Multifunctional peptide composition of cell implant and collagen-like protein and application of multifunctional peptide composition

Abstract

The invention relates to a cell implant delivery system, which comprises a cell implant combined with a novel bifunctional peptide (BiFP), has a tissue targeting function and a cell differentiation instruction function, and is used for delivering the cell implant to a target tissue. The bifunctional peptide (BiFP) is composed of a targeting sequence of XII-type collagen with a function of mediating tissue target and a binding motif sequence of integrin / DDR with a function of mediating cell binding and cell differentiation instruction.

Description

Technical Field

[0001] The present invention relates to the use of a cell graft and its delivery system in regenerative medicine. More specifically, the present invention relates to the use of a stem cell graft delivery system, wherein the cell graft delivery system is constructed from a divalent peptide comprising collagen-like peptides (CLPs) having tissue targeting and cell fate determination functions. Background Art

[0002] Collagen is the most abundant extracellular matrix (ECM) protein in the body and has been widely used in biomedical research and clinical practice. However, animal-derived collagen is limited by its high immunogenicity and pathogenicity (Lynn, A.K., et al. J Biomed Mater Res B Appl Biomater 71, 343-354, 2004). Conversely, the development of synthetic and performance- and structure-optimized collagen-like materials derived from supramolecular assembly of building blocks, such as collagen-like peptides (CLPs), has been applied to 3D cell culture and tissue engineering (Prince, E. & Kumacheva, E. Nature Reviews Materials 4, 99-115, 2019). The fibrous structure of these materials mimics the filamentous structure of the extracellular matrix, determining their biomechanical properties, signal transduction functions, and cell instructive cues (Chau, M., et al. Advances in Polymer Science 268, 167-208, 2015). In addition to fiber morphology and biophysical properties, the arrangement of adhesion ligands also affects the way cells interact with these synthetic materials, potentially accelerating regenerative medicine (Boekhoven, J. & Stupp, S.I. Adv Mater 26, 1642-1659, 2014). Another contribution of synthetic collagen-like materials is to direct transplanted cells to target tissues for successful cell therapy and regenerative medicine, such as their application in controlled drug delivery or gene editing technologies (Li, J. & Mooney, D.J. Nature Reviews Materials 1, 16071, 2016; Tong, S., et al. Nature Reviews Materials 4, 726-737, 2019). However, the needs in this field remain unmet.

[0003] The present invention develops a bifunctional peptide (BiFP) for regenerative medicine of osteoarthritis (OA) and corneal epithelial defects. It is a collagen-like peptide (CLP) with osteoarthritis (OA) cartilage targeting, corneal stroma targeting, and cell fate determination functions. The osteoarthritis (OA) cartilage targeting and corneal stroma targeting functions are mediated by specific amino acid sequences targeting type XII collagen, which is an extracellular matrix specifically expressed in osteoarthritis (OA) cartilage and corneal stroma. The cell differentiation instruction function is mediated by the binding motifs of integrin or DDR, which have been found to induce chondrogenic differentiation of human mesenchymal stem cells (hMSCs) in the past.

[0004] In our previous patent application TW202128730, we demonstrated a targeting peptide for type XII collagen (Col12-TP), which can deliver mesenchymal stem cells (MSCs) to damaged articular cartilage when conjugated with hyaluronic acid (HA). However, this method requires methylacrylation of HA at an incorporation rate of approximately 28%, followed by conjugation with Col12-TP through a Michael addition reaction, and then MSCs bind to HA through the expression of its surface CD44 protein. This strategy requires high chemical conjugation efficiency and relies on the expression of CD44 protein.

[0005] Therefore, the present invention aims to design a bifunctional peptide (BiFP) comprising a trimer of collagen-like peptide (CLP) sequences and integrin or DDR binding motifs sandwiched by Col12-TP sequences, which can deliver cell implants to target tissues when binding to cell implants. Summary of the Invention

[0006] Based on the above objectives, the present invention demonstrates that the designed bifunctional peptide (BiFP) can bind to mesenchymal stem cells (MSCs) and deliver them to target tissues, such as osteoarthritis (OA) cartilage and corneal stroma, to promote osteoarthritis (OA) and corneal epithelial regeneration. In addition, the binding of the bifunctional peptide (BiFP) enhances the cell viability, proliferation, and chondrogenic differentiation of mesenchymal stem cells (MSCs) in a dose-dependent manner.

[0007] Therefore, one aspect of the present invention relates to a cell implant or cell implant composition delivered in a delivery system, which comprises a cell implant bound to a bifunctional peptide (BiFP) for delivering the cell implant to a target tissue, wherein the bifunctional peptide (BiFP) is composed of a tissue targeting sequence and a cell binding motif sequence sandwiched by repeated GPO. In some embodiments, the target tissue expresses type XII collagen, including osteoarthritis (OA) or degenerative intervertebral discs, corneal epithelium, infarcted heart tissue, skin dermis, and perifollicular tissue.

[0008] In some embodiments, the tissue targeting sequence is a targeting sequence for osteoarthritis (OA) or degenerative intervertebral discs. In some embodiments, the cell binding sequence is a binding motif of integrin or discoidin domain receptor family member 2 (DDR).

[0009] In certain embodiments of the present invention, the bifunctional peptide (BiFP) comprises a target sequence of type XII collagen, a binding motif sequence of integrin α2β1 or discoidin domain receptor family member 2 (DDR), and at least three or more repeats of GPO sandwiching the binding motif of integrin α2β1 or discoidin domain receptor family member 2 (DDR).

[0010] In certain embodiments of the present invention, the integrin motif is a binding motif of integrin α2β1. In one embodiment, the binding motif of integrin α2β1 has the amino acid sequence of GFOGER.

[0011] In other embodiments of the present invention, the cell binding motif is a binding motif of discoidin domain receptor family member 2 (DDR). In one embodiment, the binding motif of discoidin domain receptor family member 2 (DDR) has the amino acid sequence of GVMGFO.

[0012] In one embodiment, the bifunctional peptide (BiFP) has the amino acid sequence of GPOGPOGPOGPOGFOGERGPOGPOGPOGPODLQYWYPIWDTH. In another embodiment, the bifunctional peptide (BiFP) has the amino acid sequence of GPOGPOGPOGPOGVMGFOGPOGPOGPOGPODLQYWYPIWDTH.

[0013] In some embodiments of the present invention, the cell implant includes but is not limited to mesenchymal stem cells (MSCs), progenitor or differentiated cells of the musculoskeletal system, and corneal cells or their progenitors. In one embodiment, the cell implant is an autologous mesenchymal stem cell implant. In another embodiment, the cell implant is an allogeneic mesenchymal stem cell implant.

[0014] On the other hand, the present invention relates to a method for treating a disease associated with type XII collagen expression, comprising administering to a subject in need thereof a therapeutically effective dose of a cell implant delivered by a bifunctional peptide (BiFP), wherein the bifunctional peptide (BiFP) is composed of a tissue targeting sequence and a cell binding motif sequence sandwiched by repeated GPO. In some embodiments of the present invention, the diseases associated with type XII collagen expression include but are not limited to musculoskeletal diseases, suppurative or aseptic keratitis, dry eye syndrome, heart diseases, skin defects, or hair follicle-related diseases.

[0015] In some embodiments of the present invention, musculoskeletal diseases include sprains, strains and tears of ligaments, tendons, muscles and cartilage, tendinitis, tenosynovitis, fibromyalgia, osteoarthritis, rheumatoid arthritis, disc disease, polymyalgia rheumatica, bursitis, acute and chronic back pain, osteoporosis, carpal tunnel syndrome, de Quervain's disease, trigger finger, tennis elbow, rotator cuff, ganglion cyst, osteogenesis imperfecta, Duchenne muscular dystrophy, Haller's and Hunter's syndromes, and combinations thereof. In one embodiment, the musculoskeletal disease is osteoarthritis (OA).

[0016] In other embodiments of the present invention, the heart disease is myocardial infarction. In other embodiments of the present invention, the skin defect is a burn injury.

[0017] Another aspect of the present invention relates to a cell implant composition comprising a cell implant delivered by a bifunctional peptide (BiFP), wherein the bifunctional peptide (BiFP) is composed of a tissue-targeting sequence and a cell-binding motif sequence flanked by repeating GPOs.

[0018] In some embodiments of the present invention, the cell implant composition can be used to induce new cartilage regeneration in osteoarthritis (OA) knees. In some embodiments of the present invention, the cell implant composition can be used to induce corneal epithelial regeneration in the area of corneal defects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1a It is a circular dichroism (CD) spectrogram of a peptide with a representative secondary structure. The triple-helix structures of the bifunctional peptide (BiFP), collagen, or denatured collagen peptide were measured at a concentration of 0.2 mg / ml. Figure 1b It is a particle size distribution diagram of the bifunctional peptide (BiFP) at a concentration of 0.2 mg / ml in PBS at 25 °C, which was detected by dynamic light scattering (DLS). The data shows a uniform particle size distribution (∼1.914 nm).

[0020] Figure 2a and Figure 2b It is the analysis result of the binding of the bifunctional peptide (BiFP) to human mesenchymal stem cells (hMSCs). Hochest 33258-labeled human mesenchymal stem cells (hMSCs) were co-cultured with FITC-labeled bifunctional peptide (BiFP) for 1 hour, and imaging flow cytometry was performed with an Amnis imaging flow cytometer. The left is the analysis image (scale = 10 μm), and the right is the quantitative percentage diagram of human mesenchymal stem cells (hMSCs) bound to the bifunctional peptide (BiFP).

[0021] Figure 3aFor the cell viability analysis of human mesenchymal stem cells (hMSCs), there were two groups treated with the targeting peptide of type XII collagen (Col12-TP) and the bifunctional peptide (BiFP), respectively. Human mesenchymal stem cells (hMSCs) were co-cultured with the targeting peptide of type XII collagen (Col12-TP) or the bifunctional peptide (BiFP) for 1 hour, stained with calcein-AM (green) and ethidium homodimer (magenta), and observed under a confocal microscope. The left is the analysis image (scale = 50 μm), and the right is the quantitative percentage graph of live cells. Figure 3b Growth curves of human mesenchymal stem cells (hMSCs) conjugated with the targeting peptide of type XII collagen (Col12-TP) or the bifunctional peptide (BiFP), analyzed by the WST-1 cell proliferation assay. Figure 3c Specific gene mRNA levels of human mesenchymal stem cells (hMSCs) conjugated with the targeting peptide of type XII collagen (Col12-TP) or the bifunctional peptide (BiFP), analyzed by real-time RT-PCR. Figure 3d Production of sulfated glycosaminoglycan (sGAG) in human mesenchymal stem cells (hMSCs) conjugated with the targeting peptide of type XII collagen (Col12-TP) or the bifunctional peptide (BiFP) at 14 days. Human mesenchymal stem cells (hMSCs) were stained with 1,9-dimethylmethylene blue (DMB) and analyzed at OD 525 nm, and the results were normalized by PicoGreen dsDNA quantification. Data are expressed as mean ± SD, and significant differences were determined by t-test (2 groups) or one-way ANOVA (≥3 groups) statistical analysis, *p < 0.05, **p < 0.01, ***p < 0.0001.

[0022] Figure 4a and Figure 4b In a rat model of papain / cysteine-induced osteoarthritis (OA), fluorescein isothiocyanate (FITC)-fluorescently labeled BiFP with / without blocking peptide ( Figure 4a ) and DilC18-fluorescently labeled rat mesenchymal stem cells with / without bifunctional peptide (BiFP) delivery (BiFP+rMSCs) ( Figure 4b ) were intra-articularly injected, respectively. At 24 hours after injection, the entire joint capsule was removed, and the articular surface of the distal femur was observed under a two-photon microscope. The left is the representative graph of the second harmonic generation (SHG) signal of the bifunctional peptide (BiFP) and the fluorescent signal of the bifunctional peptide (BiFP) ( Figure 4a ) and the representative graph of the fluorescent signal of rat mesenchymal stem cells (rMSCs) ( Figure 4b ), and the right is the quantitative analysis graph of the total fluorescent area. Figure 4c and Figure 4dAfter 8 weeks of treatment with or without transplantation and with bifunctional peptide (BiFP), rat mesenchymal stem cells (rMSCs), and rMSCs delivered with BiFP (BiFP+rMSCs), the distal femurs of the sham control group or osteoarthritis (OA) group were collected and subjected to hematoxylin-eosin staining (H&E stain)( Figure 4c ) and representative images of safranin-O / fast green staining( Figure 4d ). Figure 4e The analysis results of the degree of osteoarthritis (OA) were based on the analysis of sections stained with H&E and safranin-O / fast green (scale = 50 μm). Data are expressed as mean ± SD, and significant differences were determined by Student's t-test (for 2 groups) or one-way analysis of variance (one-way ANOVA) (for ≥3 groups), *p < 0.01 and ****p < 0.0001.

[0023] Figure 5a Schematic diagram of the experimental design for the regeneration of multilayer articular cartilage by EGFP-labeled human mesenchymal stem cells (EGFP-hMSCs) in a papain / cysteine-induced osteoarthritis (OA) rat model, divided into two groups: without bifunctional peptide (BiFP) delivery (hMSCs) and with bifunctional peptide (BiFP) delivery (BiFP+hMSCs). Rats were sacrificed at the designated time points, and immunohistochemical analysis or immunofluorescence analysis was performed on the distal femurs of the knee joints. Figure 5b Immunohistochemical analysis of EGFP, positive signals were shown in brown (DAB), and NC was the negative control group without the primary antibody (scale = 50 μm). Figure 5c Immunofluorescence analysis of EGFP, aggrecan, type II collagen, and type XII collagen in the osteoarthritis (OA) cartilage of rats treated with EGFP-hMSCs or EGFP-hMSCs delivered with bifunctional peptide (BiFP) (BiFP+hMSCs) to evaluate the expression of cartilage markers (scale = 50 μm).

[0024] Figure 6a Schematic diagram of the experimental design for the regeneration of multilayer articular cartilage by EGFP-labeled rat mesenchymal stem cells (EGFP-rMSCs) in a papain / cysteine-induced osteoarthritis (OA) rat model, divided into two groups: without bifunctional peptide (BiFP) delivery (rMSCs) and with bifunctional peptide (BiFP) delivery (BiFP+rMSCs). Rats were sacrificed at the designated time points, and immunohistochemical analysis or immunofluorescence analysis was performed on the distal femurs of the knee joints. Figure 6bFor immunohistochemical analysis of EGFP, positive signals were shown in brown (DAB). Figure 6c shows double immunofluorescence analysis of EGFP and cartilage markers such as chondroitin sulfate proteoglycan, type II collagen, and type XII collagen. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (scale = 50 μm).

[0025] Figure 7a Figure is a schematic diagram of the experimental design process for intervertebral disc (IVD) regeneration by EGFP-labeled human mesenchymal stem cells (EGFP-hMSCs) in a rat intervertebral disc injury model induced by 20G acupuncture. It was divided into four groups: PBS, bifunctional peptide (BiFP), hMSCs without BiFP delivery, and BiFP+hMSCs with BiFP delivery. Rats were sacrificed at the designated time points, and the tail tissues of the rats were subjected to X-ray, T2-weighted MRI, hematoxylin-eosin staining (H&E stain), safranin-O / fast green staining, immunohistochemistry, and immunofluorescence analysis. Figure 7b Figure

[0025] is a radiographic image of bone structure, while Figure 7c Figure Figure 7a is a T2-weighted MRI image of the nucleus pulposus (NP) that received intervertebral injury (the sham operation group did not receive intervertebral injury) in the caudal vertebral joint. Different treatments were performed two weeks later, and radiographic analysis was used. Figure 7d and Figure 7e Figures Figure 7b and Figure 7c are representative images of H&E staining and safranin-O / fast green staining of nucleus pulposus (NP) tissue. Figure 7f For immunohistochemical analysis of EGFP, positive signals were shown in brown (DAB). Figure 7g Figure Figure 7e shows double immunofluorescence analysis of EGFP and cartilage markers such as chondroitin sulfate proteoglycan and type II collagen. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (scale = 50 μm).

[0026] Figure 8a Figure Figure 7f is a schematic diagram of the experimental design process for corneal epithelial regeneration by EGFP-labeled human mesenchymal stem cells (EGFP-hMSCs) in a rat corneal epithelial injury model. It was divided into two groups: hMSCs without BiFP delivery and BiFP+hMSCs with BiFP delivery. Figure 8b Figure Figure 7g shows corneal epithelial defects (CEDs) induced by n-heptanol injury. After fluorescein staining at the designated time points after the start of treatment, the treatment included the use of PBS, BiFP (40 μM), EGFP-hMSCs (10 5Eye drops (20 μl) of EGFP-hMSCs delivered by single-functional peptides (SFPs) or bifunctional peptides (BiFPs). The left shows representative fluorescein-stained corneas at the indicated time points, and the right shows the quantitative results of calculating the percentage of the healing area using ImageJ Fiji software. Significant differences were determined by one-way ANOVA statistical analysis, *p < 0.05, **p < 0.01, and ***p < 0.001. Figure 8c Corneal tissue morphology detected by H&E staining at 14 days for each group (scale = 50 μm). Figure 8d Immunohistochemical staining for EGFP, with positive signals shown in brown (DAB), and NC as the negative control group (scale = 50 μm). Detailed implementation mode

[0027] Other features and advantages of the present invention will be further elaborated and described in detail in the following examples, which are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0028] Example 1: Preparation of a bifunctional peptide (BiFP) cell implant delivery system

[0029] In the present invention, a bifunctional peptide (BiFP) was synthesized to avoid complex chemical steps and improve efficiency. A 42-residue bifunctional peptide (BiFP) with the sequence GPOGPOGPOGPOGFOGERGPOGPOGPOGPODLQYWYPIWDTH, where O represents hydroxyproline, was prepared by the Biomedical Translation Research Center in Taiwan, China, in PBS (pH 7.4, 25 °C). The triple-helical structures of the bifunctional peptide (BiFP), collagen, or denatured collagen peptides were measured at a concentration of 0.2 mg / ml, respectively. The particle size distribution of the bifunctional peptide (BiFP) at a concentration of 0.2 mg / ml in PBS at 25 °C was detected by dynamic light scattering (DLS). Circular Dichroism (CD) spectroscopy confirmed that the peptide exhibited a stable triple-helical conformation in solution ( Figure 1a ). A dynamic light scattering instrument, Malvern ZS90 zetasizer (Malvern Instruments Corp, Malvern, UK), was used to evaluate the size, and the dynamic light scattering data showed its uniform size distribution in PBS, with a main size of 1.914 nm ( Figure 1b ).

[0030] The FITC-labeled bifunctional peptide (BiFP) or the rhodamine-labeled type XII collagen targeting peptide (Col12-TP) was chemically synthesized by BiomerTech (USA) or ABI (USA), and the peptide binding was identified by confocal microscopy. Briefly, for the treatment of FITC-BiFP or rhodamine Col12-TP, 1x10 6 human mesenchymal stem cells (hMSCs) were cultured with 1 μM DilC18 at 37 °C for 10 minutes, and then cultured in 200 μl of PBS containing 0, 2.5, 10, 40 μM fluorescent peptides at 37 °C for 30 minutes, gently mixed every 10 minutes. The cells were counterstained with a mounting medium containing DAPI, and the fluorescence of the peptides was photographed using an ImageXpress Micro Confocal High Content Imaging system (Molecular Devices, Sunnyvale, CA, USA).

[0031] First, we showed that the type XII collagen targeting peptide (Col12-TP) could not bind to human mesenchymal stem cells (hMSCs), while the bifunctional peptide (BiFP) could bind to human mesenchymal stem cells (hMSCs) in a dose-dependent manner. Imaging flow cytometry showed similar results to microscopic observation and further revealed that when the concentration of the bifunctional peptide (BiFP) increased from 2.5 μM to 40 μM, it would lead to an increase in the percentage of cells surrounded by FITC-BiFP ( Figure 2a ). Similarly, human mesenchymal stem cells (hMSCs) did not express type XII collagen, while the chondrocyte cell line hiP cells expressed type XII collagen and were able to bind to the type XII collagen targeting peptide (Col12-TP).

[0032] In addition, another 42-residue bifunctional peptide (BiFP) with the sequence GPOGPOGPOGPOGVMGFOGPOGPOGPOGPODLQYWYPIWDTH was prepared, where O represents hydroxyproline. One type of collagen peptide (CLP) with the amino acid sequence GVMGFO containing a DDR binding motif, flanked by repeated GPOs, was combined into a type XII collagen targeting peptide. As Figure 2b shown, the bifunctional peptide (BiFP) could also bind to human mesenchymal stem cells (hMSCs). Similarly, when the concentration of the bifunctional peptide (BiFP) increased from 2.5 μM to 40 μM, it would lead to an increase in the percentage of cells surrounded by FITC-BiFP ( Figure 2b ). These data indicate that the bifunctional peptide (BiFP) containing the GVMGFO amino acid sequence with a DDR binding motif can also be designed for the development of a regenerative cell implant delivery system.

[0033] Example 2: Dual-functional peptide (BiFP) binding enhances the survival and chondrogenic differentiation of mesenchymal stem cells (MSCs)

[0034] Regarding the treatment with FITC-labeled dual-functional peptide (BiFP) or rhodamine-labeled type XII collagen-targeting peptide (Col12-TP), 1x10 6 human mesenchymal stem cells (hMSCs) were cultured in 200 μl final volume of PBS containing 0, 2.5, 10, 40 μM fluorescent peptide at 37 °C for 30 minutes, with gentle mixing every 10 minutes. Human mesenchymal stem cells (hMSCs) were stained for live cells with calcein AM (ThermoFisher) and for dead cells with ethidium homodimer (Life Technologies), and the labeled human mesenchymal stem cells (hMSCs) were visualized using an ImageXpress Micro Confocal High Content Imaging system (Molecular Devices, Sunnyvale, CA, USA). Three independent images were taken for each group to count and quantify live human mesenchymal stem cells (hMSCs).

[0035] Compared to the type XII collagen-targeting peptide (Col12-TP), culturing human mesenchymal stem cells (hMSCs) with the addition of dual-functional peptide (BiFP) increased cell survival ( Figure 3a ), cell proliferation ( Figure 3b ), and differentiation into chondrogenic cells. The expression of chondrogenic genes such as sox9, col2a1, and aggrecan ( Figure 3c ) was increased at 1 week, and the synthesis of glycosaminoglycans was enhanced at 2 weeks ( Figure 3d ). Notably, the effects of dual-functional peptide (BiFP) on cell viability and cell proliferation were more obvious at higher concentrations than at lower concentrations (Figure 3). Taken together, these data indicate that dual-functional peptide (BiFP) binding enhances the cell survival, cell proliferation, and chondrogenic differentiation of mesenchymal stem cells (MSCs) in a dose-dependent manner.

[0036] Example 3: Application of the dual-functional peptide (BiFP) cell delivery system in the regenerative medicine of osteoarthritis (OA)

[0037] Since the bifunctional peptide (BiFP) can target osteoarthritis (OA) cartilage and integrin α2β1 has the ability to induce chondrogenesis, which may lead to chondrogenesis in mesenchymal stem cells (MSCs). The bifunctional peptide (BiFP) is applied in the regenerative medicine of osteoarthritis (OA) to deliver human mesenchymal stem cells (hMSCs) to the surface of osteoarthritis (OA) cartilage. To confirm the specific targeting activity of the bifunctional peptide (BiFP) against osteoarthritis (OA), rhodamine-labeled bifunctional peptide (BiFP) (pre-cultured without / with the blocking peptide of type XII collagen) was injected into the joints of rats with osteoarthritis (OA) respectively, and fluorescence and second harmonic generation (SHG) signals were observed by two-photon microscopy.

[0038] The surface-rendered three-dimensional reconstruction images and transverse composite images of cartilage showed obvious red dots observed in the osteoarthritis (OA) cartilage injected with bifunctional peptide (BiFP) ( Figure 4a ). When type II collagen was detected by SHG, the red dots were located in the SHG signal-deficient area, corresponding to the area (pericellular area) of osteoarthritis (OA) cartilage. Conversely, no red dots were observed in the osteoarthritis (OA) bone injected with bifunctional peptide (BiFP) pre-cultured with the blocking peptide of type XII collagen ( Figure 4a ).

[0039] After mesenchymal stem cells (MSCs) were labeled with Dil active dye, they were cultured in the absence or presence of bifunctional peptide (BiFP) and injected into the knee joints of rats with osteoarthritis (OA) intra-articularly. When type II collagen was detected by SHG, red dots were observed on the surface of the articular cartilage of the knee joints of rats with osteoarthritis (OA) injected with mesenchymal stem cells (MSCs) co-cultured with bifunctional peptide (BiFP), while no red dots were observed in the knee joints of rats with osteoarthritis (OA) injected with mesenchymal stem cells (MSCs) not co-cultured with bifunctional peptide (BiFP) ( Figure 4b ).

[0040] In addition, after co-culture with bifunctional peptide (BiFP), mesenchymal stem cells (MSCs) were immediately injected into the joints of rats with osteoarthritis (OA), and the joints were histologically examined 8 weeks after transplantation. Histomorphometric analysis showed that osteoarthritis (OA) was successfully induced compared with the sham control group ( Figure 4c 、 Figure 4d ). In addition, obvious cartilage regeneration and safranin-O staining were observed in the knee joints receiving mesenchymal stem cells (MSCs) delivered by bifunctional peptide (BiFP) ( Figure 4c 、 Figure 4d) In contrast, those knees that received mesenchymal stem cells (MSCs) without the delivery of bifunctional peptides (BiFPs), only the targeting peptide of type XII collagen (Col12-TP), and only bifunctional peptides (BiFPs) still showed severe osteoarthritis (OA), with multiple cracks on the cartilage surface and loss of safranin-O staining.

[0041] The degree of osteoarthritis (OA) was quantitatively analyzed by the modified Mankin scale score, and the results showed that the osteoarthritis (OA) score of the sham control group was lower than that of the osteoarthritis (OA) group ( Figure 4e ). Similarly, the osteoarthritis (OA) treated with mesenchymal stem cells (MSCs) delivered with bifunctional peptides (BiFPs) had significantly lower modified Mankin scale scores than the osteoarthritis (OA) treated without mesenchymal stem cells (MSCs) delivered with bifunctional peptides (BiFPs), the osteoarthritis (OA) treated with only the targeting peptide of type XII collagen (Col12-TP), or the osteoarthritis (OA) treated with only bifunctional peptides (BiFPs) ( Figure 4e ), indicating better articular cartilage repair.

[0042] Human and rat mesenchymal stem cells (MSCs) were then transduced with lentivirus to express EGFP for long-term tracking, and then delivered with bifunctional peptides (BiFPs) and intra-articularly injected once a week into the rat osteoarthritis (OA) model for 3 consecutive weeks ( Figure 5a 、 Figure 6a ). Seven days after the third transplantation, the knees were collected and the chondrogenic protein expression of the transplanted mesenchymal stem cells (MSCs) was evaluated histologically to determine cell fate.

[0043] Due to the immune-privileged nature of mesenchymal stem cells (MSCs), EGFP-human mesenchymal stem cells (EGFP-hMSCs) delivered with bifunctional peptides (BiFPs) were intra-articularly injected into the rat osteoarthritis (OA) knees once a week for 3 consecutive weeks ( Figure 5a ), and then histological evaluation of cartilage regeneration was performed 1 week later. Notably, multiple layers of GFP+ neo-cartilage were observed arranged in three vertical blocks, with the cells in the lower and middle blocks having the spherical shape of mature chondrocytes, while the cells in the upper block having the fibroblast shape of mesenchymal stem cells-like ( Figure 5b ). In addition, these cells were positive for both cartilage proteoglycan and type II collagen ( Figure 5c ). Taken together, these data indicate that multiple injections of rat and human mesenchymal stem cells (MSCs) delivered with bifunctional peptides (BiFPs) can regenerate multiple layers of neo-cartilage.

[0044] Figure 6 shows the application of rat mesenchymal stem cells (rMSCs) delivered by multiple intra-articular injections of bifunctional peptide (BiFP) in the regenerative medicine of osteoarthritis (OA). Immunostaining results showed multiple layers of EGFP+, chondroitin proteoglycan+, and type II collagen+ cells in the knee joints of EGFP-rMSCs receiving bifunctional peptide (BiFP) delivery, while this phenomenon was not observed in the knee joints of EGFP-rMSCs receiving non-bifunctional peptide (BiFP) delivery( Figure 6b ). In addition, double immunofluorescence results showed the co-localization of EGFP with chondroitin proteoglycan and type II collagen (Figure 6c). Notably, multiple layers of type XII collagen+ cells were observed in the knee joints of EGFP-rMSCs receiving non-bifunctional peptide (BiFP) delivery, while only single-layer or sparse type XII collagen+ cells were observed in the knee joints of EGFP-rMSCs receiving bifunctional peptide (BiFP) delivery.

[0045] Example 4: Application of the bifunctional peptide (BiFP) cell implant delivery system in intervertebral disc regeneration

[0046] Figure 7 shows the application of human mesenchymal stem cells (hMSCs) delivered by bifunctional peptide (BiFP) in the regeneration of intervertebral discs (IVDs). X-ray, T2-weighted MRI, and histological, histochemical, and immunofluorescence analyses all showed that, compared with the sham control group, the PBS group successfully induced IVD injury( Figure 7b - 7e ). In addition, hMSCs delivered by bifunctional peptide (BiFP) maintained the IVD height( Figure 7b ), and had obvious intervertebral disc regeneration, such as discs with high T2 water content( Figure 7c ) and safranin O-positive nucleus pulposus regions( Figure 7d ); while those groups of human mesenchymal stem cells (hMSCs) without bifunctional peptide (BiFP) delivery, bifunctional peptide (BiFP) only, and PBS still showed severe IVD injury, resulting in a decrease in intervertebral disc height, and T2-weighted MRI showed loss of discs with high water content, and histological section analysis also showed loss of safranin-O staining. Immunostaining results showed multiple layers of EGPF+, chondroitin proteoglycan+, and type II collagen+ cells in the nucleus pulposus regions of EGFP-hMSCs receiving bifunctional peptide (BiFP) delivery, while this phenomenon was not observed in the nucleus pulposus regions of EGFP-hMSCs receiving non-bifunctional peptide (BiFP) delivery( Figure 7f 、 Figure 7g ).

[0047] Example 5: Application of mesenchymal stem cells (MSCs) delivered in the bifunctional peptide (BiFP) cell implant delivery system in corneal epithelial regeneration

[0048] Type XII collagen is expressed in various tissues, including the cornea, which is rich in stroma and the anterior Bowman’s layer and is exposed when corneal epithelial defects occur in the eye. When diseases affect the clarity of the cornea, corneal transplantation remains the main method for visual rehabilitation. In addition, autologous corneal epithelial stem cells are transplanted together with amniotic membrane carriers for the treatment of severe ocular surface diseases and limbal dysfunction. However, all of these techniques rely on available corneal donor tissue, which is a major limiting factor in developing countries.

[0049] To further confirm the utility of bifunctional peptides (BiFPs) as cell carriers and to expand the application of mesenchymal stem cells (MSCs) delivered by BiFPs in corneal epithelial defect regeneration, experiments were conducted using a rat model in which repeated administration of n-heptanol was used to induce severe corneal epithelial injury. In the results of measuring the corneal defect area by fluorescein sodium staining, twice-daily administration of n-heptanol for four days resulted in failure of corneal epithelium to heal after two weeks, indicating that this is a key model for corneal epithelial injury research ( Figure 8a , flow chart).

[0050] Interestingly, the corneas that received human MSCs delivered by BiFPs had no defects one week after corneal injury, while the corneas that received human MSCs or BiFPs alone still had obvious defects two weeks after injury ( Figure 8b ). Histomorphological analysis showed that compared with the control group (PBS), groups of human MSCs or BiFPs alone, human MSCs delivered by BiFPs significantly improved the healing of corneal injury ( Figure 8c ).

[0051] The results of immunohistochemical staining showed that all newly formed epithelial cells were GFP+, indicating that human MSCs were transplanted and became the new epithelial layer in the damaged cornea ( Figure 8d ). Notably, the newly formed epithelium from human MSCs morphologically resembled normal corneal epithelium, with flattened cells in the upper layer and round cells in the lower layer ( Figure 8d ). These data demonstrate the effectiveness of BiFPs as cell carriers in corneal epithelial regeneration.

[0052] In summary, the present invention first synthesized a class of collagen peptides (CLP) containing the GFOGER amino acid sequence or the GVMGFO amino acid sequence flanked by repeated GPOs on the side, and incorporated them into a targeting peptide of type XII collagen, thereby forming a bifunctional peptide (BiFP). Then, the bifunctional peptide (BiFP) was designed for the development of a cell implant delivery system for regeneration. For example, in cartilage regeneration medicine, the cell implant delivery system can address the unmet needs in injured or degenerative articular cartilage and help repair or regenerate damaged or degenerative joints, forming multi-layered neo-chondrocytes. Similarly, in corneal regeneration medicine, human mesenchymal stem cells (hMSCs) delivered via the cell implant delivery system significantly improved corneal injury healing in a rat model of corneal epithelial injury. Therefore, the cell implant delivery system can also be applied to degenerative intervertebral discs, keratitis or dry eye syndrome, and other diseases related to type XII collagen expression, such as myocardial infarction, burns, and hair loss.

Claims

1. A cell implant delivery system comprising a cell implant and a bifunctional peptide (BiFP) for delivering the cell implant to a target tissue; wherein the bifunctional peptide (BiFP) consists of a tissue targeting sequence and a cell binding motif sequence sandwiched by repeated GPOs.

2. The cell implant delivery system according to claim 1, wherein the tissue targeting sequence is a targeting sequence for osteoarthritis (OA) or degenerative intervertebral discs.

3. The cell implant delivery system according to claim 1, wherein the tissue targeting sequence is a targeting sequence for type XII collagen.

4. The cell implant delivery system according to claim 1, wherein the cell binding sequence is a binding motif for integrin or DDR.

5. The cell implant delivery system according to claim 1, wherein the bifunctional peptide (BiFP) comprises a targeting sequence for type XII collagen, a binding motif sequence for integrin α2β1 or DDR, and at least three or more repeated GPOs sandwiching the integrin or DDR binding motif.

6. The cell implant delivery system according to claim 4, wherein the motif of the integrin is the binding motif of integrin α2β1.

7. The cell implant delivery system according to claim 6, wherein the binding motif of integrin α2β1 has the amino acid sequence GFOGER.

8. The cell implant delivery system according to claim 4, wherein the cell binding motif is the binding motif of DDR.

9. The cell implant delivery system according to claim 8, wherein the binding motif of DDR has the amino acid sequence GVMGFO.

10. The cell implant delivery system according to claim 1, wherein the bifunctional peptide (BiFP) has the amino acid sequence GPOGPOGPOGPOGFOGERGPOGPOGPOGPODLQYWYPIWDTH.

11. The cell implant delivery system according to claim 1, wherein the bifunctional peptide (BiFP) has the amino acid sequence GPOGPOGPOGPOGVMGFOGPOGPOGPOGPODLQYWYPIWDTH.

12. The cell implant delivery system according to claim 1, wherein the cell implant is selected from mesenchymal stem cells (MSCs), progenitor cells or differentiated cells of the musculoskeletal system, and corneal cells or corneal progenitor cells.

13. The cell implant delivery system according to claim 1, wherein the cell implant is an autologous mesenchymal stem cell implant.

14. The cell implant delivery system as described in claim 1, wherein the cell implant is an allogeneic mesenchymal stem cell implant.

15. A method for treating a disease associated with type XII collagen expression, comprising administering to a subject in need thereof a therapeutically effective dose of a cell implant delivered by a bifunctional peptide (BiFP), wherein the bifunctional peptide (BiFP) consists of a tissue targeting sequence and a cell binding motif sequence sandwiched by repeated GPOs.

16. The method as described in claim 15, wherein the disease associated with type XII collagen expression is a musculoskeletal disease, suppurative or aseptic keratitis, dry eye syndrome, heart disease, skin defect, or hair follicle-related disease.

17. The method as described in claim 16, wherein the musculoskeletal disease includes sprains, strains, and tears of ligaments, tendons, muscles, and cartilage, tendinitis, tenosynovitis, fibromyalgia, osteoarthritis, rheumatoid arthritis, disc disease, polymyalgia rheumatica, bursitis, acute and chronic back pain, osteoporosis, carpal tunnel syndrome, de Quervain's disease, trigger finger, tennis elbow, rotator cuff, ganglion cyst, osteogenesis imperfecta, Duchenne muscular dystrophy, Haller's and Hunter's syndromes, and combinations thereof.

18. The method as described in claim 16, wherein the musculoskeletal disease includes osteoarthritis (OA) and intervertebral disc disease (IVDD).

19. The method as described in claim 16, wherein the heart disease is myocardial infarction.

20. The method as described in claim 16, wherein the skin defect is a burn.

21. A cell implant composition for regenerative medicine, comprising a cell implant delivered by a bifunctional peptide (BiFP), wherein the bifunctional peptide (BiFP) consists of a tissue targeting sequence and a cell binding motif sequence sandwiched by repeated GPOs.

22. The cell implant composition as described in claim 21, wherein the cell implant composition can be used to induce new cartilage regeneration in the knee joint of osteoarthritis (OA).

23. The cell implant composition as described in claim 21, wherein the cell implant composition can be used to induce intervertebral disc regeneration in the intervertebral disc of intervertebral disc disease (IVDD).

24. The cell implant composition as described in claim 21, wherein the cell implant composition can be used to induce corneal epithelial regeneration in the corneal defect area.

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