A beta-targeting peptide-functionalized cellulose derivative, and a preparation method and application thereof

Abstract

The application discloses a kind of peptide functionalized cellulose derivatives targeting A beta and its preparation method and application, belong to the field of biological medicine technology.The peptide functionalized cellulose derivative obtained by connecting A beta targeting peptide with hydroxypropyl cellulose through ester bond can be combined with A beta protein through molecular recognition specificity, and self-assembly is formed into core-shell structure nanomicelle, A beta oligomer is efficiently encapsulated, A beta aggregation and fiberization are inhibited, meanwhile, the interaction between A beta and endothelial cell junction protein is blocked by anti-fouling performance, APEL effect induced by amyloid is inhibited, and the integrity of blood-brain barrier is protected.In addition, the peptide functionalized cellulose derivative has good biocompatibility, can reduce A beta plaque deposition in brain, improve the cognitive function of AD patients, and provides a new type of efficient candidate drug for the treatment of Alzheimer's disease.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a peptide-functionalized cellulose derivative targeting Aβ, its preparation method, and its application. Background Technology

[0002] Alzheimer's disease (AD) is a severe neurodegenerative disease characterized by β-amyloid (Aβ) plaque deposition, neuroinflammation, synaptic dysfunction, and blood-brain barrier (BBB) ​​damage. Soluble Aβ oligomers are particularly potent neurotoxic, directly damaging neurons and inducing endothelial leakage (APEL effect) by binding to endothelial cell junction proteins (such as VE-cadherin). This disrupts the integrity of the blood-brain barrier, creating a vicious cycle of neurodegeneration and vascular damage, thus accelerating disease progression.

[0003] Currently, interventions targeting Aβ regulation mainly focus on three directions: "inhibiting aggregation, promoting degradation, and blocking toxicity." While small molecule inhibitors, antibody drugs, and nano-inhibitors have achieved some breakthroughs, their core bottlenecks stem from insufficient scientific understanding. Small molecule inhibitors are the traditional mainstream strategy, inhibiting aggregation by binding to Aβ monomers / oligomers. Representative drugs include BACE1 inhibitors and γ-secretase modulators, with over 30 already in clinical trials. However, due to insufficient understanding of the specific binding mechanisms of different Aβ aggregation states (monomers / oligomers / fibromas) and the blood-brain barrier transport patterns, they suffer from poor targeting and significant off-target effects, leading to the failure of most Phase III trials. Essentially, the regulatory mechanisms of the Aβ pathological microenvironment on drug action remain unclear. Antibody drugs, with their high targeting capabilities, have become a hot topic in recent years. They promote phagocytic degradation or block receptor binding by binding to Aβ oligomers (Aβo). Drugs such as Aducanumab and Lecanemab have been approved or entered Phase III trials and can reduce plaque burden in the brain. However, our understanding of Aβ's immune recognition epitopes and its transport mechanism across the blood-brain barrier is lacking, and the interaction mechanism between Aβ clearance and cerebral vascular endothelial cells remains unclear. This leads to insufficient Aβ regulation, low blood-brain barrier penetration, and an increased risk of adverse reactions such as amyloid-associated imaging abnormalities (ARIA). Nanoparticle inhibitors, leveraging size effects and surface modification flexibility, achieve multiple functions including targeted binding to inhibit aggregation, drug delivery, and physical / chemical effects to break up aggregation. Some have already entered critical preclinical stages. Their advantage lies in their high potential for blood-brain barrier penetration, but the core scientific gaps lie in the insufficient understanding of the structure-activity relationship of Aβ-specific binding and the selection rules of target epitopes. Furthermore, the interaction between nanomaterials and the in vivo environment and the inflammatory triggering mechanism remain unclear, resulting in insufficient Aβ targeting and a lack of scientific basis for biocompatibility assessment, thus limiting clinical translation.

[0004] In view of this, there is an urgent need to develop a peptide-functionalized cellulose derivative that targets Aβ in order to address the shortcomings of existing technologies. Summary of the Invention

[0005] The purpose of this invention is to provide a peptide-functionalized cellulose derivative targeting Aβ, its preparation method, and its applications. This addresses the problems of poor targeting and poor therapeutic efficacy found in existing drugs used to treat Alzheimer's disease.

[0006] In a first aspect, the present invention provides a peptide-functionalized cellulose derivative targeting Aβ, the peptide-functionalized cellulose derivative comprising hydroxypropyl cellulose and an Aβ-targeting peptide, wherein the hydroxyl groups in the hydroxypropyl cellulose and the carboxyl groups in the Aβ-targeting peptide are linked by ester bonds; wherein the Aβ-targeting peptide comprises at least one of KD8 peptide, JAL-TA9 peptide, Aβ1-6A2V peptide, and RW3 peptide.

[0007] In this invention, the inventors discovered that peptide-functionalized cellulose derivatives obtained by linking Aβ-targeting peptides to hydroxypropyl cellulose via ester bonds can specifically bind to Aβ proteins through molecular recognition and self-assemble into core-shell structured nanomicelles, efficiently encapsulating Aβ oligomers, inhibiting Aβ aggregation and fibrosis. Simultaneously, through antifouling properties, they block the interaction between Aβ and endothelial cell junction proteins, inhibiting the amyloid-induced endothelial leakage (APEL) effect and protecting the integrity of the blood-brain barrier. Furthermore, these peptide-functionalized cellulose derivatives can reduce Aβ plaque deposition in the brain and improve cognitive function in AD patients, providing a novel and highly effective drug candidate for the treatment of Alzheimer's disease.

[0008] It is understood that the type of Aβ-targeting peptide can be conventionally selected according to actual usage needs, as long as it can efficiently target and bind to the Aβ protein. For example, in this invention, the Aβ-targeting peptide preferably includes at least one of KD8 peptide, JAL-TA9 peptide, Aβ1-6A2V peptide, and RW3 peptide.

[0009] In some implementations, the molar ratio of hydroxypropyl cellulose to Aβ-targeting peptide is 1:(5-20).

[0010] In this invention, by controlling the molar ratio of hydroxypropyl cellulose to Aβ-targeting peptide within a specific range, the Aβ-targeting peptide can be completely linked to hydroxypropyl cellulose, thereby improving the targeting properties and therapeutic effect on AD of the prepared peptide-functionalized cellulose derivative.

[0011] In a second aspect, the present invention provides a method for preparing the above-mentioned peptide-functionalized cellulose derivative, comprising the following steps: activating an Aβ-targeting peptide solution to obtain a carboxyl-activated Aβ-targeting peptide solution; heating an aqueous solution of hydroxypropyl cellulose to a first temperature and then holding it to obtain an aqueous solution of hydroxypropyl cellulose with folded molecular chains; mixing the carboxyl-activated Aβ-targeting peptide solution with the aqueous solution of hydroxypropyl cellulose with folded molecular chains, adding DMAP, and then carrying out an esterification reaction; after the reaction is completed, cooling the reaction solution to a second temperature, and obtaining the peptide-functionalized cellulose derivative through centrifugation, dialysis, and drying; wherein the first temperature is higher than the lower critical solution temperature of hydroxypropyl cellulose, and the second temperature is lower than the lower critical solution temperature of hydroxypropyl cellulose.

[0012] In the present invention, the preparation of the HPC-Aβ-targeting peptide copolymer by local modification and grafting of hydroxypropyl cellulose (HPC) is crucial for targeting Aβ. Among them, the esterification reaction is carried out under the condition of temperature (T) > lower critical solution temperature (LCST). Due to the disruption of the hydrophilic-hydrophobic balance, HPC forms loose aggregates, and the molecular chains interact with each other through weak hydrogen bonds. The internal hydroxyl groups are wrapped, but the surface hydroxyl groups are highly enriched and oriented (the surface free energy of the aggregates is high, and the hydroxyl groups are easy to form ester bonds with the carboxyl groups of the short peptides). Relying on the high-density enrichment advantage of the surface hydroxyl groups of the HPC aggregate state, site-specific coupling of the Aβ-targeting peptide is achieved. After the reaction is completed, it can be redispersed into a stable colloid when T < LCST. Compared with the dispersed state, the high-density short peptide array can significantly improve the affinity through multivalent binding. The intact short peptide conformation and the appropriate aggregate size are retained, ensuring the specificity of protein recognition and the efficiency of close binding, and providing an efficient technical path for the precise recognition and stable encapsulation of the target protein.

[0013] In some embodiments, in the step of obtaining the carboxyl-activated Aβ-targeting peptide solution, the Aβ-targeting peptide solution is obtained by dissolving the Aβ-targeting peptide in an organic solvent, and the mass concentration of the Aβ-targeting peptide solution is 1-3%; the activation treatment includes: adding an EDC solution and then carrying out low-power ultrasonic dissolution; wherein the addition amount of the EDC solution accounts for 1-2% of the volume of the organic solvent.

[0014] In the present invention, controlling the mass concentration of the Aβ-targeting peptide solution and the addition amount of the EDC solution within a specific range facilitates the subsequent esterification reaction.

[0015] In some embodiments, the organic solvent includes N,N-dimethylformamide.

[0016] It can be understood that the type of the organic solvent can be conventionally selected according to actual use needs as long as it can efficiently dissolve the Aβ-targeting peptide. For example, in the present invention, the organic solvent preferably includes N,N-dimethylformamide.

[0017] In some embodiments, in the step of obtaining the aqueous solution of hydroxypropyl cellulose with folded molecular chains, the mass concentration of the aqueous solution of hydroxypropyl cellulose is 0.5-5%, and the incubation time is 1-3 hours.

[0018] In this invention, by controlling the mass concentration of the hydroxypropyl cellulose aqueous solution and the incubation time within a specific range, the hydroxypropyl cellulose can be fully folded and aggregated, facilitating its reaction with the Aβ-targeting peptide.

[0019] In some embodiments, in the step of obtaining peptide-functionalized cellulose derivatives, the volume ratio of the carboxyl-activated Aβ-targeting peptide solution to the molecularly folded hydroxypropyl cellulose aqueous solution is (8-10):1; the amount of DMAP added accounts for 2-4% of the mass of hydroxypropyl cellulose; the esterification reaction includes: reacting for 12-24 h at a first temperature and a pH of 6.5-7.5 in the reaction system.

[0020] In this invention, by controlling the amount of DMAP added and the parameters of the esterification reaction within a specific range, the esterification reaction can be completed, thereby improving the targeting of the prepared peptide-functionalized cellulose derivative and its therapeutic effect on AD.

[0021] In some embodiments, in the step of obtaining peptide-functionalized cellulose derivatives, dialysis includes: transferring the reaction solution into a dialysis bag, dialyzing with deionized water for 3-5 days, and changing the dialysis solution 2-4 times a day; wherein the molecular weight cutoff of the dialysis bag is 3400-3600 Da.

[0022] In this invention, dialysis can remove impurities, further improve the purity of peptide-functionalized cellulose derivatives, and thus enhance the targeting properties and therapeutic efficacy of peptide-functionalized cellulose derivatives against Alzheimer's disease (AD).

[0023] In a third aspect, the present invention provides the use of peptide-functionalized cellulose derivatives as described above or prepared by any of the above preparation methods in the preparation of medicaments for the prevention or treatment of Alzheimer's disease.

[0024] In this invention, peptide-functionalized cellulose derivatives in aqueous solution can specifically bind to Aβ monomers, oligomers, and fibrous structures through molecular recognition between Aβ-targeting peptides and Aβ proteins, and self-assemble into core-shell structured nanomicelles. The hydrophobic core encapsulates the Aβ protein, while the hydrophilic surface (the hydroxyl group of HPC) forms a hydration layer, exhibiting anti-protein adsorption properties. Its mechanism of action includes at least one of the following: 1) physically blocking intermolecular interactions of Aβ, inhibiting Aβ aggregation and fibrosis; 2) encapsulating Aβ oligomers, reducing their neurotoxicity; 3) preventing Aβ from binding to endothelial cell VE-cadherin, inhibiting phosphorylation at the Y658 site of VE-cadherin, blocking the APEL effect, and protecting the integrity of the blood-brain barrier; 4) disrupting existing Aβ plaques and promoting Aβ clearance.

[0025] In some implementations, the drug also includes a pharmaceutically acceptable carrier.

[0026] In this invention, the term "pharmaceutically acceptable carrier" refers to excipients widely used in the pharmaceutical manufacturing industry. Excipients primarily serve to provide a safe, stable, and functional pharmaceutical composition, and may also provide methods for dissolving the active ingredient at a desired rate after administration to a subject, or for promoting effective absorption of the active ingredient after administration to a subject. Pharmaceutical excipients may be inert fillers or provide a function, such as stabilizing the overall pH of the composition or preventing degradation of the active ingredient. Pharmaceutical excipients may include one or more of the following: binders, suspending agents, emulsifiers, diluents, fillers, granulators, adhesives, disintegrants, lubricants, anti-adhesion agents, flow aids, wetting agents, gelling agents, absorption delay agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents, and sweeteners.

[0027] The pharmaceutical products provided by this invention can be prepared using any method known to those skilled in the art, based on the disclosed information. Examples include, but are not limited to, conventional mixing, dissolving, granulation, emulsification, grinding, encapsulation, embedding, or lyophilization processes.

[0028] In some implementations, the dosage form of the drug includes at least one of solid dosage form, semi-solid dosage form, and liquid dosage form.

[0029] The medicaments provided by this invention can be administered in any form, including by injection (intravenous), mucosal, oral (solid and liquid formulations), inhalation, ocular, rectal, topical, or parenteral (infusion, injection, implantation, subcutaneous, intravenous, intra-arterial, intramuscular) administration. The medicaments of this invention can also be controlled-release or sustained-release dosage forms (e.g., liposomes or microspheres). Examples of solid oral formulations include, but are not limited to, powders, capsules, tablets, soft capsules, and tablets. Examples of liquid formulations for oral or mucosal administration include, but are not limited to, suspensions, emulsions, elixirs, and solutions. Examples of topical formulations include, but are not limited to, emulsions, gels, ointments, creams, patches, pastes, foams, lotions, drops, or serum preparations. Examples of parenteral formulations include, but are not limited to, solutions for injection, dry powder formulations that can be dissolved or suspended in a pharmaceutically acceptable carrier, suspensions for injection, and emulsions for injection. Examples of other suitable formulations of a drug include, but are not limited to, eye drops and other ophthalmic preparations; aerosols, such as nasal sprays or inhalers; liquid dosage forms suitable for parenteral administration; suppositories; and tablets.

[0030] In some implementations, the drug includes at least one of the following functions: A1) inhibiting the aggregation and fibrosis of Aβ protein; A2) encapsulating Aβ oligomers and reducing their neurotoxicity; A3) inhibiting amyloid-induced endothelial leakage; A4) protecting the integrity of the blood-brain barrier; A5) reducing Aβ plaque deposition in the brain; and A6) improving cognitive function in patients with Alzheimer's disease.

[0031] The beneficial effects of this invention are as follows: Unlike existing technologies, this invention connects Aβ-targeting peptides to hydroxypropyl cellulose via ester bonds to obtain peptide-functionalized cellulose derivatives that can specifically bind to Aβ proteins through molecular recognition and self-assemble into core-shell structured nanomicelles, efficiently encapsulating Aβ oligomers, inhibiting Aβ aggregation and fibrosis. Simultaneously, through its antifouling properties, it blocks the interaction between Aβ and endothelial cell junction proteins, inhibiting the amyloid-induced endothelial leakage (APEL) effect and protecting the integrity of the blood-brain barrier. Furthermore, this peptide-functionalized cellulose derivative exhibits good biocompatibility, reduces Aβ plaque deposition in the brain, and improves cognitive function in AD patients, providing a novel and highly effective candidate drug for the treatment of Alzheimer's disease. Attached Figure Description

[0032] Figure 1The following are the synthesis, assembly, and performance characterization results of the short peptide functionalized cellulose derivative HPC-KD8 in Example 1 of this invention. Among them, (a) is a schematic diagram of the synthesis of HPC-KD8 and its interaction with Aβ oligomers, (b) is a schematic diagram of the chemical structure of HPC and KD8 peptide, (c) is the FTIR spectrum of HPC and HPC-pet, (d) is the particle size distribution and solution appearance of HPC at different temperatures, (e) is the temperature cycling absorbance change result of HPC, (fh) are the SAXS spectra of HPC, HPC-pet, and HPC-pet-Aβo, respectively, (i) is the 1D scattering curve corresponding to the SAXS spectrum, and (jq) is the microstructure image of HPC-pet, HPC-pet-Aβo, and Aβ sample.

[0033] Figure 2 The results of the inhibitory effect of HPC-pet on Aβ aggregation in Example 2 of the present invention are shown below. (a) shows the particle size changes of Aβ and HPC-pet-Aβ complex at different incubation times; (b) shows the ThT fluorescence intensity changes with incubation time; (c) shows the ThT fluorescence spectrum of the Aβ and HPC-pet-Aβ complex; (de) shows the results of the binding effect of HPC-pet with different aggregated states of Aβ studied using biolayer interferometry (BLI); and (fg) shows the results of the encapsulation ability of HPC-pet for different aggregated states of Aβ studied using BLI. Figure 3The following are the in vitro experimental verification results of HPC-pet inhibiting the APEL effect in Example 3 of this invention. (a) is a schematic diagram of the Transwell experiment; (bc) are the fluorescence spectra of the lower chamber of the Transwell in different treatment groups; (d) shows the trend of fluorescence intensity change with incubation time; (e) is a schematic diagram of the endothelial leakage experiment; (f) is a confocal fluorescence image of endothelial cell junctions; (gh) shows the statistical results of endothelial cell gap size and area; and (i) shows the HPC-pet-A effect before and after FBS incubation. The particle size variation of the complex is shown in (j), which is a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) image. Lane 1 is the protein marker, lane 2 is 10% fetal bovine serum (FBS), lane 3 is HPC-pet-Aβ monomer + FBS, lane 4 is HPC-pet-Aβ oligomer + FBS, lane 5 is HPC-pet-Aβ oligomer-fibrils + FBS, and lane 6 is HPC-pet-Aβ Lane 7 shows HPC-pet-Aβ for 36 hours, followed by 48 hours of HPC-pet-Aβ treatment with FBS; Lane 8 shows HPC-pet-Aβ for 96 hours of HPC-pet-Aβ treatment with FBS; and Lane 9 shows HPC-pet-Aβ for 180 hours of HPC-pet-Aβ treatment with FBS. (k) shows the results of Western blot analysis of phosphorylated vascular endothelial cadherin p-VEC (Tyr658 site) and β-actin (internal control) in cell lysates after different aggregate states of HPC-pet-Aβ treatment. Figure 4 The results of in vivo biocompatibility and blood-brain barrier protection of HPC-pet in Example 4 of the present invention are shown. Among them, (ah) are the statistical results of hematological parameters of mice in different treatment groups, (i) are the EBD fluorescence imaging results of mouse brain tissue, and (j) are the statistical results of EBD fluorescence intensity. Figure 5 The following is a description of the therapeutic effect of HPC-pet on AD model mice in Example 5 of the present invention. (a) is a schematic diagram of the Y-maze experiment, (b) is a schematic diagram of the Y-maze device, (c) is the statistical results of the spontaneous alternation rate of mice in different treatment groups, (d) is the fluorescence imaging results of Aβ plaques in the mouse brain, and (e) is the statistical results of fluorescence intensity. Detailed Implementation

[0034] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0035] Experimental methods not specified in the examples are generally performed under conventional conditions and as described in the manual, or as recommended by the manufacturer. Unless otherwise specified, the general equipment, materials, reagents, etc. used are commercially available.

[0036] In this invention, the KD8 peptide sequence is as follows: KLVFFAED; the JAL-TA9 peptide sequence is as follows: YKGSGFRMI; the Aβ1-6A2V peptide sequence is as follows: DVEFRH; the RW3 peptide sequence is as follows: RWRWRW; all the above peptides were synthesized by the company.

[0037] In this invention, HPC-KD8 and HPC-pet are the same substance and can be used interchangeably.

[0038] Example 1: Preparation of peptide-functionalized cellulose derivative (HPC-KD8) For example, KD8 peptide and hydroxypropyl cellulose (chemical structure as shown) are selected. Figure 1 (as shown in b) An esterification reaction is carried out to obtain peptide-functionalized cellulose derivatives (the specific synthesis and its interaction with Aβ oligomers (Aβo) are as follows). Figure 1 (as shown in a).

[0039] Specifically, it includes the following steps: 1) Take 0.38g of hydroxypropyl-substituted cellulose (HPC, molecular weight approximately 1.0×10⁻⁶). 6 Add 9 mL of deionized water to the solution (g / mol), stir at room temperature for 24 hours, and after complete dissolution, obtain a 4% (w / mol) HPC aqueous solution; 2) Heat the HPC aqueous solution obtained in step 1) to 45°C and keep it at that temperature for 1 hour to obtain an HPC aqueous solution with folded molecular chains; 3) Dissolve 0.02g of KD8 peptide (molecular weight 968.1g / mol) in 1mL of N,N-dimethylformamide (DMF), add 0.012mL of EDC solution, and perform low-power sonication until all KD8 peptide is dissolved. The sonication time should not be too long to prevent KD8 peptide from self-crosslinking; a carboxyl-activated KD8 peptide solution is obtained. 4) Add the carboxyl-activated KD8 peptide solution obtained in step 3) to the HPC aqueous solution obtained in step 2) (the molar ratio of HPC to KD8 peptide in the solution is 1:5.43), and then add 0.01g of DMAP catalyst. React for 12 hours at a temperature of 45℃ and a pH of 7.0. 5) After the reaction is complete, cool to 25°C, centrifuge, and transfer the supernatant into a dialysis bag (molecular weight cutoff 3500 Da). Dialyze with deionized water for 4 days, changing the dialysis solution 3 times a day. 6) The dialysate was freeze-dried to obtain a white, fluffy HPC-pet solid product with a yield of 78%.

[0040] The HPC-pet prepared above was subjected to performance characterization tests, and the results are as follows: Figure 1 As shown.

[0041] from Figure 1 As can be seen from this, HPC-pet is in the range of 1500-2000cm. -1 The presence of a C=O stretching vibration peak indicates that HPC-pet was successfully synthesized. Figure 1 c); Particle size distribution and solution appearance of HPC at different temperatures ( Figure 1 d) and the temperature cycling absorbance change of HPC ( Figure 1 e) The results show that at temperatures above LCST, HPC molecular chains fold and become insoluble in water, while at temperatures below LCST, HPC molecules are better dispersed.

[0042] Furthermore, two-dimensional images obtained by small-angle X-ray scattering (SAXS) were used to demonstrate the evolution of the nanostructures of three samples—HPC, HPC-pet, and HPC-pet-Aβo—from a disordered state to an ordered aggregated state. Figure 1Specifically, unmodified HPC exhibits a long-range, low-order, wide-size ordered structure. Its strong scattering in the low-q region is associated with large-scale polymer aggregates (Rg = 1.6376 nm), which can form aggregates through intermolecular interactions. KD8 peptide coupling modification (HPC-pet) only induces local structural rearrangement of HPC: changes in scattering intensity and adjustment of electron density distribution in specific q-value regions, but retains a large-scale structural framework similar to native HPC (Rg = 1.6966 nm), with a particle size slightly larger than HPC. Aβ oligomer encapsulation (HPC-pet-Aβo) triggers a significant global structural rearrangement of the HPC matrix: a significant decrease in scattering intensity in the low-q region, significant fluctuations in scattering in the medium and high-q regions, and a dramatic increase in the radius of gyration (Rg = 12.5513 nm). The HPC-pet-Aβo system forms a highly ordered, dense nanocomposite with enhanced electron density heterogeneity, confirming that Aβ oligomers can serve as assembly templates, driving the transformation of HPC chains from a dispersed state to an aggregated state, ultimately forming a micellar-like complex. The structural transformation of HPC-pet-Aβo follows a phase separation pattern similar to that of thermosensitive polymers, and the binding of Aβ oligomers is the key external stimulus that triggers this structural transformation.

[0043] Furthermore, the microstructure of HPC-pet, HPC-pet-Aβo, and Aβ samples was visually presented using scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images. Figure 1 (j-1q). Specifically, the pure Aβ monomer system exhibits typical time-dependent fibrillation evolution characteristics, gradually transforming from free monomers into oligomers, flower-like protofibril aggregates, and ultimately forming an ordered linear fibrous structure rich in β-sheets. Pre-addition of HPC-pet completely blocks the maturation and fibrillation process of Aβ; even after 96 hours of incubation, no linear fibrous structure appears in the system. HPC-pet can specifically target and bind to Aβ, inhibiting its elongation and ordered assembly, promoting the formation of spherical HPC-pet-Aβo micelle complex structures (particle size 50~200 nm). TEM directly confirms that the HPC-pet-Aβo micelle complex can be successfully assembled and can effectively encapsulate Aβ amyloid aggregates within its nanostructure, leaving only a small amount of amorphous protein fragments.

[0044] Example 2: Inhibitory effect of HPC-pet on Aβ aggregation In this embodiment, the inhibitory effect of HPC-pet on Aβ aggregation was verified in vitro. Specifically, the following steps were included: 1) Prepare a 10 μM solution of Aβ monomer (Aβm) and divide it into two groups: the experimental group is treated with an equimolar amount of HPC-pet, while the control group is not treated. 2) Both solutions were incubated in a 37℃ constant temperature incubator, and samples were taken at 0, 12, 24, 36, 48, 96, and 180 hours, respectively. 3) The particle size change of the sample was detected by DLS and the degree of Aβ fibrosis was detected by ThT fluorescence method.

[0045] Test results as follows Figure 2 As shown.

[0046] from Figure 2 As can be seen, the particle size of Aβ in the control group gradually increased with incubation time, reaching 955.4 nm after 180 hours, and the ThT fluorescence intensity significantly increased, forming mature fibers; the particle size of the HPC-pet-Aβ complex in the experimental group remained at 28-615 nm, and the ThT fluorescence intensity did not increase significantly. Figure 2 a-2c); The HPC-pet-Aβ complex can significantly reduce Aβ deposition at the sensing interface by inhibiting Aβ aggregation, thereby effectively mitigating wavelength shift, and its inhibitory effect increases with the extension of complex incubation time. Figure 2 (d-2g). Specifically, the aggregation state dependence of targeted binding: HPC-pet has a strong binding effect on Aβ oligomers, oligomer-protofibril transition aggregates, and early 36h fibroblasts, with the strongest binding effect on Aβo-p; the binding ability on mature Aβ fibroblasts at 48h and 180h is significantly reduced because the mature fibroblasts are too large and easily detach after binding with HPC-pet. Differences in aggregation state of encapsulation efficiency: HPC-pet can completely encapsulate Aβ oligomers and oligomer-protofibril transition aggregates, with no Aβ protein exposed. Therefore, free HPC-pet has no significant binding effect on the pre-formed corresponding complex; the encapsulation efficiency on early 36h Aβ fibroblasts is poor, with exposed protein, allowing free HPC-pet to bind and produce a significant binding shift. Encapsulation characteristics of mature fibrils: The pre-formed HPC-pet complexes with 8h and 180h mature Aβ fibrils were easily detached from the sensor surface due to their large particle size, and no significant free HPC-pet binding signal was detected. Core evidence: The differential binding kinetics of HPC-pet with different aggregate states of Aβ directly confirms its specific affinity for Aβ oligomers and protofibril intermediate aggregates, providing molecular interaction-level evidence for its targeted action on pathogenic Aβ intermediate aggregates.

[0047] Example 3: Inhibition of APEL effect by HPC-pet In this embodiment, the inhibitory effect of HPC-pet on the APEL effect was verified in vitro. Specifically, the following steps were included: 1) Culture HUVEC cells to a confluent monolayer to construct an endothelial cell barrier model (illustrated as shown in the diagram). Figure 3 (as shown in a) 2) The experiment was divided into three groups: the control group was treated with PBS, the model group was treated with Aβ oligomer (Aβo-p), and the experimental group was treated with HPC-pet-Aβo-p complex. 3) After 24 hours of incubation, endothelial permeability was assessed using a Transwell assay (see diagram). Figure 3 (as shown in e), fluorescent staining was used to observe intercellular spaces, and Western blot was used to detect the phosphorylation level of VE-cadherin at site Y658. Test results as follows Figure 3 As shown.

[0048] from Figure 3 As can be seen, the fluorescence intensity in the lower chamber of the Transwell in the model group was significantly increased, the intercellular space was enlarged, and the phosphorylation level of VE-cadherin was increased; the above indicators in the experimental group were not significantly different from those in the control group. The results indicate that HPC-pet effectively inhibited the APEL effect. Figure 3 (a-3k). Further analysis shows that HPC-pet can form protective micelles outside Aβ oligomers / fibrils, exhibiting excellent anti-protein adsorption properties and completely inhibiting the formation of the protein crown of this type of Aβ aggregate; while HPC-pet-Aβ fibrous aggregates still show significant protein adsorption. Combined particle size analysis and electrophoresis data indicate that the core therapeutic activity of HPC-pet lies in its ability to encapsulate neurotoxic Aβ oligomers / fibrils without a protein crown, a characteristic crucial for maintaining the integrity of the vascular endothelial barrier. Western blot experiments confirmed that pure Aβ aggregates can induce phosphorylation at the Y658 site of VE-cadherin by activating Src family kinases, thereby disrupting endothelial junctions; while HPC-pet-encapsulated Aβ oligomers / fibrils significantly inhibit this phosphorylation process, with phosphorylation of oligomers-fibrils being almost completely blocked.

[0049] Example 4: In vivo biocompatibility and blood-brain barrier protection of HPC-pet In this embodiment, the in vivo biocompatibility and blood-brain barrier protective effect of HPC-pet were verified in vivo. Specifically, the following steps were included: This in vivo experiment used mice as the experimental subjects and was conducted in two independent parts: biocompatibility verification and blood-brain barrier protection verification. The specific operation steps are as follows: 1) In vivo biocompatibility verification steps of the HPC-pet and Aβ complex Experimental groups: Experimental groups were set up containing Aβ monomers (Aβm), Aβ oligomers (Aβo), Aβ oligomer-fibrils (Aβo-p), and complexes formed by HPC-pet and the above three Aβ groups (HPC-pet-Aβm, HPC-pet-Aβo, HPC-pet-Aβo-p). Each group consisted of mouse subjects. Administration method: The above experimental samples were administered to the corresponding groups of mice via tail vein injection; Sample collection: Blood samples were collected from mice in each group 24 hours after drug administration; Detection indicators and methods: Complete blood cell count analysis was performed on blood samples to detect changes in the concentration of key blood indicators such as white blood cells, lymphocytes, monocytes, neutrophils, red blood cells, hemoglobin, and platelets.

[0050] 2) Verification steps for the protective effect of HPC-pet against Aβ-induced blood-brain barrier injury Experimental grouping: Mice were divided into multiple groups, including a pure Evans Blue (EBD) solution treatment group, an EBD solution treatment group containing Aβm / Aβo / Aβo-p treatment group, and a treatment group that was injected with Aβm / Aβo / Aβo-p first and then injected with HPC-pet 1 hour later. Administration method: All experimental reagents were administered to the corresponding groups of mice via tail vein injection. The combined administration group strictly followed the administration sequence of "Aβ first, followed by HPC-pet after 1 hour". Detection method: Fluorescence imaging of the brain of mice was performed after drug administration; Quantitative indicators: The fluorescence intensity of EBD in the brain tissue of mice in each group was analyzed and compared, and the fluorescence intensity reflected the permeability and degree of damage of the blood-brain barrier.

[0051] Test results as follows Figure 4 As shown.

[0052] Figure 4 The results showed that the HPC-pet-Aβ complex exhibited good potential therapeutic value in Aβ-related diseases, not only alleviating Aβ-induced inflammatory responses at the blood level and significantly reducing Aβ accumulation and deposition in the brain, but also having no significant impact on normal hematopoietic function. Figure 4 (a-4j). Specifically, after tail vein injection of Aβ monomers, Aβ oligomers, Aβ oligomer-fibrils, and the complex formed by HPC-pet and the above three Aβ groups, key blood parameters in mice, such as white blood cells, red blood cells, and platelets, fluctuated slightly within the normal range without significant abnormalities. This result confirms that the complex formed by HPC-pet and Aβ has no significant blood toxicity and possesses good biocompatibility, laying the foundation for its in vivo application. Figure 4Further research showed that HPC-pet can effectively protect the structural and functional integrity of the blood-brain barrier: One hour after injection of Aβ oligomers / fibrils, HPC-pet treatment significantly reduced the Evans blue (EBD) fluorescence intensity in mouse brain tissue compared to the untreated group, suggesting that HPC-pet can reverse the increased blood-brain barrier permeability induced by Aβ oligomers / fibrils. Figure 4 This phenomenon also reveals that Aβ damage to the blood-brain barrier is aggregation-dependent—Aβ monomers do not disrupt the integrity of the blood-brain barrier, while Aβ oligomers and fibrils significantly increase its permeability, and the degree of damage intensifies with increasing Aβ aggregation. From a mechanistic perspective, after in vivo administration, HPC-pet can specifically capture Aβ oligomers in the mouse bloodstream: on the one hand, it reduces the harmful impact on the blood-brain barrier, and on the other hand, it prevents further Aβ aggregation, inhibiting the Aβ-induced APEL effect at its source, thereby protecting the blood-brain barrier. In summary, HPC-pet can selectively capture Aβ oligomers in vivo, effectively inhibiting Aβ-induced blood-brain barrier damage, exhibiting both good biocompatibility and targeted protective effects, fully demonstrating its potential therapeutic value in Aβ-related diseases.

[0053] Example 5: Therapeutic effect of HPC-pet on AD model mice In this embodiment, the therapeutic effect of HPC-pet on AD model mice was verified in vivo. Specifically, the following steps were included: 1) Six-month-old 5XFAD mice were selected as AD models, and wild-type (WT) mice were used as controls. The AD mice were randomly divided into model group and treatment group, with 10 mice in each group. 2) The treatment group received a tail vein injection of HPC-pet solution (2.5 μM, 100 μL) on days 5, 10, 15 and 20, while the model group and the control group received an equal volume of physiological saline. 3) Conduct the Y-maze experiment on day 0 and day 25 (experimental procedure as follows) Figure 5 As shown in Figure a, the experimental setup is as follows: Figure 5 (as shown in b), to detect the spatial working memory of mice; 4) On day 25, the Aβ-specific probe CRANAD-2 (synthesized by the company) was injected via the tail vein, and the fluorescence intensity of Aβ plaques in the brain was detected by in vivo imaging. Test results as follows Figure 5 As shown from Figure 5 It can be seen that the spontaneous alternation rate of mice in the treatment group was 12.7% higher than that in the model group. Figure 5 c), the fluorescence intensity of Aβ plaques in the brain decreased by 44% ( Figure 5 (d) The results showed that HPC-pet could significantly improve cognitive function in AD model mice.

[0054] In summary, the peptide-functionalized cellulose derivative obtained by linking Aβ-targeting peptides to hydroxypropyl cellulose via ester bonds can specifically bind to Aβ proteins through molecular recognition and self-assemble into core-shell structured nanomicelles, efficiently encapsulating Aβ oligomers, inhibiting Aβ aggregation and fibrosis. At the same time, it blocks the interaction between Aβ and endothelial cell junction proteins through its antifouling properties, inhibits the amyloid protein-induced endothelial leakage (APEL) effect, protects the integrity of the blood-brain barrier, and has good biocompatibility. It can reduce Aβ plaque deposition in the brain and improve cognitive function in AD patients.

[0055] It should be noted that all the above embodiments belong to the same inventive concept, and the descriptions of each embodiment have different focuses. Where the description in a particular embodiment is not detailed, please refer to the description in other embodiments.

[0056] The embodiments described above are merely illustrative of implementation methods of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A peptide-functionalized cellulose derivative targeting Aβ, characterized in that, The peptide-functionalized cellulose derivative includes hydroxypropyl cellulose and an Aβ-targeting peptide, wherein the hydroxyl groups in the hydroxypropyl cellulose and the carboxyl groups in the Aβ-targeting peptide are linked by ester bonds. The Aβ targeting peptide includes at least one of KD8 peptide, JAL-TA9 peptide, Aβ1-6A2V peptide, and RW3 peptide.

2. The peptide-functionalized cellulose derivative according to claim 1, characterized in that, The molar ratio of hydroxypropyl cellulose to the Aβ-targeting peptide is 1:(5-20).

3. A method for preparing a peptide-functionalized cellulose derivative as described in claim 1 or 2, characterized in that, Includes the following steps: The Aβ-targeting peptide solution was activated to obtain a carboxyl-activated Aβ-targeting peptide solution. The aqueous solution of hydroxypropyl cellulose was heated to a first temperature and then kept at that temperature to obtain an aqueous solution of hydroxypropyl cellulose with folded molecular chains. The carboxyl-activated Aβ-targeting peptide solution was mixed with the aqueous solution of hydroxypropyl cellulose with folded molecular chains, and DMAP was added to carry out an esterification reaction. After the reaction was completed, the reaction solution was cooled to a second temperature, and after centrifugation, dialysis and drying, the peptide-functionalized cellulose derivative was obtained. Wherein, the first temperature is higher than the minimum critical dissolution temperature of hydroxypropyl cellulose, and the second temperature is lower than the minimum critical dissolution temperature of hydroxypropyl cellulose.

4. The preparation method according to claim 3, characterized in that, In the step of obtaining the carboxyl-activated Aβ-targeting peptide solution, the Aβ-targeting peptide solution is obtained by dissolving the Aβ-targeting peptide in an organic solvent, and the mass concentration of the Aβ-targeting peptide solution is 1-3%. The activation treatment includes: adding an EDC solution and then performing low-power ultrasonic dissolution; wherein the amount of the added EDC solution accounts for 1-2% of the volume of the organic solvent.

5. The preparation method according to claim 3, characterized in that, In the step of obtaining the aqueous solution of hydroxypropyl cellulose with folded molecular chains, the mass concentration of the aqueous solution of hydroxypropyl cellulose is 0.5-5%, and the heat preservation time is 1-3 hours.

6. The preparation method according to claim 3, characterized in that, In the step of obtaining the peptide-functionalized cellulose derivative, the volume ratio of the carboxyl-activated Aβ-targeting peptide solution to the aqueous solution of the molecularly folded hydroxypropyl cellulose is (8-10):

1. The amount of DMAP added is 2-4% of the mass of the hydroxypropyl cellulose; The esterification reaction includes reacting for 12-24 hours at a temperature of the first temperature and a pH of 6.5-7.

5.

7. The preparation method according to claim 3, characterized in that, In the step of obtaining the peptide-functionalized cellulose derivative, the dialysis includes: transferring the reaction solution into a dialysis bag, dialyzing with deionized water for 3-5 days, and changing the dialysis solution 2-4 times a day; The molecular weight cutoff of the dialysis bag is 3400-3600 Da.

8. The use of the peptide-functionalized cellulose derivative as described in claim 1 or 2, or the peptide-functionalized cellulose derivative prepared by any one of claims 3-7, in the preparation of drugs for the prevention or treatment of Alzheimer's disease.

9. The application according to claim 8, characterized in that, The drug also includes a pharmaceutically acceptable carrier.

10. The application according to claim 8 or 9, characterized in that, The drug has at least one of the following functions: A1) Inhibits the aggregation and fibrosis of Aβ protein; A2) Encapsulates Aβ oligomers and reduces their neurotoxicity; A3) Inhibits amyloid-induced endothelial leakage; A4) Protects the integrity of the blood-brain barrier; A5) Reduces Aβ plaque deposition in the brain; A6) Improves cognitive function in Alzheimer's patients.

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