Self-assembled polypeptide-rhein conjugate, preparation as well as preparation method and application of self-assembled polypeptide-rhein conjugate

Abstract

The invention relates to the technical field of biological medicines, and provides a self-assembled polypeptide-rhein conjugate, a preparation as well as a preparation method and application of the self-assembled polypeptide-rhein conjugate. The sequence of the MMP-9 responsive self-assembled polypeptide-rhein conjugate is Rhein-GFFPLGLAGACE, and the MMP-9 responsive self-assembled polypeptide-rhein conjugate can respond to abnormally activated MMP-9 in atherosclerotic plaque and is self-assembled into a stable nanofiber structure in situ at a diseased region; the anti-inflammatory and anti-oxidation functions of rhein in the conjugate are remarkable, but the water solubility and oral bioavailability are low, the anti-inflammatory and anti-oxidation functions are remarkably improved after being coupled with the polypeptide, targeted delivery is achieved, and the residence time is prolonged; the introduction of the tripeptide ACE simulating the function of glutathione further enhances the oxidative stress resistance of the conjugate. Furthermore, the conjugate shows excellent anti-inflammatory and anti-oxidation effects, can clear ROS, inhibit inflammation pathways, regulate macrophage phenotypes and inhibit apoptosis, and provides a new strategy for treatment of atherosclerosis.

Description

Technical Field

[0001] The present invention relates to the technical field of biopharmaceuticals, and in particular to a self-assembling polypeptide-rhein conjugate, a preparation thereof, and a preparation method and application thereof. Background Art

[0002] Atherosclerosis (AS) is a chronic, progressive vascular inflammatory disease characterized by lipid deposition, endothelial dysfunction, immune inflammatory responses, and vascular remodeling. In the early stages of the disease, damaged endothelial cells release a variety of inflammatory mediators, continuously recruiting inflammatory cells such as monocytes to migrate into the vessel wall, thereby triggering an inflammatory cascade. During this process, macrophages, regulated by the local microenvironment (e.g., cytokines, lipid composition, and oxidative stress), transform into a strongly pro-inflammatory M1 phenotype. This transformation further amplifies the inflammatory response, exacerbating vascular wall damage and reducing plaque stability. During the progressive stage of the disease, the primary inflammatory process is accompanied by a secondary event, oxidative stress. Oxidative stress leads to excessive accumulation of reactive oxygen species (ROS), which in turn aggravates endothelial dysfunction and increases vascular permeability, ultimately creating a vicious cycle of inflammation and oxidative stress, further exacerbating the local inflammatory response. Therefore, developing therapeutic agents to alleviate oxidative stress and inflammatory responses within plaques is crucial for improving the therapeutic efficacy of AS.

[0003] MMP-9 is a matrix metalloproteinase that degrades collagen and the extracellular matrix (ECM). It is primarily secreted by macrophages and neutrophils. Its elevated expression exacerbates ECM degradation, inflammatory responses, and thinning of the fibrous cap, increasing the risk of plaque rupture. Studies have shown that MMP-9 expression continues to increase during AS progression, and the degree of increase is closely correlated with plaque instability and rupture risk. Therefore, MMP-9 is not only an important effector of AS progression but is also considered a potential diagnostic and intervention target.

[0004] The natural drug rhein has significant anti-inflammatory and antioxidant properties, but its clinical application is limited by its poor water solubility, low oral bioavailability, and poor targeting. Therefore, the development of efficient, targeted, and sustained-release drug carriers for rhein is a key issue for its application in AS treatment. Summary of the Invention

[0005] The present invention aims to provide a self-assembling polypeptide-rhein conjugate, formulation, preparation method, and application thereof. This polypeptide comprises an anti-inflammatory drug-terminated "Rhein," a self-assembly module "GFF," a corresponding MMP-9 (matrix metalloproteinase-9) cleavage sequence "PLGLAG," and a glutathione-like functional tripeptide "ACE" with excellent free radical scavenging ability. The resulting polypeptide-rhein conjugate can form a self-assembled nanofiber network structure after MMP-9 cleavage in a high-MMP-9 environment. The released Rhein and ACE can effectively alleviate the inflammatory response and oxidative stress in the AS microenvironment. This material can stabilize and treat vascular AS plaques and has great potential for the development of new drugs for the treatment of AS.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides an enzyme-responsive anti-inflammatory self-assembling polypeptide-rhein conjugate, the structural formula of the conjugate is:

[0008]

[0009] Preferably, the conjugate is synthesized using Fmoc-solid phase synthesis.

[0010] The present invention also provides the use of the conjugate in preparing a drug for treating chronic progressive vascular wall inflammatory diseases.

[0011] Preferably, the chronic progressive inflammatory disease of the blood vessel wall is atherosclerosis.

[0012] The present invention also provides a medicine containing the conjugate, wherein the concentration of the conjugate in the medicine is 20 to 100 μM.

[0013] Preferably, the drug is directed against chronic progressive vascular wall inflammatory diseases.

[0014] Preferably, the chronic progressive inflammatory disease of the blood vessel wall is atherosclerosis.

[0015] Peptide drug conjugates (PDCs) are an emerging molecular drug delivery framework, typically consisting of a peptide, a drug, and a linker. Because the amino acid sequence can be flexibly regulated, structural modifications can be easily introduced to support rational drug design, thereby enhancing bioavailability and binding affinity. On the one hand, PDCs can serve as scaffolds for drug delivery, improving drug solubility, stability, and controlled release. On the other hand, PDCs can be designed as covalently bound ligand peptides that target specific cell surface receptors or biomarkers, thereby producing a lasting effect, extending the time effect, and conferring ideal pharmacokinetic characteristics. Therefore, designing peptide sequences to target highly expressed parts on diseased cells and precisely deliver drugs to target organs constitutes an effective therapeutic strategy.

[0016] The present invention obtains for the first time a polypeptide-rhein conjugate named Rh-MRSP that scavenges ROS and reduces inflammatory responses through a method of synthesizing polypeptides. The conjugate effectively inhibits the production of ROS in macrophages and the expression of inflammatory factors (TNF-α, IL-1β and IL-6) by blocking the activation of the TLR4 / NF-κB signaling pathway, promotes their transformation to the M2 phenotype, and regulates the mitochondrial function of macrophages to reduce cell apoptosis, thereby playing a role in stabilizing and treating AS.

[0017] The peptide-rhein conjugate, named Rh-MRSP, has the sequence Rhein-GFFPLGLAGACE (SEQ ID NO. 1). This conjugate can respond to abnormally activated matrix metalloproteinase-9 (MMP-9) in atherosclerotic plaques and self-assemble in situ into a stable nanofiber network structure at the lesion site. Rhein has significant anti-inflammatory and antioxidant properties, but its poor water solubility and low oral bioavailability limit its clinical application. By conjugating it with a self-assembling peptide, the water solubility and bioavailability of rhein are significantly improved, achieving targeted drug delivery and prolonging its retention time at the lesion site. In addition, the introduction of the tripeptide ACE, which mimics the function of glutathione, further enhances the peptide's ability to resist oxidative stress. This conjugate demonstrated significant anti-inflammatory and anti-oxidative stress activities in both in vitro and in vivo experiments, scavenging reactive oxygen species (ROS) levels in the microenvironment, inhibiting the activation of inflammatory signaling pathways, promoting macrophage phenotypic transformation, and inhibiting apoptosis, providing a new strategy and approach for the treatment of atherosclerosis.

[0018] The beneficial effects of the present invention are:

[0019] First, Rh-MRSP has a low critical self-assembly concentration and is inexpensive to synthesize. Second, in addition to its ability to precisely accumulate in AS plaques based on the high expression of MMP-9 in the AS microenvironment, Rh-MRSP can also form self-assembled nanofibers under the catalysis of overexpressed MMP-9 in the AS plaque microenvironment, thereby increasing drug concentration and bioavailability at the lesion site. Rh-MRSP is the first peptide-rhein conjugate discovered to stabilize and treat AS plaques. In in vivo experiments, Rh-MRSP significantly reduced the distribution of AS plaques in aortic tissue, decreased the area of ​​the necrotic core within the plaques, and increased the collagen content of the plaques by inhibiting inflammation and oxidative stress, thereby achieving the goal of treating AS.

[0020] Experiments have confirmed that the polypeptide-rhein conjugate designed in this invention has a good MMP-9 response and the ability to treat AS plaques. The improvement of inflammation and oxidative stress by combining the anti-inflammatory drug "Rhein," the self-assembly initiating unit "GFF," and the tripeptide unit "ACE" with excellent free radical scavenging ability with the "PLGLAG" MMP-9 enzyme response is a major advancement in the treatment of chronic progressive inflammatory diseases of the vascular wall. Rh-MRSP addresses Rhein's poor water solubility, low oral bioavailability, and poor targeting properties, significantly prolonging its retention time at the lesion site and increasing drug concentration and bioavailability at the lesion site. In this way, Rh-MRSP can not only effectively reduce the inflammatory response in AS plaques, but also, through its anti-oxidative stress properties, inhibit plaque development and improve plaque stability, providing a new therapeutic strategy and effective technical support for the treatment of AS. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is the mass spectrum of Rh-MRSP;

[0022] Figure 2 Mass spectrum of MRSP

[0023] Figure 3 Electron microscope micromorphology of Rh-MRSP and MRSP before and after enzyme digestion;

[0024] Figure 4 is the critical self-assembly concentration before and after Rh-MRSP cleavage;

[0025] Figure 5 The effect of Rh-MRSP on the survival rate of RAW264.7 cells;

[0026] Figure 6 Laser confocal colocalization and flow cytometry were used to detect the ROS scavenging ability of Rh-MRSP;

[0027] Figure 7Laser confocal co-localization was used to detect the anti-apoptotic ability of Rh-MRSP. DETAILED DESCRIPTION

[0028] The present invention provides an enzyme-responsive anti-inflammatory self-assembling polypeptide-rhein conjugate, the structural formula of the conjugate is:

[0029]

[0030] In the present invention, the conjugate is preferably synthesized using Fmoc-solid phase synthesis method.

[0031] The present invention also provides the use of the conjugate in preparing a drug for treating chronic progressive vascular wall inflammatory diseases.

[0032] In the present invention, the chronic progressive vascular wall inflammatory disease is preferably atherosclerosis.

[0033] The present invention also provides a medicine containing the conjugate, wherein the concentration of the conjugate in the medicine is 20 to 100 μM.

[0034] In the present invention, the concentration of the conjugate in the drug is preferably 20 to 100 μM, more preferably 20 to 60 μM, and even more preferably 40 μM.

[0035] In the present invention, the drug is directed to chronic progressive vascular wall inflammatory diseases.

[0036] In the present invention, the chronic progressive vascular wall inflammatory disease is atherosclerosis.

[0037] The amino acids and dichlororesin used in the following examples of Rh-MRSP and control examples of MRSP were obtained from Shanghai Jier Biochemical Co., Ltd. All other raw reagents and materials were commercially available. Experimental methods without specific conditions are conventional methods and conditions well known in the art.

[0038] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0039] Example Synthesis of Peptide-Rhein Conjugate Rh-MRSP (Rh-GFFPLGLAGACE)

[0040] The polypeptide-rhein conjugate Rh-MRSP in this example was synthesized using the Fmoc-solid phase synthesis method, and the specific steps are as follows:

[0041] 1) Weigh 0.516 g of dichlororesin, add it to the reactor, and swell it with DCM (dichloromethane) for 20 minutes. Then remove the DCM with an ear bulb;

[0042] 2) A mixture of the first amino acid and condensing agent 1 mmol DIEA (N,N-diisopropylethylamine) was dissolved in 12 ml dichloromethane and added to the reactor for 2 h;

[0043] 3) Wash with DCM 5 times, and react with a prepared solution of DCM:MEOH (methanol):DIEA = 17:2:1 at room temperature for 15 min.

[0044] 4) Cleavage was performed using a 20% piperidine / DMF (N,N-dimethylformamide) solution for 30 min.

[0045] 5) Weigh another 1 mmol of amino acid: 2 mmol of condensing agent: 1 mmol of DIEA mixture, dissolve in 15 mL of DMF, and add to the reactor. React for 2 h, wash with DMF 5 times, and cleave with 20% piperidine (repeat this step for the amino acids added later).

[0046] 6) After the last amino acid is reacted and cut, wash five times with DMF and five times with DCM

[0047] 7) Add a cutting agent: 95% TFA (trifluoroacetic acid) + 2.5% H2O2 + 2.5% TIS (triisopropylsilane), add the cutting agent into the reactor, and cut for 45 minutes.

[0048] 8) Collect the cutting agent and evaporate it to dryness using a rotary evaporator until an oily substance is left in the bottle.

[0049] 9) Anhydrous ether was added to the evaporated material, and solid precipitated. The ether was drained and the solid was collected as a crude product.

[0050] 10) The crude product was purified by HPLC to obtain pure polypeptide powder.

[0051] The results were detected by high resolution mass spectrometry. Figure 1 .from Figure 1 It can be seen that the obtained polypeptide structure is correct, and the polypeptide structure is shown in Formula I.

[0052]

[0053] Preparation of conjugate active solution:

[0054] 1) Dissolve 1.45 mg of pure conjugate powder in 1 mL of PBS to obtain a conjugate solution, with the final concentration of the conjugate solution being 1 mM.

[0055] 2) In subsequent experiments, the prepared conjugate solution was diluted appropriately as needed.

[0056] 3) When measuring the response properties of the conjugate in vitro: the conjugate solution was mixed with MMP-9 and reacted for 24 hours to obtain a conjugate active solution, wherein the amount of MMP-9 added was 500 ng / mL.

[0057] Synthesis of the control peptide sequence MRSP (GFFPLGLAGACE)

[0058] The polypeptide sequence MRSP in this reference example was synthesized using the Fmoc-solid phase synthesis method, with the following specific steps:

[0059] 1) Weigh 0.516 g of dichlororesin, add it to the reactor, and swell it with DCM for 20 minutes, then remove the DCM with an ear bulb;

[0060] 2) A mixture of the first amino acid and 1 mmol of DIEA as a condensing agent was dissolved in 12 ml of dichloromethane and added to the reactor for 2 h;

[0061] 3) Wash with DCM 5 times, and react with a prepared solution of DCM:MEOH:DIEA=17:2:1 at room temperature for 15 min.

[0062] 4) Cleavage with a 20% piperidine / DMF solution for 30 min.

[0063] 5) Weigh another 1 mmol of amino acid: 2 mmol of condensing agent: 1 mmol of DIEA mixture, dissolve in 15 mL of DMF, and add to the reactor. React for 2 h, wash with DMF 5 times, and cleave with 20% piperidine (repeat this step for the amino acids added later).

[0064] 6) After the last amino acid is reacted and cut, wash five times with DMF and five times with DCM

[0065] 7) Add cutting agent: 95% TFA + 2.5% H2O2 + 2.5% TIS, add the cutting agent into the reactor and cut for 45 minutes.

[0066] 8) Collect the cutting agent and evaporate it to dryness using a rotary evaporator until an oily substance is left in the bottle.

[0067] 9) Anhydrous ether was added to the evaporated material, and solid precipitated. The ether was drained and the solid was collected as a crude product.

[0068] 10) The crude product was purified by HPLC to obtain pure polypeptide powder.

[0069] The results were detected by high resolution mass spectrometry. Figure 2 .from Figure 2 It can be seen that the obtained polypeptide structure is correct, and the polypeptide structure is shown in Formula II.

[0070]

[0071] Preparation of peptide active solution:

[0072] 1) Dissolve 1.1 mg of pure peptide powder in 1 mL of PBS to obtain a peptide solution with a final concentration of 1 mM.

[0073] 2) In subsequent experiments, dilute the prepared peptide solution appropriately as needed.

[0074] 3) When measuring the response properties of the polypeptide in vitro: the polypeptide solution was mixed with MMP-9 and reacted for 24 hours to obtain a polypeptide active solution, wherein the amount of MMP-9 added was 500 ng / mL.

[0075] Experimental Example 1 Transmission Electron Microscopy Experiment

[0076] Rh-MRSP was dissolved in PBS buffer (pH 7.4) to a final concentration of 1 mM. An MMP-9-treated group (500 ng / mL of MMP-9) and an untreated control group (no MMP-9) were set up. Next, two Rh-MRSP solutions, one without MMP-9 and one with MMP-9, were dripped onto a copper grid and stained with phosphotungstic acid. After sample preparation, the micromorphology of the samples was observed using a transmission electron microscope (Talos L120CG2). The results are shown in Figure 2. Figure 3 .

[0077] from Figure 3 As can be seen in the figure, the Rh-MRSP solution without MMP-9 treatment appears as discrete clusters of particles under the TME; however, after 24 hours of treatment with MMP-9, the microscopic structure of the Rh-MRSP solution transformed into a fibrous network. In the control example, at the same experimental concentration, the Rh-MRSP without MMP-9 treatment did not form a distinct microstructure, while the Rh-MRSP treated with MMP-9 formed worm-like nanostructures.

[0078] Experimental Example 2 Determination of critical micelle concentration (CMC)

[0079] Rh-MRSP was dissolved in PBS buffer (pH 7.4) to a final concentration of 1mM. An MMP-9 treatment group (MMP-9 500ng / mL) and an untreated control group (no MMP-9) were set up. A peptide concentration gradient solution was then prepared by gradient dilution. The scattered light intensity of the solution was detected using DLS technology at a constant temperature of 25°C. The CMC values ​​of the two groups of solutions were calculated by plotting a particle size-concentration logarithmic curve, taking the inflection point of the sudden increase in particle size as the CMC value, and comparing the CMC changes before and after enzyme treatment. The results are shown in Figure 2. Figure 4As shown, the CMC of Rh-MRSP without enzyme digestion was 30.41 μΜ, while that of Rh-MRSP treated with MMP-9 was significantly reduced to 11.16 μΜ. It was also confirmed that MMP-9 could promote the assembly of polypeptides, making them transform into fibers and increase the enrichment at the atherosclerotic plaque.

[0080] Experimental Example 3 Effect of Rh-MRSP on the survival rate of RAW264.7 cells

[0081] The CCK-8 (Cell Counting Kit-8) kit was used to determine the cell survival rate after different concentrations (5, 10, 20, 40, 80 μΜ) of MRSP, Rhein and Rh-MRSP were respectively co-cultured with RAW264.7 for 24 h, in order to evaluate the biocompatibility of the materials. The experimental results are shown in Figure 5 As shown, at a concentration of 40 μΜ or below, the three substances had no significant effect on the survival rate of RAW264.7 cells, indicating that they had good biocompatibility. However, when the concentration increased to 80 μΜ, free Rhein showed obvious cytotoxicity, while MRSP and Rh-MRSP had no significant effect on cell activity even at this high concentration. This result suggests that after Rhein is coupled with MMP-9 responsive self-assembly polypeptide to form a drug delivery system, the toxicity of Rhein is significantly reduced, and the biocompatibility is better.

[0082] Experimental Example 4 Laser confocal co-localization and flow cytometry detection of the ROS (reactive oxygen species) scavenging ability of Rh-MRSP

[0083] RAW264.7 cells were pretreated with MRSP, Rhein and Rh-MRSP at a concentration of 40 μΜ for 4 h, and then the pretreated RAW264.7 cells were added to the LPS-containing medium at a concentration of 1 μg / mL for 24 h, washed with PBS for 3 times, and then added to the serum-free culture solution containing 10 μΜ DCFH-DA (2', 7'-dichlorofluorescein diacetate), and incubated in the dark for 30 min. After incubation, the cells were washed with serum-free culture medium for 3 times, and each washing was carried out in the dark. The cells were observed and photographed by confocal microscope, and the change of the intracellular reactive oxygen species level was analyzed by fluorescence intensity. As shown in Figure 6As shown in the figure, the fluorescence intensity of the LPS group cells stimulated by 1 μg / mL LPS for 24 h was significantly enhanced compared with that of the normal cells, showing that a large amount of ROS was generated in the cells, while the green fluorescence intensity in the cells of the MRSP, Rhein and Rh-MRSP groups was reduced to different degrees, and the ROS scavenging ability of Rh-MRSP was the strongest, which indicated that the drug delivery of Rhein coupled with the polypeptide was more conducive to the exertion of the anti-oxidative stress effect. In order to further determine the level of ROS in the cells, flow cytometry was used for quantitative analysis, and the experimental results were consistent with the qualitative observation results of the laser confocal microscope, thereby further confirming that Rh-MRSP had a strong anti-oxidative stress ability.

[0084] Experimental Example 5 Laser confocal co-localization detection of the anti-apoptosis ability of Rh-MRSP

[0085] RAW264.7 cells were pretreated with MRSP, Rhein and Rh-MRSP at a concentration of 40 μM for 4 h in advance, and then the pretreated RAW264.7 cells were added with 200 μM H2O2 for stimulation for 8 h. After the stimulation, the cells were gently blown with PBS buffer for 3 times. AM / PI staining kit was used for live and dead staining of the cells. The stained cells were incubated in a 37℃ incubator for 30 min in the dark, so that the dye could fully penetrate and label the cells, and then the cells were observed and image acquisition was performed by a confocal microscope. The results are shown in Figure 7 As shown in the figure, the cell survival rate of the Rh-MRSP group was the highest.

[0086] It can be known from the above examples that the self-assembled polypeptide-rhein conjugate, the preparation thereof and the preparation method and application thereof are provided. The MMP-9 responsive self-assembled polypeptide-rhein conjugate of the present application has a sequence of Rhein-GFFPLGLAGACE, can respond to the abnormally activated MMP-9 in the atherosclerotic plaque, and self-assembles into a stable nanofiber structure in situ at the lesion site; the anti-inflammatory and antioxidant functions of rhein in the conjugate are significant, but the water solubility and oral bioavailability are low, which are significantly improved after coupling with the polypeptide, realizing targeted delivery and prolonging the residence time; and the introduction of the tripeptide ACE simulating the function of glutathione further enhances the anti-oxidative stress ability of the conjugate. Further, the conjugate shows excellent anti-inflammatory and antioxidant effects, can scavenge ROS, inhibit inflammation and inhibit macrophage apoptosis, and provides a new strategy for atherosclerosis treatment.

[0087] The above only describes the preferred embodiments of the present application, and it should be noted that, for those skilled in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. An enzyme-responsive anti-inflammatory self-assembling polypeptide-rhein conjugate, characterized in that: The structural formula of the conjugate is:

2. The conjugate according to claim 1, characterized in that The conjugate was synthesized using Fmoc-solid phase synthesis method.

3. Use of the conjugate according to any one of claims 1 to 2 in the preparation of a drug for treating chronic progressive vascular wall inflammatory diseases.

4. The use according to claim 3, characterized in that The chronic progressive vascular wall inflammatory disease is atherosclerosis.

5. A drug containing the conjugate according to any one of claims 1 to 2, characterized in that: The concentration of the conjugate in the drug is 20-100 μM.

6. The drug according to claim 5, characterized in that The drug targets chronic progressive inflammatory diseases of the blood vessel walls.

7. The drug according to claim 6, characterized in that The chronic progressive vascular wall inflammatory disease is atherosclerosis.

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