Preparation method and application of POCD nano probe

Abstract

According to the preparation method and application of the POCD nano-probe, the probe has MRI (Magnetic Resonance Imaging) capability and drug loading capability and can promote drug release, so that the treatment effect is achieved. The method comprises the following steps: (1) preparing Fe3O4 nanoparticles; (2) preparing Fe3O4 (at) mPDA nano particles; (3) preparing an LF-Fe3O4 (at) mPDA nano particle; and (4) preparation of LF-RSV-Fe3O4-coated mPDA nanoparticles: RSV is dissolved in a 50% ethanol solution, then the RSV and the LF-Fe3O4-coated mPDA nanoparticles are ultrasonically mixed uniformly according to a ratio of 5: 1, the mixture is stirred at room temperature for 24 hours, centrifugally collected and washed three times with deionized water, and the LF-RSV-Fe3O4-coated mPDA nanoparticles are obtained.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical detection and medical image processing, and in particular relates to a method for preparing a POCD nanoprobe, and application of the nanoprobe in preparing a POCD nanoprobe for treating postoperative cognitive dysfunction. Background Art

[0002] Postoperative cognitive dysfunction (POCD) refers to acute or early brain dysfunction that occurs after surgery even though the patient does not have a mental illness before surgery. It is generally affected by surgery and is a complication that occurs after surgery. Postoperative cognitive dysfunction has a high prevalence in the elderly population and is characterized by a series of negative effects such as impaired memory, decreased information processing ability, and decreased attention, which seriously reduces the patient's quality of life.

[0003] The exact mechanism of the etiology and pathophysiology of POCD is not fully understood, and it may be closely related to the following factors: anesthesia, surgical trauma, inflammation, and neurovascular changes. Anesthesia-related factors (such as the type and duration of anesthesia) and surgery-related factors (such as the invasiveness and duration of surgery) play an important role. Studies have shown that various factors may increase the risk of POCD, such as advanced age, pre-existing cognitive impairment, various complications such as cardiovascular disease and diabetes, genetic factors, and perioperative factors. At present, the diagnostic criteria for POCD are constantly evolving, but the commonly used method is still neuropsychological testing, which is used to assess cognitive function before and after surgery. However, this traditional method has great challenges in implementation. First, in clinical work, it is necessary to make a comprehensive selection based on different cognitive domains and lobes. Various scales increase the difficulty of medical workers in choosing; secondly, the diversity of scales leads to longer questioning time; finally, it is difficult for patients with low education level, which may lead to inaccurate data results. In addition, the treatment of POCD is mainly prevention, including optimizing perioperative care, reducing anesthesia exposure, effectively controlling pain, and effective cognitive stimulation training after surgery. There is currently a lack of effective therapeutic drugs. Therefore, finding new diagnostic and treatment options for POCD patients is an important research goal of this topic.

[0004] Dysfunction of the blood-brain barrier is one of the important factors causing POCD. In recent years, many discoveries have been made on the mechanism of POCD, including the inflammatory response of the peripheral circulatory system caused by surgery, the inflammatory response of the central nervous system, the activation of glial cells, and the effect of oxidative stress on neuronal integrity. The blood-brain barrier is a natural protective barrier in the brain. Due to its highly selective permeability, the BBB has also become a huge obstacle for drugs to successfully enter the brain. Neither macromolecular drugs nor most anti-tumor drugs can pass through the BBB, which hinders the effective treatment of brain-related diseases. With the rapid development of brain drug transport research, nanoparticle drug delivery systems have shown great potential in the treatment of brain diseases. Nanoparticles including liposomes, micelles, inorganic nanoparticles, hybrid nanoparticles and exosomes have been used in preclinical studies for the treatment of brain diseases. More and more research evidence shows that blood-brain barrier dysfunction is an important factor causing POCD, but it is also because of the existence of the blood-brain barrier that the bioavailability of many macromolecular drugs is extremely low and the therapeutic effect of drugs is poor.

[0005] Lactoferrin is a widely used target substance in brain drug delivery. Lactoferrin is a natural glycoprotein and a member of the transferrin family. It has antiviral, anti-inflammatory, antioxidant, anti-cancer and immunostimulatory effects. Because the receptors of lactoferrin are overexpressed on many cell surfaces, it has active targeting potential and can bind to the TF receptor (TFR) and LF membrane internalization receptor (LFR) highly expressed on the surface of highly proliferating cancer cells and endothelial cells of the blood-brain barrier. LF can cross the blood-brain barrier and has been proven to be a good candidate for the manufacture of nanocarriers. Resveratrol is a natural chemical extracted from plants with anti-inflammatory effects. Its role in improving cognitive function is increasingly valued and has a certain improvement effect on postoperative cognitive dysfunction.

[0006] Photothermal therapy (PTT) has been recognized as a valuable treatment method in the biomedical field. However, it also faces several key problems that need to be solved. A major problem is that overheating can cause thermal resistance in the treated cells, which may significantly reduce the therapeutic effect of PTT. Another problem is that the heat cannot be effectively controlled and may spread to the surrounding healthy tissue, posing a potential threat to it. Therefore, how to optimize PTT to solve these problems is the focus of current research. Summary of the invention

[0007] In order to overcome the defects of the prior art, the technical problem to be solved by the present invention is to provide a method for preparing a POCD nanoprobe, which has MRI imaging capability and drug loading capacity and can promote drug release, thereby achieving a therapeutic effect.

[0008] The technical solution of the present invention is: a method for preparing a POCD nanoprobe, comprising the following steps:

[0009] (1) Preparation of Fe3O4 nanoparticles;

[0010] (2) Preparation of Fe3O4@mPDA nanoparticles: The prepared Fe3O4 nanoparticles were dissolved in TMB solution and ultrasonically dispersed to form an emulsion oily liquid. P123, F127 and dopamine hydrochloride were dissolved in ethanol solution and ultrasonically dissolved completely. The TMB solution containing Fe3O4 was added to the above solution system, mixed and ultrasonicated for 1 hour, and a mechanical stirring device was installed. After stirring evenly, ammonia water was quickly added to the reaction system, reacted for 4 hours, and collected after centrifugation.

[0011] Fe3O4@mPDA was washed alternately with water and ethanol three times, and the obtained product was freeze-dried and stored;

[0013] (3) Preparation of LF-Fe3O4@mPDA nanoparticles: LF was dissolved in PBS, EDC:NHS was added to the LF solution, mixed and placed in a 4°C refrigerator for activation for 4 h, and then Fe3O4@mPDA was added to the activated LF solution. The pH value of the mixed solution was adjusted to 8.5 with ammonia water, stirred at room temperature for 12 h, collected by centrifugation, and washed with deionized water.

[0014] Wash, freeze-dry and store at -20℃ for later use;

[0015] (4) Preparation of LF-RSV-Fe3O4@mPDA nanoparticles: RSV was dissolved in 50% ethanol solution, and then ultrasonically mixed with LF-Fe3O4@mPDA nanoparticles in a ratio of 5:1, stirred at room temperature for 24 hours, collected by centrifugation, and washed three times with deionized water to obtain LF-RSV-Fe3O4@mPDA nanoparticles.

[0016] The present invention successfully constructed a new nanoprobe LF-RSV-Fe3O4@mPDA, which has excellent MRI imaging capability and photothermal effect, has targeting performance and high biosafety, and has a drug-loading function capable of experimental drug transport; at the cellular level, it was confirmed that the new LF-RSV-Fe3O4@mPDA nanoprobe has good cell uptake ability, can achieve MRI imaging and reduce the generation of ROS at the cellular level.

[0017] Also provided is the application of the POCD nanoprobe, which is used in preparing a drug for treating postoperative cognitive dysfunction. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is the research technology roadmap of the present invention.

[0019] Figure 2 Schematic diagram of the synthesis of POCD nanoprobe according to the present invention.

[0020] Figure 3 TEM images of nanoprobes. (a) TEM image of Fe3O4, (b)

[0021] TEM image of Fe3O4@mPDA.

[0022] Figure 4 Microscopic infrared images of Fe3O4@mPDA, RSV-Fe3O4@mPD, and LF-RSV-Fe3O4@mPDA.

[0023] Figure 5 The photothermal properties of LF-RSV-Fe3O4@mPDA. (a) Temperature-time curve after 808 laser irradiation for 10 minutes at different power densities. (b) The power density is 1.0 W cm -2 Temperature-time curve after 808 laser irradiation for 10 minutes. (c) Real-time thermal image corresponding to Figure Ⅰa, and real-time thermal image corresponding to Figure Ⅱb (d) Photothermal stability of the nanoprobe.

[0024] Figure 6 Drug release of LF-RSV-Fe3O4@mPDA nanoprobe under NIR response.

[0025] Figure 7 MRI imaging performance of LF-RSV-Fe3O4@mPDA nanoprobe (a) Relaxation fitting curve of LF-RSV-Fe3O4@mPDA nanoprobe: transverse relaxation image 1 / T2; (b) T2-weighted images of LF-RSV-Fe3O4@mPDA nanoprobes with different concentrations.

[0026] Figure 8 The effect of different concentrations of H2O2 on the viability of SH-SY5Y cells, n=4,

[0027] ***p<0.001.

[0028] Fig. 9 The effects of different concentrations of LF-RSV-Fe3O4@mPDA nanoprobes on the activity of SH-SY5Y cells and bEnd.3 cells.

[0029] Fig.10 T2-weighted images of LF-RSV-Fe3O4@mPDA nanoprobe at the cellular level under different concentration gradients.

[0030] Fig.11Evaluation of the photothermal performance of nanoprobes at the cellular level (a) Effect of different NIR irradiation times on cell viability. (b) Cell viability treated with H2O2, free RSV, and nanoprobes.

[0031] (n=3).

[0032] Fig.12 Figure 2 shows the evaluation of cell phagocytosis and targeting of nanoprobes. (a) Microscope images of cells incubated with Fe3O4@mPDA and LF-Fe3O4@mPDA for 2 and 4 hours; blue fluorescence is the cell nucleus labeled with DAPI, and red fluorescence is the nanoprobe loaded with RhB; (b) Statistical graph of data analysis of rhodamine B fluorescence intensity using ImageJ software (n=3).

[0033] Fig.13 Nanoprobe ROS detection: (a) control group; (b) fluorescence image of LF-RSV-Fe3O4@mPDA group; (c) fluorescence image of H2O2 group; (d) fluorescence image of LF-RSV-Fe3O4@mPDA+H2O2 group; (e) statistical graph of average fluorescence intensity of each group (n=3, ***p<0.001).

[0034] Fig.14 Figure 1 shows the evaluation of model building. (a) The time mice spend in the new arm of the Y-maze; (b) The level of IL-6 in the blood samples of mice, statistical processing results, *p<0.05, **p<0.01, ***p<0.001.

[0035] Fig.15 The in vivo biosafety assessment of nanoprobes. (a) Body weight change curve within 15 days after the mice were injected with nanoprobes through the tail vein; (b) Blood routine indexes after the mice were injected with different nanoprobes through the tail vein (n=4); (c) Analysis of tissue sections of major organs after the mice were injected with different nanoprobes through the tail vein (HE, 200×). DETAILED DESCRIPTION

[0036] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0037] In order to make the description of the present disclosure more detailed and complete, the following provides an illustrative description of the implementation and specific embodiments of the present invention; however, this is not the only form of implementing or using the specific embodiments of the present invention. The implementation covers the features of multiple specific embodiments and the method steps and sequences used to construct and operate these specific embodiments. However, other specific embodiments can also be used to achieve the same or equivalent functions and step sequences.

[0038] like Figure 1 , 2 As shown, a method for preparing a POCD nanoprobe comprises the following steps:

[0039] (1) Preparation of Fe3O4 nanoparticles;

[0040] (2) Preparation of Fe3O4@mPDA nanoparticles: The prepared Fe3O4 nanoparticles were dissolved in

[0041] In TMB solution, ultrasonic dispersion is performed to form an emulsion oily liquid. P123, F127 and dopamine hydrochloride are dissolved in ethanol solution and ultrasonically dissolved completely. TMB solution with Fe3O4 is added to the above solution system, mixed with ultrasound for 1 hour, equipped with a mechanical stirring device, and after stirring evenly, ammonia water is quickly added to the reaction system, reacted for 4 hours, and Fe3O4@mPDA is collected after centrifugation, washed with water and ethanol alternately for 3 times, and the obtained product is freeze-dried for storage;

[0042] (3) Preparation of LF-Fe3O4@mPDA nanoparticles: LF was dissolved in PBS, EDC:NHS was added to the LF solution, mixed and placed in a 4°C refrigerator for activation for 4 h, and then Fe3O4@mPDA was added to the activated LF solution. The pH value of the mixed solution was adjusted to 8.5 with ammonia water, stirred at room temperature for 12 h, collected by centrifugation, washed with deionized water, freeze-dried and stored at -20°C for later use;

[0043] (4) Preparation of LF-RSV-Fe3O4@mPDA nanoparticles: RSV was dissolved in 50% ethanol solution, and then ultrasonically mixed with LF-Fe3O4@mPDA nanoparticles in a ratio of 5:1, stirred at room temperature for 24 hours, collected by centrifugation, and washed three times with deionized water to obtain LF-RSV-Fe3O4@mPDA nanoparticles.

[0044] The present invention successfully constructed a new nanoprobe LF-RSV-Fe3O4@mPDA, which has excellent MRI imaging capability and photothermal effect, has targeting performance and high biosafety, and has a drug-loading function capable of experimental drug transport; at the cellular level, it was confirmed that the new LF-RSV-Fe3O4@mPDA nanoprobe has good cell uptake ability, can achieve MRI imaging and reduce the generation of ROS at the cellular level.

[0045] Preferably, in the step (1), in the synthesis of the iron oleate complex, 10.8 g of ferric chloride and 36.5 g of sodium oleate are dissolved in a mixed solvent consisting of ethanol, distilled water and n-hexane, and heated to 70° C. for 4 hours. After the reaction is completed, the upper organic layer containing the iron oleate complex is washed with distilled water in a separatory funnel. After washing, the hexane is evaporated to obtain the iron oleate complex. Then, 36 g of the iron oleate complex and 5.7 g of oleic acid are dissolved in 200 g of 1-octadecene. The reaction mixture is heated to 320° C. and reacted for 30 minutes. The initial transparent solution becomes turbid brown-black. The solution is cooled to room temperature, and 500 ml of ethanol is added to the solution. After obtaining oleic acid-stabilized iron nanoparticles by centrifugation, OA-stabilized Fe3O4 is collected.

[0046] Preferably, in the step (2), the prepared Fe3O4 powder is redissolved in TMB solution, and ultrasonic dispersion is performed to form an emulsion oily 1 mg / ml liquid. 300 mg P123, 750 mg F127 and 1.5 g dopamine hydrochloride are dissolved in 100 ml of 40% ethanol solution and ultrasonically dissolved completely. The TMB solution containing Fe3O4 is added to the solution system, mixed and ultrasonically performed for 1 hour, and a mechanical stirring device is installed. After stirring evenly, 3.2 ml of 30% ammonia water is quickly added to the reaction system, reacted for 4 hours, and Fe3O4@mPDA is collected after centrifugation.

[0047] Preferably, in step (3), LF is dissolved in 10 mg / ml PBS, and EDC:NHS is added to the LF solution in a ratio of 4:3.

[0048] Preferably, the method also includes step (5), using a transmission electron microscope to observe the microscopic morphology of the mesopores and measure the particle size of the nanoprobe; using a spectrophotometer to detect the ultraviolet-visible light absorption spectrum of the drug carried by the nanoprobe; using Fourier transform infrared microspectroscopy to study the infrared spectral characteristics of the nanoprobe and analyze the formation of chemical bond peaks; using an 808nm semiconductor laser and an infrared thermal imaging system to evaluate the photothermal efficiency and stability of the nanoprobe in vitro; finally, using an MRI imaging system with a magnetic field strength of 1.5T to study the MR imaging and relaxation properties of the nanoprobe.

[0049] Preferably, the method further comprises step (6), testing the photothermal efficiency and stability of LF-RSV-Fe3O4@mPDA, firstly adding nanoprobes of different concentrations into a cuvette, and then irradiating with 808nm NIR of different power densities; at the same time, using an infrared thermal imaging system to monitor and record the temperature of the LF-RSV-Fe3O4@mPDA nanoprobe solution, setting the parameters to record once every 10 seconds for a total duration of 10 minutes, and also recording the real-time thermal image corresponding to the nanoprobe solution; the experiment uses power densities of 0.5, 1.0, 1.5 and 2.0 W cm -2 808 nm laser irradiation of 1 ml of nanoprobe solution for 10 min, Fe concentration of 50 μg mL -1 ; According to the temperature change, the power is selected as 1.0W cm -2 The nanoparticles were irradiated with 808 nm laser for 10 min at different Fe concentrations of 0, 25, 50, 75, 100, and 200 μg mL -1 ; Use 1.0W cm -2 The 808nm laser was switched on and off every 5 minutes, and the temperature changes were recorded. The images and experimental data obtained from the infrared thermal imaging system were collected and fitted into a time-temperature curve.

[0050] Preferably, the method further comprises step (7), evaluating the drug loading rate of the LF-RSV-Fe3O4@mPDA nanoprobe, stirring the probe and RSV in a ratio of 5:1, measuring the drug absorbance using an ultraviolet spectrophotometer and an enzyme reader, and obtaining the loading rate and encapsulation rate of the nanoprobe based on the fitted drug concentration standard curve; performing a drug release experiment, respectively placing the LF-RSV-Fe3O4@mPDA into two dialysis bags, each bag containing 1 mg mL -1 LF-RSV-Fe3O4@mPDA was prepared. 1 ml of nanoprobe solution was filled in the dialysis bag. Then, the two dialysis bags were immersed in a pH 7.4 solution with a total volume of 20 ml. The bags were shaken at 100 rpm. 1 ml of dialysate was taken out at 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 15 h, 17 h, 24 h, 28 h, 36 h, and 48 h. At the same time, an equal amount of buffer was added to keep the total volume equal. Before each collection of the dialysate, 1 W cm -2 The NIR laser was irradiated with a PBS buffer solution with a pH value of 7.4 for 10 minutes. Finally, the RSV concentration of the released drug was measured by a microplate reader according to the RSV absorption wavelength, and the amount of RSV released was calculated according to the RSV dose absorption curve.

[0051] Preferably, the method also includes step (8), evaluating the relaxation performance of the nanoprobe, first using ICP to determine the concentration of iron ions in LF-RSV-Fe3O4@mPDA, preparing LF-RSV-Fe3O4@mPDA into solutions of different concentrations of 0, 10, 25, 50, 75, and 100 μg / ml, placing the solutions in 1.5 ml centrifuge tubes, placing the centrifuge tubes on a centrifuge tube rack filled with water, and using an MRI magnetic resonance scanner to scan to obtain the T2 relaxation time of the nanoprobe at different concentrations, converting the units according to the measured iron particle concentration by ICP, drawing the corresponding relaxation rate fitting curve, and analyzing the curve to evaluate the MRI imaging performance of LF-RSV-Fe3O4@mPDA; the specific scanning parameters are as follows: T2 3D sequence, TR=2000ms, TE=50ms, FOV=140mm×140mm.

[0052] Also provided is the application of the POCD nanoprobe, which is used in preparing a drug for treating postoperative cognitive dysfunction.

[0053] Preferably, it is used to target the blood-brain barrier.

[0054] The integrated diagnosis and treatment nanoprobe LF-RSV-Fe3O4@mPDA was synthesized to have good targeting performance, drug loading performance and MRI imaging performance. First, LF was used to target the LF receptor of the blood-brain barrier. Secondly, the large specific surface area and good photothermal performance of the polydopamine mesoporous layer were used to achieve the precise release of RSV and the function of low-temperature photothermal therapy. Finally, the MRI imaging performance of Fe3O4 was used to achieve the imaging of the blood-brain barrier and the evaluation of the therapeutic effect. In the synthesis process of the nanoprobe, oleic acid-stabilized Fe3O4 was used as the core, and mesoporous dopamine was coated on the outside as the drug loading system. LF was covalently linked by EDC / NHS to give it good targeting performance. However, there are many influencing factors in the synthesis process. The control of the morphology, particle size, etc. of mesoporous dopamine and the conversion of the aqueous phase all affect the final experimental results.

[0055] Figure 3 TEM images of nanoprobes. (a) TEM image of Fe3O4, (b)

[0056] TEM image of Fe3O4@mPDA.

[0057] Figure 4 Microscopic infrared images of Fe3O4@mPDA, RSV-Fe3O4@mPD, and LF-RSV-Fe3O4@mPDA.

[0058] Figure 5The photothermal properties of LF-RSV-Fe3O4@mPDA. (a) Temperature-time curve after 808 laser irradiation for 10 minutes at different power densities. (b) The power density is 1.0 W cm -2 Temperature-time curve after 808 laser irradiation for 10 minutes. (c) Real-time thermal image corresponding to Figure Ⅰa.

[0059] Real-time thermal image corresponding to Figure Ⅱb (d) Photothermal stability performance of the nanoprobe.

[0060] Figure 6 Drug release of LF-RSV-Fe3O4@mPDA nanoprobe under NIR response.

[0061] Figure 7 MRI imaging performance of LF-RSV-Fe3O4@mPDA nanoprobe (a) Relaxation fitting curve of LF-RSV-Fe3O4@mPDA nanoprobe: transverse relaxation image 1 / T2; (b) T2-weighted images of LF-RSV-Fe3O4@mPDA nanoprobes with different concentrations.

[0062] Figure 8 The effect of different concentrations of H2O2 on the viability of SH-SY5Y cells, n=4,

[0063] ***p<0.001.

[0064] Fig. 9 The effects of different concentrations of LF-RSV-Fe3O4@mPDA nanoprobes on the activity of SH-SY5Y cells and bEnd.3 cells.

[0065] Fig.10 T2-weighted images of LF-RSV-Fe3O4@mPDA nanoprobe at the cellular level under different concentration gradients.

[0066] Fig.11 Evaluation of the photothermal performance of nanoprobes at the cellular level (a) Effect of different NIR irradiation times on cell viability. (b) Cell viability treated with H2O2, free RSV, and nanoprobes.

[0067] (n=3).

[0068] Fig.12 Figure 2 shows the evaluation of cell phagocytosis and targeting of nanoprobes. (a) Microscope images of cells incubated with Fe3O4@mPDA and LF-Fe3O4@mPDA for 2 and 4 hours; blue fluorescence is the cell nucleus labeled with DAPI, and red fluorescence is the nanoprobe loaded with RhB; (b) Statistical graph of data analysis of rhodamine B fluorescence intensity using ImageJ software (n=3).

[0069] Fig.13 Nanoprobe ROS detection: (a) control group; (b) fluorescence image of LF-RSV-Fe3O4@mPDA group; (c) fluorescence image of H2O2 group; (d) fluorescence image of LF-RSV-Fe3O4@mPDA+H2O2 group; (e) statistical graph of average fluorescence intensity of each group (n=3, ***p<0.001).

[0070] Fig.14 Figure 1 shows the evaluation of model building. (a) The time mice spend in the new arm of the Y-maze; (b) The level of IL-6 in the blood samples of mice, statistical processing results, *p<0.05, **p<0.01, ***p<0.001.

[0071] Fig.15 The in vivo biosafety assessment of nanoprobes. (a) Body weight change curve within 15 days after the mice were injected with nanoprobes through the tail vein; (b) Blood routine indexes after the mice were injected with different nanoprobes through the tail vein (n=4); (c) Analysis of tissue sections of major organs after the mice were injected with different nanoprobes through the tail vein (HE, 200×).

[0072] This study used the soft template method. Compared with the hard template method, the soft template method is a method for synthesis at the molecular level. The chemical reaction between the template agent of the soft template and the carbon precursor is an important factor in the synthesis process. The synthesis process is relatively simple and has less pollution to the environment. In order to further simplify the synthesis process, the double soft template method was used. Fe3O4 was redissolved in TMB, ultrasonically treated to form an emulsion, and then added to a block copolymer (P123 and F127) solution with a certain concentration of ethanol as a solvent. After adding a certain proportion of dopamine hydrochloride, ultrasonically uniform, a certain amount of ammonia water was added under mechanical stirring to provide alkaline conditions. In this synthesis process, we found that consistent with existing literature reports, by finely adjusting the mass ratio of P123 to F127 in the reaction system, the filling parameters of the surfactant can be accurately controlled. At the same time, the amount of TMB also has a great influence. The amount of dopamine hydrochloride is also related to the thickness of the mesoporous dopamine. The temperature and the concentration of ethanol in the reaction system have a great influence on the morphology of mesoporous dopamine. Guan BY et al. synthesized mesoporous dopamine using a dual template method. When the mass ratio of P123 to F127 reached 1:1 and 5:3, the morphology of mesoporous dopamine became non-porous wrinkled nanosheets and nanodisks. By continuously increasing the volume fraction of ethanol from 30% to 40%, and then to 45%, the mesoscopic dopamine structure formed can be changed from a mixture of nanospheres and bowl-shaped nanoparticles to dendritic nanoparticles, and then to walnut-shaped nanoparticles. When the volume fraction of ethanol is further increased to 55%, non-porous particles with wrinkled sheet morphology are formed. Therefore, this experiment continuously adjusts the mass ratio of each substance during the synthesis process to control the temperature and reaction system.

[0073] The particle size of carriers involved in brain drug transport is generally below 200nm, which facilitates their endocytosis and smooth entry into the BBB. Therefore, the nanoprobe constructed in this experiment controls the amount of dopamine hydrochloride and the reaction time. The particle size displayed under a transmission electron microscope is about 100nm, which can better pass through the BBB. However, the proportion of the mesoporous morphology of the nanoprobe should be more accurately controlled to form a microporous structure, which may be more conducive to improving the drug loading rate.

[0074] Imaging examinations play an important role in disease diagnosis, treatment, and monitoring. They are non-invasive, can provide detailed structural and functional information, and can achieve early diagnosis and monitor disease progression. MRI, which is non-invasive and has no ionizing radiation, has great development potential. In order to increase the contrast between tissues, exogenous contrast agents are introduced during the examination. Common MRI contrast agents are gadolinium contrast agents. Gadolinium agents stay in the body for a long time, are excreted slowly, and have potential nephrotoxicity, especially for patients with renal insufficiency. Studies have prepared contrast agents based on iron nanoparticles. Iron contrast agents are relatively safe and cause less damage to the kidneys. Currently, five types of superparamagnetic iron oxide nanoparticles (SPIONs) have been designed and clinically tested as magnetic resonance contrast agents, but they have only been put into use in a few countries.

[38] The unique advantages of SPION have great research potential in the field of molecular imaging.

[0075] In this study, Fe3O4 was used as an imaging material, which showed good T2 imaging ability under MRI. The particle size of the synthesized Fe3O4 nanoparticles was about 12nm. The synthesized Fe3O4@mPDA was found to contain Fe3O4 particles in the mesoporous structure through transmission electron microscopy. The MRI results showed that the r2 value was 5.704mM -1 s -1 , indicating that LF-RSV-Fe3O4@mPDA nanoprobe is a good T2-weighted negative contrast agent.

[0076] This study tested the photothermal conversion performance and photothermal stability of LF-RSV-Fe3O4@mPDA in vitro. It is known from relevant literature that the environment of 41-43°C can promote the permeability of the blood-brain barrier, increase the accumulation of drugs in the brain, and produce a better therapeutic effect on neurodegenerative diseases. In this experiment, a power density of 1.0 W cm -2 The 808 nm laser was used to treat 50 μg mL -1The temperature of the LF-RSV-Fe3O4@mPDA nanoprobe solution can reach 40.51°C after 10 minutes of irradiation, indicating that the nanoprobe has strong photothermal conversion ability. When the near-infrared light is switched on and off within one hour, the temperature rise and fall of the nanoprobe are similar, indicating that LF-RSV-Fe3O4@mPDA has good photothermal stability. The results of the comprehensive in vitro photothermal test and the photothermal performance evaluation at the cell level verified that the nanoprobe has good photothermal conversion performance and photothermal stability, and is a good photothermal response material.

[0077] The blood-brain barrier is a highly selective dynamic interface between the blood and the brain, which can protect central neurons from being damaged. The existence of the blood-brain barrier makes it difficult for most central nervous system drugs to reach the brain parenchyma, blocking the entry of almost all macromolecular drugs and nearly 98% of small molecule drugs, making the bioavailability of many drugs extremely low. Therefore, in the central nervous system, targeted therapy is extremely important. One of the most important links in achieving targeted therapy of the CNS is to cross the biological barrier. In recent years, nanoparticles have been widely studied in brain drug transport due to their superior performance. At present, among many CNS targeting, receptor-mediated targeting is widely studied. Through literature review, it is known that LF is a glycoprotein belonging to the transferrin (TF) family, which can bind to TF receptors (TFRs) and LF membrane internalization receptors (LFRs) highly expressed on the surface of highly proliferative cancer cells and blood-brain barrier cells, thereby promoting its approach to the cell nucleus. This advantage can be used to develop active targeted drug delivery systems, which can easily cross the blood-brain barrier, allowing drugs to reach the site of injury and achieve good therapeutic effects.

[0078] In this study, LF was coupled by covalent binding of EDC / NSH. The characteristic peak of amide bond appeared through microscopic infrared spectrum, which proved that LF was successfully connected to the surface of nanoprobe. At the cellular level, the targeted probe was co-incubated with non-targeted probe and cells, and the targeted nanoprobe could be taken up more by cells, which proved that the synthesized nanoprobe had good targeting performance. In previous studies, it has been verified at the animal level that nanoprobes connected to LF can reach the brain more than those not connected to LF, which proves that LF is a good targeting substance.

[0079] More and more studies have shown that drugs must have a high loading rate and good drug release capacity at the same time, so that they can be released after being transported to the site of injury and achieve a good release effect. Most of the current research is devoted to the development of stimulus-responsive drug release systems, which can remain stable under extracellular conditions and respond to intracellular stimuli after internalization, including acidic pH environment, redox, reactive oxygen, NIR photothermal stimulation, etc. to trigger rapid drug release.

[0080] In this study, the release of drugs under photothermal response was discussed. A pH of 7.4 was used in vitro to simulate the brain environment. The results showed that the nanoprobe can release drugs. Under NIR irradiation conditions, the nanodrug delivery system can release more drugs at the same time, indicating that the probe has photothermal responsive drug release capabilities. In previous studies, the research team also synthesized a black TiO2 nanoprobe for pancreatic tumors. The probe achieves MRI-guided photothermal chemotherapy by targeting IGF1 receptors. Experimental results show that the nanoprobe can promote the release of chemotherapy drugs through photothermal effects and an acidic environment of pH 5 to achieve tumor destruction, providing new strategies and methods for the treatment of pancreatic cancer.

[0081] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any simple modification, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention are still within the protection scope of the technical solution of the present invention.

Claims

1. A method for preparing a POCD nanoprobe, characterized in that: It includes the following steps: (1) Preparation of Fe3O4 nanoparticles; (2) Preparation of Fe3O4@mPDA nanoparticles: The prepared Fe3O4 nanoparticles were dissolved in TMB solution and dispersed by ultrasonic to form an emulsion oily liquid. P123, F127 and dopamine hydrochloride were dissolved in ethanol solution and dissolved by ultrasonic. The TMB solution containing Fe3O4 was added to the above solution system and mixed by ultrasonic for 1 hour. A mechanical stirring device was installed and after stirring evenly, ammonia water was quickly added to the reaction system and reacted for 4 hours. Fe3O4@mPDA was collected after centrifugation and washed with water and ethanol alternately for 3 times. The obtained product was freeze-dried for storage. (3) Preparation of LF-Fe3O4@mPDA nanoparticles: LF was dissolved in PBS and Add EDC:NHS to the solution, mix well and place in a 4℃ refrigerator for 4h for activation, then add Fe3O4@mPDA to the activated LF solution, adjust the pH value of the mixed solution to 8.5 with ammonia water, stir at room temperature for 12h, collect by centrifugation, wash with deionized water, freeze-dry and store at -20℃ for later use; (4) Preparation of LF-RSV-Fe3O4@mPDA nanoparticles: RSV was dissolved in 50% ethanol solution, and then ultrasonically mixed with LF-Fe3O4@mPDA nanoparticles in a ratio of 5:1, stirred at room temperature for 24 hours, collected by centrifugation, and washed three times with deionized water to obtain LF-RSV-Fe3O4@mPDA nanoparticles.

2. The method for preparing the POCD nanoprobe according to claim 1, characterized in that: In the step (1), in the synthesis of the iron oleate complex, 10.8 g of ferric chloride and 36.5 g of sodium oleate are dissolved in a mixed solvent consisting of ethanol, distilled water and n-hexane, and heated to 70° C. for 4 hours. After the reaction is completed, the upper organic layer containing the iron oleate complex is washed with distilled water in a separatory funnel. After washing, the hexane is evaporated to obtain the iron oleate complex. Then, 36 g of the iron oleate complex and 5.7 g of oleic acid are dissolved in 200 g of 1-octadecene. The reaction mixture is heated to 320° C. and reacted for 30 minutes. The initial transparent solution becomes turbid brown-black. The solution is cooled to room temperature, and 500 ml of ethanol is added to the solution. After obtaining oleic acid-stabilized iron nanoparticles by centrifugation, OA-stabilized Fe3O4 is collected.

3. The method for preparing the POCD nanoprobe according to claim 2, characterized in that: In the step (2), the prepared Fe3O4 powder is redissolved in TMB solution, and ultrasonic dispersion is performed to form an emulsion oily 1 mg / ml liquid. 300 mg P123, 750 mg F127 and 1.5 g dopamine hydrochloride are dissolved in 100 ml of 40% ethanol solution and ultrasonically dissolved completely. The TMB solution containing Fe3O4 is added to the solution system, mixed with ultrasound for 1 hour, and a mechanical stirring device is installed. After stirring evenly, 3.2 ml of 30% ammonia water is quickly added to the reaction system, reacted for 4 hours, and Fe3O4@mPDA is collected after centrifugation.

4. The method for preparing the POCD nanoprobe according to claim 3, characterized in that: In the step (3), LF is dissolved in 10 mg / ml PBS, and EDC:NHS is added to the LF solution at a ratio of 4:

3.

5. The method for preparing the POCD nanoprobe according to claim 4, characterized in that: The method also includes step (5), using a transmission electron microscope to observe the microscopic morphology of the mesopores and measure the particle size of the nanoprobe; using a spectrophotometer to detect the ultraviolet-visible light absorption spectrum of the drug carried by the nanoprobe; using Fourier transform infrared microspectroscopy to study the infrared spectral characteristics of the nanoprobe and analyze the formation of chemical bond peaks; using an 808nm semiconductor laser and an infrared thermal imaging system to evaluate the photothermal efficiency and stability of the nanoprobe in vitro; and finally, using an MRI imaging system with a magnetic field strength of 1.5T to study the MR imaging and relaxation properties of the nanoprobe.

6. The method for preparing the POCD nanoprobe according to claim 5, characterized in that: The method also includes step (6), testing the photothermal efficiency and stability of LF-RSV-Fe3O4@mPDA, firstly adding nanoprobes of different concentrations into a cuvette, and then irradiating with 808nm NIR of different power densities; at the same time, using an infrared thermal imaging system to monitor and record the temperature of the LF-RSV-Fe3O4@mPDA nanoprobe solution, setting the parameters to record once every 10 seconds, the total duration is 10 minutes, and also recording the real-time thermal image corresponding to the nanoprobe solution; the experiment uses power densities of 0.5, 1.0, 1.5 and 2.0Wcm -2 808 nm laser irradiation of 1 ml of nanoprobe solution for 10 min, Fe concentration of 50 μg mL -1 ; According to the temperature change, the power is selected as 1.0W cm -2 The nanoparticles were irradiated with 808 nm laser for 10 min at different Fe concentrations of 0, 25, 50, 75, 100, and 200 μg mL -1 ; Use 1.0W cm -2 The 808nm laser was switched on and off every 5 minutes, and the temperature changes were recorded. The images and experimental data obtained from the infrared thermal imaging system were collected and fitted into a time-temperature curve.

7. The method for preparing the POCD nanoprobe according to claim 6, characterized in that: The method also includes step (7), evaluating the drug loading rate of the LF-RSV-Fe3O4@mPDA nanoprobe, stirring the probe and RSV in a ratio of 5:1, measuring the drug absorbance using an ultraviolet spectrophotometer and an enzyme reader, and obtaining the loading rate and encapsulation rate of the nanoprobe based on the fitted drug concentration standard curve; performing a drug release experiment, LF-RSV-Fe3O4@mPDA was loaded into two dialysis bags, each containing 1 mg mL -1 LF-RSV-Fe3O4@mPDA was prepared. 1 ml of nanoprobe solution was filled in the dialysis bag. Then, the two dialysis bags were immersed in a pH 7.4 solution with a total volume of 20 ml. The bags were shaken at 100 rpm. 1 ml of dialysate was taken out at 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 15 h, 17 h, 24 h, 28 h, 36 h, and 48 h. At the same time, an equal amount of buffer was added to keep the total volume equal. Before each collection of the dialysate, 1 W cm -2 The NIR laser was irradiated with a PBS buffer solution with a pH value of 7.4 for 10 minutes. Finally, the RSV concentration of the released drug was measured by a microplate reader according to the RSV absorption wavelength, and the amount of RSV released was calculated according to the RSV dose absorption curve.

8. The method for preparing the POCD nanoprobe according to claim 7, characterized in that: The method further comprises step (8), evaluating the relaxation performance of the nanoprobe, firstly using ICP to determine the concentration of iron ions in LF-RSV-Fe3O4@mPDA, and then Solutions with different concentrations of 0, 10, 25, 50, 75, and 100 μg / ml were prepared and placed in 1.5 ml centrifuge tubes. The centrifuge tubes were placed on a centrifuge tube rack filled with water, and an MRI magnetic resonance scanner was used to obtain the T2 relaxation time of the nanoprobes at different concentrations. The units were converted according to the measured iron particle concentration by ICP, and the corresponding relaxation rate fitting curve was drawn and analyzed to evaluate the MRI imaging performance of LF-RSV-Fe3O4@mPDA. The specific scanning parameters are as follows: T2 3D sequence, TR=2000ms, TE=50ms, FOV=140mm×140mm.

9. Application of POCD nanoprobe, characterized by: The invention is used in preparing medicine for treating postoperative cognitive dysfunction.

10. The use of the POCD nanoprobe according to claim 9, characterized in that: Its application is in targeting the blood-brain barrier.

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