Nerve growth factor-loaded bionic prussian blue nano preparation as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medicines, and discloses a nerve growth factor-loaded bionic prussian blue nano preparation which is of a shell-core structure, the core of the preparation is prussian blue nano particles coated with a polydopamine coating, and a nerve growth factor is loaded on the surface of the polydopamine coating; and the shell is a bionic hybrid membrane. By utilizing the characteristic of prolonging the half-life period of the bionic hybrid membrane, the enrichment and slow release of the medicine at the diseased region are realized; the anti-osteoporosis effects of two different mechanisms of bone formation promotion of nerve growth factors and oxidation resistance of Prussian blue are utilized, and a nano-carrier and a bionic system are used for assistance, so that the prevention and treatment effect of osteoporosis is maximized. The invention also discloses a preparation method of the compound and application of the compound in preparation of medicines for preventing and / or treating osteoporosis. The basic problem of osteogenic ability impairment in osteoporosis is solved, high drug enrichment of osteoporosis parts is achieved, and new theoretical support is provided for development of novel drugs for preventing and treating osteoporosis and related prevention and treatment.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and particularly relates to a biomimetic Prussian blue nano - preparation loaded with nerve growth factor, its preparation method and application. Background Art

[0002] Osteoporosis (OP) is the most common orthopedic disease in the elderly and is also a major cause of age - related death and disability. However, the currently commonly used first - line drugs for treating OP are anti - resorptive drugs such as bisphosphonates and denosumab. These drugs not only cannot solve the fundamental problem of impaired osteogenic ability but also increase the risk of atypical femoral fractures and osteonecrosis of the jaw after long - term use. Therefore, there is an urgent need to develop a safe and highly effective anti - osteoporosis drug.

[0003] Nerve Growth Factor (NGF) is one of the earliest discovered neurotrophic factors (NTs). It plays an important regulatory role in the development, differentiation, growth, regeneration, and expression of functional characteristics of central and peripheral neurons. In recent years, many scholars have confirmed that NGF promotes bone regeneration. However, NGF is a macromolecular water - soluble protein with a molecular weight of about 13KD. It is easily hydrolyzed by enzymes in the body and has a short half - life, so it is difficult to accumulate at the disease site. In addition, NGF cannot solve the problem of high oxidative stress levels in senile osteoporosis. Therefore, developing a new NGF composite preparation to achieve in - vivo delivery of NGF, promoting bone formation while antioxidizing, so as to prevent and treat osteoporosis.

[0004] In recent years, with the development of nanotechnology, nanomaterials have shown great advantages in drug delivery in the form of nano - preparations. They can effectively increase the circulating half - life of drugs and achieve the enrichment of drugs at the disease site, providing a potential alternative for the prevention and treatment of OP. Many scholars have used modern nanomaterials to load exogenous NGF to achieve controlled release of NGF at the bone defect site and improve the bioavailability of NGF. Prussian Blue (PB) is a nano - material (Nanoparticles, NPs) with an iron - centered organic metal framework structure that has been certified by the US Food and Drug Administration (FDA). Due to its excellent antioxidant activity, drug - loading capacity, and biocompatibility, it has received increasing attention. However, since the human immune system recognizes nano - preparations as foreign substances and rapidly clears them, the nano - preparations have a half - life of only a few hours or shorter. Therefore, how to extend the blood circulation time of nano - preparations and increase their aggregation ability at the osteoporosis site is the main challenge faced by current nano - preparations for the prevention and treatment of OP during the delivery process. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to develop a safe and efficient biomimetic Prussian blue nanoplatform loaded with nerve growth factor in view of the defects that nerve growth factor is easily hydrolyzed by enzymes, has a short half-life, and does not have antioxidant activity, as well as the deficiency that existing nanodrugs are easily metabolized and cleared rapidly. This nanoplatform has a long blood circulation half-life and biocompatibility, and can synergistically play the roles of promoting bone formation and antioxidant through nerve growth factor and Prussian blue, fundamentally solve the problem of impaired osteogenic ability, and thus treat osteoporosis.

[0006] To solve the above technical problems, the technical solution proposed by the present invention is as follows: A biomimetic Prussian blue nanoplatform loaded with nerve growth factor, which has a core-shell structure. Its core is Prussian blue nanoparticles coated with a polydopamine layer, and nerve growth factor is loaded on the surface of the polydopamine layer; its shell is a biomimetic hybrid membrane.

[0007] In the above-mentioned biomimetic Prussian blue nanoplatform loaded with nerve growth factor, preferably, the biomimetic hybrid membrane is prepared by mixing osteoprogenitor cell membranes and erythrocyte membranes in a mass ratio of 1:1 - 2 and then breaking them.

[0008] Cells are the most basic units that make up the body, and there are characteristic proteins on their membranes to help them adapt to the complex internal environment of the body. Erythrocytes are the most numerous type of blood cells in the blood, and their cell membranes help the nanoplatform adapt to the blood environment. Osteoprogenitor cells are the key cells responsible for bone formation in the bone, and their cell membranes help the nanoplatform adapt to the internal environment of the bone. In the present invention, the cell membranes of these two types of cells are selected to construct the biomimetic nanoplatform to avoid clearance by the body's immune system, extend the blood circulation half-life of the nanoplatform, and further accumulate at the osteoporosis site to fully exert the anti-osteoporosis effect of the nanoplatform. This "top-down" technology retains the complete physicochemical properties of the core of the nanoparticles while utilizing the complex physiological functions of the cell membranes, which are difficult to replicate by individual nanomaterials.

[0009] Preferably, the mass ratio of the Prussian blue nanoparticles to the biomimetic hybrid membrane is 1 - 2:1, the mass ratio of the nerve growth factor to the Prussian blue nanoparticles is 1:200 - 300, and the mass ratio of the Prussian blue nanoparticles to the polydopamine layer is 1 - 0.5:1.

[0010] Preferably, the nanoparticles of the nanoplatform are cube-shaped, with an average particle size of 200 ± 1 nm, more preferably 202.4 ± 0.8021 nm.

[0011] Based on a general inventive concept, the present invention also provides a method for preparing a biomimetic Prussian blue nanopreparation loaded with nerve growth factor, comprising the following steps: S1. Prepare a biomimetic hybrid membrane solution; S2. Mix an FeCl 3 solution with a citric acid as a dissolution medium and a K 4 [Fe(CN) 6 solution, and stir in a water bath to obtain a Prussian blue nanoparticle solution, i.e., a PB solution; S3. Mix a hydrochloric acid dopamine solution with the PB solution prepared in step S2, and after water bath ultrasonic treatment, stirring, centrifugation and dispersion, obtain a Prussian blue nanoparticle dispersion coated with a polydopamine coating, i.e., a PB@PDA dispersion; S4. Mix the PB@PDA dispersion prepared in step S3 with a nerve growth factor solution, and after stirring, centrifugation and dispersion, obtain a PB@PDA dispersion loaded with nerve growth factor, i.e., a PB@PDA@NGF dispersion; S5. Mix the PB@PDA@NGF dispersion prepared in step S4 with the biomimetic hybrid membrane solution prepared in step S1, and repeatedly extrude, and after centrifugation and dispersion, obtain a PB@PDA@NGF dispersion coated with a biomimetic hybrid membrane coating, i.e., obtain a biomimetic Prussian blue nanopreparation loaded with nerve growth factor.

[0012] In the above preparation method, preferably, in step S1, the specific preparation process of the biomimetic hybrid membrane is: mix an osteoprogenitor cell membrane and an erythrocyte membrane according to a mass ratio of 1:1 to 2, and perform ultrasonic fragmentation in a water bath at 2 to 8 °C and a power of 80 to 100 W for 1 to 2 min, and then stir in a water bath at 30 to 40 °C and a rotation speed of 500 to 800 rpm in a phosphate buffer solution for 1 to 2 h.

[0013] Preferably, in step S2, the molar mass ratio of citric acid, FeCl 3 and K 4 [Fe(CN) 6 is 20 to 30:1:1, the water bath temperature is 50 to 70 °C, the stirring rotation speed is 500 to 800 rpm, and the water bath time is 20 to 30 min.

[0014] Preferably, in step S3, the solvent of the dopamine hydrochloride solution is a Tris-HCl solution with a concentration of 8-10 mM and a pH value of 7-9; the mass ratio of Prussian blue to dopamine hydrochloride is 1-0.5:1, preferably 4:3; the temperature of the water bath ultrasound is 20-30 °C, the power is 80-100 W, and the time is 10-30 min, preferably 15 min; the stirring speed is 500-800 rpm, the stirring temperature is 30-40 °C, and the stirring time is 0.5-2 h, preferably 1 h; the centrifugation speed is 12,000-14,000 rpm, and the centrifugation time is 10-20 min.

[0015] Preferably, in step S4, the mass ratio of nerve growth factor to Prussian blue is 1:200-300, preferably 1:250, the stirring speed is 400-800 rpm, the stirring temperature is 2-8 °C, and the stirring time is 12-24 h, preferably 20 h; the centrifugation speed is 12,000-14,000 rpm, and the centrifugation time is 10-20 min.

[0016] Preferably, in step S5, the mass ratio of Prussian blue to the biomimetic hybrid membrane is 1-2:1, and the repeated extrusion means repeated extrusion at least 10 times with a micro-extruder with a pore size of 200 nm.

[0017] Based on a general inventive concept, the present invention also provides an application of a biomimetic Prussian blue nanoformulation loaded with nerve growth factor in the preparation of a drug for preventing and / or treating osteoporosis, and the osteoporosis is preferably senile osteoporosis.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The present invention designs a biomimetic nanoformulation with high safety and biodegradability. The formulation is loaded with nerve growth factor by Prussian blue nanoparticles coated with a polydopamine coating, and a biomimetic hybrid membrane is camouflaged on the outermost layer of the nanocomposite. Utilizing the characteristic of the biomimetic hybrid membrane to extend the half-life, the enrichment and slow release of the drug at the lesion site are achieved.

[0019] 2. The biomimetic Prussian blue nanoformulation loaded with nerve growth factor provided by the present invention utilizes the anti-osteoporosis effects of two different mechanisms of nerve growth factor promoting bone formation and Prussian blue antioxidant, assisted by a nanocarrier and a biomimetic system, to maximize the prevention and treatment effects of osteoporosis.

[0020] 3. In addition to loading nerve growth factor, the present invention can also be used as an alternative platform for the treatment of other diseases, such as tumors, rheumatoid arthritis, and atherosclerosis, by replacing different types of biomimetic hybrid membranes and drugs. Therefore, the present invention combines the advantages of the biomimetic hybrid membrane coating and the nanocomposite, and can provide multiple functions and advantages for the treatment of various diseases.

[0021] 4. The preparation method of the present invention optimizes various parameter conditions and successfully prepares a biomimetic Prussian blue nanoplatform loaded with nerve growth factor in the shape of a uniform and dispersed cube with a "core-shell" structure. It has excellent antioxidant capacity and good biosafety, can effectively promote bone formation, and can increase the enrichment of the nanoplatform in organs by prolonging the blood circulation time.

[0022] 5. The present invention can improve the clinical application defects of nerve growth factor, solve the fundamental problem of impaired osteogenic ability in osteoporosis, can effectively target the osteoporosis site, achieve high drug enrichment at the osteoporosis site, and provide new theoretical support for the development of new drugs for the prevention and treatment of osteoporosis and related prevention and treatment, which has important scientific significance, use value and economic value. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] 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 following drawings are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0024] Figure 1 It is an optimized preparation process diagram of the biomimetic Prussian blue nanoplatform loaded with nerve growth factor of the present invention, where: A is the particle size of PB@PDA nanoparticles prepared under different ultrasonic times (15 min, 30 min) and stirring times (1 h, 2 h); B is the encapsulation efficiency of NGF under different mass ratios of NGF to PB (1:200, 1:250, 1:300) and stirring times (16 h, 20 h). Figure 2 It is a characterization diagram of the biomimetic Prussian blue nanoplatform loaded with nerve growth factor of the present invention, where: A is the fluorescence image of cell membrane fusion of red blood cell membrane and osteoblast precursor cell membrane; B is the transmission electron microscopy images of PB, PB@PDA, M@PB@PDA@NGF; C-D are the particle size and potential of PB, PB@PDA, PB@PDA@NGF, M@PB@PDA@NGF in sequence; E-G are the elemental analysis spectra of PB, PB@PDA, PB@PDA@NGF in sequence; in the figure, P represents PB, PD represents PB@PDA, PDN represents PB@PDA@NGF, and MPDN represents M@PB@PDA@NGF; Figure 3Characteristics of the bionic Prussian blue nanoplatform loaded with nerve growth factor (NGF) for antioxidation and osteogenesis promotion of the present invention. In the figures: A is the fluorescence image of reactive oxygen species (ROS) in pre-osteoblasts; B is the flow cytometry quantification of ROS in pre-osteoblasts; C is the DPPH assay quantification (total antioxidant capacity); D is the alkaline phosphatase staining image (osteogenesis promotion performance); E is the alizarin red staining image (osteogenesis promotion performance); in the figures, NGF represents nerve growth factor, MPD represents M@PB@PDA, and MPDN represents M@PB@PDA@NGF; Figure 4 Blood half-life of the bionic Prussian blue nanoplatform loaded with nerve growth factor (M@PB@PDA@NGF) of the present invention in C57BL / 6 mice and biodistribution in senile osteoporosis (SAMP6) mice. In the figures: A-B are the in vitro fluorescence images and fluorescence intensity statistical charts of mouse blood at different time points in sequence; C-D are the fluorescence localization images and fluorescence intensity statistical charts of the lower limb bones of mice in sequence; E-F are the fluorescence localization images and fluorescence intensity statistical charts of the heart, liver, spleen, lungs, and kidneys of mice in sequence; Figure 5 Therapeutic efficacy diagrams of different treatment groups for osteoporosis of the present invention. In the figures: A-D are the representative micro-CT images of femurs and quantitative analysis diagrams of related parameters in sequence; E-F are the H&E staining images and Masson staining images of the tibias of senile osteoporosis mice treated with different treatment groups in sequence; in the figures, NGF represents nerve growth factor, MPD represents M@PB@PDA, and MPDN represents M@PB@PDA@NGF; Figure 6 H&E staining images of the heart, liver, spleen, lungs, and kidneys of senile osteoporosis mice treated with different treatment groups of the present invention; in the figures, NGF represents nerve growth factor, MPD represents M@PB@PDA, and MPDN represents M@PB@PDA@NGF. Detailed implementation manners

[0025] To facilitate the understanding of the present invention, the present invention will be described more comprehensively and in detail below in conjunction with the specification drawings and preferred embodiments, but the protection scope of the present invention is not limited to the following specific embodiments.

[0026] Unless otherwise defined, all professional terms used hereinafter have the same meaning as commonly understood by those skilled in the art. The professional terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the protection scope of the present invention.

[0027] Unless otherwise specifically stated, various raw materials, reagents, instruments, and equipment used in the present invention can be obtained through market purchases or can be prepared by existing methods.

[0028] Example 1: A biomimetic Prussian blue nano - preparation loaded with nerve growth factor, which has a core - shell structure. Its core is Prussian blue nanoparticles coated with a polydopamine coating, and nerve growth factor is loaded on the surface of the polydopamine coating; its shell is a biomimetic hybrid membrane.

[0029] (1)Preparation of a biomimetic Prussian blue nano - preparation loaded with nerve growth factor S1. Preparation of a biomimetic hybrid membrane dispersion: Take the whole blood of C57BL / 6 mice, wash it with PBS (pH = 7.4), and centrifuge at 3000 rpm for 5 min to remove plasma. Resuspend the red blood cells in 0.25×PBS (pH 7.4) and lyse them at 4℃ for 6 - 8 h. Then centrifuge the lysate at 12000 rpm for 10 min to obtain RBCm and wash it with 0.25×PBS (pH 7.4) until the supernatant becomes colorless. Collect the washed RBCm.

[0030] The preparation of the membrane of pre - osteoblasts (MC3T3 - E1 cells) is carried out according to the instructions of the membrane protein extraction kit. When the MC3T3 - E1 cells in the cell culture dish are confluent, scrape the cells with a cell scraper and centrifuge at 800 rpm for 5 min. The collected cells are washed twice with PBS (pH 7.4) at 4℃, and then suspended in membrane protein extractant A containing PMSF (1 mM). The mixture is lysed in an ice bath for 30 min and sonicated in a 4℃ water bath for 10 min (80W, on for 0.3 s, off for 0.5 s). Freeze - thaw repeatedly at 37℃ and - 80℃ for 4 times, 20 - 30 min each time. Then centrifuge at 4℃, 800 rpm for 10 min. Take the supernatant and continue to centrifuge at 4℃, 13000 rpm for 30 min to obtain the pre - osteoblast cell membrane.

[0031] Use the BCA protein assay kit to determine the protein concentrations of the red blood cell membrane (RBCm) and the pre - osteoblast cell membrane (MC3T3m). The mass of the membrane is twice the mass of the membrane protein. Mix RBCm and MC3T3m at a mass ratio of 1:1, sonicate in a 4℃ water bath for 1 min (100W, on for 0.5 s, off for 0.5 s), and stir in a 37℃ water bath at a speed of 600 rpm for 1 h to complete membrane fusion; S2. Preparation of a PB nanoparticle solution: Mix the FeCl 3 solution with the K 4 [Fe(CN) 6 solution. The molar mass ratio of citric acid, FeCl 3 and K 4 [Fe(CN) 6 is 25:1:1. The water bath temperature is 60℃, the stirring speed is 800 rpm, and the water bath time is 30 min to obtain a PB nanoparticle solution; S3. Preparation of PB dispersion coated with polydopamine coating: After mixing 1.5 mg of dopamine hydrochloride dissolved in 2 mL of Tris-HCl solution (10 mM, pH = 8.5) with 500 μL of PB solution (4 mg / mL), ultrasonic bath at 25 °C (100 W) for 15 min, stir at 35 °C and 800 rpm for 1 h, centrifuge at 12,000 rpm for 20 min, and redissolve with 1 mL of ddH 2 O, then ultrasonic bath (50 W) for 5 min to obtain PB (PB@PDA, PD) dispersion coated with polydopamine coating; S4. Preparation of PB composite loaded with NGF: Mix the PD dispersion prepared in S3 and NGF solution at a mass ratio of 200:1, stir at 4 °C and 800 rpm for 20 h, centrifuge at 12,000 rpm for 20 min, and redissolve with 1 mL of ddH 2 O, then ultrasonic bath (50 W) for 5 min to obtain PB@PDA (PB@PDA@NGF, PDN) dispersion loaded with NGF; S5. Preparation of biomimetic PB nanocomposite loaded with NGF: Centrifuge the PDN dispersion in S4 at 12,000 rpm for 20 min, redissolve the obtained PDN precipitate in PBS solution, mix it with the biomimetic hybrid membrane (2 mL, 1 mg / mL) after ultrasonic bath treatment at a mass ratio of 1:1, and repeatedly extrude through a micro extruder with a pore size of 200 nm at least 10 times to obtain a biomimetic Prussian blue nano preparation loaded with nerve growth factor (M@PB@PDA@NGF, MPDN).

[0032] (2) Preparation process optimization and characterization of biomimetic Prussian blue nano preparation loaded with nerve growth factor Taking the biomimetic Prussian blue nano preparation loaded with nerve growth factor prepared in Example 1 as an example for optimization and characterization.

[0033] The results are as Figure 1 shown, Figure 1 The particle size results of A show that the PB@PDA nanoparticles obtained after ultrasonic treatment for 15 min and stirring for 1 h of the mixed solution of PB and DA have the smallest particle size; Figure 1 The measurement results of the NGF encapsulation efficiency of B confirm that when the mass ratio of NGF to PB is 1:250 and the stirring time is 20 h, the NGF encapsulation efficiency is the highest. The above results comprehensively show that the optimized preparation conditions of PB@PDA are ultrasonic bath for 15 min and stirring for 1 h; the optimized preparation conditions of PB@PDA@NGF are the mass ratio of NGF to PB of 1:250 and the stirring time of 20 h.

[0034] After the preparation conditions are optimized, the characterization results of the biomimetic Prussian blue nano preparation loaded with nerve growth factor are as Figure 2 shown,Figure 2 The laser confocal fluorescence image of A indicates the successful fusion and preparation of the bionic hybrid film. Figure 2 The transmission electron microscope image of B and Figure 2 The DLS particle size and potential measurement results of C and D indicate that the MPDN composite preparation was successfully prepared, showing a uniformly dispersed cubic shape. The bionic hybrid film was wrapped around the outer layer of PDN, presenting an obvious "core-shell" structure, with an average particle size of 202.4 ± 0.8021 nm and an average potential of -19.20 ± 1.572 mV. Figure 2 The elemental analysis spectra of E, F, and G indicate that compared with PD, the content ratio of sulfur element in PDN increased from 0.53% to 2.82%, indirectly indicating the successful loading of NGF.

[0035] (3) Effects of the bionic Prussian blue nano - preparation loaded with nerve growth factor in vitro antioxidant and osteogenesis promotion Taking the bionic Prussian blue nano - preparation loaded with nerve growth factor prepared in Example 1 as an example, this effect is described.

[0036] The results are as Figure 3 shown, Figure 3 The intracellular ROS fluorescence map of A and Figure 3 The flow cytometry quantitative map of B both indicate that MPDN can significantly reduce the intracellular reactive oxygen species (ROS) level; Figure 3 C shows that MPDN has excellent antioxidant ability; Figure 3 The alkaline phosphatase staining of D and Figure 2 The alizarin red staining map of D show that MPDN can effectively promote osteogenesis.

[0037] (4) Bionic performance of the bionic Prussian blue nano - preparation loaded with nerve growth factor in vivo Continuing with the bionic Prussian blue nano - preparation loaded with nerve growth factor prepared in Example 1, the blood half - life and in - vivo biodistribution of the M@PB@PDA@NGF bionic nano - preparation were determined by semi - quantitative means of detecting fluorescence intensity.

[0038] Experiment 1: C57BL / 6 mice were intravenously injected with 200 μL of a dose concentration of 2.5 mg / kg of chlorin e6 (Ce6), PD Ce6 @HM, and blood samples were collected at different time points for fluorescence intensity measurement.

[0039] Among them, PD Ce6 @HM was prepared according to the following method: First, 50 mg of EDC and 10 mg of Ce6 were dissolved in ddH 2In O, stir at 800 - 1000 rmp at room temperature for 30 min - 1 h. Then, add 50 mg of NHS to the above solution and stir at 800 - 1000 rpm at room temperature for 1.5 h. Next, add 5 mg of PD to the above solution and stir at room temperature for 6 h. Then, centrifuge the above mixed solution at 12000 rpm at room temperature for 20 min to remove free EDC, NHS, and Ce6. Finally, redissolve the PD precipitate obtained by the above centrifugation in PBS, and add the biomimetic hybrid membrane (composed of red blood cell membrane RBCm and pre - osteoblast cell membrane MC3T3m after water - bath ultrasonic treatment, the method is as in step S1) (5 mL, 1 mg / mL), and repeatedly extrude through a micro - extruder with a pore size of 200 nm at least 10 times to obtain the PD@HM solution, and place it in a refrigerator at 4 °C for later use. Ce6 Precipitate was redissolved in PBS, and the biomimetic hybrid membrane (composed of red blood cell membrane RBCm and pre - osteoblast cell membrane MC3T3m after water - bath ultrasonic treatment, the method is as in step S1) (5 mL, 1 mg / mL) was added, and repeatedly extruded through a micro - extruder with a pore size of 200 nm at least 10 times to obtain the PD Ce6 @HM solution, and placed in a refrigerator at 4 °C for later use.

[0040] Experiment 2: After feeding senile osteoporosis mice with normal diet for 2 months, inject 200 μL of Ce6, PD Ce6 @HM (the same as in Experiment 1) with a dose concentration of 2.5 mg / kg via the tail vein. After 24 h, take the lower limb bones and main organs of the mice for fluorescence imaging.

[0041] The results are as Figure 4 shown, Figure 4 A - B shows that compared with the free Ce6 group, the blood circulation period of the PD Ce6 @HM group was significantly prolonged, which was 1.57 times that of the free Ce6 group (0.669 h vs 0.426 h). It shows that the nano - preparation camouflaged by the biomimetic hybrid membrane is beneficial to prolong the blood circulation time. Figure 4 C - D shows that compared with the free Ce6 group, the PD Ce6 @HM group was significantly enriched at the femur and tibia, indicating that the biomimetic Prussian blue nano - preparation loaded with nerve growth factor prepared in Example 1 can effectively target the osteoporosis site. Figure 4 E - F shows that compared with the free Ce6 group, the PD Ce6 @HM group had a higher enrichment degree in the main organs such as the heart, liver, spleen, lungs, and kidneys. Combining with Figure 4 A - B, it shows that the nano - preparation camouflaged by the biomimetic hybrid membrane can increase the enrichment of the nano - preparation in organs by prolonging the blood circulation time.

[0042] Example 2: Application of a biomimetic Prussian blue nano - preparation loaded with nerve growth factor in the preparation of drugs for preventing and / or treating osteoporosis. The HA - M@AT@GP biomimetic nano - preparation prepared in Example 1 was used for the treatment of senile osteoporosis.

[0043] Mouse treatment experiment: After 1 week of adaptive feeding, 4-month-old senescence-accelerated male mice (SAMP6, which can be used as an animal model for senile osteoporosis) and normal senescent male mice of the same age (SAMR1, used as a normal control for SAMP6) were administered drugs. The drugs were administered once every two days for 6 consecutive weeks, all by tail vein injection. The treatment groups were the single NGF (10 mg / kg) group, the MPD (2.5 mg / kg) group, and the MPDN (2.5 mg / kg) group. Three days after the end of the treatment, the femurs and tibias of the mice were dissected for micro-CT detection, H&E staining, and Masson staining to investigate the anti-osteoporosis effects of different treatment groups.

[0044] The results are as Figure 5 shown. Compared with the NGF and MPD groups, the MPDN treatment group significantly improved the bone microstructure of the mice in the osteoporosis group and promoted bone regeneration, indicating that the biomimetic nanoformulation constructed in the present invention can effectively treat senile osteoporosis.

[0045] In vivo biosafety experiment: Using the treatment protocol adopted in Example 2, after the treatment of senile osteoporosis mice, the main organs (heart, liver, spleen, lung, and kidney) of the mice in each group were collected for H&E staining to investigate the biosafety of different treatment groups.

[0046] The results are as Figure 6 shown. No obvious organic lesions were found in the organs of the mice in different treatment groups, indicating that the biomimetic nanoformulation MPDN constructed in the present invention has good biosafety.

Claims

1. A bionic Prussian blue nano preparation loaded with nerve growth factor, characterized in that: The invention is a core-shell structure, wherein the core is a Prussian blue nanoparticle coated with a polydopamine coating, and the surface of the polydopamine coating is loaded with a nerve growth factor; and the shell is a bionic hybrid membrane.

2. The bionic Prussian blue nano preparation loaded with nerve growth factor according to claim 1, characterized in that: The bionic hybrid membrane is prepared by mixing osteoblast precursor cell membrane and erythrocyte membrane in a mass ratio of 1:1-2 and then crushing them.

3. The bionic Prussian blue nano preparation loaded with nerve growth factor according to claim 1, characterized in that: The mass ratio of the Prussian blue nanoparticles to the bionic hybrid membrane is 1-2:1, the mass ratio of the nerve growth factor to the Prussian blue nanoparticles is 1:200-300, and the mass ratio of the Prussian blue nanoparticles to the polydopamine coating is 1-0.5:

1.

4. The bionic Prussian blue nanoparticle preparation loaded with nerve growth factor according to any one of claims 1 to 3, characterized in that: The particles of the nanoformulation are in a cubic shape and have an average particle size of 200±1 nm.

5. A method for preparing a bionic Prussian blue nano preparation loaded with nerve growth factor, characterized in that: The steps include: S1, preparing a biomimetic hybrid membrane solution; S2, mixing the FeCl3 solution and K4[Fe(CN)6] solution using citric acid as the dissolving medium, and stirring to obtain a Prussian blue nanoparticle solution, i.e., a PB solution; S3, mixing the dopamine hydrochloride solution with the PB solution prepared in step S2, and obtaining a Prussian blue nanoparticle dispersion coated with a polydopamine coating, i.e., a PB@PDA dispersion, after ultrasonication, stirring, centrifugation and dispersion; S4, mixing the PB@PDA dispersion prepared in step S3 with the nerve growth factor solution, stirring, centrifuging and dispersing to obtain a PB@PDA dispersion loaded with nerve growth factor, i.e., a PB@PDA@NGF dispersion; S5. The PB@PDA@NGF dispersion prepared in step S4 and the bionic hybrid membrane solution prepared in step S1 are mixed and squeezed repeatedly, and the mixture is dispersed by centrifugation to obtain a PB@PDA@NGF dispersion coated with a bionic hybrid membrane coating, i.e., a bionic Prussian blue nanopreparation loaded with nerve growth factor is obtained.

6. The preparation method according to claim 5, characterized in that: In step S1, the specific preparation process of the bionic hybrid membrane is: the osteoblast precursor cell membrane and the red blood cell membrane are mixed in a mass ratio of 1:1-2, broken by water bath ultrasound at 2-8°C and a power of 80-100W for 1-2 minutes, and then stirred in a phosphate buffer at 30-40°C and a speed of 500rpm-800rpm for 1-2 hours.

7. The preparation method according to claim 5, characterized in that: In step S2, the molar mass ratio of citric acid, FeCl3 and K4[Fe(CN)6] is 20-30:1:1, the water bath temperature is 50-70°C, the stirring speed is 500-800 rpm, and the water bath time is 20-30 min.

8. The preparation method according to claim 5, characterized in that: In step S3, the solvent of the dopamine hydrochloride solution is a Tris-HCl solution, the concentration of which is 8-10 mM and the pH value is 7-9; the mass ratio of Prussian blue to dopamine hydrochloride is 1-0.5:1; the temperature of water bath ultrasound is 20-30°C, the power is 80-100 W, and the time is 10-30 min; the stirring speed is 500-800 rpm, the stirring temperature is 30-40°C, and the stirring time is 0.5-2 h; the centrifugal speed is 12000-14000 rpm, and the centrifugal time is 10-20 min.

9. The preparation method according to claim 5, characterized in that: In step S4, the mass ratio of nerve growth factor to Prussian blue is 1:200-300, the stirring speed is 400-800 rpm, the stirring temperature is 2-8°C, and the stirring time is 12-24 hours; the centrifugal speed is 12000-14000 rpm, and the centrifugal time is 10-20 minutes; in step S5, the mass ratio of Prussian blue to bionic hybrid membrane is 1-2:1, and the repeated extrusion refers to repeated extrusion at least 10 times using a micro extruder with a pore size of 200 nm.

10. Use of the bionic Prussian blue nanoformulation loaded with nerve growth factor according to any one of claims 1 to 4 or the bionic Prussian blue nanoformulation loaded with nerve growth factor prepared by the preparation method according to any one of claims 5 to 9 in the preparation of drugs for preventing and / or treating osteoporosis.

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