Preparation method and application of composite drug-loaded nanoparticles targeting blood brain barrier

Abstract

The embodiment of the invention provides a preparation method and application of composite drug-loaded nanoparticles targeting a blood brain barrier, and the composite drug-loaded nanoparticles are prepared by taking mesoporous polydopamine (MPDA) as a carrier and covalently coupling a blood brain barrier targeting peptide RPP-12, a microglial cell targeting peptide MG-1 and an MMP-2 responsive connecting peptide Pep (Ac-CSSSGPLGIAGQSSS). The multi-stage precise delivery of blood brain barrier penetration-inflammatory cell targeting-microenvironment response release is realized. In-vivo animal experiments show that the nanoparticles can significantly reduce the level of pro-inflammatory factors (IL-1, IL-6 and TNF-alpha) of craniocerebral trauma model mice, promote polarization of pro-inflammatory microglial cells to anti-inflammatory phenotypes, promote survival of nerve cells and improve neurological functions (sucrose preference experiments, forced swimming experiments and the like). According to the application, by integrating a multi-stage targeting strategy and a microenvironment response release mechanism, the bottleneck that glucocorticoid is low in intracerebral delivery efficiency and large in systemic side effect is broken through, and an efficient and safe new treatment strategy is provided for central nervous system diseases such as traumatic brain injury.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a preparation method and application of a composite drug-loaded nanoparticle targeting the blood-brain barrier. BACKGROUND

[0002] The treatment of central nervous system (CNS) inflammatory diseases (such as traumatic brain injury, neurodegenerative diseases) faces multiple challenges, including the physical barrier of the blood-brain barrier (BBB), insufficient drug targeting, and the complexity of the inflammatory microenvironment. Although synthetic glucocorticoids (such as dexamethasone) have strong anti-inflammatory effects, the BBB permeability is less than 5%, and a large dose of systemic administration is required to maintain an effective concentration in the brain, resulting in serious side effects such as immunosuppression and osteoporosis. Although existing nanocarriers can enhance brain targeting through surface modification, most of them lack the ability to respond to the release of the lesion microenvironment (such as matrix metalloproteinase-2, MMP-2), resulting in low drug utilization. In addition, the overactivation of pro-inflammatory cytokines (such as IL-17) and NLRP3 / STAT3 signaling pathways is a key pathological mechanism of CNS inflammation, but existing treatment methods are difficult to achieve targeted delivery and pathway regulation at the same time.

[0003] Mesoporous polydopamine (MPDA) has become a research hotspot in the field of drug delivery due to its high drug loading capacity, biocompatibility, and easy surface modification. However, the targeting and responsiveness of single MPDA nanoparticles still need to be optimized. SUMMARY

[0004] The embodiments of the present application aim to provide a preparation method and application of a composite drug-loaded nanoparticle targeting the blood-brain barrier, which can achieve precise drug delivery and controlled release.

[0005] The technical solution of the present application is as follows: In a first aspect, the embodiments of the present application provide a preparation method of a composite drug-loaded nanoparticle targeting the blood-brain barrier, which comprises the following steps: Step (1) Synthesis of mesoporous polydopamine (MPDA) nanoparticles: 100 mg of mesoporous silica (MSN) is dispersed in 10 mM Tris-HCl buffer solution at pH 8.5, and 2 mg / mL of dopamine hydrochloride is added. Stirring at 40°C for 24 hours allows dopamine to polymerize on the surface of MSN to form a polydopamine (PDA) coating. PDA@MSN is collected by centrifugation at 12,000 rpm for 15 minutes, washed with ultrapure water for 3 times, and then etched with 5% hydrofluoric acid to remove the MSN template. MPDA is obtained by centrifugal purification, and the mesoporous structure is verified by TEM. The particle size is determined by DLS to be 150±20 nm. Step (2) Synthesis of FLUDRO-Pep-MPDA nanoparticles: 20 mg MMP-2 responsive peptide Pep (Ac-CSSSGPLGIAGQSSS) was dissolved in 2 mL 0.1 M, pH 5.2 MES buffer, 21.2 mg EDC and 78 mg sulfo-NHS activated carboxyl were added, and reacted with 29.2 mg fludrocortisone (FLUDRO) for 24 hours, and then purified by MWCO ~1000 Da dialysis bag and freeze-dried for 48 hours to obtain Pep-FLUDRO conjugate; which was added to 2 mg / mL of mesoporous polydopamine basic solution, and magnetically stirred for 24 hours; Step (3) Double-targeted peptide coupling: 10 mg PEG-NHS ester polymer (molecular weight ~5000 Da) was reacted with 66 mg RPP-12 peptide in 2 mL 0.2M Na2CO3 solution for 24 hours, and then combined with the drug-loaded nanoparticles obtained in step (2) in bicine buffer at pH 8.5 to obtain composite drug-loaded nanoparticles FLUDRO-Pep-MPDA@RAP12 / MG-1.

[0006] In the above scheme, the mass ratio of PEG-NHS ester polymer to RPP-12 peptide in step (3) is 1:6.6, and the mass ratio of pegylated RAP-12 to drug-loaded nanoparticles is (1-3):10.

[0007] In the above scheme, the mesoporous pore size of the MPDA nanoparticles is 2-5 nm, the specific surface area is 100-300 m² / g, the heavy water hydration temperature is 20-25℃, the ultrasonic treatment time is 5-15 min, and the frequency is 25-35 kHz.

[0008] In a second aspect, the embodiments of the present application provide a composite drug-loaded nanoparticle targeting the blood-brain barrier, wherein the composite drug-loaded nanoparticle FLUDRO-Pep-MPDA@RAP12 / MG-1 is prepared by the preparation method of the first aspect, and the composite drug-loaded nanoparticle FLUDRO-Pep-MPDA@RAP12 / MG-1 has a core-shell structure, the inner core is mesoporous polydopamine, and the shell layer comprises Pep-FLUDRO conjugate, RPP-12 peptide and MG-1 peptide; the particle size is 150±20 nm, the Zeta potential is -15 to -25 mV, the drug loading capacity β is ≥80%, and the drug release rate is ≥75% in 24 hours when the MMP-2 concentration is ≥5 U / mL.

[0009] In the above scheme, the RPP-12 peptide is coupled to the surface of the nanoparticle by PEGylation modification, and the MG-1 peptide is covalently anchored to the surface of the polydopamine by carboxyl-amino condensation reaction.

[0010] In the above scheme, the composite nanoparticle is applied to treat inflammatory diseases of the central nervous system.

[0011] In the above scheme, the disease includes traumatic brain injury (TBI), Alzheimer's disease or multiple sclerosis.

[0012] In the above scheme, the drug is administered by tail vein injection, and the dosage is 1 / 5-1 / 3 of the traditional systemic administration dosage of glucocorticoids.

[0013] In the third aspect, the embodiments of the present application provide a pharmaceutical composition for treating traumatic brain injury, which comprises the composite nanoparticle of the second aspect, a pharmaceutically acceptable carrier, and optionally an anti-inflammatory auxiliary component.

[0014] In the above scheme, the carrier is physiological saline or phosphate buffer solution (PBS) with pH 7.4, and the concentration of the nanoparticle in the pharmaceutical composition is 0.1-100 μg / mL.

[0015] The embodiment of the present application provides a preparation method and application of a composite drug-loaded nanoparticle targeting the blood-brain barrier, and the preparation method of the composite drug-loaded nanoparticle targeting the blood-brain barrier comprises the following steps: (1) synthesis of mesoporous polydopamine (MPDA) nanoparticles: 100 mg of mesoporous silica (MSN) is dispersed in 10 mM of Tris-HCl buffer solution with pH 8.5, 2 mg / mL of dopamine hydrochloride is added, and the mixture is stirred at 40°C for 24 hours, so that dopamine is polymerized on the surface of the MSN to form a polydopamine (PDA) coating; the PDA@MSN is collected by centrifugation at 12,000 rpm for 15 minutes, washed with ultrapure water for 3 times, and then etched with 5% hydrofluoric acid to remove the MSN template, and the MPDA is purified by centrifugation, the mesoporous structure of which is verified by TEM, and the particle size is determined by DLS to be 150±20 nm; (2) synthesis of FLUDRO-Pep-MPDA nanoparticles: 20 mg of MMP-2 responsive peptide Pep (Ac-CSSSGPLGIAGQSSS) is dissolved in 2 mL of 0.1M MES buffer solution with pH 5.2, 21.2 mg of EDC and 78 mg of sulfo-NHS are added to activate the carboxyl group, and the mixture is reacted with 29.2 mg of fludrocortisone (FLUDRO) for 24 hours, and then the Pep-FLUDRO combination is purified by a MWCO~1000Da dialysis bag and freeze-dried for 48 hours; the combination is added into 2 mg / mL of a basic solution of mesoporous polydopamine, and the mixture is magnetically stirred and reacted for 24 hours; (3) double-targeting peptide coupling: 10 mg of PEG-NHS ester polymer (molecular weight~5000Da) is reacted with 66 mg of RPP-12 peptide in 2 mL of 0.2M Na2CO3 solution for 24 hours, and then the mixture is purified by a MWCO~3500Da dialysis bag, and then combined with the drug-loaded nanoparticles obtained in step (2) in a bicine buffer solution with pH 8.5 to obtain the composite drug-loaded nanoparticles FLUDRO-Pep-MPDA@RAP12 / MG-1. By using the above scheme, the FLUDRO-Pep-MPDA@RAP12 / MG-1 composite nanoparticles are constructed, the double functionalization of the MMP-2 responsive peptide and the brain-targeting peptide is combined, precise delivery and controllable release of the drug are realized, and a new strategy is provided for the treatment of CNS inflammatory diseases.

[0016] The beneficial effects of the present application are: Multi-stage targeting synergism: the blood-brain barrier penetration (RPP-12) and microglial cell targeting (MG-1) are integrated, so that the brain accumulation of the drug is increased by 5 times, the drug concentration in the inflammatory area is 8 times that of the traditional drug delivery method, and the problem of poor targeting of the traditional drug is solved.

[0017] Intelligent response release reduces toxicity: MMP-2 triggers local drug release, reduces systemic exposure, avoids immune suppression and osteoporosis caused by high-dose glucocorticoids, and realizes "on-demand drug delivery at the lesion site".

[0018] Multi-dimensional therapeutic effect: not only inhibits neuroinflammation, but also promotes nerve regeneration, improves synaptic function and regulates microglia polarization (CD206+ / Iba-1+ anti-inflammatory type ratio increases by 30%), showing comprehensive treatment potential for TBI and providing a new treatment paradigm for central nervous system diseases. BRIEF DESCRIPTION OF DRAWINGS

[0019] The drawings incorporated into the specification and forming part of the specification, these drawings show embodiments consistent with the present application, and together with the specification, serve to explain the technical solutions of the present application. Obviously, the drawings in the following description are only some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained from these drawings without creative labor.

[0020] The flowchart shown in the drawings is only an exemplary illustration, and is not necessarily to include all contents and operations / steps, nor is it necessarily executed in the order described. For example, some operations / steps can be further decomposed, and some operations / steps can be combined or partially combined, so the actual execution order may be changed according to the actual situation.

[0021] Figure 1 An optional flowchart for a preparation method of a composite drug-loaded nanoparticle targeting the blood-brain barrier is provided for the embodiments of the present application; Figure 2 A schematic diagram for the synthesis of FLUDRO-Pep-MPDA@RAP12 / MG-1.

[0022] Figure 3 A scanning electron microscope image and DLS result schematic diagram of FLUDRO-Pep-MPDA@RAP12 / MG-1 nanoparticles.

[0023] Figure 4 A schematic diagram of the zeta potential value of FLUDRO-Pep-MPDA@RAP12 / MG-1 nanoparticles.

[0024] Figure 5 A schematic diagram of the in vitro release and biocompatibility experiment of FLUDRO-Pep-MPDA@RAP12 / MG-1 nanoparticles.

[0025] Figure 6 A schematic diagram of the in vivo biocompatibility experiment of FLUDRO-Pep-MPDA@RAP12 / MG-1 nanoparticles.

[0026] Figure 7 A schematic diagram showing the effect of FLUDRO-Pep-MPDA@RAP12 / MG-1 nanoparticles on promoting the recovery of neurological function in mice after craniocerebral trauma.

[0027] Figure 8 A schematic diagram showing the effect of FLUDRO-Pep-MPDA@RAP12 / MG-1 nanoparticles on promoting the recovery of neurological function in mice after craniocerebral trauma. DETAILED DESCRIPTION

[0028] In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the following will further describe the specific technical solutions of the present application with reference to the drawings in the embodiments of the present application. The following embodiments are used to illustrate the present application, but are not used to limit the scope of the present application.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description herein is for describing the embodiments of the present application only and is not intended to limit the present application.

[0030] In the following description, the terms "some embodiments", "this embodiment", "the present embodiment", "embodiments of the present application" and the like describe a subset of all possible embodiments, but it is understood that "some embodiments" can be the same subset or different subsets as each other and can be combined with each other without conflict.

[0031] If the application file contains similar descriptions of "first / second", the following explanations are added. In the following description, the terms "first\second\third" referred to only distinguish similar objects, and do not represent a specific order of the objects. It is understood that "first\second\third" can be interchanged in a specific order or sequence as allowed, so that the embodiments of the present application described herein can be implemented in an order other than that illustrated or described herein.

[0032] The treatment of central nervous system (CNS) inflammatory diseases (such as traumatic brain injury, neurodegenerative diseases) faces multiple challenges, including the physical barrier of the blood-brain barrier (BBB), insufficient drug targeting, and the complexity of the inflammatory microenvironment. Although synthetic glucocorticoids (such as dexamethasone) have strong anti-inflammatory effects, the BBB permeability is less than 5%, and large doses of systemic administration are required to maintain an effective concentration in the brain, leading to serious side effects such as immunosuppression and osteoporosis. Although existing nanocarriers can enhance brain targeting through surface modification, most lack the ability to respond to the lesion microenvironment (such as matrix metalloproteinase-2, MMP-2), resulting in low drug utilization. In addition, the overactivation of pro-inflammatory cytokines (such as IL-17) and the NLRP3 / STAT3 signaling pathway is a key pathological mechanism of CNS inflammation, but existing treatment methods are difficult to achieve simultaneous targeted delivery and pathway regulation.

[0033] Mesoporous polydopamine (MPDA) has become a research hotspot in the field of drug delivery due to its high drug loading capacity, biocompatibility, and easy surface modification. However, the targeting and responsiveness of single MPDA nanoparticles still need to be optimized. The present application constructs FLUDRO-Pep-MPDA@RAP12 / MG-1 composite nanoparticles, combines the dual functionalization of MMP-2 responsive peptides and brain targeting peptides, and realizes precise drug delivery and controlled release, providing a new strategy for the treatment of CNS inflammatory diseases.

[0034] Based on this, the present application provides a preparation method of a composite drug-loaded nanoparticle targeting the blood-brain barrier, Figure 1 To provide an optional flowchart of a preparation method of a composite drug-loaded nanoparticle targeting the blood-brain barrier for the embodiments of the present application, the steps shown will be described. Figure 1 The steps shown will be described.

[0035] S101, synthesis of mesoporous polydopamine (MPDA) nanoparticles: 100 mg of mesoporous silica (MSN) is dispersed in 10 mM Tris-HCl buffer at pH 8.5, 2 mg / mL dopamine hydrochloride is added, and stirring is carried out at 40°C for 24 hours to allow dopamine to polymerize on the surface of MSN to form a polydopamine (PDA) coating; PDA@MSN is collected by centrifugation at 12,000 rpm for 15 minutes, washed with ultrapure water for 3 times, and then etched with 5% hydrofluoric acid to remove the MSN template, and MPDA is purified by centrifugation, the mesoporous structure is verified by TEM, and the particle size is determined by DLS to be 150±20 nm.

[0036] In some embodiments of the present application, the mesoporous pore size of the MPDA nanoparticles is 2-5 nm, the specific surface area is 100-300 m² / g, the heavy water hydration temperature is 20-25°C, the ultrasonic treatment time is 5-15 min, and the frequency is 25-35 kHz.

[0037] S102, Synthesis of FLUDRO-Pep-MPDA nanoparticles: 20 mg MMP-2 responsive peptide Pep (Ac-CSSSGPLGIAGQSSS) was dissolved in 2 mL 0.1 M, pH 5.2 MES buffer, 21.2 mg EDC and 78 mg sulfo-NHS activated carboxyl were added, and reacted with 29.2 mg fludrocortisone (FLUDRO) for 24 hours, purified by MWCO ~1000 Da dialysis bag and freeze-dried for 48 hours to obtain Pep-FLUDRO conjugate; which was added to 2 mg / mL mesoporous polydopamine basic solution, and magnetically stirred for 24 hours.

[0038] S103, Double-targeting peptide coupling: 10 mg PEG-NHS ester polymer (molecular weight ~5000 Da) was reacted with 66 mg RPP-12 peptide in 2 mL 0.2 M Na2CO3 solution for 24 hours, and after purification by MWCO ~3500 Da dialysis bag, it was combined with the drug-loaded nanoparticles obtained in step (2) in bicine buffer at pH 8.5 to obtain composite drug-loaded nanoparticles FLUDRO-Pep-MPDA@RAP12 / MG-1.

[0039] In some embodiments of the present application, the mass ratio of PEG-NHS ester polymer to RPP-12 peptide is 1:6.6, and the mass ratio of pegylated RAP-12 to drug-loaded nanoparticles is (1-3):10.

[0040] Characteristics of composite nanoparticles Physical structure: core-shell structure, the inner core is mesoporous polydopamine, the shell layer contains Pep-FLUDRO conjugate, RPP-12 peptide and MG-1 peptide, the particle size is 150±20 nm, the polydispersity index (PDI) is <0.2, and it is suitable for penetrating the blood-brain barrier by endocytosis.

[0041] Response mechanism: under the action of MMP-2 (≥5 U / mL) highly expressed in inflammatory microenvironment, Pep-FLUDRO conjugate is broken, and the drug release rate is 78% in 24 hours, which is significantly higher than that of the non-response group (P<0.05).

[0042] Targeting ability: RPP-12 peptide mediates blood-brain barrier penetration, and in the bEnd.3 cell model cultured in Transwell, the accumulation of fluorescently labeled particles on the basal side is 2.3 times higher than that of the unmodified group (P<0.01); MG-1 peptide targets activated microglia, and the uptake amount is increased by 45% when co-cultured with BV2 cells (observed by CLSM).

[0043] The application provides a composite drug-loaded nanoparticle targeting the blood-brain barrier, the composite drug-loaded nanoparticle FLUDRO-Pep-MPDA@RAP12 / MG-1 is prepared by the preparation method, the composite drug-loaded nanoparticle FLUDRO-Pep-MPDA@RAP12 / MG-1 is a core-shell structure, the inner core is mesoporous polydopamine, the shell layer comprises a Pep-FLUDRO conjugate, an RPP-12 peptide and an MG-1 peptide; the particle size is 150±20 nm, the Zeta potential is-15 to-25 mV, the drug loading amount β is greater than or equal to 80 %, and the drug release rate is greater than or equal to 75 % in 24 hours when the MMP-2 concentration is greater than or equal to 5 U / mL.

[0044] In some embodiments of the application, the RPP-12 peptide is coupled with the nanoparticle surface PEG through pegylation modification, and the MG-1 peptide is covalently anchored on the polydopamine surface through carboxyl-amino condensation reaction.

[0045] In some embodiments of the application, the composite nanoparticle is applied to treat inflammatory diseases of the central nervous system. The diseases include traumatic brain injury (TBI), Alzheimer's disease or multiple sclerosis. The drug is administered by tail vein injection, and the administration dose is 1 / 5-1 / 3 of the traditional glucocorticoid systemic administration dose.

[0046] The application provides a pharmaceutical composition for treating traumatic brain injury, the composite nanoparticle, a pharmaceutically acceptable carrier and optionally an anti-inflammatory auxiliary ingredient.

[0047] In some embodiments of the application, the carrier is physiological saline or phosphate buffer solution (PBS) with pH 7.4, and the nanoparticle concentration in the pharmaceutical composition is 0.1-100 μg / mL.

[0048] A preparation method of a composite drug-loaded nanoparticle targeting the blood-brain barrier, comprising: I. Materials and methods Reagents and instruments Mesoporous silica (MSN), dopamine hydrochloride, EDC, sulfo-NHS, fluorinated steroidal anti-inflammatory drug (FLUDRO), RAP-12 peptide, MG-1 peptide, PEG-NHS ester polymer (MW 5000 Da).

[0049] Transmission electron microscope (TEM), dynamic light scattering instrument (DLS), high performance liquid chromatograph (HPLC), laser confocal scanning microscope (CLSM), Transwell culture plate.

[0050] Synthesis of MPDA nanoparticles MSN was dispersed in 100 mL Tris-HCl buffer (10 mM, pH 8.5) and 2 mg / mL dopamine hydrochloride was added. The mixture was stirred at 40 °C for 24 h. PDA@MSN was collected by centrifugation (12,000 rpm, 15 min) and washed with ultrapure water for 3 times. MPDA was obtained by etching with 5% hydrofluoric acid for 12 h and centrifugation purification. TEM was used to observe the mesoporous structure and DLS was used to determine the particle size (150 ± 20 nm).

[0051] The synthesis of FLUDRO-Pep-MPDA nanoparticles was as shown in Figure 2 and realized as follows: Pep-FLUDRO conjugate preparation: 20 mg Pep was dissolved in 2 mL 0.1 M MES buffer (pH 5.2), and 21.2 mg EDC and 78 mg sulfo-NHS were added. After activation for 30 min, 29.2 mg FLUDRO was added and the reaction was allowed to proceed for 24 h. The product was purified by dialysis (MWCO 1000 Da) and lyophilized to obtain Pep-FLUDRO.

[0052] Drug-loaded nanoparticle preparation: 29.2 mg Pep-FLUDRO was added to 2 mL MPDA alkaline solution (2 mg / mL) and magnetically stirred for 24 h. FLUDRO-Pep-MPDA was collected by centrifugation (10,000 g, 20 min).

[0053] Brain-targeting functional modification PEGylated RAP-12 preparation: 10 mg PEG-NHS ester was reacted with 66 mg RAP-12 in 2 mL 0.2 M Na2CO3 solution for 24 h, and the product was purified by dialysis (MWCO 3500 Da).

[0054] Targeted nanosystem construction: PEGylated RAP-12 was mixed with FLUDRO-Pep-MPDA in bicine buffer at pH 8.5 and the reaction was allowed to proceed for 24 h to obtain FLUDRO-Pep-MPDA@RAP12 / MG-1.

[0055] II. Characterization and functional verification Physical characterization DLS: The particle size (150 ± 20 nm) and Zeta potential (-20 ± 5 mV) of the nanoparticles were determined.

[0056] TEM: observe the mesoporous structure and "core-shell" morphology of MPDA, MPDA is a porous core, and the surface is uniformly coated with Pep-FLUDRO and targeting peptide.

[0057] Drug loading detection: HPLC analysis of unbound FLUDRO in the supernatant, calculate the drug loading β=(C0-C s ) / C n , the results show that β>80%.

[0058] Targeting and release function verification In vitro BBB model: Transwell culture bEnd.3 cells, add fluorescently labeled nanoparticles, detect the fluorescence intensity on the basal side after 24 hours, find that the fluorescence intensity of the modified group is significantly higher than that of the unmodified group (P<0.05), which proves the brain targeting.

[0059] MMP-2 responsive release: incubate nanoparticles with different concentrations of MMP-2, HPLC detection shows that the drug release rate increases with the increase of MMP-2 concentration, and reaches 75% at 10 U / mL, which verifies the responsiveness.

[0060] Microglia targeting: CLSM observation shows that after co-culture with BV2 cells, the uptake of nanoparticles in the modified group increases significantly (P<0.01), and the expression of pro-inflammatory factor IL-1β is down-regulated (P<0.05), which proves the inhibition of microglia polarization.

[0061] Figure 3 The scanning electron micrograph of FLUDRO-Pep-MPDA@RAP12 / MG-1 nanoparticles and the DLS result schematic diagram, Figure 3 show the particle size of the nanoparticles, show the core-shell structure and uniform dispersibility, the particle size is about 100-150 nm, which meets the design target. Figure 4 The potential value schematic diagram of FLUDRO-Pep-MPDA@RAP12 / MG-1 nanoparticles, Figure 4 shows that the drug and nanoparticles are successfully loaded.

[0062] Biocompatibility evaluation Figure 5 The in vitro release and biocompatibility experiment schematic diagram of FLUDRO-Pep-MPDA@RAP12 / MG-1 nanoparticles is shown in Figure 5 , CCK-8 experiment shows that the nanoparticles have no significant effect on the survival rate of PC12 and BV2 cells in the concentration range of 0.1-100 μg / mL (P>0.05), which indicates that the MMP-2 responsive release and good biocompatibility. Figure 6Schematic diagram of in vivo biocompatibility experiment of FLUDRO-Pep-MPDA@RAP12 / MG-1 nanoparticles, as shown in Figure 6 The results show that the nano-drug particles have good in vivo biocompatibility.

[0063] Blood biochemistry and routine blood analysis show that the dosage of the nanoparticles is only 1 / 5-1 / 3 of that of traditional glucocorticoids, the liver and kidney function indicators are normal, and the incidence of systemic side effects is reduced by 70% compared with traditional glucocorticoids.

[0064] Indications: It is suitable for central nervous system diseases with neuroinflammation as the core mechanism, such as traumatic brain injury, Alzheimer's disease, multiple sclerosis, etc.

[0065] Administration method: intravenous injection, the nanoparticles can be dispersed in physiological saline or pH 7.4 phosphate buffer solution (PBS), the recommended concentration is 0.1-100 μg / mL, and the dosage is 1 / 5-1 / 3 of that of traditional therapy.

[0066] Examples of treating central nervous system inflammatory diseases are as follows: Traumatic brain injury (TBI) model verification: in the midline hydraulic impact C57 mouse model, tail vein injection of FLUDRO-Pep-MPDA@RAP12 / MG-1 (2 mg / kg) can significantly reduce the levels of pro-inflammatory factors IL-1β, IL-6, and TNF-α in the prefrontal cortex and hippocampus (40%-60% reduction), while increasing the levels of anti-inflammatory factors IL-10 and TGF-β (2.5 times increase, ELISA detection, P<0.05).

[0067] As shown in the example, Figure 7 As shown in the example, it is shown that FLUDRO-Pep-MPDA@RAP12 / MG-1 nanoparticles inhibit pro-inflammatory microglia cells in the cortex around the traumatic lesion after craniocerebral trauma, and increase the number of neuron stocks. Figure 8 As shown in the example, it is shown that FLUDRO-Pep-MPDA@RAP12 / MG-1 nanoparticles promote the recovery of neurological function in craniocerebral trauma mice.

[0068] Neuroprotection and repair: promote hippocampal dentate gyrus neural stem cell proliferation (28% increase in SOX2+ / BrdU+ double positive cells), differentiation (increase in DCX+ / NEUN+ cells), and synaptic remodeling (41% up-regulation of PSD95 protein expression, western blot detection, P<0.05), and improve the motor coordination ability and spatial memory of mice (sucrose preference rate increased to 65%, and the immobility time in forced swimming was shortened by 35%, P<0.01).

[0069] Advantages of the present application: Multi-stage targeting synergy: Integrating blood-brain barrier penetration (RPP-12) and microglia targeting (MG-1), the drug accumulation in the brain is increased by 5 times, and the drug concentration in the inflammatory area is 8 times that of traditional drug delivery methods, solving the problem of poor targeting of traditional drugs.

[0070] Intelligent response release reduces toxicity: MMP-2 triggers local drug release, reducing systemic exposure, avoiding immune suppression and osteoporosis caused by high-dose glucocorticoids, and achieving "on-demand drug delivery at the lesion site".

[0071] Multi-dimensional therapeutic effect: Not only inhibits neuroinflammation, but also promotes neuroregeneration, improves synaptic function, and regulates microglia polarization (CD206+ / Iba-1+ anti-inflammatory type ratio increases by 30%), showing comprehensive treatment potential for TBI, and providing a new treatment paradigm for central nervous system diseases.

[0072] It should be understood that the "one embodiment" or "an embodiment" or "some embodiments" mentioned throughout the specification means that the specific features, structures or characteristics related to the embodiment are included in at least one embodiment of the present application. Therefore, "in one embodiment" or "in an embodiment" or "in some embodiments" appearing throughout the specification does not necessarily refer to the same embodiment. In addition, these specific features, structures or characteristics can be combined in one or more embodiments in any suitable manner. It should be understood that in various embodiments of the present application, the size of the sequence number of the above processes does not mean the order of execution, and the execution order of the processes should be determined according to its function and inherent logic, and should not constitute any limitation on the implementation process of the embodiments of the present application. The sequence number of the above embodiments of the present application is only for description, not representing the advantages and disadvantages of the embodiments. The above description of each embodiment tends to emphasize the differences between each embodiment, and the same or similar parts can be referred to each other. For the sake of brevity, this paper will not repeat here.

[0073] The above modules described as separate components can or can not be physically separated, and the components shown as modules can or can not be physical modules; they can be located in one place or distributed on multiple network units; part or all of the modules can be selected according to actual needs to achieve the purpose of the present embodiment.

[0074] In addition, each functional module in each embodiment of the present application can be integrated in one processing unit, or each module can be a separate unit, or two or more modules can be integrated in one unit; the above integrated modules can be realized in the form of hardware or hardware plus software function unit.

[0075] Those skilled in the art can understand that all or part of the steps of the foregoing method embodiments can be completed by relevant hardware of program instructions, and the foregoing program can be stored in a computer readable storage medium. When the program is executed, the program executes the steps of the foregoing method embodiments. The foregoing storage medium includes a mobile storage device, a read only memory (ROM), a magnetic disc or an optical disc, and various media that can store program codes.

[0076] The methods disclosed in the several method embodiments provided by the embodiments of the present application can be combined arbitrarily without conflict to obtain new method embodiments.

[0077] The features disclosed in the several product embodiments provided by the embodiments of the present application can be combined arbitrarily without conflict to obtain new product embodiments.

[0078] The features disclosed in the several method or device embodiments provided by the embodiments of the present application can be combined arbitrarily without conflict to obtain new method or device embodiments.

[0079] The foregoing is only a manner of implementing the embodiments of the present application, but the protection scope of the embodiments of the present application is not limited to this. Any person skilled in the art can easily think of changes or replacements within the technical range disclosed by the present application, which should be covered in the protection scope of the embodiments of the present application. Therefore, the protection scope of the embodiments of the present application should be subject to the protection scope of the claims.

Claims

1. A method for preparing composite drug-loaded nanoparticles targeting the blood-brain barrier, characterized in that, The method includes: Step (1) Synthesis of mesoporous polydopamine (MPDA) nanoparticles: 100 mg of mesoporous silica (MSN) was dispersed in 10 mM Tris-HCl buffer at pH 8.5, and 2 mg / mL dopamine hydrochloride was added. The mixture was stirred at 40 °C for 24 hours to allow dopamine to polymerize on the MSN surface to form a polydopamine (PDA) coating. PDA@MSN was collected by centrifugation at 12,000 rpm for 15 minutes. After washing three times with ultrapure water, the MSN template was removed by etching with 5% hydrofluoric acid. MPDA was obtained by centrifugation and purification. Its mesoporous structure was verified by TEM, and the particle size was measured to be 150 ± 20 nm by DLS. Step (2) Synthesis of FLUDRO-Pep-MPDA nanoparticles: 20 mg of MMP-2 responsive peptide Pep (Ac-CSSSGPLGIAGQSSS) was dissolved in 2 mL of 0.1 M, pH 5.2 MES buffer, and 21.2 mg of EDC and 78 mg of sulfo-NHS were added to activate the carboxyl group. The mixture was reacted with 29.2 mg of fludrocortisone (FLUDRO) for 24 hours, purified by dialysis using a MWCO~1000 Da dialysis bag, and lyophilized for 48 hours to obtain the Pep-FLUDRO conjugate. The conjugate was then added to a 2 mg / mL mesoporous polydopamine alkaline solution and magnetically stirred for 24 hours. Step (3) Dual-targeting peptide conjugation: 10 mg of PEG-NHS ester polymer (molecular weight ~5000 Da) and 66 mg of RPP-12 peptide were reacted in 2 mL of 0.2 M Na2CO3 solution for 24 hours. After purification by MWCO~3500 Da dialysis bag, it was combined with the drug-loaded nanoparticles obtained in step (2) in bicine buffer at pH 8.5 to obtain the composite drug-loaded nanoparticles FLUDRO-Pep-MPDA@RAP12 / MG-1.

2. The preparation method according to claim 1, characterized in that, In step (3), the mass ratio of PEG-NHS ester polymer to RPP-12 peptide is 1:6.6, and the mass ratio of polyethylene glycolated RAP-12 to drug-loaded nanoparticles is (1-3):

10.

3. The preparation method according to claim 1, characterized in that, The MPDA nanoparticles have a mesopore size of 2-5 nm, a specific surface area of ​​100-300 m² / g, a rehydration temperature of 20-25 °C, and an ultrasonic treatment time of 5-15 min with a frequency of 25-35 kHz.

4. A composite drug-loaded nanoparticle targeting the blood-brain barrier, characterized in that, The composite drug-loaded nanoparticles FLUDRO-Pep-MPDA@RAP12 / MG-1 are prepared by the preparation method according to any one of claims 1-3. The composite drug-loaded nanoparticles FLUDRO-Pep-MPDA@RAP12 / MG-1 have a core-shell structure, with the core being mesoporous polydopamine and the shell containing Pep-FLUDRO conjugate, RPP-12 peptide, and MG-1 peptide. The particle size is 150±20 nm, the zeta potential is -15 to -25 mV, the drug loading β is ≥80%, and the drug release rate is ≥75% after 24 hours when the MMP-2 concentration is ≥5 U / mL.

5. The composite nanoparticles according to claim 4, characterized in that, The RPP-12 peptide is coupled to the PEG surface of nanoparticles through polyethylene glycol modification, and the MG-1 peptide is covalently anchored to the polydopamine surface through a carboxyl-amino condensation reaction.

6. The application of a composite drug-loaded nanoparticle targeting the blood-brain barrier, characterized in that, The composite nanoparticles described in claim 4 or 5 are used to treat inflammatory diseases of the central nervous system.

7. The application according to claim 6, characterized in that, The diseases mentioned include traumatic brain injury (TBI), Alzheimer's disease, or multiple sclerosis.

8. The application according to claim 6, characterized in that, The drug is administered via tail vein injection at a dose that is 1 / 5 to 1 / 3 of the dose administered systemically as a traditional glucocorticoid.

9. A pharmaceutical composition for treating traumatic brain injury, characterized in that, It comprises the composite nanoparticles as described in claim 4 or 5, a pharmaceutically acceptable carrier, and optionally an anti-inflammatory adjuvant.

10. The pharmaceutical composition according to claim 9, characterized in that, The carrier is physiological saline or phosphate-buffered saline (PBS) at pH 7.4, and the concentration of nanoparticles in the drug composition is 0.1-100 μg / mL.

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