Construction method and application of ac-sdkp-pevs drug delivery system

Abstract

The application discloses a construction method and application of an Ac-SDKP-PEVs drug delivery system, and belongs to the technical field of biological medicines. In the application, PEVs are used as a biomimetic nano delivery carrier, the C-terminal polyhistidine tag of Ac-SDKP is used in combination with a cholesteric anchoring strategy, Ac-SDKP is constructed into an Ac-SDKP-His6-CHOL derivative and is loaded on the PEVs to form the Ac-SDKP-PEVs drug delivery system, so that the anti-inflammatory tetrapeptide has higher drug loading efficiency, is more stable in nanometer scale, has lower pro-inflammatory risk and has enhanced endothelial anti-inflammatory effect.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to an Ac-SDKP-PEVs drug delivery system for delivering the anti-inflammatory peptide Ac-SDKP via platelet-derived extracellular vesicles (PEVs) with low pro-inflammatory risk, a method for constructing the system, and its application in the preparation of drugs for Kawasaki disease. Background Technology

[0002] Kawasaki disease is an acute systemic vasculitis that primarily affects children, with serious complications including coronary artery dilation, aneurysm, stenosis, and thrombosis. Current first-line treatment often involves intravenous immunoglobulin combined with systemic anti-inflammatory or antiplatelet regimens such as aspirin. However, some patients still experience immunoglobulin non-responsiveness, increasing the risk of coronary artery disease. Traditional systemic anti-inflammatory therapies lack the ability to target inflamed blood vessels, especially localized inflammatory lesions in the coronary arteries, resulting in significant drug exposure in non-target tissues. While traditional antiplatelet therapy can inhibit aggregation, it cannot effectively reverse vascular wall immune inflammation and may pose safety risks such as bleeding.

[0003] N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is an endogenous tetrapeptide with anti-inflammatory, anti-fibrotic, and cardioprotective effects, making it suitable for intervention in inflammatory vascular diseases. However, free Ac-SDKP is easily degraded rapidly by angiotensin-converting enzyme in vivo, has a short half-life, and lacks targeting specific tissues or inflammatory sites, making direct administration difficult to fully exert its therapeutic effect. Summary of the Invention

[0004] The purpose of this invention is to address the problems of short half-life, lack of inflammatory vascular targeting, and insufficient treatment efficiency of free Ac-SDKP in the prior art, and to provide a method for constructing and applying an Ac-SDKP-PEVs drug delivery system.

[0005] The technical solution adopted in this invention is as follows:

[0006] A method for constructing an Ac-SDKP-PEVs drug delivery system includes the following steps:

[0007] S1. Ac-SDKP is modified with a C-terminal polyhistidine tag and cholesterol to form the drug molecule to be loaded, Ac-SDKP-HHHHHH-CHOL.

[0008] S2. The Ac-SDKP-HHHHHH-CHOL and platelet-derived extracellular vesicles (PEVs) are mixed in sterile phosphate buffer to prepare a mixture.

[0009] S3. The mixture is incubated at a constant temperature. After incubation, it is centrifuged at high speed, and the precipitate is collected to obtain the Ac-SDKP-PEVs drug delivery system.

[0010] Preferably, in step S2, the method for preparing platelet-derived extracellular vesicles (PEVs) includes the following steps:

[0011] a. Collect fresh whole blood from C57BL / 6J mice and place it in pre-cooled phosphate buffer containing ethylenediaminetetraacetic acid and prostaglandin E1;

[0012] b. Centrifuge the blood at 4°C and 100×g for 15 minutes and collect the supernatant; centrifuge again for 5 minutes under the same conditions.

[0013] c. Centrifuge the supernatant at 4℃ and 800×g for 20 minutes, discard the supernatant to obtain platelet precipitate; wash with phosphate buffer and resuspend, then adjust the platelet concentration to 2×10^8 cells / mL.

[0014] d. Add bovine plasma-derived thrombin to the platelet suspension to a final concentration of 2 U / mL, incubate at room temperature for 30 minutes to induce platelets to release extracellular vesicles;

[0015] e. After activation, centrifuge at 4℃ and 800×g for 15 minutes and collect the supernatant containing PEVs.

[0016] f. Centrifuge the supernatant at 4℃ and 120000×g for 90 minutes, discard the supernatant, and the resulting precipitate is the purified PEVs; resuspend in 50-100 μL of sterile phosphate buffer and store on ice for a short period or at -80℃ for a long period.

[0017] Preferably, in S2, the mass ratio of Ac-SDKP-HHHHHH-CHOL to PEVs is 20-80:200; the total volume of the mixture is 300 μL.

[0018] Preferably, in step S2, the pH of the sterile phosphate buffer solution is 7.4.

[0019] Further preferably, in step S3, the conditions for constant temperature incubation of the mixture are: constant temperature incubation at 25°C and 150 rpm for 60 minutes.

[0020] Further preferably, in S3, the conditions for ultracentrifugation are: centrifugation at 4°C and 100,000 × g for 60 minutes.

[0021] The present invention also provides an Ac-SDKP-PEVs drug delivery system constructed by the above construction method, wherein the drug delivery system has a drug delivery efficiency of greater than 93% and a drug delivery amount of 9.70%-37.48%.

[0022] The present invention also provides the application of the above-mentioned Ac-SDKP-PEVs drug delivery system in the preparation of drugs for the treatment of Kawasaki disease.

[0023] Platelet-derived extracellular vesicles (PEVs) inherit some of the membrane proteins of platelets, such as CD41 and P-selectin CD62P, and have the potential to recognize activated endothelial cells and sites of inflammation. At the same time, they do not have complete organelles and the ability to synthesize proteins de novo. Theoretically, they can retain the inflammatory tendency or "navigation" ability of platelets, thereby reducing the risks of pro-inflammatory secretion, thrombosis and cell infusion that may be caused by intact platelets.

[0024] This invention prepares PEVs with CD41 and CD62P expression and low pro-inflammatory risk by thrombin-activated platelets, differential centrifugation, and ultracentrifugation. PEVs themselves do not induce significant release of pro-inflammatory cytokines such as TNF-α, IL-6, and IL-1β, and do not significantly affect hemostasis time, liver and kidney function, or the structure of major organs and tissues in vivo. Simultaneously, Ac-SDKP is modified with a C-terminal polyhistidine tag and cholesterol to form Ac-SDKP-HHHHHH-CHOL. Finally, PEVs are used as biomimetic nanodelivery carriers, and Ac-SDKP-HHHHHH-CHOL is used as the drug molecule to be loaded onto PEVs. The polyhistidine tag enhances the coordination or electrostatic interaction with the surface components of PEVs, and cholesterol is inserted into the lipid bilayer membrane of PEVs, thereby forming the Ac-SDKP-PEVs drug delivery system. This enables the anti-inflammatory tetrapeptide to achieve higher drug loading efficiency, more stable nanoscale, low pro-inflammatory risk, and enhanced endothelial anti-inflammatory effect.

[0025] The beneficial effects of this invention are as follows:

[0026] 1. High drug loading efficiency: Ac-SDKP-PEVs can achieve drug loading efficiency of over 93% under different feed ratios, with a mass ratio of 200:20 reaching approximately 97.01%.

[0027] 2. Adjustable drug loading: As the amount of Ac-SDKP added increases, the drug loading increases linearly from about 9.70% to about 37.48%, which makes it easy to adjust the drug loading level according to different therapeutic dosage requirements.

[0028] 3. Stable physicochemical properties after drug loading: The loading of Ac-SDKP did not significantly change the particle size and zeta potential of PEVs, which is beneficial to maintaining the original nanodelivery characteristics of PEVs.

[0029] 4. Low pro-inflammatory risk: PEVs do not release or release significantly low levels of pro-inflammatory cytokines such as TNF-α, IL-6, and IL-1β under thrombin stimulation, thus reducing the risk of additional inflammation compared to intact platelets.

[0030] 5. Good biocompatibility: PEVs at concentrations up to 40 μg / mL and Ac-SDKP-PEVs at concentrations up to 1.6 μg / mL have no significant toxicity to endothelial cells; intravenous injection of 20 mg / kg PEVs into mice does not prolong bleeding time and does not affect liver and kidney function or the structure of lung, liver, spleen and kidney tissues.

[0031] 6. Enhanced anti-inflammatory efficacy: Ac-SDKP-PEVs can significantly inhibit lipopolysaccharide-induced secretion of TNF-α, IL-6, and IL-1β in endothelial cells, with better effects than an equal dose of free Ac-SDKP.

[0032] 7. Potential for targeting inflammatory blood vessels: PEVs express platelet-associated membrane proteins such as CD41 and CD62P on their surface, which can be used to identify activated endothelial cells and the inflammatory microenvironment, providing a basis for targeted therapy of inflammatory vascular diseases such as Kawasaki disease. Attached Figure Description

[0033] Figure 1 Preparation and characterization of platelet-derived extracellular vesicles (PEVs). The results include: (A) PEV preparation process; (B) Transmission electron microscopy (TEM) images and magnified views of PEVs; (C) Size distribution of resting platelets; (D) Size distribution of thrombin-activated platelets; (E) Size distribution of purified PEVs; (F) Zeta potential of resting platelets and PEVs; (G) Size stability of PEVs stored at −80℃ for 0, 3, 5, and 7 days; (H) Western blot results of Na⁺ / K⁺-ATPase, CD41, and CD62 in resting platelets, activated platelets, and PEVs; (I) Relative expression level of CD41; and (J) Relative expression level of CD62.

[0034] Figure 2 The construction and performance testing of the Ac-SDKP-PEVs drug delivery system were carried out. The results included: (A) Ac-SDKP molecular structure; (B) Ac-SDKP-PEVs preparation process; (C) drug loading efficiency at different feed mass ratios; (D) drug loading amount at different feed mass ratios; (E) median particle size of PEVs and Ac-SDKP-PEVs; and (F) zeta potential of PEVs and Ac-SDKP-PEVs.

[0035] Figure 3 The biocompatibility of PEVs and Ac-SDKP-PEVs was evaluated. Specifically, (A) the effect of different concentrations of PEVs on the viability of HCAECs and HUVECs; (B) the effect of different concentrations of Ac-SDKP-PEVs on the viability of HCAECs and HUVECs; (C) the tail bleeding time in the control group, platelet group, and PEVs group; and (D) the serum ALT, AST, BUN, and Cr levels in mice on days 0, 1, and 7 after PEV injection.

[0036] Figure 4 This is an in vivo pathological safety evaluation of PEVs. The columns in the figure, from left to right, represent the lung, liver, spleen, and kidney; the rows represent pre-injection, day 1 post-injection, and day 7 post-injection. The top image for each organ is a low-magnification panoramic view, and the bottom image is a high-magnification magnified view of the selected area.

[0037] Figure 5 The low pro-inflammatory risk of PEVs and the in vitro anti-inflammatory effect of Ac-SDKP-PEVs. Among them, (A) the level of TNF-α released by platelets and PEVs before and after thrombin stimulation; (B) the level of IL-6 release; (C) the level of IL-1β release; (D) the effect of different treatments on the secretion of TNF-α by LPS-induced HCAECs and HUVECs; (E) the level of IL-6 secretion; (F) the level of IL-1β secretion. Detailed Implementation

[0038] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0039] Example 1: Preparation of Ac-SDKP-PEVs Drug Delivery System

[0040] 1. Preparation of PEVs

[0041] Fresh whole blood from C57BL / 6J mice was collected and placed in pre-chilled PBS containing EDTA and PGE1. The mixture was centrifuged at 100×g for 15 minutes at 4°C, and the platelet-rich supernatant was collected. This supernatant was then centrifuged again under the same conditions for 5 minutes to remove residual red blood cells. The resulting supernatant was centrifuged at 800×g for 20 minutes at 4°C to obtain platelet pellet. After washing with PBS, the pellet was resuspended and adjusted to approximately 2×10^8 cells / mL. Thrombin was added to a final concentration of 2 U / mL, and the pellet was incubated at room temperature for 30 minutes. Subsequently, the pellet was centrifuged at 800×g for 15 minutes at 4°C to remove cells and debris. The supernatant was then ultracentrifuged at 120000×g for 90 minutes at 4°C. The resulting pellet was resuspended in 50-100 μL of sterile PBS to obtain PEVs. The preparation and characterization of PEVs are as follows: Figure 1 As shown.

[0042] 2. Preparation of Ac-SDKP-PEVs

[0043] Ac-SDKP was modified with a C-terminal polyhistidine tag and cholesterol to form the drug molecule Ac-SDKP-HHHHHH-CHOL.

[0044] PEVs were mixed with Ac-SDKP-HHHHHH-CHOL at mass ratios of 200:20, 200:40, 200:60, and 200:80 in sterile PBS at pH 7.4, with a total volume of 300 μL. The mixtures were incubated at 25°C and 150 rpm for 60 minutes. After incubation, the mixture was ultracentrifuged at 4°C and 100,000 × g for 60 minutes. The supernatant containing free drug was discarded, and the precipitate was Ac-SDKP-PEVs.

[0045] Example 1 prepared Ac-SDKP-PEVs drug delivery systems in multiple ratios (PEVs to Ac-SDKP-HHHHHH-CHOL mass ratios of 200:20, 200:40, 200:60, and 200:80, respectively).

[0046] Example 2 Performance Testing of Ac-SDKP-PEVs Drug Delivery System

[0047] 1. Drug loading effect test

[0048] The supernatant from step 2 of Example 1 was collected, and the free Ac-SDKP content was detected using an Ac-SDKP enzyme-linked immunosorbent assay kit. The bound drug mass B=TF was calculated based on the total drug dosage T and the free drug mass F, and the drug loading efficiency LE=(B / T)×100% and drug loading DLC=[B / (PEVs mass+B)]×100% were calculated. Experimental results showed that the drug loading efficiencies at mass ratios of 200:20, 200:40, 200:60, and 200:80 were approximately 97.01%, 96.37%, 96.11%, and 93.69%, respectively; and the drug loadings were approximately 9.70%, 19.27%, 29.02%, and 37.48%, respectively. Figure 2 ).

[0049] 2. Physicochemical properties and characterization by biomarkers

[0050] like Figure 1 As shown, the morphology of PEVs was observed using transmission electron microscopy, confirming them as spherical or near-spherical vesicles. Dynamic light scattering was used to detect the particle size of PEVs, which was mainly concentrated in the range of 90-300 nm, with a peak of approximately 141.8 nm. The median particle size before and after drug loading was approximately 135.6±4.9 nm and 134.6±8.5 nm, respectively, and the zeta potential was approximately -12.7±0.3 mV and -12.9±0.7 mV, respectively, with no significant difference between the two. Western blotting was used to detect CD41, CD62P, and Na+ / K+-ATPase, confirming that PEVs originated from platelets and that CD62P expression did not show a significant over-increase.

[0051] 3. Biocompatibility evaluation

[0052] HCAECs and HUVECs were co-cultured with different concentrations of PEVs or Ac-SDKP-PEVs (prepared in Example 1 with a PEVs to Ac-SDKP-HHHHHH-CHOL mass ratio of 200:60) for 24 hours, and cell viability was detected using the CCK-8 assay. The results showed ( Figure 3 , Figure 4 When PEVs concentrations reached as high as 40 μg / mL and Ac-SDKP-PEVs concentrations reached as high as 1.6 μg / mL, cell viability did not decrease significantly compared to the control group. In healthy mice, tail vein injection of PEVs at 20 mg / kg did not significantly prolong tail bleeding time; ALT, AST, UREA, and CREA levels were normal on days 1 and 7 post-injection, and no obvious toxic damage or inflammatory changes were observed in lung, liver, spleen, and kidney tissue sections.

[0053] 4. Evaluation of low pro-inflammatory risk and anti-inflammatory effect

[0054] Platelets and PEVs were adjusted to the same protein concentration and stimulated with thrombin for 24 hours. TNF-α, IL-6, and IL-1β levels in the supernatant were then measured. Results showed ( Figure 5 Compared to intact platelets, PEVs did not induce significant release of pro-inflammatory factors. Further, in lipopolysaccharide-induced inflammation models of HCAECs and HUVECs (200 ng / mL), treatment with Ac-SDKP-PEVs (prepared in Example 1 with a PEVs to Ac-SDKP-HHHHHH-CHOL mass ratio of 200:60; equivalent to 0.4 μg / mL Ac-SDKP), equal doses of free Ac-SDKP, or PEVs was performed. Results showed that Ac-SDKP-PEVs significantly reduced TNF-α, IL-6, and IL-1β secretion, exhibiting superior anti-inflammatory effects compared to free Ac-SDKP.

[0055] The specification and drawings of this invention are intended to be illustrative rather than restrictive. Based on this invention, those skilled in the art can make substitutions and modifications to some of the technical features without creative effort, and all such modifications are within the scope of protection of this invention.

Claims

1. A method for constructing an Ac-SDKP-PEVs drug delivery system, characterized in that, Includes the following steps: S1. Ac-SDKP is modified with a C-terminal polyhistidine tag and cholesterol to form the drug molecule to be loaded, Ac-SDKP-HHHHHH-CHOL. S2. The Ac-SDKP-HHHHHH-CHOL and platelet-derived extracellular vesicles (PEVs) are mixed in sterile phosphate buffer to prepare a mixture. S3. The mixture is incubated at a constant temperature. After incubation, it is centrifuged at high speed, and the precipitate is collected to obtain the Ac-SDKP-PEVs drug delivery system.

2. The construction method according to claim 1, characterized in that, In step S2, the preparation method of platelet-derived extracellular vesicles (PEVs) includes the following steps: a. Collect fresh whole blood from C57BL / 6J mice and place it in pre-cooled phosphate buffer containing ethylenediaminetetraacetic acid and prostaglandin E1; b. Centrifuge the blood at 4°C and 100×g for 15 minutes and collect the supernatant; centrifuge again for 5 minutes under the same conditions. c. Centrifuge the supernatant at 4℃ and 800×g for 20 minutes, discard the supernatant to obtain platelet precipitate; wash with phosphate buffer and resuspend, then adjust the platelet concentration to 2×10^8 cells / mL. d. Add bovine plasma-derived thrombin to the platelet suspension to a final concentration of 2 U / mL, incubate at room temperature for 30 minutes to induce platelets to release extracellular vesicles; e. After activation, centrifuge at 4℃ and 800×g for 15 minutes and collect the supernatant containing PEVs. f. Centrifuge the supernatant at 4℃ and 120000×g for 90 minutes, discard the supernatant, and the resulting precipitate is the purified PEVs; resuspend in 50-100 μL of sterile phosphate buffer and store on ice for a short period or at -80℃ for a long period.

3. The construction method according to claim 1, characterized in that, In S2, the mass ratio of Ac-SDKP-HHHHHH-CHOL to PEVs is 20-80:200; the total volume of the mixture is 300 μL.

4. The construction method according to claim 1, characterized in that, In S2, the pH of the sterile phosphate buffer solution is 7.

4.

5. The construction method according to claim 1, characterized in that, In step S3, the conditions for constant temperature incubation of the mixture are: constant temperature incubation at 25°C and 150 rpm for 60 minutes.

6. The construction method according to claim 1, characterized in that, In S3, the conditions for ultracentrifugation are: centrifugation at 4°C and 100,000 × g for 60 minutes.

7. The Ac-SDKP-PEVs drug delivery system constructed by the construction method according to any one of claims 1-6, characterized in that, The drug loading efficiency of the drug loading system is greater than 93%; the drug loading capacity is 9.70%-37.48%.

8. The use of the Ac-SDKP-PEVs drug delivery system as described in claim 7 in the preparation of drugs for treating Kawasaki disease.

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