Amifostine-loaded nanoparticles and their preparation method and application

Abstract

The invention discloses a kind of nanoparticles of load amifostine and its preparation method and application, belong to the field of pharmaceutical technology; The nanoparticles of load amifostine of the present invention are spherical shell-core structure, including PLGA core and DSPE PEG AMF and PS 80 surrounding the outside of PLGA core, and the particle diameter of the nanoparticles is 50 500nm. The nanoparticles of load amifostine provided by the present invention can enter the brain through the blood-brain barrier after ophthalmic vein injection, are converted into active form after metabolism by alkaline phosphatase on the surface of normal brain cells, play the role of scavenging oxygen free radicals (ROS), so as to prevent the occurrence of radiation brain injury, the nanoparticles of load amifostine obtained are significantly better than original drug amifostine in the preventive effect on brain injury, can be applied to the preparation of medicines for preventing radiation brain injury (RIBI); and the preparation method provided by the present invention is simple, which is conducive to actual production.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicines, and in particular relates to nanoparticles loaded with amifostine, a preparation method thereof and an application thereof. Background Art

[0002] Radiation-induced brain injury (RBI) is a serious, irreversible, and even life-threatening complication of radiotherapy for patients with head and neck cancer. Clinical manifestations primarily include increased intracranial pressure due to cerebral edema and localized symptoms or signs caused by necrotic brain tissue. Currently, the primary treatment for RBI is glucocorticoids, which can inhibit the onset and progression of inflammatory responses in the brain, reduce the severity of neurological deficits, and reduce lesion size, thereby improving long-term prognosis. Commonly used RBI RBI steroids include dexamethasone, methylprednisolone, and prednisone, with methylprednisolone being the most widely used. However, steroid therapy is associated with side effects such as Cushing's-like syndrome, peptic ulcers, osteoporosis, immunosuppression, and psychiatric disorders, and only 35% of patients are sensitive to steroid therapy. Other adjunctive therapies, such as antiplatelet therapy, anticoagulation, hyperbaric oxygen therapy, and circulatory stimulation, have been shown to improve RBI. Anticoagulants such as heparin and warfarin can improve clinical symptoms of RBI, possibly by reversing capillary endothelial damage. Hyperbaric oxygen therapy aims to increase the oxygen partial pressure in tissue cells to stimulate vascular repair mechanisms, reduce vascular permeability, and mitigate radiation-induced necrosis of normal surrounding tumor tissue. Studies have shown that hyperbaric oxygen can reduce cerebral edema. Craniotomy and resection of radiation-induced lesions are currently reserved for patients with refractory brain injuries or those who have failed conservative treatment. However, conventional treatment regimens, primarily based on hormones, have reported efficacy rates of only approximately 20-35%, indicating that there is currently no satisfactory preventive or effective treatment.

[0003] Amifostine (AMF), also known as Amifostine, is a white solid chemical with the chemical name 2-(3-aminopropylamino)-ethanethiol phosphate and the molecular formula C5H 15 N2O3PS has a molecular weight of 325.4242, a melting point of 160-161°C, and is readily soluble in water. Amifostine is an effective free radical scavenger and a protective agent for normal cells. It is clinically used primarily as an adjuvant therapy for various cancers and has been shown to prevent radiation-related toxicity. However, amifostine is highly water-soluble and therefore cannot cross the blood-brain barrier, hindering its application in preventing radiation-induced brain injury. Summary of the Invention

[0004] The purpose of the present invention is to overcome the deficiencies of the above-mentioned prior art and provide a nanoparticle loaded with amifostine that can target the blood-brain barrier and effectively achieve brain radiation protection, as well as a preparation method and application thereof.

[0005] To achieve the above object, the technical solution adopted by the present invention is: a nanoparticle loaded with amifostine, the nanoparticle has a spherical shell-core structure, including a PLGA core and DSPE-PEG-AMF and PS 80 surrounding the PLGA core, and the particle size of the nanoparticle is 50-500nm.

[0006] The present invention provides nanoparticles loaded with amifostine, which are formed by co-assembling PLGA, DSPE-PEG-AMF, and PS 80 to form nanoparticles of a specific particle size. The resulting nanoparticles can enter the brain through the blood-brain barrier after ophthalmic vein injection, and are converted into an active form after metabolism by alkaline phosphatase (ALP) on the surface of normal brain cells, which volatilizes and scavenges oxygen free radicals, thereby preventing the occurrence of radiation-induced brain damage. The obtained nanoparticles loaded with amifostine have a significantly better preventive effect on brain damage than the original drug Amifostine (trade name, Amifostine for Injection). Specifically, in the nanoparticles loaded with amifostine provided by the present invention, PEG can protect the nanoparticles from being cleared by the blood of the reticuloendothelial system and prolong drug circulation; PS 80 surrounds the PLGA core to form a part of the shell structure of the spherical structure, which is used for brain-targeted drug delivery.

[0007] The particle size range of the nanoparticles of the present invention is determined by using dynamic light scattering technology to test the particle size of the nanoparticles for quality control, and the particle size range is 50-500 nm after weighted statistics based on the number of particles.

[0008] In addition, the present invention also provides a method for preparing the amifostine-loaded nanoparticles, which comprises the following steps:

[0009] (1) Synthesis of DSPE-PEG-AMF: A methanol solution of DSPE-PEG-NHS was added to an aqueous solution of amifostine in triethylamine, followed by addition of PBS buffer and reaction under inert gas protection. After the reaction, ether was added for precipitation, followed by filtration and washing. The residue was collected and purified, and then lyophilized to obtain DSPE-PEG-AMF.

[0010] (2) Preparation of nanoparticles loaded with amifostine: A dimethylformamide solution of PLGA, a dimethylformamide solution of DSPE-PEG-AMF and an aqueous solution of PS 80 were mixed and added to water and stirred, followed by centrifugation and washing to obtain nanoparticles loaded with amifostine.

[0011] As a preferred embodiment of the preparation method of the present invention, in step (1), the molar ratio of DSPE-PEG-NHS, amifostine and triethylamine is DSPE-PEG-NHS: amifostine: triethylamine = 1: (0.8-1.0): (1.2-1.8).

[0012] As a preferred embodiment of the preparation method of the present invention, in step (1), the reaction temperature is 20-30°C, the reaction time is 10-14h, and the pH value of the PBS buffer is 7.

[0013] As a preferred embodiment of the preparation method of the present invention, in the step (1), the mass concentration of DSPE-PEG-NHS in the methanol solution of DSPE-PEG-NHS is 12.5-37.5 mg / ml; the mass concentration of amifostine in the triethylamine aqueous solution of amifostine is 13.89-41.67 mg / ml, and the mass concentration of triethylamine is 9.84-29.52 mg / ml.

[0014] As a preferred embodiment of the preparation method of the present invention, in step (1), the volume ratio of the added amount of diethyl ether to the reaction liquid after the reaction is diethyl ether: reaction liquid = (18-22):1.

[0015] As a preferred embodiment of the preparation method of the present invention, in the step (1), the purification method is dialysis, and the dialysis is performed using MWCO 2000.

[0016] As a preferred embodiment of the preparation method of the present invention, if fluorescence imaging is required, DiD (1,1-dioctadecyl-3,3,3,3-tetramethyldodecanocyanine 4-chlorobenzenesulfonate) dye can be added in step (2); specifically, if step (2) of adding DiD dye is: a dimethylformamide solution of PLGA, a dimethylformamide solution of DSPE-PEG-AMF, a PS 80 aqueous solution and a dimethylformamide solution of DiD dye are mixed, added to water and stirred, and then centrifuged and washed to obtain nanoparticles loaded with amifostine.

[0017] As a preferred embodiment of the preparation method of the present invention, the mass concentration of the DiD dye in the dimethylformamide solution of the DiD dye is 9.5-10.5 μg / mL.

[0018] As a preferred embodiment of the preparation method of the present invention, in step (2), the mass ratio of PLGA, DSPE-PEG-AMF and PS 80 is PLGA:DSPE-PEG-AMF:PS 80=5:(5.5-6.5):(29-31).

[0019] As a preferred embodiment of the preparation method of the present invention, the mass ratio of PLGA to DiD dye is 500:(0.8-1.2).

[0020] As a preferred embodiment of the preparation method of the present invention, in the step (2), the mass concentration of PLGA in the dimethylformamide solution is 4.5-5.5 mg / mL; the mass concentration of DSPE-PEG-AMF in the dimethylformamide solution is 5.5-6.5 mg / mL; and the mass concentration of PS 80 in the PS 80 aqueous solution is 1.5-2.5 mg / mL.

[0021] As a preferred embodiment of the preparation method of the present invention, in the step (2), the volume ratio of dimethylformamide to water is 1:(14-16).

[0022] As a preferred embodiment of the preparation method of the present invention, in step (2), the centrifugation is carried out in a centrifugal filter with a molecular weight cut-off value of 100 KDa.

[0023] In addition, the present invention also provides the use of the nanoparticles loaded with amifostine in preparing drugs for preventing radiation-induced brain damage.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The present invention provides amifostine-loaded nanoparticles, which are prepared by co-assembling PLGA, DSPE-PEG-AMF and PS 80 to form nanoparticles of a specific particle size. The obtained nanoparticles can enter the brain through the blood-brain barrier after being injected into the ophthalmic vein, and are converted into an active form after being metabolized by alkaline phosphatase on the surface of normal brain cells, and volatilize to scavenge oxygen free radicals (ROS), thereby preventing the occurrence of radiation-induced brain injury. The obtained amifostine-loaded nanoparticles have a significantly better preventive effect on brain injury than the original drug amifostine, and can be used in the preparation of drugs for preventing radiation-induced brain injury (RIBI). In addition, the preparation method provided by the present invention is simple and is conducive to actual production. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 A is a schematic diagram of the preparation process of DAPP NPs and the schematic diagram of the onset process in Example 1;

[0027] Figure 1 Figure B is a schematic diagram of the onset of action of DAPP NPs after intravenous injection in Example 1;

[0028] Figure 2 Figure A is the MALDI-TOF spectrum result of DSPE-PEG-NHS in Example 1;

[0029] Figure 2 Figure B is the MALDI-TOF spectrum result of DSPE-PEG-AMF in Example 1;

[0030] Figure 2 Middle C is the isotope profile of peaks 2423.44, 2467.46, and 2511.49 of the MALDI-TOF spectrum of DSPE-PEG-AMF in Effect Example 1;

[0031] Figure 3 A is the structural formula of DSPE-PEG-NHS and DSPE-PEG-AMF in Example 1;

[0032] Figure 3 Middle B is a magnified comparison of the hydrogen spectra of DSPE-PEG-NHS and DSPE-PEG-AMF in Example 1;

[0033] Figure 3 Figure C is a magnified comparison of the phosphorus spectra of DSPE-PEG-NHS and DSPE-PEG-AMF in Example 1;

[0034] Figure 3 D is a magnified view of the hydrogen spectrum of DSPE-PEG-AMF in Example 1;

[0035] Figure 4 This is the electron microscopy image of DAPP NPs in effect example 1;

[0036] Figure 5 This is the NTA measurement result of DAPP NPs in Effect Example 1;

[0037] Figure 6 This is the size change diagram of DAPP NPs in PBS solution in effect example 1 (10-day stability);

[0038] Figure 7 Figure A is the UPLC results of DAPP NPs and DSPE-PEG-AMF;

[0039] Figure 7 Figure B is the standard curve of DSPE-PEG-AMF solution;

[0040] Figure 8 Middle A is a picture of primary neurons incubated with DAPP NPs at 37°C for 1 hour in Effect Example 2 (illustration of DAPP NPs uptake by primary neurons observed under a fluorescence microscope);

[0041] Figure 8 Middle B is the flow cytometric image of primary neurons incubated with DAPP NPs for 30 min and 1 h in effect example 2 (flow cytometry detection of DAPP NPs uptake by primary neurons);

[0042] Figure 8Middle C is the average fluorescence intensity (n=3) of primary neurons incubated with DAPP NPs for 30 min and 1 h in effect example 2 (statistical graph of flow cytometry results);

[0043] Figure 8 Figure D shows the results of incubating BV2 cell lines with DAPP NPs at 37°C for 1 hour in Example 2; (BV2 cells were observed for DAPP NPs uptake using a fluorescence microscope)

[0044] Figure 8 Middle E is the flow cytometric image of BV2 cells incubated with DAPP NPs for 30 minutes and 1 hour in effect example 2. Flow cytometry was used to detect the uptake of DAPP NPs by primary neurons;

[0045] Figure 8 Middle F is the average fluorescence intensity (n=3) of BV2 cells incubated with DAPP NPs for 30 minutes and 1 hour in effect example 2 (flow cytometry results statistics);

[0046] Figure 8 Middle G is the picture of cell apoptosis detected by flow cytometry in effect example 2;

[0047] Figure 8 Middle H is a histogram of the results of cell apoptosis detected by flow cytometry in Effect Example 2 (flow cytometry apoptosis statistics graph);

[0048] Figure 9 A in the middle is the result of detecting the viability of BV2 cells using CCK-8 after incubation of DAPP NPs with AMF at different concentrations for 24 hours in Example 3;

[0049] Figure 9 Middle B is the result of viability of BV2 cells irradiated with 10 Gy X-rays after incubation of DAPP NPs and AMF at different concentrations for 1 hour in Effect Example 3;

[0050] Figure 10 Middle A is the result of crystal violet staining of surviving cell colonies in different treatment groups of BV2 cells and S18 cells irradiated with 4 Gy X-rays in Effect Example 4;

[0051] Figure 10 Middle B is a quantitative result diagram of the surviving cell colonies in different treatment groups of BV2 cells irradiated with 4 Gy X-rays in Effect Example 4;

[0052] Figure 10 Middle C is a quantitative result graph of the surviving cell colonies in different treatment groups of S18 cells irradiated with 4 Gy X-rays in Effect Example 4;

[0053] Figure 11Middle A is the r-H2AX immunofluorescence assay in effect example 5 to detect the degree of DNA double-strand damage in primary neurons after different treatments;

[0054] Figure 11 Middle B is the fluorescence histogram of the results of r-H2AX immunofluorescence detection of DNA double-strand damage in primary neurons after different treatments in Example 5 ( Figure 11 Quantitative plot of A);

[0055] Figure 11 Middle C shows the degree of DNA double-strand damage in BV2 cells after different treatments as measured by r-H2AX immunofluorescence in Effect Example 5;

[0056] Figure 11 D is the fluorescence histogram of DNA double-strand damage results of BV2 cells after different treatments by r-H2AX immunofluorescence method in effect example 5 ( Figure 11 Quantitative plot of C);

[0057] Figure 12 Middle A is the fluorescence image of ROS generation in primary neuronal cells after different treatments in Effect Example 6;

[0058] Figure 12 Middle B is a fluorescence histogram of ROS generation in primary neuronal cells after different treatments in Effect Example 6;

[0059] Figure 12 Middle C is a flow cytometric graph of ROS generation in primary neuronal cells after different treatments in Effect Example 6;

[0060] Figure 12 Middle D is a flow cytometry histogram of ROS generation in primary neuronal cells after different treatments in Effect Example 6;

[0061] Figure 12 Middle E is the fluorescence image of ROS generation in BV2 cells after different treatments in Effect Example 6;

[0062] Figure 12 Middle F is the fluorescence histogram of ROS generation in BV2 cells after different treatments in Effect Example 6;

[0063] Figure 12 Middle G is the flow cytometric graph of ROS generation in BV2 cells after different treatments in Effect Example 6;

[0064] Figure 12 Middle H is a flow cytometry histogram of ROS generation in BV2 cells after different treatments in Effect Example 6;

[0065] Figure 13 Center A shows fluorescence imaging of the mouse brain after intravenous injection of DAPP NPs or PBS;

[0066] Figure 13 Middle B is the fluorescence imaging of the mouse brain slice in Example 7;

[0067] Figure 13 Middle C is the co-localization result of DAPP NPs and NeuN in effect example 7 (uptake of DAPPNPs by primary neurons in the brain);

[0068] Figure 13 Middle D is the co-localization result of DAPP NPs and IBA1 in effect example 7 (uptake of DAPPNPs by microglia in the brain);

[0069] Figure 13 Middle E is the co-localization result of DAPP NPs and GFAP in effect example 7 (uptake of DAPPNPs by astrocytes in the brain);

[0070] Figure 14 Middle A is the weight curve of mice (n=5) after continuous administration of DAPP NPs or PBS for 14 days in Effect Example 7;

[0071] Figure 14 Middle B is an H&E-stained section of the main organs of mice collected 14 days after intravenous administration in Effect Example 7;

[0072] Figure 14 Middle C is the blood routine analysis results of mice 14 days after intravenous administration in Effect Example 7;

[0073] Figure 14 Middle D is the result of blood biochemical analysis of mice 14 days after intravenous administration in Effect Example 7;

[0074] Figure 15 Schematic diagram of the timeline for the experimental design of the brain protective function of DAPP NPs in Example 7;

[0075] Figure 16 This is the dose distribution diagram of the target area of ​​X-knife brain radiotherapy in mice;

[0076] Figure 17 Middle A is the fluorescence image of ROS generation in the right thalamus tissue of mice after different treatments in Effect Example 7;

[0077] Figure 17 Middle B is a histogram of fluorescence data of ROS generation in the right thalamus tissue of mice after different treatments in Effect Example 7;

[0078] Figure 17 Middle C is the MRI image of the mouse brain after different treatments in effect case 7

[0079] Figure 17 Middle D is the quantitative image of brain lesions of mice after different treatments in effect example 7;

[0080] Figure 17 Middle E is a representative image of H&E-stained brain sections of mice with lesions in the right thalamus after different treatments in Effect Example 7;

[0081] Figure 17 Middle F is the immunohistochemical staining of CD34 in the right thalamus of mice after different treatments in Example 7;

[0082] Figure 17 Middle G is the histogram of the optical density (OD) values ​​of CD34 in the right thalamus of mice after different treatments in Example 7;

[0083] Figure 18 Middle A is a representative fluorescence image of NeuN staining in the right thalamus of mice after different treatments in Effect Example 7;

[0084] Figure 18 Middle B is a representative fluorescence histogram of NeuN staining in the right thalamus of mice after different treatments in Example 7

[0085] Figure 18 Middle C is a representative fluorescence image of NeuN staining in the right hippocampus (DG region) of mice after different treatments in Effect Example 7;

[0086] Figure 18 Middle D is a representative fluorescence histogram of NeuN staining in the right hippocampus (DG region) of mice after different treatments in Effect Example 7;

[0087] Figure 18 Middle E is a representative fluorescence image of NeuN staining in the right hippocampus (CA1 region) of mice after different treatments in Effect Example 7;

[0088] Figure 18 Middle F is a representative fluorescence histogram of NeuN staining in the right hippocampus (CA1 region) of mice after different treatments in Effect Example 7;

[0089] Figure 18 Middle G is a representative fluorescence image (CA3) of NeuN staining in the right hippocampus of mice after different treatments in Effect Example 6;

[0090] Figure 18 Middle H is a representative fluorescence histogram of NeuN staining in the right hippocampus (CA3 area) of mice after different treatments in Effect Example 7;

[0091] Figure 19 Middle A is a representative fluorescence image of IBA1 staining in the right thalamus of mice after different treatments in Effect Example 7;

[0092] Figure 19 Middle B is a representative fluorescence histogram of IBA1 staining in the right thalamus of mice after different treatments in Effect Example 7;

[0093] Figure 19 Middle C is a representative fluorescence image of GFAP staining in the right thalamus of mice after different treatments in Effect Example 7;

[0094] Figure 19 Middle D is a representative fluorescence histogram of GFAP staining in the right thalamus of mice after different treatments in Effect Example 7. DETAILED DESCRIPTION

[0095] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0096] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0097] Example 1

[0098] An embodiment of the present invention provides nanoparticles loaded with amifostine. The preparation method of the nanoparticles loaded with amifostine comprises the following steps:

[0099] (1) Synthesis of DSPE-PEG-AMF: DSPE-PEG-NHS (#PS2-HE1-2K, Pengshuo, Shanghai, China, 50 mg, 0.051 mmol) [1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-n-[poly(ethyleneglycol)]-hydroxy succinimid, PEG M 2000] was dissolved in 2 mL of methanol solution to obtain a methanol solution of DSPE-PEG-NHS; amifostine (#HY-B0639, MedChemExpress, NJ, USA, 11.11 mg, 0.047 mmol) and triethylamine (7.89 mg, 0.078 mmol) were added to 400 μL of ultrapure water to obtain a triethylamine aqueous solution of amifostine; the DSPE-PEG-NHS methanol solution was then added to the triethylamine aqueous solution of amifostine, and PBS buffer was subsequently added to a total solvent volume of 20 mL (pH 7) and reacted at 25°C under nitrogen protection for 12 h. After the reaction, 400 mL of ether was added for precipitation, followed by filtration and washing. The filter residue was collected and dialyzed for dehydration, and the product after dialysis and dehydration was lyophilized to obtain DSPE-PEG-AMF;

[0100] (2) Preparation of amifostine-loaded nanoparticles (DAPP NPs): 1 mL of organic phase (5 mg of D,L-lactide-co-glycolide (PLGA), 6 mg of DSPE-PEG-AMF, and 10 μg of DiD dye dissolved in DMF (dimethylformamide)) was prepared, and 15 mL of aqueous phase (30 mg of PS 80 dissolved in ultrapure water) was prepared. The organic phase was slowly added dropwise to the aqueous phase under stirring. The mixture was then centrifuged using a centrifugal filter with a molecular weight cutoff of 100 kDa, washed, and dried to obtain DAPP NPs.

[0101] The schematic diagram of the preparation process of DAPP NPs and the schematic diagram of the onset process are as follows: Figure 1 As shown in A and B, Figure 1 Figure A shows that the synthesized DSPE-PEG-AMF, OLGA and PS 80 are self-assembled into nanoparticles, which are then converted into active forms under ALP metabolism, thereby volatilizing and scavenging oxygen free radicals. Figure 1 Middle B shows that the synthesized DAPP NPs cross the blood-brain barrier intravenously and are converted into free thiol form under the action of ALP, scavenging ROS and preventing RIBI.

[0102] Example 2

[0103] The embodiment of the present invention provides nanoparticles loaded with amifostine. The only difference between the preparation method of the nanoparticles loaded with amifostine and that of Example 1 is that DiD dye is not added.

[0104] Effect Example 1

[0105] This effect example verifies the characterization of the raw materials and products in the preparation process of DAPP NPs in Example 1. In order to characterize the synthesis results, the mass distribution of DSPE-PEG-AMF was detected by MALDI-TOF and compared with DSPE-PEG-NHS. The results are as follows: Figure 2 As shown in A (spectrum of DSPE-PEG-NHS) and B (spectrum of DSPE-PEG-AMF), Figure 2 As can be seen in Figures A and B, the main peak distribution of m / z is regularly spaced at 44 (approximately the mass of PEG monomer). The regular spacing of m / z indicates that there are no visible side reactions or impurities during the entire synthesis process; the m / z distribution of DSPE-PEG-AMF is higher than that of DSPE-PEG-NHS; in addition, Figure 2As can be seen from Figure C, the measured m / z of DSPE-PEG-AMF are approximately 2423.384, 2467.391, and 2511.395, which are highly correlated with the theoretical m / z of the corresponding DSPE-PEG-AMF (2423.44, 2467.46, and 2511.49); that is, MALDI-TOF analysis showed that DSPE-PEG-AMF was successfully bound and purified; further analysis was performed using 1 H and 31 PNMR characterization of the structure of DSPE-PEG-AMF (structural characterization as shown in Figure 3 A), from Figure 3 Part of the characteristic hydrogen of DSPE-PEG-AMF can be confirmed in B, and from the 1 The characteristic hydrogen H of the NHS group could not be detected in the H NMR spectrum. d , indicating that all NHS groups are replaced by AMF groups; Figure 3 As can be seen in Figure C, compared with DSPE-PEG-NHS, the 31 A new characteristic peak belonging to AMF can be seen in the P spectrum, which further confirms the grafting of AMF; Figure 3 As can be seen in D, through the characteristic hydrogen H b and H c After calibrating the integrated area of ​​the hydrogen spectrum, the H e Based on the calculation of the integrated area, the degree of polymerization (DPn) of the PEG segment is about 27, so the number average molecular weight (Mn) of DSPE-PEG-AMF is about 2250;

[0106] The synthesized DAPP NPs were then characterized. The synthesized DAPP NPs were dispersed in PBS solution and negatively stained with 2 wt% uranyl acetate aqueous solution on a luminescently discharged formaldehyde-coated copper grid. The nanoparticles were observed using a TECNAI G2 F20 transmission electron microscope. Figure 4 It can be seen from the electron microscopy that DAPP NPs are spherical with a diameter of about 110 nm. Figure 5 As can be seen from the figure, the hydrodynamic diameter of DAPP NPs is concentrated in the range of 108-135nm and the zeta potential is -19.300±0.289 as measured by NanoSight NS300 nanoparticle tracking analysis (NTA), indicating that the charge repulsion between the nanoparticles is sufficient and has good shelf stability. DAPP NPs were further placed in PBS solution and the changes in nanoparticle size were tested. The results are shown in the figure. Figure 6 As shown, from Figure 6As can be seen in the figure, DAPP NPs showed good colloidal stability in PBS solution, as no change in size distribution was observed within 10 days;

[0107] Finally, UPLC experiment was used to quantify the content of DSPE-PEG-AMF in DAPP NPs. The UPLC experimental method was as follows: the UPLC experiment used an ACQUITY UPLC H-Class (Waters) system equipped with an ACQUITY UPLC BEH C18 reversed-phase column (Waters, 1.7 μm, 2.1 mm × 50 mm), column temperature 40°C, eluent A: ultrapure water, eluent B: acetonitrile; all eluents were supplemented with 0.1% by volume trifluoroacetic acid as an auxiliary agent, and the eluent used the elution gradient with a flow rate of 0.4 mL / min as shown in Table 1;

[0108] Table 1

[0109] Time(min) A％ B％ 0 80 20 10 20 80 12 20 80 12.5 80 20 15 80 20

[0110] The specific process is: using DMF as a solvent, 1.05 mg / mL, 0.75 mg / mL, 0.45 mg / mlL, 0.15 mg / mL, 0.075 mg / mL and 0.015 mg / mL DSPE-PEG-AMF standard solutions were prepared for UPLC analysis and a standard curve was established: 10 μL of the standard solution was injected into the UPLC; then the peak in the 5.75-5.95 min interval was integrated, and a standard curve of the peak area corresponding to the initial concentration of the sample was established; the nanoparticles were prepared according to the above method (0.060 mL of organic phase, 0.900 mL of aqueous phase), after ultracentrifugation, the concentrated sample was recovered and freeze-dried, and redissolved with 100 μL of DMF, and then the 10 μL redissolved sample was diluted to 100 μL with DMF; 10 μL of the diluted sample was injected into the UPLC analysis, and the concentration of DSPE-PEG-AMF in the sample was determined according to the standard curve. The results are as follows Figure 7 As shown, DSPE-PEG-AMF in the nanoparticles was identified and quantified by the characteristic peak at 5.85 min ( Figure 7 A); the retention efficiency (RE%) of DSPE-PEG-AMF in nanoparticles is 100%*m nanoparticles / m starting solution, which is 93.88% ( Figure 7 Middle B); M determined by NMR n , the equivalent dose of AMF in the nanoparticles can be obtained.

[0111] Effect Example 2

[0112] This effect example conducts a neuronal cell uptake experiment on the DAPP NPs prepared in Example 1, specifically comprising the following steps:

[0113] (1) 2×10 4 BV2 cells (microglial cell line) were seeded onto a 24-well plate slide at a density of 1:1. After 24 hours, the BV2 cells were treated with the DAPP NPs prepared in Example 1, incubated at 37°C for 1 hour, rinsed twice with cold PBS buffer, fixed with 4% paraformaldehyde for 15 minutes, and stained with DAPI; fluorescence imaging of the cells was performed on a fluorescence microscope (Leica DM4B, Germany);

[0114] (2) Flow cytometry was used to quantify the uptake of nanoparticles: BV2 cells were plated at 8×10 4 The cells were seeded at a density of 100 μg / ml in a 6-well plate. After 24 hours, the BV2 cells were treated with the DAPP NPs prepared in Example 1. After 30 minutes and 1 hour, the cells were washed twice with PBS buffer, and the cells were collected by trypsin treatment for quantitative analysis by flow cytometry.

[0115] The results obtained are as follows Figure 8 shown, specifically, Figure 8 Middle A is a picture of primary neurons incubated with DAPP NPs loaded with DiD dye (magenta signal) at 37°C for 1 hour, and the cell nuclei were stained with DAPI (blue signal); Figure 8 Middle B and C are the flow cytometry profiles and mean fluorescence intensity of primary neurons incubated with DAPP NPs for 30 min and 1 h, respectively (n=3); Figure 8 Middle D is an image of BV2 cells incubated with DAPP NPs loaded with DiD dye (magenta signal) at 37°C for 1 h, and the cell nuclei were stained with DAPI (blue signal); Figure 8 Middle E and F are the flow cytometric profiles and mean fluorescence intensity of BV2 cells incubated with DAPP NPs for 30 min and 1 h, respectively (n=3); Figure 8 In the figure, BV2 cells were irradiated with 10 Gy x-rays, and then incubated with DAPP NPs (corresponding to 10 μg / mL AMF) and AMF (10 μg / mL) for 1 hour, and then cell apoptosis was detected by flow cytometry; Figure 8 It can be seen that compared with AMF, DAPP NPs have efficient cell uptake ability and lower toxicity, and have a stronger protective effect on normal nerve cells.

[0116] Effect Example 3

[0117] This effect example uses the CCK-8 method to detect the cytotoxicity and radiation protection properties of the DAPP NPs prepared in Example 1, which specifically includes the following steps:

[0118] (1) Cytotoxicity assay: BV2 cells were seeded into 96-well culture plates (8 × 10 cells per well). 3 ) were treated with different concentrations of amifostine original drug (concentration gradient: 0 μg / mL, 1 μg / mL, 2.5 μg / mL, 5 μg / mL, 12 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL) and DAPP NPs (concentration equivalent to: 0 μg / mL, 1 μg / mL, 2.5 μg / mL, 5 μg / mL, 12 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL of amifostine). After 24 hours, BV2 cells were washed with PBS and incubated with 100 μL of DMEM medium containing 10% CCK-8 (Dojindo Laboratories, Kumamoto, Japan) for 1 hour. The absorbance of the supernatant at 450 nm was detected using a microplate reader.

[0119] (2) Radiation protection performance test: In order to evaluate the radiation protection effect, 8×10 3 BV2 cells were seeded in 96-well plates. After 24 hours, the cells were treated with Ham's F12K DMEM containing PBS, amifostine (0 μg / mL, 1 μg / mL, 2.5 μg / mL, 5 μg / mL, 12 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL), or DAPP NPs (concentrations equivalent to: 0 μg / mL, 1 μg / mL, 2.5 μg / mL, 5 μg / mL, 12 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL of amifostine). After 1 hour, the cells were irradiated with 10 Gy (X-ray). After 24 hours, the cell viability was determined by CCK-8 assay.

[0120] The results obtained are as follows Figure 9 As shown, Figure 9 Middle A shows the viability of BV2 cells detected by CCK-8 after incubation with different concentrations of DAPP NPs and AMF for 24 h; Figure 9 B shows the viability of BV2 cells after incubation with different concentrations of DAPP NPs and AMF for 1 hour and irradiation with 10Gy X-rays; Figure 9It can be seen that compared with the original drug amifostine, at the same concentration, DAPP NPs showed better in vitro safety; in terms of radiation protection of the brain, DAPP NPs showed better radiation protection than the original drug amifostine. At the same time, the flow cytometry apoptosis experiment also showed that DAPP NPs more effectively inhibited radiation-induced BV2 cell apoptosis than the original drug amifostine.

[0121] Effect Example 4

[0122] This effect example performs a clone formation experiment on the DAPP NPs prepared in Example 1, specifically comprising the following steps:

[0123] BV2 and S18 cells (nasopharyngeal carcinoma cell lines) were seeded into 6-well culture plates (1.0 × 10 3 ), 24 h later, BV2 cells were treated with DAPP NPs (at a concentration equivalent to 10 μg / mL amifostine), amifostine (10 μg / mL), or PBS for 1 h and then irradiated with X-rays (4 Gy). Ten days later, BV2h and S18 cells were washed with PBS, stained with crystal violet, and the number of colonies was counted.

[0124] The results obtained are as follows Figure 10 As shown, Figure 10 A to C are crystal violet staining (A) and quantification (B, C) of surviving cell colonies in different treatment groups (control group, PBS, AMF, and DAPP NPs) of BV2 cells and S18 cells irradiated with 4 Gy X-rays; Figure 10 It can be seen that the protective effect of DAPP NPs on BV2 cells is better than that of amifostine. On the contrary, nasopharyngeal carcinoma cells (S18 cells) are not protected by amifostine and DAPP NPs, showing the selective effect of amifostine and DAPP NPs on normal cells.

[0125] Effect Example 5

[0126] This effect example performs r-H2AX staining on the DAPP NPs prepared in Example 1, specifically comprising the following steps:

[0127] BV2 cells or primary neurons were seeded on 24-well slides (2*10 4), cells were treated with DAPP NPs (at a concentration equivalent to 10 μg / mL amifostine), amifostine (10 μg / mL), or PBS. One hour later, cells were irradiated with 10 Gy of X-rays. Three hours later, cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature. The membranes were then permeabilized with 0.1% Triton X-100 for 30 minutes and blocked with 3% BSA for 1 hour. Cells were then incubated with rabbit r-H2AX (1:1000, #ab11174, Abcam, Cambridge, MA, USA) at 4°C overnight. Cells were then incubated with anti-rabbit IgG (1:5000, #E030140-01, Earthox, Burlingame, CA, USA) at 37°C for 1 hour and stained with DAPI for 5 minutes. Finally, cells were imaged using a fluorescence microscope (Leica DM4B, Germany).

[0128] The results obtained are as follows Figure 11 As shown, Figure 11 A and B in the middle are r-H2AX immunofluorescence assay (red signal) to detect DNA double-strand damage in primary neurons after different treatments. Figure 11 C and D are r-H2AX immunofluorescence assay (red signal) to detect DNA double-strand damage in BV2 cells after different treatments; Figure 11 As can be seen, neither the primary neurons nor the BV2 cells in the control group or the DAPP NPs-only treatment group showed obvious red fluorescence signals, corresponding to negligible DNA breaks. In contrast, cells treated with IR+PBS showed obvious red fluorescence, indicating severe radiation-induced DNA damage. Treatment with amifostine or DAPP NPs significantly attenuated the red fluorescence signal of irradiated cells. In addition, compared with the original drug amifostine, DAPP NPs caused a further decrease in DSBs. These results indicate that compared with amifostine, DAPP NPs can effectively protect DNA from radiation-induced damage.

[0129] Effect Example 6

[0130] This effect example detects the cellular ROS level of the DAPP NPs prepared in Example 1, specifically comprising the following steps:

[0131] (1) Fluorescence imaging to detect cellular ROS levels

[0132] BV2 cells or primary neurons were seeded on cell slides in 24-well plates (2*10 4After 24 hours, cells were incubated with DAPP NPs (at a concentration equivalent to 10 μg / mL amifostine), amifostine (10 μg / mL), or PBS. 30 minutes later, cells were incubated with a mixture of DAPI and dihydroethidium (DHE) probe (#C260, ABP Biosciences, USA) at 37°C for 20 minutes. The cells were then rinsed with PBS and irradiated with 10 Gy of X-rays. Finally, the cells were imaged under a fluorescence microscope (Leica DM4B, Germany). Fluorescence intensity was quantified in the acquired digital images using ImageJ software.

[0133] (2) Flow cytometry detection of cellular ROS levels

[0134] BV2 cells were seeded in six-well culture plates (1×10 5 After 24 hours, cells were treated with DAPP NPs (at a concentration equivalent to 10 μg / mL amifostine), amifostine (10 μg / mL), or PBS. After 30 minutes, cells were incubated with a dihydroethidium (DHE) probe at 37°C for 20 minutes. The cells were then rinsed with PBS and irradiated with 10 Gy of X-rays. After 30 minutes, the cells were rinsed twice with PBS buffer and harvested with trypsin for flow cytometry analysis.

[0135] (3) Fluorescence imaging to detect tissue ROS levels

[0136] Anesthetized mice were perfused with PBS via the cardiac vein, and brains were removed. Brain tissue was cut into 8-μm coronal sections using a freezing microtome. Brain sections were incubated with a 5 μM DHE solution containing a NOX-2 neutralizing antibody at 37°C for 30 minutes. After incubation, the slides were rinsed with PBS, and images of the right thalamic region were captured using a fluorescence microscope (Leica DM4B, Germany). Fluorescence intensity of the acquired digital images was quantified using ImageJ software.

[0137] The results obtained are as follows Figure 12 As shown, Figure 12 A and B are fluorescence images of ROS generation (red signal) in primary neuronal cells after different treatments, C and D are flow cytometric maps of ROS generation in primary neuronal cells after different treatments, E and F are fluorescence images of ROS generation in BV2 cells after different treatments (red signal), G and H are flow cytometric maps of ROS generation in BV2 cells after different treatments; Figure 12As can be seen in A and B as well as E and F, a clear red fluorescence signal was detected in the IR+PBS group, representing a large amount of ROS production. In addition, the control group or the group treated with DAPP NPs alone showed only a weak fluorescence signal, suggesting a small amount of ROS. However, when irradiated cells were treated with amifostine or DAPP NPs, the red fluorescence was significantly reduced; in addition, compared with the original drug amifostine, DAPP NPs treatment led to a further weakening of the fluorescence signal, which was consistent with the fluorescence imaging analysis. Flow cytometry analysis also showed a similar trend ( Figure 12 (C and D, G and H). These results indicate that DAPP NPs have a stronger ROS scavenging ability in vitro than the original drug amifostine.

[0138] Effect Example 7

[0139] This study used adult male C57BL / 6 mice (7-8 weeks old) (Number: #N000013-1, Guangdong Yaokang, China) to conduct animal experiments on the DAPP NPs prepared in Example 1. The mice were housed under standard conditions of 25°C, 55-60% humidity, and 12 hours of light per day. All experimental procedures were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals and approved by the Animal Research Ethics Committee of Sun Yat-sen University.

[0140] Specifically, the following experiments are included:

[0141] (1) Blood-brain barrier permeability test

[0142] The DAPP NPs prepared in Example 1 (at a dose equivalent to 250 mg / kg amifostine) were injected into the retroorbital sinus of healthy C57BL / 6 mice. After 30 minutes or 1 hour, the mice were sacrificed and perfused with PBS and 4% PFA via the cardiac vein. The brains were harvested and imaged using an IVIS Spectrum Imaging System (PerKinElmer). The brains were then embedded in OCT and cryosectioned into 20 μm thick frozen sections, and the nuclei were stained with DAPI. Finally, the slides were imaged under a fluorescence microscope (Leica DM4B, Germany).

[0143] The results obtained are as follows Figure 13 As shown, Figure 13 Middle A is an in vivo imaging system (IVIS) image of the healthy mouse brain, showing fluorescence imaging at 30 and 60 minutes after intravenous injection of DiD dye-loaded DAPP NPs (corresponding to 250 mg / kg AMF). Figure 13 B is a representative fluorescence microscopy image of mouse brain slices. Figure 13Figure CE shows the colocalization of DAPP NPs (magenta signal) with different cells (NeuN (neurons), IBA1 (microglia), GFAP (astrocytes)). Brain sections were stained with antibodies against different cell types and secondary ab 488 (green). Cell nuclei were stained with DAPI (blue). Figure 13 It can be seen that DAPP NPs accumulate in brain tissue, indicating that DAPP NPs have the ability to cross the blood-brain barrier; it also confirms that intravenously injected DAPP NPs can obviously accumulate in the brain parenchyma and then distribute in neurons, microglia and astrocytes.

[0144] (II) In vivo biosafety determination of DAPP NPs

[0145] Ten healthy C57B6 / L male mice were divided into two groups: PBS group; DAPP NPs group. Mice in the DAPP NPs group were given DAPP NPs (dose equivalent to amifostine 250 mg / kg) daily for 14 consecutive days. Mice in the PBS group were given the same volume of PBS as the DAPP NPs group. Body weight was recorded every day for 14 days. After 14 days, the mice were euthanized and whole blood was collected for blood routine and blood biochemical analysis. Major organs were taken for H&E staining. Finally, images were taken under a microscope (Leica DM4B, Germany). The results obtained are shown in Figure 2. Figure 14 As shown, Figure 14 A is the body weight curve of mice treated with DAPP NPs or PBS (n=5), B is the H&E-stained sections of the main organ tissues of mice collected 14 days after intravenous administration, including heart, liver, spleen, lung and kidney, C and D are the hematological and serum biochemical analyses of mice in each group 14 days after intravenous administration; Figure 14 As can be seen in the figure, the survival rate of mice in the DAPP NPs-treated and untreated groups remained 100%, and the weight changes of mice in the two groups were minimal (A). Histological analysis showed that all organs of mice treated with DAPP NPs were normal, and no tissue or cell lesions were observed (B). Further testing of blood routine and blood biochemical indicators showed (C and D) that there was no significant difference in the levels of representative blood cell counts, including white blood cell (WBC), red blood cell (RBC) and platelet (PLT). The blood biochemical indicators of the two groups, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin (ALB) and urea nitrogen (BUN), were all normal; that is, DAPP NPs had no obvious harm to the physiological health of mice.

[0146] (3) Animal experiments

[0147] 1. Construction of mouse brain transplantation model

[0148] C57BL / 6 mice were anesthetized intraperitoneally with 1% pentobarbital and then immobilized using a custom-made positional immobilization device. Conventional Siemens wide-aperture CT scans were performed with a 0.6mm slice thickness. To increase the superficial dose to the head, a 1cm compensation film was applied to the mouse head. Scanned images were transferred to the Monaco version 5.11.03 planning system (Elekta, Stockholm, Sweden). The target volume and organs at risk (OAR) were delineated by the same experienced radiation oncologist. The target volume was a 2mm diameter circular area in the right cerebral cortex and hippocampus, and the OAR was the left hemisphere. All plans were completed using the Monaco system by the same experienced physicist. The prescribed dose was 50Gy (50% dose curve). A specific immobilization device was used to position the mouse's head and feet perpendicular to the bed entry and exit direction. Five to six stereotactic cones with a 5mm diameter were used for irradiation, with field angles of approximately 330, 0, 30, 150, 180, and 210 degrees. The irradiation energy was 6 MV FFF. All irradiations were performed on an Elekta Infinity linear accelerator (160 multileaf collimators, grating thickness 0.5 cm).

[0149] 2. Detection of the brain protective function of DAPP NPs

[0150] Twenty-eight healthy C57BL / 6 male mice were divided into four groups (7 mice in each group): (i) control group, (ii) X-ray + PBS, (iii) X-ray + AMF (250 mg / kg), and (iv) X-ray + DAPP NPs (the dose is equivalent to 250 mg / kg amifostine). Mice in groups (ii), (iii), and (iv) were intravenously injected with PBS, amifostine, or DAPP NPs. One hour later, the mice were exposed to 50 Gy (50% equivalent dose) of X-knife. Three mice in each group were killed 4 hours after irradiation, and the ROS level in brain tissue was detected. The remaining 16 mice (4 mice in each group) were continued to be raised for 8 weeks after irradiation and then killed (the timeline diagram of the experimental design is shown in the figure). Figure 15 The volume of delayed brain necrosis, neuronal loss, and glial cell activation were assessed.

[0151] Among them, the schematic diagram of the application of X-knife in the detection of DAPP NPs brain protection function is as follows Figure 16 As shown, Figure 16 Shown are X-ray knife dose contours superimposed on a CT image of a mouse. The three panels show the sagittal, transverse, and coronal planes, respectively. The three contour lines shown are 100% (red), 75% (magenta), and 50% (yellow) isodose.

[0152] 3. Mouse MRI Imaging and Image Analysis

[0153] Mouse brains were imaged using a 3.0T MRI (Siemens Magnetom Vida 64-channel / 2018, Simens Healthineers, Erlangen, Germany). Mice were anesthetized with isoflurane / O₂ (3% [vol / vol]) and maintained with isoflurane / O₂ (1% [vol / vol]) throughout the experiment. Mice were secured in a laboratory-built headrest. Body temperature was maintained at 37°C using a warming pad with circulating warm water. The contrast agent Omniscan (50 μg / mL) was injected intraperitoneally. Necrotic volume was assessed using T1-weighted images. MR image analysis was performed using ITK-SNAP software. Regions of interest were extracted from the entire brain, specifically the right hemisphere of the mouse brain, on several consecutive image slices, involving all high-intensity foci. Data on all necrotic volumes were recorded and analyzed using rapid and fine contrast adjustment (stepwise threshold adjustment) and the progressive loading of segmented image files.

[0154] 4. Immunofluorescence staining

[0155] Mice were anesthetized and perfused intracardially with 4% paraformaldehyde (PFA) in 0.1 M PBS, after which brains were removed. Brain samples were cut into 20-μm coronal sections using a freezing microtome. The sections were blocked with 10% normal goat or donkey serum in 3% BSA / PBS for 1 hour at room temperature and then incubated overnight at 4°C with the following primary antibodies: anti-NeuN (1:200, #ab104224, Abcam), anti-Iba1 (1:200, #019-19741, Wako), and anti-GFAP (1:2000, #3670S, CST). After three washes with PBS, the sections were incubated with specific Alexa Fluor-conjugated secondary antibodies for 1 hour at room temperature. Nuclei were stained with DAPI. Finally, brain sections were imaged under a fluorescence microscope. Fluorescence intensity was quantified in acquired digital images using ImageJ software.

[0156] 5. Statistical analysis

[0157] Statistical analysis and graphics were performed using GraphPad Prism software. Comparisons between two groups were performed using the Student's t-test, and comparisons between more than two groups were performed using one-way analysis of variance (ANOVA) with Tukey's post-hoc analysis. Statistical significance was determined when P < 0.05.

[0158] 6. Results Analysis

[0159] (1) Brain tissue ROS levels and brain necrosis: The results are as follows Figure 17 As shown, Figure 17 A and B are fluorescence images (red signals) of ROS generation in the right thalamic region of mice after different treatments; C and D are representative MRI images of mice after different treatments; E is a representative image of H&E-stained brain sections of lesions in the right thalamic region of mice after different treatments, with yellow arrows indicating microbleeds, red arrows indicating vascular proliferation and congestion, and green arrows indicating vacuolated cells; F and G are immunohistochemical staining and optical density (OD) values ​​of CD34 in the right thalamic region of mice after different treatments. Figure 17 As can be seen in A and B, the fluorescence signal in the right thalamus of the control mice was negligible, while the mice treated with IR+PBS (radiotherapy+PBS) showed a strong DHE red fluorescence signal, which corresponds to a high ROS level. Compared with the IR+PBS treatment group, the fluorescence signal was significantly weakened when the irradiated mice were treated with DAPP NPs. These results indicate that DAPP NPs can effectively reduce the production of ROS in brain tissue in the early stage after irradiation. In contrast, IR+AMF (radiotherapy + Amifostine ) ROS levels in the IR+PBS group were similar to those in the X-ray+PBS group (C, D). Brain MRI was performed 8 weeks after irradiation, demonstrating that DAPP NPs also effectively prevented radiation-induced delayed brain necrosis, whereas the original drug, amifostine, showed no significant protective effect. Hematoxylin and eosin (H&E) staining was used to observe brain morphological changes after different treatments. As shown in E, compared with the control group, the right thalamus in the IR+PBS group was significantly damaged, with disorganized structure, microbleeds, vascular hyperplasia, and vacuolated cells. Compared with the IR+PBS and IR+AMF groups, the IR+DAPP NPs group showed improved brain tissue pathology, with organized brain tissue, reduced microbleeds, and decreased vascular hyperplasia. Furthermore, immunohistochemical staining for CD34 was used to evaluate vascular proliferation. As shown in F and G, CD34 IHC staining revealed that DAPP NPs treatment significantly reduced the number of blood vessels in the right thalamus compared with the IR+PBS and IR+AMF groups. In summary, DAPP NPs have a superior protective effect against RIBI when administered intravenously compared with AMF.

[0160] (2) Brain neuron loss: The results are as follows Figure 18 As shown, Figure 18 A and B are representative fluorescence images of NeuN staining (red signal) in the right thalamus of mice after different treatments, and CH are representative fluorescence images of NeuN staining (red signal) in the right hippocampus of mice after different treatments; Figure 18As can be seen in the IR+PBS group, the number of NeuN-positive cells in the lesion area was significantly reduced, indicating significant neuronal loss after irradiation. When mice were treated with DAPP NPs, the number of NeuN-positive cells increased significantly, while no significant increase was observed in the IR+AMF group. These results suggest that DAPP NPs exhibit superior radioprotection against neuronal loss compared to the original drug, amifostine.

[0161] (3) Glial cell activation: The results are as follows Figure 19 As shown, Figure 19 A and B are representative fluorescence images of IBA1 staining in the right thalamus of mice after different treatments, and C and D are representative fluorescence images of GFAP staining (green signal) in the right thalamus of mice after different treatments; Figure 19 As can be seen in the IR+PBS group, compared with the control group, significantly more IBA1-positive and GFAP-positive cells were observed, indicating that microglia and astrocytes were significantly activated after irradiation. The application of DAPP NPs significantly reduced the activation of microglia and astrocytes, as shown by a decrease in cell size and the proportion of IBA1-positive and GFAP-positive cells, while the use of amifostine did not lead to a significant decrease in cell size or the proportion of IBA1-positive and GFAP-positive cells. Overall, our results confirm that DAPP NPs exhibit a superior protective effect against radiation-induced glial cell activation compared to the original drug amifostine.

[0162] Finally, it should be noted that the above embodiments are intended to illustrate the technical solutions of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A nanoparticle loaded with amifostine, characterized in that: The nanoparticles have a spherical core-shell structure, including a PLGA core surrounded by DSPE-PEG-AMF and PS 80. The particle size of the nanoparticles is 110 nm. The hydrodynamic diameter of the nanoparticles is concentrated in the range of 108-135 nm as measured by Nano Sight NS300 nanoparticle tracking analysis. The nanoparticles loaded with amifostine are ophthalmic intravenous injection preparations; the preparation method of the nanoparticles loaded with amifostine comprises the following steps: (1) Synthesis of DSPE-PEG-AMF: DSPE-PEG-NHS methanol solution was added to amifostine triethylamine aqueous solution, followed by addition of PBS buffer and reaction under inert gas protection. After the reaction, ether was added for precipitation, followed by filtration and washing. The residue was collected and purified, and then lyophilized to obtain DSPE-PEG-AMF. (2) Preparation of nanoparticles loaded with amifostine: PLGA dimethylformamide solution, DSPE-PEG-AMF dimethylformamide solution and PS 80 aqueous solution were mixed and added to water and stirred, followed by centrifugation and washing to obtain nanoparticles loaded with amifostine; In the step (1), the molar ratio of DSPE-PEG-NHS, amifostine and triethylamine is DSPE-PEG-NHS: amifostine: triethylamine = 1: (0.8-1.0): (1.2-1.8); In the step (2), the mass ratio of PLGA, DSPE-PEG-AMF and PS 80 is PLGA:DSPE-PEG-AMF:PS 80=5:(5.5-6.5):(29-31); In step (1), the reaction temperature is 20-30°C, the reaction time is 10-14 hours, and the pH value of the PBS buffer is 7; In the step (2), the centrifugation is performed in a centrifugal filter with a molecular weight cut-off value of 100 KDa.

2. The nanoparticles loaded with amifostine according to claim 1, characterized in that In the step (1), in the DSPE-PEG-NHS methanol solution, the mass concentration of DSPE-PEG-NHS is 12.5-37.5 mg / mL; in the triethylamine aqueous solution of amifostine, the mass concentration of amifostine is 13.89-41.67 mg / mL, and the mass concentration of triethylamine is 9.84-29.52 mg / mL.

3. The amifostine-loaded nanoparticles according to claim 1, characterized in that In the step (2), the mass concentration of PLGA in the dimethylformamide solution is 4.5-5.5 mg / mL; the mass concentration of DSPE-PEG-AMF in the dimethylformamide solution is 5.5-6.5 mg / mL; and the mass concentration of PS 80 in the PS 80 aqueous solution is 1.5-2.5 mg / mL.

4. The amifostine-loaded nanoparticles according to claim 1, characterized in that In the step (2), the volume ratio of dimethylformamide to water is 1:(14-16).

5. Use of the amifostine-loaded nanoparticles according to claim 1 in preparing a drug for preventing radiation-induced brain injury.

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