Polypeptide with cfDNA capturing and ROS removing functions and preparation and application thereof

Abstract

The invention relates to a polypeptide with cfDNA capture and ROS removal functions as well as preparation and application of the polypeptide. According to the invention, the GPXP and PCTP functional modules are optimized, and a polypeptide drug system of GPXP and RES-PCTP (responsive cationic peptide, RES-PCTP) is constructed, i.e., the RES-PCTP-coated GPXP is constructed, so that the GPXP and PCTP-coated RES-PCTP-coated RES-PCTP-coated RES-PCTP-coated RES-PCTP-coated RES-PCTP is obtained. Polycation peptide PCTP (FAAVV-GG-GPLGLAG-RKRKRKRK) and GPXP (FAAVV-UKUNLN-GG-LEVSRKN) are jointly assembled into a polypeptide drug system (FAAVV-GG-GPLGLAG-RKRKRK / FAAVV-UKUNLN-GG-LEVSRKN), after nasal cavity drug delivery is conducted, the polypeptide drug system is sensitive to overexpressed MMP-2, the GPLGLAG-RKRKK / FAAVV-UKUNLN-GG-LEVSRKN is broken, RKRKRKRKRK polycations are released, cfDNA can be captured outside cells, activation of TLR-9 by the In addition, LEVSRKN is targeted to neuronal cells in apoptosis, and enters the interior of focus neurons, so that the GPX mimic peptide function of the UKUNLN sequence is exerted, and oxidative stress and neuronal ferroptosis are inhibited. And the two synergistically regulate and control the function of a nerve-colloid unit, so that the SBI after SAH is efficiently relieved at multiple targets.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and more specifically relates to a polypeptide with cfDNA capture and ROS scavenging functions and the preparation and application of the polypeptide. Background Art

[0002] 80% of spontaneous subarachnoid hemorrhage (SAH) is caused by ruptured intracranial aneurysms. The incidence rate is 6-7 per 100,000 people per year, and in some countries it is as high as 20 per 100,000 people. The mortality rate of SAH is 24h (37%), 7d (75%), and 28d (41.7%) after onset. 50% of the survivors of SAH will suffer long-term neuropsychological effects and decreased quality of life. The main reason for the high mortality and disability rate of SAH is secondary brain injury (SBI).

[0003] The causes of SBI after SAH may involve multiple aspects, which interact with each other and jointly lead to further damage to brain tissue. After SAH, a large amount of blood flows into the subarachnoid space, resulting in a sharp increase in intracranial pressure (ICP). This increase in pressure may be further aggravated by the obstruction of cerebrospinal fluid circulation, forming hydrocephalus, which further increases ICP. The increase in ICP will reduce cerebral perfusion pressure (CPP), that is, the difference between mean arterial pressure (MAP) and ICP. The decrease in CPP will reduce cerebral blood flow (CBF), causing global cerebral ischemia. This is one of the important causes of secondary brain injury after SAH. After SAH, the blood flowing into the subarachnoid space will mechanically stimulate the cerebral blood vessels and induce cerebral vasospasm. This spasm may last for days to weeks, leading to stenosis of cerebral arteries and further reducing CBF. In addition, blood degradation products such as hemoglobin and methemoglobin may also promote the occurrence of cerebral vasospasm. Cerebral vasospasm not only aggravates cerebral ischemia, but may also cause serious consequences such as cerebral infarction. After SAH, red blood cell degradation products can activate central nervous system immune cells, such as astrocytes and microglia, which release a large number of inflammatory factors through the NF-κB and mitogen-activated protein kinase pathways, causing extensive inflammation. The inflammatory response not only aggravates cerebral vasospasm, but may also lead to complications such as meningitis. In addition, the inflammatory response may also damage the blood-brain barrier (BBB), allowing harmful substances in the blood to enter the brain tissue, further aggravating brain damage. After SAH, brain tissue may edema due to factors such as increased intracranial pressure, cerebral vasospasm and inflammatory response. Brain edema is divided into two types: vasogenic edema and cytotoxic edema. Vasogenic edema is caused by the infiltration of plasma components into brain tissue due to BBB destruction, while cytotoxic edema is intracellular edema caused by metabolic disorders of brain cells. Brain edema will further increase ICP, reduce CPP, and aggravate cerebral ischemia and brain damage. After SAH, red blood cells in the blood rupture to release hemoglobin, which is further degraded into toxic substrates in brain tissue, such as iron ions and bilirubin. These toxic substrates can catalyze the production of reactive oxygen species (ROS), activate neuroinflammatory responses, destroy microcirculation and the blood-brain barrier, and lead to cerebral edema and acute neurological dysfunction. In addition, the degradation products of hemoglobin may also promote cerebral vasospasm and cerebral thrombosis, further aggravating brain damage. In addition to the above factors, secondary brain damage after SAH may also involve other factors, such as electrolyte disorders, neuronal death, and glial cell destruction. These factors may be intertwined and mutually reinforcing, leading to severe disability and death in patients after SAH.

[0004] The complex pathological mechanism of secondary brain injury after SAH makes neuroprotective agents a hot topic and a difficult point in research. Since 2006, more than 1,000 neuroprotective drugs have been launched, more than 900 have entered clinical animal experiments, and 114 have entered clinical research clinics, but very few have really made it to the end, and no neuroprotective drug has been successfully transformed into clinical practice so far. The uncontrollable neuroinflammatory cascade and oxidative stress triggered by neuro-glial units are one of the main reasons for the failure of neuroprotective drugs in clinical trials. Single-target therapy and traditional multi-target therapy consisting of a simple combination of a single single-target drug have limited efficacy in solving these interrelated events due to the spatiotemporal heterogeneity of the effects of single-target components. In view of the complex and changeable pathological characteristics of SBI, it is urgent to develop new therapeutic targets, multi-target intervention drugs and new drug delivery systems.

[0005] In recent years, cfDNA capture materials have been widely used in preclinical studies for the treatment of cfDNA-related diseases. For example, deoxyribonuclease I (DNase I) has shown good prospects for reducing cfDNA levels by directly cutting DNA chains, but its lack of specificity and related side effects limit its clinical application. Cationic polymers can neutralize negatively charged cfDNA and inhibit its recognition of the activation of TLR9 receptors, showing good therapeutic prospects in the treatment of certain cfDNA-related diseases (abdominal trauma, sepsis, etc.). However, known cationic materials still have basic defects such as severe toxicity and immunogenicity caused by high positive charges, and cannot capture cfDNA in a targeted manner at the lesion site. At the same time, due to the strict restrictions of the blood-brain barrier, the bioavailability is extremely low, which makes it impossible to effectively promote clinical applications. Summary of the invention

[0006] In view of the above technical problems, the present invention provides a polypeptide with cfDNA capture and ROS scavenging functions and its preparation and application, so as to solve the problems in the prior art of few SBI treatment targets and the failure of neuroprotective drugs caused by the neuroinflammatory cascade reaction and oxidative stress reaction triggered by microglia and neurons, and aims to provide a safe and effective drug targeting cfDNA capture / ROS scavenging for effectively inhibiting neuroinflammation in SBI after SAH.

[0007] To achieve the above objectives, in a first aspect, the present invention provides a polypeptide having cfDNA capture and ROS scavenging functions, comprising a polycationic peptide and a GPX (Glutathione Peroxidase) mimetic peptide, wherein the polycationic peptide comprises a hydrophobic series, an MMP-2 responsive series and a cationic series, and the GPX mimetic peptide comprises a hydrophobic series, a seleno series and a neuron targeting series.

[0008] The polypeptide of the present invention promotes the mutual aggregation of hydrophobic residues in the peptide chain through the hydrophobic effect of the hydrophobic series, buries the hydrophobic residues inside the molecule, and helps to form a stable polypeptide tertiary structure. After the polycationic peptide and the GPX mimetic peptide are assembled into a polypeptide system, they are sensitive to MMP-2, which promotes the rupture of the polycationic peptide to release polycations, which can capture negatively charged cfDNA outside the cell, inhibit cfDNA from activating TLR-9, and thus inhibit the NF-kB pathway to achieve the purpose of inhibiting the production of proinflammatory cytokines. In addition, the GPX mimetic peptide is targeted to apoptotic neuronal cells and enters the lesion neurons, exerting the function of the GPX mimetic peptide targeting the neuron series, and inhibiting oxidative stress and neuronal ferroptosis. The two synergistically regulate the function of the neuro-glial unit to achieve multi-target efficient reduction of SBI after SAH.

[0009] In a further optimized embodiment, the polycationic peptide has an amino acid sequence as shown in SEQ ID NO.1.

[0010] Among them, the amino acid sequence of SEQ ID NO.1 is FAAVV-GG-GPLGLAGG-RKRKRKRK.

[0011] In some further optimized embodiments of the above two schemes, the GPX mimetic peptide has an amino acid sequence as shown in SEQ ID NO.2.

[0012] Among them, the amino acid sequence of SEQ ID NO.2 is FAAVV-UKUNLN-GG-LEVSRKN.

[0013] Another object of the present invention is to provide a composition for preparing a drug for treating SBI, wherein the composition comprises the polypeptide described in the first aspect of the present invention.

[0014] In a preferred embodiment, the drug composition also contains other drugs with neuroprotective or antioxidant effects, such as but not limited to vitamin E, coenzyme Q10, etc., to further enhance the therapeutic effect of the polypeptide and improve the survival rate and quality of life of patients.

[0015] The third object of the present invention is to provide a drug for treating, inhibiting or alleviating SBI after SAH, comprising the polypeptide with cfDNA capture and ROS scavenging functions as described in the first aspect of the present invention.

[0016] In some more preferred embodiments, the above-mentioned drugs for treating, inhibiting or alleviating SBI after SAH further comprise some common carriers in the art.

[0017] In a more preferred embodiment, the drug for treating, inhibiting or alleviating SBI after SAH is a nasal preparation. There is a direct pathway between the nasal cavity and the brain, and nasal administration can bypass the blood-brain barrier, allowing the drug of the present invention to enter the brain directly through the olfactory epithelial cells and the trigeminal nerve pathway, reducing systemic side effects and reducing the total dose required for the drug, thereby achieving the treatment of brain diseases. In addition, drugs administered nasally can usually take effect quickly and can also avoid the infection risks that may be caused by injection administration.

[0018] Different from the existing technology, the above technical solution is based on the newly discovered new target cfDNA in clinical practice. It aims at the key scientific problems faced by SBI treatment, namely how to solve the single target problem to improve SBI and how to achieve efficient and precise targeting of target cells - microglia and neurons. This project "Active peptide-mediated cfDNA capture and ROS clearance for secondary brain injury after subarachnoid hemorrhage" is proposed. It is planned to optimize the GPXP and PCTP functional modules and construct a polypeptide drug system of GPXP and responsive cationic peptide (RES-PCTP), namely RES-PCTP@GPXP. The polycationic peptide PCTP (FAAVV-GG-GPLGLAG-RKRKRKRK) and GPXP (FAAVV-UKUNLN-GG-LEVSRKN) were co-assembled into a peptide drug system (FAAVV-GG-GPLGLAG-RKRKRKRK / FAAVV-UKUNLN-GG-LEVSRKN). After nasal administration, the peptide drug system was sensitive to overexpressed MMP-2, GPLGLAG was broken, and RKRKRKRK polycation was released, which could capture cfDNA outside the cell, inhibit cfDNA activation of TLR-9, thereby inhibiting the NF-kB pathway and the production of proinflammatory cytokines. In addition, LEVSRKN was targeted to apoptotic neuronal cells and entered the lesion neurons, exerting the GPX mimetic peptide function of the UKUNLN sequence to inhibit oxidative stress and neuronal ferroptosis. The two synergistically regulated the function of the neuro-glial unit to achieve multi-target and efficient reduction of SBI after SAH.

[0019] The above-mentioned records related to the invention content are only an overview of the technical solution of the present application. In order to enable ordinary technicians in the field to more clearly understand the technical solution of the present application, and then implement it according to the text of the specification and the contents recorded in the drawings, and to make the above-mentioned purpose and other purposes, features and advantages of the present application easier to understand, the following is an explanation in combination with the specific implementation mode and drawings of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The drawings are only used to illustrate the principles, implementation methods, applications, characteristics and effects of the specific embodiments of the present application and other related contents, and shall not be considered as limitations of the present application.

[0021] In the drawings of the specification: Figure 1 The data are the correlation between serum free cell DNA (cfDNA) levels and initial clinical conditions, among which A is the comparison of cfDNA levels between healthy people and patients with aSAH (aneurysmal Subarachnoid Hemorrhage); B is the correlation analysis between cfDNA and initial clinical status; C is the difference in serum cfDNA levels between patients with mild SAH and severe SAH; D is the relationship between serum cfDNA and mFisher score (severity of bleeding); E is the receiver operating characteristic curve (ROC) analysis of cfDNA levels predicting severe SAH; F is the comparison of serum cfDNA between patients with good prognosis and patients with poor prognosis in aSAH; G is the correlation between cfDNA and poor prognosis (mRS 3-6); H is the receiver operating characteristic curve (ROC) analysis of cfDNA levels predicting poor prognosis in patients with aSAH; Figure 2 Diagram of the SAH mouse model established by intravascular puncture; Figure 3 This is a schematic diagram of the synthesis of the RES-CTP@GPXP polypeptide drug system in Example 2; Figure 4 The microscopic morphology of the polypeptide RES-CTP@GPXP provided in Example 2 observed by transmission electron microscopy (TEM), scale bar: 200 nm; Figure 5 is the selenium (Se) content detected by inductively coupled plasma mass spectrometry (ICP-MS); Figure 6 The Z-average particle size and zeta potential were measured in phosphate buffered saline (PBS) medium using Zeta sizer Nano ZS. Figure 7 Results of quantitative assessment of sensorimotor function using mGS (n=12 independent samples); Figure 8 Total walking distance in a 10-min open field test on day 7 after subarachnoid hemorrhage (SAH) (n = 6 independent samples); Fig. 9 The mean speed in the 10-min open field test on day 7 after subarachnoid hemorrhage (SAH) (n = 6 independent samples); Fig.10Representative immunofluorescence staining analysis results of matrix metalloproteinase-2 (MMP-2, red) / DAPI (blue) in the ipsilateral basal cortex 24 hours after subarachnoid hemorrhage (SAH), scale bar: 100 μm (n = 5 independent samples); Fig.11 Quantitative analysis of mean fluorescence intensity (MFI) of matrix metalloproteinase-2 24 h after subarachnoid hemorrhage (SAH), scale bar: 100 μm (n = 5 independent samples); Fig.12 is the cumulative leucine-enkephalin (LK) release of RES-CTP@GPXP treated or untreated with matrix metalloproteinase-2 (MMP-2); Fig.13 The ζ potential changes of RES-CTP@GPXP with the continuous addition of matrix metalloproteinase-2 (MMP-2) and calf thymus DNA; Fig.14 is the binding affinity of RES-CTP@GPXP treated or untreated with matrix metalloproteinase-2 (MMP-2) to DNA; Fig.15 DPPH scavenging efficiency was used to evaluate the total antioxidant performance; Fig.16 To detect neuronal survival rate after peptide nmparticle treatment using CCK-8 assay; Fig.17 Fluorescent images of the mouse brain were taken at different time points after Cy7-GPXP, Cy7-CTP@GPXP, and Cy7-RES-CTP@GPXP were administered intranasally to the mice; Fig.18 To quantitatively analyze the total radiation efficiency of the brain after intranasal administration of Cy7-GPXP, Cy7-CTP@GPXP, and Cy7-RES-CTP@GPXP to mice (n=3 independent samples); Fig.19 To perform ex vivo fluorescence imaging of the isolated brain and other major organs at different time points after treatment with different peptide nanoparticles; Fig. 20 To quantitatively analyze the total radiation efficiency after RES-CTP@GPXP treatment in in vitro experiments (n=3 independent samples); Fig.21A is a representative immunofluorescence staining image of the ipsilateral basal cortex on day 3 after subarachnoid hemorrhage (SAH), including TUNEL (red) / NeuN (green) / DAPI (blue), MDA (red) / NeuN (green) / DAPI (blue), and 4-HNE (red) / NeuN (green) / DAPI (blue), scale bar: 100 μm; (n=5 independent samples); B is the quantitative analysis of TUNEL-positive cells in total DAPI-positive cells, (n=5 independent samples); C is the quantitative analysis of NeuN-positive cells in total DAPI-positive cells, (n=5 independent samples); Fig. 22 D in the middle is the iron content of the ipsilateral basal cortex detected by an iron assay kit on the 3rd day after subarachnoid hemorrhage (SAH) (n=5 independent samples); E is the GSH content of the ipsilateral basal cortex detected by a glutathione (GSH) assay kit on the 3rd day after SAH (n=5 independent samples); F and G are quantitative analysis of the mean fluorescence intensity (MFI) of MDA and 4-HNE, respectively (n=5 independent samples); H is the mitochondrial morphology of the ipsilateral basal cortex observed by transmission electron microscopy (TEM) on the 3rd day after SAH, scale bar: 2μm (n=5 independent samples). DETAILED DESCRIPTION

[0022] In order to explain in detail the possible application scenarios, technical principles, specific schemes that can be implemented, and the purposes and effects that can be achieved, the following is a detailed description of the specific embodiments listed in conjunction with the accompanying drawings. The embodiments described herein are only used to more clearly illustrate the technical solutions of the present application, and are therefore only used as examples, and cannot be used to limit the scope of protection of the present application.

[0023] Reference to "embodiment" herein means that the specific features, structures or characteristics described in conjunction with the embodiment may be included in at least one embodiment of the present application. The term "embodiment" appearing in various places in the specification does not necessarily refer to the same embodiment, nor does it particularly limit its independence or association with other embodiments. In principle, in the present application, as long as there is no technical contradiction or conflict, the various technical features mentioned in the embodiments can be combined in any way to form a corresponding implementable technical solution.

[0024] Unless otherwise defined, the technical terms used in this document have the same meanings as those generally understood by those skilled in the art to which this application belongs; the use of relevant terms in this document is only for describing specific embodiments and is not intended to limit this application.

[0025] In the description of this application, the term "and / or" is an expression used to describe the logical relationship between objects, indicating that three relationships may exist, for example, A and / or B, which means: A exists, B exists, and A and B exist at the same time. In addition, the character " / " in this article generally indicates that the objects before and after are in an "or" logical relationship.

[0026] In the present application, terms such as “first” and “second” are merely used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship of quantity, priority or sequence between these entities or operations.

[0027] Without further limitations, in this application, the words "include", "comprises", "has" or other similar expressions used in the sentences are intended to cover non-exclusive inclusion. These expressions do not exclude the presence of additional elements in the process, method or product including the elements, so that the process, method or product including a series of elements may include not only those limited elements, but also other elements not explicitly listed, or also include elements inherent to such process, method or product.

[0028] Similar to the understanding in the Examination Guidelines, in this application, expressions such as "greater than", "less than", "exceed" and the like are understood to exclude the number itself; expressions such as "above", "below", "within" and the like are understood to include the number itself. In addition, in the description of the embodiments of this application, "multiple" means more than two (including two), and similar expressions related to "multiple" are also understood in this way, such as "multiple groups", "multiple times", etc., unless otherwise clearly and specifically limited.

[0029] In the description of the embodiments of the present application, space-related expressions used, such as "center", "longitudinal", "lateral", "length", "width", "thickness", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "vertical", "top", "bottom", "inside", "outside", "clockwise", "counterclockwise", "axial", "radial", "circumferential", etc., indicate the orientation or position relationship based on the orientation or position relationship shown in the specific embodiments or drawings, and are only for the convenience of describing the specific embodiments of the present application or facilitating the reader's understanding, and do not indicate or imply that the referred device or component must have a specific position, a specific orientation, or be constructed or operated in a specific orientation, and therefore cannot be understood as a limitation on the embodiments of the present application.

[0030] Unless otherwise expressly specified or limited, in the description of the embodiments of the present application, the terms such as "install", "connect", "connect", "fix", "set", etc. used should be understood in a broad sense. For example, the "connection" can be a fixed connection, a detachable connection, or an integrated setting; it can be a mechanical connection, an electrical connection, or a communication connection; it can be a direct connection, or it can be indirectly connected through an intermediate medium; it can be the internal connection of two elements or the interaction relationship between two elements. For technicians in the technical field to which the present application belongs, the specific meanings of the above terms in the embodiments of the present application can be understood according to the specific circumstances.

[0031] Cerebrovascular disease is a major health problem worldwide. Among them, subarachnoid hemorrhage (SAH) is a serious cerebrovascular disease with extremely high mortality and disability rates. After SAH, brain ischemia and reperfusion will cause brain damage, in which microglia in the brain will be activated and release a large number of reactive oxidative species (ROS), which will further damage brain tissue, forming a vicious cycle. At the same time, the level of cell-free DNA (cfDNA) in the cerebrospinal fluid (CSF) will also increase significantly. These cfDNAs contain a large number of damage-associated molecular patterns (DAMPs), triggering some new inflammatory driving mechanisms, further aggravating brain damage. Among them, pathological cell-free DNA (cfDNA) triggers pro-inflammatory responses through the activation of toll-like receptors on immune cells. This mechanism plays a very important role in the occurrence and development of certain inflammatory diseases, but there is no literature report on the role of cfDNA in SBI after SAH. Please refer to Figure 1 Based on the data from clinical studies, we found a positive correlation between serum cfDNA levels and the severity of SAH, and serum cfDNA was correlated with poor prognosis three months after SAH.

[0032] Peptide drugs have the advantages of high specificity, low toxicity, low immunogenicity, easy modification, and biocompatibility, and are expected to become leading molecules and alternative drugs for the treatment of neurological diseases. Glucagon-like peptide-1-liraglutide (clinically used to control blood sugar) showed good neuroprotective effects in animal experiments to treat SAH; apoE-related peptides (Apolipoprotein E, a mimetic peptide or active fragment of apolipoprotein E) also showed certain anti-inflammatory and antioxidant effects. In addition, existing drugs can often only treat one aspect and cannot comprehensively solve the problem of brain damage. For example, antioxidants can remove ROS but cannot reduce the level of cfDNA, while polycationic peptides can capture cfDNA but cannot remove ROS. Secondly, the side effects of existing drugs are large and may impose additional burdens on patients. Finally, existing drugs are not effective in crossing the blood-brain barrier, which limits their application in the treatment of brain damage. Therefore, the development of a peptide drug with multiple functions, low side effects and the ability to effectively cross the blood-brain barrier is an urgent problem to be solved in this field.

[0033] The present invention constructs peptides based on clinical targets and designs an MMP-2 responsive peptide to exert the multi-target effect of the peptide, and treats SBI based on cfDNA / ROS dual-clearing drugs targeting neuro-glial units. Two representative targets, cfDNA and ROS, were selected as model combination modes for drug design, and a drug treatment strategy of "in situ proliferation and single targeted release" was proposed, in which the key drug components were targeted and released on the lesions to improve the efficacy of the peptide drug.

[0034] MMP-2, or Matrix Metallopeptidase -2, is a gene located on chromosome 16 and is a member of the matrix metalloproteinase (MMP) gene family. The protein encoded by MMP-2 is a collagenase A, type IV collagenase that has the ability to cleave extracellular matrix components and participate in signal transduction molecules. Activation of this protein can occur on the cell membrane and can be activated extracellularly by proteases or intracellularly by S-glutathione without the need for proteolytic removal of the pro-domain. MMP-2 is thought to be involved in multiple pathways, including a role in the nervous system, endometrial menstrual breakdown, regulation of vascularization, and metastasis. Notably, mutations in this gene have been associated with Winchester syndrome and nodular arthropathy osteolysis (NAO) syndrome.

[0035] In terms of the relationship between SAH and MMP-2, although there is no direct evidence that SAH causes overexpression of MMP-2, studies have shown that after subarachnoid hemorrhage, a variety of biomolecules and signaling pathways in brain tissue change, and these changes may include changes in the expression and activity of MMP-2. However, the specific mechanism still needs further research to clarify. The present invention designs an MMP-2 responsive peptide, based on cfDNA / ROS dual-clearing drug targeting neuro-glial unit to treat SBI, selects two representative targets cfDNA and ROS as model combination modes for drug design, and proposes a "in situ proliferation and single targeted release" drug treatment strategy, in which the key drug ingredients are targeted and released on the lesion to improve the efficacy of the peptide drug. Specifically, active peptide-mediated cfDNA capture and ROS clearance are proposed for secondary brain injury after subarachnoid hemorrhage, and the GPXP and CTP functional modules are optimized to construct a GPXP and responsive polycationic peptide (RES-PCTP) peptide drug system, namely RES-PCTP@GPXP.

[0036] The polypeptide used in the present invention was purchased from Anhui Hefei Kesheng Jingtai Biotechnology Co., Ltd. and prepared by solid phase synthesis with a purity of 98%.

[0037] All ethical issues of animal experiments in the following examples of the present invention complied with the Animal Care Guidelines of Fujian Medical University (IACUC FJMU 2024-Y-0857), and also complied with the “Guide for the Care and Use of Laboratory Animals” of the National Institutes of Health of the United States.

[0038] Unless otherwise specified, the reagents, instruments and methods used in the following examples of the present invention are generally known in the art.

[0039] Unless otherwise specified, the data of the following embodiments of the present invention are expressed in the form of mean ± standard deviation (mean ± SD). Statistical analysis was performed using GraphPad Prism software (version 10.0, San Diego, California, USA) and MedCalc software (version 20.0.4, Ostend, Belgium). The Mann-Whitney test (two-tailed) was used to compare the differences between two unpaired groups, and the one-way analysis of variance (ANOVA) test was used to compare the differences between multiple groups of data. The performance of serum free deoxyribonucleic acid (cfDNA) in predicting brain damage in patients with aneurysmal subarachnoid hemorrhage (aSAH) was evaluated by the receiver operating characteristic curve (ROC) and the area under the ROC curve (AUC). Statistical analysis results were analyzed by one-way analysis of variance (ANOVA) or Student's t-test when applicable. When p<0.05, p<0.01,When p<0.001, there was a significant difference between the data.

[0040] Example 1

[0042] See also Figure 2 The SAH mouse model was established by intravascular puncture. Specifically, the following steps were included: 1. Select 10-week-old male C57BL / 6 mice (weight 20-25g); 2. Anesthesia was performed by intraperitoneal injection of 0.2 ml / 10 g tribromoethanol (Yoshida Biotechnology, China), followed by shaving of hair 5 min later and disinfection with 2% chlorhexidine; 3. Place the mouse in supine position and fix it on the operating table. Make a midline incision on the neck, separate and expose the left common carotid artery, left internal carotid artery, and left external carotid artery under a stereomicroscope, electrocoagulate and cut off the branches of the external carotid artery, and temporarily block the blood flow of the common carotid artery with an artery clamp; 4. Ligate and cut off the external carotid artery 5 mm distal to the bifurcation of the carotid artery to form a stump of about 5 mm in length. Clamp the stump distal to the stump and cut a small "V"-shaped incision. Introduce a 2-cm-long 5-0 puncture line and carefully move it toward the distal end until you feel resistance. It is estimated that you have reached the intracranial segment of the internal carotid artery. Then move it slightly distally until you estimate that you have pierced the bifurcation of the internal carotid artery. Quickly withdraw the nylon line, ligate the cut stump to stop bleeding, and remove the artery clamp at the common carotid artery to restore blood perfusion. 5. Reposition the neck muscles in turn, and suture the subcutaneous tissue and skin in layers. Monitor vital signs throughout the operation. In the sham group (Sham), only the nylon thread was introduced until resistance was felt, and no force was applied to puncture the distal end. The rest of the surgical procedures were consistent with the above method. 6. Closely monitor the vital signs of experimental animals after surgery and keep them in separate cages.

[0043] Mice in the sham-operated group also underwent surgical manipulation, but without puncturing of blood vessels. Example 2

[0044] A peptide with cfDNA capture and ROS scavenging functions: RES-CTP@GPXP

[0045] This embodiment provides a polypeptide RES-CTP@GPXP with cfDNA capture and ROS scavenging functions, comprising a responsive polycationic peptide (RES-CTP) and a GPX mimetic peptide (GPXP), wherein the responsive polycationic peptide comprises a hydrophobic series, an MMP-2 responsive series and a cationic series, and the GPX mimetic peptide comprises a hydrophobic series, a selenogene series and a neuron targeting series. Their design principles are as follows: 1) The design of the polycationic peptide RES-CTP includes a hydrophobic sequence (FAAVV) to drive self-assembly, a matrix metalloproteinase-2 (MMP-2) cleavable sequence (GPLGLA) to respond to the elevated MMP-2 level in the lesion site, and a cationic sequence (RKRKRKRK) to clear pathological extracellular free DNA (cfDNA); 3) The design of GPXP includes a hydrophobic sequence (FAAVV) to drive self-assembly, a selenium-containing glutathione peroxidase mimetic sequence (UKUNLN) to reduce oxidative damage, and a neuron targeting sequence (LEVSRKN) to increase its uptake in damaged neurons.

[0046] Specifically, the polycationic peptide contains a FAAVV hydrophobic sequence, an MMP-2 responsive series -GG-GPLGLAGG- and a cationic series RKRKRKRK, and the amino acid sequence of the polycationic peptide is FAAVV-GG-GPLGLAGG-RKRKRKRK. The GPX mimetic peptide contains a FAAVV hydrophobic sequence, a seleno series -(SeCys)-K-(SeCys)-NLNGG and a neuron targeting series LEVSRKN, and the amino acid sequence of the GPX mimetic peptide is FAAVV-UKUNLN-GG-LEVSRKN.

[0047] like Figure 3 As shown, the preparation method of the above polypeptide RES-CTP@GPXP is: Step 1: Peptide synthesis. First, according to the amino acid sequences shown in SEQ ID NO.1 and SEQ ID NO.2, the responsive polycationic peptide and GPX mimetic peptide were synthesized by solid phase synthesis. During the synthesis, the Fmoc protection strategy was used to gradually couple each amino acid to finally obtain the target polypeptide. The amino acid sequence shown in SEQ ID NO.1 is FAAVV-GG-GPLGLAGG-RKRKRKRK, and the amino acid sequence shown in SEQ ID NO.2 is FAAVV-UKUNLN-GG-LEVSRKN.

[0048] Step 2: Peptide purification. The synthesized peptide was purified by reverse phase high performance liquid chromatography (RP-HPLC) to obtain a high purity peptide. The chromatographic conditions were: the mobile phase was a mixed solvent of acetonitrile and water, gradient elution, a flow rate of 1.0 mL / min, and a detection wavelength of 214 nm.

[0049] Step 3: Peptide compounding. The purified responsive polycationic peptide RES-CTP and GPX analog peptide GPXP lyophilized powder were dissolved in PBS (pH=7.4) at a concentration of 5 mM, and then incubated at 25°C for 30 minutes to form a peptide with cfDNA capture and ROS scavenging functions - RES-CTP@GPXP peptide drug system. During the co-assembly process, the ratio of the two was changed to investigate their effects on the treatment of brain injury, and the optimal co-assembly ratio was finally determined.

[0050] RES-CTP and GPXP are amphiphilic structures, and their shared FAAVV hydrophobic sequence can drive co-assembly in aqueous solution. Therefore, we further studied the co-assembly behavior of the RES-CTP@GPXP system. It was observed that RES-CTP and GPXP co-assembled to form uniform spherical particles with a particle size of about 30 nm (see Figure 4 We then measured the selenium content using inductively coupled plasma mass spectrometry (ICP-MS) and found that GPXP had a high selenium content of approximately 6.67%, whereas after co-assembly with the selenium-free RES-CTP, the selenium content decreased to approximately 3.47% (see Figure 5 ). It can be seen that RES-CTP and GPXP can be effectively co-assembled in aqueous solution to form RES-CTP@GPXP.

[0051] Next, the peptide drug system RES-CTP@GPXP with cfDNA capture and ROS scavenging functions was dissolved in phosphate buffer (PBS, 10 mM, pH=7.4), and its surface charge properties and particle size were tested using Zeta sizer Nano ZS. Figure 6 As shown, the results show that the Z-average particle size of GPXP is about 39.13nm and the zeta potential is about +5.01mv; while RES-CTP@GPXP has a similar Z-average particle size, but the zeta potential increases to about 19.83mv, which is attributed to its co-assembly with the positively charged RES-CTP with a high zeta potential (30.47mV). In addition, we found that the particle size and zeta potential of RES-CTP@GPXP showed good stability within 72h, which proves that they are suitable for in vitro and in vivo applications.

[0052] Example 3

[0053] After modeling, the peptide group was immediately given 4 mg / kg (RES-CTP@GPXP peptide drug system), 10 ul / mouse; the edaravone / dexamethasone group (purchased from Nanjing Xiansheng Pharmaceutical Co., Ltd.) was given 8 ul / mouse, and the saline group was given 10 ul / mouse saline. For intranasal administration, the mice were placed supine under anesthesia. Each nostril was alternately administered 10 μL each time, with a total dose of 4 mg / kg.

[0054] To evaluate the improvement of brain injury in vivo, hematoxylin and eosin (H&E) staining and Nissl staining were performed. The mouse brain was perfused with 10% formaldehyde solution and then immersed in 4% paraformaldehyde solution at 4°C for 72 h. The brain tissue of the ipsilateral basal cortex was embedded in 30% paraffin, cut into 20 μm thick sections, and dewaxed with xylene and graded alcohol. Finally, H&E staining was performed using a hematoxylin and eosin staining kit, and Nissl staining was performed using toluidine blue. The sections were observed and photographed under an optical microscope.

[0055] To characterize the effects of different treatments on the recovery of neurological deficits after subarachnoid hemorrhage (SAH), we performed neurological assessments and behavioral tests. First, we assessed sensorimotor function using the modified Garcia score (mGS). Figure 7 , the mGS of the saline treatment group (Saline) was lower, indicating that SAH caused severe sensory and motor deficits. However, treatment with the peptide RES-CTP@GPXP with cfDNA capture and ROS scavenging functions provided by the present application can increase the mGS to about 16 points, showing a significant recovery of neurological deficits.

[0056] In addition, we also conducted behavioral assessments, including an open field test and a Y-maze spontaneous alternation task. The open field test mainly evaluates motor function and physical activity by measuring the total walking distance and speed. The test results showed that the total walking distance and average speed of the RES-CTP@GPXP treatment group increased compared with the other treatment groups, indicating improved motor activity and recovery of physical function (see Figure 8 , Fig. 9 ). Anxiety-like behavior, assessed by the percentage of time spent in the center area of ​​the open field test, was also reduced after RES-CTP@GPXP treatment, and these mice spent more time in the center area. Finally, we assessed spatial working memory, one of the neurological functions commonly impaired after SAH, using the Y-maze spontaneous alternation task. RES-CTP@GPXP-treated mice showed a higher alternation rate, indicating improved working memory function. All of the above experimental results indicate that as an advanced multi-target therapeutic platform, RES-CTP@GPXP can significantly enhance neurobehavioral outcomes after SAH. Example 4

[0057] See also Fig.10 , Fig.11 , in vitro immunofluorescence staining results showed that MMP-2 expression in microglia was significantly increased after heme treatment. In addition, further subarachnoid hemorrhage (SAH) surgery on mice also increased the level of MMP-2 in the ipsilateral basal cortex compared with the sham group. These results indicate that MMP-2 is overexpressed in SAH brain injury, which provides a pathological signal for triggering the in situ cleavage of RES-CTP@GPXP. Previous work has demonstrated that the peptide bond (GPLGLAG) can be cleaved in the presence of MMP-2. Please refer to Fig.12 , after incubation with MMP-2, the cationic sequence LAGRKRKRKRK (referred to as LK peptide) was detected to be released from the RES-CTP@GPXP system. In the presence of MMP-2, the cumulative release rate of LK peptide reached about 72.13%, while in the absence of MMP-2, the cumulative release rate of LK peptide from the RES-CTP@GPXP system was only about 15.40%. For the CTP@GPXP system, the cumulative release rate of LK peptide was less than 16% regardless of the presence or absence of MMP-2. In addition, after dialysis and freeze-drying after 48h of MMP-2 incubation, the released LK peptide was detected by electrospray ionization mass spectrometry (ESI-MS), and the detection results showed that MMP-2 could induce the cleavage of the GPLGLAG bond and the release of the cationic LK peptide. In addition, we monitored the changes in colloidal properties after the addition of MMP-2 and found that the Z-average particle size decreased from about 37.15 nm to about 25.05 nm and the zeta potential decreased from about +19.83 mv to about +14.2 mv (see Fig.13 ). This indicates that the LK peptide is separated from the co-assembly system RES-CTP@GPXP, resulting in a decrease in its particle size and surface positive charge. The above test results show that the RES-CTP@GPXP polypeptide drug system provided by the present invention can remain stable under normal conditions, but splits into LK peptide and remaining GPXP in the presence of pathological MMP-2. Example 5

[0058] DNA binding ability was assessed using the Quant-iT PicoGreen double-stranded DNA (dsDNA) kit. Calf thymus DNA was mixed with PicoGreen reagent in Tris-EDTA buffer and incubated in the dark for 10 min to ensure optimal fluorescence conditions. Then, RES-CTP@GPXP was pre-incubated for 24 h in the presence or absence of matrix metalloproteinase-2 (MMP-2) before being introduced into the calf thymus DNA solution and incubated for an additional 30 min in the dark. Subsequently, the fluorescence intensity was measured at 520 nm to quantify the DNA binding affinity.

[0059] The DNA binding affinity of the RES-CTP@GPXP system was investigated by competitive binding experiments between double-stranded DNA (dsDNA) and PicoGreen reagent. Fig.14 , it was observed that the DNA binding rate of RES-CTP@GPXP increased with the increase of peptide / DNA mass ratio, reaching about 54.94% at a mass ratio of 1.0 and about 75.58% at a mass ratio of 10.0. However, its DNA binding ability was still weak compared with the RES-CTP group. This was mainly because after co-assembly, the neutral GPXP diluted the positive charge from RES-CTP. However, after incubation with MMP-2, the cationic LK peptide could be released from the co-assembly system and restore its positive charge effect, thereby further enhancing the DNA binding ability.

[0060] In addition, we further investigated the DNA clearance properties. Fig.13 As shown, after incubation with MMP-2, calf thymus DNA (ctDNA, a typical double-stranded DNA) was added sequentially, resulting in an increase in the Z-average particle size to about 91.68 nm and a decrease in the zeta potential to about -13.70 mv. This indicates that the cationic LK peptide can remove double-stranded DNA by electrostatic adsorption to form a nm-scale complex, which is beneficial to inhibit the binding of cytosolic free DNA (cfDNA) to TLR9. Example 6

[0061] This example conducted a diphenyl-1-trinitrophenylhydrazine (DPPH) experiment to detect its total antioxidant capacity. Fig.15As shown in the figure, RES-CTP@GPXP can inhibit the generation of DPPH free radicals to varying degrees. The scavenging efficiency of RES-CTP@GPXP for DPPH is over 50% at a concentration of about 80µg / mL. In addition, a cell model of neuronal injury induced by hemorrhage was established after treatment with 60μM heme, in which a large amount of reactive oxygen species (ROS) was produced in mouse hippocampal neuronal cells (HT22 cells). Treatment with the RES-CTP@GPXP peptide drug system can effectively reduce the intracellular ROS level from about 100% to about 14.43%, thereby alleviating the oxidative stress of HT22 cells to a great extent.

[0062] In summary, the key functions of the RES-CTP@GPXP polypeptide drug system provided by the present invention have been confirmed, including responsiveness to matrix metalloproteinase-2 (MMP-2), high binding affinity to cytoplasmic free DNA (cfDNA) and the ability to scavenge reactive oxygen species (ROS), which indicate that it has the potential to achieve in situ proliferation and dual clearance of pathological cfDNA / ROS in further in vivo experiments. Example 7

[0063] In order to evaluate the neuroprotective effect and safety of the RES-CTP@GPXP peptide drug system, we conducted in vitro cell experiments. First, a cell model of subarachnoid hemorrhage (SAH) was established by treating HT22 cells with 60μM heme, at which time the cell survival rate was reduced to about 50.07%. Then, to illustrate the superiority of the matrix metalloproteinase-2 (MMP-2)-mediated in situ proliferation strategy, we set up a single-target treatment group (RES-CTP and GPXP alone), a simple combination of CTP@GPXP without MMP-2 responsiveness as a traditional multi-target treatment group, and RES-CTP@GPXP as a flexible multi-target treatment group to treat HT22 cells. The results showed that the RES-CTP@GPXP treatment group had the highest cell survival rate of about 91.81% at a concentration of 10μg / mL (see Fig.16 ), whose in vitro neuroprotective effect was stronger than that of other treatment groups, which was attributed to its more effective dual clearance of cytoplasmic free DNA (cfDNA) and reactive oxygen species (ROS). In addition, we also used a standard cell counting kit-8 (CCK-8) experiment to evaluate their cytotoxicity. Different concentrations of CTP@GPXP and RES-CTP@GPXP were co-cultured with glial unit-related cells (including HT22 cells and mouse microglial BV2 cells). After 24h of incubation, the survival rate of these cells exceeded 95% even when the cells were exposed to a high concentration of 50μg / mL. Therefore, RES-CTP@GPXP showed a strong inhibitory effect on neuronal cell death.

[0064] In contrast, polyamidoamine dendrimer G3 (PAMAM-G3), a traditional material for removing cfDNA, showed increased cytotoxicity. It can be seen that the RES-CTP@GPXP peptide drug system has effective in vitro neuroprotective effects, safety and therapeutic potential. Example 8

[0065] The blood-brain barrier (BBB), composed of tight junctions between brain microvascular endothelial cells, has limited the development of therapeutic drugs for central nervous system (CNS) diseases. Although subarachnoid hemorrhage (SAH) can temporarily disrupt the integrity of the BBB, allowing some drugs to enter the brain, the time window for this effect is limited. Delivery systems for macromolecules and nanomedicines administered by traditional routes (such as intravenous injection) still need to overcome the BBB, which may need to be achieved through specific surface modification strategies.

[0066] The nose-brain pathway has attracted increasing attention as a new and efficient central nervous system drug delivery route. This pathway mainly utilizes the olfactory nerve and trigeminal nerve pathways to achieve direct drug delivery to the brain and bypass the BBB. Its potential for non-invasive central nervous system drug delivery makes nasal administration very promising in clinical translation.

[0067] In this example, Cy7-labeled peptide nanoparticles (including Cy7-GPXP, Cy7-CTP@GPXP, and Cy7-RES-CTP@GPXP) were delivered intranasally to mice with subarachnoid hemorrhage (SAH) and monitored using an in vivo fluorescence imaging system (IVIS). In the Cy7-RES-CTP@GPXP group, a weak fluorescence signal was detected in the brain within 10 minutes after administration, and the signal was significantly enhanced at 30 minutes and lasted for at least 12 hours. During this period, the fluorescence intensity of RES-CTP@GPXP reached a peak (see Fig.17 , Fig.18 ). The transport efficiency of Cy7-RES-CTP@GPXP and Cy7-CTP@GPXP from the nose to the brain was roughly similar, while that of Cy7-GPXP was lower. This indicates that Cy7-RES-CTP@GPXP and Cy7-CTP@GPXP significantly improved the transport efficiency to the brain.

[0068] The enhanced nose-to-brain delivery efficiency of Cy7-RES-CTP@GPXP suggests that it has the potential to rapidly reach therapeutic levels in the brain, making it an ideal candidate for emergency treatment of SAH. Ex vivo fluorescence imaging of major organs, including the liver and kidneys, showed that Cy7-RES-CTP@GPXP, Cy7-CTP@GPXP, and Cy7-GPXP were also distributed in these organs, which may be due to their partial absorption into the systemic circulation through the capillary network of the nasal mucosa. See Fig.19 , Fig. 20 , the fluorescence signals in the liver and kidneys decreased significantly after 24 h, indicating that they were cleared through liver metabolism or renal excretion. These results indicate that intranasal administration of Cy7-RES-CTP@GPXP can promote rapid and sustained brain delivery while minimizing systemic exposure. This delivery method has great potential in the treatment of acute neurological diseases such as SAH, which require timely and targeted therapeutic intervention. Example 9

[0069] Oxidative stress is a key factor in neuronal damage, which is involved in the initiation of neuroinflammation and exacerbates the development of neuroinflammation. After subarachnoid hemorrhage (SAH), neuronal oxidative damage was found, mainly manifested by increased levels of 8-hydroxyguanine (8-OHG, a DNA / RNA oxidation biomarker), which was mainly localized in damaged neurons. RES-CTP@GPXP treatment can effectively reduce oxidative damage through the ROS elimination function of GPXP peptide, and its effect is better than CTP@GPXP treatment.

[0070] When reaching the lesion site, the RES-CTP@GPXP system can utilize matrix metalloproteinase 2 (MMP-2)-mediated in situ proliferation to decompose into individual CTP and GPXP components. Among them, CTP can clear pathological circulating free DNA (cfDNA) and block the activation of TLR9, thereby reducing neuroinflammation; while GPXP can alleviate the oxidative damage of neurons. Therefore, this flexible strategy can achieve better results in restoring glial unit dysfunction. Example 10

[0071] This example studies the inhibitory effect of the RES-CTP@GPXP peptide drug system on neuronal programmed cell death. Subarachnoid hemorrhage (SAH) can induce neuroinflammation and oxidative damage, both of which can drive different types of programmed cell death, among which neuronal apoptosis and ferroptosis are particularly important. First, we performed NeuN / TUNEL staining on the third day after SAH, and the results showed that the number of TUNEL-positive neurons in the saline-treated group increased significantly, indicating that there was extensive neuronal apoptosis in the ipsilateral basal cortex (see Fig.21). However, compared with other treatment groups, RES-CTP@GPXP treatment significantly reduced the proportion of apoptotic cells. The above experimental results indicate that RES-CTP@GPXP can effectively alleviate neuronal apoptosis after SAH. Embodiment 11

[0072] The main features of ferroptosis are iron-dependent lipid peroxidation and mitochondrial abnormalities, including outer membrane disruption and reduced cristae. In the context of subarachnoid hemorrhage (SAH), this programmed cell death is increasingly recognized as a major cause of neuronal injury, driven by dysregulated iron metabolism and excessive oxidative stress. Therefore, we investigated the typical features of ferroptosis, including increased iron levels and decreased glutathione (GSH) levels, which implicate impaired cellular antioxidant defenses. SAH-induced ferroptosis resulted in increased iron levels and decreased GSH levels after SAH compared with sham controls (see Figure 5). Fig. 22 In addition, malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) are important markers of ferroptosis. They are byproducts of lipid peroxidation, and their accumulation indicates oxidative damage and increased lipid peroxidation, marking the occurrence of ferroptosis. Fig.21 Middle A and Fig. 22 As shown in F and G, the levels of MDA and 4-HNE in the saline group increased significantly. In addition, transmission electron microscopy (TEM) images showed changes in mitochondrial morphology after SAH, mainly involving mitochondrial membrane rupture and reduced cristae, indicating the induction of neuronal ferroptosis (see Fig. 22 After treatment, it was observed that RES-CTP@GPXP showed a strong ability to restore these abnormal changes, and its inhibitory effect on neuronal ferroptosis was superior to that of the CTP@GPXP group without matrix metalloproteinase 2 (MMP-2)-mediated in situ proliferation. Example 12

[0073] In vivo biosafety is crucial for the clinical application of therapeutic drugs. We systematically evaluated the in vivo biosafety of RES-CTP@GPXP at the blood and organ levels. First, blood samples were collected through the angular vein on day 7 after administration, and hematological examinations and biochemical tests were performed. There were no statistically significant differences between the RES-CTP@GPXP-treated group and the sham operation group in hematological indices and alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (CRE) levels, indicating that RES-CTP@GPXP did not affect blood cell counts or liver and kidney function. In addition, we further studied the potential organ toxicity by sacrificing mice and collecting these key organs (brain, heart, liver, spleen, lungs, and kidneys) for hematoxylin and eosin (H&E) staining. By Fig.19 and Fig. 20 The results showed that the use of therapeutic doses of RES-CTP@GPXP did not cause observable morphological changes and damage to these organs.

[0074] Compared with the prior art, the technical solution of this application mainly solves the following technical problems: 1. Comprehensive solution to the problem of brain damage: Existing drugs can often only treat one aspect and cannot comprehensively solve the problem of brain damage. The polypeptide drug of this technical solution can capture cfDNA and remove ROS by containing the polycationic peptide RES-CTP and the GPX analog peptide GPXP, thereby comprehensively reducing brain damage from multiple aspects. 2. Reduce side effects: Existing drugs have large side effects and may impose an additional burden on patients. The polypeptide drug of this technical solution reduces the side effects of the drug and improves the safety of patient treatment by optimizing the amino acid sequence. 3. Effectively cross the blood-brain barrier: Existing drugs are not effective in crossing the blood-brain barrier, which limits their application in the treatment of brain injuries. The polypeptide drug of this technical solution enhances its ability to penetrate the blood-brain barrier by optimizing the sequence, thereby improving its application effect in the treatment of brain injuries.

[0075] The beneficial effects of the technical solution of the present application are as follows: 1. Multifunctionality: A polypeptide with cfDNA capture and ROS scavenging functions of the present invention has the functions of polycationic peptide and GPX mimetic peptide at the same time. Among them, polycationic peptide can capture cfDNA and reduce brain damage; GPX mimetic peptide can remove ROS and reduce oxidative stress. This multifunctional design enables the present invention to comprehensively solve the problem of brain damage after SAH, and the effect is more comprehensive and more effective than the existing drugs that can only treat one aspect. 2. Low side effects: The polypeptide drug of the present invention is composed of natural amino acids, and its side effects are smaller than those of existing chemically synthesized drugs, and the physical burden on patients is lighter. At the same time, since the polypeptide drug of the present invention can effectively cross the blood-brain barrier, it can directly act on the brain injury area, reducing the distribution and metabolic process of the drug in the body, and further reducing the possibility of side effects. 3. Efficiently cross the blood-brain barrier: The polypeptide drug of the present invention has good lipophilicity and hydrophilicity by optimizing the sequence design, and can effectively cross the blood-brain barrier. Compared with existing drugs, the polypeptide drug of the present invention has a better effect in crossing the blood-brain barrier, can act on the brain injury area more quickly and accurately, and improves the therapeutic effect. 4. Combined application: The polypeptide drug of the present invention can be used in combination with other drugs with neuroprotective or antioxidant effects to further enhance the therapeutic effect. This combination application method can achieve the synergistic effect of the drugs and improve the therapeutic effect. At the same time, it can also reduce the amount of each drug used and reduce side effects. 5. Wide scope of application: The polypeptide drug of the present invention can not only be used to treat SBI after SAH, but also can be used to treat brain damage caused by other reasons, such as brain trauma, cerebral ischemia, etc. Its wide range of application makes it have broad application prospects in the field of brain injury treatment.

[0076] Due to the advanced nature of the technical solution of the present application, it can be widely used in the fields of biotechnology, molecular diagnosis, and drug therapy. In the field of biotechnology, the polypeptide of the present invention can be used to study the pathogenesis of cerebrovascular diseases, especially in the activation of microglia in the brain and the release of reactive oxidative species. In addition, the polypeptide of the present invention can also be used to develop new drugs, especially drugs for cerebrovascular diseases. In the field of molecular diagnosis, the polypeptide of the present invention can be used to detect the level of cell-free DNA in cerebrospinal fluid and the content of damage-related molecular patterns in these cfDNA. This will help in the early diagnosis of cerebrovascular diseases, especially in the assessment of brain damage after subarachnoid hemorrhage. In the field of drug therapy, the polypeptide of the present invention can be used to treat and prevent brain damage after subarachnoid hemorrhage. Since the polypeptide of the present invention has cfDNA capture and ROS scavenging functions, it can comprehensively solve the problem of brain damage. At the same time, due to its low side effects and high ability to cross the blood-brain barrier, it can reduce the side effects of drugs and improve its application in the treatment of brain damage. In general, the polypeptide of the present invention has broad application prospects in the fields of biotechnology, molecular diagnosis, and drug therapy, and is expected to bring new breakthroughs in the treatment of cerebrovascular diseases.

[0077] Finally, it should be noted that although the above embodiments have been described in the specification and drawings of this application, this does not limit the scope of patent protection of this application. All technical solutions generated by replacing or modifying equivalent structures or equivalent processes based on the essential concept of this application using the contents recorded in the specification and drawings of this application, as well as directly or indirectly implementing the technical solutions of the above embodiments in other related technical fields, are included in the scope of patent protection of this application.

Claims

1. A polypeptide having cfDNA capture and ROS scavenging functions, characterized in that: It comprises a polycationic peptide and a GPX mimetic peptide, wherein the polycationic peptide comprises a hydrophobic series, an MMP-2 responsive series and a cationic series, and the GPX mimetic peptide comprises a hydrophobic series, a selenogene series and a neuron targeting series.

2. The polypeptide according to claim 1, characterized in that The polycationic peptide has an amino acid sequence as shown in SEQ ID NO.

1.

3. The polypeptide according to claim 1 or 2, characterized in that The GPX mimetic peptide has an amino acid sequence as shown in SEQ ID NO.

2.

4. A composition for preparing a drug for treating SBI, characterized in that: Comprising the polypeptide according to any one of claims 1 to 3.

5. The composition according to claim 4, characterized in that Other drugs with neuroprotective or antioxidant effects are also included.

6. A drug for treating, inhibiting or alleviating SBI after SAH, characterized in that: Comprising the polypeptide according to any one of claims 1 to 3.

7. The drug according to claim 6, characterized in that Also included is a carrier.

8. The drug according to claim 6, characterized in that The medicine is a nasal preparation.

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