Self-assembled polypeptide-rheic acid conjugates, formulations, preparation methods and applications

Abstract

This invention relates to the field of biopharmaceutical technology, providing a self-assembled peptide-rhein conjugate, its formulation, preparation method, and applications. The MMP-9-responsive self-assembled peptide-rhein conjugate of this invention, with the sequence Rhein-GFFPLGLAGACE, can respond to abnormally activated MMP-9 in atherosclerotic plaques, self-assembling in situ into a stable nanofiber structure at the lesion site. The rhein in this conjugate exhibits significant anti-inflammatory and antioxidant functions, but its water solubility and oral bioavailability are low. These are significantly improved after conjugation with the peptide, achieving targeted delivery and prolonging retention time. The introduction of the tripeptide ACE, which mimics the function of glutathione, further enhances the conjugate's ability to resist oxidative stress. Furthermore, this conjugate exhibits excellent anti-inflammatory and antioxidant effects, scavenging ROS, inhibiting inflammatory pathways, regulating macrophage phenotype, and inhibiting apoptosis, providing a new strategy for the treatment of atherosclerosis.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, and in particular to self-assembled polypeptide-rheic acid conjugates, formulations, preparation methods, and applications thereof. Background Technology

[0002] Atherosclerosis (AS) is a chronic, progressive inflammatory disease of the vascular wall characterized by lipid deposition, endothelial dysfunction, immune inflammatory response, and vascular remodeling. In the early stages of the disease, damaged endothelial cells release various inflammatory mediators, continuously recruiting inflammatory cells such as monocytes to migrate to the vessel wall, thereby triggering an inflammatory cascade. During this process, macrophages, under the regulation of the local microenvironment (such as cytokines, lipid components, and oxidative stress), transform into the M1 phenotype with strong pro-inflammatory properties. This transformation further amplifies the inflammatory response, exacerbates vascular wall damage, and reduces plaque stability. In the disease progression stage, the primary inflammatory process is accompanied by the tertiary event of oxidative stress. Oxidative stress leads to excessive accumulation of reactive oxygen species (ROS) in the body, which in turn aggravates endothelial cell dysfunction, increases vascular permeability, and ultimately forms a vicious cycle of inflammation and oxidative stress, further exacerbating the local inflammatory response. Therefore, developing AS therapeutics to alleviate oxidative stress and inflammatory responses within plaques is crucial for improving the efficacy of AS treatment.

[0003] MMP-9 is a matrix metalloproteinase that degrades collagen and extracellular matrix (ECM). Primarily secreted by macrophages and neutrophils, its elevated levels exacerbate ECM degradation, inflammatory responses, and thinning of the fibrous cap, increasing the risk of plaque rupture. Studies have shown that MMP-9 expression continuously increases during AS progression, and the degree of elevation is closely related to plaque instability and rupture risk. Therefore, MMP-9 is not only an important effector of AS progression but also considered a potential diagnostic and interventional target.

[0004] Rhein, a natural drug, possesses significant anti-inflammatory and antioxidant properties. However, its poor water solubility, low oral bioavailability, and poor targeting limit its clinical application. Therefore, developing efficient, targeted, and sustained-release drug carriers is crucial for better utilizing rhein in the treatment of ankylosing spondylitis (AS). Summary of the Invention

[0005] The purpose of this invention is to provide a self-assembled polypeptide-rhein conjugate, its formulation, preparation method, and applications. This polypeptide possesses an anti-inflammatory drug terminator "Rhein," a self-assembling module "GFF," the corresponding MMP-9 (matrix metalloproteinase-9) cleavage sequence "PLGLAG," and a glutathione-like functional tripeptide "ACE" with good free radical scavenging ability. The fused 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 novel drugs for the treatment of AS.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides an enzyme-responsive anti-inflammatory self-assembling peptide-rheic acid conjugate, the structural formula of which is:

[0008]

[0009] Preferably, the coupling compound is synthesized using the Fmoc-solid phase synthesis method.

[0010] The present invention also provides the use of the aforementioned conjugate in the preparation of medicaments for treating chronic progressive inflammatory diseases of the vascular wall.

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

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

[0013] Preferably, the drug targets chronic progressive inflammatory diseases of the vascular wall.

[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 composed of a peptide, a drug, and a linker. Due to the flexible tunability of their amino acid sequences, structural modifications can be easily introduced to support rational drug design, thereby enhancing bioavailability and binding affinity. On 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, resulting in sustained effects, extended time-dependent effects, and desirable pharmacokinetic characteristics. Therefore, designing peptide sequences to target highly expressed regions on diseased cells for precise drug delivery to target organs constitutes an effective therapeutic strategy.

[0016] This invention is the first to synthesize a polypeptide-rheic acid conjugate named Rh-MRSP, which clears ROS and reduces inflammatory response, through a polypeptide synthesis method. This 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 the transformation of macrophages to the M2 phenotype, and regulates macrophage mitochondrial function to reduce apoptosis, thereby playing a role in stabilizing and treating the AS plate.

[0017] A peptide-rhein conjugate named Rh-MRSP, with the sequence Rhein-GFFPLGLAGACE (SEQ ID NO. 1), responds to abnormally activated matrix metalloproteinase-9 (MMP-9) in atherosclerotic plaques, undergoing in-situ self-assembly into a stable nanofiber network structure at the lesion site. Rhein possesses significant anti-inflammatory and antioxidant functions, but its poor water solubility and low oral bioavailability limit its clinical application. Conjugation with the self-assembling peptide significantly improves the water solubility and bioavailability of rhein, enabling targeted drug delivery and prolonging its residence time at the lesion site. Furthermore, the introduction of a tripeptide ACE mimicking glutathione function further enhances the peptide's antioxidant stress resistance. This conjugate exhibits significant anti-inflammatory and antioxidant stress resistance in both in vitro and in vivo experiments, effectively 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 method for the treatment of atherosclerosis.

[0018] The beneficial effects of this invention are as follows:

[0019] First, Rh-MRSP has a low critical self-assembly concentration and is inexpensive to synthesize. Second, in addition to accurately enriching itself at AS plaque sites due to the high expression of MMP-9 in the AS microenvironment, Rh-MRSP can also form self-assembled nanofibers under the catalysis of MMP-9 overexpression in the AS plaque microenvironment, improving drug concentration and bioavailability at the lesion site. Rh-MRSP is the first peptide-rheic acid conjugate discovered that can 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 ​​necrotic cores within plaques, and increased plaque collagen content by inhibiting inflammation and oxidative stress, thereby achieving the goal of treating AS.

[0020] Experiments have confirmed that the peptide-rhein conjugate designed in this invention exhibits excellent MMP-9 response and therapeutic ability for AS plaques. The combination of the anti-inflammatory drug "Rhein," the self-assembly initiating unit "GFF," and the tripeptide unit "ACE" with good free radical scavenging ability, along with the "PLGLAG" MMP-9 enzyme response to improve inflammation and oxidative stress, represents a significant advancement in the treatment of chronic progressive vascular wall inflammatory diseases. Rh-MRSP overcomes the limitations of Rhein, such as poor water solubility, low oral bioavailability, and poor targeting, significantly prolonging its residence time at the lesion site and increasing drug concentration and bioavailability. In this way, Rh-MRSP not only effectively reduces the inflammatory response in AS plaques but also inhibits plaque development and improves plaque stability through its anti-oxidative stress properties, providing a novel treatment strategy and effective technical support for AS treatment. Attached Figure Description

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

[0022] Figure 2 Mass spectrum of MRSP

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

[0024] Figure 4 The critical self-assembly concentration before and after Rh-MRSP digestion;

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

[0026] Figure 6 To detect the ROS scavenging ability of Rh-MRSP using laser confocal microscopy and flow cytometry;

[0027] Figure 7To detect the anti-apoptotic ability of Rh-MRSP using laser confocal co-localization. Detailed Implementation

[0028] This invention provides an enzyme-responsive anti-inflammatory self-assembling peptide-rheic acid conjugate, the structural formula of which is:

[0029]

[0030] In this invention, the coupling compound is preferably synthesized using the Fmoc-solid phase synthesis method.

[0031] The present invention also provides the use of the aforementioned conjugate in the preparation of medicaments for treating chronic progressive inflammatory diseases of the vascular wall.

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

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

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

[0035] In this invention, the drug targets chronic progressive inflammatory diseases of the vascular wall.

[0036] In this invention, the chronic progressive inflammatory disease of the blood vessel wall is atherosclerosis.

[0037] The amino acids and dichlororesin materials involved in the following Rh-MRSP and control MRSP examples were obtained from Shanghai Jier Biochemical Co., Ltd., and all other original reagents and materials were available through commercial channels. Experimental methods without specific conditions were conventional methods and conditions well known in the field.

[0038] The technical solutions provided by the present invention will be 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 the peptide-rhein conjugate Rh-MRSP (Rh-GFFPLGLAGACE)

[0040] In this embodiment, the polypeptide-rheic acid conjugate Rh-MRSP was synthesized using the Fmoc-solid phase synthesis method. The specific steps are as follows:

[0041] 1) Weigh 0.516g of dichloromethane resin, add it to the reactor, and swell it with DCM (dichloromethane) for 20 minutes. Then remove the DCM with a syringe.

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

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

[0044] 4) Cut with a 20% piperidine / DMF (N,N-dimethylformamide) solution for 30 min.

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

[0046] 6) After the last amino acid has reacted and been completely cleaved, wash five times with DMF and five times with DCM.

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

[0048] 8) After collecting the cutting flux, evaporate it to dryness using a rotary evaporator until the liquid in the bottle is oily.

[0049] 9) Add anhydrous ether to the evaporated substance. A solid will precipitate out. Dry the ether and collect the solid as the crude product.

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

[0051] The results of high-resolution mass spectrometry analysis are shown below. 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 active solution of coupling agent:

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

[0055] 2) In subsequent experiments, the prepared coupling solution should be diluted appropriately as needed.

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

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

[0058] The polypeptide sequence MRSP in this comparative example was synthesized using the Fmoc-solid phase synthesis method. The specific steps are as follows:

[0059] 1) Weigh 0.516g of dichlororesin, add it to the reactor, and swell it with DCM for 20 minutes. Then remove the DCM with a rubber bulb.

[0060] 2) The first amino acid and 1 mmol of condensing agent DIEA were dissolved in 12 ml of dichloromethane and added to the reactor, and the reaction was carried out for 2 h;

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

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

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

[0064] 6) After the last amino acid has reacted and been completely cleaved, 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 to the reactor and cut for 45 minutes.

[0066] 8) After collecting the cutting flux, evaporate it to dryness using a rotary evaporator until the liquid in the bottle is oily.

[0067] 9) Add anhydrous ether to the evaporated substance. A solid will precipitate out. Dry the ether and collect the solid as the crude product.

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

[0069] The results of high-resolution mass spectrometry analysis are shown below. Figure 2 .from Figure 2 The obtained polypeptide structure is correct, as shown in Formula II.

[0070]

[0071] Preparation of peptide active solution:

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

[0073] 2) In subsequent experiments, the prepared polypeptide solution should be diluted appropriately as needed.

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

[0075] Experiment Example 1: Transmission Electron Microscopy Experiment

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

[0077] from Figure 3 As can be seen, the Rh-MRSP solution without MMP-9 treatment exhibits discrete clustered particles under TME; while the Rh-MRSP solution treated with MMP-9 transforms into a fibrous network structure under the microscope after 24 hours. 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 a worm-like nanostructure.

[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 1 mM. MMP-9 treatment groups (500 ng / mL of MMP-9 added) and an untreated control group (no MMP-9 added) were established. Subsequently, a gradient of peptide concentration solutions was prepared using a serial dilution method. The intensity of scattered light in the solutions was measured using DLS technology at a constant temperature of 25°C. A logarithmic particle size-concentration curve was plotted, and the inflection point of a sudden increase in particle size was taken as the CMC value. The CMC values ​​of the two groups were calculated by comparing the changes in CMC before and after enzyme treatment. The results are as follows: Figure 4As shown, the CMC of undigested Rh-MRSP was 30.41 μM, while after treatment with MMP-9, its CMC significantly decreased to 11.16 μM. This also confirms that MMP-9 can promote the assembly of peptides, converting them into fibrous structures and increasing their accumulation at atherosclerotic plaques.

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

[0081] Cell viability was assessed by co-culturing RAW264.7 cells with different concentrations (5, 10, 20, 40, 80 μM) of MRSP, Rhein, and Rh-MRSP for 24 h using the CCK-8 (Cell Counting Kit-8) assay to evaluate the biocompatibility of the materials. The experimental results are as follows: Figure 5 As shown, at concentrations of 40 μM and below, these three substances had no significant effect on the survival rate of RAW264.7 cells, indicating good biocompatibility. However, when the concentration increased to 80 μM, free Rhein exhibited significant cytotoxicity, while MRSP and Rh-MRSP, even at this high concentration, had no significant effect on cell viability. This result suggests that conjugating Rhein with MMP-9-responsive self-assembling peptides to form a drug delivery system significantly reduces Rhein toxicity and provides better biocompatibility.

[0082] Experiment 4: Laser confocal microscopy and flow cytometry were used to detect the scavenging ability of Rh-MRSP against ROS (reactive oxygen species).

[0083] RAW264.7 cells were pretreated with 40 μM MRSP, Rhein, and Rh-MRSP for 4 h. The pretreated cells were then stimulated in LPS medium containing 1 μg / mL for 24 h, washed three times with PBS, and subsequently incubated in serum-free medium containing 10 μM MCFH-DA (2',7'-dichlorofluorescein diacetate) for 30 min in the dark. After incubation, the cells were washed three more times with serum-free medium, each wash in the dark. Cells were observed and photographed using a confocal microscope, and changes in intracellular reactive oxygen species levels were analyzed by fluorescence intensity. Figure 6As shown, the fluorescence intensity of cells stimulated with 1 μg / mL LPS for 24 h was significantly enhanced compared to normal cells, indicating a large amount of intracellular ROS production. The green fluorescence intensity in cells stimulated with MRSP, Rhein, and Rh-MRSP all decreased to varying degrees, with Rh-MRSP exhibiting the strongest ROS scavenging ability. This suggests that conjugating Rhein to peptides for drug delivery is more conducive to its anti-oxidative stress effect. To further determine the intracellular ROS level, flow cytometry was used for quantitative analysis. The experimental results were consistent with the qualitative observations obtained by laser confocal microscopy, further confirming the strong anti-oxidative stress capacity of Rh-MRSP.

[0084] Experiment 5: Laser confocal co-localization detection of the anti-apoptotic ability of Rh-MRSP

[0085] RAW264.7 cells were pretreated with 40 μM MRSP, Rhein, and Rh-MRSP for 4 h. The pretreated RAW264.7 cells were then stimulated with 200 μM H2O2 for 8 h. After stimulation, the cells were gently washed three times with PBS buffer. Cell viability was determined using an AM / PI staining kit. The stained cells were incubated at 37°C in the dark for 30 min to allow for complete dye penetration and labeling. Cell observation and image acquisition were then performed using a confocal microscope. Results are as follows: Figure 7 As shown, the Rh-MRSP group had the highest cell survival rate.

[0086] As can be seen from the above embodiments, the present invention provides a self-assembled peptide-rhein conjugate, its formulation, preparation method, and application. The MMP-9-responsive self-assembled peptide-rhein conjugate of the present invention, with the sequence Rhein-GFFPLGLAGACE, can respond to abnormally activated MMP-9 in atherosclerotic plaques and self-assemble into a stable nanofiber structure in situ at the lesion site. The rhein in this conjugate has significant anti-inflammatory and antioxidant functions, but its water solubility and oral bioavailability are low. These are significantly improved after conjugation with the peptide, achieving targeted delivery and prolonging retention time. The introduction of the tripeptide ACE, which mimics the function of glutathione, further enhances the conjugate's ability to resist oxidative stress. Furthermore, this conjugate exhibits excellent anti-inflammatory and antioxidant effects, capable of scavenging ROS, inhibiting inflammation, and suppressing macrophage apoptosis, providing a new strategy for the treatment of atherosclerosis.

[0087] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An enzyme-responsive anti-inflammatory self-assembling polypeptide-rheic acid conjugate, characterized in that, The structural formula of the coupling is: Formula I.

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

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