A polypeptide, a nanoprobe targeting new blood vessels of osteoarthritis H type and application

Abstract

This invention belongs to the field of biomedical technology and provides a polypeptide, a nanoprobe, and their applications for targeting H-type angiogenesis in osteoarthritis. The amino acid sequence of the polypeptide is shown in SEQ ID NO: 1. The polypeptide and nanoprobe provided by this invention can specifically recognize H-type angiogenesis and overcome the bone tissue delivery barrier, opening up new pathways for regulating the interaction between the bone and blood vessel microenvironment, and has significant scientific and clinical translational value.

Description

A peptide, nanoprobe, and its application targeting H-type angiogenesis in osteoarthritis. Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a polypeptide, nanoprobe, and application of targeting H-type angiogenesis in osteoarthritis. Background Technology

[0002] Osteoarthritis (OA) is a panarthritis characterized by degeneration of articular cartilage, abnormal remodeling of subchondral bone, osteophyte formation, and synovitis. Its harm is not limited to the joints themselves, but also seriously affects the patient's overall health and quality of life.

[0003] Recent studies have shown that osteoarthritis (OA) is not simply a cartilage lesion, but rather a disease involving the entire joint. Subchondral bone neovascularization plays a crucial driving role in the occurrence and development of OA. In the early stages of OA, abnormal angiogenesis (such as H-shaped vessels) occurs in the subchondral bone region. These neovascularized vessels are incomplete in structure and highly permeable, not only disrupting the normal bone-cartilage interface barrier but also providing pathways for inflammatory cells and various pro-degradative factors (such as matrix metalloproteinases and vascular endothelial growth factor) to invade the cartilage, accelerating cartilage degradation. Simultaneously, they accompany sensory nerve fibers, invading the joint, directly exposing pain nerve endings to a disease environment filled with inflammatory factors and high pressure. This is a key anatomical basis for rest pain and nocturnal pain in OA. Therefore, precisely inhibiting pathological H-shaped neovascularization in the subchondral bone region is considered a promising therapeutic strategy to delay cartilage destruction and modify disease progression.

[0004] While targeting pathological angiogenesis has become an important strategy for treating various diseases, existing treatment strategies are primarily designed to target rapidly proliferating panvascular endothelial cells. For example, strategies targeting the vascular endothelial growth factor / vascular endothelial growth factor receptor (VEGF / VEGFR) signaling pathway are widely used in oncology and other fields. These strategies mainly involve directly inhibiting angiogenesis signals through macromolecular drugs or monoclonal antibodies, thereby blocking the instructions driving angiogenesis. However, these strategies have revealed limitations in the treatment of angiogenesis in osteoarthritis (OA). First, VEGFR plays a crucial physiological role in maintaining systemic vascular homeostasis. Systemic inhibition inevitably leads to serious side effects such as hypertension and proteinuria, which is unacceptable in terms of risk-benefit ratio for OA patients requiring long-term intervention. Second, if intra-articular local administration is used to avoid systemic toxicity, existing monoclonal antibodies and small molecule inhibitors have short intra-articular half-lives and rapid clearance rates, making it difficult to maintain effective drug concentrations. This leads to repeated dosing and increases the risk of infection. Furthermore, non-selective and comprehensive blocking of VEGF signaling may interfere with the physiological repair role of this pathway in chondrocyte survival and endochondral ossification, potentially exacerbating joint structural damage. Another approach involves using RGD peptides (peptides containing the arginine-glycine-aspartic acid sequence) to specifically target integrin (αvβ3) highly expressed on neovascular endothelial cells, delivering conjugated therapeutic drugs to the lesion site. However, the expression of integrin αvβ3 is not unique to pathological vascular endothelial cells; it is also distributed in normal cells such as osteoclasts and activated leukocytes. This leads to insufficient targeting specificity of this strategy, posing a risk of off-target effects, interfering with bone metabolism, or triggering unexpected inflammatory responses. Moreover, H-type neovascularization in osteoarthritis is deeply embedded within dense bone tissue, situated in an exceptionally complex sclerotic microenvironment composed of osteocytes, osteoblasts, osteoclasts, and inflammatory cells. Large molecule drugs or non-targeted agents struggle to effectively penetrate the bone matrix and accumulate around H-type neovascularization, resulting in insufficient drug exposure at the target site and significantly reduced efficacy.

[0005] Therefore, there is an urgent need for a new strategy that can overcome tissue specificity, targeting precision, and delivery efficiency. Summary of the Invention

[0006] Based on this, the present invention aims to provide a polypeptide, nanoprobe, and application for targeting H-type angiogenesis in osteoarthritis. It can specifically identify H-type angiogenesis and overcome the bone tissue delivery barrier, opening up new avenues for regulating the interaction between the bone and blood vessel microenvironment, and has important scientific significance and clinical translational value.

[0007] The technical solution adopted by this invention to achieve its technical objectives is as follows:

[0008] This invention provides a polypeptide that targets H-type angiogenesis in osteoarthritis, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0009] Furthermore, the polypeptide is composed of an α-helical polypeptide, a linker sequence, and a CendR polypeptide linked together by covalent bonds. The amino acid sequence of the α-helical polypeptide is shown in SEQ ID NO: 2, the amino acid sequence of the linker sequence is GSG, and the amino acid sequence of the CendR polypeptide is shown in SEQ ID NO: 3.

[0010] The present invention also provides a lipid nanoprobe (HT-pLNP) for targeting H-type vessel angiogenesis in osteoarthritis, comprising the above-mentioned peptide as well as phospholipids and cholesterol esters.

[0011] Preferably, the phospholipid comprises 1,2-dimyristic-sn-glycerol-3-phosphocholine.

[0012] Preferably, the lipid nanoprobe is loaded with the lipid-soluble optical probe DiR-BOA or the drug mangiferin (MGF).

[0013] Preferably, the lipid nanoprobe has an average particle size of 10-20 nm.

[0014] The present invention further provides the use of the above-mentioned polypeptide or lipid nanoprobe in the preparation of medicaments or diagnostic reagents for the prevention, diagnosis or treatment of osteoarthritis.

[0015] Preferably, the lipid nanoprobe can efficiently target HUVEC cells.

[0016] Preferably, the lipid nanoprobe can penetrate the synovium and cartilage matrix of the joint, reach the deep subchondral bone lesion area, and efficiently target H-type neovascularization in osteoarthritis.

[0017] Preferably, the drug comprises a combination therapy for osteoarthritis, wherein the combination therapy uses a lipid nanoprobe to load MGF drug to effectively maintain the morphology of cartilage tissue, thereby playing a cartilage-protective role and alleviating the progression of osteoarthritis.

[0018] The beneficial effects of this invention are as follows:

[0019] (1) High specificity targeting: The present invention designs a novel polypeptide sequence that can bind to pathological angiogenesis in OA with high affinity and high specificity. At the cellular level, the nanoprobe HT-pLNP of the present invention can be effectively taken up by HUVECs. At the animal level, the HT-pLNP of the present invention can effectively target H-type angiogenesis in the subchondral bone of OA, thereby achieving targeted drug delivery and thus achieving the purpose of treating OA.

[0020] (2) Excellent tissue permeability: The nanoprobe constructed in this invention has a particle size strictly controlled between 10 and 20 nm, which can ensure that the probe can freely penetrate the dense joint synovium and cartilage matrix and reach the subchondral bone lesion area that traditional drugs cannot effectively reach, while avoiding the problem of excessively small particles (less than 10 nm) being quickly cleared.

[0021] (3) Good safety: Treatment is performed by intra-articular injection, avoiding systemic exposure and non-specific effects, and reducing side effects.

[0022] (4) Good biocompatibility: The raw materials for the preparation of this nanoprobe are phospholipids, cholesterol esters and polypeptides, etc. These raw materials have been used in clinical trials and have good biocompatibility.

[0023] (5) The preparation process is simple and easy to scale up production.

[0024] (6) The nanoprobe can selectively encapsulate water-soluble or lipid-soluble drugs according to the specific needs of the experiment, thereby enhancing the therapeutic potential of the drug. Attached Figure Description

[0025] Figure 1. FPLC purification diagram of HT-pLNP.

[0026] Figure 2. HT-pLNP particle size distribution measured using a dynamic light scattering (DLS) system.

[0027] Figure 3. Laser confocalization results of (DiR-BOA)pLNP and (DiR-BOA)HT-pLNP nanoprobes after incubation with HUVEC cells for 1 h.

[0028] Figure 4. Flow cytometry results of (DiR-BOA)pLNP and (DiR-BOA)HT-pLNP nanoprobes after incubation with HUVEC cells for 1 h.

[0029] Figure 5 shows the overall fluorescence imaging results at 12 h, 24 h, 48 h, 72 h, 120 h, and 168 h after intra-articular injection.

[0030] Figure 6. Confocal imaging of frozen sections of the knee joint 24 h after intra-articular injection of (DiR-BOA) HT-pLNP.

[0031] Figure 7. Safranin O-Fix Green staining of the knee joints of the three groups of mice after treatment.

[0032] Figure 8. Statistical chart of OARSI scores of safranin O-fast green stained sections of mice in the three groups after treatment. Detailed Implementation

[0033] The present invention will now be described in detail with reference to specific embodiments. The following specific embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any way.

[0034] Example

[0035] This invention designs a polypeptide capable of targeting H-type angiogenesis in osteoarthritis. This polypeptide is composed of an α-helical polypeptide, a linker sequence, and a CendR polypeptide linked together by covalent bonds. The amino acid sequence of the α-helical polypeptide is DWFKAFYDKVAEKFKEAF (SEQ ID NO: 2), the amino acid sequence of the linker sequence is GSG, the amino acid sequence of the CendR polypeptide is RGERPPR (SEQ ID NO: 3), and the amino acid sequence of the CendR polypeptide is DWFKAFYDKVAEKFKEAFGSGRGERPPR (SEQ ID NO: 1). This invention also constructs a lipid nanoprobe HT-pLNP capable of targeting H-type angiogenesis in osteoarthritis. It is composed of a targeting polypeptide, phospholipids, and cholesterol esters, and can be loaded with the lipid-soluble optical probe DiR-BOA and the drug mangiferin (MGF) as needed. Main components include:

[0036] 1. Synthesis of HT-pLNP nanoprobes:

[0037] 3 μmol of 1,2-dimyristoyl-sn-glycerol-3-phosphocholine (1,2-DIMYRISTOYL-SN-GLYCERO-3-PHOSPHOCHOLINE, DMPC, purchased from Avanti Polar Lipids, catalog number P2663) and 0.2 μmol of cholesterol ester (Cholesteryl oleate, CO, purchased from Sigma-Aldrich, catalog number 151114) were dissolved in 400 μL of chloroform (purchased from Xilong Technology, catalog number 13700901). After mixing, the solution was transferred to a round-bottom flask, and the mouth of the flask was sealed with sealing film. The flask was then placed in a nitrogen evaporator and dried with a stable stream of nitrogen. After drying, a thin film formed on the bottom of the flask. The flask was then placed in a vacuum desiccator and dried at room temperature for 1 h. Then, 1 mL of 1×PBS buffer (pH 1500-10000) was added. 7.4) The dried mixture was thoroughly mixed using a vortex mixer, and the round-bottom flask was placed in a 48°C water bath and sonicated for 1 h. Then, 1 mL of an aqueous solution of a 2 mg / mL peptide targeting H-type angiogenesis in osteoarthritis (DWFKAFYDKVAEKFKEAFGSGRGERPPR) (all peptides mentioned in this application were synthesized by Shanghai Chutai Biotechnology Co., Ltd.) was added to the round-bottom flask, mixed well, sealed, and incubated at 4°C overnight.

[0038] The following day, the overnight mixture was concentrated to 500 μL using a 30 kD centrifuge tube at 4 °C and 2500 rpm. The nanoprobe was then purified using a Hiload Superdex 16 / 600 200 pg column via FPLC. The purification results are shown in Figure 1, indicating that the nanoprobe was successfully synthesized. The solution rich in HT-pLNP nanoprobe was collected for later use.

[0039] The particle size of the collected nanoprobes was measured using a dynamic light scattering (DLS) system. The results are shown in Figure 2. The average particle size was 12 nm, and the particles were uniform and well dispersed.

[0040] 2. In vitro targeting of HT-pLNP nanoprobes:

[0041] To verify the ability of the prepared nanoprobe HT-pLNP to target angiogenesis, the selected fluorescent dye was the lipid-soluble optical probe 1,1'-dioctadecyl-3,3,3',3'-tetramethylin-dotricarbocyanine iodide bisoleate (DiR-BOA). The formulation ratio of 3 μmol DMPC, 0.1 μmol CO, and 0.2 μmol DiR-BOA was dissolved together in 400 μL of chloroform, and the nanoprobe was synthesized according to the synthesis method of HT-pLNP nanoprobe in step 1. In the step of adding peptides, 2 mg of both α-helical peptide and peptide targeting OA angiogenesis were added to construct nanoprobes (DiR-BOA)pLNP and (DiR-BOA)HT-pLNP containing DiR-BOA fluorescent dye, with (DiR-BOA)pLNP as a control.

[0042] To investigate the ability of HT-pLNP to target endothelial cells, we seeded human umbilical vein endothelial cells (HUVECs) in confocal culture dishes, with 1 × 10⁶ cells per dish. 5 Cells were cultured at 37℃ and 5% CO2 (all cell culture conditions were based on this) for 24 h. The nanoprobes (DiR-BOA)pLNP and (DiR-BOA)HT-pLNP were diluted to 10 μM in 400 μL of HUVEC medium (purchased from ScienCell, catalog number 1001) and incubated for 1 h. Confocal imaging was then performed. The results showed that the fluorescence intensity of DiR-BOA in HUVEC cells treated with (DiR-BOA)HT-pLNP was significantly stronger than that of (DiR-BOA)pLNP (Figure 3). To quantitatively determine the ability of HUVEC cells to take up nanoprobes, HUVEC cells were seeded in 96-well plates, with 1 × 10⁶ cells seeded in each well. 4 HUVEC cells were cultured for 24 h, and then the nanoprobes (DiR-BOA)pLNP and (DiR-BOA)HT-pLNP were diluted to the corresponding concentrations using HUVEC medium and incubated for 1 h. Flow cytometry results showed that HUVECs could take up (DiR-BOA)HT-pLNP in a concentration-dependent manner, and the fluorescence signal was stronger than that observed in the (DiR-BOA)pLNP group (Figure 4). This indicates that the nanoprobe HT-pLNP can efficiently target HUVEC cells.

[0043] 3. In vivo targeting evaluation of HT-pLNP nanoprobes:

[0044] To verify the retention time of the nanoprobe in mice and its ability to target angiogenesis in osteoarthritis (OA), an OA model was first established by performing DMM (medial meniscus instability) surgery on 10-week-old C57BL / 6 mice. Four weeks after modeling, 10 μL of the HT-pLNP nanoprobe carrying DiR-BOA dye, synthesized in step 2, was injected intra-articularly at a concentration of 80 μM. Whole-body fluorescence imaging was performed using a small animal in vivo imaging system at different time points: 12 h, 24 h, 48 h, 72 h, 120 h, and 168 h post-injection. The results, shown in Figure 5, indicate that the nanoprobe can remain in the joint cavity for a prolonged period, providing a reliable reference for subsequent treatment.

[0045] To observe the specific distribution of the nanoprobe in the knee joint, 24 h after intra-articular injection of the drug into model mice (DMM) and the same-age sham-operated group (Sham), which had been modeled for 4 weeks, the joints were dissected. The knee joints were fixed with 4% PFA (Paraformaldehyde, purchased from Maclean's, catalog number P804537) for 24 h, and then decalcified with 0.5 M EDTA (ethylenediaminetetraacetic acid, purchased from Biotopped, catalog number E6030G) solution for 3 days. The EDTA solution was changed every 24 h. After decalcification, the joints were transferred to 30% sucrose (purchased from Maclean's, catalog number S6212) solution for dehydration. After the knee joints sank to the bottom, frozen sections and confocal imaging were performed to clarify the targeting ability of (DiR-BOA)HT-pLNP for OA neovascularization. As shown in Figure 6, the results indicate that mice undergoing DMM surgery showed significant angiogenesis 4 weeks after the lesion. Furthermore, DiR-BOA was able to co-localize with CD31 (also known as PECAM-1, platelet endothelial cell adhesion molecule, antibody purchased from Santa Cruz, catalog number sc-376764) and EMCN (Endomucin, endothelial mucin, antibody purchased from Santa Cruz, catalog number sc-65495) double-positive neovascularization in OA, indicating that this nanoprobe can effectively penetrate the synovium and cartilage matrix of joints, reaching the deep subchondral bone lesion area and efficiently targeting H-type neovascularization in OA.

[0046] 4. Therapeutic effects of HT-pLNP nanoprobes:

[0047] MGF (purchased from MCE, catalog number HY-N0290) has good anti-inflammatory and anti-proliferative pharmacological effects. Its proliferative activity can inhibit the abnormal proliferation of vascular endothelial cells, and its anti-inflammatory effect can downregulate VEGF (vascular endothelial growth factor) and other pro-vascular endothelial growth factors. Therefore, it was used as a drug for encapsulation. 3 μmol DMPC, 0.1 μmol CO, and 0.2 μmol MGF were dissolved together in 400 μL chloroform. Following the synthesis method of HT-pLNP nanoprobe in step 1, the MGF-containing nanoprobe MGF@HT-pLNP was synthesized, which can achieve the effect of synergistic targeted combined therapy for OA angiogenesis.

[0048] To verify the therapeutic effect of the nanoprobe MGF@HT-pLNP on OA, we first constructed an OA model by performing DMM surgery on 10-week-old C57BL / 6 mice. Four weeks after modeling, the mice were randomly divided into a Sham group, a DMM group, and an MGF@HT-pLNP treatment group. The mice were administered the drugs once every 7 days. The Sham and DMM groups received intra-articular injections of sterile PBS at a dose of 10 μL per mouse, while the MGF@HT-pLNP treatment group received intra-articular injections of the MGF@HT-pLNP nanoprobe at a dose of 0.5 mg / kg MGF. After 28 days of continuous administration, the knee joints were removed. To evaluate the therapeutic effect of the nanoprobe on OA mice, the knee joints of different treatment groups were stained with Safranin O-Fix Green. The results are shown in Figure 7. In the Sham group, the cartilage thickness was uniform, the cell morphology was clear, and the matrix staining was uniform. In the DMM group, the cartilage was thinner, and the number of superficial chondrocytes decreased or disappeared. In contrast, the MGF@HT-pLNP group had relatively uniform cartilage thickness, intact chondrocyte morphology, and slightly lighter cartilage matrix staining compared to the Sham group. The OARSI scoring criteria of the Osteoarthritis Research Society International (OARSI) were used to score the Safranin O-Fix Green stained sections. The results are shown in Figure 8. Compared with the Sham group, the score of the DMM group was significantly higher, while the score of the MGF@HT-pLNP group was significantly lower than that of the DMM group. The difference was statistically significant, indicating that the nanoprobe loaded with MGF can effectively maintain the morphology of cartilage tissue, play a cartilage-protective role, and thus alleviate the progression of OA.

[0049] Through experimental verification of the scheme, the nanoprobe constructed using the peptides designed in this invention can maintain a stable particle size of about 12 nm, which can penetrate the cartilage matrix and reach the subchondral bone lesion site. It has good HUVEC cell targeting, can remain in the lesion site of OA mice for a long time and efficiently target H-type neovascularization in the subchondral bone of OA. At the same time, by encapsulating the drug MGF, it can effectively alleviate the progression of OA, making it an innovative precision treatment approach.

[0050] Obviously, the above embodiments of the present invention are merely examples to illustrate the present invention more clearly, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is impossible to exhaustively list all implementation methods here. Any obvious variations or modifications derived from the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. A polypeptide targeting H-type angiogenesis in osteoarthritis, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO:

1.

2. The polypeptide according to claim 1, characterized in that, The polypeptide is composed of an α-helical polypeptide, a linker sequence, and a CendR polypeptide linked together by covalent bonds. The amino acid sequence of the α-helical polypeptide is shown in SEQ ID NO: 2, the amino acid sequence of the linker sequence is GSG, and the amino acid sequence of the CendR polypeptide is shown in SEQ ID NO:

3.

3. A lipid nanoprobe targeting H-type angiogenesis in osteoarthritis, characterized in that, It includes the polypeptide of claim 1, as well as phospholipids and cholesterol esters.

4. The lipid nanoprobe according to claim 3, characterized in that, The phospholipids include 1,2-dimyristic-sn-glycerol-3-phosphocholine.

5. The lipid nanoprobe according to claim 3, characterized in that, The lipid nanoprobe is loaded with the lipid-soluble optical probe DiR-BOA or the drug mangiferin MGF.

6. The lipid nanoprobe according to claim 3, characterized in that, The lipid nanoprobes have an average particle size of 10-20 nm.

7. The use of the polypeptide of claim 1 or 2 or the lipid nanoprobe of any one of claims 3 to 6 in the preparation of a medicament for treating osteoarthritis.

8. The application according to claim 7, characterized in that, The lipid nanoprobes can efficiently target HUVEC cells.

9. The application according to claim 7, characterized in that, The lipid nanoprobe can penetrate the synovium and cartilage matrix of the joint, reach the deep subchondral bone lesion area, and efficiently target the H-type neovascularization in osteoarthritis.

10. The application according to claim 7, characterized in that, The drug includes a combination therapy for osteoarthritis, which uses lipid nanoprobes loaded with MGF drugs to effectively maintain the morphology of cartilage tissue, play a cartilage-protective role, and thus alleviate the progression of osteoarthritis.

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