Polypeptide-drug conjugate (PDC) nano prodrug with function of repairing pericerebral cells

Abstract

The invention belongs to the technical field of pharmaceutical preparations, relates to a PDC (Polycrystalline Diamond Compact) nano prodrug with a function of repairing pericerebral cells, and in particular relates to a polypeptide-drug conjugate (PDC) nano prodrug with a function of repairing pericerebral cells as well as a preparation method and application thereof. The PDC nano prodrug is formed by coupling a fusion peptide and a polyphenol drug through a phenylboronic acid ester bond and further self-assembling, and the PDC nano prodrug has the advantages that preparation is simple, only water bath ultrasonic treatment is needed, and production amplification is easy; the material-free co-loading of the polypeptide and the small molecule medicine can be realized, the potential toxicity of a carrier material is avoided, and meanwhile, the medicine loading capacity is greatly improved; precise delivery and response release of a focus part are realized, and a complex pathological mechanism of brain diseases is regulated and controlled in multiple links; a more economical and effective treatment mode can be provided for prevention and treatment of related brain diseases, and the application prospect is wide.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical formulation technology, and relates to PDC nanoprodrugs with the function of repairing brain pericytes, specifically to a polypeptide-drug conjugate (PDC) nanoprodrug with the function of repairing brain pericytes, its preparation method and application. Technical Background

[0002] Reports indicate that with an aging population, increasing life pressures, and worsening environmental pollution, the incidence of brain diseases is rising annually, seriously threatening human life and health. Currently, most brain diseases remain incurable, primarily due to the complexity of their causes and the limitations imposed by the blood-brain barrier (BBB) ​​on drug delivery to the brain. Therefore, efficiently delivering drugs into the central nervous system and effectively regulating complex pathogenic factors is a key focus of research in brain disease treatment.

[0003] Pericytes, also known as wall cells, are structural cells widely distributed throughout the walls of microvessels throughout the body. In the central nervous system, pericytes, along with vascular endothelial cells, the basement membrane, glial cells, and adjacent neurons, constitute the neurovascular unit. Within the neurovascular unit, pericytes occupy a strategic core position, primarily responsible for the construction and maintenance of the blood-brain barrier (BBB) ​​and the regulation of cerebral blood flow. Pericytes also participate in physiological processes such as angiogenesis and neurogenesis, neuroinflammatory responses, and waste removal. Therefore, healthy pericytes are crucial for maintaining central homeostasis. However, increasing evidence suggests that pericyte dysfunction is present in various brain diseases. These diseases include Alzheimer's disease (AD), multiple sclerosis (MS), Parkinson's disease (PD), stroke, traumatic brain injury (TBI), amyotrophic lateral sclerosis (ALS), and brain tumors. When pericytes malfunction or are lost, cerebral blood flow decreases, leading to insufficient brain energy supply. Simultaneously, the integrity of the blood-brain barrier (BBB) ​​is disrupted, allowing blood-borne toxic proteins and immune cells to infiltrate the brain, triggering neuroinflammation and causing neuronal damage. Furthermore, damaged pericytes generate large amounts of reactive oxygen species (ROS), causing oxidative stress, and secrete matrix metalloproteinase 9 (MMP9) to degrade tight junction proteins, further exacerbating BBB leakage and insufficient blood perfusion, creating a vicious cycle that continuously worsens the condition. Therefore, reducing pericyte loss and restoring its function is crucial for maintaining the normal physiological function of the central nervous system, and thus holds promise as a new direction for the treatment of pericyte-related brain diseases.

[0004] To address the dysfunction and loss of pericytes, researchers have attempted exogenous pericyte transplantation. While this therapy shows promising therapeutic potential, the scarcity and complex preparation of pericytes for transplantation result in significant economic and time costs. Furthermore, inherent problems such as non-replicability, epigenetic variation, genetic instability, and transplant rejection severely limit its application in the treatment of brain diseases. In contrast, targeted regulation of pericytes through medication to promote the generation of endogenous pericytes and alleviate pericyte damage represents a more economical and effective solution for the prevention and treatment of pericyte-related brain diseases.

[0005] Given the complexity of pericyte pathological damage, combined drug therapy to simultaneously regulate multiple pathological mechanisms is an effective strategy for improving pericyte dysfunction. MMP9 inhibitory peptides can block the pro-inflammatory signaling pathway CypA-MMP9 in pericytes, inhibiting MMP9 secretion and activity, thus promoting the repair of tight junctions in the blood-brain barrier (BBB) ​​and potentially maintaining BBB integrity. Polyphenolic drugs possess excellent antioxidant, anti-inflammatory, and autophagy-enhancing activities, and are expected to alleviate oxidative stress and neuroinflammation, promote the degradation of intracellular toxic proteins, help reshape the pericyte microenvironment, and enhance pericyte activity.

[0006] Because peptide drugs are highly hydrophilic, they have poor in vivo stability and are rapidly eliminated; while most polyphenol drugs are poorly soluble in water, and neither can easily cross the blood-brain barrier (BBB). Therefore, to overcome these drawbacks and ensure good synchronicity of the two drugs for better combined therapeutic effects, a suitable co-delivery system must be designed. However, how to efficiently co-load these two classes of drugs with significantly different physicochemical properties (traditional nanosystems typically have a drug loading capacity of less than 1% for peptides) and improve their targeting efficiency to diseased pericytes are key issues that urgently need to be addressed in constructing co-delivery systems. Nanoparticle prodrugs, which combine the advantages of both prodrugs and nanoformulations, have become a research hotspot in pharmaceutics in recent years. Among them, multifunctional nanoparticle prodrugs based on peptide-drug conjugates (PDCs) have attracted widespread attention. PDC nanoparticle prodrugs use peptides as the hydrophilic end and small molecule drugs as the hydrophobic end, coupled through breakable / unbreakable linkers. When the hydrophilic and hydrophobic ends reach an amphiphilic balance, PDCs can self-polymerize in aqueous solution to form nanostructures. PDC nanoprodrugs not only significantly increase the drug loading capacity of peptides and small molecule drugs, reducing the risk of premature drug leakage, but their nanostructure also helps enhance the in vivo stability of both drugs and prolong blood circulation time. Furthermore, the peptide terminus of PDC nanoprodrugs can be augmented with specific targeting groups, tunable domains, and stimulus-responsive peptide sequences or chemical bonds, endowing the nanoprodrug system with excellent targeted delivery and responsive drug release capabilities. Therefore, PDC nanoprodrugs provide an effective solution for the combined application of MMP9 inhibitory peptides and polyphenolic drugs.

[0007] Based on the current state of the technology, the inventors of this application intend to provide a polypeptide-drug conjugate (PDC) nanoprodrug with the function of repairing brain pericytes, its preparation method and application. Summary of the Invention

[0008] The purpose of this invention is to address the significant lack of research on targeted regulation of pericyte dysfunction to improve the treatment of brain diseases, given the current state of existing technologies. This invention provides a multifunctional PDC nanoprodrug based on MMP9 inhibitory peptides and polyphenolic drugs, designed to enhance the function of neurovascular units by repairing damaged pericytes, restoring central homeostasis, thereby delaying the progression of pericyte-related encephalopathy or improving prognostic efficacy.

[0009] This nanoprodrug incorporates a fusion peptide with both targeting and therapeutic functions, consisting of a cell adhesion molecule targeting peptide and a matrix metalloproteinase 9 (MMP-9) inhibitory peptide linked by a flexible linker. By screening the amino acid composition and sequence length of the flexible linker and optimizing the number of phenylboronic acid ester bonds, the PDC nanoprodrug can be endowed with excellent pericyte targeting performance, reactive oxygen species-responsive drug release performance, and synergistic enhancement in improving pericyte dysfunction. The PDC nanoprodrug is simple to prepare, requiring only water bath sonication, and is easily scaled up for production. It enables the co-loading of peptides and small molecule drugs without materialization, avoiding the potential toxicity of carrier materials while significantly increasing drug loading. It achieves precise delivery and responsive release at the lesion site, regulating the complex pathological mechanisms of brain diseases at multiple stages. Overcoming the serious drawbacks of existing pericyte transplantation therapies, it provides a more economical and effective treatment method for the prevention and treatment of pericyte-related brain diseases, demonstrating excellent clinical translational potential.

[0010] Specifically,

[0011] The PDC nanoprodrug of the present invention is formed by coupling a fusion peptide and a polyphenolic drug through a phenylboronic acid ester bond and then further self-assembling; wherein the fusion peptide is formed by linking a cell adhesion molecule targeting peptide and a matrix metalloproteinase 9 inhibitory peptide through a flexible linker.

[0012] More specifically,

[0013] The polypeptide terminus of the PDC of the present invention is composed of a multifunctional fusion peptide obtained by fusing a targeting peptide and a therapeutic peptide. Based on the fact that vascular endothelial cells and pericytes of damaged BBB highly express cell adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), the targeting peptide with high affinity for cell adhesion molecules is fused with an MMP9 inhibitory peptide. The resulting fusion peptide can not only endow the PDC nanoprodrug with the ability to penetrate the inflammatory BBB endothelium and concentrate in damaged pericytes, but also retain or enhance the pharmacological activity of the MMP9 inhibitory peptide.

[0014] The multifunctional fusion peptide of this invention, wherein the MMP9 inhibitory peptide is selected from COG133 peptide (Ac-LRVRLASHLRKLRKRLL-NH2), COG1410 peptide (Ac-AS-Aib-LRKL-Aib-KRLL-NH2), CN-105 peptide (Ac-VSRRR-NH2), Ac-hE18A-NH2 peptide (Ac-LRKLRKRLLR-DWLKAFYDKVAEKLKEAF-NH2), mR18L peptide (Ac-GFRRFLGSWARIYRAFVG-NH2), ApoE (141-155) peptide (Ac-LRKLRKRLLRDADDLLRKLRKRLLRDADDL), ApoEdp peptide (Ac-LRKLRKRLLLRKLRKRLL-NH2), RSH-12 peptide (MHPNAGHGS). One of the following: LMR, CTT peptide (CTTHWGFTLC), KAH peptide (KAHWGFTLD), M3 peptide (TF KEPVPDLC), CTF peptide (CTFKEIVPDLC), VPG peptide (VPGALAAA), and C6 peptide (KAHWGFTLD).

[0015] The multifunctional fusion peptide of the present invention comprises a cell adhesion molecule targeting peptide selected from one of the following: VH4 peptide (VH PK), VH7 peptide (VHPKQHR), VH13 peptide (VHPKQHRGGSKGC), VHS peptide (VHSPNKK), CNN peptide (CNNSKSHTC), CQI peptide (CQIDSPC), cLAB.L peptide (Pen-ITDGEATDSGC), NNQ peptide (NNQKIVNLKEKVAQLEA), GRG peptide (GRGEFRGRDNSVSVV), and FEG peptide (FEGFSFLAFEDFVSSI).

[0016] The multifunctional fusion peptide of this invention comprises a cell adhesion molecule-targeting peptide and an MMP9 inhibitory peptide linked by a flexible linker. The flexible linker is selected from one of the following: glycine (G)n, alanine (A)n, histidine (H)n, aspartic acid (D)n, threonine-aspartic acid (TD)n, and serine-aspartic acid (SD)n, where n represents the number of amino acid repeats, ranging from 1 to 8. The amino acid composition and length (i.e., the n value) of the flexible linker are crucial for maintaining the activity of both the cell adhesion molecule-targeting peptide and the MMP9 inhibitory peptide, as well as for the stability and safety of the PDC nanoparticle prodrug. This invention emphasizes that linker selection is indispensable in the construction of the fusion peptide.

[0017] The multifunctional fusion peptide described in this invention is synthesized via a solid-phase peptide synthesis method, wherein the cell adhesion molecule targeting peptide is at the N-terminus and the MMP9 inhibitory peptide is at the C-terminus (the direction of peptide synthesis is from the N-terminus to the C-terminus). Through rational design, the multifunctional fusion peptide exhibits stronger stability, brain targeting, and pharmacological activity than the original targeting peptide and therapeutic peptide.

[0018] The PDC nanoprodrug of this invention comprises a polyphenolic drug selected from one of the following: quercetin, rutin, curcumin, catechin, epigallocatechin gallate, epigallocatechin gallate (EGCG), gallic acid, caffeic acid, ellagic acid, cyanidin, α-naphthoquinone, γ-naphthoquinone, proanthocyanidin B1, proanthocyanidin B3, hesperidin, naringenin, luteolin, apigenin, oleuropein, hydroxytyrosol, chlorogenic acid, punicin, nordihydroguaiac acid, baicalin, vitexin, and hesperidin. Since the polyphenolic drug constitutes the hydrophobic end of the PDC, in order to self-assemble into nanoparticles with the hydrophobic core, the octanol-water partition coefficient (Log P) of the polyphenolic drug needs to be greater than 1.5.

[0019] The PDC nanoprodrug of this invention comprises a fusion peptide linked to a polyphenolic drug via a ROS-sensitive chemical bond. Based on the pathological characteristic of ROS-rich pericytes and their microenvironment, selecting a ROS-sensitive chemical bond as the linker between the fusion peptide and the polyphenolic drug facilitates the rapid release of the PDC nanoprodrug in response to high concentrations of ROS both inside and outside the diseased pericytes, thereby simultaneously regulating multiple pathological mechanisms and enhancing the combined therapeutic effect of the two drugs.

[0020] The PDC nanoprodrug of this invention contains a ROS-sensitive chemical bond that is a phenylboronic acid ester bond. The introduction method is as follows: the carboxyl group of 4-carboxyphenylboronic acid reacts with the primary amino group of the lysine and / or arginine side chain in the fusion peptide sequence to form an amide bond, thereby introducing the phenylboronic acid group onto the fusion peptide; the phenylboronic acid group further reacts with the active hydroxyl group of the polyphenol drug to form a phenylboronic acid ester bond.

[0021] The PDC nanoprodrug of this invention, wherein the number of phenylboronic acid ester bonds introduced into the fusion peptide varies depending on the number of lysine and / or arginine contained in the fusion peptide sequence. It should be emphasized that the number of phenylboronic acid ester bonds directly affects the ROS responsiveness, drug loading capacity, and stability of the PDC nanoprodrug, and is optimized to 1–10.

[0022] The PDC nanoprodrug of this invention is prepared by dissolving a fusion peptide with a side chain modified with phenylboronic acid groups in an aqueous phase, dissolving a polyphenolic drug in an organic phase, vortexing the two phases for 30 s to 2 min, and then sonicating in a water bath (temperature: 15℃ to 25℃, power: 50W to 120W, time: 5 to 60 min) to obtain the PDC nanoprodrug. The organic phase solvent is selected from ethanol, dimethyl sulfoxide, and N,N-dimethylformamide; the aqueous phase solvent is selected from deionized water, PBS buffer, and Tris buffer; the volume ratio of the organic phase to the aqueous phase is 1:5 to 1:20 (v / v).

[0023] The PDC nanoprodrug prepared in this invention has a particle size of 80–300 nm, a polydispersity index (PDI) of 0.103–0.257, a zeta potential of -11.43–+30.97 mV, a drug loading of 23.33%–42.57% for the MMP9 inhibitory peptide, and a drug loading of 10.63%–49.23% for the polyphenolic drug. The nanoprodrug is stable in PBS for at least two weeks and exhibits good ROS-responsive drug release capability. At the tested concentration, it shows no cytotoxicity (cell viability >80%) or hemolysis (hemolysis rate <5%).

[0024] The PDC nanoprodrug prepared in this invention, according to pharmacokinetic studies in rats, has a blood half-life (t... 1 / 2 Compared with polyphenol drugs alone, the clearance rate (CL) was significantly reduced, indicating that the preparation of nano-prodrugs can slow down clearance and prolong the drug circulation time in the blood.

[0025] The PDC nanoprodrug described in this invention can be administered via intravenous injection, subcutaneous injection, intramuscular injection, or nasal administration. After intravenous and nasal administration, the nanoprodrug rapidly enters the brain, reaching peak concentration in approximately 15 minutes to 1 hour. After subcutaneous and intramuscular injection, the time to peak concentration in the brain is delayed, but the drug's residence time in the brain is prolonged.

[0026] After administration of the PDC nanoprodrug prepared in this invention to mice, immunofluorescence staining of brain slices revealed that the distribution of the nanoprodrug in the lesion brain region of the brain disease model mice was significantly greater than that in the normal mouse brain, and it was mainly concentrated in the endothelial cells and pericytes of the damaged BBB, laying the foundation for its role in regulating pericyte function.

[0027] The PDC nanoprodrug prepared in this invention can effectively inhibit the CypA-MMP9 pathway in pericytes, reduce MMP9 activity, alleviate ROS and inflammatory factor levels, promote the excretion and clearance of waste by pericytes, and demonstrate a good effect on repairing damaged pericytes.

[0028] The PDC nanoprodrug prepared in this invention, when administered to model mice or transgenic mice, can promote pericyte regeneration, restore pericyte coverage and cerebral blood flow in brain blood vessels, and maintain the integrity of BBB structure and function. At the same time, it can reduce oxidative stress and neuroinflammation, inhibit the excessive activation of microglia and astrocytes, achieve a benign cycle of neurovascular unit homeostasis, thereby protecting neurons and effectively alleviating the progression of pericyte-related brain diseases.

[0029] The beneficial effects of this invention are mainly reflected in:

[0030] (1) By using a fusion peptide with both targeting and therapeutic functions as the peptide terminus of PDC nanoprodrugs, the limitation of the peptide terminus in the traditional PDC model having only targeting function is overcome. This allows PDC nanoprodrugs to be used in combination therapy for diseases with complex etiologies, expanding their application scope. Furthermore, the designed multifunctional fusion peptide exhibits stronger stability, brain targeting, and pharmacological activity than the original targeting peptide and therapeutic peptide, enhancing the feasibility of transforming and applying PDC nanoprodrugs.

[0031] (2) By utilizing the component characteristics of PDC nanoprodrugs, the difficulty in constructing a co-delivery system—characterized by the significant differences in the physicochemical properties of MMP9 inhibitory peptides and polyphenolic drugs—is cleverly transformed into a usable nanostructure construction characteristic. Compared to traditional co-delivery systems, this significantly increases the drug loading capacity of both drugs and eliminates the need for carrier materials, thus avoiding the safety issues associated with them. Furthermore, the PDC nanoprodrug preparation process is simple and easy to scale up, demonstrating excellent transformation potential.

[0032] (3) The fusion peptide and polyphenolic drugs are coupled through phenylboronic acid ester bonds. By adjusting the number of phenylboronic acid ester bonds, the two drugs can be delivered to the diseased pericytes at a preset therapeutic ratio. In addition, the two drugs are released in response to high concentrations of ROS, thereby simultaneously regulating multiple pathological pathways and synergistically enhancing the improvement effect on pericyte dysfunction.

[0033] (4) This provides a novel approach to treating brain diseases by restoring neurovascular unit homeostasis through targeted regulation of pericyte function via combined drug therapy. This approach overcomes the serious drawbacks of existing pericyte transplantation therapies, such as time consumption, high cost, epigenetic variation, gene instability, and transplant rejection, providing a more economical and effective solution for improving the treatment of pericyte-related brain diseases. Attached Figure Description

[0034] Figure 1 The inhibitory effect of the fusion peptide on the CypA-MMP9 pathway.

[0035] Figure 2 Evaluation of the brain targeting properties of fusion peptides.

[0036] Figure 3 Evaluation of the hemolytic activity of the fusion peptide.

[0037] Figure 4 : The 1H NMR spectrum of the phenylboronic acid-modified fusion peptide.

[0038] Figure 5 Transmission electron microscopy image of PDC nanoprodrug.

[0039] Figure 6 ROS-responsive drug release characteristics of PDC nanoprodrugs: (A) Particle size change in H2O2 environment; (B) Morphological characteristics when ROS response dissipates; (C) Cumulative drug release curves of nanoprodrugs containing different phenylboronic acid ester bonds.

[0040] Figure 7 Blood concentration-time curve of PDC nanoprodrug.

[0041] Figure 8 Evaluation of the brain-targeting properties of PDC nanoprodrugs.

[0042] Figure 9 Evaluation of the pericellular targeting of PDC nanoprodrugs.

[0043] Figure 10 : Regulation of pericyte functional proteins by PDC nanoprodrugs.

[0044] Figure 11 PDC nanoprodrugs inhibit abnormal activation of microglia and astrocytes.

[0045] Figure 12 PDC nanoprodrugs improve brain pericyte dysfunction: (A) Evaluation of Evans blue BBB permeability; (B) Evaluation of amyloid plaque clearance ability.

[0046] Figure 13 The protective effect of PDC nanoprodrugs on neurons. Detailed Implementation

[0047] The present invention is illustrated by the following description and embodiments. The following description is non-limiting and does not limit the scope of the claims of the present invention.

[0048] Example 1: Construction of fusion peptide

[0049] This invention synthesizes a series of fusion peptides using a solid-phase synthesis method and purifies them by HPLC. The composition of the obtained fusion peptides includes, but is not limited to, the contents shown in Table 1.

[0050] Table 1. Composition of various fusion peptides (from N-terminus to C-terminus)

[0051]

[0052] Example 2: Inhibitory effect of fusion peptide on MMP9 activity

[0053] The MMP9 inhibitory peptide mR18L synthesized in Example 1, along with the fusion peptides FTm and FGm (5 μM), were added to pericytes damaged by lipopolysaccharide (LPS, 100 ng / mL). After incubation for 24 h, total protein was extracted from each group of cells. The expression levels of CypA and MMP9 were detected by Western blot. The results are attached. Figure 1 As shown, compared with the normal control, the expression of CypA and MMP9 in LPS-stimulated pericytes was significantly upregulated. After treatment with FTm or FGm, the expression of both CypA and MMP9 was significantly reduced, with FTm showing a stronger inhibitory effect. While the levels of these two proteins in the mR18L treatment group were lower than those in the LPS group, there was no significant difference between the two groups. These results indicate that the fusion peptide enhances the inhibitory effect of mR18L on the CypA-MMP9 pathway.

[0054] Example 3: Binding force of fusion peptides to cell adhesion molecules

[0055] The binding affinity between the fusion peptides and VCAM-1 was determined using biomembrane interferometry. The fusion peptides VGA-1, VGA-2, VGA-3, CDA, and CSA synthesized in Example 1, along with their respective target peptides VH4 and CNN, were biotinylated and immobilized using a streptavidin sensor. The results were then analyzed using an Octet Red 96 high-throughput biomolecular interaction analyzer. The results are shown in Table 2. The apparent dissociation constants (Kd) between VGA-1, VGA-2, and CSA and the receptor molecule were significantly smaller than those of the original target peptides. The Kd value of VGA-3 was slightly smaller than that of the original target peptide, while the Kd value of CDA was larger than that of the original target peptide. This highlights the importance of selecting a suitable flexible linker to impart stronger receptor affinity to the fusion peptides compared to the original target peptides.

[0056] Table 2. Results of the determination of the apparent dissociation constant (Kd) between the peptide and VCAM-1.

[0057]

[0058] Example 4: Brain-targeting properties of fusion peptides

[0059] The targeting peptide NNQ and the fusion peptides NGK and NHK synthesized in Example 1 were modified with N-terminal Cy5 and injected via tail vein into LPS-induced neuroinflammation model mice (dose of 1.5 μCy5 for each peptide). After 30 min, the mice were perfused with their hearts, and brain tissue was harvested. In vitro imaging of the brain was performed using an IVIS Spectrum imaging system, and fluorescence intensity was analyzed. The results are attached. Figure 2As shown, the brain fluorescence of both fusion peptide groups was significantly stronger than that of the original targeting peptide group NNQ. Among them, the fusion peptide NHK had a stronger ability to penetrate into the brain, which confirms that by rationally designing fusion peptides, their brain targeting can be significantly better than that of the original targeting peptides.

[0060] Example 5: Stability evaluation of the fusion peptide

[0061] The target peptide CQI, MMP9 inhibitory peptide Ac-hE18A-NH2, fusion peptides CTA and CAA synthesized in Example 1 were dissolved in PBS to prepare peptide solutions with a concentration of 1 mg / mL. 10 μL of each peptide solution was mixed with 90 μL of 50% mouse serum and incubated at 37°C. Samples were taken at preset time points, and the residual peptide content was determined by HPLC. The results showed that at 1 h, the residual rate of CTA peptide was the highest, at 93.47%; followed by CAA, with a residual rate of 85.69%; the residual rate of Ac-hE18A-NH2 was 70.36%; while the residual rate of CQI was the lowest, at 35.43%, indicating that the fusion of peptides enhanced their plasma stability.

[0062] Example 6: Evaluation of the hemolytic activity of the fusion peptide

[0063] The MMP9 inhibitory peptide COG133 synthesized in Example 1, along with the fusion peptides LGC and LDC, were mixed with PBS solution containing 2% (v / v) red blood cells and incubated at 37°C for 4 h. The mixtures were then centrifuged (4°C, 2000 rpm, 5 min), and the appearance of the supernatant was observed. Absorbance values ​​were measured, and the hemolysis rate was calculated. Results are attached. Figure 3 As shown, the hemolysis rates of COG133, LGC, and LDC were 7.59%, 14.75%, and 0.94%, respectively, indicating that COG133 itself has a certain risk of hemolysis. The fusion peptide LGC linked by (G)4linker amplifies the hemolytic properties of COG133. In contrast, the fusion peptide LDC linked by (D)4linker improves the hemolytic defects of the original therapeutic peptide and enhances biocompatibility.

[0064] Example 7: Cytotoxicity evaluation of the fusion peptide

[0065] The MMP9 inhibitory peptides COG1410 and RSH-12 synthesized in Example 1, as well as the fusion peptides VGC, VSC, VTR, and VHR, were incubated with primary pericytes at 37°C for 24 h. The culture medium was then discarded, and fresh medium containing 10% CCK-8 was added for an additional 1 h of incubation. Absorbance values ​​were measured using a microplate reader, and cell viability was calculated. The results showed that when the concentrations of the six peptides were in the range of 5–20 μM, the survival rate of pericytes in each group exceeded 90%, indicating that the fusion peptides had good safety.

[0066] Example 8: Synthesis and characterization of fusion peptides with side-chain modified phenylboronic acid groups

[0067] 4-Carboxyphenylboronic acid (CBA) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide (EDC / NHS, 10 / 2 eq.) were dissolved in anhydrous N,N-dimethylformamide, stirred at room temperature for 24 h under nitrogen protection to prepare the active ester of 4-carboxyphenylboronic acid (CBA-NHS). Subsequently, the fusion peptide synthesized in Example 1 was added and mixed with CBA-NHS (5–25 eq.), stirred at room temperature for 24 h under nitrogen protection, dialyzed against deionized water for 48 h, and lyophilized to obtain fusion peptides with different numbers of phenylboronic acid groups modified on the side chains. The 1H NMR spectrum was then analyzed. 1 Characterization by H-NMR. Results are attached. Figure 4 As shown, the characteristic peak of phenylboronic acid appears in the product at δ = 7.80–7.96 ppm, indicating that the modification was successful.

[0068] Example 9: Preparation of PDC nanoprodrugs

[0069] The fusion peptide containing phenylboronic acid groups prepared in Example 8 was dissolved in deionized water or PBS / Tris buffer, and the polyphenolic drug was dissolved in the organic phase. The organic solvent could be ethanol, dimethyl sulfoxide, or N,N-dimethylformamide. The two phases were vortexed at a volume ratio of 1:5 to 1:20 and ultrasonicated in a water bath at room temperature (power: 50W to 120W, time: 5 to 60 min). Under ultrasonication, the drug covalently linked with the phenylboronic acid groups to form phenylboronic esters, and then self-polymerized with the hydrophobic drug as the core to form nanoparticles, which are the PDC nanoprodrugs.

[0070] The study found that the octanol-water partition coefficient (Log P) of polyphenol drugs needs to be greater than 1.5 to form PDC nanoprodrugs; otherwise, they can only form PDCs and cannot self-assemble into nanoparticles.

[0071] Example 10: Characterization of PDC nanoprodrugs

[0072] The particle size of the PDC nanoprodrug prepared in Example 9 was measured to be 80–300 nm using a Malvern particle size analyzer. The polydispersity index (PDI) was 0.103–0.257, and the zeta potential was -11.43–+30.97 mV. Transmission electron microscopy revealed that the PDC nanoprodrug exhibited a perfectly spherical shape. (See attached image) Figure 5 ).

[0073] With increasing numbers of phenylboronic acid groups modified on the side chains of the fusion peptide (n = 1–10), the drug loading capacity of PDC nanoprodrugs for polyphenols significantly increased. The final PDC nanoprodrugs contained 23.33%–42.57% MMP9 inhibitory peptide and 10.63%–49.23% polyphenolic drugs. The drug loading amounts can be adjusted according to clinical needs to achieve optimal synergistic therapeutic effects.

[0074] Example 11: Stability of PDC nanoprodrugs

[0075] Following the method described in Example 9, fusion peptides CDA (CDA-(CBA)n) containing different numbers of phenylboronic acid groups were reacted with quercetin (Que) to prepare PDC nanoprodrugs CDA-(CBA)n@Que-NPs with a particle size of approximately 140 nm. These nanoprodrugs were stored at 4°C in the dark, and particle size changes were measured periodically. The results showed that when the number of phenylboronic acid groups was low (n = 1–3), the particle size of CDA-(CBA)n@Que-NPs increased slightly within two weeks; while when n was 4–10, the particle size of CDA-(CBA)n@Que-NPs remained unchanged within three weeks. This indicates that the PDC nanoprodrugs can be stably stored at 4°C for at least two weeks, and that increasing the number of phenylboronic acid groups contributes to the stability of the nanoprodrugs.

[0076] Example 12: ROS-responsive drug release of PDC nanoprodrugs

[0077] The CDA-(CBA)3@Que-NPs (n=3) prepared in Example 11 were dispersed in PBS or PBS containing 100 μM hydrogen peroxide (H2O2), placed in a shaker (37℃, 100 r / min), and removed after 2 h to detect changes in particle size and morphology. The results are attached. Figure 6 As shown in AB, when dispersed in 100 μM H2O2, the particle size of CDA-(CBA)3@Que-NPs increased sharply from 142 nm to 1514 nm within 2 h, and its morphology also changed from a round sphere to a scattered fragment. This indicates that in a high ROS environment, the phenylboronic acid ester bonds in PDC nanoprodrugs can break rapidly, leading to the destruction of the nanostructure.

[0078] CDA-(CBA)n@Que-NPs (n = 3, 6, 9) were uniformly dispersed in PBS or PBS containing 100 μM H₂O₂ and divided into 24 equal portions. Each portion was shaken in a shaker (37℃, 100 rpm), and three portions were taken at regular intervals, centrifuged, and the supernatant was analyzed by HPLC to determine the Que content in the release medium at each time point and calculate the cumulative release rate. (See attached image) Figure 6 As shown in Figure C, Que is more readily released from CDA-(CBA)n@Que-NPs under H2O2 stimulation. Among them, CDA-(CBA)9@Que-NPs had the highest cumulative release rate, reaching 4.56 times that of the PBS control group, indicating that as the n value increases, the responsiveness of the nanoprodrug to ROS is enhanced, and the drug release is faster.

[0079] Example 13: Cytotoxicity and hemolytic activity of PDC nanoprodrugs

[0080] According to the method in Example 9, the fusion peptide LGC or LDC containing phenylboronic acid group was reacted with α-dextrin (α-MG) to prepare PDC nanoprodrugs LGC-(CBA)5@MG-NPs or LDC-(CBA)5@MG-NPs.

[0081] The cytotoxicity of PDC nanoprodrugs to primary pericytes and brain capillary endothelial cells (bEnd.3) was evaluated according to the method in Example 7. In the experiment, nanoparticle solutions were used instead of the original peptide solutions, with concentrations ranging from 5 to 30 μM. The results showed that neither nanoprodrug caused significant toxicity to pericytes or bEnd.3 cells at the tested concentrations, and cell viability exceeded 80%.

[0082] The hemolytic activity of the PDC nanoprodrug was evaluated according to the method in Example 6, with the nanoparticle solution replacing the original peptide solution in the experiment. The results showed that the hemolytic rate of LGC-(CBA)5@MG-NPs was as high as 28.94%, while that of LDC-(CBA)5@MG-NPs was only 3.15%, indicating that the hemolytic activity of the PDC nanoprodrug is related to the flexible linker composition at the fusion peptide end.

[0083] Example 14: Pharmacokinetic evaluation of PDC nanoprodrugs

[0084] Nine SD rats were randomly divided into two groups of three. Free curcumin solution (Cur), PDC nanoparticle prodrugs NHK-CBA@Cur-NPs, and VTR-CBA@Cur-NPs were administered via tail vein injection at a dose of 5 mg Cur / kg body weight. Following administration, blood samples were collected from the orbital cavity at 2 min, 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, and 12 h. Plasma was collected by centrifugation (4℃, 4000 rpm, 15 min), and the concentration of Cur in the blood was determined using LC-MS / MS. Blood concentration-time curves were plotted, and pharmacokinetic parameters were calculated (Table 3). [Following the attached...] Figure 7 As shown in Table 3, free Cur is easily degraded in blood, with an elimination half-life of t. 1 / 2 The retention time was 2.85 hours. However, Cur, administered as a nano-prodrug, showed a significantly prolonged blood retention, increasing the likelihood of drug circulation into the brain. The two nano-prodrugs... 1 / 2 The difference may be due to the fact that VTR-CBA@Cur-NPs carry less positive charge than NHK-CBA@Cur-NPs.

[0085] Table 3. Main pharmacokinetic parameters (n=3)

[0086]

[0087] *p<0.05,**p<0.01 compared with Cur solution.P values ​​were calculated through unpaired two-tailedtest.

[0088] Example 15: Brain distribution of PDC nanoprodrugs administered via different routes

[0089] The VTR-CBA@Cur-NPs prepared in Example 14 were administered to mice via tail vein injection, subcutaneous injection, intramuscular injection, and intranasal administration, at a dose of 2 mg Cur / kg body weight. After administration for 5 min, 15 min, 30 min, 1 h, 2 h, and 4 h, the mice were cardiac perfused, brain tissue was dissected, and brain homogenates were prepared. The concentration of Cur in the brain tissue was measured using LC-MS / MS. The results showed that in the intravenous injection group, the Cur concentration in the mouse brain peaked at 30 min; in the intranasal administration group, the peak concentration was reached between 15 and 30 min. In contrast, subcutaneous and intramuscular injections both peaked at 2 h, but their brain drug concentrations at 4 h were higher than those in the intravenous and intranasal administration groups, exhibiting a prolonged drug retention in the brain. These results demonstrate that PDC nanoprodrugs can achieve intrabrain delivery through multiple administration routes, showcasing their broad applicability in various clinical applications.

[0090] Example 16: Evaluation of the brain-targeting properties of PDC nanoprodrugs

[0091] Fluorescently labeled PDC nanoprodrugs (FTm-(CBA)2@AP-NPs / DiR) were prepared by co-dissolving the fluorescent dye DiR and apigenin (AP) in an organic solvent, following the method described in Example 9. Free DiR, FTm-(CBA)2@AP-NPs / DiR, and DiR-encapsulated polyethylene glycol-polylactic acid nanoparticles (PEG-PLA-NPs / DiR) were injected intravenously into LPS-induced neuroinflammatory model mice (dose: 5 mg / kg DiR). After 30 minutes, the mice were sacrificed by cardiac perfusion, and their brains were dissected for in vitro imaging. Results are attached. Figure 8 As shown, the fluorescence intensity of FTm-(CBA)2@AP-NPs / DiR in the brain was 22.66 times that of free DiR and 3.75 times that of PEG-PLA-NPs / DiR, indicating that the PDC nanoprodrug not only retains the good brain targeting of the fusion peptide, but also has a significantly stronger brain penetration ability than ordinary nanoparticles.

[0092] Example 17: Evaluation of pericellular targeting of PDC nanoprodrugs

[0093] Fluorescent dye BODIPY was co-dissolved with rutin (RU) in an organic solvent to prepare fluorescently labeled PDC nanoprodrug CSA-(CBA)6@RU-NPs / BODIPY according to the method in Example 9. Healthy mice and APP / PS1AD mice were injected via tail vein (dose: 2 mg BODIPY / kg body weight). Mice were anesthetized 30 min after injection, and their hearts were perfused with physiological saline and 4% paraformaldehyde. Brain tissue was harvested, prepared into vibratory sections, and immunofluorescently stained with pericytes (PDGFRβ antibody), vascular endothelial cells (CD31 antibody), and astrocytes (GFAP antibody). Images were observed and analyzed using a confocal microscope. Results are attached. Figure 9 As shown, the accumulation of CSA-(CBA)6@RU-NPs in the brain regions of diseased mice was significantly higher than that in normal mice. Further immunofluorescence analysis of brain slices from diseased mice revealed that CSA-(CBA)6@RU-NPs exhibited significant co-localization with vascular endothelial cells and pericytes, but less co-localization with astrocytes, indicating that the nanoprodrug mainly concentrated in the endothelial cells and pericytes of damaged BBBs, which is beneficial for its regulation of pericyte function.

[0094] Example 18: Regulation of pericyte functional proteins by PDC nanoprodrugs

[0095] Primary pericytes were seeded in 6-well plates and cultured for 24 h. The culture medium was then aspirated, and 100 ng / mL LPS and one of the following formulations (Que, COG133, COG133+Que, LDC-(CBA)5, LDC-(CBA)5+Que, or LDC-(CBA)5@Que-NPs) were added to each well at a concentration of 5 μM. The positive control group received only serum-free medium, while the negative control group received only LPS. After another 24 h of incubation, total protein was extracted from each group of cells, and the expression levels of CypA, MMP9, PDGFRβ, and ET-A were detected by Western blot. Results are attached. Figure 10 As shown, after primary pericytes were stimulated with LPS for 24 hours, PDGFRβ expression was significantly downregulated, accompanied by abnormal activation of CypA, MMP9, and ET-A. LDC-(CBA)5@Que-NPs treatment showed a stronger regulatory effect on pericyte functional proteins than other agents, restoring PDGFRβ expression to normal levels and significantly downregulating the expression of CypA, MMP9, and ET-A.

[0096] Example 19: Evaluation of the reduction of oxidative stress levels in the brains of Parkinson's disease model rats by PDC nanoprodrugs

[0097] C57 mice were randomly divided into four groups of six each. Three groups received intraperitoneal injections of 20 mg MPTP / kg / day for seven consecutive days to obtain MPTP-induced PD model mice. The remaining group of healthy mice served as the normal control group. Starting from day 8 of modeling, mice were treated subcutaneously for two consecutive weeks: the normal control group and PD group received saline, while the treatment groups received VSC-(CBA)3+EGCG or VSC-(CBA)3@EGCG-NPs. Two weeks later, the mice were sacrificed, and total protein was extracted from their brains. The levels of malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-PX) were measured according to the corresponding kit instructions. The results showed that the MDA level in the brain tissue of the PD group increased to 183.33% of that in the normal control group, while the activities of GSH-PX and SOD decreased by 48.45% and 54.67%, respectively, indicating severe oxidative stress damage in the brains of PD mice. The activities of MDA, GSH-PX and SOD enzymes in the brain of the nanoprodrug group were regulated to near normal levels, indicating that VSC-(CBA)3@EGCG-NPs have strong antioxidant capacity.

[0098] Example 20: PDC nanoprodrugs inhibit inflammatory responses in multiple sclerosis model mice

[0099] C57 mice were randomly divided into three groups of six each. Multiple sclerosis (MS) models were established by gavage administration of dicyclohexanone oxaloyl dihydrazone (CPZ) suspension at 300 mg / kg / day for three consecutive weeks. A separate group of healthy mice served as the normal control group. After successful modeling, a three-week treatment regimen was initiated: the normal control group and the MS group received saline, while the treatment groups received either LDC-(CBA)6+MG (γ-dextrin) or LDC-(CBA)6@MG-NPs, respectively. After three weeks, mouse brain tissue was collected, and the mRNA levels of TNF-α, IL-1β, IL-6, IFNγ, and CX3CL1 were measured using PCR. Simultaneously, microglia (Iba1 antibody) and astrocytes (GFAP antibody) were stained with immunofluorescence. The results showed that the mRNA levels of inflammatory factors TNF-α, IL-1β, IL-6, and IFNγ in the brains of mice in the LDC-(CBA)6@MG-NPs group were most significantly downregulated, while the level of the anti-inflammatory factor CX3CL1 was significantly increased. All indicators were comparable to those in the normal control group. Furthermore, the nano-prodrug significantly inhibited the overactivation of microglia and astrocytes (see appendix). Figure 11 These results indicate that PDC nanoprodrugs can effectively enhance the homeostatic regulation of neuroinflammation by pericytes.

[0100] Example 21: PDC nanoprodrugs promote pericyte regeneration in a mouse stroke model

[0101] SD rats were randomly divided into three groups of six each. Two groups were used to establish a stroke model via cerebral artery occlusion (MCAO), while the remaining group of healthy rats served as a normal control group. Following MCAO surgery, rats underwent 28 days of continuous treatment: the normal control and MCAO groups received saline, while the treatment groups received CTA-(CBA)4+Cur or CTA-(CBA)4@Cur-NPs. On day 28, rat brain tissue was harvested and prepared into vibratory sections. Pericytes (PDGFRβ antibody) and cerebral blood vessels (CD31 antibody) were stained with immunofluorescence, observed using a confocal microscope, and the pericyte coverage of each brain section was calculated using the following formula:

[0102]

[0103] The results showed that the MCAO model mice suffered severe pericyte loss, with a coverage rate of only 55.46% of the normal control group. However, treatment with CTA-(CBA)4@Cur-NPs promoted pericyte regeneration, restoring the pericyte coverage rate of this group of mice to 87.18% of the normal control group.

[0104] Example 22: PDC nanoprodrugs improve pericytic dysfunction in Alzheimer's disease mice

[0105] Fourteen-month-old APP / PS1 transgenic mice were randomly divided into three groups (n=12 per group), with wild-type mice of the same age and background serving as normal controls (Wild-type, WT). A treatment regimen of tail vein administration for 28 consecutive days was adopted, specifically: the WT and AD groups were given saline, while the treatment groups were given VHR-(CBA)2+Lut (luteolin) or VHR-(CBA)2@Lut-NPs. On day 29, the following experiments were conducted: (1) Cerebral blood flow measurement: Mice were anesthetized by inhaling 1%–1.5% isoflurane, and their heads were fixed using a stereotaxic device. The scalp was cut open to expose the skull, and cerebral blood flow in each group was monitored using a laser speckle blood flow imaging system; (2) BBB leakage evaluation: Each group of mice was injected via tail vein with 2% Evans blue dye solution at a dose of 5 mL / kg. Two hours later, the mice underwent cardiac perfusion, and the brain tissue was dissected to make frozen sections. The distribution of Evans blue fluorescence in the brain tissue was observed under a confocal microscope. (3) Evaluation of the ability to clear amyloid plaques: Mouse brain tissue was taken, paraffin sections were made, amyloid plaques were stained with immunohistochemistry, and the images were observed and analyzed under an optical microscope.

[0106] Cerebral blood flow measurements showed that the blood perfusion volume of AD mice was only 65.24% of that of the WT group. However, after AD mice were treated with VHR-(CBA)2@Lut-NPs, their cerebral blood flow reached 97.12% of that of the WT group, which is close to the normal level, indicating that VHR-(CBA)2@Lut-NPs can effectively improve the diastolic function of pericytes.

[0107] The results of the BBB leakage test are attached. Figure 12 As shown in Figure A, AD mice exhibit significant BBB leakage, with abundant Evans blue-albumin complex fluorescence (magenta) in their brain tissue. VHR-(CBA)2+Lut treatment somewhat alleviated BBB leakage. However, treatment with VHR-(CBA)2@Lut-NPs significantly inhibited BBB leakage, with the amount of the complex in the brain tissue being only 32.08% of that in AD mice. This indicates that VHR-(CBA)2@Lut-NPs enhances BBB structural integrity by regulating pericytes.

[0108] The results of amyloid plaque removal are shown in the attached figure. Figure 12 As shown in Figure B, compared to the AD group, the area of ​​amyloid plaques in the brain of the VHR-(CBA)2@Lut-NPs group was reduced by 82.43%, indicating that it can accelerate the clearance of toxic proteins in the brain by repairing damaged pericytes.

[0109] Example 23: Neuroprotective effect of PDC nanoprodrugs

[0110] Coronal paraffin sections of brain tissue from mice treated in Example 22 were prepared and stained with hematoxylin and eosin (H&E). Images were acquired and analyzed using a VS200 slide scanner. Results are attached. Figure 13 As shown, the AD group contained a large number of deeply stained, pyknotted degenerated neurons. In the VHR-(CBA)2@Lut-NPs group, most neurons were neatly arranged, with full cell morphology and uniform staining. The number of degenerated neurons in this group was only 18.15% of that in the AD group, indicating that VHR-(CBA)2@Lut-NPs improved neuronal survival by regulating pericyte dysfunction.

Claims

1. A polypeptide-drug conjugate (PDC) nanoprodrug having a repair pericyte function, characterized in that, The PDC nano prodrug is coupled by phenyl borate ester bond between a fusion peptide and a polyphenol drug and further self-assembled to form; wherein, The fusion peptide is connected by a flexible linker between a cell adhesion molecule targeting peptide and a matrix metalloproteinase 9 inhibiting peptide.

2. The PDC nanoprodrug of claim 1, wherein, The cell adhesion molecule targeting peptide is selected from one of VH4 peptide (VHPK), VH7 peptide (VHPKQHR), VH13 peptide (VHPKQHRG GSKGC), VHS peptide (VHSPNKK), CNN peptide (CNNSKSHTC), CQI peptide (CQIDSPC), cLAB.L peptide (Pen-ITDGEATDSGC), NNQ peptide (NNQKIVNLKEKVAQ LEA), GRG peptide (GRGEFRGRDNSVSVV), and FEG peptide (FEGFSFLAFEDFVSSI).

3. The PDC nanoprodrug of claim 1, wherein, The matrix metalloproteinase 9 inhibiting peptide is selected from one of COG133 peptide (Ac-LRVRLASHLRKLRKRLL-NH2), COG1410 peptide (Ac-AS-Aib-LRKL-Aib-KRLL-NH2), CN-105 peptide (Ac-VSRRR-NH2), Ac-hE18A-NH2 peptide (Ac-LRKLRKRLLR-DWLKAFYDKVAEKLKEAF-NH2), mR18L peptide (Ac-GFRRFLGSWARIYRAFVG-NH2), ApoE (141-155) peptide (Ac-LRKLRKRLLRDADDLLRKLRKRLLRDADDL), ApoEdp peptide (Ac-LRKLRKRLLLRKLRKRLL-NH2), RSH-12 peptide (MHPNAGHGSLMR), CTT peptide (CTTHWGFTLC), KAH peptide (KAHWGFTLD), M3 peptide (TFKEPVPDLC), CTF peptide (CTFKEIVPDLC), VPG peptide (VPGALAAA), and C6 peptide (KAHWGFTLD).

4. The PDC nanoprodrug of claim 1, wherein, The flexible linker is selected from one of glycine (G)n, alanine (A)n, histidine (H)n, aspartic acid (D)n, threonine-aspartic acid (TD)n, and serine-aspartic acid (SD)n, wherein n represents the number of amino acid repeats, and n is 1-8.

5. The PDC nanoprodrug of claim 1, wherein, The polyphenol drug is selected from one of quercetin, rutin, curcumin, catechin, epicatechin gallate, epigallocatechin gallate (EGCG), gallic acid, caffeic acid, ellagic acid, cyanidin, alpha-bisabolol, gamma-bisabolol, procyanidin B1, procyanidin B3, hesperetin, naringenin, luteolin, apigenin, oleuropein, hydroxytyrosol, chlorogenic acid, punicalagin, nordihydroguaiaretic acid, baicalin, vitexin, and hesperidin.

6. The PDC nanoprodrug of claim 1, wherein, The benzene borate bond is introduced by reacting the carboxyl group of 4-carboxyphenyl boronic acid with the primary amine group of the side chain of lysine and / or arginine in the fusion peptide sequence to introduce the benzene borate group into the fusion peptide; and further reacting the benzene borate group with the active hydroxyl group of the polyphenol drug to form the benzene borate bond.

7. The PDC nanoprodrug of claim 6, wherein, The number of benzene borate bonds in the PDC nano-prodrug is 1-10.

8. The PDC nanoprodrug of claim 1, wherein, The preparation method of the nano-prodrug is as follows: dissolving the fusion peptide in an aqueous phase, dissolving the polyphenol drug in an organic phase, vortex mixing the two phases for 30 s-2 min, and then ultrasonically treating in a water bath, wherein the temperature is 15-25 DEG C, the power is 50-120 W, and the time is 5-60 min, to obtain the nano-prodrug. In the preparation method, The aqueous phase solvent is selected from one of deionized water, PBS buffer and Tris buffer; The organic phase solvent is selected from one of ethanol, dimethyl sulfoxide and N,N-dimethylformamide; The volume ratio of the organic phase to the aqueous phase is 1:5-1:20 (v / v).

9. Use of the PDC nano-prodrug of claim 1 in the preparation of a drug for preventing and treating pericyte-related brain diseases.

10. Use according to claim 9, characterized in that, The PDC nano-prodrug is applied by intravenous injection, subcutaneous injection, intramuscular injection or nasal administration.

Images