Cyclic polypeptide as well as preparation method and application thereof

Abstract

The invention provides a cyclic polypeptide, which is characterized in that a neuronal targeting peptide Tet1 is used as a basic peptide chain, cysteine is respectively introduced to the C terminal and the N terminal of the basic peptide chain, and sulfydryl of the cysteine reacts with a cross-linking agent to form a cyclic structure; the cross-linking agent is selected from one of 4, 4 '-di (bromomethyl) biphenyl, decafluorobenzene or a maleimide functional aliphatic derivative. The invention also provides a preparation method of the cyclic polypeptide and application of the cyclic polypeptide in preparation of a drug delivery carrier for treating neurodegenerative diseases. Cysteine is introduced to the C end and the N end of the neuron targeting peptide Tet1, sulfydryl reacts with different cross-linking agents to construct an annular polypeptide library with the lipophilicity regulation and control function, an endocytosis path mediated by a cavette is triggered to break through age-dependent blood-brain barrier delivery obstacles, blood-brain barriers are efficiently penetrated, neurons are accurately targeted, and the neuronal targeting peptide Tet1 has the advantages of being high in targeting ability, good in targeting effect and the like. And a novel drug delivery carrier is provided for treatment of neurodegenerative diseases.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a cyclic polypeptide, its preparation method, and its uses. Background Technology

[0002] In recent years, the global incidence of neurodegenerative diseases (NDs) has continued to rise significantly, with the total number of patients exceeding 50 million, posing a severe challenge to global public health systems. In the development of treatments for these diseases, the blood-brain barrier (BBB) ​​has consistently been a critical bottleneck. The BBB, composed of tightly connected endothelial cells, pericytes, astrocyte terminale, and neurons, is a precise and dynamic physiological interface. Its core function is to strictly regulate the entry and exit of substances between the blood and the central nervous system, maintaining the stability of the internal environment of the central nervous system while resisting the invasion of exogenous harmful substances and neurotoxins, thus playing a vital role in protecting brain function.

[0003] However, the high selectivity of the blood-brain barrier also presents serious problems: the vast majority of drugs used to treat neurodegenerative diseases cannot effectively penetrate this barrier to reach brain tissue and exert their effects. To address this challenge, supraphysiological doses are often used in clinical practice, but this not only fails to achieve the desired therapeutic effect but also causes severe systemic toxicity, further limiting the application of existing treatment options.

[0004] To overcome the blood-brain barrier, peptide-based blood-brain barrier targeted delivery strategies have received widespread attention in recent years. Traditional peptide delivery strategies mainly rely on endogenous receptors (such as transferrin receptors) highly expressed on brain endothelial cells to achieve drug penetration across the blood-brain barrier through receptor-mediated transcytosis. However, related studies have shown that the substance transport mechanisms of brain endothelial cells undergo significant remodeling with age: in young individuals, receptor-mediated transcytosis is the dominant transport mechanism, while with age, the transport mechanism gradually shifts towards non-selective pituitary-mediated transport. This age-related change in transport mechanisms leads to reduced efficiency of traditional peptide delivery systems that rely on specific endogenous receptors in elderly individuals, failing to meet the treatment needs of elderly patients with neurodegenerative diseases.

[0005] Therefore, there is an urgent need to develop a novel peptide delivery technology that can effectively utilize the small-cell transport pathway and is suitable for different age groups. Summary of the Invention

[0006] The technical problem to be solved by this invention is to provide a cyclic polypeptide that does not depend on endogenous receptors in brain endothelial cells, adapts to age-related changes in blood-brain barrier transport mechanisms, and has both efficient blood-brain barrier penetration and neuronal targeting functions, as well as its preparation method and uses. By introducing cysteine ​​residues at the C and N ends of the neuronal targeting peptide Tet1, and using thiol groups to react with different cross-linking agents to construct a cyclic polypeptide library with regulated lipophilicity, the invention triggers the pituitary-mediated endocytosis pathway to overcome age-dependent blood-brain barrier delivery barriers, efficiently penetrates the blood-brain barrier and precisely targets neurons, providing a novel drug delivery carrier for the treatment of neurodegenerative diseases.

[0007] The present invention solves the above-mentioned technical problems by adopting the following technical solutions: A cyclic polypeptide is based on the neuronal targeting peptide Tet1 with the amino acid sequence HLNILSTLWKYR. Cysteine ​​residues are introduced at the C-terminus and N-terminus of the basic peptide chain, respectively. The thiol groups of the cysteine ​​residues react with a cross-linking agent to form a cyclic structure. The cross-linking agent is selected from 4,4'-bis(bromomethyl)biphenyl, decafluorobenzene, or maleimide-functionalized aliphatic derivatives.

[0008] As one of the preferred embodiments of the present invention, when the crosslinking agent is 4,4'-bis(bromomethyl)biphenyl, the thiol group of the cysteine ​​reacts with the 4,4'-bis(bromomethyl)biphenyl via S... N 2. Nucleophilic substitution reaction forms an aromatic cross-linked cyclic polypeptide (cTP-Ar).

[0009] As one of the preferred embodiments of the present invention, when the crosslinking agent is decafluorobenzene, the thiol group of cysteine ​​is substituted with the decafluorobenzene via nucleophilic aromatic substitution (S... N Ar) reaction forms a perfluorinated aromatic cross-linked cyclic polypeptide (cTP-Ar) F ).

[0010] As one of the preferred embodiments of the present invention, when the crosslinking agent is a maleimide-functionalized aliphatic derivative, the thiol group of cysteine ​​reacts with the derivative via a Michael addition reaction to form an aliphatic crosslinked cyclic polypeptide (cTP-Alk) or a perfluorinated aliphatic crosslinked cyclic polypeptide (cTP-Alk). F -1、cTP-Alk F -2).

[0011] As one of the preferred embodiments of the present invention, the cyclic polypeptide is further fluorescently labeled with fluorescein isothiocyanate (FITC) or indocyanine green (ICG); the fluorescent labeling is used for in vitro characterization or in vivo tracing of the cyclic polypeptide.

[0012] A method for preparing the above-mentioned cyclic polypeptide includes the following steps: S1. Solid-phase polypeptide synthesis: After swelling the Rink Amide-MBHA resin, the Fmoc protecting group was removed using a deprotection solution. A linear peptide chain containing C-terminal and N-terminal cysteine ​​was synthesized through the coupling reaction of Fmoc-protected amino acids. The linear peptide was obtained by cleavage and precipitation. S2, macrocyclic peptide synthesis: Linear peptides are mixed with cross-linking agents and cyclized in a corresponding buffer system to form cyclic structures; S4. Purification: The cyclization reaction product was separated and purified by high performance liquid chromatography to obtain the cyclic polypeptide.

[0013] As one of the preferred embodiments of the present invention, in step S1: The resin swelled in DMF for 30 min; the deprotection solution was a mixture of morpholine and N,N-dimethylformamide (DMF) in a volume ratio of 7:3; in the coupling reaction, the molar ratio of Fmoc-protected amino acid, HATU, and DIPEA relative to the resin was 3:3:6, and each amino acid was coupled twice, each time under a nitrogen atmosphere for 1 h; after the reaction, the resin was washed sequentially with DMF and dichloromethane (DCM); the cutting fluid used was a mixture of 95% trifluoroacetic acid (TFA), 2.5% triisopropylsilane (TIS), and 2.5% water, which was concentrated under reduced pressure after 2 h of reaction, and the linear peptide was obtained by precipitation with ice-cold diethyl ether.

[0014] As one of the preferred embodiments of the present invention, in step S2: The crosslinking agent was 4,4'-bis(bromomethyl)biphenyl, decafluorobenzene, or maleimide-functionalized aliphatic derivatives, with a molar ratio of 1:1 to the linear peptide. When the crosslinking agent was 4,4'-bis(bromomethyl)biphenyl, the buffer system was a DMF / NH4HCO3 mixed solution with a final NH4HCO3 concentration of 50 mM, and the reaction was carried out at room temperature with stirring for 24 h. When the crosslinking agent was decafluorobenzene, the buffer system was a DMF / Tris solution with a final Tris concentration of 30 mM, and the reaction was carried out at room temperature with stirring for 24 h. When the crosslinking agent was a maleimide-functionalized aliphatic derivative, the buffer system was a DMF / pH7.0 buffer solution, and the reaction was carried out at room temperature with stirring for 6 h.

[0015] The use of the above-mentioned cyclic polypeptide in the preparation of a drug delivery carrier for the treatment of neurodegenerative diseases.

[0016] As one of the preferred embodiments of the present invention, the cyclic polypeptide is a perfluorinated aliphatic cross-linked cyclic polypeptide (cTP-Alk). F -1、cTP-Alk F -2), which can efficiently penetrate the blood-brain barrier and target neurons, and still maintains efficient delivery capability in aging individuals.

[0017] Design concept and principles: The core starting point of this invention stems from a focus on a key challenge in the treatment of neurodegenerative diseases (NDs)—achieving effective drug delivery across the brain's blood-brain barrier (BBB) ​​has always been a critical bottleneck in this field. Existing technologies largely rely on transcytosis mediated by endogenous receptors in BBB endothelial cells (such as transferrin receptor and low-density lipoprotein receptor-associated protein) to overcome the barrier. However, this strategy has significant drawbacks: with aging, the expression of key transport receptors in brain endothelial cells is downregulated, leading to a gradual decline in receptor-mediated transcytosis efficiency. Furthermore, broader biological changes in the BBB during aging further limit the applicability of this strategy, ultimately failing to meet the needs of elderly patients for continuous and efficient drug delivery. Therefore, developing novel receptor-independent delivery methods has become an urgent problem to be solved.

[0018] Based on this technical challenge, and combining the structural advantages and tunable properties of macrocyclic peptides compared to linear peptides, this invention is designed as follows: Due to their cyclic structure, macrocyclic peptides naturally possess superior conformational stability and stronger membrane permeability. By introducing cross-linking agents with different properties, the physicochemical properties of macrocyclic peptides can be further precisely controlled. In particular, the introduction of hydrophobic cross-linking structures can significantly enhance the interaction between peptides and cell membrane lipid components, enabling macrocyclic peptides to have a specific affinity for membrane microdomains rich in cholesterol and sphingolipids (i.e., the pit structure basis). Based on this, the study hypothesizes that by rationally designing to enhance the affinity of macrocyclic peptides for lipids, the pit-mediated endocytosis pathway can be triggered, thereby achieving efficient and age-independent BBB permeation. This is also the core basis of the invention's "bypassing receptor-mediated transport" strategy—using macrocyclic peptides to promote pit-mediated transport, thereby overcoming the drug delivery barrier caused by the downregulation of BBB receptors due to aging.

[0019] Meanwhile, considering the need to develop multifunctional carriers with both efficient BBB crossing and precise lesion localization in NDs treatment, this invention further designed a bifunctional macrocyclic peptide platform, aiming to simultaneously overcome the age-related decline in BBB permeability and enhance neuronal-specific delivery capabilities. By systematically regulating the cross-linking structure to finely adjust the lipophilicity of macrocyclic peptides, the macrocyclic peptides constructed on this platform exhibit significantly higher absorption efficiency in the lipid-rich octanol phase compared to linear peptides. This improved property stems from the introduction of hydrophobic cross-linking structures and the limitation of peptide conformational flexibility imposed by macrocyclization, which both shields the polar amide backbone and promotes the full exposure of nonpolar side chains. Notably, fluorinated macrocyclic peptides exhibit stronger octanol phase partitioning ability compared to non-fluorinated peptides. This enhancement effect is not only due to the higher hydrophobicity of the CF2 group compared to the CH2 group, but also relies on the unique physicochemical properties of fluorine atoms (high electronegativity, strong dipole interaction, and significant amphiphilicity), which can more effectively promote the binding between macrocyclic peptides and lipid alkyl chains.

[0020] Based on the above findings, this invention ultimately uses the Tet1 peptide, which has neuronal targeting capabilities, as a base to construct a series of macrocyclic peptide libraries with different lipophilicities by introducing different types of cross-linking agents (including fluorinated cross-linking agents). The aim is to rationally regulate the lipid affinity of peptides through a combination of fluorination modification and macrocyclization strategies. This achieves efficient, receptor-independent BBB penetration through pituitary-mediated transport mechanisms, while relying on the targeting properties of the Tet1 peptide to achieve precise neuronal localization. Ultimately, a bifunctional shuttle peptide with both BBB penetration and neuronal targeting functions is obtained, solving the multiple problems of existing vectors that are difficult to balance penetration efficiency, age adaptability, and targeting accuracy, and providing a novel delivery solution for age-related BBB permeability disorders.

[0021] The advantages of this invention compared to the prior art are: (1) Overcoming the age-related blood-brain barrier delivery bottleneck to meet the needs of elderly patients Existing technologies rely on transcytosis mediated by endogenous receptors in brain endothelial cells. With age, receptors are downregulated and biological changes occur in the BBB, leading to a decline in delivery efficiency and failing to meet the needs of the elderly population with a high incidence of NDs. This invention, through a design of "macrocyclicization + differentiated cross-linking modification", enables macrocyclic peptides to trigger pituitary-mediated endocytosis pathways. This pathway does not depend on endogenous receptors and can still maintain efficient BBB penetration and neuronal targeting capabilities in aging individuals, fundamentally solving the core problem of "age-dependent delivery failure".

[0022] (2) Achieve dual-function synergy of "BBB penetration-neuronal targeting" and overcome the limitations of existing carriers. Existing vectors often struggle to balance BBB penetration efficiency with lesion targeting precision, frequently resulting in "retention in the extracellular space or non-target cells after penetration." This invention uses the Tet1 peptide, which has neuronal targeting capabilities, as its basic framework, and combines macrocyclization and hydrophobic crosslinking modifications to enhance membrane affinity and improve pituitary-mediated BBB penetration efficiency. At the same time, relying on the natural targeting properties of the Tet1 peptide, it ensures that after penetrating the BBB, it is preferentially taken up by neurons, reducing non-specific binding to other brain cells, truly achieving a dual-function synergy of "highly efficient penetration + precise targeting."

[0023] (3) Balance delivery efficiency and biocompatibility to reduce clinical risks. While some existing lipophilic modified carriers can enhance membrane permeability, they easily disrupt the orderly arrangement of the cell membrane lipid bilayer, leading to cytotoxicity. This invention precisely controls the lipophilicity of macrocyclic peptides through the selection of differentiated cross-linking agents: perfluorinated aliphatic cross-linked cyclic peptides, due to the conformational flexibility of perfluorinated aliphatic chains, can adapt to cell membrane structures and avoid membrane stability damage, improving delivery efficiency while exhibiting negligible toxicity to brain endothelial cells, neurons, and microglia; while perfluorinated aromatic cross-linked cyclic peptides exhibit significant cytotoxicity and membrane damage. The design of this invention effectively avoids this defect, balancing efficiency and safety.

[0024] (4) The preparation is controllable and supports precise characterization and tracing. Existing carrier preparation processes are often vague and their performance is difficult to trace. This invention adopts the mature Fmoc solid-phase peptide synthesis strategy, with clearly defined key operating parameters (such as resin swelling, deprotection solution ratio, and coupling reagent dosage). The overall process is highly reproducible and easy to scale up for production. Meanwhile, the structure and purity of all synthesized peptides are confirmed by high-resolution mass spectrometry (HRMS) and high-performance liquid chromatography (HPLC), and support fluorescein isothiocyanate (FITC) or indocyanine green (ICG) labeling, which can meet the requirements of in vitro characterization and in vivo tracing, providing reliable support for subsequent pharmaceutical research and application, and reducing the cost of technology transfer. Attached Figure Description

[0025] Figure 1 This is a schematic diagram of solid-phase polypeptide synthesis in Example 1; Figure 2 This is a schematic diagram of the linear polypeptide amidation synthesis in Example 1; Figure 3 This is a schematic diagram of the synthesis of the cyclic polypeptide in Example 1; Figure 4 The structure and characterization of the TP compound in Example 1 are shown in the figure (Figure a shows the chemical structure of the TP compound; Figure b shows the HPLC spectrum of the TP compound). Figure 5 It is cTP-Alk in Example 1 F -1 Structure and characterization of compound (Figure a shows cTP-Alk) F -1 Chemical structure of compound; b Figure shows cTP-Alk F HPLC spectrum of compound -1). Figure 6 It is cTP-Ar in Example 1 F Structure and characterization of the compound (Figure a shows cTP-Ar) F The chemical structure of the compound; Figure b shows cTP-Ar F (HPLC spectrum of the compound). Figure 7It is cTP-Alk in Example 1 F -2 Structure and characterization of compound (Figure a shows cTP-Alk) F -2 Chemical structure of compound; Figure b shows cTP-Alk F (HPLC spectrum of compound -2) Figure 8 The structure and characterization of the FITC-TP compound in Example 1 are shown in the figure (Figure a shows the chemical structure of the FITC-TP compound; Figure b shows the HPLC spectrum of the FITC-TP compound). Figure 9 The structure and characterization of the FITC-cTP-Ar compound in Example 1 are shown in the figure (Figure a shows the chemical structure of the FITC-cTP-Ar compound; Figure b shows the HPLC spectrum of the FITC-cTP-Ar compound). Figure 10 The structure and characterization of the FITC-cTP-Alk compound in Example 1 are shown in the figure (Figure a shows the chemical structure of the FITC-cTP-Alk compound; Figure b shows the HPLC spectrum of the FITC-cTP-Alk compound). Figure 11 It is FITC-cTP-Alk in Example 1 F -1 Structure and characterization of compound (Figure a shows FITC-cTP-Alk) F -1 Chemical structure of compound; b Figure shows FITC-cTP-Alk F HPLC spectrum of compound -1). Figure 12 It is FITC-cTP-Ar in Example 1 F Structure and characterization of the compound (Figure a shows FITC-cTP-Ar) F Chemical structure of the compound; Figure b shows FITC-cTP-Ar F (HPLC spectrum of the compound). Figure 13 It is FITC-cTP-Alk in Example 1 F -2 Structure and characterization of the compound (Figure a shows FITC-cTP-Alk) F -2 Chemical structure of compound; Figure b shows FITC-cTP-Alk F (HPLC spectrum of compound -2) Figure 14 The structure and characterization of the ICG-TP compound in Example 1 are shown in the figure (Figure a shows the chemical structure of the ICG-TP compound; Figure b shows the HPLC spectrum of the ICG-TP compound). Figure 15The structure and characterization of the ICG-cTP-Ar compound in Example 1 are shown in the figure (Figure a shows the chemical structure of the ICG-cTP-Ar compound; Figure b shows the HPLC spectrum of the ICG-cTP-Ar compound). Figure 16 The structure and characterization of the ICG-cTP-Alk compound in Example 1 are shown in the figure (Figure a shows the chemical structure of the ICG-cTP-Alk compound; Figure b shows the HPLC spectrum of the ICG-cTP-Alk compound). Figure 17 It is ICG-cTP-Alk in Example 1 F -1 Structure and characterization of compound (Figure a shows ICG-cTP-Alk) F -1 Chemical structure of compound; b Figure shows ICG-cTP-Alk F HPLC spectrum of compound -1). Figure 18 It is ICG-cTP-Ar in Example 1 F Structure and characterization of the compound (Figure a shows ICG-cTP-Ar) F Chemical structure of the compound; Figure b shows ICG-cTP-Ar F (HPLC spectrum of the compound). Figure 19 It is ICG-cTP-Alk in Example 1 F -2 Structure and characterization of compound (Figure a shows ICG-cTP-Alk) F -2 Chemical structure of compound; Figure b shows ICG-cTP-Alk F (HPLC spectrum of compound -2) Figure 20 The structure and characterization of the ICG-T7 compound in Example 1 are shown in the figure (Figure a shows the chemical structure of the ICG-T7 compound; Figure b shows the HPLC spectrum of the ICG-T7 compound). Figure 21 The structure and characterization of the ICG-ANG compound in Example 1 are shown in the figure (Figure a shows the chemical structure of the ICG-ANG compound; Figure b shows the HPLC spectrum of the ICG-ANG compound). Figure 22 The results of peptide lipid affinity analysis in Example 2 are shown in Figure 2 (Figure 1a shows the distribution of ICG-labeled peptides in an octanol / PBS system containing DP and CHOL; Figure 2b shows the comparison of the relative absorbance of each peptide in the octanol phase). Figure 23This is the analysis result of the uptake characteristics and endocytosis mechanism of FITC-labeled peptides on human brain microvascular endothelial cells (hCMEC / D3) in Example 2 (Figure a shows the fluorescence imaging of hCMEC / D3 cells by FITC-labeled peptides, scale bar: 25 μm; Figure b shows the fluorescence intensity distribution in hCMEC / D3 cells, scale bar: 25 μm; Figure c shows the quantitative analysis of fluorescence values ​​by flow cytometry; Figures d and e show the FITC-cTP-Ar) F With FITC-cTP-Alk F -2 cellular uptake pathway analysis); Figure 24 The results of the analysis of the uptake characteristics of FITC-labeled peptides in PC12 cells in Example 2 are shown in Figure 2 (Figure a shows the fluorescence imaging of FITC-labeled peptides in PC12 cells, scale bar: 40 μm; Figure b shows the average fluorescence intensity of FITC-labeled peptides in PC12 cells as measured by flow cytometry). Figure 25 The results of the cytotoxicity and lactate dehydrogenase release rate assays in Example 2 are as follows (in the figure, figures a, b, and c show the cytotoxicity results of bEnd.3, PC12, and BV2 cells after 24 hours of treatment with the peptide, measured by CCK-8 assay; figure d shows the effect of the peptide on the survival rate of bEnd.3 cells, measured by lactate dehydrogenase release assay; the labels 1, 2, 3, and 4 represent the first, second, third, and fourth columns of each group in the figure, respectively). Figure 26 The results of fluorescence signal and quantitative analysis of ICG-labeled peptides in isolated brain tissue of young C57BL / 6 mice in Example 2 are shown in Figure 2. (Figure a shows the fluorescence signal of ICG-labeled peptides in isolated brain tissue of young mice detected by an in vivo imaging system, ANG: low-density lipoprotein receptor-1 targeting peptide, T7: transferrin targeting peptide; Figure b shows the quantitative analysis of fluorescence signal). Figure 27 This is two-photon imaging of the blood-brain barrier permeability of ICG-labeled peptides in the brain tissue of young C57BL / 6 mice in Example 2; Figure 28 The results of two-photon imaging and permeability index statistics of ICG-labeled peptides in the brain tissue of aged C57BL / 6 mice in Example 2 are as follows (Figure a shows the BBB permeability of ICG-labeled peptides in the brain tissue of aged mice detected by a two-photon system, scale bar: 1000 μm; Figure b shows the BBB permeability index statistics). Figure 29 This is the result of immunofluorescence staining and quantitative analysis of ICG-cTP-AlkF-2 targeting neurons in the brain tissue of young and aged C57BL / 6 mice in Example 2 (Figure a shows the evaluation of ICG-cTP-AlkF-2 by immunofluorescence staining). FFigure 1 shows the neuronal targeting of MAP-2 in young and aging mice (MAP-2: neuronal marker, scale bar: 50 μm). Figure 2b shows the quantitative fluorescence analysis performed using ImageJ. Detailed Implementation

[0026] The embodiments of the present invention are described in detail below. These embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments. Furthermore, unless otherwise specified, the reagents used in the following embodiments are all conventional reagents in the art; the experimental conditions and methods used, unless otherwise specified, are all conventional conditions and methods in the art and will not be described again.

[0027] Example 1: Synthesis and Characterization of Cyclic Peptides: I. Solid-phase polypeptide synthesis ( Figure 1 ) The basic peptide chain is based on the neuronal-targeting Tet1 peptide, with the amino acid sequence HLNILSTLWKYR. To achieve the subsequent cyclization reaction, one cysteine ​​residue is introduced at the N-terminus and C-terminus of the basic peptide chain to form a linear peptide sequence of "cysteine-Tet1-cysteine". All subsequent operations in this part revolve around the synthesis of this target linear peptide.

[0028] 1. Resin swelling and deprotection: Rink Amide-MBHA resin was placed in DMF to swell for 30 min; the Fmoc protecting group was removed using a deprotection solution (morpholine:DMF=7:3, v / v).

[0029] 2. Amino acid coupling: Dissolve the Tet1 peptide core sequence and the Fmoc protected amino acids corresponding to the two cysteine ​​residues, HATU, and DIPEA in DMF at a molar ratio of 3:3:6 relative to the resin to prepare an amino acid coupling solution; add the coupling solution to the above deprotected resin and react with shaking at room temperature under a nitrogen atmosphere for 1 hour to complete the first coupling; repeat the above coupling operation for the second coupling; after coupling is completed, wash the resin three times each with DMF and DCM to remove unreacted reagents.

[0030] 3. Fluorescent labeling (optional): If fluorescently labeled peptides are to be prepared, fluorescent molecules are grafted after all amino acid conjugation is completed.

[0031] FITC labeling: DMF, DIPEA (4 molar equivalents relative to the resin) and FITC (2 molar equivalents relative to the resin) were added to the resin loaded with peptides. The reaction was carried out under nitrogen protection and shaking for 6 hours at room temperature. After the reaction was completed, the resin was washed 3 times with DMF and DCM alternately.

[0032] ICG labeling: DMF, ICG (2 molar equivalents relative to the resin), HATU (2 molar equivalents relative to the resin), and DIPEA (4 molar equivalents relative to the resin) were added to the resin loaded with peptides. The reaction was carried out under nitrogen protection and shaking for 6 hours at room temperature. After the reaction was completed, the resin was washed 3 times with DMF and DCM alternately.

[0033] 4. Cutting and precipitation: Add cutting solution (95% TFA, 2.5% TIS, 2.5% water, v / v / v) to the washed resin to remove the peptide side chain protecting groups and resin. The reaction lasts for 2 hours. After the reaction, concentrate the mixture under reduced pressure and precipitate the peptide product with ice-cold diethyl ether to obtain crude linear peptide, which can be used directly in subsequent steps without purification.

[0034] II. Linear peptide amidation synthesis ( Figure 2 ) The amidated linear peptide synthesized in this step is only used as a linear peptide control for subsequent experiments.

[0035] 1. Preparation of reaction system: Dissolve the above crude linear peptide and bromoacetamide in DMF / NH4HCO3 aqueous solution and stir until completely dissolved, so that the final concentrations of each component in the reaction system are: linear peptide 1mM, bromoacetamide 2mM, NH4HCO3 50mM.

[0036] 2. Amide reaction and purification: The reaction system was stirred at room temperature for 24 h; after the reaction, it was concentrated under reduced pressure, and ice-cold diethyl ether was added to precipitate the peptide product. The product was dissolved in a mixed solvent of ACN / water (ACN:water = 1:1, v / v) and purified by semi-preparative high performance liquid chromatography to obtain the amidated linear peptide.

[0037] III. Macrocyclic peptide synthesis ( Figure 3 ) Depending on the type of cross-linking agent, the following three synthesis methods are used, all of which use the crude linear peptide obtained in step one as raw material: Synthesis of aromatic cross-linked cyclic polypeptides (cTP-Ar): A linear peptide and 4,4'-di(bromomethyl)biphenyl were dissolved in a DMF / NH4HCO3 mixed solution and stirred until homogeneous, achieving a final concentration of 1 mM for the linear peptide, 1 mM for 4,4'-di(bromomethyl)biphenyl, and 50 mM for NH4HCO3. The reaction was carried out at room temperature for 24 h. After the reaction, the solution was concentrated, and ice-cold diethyl ether was added to precipitate the product. The crude cyclic polypeptide was collected by centrifugation. During this process, the thiol groups of the cysteine ​​residues at both ends of the linear peptide and the bromomethyl group of 4,4'-di(bromomethyl)biphenyl cross-linked via S-coupling. N 2. Nucleophilic substitution reaction, ultimately constructing an aromatic cross-linked cyclic structure.

[0038] Perfluorinated aromatic cross-linked cyclic polypeptides (cTP-Ar) FSynthesis: The linear peptide and decafluorobenzene were dissolved in DMF / Tris solution and stirred until homogeneous, resulting in a final concentration of 1 mM for the linear peptide, 1 mM for the decafluorobenzene, and 30 mM for the Tris. The reaction was carried out at room temperature for 24 h. Subsequent precipitation was performed as described above for aromatic cross-linked cyclic peptides. During this process, the thiol groups of the cysteine ​​residues at both ends of the linear peptide were nucleophilically substituted with the decafluorobenzene (S... N Ar) reaction, ultimately constructing a perfluorinated aromatic cross-linked cyclic structure.

[0039] Aliphatic / perfluorinated aliphatic cross-linked cyclic peptides (cTP-Alk / cTP-Alk) F -1 / cTP-Alk F -2) Synthesis: The linear peptide is functionalized with the corresponding maleimide aliphatic derivative (Alk / Alk) F -1 / Alk F -2) Dissolve in DMF / pH7.0 buffer (volume ratio 5:1), stir until homogeneous, and bring the final concentration of the linear peptide and the maleimide-functionalized derivative to 1 mM. Stir at room temperature for 6 h, and perform subsequent precipitation as above. During this process, the thiol groups of the cysteine ​​residues at both ends of the linear peptide react with the maleimide-functionalized aliphatic derivative to form aliphatic cross-linked or perfluorinated aliphatic cross-linked cyclic structures via Michael addition reaction.

[0040] IV. Purification: The crude cyclic peptides collected above were dissolved in an ACN / water mixed solvent (ACN:water = 1:1, v / v) and purified by semi-preparative HPLC to obtain pure cyclic peptides.

[0041] V. Structure and Purity Characterization: The molecular structure of the pure cyclic peptide obtained above was confirmed by high-resolution mass spectrometry (HRMS), and the correctness of the structure was verified by comparing the measured molecular weight with the theoretically calculated molecular weight. The purity of the cyclic peptide was analyzed by high-performance liquid chromatography (HPLC) to ensure that the purity of the target product meets the requirements of subsequent experiments and applications.

[0042] The specific chemical formulas, theoretically calculated molecular weights (Calculated m / z), and measured molecular weights (Found m / z) of the key cyclic peptides synthesized in this invention (including amidated linear peptide control and fluorescently labeled peptide) are shown in Table 1 below, further confirming the correctness of the target cyclic peptide structure.

[0043] Table 1. Chemical formulas and mass spectrometry analysis of peptides

[0044] Meanwhile, the purity and structure of the above-mentioned peptides were verified by HPLC and HRMS spectra. Figures 4-21As shown in the figure, the target peaks in each spectrum are symmetrical and free of obvious impurities, and the measured m / z values ​​are in high agreement with the theoretical calculation values ​​in Table 1. This together confirms that the linear peptides, cyclic polypeptides and fluorescently labeled peptides synthesized in this invention have correct structures and meet the purity requirements, which can meet the needs of subsequent experiments and applications.

[0045] Example 2, Key Performance Verification: I. Peptide-lipid affinity experiment Dodecyl monophosphate (DP) and cholesterol (CHOL) were dissolved in octanol to prepare a 200 μM mixed solution. This mixed solution was then mixed with PBS buffer (pH 7.4) at a 1:1 volume ratio. An ICG-labeled target cyclic peptide (or linear peptide control) was added to the mixture, and the solution was incubated at 100 rpm for 24 h in a 37°C constant-temperature shaker. After incubation, the mixture was rapidly centrifuged (1000 rpm, 1 min), and the absorbance of the octanol solution was measured at 780 nm using a microplate reader. The lipid affinity of different peptides was evaluated by comparing the absorbance values.

[0046] The results are as follows Figure 22 As shown, this invention constructs cyclic polypeptides (especially perfluorinated aliphatic cross-linked cyclic polypeptides, such as cTP-Alk). F -1、cTP-Alk F -2) The absorbance in the octanol phase is significantly higher than that of the linear peptide TP, and the absorbance of the fluorinated cyclic peptide is better than that of the non-fluorinated cyclic peptide. Among them, the perfluorinated aliphatic cross-linked cyclic peptide has the strongest octanol phase partitioning ability. This advantage comes from the high hydrophobicity of the CF2 group and the strong dipole interaction of the fluorine atom, which can more efficiently promote the binding of the peptide to the lipid alkyl chain, laying the foundation for subsequent triggering of the pit-mediated endocytosis pathway.

[0047] II. Characterization of the uptake capacity of brain endothelial cells and neurons PC12 cells (highly differentiated) were combined with human brain microvascular endothelial cells hCMEC / D3 (2×10⁻⁶ cells). 5 Cells were seeded in confocal culture dishes and cultured for 24 h. They were then co-incubated with a FITC-labeled peptide solution (5 μM) for 4 h. After washing three times with PBS buffer, Hoechst staining was added to the nuclei for 15 min. PC12 cells were fixed with 4% paraformaldehyde at room temperature for 10 min, washed with PBS, and then examined. hCMEC / D3 cells were incubated with DID fluorescent dye for 10 min. The distribution of peptides within the cells was observed using a fluorescence microscope. Simultaneously, the fluorescence intensity of intracellular peptides was quantitatively detected by flow cytometry.

[0048] The results are as follows Figure 23 a~c and Figure 24As shown, in hCMEC / D3 cells, perfluoroaliphatic cross-linked cyclic peptides (such as cTP-Alk) are present. F The fluorescence intensity of -2) was significantly higher than that of linear peptide TP and perfluorinated aromatic cross-linked cyclic peptides (such as cTP-Ar). F The average fluorescence intensity was 7.73 times that of linear peptides and 1.81 times that of perfluorinated aromatic cross-linked peptides, respectively, and the fluorescence signal was mainly localized in the cytoplasmic region (e.g., Figure 23 As shown in a~c), this indicates efficient internalization and cytoplasmic delivery capabilities; in PC12 cells, the uptake of this type of perfluorinated aliphatic cross-linked cyclic peptide is also optimal, with an average fluorescence intensity 7.71 times that of linear peptide TP and 1.46 times that of perfluorinated aromatic cross-linked cyclic peptides (as shown in a~c). Figure 24 As shown in the figure, it confirms that it has the dual functional characteristics of efficient endothelial cell penetration and neuronal targeted uptake.

[0049] III. Research on the Mechanism of Cellular Endocytosis hCMEC / D3 cells (2×10) 5 Cells were seeded in confocal culture dishes and cultured for 24 h. The culture medium was discarded, and cells were treated with the endocytosis inhibitor genistein (700 μM), cytochalasin D (10 μM), and chlorpromazine (20 μM) at 37°C for 2 h. Subsequently, the inhibitors were discarded, and FITC-labeled cyclic peptides (5 μM) were added for further 4 h of culture. Cells were washed three times with PBS, and trypan blue (0.2 mg / mL) was added to quench cell membrane fluorescence. Finally, fluorescence intensity was detected by flow cytometry.

[0050] The results are as follows Figure 23 As shown in d and e, the perfluorinated aliphatic cross-linked cyclic polypeptides (such as cTP-Alk) after genistein pretreatment are shown to be more efficient. F -2) and perfluorinated aromatic cross-linked cyclic peptides (such as cTP-Ar) F The intracellular fluorescence intensity of the cytochalasin D treatment was significantly reduced; treatment with cytochalasin D also led to a decrease in fluorescence intensity, but treatment with chlorpromazine had a smaller effect on fluorescence intensity; at the same time, the fluorescence intensity of the 4°C incubation group (inhibiting energy-dependent endocytosis) was significantly lower than that of the 37°C group. The above results confirm that the cyclic polypeptides of the present invention (especially the perfluorinated aliphatic cross-linked type) mainly rely on pit-mediated endocytosis and macropinocytosis to enter hCMEC / D3 cells, and that this internalization process is energy-dependent, further explaining its receptor-independent, BBB-adapted BBB penetration mechanism for aging individuals.

[0051] IV. Cytotoxicity and Lactate Dehydrogenase (LDH) Release Rate Assay 1. bEnd.3 (brain endothelial cells), PC12 (neurons), and BV2 cells (microglia) were sized at a ratio of 5 × 10⁻⁶. 3Cells were seeded at a density of 1 / well in 96-well plates and cultured for 24 h. Subsequently, peptides were added to the cells for 24 h. After washing the cells three times with PBS buffer, 10% (v / v) of CCK-8 reagent prepared with fresh culture medium was added to each well, and the cells were incubated at 37°C in the dark for 2 h. The absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated using the following formula: Cell viability = (Experimental group absorbance - Blank group absorbance) / (Control group absorbance - Blank group absorbance) × 100%.

[0052] 2. After resuspending the bEnd.3 cells, use 1×10 5 Cells were seeded at a density of 1:1 in 96-well plates and cultured at 37°C with 5% CO2 for 24 h to allow cell adhesion. The medium was then replaced with serum-free medium containing different concentrations of peptide samples and cultured for another 24 h. One h before the end of the treatment, LDH release reagent (10% of the original medium volume) was added to the pre-selected positive control wells. After incubation, 120 μL of the supernatant was transferred to new clear 96-well plates, and 60 μL of LDH detection working solution was added to each well. The plates were then incubated on a horizontal shaker in the dark for 30 min. The absorbance at 490 nm was measured using a microplate reader, and the LDH release rate, reflecting cytotoxicity, was calculated using the following formula: LDH release rate = (Experimental group absorbance - Blank group absorbance) / (Positive control group absorbance - Blank group absorbance) × 100%.

[0053] The results are as follows Figure 25 As shown, at a concentration of 120 μM, the perfluoroaromatic cross-linked cyclic peptide reduced the viability of bEnd.3, PC12, and BV2 cells to below 30%, and significantly increased the LDH release rate, indicating significant cytotoxicity and cell membrane damage. In contrast, at the same concentration, the perfluoroaliphatic cross-linked cyclic peptide maintained high viability in all three cell types, and the LDH release rate was not significantly different from the blank control group, confirming its superior biocompatibility and effectively avoiding the toxic risks associated with lipophilic modifications.

[0054] V. Near-infrared fluorescence imaging and two-photon imaging of fluorescently labeled peptides in young / aged mice To investigate the accumulation of peptides in the brain tissue of C57BL / 6 mice with age, ICG-labeled peptides (100 μM, 100 μL) were injected into C57BL / 6 mice via the tail vein. Maestro™ EX in vivo imaging was performed on anesthetized mice at different time points after injection. Mice were sacrificed 24 hours after injection, and ex vivo tissue was collected for fluorescence imaging. Additionally, ICG-labeled peptides (100 μM, 100 μL) were injected into C57BL / 6 mice of different ages via the tail vein. Three hours later, FITC-glucan (2000 kDa) was injected, and mice were sacrificed 5 minutes later, with brain tissue collected for two-photon imaging analysis.

[0055] The results are as follows Figure 26 , 27 As shown in Figures 28 and 29, in near-infrared fluorescence imaging, the fluorescence intensity of ICG-labeled perfluorinated aliphatic cross-linked cyclic peptides (such as cTP-AlkF-2) in the brains of young rats was more than 2.36 times and 1.4 times that of linear peptide TP and receptor-mediated peptides (ICG-T7, ICG-ANG), respectively, and they exhibited stronger retention capacity in the brain. Figure 26 As shown); two-photon imaging further confirmed that the peptide exhibited highly efficient BBB penetration in young mice, with a penetration index significantly higher than that of linear peptide TP and perfluorinated aromatic cross-linked cyclic peptides (as shown). Figure 27 As shown), it is comparable to the RMT blood-brain barrier shuttle peptide; and in two-photon imaging of aging mice, the BBB penetration efficiency of this peptide is further increased by 1.41 times compared with that of young mice, and is much higher than that of receptor-mediated peptides whose penetration ability is significantly reduced in aging mice. Figure 28 As shown); simultaneously, MAP2 immunofluorescence co-localization analysis showed that the accumulation of this peptide in the neuronal regions of aging mice was 1.85 times higher than that in young mice (as shown). Figure 29 (As shown). The above results confirm that this peptide not only penetrates the BBB efficiently in young individuals, but also further enhances penetration efficiency and neuronal targeting in aging individuals, thus meeting the treatment needs of the elderly population with a high incidence of neurodegenerative diseases.

[0056] Accordingly, the cyclic peptides (especially perfluorinated aliphatic cross-linked cyclic peptides) prepared in this invention possess highly efficient blood-brain barrier penetration, precise neuronal targeting, and excellent biocompatibility. Furthermore, they maintain stable delivery performance in aging individuals, effectively overcoming the age-dependent bottleneck of existing delivery systems. Based on these core characteristics, these cyclic peptides can be used to prepare drug delivery carriers for the treatment of neurodegenerative diseases.

[0057] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A cyclic polypeptide, characterized in that, The neuronal targeting peptide Tet1 with the amino acid sequence HLNILSTLWKYR is used as the basic peptide chain. Cysteine ​​residues are introduced at the C-terminus and N-terminus of the basic peptide chain, respectively. The thiol group of the cysteine ​​reacts with a cross-linking agent to form a cyclic structure. The cross-linking agent is selected from one of 4,4'-bis(bromomethyl)biphenyl, decafluorobenzene, or maleimide-functionalized aliphatic derivatives.

2. The cyclic polypeptide according to claim 1, characterized in that, When the crosslinking agent is 4,4'-bis(bromomethyl)biphenyl, the thiol group of the cysteine ​​reacts with the 4,4'-bis(bromomethyl)biphenyl via S... N 2. Nucleophilic substitution reaction forms aromatic cross-linked cyclic polypeptides.

3. The cyclic polypeptide according to claim 1, characterized in that, When the crosslinking agent is decafluorobenzene, the thiol group of cysteine ​​reacts with decafluorobenzene through a nucleophilic aromatic substitution reaction to form a perfluoroaromatic crosslinked cyclic polypeptide.

4. The cyclic polypeptide according to claim 1, characterized in that, When the crosslinking agent is a maleimide-functionalized aliphatic derivative, the thiol group of cysteine ​​reacts with the derivative via a Michael addition reaction to form an aliphatic crosslinked cyclic polypeptide or a perfluorinated aliphatic crosslinked cyclic polypeptide.

5. The cyclic polypeptide according to any one of claims 1 to 4, characterized in that, The cyclic polypeptide is further fluorescently labeled with fluorescein isothiocyanate or indocyanine green; the fluorescent labeling is used for in vitro characterization or in vivo tracing of the cyclic polypeptide.

6. A method for preparing a cyclic polypeptide as described in any one of claims 1 to 4, characterized in that, Includes the following steps: S1. Solid-phase polypeptide synthesis: After swelling the Rink Amide-MBHA resin, the Fmoc protecting group was removed using a deprotection solution. A linear peptide chain containing C-terminal and N-terminal cysteine ​​was synthesized through the coupling reaction of Fmoc-protected amino acids. The linear peptide was then obtained by cleavage and precipitation. S2, macrocyclic peptide synthesis: Linear peptides are mixed with cross-linking agents and cyclized in a corresponding buffer system to form cyclic structures; S3, Purification: The cyclization reaction product was separated and purified by high performance liquid chromatography to obtain the cyclic polypeptide.

7. The preparation method according to claim 6, characterized in that, In step S1: The resin swelled in DMF for 30 min; the deprotection solution was a mixture of morpholine and DMF at a volume ratio of 7:3; in the coupling reaction, the molar ratio of Fmoc-protected amino acids, HATU, and DIPEA relative to the resin was 3:3:6, and each amino acid was coupled twice, each time under a nitrogen atmosphere for 1 h; after the reaction, the resin was washed sequentially with DMF and dichloromethane; the cutting fluid used was a mixture of 95% trifluoroacetic acid, 2.5% triisopropylsilane, and 2.5% water, which was concentrated under reduced pressure after 2 h of reaction, and the linear peptide was obtained by precipitation with ice-cold diethyl ether.

8. The preparation method according to claim 6, characterized in that, In step S2: The crosslinking agent was 4,4'-bis(bromomethyl)biphenyl, decafluorobenzene, or maleimide-functionalized aliphatic derivatives, with a molar ratio of 1:1 to the linear peptide. When the crosslinking agent was 4,4'-bis(bromomethyl)biphenyl, the buffer system was a DMF / NH4HCO3 mixed solution with a final NH4HCO3 concentration of 50 mM, and the reaction was carried out at room temperature with stirring for 24 h. When the crosslinking agent was decafluorobenzene, the buffer system was a DMF / Tris solution with a final Tris concentration of 30 mM, and the reaction was carried out at room temperature with stirring for 24 h. When the crosslinking agent was a maleimide-functionalized aliphatic derivative, the buffer system was a DMF / pH7.0 buffer solution, and the reaction was carried out at room temperature with stirring for 6 h.

9. Use of a cyclic polypeptide as described in any one of claims 1 to 4 in the preparation of a drug delivery carrier for the treatment of neurodegenerative diseases.

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