Polypeptide-modified ROS (reactive oxygen species)-removing nano-particles and application thereof

Abstract

The invention discloses a polypeptide modified nano-particle. The nano-particle is a nano-particle capable of removing or consuming active oxygen. The polypeptide can adsorb or combine TSP-1, and the amino acid sequence of the polypeptide is as shown in SEQ ID NO: 1. The nano-particles prepared by the invention are stable in characterization, can specifically adsorb TSP-1 in vivo and in vitro, remove ROS, significantly inhibit TSP-1 increase and cell apoptosis in IRI organs and significantly enhance the protection effect on the IRI organs, are convenient to prepare, and have good application prospects in the fields of organ IRI, organ transplantation treatment and protection, cardiovascular surgery and the like.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of nanomedicine, drugs and biomedical polymer materials, and mainly relates to a polypeptide modified ROS scavenging nanoparticle and application thereof. BACKGROUND

[0002] Ischemia-reperfusion injury (IRI) includes the interruption of blood flow caused by the blockage of blood vessels, and reperfusion injury refers to the damage of the cells or tissues in the blood supply area after the blood vessels in this part recover to normal blood flow, or new damage, etc., which is an irreversible damage. IRI is relatively common in clinical practice and can lead to the occurrence of various pathological diseases, including myocardial infarction, ischemic stroke, acute kidney injury, etc. It is caused by various factors, including heart surgery, vascular surgery, acute heart failure and shock; and cannot be avoided during transplantation, leading to delayed graft function or even transplantation failure. However, there is no appropriate method to effectively inhibit IRI.

[0003] Thrombospondin-1 (TSP-1) is a 450 kDa multifunctional homotrimeric glycoprotein secreted by platelets, vascular endothelial cells, vascular smooth muscle cells and non-vascular cells. As a vascular endothelial growth factor suppressor protein, TSP-1 has multiple domains and can bind to various cell surface receptors, such as CD47 and CD36, to inhibit cell cycle progression and induce vascular endothelial cell, tissue cell apoptosis and necrosis, inflammation, etc., and also can inhibit the NO pathway of endothelial cells, thereby inhibiting vasodilation and even causing microcirculation blood flow interruption, etc. In addition, TSP-1 can also inhibit angiogenesis by antagonizing the effect of VEGF and activate TGF-β to cause kidney damage and fibrosis. After IRI occurs, the content of TSP-1 in the body rapidly increases and is considered to be a key mediator of IRI. Therefore, TSP-1 can be used as a potential target for treating IRI, and blocking the activity of TSP-1 can be expected to prevent and treat IRI.

[0004] Peptides that bind to the TSP-1 sequence (e.g., leucine-serine-lysine-leucine (LSKL, SEQ ID NO: 1)) can act as TSP1 antagonists, consuming TSP-1 activity and inhibiting its binding to TGF-β, CD47 signaling, etc., thereby reducing the disease-causing activity of TSP-1 and potentially inhibiting the occurrence of IRI and protecting organ function. However, due to the low efficiency of peptide molecules reaching target tissues and their susceptibility to enzymatic degradation and inactivation, peptide drugs have short half-lives and low bioavailability, making them less than ideal for the treatment of IRI. Therefore, the rational design of peptide drugs is essential to address these shortcomings.

[0005] In the rescue and treatment of ischemic diseases, medical researchers have gradually discovered that the main factor causing tissue damage is not ischemia itself, but reperfusion injury that occurs after blood supply is restored. During reperfusion, electrons escape from the mitochondrial respiratory chain, and superoxide anions, hydroxyl radicals (singlet oxygen, hydrogen peroxide), etc., cannot be consumed and are converted into reactive oxygen species (ROS). This induces an increase in oxidative stress, exceeding the cell's antioxidant capacity. This leads to damage to the cell membrane structure, inactivation of cellular DNA and proteins, apoptosis and necrosis, inflammatory infiltration, etc., ultimately resulting in irreparable ischemic damage. Therefore, oxidative stress is another major pathogenesis of ischemic inflammatory disease (IRI). Blocking TSP-1 activity while simultaneously scavenging or inhibiting ROS activity holds promise for improving the efficacy of drugs in preventing and treating IRI.

[0006] Enzymes are a special class of proteins that act as biological catalysts, playing a crucial role in living organisms. They accelerate the rate of biochemical reactions by lowering the activation energy of chemical reactions, without being consumed in the process. Catalase is a common biological enzyme that efficiently breaks down harmful hydrogen peroxide into harmless water and oxygen, thus protecting cells from oxidative stress damage. The advantages of catalase lie in its high efficiency and specificity. They can operate under mild conditions without damaging other biomolecules, while exhibiting high substrate specificity, catalyzing only specific reactions. Furthermore, catalase activity can be precisely regulated by cells to adapt to different physiological needs. Catalase has enormous application potential in medicine, industry, and environmental science, demonstrating unique technical and economic advantages in disease treatment.

[0007] Compared to chemical drugs, bioenzymes and peptides offer advantages such as higher efficiency, safety, and tolerability, as well as greater selectivity and less likelihood of accumulation in the body. However, their disadvantages are also significant: they are physicochemically unstable, easily oxidized and hydrolyzed, have short half-lives, and are rapidly eliminated. Therefore, rational drug design, taking into account the advantages and disadvantages of bioenzymes and peptides, is essential. Nanotechnology is an emerging scientific technology with wide applications in medicine, materials science, electronics, and other fields. In pharmaceutics, nanoparticles (NPs) are defined by a size between 1 and 1000 nanometers. NPs can improve drug targeting, allowing drugs to reach lesion sites more accurately, thereby improving efficacy; NPs can achieve controlled drug release, releasing drugs at specific times and locations to maintain stable blood drug concentrations and alter the blood circulation half-life; nanomedicine formulations can enhance drug bioavailability; NPs enable targeted and controlled drug release, reducing damage to normal tissues and thus lowering drug side effects. Therefore, NPs have become one of the most promising and effective targeted and controlled drug delivery technologies. It can serve as one of the most promising delivery carriers for bioenzyme drugs and peptide drugs. Summary of the Invention

[0008] This invention develops a peptide-modified ROS-scavenging nanoparticle and its applications. The nanoparticle utilizes a nanotechnology strategy to consume TSP-1 and scavenge ROS, thereby developing a combined approach to prevent or treat IRI.

[0009] The present invention mainly solves the above-mentioned technical problems through the following technical solutions.

[0010] A first aspect of the present invention provides a polypeptide-modified nanoparticle, wherein the nanoparticle is capable of scavenging or consuming reactive oxygen species (ROS); the polypeptide is capable of adsorbing or binding TSP-1, and its amino acid sequence is shown in SEQ ID NO:1.

[0011] In some embodiments, the peptide specifically binds to TSP-1, and the nanoparticles have ROS scavenging capabilities (which may be simply referred to as (ROS-scavenged NPs)@Peptide).

[0012] In some embodiments, the nanoparticles are nanomaterials with the function of scavenging or consuming reactive oxygen species.

[0013] In some embodiments, the nanoparticles carry a drug that can scavenge or consume reactive oxygen species.

[0014] In some embodiments, the nanoparticles also include a polymer coated on the surface.

[0015] In some embodiments, the polymer is a crosslinked polymer, wherein the polymerizing monomer is selected from acrylamide, N-(3-aminopropyl)methacrylamide, 2-acrylamido-2-methylpropanesulfonic acid, and 2-methacryloyloxyethyl phosphocholine; and the crosslinking agent is N,N'-methylenebisacrylamide.

[0016] In some specific implementations, the polymer is a cross-linked polymer, and the polymerizing monomer is 2-methacryloyloxyethyl phosphocholine.

[0017] In some specific embodiments, the crosslinked polymer also includes minor monomers.

[0018] In some specific embodiments, the secondary monomer is PEG succinimide carboxymethyl ester.

[0019] In some specific implementations, the molar ratio of the secondary monomer to the polymeric monomer is 1:10;

[0020] In some specific implementations, the drug is catalase (CAT).

[0021] In some embodiments, the polypeptide binds to functional groups modified on the surface of nanoparticles via its amino or side chain groups.

[0022] In some specific embodiments, the functional group is a functional group capable of undergoing a click reaction with the amino group or the side chain group.

[0023] In some specific embodiments, the functional group is N-hydroxysuccinimide.

[0024] A second aspect of the present invention provides a method for preparing nanoparticles as described in any one of the first aspects of the present invention, comprising the following steps:

[0025] The functional group-modified nanoparticles are mixed with a polypeptide containing the amino acid sequence shown in SEQ ID NO: 1 to obtain the product.

[0026] In some embodiments, the preparation of the polypeptide and / or the modification of the nanoparticles with functional groups are further included prior to mixing.

[0027] In some specific implementations, the method includes one or more of the following steps:

[0028] (1) Protein modification: NAS was modified on the surface of CAT to obtain a CAT solution modified with NAS; wherein the molar ratio of CAT to NAS was 1:30.

[0029] (2) Polymer modification: 2-methacryloyloxyethyl phosphocholine, PEG succinimide carboxymethyl acrylate N,N'-methylenebisacrylamide, TEMED and APS were added to the CAT solution modified with NAS obtained in step (1) to react and obtain CAT nanoparticles-NHS.

[0030] (3) Peptide modification: The peptide is added to the solution containing CAT nanoparticles-NHS obtained in step (2), mixed, and incubated to obtain CAT nanoparticles-LSKL.

[0031] In some specific implementations, the molar ratio of 2-methacryloyloxyethyl phosphocholine, PEG succinimide carboxymethyl acrylate N,N'-methylenebisacrylamide, TEMED, and APS in step (2) of the method is 10:1:1:4:1.

[0032] In some specific implementations, the molar ratio of 2-methacryloyloxyethyl phosphocholine to CAT in step (2) of the method is 25000:1.

[0033] In some specific implementations, the molar ratio of the CAT nanoparticles-NHS to the polypeptide is (1~2):1.

[0034] In some specific implementations, the molar ratio of the CAT nanoparticles-NHS to the polypeptide is 2:1.

[0035] A third aspect of the present invention provides a medicament for treating or preventing ischemia-reperfusion injury, comprising polypeptide-modified nanoparticles as described in any of the first aspects of the present invention.

[0036] In some specific implementations, the ischemia-reperfusion injury is renal ischemia-reperfusion injury.

[0037] A fourth aspect of the invention provides the use of peptide-modified nanoparticles as described in any of the first aspects of the invention in the preparation of medicaments for treating or preventing ischemia-reperfusion injury.

[0038] In some specific implementations, the ischemia-reperfusion injury is renal ischemia-reperfusion injury.

[0039] In this invention, the unmodified nanoparticles capable of scavenging or consuming ROS can be common bioenzyme nanoparticles such as catalase, or nanoparticles carrying drugs with ROS scavenging functions; for example, PLGA and liposomes. These materials have good biocompatibility and are easy to modify.

[0040] The preparation method of the peptide-modified ROS-scavenging nanoparticles described in this invention can be conventional in the field and has no specific requirements. Any preparation method that can link the ROS-consuming nanoparticles together with the TSP-1 binding peptide can be applied to this invention.

[0041] The combined use of TSP-1 binding peptides and ROS-scavenging nanoparticles known in the art can be used in the preparation of the (ROS-scavenged NPs)@Peptide of this invention.

[0042] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0043] The reagents and raw materials used in this invention are all commercially available.

[0044] The positive and progressive effects of this invention are as follows:

[0045] This invention provides a (ROS-scavenged NPs)-Peptide prepared by conjugating a targeted drug delivery carrier based on ROS-scavenging nanoparticles, such as CAT nanoparticles, with a peptide (specifically binding TSP-1). This type of nanomedicine is characterized as stable and can specifically adsorb TSP-1 in vitro and in vivo while scavenging ROS. Combined therapy significantly improves the effect of inhibiting IRI.

[0046] It specifically binds to TSP-1 in vitro, achieves in vivo distribution and organ enrichment of peptide and ROS scavenging drugs, significantly inhibits the increase of TSP-1 in IRI organs, and has a protective effect against organ IRI damage.

[0047] The (ROS-scavenged NPs)-Peptide of this invention is easy to prepare and has good application prospects in the fields of organ IRI, organ transplantation treatment and protection, and cardiovascular surgery. Attached Figure Description

[0048] Figure 1 The nanoscale characterization results are for CAT NPs and CAT NPs@Peptide in Example 1.

[0049] Figure 2 The results show the adsorption of TSP-1 protein by PBS, LSKL, and the analogues Au NPs and Au NPs@Peptide in Example 2.

[0050] Figure 3 The results show the in vivo distribution of CAT NPs and CAT NPs@Peptide in kidney IRI mice in Example 3.

[0051] Figure 4 The results show the enrichment of CAT NPs and CAT NPs@Peptide in IRI kidneys in Example 4.

[0052] Figure 5 The results in Example 5 showed that PBS, CAT NPs, Peptide, BSA NPs@Peptide, and CAT NPs@Peptide reduced TSP-1 levels in IRI kidneys to varying degrees.

[0053] Figure 6 The results in Example 6 show that PBS, CAT NPs, Peptide, BSA NPs@Peptide, and CAT NPs@Peptide reduced ROS levels in IRI kidneys to varying degrees.

[0054] Figure 7 The results in Example 7 show that PBS, CAT NPs, Peptide, BSA NPs@Peptide, and CAT NPs@Peptide reduced IRI renal structural damage to varying degrees.

[0055] Figure 8 This is a schematic diagram illustrating the mechanism of action of CAT NPs@Peptide. Detailed Implementation

[0056] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0057] This invention is better understood through the following experimental examples, which illustrate the synthesis, characterization, in vitro and post-IRI adsorption of TSP-1 by the (ROS-scavenged NPs)-peptide of this invention, and its effects on the prevention and treatment of renal injury due to ischemia-reperfusion injury. These examples are merely illustrative and do not represent the entirety of the invention.

[0058] The CAT NPs@Peptide used in this invention can be prepared by free radical polymerization: first, CAT nanoparticles modified with N-hydroxysuccinimide (NHS) functional groups are prepared to form a CAT NPs@NHS mixture, and then the peptides are linked together with the prepared particles, such as by reacting the amino group (NH2) of an amino acid with NHS to form a stable CAT NPs@Peptide.

[0059] Example 1: Preparation and characterization of CAT NPs@Peptide

[0060] Taking CAT NPs@Peptide as an example:

[0061] (1) Protein modification: N-hydroxysuccinimide acrylate (NAS) was modified on the CAT surface at a molar ratio of 1:30;

[0062] (2) Polymer modification: 2-methacryloyloxyethyl phosphocholine, PEG succinimide carboxymethyl acrylate, N,N'-methylenebisacrylamide, N,N,N',N'-tetramethylethylenediamine (TEMED), and ammonium persulfate (APS) (molar ratio of 10:1:1:4:1, molar ratio of 2-methacryloyloxyethyl phosphocholine to CAT of 25000:1) were added to the CAT solution modified with NAS. After reacting for 30 minutes, CAT nanoparticles-NHS were obtained.

[0063] (3) Peptide modification: The peptide and the solution of CAT nanoparticle-NHS are mixed with LSKL and incubated for 1.5 hours to obtain CAT nanoparticle-LSKL; wherein the molar ratio of CAT nanoparticle-NHS to the peptide is 2:1.

[0064] The size of nanoparticles was determined by non-dynamic light scattering at 25°C, with each sample being tested at least three times. Figure 1 The characterization of CAT NPs and CAT NPs@Peptide is shown. CAT NPs and CAT NPs@Peptide exhibit a regular spherical morphology with particle sizes of approximately 7.14 nm and 17.66 nm, respectively, and demonstrate stable physicochemical properties and good dispersibility.

[0065] Example 2: NPs-Peptide has an adsorption effect on TSP1 protein.

[0066] After TSP-1 binds to the fluorescent dye AF647 (Alexa Flour647) overnight, TSP-1-AF647 is mixed with free peptide, Au NPs, Au NPs@Peptide, and an equal volume of PBS (as a control). After incubation overnight at 4°C, the mixture is centrifuged at 30,000 g for 30 minutes. The fluorescence intensity of AF647 in the collected precipitate is measured using a high-throughput small animal in vivo optical imaging system. Figure 2 This indicates that Au NPs@Peptide has a strong adsorption effect on TSP-1 protein; however, the adsorption effects of PBS control, free Peptide, and Au NPs are all significantly lower than those of Au NPs@Peptide.

[0067] Example 3: Peptide conjugation increased the accumulation of CAT NPs in the IRI kidney.

[0068] BALB / c mice were anesthetized and placed on a heated pad to maintain a body temperature of approximately 37 °C. Reperfusion injury (IRI) was induced by surgically clamping the left renal pedicle and then slowly releasing the clamp, followed by suturing the incision. Three hours after reperfusion, PBS, FITC-labeled CAT NPs, and CAT NPs@Peptide (CAT NPs / FITC and CAT NPs@Peptide / FITC, respectively) were injected via the tail vein. Mice were sacrificed 24 hours later, and tissues from the brain, heart, lungs, liver, spleen, and kidneys were collected for fluorescence imaging. CAT NPs / FITC were found to be significantly enriched in the liver and the kidneys of the IRI. The peptide conjugation altered the metabolic behavior of CAT NPs / FITC in the mouse kidneys, increasing the specific accumulation of CAT NPs in the IRI kidneys. Compared to the untreated contralateral kidney, CAT NPs@Peptide / FITC showed more significant accumulation in the IRI kidney. Figure 3 and Figure 4 ).

[0069] Example 4: CAT NPs@Peptide reduced the level of TSP-1 in the kidneys of mice after ischemia-reperfusion injury.

[0070] After fixation overnight with IRI kidney tissue in the sham group, the fixative was removed by immersion in 30% sucrose solution. Sections were then counterstained with anti-TSP-1 antibody and goat anti-rabbit IgG secondary antibody, and mounted with DAPI. Images were captured using confocal laser scanning microscopy, and all images were analyzed using ZEN Blue. Results showed that compared to the Sham group, TSP-1 levels were significantly increased in the PBS, Peptide, and CAT NPs treatment groups. However, only the Peptide-carrying nanomaterial treatment groups (BSA NPs@Peptide and CAT NPs@Peptide) did not show a significant increase in TSP-1 levels in IRI kidneys after treatment, indicating that Peptide-carrying nanomaterials can reduce TSP-1 levels in IRI kidneys in vivo. Figure 5 ).

[0071] Example 5: CAT NPs@Peptide reduced oxidative stress levels in mouse kidneys after ischemia-reperfusion injury.

[0072] After sham surgery and fixation with IRI kidney tissue, followed by glycosylation, sections were prepared, counterstained with anti-NHE antibody and goat anti-mouse IgG secondary antibody, and mounted with DAPI. Images were captured using confocal laser scanning microscopy, and all images were analyzed using ZENBlue. Results showed that compared to the PBS, Peptide, and BSA NPs@Peptide treatment groups, the CAT-carrying nanomaterial treatment groups (CAT NPs and CAT NPs@Peptide) significantly suppressed ROS levels in IRI kidneys, especially the CAT NPs@Peptide combination therapy group. This indicates that CAT-carrying nanomaterials can reduce ROS levels in IRI kidneys in vivo. Figure 6 ).

[0073] Example 6: NPs-Peptide has a protective effect on IRI kidneys.

[0074] The sham surgery group (Sham) had kidney tissue from patients with renal incision (IRI) fixed in 4% paraformaldehyde, followed by dehydration, embedding, and sectioning. These sections were stained with hematoxylin and eosin (H&E). All sections were observed and photographed using an optical microscope. Compared to the PBS and Peptide groups, CAT NPs and BSA NPs@Peptide reduced the degree of kidney damage in IRI. However, only the combined treatment with CAT NPs@Peptide significantly reduced the degree of vacuolar degeneration, cell casts, and cell necrosis in the kidney tissue after IRI; the treatment effect was the most significant, demonstrating that the combined treatment has a significant ability to reduce the increase of TSP-1 and ROS in the kidneys of patients with IRI and has a strong protective effect on the kidneys of patients with IRI. Figure 7 The mechanism by which CAT NPs@Peptide reduces TSP-1 and clears ROS is illustrated in the diagram below. Figure 8 As shown.

Claims

1. A polypeptide-modified nanoparticle, characterized in that, The nanoparticle is a nanoparticle capable of scavenging or consuming reactive oxygen species; the polypeptide is capable of adsorbing or binding TSP-1, and the amino acid sequence is shown as SEQ ID NO:

1.

2. The nanoparticle of claim 1, wherein, The nanoparticle is a nanoparticle having the function of scavenging or consuming reactive oxygen species, or the nanoparticle carries a drug capable of scavenging or consuming reactive oxygen species.

3. The nanoparticle of claim 2, wherein, The nanoparticle further comprises a polymer wrapped on the surface; the polymer is preferably a cross-linked polymer, and the polymerized monomer is selected from acrylamide, N-(3-aminopropyl) methacrylamide, 2-acrylamido-2-methylpropanesulfonic acid, 2-methacryloyloxyethylphosphocholine, and preferably 2-methacryloyloxyethylphosphocholine; the cross-linked polymer further comprises a secondary monomer, and the secondary monomer is acrydite, and the molar ratio of the secondary monomer to the polymerized monomer is preferably 1:10; and the cross-linking agent is N,N'-methylenebisacrylamide. And / or, the drug is CAT.

4. The nanoparticle of any one of claims 1-3, wherein, The polypeptide is combined with the functional group modified on the surface of the nanoparticle through its amino group or side chain group; preferably, the functional group is a functional group capable of click reaction with the amino group or side chain group; more preferably, the functional group is N-hydroxysuccinimide.

5. A method of preparing the nanoparticle according to any one of claims 1 to 4, characterized in that, The method comprises the following steps: Mixing the functional group modified nanoparticle with the polypeptide having the amino acid sequence shown as SEQ ID NO: 1, and obtaining the mixture; Preferably, the method further comprises the steps of preparing the polypeptide and / or modifying the nanoparticle with the functional group before mixing.

6. The method of claim 5, wherein, The method comprises one or more of the following steps: (1) Protein modification: modifying NAS on the surface of CAT to obtain a CAT solution modified with NAS; wherein the reaction molar ratio of CAT to NAS is 1:30; (2) Polymer modification: adding 2-methacryloyloxyethylphosphocholine, acrydite, N,N'-methylenebisacrylamide, TEMED and APS to the CAT solution modified with NAS obtained in step (1) at a molar ratio of 10:1:1:4:1, and preferably the molar ratio of 2-methacryloyloxyethylphosphocholine to CAT is 25000:1, to obtain CAT nanoparticle-NHS by reaction; (3) Polypeptide modification: adding the polypeptide to the solution containing CAT nanoparticle-NHS obtained in step (2), mixing uniformly, and incubating to obtain CAT nanoparticle-LSKL; Preferably, the molar ratio of CAT nanoparticle-NHS to the polypeptide is (1-2):

1.

7. A drug for treating or preventing ischemia-reperfusion injury, comprising the polypeptide modified nanoparticle according to any one of claims 1-4.

8. The medicament according to claim 7, wherein The ischemia-reperfusion injury is renal ischemia-reperfusion injury.

9. Use of the polypeptide modified nanoparticle according to any one of claims 1-4 in the preparation of a drug for treating or preventing ischemia-reperfusion injury.

10. The use according to claim 9, wherein the compound is ###00003### or a pharmaceutically acceptable salt thereof. The ischemia-reperfusion injury is renal ischemia-reperfusion injury.

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