740-760 °C in a hydrogen atmosphere to obtain highly active iron powder, which is filled into a high-pressure synthesis reactor after isolating oxygen; S2. Press carbon monoxide gas to 290-310 MPa by a high-pressure compressor and introduce it into the reactor. At the same time, heat the reactor body to 190-210 °C to carry out a high-pressure synthesis reaction to generate polycarbonyl iron, which is gradually depressurized and cooled to a liquid; S3. After tempering at 150-160 °C for multiple times, annealing under reduced pressure at 9-11 MPa, and calcining and passivating under a N2 protection atmosphere, nano-carbonyl iron powder is obtained.

4. The preparation method of the optothermal-gas cascade biomimetic nanoreactor with optomagnetic in-situ self-assembly according to claim 2, wherein, In step 2), the preparation of the CIP@Fe3O4 nano-reactor includes the following steps: Deposit and grow Fe3O4 on the surface of the nano-carbonyl iron powder by in-situ co-precipitation method with ammonia water, ferrous salt and ferric salt solutions at 80-90 °C and pH = 10 to prepare a CIP@Fe3O4 core-shell structure nano-reactor with nano-carbonyl iron powder as the core and Fe3O4 as the shell layer.

5. The preparation method of the photo-thermal gas cascade biomimetic nano-reactor with photo-magnetic in-situ self-assembly according to claim 2, characterized in that, In step 3), the preparation of CIP@Fe3O4@PM includes the following steps: Collect whole blood with an ACD vacuum anticoagulant tube and centrifuge it at a centrifugal force of 100-200 g for 20 min at room temperature; carefully aspirate the upper white platelet-rich plasma (PRP) and transfer it to another EP tube, and add a PGE1 dilution solution with a final concentration of 1 μM; centrifuge the PRP at a centrifugal force of 800-1000 g for 20 min to precipitate platelets, discard the supernatant plasma, carefully add 0.5 mL of PBS containing 1-3 mM PMSF along the wall of the EP tube, resuspend the purified platelets, and place them in a -80 °C refrigerator for 3 repeated freeze-thaw cycles, then wash and centrifuge with PBS. The obtained platelet membrane is resuspended in PBS and stored in a -80 °C refrigerator; The extracted platelet membrane is wrapped around the surface of the CIP@Fe3O4 prepared in step 2) by ultrasonic and polycarbonate membrane extrusion methods to prepare CIP@Fe3O4@PM.

6. Use of the photo-thermal-gas cascade biomimetic nanoreactor prepared by photo-magnetic in-situ self-assembly as described in claim 1 or the photo-thermal-gas cascade biomimetic nanoreactor prepared by the method as described in claim 2 in the preparation of anti-tumor drugs.

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