Oligopeptide, its test kit, and its pharmaceutical composition

Abstract

The present disclosure relates to an oligopeptide. The oligopeptide comprises an amino acid sequence. The amino acid sequence comprises a binding motif, and the binding motif has a specific amino acid sequence. The present disclosure also relates to a test kit comprising an oligopeptide and a pharmaceutical composition comprising an oligopeptide.

Description

Technology Field

[0001] Technology field

[0002] delete

[0003] delete

[0004] The present disclosure relates to oligopeptides, medical test kits, and pharmaceutical compositions. More specifically, the present disclosure relates to oligopeptides specific to collagen XII, test kits thereof, and pharmaceutical compositions. Background Technology

[0005] Because articular cartilage lacks the ability to self-repair, the incidence of osteoarthritis (OA) is increasing, particularly among people over the age of 60. Drug therapy using anti-inflammatory drugs, intra-articular injections using lubricants, and surgeries including microfractures and saccyplasty remain the current methods of OA treatment, but they only alleviate symptoms. There is no disease-modifying agent for OA. Cell-based therapies using autologous chondrocyte transplantation have been effective only for treating localized articular cartilage defects. Stem cell or progenitor cell transplantation has now emerged as an alternative to chondrocytes in the treatment of OA and osteochondral defects, particularly large lesions.

[0006] Mesenchymal stem cells (MSCs), which possess self-renewing and pluripotent differentiation capabilities, are used not only to repair mesenchymal tissues but also for tissue engineering of cartilage and bone. The long-term safety of intra-articular injection of MSCs has been demonstrated in 41 patients with knee OA. Furthermore, the clinical efficacy and safety of MSC transplantation for the treatment of OA have been demonstrated by a meta-analysis involving 11 eligible trials and 582 patients with knee OA.

[0007] A two-year follow-up study regarding the efficacy of intra-articular injections for the treatment of knee OA revealed potential concerns regarding the sustainability of clinical and structural outcomes in the low- and intermediate dose groups of treatment, suggesting the need for further research. Intra-articular injection of MSCs using hyaluronic acid (HA) as a vehicle demonstrated superior efficacy compared to HA alone for the treatment of OA induced by anterior cruciate ligament (ACL) transection. This study and others have highlighted the importance of developing methods to enhance the local delivery of cells to damaged articular cartilage through the non-specific binding of MSCs to the synovium, meniscus, and ligamentous tissue. However, there are few studies, if any, that have focused on this. Magnetically labeled MSCs have been applied for articular cartilage repair. Although magnetically labeled MSCs do not indicate a deterioration of cartilage differentiation, there are concerns regarding iron absorption by the tissue.

[0008] In the current study, the inventors identified OA-targeting peptides through bio-panning of a phage display peptide library using human OA specimens. The application of OA-targeting peptides as diagnostic agents, lubricating aids, and delivery of MSCs to the joint surface was further investigated in an enzyme-induced OA rat model and an ACL-transcended OA pig model.

[0009] According to one aspect of the present disclosure, an oligopeptide comprises an amino acid sequence containing a binding motif, wherein the binding motif is represented by formula (i), WX1PX2W (i), wherein W is tryptophan, P is proline, X1 and X2 are each amino acids, and X1 and X2 are the same or different from each other; or the binding motif is represented by formula (ii), DTH (ii), wherein D is aspartic acid, T is threonine, and H is histidine.

[0010] According to another aspect of the present disclosure, a test kit comprises an oligopeptide of the aforementioned aspect.

[0011] According to another aspect of the present disclosure, a pharmaceutical composition comprises an oligopeptide of the aforementioned aspect and a therapeutic molecule or stem cell that binds to the oligopeptide.

[0012] delete Brief explanation of the drawing

[0013] The present disclosure can be more fully understood by reading the detailed description of the following embodiments with reference to the accompanying drawings: Figure 1 shows the results of in vivo imaging demonstrating the binding ability of C5-24 peptide to OA cartilage. Figure 2 shows the results of applying C5-24 peptide in the initial diagnosis of OA. Figure 3 shows the results of applying C5-24 peptide in joint lubrication. Figure 4 shows the results of applying C5-24 peptide in OA regenerative medicine. Figure 5 shows the results of Prussian blue staining for MRI analysis and MSC tracking. Figure 6 shows the results of confirming the binding protein of the C5-24 peptide. Specific details for implementing the invention

[0014] The present disclosure will be further illustrated by the following specific embodiments to facilitate the full utilization and practice of the present disclosure by a person skilled in the art without excessive interpretation and excessive experimentation. However, these practical details are used to describe how to practice the materials and methods of the present disclosure and are not strictly necessary.

[0015] I. Results

[0016] <OA-표적화 펩티드의 확인>

[0017] Using a phage display peptide library, the inventors examined OA articular cartilage excised from the subchondral bone of the knee joint from patients who had undergone total knee replacement. The OA cartilage specimens were homogenized to obtain tissue lysates or cut into 5 mm x 5 mm square tissues. Through five rounds of selection of phage display peptides (biopanning) binding to tissue lysates and tissue fragments, the titers of the bound phages were significantly increased to 388-fold and 864-fold, respectively. ELISA screening was further applied to phage clones collected from the fifth round of biopanning, and clones with high affinity for tissue lysates or fragments were selected, sequenced, and aligned. Finally, the inventors identified five groups of targeted phages sharing distinct common motifs. The binding ability of the selected phage clones was verified in the human chondrocyte fluorescence cell line, hPi-GL10, by immunofluorescence staining. All identified phage clones labeled with M13-PE (antibody conjugated to a fluorescent dye) bound to hPi-GL in a dose-dependent manner. In particular, C5-24 and C5-91 peptides exhibited specific and significant binding scenarios in hPi-GL. To investigate phage clones that bind specifically to OA cartilage rather than other soft tissues, such as the synovium and meniscus, human OA tissue sections were immunostained using horseradish peroxidase (HRP)-labeled phage clones, followed by semi-quantification of the deposited 3,3-diaminobenzidine (DAB) intensity (- to +++). In particular, C5-24 (presented as the amino acid sequence of SEQ ID NO. 1) and C5-91 (presented as the amino acid sequence of SEQ ID NO. 2) peptides exhibited excellent binding activity to cartilage but did not exhibit binding activity to the meniscus and synovium. Furthermore, the C5-24 peptide demonstrated the best specificity for targeting the territorial region of OA cartilage and was selected for subsequent studies.

[0018] <OA 표적화의 생체내 영상>

[0019] To demonstrate the OA-specific targeting activity of C5-24 peptides, rhodamine-labeled C5-24 peptides and scrambled peptides were separately injected into OA joints in a rat model for two-photon microscopy observation of fluorescence and second harmonic generation (SHG) signals. The scrambled peptides contained all the same amino acids as the original peptides but in a new, random sequence. Surface-rendered 3D reconstruction images and transversal composite images of the cartilage revealed rare red spots randomly present in the control cartilage injected with C5-24 peptides, the control cartilage injected with scrambled peptides, and the OA cartilage injected with scrambled peptides. Conversely, red spots were observed in the OA cartilage injected with C5-24 peptides. When type II collagen was irradiated with SHG, the red spots localized in areas without SHG signals, corresponding to the occupied regions of the OA cartilage (Fig. 1a). From the z-axis plane, it was measured that the C5-24 peptide reached a depth of at least 50 μm in the OA cartilage (Fig. 1a). In addition, the total fluorescent peptide binding area (Fig. 1b) and binding strength (Fig. 1c) were further calculated in all slices, showing a significant difference in C5-24 peptide targeting between OA and control cartilage. These data demonstrate the differentiated recognition capability and specificity of the C5-24 peptide for targeting the occupied area of ​​OA cartilage.

[0020] <Application in Initial OA Diagnosis>

[0021] To demonstrate the applicability of OA-targeting peptides in various diagnostic agents for the early diagnosis of OA, C5-24 and scrambled peptides were conjugated with superparamagnetic iron oxide (SPIO) (Fig. 2a). Fourier-transform infrared spectroscopy (FTIR) showed an increased NH band / CO band ratio, indicating successful installation of SPIO in the form of C5-24 and scrambled peptides injected intra-articularly into the OA joints of a rat model established by enzyme digestion (Fig. 2b). Magnetic resonance imaging (MRI) of the OA knee joint without peptide-conjugated SPIO injection showed no difference compared to the sham control knee joint, indicating a limitation of MRI for the early diagnosis of OA when articular cartilage has not yet been severely worn away. Similarly, scrambled peptide-conjugated SPIO that did not bind to OA cartilage and reduced MRI signal also failed to distinguish early OA from the mock control. Conversely, C5-24 peptide-conjugated SPIO bound to OA cartilage and caused reduced MRI signal in OA cartilage, but not in healthy cartilage (Fig. 2c). To bring us one step closer to a clinical setting, the feasibility of C5-24 peptide-conjugated SPIO for the diagnosis of early OA was further confirmed in a large animal OA model established by ACL-truncation in Lanyu minipigs.Two months after ACL-resection, mock control knee joints that received or did not receive C5-24 peptide-conjugated SPIO, or OA knee joints that did not receive C5-24 peptide-conjugated SPIO, showed no difference in T1- and T2-weighted MR images (Fig. 2d), indicating difficulties in using MRI for early OA diagnosis. However, OA knees that received C5-24 peptide-conjugated SPIO showed enhanced signal reduction in T1- and T2-weighted MR images, demonstrating the sensitivity of C5-24 peptide-conjugated SPIO for early OA diagnosis. Taken together, these data suggest that imaging contrast agents, such as SPIO, can be applied to early OA diagnosis in conjunction with MR imaging systems when conjugated with C5-24 peptide.

[0022] Applications in Joint Lubrication

[0023] To investigate the potential of C5-24 peptides to deliver HA to OA cartilage for lubrication, C5-24 peptides or scrambled peptides were conjugated with HA and referred to herein as C5-24-HA and scrambled-HA, respectively (Fig. 3a). Methacrylization of HA-MA 1 It was measured to be approximately 28.1% by H photon-NMR (nuclear magnetic resonance) and was used as an intermediate for the subsequent C5-24 peptide (Fig. 3b) and scrambled peptide conjugation. The static friction coefficient (μ) s ) and kinetic friction coefficient (μ kRheological lubrication properties, including ), were evaluated using a rotational test protocol modified from a previous report and compared among paired human OA cartilage cylinder discs (collected from 13 individuals) treated with non-modified HA, scrambled HA, or C5-24-HA. The total friction coefficients for non-modified HA, scrambled HA, and C5-24-HA in the 1.2s relaxation scenario were: μ, respectively. s at 0.065, 0.073, and 0.044 and μ k At 0.045, 0.052, and 0.034, and were reduced by 32.3% and 24.4% in C5-24-HA compared to unmodified HA; in the 12s relaxation scenario: μ, respectively s at 0.072, 0.075, and 0.043 and μ k These values ​​were 0.045, 0.052, and 0.033, representing a 40.3% and 26.7% reduction in C5-24-HA compared to unmodified HA; in the 120s relaxation scenario: μ, respectively s at 0.077, 0.079, and 0.044 and μ k At 0.048, 0.055, and 0.034, and were reduced by 42.9% and 29.2% in C5-24-HA compared to unmodified HA; in the 1200s relaxation scenario: μ, respectively s at 0.094, 0.102, and 0.066 and μ kThe values ​​were 0.060, 0.067, and 0.042, representing a 29.8% and 30% reduction for C5-24-HA compared to non-deformed HA (Fig. 3c). Briefly, C5-24-HA exhibited statistically significantly superior static and kinetic friction characteristics compared to non-deformed HA and scrambled-HA across all relaxation phases, demonstrating superior lubrication. Furthermore, C5-24-HA demonstrated better lubrication than non-deformed HA and scrambled-HA in the rheological preconditioning phase and torque measurements. Representative individual patient data are included in the supplementary information, showing the same scenario where cartilage disc height was gradually lost during the preconditioning phase at a relaxation time of 3600 s; however, it returned to a consistent cartilage disc height in the subsequent four phases of the relaxation period, which reduced the factors affecting friction measurements. Additionally, these data suggest the applicability of C5-24 peptides in the development of novel and effective joint lubricants for OA.

[0024] <OA 재생 의학에서의 적용>

[0025] C5-24-HA can be applied to MSC regenerative medicine by binding to CD33, an HA receptor widely expressed on the surface of MSC cells, and delivering MSCs to the surface of OA cartilage. Furthermore, the chondrogenic activity of HA has the potential to induce MSC chondrogenesis, as previously demonstrated. To verify this, rat MSCs were supplied with SPIO for follow-up and incubated with fluorescently conjugated C5-24-HA or Scrambled-HA (Fig. 4a). Fluorescence microscopy revealed that the MSCs were closely surrounded by green fluorescence (Fig. 4b, illustrated in a black-and-white schema). Furthermore, after incubation with C5-24-HA or Scrambled-HA, the MSCs were immediately injected into the OA joints of the rat model, and histological examination was performed 8 weeks after implantation. Histomorphometric analysis indicated successful induction of OA when comparing the mock control group with the OA group (Figs. 4c, 4d). Furthermore, knee joints receiving MSCs delivered by C5-24-HA showed clear cartilage regeneration and safranin-O staining (Fig. 4c), whereas those receiving MSCs delivered by Scrambled-HA still exhibited severe OA, showing multiple fissures on the cartilage surface along with a loss of safranin-O staining. Quantification of the degree of OA by the modified Mankin score also indicated that the former had better improvement in OA than the latter (Fig. 4d). To track the cells of SPIO-supplied MSCs transplanted into OA joints, MRI scanning (Fig. 5a) and Prussian blue staining (Fig. 5b) were performed 3 days after transplantation in rats, and specific homing of MSCs into OA cartilage was observed in the C5-24-HA-assisted group, but not in the Scrambled-HA-assisted group.These data suggest the applicability of C5-24-HA in enhancing MSC regenerative medicine.

[0026] <Identification of Binding Proteins>

[0027] To identify the putative target protein derived from human OA cartilage tissue that binds to the C5-24 peptide, the inventors identified the binding target using biotinylated C5-24 peptide combined with the chemical cross-linker 3,3'-dithiobis(sulfosuccinimidylpropionate) (DTSSP), followed by liquid chromatography (LC-MS / MS) with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and tandem mass spectrometry (Fig. 6a). Silver staining revealed several sharp bands, such as coimmunoprecipitated proteins COIP-1, COIP-3, and COIP-5 (Fig. 6b), which were collected separately, digested with trypsin, and analyzed by LC-MS / MS. The fragments were identified by searching the Swiss Protein Database using algorithms employing the MASCOT and TurboSequest search engines. The inventors discovered several candidate proteins, including the collagen alpha-1 (XII) and collagen alpha-3 (VI) fragments, based on probability scores, indicating a probability that these peptides belong to proteins up to 850 and 372, respectively, which is higher than most other identified peptides. To further identify these protein fragments as target proteins for C5-24 peptides, the inventors investigated the mutual binding activity between the target proteins and biotinylated C5-24 peptides using ELISA. The inventors first pre-coated ELISA plates with a specific collagen concentration to determine the optimal collagen concentration for peptide binding (Fig. 6c), and subsequently investigated peptide binding using collagen alpha-1 (XII) at 3.3 μg / mL (Fig. 6d).The inventors found that the biotin-C5-24 peptide bound to collagen alpha-1 (XII) and collagen alpha-3 (VI) fragments, but the biotin-scrambled peptide did not. However, mutual dose-dependent binding was observed only between collagen alpha-1 (XII) and the biotin-C5-24 peptide (Figs. 6c, 6d). Furthermore, there was no difference in the binding of the biotin-C5-24 peptide and the biotin-scrambled peptide to bovine serum albumin (BSA). Additionally, these data suggest that collagen alpha-1 (XII) is the target protein of the C5-24 peptide.

[0028] To predict the structure of the protein-peptide complex, protein-peptide docking was approached through the establishment of several reliable structural models targeting human collagen XII, based on homology modeling and searches for sequence similarity. These structural models were subsequently applied to calculate possible molecular docking poses with C5-24 and C5-91 peptide chains, which are the most promising peptide chains and can be selected for subsequent experiments in the inventors' study. The protein-peptide docking models were based on algorithms that follow the lowest Gibbs free energy and chemical thermodynamics after peptide chain binding with the target protein. The inventors' data showed that both C5-24 and C5-91 peptide chains were targeted to the pocket site of collagen XII in the L1385–S2285 region as poses 125 and 68, respectively, and to the C-terminus of S2506–P2724 as poses 34 and 42, which share the same docking site for C5-24 and C5-91 with the highest pose frequency (Fig. 6e). Furthermore, these predicted poses share WXPXW, an important common binding motif that can govern the major docking affinity between the peptide chain and collagen XII. In addition, the sequence homology of collagen XII among humans, pigs, rabbits, rats, and mice reached 90.3% similarity and 83.7% identity, and phylogenetic analysis revealed high genetic correlation of collagen XII among these five species. C5-24 peptides were highly reliable in rodent, rabbit, and porcine OA model investigations. Furthermore, according to the common domains in Table 1, peptide sequences in the same group shared important and identical motifs, such as FVEW and DTH in groups 1 and 3, respectively.

[0029]

[0030] Finally, the inventors demonstrated the exclusive expression of Collagen XII in OA articular cartilage. The expression of Collagen XII was observed only in rat OA cartilage and not in normal articular cartilage. Furthermore, the expression of Collagen XII was observed only in human OA cartilage but not in non-OA cartilage. Consistent with the region where the C5-24 peptide binds (Fig. 1a), Collagen XII is expressed primarily in the region occupied by clustered chondrocytes. These data are also supported by a cohort study including 161 OA patients and 29 non-OA patients. Preliminary analysis showed a significant increase in COL12A1 mRNA levels in combined OA hip and OA knee cartilage compared to non-OA cartilage.

[0031] Application in disease-modifying OA drugs (DMOADs)

[0032] There are currently no approved DMOADs. Therefore, OA may be a serious disease with an unmet medical need for therapies that modify underlying pathophysiology and translate into long-term, clinically relevant benefits. Currently, there are several drugs in Phase 2, Phase 3, or preclinical stages, including fibroblast growth factor-18 (Sprifermin), which targets cartilage regeneration, and Kartogenin, which stimulates chondrogenesis by promoting the degradation and nuclear internalization of core binding factor beta (CBFβ). All of these developing DMOADs can be further aided by the OA-targeted peptide developed in this study to accelerate delivery to OA tissues. Furthermore, most of these drugs focus on the intra-articular route of administration, as opposed to systemic drug therapies, and aim to improve safety profiles by enhancing local bioavailability, bypassing conventional barriers, minimizing systemic toxicity, and reducing off-target effects. However, it is important to recognize that the indicated placebo effect from local intra-articular administration makes the evaluation of efficacy more difficult. Improvements in the accuracy of the technology applied to deliver therapeutic agents to the OA site, such as the peptide discovered in this study, could lead to the successful development of effective treatments for OA. Future efforts should be directed toward delivering disease-modifying drugs with sophisticated carriers equipped with OA-targeting peptides to enhance the development of DMOADs.

[0033] The inventors identified several phage-encoded peptide motifs (WXPXW and DTH) that selectively return to OA joints without any significant targeting of synovial tissues, menisci, and other joint soft tissues, including ligaments. Furthermore, the inventors identified C5-24 and C5-91 peptides that specifically bind to chondrocyte-occupied regions in OA joints. C5-24 was successfully conjugated to SPIO and HA, respectively, for OA diagnostic and lubricating purposes. Although C5-91 peptide was not confirmed to deliver diagnostic or lubricating agents to the articular surface of OA joints, C5-91 peptide was believed to have the same function because it has the same size as C5-24 peptide and shares the same motif.

[0034] Although collagen II is the basis of hyaline cartilage, which constitutes 85-90% of all proteins in articular cartilage, aging or OA leads to damage that begins around the chondrocytes (occupancy area) on the joint surface and extends to the entire cartilage with progressive degeneration. Given that collagen II is not specifically expressed in OA, collagen II-targeting peptides cannot be applied to the diagnosis, treatment, lubrication, and regenerative medicine of OA. In contrast, OA-targeting peptides sharing the binding motif WXPXW have been experimentally and in silico-proven to selectively hom to the occupancy area and bind to collagen XII, which is exclusively expressed in OA cartilage, as demonstrated in this study. Immunofluorescence studies using antibodies have demonstrated that collagen XII is localized in collagen I-containing dense connective tissue structures such as tendons, ligaments, perichons, and the periosteum of embryonic tissues, suggesting its appearance during joint degeneration and regeneration (Type XII collagen is also expressed in the tissues of the cornea, intervertebral discs, and organs). Further research is needed to clarify the role of collagen XII in OA regeneration. In conclusion, the inventors have developed a novel delivery platform targeting collagen XII for the improvement of OA lubrication, diagnosis, treatment, and regenerative medicine.

[0035] Although peptides binding to collagen XII have been developed for the diagnosis, lubrication, and regenerative medicine of OA, they may also be suitable for other conditions, such as corneal ulcers and perforations that may occur in severe dry eye, and for damage or degenerative diseases associated with other tissues containing hyaline cartilage, such as intervertebral discs and tracheal cartilage. For example, collagen XII expressed in Bowman's layer of cornea is overexpressed during corneal ulcers and scar formation; therefore, functionalized collagen XII-targeting peptides can assist in the delivery of lubricants, anti-inflammatory drugs, and stem cells for the treatment of corneal ulcers. In conclusion, the inventors have developed a novel delivery platform targeting collagen XII for the improvement of OA lubrication, diagnosis, treatment, and regenerative medicine. The platform may also be used to treat other conditions, such as eye ulcers and diseases associated with other tissues containing hyaline cartilage.

[0036] Materials and Methods

[0037] The aforementioned embodiments of the present application are carried out based on the following methods and materials, and their detailed description is as follows.

[0038] <Preparation of Cartilage Specimens for Biopanning and ELISA Screening>

[0039] To avoid interference from individual differences between patients, the inventors used surgical articular cartilage specimens from the same OA patient for five biopannings in the phage display experiment. To ensure consistency in the particle size composition of the cartilage used for the five biopannings, the following process was used. A human surgical OA specimen weighing 3.2 g was added to 2 volumes of phosphate-buffered saline (PBS) and homogenized. The cartilage homogenate was centrifuged at 800 xg and 4°C for 10 minutes, and the precipitate was collected as the "large particle cartilage sample (C1)." The supernatant was added to a new centrifuge tube, centrifuged at 1,500 xg and 4°C for 10 minutes, and the pellet was collected as the "medium particle cartilage sample (C2)." The supernatant was centrifuged again at 2,000 xg and 4°C for 10 minutes, and the precipitate was collected as "small particle cartilage sample (C3)." The supernatant was then collected separately as "cartilage tissue lysate" for another 5 biopanning cycles, different from the biopanning performed on "cartilage tissue pieces." For the "cartilage tissue lysate" biopanning, C1, C2, and C3 were weighed and each was subsampled into 5 equal parts. For each biopanning cycle, a mixture of the subsamples of C1, C2, and C3 was used for 5 cycles.

[0040] For "cartilage tissue fragment" biopanning, cartilage specimens were also cut into square pieces (5 x 5 mm in size) and attached to a 96-well ELISA plate with nail polish, one piece per well for chondrocyte binding screening.

[0041] <OA 연골 조직 용해물 및 조각을 표적화하는 파지 클론의 바이오 패닝>

[0042] For "chondrocyte lysate" biopanning, the tissue lysate supernatant was diluted 10-fold with coating buffer [0.1 M NaHC O3, pH 8.6] and coated onto new 10 cm Petri dishes for biopanning (and onto 96-well ELISA plates for screening) at 4°C for 24 hours prior to use. The tissue lysate-coated plates were blocked overnight at 4°C with 1% BSA in PBS, 10 pfu of Ph.D.-12™ phage (New England BioLabs, Ipswich, MA, USA) display peptide library was added, and the plates were incubated at 4°C for 1 hour. After washing, the bound phages were eluted with 1 ml of log-phase ER2738 culture at 37°C for 20 minutes while shaking at 100 rpm. This eluted phage pool was amplified and titrated with the ER2738 overnight culture. The recovered phages were used as inputs for the next cycle of panning, and a total of 130 phage clones were randomly selected from the 5th cycle of biopanning to be cultured for ELISA screening.

[0043] The treated cartilage specimens were blocked with 1% bovine serum albumin (BSA) in PBS at 4°C for 1 hour for "cartilage tissue fragment" biopanning in each session. A Ph.D.-12™ (New England BioLabs, Ipswich, MA, USA) phage display peptide library containing 10 plaque-forming units (pfu) was added initially and incubated at 4°C for 1 hour. After washing, the bound phages were eluted with 1 ml of log-phase E. coli ER2738 culture (New England BioLabs) at 37°C for 30 minutes while shaking at 100 rpm. This eluted phage collection was amplified and titrated with the ER2738 overnight culture. The recovered phages were used as inputs for the panning of the next session, and a total of 95 phage clones were randomly selected from the 5th session of biopanning to be cultured for ELISA screening.

[0044] <OA 연골을 표적화하는 아미노산 서열 모티프의 확인>

[0045] The binding activity of selected phage clones to chondrocyte lysates and chondrocyte fragments was investigated by ELISA. Phage clones with the highest binding affinity (A490 values ​​>0.15 for chondrocyte lysates and >2.0 for chondrocyte fragments) were selected and sequenced. The inventors identified five distinct groups with different common motifs by amino acid sequence alignment (presented in Table 1).

[0046] <hPi-GL 연골세포 세포주의 면역형광을 이용하는 OA 연골을 표적화하는 펩티드의 검증>

[0047] For the investigation of these phage clones, slide-cultured hPi-GL cells were fixed in 4% paraformaldehyde in PBS for 15 minutes at room temperature, washed with PBS, permeated with 0.1% Triton X-100 for 30 minutes at room temperature, and blocked for non-specific binding with 1% BSA / PBST. Slide-cultured hPi-GL cells were 4 x 10 8 pfu, 8 x 10 8 pfu, and 10 9 Selected phage clones of pfu were incubated separately at 4°C for 1 hour. After removing unbound phages by washing, cells were incubated with anti-M13 mouse mAb (GE Healthcare, Milwaukee, WI, USA) as the primary antibody and R-phycoerythrin-Affinipure F(ab')2 fragment goat anti-mouse IgG (Jackson ImmunoResearch Inc.) as the secondary antibody, respectively, at room temperature for 1 hour. Subsequently, the cells were washed with PBST and counterstained with Hoechst 33258 (1 μg / ml; Sigma-Aldrich) at room temperature for 10 minutes. Cells were analyzed for phage binding and localization by fluorescence using confocal microscopy (Zeiss LSM 700).

[0048] Selection of Peptides Targeting OA Cartilage Rather Than Synovium and Meniscus

[0049] To investigate the localization targeting phages bound to joint tissue, human OA cartilage specimens were used for the investigation. Paraffin-embedded human OA tissue, synovial, and meniscal sections were retrieved from specimens of human OA surgical treatment under the approval of the Institutional Review Board of Chinese Medical University Hospitals (IRB no. CMUH108-REC1-046 and T-CMU-23728). Written informed consent was obtained, and all human tissue samples were coded for anonymity. All sections were dried, paraffin removed, and rehydrated according to standard protocols, followed by C5-87, C5-66, C5-83, C5-91, C5-24, E5-8, and C5-46 phage clones, or control phages (5 x 10⁶). 8 The sections were incubated with pfu / μl. After washing, the sections were treated with anti-M13 mouse mAb (GE Healthcare) for 1 hour at room temperature. After several washing steps, immunoreactivity was detected using a biotin-free ultra-sensitive polymer-HRP detection system (Biogenex, Fremont, CA, USA). The slides were lightly counterstained with hematoxylin, mounted on an Aquatex (Merck, Darmstadt, Germany), and examined under a light microscope. Peptide sequences found on phage clones exhibiting permanent binding to chondrocytes rather than synovial or meniscal cartilage were selected and synthesized for subsequent studies.

[0050] Establishment of the RAT OA Model

[0051] In the rat model, OA was established with slight modifications as previously described. Briefly, male SD rats weighing approximately 300 grams were used in this study. All animal experiments were approved by the Animal Use and Care Committee of Chinese Medical University. Rats were maintained under standard laboratory conditions (temperature 24°C, 12-hour light-dark cycle), fed a standard diet, and given tap water. Rats were anesthetized with 2.5% isoflurane (Abott, USA) at a flow rate of 70 ml / min prior to each injection. Rat joint OA was induced in each group by injecting 0.2 ml of 4% papain solution (Sigma-Aldrich, USA) along with 0.1 ml of 0.03 M cysteine ​​(Sigma-Aldrich, USA) as an activator into the right knee. The same amount of saline was injected into the left knee in each group. Injections were repeated on days 4 and 7, respectively, and rat knees were removed for histological analysis 2 weeks after the last papain injection to confirm the formation of OA. The OA model established in rats was used in subsequent experiments for additional intra-articular injections.

[0052] <Preparation of Rhodamine-Labeled C5-24 Peptide and 2-Photon Microscopy Observation>

[0053] To demonstrate the OA-specific targeting activity of the C5-24 peptide, the DYLWQYPDITWH peptide, which cannot bind to OA cartilage, was used as the scrambled peptide. Rhodamine-labeled C5-24 and scrambled peptides were injected separately into the joints of rats with or without enzyme-induced OA (control group). The C5-24 and scrambled peptides were chemically synthesized (ABI, USA), modified via a click reaction with a biotin-PEG2-iodoacetyl crosslinker (Thermo Fisher Scientific, USA) in HEPES buffer pH 8.0, additionally linked with avidin-labeled rhodamine (Jacksonlmmuno, USA), dialyzed in ddH2O with a molecular weight 4 K cutoff to remove unlabeled rhodamine, and further lyophilized and stored at -20°C. A subsample of 1 μg of rhodamine-labeled peptide in 40 μl of PBS was used for intra-articular injection using a 30 G syringe.

[0054] One day after injection, rat knees were removed, and both femoral condyles and tibias were thoroughly cleaned, immersed in PBS, and finely attached to a 3.5 cm dish for 2-photon microscopy observation. For imaging, the microscope system was operated using a near-infrared femtosecond laser (Mira 900, Coherent, USA) at a center wavelength of 810 nm, a pulse repetition rate of 76 MHz, and a pulse width of 200 fs. The laser power was controlled to 20 mW, which is sufficient to generate SHG and TPEF and also prevent photodamage during continuous illumination. Therefore, the wavelength of SHG from collagen fibers is 405 nm, while the TPEF from collagen, elastin, FAD, and NADH is approximately 450 to 650 nm. All images were obtained using a laser scanning unit (Fluoview 300, Olympus, Japan), a pair of two objective lenses (UPlanSApo20x / 0.75, Olympus, Japan) for both laser focusing and photon collection, and two photomultiplier tubes for SHG and TPEF detection, respectively. SHG and TPEF were filtered from the intense excitation laser background by a combination of a band-pass filter (FF01-405 / 10, Semrock, USA) and colored glass (BG39, Schott, Germany). Then, they were split by a dichroic mirror (FF435-Di01, Semrock, USA) and detected forward. It is known that the inventors used a cube polarization beam splitter (GT10-B, Thorlabs, USA) combined with half (AHWP05M-980, Thorlabs, USA) and quarter (AQWP05M-980, Thorlabs, USA) waveplates, respectively, to demonstrate LP and CP imaging.After focusing the objective lens, only linear polarization extinction ratios greater than 50:1 and circular polarization ellipticity of less than 1.1 (Imax / lmin) could be used for subsequent 2-photon imaging. The acquired images were primarily processed and analyzed using ImageJ / FiJi software (National Institutes of Health, Bethesda, MD, USA). The type II collagen structure reconstructed via the second harmonic generation image (Fig. 1a) revealed a porous collagen fiber interconnected structure (green) surrounding overlapping chondrocytes (black area).

[0055] < Preparation and IR Spectroscopy of C5-24 Peptide-Conjugated Superparamagnetic Iron Oxide (SPIO) >

[0056] While chemically synthesizing C5-24 and scrambled peptides, aminosilane-modified SPIO particles with a diameter of 50 nm (Chemicell GmBH, Germany) were first crosslinked with succinimidyl-[(N-maleimidopropionamidc)-tetraethyleneglycol]ester (Thermo Fisher Scientific, USA) in sodium bicarbonate buffer at pH 8.5 to form amide bonds, subsequently interacting with the sulfhydryl groups on the cysteine ​​phase of the peptide at pH 7.2 to form stable thioether bonds, and then dialysis was performed in ddH2O with a molecular weight 10 K cutoff to remove the free-form peptide, crosslinking linker, salt, and leaving group. The mixture was further concentrated under reduced pressure, resuspended in PBS, and stored at 4°C for experiments within 2 weeks. To analyze the installation of peptides on SPIO, a portion of the prepared SPIO was freeze-dried, thoroughly ground with potassium bromide (KBr) at a 1:100 wt. / wt. ratio, and milled at 200 pound / inch 2 It was compressed to form a thin pellet for subsequent infrared radiation spectroscopy (Perkin Elmer, USA). Infrared radiation was scanned at frequencies of 400–4000 1 / cm to record characteristic groups of molecules in the transmission mode and fingerprint regions, respectively.

[0057] <Magnetic Resonance Imaging (MRI) Analysis of OA in a Rat Model>

[0058] As indicated in the presented results, rats were anesthetized by inhalation at the indicated time points, and MRI scanning was performed. MRI scans were performed at the Institute of Biomedical Sciences, Academia Sinica in Taiwan using a 4.7T MR scanning system (Bruker BioSpin, Germany). T1-weighted and T2-weighted sagittal sections were prepared using the following settings: a fast spin echo sequence with a repetition time of 2000 ms and an echo time of 72 ms; a slice thickness of 1 mm; a 1 mm inter-slice gap; a matrix of 256; TE 60; TR 2000; a field of view of 60 mm; and a mean of 2. Image resolution and quality were maximized using a 60 mm volume resonator and a 2 cm diameter surface receiving coil. The tomographic DICOMs of the MRI were analyzed by Osirix MD (Osirix Ltd., USA).

[0059] <OA in Mini-pig Model, Intra-articular Injection of C5-24 Peptide-Conjugated SPIO and 3T-MRI Analysis>

[0060] For anterior cruciate ligament (ACL) transverse transectomy to establish OA, Taiwan Lan-Yu minipigs (9 months old, weighing approximately 50-60 kg) were anesthetized by an intramuscular (im) injection of a combination of Stressil (20 mg / kg) and atropine sulfate (0.02 mg / kg), followed 15 minutes later by an intramuscular injection of Zoletil® 50 (4 mg / kg, Virbac Animal Health, France). To obtain a more homogeneous knee joint group, only female pigs were included in this study. During surgery, the animals were continuously anesthetized with oxygen (flow rate 1.5 L / min), nitrous oxide (flow rate 1 L / min), and a gas containing 1% isoflurane. The right hind leg was washed and sterilely covered. After intravenous administration of cefazolin (2 g), an incision of approximately 7 cm was made in the skin of the right knee from the patella to the tibial tuberosity. The joint was then opened medially to the patellar ligament, and the patella was partially dislocated. The ACL was then fixed with a clamp, and the distal end was amputated using a scalpel. To prevent spontaneous healing of the ACL following this transection, a proximal resection was additionally performed using an electrical arthrosector. After successful rinsing with sterile 0.9% saline solution, the skin incision was closed in layers using 1-0 VICRYL® sutures (Ethicon, USA). The minipig was able to walk and move normally after this procedure. MRI scans were performed at the indicated time using a 3T MR scanning system (Achieva x 3.0, Philips, Germany) at NARLab, Instrument Technology Research Center in Taiwan.T1-weighted and T2-weighted sagittal slices were prepared using the following settings: a repetition time of 2000 ms and an echo time of 72 ms; a slice thickness of 3 mm; a matrix of 512; TE 200; TR 3500; a field of view of 60 mm; and a mean of 2. The tomographic DICOMs of the MRI were analyzed by Osirix MD (Osirix Ltd., USA).

[0061] <C5-24 및 스크램블 펩티드 콘쥬게이션된 히알루론산(HA)의 제조>

[0062] Peptide-conjugated HA was synthesized with slight modifications as previously described. Briefly, MeHA was first synthesized by reacting methacrylic anhydride (94%, MW 154.17; Sigma) with 1% (wt / vol) HA (sodium hyaluronate powder, molecular weight approximately 110-150 kDa; Kikkoman, Japan) in deionized water at pH 8, purified by dialysis (molecular weight cutoff 6-8 kDa), and then freeze-dried. The methacrylation efficiency of the intermediate MeHA macromer 1 It was evaluated by ¹H NMR. C5-24 and scrambled peptides containing cysteine ​​residues at the C-terminus allow the sulfhydryl group to react with MeHA via Michael addition. The MeHA macromer and peptide were dissolved in triethanolamine-buffered saline (TEOA buffer, 0.2 M TEOA, 0.3 M total osmolarity, pH 8.0) and maintained overnight at 37°C for peptide bonding. The peptide-conjugated HA was dialyzed in ddH2O with a molecular weight 12 K cutoff to remove free forms of peptide, TEOA, salt, and MA, and was further lyophilized and stored at room temperature. The peptide-conjugated HA was dissolved in 0.1 M acid and 1 The conjugation efficiency of the peptide was evaluated by performing 1H NMR.

[0063] Lubricant Performance Analysis

[0064] Human articular cartilage samples collected from the femoral condyles of cartilage were prepared for a lubrication test slightly modified from previous publications. Human articular cartilage samples were sectioned from patients who underwent total knee arthroplasty under the strict supervision of the IRB committee from Chinese medical university hospitals (IRB No.: CMUH108-REC1-046, and T-CMU-23728). Care was taken to avoid damaging the articular surfaces during dissection. The superficial layer of the OA cartilage from individual patients was preserved intact, and cylindrical discs with diameters of 8.0 mm and 6.0 mm were obtained by punch cutting, respectively. Only the deep layer of the cartilage was cut to obtain flat discs for adhesion to the metal counter-surface of a specially designed test module while performing friction measurements on a rheometer. Protease inhibitors were added to ensure the cartilage was used fresh without freezing and to avoid altering surface lubrication properties. After vigorously washing the samples overnight in PBS to deplete any residual synovial fluid from the cartilage surface, they were separated into at least three groups. As indicated in the results for the binding of unmodified HA or peptide-modified HA with cartilage discs, the cartilage discs were pre-incubated for 2 hours in 1 ml of original HA or peptide-modified HA (1% HA in PBS), then immersed in 10 ml of PBS in a test module and mounted on a rheometer (HR-1, TA Instrument Ltd., USA) for friction measurement.

[0065] The rheometer was initially set to zero using a standard protocol following the manufacturer's instructions. Subsequently, the inventors calculated the initial height of the cartilage sample using electronic calipers and then loaded the sample onto the rheometer. The sample was bonded to the top and bottom rheometer fixtures of a parallel plate configuration using cyanoacrylate adhesive. Only a thin layer of adhesive bonded the cartilage and metal fixture surfaces. A 6.0 mm sample surface was placed on top of an 8.0 mm surface. To avoid insufficient contact between the sample surfaces, load value fluctuations, and minimize errors in height measurement, the top sample was lowered and pressed against the bottom sample until the load value reached ~0.01 N. The corresponding recorded height, automatically detected by the rheometer, was used for strain calculation. The instrument was programmed to record the total cartilage thickness and calculate the height for approximately 14% compression. The total thickness of the human OA cartilage samples ranged from approximately 2.5 to 3.5 mm, and they were tested in a bath of HA / PBS solution (10 mL) covered with a protective lid to prevent drying. Each sample was verified for proper alignment and surface irregularities, and experiments were performed on samples with flat surfaces. The samples were immersed in the test lubricant, compressed to 86% of their original combined height, and the effective radius R of the angular velocity multiplied by the ring eff = 2 / 3[(Ro 3 -Ri 3 ) / (Ro 2 -Ri 2Preconditioning was performed by rotating two full turns in each direction at an effective sliding speed of 0.3 mm / s, defined as )]. After repeating this preconditioning two more times, the fluid pressure in the compressed cartilage was allowed to completely subside during a stress-relaxation period of 3,600 seconds. Equilibrium normal stress data were recorded and measured for each experimental group. The lubrication test was performed in 14 stages. The first two stages were considered negligible and were used as a cleaning or pre-shear stage. Stages 3, 6, 9, and 12 were performed to analyze the effects of different relaxation periods. Samples were allowed to relax for 1,200, 120, 12, and 1.2 seconds between tests. Lubrication data were recorded during stages 4-5, 7-8, 10-11, and 13-14; each stage was at a different rotational direction and a constant shear rate. During each test, torque (τ) and axial force (N) were measured, and the coefficient of kinetic friction μ k The instantaneous measurement of was determined from the following equation: μ k = τ / (R eff x N). Moment μ k The average μ used to compare the values k To generate, the average was calculated for the second rotation in each direction. The static friction coefficient is the instantaneous μ at the maximum torque value found during the test start period. s = τmax / (R eff It was calculated as x N). After the experiment, approximately 14% central indentation due to compression on the cartilage surface was confirmed.

[0066] <Rat MSC Isolation and Labeling with SPIO, and Delivery via C5-24 Peptide Conjugated HA>

[0067] Rat MSCs were isolated and inflated as previously described. Briefly, femurs and soft tissues were aseptically isolated from two female Sprague-Dawley rats aged 8 to 10 weeks (BioLASCO Taiwan Co Ltd, Taipei, Taiwan). Bone marrow mononuclear cells were isolated by density gradient centrifugation and suspended in complete culture medium (CCM: α-MEM supplemented with 16.6% fetal bovine serum, 100 U / mL penicillin, 100 μg / mL streptomycin, and 2 mM L-glutamine), then placed in a culture dish at a volume of 1 x 10⁶ 5 / cm 2 Seeding was performed at a density of [value]. Non-adherent cells were removed by washing and changing the medium after 24 hours. When the cells reached sub-confluence, cells (pass 0) were harvested for subsequent subcultures. Subsequently, the cells were set at 100 cells / cm². 2 Seeded at a density and grown in CCM with the medium changed twice a week. The MSCs used in this study were passages 3-4.

[0068] For MSC labeling with superparamagnetic iron oxide nanoparticles (SPIO), 50 μg / mL SPIO (Chemicell GmbH, Germany) was pre-mixed with 0.75 μg / mL poly-L-lysine (Sigma Aldrich, USA) in culture medium at room temperature for 1 hour. For endocytosis of SPIO nanoparticles, 4 x 10⁶ MSCs were placed in a 6-well plate. 4 Seed at a density of 1 / well and grown for 24 hours, then thoroughly washed with PBS. Subsequently, MSCs were collected in microtubes and cultured at a density of 1 x 10⁶ in serum-free medium with 2% C5-24 peptide-conjugated HA. 6Cells were incubated at a concentration of 200 μl at 37°C for 30 minutes. For intra-articular injection, the volume of HA-encapsulated MSCs was 1 x 10⁶ 6 The volume was reduced to 25 μl containing cells and precisely injected into the synovium capsule of the knee joint of OA rats.

[0069] Histological, immunofluorescence, and immunohistochemical analysis and confocal microscopy observation

[0070] For the histological analysis of HA-encapsulated MSC transplants, rats were sacrificed at the post-transplant time as indicated in the results, and the entire knee joint was removed, fixed in 4% paraformaldehyde (PFA) in PBS, decalcified in 0.5 M EDTA for 2 weeks, embedded in paraffin, and serially sectioned to a thickness of 5 μm in the sagittal direction. For H&E staining, Prussian blue staining, and Safranin-0 staining, serial sections were prepared from the mid-femoral zone according to standard protocols and observed using a phase-contrast microscope (Carl Zeiss). For H&E staining, the deparaffinized slides were serially rehydrated, stained with Lillie Mayer hematoxylin (Sigma Aldrich, USA) for 10 minutes, stained with Eosin Y (Sigma Aldrich, USA) for 30 seconds, finished with serial dehydration, washed, and mounted. For Prussian blue staining, slides were prepared similarly to H&E, rehydrated slides were stained with 5% potassium ferrocyanide in 10% HCl solution (Sigma Aldrich, USA) for 20 minutes, counterstained with Fast Red, finished with dehydration, washed, and mounted with resin gel and coverslips. For safranin-O staining, rehydrated slides were stained with 0.05% Fast Green solution for 3 minutes, stained with 0.1% safranin-O solution for 5 minutes, finished with washing, and mounted with resin gel.

[0071] For confocal microscopy observation of HA-encapsulated MSCs, HA was methacrylated, conjugated with the Alexa-488 fluorescent dye, and prepared at 2% in PBS. MSCs were collected in microtubes, labeled with the Dil3 fluorescent dye (Invitrogen, USA) according to the manufacturer's instructions, and incubated with the HA solution at 37°C for 30 minutes. Subsequently, they were dropped onto slides and immediately observed using a confocal microscope (Leica), and 3D images were reconstructed using ImageJ Fiji (NIH).

[0072] <Identification of Target Proteins of C5-24 Peptides by Affinity Trapping, Liquid Chromatography-Tandem Mass Spectrometry (LC-MS / MS), and ELISA>

[0073] To identify the binding targets of the C5-24 peptide chain, human OA cartilage specimens were homogenized for affinity trapping. 1 mg / ml of biotinylated C5-24 peptide in PBS was added to the cartilage homogenate and incubated at 4°C for 1 hour. After washing, DTSSP solution was added to a final concentration of 2 mM for peptide-target protein cross-linking. The reaction mixture was incubated and rotated at room temperature for 30 minutes. The reaction was stopped using 1 M Tris base. Chondrocytes were lysed in the first lysis buffer (100 mM Tris acetate, 1 M NaCl in pH 8.0) at 4°C for 24 hours, the lysate was centrifuged, and the pellet was retreated with the second lysis buffer (50 mM sodium acetate, 4 M guanidine HCl in pH 5.8, 65 mM DTT, 10 mM EDTA) at 4°C for another 24 hours. After centrifugation, the guanidine extract was mixed with 100% ethanol (5:1 volume ratio) at -20°C for 16 hours to ensure the removal of residual guanidine HCl. The target protein fraction was centrifuged at 16,000 xg at 4°C for 45 minutes to form a precipitate, the pellet was washed with 90% ethanol, dried, and redissolved in 100 mM acetic acid containing 100 μg / ml pepsin. MyOne Streptavidin C1 Dynabeads (MyOne Streptavidin C1 Dynabeads, Invitrogen, Carlsbad, CA, USA) were added to the protein lysate and mixed thoroughly for 1 hour. The peptide-protein complex was pulled down using immuno-magnetic separation. Finally, the purified proteins were separated by a dodecyl sodium sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Bio-Rad) gradient and silver stained using the SilverQuest silver staining kit (Invitrogen).

[0074] The stained protein bands were cut into small fragments and washed three times for 5 minutes with 10 mM ammonium bicarbonate containing 50% ACN (ABC, Sigma, St Louis, MO). The gel fragments were dehydrated with 100% CAN, rehydrated with 25 mM ABC (pH 8.2) solution containing 1 ng / μl trypsin (Promega, Madison, Wl), and incubated overnight at 37°C. After digestion, the trypsin-treated peptides were extracted from the gel using 1% FA in 50% ACN and dried using a centrifuge. The peptide fragments were identified by LC-MS / MS. LC-MS / MS was performed using an ion trap mass spectrometer (HCTultra PTM discovery, Bruker, Billerica, MA) coupled online with an Ultimate 3000 nanoLC system (Dionex, Sunnyvale, CA). Samples were injected into a trap column (C18, 5 μm, 1 mm x 5 mm, Dionex, Sunnyvale, CA) and separated online using a reverse-phase column (Atlantis C18, 3 μm, 75 μm x 150 mm, Waters, Milford, MA) at a flow rate of 300 nl / min. Peptides were eluted within 6 minutes with an H2O / ACN gradient of 2 to 40% solvent B (100% ACN, 0.1% FA) and within 24 minutes with a B gradient of 40 to 70%. MS and MS / MS scan ranges were 400–1600 m / z and 100–2500 m / z, respectively. Protein candidates were identified by searching the Swiss protein database using MASCOT (Matrix Science, London, UK) and the TurboSequest search engine (Thermo Fisher Scientific, Waltham, MA, USA), and then validated by ELISA.

[0075] Initially, ELISA plates were coated with collagen alpha-3(VI) and collagen alpha-1(XII) in coating buffer (0.5 M NaHC O3) at room temperature for 2 hours and blocked overnight at 4°C with 5% milk / TBST. Biotinylated peptides were added to the ELISA plates and incubated at room temperature for 1 hour. The plates were washed with PBS, and the biotinylated peptides were probed with HRP-conjugated mouse anti-MIS antibody (GE Healthcare Biosciences). Binding of the biotinylated peptides to recognized collagen alpha-3(VI) or collagen alpha-1(XII) was detected by HRP-conjugated streptavidin (Thermo Pierce Biotechnology Scientific). The plates were washed with PBS and subsequently incubated with the peroxidase substrate ophenylenediamine dihydrochloride (OPD; Sigma). The reaction was terminated with 3 N HCl, and the absorbance at 490 nm was measured using an ELISA reader.

[0076] <C5-24 펩티드 도킹 표적의 상동성 모델링>

[0077] Molecular modeling to further identify the binding targets of selected phage clones on cartilage tissue was performed using the Dassault system (BIOVIA, Discovery Studio Modeling Environment, Release 2019, San Diego, USA) according to the developer's instructions. Briefly, the standard sequence codes for human, mouse, and porcine ColXII retrieved from the Uniprot database were Q99715, Q60847, and F1RQI0, respectively. Three distinct parts of the human ColXII homology model were constructed using MODELER based on templates (PDB codes: 1FNF, 2B2X, 2UUR) derived from BLAST results. The length of the first human ColXII model was from L1385 to S2285, and it was 30% identical to the template 1FNF; this represented a fibronectin structure and could be used for model establishment due to its highly conserved structural topology. The second and third human ColXII models ranged from K2321 to L2513 and S2506 to P2724, respectively, and possessed 31% and 36% sequence identity with templates 2B2X and 2UUR. All homologous models were first verified using PDF total energy, Discrete Optimized Protein Energy (DOPE), and score confirmation, as well as Ramachandran plots and enhanced structures to obtain valid backbone and sidechain conformations. The most representative protein templates were used to predict binding sites and to determine the poses of C5-24 and C5-91 peptide chains based on the most promising results in IHC. Subsequently, protein-peptide docking using ZDOCK was performed to search for potential binding regions. The docking capability and accuracy between the peptide and the target protein template were validated using Z_Dock scores and E_R_Dock scores.

[0078] <Statistical Analysis>

[0079] Data are presented as mean ± SD. Statistical comparisons were performed using Student's t-tests or one-way analysis of variance (ANOVA), and a p-value of <0.05 was considered significant. All calculations were performed using the statistical analysis system (SAS) authorized for Chinese medical universities. All in vivo data represent at least three independent experiments as indicated.

[0080] Although the present disclosure has been described in considerable detail with reference to specific embodiments, other embodiments are possible. Therefore, the spirit and scope of the appended claims should not be limited to the description of the embodiments contained herein.

[0081] It will be apparent to those skilled in the art that various modifications and variations may be made to the structure of the present disclosure without departing from the scope or spirit of the present disclosure. In light of the foregoing description, the present disclosure is intended to include modifications and variations of the present disclosure if such modifications and variations fall within the scope of the following claims.

Claims

Claim 1 An oligopeptide having an amino acid sequence containing a binding motif, wherein the binding motif is represented by formula (i), WX1PX2W (i), where W is tryptophan, P is proline, X1 and X2 are each amino acids, and X1 and X2 are the same or different from each other; or an oligopeptide having at least 90% identity with at least one of the full-length amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO:

4. Claim 2 An oligopeptide according to claim 1, wherein the amino acid sequence is identical to at least one of the full-length amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO:

4. Claim 3 In claim 1, the binding target of the oligopeptide is collagen XII, the oligopeptide has binding specificity for cartilage tissue derived from a patient diagnosed with osteoarthritis, and the cartilage tissue is characterized by the expression of collagen XII. Claim 4 A test kit for diagnosing osteoarthritis comprising the oligopeptide of claim 1. Claim 5 A test kit according to paragraph 4, wherein the oligopeptide is combined with superparamagnetic iron oxide (SPIO) or an image developer. Claim 6 A pharmaceutical composition for treating osteoarthritis, spinal disease, eye disease, or degenerative disease, comprising the oligopeptide of claim 1; and a therapeutic molecule or stem cell that binds to the oligopeptide. Claim 7 A pharmaceutical composition according to claim 6, wherein the therapeutic molecule is a drug for treating osteoarthritis, a drug for treating spinal diseases, a drug for treating eye diseases, hyaluronic acid, chondrocyte growth factor, or a composition thereof, and the stem cell is a mesenchymal stem cell (MSC). Claim 8 delete Claim 9 delete Claim 10 delete Claim 11 delete Claim 12 delete Claim 13 delete Claim 14 delete Claim 15 delete Claim 16 delete Claim 17 delete Claim 18 delete Claim 19 delete

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