A pathological blood-brain barrier targeting nano-preparation for treating Alzheimer's disease and a preparation method thereof

Abstract

The application discloses a pathological blood-brain barrier targeting nano-preparation for treating Alzheimer's disease and a preparation method thereof, and belongs to the technical field of biological medicine. The nano-preparation takes nanoparticles assembled by an amphiphilic block copolymer as a carrier, and is co-modified with RAP peptides and RGD peptides on the surface. The RAP peptides can specifically recognize and bind to the RAGE receptor which is highly expressed by vascular endothelial cells in a pathological state, so that precise targeting is realized, and the RAP peptides also have the activity of blocking the RAGE-AB pathological pathway. The RGD peptides serve as an integrin binding motif, can significantly enhance the endocytosis efficiency of the nanoparticles through the interaction with the cell surface integrin receptor, and promote the transportation of the nanoparticles to lysosomes after endocytosis. Through the synergistic effect of the two ligands, the nanoparticles can not only be efficiently targeted to the diseased brain vascular endothelium expressing RAGE, but also be effectively internalized and guided to lysosomes, so that the targeted drug delivery is realized, the degradation of the RAGE protein is enhanced by means of the lysosome pathway, and the active intervention on the key pathological pathway is realized.

Description

Technical Field

[0001] This invention relates to a pathological blood-brain barrier-targeting nanoparticle formulation for the treatment of Alzheimer's disease and its preparation method. Specifically, it relates to a nanoparticle formulation that can specifically target the damaged blood-brain barrier in the course of Alzheimer's disease, and a method for preparing the nanoparticle formulation, belonging to the field of biomedical technology. Background Technology

[0002] Alzheimer's disease (AD) is a neurodegenerative disease characterized primarily by progressive cognitive impairment. Its pathological features include senile plaques formed by the deposition of β-amyloid (Aβ) protein and neurofibrillary tangles resulting from the hyperphosphorylation of tau protein. Currently, drug treatments for AD have very limited efficacy, a key bottleneck being the blood-brain barrier (BBB), which severely restricts the entry of therapeutic drugs from the bloodstream into brain tissue.

[0003] The blood-brain barrier (BBB) ​​is a highly selective semi-permeable membrane barrier structure composed of brain capillary endothelial cells and their tight junctions, astrocyte terminale, pericytes, and other components. Under physiological conditions, it effectively protects the central nervous system, but also prevents over 98% of small molecule drugs and almost all large molecule drugs from entering the brain. To overcome this barrier, nanoparticles have been extensively studied as a promising brain-targeted delivery system. Existing technologies mainly focus on utilizing the physiological characteristics of the BBB for drug delivery. For example, current technologies often design nanoparticles to mimic natural ligands (such as transferrin and lactoferrin), allowing them to cross the healthy BBB via receptor-mediated endocytosis (RMT). However, these strategies based on BBB physiological receptors have a fundamental limitation: they cannot effectively distinguish between the pathological BBB in disease states and the normal physiological BBB. Specifically, existing technologies have the following shortcomings:

[0004] (1) Lack of specific recognition ability for lesion areas: Existing technologies, such as Chinese patents CN120694949A and CN120241645A, mainly rely on receptors that are uniformly highly expressed in the whole brain brain (such as transferrin receptor TfR and lactoferrin receptor). Although studies have confirmed that the expression of such receptors may not decrease during the course of AD, this "universal" targeting strategy leads to the widespread distribution of nanoparticles throughout the brain, rather than specific enrichment in the core lesion area. As a result, it is difficult to achieve effective therapeutic drug concentrations locally at the lesion site, and the drug carriers distributed in large quantities in normal brain areas outside the lesion may bring significant neurotoxicity risks, limiting their therapeutic window.

[0005] (2) Failure to utilize specific biomarkers of pathological BBB: Studies have shown that during AD progression, cerebral vascular endothelial cells in the affected brain region are activated and specifically upregulated in the expression of novel biomarkers such as the receptor for advanced glycoprotein end products (RAGE). These biomarkers are expressed at extremely low levels in normal brain capillary endothelium. However, existing targeting strategies (such as targeting TfR) completely ignore this pathological feature, and their targeting mechanisms are disconnected from the disease progression status, failing to achieve precise drug delivery "at the right time and in the right place".

[0006] (3) The contradiction between systemic delivery and lesion delivery efficiency: Due to the above-mentioned non-selectivity, in order to accumulate sufficient drug in the lesion area, the systemic dosage must be increased. This leads to the large-scale clearance of nano-formulations in mononuclear phagocytic organs such as the liver and spleen, increasing the systemic exposure burden and metabolic stress of the carrier material, resulting in a waste of drug resources and exacerbating the potential risk of systemic side effects.

[0007] Therefore, there is an urgent need in this field for a novel nanoparticle formulation that can actively recognize and target molecules specifically highly expressed on the blood-brain barrier in the pathological state of Alzheimer's disease (AD). RAGE, as one of the key transport receptors for β-amyloid (Aβ), not only mediates its influx across the BBB after binding to Aβ, but also activates downstream signaling pathways such as nuclear factor κB (NF-κB), triggering oxidative stress and inflammatory cascade responses.

[0008] In the prior art, Chinese patent CN110652594B discloses a multi-target therapeutic micelle and its preparation method for regulating the microenvironment of Alzheimer's disease. This formulation constructs nanomicelles using polymer materials modified with Ab peptides. Utilizing the affinity of Ab peptides for RAGE receptors, the micelles can target AD lesion areas. Simultaneously, the phenylboronic acid ester structure in the system can responsively degrade in the inflammatory microenvironment, promoting drug release and thus enhancing drug accumulation and efficacy in brain tissue. The core mechanism of this method is to utilize targeting peptides (specific ligands of RAGE receptors—Ab peptides) to achieve spatial enrichment of the carrier. Its function is mainly limited to "binding-delivery," aiming to increase local drug concentration to enhance the efficacy of AD treatment.

[0009] In addition, Chinese patent CN112826794B discloses a nanocomposite for targeted repair of neurovascular lesions and its preparation and application. The composite contains lipids, apolipoproteins, and a targeting peptide. The targeting peptide is formed by a bridging structure that covalently links the nanocarrier end and the peptide chain that specifically binds to the RAGE site of cerebrovascular lesions. It can actively target and bind to the cerebral vascular endothelium, repair vascular endothelial cells, promote the clearance of perivascular Aβ plaques, and thus repair cerebral blood vessels and neurovascular unit components such as microglia, astrocytes, and neurons. Through the combined repair of multiple components, the function of cerebral blood vessels and cerebral blood flow are restored, thereby achieving the purpose of improving cognitive impairment. At the same time, the composite can also carry drugs, realizing the dual function of treatment and delivery. The targeting peptide sequence used is derived from Aβ amyloid protein. Its design concept is to utilize the known pathological interaction between Aβ and RAGE to achieve targeting and penetration of the lesion site.

[0010] In summary, existing technologies have attempted to utilize RAGE as a target to design nanosystems capable of delivering drugs to brain regions affected by Alzheimer's disease (AD), achieving, to some extent, targeted enrichment of the carrier or its repair function. However, these approaches still have the following limitations: First, their targeting mechanisms mainly rely on simple ligand-receptor binding, with functions limited to spatial localization and failing to further intervene in key pathological signaling pathways mediated by RAGE. Second, the targeting ligands used (such as Aβ-derived peptides) are themselves closely related to disease progression, posing potential biosafety risks, and their mechanism of action remains a passive utilization of existing pathological connections, lacking the ability to actively regulate the RAGE-Aβ positive feedback loop. Therefore, developing a novel drug delivery system that can both achieve efficient drug enrichment in pathological brain regions by targeting RAGE and directly intervene in the RAGE-Aβ pathway to break the vicious cycle of disease remains a key unsolved problem in the field. Summary of the Invention

[0011] This invention aims to overcome the shortcomings of existing brain-targeting technologies, such as poor selectivity for lesion regions and inability to intervene in key pathological pathways. It provides a pathological blood-brain barrier-targeting nanoparticle formulation for the treatment of Alzheimer's disease and its preparation method. This nanoparticle formulation uses nanoparticles assembled from amphiphilic block copolymers as carriers, with a targeting ligand (RAP peptide) and a functional ligand (RGD peptide) co-modified on their surface. The RAP peptide specifically recognizes and binds to RAGE receptors highly expressed in vascular endothelial cells under pathological conditions, achieving precise targeting and possessing the activity to block the RAGE-AB pathological pathway. The RGD peptide, as an integrin-binding motif, significantly enhances the endocytosis efficiency of the nanoparticles through interaction with cell surface integrin receptors and promotes their transport to lysosomes after endocytosis. Through the synergistic effect of the two ligands, the nanoparticles can efficiently target diseased brain vascular endothelium expressing RAGE and be effectively internalized and guided to lysosomes. This achieves targeted drug delivery while simultaneously enhancing the degradation of RAGE proteins via the lysosomal pathway, thus enabling active intervention in key pathological pathways.

[0012] This invention is achieved through the following technical solution: a pathological blood-brain barrier-targeting nano-formulation for treating Alzheimer's disease, wherein the nano-formulation comprises at least the following components:

[0013] (1) Carrier nanoparticles assembled from amphiphilic block copolymers, wherein the amphiphilic block copolymers comprise hydrophobic blocks and hydrophilic blocks;

[0014] (2) A targeting ligand modified on the surface of the carrier nanoparticles, wherein the targeting ligand is a RAP peptide with the amino acid sequence CELKVLMEKEL;

[0015] (3) A functional ligand modified on the surface of the carrier nanoparticles, wherein the functional ligand is an RGD peptide with the amino acid sequence c(RGDfK).

[0016] The nanoformulation also includes a hydrophobic drug loaded in the carrier nanoparticles.

[0017] The hydrophobic drug is selected from at least one of curcumin, resveratrol, pioglitazone, and simvastatin.

[0018] The hydrophobic block is a polylactic acid-glycolic acid copolymer with a molecular weight of 2000-20000; the hydrophilic block is polyethylene glycol with a molecular weight of 500-5000.

[0019] In the amphiphilic block copolymer, the block ratio of polylactic acid-glycolic acid copolymer to polyethylene glycol is 50:50 to 10:90.

[0020] The hydrophilic block is connected to a reactive functional group at its end, and the targeting ligand or functional ligand is covalently modified on the surface of the carrier nanoparticle through the reactive functional group.

[0021] The reactive functional group is selected from one of maleimide (MAL), carboxyl (-COOH), amino (-NH2), and azide (-N3).

[0022] The terminal of the functional ligand contains a thiol, amino, carboxyl, or alkynyl group covalently linked to the reactive functional group.

[0023] This invention also provides a method for preparing a pathological blood-brain barrier-targeting nanoparticle formulation for treating Alzheimer's disease, comprising the following steps:

[0024] S1. The amphiphilic block copolymer and the targeting ligand are dissolved in organic solvents to obtain polymer solution and targeting ligand solution respectively. Under inert gas protection, the polymer solution and the targeting ligand solution are mixed to carry out covalent linkage reaction to obtain the first reaction mixture. After purification and freeze drying, a polymer carrier material with the targeting ligand modified on the surface is obtained.

[0025] S2. The amphiphilic block copolymer and the functional ligand are dissolved in organic solvents to obtain polymer solution and functional ligand solution respectively. Under inert gas protection, the polymer solution and functional ligand solution are mixed to carry out covalent linkage reaction to obtain a second reaction mixture. After purification and freeze drying, a polymer carrier material with functional ligand modified on the surface is obtained.

[0026] S3. Disperse the above two polymer carrier materials in a solvent, or disperse the above two polymer carrier materials and hydrophobic drugs in a solvent, and assemble them into carrier nanoparticles by ultrasonic treatment. Remove the unencapsulated hydrophobic drugs, and after purification, obtain the pathological blood-brain barrier targeted nano-formulation.

[0027] The molar ratio of the amphiphilic block copolymer to the targeting ligand is 1:1.1 to 1:1.9; the molar ratio of the amphiphilic block copolymer to the functional ligand is 1:1.1 to 1:1.9.

[0028] The covalent bonding reaction was carried out in a water bath at 25–55°C for 12–48 hours.

[0029] In the solvent, the molar ratio of polymer carrier material with surface-modified targeting ligands to polymer carrier material with surface-modified functional ligands is 1:9 to 9:1.

[0030] Alternatively, in the solvent, the mass ratio of the polymer carrier material with surface-modified targeting ligands, the polymer carrier material with surface-modified functional ligands, and the hydrophobic drug is 1:9:10 to 9:1:1.

[0031] The ultrasonic treatment is performed at a power of 50-200W for 1-10 minutes, with a pause of 1-10 seconds after each 1-10 seconds of ultrasonic treatment.

[0032] The organic solvent is selected from at least one of N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), methanol, tetrahydrofuran, and acetonitrile.

[0033] The solvent is selected from at least one of dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), methanol, tetrahydrofuran, and acetonitrile.

[0034] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0035] (1) The nano-formulation of the present invention can achieve precise and efficient targeted delivery to pathological brain regions. The nano-formulation specifically recognizes and targets the RAGE receptor highly expressed in the lesion site of Alzheimer's disease through surface-modified RAP peptides. It can actively recognize and accumulate in the lesion area, significantly improve the local drug concentration in the lesion, and effectively overcome the problems of insufficient drug concentration in the lesion and high toxicity risk in normal brain regions caused by the non-selective and widespread distribution of the entire brain in the prior art.

[0036] (2) The nano-formulation of the present invention has both targeted delivery and disease modification functions. The RAP peptide can not only achieve specific targeted binding, but also block the vicious cycle of neuroinflammation and Aβ deposition by intervening in the RAGE-Aβ pathological pathway, thereby achieving the organic combination of "targeted delivery" and "disease modification" from the source. It is expected to inhibit neuroinflammation, reduce Aβ deposition and promote the recovery of blood-brain barrier function.

[0037] (3) The nano-formulation of the present invention adopts the dual-ligand synergistic mechanism of RAP peptide and RGD peptide. RAP peptide is responsible for accurately recognizing RAGE receptor, and RGD peptide, as a cell uptake enhancement motif, can significantly promote the endocytosis of nanoparticles and guide them to lysosomes. The synergistic effect of the two ligands can improve the targeting efficiency and effectively promote the degradation of RAGE protein via the lysosomal pathway, thereby realizing a complete pathological intervention chain from "recognition" to "clearance".

[0038] (4) In the nano-formulation of the present invention, the RAP peptide has an activity regulation function for binding to RAGE, and its targeting efficiency is positively correlated with the severity of the pathology. During the active phase of the disease, high expression of RAGE guides the nanoparticles to accumulate in large quantities and exert a therapeutic effect; as the treatment takes effect and RAGE expression is downregulated, the delivery efficiency of the nanoparticles decreases accordingly, thereby achieving adaptive adjustment of the therapeutic dose, improving the efficacy while optimizing the in vivo distribution and significantly improving the safety of treatment.

[0039] (5) The nano-formulation of the present invention uses PLGA-PEG as a carrier, which can efficiently encapsulate a variety of hydrophobic therapeutic drugs (such as curcumin), improve their solubility and delivery efficiency. At the same time, the loaded drugs can synergize with the RAGE targeting-regulation-degradation mechanism of the nanoparticles themselves, thus providing an ideal delivery platform for realizing the combined treatment strategy of Alzheimer's disease.

[0040] (6) The nano-formulation of the present invention utilizes the lesion-specific enrichment and intelligent regulation mechanism to significantly reduce the unnecessary distribution of drugs in normal tissues, thereby reducing systemic exposure and related toxicity risks, and achieving a better safety window while achieving potent treatment. Attached Figure Description

[0041] Figure 1 The matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF) results are for the raw material PLGA (50:50) 5000-PEG2000-MAL.

[0042] Figure 2 The matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF) results for PLGA (50:50) 5000-PEG2000-RAP are shown.

[0043] Figure 3 The matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF) results for PLGA (50:50) 5000-PEG2000-RGD are shown.

[0044] Figure 4 The hydrated particle size distribution and transmission electron microscopy image of cur@RRNP (scale bar is 100 nm).

[0045] Figure 5 The particle size stability of RRNP in 5% glucose (N=3).

[0046] Figure 6 The particle size stability of cur@RRNP in 5% glucose (N=3).

[0047] Figure 7 Results of bEnd.3-RAGE cell uptake of different nanoparticles (NP and RRNP) (N=3).

[0048] Figure 8 Results of affinity for different nanoparticles (NP and RRNP) with bEnd.3-RAGE cells (N=3).

[0049] Figure 9 Results of co-localization of different nanoparticles (NP and RRNP) with lysosomes.

[0050] Figure 10 The expression level of RAGE tag protein after RRNP administration.

[0051] Figure 11 Leakage in an in vitro BBB model after administration of different formulations.

[0052] Figure 12 Immunofluorescence imaging results of ZO-1, a tight junction protein in bEnd.3-RAGE cells, after administration of different formulations.

[0053] Figure 13 Results of bEnd.3-RAGE cell viability assay after administration of different formulations.

[0054] Figure 14 ELISA results of anti-inflammatory factor IL-6 in bEnd.3-RAGE cells administered with different formulations.

[0055] Figure 15 ELISA results of anti-inflammatory factor IL-10 in bEnd.3-RAGE cells after administration of different formulations.

[0056] Figure 16 ROS test results for BV2 after administration of different formulations.

[0057] Figure 17 Signal distribution in the brains of healthy mice and AD model mice after administration of DiD@RRNP.

[0058] Figure 18 Signal distribution in the isolated brains of healthy mice and AD model mice after administration of DiD@RRNP.

[0059] Figure 19 Co-localization of DiD@RRNP with RAGE protein in the brains of healthy mice and AD model mice after administration.

[0060] Figure 20 Nesting behavior scores of mice after treatment with different formulations (N=14).

[0061] Figure 21 The number of times AD model mice crossed the platform in the water maze after treatment with different formulations (N=14).

[0062] Figure 22 The cumulative time spent on the platform in the water maze behavior of AD model mice after treatment with different formulations (N=14).

[0063] Figure 23 The cumulative time spent on the platform in the water maze behavior of AD model mice after treatment with different formulations (N=14).

[0064] Figure 24 The alternation rate of AD model mice in the Y-maze behavioral test after treatment with different formulations (N=14).

[0065] Figure 25 Immunofluorescence staining of the brains of AD model mice after treatment with different dosage forms.

[0066] Figure 26 The leakage of cerebral blood vessels in mice after treatment with different formulations (qualitative).

[0067] Figure 27 The leakage of cerebral blood vessels in mice after treatment with different formulations (quantitative).

[0068] Figure 28 The leakage of cerebral blood vessels in mice after treatment with different formulations (quantitative).

[0069] Figure 29 The expression of tight junction proteins in the cerebral blood vessels of mice after treatment with different formulations.

[0070] Figure 30 The perfusion of cerebral blood vessels in mice after treatment with different formulations.

[0071] Figure 31 The expression levels of the inflammatory factor TNF-α in mouse brain tissue after treatment with different formulations are shown.

[0072] Figure 32 The expression levels of the inflammatory factor IFN-γ in mouse brain tissue after treatment with different formulations.

[0073] Figure 33 The expression levels of the inflammatory factor NF-κB in mouse brain tissue after treatment with different formulations.

[0074] Figure 34 The expression of BDNF in the brains of mice after treatment with different formulations.

[0075] Figure 35 The expression of SYN protein, a synaptophysin, in the brains of mice after treatment with different formulations. Detailed Implementation

[0076] The invention's objective, technical solution, and beneficial effects will be further explained in detail below.

[0077] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the claimed invention. Unless otherwise stated, 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 invention pertains.

[0078] This invention provides a pathological blood-brain barrier targeted nanoparticle for the treatment of Alzheimer's disease and its preparation method, which has comprehensive advantages in terms of targeting precision, pathological intervention depth, system intelligence and clinical translation potential. Its core is: (1) a nanocarrier is formed by self-assembly of a biocompatible amphiphilic block copolymer (PLGA-PEG); (2) the targeting ligand RAP peptide and the functional ligand RAP peptide are co-modified on the surface of the carrier. The RAP peptide achieves precise binding to the receptor for advanced glycoprotein end products (RAGE) that is highly expressed in vascular endothelial cells under pathological conditions. The RGD peptide can enhance the endocytosis efficiency of the nanocarrier by target cells and guide it into the lysosomal pathway, thereby synergistically promoting the lysosomal degradation of RAGE protein; (3) the carrier is simultaneously loaded with hydrophobic therapeutic drugs (such as curcumin, resveratrol, etc.), and the RAP peptide itself has the activity of blocking the RAGE-AB pathological pathway; finally, a multi-therapeutic effect of "targeted delivery-pathway regulation-drug synergy" is achieved.

[0079] The following is a detailed overview of the technical solution of this invention:

[0080] (I) Construction of carrier nanoparticles

[0081] The present invention uses carrier nanoparticles assembled from amphiphilic block copolymers, wherein the amphiphilic block copolymers contain hydrophobic blocks and hydrophilic blocks.

[0082] Hydrophobic blocks: such as polylactic acid-glycolic acid copolymer (PLGA), polycaprolactone (PCL), polystyrene (PS), or polylactic acid (PLA), with a molecular weight range of 2,000 to 20,000, are used to form a dense hydrophobic core to encapsulate therapeutic drugs (such as curcumin) and isolate them from the external environment, thereby significantly enhancing their chemical stability.

[0083] Hydrophilic blocks: such as polyethylene glycol (PEG), polyethylene oxide (PEO), polylysine (PLL), polyacrylic acid (PAA), or polyglycolic acid (PGA), with a molecular weight range of 500 to 5000, are used to form a hydration layer, giving nanoparticles "stealth" properties, greatly prolonging blood circulation time, creating the necessary conditions for targeted accumulation, and thus simultaneously achieving "long circulation" and "stable drug delivery".

[0084] Block ratio: The block ratio of PLGA to PEG is 50:50 to 10:90, which is used to precisely control the self-assembly behavior of the material in solution and its final properties.

[0085] (II) Design and modification of targeted ligands and functional ligands

[0086] This invention uses a RAP peptide as a targeting ligand. The amino acid sequence of this peptide is CELKVLMEKEL, as shown in SEQ ID NO: 1. It is an antagonistic peptide obtained through rational design based on the structural characteristics of the endogenous ligand S100P protein of RAGE. Compared with existing technologies that directly use the Aβ fragment (KLVFFAED) or Aβ-derived peptides (such as AC-FAEKFKEAVKDYFAKFWD-GSG-ELKVLMEKEL) as ligands, the RAP peptide has the following advantages:

[0087] High specificity and targeting capability: It can specifically recognize and bind to RAGE receptors, achieving precise targeting of vascular endothelial cells at the lesion site;

[0088] Pathological intervention function: It not only achieves spatial targeting and penetration of the pathological blood-brain barrier, but also competitively inhibits the binding of various endogenous ligands such as S100P, S100A4 and HMGB1 to RAGE, thereby blocking the activation of downstream signaling pathways such as NF-κB mediated by RAGE, and inhibiting neuroinflammation and Aβ influx across the blood-brain barrier from the upstream.

[0089] Safety: It avoids the immunogenicity and potential biosafety risks that may arise from directly using Aβ-related sequences.

[0090] In terms of specific modification methods, reactive functional groups, including but not limited to maleimide, carboxyl, amino, or azide groups, are pre-attached to the ends of the PEG chain of the carrier polymer. The RAP peptide is covalently linked to the above-mentioned reactive functional groups through the corresponding reactive groups (such as thiol, amino, etc.) at its ends, thereby being stably modified on the surface of the nanoparticles.

[0091] Meanwhile, this invention uses RGD peptide as a functional ligand with an amino acid sequence of C (RGDfK), as shown in SEQ ID NO: 2, i.e., a ring (Arg-Gly-Asp-D-Phe-Lys), and its C-terminus is modified with thiol (-SH), amino, carboxyl, azide, etc., for covalent connection with the reactive functional groups at the end of the PEG chain, so as to achieve its stable modification on the surface of nanoparticles.

[0092] Specifically, the role of the RGD peptide in this invention is as follows: As a cell adhesion and endocytosis-promoting motif, the RGD peptide can specifically recognize integrin receptors on the cell surface, significantly enhancing the cellular uptake efficiency of nanoparticles and guiding endocytosed nanoparticles into the lysosomal transport pathway. It works synergistically with the RAP peptide, which specifically recognizes and binds to highly expressed RAGE proteins at lesion sites for targeted anchoring; the RGD peptide further promotes nanoparticle endocytosis and intracellular lysosomal guidance, thereby jointly enhancing the delivery efficiency of nanoparticles to diseased cells and creating the necessary conditions for subsequent degradation of RAGE proteins via the lysosomal pathway. This dual-ligand synergy, while enhancing targeting precision, effectively promotes intracellular clearance of pathological proteins, achieving a deep integration of targeted delivery and disease modification.

[0093] (III) Drug selection and loading

[0094] The nano-formulation of the present invention can efficiently encapsulate a variety of hydrophobic drugs with anti-AD activity, including but not limited to: curcumin, resveratrol, pioglitazone, simvastatin and other anti-inflammatory drugs.

[0095] The drug is loaded into the hydrophobic core of the nanoparticle through physical encapsulation. The mass ratio of polymer carrier material to drug is 100:1 to 10:1, ensuring high encapsulation efficiency and drug loading.

[0096] (iv) Preparation method

[0097] The preparation method of the present invention mainly includes two steps:

[0098] Step 1: Synthesis of the polymer support

[0099] PLGA-PEG polymers with terminal reactive functional groups and RAP peptides were dissolved separately in organic solvents (such as DMF, DMSO, methanol, tetrahydrofuran, or acetonitrile). Under inert gas protection and gentle heating (25–55 °C), the mixture was reacted for 12–48 hours to covalently link the RAP peptides to the PLGA-PEG polymer. After the reaction was complete, the polymers were purified by dialysis or precipitation, and then freeze-dried to obtain polymer carrier materials with surface-modified targeting ligands.

[0100] PLGA-PEG polymers with terminal reactive functional groups and RGD peptides were dissolved separately in organic solvents (such as DMF, DMSO, methanol, tetrahydrofuran, or acetonitrile). Under inert gas protection and gentle heating (25–55 °C), the mixture was reacted for 12–48 hours to covalently link the RGD peptides to the PLGA-PEG polymer. After the reaction was complete, the polymers were purified by dialysis or precipitation, and then freeze-dried to obtain polymer carrier materials with surface-modified functional ligands.

[0101] In the above process, the molar ratio of PLGA-PEG polymer to RAP peptide is controlled at 1:1.1 to 1:1.9, and the molar ratio of PLGA-PEG polymer to RGD peptide is controlled at 1:1.1 to 1:1.9, to ensure that the terminal functional groups of PEG are fully modified.

[0102] Step 2: Assembly and drug delivery of nano-formulations

[0103] The two polymer carrier materials obtained in step one are dissolved or dispersed together with the hydrophobic drug in a solvent (the mass ratio of polymer carrier material with surface-modified targeting ligand, polymer carrier material with surface-modified functional ligand, and hydrophobic drug is 1:9:10 to 9:1:1). The polymer self-assembles into drug-loaded nanoparticles by using probe ultrasound (power 50-200W, pulse mode: ultrasound 1-10s, pause 1-10s, total duration 1-10min). Subsequently, the unencapsulated free drug is removed by centrifugation, filtration, or dialysis. After purification, the final pathological blood-brain barrier targeted nanoformulation is obtained.

[0104] In summary, based on the above technical solutions, the pathological blood-brain barrier targeted nano-formulation of the present invention is obtained. The principle of its pathological blood-brain barrier targeting and intelligent regulation mechanism is as follows:

[0105] In the pathological state of Alzheimer's disease (AD), cerebral vascular endothelial cells in the affected brain region highly express the receptor for advanced glycoprotein advanced products (RAGE). Nanoparticles modified with RAP peptides can reach the lesion area through blood circulation, and can achieve targeted localization by leveraging the specific recognition and binding of RAP peptides to RAGE. Furthermore, they can penetrate the blood-brain barrier in the pathological state through receptor-mediated endocytosis and selectively accumulate in the lesion area.

[0106] Based on the achievement of targeted enrichment, the nano-formulation of the present invention exerts its therapeutic effect through multiple pathways: on the one hand, the hydrophobic drug loaded on the nanoparticles can achieve local high-concentration release in the lesion area and directly exert pharmacological effects; on the other hand, the RAP peptide modified on the surface of the nanoparticles has the function of inhibiting RAGE activity, competitively blocking the binding of endogenous ligands to RAGE, interfering with the RAGE-Aβ pathological positive feedback loop, thereby reducing Aβ influx, inhibiting neuroinflammatory response and ultimately promoting the downregulation of RAGE expression, thus intervening in the disease process at the molecular level.

[0107] More importantly, the aforementioned RAGE expression downregulation process and the targeting efficiency of the drug delivery system of this invention form a dynamic balance mechanism. During disease progression, high RAGE expression can guide more nanoparticles to be delivered to the lesion site; as treatment takes effect and RAGE expression gradually decreases, the targeted delivery efficiency of the nanoparticles also weakens accordingly. This self-regulating characteristic achieves dynamic matching between the treatment process and the disease state, which not only enhances the precise intervention in the lesion area but also reduces unnecessary exposure to normal tissues, thereby optimizing the overall treatment effect and improving treatment safety.

[0108] Therefore, this invention integrates active targeting, pathological intervention, and self-regulation to construct an intelligent drug delivery system that can respond to disease states in both time and space, providing a new strategy for the treatment of Alzheimer's disease that combines precision and safety.

[0109] The specific implementation of the present invention will be described below with reference to the embodiments. Of course, the scope of protection of the present invention is not limited to the following embodiments.

[0110] Example 1: RAGE Targeted Nanoparticles (RRNP)

[0111] 35.0 mg of PLGA (50:50) 5000-PEG2000-MAL (purchased from Huzhou Carbohydrate Pharmaceutical Technology Co., Ltd.) was weighed and dissolved in 3 mL of dry DMF. 10.0 mg of RAP peptide (amino acid sequence CELKVLMEKEL) was weighed and dissolved in 1 mL of DMF. The two were mixed in a 25 mL round-bottom flask, sealed, and reacted under nitrogen protection in a 40°C water bath for 24 h. The molar ratio of PLGA (50:50) 5000-PEG2000-MAL to RAP was 1:1.5. After the reaction was complete, the reaction mixture was transferred to a dialysis bag with a molecular weight cutoff of 3500 Da and purified by dialyzing in ultrapure water for 24 h, with fresh dialysis medium replaced several times during the process. Finally, the purified product was freeze-dried and stored at -20℃ to obtain PLGA5000-PEG2000-RAP. The relative molecular mass shift relative to the starting material PLGA(50:50)5000-PEG2000-MAL was verified by time-of-flight mass spectrometry, indicating that the synthesis was successful.

[0112] 35.0 mg of PLGA (50:50) 5000-PEG2000-MAL was weighed and dissolved in 3 mL of dry DMF. 10.0 mg of RGD peptide was weighed and dissolved in 1 mL of DMF. The two were mixed in a 25 mL round-bottom flask, sealed, and reacted under nitrogen protection in a 40°C water bath for 24 h. The molar ratio of PLGA (50:50) 5000-PEG2000-MAL to RGD was 1:1.5. After the reaction was complete, the reaction mixture was transferred to a dialysis bag with a molecular weight cutoff of 3500 Da and purified by dialyzing in ultrapure water for 24 h, with fresh dialysis medium replaced several times during this period. Finally, the purified product was freeze-dried and stored at -20℃ to obtain PLGA(50:50)5000-PEG2000-RGD. The relative molecular mass shift relative to the starting material PLGA(50:50)5000-PEG2000-MAL was verified by time-of-flight mass spectrometry, indicating that the synthesis was successful.

[0113] Weigh 10 mg of PLGA (50:50) 5000-PEG2000-RAP and 10 mg of PLGA (50:50) 5000-PEG2000-RGD into 2 mL EP tubes, and add 1 mL of DMSO to each tube to dissolve them as stock solutions. Mix 120 μL of PLGA (50:50) 5000-PEG2000-RAP and 80 μL of PLGA (50:50) 5000-PEG2000-RGD thoroughly, add 4 mL of ultrapure water, vortex to mix, and then sonicate using a cell disruptor at 100 W for 5 min under ice bath conditions, pausing for 5 seconds every 5 seconds. Purify by ultrafiltration at 4 °C to remove DMSO, yielding RRNP.

[0114] Example 2: RAGE-targeted nanoparticles loaded with curcumin (cur@RRNP)

[0115] PLGA(50:50)5000-PEG2000-RAP and PLGA(50:50)5000-PEG2000-RGD were prepared in the same manner as in Example 1.

[0116] Weigh 10 mg of PLGA (50:50) 5000-PEG2000-RAP, 10 mg of PLGA (50:50) 5000-PEG2000-RGD, and 10 mg of curcumin into 2 mL EP tubes, and add 1 mL of DMSO to each tube to dissolve them and prepare stock solutions. Mix 120 μL of PLGA (50:50) 5000-PEG2000-RAP, 80 μL of PLGA (50:50) 5000-PEG2000-RGD, and 10 μL of curcumin stock solution thoroughly, add 4 mL of ultrapure water, and sonicate using a cell disruptor at 100 W for 5 min under ice bath conditions, pausing for 5 seconds every 5 seconds. Centrifuge at 6000 rpm for 30 min at 4 °C to remove unencapsulated curcumin, then place in an ultrafiltration tube for ultrafiltration purification to remove DMSO, yielding cur@RRNP.

[0117] The following experiments were conducted using the raw materials and nanoparticles from Example 1 above:

[0118] (a) Time-of-flight mass spectrometry results

[0119] The M / Z ratio of the raw material PLGA(50:50)5000-PEG2000-MAL was examined on a time-of-flight instrument as a reference for the subsequent synthesis of PLGA(50:50)5000-PEG2000-RAP and PLGA(50:50)5000-PEG2000-RGD products, in order to verify whether the two were successfully synthesized.

[0120] The evaluation method is as follows: the raw material or synthetic product is dispersed in ultrapure water, dropped onto a sample plate and dried, and then a sample matrix (15 mg / mL gallic acid dissolved in acetonitrile solution containing 1‰ trifluoroacetic acid, wherein the volume of acetonitrile:water is 1:1) is added, dried, and then measured using a matrix-assisted laser desorption / ionization time-of-flight mass spectrometer (MALDI-TOF).

[0121] The determination results of raw material PLGA (50:50) 5000-PEG2000-MAL are shown below. Figure 1 As shown. Figure 2 and Figure 3 The results are for different synthetic products (PLGA(50:50)5000-PEG2000-RAP and PLGA(50:50)5000-PEG2000-RGD).

[0122] Combination Figure 1 and Figure 2It can be seen that, compared with the raw material PLGA(50:50)5000-PEG2000-MAL, the M / Z result of PLGA(50:50)5000-PEG2000-RAP is significantly improved, which verifies the successful synthesis of PLGA(50:50)5000-PEG2000-RAP.

[0123] Combination Figure 1 and Figure 3 It can be seen that, compared with the raw material PLGA(50:50)5000-PEG2000-MAL, the M / Z result of PLGA(50:50)5000-PEG2000-RGD is significantly improved, which verifies the successful synthesis of PLGA(50:50)5000-PEG2000-RGD.

[0124] (ii) Nanoparticle size

[0125] The particle size distribution of the cur@RRNP nanoparticles was examined to evaluate their dispersion performance.

[0126] The evaluation method was as follows: the synthesized nanoparticles were measured using a Brookhaven 90Plus PALS nanoparticle size analyzer and observed using a transmission electron microscope. The equipment used was a Hitachi H-600 transmission electron microscope with a magnification of 10 million times.

[0127] See test results Figure 4 As shown.

[0128] Depend on Figure 4 It is evident that the nanoparticles cur@RRNP have a uniform particle size distribution, an appropriate particle size, and a microstructure that shows well-dispersed and uniform spherical nanoparticles.

[0129] (III) Stability of nanoparticles

[0130] The stability of RRNP nanoparticles and cur@RRNP was investigated to evaluate their ability to deliver drugs in the physiological environment in vivo.

[0131] The testing method is as follows: nanoparticles are dispersed in 5% glucose, and their particle size is measured at specific time points.

[0132] See test results Figure 5 and Figure 6 As shown.

[0133] Depend on Figure 5 As can be seen, the particle size and PDI of the RRNP nanoparticles did not change significantly within 72 h, demonstrating the stability of the RRNP nanoparticles in 5% glucose. Figure 6As can be seen, the particle size and PDI of the cur@RRNP nanoparticles did not change significantly within 72 h, proving that the cur@RRNP nanoparticles have good stability in 5% glucose.

[0134] (III) Cell targeting

[0135] Using nanoparticles NP as a control, the uptake capacity of nanoparticles RRNP on the cell line bEnd.3-RAGE, which highly expresses RAGE, was investigated to evaluate its targeting ability to RAGE lesions.

[0136] Nanoparticles (NPs): Weigh 10 mg of PLGA (50:50) 5000-mPEG5000 into a 2 mL EP tube, add 1 mL of DMSO, and dissolve to prepare a stock solution. Mix 200 μL of PLGA (50:50) 5000-mPEG5000 thoroughly, add to 4 mL of ultrapure water, vortex to mix, and then sonicate using a cell disruptor at 100 W for 5 min under ice bath conditions, pausing for 5 seconds every 5 seconds. Purify by ultrafiltration at 4 °C to remove DMSO, yielding nanoparticles (NPs).

[0137] The evaluation method was as follows: luciferin cou6 was used to modify unmodified nanoparticles NP and RRNP, respectively, to obtain cou6@NP and cou6@RRNP nanoparticles. After incubating the nanoparticles in bEnd.3-RAGE cells for a period of time, the uptake of the nanoparticles was examined by flow cytometry.

[0138] See test results Figure 7 As shown.

[0139] Depend on Figure 7 It is evident that, compared to the control nanoparticle NP, the cell line bEnd.3-RAGE with high RAGE expression exhibits better cellular uptake of nanoparticle RRNP, indicating that nanoparticle RRNP has better lesion targeting ability.

[0140] (iv) Cell affinity

[0141] Using nanoparticles NP as a control, the affinity of nanoparticles RRNP for the cell line bEnd.3-RAGE, which highly expresses RAGE, was investigated to evaluate its targeting ability for RAGE lesions.

[0142] The evaluation method was as follows: PLGA nanoparticles NP and RRNP without target modification were modified with fluorescein cou6 to obtain cou6@NP and cou6@RRNP nanoparticles, respectively. The nanoparticles were incubated with bEnd.3-RAGE cells at 4℃ for 90 min, and the fluorescence signal was collected by flow cytometry. The linear fitting curves were fitted using the One-sitetotal mode.

[0143] See test results Figure 8 As shown.

[0144] Depend on Figure 8 It is evident that, compared to the control nanoparticle NP, the nanoparticle RRNP exhibits better cell affinity for the cell line bEnd.3-RAGE, which has high RAGE expression, thus further demonstrating that the nanoparticle RRNP has better lesion targeting ability.

[0145] (v) Lysosomal targeting ability

[0146] Using NP nanoparticles as a control, the targeting and enrichment efficiency of RRNP nanoparticles to lysosomes after being taken up by cells was investigated by lysosomal colocalization experiments, so as to evaluate its ability to achieve targeted delivery to lysosomes.

[0147] The evaluation method was as follows: PLGA nanoparticles without target modification, NP and RRNP nanoparticles, were modified with fluorescein cou6 to obtain cou6@NP and cou6@RRNP nanoparticles, respectively. These nanoparticles were then co-cultured with bEnd.3-RAGE cells for 6 hours. Subsequently, lysosomes were labeled with 50 nM LysoTracker Red DND-99 for 45 minutes, and the cells were fixed with 4% paraformaldehyde. Co-localization was analyzed using confocal laser scanning microscopy (CLSM).

[0148] See test results Figure 9 As shown. Figure 9 In the diagram, A shows the co-localization results of cou6@NP with lysosomes, B shows the co-localization curve of cou6@NP with lysosomes, C shows the co-localization results of cou6@RRNP with lysosomes, and D shows the co-localization curve of cou6@RRNP with lysosomes.

[0149] Depend on Figure 9 It is evident that, compared to the control nanoparticle NP, the nanoparticle RRNP exhibits better co-localization with lysosomes.

[0150] This indicates that RRNP nanoparticles can be delivered to lysosomes more effectively. This result demonstrates that RRNP nanoparticles have good lysosomal targeted delivery capabilities, providing important subcellular distribution evidence for further enhancing the degradation of RAGE proteins through the lysosomal pathway.

[0151] (vi) Protein expression level

[0152] Using the Ctrl group as a control, the expression level of RAGE protein in cells treated with different concentrations of nanoparticle RRNP was detected by Western blot to evaluate the regulatory ability of nanoparticle RRNP on the key target protein RAGE under pathological conditions.

[0153] The other nanoparticles were diluted in complete culture medium and incubated with cells. The Ctrl group was directly added with complete culture medium, which was DMEM medium containing 10% fetal bovine serum and 1% penicillin antibiotics.

[0154] The evaluation method was as follows: different concentrations of RRNP nanoparticles were prepared, and different concentrations of RRNP nanoparticles were co-incubated with bEnd.3-RAGE cells (a constructed cell line with high RAGE expression) for 24 h. The total protein was then extracted, and the expression level of the target protein RGAE-FLAG was examined using Western blot technology.

[0155] See test results Figure 10 As shown.

[0156] Depend on Figure 10 As can be seen, after incubating bEnd.3-RAGE cells with different concentrations of RRNP nanoparticles, the expression level of the target protein RAGE-FLAG was reduced compared to the Ctrl group, demonstrating that RRNP nanoparticles have the effect of reducing the expression of pathological RAGE proteins. Combined with the aforementioned experimental results on "lysosomal targeting ability," this demonstrates that RRNP nanoparticles have the ability to move from "organelle targeting" to "final protein degradation," achieving their efficacy of "targeted delivery-regulated degradation."

[0157] Furthermore, it should be noted that in the experiments described in (iii) to (vi) above, only the nanoparticles RRNP were tested. The reason for this is that cellular uptake mainly depends on the functional group modification on the surface of the nanoparticles. Whether or not the nanoparticles are loaded with drugs has little effect on their intracellular behavior. Therefore, by demonstrating the lysosomal co-localization characteristic of the nanoparticles RRNP, it can be shown that RRNP has better lysosomal targeting ability and thus can also serve as a drug carrier to improve the localization of drugs at lysosomes.

[0158] (vii) Permeability assessment of in vitro blood-brain barrier model

[0159] This study aimed to evaluate the effects of different formulations (nanoparticle RRNP and cur@RRNP) on Aβ-induced pathological changes in blood-brain barrier permeability.

[0160] Experimental Methods: The integrity of an in vitro blood-brain barrier model was assessed by quantitatively measuring paracellular channel leakage of sodium fluorescein in an endothelial cell monolayer. Specifically, bEnd.3 cells were seeded at a density of 3000 cells per well in 3 μm polycarbonate membrane Transwell chambers. Transepithelial resistance (TEER) was monitored daily to assess barrier formation. Once TEER reached a stable plateau (indicating the formation of a mature, tightly packed BBB model), cells were exposed to 10 μM amyloid-β protein (Aβ) for 24 hours to induce barrier dysfunction. Following Aβ treatment, cells underwent an additional 24-hour experimental intervention. For permeability testing, HBSS was injected into the basal-side chamber, while the culture medium in the apical chamber was replaced with 0.2 mL of HBSS detection buffer containing 10 μg / mL sodium fluoride. After 30 minutes of incubation, basal-side HBSS was collected, diluted, and analyzed using a microplate reader at an excitation wavelength of 473 nm and an emission wavelength of 535 nm.

[0161] The experimental results are shown in Figure 11 As shown.

[0162] Depend on Figure 11 It is evident that, compared to the Aβ-damaged group, the sodium fluorescein permeability was significantly reduced in the model treated with nanoparticles RRNP and cur@RRNP, indicating that both nanoparticles RRNP and cur@RRNP can alleviate Aβ-induced blood-brain barrier leakage to varying degrees and have the effect of improving the integrity of the blood-brain barrier under pathological conditions.

[0163] (viii) In vitro assessment of blood-brain barrier function repair

[0164] This study evaluated the effects of different formulations (nanoparticle RRNP and cur@RRNP) on the repair of tight junction protein expression after Aβ injury.

[0165] Experimental methods: bEnd.3-RAGE cells were incubated with 10 μM Aβ for 24 h to induce ZO-1 damage. After incubation with the drug preparation for 24 h, the cells were fixed with 4% paraformaldehyde. The cells were then subjected to ZO-1 immunofluorescence staining, and finally the cell nuclei were stained with DAPI. After adding an anti-fluorescence quencher, the cells were mounted and imaged using a Leica inverted fluorescence microscope.

[0166] See the experimental results. Figure 12 As shown.

[0167] Depend on Figure 12 It is evident that, compared with the Aβ-damaged group, both the RRNP and cur@RRNP treatment groups showed varying degrees of ZO-1 restoration, indicating that both nanoparticles can effectively salvage the loss of tight junction proteins caused by Aβ, thus verifying their regulatory effect on pathological BBB.

[0168] (ix) MTT cell viability assay

[0169] This study evaluated the protective effects of different formulations (nanoparticle RRNP and cur@RRNP) against Aβ-induced cerebral vascular endothelial cell toxicity.

[0170] Experimental methods: bEnd.3-RAGE cells were incubated with the preparation for 24 h, and then incubated with 10 μM Aβ for 24 h to induce cell damage. 10 μL of 5 mg / mL MTT solution was added, and the cells were incubated in the dark for 4 h. The absorbance value at 490 nm was then scanned using an ELISA reader.

[0171] See the experimental results. Figure 13 As shown.

[0172] Depend on Figure 13 It is evident that, compared with the Aβ-damaged group, bEnd.3-RAGE cells treated with nanoparticles RRNP and cur@RRNP showed varying degrees of cell viability recovery, indicating that both nanoparticle formulations can effectively alleviate the toxic damage of Aβ to vascular endothelial cells and have a regulatory effect on pathological BBB.

[0173] (x) Enzyme-linked immunosorbent assay (ELISA)

[0174] This study evaluated the inhibitory effects of different formulations (nanoparticle RRNP and cur@RRNP) on Aβ-induced inflammatory responses in cerebral vascular endothelial cells.

[0175] Experimental methods: bEnd.3-RAGE cells were incubated with 10 μM Aβ for 24 h to induce cell damage. After incubating the cells with the preparation for another 24 h, the supernatant was collected, and the levels of IL-6 and IL-10 in the supernatant were measured by ELISA.

[0176] See the experimental results. Figure 14 , Figure 15 As shown.

[0177] Depend on Figure 14 and Figure 15 It is evident that, compared with the Aβ-damaged group, the supernatant of bEnd.3-RAGE cells treated with nanoparticles RRNP and cur@RRNP showed varying degrees of increased levels of anti-inflammatory factors IL-6 and IL-10, indicating that both nanoparticle formulations can effectively inhibit Aβ-triggered endothelial cell inflammatory responses and possess certain anti-inflammatory effects.

[0178] (xi) Detection experiment of reactive oxygen species (ROS)

[0179] This study evaluated the protective effects of different formulations (nanoparticle RRNP and cur@RRNP) against LPS-induced oxidative stress damage in microglia.

[0180] Experimental methods: BV2 cells were incubated with 1 μg / mL LPS for 24 h to induce cell damage. Then, different preparations were added and the cells were incubated for 24 h. Finally, DCFH-DA reactive oxygen species probe was added and the cells were incubated for 30 min. Flow cytometry was used to measure the ROS signal of BV2 cells.

[0181] See the experimental results. Figure 16 As shown.

[0182] Depend on Figure 16 It is evident that, compared with the LPS-damaged group, the reactive oxygen species (ROS) fluorescence signal intensity of BV2 cells treated with nanoparticles RRNP and cur@RRNP was reduced to varying degrees, indicating that both nanoparticle formulations can alleviate LPS-induced oxidative stress in microglia and have a certain inhibitory activity against inflammation-related oxidative damage.

[0183] (xii) In vivo targeting verification experiment in mouse model

[0184] To systematically evaluate the targeted delivery efficiency and mechanism of RRNP nanoparticles in an Alzheimer's disease model, the following experiments were conducted using AD model mice:

[0185] In vivo dynamic imaging experiment: DiD@RRNP nanoparticles were obtained by labeling RRNP nanoparticles with DiD. Small animal in vivo imaging was used to monitor the signals of DiD@RRNP nanoparticles in the brains of healthy mice and AD model mice at different time points after drug administration.

[0186] In vitro brain tissue imaging experiment: DiD-labeled RRNP nanoparticles were obtained to obtain DiD@RRNP nanoparticles. After administration for 4 h, mice were dissected and perfused. In vitro brain tissue of mice was taken and fixed in paraformaldehyde for 2 h. Fluorescence signals in the brains of healthy mice and model AD mice were monitored using small animal in vivo imaging pairs.

[0187] Immunofluorescence colocalization analysis of brain slices: DiD-labeled RRNP nanoparticles were used to obtain DiD@RRNP nanoparticles. Mice were dissected 4 h after drug administration. After perfusion, isolated brain tissue from mice was collected and fixed in paraformaldehyde for 2 h. The brain tissue was then dehydrated with 15% and 30% sucrose solutions, respectively, and then frozen sectioned. After frozen sectioning, the brain slices were stained with RAGE antibody and imaged using Leica inverted fluorescence microscopy. Image J was used to analyze the colocalization coefficients.

[0188] See the experimental results. Figures 17 to 19As shown.

[0189] Figure 17 This study was used to verify the dynamic distribution of DiD@RRNP in the brains of in vivo AD model mice. Figure 17 It is evident that, compared with healthy mice, DiD@RRNP exhibits a stronger signal distribution in the pathological brain of AD model mice, thus verifying the targeting ability of DiD@RRNP for lesions.

[0190] Figure 18 This study was used to verify the static distribution intensity of DiD@RRNP in the isolated AD model mouse brain. Figure 18 It is evident that, compared with healthy mice, DiD@RRNP exhibits a stronger signal distribution in the pathological brain of AD model mice, thus verifying the targeting ability of DiD@RRNP for lesions.

[0191] Figure 19 Spatial localization of DiD@RRNP cell / tissue distribution in the brain of AD model mice relative to RAGE receptor expression. Figure 19 It is evident that, compared with healthy mice, DiD@RRNP exhibits stronger signal distribution in the pathological brain of AD model mice and stronger co-localization with RAGE, indicating that DiD@RRNP can achieve lesion targeting through higher affinity for RAGE.

[0192] Figure 19 In the diagram, A represents a healthy mouse and B represents an AD model mouse. In A and B, (1) is a single-channel diagram of DAPI; (2) is a single-channel diagram of RAGE immunofluorescence staining; (3) is a single-channel diagram of DID; (4) is a three-channel combined diagram; and (5) is a Pearson colocation coefficient diagram.

[0193] (xiii) Behavioral Experiments

[0194] To systematically evaluate the improvement of cognitive function in AD model mice after treatment with different formulations (nanoparticle RRNP and cur@RRNP), the following experiments were conducted to comprehensively evaluate the treatment's effect on the recovery of cognitive impairment.

[0195] Nesting behavior experiment: The nesting behavior of mice was studied after treatment with different formulations. After mice were acclimatized to corn cob bedding for 24 hours, fifteen sheets of paper towels were neatly laid out in a fixed position in the cage. After a 6-hour observation period, the condition of the paper towels was photographed and examined to assess the degree of tearing and the nesting area. Subsequently, a standardized scoring system of 0 to 5 points was used to score the nesting ability of the mice.

[0196] Behavioral water maze experiment: A circular water maze was divided into four quadrants. One quadrant was selected as the target quadrant, and a circular platform was placed there. Tap water was added to the platform, and a navigation training experiment was conducted for the first four days. Mice were gently placed facing the pool wall in the middle of each training quadrant and allowed to swim freely for 60 seconds. If the mouse independently found the platform and stayed there for 10 seconds, the system automatically stopped monitoring. If it failed to find the platform, it needed to be manually guided to the platform and stay there for 10 seconds. After four days of training, a spatial exploration experiment was conducted. The platform in the target quadrant was removed, and the mice were allowed to swim freely for 60 seconds. The number of times the mouse crossed the original platform, the cumulative time the mouse stayed at the original platform location, and the latency period when the mouse first found the original platform location were recorded.

[0197] Behavioral Y-maze Experiment: The Y-maze test was used to assess the spatial exploration and memory abilities of mice after treatment. The maze consisted of three identical arms (each approximately 13 cm long, 6.5 cm wide, and 6 cm high), arranged in a "Y" shape. The three arms were connected at the central point, forming a 120° angle between each arm. Mice were released to the fixed arms and allowed to explore freely for 5 minutes. The order in which mice entered and left each arm of the maze within 5 minutes was recorded and analyzed using the EthoVision XT system. The spontaneous alternation rate for each mouse was calculated using the following formula: Spontaneous alternation rate = (Actual alternations / Theoretical maximum alternations) × 100.

[0198] See the experimental results. Figures 20 to 24 As shown.

[0199] Figure 20 Used to validate executive function and daily living abilities in mice. Figure 20 It is evident that mice treated with the nanoparticle cur@RRNP showed the highest nesting scores, indicating that this nanoparticle formulation can effectively restore executive function and daily activities in AD model mice.

[0200] Figures 21 to 23 Used to verify the spatial learning and memory abilities of mice. Figure 21 It is evident that the number of times mice treated with the cur@RRNP nanoparticles crossed the platform was significantly increased compared to the AD model group, indicating that this nanoparticle formulation can effectively restore the spatial memory retention ability of AD model mice; Figure 22 It is evident that the cumulative time spent at the platform spatial location in mice treated with cur@RRNP was significantly increased compared to the AD model group, indicating that this nano-formulation can effectively restore the accuracy of spatial memory in AD model mice; Figure 23It is evident that the latency period for mice to first locate the platform after treatment with the nanoparticle cur@RRNP was significantly shortened compared to the AD model group, indicating that this nanoparticle formulation can effectively restore the speed of spatial learning and memory retrieval in AD model mice.

[0201] Figure 24 This was used to verify spatial working memory and exploratory willingness in mice. Figure 24 It is evident that mice treated with the nanoparticle cur@RRNP showed a significant increase in their autonomous alternation rate in the Y maze, demonstrating that this nanoparticle formulation can effectively restore the impaired memory and spatial exploration abilities of AD model mice.

[0202] (xiv) In vivo verification tests of pathological histology and function

[0203] To systematically evaluate the effects of different formulations (nanoparticle RRNP and cur@RRNP) on the improvement of brain pathology, blood-brain barrier function and cerebral blood flow in AD model mice, the intervention effects of the treatment on AD-related pathological processes were comprehensively evaluated through the following experiments.

[0204] Immunofluorescence detection of Aβ plaques in brain slices: Brains of mice after treatment were taken, frozen sections were prepared, and primary and secondary antibodies were incubated sequentially with immunofluorescence staining. Finally, imaging was performed using a fluorescence microscope.

[0205] Evans blue leakage assay: 5% (w / v) Evans blue solution was prepared in PBS and injected intraperitoneally at a dose of 10 μL per 10 g body weight. After a 3-hour circulation period to promote dye distribution, mice were sacrificed and perfused with 20 mL of PBS to completely remove the dye from the blood vessels. Intact brain tissue was then harvested for photographing to qualitatively assess dye extravasation. The right hemisphere of each brain tissue was then separated and homogenized in 50% (w / v) TCA solution at a ratio of 30 μL per 10 mg of tissue. The homogenate was centrifuged at 12,000 rpm for 20 minutes to precipitate cell debris, and the supernatant was carefully collected. 100 μL of the supernatant was aliquoted into 96-well plates, and the absorbance was measured at 610 nm and 620 nm using a microplate reader to quantitatively determine the amount of Evans blue extravasation.

[0206] Two-photon in vivo imaging for vascular permeability detection: To detect blood-brain barrier permeability, mice were anesthetized with tribromoethanol (20 μL / 10 g), fixed in a stereotactic frame, and placed under a Nikon A1plus confocal microscope equipped with a Plan Apo 4x objective (numerical aperture: 0.2, refractive index: 1.000). Laser scanning confocal imaging was performed using a resonant scanner and a DU4 gallium arsenide phosphide detector. The dye mixture contained a dye mixture of 20 mg / mL FITC-labeled dextran (molecular weight 500 kDa, MKBio, China) (20 mg / mL in PBS) and 10 mg / mL TRITC-labeled dextran (molecular weight 40 kDa, MKBio, China) dissolved in PBS and injected via the tail vein. In vivo image sequences were acquired 20 minutes after injection. Two independent imaging planes were used: one with FITC excitation wavelength of 488.0 nm, laser power of 7.0%, PMT voltage of 75 V, and aperture of 226.05 μm; the other was imaged using an Alexa Fluor 594 (Alx594) with excitation at 561.0 nm, laser power of 18.0%, PMT voltage of 75 V, and the same aperture size. Imaging parameters included scanner zoom of 2,000, scan speed of 15, unidirectional scan direction, and no averaging or integration processing. To ensure comprehensive data acquisition, a time loop consisting of 271 equally spaced frames was executed within a 10,000 ms period.

[0207] Immunofluorescence detection of tight junction protein in brain slices: To detect the expression of tight junction protein in the blood-brain barrier, brain tissue was taken from treated mice, fixed and dehydrated, and then frozen sections were prepared. The sections were stained with primary and secondary antibodies, and then stained with DAPI and imaged using inverted fluorescence microscopy.

[0208] Laser speckle contrast imaging for cerebral blood flow perfusion assessment: Mice were anesthetized with tribromoethanol and fixed in a stereotactic apparatus (RWD Life Sciences, China). A fine incision was made along the midline of the mouse scalp to expose the skull, which was then flushed with saline to ensure a clear view. Cerebral blood flow was assessed using laser speckle contrast imaging (RWD Life Sciences, China).

[0209] See the experimental results. Figures 25 to 30 As shown.

[0210] Figure 25 This study aimed to verify the effect of nano-formulations on clearing or reducing Aβ pathological deposits in the brains of AD model mice. Figure 25 It is evident that the deposition of Aβ plaques in the brains of mice treated with cur@RRNP was significantly reduced, thus demonstrating that the nano-formulation has the effect of reducing Aβ deposition and thereby exerting a therapeutic effect.

[0211] Figure 26 and Figure 27 This study was used to verify the restorative effect of nano-formulation on the blood-brain barrier integrity in AD model mice (BBB permeability was reflected by detecting the degree of extravascular dye leakage). As shown in the figure, the leakage of cerebral blood vessels in mice treated with cur@RRNP was reduced (see [link to data on visual leakage range]). Figure 26 In the diagram, A is the top view of the brain tissue, and B is the bottom view; for the amount of Evans blue exudate in the brain tissue, please refer to [reference needed]. Figure 27 This demonstrates that the nano-formulation can effectively restore the damaged BBB in AD model mice.

[0212] Figure 28 This study aimed to verify the real-time ameliorative effect of nano-formulations on cerebral vascular permeability in AD model mice under in vivo conditions (dynamically reflecting BBB integrity through extravasation of fluorescent dextran of different molecular weights). (From...) Figure 28 It is evident that dye leakage in the cerebral blood vessels of mice treated with cur@RRNP was reduced, demonstrating that this nano-formulation can effectively restore the damaged BBB in AD model mice.

[0213] Figure 29 This study aimed to validate the repair effect of nano-formulations on the tight junction structure of cerebral vascular endothelium in AD model mice (by detecting the expression and distribution of tight junction proteins to reflect the integrity of the BBB structure). (The study was conducted using nano-formulations.) Figure 29 It is evident that the expression of tight junction protein in the cerebral blood vessels of mice treated with cur@RRNP was restored, demonstrating that this nano-formulation can effectively restore the damaged BBB in AD model mice.

[0214] Figure 30 This study was used to verify the effect of nano-formulation on improving cerebral blood flow perfusion in AD model mice. Figure 30 It is evident that cerebral blood flow was restored in mice treated with cur@RRNP, demonstrating that this nano-formulation can effectively restore the function of damaged cerebral blood vessels in AD model mice.

[0215] (XV) Detection of neuroinflammatory factors

[0216] To evaluate the improvement of neuroinflammation levels in the brains of AD model mice after treatment with different nanoparticles (RRNP nanoparticles and cur@RRNP), the following experiments were conducted to verify the inhibitory effect of the nanoparticles on AD-related neuroinflammation.

[0217] TNF-α level detection in brain tissue: Total protein was extracted from mouse brains, and the level of the inflammatory factor TNF-α in the brain was measured by ELISA.

[0218] IFN-γ level detection in brain tissue: Total protein was extracted from mouse brains, and the level of the inflammatory factor IFN-γ in the brain was measured by ELISA.

[0219] Detection of NF-κB protein expression in brain tissue: Total protein was extracted from mouse brains, and the level of the inflammatory factor NF-κB in the brain was measured by Western blotting.

[0220] See the experimental results. Figures 31 to 33 As shown.

[0221] Depend on Figure 31 It is evident that the level of the inflammatory factor TNF-α in the brain tissue of mice treated with cur@RRNP decreased significantly, indicating that this nano-formulation can effectively alleviate oxidative stress in the mouse brain.

[0222] Depend on Figure 32 It is evident that the level of the inflammatory factor IFN-γ in the brain tissue of mice treated with cur@RRNP decreased significantly, indicating that this nano-formulation can effectively alleviate oxidative stress in the mouse brain.

[0223] Depend on Figure 33 It is evident that the level of the inflammatory factor NF-κB in the brain tissue of mice treated with cur@RRNP decreased significantly, indicating that this nano-formulation can effectively alleviate oxidative stress in the mouse brain by inhibiting the NF-κB pathway.

[0224] (xvi) Biomarker detection experiment

[0225] To evaluate the improvement of neural plasticity and synaptic integrity in the brains of AD model mice after treatment with different nanoparticles (RRNP nanoparticles and cur@RRNP), the following experiments were conducted to verify the promoting effects of the nanoparticle treatment on the survival of AD-related neurons, synaptic regeneration, and neural network repair.

[0226] BDNF protein expression in brain tissue: Total protein was extracted from mouse brains, and the level of BDNF in the brain was measured by Western blotting.

[0227] Brain tissue synaptophysin immunofluorescence assay: Brain tissue was collected from treated mice, fixed and dehydrated, and then frozen sections were prepared. The sections were stained with primary and secondary antibodies, and then stained with DAPI. Imaging was performed using inverted fluorescence microscopy.

[0228] See the experimental results. Figure 34 and Figure 35 As shown.

[0229] Figure 34 This study aimed to verify the effect of nanoformulations on the expression level of brain-derived neurotrophic factor (BDNF) in the brains of AD model mice. Figure 34It is evident that the expression level of BDNF protein in the brain tissue of mice treated with cur@RRNP was restored, demonstrating that this nanoformulation can help support the survival of existing neurons and promote the growth, regeneration, and creation of new neurons and synapses by increasing the level of brain-derived neurotrophic factor.

[0230] Figure 35 This study aimed to verify the effects of nanoformulations on synaptic density and integrity in the hippocampus of AD model mice. Figure 35 It is evident that the expression level of synaptophysin SYN protein in the hippocampus of mice treated with cur@RRNP was restored, demonstrating that this nanoformulation can promote the growth, regeneration, and creation of neurons and synapses.

[0231] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications or equivalent changes made to the above embodiments based on the technical essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. A pathological blood-brain barrier-targeting nano-formulation for the treatment of Alzheimer's disease, characterized in that: The nano-formulation contains at least the following components: (1) Carrier nanoparticles assembled from amphiphilic block copolymers, wherein the amphiphilic block copolymers comprise hydrophobic blocks and hydrophilic blocks; (2) A targeting ligand modified on the surface of the carrier nanoparticles, wherein the targeting ligand is a RAP peptide with the amino acid sequence CELKVLMEKEL; (3) A functional ligand modified on the surface of the carrier nanoparticles, wherein the functional ligand is an RGD peptide with the amino acid sequence c(RGDfK).

2. The pathological blood-brain barrier-targeting nano-formulation for treating Alzheimer's disease according to claim 1, characterized in that: The nanoformulation also includes a hydrophobic drug loaded in the carrier nanoparticles.

3. The pathological blood-brain barrier targeted nano-formulation for treating Alzheimer's disease according to claim 2, characterized in that: The hydrophobic drug is selected from at least one of curcumin, resveratrol, pioglitazone, and simvastatin.

4. The pathological blood-brain barrier targeted nano-formulation for treating Alzheimer's disease according to claim 1, characterized in that: The hydrophobic block is a polylactic acid-glycolic acid copolymer with a molecular weight of 2000-20000; the hydrophilic block is polyethylene glycol with a molecular weight of 500-5000.

5. A pathological blood-brain barrier-targeting nano-formulation for treating Alzheimer's disease according to claim 4, characterized in that: In the amphiphilic block copolymer, the block ratio of polylactic acid-glycolic acid copolymer to polyethylene glycol is 50:50 to 10:

90.

6. The pathological blood-brain barrier targeted nano-formulation for treating Alzheimer's disease according to claim 1, characterized in that: The hydrophilic block is connected to a reactive functional group at its end, and the targeting ligand or functional ligand is covalently modified on the surface of the carrier nanoparticle through the reactive functional group.

7. A pathological blood-brain barrier-targeting nano-formulation for treating Alzheimer's disease according to claim 6, characterized in that: The reactive functional group is selected from maleimide, carboxyl, amino, and azide groups.

8. A pathological blood-brain barrier-targeting nano-formulation for treating Alzheimer's disease according to claim 7, characterized in that: The terminal of the functional ligand contains a thiol, amino, carboxyl, or alkynyl group covalently linked to the reactive functional group.

9. The method for preparing a pathological blood-brain barrier-targeting nanoparticle formulation for treating Alzheimer's disease as described in any one of claims 1 to 8, characterized in that: Includes the following steps: S1. The amphiphilic block copolymer and the targeting ligand are dissolved in organic solvents to obtain polymer solution and targeting ligand solution respectively. Under inert gas protection, the polymer solution and the targeting ligand solution are mixed to carry out covalent linkage reaction to obtain the first reaction mixture. After purification and freeze drying, a polymer carrier material with the targeting ligand modified on the surface is obtained. S2. The amphiphilic block copolymer and the functional ligand are dissolved in organic solvents to obtain polymer solution and functional ligand solution respectively. Under inert gas protection, the polymer solution and functional ligand solution are mixed to carry out covalent linkage reaction to obtain a second reaction mixture. After purification and freeze drying, a polymer carrier material with functional ligand modified on the surface is obtained. S3. Disperse the above two polymer carrier materials in a solvent, or disperse the above two polymer carrier materials and hydrophobic drugs in a solvent, and assemble them into carrier nanoparticles by ultrasonic treatment. Remove the unencapsulated hydrophobic drugs, and after purification, obtain the pathological blood-brain barrier targeted nano-formulation.

10. The preparation method according to claim 9, characterized in that: The molar ratio of the amphiphilic block copolymer to the targeting ligand is 1:1.1 to 1:1.9; the molar ratio of the amphiphilic block copolymer to the functional ligand is 1:1.1 to 1:1.

9.

11. The preparation method according to claim 9, characterized in that: The covalent bonding reaction was carried out in a water bath at 25–55°C for 12–48 hours.

12. The preparation method according to claim 9, characterized in that: In the solvent, the molar ratio of polymer carrier material with surface-modified targeting ligands to polymer carrier material with surface-modified functional ligands is 1:9 to 9:

1.

13. The preparation method according to claim 9, characterized in that: In the solvent, the mass ratio of the polymer carrier material with surface-modified targeting ligands, the polymer carrier material with surface-modified functional ligands, and the hydrophobic drug is 1:9:10 to 9:1:

1.

14. The preparation method according to claim 9, characterized in that: The ultrasonic treatment is performed at a power of 50-200W for 1-10 minutes, with a pause of 1-10 seconds after each 1-10 seconds of ultrasonic treatment.

15. The preparation method according to claim 9, characterized in that: The organic solvent is selected from at least one of N,N-dimethylformamide, dimethyl sulfoxide, methanol, tetrahydrofuran, and acetonitrile.

16. The preparation method according to claim 9, characterized in that: The solvent is selected from at least one of dimethyl sulfoxide, N,N-dimethylformyl, tetrahydrofuran, and acetonitrile.

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