Mouse nerve growth factor nano drug-loaded material and preparation method thereof

Abstract

The invention discloses a mouse nerve growth factor nano drug-loaded material and a preparation method thereof in the technical field of nano drug-loaded materials, and aims to solve the problem of low blood-brain barrier penetration rate. PBCA is subjected to in-situ modification, a hyperbranched amino structure is introduced into a poly (n-butyl cyanoacrylate) molecule, and through introduction of amino, the drug-loaded nano drug-loaded material is prepared. The electropositivity of the nano drug-loaded particles can be increased, so that the capacity of the drug for penetrating through the blood brain barrier during transport can be improved through an adsorption mediation way.

Description

Technical Field

[0001] The invention belongs to the technical field of nano drug-carrying materials, and specifically refers to a mouse nerve growth factor nano drug-carrying material and a preparation method thereof. Background Art

[0002] Traumatic brain injury (TBI) can be defined as a disease in which a severe head injury caused by external force interferes with normal brain function. The main causes of brain trauma include falls, motor vehicle collisions, and physical contact sports. TBI can affect the structure and function of the brain, leading to changes in cognition, emotion, behavior, etc., and even life-threatening in severe cases. Mild traumatic brain injury (mild concussion) usually refers to a patient's temporary loss of consciousness or symptoms such as headache and dizziness, which recovers quickly and is usually not accompanied by significant long-term neurological damage; moderate traumatic brain injury: patients may experience loss of consciousness for several hours, accompanied by neurocognitive dysfunction such as memory loss and difficulty concentrating, and recovery is slow. Imaging examinations often show mild to moderate brain structural changes, such as brain edema and hematoma; severe traumatic brain injury: patients lose consciousness for a long time and may fall into a coma or vegetative state, often accompanied by long-term neurological deficits, requiring long-term treatment and rehabilitation; imaging examinations show significant brain parenchymal damage or extensive brain edema, and surgical intervention may be required (such as removing hematomas, repairing ruptured blood vessels, etc.). Nerve growth factor (NGF), which has the function of promoting nerve damage and repair, is an important nerve growth factor as a nerve regeneration inducer. It can promote the growth, differentiation, survival and repair of neurons, especially the damage and repair of peripheral nerves and central nervous system. NGF activates the intracellular signal transduction pathway by binding to its receptors TrkA (tropomyosin receptor kinase A) and p75NTR (low-affinity nerve growth factor receptor), promotes the survival, regeneration and differentiation of neurons, and has been widely studied for the repair of peripheral nerve damage and central nervous system damage, especially for the treatment of Alzheimer's disease and other neurodegenerative diseases. The blood-brain barrier is a special barrier formed by tight junctions between the endothelial cells of the brain blood vessels. Its function is to protect the brain from harmful substances and pathogens, and it also restricts many drugs and large molecules (such as proteins, antibodies, etc.) from entering the brain. NGF is a polypeptide composed of 118 amino acids with a molecular weight of about 13-14 kDa. Although it is relatively small, it is still a large molecule. The permeability of the blood-brain barrier to large molecules is very limited, especially for protein substances. At the same time, NGF is water-soluble and lacks the lipophilicity to effectively cross the blood-brain barrier. The endothelial cell membrane of the blood-brain barrier is composed of a phospholipid bilayer. Lipophilic molecules can more easily pass through this lipid structure, while water-soluble molecules such as NGF are difficult to pass freely. Summary of the invention

[0003] In view of the above situation, in order to overcome the defects of the prior art, the present invention provides a mouse nerve growth factor nano drug delivery material and a preparation method thereof. In order to solve the problem of low blood-brain barrier penetration rate, the present invention introduces a hyperbranched amino structure into the polybutyl cyanoacrylate molecule by in-situ modification of PBCA. The introduction of the amino group can increase the positive charge of the nano drug delivery particles, thereby improving the ability of drug transport to penetrate the blood-brain barrier through an adsorption-mediated pathway.

[0004] In order to achieve the above object, the technical solution adopted by the present invention is as follows: The present invention also provides a method for preparing a mouse nerve growth factor nano drug-carrying material, which specifically comprises the following steps:

[0005] S1. Dissolve ethylenediamine in methanol, add methyl acrylate and n-butyl α-cyanoacrylate under stirring at 200-250 rpm, place at room temperature, stir and react for 48 hours, remove methanol from the reaction system, and obtain a PBCA modified polymer;

[0006] Preferably, in step S1, the raw material components for preparing the PBCA-modified polymer include, by weight, 1 part of ethylenediamine, 0.4-0.7 parts of methyl acrylate, and 1.2-1.8 parts of n-butyl α-cyanoacrylate;

[0007] Preferably, in step S1, the mass concentration of ethylenediamine in methanol is 5-10 g / L;

[0008] S2, dissolving the PBCA modified polymer prepared in step S1 in the dextran solution, adding chloroauric acid, stirring continuously at room temperature at a speed of 300-320 rpm overnight, adding sodium citrate, heating to 40-50° C. and stirring for 30-40 min, after the reaction system is cooled, centrifuging at 8000 rpm for 20 min, filtering through a 3 kDa filter membrane, washing with anhydrous ethanol, and freeze-drying to obtain the Au-PBCA modified polymer;

[0009] Preferably, in step S2, the dextran solution is a solution of dextran dissolved in deionized water, and the mass fraction of dextran is 5%-7%; the mass concentration of the PBCA-modified polymer in the dextran solution is 2-4 g / L;

[0010] Preferably, in step S2, the mass ratio between chloroauric acid and sodium citrate is 10:5-7;

[0011] Preferably, in step S2, the mass ratio between the chloroauric acid and the PBCA-modified polymer is 1:4-5;

[0012] S3, dissolving the Au-PBCA modified polymer prepared in step S2 in physiological saline and adjusting the pH to 7.0 with 1 mol / L NaOH solution, filtering with 0.8 μm single-layer filter paper, centrifuging at 9000-10000 rpm for 40-50 min, discarding the supernatant, resuspending with ultrapure water, centrifuging again, repeating the resuspending, centrifuging, and freeze-drying 2-3 times, freeze-drying the precipitate, repeating the operation 2-3 times, dissolving the precipitate in physiological saline, adding mNGF, and standing at 4°C for 5-6 hours to obtain mNGF carrier polymer;

[0013] Preferably, in step S3, the mass concentration of the precipitate in physiological saline after freeze-drying is 10-20 mg / mL;

[0014] Preferably, in step S3, the volume fraction of mNGF in physiological saline is 10%-12%;

[0015] S4, dissolving SMCC (4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimidyl ester) in DMSO solvent, adding the mNGF carrier polymer prepared in step S3, shaking and mixing for 30-50 minutes, placing at 4°C, and when the reaction system is cooled to 4°C, adding the permeable polypeptide solution to the reaction system, stirring overnight, filtering with an ultrafiltration centrifuge tube, and freeze-drying to obtain a mouse nerve growth factor nano drug delivery material;

[0016] Preferably, in step S4, the mass concentration of SMCC in DMSO solvent is 1-2 mg / mL;

[0017] Preferably, in step S4, the added mass of SMCC is 1%-2% of the mass of the mNGF carrier polymer;

[0018] Preferably, in step S4, the permeable polypeptide solution is a solution of Penetratin polypeptide dissolved in PBS buffer solution, and the added mass of Penetratin polypeptide is 4%-6% of the mass of mNGF carrier polymer.

[0019] The invention also provides a mouse nerve growth factor nano drug-carrying material prepared according to the method.

[0020] The beneficial effects achieved by the present invention are as follows:

[0021] The present invention provides a mouse nerve growth factor nano drug-carrying material and a preparation method thereof. The present invention introduces a hyperbranched amine structure into a poly(n-butyl cyanoacrylate) molecule by in-situ modification of PBCA. The introduction of the amine group can increase the active groups on the surface of the nano drug-carrying particles, load Au nanoparticles through the active sites, improve the positive charge of the drug-carrying particles, and improve the ability of drug transport to penetrate the blood-brain barrier through an adsorption-mediated pathway. At the same time, the nano drug-carrying particles modified with penetratin polypeptide can be surface-modified through intermolecular interactions such as hydrophobic interaction and ion binding, and the surface of the nano drug-carrying particles can be modified by using The membrane protein binding method disturbs the phosphate molecular layer to form reverse micelles, thereby improving the ability of drug-loaded particles to penetrate biological membranes and physiological barriers, and achieving better absorption of drugs. The present invention mixes ethylenediamine, methyl acrylate and α-cyanoacrylate n-butyl to react to generate a hyperbranched polymer structure, uses abundant amino groups as active groups, reacts with gold chlorate and sodium citrate, and loads gold nanoparticles on the polymer surface in an in-situ modified manner. The Penetratin polypeptide is derived from one of the side chains of the cell penetrating peptide, and improves the ability to penetrate the blood-brain barrier through the CPP-mediated delivery method. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 TEM image of the precipitate prepared in step S3 of Example 1;

[0023] Figure 2 The repair results of the nano drug-carrying materials prepared in Examples 1-3 and Comparative Examples 1-2 are shown;

[0024] Figure 3 The EB leakage test results of the treatment group in Example 1 and other groups are shown in FIG.

[0025] Figure 4 This is a graph showing cerebral blood flow monitoring of mice in the treatment group of Example 1 and other groups.

[0026] The accompanying drawings are used to provide further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention. DETAILED DESCRIPTION

[0027] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0028] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein may be applied to the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only and are not intended to limit the content of this application.

[0029] The experimental methods in the following examples are conventional methods unless otherwise specified; the experimental materials and test strains used in the following examples are purchased from commercial channels unless otherwise specified.

[0030] The sources of materials used in the present invention are as follows:

[0031] mNGF (mouse nerve growth factor) is Enjingfu mouse nerve growth factor for injection, with a specification of 18ug (9000U) / bottle. Add it to 2mL of water for injection and dissolve it for later use;

[0032] Penetratin polypeptide (CasNo: 214556-79-3) was purchased from Hangzhou Peptidejia Biotechnology Co., Ltd.

[0033] Example 1

[0034] This embodiment provides a method for preparing a mouse nerve growth factor nano drug-carrying material, which specifically comprises the following steps:

[0035] S1. Accurately weigh 1.1 mL of ethylenediamine and place it in a three-necked flask, add 50 mL of methanol solvent to fully dissolve it, place it on a magnetic stirrer and stir it at a speed of 200 rpm, add 0.42 mL of methyl acrylate and 1.15 mL of n-butyl α-cyanoacrylate, place it at room temperature and stir it for 48 hours, then remove the methanol solvent by vacuum distillation to obtain a PBCA modified polymer;

[0036] S2, accurately weigh 5 g of dextran and dissolve it in 100 mL of ultrapure water. After stirring evenly, add 0.4 g of the PBCA-modified polymer prepared in step S1 to the dextran solution and mix thoroughly. Then, take 0.1 g of chloroauric acid, add it at room temperature and stir it at 300 rpm overnight, add 5 mL of 1 wt% sodium citrate solution, heat it to 40 ° C and stir it for 40 min, wait for the reaction system to cool to room temperature, centrifuge it at 8000 rpm for 20 min, filter it through a 3 kDa filter membrane, wash it with anhydrous ethanol, repeat three times, and freeze-dry it to obtain an Au-PBCA-modified polymer.

[0037] S3, placing the Au-PBCA modified polymer prepared in step S2 in a flask, adding physiological saline to fully dissolve it, adjusting the pH to 7.0 with 1 mol / L NaOH solution, filtering with a 0.8 μm single-layer filter paper, centrifuging at 9000 rpm for 40 min, discarding the supernatant, resuspending with ultrapure water, centrifuging at 9000 rpm for 40 min, freeze-drying the precipitate, resuspending with ultrapure water again, centrifuging at 9000 rpm again, discarding the supernatant after 40 min, freeze-drying the precipitate, taking 0.1 g of the precipitate and dissolving it in 10 mL of physiological saline, mixing well, adding 1 mL of mNGF, mixing well, and standing at 4°C for 6 h to obtain an mNGF carrier polymer;

[0038] The morphology was observed by using a transmission electron microscope. The precipitate after multiple resuspension with ultrapure water, centrifugation, and freeze-drying in step S3 was placed under a transmission electron microscope for observation. Figure 1 This is a TEM image of the precipitate prepared in step S3 of Example 1. The precipitate is a blank nanocarrier particle of mNGF. As shown in the figure, the nanocarrier particle prepared in this example has a spherical structure and is hollow inside, and can be used to encapsulate mNGF;

[0039] S4. Dissolve the mNGF carrier polymer prepared in step S3 in 10 mL of PBS buffer solution to obtain an mNGF carrier polymer solution. Accurately weigh 0.1 g of SMCC, add it to 100 mL of DMSO solvent, take 1 mL and add it to the mNGF carrier polymer solution. After oscillating and mixing for 50 min, place it at 4° C., wait for the reaction system to cool to 4° C. for use. Dissolve 10 mg of Penetratin polypeptide in 1 mL of PBS buffer solution, take 0.4 mL and add it to the reaction system. After stirring overnight, place it in an ultrafiltration centrifuge tube for filtration, remove the filtrate, and freeze-dry to obtain a mouse nerve growth factor nano-drug delivery material.

[0040] This embodiment provides a mouse nerve growth factor nano drug-carrying material prepared according to the above method.

[0041] Example 2

[0042] This embodiment provides a method for preparing a mouse nerve growth factor nano drug-carrying material, which specifically comprises the following steps:

[0043] S1. Accurately weigh 1.1 mL of ethylenediamine and place it in a three-necked flask, add 75 mL of methanol solvent to fully dissolve it, place it on a magnetic stirrer and stir it at a speed of 230 rpm, add 0.58 mL of methyl acrylate and 1.43 mL of n-butyl α-cyanoacrylate, place it at room temperature and stir it for 48 hours, then remove the methanol solvent by vacuum distillation to obtain a PBCA modified polymer;

[0044] S2, accurately weigh 6 g of dextran and dissolve it in 100 mL of ultrapure water. After stirring evenly, add 0.2 g of the PBCA modified polymer prepared in step S1 to the dextran solution and mix thoroughly. Then, take 40 mg of chloroauric acid, stir at 310 rpm overnight at room temperature, add 2.8 mL of 1 wt% sodium citrate solution, heat to 50 ° C and stir for 35 min, wait for the reaction system to cool to room temperature, centrifuge at 8000 rpm for 20 min, filter through a 3 kDa filter membrane, wash with anhydrous ethanol, repeat three times, and freeze-dry to obtain Au-PBCA modified polymer;

[0045] S3, placing the Au-PBCA modified polymer prepared in step S2 in a flask, adding physiological saline to fully dissolve it, adjusting the pH to 7.0 with 1 mol / L NaOH solution, filtering with a 0.8 μm single-layer filter paper, centrifuging at 9000 rpm for 40 min, discarding the supernatant, resuspending with ultrapure water, centrifuging at 9000 rpm for 40 min, freeze-drying the precipitate, resuspending with ultrapure water, centrifuging again at 9000 rpm, discarding the supernatant after 40 min, freeze-drying the precipitate, taking 0.15 g of the precipitate and dissolving it in 10 mL of physiological saline, mixing well, adding 1.2 mL of mNGF, mixing well, and standing at 4°C for 6 h to obtain an mNGF carrier polymer;

[0046] S4. Dissolve the mNGF carrier polymer prepared in step S3 in 10 mL of PBS buffer solution to obtain an mNGF carrier polymer solution. Accurately weigh 0.1 g of SMCC, add it to 100 mL of DMSO solvent, take 2 mL and add it to the mNGF carrier polymer solution. After oscillating and mixing for 40 minutes, place it at 4°C, wait for the reaction system to cool to 4°C for use, take 10 mg of Penetratin polypeptide and dissolve it in 1 mL of PBS buffer solution, take 0.6 mL and add it to the reaction system. After stirring overnight, place it in an ultrafiltration centrifuge tube for filtration, remove the filtrate, and freeze-dry to obtain a mouse nerve growth factor nano-drug delivery material.

[0047] This embodiment provides a mouse nerve growth factor nano drug-carrying material prepared according to the above method.

[0048] Example 3

[0049] This embodiment provides a method for preparing a mouse nerve growth factor nano drug-carrying material, which specifically comprises the following steps:

[0050] S1. Accurately weigh 1.1 mL of ethylenediamine and place it in a three-necked flask, add 100 mL of methanol solvent to fully dissolve it, place it on a magnetic stirrer and stir it at a speed of 240 rpm, add 0.75 mL of methyl acrylate and 1.70 mL of n-butyl α-cyanoacrylate, place it at room temperature and stir it for 48 hours, then remove the methanol solvent by vacuum distillation to obtain a PBCA modified polymer;

[0051] S2, accurately weigh 7 g of dextran and dissolve it in 100 mL of ultrapure water. After stirring evenly, add 0.3 g of the PBCA modified polymer prepared in step S1 to the dextran solution and mix thoroughly. Then, take 75 mg of chloroauric acid, stir at 320 rpm overnight at room temperature, add 4.5 mL of 1 wt% sodium citrate solution, heat to 45 ° C and stir for 30 min, wait for the reaction system to cool to room temperature, centrifuge at 8000 rpm for 20 min, filter through a 3 kDa filter membrane, wash with anhydrous ethanol, repeat three times, and freeze-dry to obtain Au-PBCA modified polymer;

[0052] S3, placing the Au-PBCA modified polymer prepared in step S2 in a flask, adding physiological saline to fully dissolve it, adjusting the pH to 7.0 with 1 mol / L NaOH solution, filtering with a 0.8 μm single-layer filter paper, centrifuging at 9000 rpm for 40 min, discarding the supernatant, resuspending with ultrapure water, centrifuging at 9000 rpm for 40 min, freeze-drying the precipitate, resuspending with ultrapure water, centrifuging again at 9000 rpm, discarding the supernatant after 40 min, freeze-drying the precipitate, taking 0.1 g of the precipitate and dissolving it in 10 mL of physiological saline, mixing well, adding 1 mL of mNGF, mixing well, and standing at 4°C for 6 h to obtain an mNGF carrier polymer;

[0053] S4. Dissolve the mNGF carrier polymer prepared in step S3 in 10 mL of PBS buffer solution to obtain an mNGF carrier polymer solution. Accurately weigh 0.1 g of SMCC, add it to 50 mL of DMSO solvent, take 1 mL and add it to the mNGF carrier polymer solution. After oscillating and mixing for 30 minutes, place it at 4°C, wait for the reaction system to cool to 4°C for use, take 10 mg of Penetratin polypeptide and dissolve it in 1 mL of PBS buffer solution, take 0.5 mL and add it to the reaction system. After stirring overnight, place it in an ultrafiltration centrifuge tube for filtration, remove the filtrate, and freeze-dry to obtain a mouse nerve growth factor nano-drug delivery material.

[0054] This embodiment provides a mouse nerve growth factor nano drug-carrying material prepared according to the above method.

[0055] Comparative Example 1

[0056] This comparative example provides a nano drug-carrying material and a preparation method thereof, which is different from Example 1 only in that ethylenediamine and methyl acrylate are not included in the components, and the remaining components and component contents are the same as those in Example 1.

[0057] Comparative Example 2

[0058] This comparative example provides a nano drug-carrying material and a preparation method thereof, which is different from Example 1 only in that α-cyanoacrylate is not included in the components, and the remaining components and component contents are the same as those in Example 1.

[0059] Experimental Example 1

[0060] This example uses a mouse model to test the TBI treatment effect of the nano drug-carrying materials prepared in the examples and comparative examples:

[0061] Experimental animals: 70 4-week-old SPF male mice were raised with free access to food and water, 12h / 12h day and night lighting, and a temperature and humidity of 20~26℃ and 40%~70%.

[0062] Experimental method: The mice were divided into 7 groups, with Examples 1-3 and Comparative Examples 1-2 corresponding to one group respectively. In addition, a blank group and a control group were set up. The control group was treated directly with mNGF, and the mice were anesthetized by gas anesthesia. The method selected for TBI modeling was controlled cortical impact (CCI), and the main parameters selected were as follows: depth: -2.2 mm; impact speed: 5 m / s; the impact site was 1.5 mm behind the coronal suture of the mouse and 2.5 mm to the right of the midline. A bone window was opened here before the impact without damaging the dura mater; after the impact, the mouse was left with 5-10 minutes of bleeding time, and after the bleeding of the injury site coagulated, it was disinfected with iodine tincture, the scalp was sutured, and the mouse was kept warm until it recovered.

[0063] Dosage method: intramuscular injection is used for administration. The syringe is selected as an insulin needle for injection. The injection site is selected as the plump part of the left hind limb muscle of the TBI mouse. The injection dose is 100μl. The specific operation method is as follows: the insulin needle draws 100μl of the drug delivery system and exhausts the air; gently pulls the left hind limb muscle of the mouse to make it tight, inserts the needle, and draws back no blood; slowly injects the drug into the muscle, and the injection time is kept at about 5s; stay for 10s after the injection is completed, and then rotate the needle 180° to withdraw the injected mouse hind limb slightly swells, and no liquid seeps out, and the drug delivery is completed.

[0064] NeuN (Neuron-specific Nuclear Antigen) is a protein mainly expressed in nerve cells and is widely used as a neuron marker in neuroscience research. In this experiment, the repair performance of the nano-drug-carrying material was determined by measuring the relative expression of NeuN. Figure 2 This is a graph showing the repair results of the nano-drug carrier materials prepared in Examples 1-3 and Comparative Examples 1-2. As shown in the figure, the neuron repair level of the Example treatment group is significantly higher than that of the control group, and is also higher than that of the Comparative Example 1 treatment group and the Comparative Example 2 treatment group. Since the nano-drug carrier materials prepared in the Examples have improved the ability to penetrate the blood-brain barrier for drug delivery, the drug carrier materials prepared in the Examples of the present invention have a higher repair level for neurons.

[0065] Experimental Example 2 This experimental example uses a mouse model to test the therapeutic effect of the nano drug-carrying material prepared in Example 1: Experimental animals: 40 4-week-old SPF male mice were raised with free access to food and water, 12h / 12h day and night lighting, and a temperature and humidity of 20~26℃ and 40%~70%.

[0066] Experimental method: The mice were divided into 4 groups, namely the treatment group of Example 1, a blank group, a control group and a TBI treatment group. The blank group was healthy mice that had not been treated with TBI, the control group was treated directly with mNGF, and the TBI treatment group was mice that had been treated with TBI but not treated by any other means. The mice were anesthetized by gas anesthesia, and the method used for TBI modeling was controlled cortical impact (CCI). The main parameters selected were as follows: depth: -2.2 mm; impact speed: 5 m / s; the impact site was 1.5 mm behind the coronal suture of the mouse and 2.5 mm to the right of the midline. A bone window was opened here before the impact without damaging the dura mater; after the impact, the mouse was left with 5-10 minutes of bleeding time. After the bleeding of the injury site coagulated, it was disinfected with iodine, the scalp was sutured, and the mouse was kept warm until it recovered.

[0067] Administration method: Intramuscular injection is used for administration, and the syringe is selected as an insulin needle for injection. The injection site is selected as the plump part of the left hind limb muscle of the mice in the treatment group and the control group in Example 1, and the injection dose is 100 μl. The specific operation method is as follows: 100 μl of the drug administration system is extracted with the insulin needle, and the air is exhausted; the muscle of the left hind limb of the mouse is gently pulled to make it tight, and the needle is inserted, and no blood is withdrawn; the drug is slowly injected into the muscle, and the injection time is kept at about 5 seconds; after the injection is completed, stay for 10 seconds, and then rotate the needle 180° to withdraw the injected mouse hind limb. The injected mouse hind limb is slightly swollen, and no liquid exudates, and the drug administration is completed.

[0068] Figure 3The figures are the results of the EB leakage test of the treatment group of Example 1 and other groups, wherein A is the brain image of mice in the blank group, B is the brain image of mice with traumatic brain injury but not treated, C is the brain image of mice after mNGF treatment, and D is the brain image of mice after treatment with the nano drug carrier material prepared in Example 1 of the present invention; as shown in the figure, there is no EB leakage in the brain of mice in the blank group, the staining range of mice in the TBI treatment group is large and the color is dark, and the staining range of mice treated only with mNGF is larger than that of the treatment group in Example 1, and the color is also darker, indicating that the nano drug carrier material loaded with mNGF prepared by the present invention has a significant effect on the treatment of TBI in mice.

[0069] Figure 4 The figures are cerebral blood flow monitoring diagrams of mice in the treatment group of Example 1 and other groups, wherein A is a brain image of mice in the blank group, B is a brain image of mice that have been subjected to traumatic brain injury but not treated, C is a brain image of mice in the control group after mNGF treatment, and D is a brain image of mice treated with the nano drug carrier material prepared in Example 1 of the present invention; as shown in the figure, the area shown in area 1 is the brain injury range after TBI treatment, and the color from blue to red (range 0-400) indicates the intensity of cerebral blood flow, the blue end indicates low flow, and the red end indicates high flow. The cerebral blood flow in area 1 of the brain image of mice after TBI treatment is low. After treatment with mNGF and the nano drug carrier material prepared in Example 1 of the present invention, the cerebral blood flow is significantly increased, and the increase in cerebral blood flow in the treatment group of Example 1 is more obvious.

[0070] according to Figure 3 and Figure 4 It can be clearly seen that the nano drug-carrying material prepared in Example 1 has a significant repair effect on the neurons of TBI mice.

[0071] While the embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that many changes, modifications, substitutions and alterations may be made to the embodiments without departing from the principles and spirit of the invention.

[0072] The present invention and its implementation methods are described above, which is not restrictive. The drawings are only one of the implementation methods of the present invention, and the actual application is not limited thereto. In short, if ordinary technicians in the field are inspired by it and design methods and embodiments similar to the technical solution without creativity without departing from the purpose of the invention, they should all fall within the protection scope of the present invention.

Claims

1. A method for preparing a mouse nerve growth factor nano drug-carrying material, Features: The specific steps include: S1. Dissolve ethylenediamine in methanol, add methyl acrylate and n-butyl α-cyanoacrylate under stirring at 200-250 rpm, place at room temperature, stir and react for 48 hours, remove methanol from the reaction system, and obtain a PBCA modified polymer; S2, dissolving the PBCA modified polymer prepared in step S1 in the dextran solution, adding chloroauric acid, stirring continuously at room temperature at a speed of 300-320 rpm overnight, adding sodium citrate, heating to 40-50° C. and stirring for 30-40 min, after the reaction system is cooled, centrifuging at 8000 rpm for 20 min, filtering through a 3 kDa filter membrane, washing with anhydrous ethanol, and freeze-drying to obtain the Au-PBCA modified polymer; S3, dissolving the Au-PBCA modified polymer prepared in step S2 in physiological saline and adjusting the pH to 7.0 with 1 mol / L NaOH solution, filtering with 0.8 μm single-layer filter paper, centrifuging at 9000-10000 rpm for 40-50 min, discarding the supernatant, resuspending with ultrapure water, centrifuging again, freeze-drying the precipitate, repeating resuspending, centrifuging, and freeze-drying 2-3 times, dissolving the precipitate in physiological saline, adding mNGF, and standing at 4°C for 5-6 hours to obtain mNGF carrier polymer; S4. Dissolve 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide ester in DMSO solvent, add the mNGF carrier polymer prepared in step S3, shake and mix for 30-50 minutes, place at 4°C, and when the reaction system is cooled to 4°C, add the permeable polypeptide solution to the reaction system, stir overnight, filter with an ultrafiltration centrifuge tube, and freeze-dry to obtain the mouse nerve growth factor nano-drug carrier material.

2. The method for preparing a mouse nerve growth factor nano drug-carrying material according to claim 1, Features: In step S1, the raw material components for preparing the PBCA modified polymer include, by weight, 1 part of ethylenediamine, 0.4-0.7 parts of methyl acrylate, and 1.2-1.8 parts of n-butyl α-cyanoacrylate; the mass concentration of the ethylenediamine in methanol is 5-10 g / L.

3. The method for preparing a mouse nerve growth factor nano drug-carrying material according to claim 2, Features: In step S2, the dextran solution is a solution of dextran dissolved in deionized water, and the mass fraction of dextran is 5%-7%; the mass concentration of the PBCA-modified polymer in the dextran solution is 2-4 g / L.

4. The method for preparing a mouse nerve growth factor nano drug-carrying material according to claim 3, Features: In step S2, the mass ratio of the chloroauric acid to sodium citrate is 10:5-7; the mass ratio of the chloroauric acid to the PBCA-modified polymer is 1:4-5.

5. The method for preparing a mouse nerve growth factor nano drug-carrying material according to claim 4, Features: In step S3, the mass concentration of the precipitate after freeze-drying in physiological saline is 10-20 mg / mL.

6. The method for preparing a mouse nerve growth factor nano drug-carrying material according to claim 5, Features: In step S3, the volume fraction of mNGF in physiological saline is 10%-12%.

7. The method for preparing a mouse nerve growth factor nano drug-carrying material according to claim 6, Features: In step S4, the mass concentration of the 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide ester in the DMSO solvent is 1-2 mg / mL.

8. The method for preparing a mouse nerve growth factor nano drug-carrying material according to claim 7, Features: In step S4, the added mass of 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide ester is 1%-2% of the mass of the mNGF carrier polymer.

9. The method for preparing a mouse nerve growth factor nano drug-carrying material according to claim 8, Features: In step S4, the permeable polypeptide solution is a solution of Penetratin polypeptide dissolved in PBS buffer solution, and the added mass of Penetratin polypeptide is 4%-6% of the mass of mNGF carrier polymer.

10. A mouse nerve growth factor nano drug-carrying material, Features: Prepared according to the preparation method according to any one of claims 1 to 9.

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