A cationic peptide compound targeting cartilage, its preparation method and application

Abstract

This invention discloses a cationic peptide compound targeting cartilage, its preparation method, and its application. The cationic peptide compound has the following general structural formula: R-(X a -Nlys-Y b ) n In this compound, R represents H, a drug-active molecule, or a group containing a signal marker; X and Y are independently selected from glycine residues and imine residues; Nlys is an N-(4-aminobutyl)glycine residue; a and b are independently selected from any integer between 0 and 3; and n is any positive integer between 3 and 10. This compound can target glycosaminoglycan chains in cartilage through electrostatic interactions and exhibits anti-protease degradation activity, which helps prolong its circulation time in vivo. This compound supports systemic administration and can be used to label cartilage throughout the body via intravenous injection for real-time monitoring of cartilage development, aging, and lesions. It has broad application potential in molecular imaging, drug delivery, and biomaterials for treating cartilage-related diseases.

Description

Technical Field

[0001] This invention relates to the field of biochemistry, and in particular to a cationic peptide compound that targets cartilage, its preparation method, and its application. Background Technology

[0002] Cartilage is an avascular tissue composed of collagen fibers and negatively charged glycosaminoglycans (GAG). It plays crucial structural and mechanical roles in various organ systems, such as cushioning joints and intervertebral spaces, shaping the ears and nose, and maintaining the trachea. Cartilage is a tissue that is difficult to repair itself; structural damage and functional impairment are core pathological changes in musculoskeletal degenerative diseases (such as knee and facial osteoarthritis), affecting the health and quality of life of the global elderly population. It is also a key factor in the disability caused by autoimmune diseases such as rheumatoid arthritis (RA) and ankylosing spondylitis. However, sensitive radiological detection methods for cartilage damage are currently lacking. Traditional X-ray imaging cannot visualize cartilage, so doctors can only infer the extent of cartilage damage based on bone spacing, and arthritic joints with a disease course of less than 6 months may appear normal on X-rays.

[0003] In related technologies, the dense matrix of cartilage is composed of collagen fibers and anionic GAG networks. Several cartilage-targeting molecules have been reported in recent years using these as targets. Examples include extracting chondrocyte membranes as natural cartilage-targeting materials, designing and screening synthetic cartilage collagen-targeting molecules (such as collagen-II binding peptide WYRGRL), and GAG-binding cationic fluorescent dyes, peptides, and polymers. GAG-targeting molecules are widely used due to their ease of preparation, single component (compared to cell membranes extracted from chondrocytes), and direct targeting of cartilage GAG ​​components (unlike targeting molecules for other cartilage components such as type II collagen peptides). However, these GAG-targeting molecules are often easily cleared or degraded in the bloodstream due to their large size or unstable natural sequences, making them primarily suitable for in vitro applications or local administration (i.e., intra-articular injection), rather than intravenous systemic administration. Moreover, since cartilage is an avascular tissue, the extracellular matrix (ECM) in the deep layers of cartilage tissue becomes increasingly dense, which restricts the diffusion and penetration of drugs, making it difficult for drugs to reach the cartilage lesion site and thus exert their therapeutic or indicative effects.

[0004] Therefore, there is an urgent need to find a peptide compound with high cartilage specificity and cartilage matrix permeability, high serum stability, and suitable for in vivo systemic administration, so as to provide technical support for the future development and clinical translation of molecular imaging contrast agents, targeted drug delivery, and joint lubrication materials targeting cartilage GAGs. Summary of the Invention

[0005] This invention aims to address at least one of the technical problems existing in the prior art. To this end, this invention proposes a cationic peptide compound that targets cartilage, enabling it to penetrate to millimeter-deep depths via electrostatic interactions and GAG chains within the cartilage. Furthermore, this cationic peptide compound contains the non-natural amino acid peptide Nlys (N-(4-aminobutylglycine), CAS No.: 13051-67-7) residues, which are not recognized or degraded by common proteases, exhibiting anti-protease degradation activity and contributing to prolonged circulation time to support systemic drug administration. Therefore, intravenous injection of live-cell cartilage can be used to monitor cartilage development, aging, and lesions in real time.

[0006] The present invention also proposes a method for preparing cationic peptide compounds that target cartilage.

[0007] This invention also proposes a cartilage-targeting probe.

[0008] The present invention also proposes a method for preparing a cartilage-targeting probe.

[0009] The present invention also proposes a reagent kit for detection.

[0010] This invention also proposes the application of cartilage-targeting cationic peptide compounds and / or cartilage-targeting probes in the preparation of cartilage-targeting related products.

[0011] In a first aspect, the present invention provides a cationic peptide compound targeting cartilage, having the following general structural formula: R-(X a -Nlys-Y b ) n ; Wherein, R is H, a drug active molecule, or a group containing a signal marker; X and Y are independently selected from either glycine residues or imino acid residues; Nlys is an N-(4-aminobutyl)glycine residue; a and b are independently selected from any integer between 0 and 3; and n is any positive integer between 3 and 10.

[0012] The cationic peptide compounds according to embodiments of the present invention have at least the following beneficial effects: (1) The cationic peptide compounds of the present invention have excellent cartilage affinity and can achieve efficient binding through electrostatic interaction with anionic glycosaminoglycans (GAGs) in the cartilage matrix. For example, the cationic peptide compound (NlysO)7 has a penetration depth of 1 mm in in vitro experiments, and the signal distribution is consistent with the GAG ​​density. In in vivo imaging, it can specifically label hyaline cartilage, fibrocartilage and elastic cartilage (such as joints, ear cartilage, spine, etc.), with strong and persistent signals.

[0013] (2) The cationic peptide compounds of the present invention have excellent serum stability. Because they contain peptide residues Nlys (non-natural amino acids), they are not easily recognized and degraded by common proteases, and have anti-protease degradation effect, which helps to prolong the circulation time to support systemic administration.

[0014] (3) The cationic peptide compounds of the present invention exhibit high biocompatibility. Cytotoxicity experiments show that the Nlys-containing cationic peptide compounds of the present invention are non-toxic to chondrocytes at concentrations ≤40 μM, and their cytotoxicity is significantly reduced compared to the widely accepted natural cationic polypeptide R8 with cartilage-targeting capabilities. Furthermore, in in vivo experiments, no abnormalities were observed in mouse blood parameters and organ histology after multiple injections, indicating excellent biocompatibility and supporting their potential clinical application as in vivo imaging agents or drug carriers.

[0015] In some embodiments of the present invention, the structural formula of the Nlys is as follows: ; In the formula, the wavy line indicates that the atom is a bonded atom.

[0016] In some embodiments of the present invention, a and b are independently selected from any integer between 0 and 3. For example, a can be 0, 1, 2 or 3, where when a is 0, it indicates that X does not exist; b can be 0, 1, 2 or 3, where when b is 0, it indicates that Y does not exist.

[0017] In some embodiments of the present invention, X is a glycine residue; Y is an imine residue; And / or, n is any positive integer between 5 and 9. For example, n can be 5, 6, 7, 8, or 9.

[0018] In some embodiments of the present invention, the imine residues include proline residues (Pro, P) or hydroxyproline residues (Hpy, O).

[0019] In some embodiments of the present invention, the molar ratio of the peptide residue Nlys to the imine residue is 1:1.

[0020] In some embodiments of the present invention, the cationic peptide compound comprises the structural formulas shown in formulas (I) to (VI): ; Wherein, R is independently selected from H, a pharmaceutically active molecule, or a group containing a signal marker; n is independently taken from any positive integer between 5 and 9. For example, n can be 5, 6, 7, 8, or 9.

[0021] In some preferred embodiments of the present invention, the cationic peptide compound has the structural formula (NlysO)n, where O is a hydroxyproline residue and n is independently taken from any positive integer between 5 and 9. For example, n can be 5, 6, 7, 8, or 9.

[0022] In some embodiments of the present invention, the pharmaceutically active molecule includes a pharmaceutically active molecule having therapeutic functions for cartilage damage and / or lesions. It is understood that cationic peptide compounds containing said pharmaceutically active molecule can exert their effects by targeted injection into cartilage.

[0023] In a second aspect, the present invention provides a method for preparing the cationic peptide compound targeting cartilage described in the first aspect, comprising preparation by solid-phase synthesis.

[0024] The preparation method according to the embodiments of the present invention has at least the following beneficial effects: the cationic peptide compounds of the present invention have strong structural designability, and the peptide residue Nlys can be synthesized on resin by submonomer method during the preparation process, while combining with other amino acid residues for solid-phase synthesis. The preparation method is simple and suitable for industrial production.

[0025] In some embodiments of the present invention, the solid-phase synthesis method includes Fmoc chemical solid-phase synthesis and / or peptide submonomer solid-phase synthesis.

[0026] Preferably, the solid-phase synthesis method is the solid-phase synthesis method of Fmoc and peptide submonomers.

[0027] A third aspect of the present invention provides a cartilage-targeting probe comprising the cartilage-targeting cationic peptide compound described in the first aspect, wherein R is a group containing a signal marker.

[0028] The cartilage-targeting probe according to embodiments of the present invention has at least the following beneficial effects: (1) Compared to easily degraded and metabolized cartilage-targeting natural peptides (e.g., R8), the Nlys-containing cartilage-targeting probes of the present invention [e.g., (Nlys)7, (GNlys)7, (GNlysO)7, and (NlysO)7] exhibit high stability against protease degradation, allowing for a wider in vivo metabolic time window, thereby enabling intravenous systemic administration and sufficient cartilage delivery. Secondly, the Nlys-containing cartilage-targeting probes of the present invention have relatively low molecular weight and size, which facilitates penetration of the physical barrier of the cartilage matrix and allows for in vivo cartilage targeting.

[0029] (2) The cartilage-targeting probes of the present invention [e.g., Cy5-(NlysO)7] can be used to label all types of cartilage tissue and anatomical structures throughout zebrafish or mice via systemic administration. These findings will provide an unprecedentedly convenient tool for in vivo observation and assessment of the development (e.g., endochondral ossification), aging, and injury (e.g., fracture healing) of the musculoskeletal system.

[0030] In some embodiments of the present invention, the group containing the signal marker comprises a connector and a signal marker. It is understood that the group containing the signal marker utilizes the connector to attach the signal marker to the "-(X"). a -Nlys-Y b ) n "connect.

[0031] In some embodiments of the present invention, the linker is an amino acid and / or an amino acid derivative.

[0032] In some embodiments of the present invention, the amino acid derivative includes 6-aminohexanoic acid.

[0033] In some embodiments of the present invention, the signal marker includes at least one of fluorescent dyes, magnetic resonance contrast agents, radioactive isotopes, gold nanoparticles, magnetic nanoparticles, and quantum dots.

[0034] In some embodiments of the present invention, the fluorescent dye is selected from at least one of anthocyanins, rhodamine, BODIPY, FITC, erythrosine, phthalocyanine, phycocyanin, phycoerythrin, and Alexa Fluor.

[0035] In some embodiments of the present invention, the anthocyanin is selected from any one of sulfo-Cy3, sulfo-Cy5, sulfo-Cy5.5, sulfo-Cy7, and sulfo-Cy7.5.

[0036] In some embodiments of the present invention, the magnetic resonance contrast agent includes, but is not limited to, gadolinium chelates and europium chelates.

[0037] In some embodiments of the present invention, the radioactive isotopes include, but are not limited to, those mentioned above. 3 H, 125 I, 131 I, 14 C 18 At least one of F.

[0038] In some embodiments of the present invention, taking sulfo-Cy5 as a fluorescent dye as the signal marker and 6-aminohexanoic acid as the linker as an example, the cartilage-targeting probe includes any one of the compounds shown in formulas (Ia) to (VI-a): ; ; ; ; The n is independently taken from any positive integer between 3 and 10. For example, n can be 3, 4, 5, 6, 7, 8, 9, or 10.

[0039] It is understandable that, in practical applications, the fluorescent dye label sulfo-Cy5 can be replaced with other signal markers depending on the application scenario, as it has considerable cartilage targeting function.

[0040] In some embodiments of the present invention, n is taken from any positive integer between 5 and 9. For example, n can be 5, 6, 7, 8, or 9.

[0041] A fourth aspect of the present invention provides a method for preparing the cartilage-targeting probe described in the third aspect, comprising the following steps: S1. A cationic peptide compound containing the aforementioned connector for targeting cartilage is prepared using a solid-phase synthesis method; S2. The product of step S1 is reacted with the signal marker and purified to obtain the final product.

[0042] In a fifth aspect, the present invention provides a kit for detection comprising a cationic peptide compound targeting cartilage as described in the first aspect, and / or a cartilage-targeting probe as described in the third aspect.

[0043] The kit according to embodiments of the present invention has at least the following beneficial effects: Traditional histopathological techniques for detecting GAG levels in cartilage (such as safranin-fixing and fast green staining) are cumbersome and difficult to quantify, making it hard to reflect subtle changes in cartilage GAG ​​content. However, the detection kit developed in this invention, containing cationic peptide compounds or cartilage-targeting probes, can accurately reflect the GAG ​​content and changes in cartilage tissue through histological staining and quantitative fluorescence imaging analysis. More importantly, the cartilage-targeting probe of this invention [e.g., Cy5-(NlysO)7] can be used to in vivo visualize subtle changes such as GAG loss in mouse cartilage caused by aging and RA arthritis.

[0044] A sixth aspect of the invention provides a cationic peptide compound targeting cartilage as described in the first aspect, and / or the use of a cartilage-targeting probe as described in the third aspect in any of the following: A) Products for preparing cartilage imaging; B) Preparation of drugs or carriers targeting cartilage; C) Prepare products for detecting cartilage development, aging, damage and / or lesions; D) Products for preparing animal models related to cartilage imaging; E) Preparation of biomaterials targeting cartilage; F) Prepare medical devices with cartilage targeting and / or cartilage fluorescence imaging functions.

[0045] In clinical or pre-clinical procedures, intra-articular injection is the primary route of drug delivery to cartilage. However, most drugs are rapidly cleared from the joint cavity via the lymphatic system and capillaries (e.g., nonsteroidal anti-inflammatory drugs have an average half-life of only 1-4 hours in synovial fluid). This short target tissue half-life limits the duration of treatment and significantly impacts overall efficacy. Furthermore, joint diseases often lead to osteophyte formation and synovial fluid accumulation, causing joint cavity narrowing and making intra-articular injection difficult. The use of the cartilage-targeting cationic peptide compounds of this invention as drug carriers can improve cartilage penetration and help prolong drug retention time.

[0046] In some embodiments of the present invention, the cartilage includes, but is not limited to, hyaline cartilage (such as articular cartilage), fibrocartilage (such as meniscus), and elastic cartilage (such as ear).

[0047] In some embodiments of the present invention, the cartilage lesion includes arthritis.

[0048] Other features and advantages of the present invention will be set forth in the following description. Attached Figure Description

[0049] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1The chemical structures of some polypeptides or peptide-like compounds of the present invention and their MALDI-TOF MS and circular dichroism (CD) spectra are shown, where a is Cy5-R8; b is Cy5-(GKO)7; c is Cy5-(GNlysO)7; d is Cy5-(NlysO)7; e is Cy5-(GNlys)7; f is Cy5-Nlys7; and g is Cy5-(GO)7.

[0050] Figure 2 The chemical structures and MALDI-TOF MS spectra of another part of the peptide compounds of this invention are shown, where a is Cy5-(NlysP)7; b is Cy5-(NlysG)7; c is Cy5-(NlysO)5; d is Cy5-(NlysO)9; e is Cy5-(ONlys)7; f is Cy5-Nlys5; and g is Cy5-Nlys9.

[0051] Figure 3 The results of cartilage frozen sections stained with cartilage probes from Examples 1-4 and Comparative Examples 1-3 of the present invention are shown. In the figures, a is after washing with 1×PBS buffer; b is after washing with 10×PBS buffer; and c is after pretreatment with chondroitin ABC (ChABC) (washed with 1×PBS buffer).

[0052] Figure 4 The results are based on the staining of frozen cartilage sections using probes from Examples 5-11 and Comparative Example 1 of this invention, where a is Cy5-Nlys5 and Cy5-(NlysO)5 cartilage probes, b is (NlysG)7, (ONlys)7 and (NlysP)7 cartilage probes, and c is Nlys9 and (NlysO)9 cartilage probes.

[0053] Figure 5 The following are in vitro experimental results on cartilage uptake, retention, and permeation according to the present invention: a) shows the fluorescence intensity changes of Cy5-R8, Cy5-(GKO)7, Cy5-(GO)7, and Cy5-(NlysO)7 after 24 h of incubation with porcine cartilage slices; b) shows the fluorescence intensity changes of Cy5-R8, Cy5-(GKO)7, and Cy5-(NlysO)7 after desorption; c) shows representative confocal microscopy images and spatial fluorescence quantitative detection results (scale bar: 200 μm); d) shows the results of Cy5-(GNlys)7, Cy5-Nlys7, Cy5-(GNlysO)7, and Cy5-(NlysO)7 after 24 h of incubation with porcine cartilage slices. h represents the fluorescence intensity change; e represents the fluorescence intensity change during the desorption process of Cy5-(GNlys)7, Cy5-Nlys7, Cy5-(GNlysO)7 and Cy5-(NlysO)7. Data: mean + standard error (n = 6 independent samples).

[0054] Figure 6 The results of serum stability testing for this invention are shown in Figure a, where a is a normalized HPLC result of Cy5-R8, Cy5-(GKO)7, Cy5-(GO)7, and Cy5-(NlysO)7 after incubation in 25% mouse serum for 10 min, 2 h, and 24 h; b is a bar chart showing the percentage of the area under the liquid phase (ABR) of the intact compounds after incubation in serum for 10 min, 2 h, and 24 h; and c is a bar chart showing the stability of Cy5-(GNlys)7 and Cy5-NlysO. 7. Normalized HPLC results of Cy5-(GNlysO)7 after incubation in 25% mouse serum for 10 min, 2 h, and 24 h are represented by plots; d is a bar chart showing the percentage of the area under the liquid chromatography (LC) peaks of intact compounds Cy5-(GNlys)7, Cy5-Nlys7, Cy5-(GNlysO)7, and Cy5-(NlysO)7 after incubation in serum for 10 min, 2 h, and 24 h; e is the MALDI-MS peak of the degradation compound fragments of Cy5-R8, Cy5-(GKO)7, Cy5-(GO)7, and Cy5-Nlys7. * in a and c represents degradation peaks; * in b and d represents undetected peaks. Data: mean + standard error (n=3 samples).

[0055] Figure 7 These are in vivo fluorescence images of nude mice (8 weeks old) before (0 hours) and at different time points after intravenous injection of 1 nmol of each Cy5 fluorescently labeled compound, representing the results of this invention.

[0056] Figure 8 The images show the fluorescence of organs isolated from nude mice 2 hours after intravenous injection of 1 nmol of each Cy5 fluorescently labeled compound.

[0057] Figure 9 Fluorescence images of each Cy5 fluorescently labeled compound taken up by cartilage throughout nude mice.

[0058] Figure 10 The images show representative fluorescence images of newborn nude mice (1 week old) injected with Cy5-(NlysO)7 according to the present invention. Among them, a is a representative in vivo fluorescence image; b is a bright-field photograph of representative whole-body tissues after being cleared with organic solution; c is a representative fluorescence image. Scale bar: 0.5 cm.

[0059] Figure 11This is the in vivo cartilage imaging result of the present invention, where a is a representative in vivo imaging image of 16-week-old nude mice 2 hours after a tail vein injection of 1 nmol of Cy5-R8 or Cy5-(NlysO)7 probe; b is an in vivo imaging image of group a after an injection of Cy5-R8 or Cy5-(NlysO)7 probe (1 nmol). Representative fluorescence images of cartilage-rich tissue collected from mice 2 hours after Cy5-(NlysO)7 injection; c shows light-panel fluorescence microscopy of the paws, knees, and spines of mice from a and b, demonstrating the localization of Cy5-(NlysO)7 uptake in in vivo; d shows paraffin sections of cartilage tissue from mice injected with Cy5-(NlysO)7 (H&E, Safranin-Fix-Green staining, and DAPI counterstaining) compared to in situ fluorescence (yellow); e shows a whole-body light-panel fluorescence microscopy scan (3D) of 1-week-old newborn mice injected with Cy5-(NlysO)7; f shows light-panel fluorescence microscopy images of local anatomical structures (2D); g shows fluorescence images (arrows: injection sites) of live zebrafish (5 days old, n = 6 fish) 2 hours after injection of 0.1 pmol Cy5-(NlysO)7 and a representative image comparison with alexandrite blue staining (bottom). Scale bar: 1 mm (c, f), 275 μm (d), 5 mm (e), 500 μm (g).

[0060] Figure 12 This invention presents the results of in vivo detection of GAG loss in aging knee and inflamed ankle joints using Cy5-(NlysO)7. In this study, a) shows the in vivo fluorescence images and signal quantification of the knee joints of young (12-week-old) and aged (74-week-old) Balb / c mice 2 hours after intravenous injection of 2 nmol Cy5-(NlysO)7 (n=4, Mann-Whitney U test); b) is a light-panel fluorescence microscopy image (sagittal section, 3D reconstruction shown in the upper right corner); c) is a comparison of histological staining (H&E, safranin-fast green, toluidine blue) and in situ fluorescence of paraffin sections of the knee joint (yellow, blue for DAPI counterstaining); d) is the in vivo fluorescence image and signal quantification of the ankle joint in a mouse model of unilateral collagen antibody-induced arthritis (CAIA) (n= Six mice were used (paired t-test); e is a 3D radiograph of the hind paw of a CAIA model mouse; f is a comparison of histological staining (H&E, safranin-fixing green, toluidine blue) and in situ fluorescence (yellow, blue for DAPI counterstaining) of the inflammatory ankle joint. Scale bar: 1000 μm (b); 275 μm (c, f); 2000 μm (e).

[0061] Figure 13 The results of knee joint fluorescence imaging (n = 4 mice) 72 hours after intravenous injection of 2 nmol Cy5-(NlysO) according to the present invention.

[0062] Figure 14This image shows the in vivo Cy5-(NlysO)7 uptake in the ankle joint of unilateral CAIA mice with inflammatory RA. Image a shows in vivo and in vitro fluorescence images of the hind paws of all unilateral CAIA model mice (n = 6 mice, numbered 1 to 6); image b shows representative fluorescence micrographs of frozen sections of inflamed and non-inflammatory (control) ankle joints stained with Cy5-(NlysO)7. Scale bar: 100 μm.

[0063] Figure 15 Results of chondrocyte toxicity assays. a represents Cy5-(GO)7; b represents Cy5-(GKO)7; c represents Cy5-(GNlysO)7; d represents Cy5-(GNlys)7; e represents Cy5-Nlys7; f represents Cy5-R8; g represents Cy5-(NlysO)7. The horizontal axis represents the concentration of each compound in different cell cultures. Data are shown as mean ± standard error (data points: n = 4 independent wells).

[0064] Figure 16 Routine blood analysis results for Cy5-Nlys7, Cy5-(NlysO)7, Cy5-(GNlysO)7, and Cy5-(GNlys)7 mice are given, where a represents white blood cell count; b represents lymphocyte percentage; c represents red blood cell count; d represents hemoglobin content; e represents hematocrit; f represents mean corpuscular volume (MCV); g represents mean corpuscular hemoglobin (MCH); h represents MHC concentration; i represents the standard deviation of red blood cell distribution width (RDW); j represents platelet count; k represents mean platelet volume; and l represents platelet distribution width. n = 5 mice.

[0065] Figure 17 The images show representative H&E staining results of major organs in mice used in the in vivo safety experiment of Cy5-Nlys7, Cy5-(NlysO)7, Cy5-(GNlysO)7 and Cy5-(GNlys)7 (control: PBS) of this invention (n = 5 mice), scale bar: 200 μm. Detailed Implementation

[0066] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0067] The terms "preferred," "more preferably," etc., used in this invention refer to embodiments of the invention that provide certain beneficial effects under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the description of one or more preferred embodiments does not imply that other embodiments are unavailable, nor is it intended to exclude other embodiments from the scope of this invention.

[0068] When a numerical range is disclosed herein, the range is considered continuous and includes the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0069] In the description of this invention, the reference term "and / or" includes all and any combination of one or more of the associated listed items.

[0070] In the description of this invention, the terms "one embodiment," "some embodiments," "illustrative embodiment," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0071] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0072] Example 1: (GNlysO)7 1. Cationic peptide compounds (GNlysO)7 This embodiment provides a cartilage-targeting cationic peptide compound (GNlysO)7, wherein G is a glycine residue, O is a hydroxyproline residue, and Nlys is an N-(4-aminobutyl)glycine residue, with the following structural formula: .

[0073] The structural formula of the cationic peptide compound (GNlysO)7 is shown below: .

[0074] 2. Peptide-like probe Cy5-(GNlysO)7 This embodiment also provides a cartilage-targeting peptide-like probe Cy5-(GNlysO)7, which is obtained by linking a fluorescent group Cy5 (which can be replaced with other fluorescent groups as needed) to the N-terminus of the above-mentioned cationic peptide compound (GNlysO)7 via Ahx. The chemical structure of this peptide-like probe is shown below: .

[0075] The preparation method of the aforementioned peptide-like probe Cy5-(GNlysO)7 specifically includes the following: (1) Amino acids: including Fmoc-Arg(pbf)-OH (Macklin, F809667), Fmoc-Hyp(tBu)-OH (Aladdin, F117058), Fmoc-Gly-OH (Aladdin, F103019), Fmoc-Lys(Boc)-OH (GL Biochem, 36802), Fmoc-Ahx-OH (Aladdin, F117709), etc., all of which were coupled according to the standard Fmoc procedure. All materials and reagents were purchased from commercial sources and used directly without further purification.

[0076] (2) Solid-phase synthesis of Nlys: Nlys peptide residues were synthesized on resin by a submonomer method, specifically including: acylation in DMF using N,N'-diisopropylaminomethane (DIC, Aladdin, D106162) and bromoacetic acid (Macklin, B802565) (resin:DIC:bromoacetic acid = 1:9.8:10), reaction for 30 minutes, repeated twice; after washing the resin three times with DMF, 20 molar equivalents of N-Boc-1,4-diaminobutane (Macklin, N802045, 0.1 M in DMF) were added, the reaction mixture was stirred at 50 °C for 60 minutes, then washed and the next residue was attached.

[0077] (3) Amino acid solid-phase coupling: Sequences were prepared on a PurePep Chorus peptide synthesizer using Rink Amide AM resin (HECHENG, loading: 0.37 mmol / g). The resin was swelled in DMF (Aladdin, D112002) for 10 min. Fmoc deprotection was performed in DMF at 50 °C with 20% (v / v) piperidine (Detian fine chemicals, 20191105) for 90 s. Coupling of all amino acids was performed in DMF using HATU (Aladdin, H109327), HOAT (Macklin, H811122), and DIEA (EMDMillipore, S1807494945) (resin:amino acid:HATU:HOAT:DIEA = 1:5:5:5:10), reacted at 50 °C for 5 min, and repeated twice.

[0078] (4) Fluorescent labeling: The fluorescent labeling of the sequence was achieved by reacting peptide / peptide binding resin (2.5 molar equivalents) with sulfoCyanine5-NHS (1 molar equivalent, designed as Cy5, Lumiprobe, 63220) and DIEA (3 molar equivalents) in dimethyl sulfoxide (DMSO, Aladdin, D106264) at room temperature in the dark for more than 24 hours.

[0079] (5) Separation and Purification: The final cleavage of the synthesized peptides from the solid phase was accomplished by treating the resin with a mixture of TFA (Macklin, T818782-500 ml) / TIS / water (95:2.5:2.5) for 3 hours. The TFA lysis solution was collected and evaporated to approximately 0.5 mL under a nitrogen stream. The crude product was precipitated by adding excess cold diethyl ether to the TFA solution and then centrifuging at 4000 rpm for 4 minutes at 4 °C. The crude peptide or peptide compounds were purified by reversed-phase high-performance liquid chromatography (RP-HPLC) on a semi-preparative column (Agilent ZORBAX StableBond 300 C18) using a linear gradient of acetonitrile from 5% to 50% over 25 minutes at room temperature. The purified fraction was collected, lyophilized, and reconstituted in Milli-Q water to form a stock solution.

[0080] Example 2: (NlysO)7 1. Cationic peptide compounds (NlysO)7 This embodiment provides a cartilage-targeting cationic peptide compound (NlysO)7, wherein Nlys is an N-(4-aminobutyl)glycine residue and O is a hydroxyproline residue. The structural formula of the cationic peptide compound (NlysO)7 is shown below: .

[0081] 2. Peptide-like probe Cy5-(NlysO)7 This embodiment also provides a cartilage-targeting peptide-like probe Cy5-(NlysO)7, which is obtained by linking a fluorescent group Cy5 (which can be replaced with other fluorescent groups as needed) to the N-terminus of the above-mentioned cationic peptide compound (NlysO)7 via Ahx. The chemical structure of this peptide-like probe is shown below: .

[0082] The above-mentioned peptide probe Cy5-(NlysO)7 was prepared by referring to the preparation method in Example 1 above.

[0083] Example 3: (GNlys)7 1. Cationic peptide compounds (GNlys) 7 This embodiment provides a cationic peptide compound (GNlys)7 targeting cartilage, wherein G is an n-substituted glycine and Nlys is an N-(4-aminobutyl)glycine residue. The structural formula of the cationic peptide compound (GNlys)7 is shown below: .

[0084] 2. Peptide-like probe Cy5-(GNlys)7 This embodiment also provides a cartilage-targeting peptide-like probe Cy5-(GNlys)7, which is obtained by linking a fluorescent group Cy5 (which can be replaced with other fluorescent groups as needed) to the N-terminus of the above-mentioned cationic peptide compound (GNlys)7 via Ahx. The chemical structure of this peptide-like probe is shown below: .

[0085] The above-mentioned peptide probe Cy5-(GNlys)7 was prepared by referring to the preparation method in Example 1 above.

[0086] Example 4: Nlys7 1. Cationic peptide compound Nlys7 This embodiment provides a cartilage-targeting cationic peptide compound, Nlys7, wherein Nlys is an N-(4-aminobutyl)glycine residue. The structural formula of this cationic peptide compound GNlys7 is shown below: .

[0087] 2. Peptide-mimicking probe Cy5-Nlys7 This embodiment also provides a cartilage-targeting peptide probe Cy5-Nlys7, which is obtained by linking a fluorescent group Cy5 (which can be replaced with other fluorescent groups as needed) to the N-terminus of the above-mentioned cationic peptide compound Nlys7 via Ahx. The chemical structure of this peptide probe is shown below: .

[0088] The preparation method of the above-mentioned peptide probe Cy5-Nlys7 is the same as that in Example 1 above. In addition, no amino acid monomer (such as Fmoc-Gly-OH) needs to be added during the preparation process.

[0089] Example 5: (NlysG)7 1. Cationic peptide compounds (NlysG) 7 This embodiment provides a cationic peptide compound (NlysG)7 that targets cartilage, wherein Nlys is an N-(4-aminobutyl)glycine residue and G is a glycine residue.

[0090] 2. Peptide-like probe (NlysG) 7 This embodiment also provides a cartilage-targeting peptide probe Cy5-(NlysG)7, which is obtained by linking a fluorescent group Cy5 (which can be replaced with other fluorescent groups as needed) to the N-terminus of the above-mentioned cationic peptide compound (NlysG)7 via Ahx. The preparation method of the above-mentioned peptide probe Cy5-(NlysG)7 is the same as that in Example 1 above.

[0091] Example 6: (ONlys)7 1. Cationic peptide compounds (ONlys) 7 This embodiment provides a cationic peptide compound (ONlys)7 that targets cartilage, wherein Nlys is an N-(4-aminobutyl)glycine residue and O is a hydroxyproline residue.

[0092] 2. Peptide-like probe (ONlys) 7 This embodiment also provides a cartilage-targeting peptide probe Cy5-(ONlys)7, which is obtained by linking a fluorescent group Cy5 (which can be replaced with other fluorescent groups as needed) to the N-terminus of the above-mentioned cationic peptide compound (ONlys)7 via Ahx. The preparation method of the above-mentioned peptide probe Cy5-(ONlys)7 is the same as that in Example 1 above.

[0093] Example 7: (NlysP)7 1. Cationic peptide compounds (NlysP)7 This embodiment provides a cationic peptide compound (NlysP)7 that targets cartilage, wherein Nlys is an N-(4-aminobutyl)glycine residue and P is a proline residue.

[0094] 2. Peptide-like probe (NlysP) 7 This embodiment also provides a cartilage-targeting peptide probe Cy5-(NlysP)7, which is obtained by linking a fluorescent group Cy5 (which can be replaced with other fluorescent groups as needed) to the N-terminus of the above-mentioned cationic peptide compound (NlysP)7 via Ahx. The preparation method of the above-mentioned peptide probe Cy5-(NlysP)7 is the same as that in Example 1 above.

[0095] Example 8: Nlys5 1. Cationic peptide compound Nlys5 This embodiment provides a cationic peptide compound Nlys5 that targets cartilage, wherein Nlys is an N-(4-aminobutyl)glycine residue.

[0096] 2. Peptide-mimicking probe Cy5-Nlys5 This embodiment also provides a cartilage-targeting peptide probe Cy5-Nlys5, which is obtained by linking a fluorescent group Cy5 (which can be replaced with other fluorescent groups as needed) to the N-terminus of the above-mentioned cationic peptide compound Nlys5 via Ahx.

[0097] The above-mentioned peptide probe Cy5-Nlys5 was prepared by referring to the preparation method in Example 1 above.

[0098] Example 9: (NlysO)5 1. Cationic peptide compounds (NlysO)5 This embodiment provides a cationic peptide compound (NlysO)5 that targets cartilage, wherein Nlys is an N-(4-aminobutyl)glycine residue and O is a hydroxyproline residue.

[0099] 2. Peptide-like probe Cy5-(NlysO)5 This embodiment also provides a cartilage-targeting peptide probe Cy5-(NlysO)5, which is obtained by linking a fluorescent group Cy5 (which can be replaced with other fluorescent groups as needed) to the N-terminus of the above-mentioned cationic peptide compound Nlys5 via Ahx.

[0100] The preparation method of the above-mentioned peptide probe Cy5-(NlysO)5 is the same as that in Example 1 above. In addition, no amino acid monomer (such as Fmoc-Gly-OH) needs to be added during the preparation process.

[0101] Example 10: Nlys9 1. Cationic peptide compound Nlys9 This embodiment provides a cationic peptide compound Nlys9 that targets cartilage, wherein Nlys is an N-(4-aminobutyl)glycine residue.

[0102] 2. Peptide-mimicking probe Cy5-Nlys9 This embodiment also provides a cartilage-targeting peptide probe Cy5-Nlys9, which is obtained by linking a fluorescent group Cy5 (which can be replaced with other fluorescent groups as needed) to the N-terminus of the above-mentioned cationic peptide compound Nlys5 via Ahx.

[0103] The above-mentioned peptide probe Cy5-Nlys9 was prepared by referring to the preparation method in Example 1 above.

[0104] Example 11: (NlysO)9 1. Cationic peptide compounds (NlysO)9 This embodiment provides a cationic peptide compound (NlysO)9 that targets cartilage, wherein Nlys is an N-(4-aminobutyl)glycine residue and O is a hydroxyproline residue.

[0105] 2. Peptide-like probe Cy5-(NlysO)9 This embodiment also provides a cartilage-targeting peptide probe Cy5-(NlysO)9, which is obtained by linking a fluorescent group Cy5 (which can be replaced with other fluorescent groups as needed) to the N-terminus of the above-mentioned cationic peptide compound Nlys9 via Ahx.

[0106] The preparation method of the above-mentioned peptide probe Cy5-(NlysO)9 is the same as that in Example 1 above. In addition, no amino acid monomer (such as Fmoc-Gly-OH) needs to be added during the preparation process.

[0107] Comparative Example 1: (GO)7 1. Cationic peptide compounds (GO)7 This comparative example provides a peptide compound (GO)7, wherein G is an n-substituted glycine and O is a hydroxyproline residue. The structural formula of the peptide compound (GO)7 is shown below: .

[0108] 2. Probe Cy5-(GO)7 This comparative example also provides a probe Cy5-(GO)7, which is obtained by linking a fluorescent group Cy5 to the N-terminus of the above-mentioned peptide compound (GO)7 via Ahx. The chemical structure of this probe is shown below: .

[0109] The preparation method of the probe Cy5-(GO)7 is as described in Example 1.

[0110] Comparative Example 2: (GKO)7 1. Cationic peptide compounds (GKO) 7 This comparative example provides a cartilage-targeting cationic peptide compound (GKO)7, where G is an n-substituted glycine, K is a lysine residue, and O is a hydroxyproline residue. This cationic peptide compound (GKO)7 is a natural polypeptide isomer of (GNlysO)7, differing only in the position of the Lys side chain on the peptide backbone. Its structural formula is shown below: .

[0111] 2. Cartilage probe Cy5-(GKO)7 This comparative example also provides a natural cartilage probe Cy5-(GKO)7, which is obtained by linking a fluorescent group Cy5 to the N-terminus of the above-mentioned cationic peptide compound (GKO)7 via Ahx. The chemical structure of this cartilage probe is shown below: .

[0112] The preparation method of the above-mentioned cartilage probe Cy5-(GKO)7 is as described in Example 1.

[0113] Comparative Example 3: R8 1. Cationic peptide compound R8 This comparative example provides a cartilage-targeting cationic peptide compound R8, where R is an arginine residue. This cationic peptide compound R8 is a widely accepted natural cationic polypeptide with cartilage-targeting capabilities, and its structural formula is shown below: .

[0114] 2. Cartilage probe Cy5-R8 This comparative example also provides a cartilage probe Cy5-R8, which is obtained by linking a fluorescent group Cy5 to the N-terminus of the above-mentioned cationic peptide compound R8 via Ahx. The chemical structure of this cartilage probe is shown below: .

[0115] The preparation method of the above-mentioned cartilage probe Cy5-R8 is as described in Example 1.

[0116] Detection Example 1: MALDI-MS and Circular Dichroism Spectroscopy Analysis This test example verifies the cartilage probes prepared in Examples 1-4 and Comparative Examples 1-3 by mass spectrometry and circular dichroism (CD) spectroscopy. The specific methods are as follows: (1) Mass spectrometry verification: The cartilage probes prepared in Examples 1-4 and Comparative Examples 1-3 were verified using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS, Shimadzu 8020). The concentration of the stock solution was determined by measuring the concentration at 646 nm wavelength (extinction coefficient: 271000 M). -1 cm -1 The absorbance was determined using an Implen NP80 spectrophotometer.

[0117] (2) Circular dichroism spectroscopy analysis: CD spectra were measured using a JASCO J-1500 CD spectrometer. The stock solutions of the prepared cartilage probes were diluted to 50 μM using 1×PBS buffer. Samples were scanned in the wavelength range of 200 to 260 nm using a quartz cuvette with a 0.1 cm path length at 25 °C. The mean residue ellipticity (MRE, [θ]) was determined using the following formula: [θ] = (θ × m) / (c × l × n); Where θ is the measured ellipticity (mdeg); n is the number of residues; m is the molecular weight (g / mol); c is the concentration (mg / mL); and l is the optical path length of the cuvette (mm).

[0118] The CD spectra for each report are the average of two independent scans after background correction with blank PBS buffer signal. CD parameters: bandwidth, 5 nm; digital integration time, 16 seconds; scan speed, 20 nm / min; data step size, 0.1 nm.

[0119] Figure 1 and Figure 2 The chemical structures of all probes and MALDI-TOF MS results are presented, with the mass spectrometry results showing that the structure of the prepared cartilage probe is consistent with expectations. Figure 1The circular dichroism (CD) spectroscopy results show that the CD spectra of these Cy5-labeled probe sequences in PBS solution mostly exhibit a randomly coiled conformation (e.g., Cy5-(GKO)7, Cy5-(GO)7, Cy5-(GNlysO)7, and Cy5-R8). Cy5-(NlysO)7 exhibits the most typical type II polyproline helix CD curve, showing the highest peak at 225 nm. This may be related to its higher proportion of hydroxyproline (Hyp, O) and peptide residues (Nlys). Cy5-(GNlys)7 and Cy5-Nlys7 show no structural features at all in the CD spectra, possibly due to the lack of a chiral center in the entire molecule.

[0120] Test Example 2: Cartilage Affinity Test In this test example, cartilage affinity was detected by fluorescent staining of frozen cartilage sections on the cartilage probes prepared in Examples 1-11 and Comparative Examples 1-3. The specific method is as follows: Porcine cartilage tissue was longitudinally frozen and cut into 10 μm thick sections from the joint surface to the subchondral bone side. The sections were attached to standard glass slides and incubated overnight at 4°C in PBS solution containing 10 μM Cy5-labeled peptide compound (cartilage probe). The sections were then washed with 1× PBS buffer and contrast stained with DAPI.

[0121] To test the binding specificity for GAG, a set of cartilage sections were pretreated with chondroitin sulfate ABC (ChABC; Sigma, C3667, 0.25 U / mL, 200 μL per section) at 37°C for 24 hours to remove GAG ​​content before probe staining. After cartilage staining, a set of slides were thoroughly washed with 10× PBS buffer at room temperature to test the binding capacity of the probes in high ionic strength solutions. All sections were imaged using an EVOS M7000 imaging system (ThermoFisher, optical filters: Cy5 and DAPI, objectives: 4×). Based on the above method, adjacent frozen sections of cartilage on the tibial surface of the porcine knee joint were stained, washed, and fluorescently imaged using Cy5-labeled cartilage probes.

[0122] Figure 3 The results of cartilage frozen section staining detection using cartilage probes from Examples 1-4 and Comparative Examples 1-3 are presented. The results show that Cy5-labeled (GKO)7, (GNlysO)7, (NlysO)7, (GNlys)7, and Nlys7 can all significantly bind to cartilage tissue and exhibit similar fluorescence intensities. Furthermore, the fluorescence signal gradually increases with depth from the cartilage surface (…). Figure 3(As shown in a); this is consistent with the increase in GAG chain density in cartilage with increasing tissue depth. In stark contrast to these cationic sequences, Cy5-labeled (GO)7 did not show cartilage affinity ( Figure 3 As shown in Figure a), the electrostatic interaction between the cationic side chains of peptide compounds and the anions in the cartilage matrix is ​​likely the driving factor for their binding to cartilage. Furthermore, when the stained cartilage sections were washed with a solution of higher ion concentration (10×PBS), the fluorescence signal in the tissue was significantly reduced. Figure 3 (As shown in b). This result supports the hypothesis that electrostatic attraction mediates affinity, and that electrostatic interactions are weakened in solutions with high ion concentrations. Furthermore, after pre-degrading and removing the GAG ​​component with chondroitin sulfate (ChABC) before fluorescent staining, these cationic sequences lost their cartilage affinity. Figure 3 As shown in Figure c), this indicates that these Cy5-labeled peptide compounds have GAG ​​targeting specificity.

[0123] Figure 4 The results of cartilage frozen section staining detection of the probes in Examples 5-11 and Comparative Example 1 are shown. The results show that regardless of changing the sequence length (e.g., changing Nlys7 to Nlys5 or Nlys9, or (NlysO)7 to (NlysO)5 or (NlysO)9), changing the structure of the residues (e.g., changing hydroxyproline O to proline P, such as (NlysP)7 and (NlysO)7), or changing the order of residues within the repeating unit (e.g., changing (GNlys)7 to (NlysG)7, or (NlysO)7 to (ONlys)7), the good affinity of the probes for cartilage tissue can be maintained.

[0124] The above results indicate that the cationic peptide compounds of the present invention (such as (GNlysO)7, (NlysO)7, (GNlys)7, Nlys7, Nlys5, (NlysO)5, Nlys9, (NlysO)9, (NlysG)7, (NlysP)7 or (ONlys)7) can all bind to GAG in cartilage through electrostatic interactions, exhibiting GAG targeting specificity.

[0125] Test Example 3: Cartilage uptake, retention, and permeation test This test example examined the cartilage uptake, retention, and tissue penetration capabilities of the cartilage probes prepared in Examples 1-4 and Comparative Examples 1-3. The specific experimental methods are as follows: (1) Slices of pork cartilage disc Porcine cartilage was freshly collected from the articular cartilage of the knee joint of an adult pig obtained from a local slaughterhouse. The porcine cartilage disc (diameter: 6 mm) was removed using an osteotomy drill and continuously rinsed in PBS buffer to maintain cooling. In experiments using unfixed porcine cartilage samples, all solutions contained 100 U / mL penicillin and 0.1 mg / mL streptomycin (Solarbio, P1400) to prevent bacterial growth.

[0126] (2) In vitro cartilage uptake and desorption The obtained porcine cartilage blocks were cut into small samples of approximately 10 mg (wet weight) and incubated at room temperature in 96-well plates (n = 6 samples) with a Cy5-labeled peptide compound solution (10 μM per sample, 200 μL 1× PBS buffer). The fluorescence FN1 of the culture medium was measured at specified time points (N1 = 0.5, 1, 2, 4, 6, 8, 10, 12, 24 hours) using a microplate reader (PerkinElmer EnVision; excitation wavelength: 646 nm, emission wavelength: 662 nm) to estimate the probe absorption. The fluorescence level of the compound solution measured before incubation was recorded as Fpre. The percentage of unabsorbed material was calculated as FN1 / Fpre × 100%, and the final fluorescence measurement after 24 hours of adsorption was recorded as F24h.

[0127] Desorption experiments were performed 24 hours after incubation of cartilage samples with the probe. Each sample was briefly rinsed with 1×PBS buffer to remove surface-bound probe solution and then transferred to wells containing 200 μL of blank 1×PBS buffer and incubated in 96-well plates at room temperature. The fluorescence FN2 of the wash buffer in each well was measured using a microplate reader at specified time points (N2 = 0.5, 1, 2, 4, 6, 8, 10, 12, 24 hours). The desorption percentage was calculated as FN2 / Fabsorbed × 100%, where Fabsorbed is the average fluorescence change of each compound throughout the adsorption experiment (Fabsorbed = Fpre - F24h).

[0128] (3) Confocal fluorescence microscopy Porcine cartilage discs (6 mm in diameter) were incubated for 24 hours in 200 μL of Cy5-R8 or Cy5-(NlysO)7 solution (10 μM in 1× PBS buffer), followed by three washes with 1× PBS buffer. A thin slice (X: 100–200 μm, Y: 6 mm, Z: 1000 μm) was cut from the center of each cartilage disc and scanned using a confocal microscope (Zeiss 880). Imaging was acquired at 10x magnification, and only Z-stack scans (step depth: 12 μm) on the central plane of the sample were recorded.

[0129] Test results as follows Figure 5 As shown, Cy5-labeled R8 can be absorbed very rapidly by cartilage tissue, with only about 20% of R8 remaining in the supernatant 30 minutes after cartilage is placed in the solution; due to the lack of cartilage affinity, the concentration of (GO)7 in the supernatant remains almost unchanged; the concentration of (NlysO)7 in the solution gradually decreases in the first 10 hours and appears to reach saturation or equilibrium at 24 hours. Figure 5 (as shown in a). Other Nlys-containing peptides also follow a similar trend ( Figure 5 (As shown in d). It is worth noting that in this experiment, the Cy5 concentration in the supernatant of R8 and (GKO)7 solutions gradually increased after decreasing. Figure 5 As shown in a), this suggests that after binding, the two peptides do not appear to be stably retained in the tissue or may be digested by residual proteases in the tissue and then released back into the incubation solution.

[0130] After 24 hours of probe adsorption, cartilage strips soaked in the probe solution for 24 hours were immersed in 200 μL of blank PBS solution. Cy5 fluorescence intensity in the supernatant was periodically measured to monitor the desorption of the cationic compounds. Measurements showed that the dissociation rates of (NlysO)7 and R8 from cartilage were almost equally slow, and significantly lower than that of (GKO)7. Figure 5 (as shown in b); among all Nlys-containing probes, (GNlys)7 and Nlys7 have slightly slower desorption and release rates than other probes ( Figure 5 (as shown in e).

[0131] To visualize the cartilage infiltration depth of the probe, a 6 mm diameter, 1 mm high cartilage disc was immersed in 200 μL of Cy5-R8 and (NlysO)7 solution for 24 hours at room temperature. Confocal imaging was then performed on a symmetrical cross-section of the central part of the cartilage disc. It was found that Cy5-(NlysO)7 penetrated to a depth of 1 mm into the central tissue and was distributed within the cartilage matrix. Furthermore, its fluorescence signal gradually increased with increasing cartilage depth. Figure 5 (as shown in c). In contrast, although the fluorescence signal of R8 is also widely distributed in the cross section, it does not show a spatial distribution consistent with the GAG ​​density, but rather co-localizes more obviously with chondrocytes. R8 has significantly poorer specificity for the cartilage GAG ​​matrix. This spatial distribution is related to R8's ability to penetrate the cell membrane.

[0132] The above results indicate that, compared to natural peptide probes, Nlys-containing peptide probes exhibit better penetration into the cartilage matrix, as well as more stable GAG ​​binding and retention.

[0133] Example 4: Serum stability test This test example used high-performance liquid chromatography (HPLC) to detect the stability of the cartilage probes prepared in Examples 1-4 and Comparative Examples 1-3 after incubation with mouse serum. The specific experimental methods are as follows: Cy5-labeled cartilage probes (10 nmol) were incubated at 37°C with 25% v / v mouse serum (Biosharp, BL1053A diluted in 1× PBS). Samples (200 μL) were taken periodically at 0 h (dead time: approximately 10 min), 2 h, and 24 h, and 400 μL of 3.75% trichloroacetic acid in acetonitrile was added at 4°C. All samples were then centrifuged at 13,800 g for 10 min at 4°C to remove precipitated serum proteins. The supernatant (150 μL) was mixed with 550 μL of water containing 0.1% TFA and centrifuged at 2,630 g at 4°C for 5 min. 5 μL of the supernatant was mixed with 200 μL of water containing 0.1% TFA as the final sample for HPLC analysis at room temperature, with a monitoring wavelength of 646 nm. The integrated area of ​​the complete fraction was recorded. The peak area at the “zero” minute time point was designated as 100% complete, and the peak area at each subsequent time point was normalized to the zero-minute peak to calculate the percentage of remaining compound. All experiments were repeated three times.

[0134] Test results as follows Figure 6 As shown, a) is a representative HPLC normalized result graph of Cy5-R8, Cy5-(GKO)7, Cy5-(NlysO)7, and Cy5-(GO)7 after incubation in 25% mouse serum for 10 min, 2 h, and 24 h. b) is a bar chart of the percentage of the area under the liquid phase (AUM) of the intact compounds after incubation in serum for 10 min, 2 h, and 24 h. c) is a representative HPLC normalized result graph of Cy5-(GNlys)7, Cy5-Nlys7, and Cy5-(GNlysO)7 after incubation in 25% mouse serum for 10 min, 2 h, and 24 h. Table d shows the percentage of the HPLC peak area of ​​the intact compounds Cy5-(GNlys)7, Cy5-Nlys7, Cy5-(GNlysO)7, and Cy5-(NlysO)7 after incubation in serum for 10 min, 2 h, and 24 h. Table e shows the MALDI-MS peaks of the calculated full-length sequence MS (exact mass) and the eluted Cy5-R8, Cy5-(GKO)7, Cy5-(GO)7, and Cy5-Nlys7 degradation compound fragments from the measured HPLC peaks. In a and c, * represents degradation peaks; in b and d, * represents undetected peaks. Data: mean + standard error (n=3 samples).

[0135] Figure 6The results showed that the natural cartilage probe Cy5-R8 was extremely unstable. Even less than 10 minutes after mixing with serum (the peptide was separated immediately after adding serum; the necessary experimental dead time was ≤10 min), the Cy5-R8 sample could not produce an elution peak of the intact peptide in HPLC analysis; only MALDI-MS detected the degradation fragment. Meanwhile, approximately 25% of the Cy5-(GO)7 and Cy5-(GKO)7 cartilage probes were degraded within 24 hours. Conversely, the Cy5-(NlysO)7, Cy5-(GNlysO)7, and Cy5-(GNlys)7 probes exhibited high stability and were almost unaffected by serum proteases. Even the most unstable sequence among the four Nlys compounds, Cy5-Nlys7, showed significantly better serum stability than the natural peptide Cy5-R8. Even though these sequences are rich in cations, unlike Cy5-R8, their sequences rich in non-natural peptide-like residues and non-classical amino acid Hyp residues likely protect them from serum protein recognition and degradation.

[0136] The above results indicate that, compared to the natural peptide probe Cy5-R8, the Nlys-containing peptide probe of this invention is more effective in protecting the probe from recognition and degradation by serum proteins, and has relatively better serum stability.

[0137] Test Example 5: Detection of Targeting Capability in Living Cartilage Based on the high serum stability of the Nlys cationic peptides contained in this invention, this test was conducted using mice and zebrafish as experimental subjects to further test whether the probe could achieve in vivo targeting and fluorescence visualization of cartilage through systemic administration. The specific methods are as follows: (1) Obtaining animal models Mice: All mouse housing and experimental procedures strictly followed the policies of the Laboratory Animal Ethics Committee of the Fifth Affiliated Hospital of Sun Yat-sen University (Agreement Nos.: 00361, 00436). Male Balb / c nu mice (16–18 weeks old) were used for general in vivo imaging experiments. Newborn Balb / c nu mice (7 days old) were used for neonatal mouse experiments. 74-week-old female Balb / c mice (compared to 12-week-old mice) were used for aged joint imaging experiments.

[0138] Zebrafish: Wild-type AB zebrafish strains (from the China Zebrafish Resource Center) were maintained under standard conditions (28.5°C, 14 hours light and 10 hours darkness) via an automated lighting system (marine). All procedures involving zebrafish strictly followed the policies of the Laboratory Animal Ethics Committee of the Fifth Affiliated Hospital of Sun Yat-sen University (Agreement No.: Z00005). Zebrafish embryos were collected via natural spawning and preserved in E3 medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2 and 0.33 mM MgSO4) containing 0.003% N-phenylthiourea (PTU, Sigma, P7629) to reduce melanin deposition.

[0139] (2) In vivo fluorescence imaging in mice Fluorescent probes were prepared at a dose of 1 nmol (10 μM in 100 μL of 1× PBS buffer) and injected into the tail vein of each mouse 2 hours before fluorescence imaging using an IVIS Spectrum imaging system (PerkinElmer Lumina III). Figure 13 and Figure 14-15 For joint aging and RA, each mouse was intravenously injected with 2 nmol of Cy5-(NlysO)7 (20 μM in 100 μL of 1× PBS buffer). For 1-week-old newborn mice, each mouse was intravenously injected with 1 nmol of Cy5-(NlysO)7 (20 μM in 50 μL of 1× PBS buffer). All in vivo imaging was performed 2 hours after probe injection using an IVIS Spectrum imager (PerkinElmer, fluorescence: ex / em: 620 / 670 nm, exposure: 1 sec, bing: 4, F / Stop: 2; field of view: A / B / C / Z). During image acquisition, mice were anesthetized with 3% isoflurane (RWD, R510-22-10). All acquired fluorescence images were analyzed using Living Image software (PerkinElmer). The fluorescence signal of each sample was measured by quantifying the average radiative efficiency within a selected elliptical region of interest.

[0140] (3) Histology and microscopy Paraffin-embedded tissues were cut to a thickness of 5 micrometers using a microtome. Paraffin was removed by two 5-minute cycles in each solvent: xylene, 100% ethanol, 95% ethanol, 85% ethanol, 75% ethanol, 50% ethanol, and PBS buffer. For histological staining, mouse knee joint sections were stained with safranin O (Solarbio, G1371), toluidine blue (Servicebio, G1032), or hematoxylin-eosin (H&E, Phygene, PH0516) according to the manufacturer's instructions. All histological sections were prepared by Servicebio (Wuhan, China).

[0141] Frozen sections of decalcified mouse ankles were incubated with Cy5-(NlysO)7 (200 μL, 1 μM in 1× PBS buffer) at 4°C for 4 hours after removal of OCT compounds. After washing, the sections were mounted in anti-fading mounting media. All fluorescently stained tissue sections were imaged or scanned using an EVOS M7000 imaging system (optical filters: Cy5, DAPI; objective lens: 10×). Non-fluorescent tissue sections were scanned using a Pannoramic 250 Flash III scanner (3DHISTECH, 20×).

[0142] (4) Tissue clearing and light slide fluorescence microscopy Following injection of 1 or 2 nmol of Cy5-(NlysO)7 and fluorescence imaging 2 hours post-injection, mice were perfused with 0.02% m / v heparin (in 1× PBS buffer) and blank PBS to remove blood. Samples were then collected and washed according to the PEGASOS method. After fixation with 4% PFA for 24 hours at room temperature, samples were decalcified with 20% EDTA (pH 7.0) solution for at least 4 days at room temperature. Decalcified samples were washed three times with PBS buffer and then destained with 25% Quadrol (Sigma, 122262) for 2 days. After washing three times with PBS buffer, samples were defatted with tert-butanol (Sigma, 360538) and dehydrated with 70% v / v tert-butanol, 27% v / v PEGMEMA500 (Sigma, 447943), and 3% w / v Quadrol for 2 days. Finally, the samples were immersed in a cleaning medium (BB-PEG) consisting of 75% benzyl benzoate (Sigma, W213802) by volume, 22% PEGMEMA500 by volume, and 3% Quadrol by weight for at least 2 days. All steps were performed in the dark.

[0143] Each sharpened sample was imaged on a LaVisionBiotec Ultramicroscope II equipped with an sCMOS camera (excitation / emission wavelengths: 630 / 680 nm). The tissue was immersed in an imaging chamber filled with BB-PEG media. Each sharpened sample was scanned on both sides at 4x magnification, with three light sheet beams per side and a Z-axis step size of 5 μm. Images were acquired via sequential light sheet scans and stitched together on both sides using a blending algorithm. Images were acquired by ImSpector (LaVisionBioTec), saved as 16-bit grayscale TIFF images for each channel, and reconstructed using Imaris software. Video was produced at 25 frames per second.

[0144] (5) Confocal fluorescence microscopy Five days after fertilization, zebrafish (dpf) were anesthetized with 0.003% trifluorochloroacetic acid (Solarbio, T8910) and injected with Cy5-(NlysO)7 (100 μM, 1 nL, 0.1 pmol). All zebrafish were anesthetized 2 hours after injection for imaging using an EVOS M7000 (optical filter: Cy5; objective: 4×) or a confocal microscope (10×). For confocal imaging, zebrafish were fixed in 1% agarose (Macklin, A6338). They were imaged at 10x magnification with Z-stack scans obtained at a step depth of 3 μm. Simultaneously, some 5 dpf zebrafish were embedded in glycerol, stained with Alcian Blue (Sigma, 75881-23-1) according to the manufacturer's instructions, and imaged using a Leica S9D microscope.

[0145] Figure 7 Fluorescence images of nude mice were obtained from 0 to 24 hours after a single tail vein injection of 1 nmol of different probes. The results showed that Nlys-rich peptide probes (such as Cy5-(NlysO)7, Cy5-(GNlysO)7, Cy5-(GKO)7, Cy5-(GNlys)7, and Cy5-Nlys7) exhibited stronger fluorescence signals at each time point than Cy5-R8 and Cy5-(GKO)7 in the knee joint and costal cartilage regions of the thoracic cavity, and were still detectable 8–12 hours post-injection. In contrast, the signal of Cy5-R8 became difficult to detect 2 hours post-injection. These in vivo observations are consistent with the aforementioned in vitro serum stability experiments.

[0146] Figure 8Fluorescence imaging of various organs in dissected nude mice showed that the cationic peptide probe was mainly cleared through the kidneys and liver (in small amounts). Two hours after tail vein injection, in vivo imaging showed that probe Cy5-(NlysO)7 was enriched in the cartilage-rich knee joint, ears, nose, ribs, spine, and ankle joints of nude mice, while Cy5-R8 was absent. Figure 9 and Figure 11 (a) Magnified local observation revealed that all cartilaginous anatomical structures were clearly fluorescently labeled with Cy5-(NlysO)7, including the cricoid cartilage of the trachea, ankle joint, ears, paws, knee joint, tail, spine, and costal cartilage. Figure 11 (b) in the middle.

[0147] Three-dimensional light-film imaging of transparent ex vivo samples clearly shows that Cy5-(NlysO)7 can be taken up in vivo in the interphalangeal tuberosities, articular cartilage, growth plates, and menisci of the knee joint, as well as the vertebral endplates of the spine (and weakly taken up in the intervertebral discs). Figure 11 (c) Moreover, the in situ fluorescence signal taken up in cartilage can even be preserved in paraffin-embedded tissue sections and perfectly matches the GAG ​​content shown by Safranin O staining of adjacent sections. Figure 11 (d in the image). These images and histopathological results indicate that the cationic peptide Cy5-(NlysO)7 can target all three types of cartilage in vivo (hyalinous cartilage: such as articular cartilage; fibrocartilage: such as meniscus; elastic cartilage: such as ear).

[0148] Using the above methods, this invention also discovered that signals in newborn mice (milk mice) are enriched in the articular cartilage throughout the body. Figure 10 and Figure 11 The difference from adult mice is that in suckling mice undergoing skeletal development, probe uptake of developing and ossifying bones is particularly evident, in addition to ordinary cartilage tissue (such as the nasal septum). This includes the vertebral bodies and transverse costal facets of developing (endochondral ossification) thoracic vertebrae, the vertebral bodies of caudal vertebrae, and the highly active, thick epiphyseal plates of the knee and ankle joints. Figure 11 (f in the text). Furthermore, this invention also discovered that intravascular injection of Cy5-(NlysO)7 can clearly mark developing zebrafish larvae in vivo (f in the text). Figure 11 The head cartilage (g) in the specimen includes the pharyngeal arch and cranial cartilage; these labeled anatomical structures are consistent with the results of alcinocyanine blue staining in the fixed specimen. Figure 11 g in (the middle part).

[0149] The above results show that the fluorescence signal of the Nlys-rich peptide probe of the present invention in the knee joint and costal cartilage region of the thoracic cavity is stronger than that of Cy5-R8 and Cy5-(GKO)7 at each time point. Among them, the cationic peptide compound (NlysO)7 has the potential to detect cartilage development in vivo in different animal models.

[0150] Example 6: Visualization of cartilage aging, degeneration, and inflammatory erosion GAG loss is a key marker and factor in cartilage aging and pathology. For example, GAG degradation mediated by metalloproteinases such as ADAMTS is an important driver of cartilage degeneration and osteoarthritis, and a core molecular event in rheumatoid arthritis that erodes and destroys cartilage. Therefore, developing a non-invasive molecular imaging tool to assess GAG loss in cartilage is crucial. In this study, Cy5-(NlysO)7 was used as an example to demonstrate its application in in vivo imaging of aging knee and inflamed ankle joints in mice. The specific imaging method is described in Case 5.

[0151] For the mouse model, the rheumatoid arthritis (RA) model was established in 6-8 week old female Balb / c mice by intraperitoneal injection of an anti-collagen antibody mixture (1.5 mg per mouse; Chondrex, 53100) and selective injection of lipopolysaccharide (LPS, 12.5 μg per mouse; Chondrex, 53100) into the left hind paw three days later.

[0152] First, Cy5-(NlysO)7 fluorescence imaging in mice was used to differentiate degenerative or inflammatory joints from normal joints. Figure 12 Two hours after tail vein injection, in vivo fluorescence imaging showed that Cy5-(NlysO)7 uptake in the knee joint of aged mice (74 weeks old) was significantly lower than that in young (12 weeks old) mice. Figure 12 a and Figure 13 The median fluorescence signal in the knee joint region of the probe was only half that of the young control. Figure 12 (b) Light slide scanning of the transparent samples revealed significantly lower GAG levels in the knee cartilage and growth plate of older mice compared to younger mice (the difference appeared to be even greater in the growth plate). Figure 12 c). The low fluorescence of the Cy5-(NlysO)7 probe preserved in paraffin-embedded sections of anatomically dissected joints also indicates significant GAG loss in the degenerated articular cartilage and meniscus of aged mice. This result was confirmed by safranin-fast green and toluidine blue staining of adjacent sections. Figure 12 (c in the text)

[0153] In a rheumatoid arthritis (RA) imaging experiment, this invention involved intravenously injecting Cy5-(NlysO)7 into a unilateral arthritis model mouse via tail vein. In vivo fluorescence imaging, in vitro light section microscopy of the joint, and histological analysis all showed that the uptake of Cy5-(NlysO)7 in the ankle cartilage of each mouse with arthritis was significantly lower than that in the ankle cartilage of non-arthritis mice. Figure 12 df and Figure 14 These findings strongly demonstrate the possibility of non-invasively examining GAG loss in joint degeneration and arthritis via imaging, rather than relying solely on histological analysis from euthanasia of euthanized animals for experimental endpoints.

[0154] The results above show that the cartilage probe Cy5-(NlysO)7 of the present invention can be used for non-invasive examination and evaluation of cartilage aging and lesions, and can visualize inflammatory erosion.

[0155] Example 7: Biosafety Testing Given that many cationic compounds are known to penetrate cell membranes and cause cell death, this test example examines the biotoxicity and in vivo biocompatibility of the cartilage probes prepared in Examples 1-4 and Comparative Examples 1-3. The specific testing methods are as follows: Cytotoxicity assay: Rat chondrocytes (Jennio, JNO-720) were seeded in 96-well plates at a density of 5000 cells per well in DMEM medium (Precell, PM150210). Cultures were maintained under standard conditions of 37°C and 5% CO2. The medium was supplemented with 10% fetal bovine serum (FBS, Precell, 164210) and penicillin-streptomycin (1:100 dilution from 100× stock solution, ThermoFisher, 15140122) to support cell growth and prevent bacterial contamination. When cell confluence reached 80%, probe concentrations (ranging from 0 to 40 µM) were gradually added to the cell culture medium (n = 4 wells per concentration). After 24 hours of incubation, the medium was removed, and cells were washed three times with PBS buffer. Cell viability was assessed using the CCK-8 assay kit (GLPBIO, GK10001-5) according to the manufacturer's instructions. After a further 4-hour incubation, the optical density (OD) of the CCK-8 solution was measured at 450 nm using a microplate reader (PerkinElmer, EnVision).

[0156] In vivo biocompatibility testing: Each C57BL / 6J mouse (6–8 weeks old) was intravenously injected with 100 μL of PBS or each peptide or peptide-mimicking probe (2 nmol per mouse, dissolved in 100 μL 1× PBS buffer) on days 0, 7, and 14. Mice were sacrificed on day 15, 24 hours after the last injection. The heart, liver, spleen, lungs, and kidneys of each mouse were collected and processed into formalin-fixed paraffin-embedded sections for histological evaluation with H&E staining. Whole blood was collected via the posterior orbital sinus. H&E and blood samples were submitted to a specialist department for preparation and analysis.

[0157] Cell viability test results as follows Figure 15 As shown (based on one-way ANOVA combined with post-hoc Tukey HSD test), Figure 15 (ae and g) and the Kruskal-Wallis test combined with Dunn's multiple comparison test ( Figure 15 Analysis of f) showed that the Cy5-R8 group exhibited significant cytotoxicity at high concentrations (20-40 μM) compared to the 0 μM control group (P<0.05). Unlike Cy5-R8, these peptide-based cationic sequences of the present invention did not show significant cytotoxicity at the tested concentrations. Routine blood tests in normal mice after three injections of these Nlys-rich peptide-mimicking probes showed no difference compared to control mice injected with PBS buffer. Figure 16 Furthermore, histological analysis of major organs in mice injected with Nlys probes, including representative H&E staining, revealed the histological morphology of the heart, liver, spleen, lungs, and kidneys in different groups of mice. No hemorrhage, inflammation, or other pathological signs were found. Figure 17 These data provide preliminary evidence of the biocompatibility of these cationic cartilage-targeting peptides.

[0158] The above results show that the Nlys-containing peptide probes of the present invention did not exhibit significant cytotoxicity at the tested concentrations, demonstrating good biocompatibility.

[0159] The embodiments of the present invention have been described in detail above. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A cationic peptide compound targeting cartilage, characterized in that, It has the following general formula: R-(X a -Nlys-Y b ) n ; Wherein, R is one of H, a drug active molecule, or a group containing a signal marker; X and Y are independently selected from any one of glycine residues, proline residues, or hydroxyproline residues; Nlys is an N-(4-aminobutyl)glycine residue; a and b are independently selected from 0 or 1; and n is any positive integer between 5 and 9.

2. The cationic peptide compound according to claim 1, characterized in that, The structural formulas of the cationic peptide compounds are shown in formulas (I) to (VI): ； Wherein, R is independently selected from H, a drug active molecule, or a group containing a signal marker; and n is independently taken from any positive integer between 5 and 9.

3. A method for preparing a cartilage-targeting cationic peptide compound as described in claim 1 or 2, characterized in that, This includes preparation using solid-phase synthesis methods.

4. The preparation method according to claim 3, characterized in that, The solid-phase synthesis method includes Fmoc chemical solid-phase synthesis and / or peptide submonomer solid-phase synthesis.

5. A cartilage-targeting probe, characterized in that, The compound comprises a cartilage-targeting cationic peptide compound as described in claim 1 or 2, wherein R is a group containing a signal marker.

6. The cartilage-targeting probe according to claim 5, characterized in that, The group containing the signal marker includes a connector and a signal marker.

7. The cartilage-targeting probe according to claim 6, characterized in that, The connector is an amino acid and / or an amino acid derivative.

8. The cartilage-targeting probe according to claim 6, characterized in that, The signal markers include at least one of fluorescent dyes, radioactive isotopes, magnetic resonance contrast agents, gold nanoparticles, magnetic nanoparticles, and quantum dots.

9. A method for preparing a cartilage-targeting probe as described in any one of claims 6 to 8, characterized in that, Includes the following steps: S1. A cationic peptide compound containing the aforementioned connector for targeting cartilage is prepared using a solid-phase synthesis method; S2. The product of step S1 is reacted with the signal marker and purified to obtain the final product.

10. A reagent kit for detection, characterized in that, The compound comprises a cationic peptide compound targeting cartilage as described in claim 1 or 2, and / or a cartilage-targeting probe as described in claims 5 to 8.

11. The cartilage-targeting cationic peptide compound of claim 1 or 2, and / or the cartilage-targeting probe of any one of claims 5 to 8, in any of the following: A) Products for preparing cartilage imaging; B) Preparation of drugs or carriers targeting cartilage; C) Prepare products for detecting cartilage development, aging, damage and / or lesions; D) Products for preparing animal models related to cartilage imaging; E) Preparation of biomaterials targeting cartilage; F) Prepare medical devices with cartilage targeting and / or cartilage fluorescence imaging functions.

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