Drug targeting delivery system for targeting joint pathology fibroblast-like synovial cells and application of drug targeting delivery system

Abstract

The invention provides a universal drug targeting delivery system for constructing pathological fibroblast-like synovial cells in osteoarthritis and application of the universal drug targeting delivery system. According to the drug targeting delivery system, vasoactive intestinal peptide is modified to the surface of polydopamine-coated large-aperture calcium ion-doped silicon dioxide; the vasoactive intestinal peptide specifically targets a VPAC1 receptor, and targeted delivery of the nano-drug to pathological fibroblast-like synovial cells in osteoarthritis is successfully realized. The vasoactive intestinal peptide provided by the invention is a polypeptide, has abundant amino groups and carboxyl groups, and can be connected to a drug delivery carrier through a simple chemical reaction, so that efficient targeted delivery of drugs and accumulation of pathological cells are realized.

Description

Technical Field

[0001] The present invention belongs to the field of osteoarthritis treatment and biomedicine technology, and in particular relates to a drug delivery system targeting pathological joint fibroblast-like synoviocytes and an application thereof. Background Art

[0002] Osteoarthritis (OA) is one of the most common degenerative diseases. According to the World Health Organization (WHO), the number of people with OA worldwide reached 528 million in 2019. As the population ages, the number of people with OA is expected to continue to rise. It is predicted that by the end of 2035, the number of people with OA worldwide will increase to 800 million, making OA the most common disabling disease in the world. This will not only significantly affect people's quality of life, but will also bring huge economic burdens to society and individuals.

[0003] In the early stages of osteoarthritis, the disease is mainly manifested by synovial inflammation and mild damage to osteochondral tissue, in which the synovial microenvironment plays an important role. There are two main types of cells in the synovium of the joint: macrophage-like synoviocytes and fibroblast-like synoviocytes (FLS). Macrophages are responsible for engulfing the remains of bacteria and apoptotic cells and maintaining a dynamic balance between pro-inflammatory and anti-inflammatory factors in the synovial fluid. On the other hand, FLS secrete a large amount of long-chain hyaluronic acid, participate in lubrication and immune regulation, and a variety of connective tissue components to maintain the homeostasis of the joint cavity. With the pathological progression of osteoarthritis, fibroblast-like synoviocytes in the synovial microenvironment undergo pathological changes accompanied by vascular invasion. This leads to the infiltration of a large number of T cells, B cells, and macrophages, which together form a vascular cloud, further damaging the adjacent cartilage matrix and subchondral bone. Recent studies have shown that during the progression of osteoarthritis, the Wnt / β-catenin signaling pathway in the joint synovium is overactivated, promoting the secretion of R-spondin 2 by pathological fibroblast-like synoviocytes. These pathological fibroblast-like synoviocytes induce macrophage polarization to the inflammatory M1 phenotype through R-spondin 2-mediated signals and promote the transformation of Sox9-positive osteochondroprogenitor cells to the osteogenic phenotype, promoting the formation of osteophytes in advanced osteoarthritis. In addition, studies have shown that in early osteoarthritis, pathological FLS exacerbate macrophage glycolysis and M1 macrophage polarization by secreting inflammatory exosomes, thereby accelerating the progression of osteoarthritis.

[0004] If effective intervention is not given at this stage, the pathological transformation of fibroblast-like synoviocytes may lead to a shift in the synovial microenvironment to an inflammatory state, thereby promoting the proliferation and infiltration of a large number of immune cells. This will lead to increased secretion of proinflammatory cytokines and proteases, further aggravating synovial inflammation and severely damaging the osteochondral matrix, ultimately leading to extensive cartilage damage and even joint deformities in patients. At present, the clinical treatment strategies for early osteoarthritis mainly focus on anti-inflammatory measures and pain relief, including the use of oral analgesics, nonsteroidal anti-inflammatory drugs, specific cyclooxygenase inhibitors, or intra-articular injections of corticosteroids. However, oral medications usually require high doses to achieve the desired therapeutic effect, and long-term overdose in conditions such as chronic arthritis may lead to a series of complications, including osteoporosis and diabetes. In addition, the unique tissue structure of the bone-joint region lacks blood vessels, nerves, and lymphatic tissues, and the frequent exchange of synovial fluid limits the absorption of injected drugs. Overall, the current clinical treatment methods for early osteoarthritis are mainly non-specific symptomatic treatments, and lack therapeutic targets for blocking the early pathological process of osteoarthritis, so it is impossible to effectively intervene in the pathological progression of the disease.

[0005] Patent document CN118178353A discloses a biomimetic nano drug delivery carrier targeting chondrocytes and its preparation method and application. A chondrocyte membrane-modified nanocarrier is developed, with high molecular polymer nanoparticles capable of loading drugs as the core, and chondrocyte membrane is coated on its surface to obtain a delivery carrier. The obtained carrier has the ability to promote chondrocyte uptake in a targeted manner for long-term treatment of osteoarthritis. However, the target of the drug delivery system in this application is chondrocytes, and for the blocking of early osteoarthritis, fibroblast-like synoviocytes are earlier and more suitable therapeutic targets. At the same time, chondrocyte membranes are used as drug carriers, and multiple steps and multiple reagents are involved in the extraction of chondrocyte membranes and the preparation of carriers, which also increases the complexity and difficulty of preparation and production costs.

[0006] Therefore, providing a technology that can effectively target and block osteoarthritis, especially effectively intervene in the further lesions of pathological fibroblast-like synoviocytes, is of great significance for effectively intervening in the pathological progression of osteoarthritis. Summary of the invention

[0007] In view of the problems existing in the prior art, the present invention provides a drug targeted delivery system targeting pathological fibroblast-like synoviocytes in osteoarthritis, and a preparation method and application thereof. The drug targeted delivery system successfully achieves the targeted accumulation of nanocarriers in osteoarthritis synovial tissue and pathological fibroblast-like synoviocytes and targeted drug delivery by modifying vasoactive intestinal peptide onto the surface of polydopamine-coated large-pore calcium ion-doped silica.

[0008] In order to achieve the above objectives, the present invention adopts the following technical solutions:

[0009] In one aspect, the present invention provides a drug targeting delivery system targeting pathological fibroblast-like synoviocytes, comprising vasoactive intestinal peptide and a drug delivery carrier, wherein the vasoactive intestinal peptide specifically targets pathological fibroblast-like synoviocytes.

[0010] The research on drug delivery systems for the treatment of osteoarthritis in the prior art mainly targets bone and joint chondrocytes or tissues, macrophages, and lacks targeted delivery technology for pathological fibroblast-like synoviocytes in osteoarthritis. However, the pathological transformation of fibroblast-like synoviocytes is a key pathological event in the early stage of osteoarthritis, which may lead to increased secretion of proinflammatory cytokines and proteases, promote inflammatory response of the synovium, and aggravate the degradation of cartilage. Therefore, the construction of a pathological fibroblast-like synoviocyte drug targeted delivery system can more effectively intervene in the pathological progression of bone and joints. Therefore, the present invention targets pathological fibroblast-like synoviocytes and studies drug delivery systems that can effectively target pathological fibroblast-like synoviocytes. The present invention first analyzes the gene maps of bone and joint patients and healthy synovial tissues through the GEO database, and finds that the expression of VPAC1 in the synovial tissue of osteoarthritis patients is significantly increased compared to healthy synovial tissues. Through research on the literature, it was found that VPAC1 is a specific receptor of vasoactive intestinal peptide in bone and joint diseases. Therefore, the present invention speculates that vasoactive intestinal peptide can achieve targeted delivery to pathological fibroblast-like synoviocytes in OA by targeting its receptor VPAC1. In some embodiments, the present invention verifies the specific expression of VPAC1 in pathological fibroblast-like synoviocytes of osteoarthritis. The results showed that the expression level of VPAC1 in fibroblast-like synoviocytes of healthy mice was very low, while the expression in pathological fibroblast-like synoviocytes was significantly increased, and VPAC1 was highly expressed only in the synovial tissue of OA mice. At the same time, VPAC1 maintains continuous high expression during the development of OA. Further, the expression verification results of synovial tissue of osteoarthritis patients were consistent with those in mouse tissues.

[0011] Therefore, the present invention innovatively proposes a method for constructing a drug delivery system targeting pathological fibroblast-like synoviocytes in OA based on vasoactive intestinal peptide, and achieves targeted accumulation of drug carriers in pathological fibroblast-like synoviocytes and drug delivery by modifying the polypeptide onto the surface of drug delivery carriers, thereby achieving targeted treatment of early osteoarthritis.

[0012] In some embodiments of the present invention, it is also verified by in vitro experiments and in vivo experiments that vasoactive intestinal peptide modification can improve the targeting ability of nanomedicines to pathological fibroblast-like synoviocytes of osteoarthritis. The results of in vitro experiments show that vasoactive intestinal peptide modification can significantly enhance the selective phagocytosis ability of pathological fibroblast-like synoviocytes of osteoarthritis to nanomedicines; the results of in vivo experiments show that vasoactive intestinal peptide modification can effectively achieve the targeted accumulation of nanocarriers to pathological fibroblast-like synoviocytes in mouse synovial tissue, and also significantly prolong the retention ability of nanomedicine carriers in bone joints, indicating that nanomedicine carriers constructed based on vasoactive intestinal peptide can effectively achieve targeted delivery of drugs to pathological fibroblast-like synoviocytes.

[0013] Furthermore, the drug delivery carrier includes any one or more of an inorganic drug carrier and an organic drug carrier.

[0014] In some embodiments, the drug delivery carrier is an inorganic nanocarrier, including but not limited to silica, liposomes, etc., and the vasoactive intestinal peptide is modified onto the surface of the inorganic nanocarrier.

[0015] Drug delivery carriers are key components of targeted drug delivery systems. Selecting a suitable drug carrier requires consideration of many factors, including the physicochemical properties of the drug, the delivery route, targeting, and the therapeutic goal. Therefore, in an embodiment of the present invention, since the loaded drug is a macromolecular protein, namely, catalase and superoxide dismutase, when this implementation method uses large-pore calcium ion-doped silica (Ca-MSN) as a drug carrier, it can effectively improve the targeting ability and therapeutic effect of the drug delivery system. On the one hand, calcium ion doping enhances the affinity of the carrier for protein drugs, and on the other hand, the large pore size of the carrier can meet the loading of the drug.

[0016] In some embodiments, the present invention verifies through experiments that only by using vasoactive intestinal peptide to modify drug delivery carriers can the targeting effect and therapeutic effect of drugs be improved. The results show that the drug delivery system constructed by using polyethyleneimine (PEI) to positively charge the drug delivery carrier is mainly accumulated in cartilage tissue, while after the drug delivery carrier is modified with vasoactive intestinal peptide, the drug is mainly accumulated in synovial cells. The main reason is that vasoactive intestinal peptide interacts with the VPAC1 receptor in the synovial tissue, thereby significantly improving the targeting of the drug delivery carrier to the synovial tissue.

[0017] In some embodiments, polydopamine is encapsulated on the surface of the Ca-MSN carrier, and the vasoactive intestinal peptide is modified on the surface of the polydopamine.

[0018] Because before vasoactive intestinal peptide modifies the Ca-MSN carrier, the Ca-MSN carrier needs to be modified or modified before the vasoactive intestinal peptide can be bound to the Ca-MSN carrier. Because the amino group and phenolic hydroxyl group on the surface of polydopamine can react with the amino group and carboxyl group of vasoactive intestinal peptide in a weakly alkaline environment, the grafting of vasoactive intestinal peptide is achieved. In addition, the drug loading performance of the Ca-MSN carrier is good, the drug loading capacity and encapsulation rate are high, and there is also good biocompatibility between the target. The present invention creatively uses polydopamine to encapsulate Ca-MSN. On the one hand, it utilizes the good biocompatibility and biodegradability of polydopamine. In drug delivery, polydopamine is used as a carrier material to reduce the risk of toxicity and immune response. On the other hand, polydopamine has phenolic hydroxyl groups and amino groups. In a weakly alkaline aqueous phase, it can form oxidized dopamine quinone through redox reaction, thereby completing the polymerization process, and then forming a thin film to enhance the adhesion between the carrier and other molecules. Further combining vasoactive intestinal peptide with the polydopamine molecule can achieve targeted delivery of the drug carrier to pathological fibroblast-like synoviocytes, further improving the targeting specificity of the drug carrier.

[0019] Furthermore, the drug targeted delivery system also includes osteoarthritis therapeutic drugs, and the drugs include any one or more of small molecule compounds, nucleic acid drugs, and macromolecular protein drugs.

[0020] In some embodiments, the small molecule drugs include but are not limited to non-steroidal anti-inflammatory drugs, steroid drugs, and analgesics; the nucleic acid drugs include but are not limited to siRNA and mRNA; the macromolecular protein drugs include but are not limited to cartilage proteoglycan antibodies and matrix metalloproteinase inhibitors.

[0021] Furthermore, the mass ratio of the vasoactive intestinal peptide to the drug delivery carrier is 0.5:1.

[0022] In some embodiments, the mass ratio of vasoactive intestinal peptide, silicon dioxide and drug (superoxide dismutase and catalase) is 1:2:2:1.

[0023] In yet another aspect, the present invention provides a drug for targeted treatment of osteoarthritis, comprising the drug targeted delivery system as described above.

[0024] It is understandable that the drug targeted delivery system provided by the present invention may also include various pharmaceutical ingredients such as wetting agents, solubilizers, osmotic pressure regulators, coating materials, colorants, pH regulators, antioxidants, antibacterial agents or buffers to prepare various drugs or pharmaceutical products within the scope of not damaging the effects of the present invention.

[0025] In another aspect, the present invention provides a method for preparing a drug targeted delivery system, wherein vasoactive intestinal peptide is modified onto the surface of a drug delivery carrier.

[0026] Furthermore, the method comprises the following steps:

[0027] S1. Drug loading on silica carrier;

[0028] S2. Polydopamine encapsulated silica;

[0029] S3. Vasoactive intestinal peptide modified drug carrier.

[0030] Furthermore, the drug in step S1 includes any one or more of small molecule compounds, nucleic acid drugs, and macromolecular protein drugs.

[0031] In some embodiments, in step S2 and step S3, a Tris-HCl buffer with a pH of 8.5 and a concentration of 10 mM is used to provide a weakly alkaline reaction environment for the self-polymerization of dopamine and the grafting of vasoactive intestinal peptide.

[0032] In yet another aspect, the present invention provides use of vasoactive intestinal peptide for preparing a targeting ligand for a drug delivery system specifically targeting pathological fibroblast-like synoviocytes in osteoarthritis.

[0033] The present invention has the following beneficial effects:

[0034] 1. The drug targeted delivery system provided by the present invention has strong targeting: vasoactive intestinal peptide can enhance the targeting of drugs by specifically binding to the receptor VIPAC1 on pathological fibroblast-like synoviocytes. This targeted delivery can effectively increase the concentration of drugs in diseased tissues and reduce the impact on healthy tissues.

[0035] 2. The vasoactive intestinal peptide provided by the present invention has good biocompatibility and biodegradability, can effectively reduce the immune response and toxicity during drug use, and is suitable for in vivo application.

[0036] 3. The vasoactive intestinal peptide provided by the present invention is rich in amino and carboxyl groups, providing a variety of chemical modification possibilities. The polypeptide can be connected to a nano-drug or a nano-delivery platform through a simple chemical reaction to achieve targeted functionalization of the nano-drug or the nano-delivery platform.

[0037] 4. Enhance drug stability: By combining drugs with peptides, the stability of drugs in the body can be improved, preventing rapid metabolism and clearance of drugs, thereby extending the biological half-life of drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to more clearly illustrate the technical solutions of the exemplary embodiments of the present invention, the following is a brief introduction to the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and should not be regarded as limiting the scope. For those of ordinary skill in the art, other relevant drawings can be obtained based on these drawings without creative work.

[0039] Figure 1 This is the tissue-specific expression of VPAC1 in pathological fibroblast-like synoviocytes of osteoarthritis of the present invention. The data of the bar graph in Figure a are expressed as mean ± standard deviation (SD), and one-way ANOVA (one-way ANOVA) is used. "*" indicates p < 0.05, and p < 0.05 is considered to be statistically significant; circles are used to intuitively display the distribution characteristics of each group of data.

[0040] Figure 2 This is to verify in vitro the targeting ability of the nanomedicine before and after vasoactive intestinal peptide modification on pathological FLS.

[0041] Figure 3 This is the in vivo verification of the retention capacity of the nanodrug in bone joints before and after vasoactive intestinal peptide modification in the present invention. The data of the bar graph in Figure b are expressed as mean ± standard deviation (SD), and one-way ANOVA (one-way ANOVA) is used. "**" indicates p < 0.01, "***" indicates p < 0.001, and "****" indicates p < 0.0001. p < 0.05 is considered to be statistically significant; circles are used to intuitively display the distribution characteristics of each group of data.

[0042] Figure 4 This is to verify in vivo the targeting ability of the nanomedicine before and after vasoactive intestinal peptide modification on pathological FLS.

[0043] Figure 5 : The fluorescence intensity values ​​of the nanomedicine in the synovial tissue before and after modification with vasoactive intestinal peptide of the present invention, wherein the data in the bar graph are expressed as mean ± standard deviation (SD), and one-way ANOVA (one-way ANOVA) is used, "*" indicates p < 0.05, "**" indicates p < 0.01, and "***" indicates p < 0.001, and p < 0.05 is considered to be statistically significant, and circles are used to intuitively display the distribution characteristics of each group of data.

[0044] Figure 6The targeting ability of the drug delivery system with different targeting modes of the present invention in osteoarthritis, wherein the data of the bar graph in Figure b are expressed as mean ± standard deviation (SD), and one-way ANOVA is used, "****" indicates p < 0.0001, and p < 0.05 is considered to be statistically significant; the circles are used to intuitively display the distribution characteristics of each group of data.

[0045] Figure 7 This is a verification of the therapeutic effect of the vasoactive intestinal peptide-modified nanodrug on osteoarthritis. The data in the bar graphs in Figures b, d, and f are expressed as mean ± standard deviation (SD). One-way ANOVA was used, "*" indicates p < 0.05, "**" indicates p < 0.01, "***" indicates p < 0.001, and "****" indicates p < 0.0001. p < 0.05 is considered to be statistically significant; circles are used to intuitively display the distribution characteristics of each group of data. DETAILED DESCRIPTION

[0046] In order to make the above-mentioned objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments and drawings. The exemplary implementation modes of the present invention and their description are only used to explain the present invention and are not intended to limit the present invention.

[0047] Unless otherwise specified, the raw materials used in the examples were purchased commercially.

[0048] Example 1: Preparation of the drug targeted delivery system provided by the present invention

[0049] 1. Hexadecyltrimethylammonium bromide (1.05 g, 2.73 mmol) and 3.5 mL of sodium hydroxide solution (2 M) were mixed in 240 mL of ddH 2 O and mixed evenly. After stirring at 80 °C for 40 min, the solution turned transparent brown.

[0050] 2. Slowly inject 5 mL of ethyl silicate (22.4 mmol) into the mixed solution (the tip of the pipette should be inserted into the liquid surface) at 1 mL / min, and stir at 150 rpm for 2.5 h at 80 °C;

[0051] 3. After centrifugation, retain the precipitate and wash with plenty of water (12000 rpm, 4℃, 8 min, centrifuge and wash 3 times);

[0052] 4. After measuring the solution concentration, the mesoporous silica (MSN) precursor was centrifuged and washed twice with methanol;

[0053] 5. Disperse 0.5 g of MSN into 100 mL of methanol solution; after uniform dispersion, add 2.3614 g of Ca(NO 3 ) 2 ·4H 2 O;

[0054] 6. After mixing evenly, stir and react at 50°C for 48 h;

[0055] 7. Centrifuge while hot to obtain the precipitate, then rinse with methanol and ddH 2 O were washed twice with water (12000 rpm, 4 °C, 8 min) to obtain Ca-MSNs;

[0056] 8. After superoxide dismutase (SOD), catalase (CAT) and large-pore calcium ion-doped silica (Ca-MSN) were stirred at room temperature for 24 hours, SOD and CAT were loaded into Ca-MSN to prepare SOD / CAT@Ca-MSN. The prepared SOD / CAT@Ca-MSN was evenly dispersed in 10mM Tris-HCl buffer (pH=8.5) to make the final concentration of SOD / CAT@Ca-MSN 1 mg / mL.

[0057] 9. Add dopamine hydrochloride (PDA) to the solution in step 8 to make the final concentration of dopamine hydrochloride 0.5 mg / mL, stir at room temperature in the dark for 24 h to obtain the nanodrug SOD / CAT@Ca-MSN-PDA (S / C@CP).

[0058] 10. Wash twice by centrifugation with water and ethanol (12000rpm, 4℃, 10 min);

[0059] 11. The obtained S / C@CP was placed in 10 mM Tris-HCl buffer (pH = 8.5) containing 1 mg / mL vasoactive intestinal peptide and stirred at 4°C in the dark for 240 min;

[0060] 12. Centrifuge (12000rpm, 4℃, 10 min) and wash with water three times to obtain the drug targeted delivery system S / C@CPV.

[0061] Example 2: VPAC1 is highly expressed in pathological FLS of osteoarthritis

[0062] In order to verify the high expression of VPAC1, a receptor of vasoactive intestinal peptide, in osteoarthritis pathological fibroblast-like synoviocytes (OA-FLS), fibroblast-like synoviocytes from healthy rats were collected in this example. According to the literature, 20ng / mL IL-1β was used to induce osteoarthritis pathological fibroblast-like synoviocytes. Cell proteins were extracted and the expression of VPAC1 in different fibroblast-like synoviocytes was detected by immunoblotting. At the same time, synovial tissues, cartilage tissues of healthy mice and OA mice and OA mouse joint samples at different time points were collected, and the expression of VPAC1 in synovial tissues and cartilage tissues was detected by immunohistochemical staining; finally, synovial tissues of osteoarthritis patients were collected, and further verification was performed by fixation, dehydration, transparency, paraffin embedding, sectioning, and immunohistochemical staining.

[0063] like Figure 1 As shown in Figure 2, VPAC1 expression is very low in healthy fibroblast-like synoviocytes, while VPAC1 expression is significantly increased in diseased fibroblast-like synoviocytes ( Figure 1 VPAC1 was highly expressed only in the synovial tissue of OA mice ( Figure 1 b). At the same time, it was found that VPAC1 maintained a continuous high expression during OA progression ( Figure 1 c). Further verification of the expression in synovial tissues of osteoarthritis patients was consistent with that in mouse tissues, with VPAC1 being highly expressed in OA synovial tissues ( Figure 1 d) in.

[0064] Example 3: In vitro evaluation of the targeting ability of nanomedicines before and after vasoactive intestinal peptide modification to pathological FLS

[0065] In order to further verify the selective phagocytic ability of fibroblast-like synoviocytes to nanomedicines before and after vasoactive intestinal peptide modification, the S / C@CP nanomedicine (not modified with vasoactive intestinal peptide) in Example 1 was used as a control in this example, and the drug targeted delivery system S / C@CPV and S / C@CP nanomedicine prepared in Example 1 were grafted with Cy5-NHS fluorescent groups, respectively. First, 10 mg of S / C@CP was placed in 2 mL of 10 mM Tris-HCl buffer (pH = 8.5) containing 0.5 mg / mL Cy5, stirred at room temperature in the dark for 12 h, and centrifuged and washed with water three times to obtain S / C@CP-Cy5; for S / C@CPV-Cy5, the obtained S / C@CP-Cy5 was placed in 10 mM Tris-HCl buffer (pH = 8.5) containing 1 mg / mL vasoactive intestinal peptide, stirred at 4°C in the dark for 240 min, centrifuged at 12000 rpm at 4°C for 10 min, and washed with water three times to obtain S / C@CPV-Cy5.

[0066] Fibroblast-like synoviocytes from healthy rats and pathological fibroblast-like synoviocytes from osteoarthritis were soaked in 75% ethanol for 12 hours and then sterilized. Then, high-glucose DMEM complete medium containing 25μg / mL S / C@CP-Cy5 and S / C@CPV-Cy5 was co-cultured with the two cells for 12 hours, and then rinsed with sterile PBS three times to wash away excess nanomedicines, and then fixed to observe the cell's selective phagocytic ability of drugs by immunofluorescence. Among them, Cy5 labeled nanomedicines, and Vimentin was the identification marker of fibroblast-like synoviocytes.

[0067] The experimental results are as follows Figure 2 As shown, compared with the control group, the Cy5-labeled fluorescence signal was significantly enhanced after OA-FLS cells were treated with vasoactive intestinal peptide-modified nanomedicines, indicating that vasoactive intestinal peptide modification can significantly increase the selective phagocytic ability of osteoarthritis pathological fibroblast-like synoviocytes for nanomedicines, but has little effect on the selective phagocytic ability of healthy pathological fibroblast-like synoviocytes for nanomedicines.

[0068] Example 4: In vivo evaluation of the effect of vasoactive intestinal peptide modification on the retention capacity of nanodrugs in bone and joints

[0069] In order to further evaluate the effect of vasoactive intestinal peptide modification on the retention ability of nanodrugs in bone joints, osteoarthritis mice were used as animal models in this example, and the drug targeted delivery system S / C@CPV and S / C@CP nanodrugs prepared in Example 1 were grafted with Cy5-NHS fluorescent groups, respectively. First, 10 mg of S / C@CP was placed in 2 mL of 10 mM Tris-HCl buffer (pH=8.5) containing 0.5 mg / mL Cy5, stirred at room temperature in the dark for 12 h, and centrifuged and washed with water three times to obtain S / C@CP-Cy5; for S / C@CPV-Cy5, the obtained S / C@CP-Cy5 was placed in 10 mM Tris-HCl buffer (pH=8.5) containing 1 mg / mL vasoactive intestinal peptide, stirred at 4°C in the dark for 240 min, centrifuged at 12000 rpm at 4°C for 10 min, and washed with water three times to obtain S / C@CPV-Cy5. Then, 25 μg of S / C@CP-Cy5 and S / C@CPV-Cy5 were injected into the joints of osteoarthritis mice, respectively, and the retention ability of the nanodrug was indicated by Cy5 labeling and in vivo fluorescence imaging of small animals.

[0070] like Figure 3 As shown, compared with the control group, the nanomedicine in the S / C@CPV group of mice still had fluorescence signals on the 14th day ( Figure 3 The fluorescence intensity value retained in the S / C@CPV group mice on day 14 was significantly higher than that in the control group ( Figure 3 (b) shows that vasoactive intestinal peptide modification can significantly prolong the joint retention ability of nanomedicine.

[0071] Example 5: In vivo evaluation of the targeting ability of nanomedicines before and after vasoactive intestinal peptide modification to pathological FLS

[0072] This example further evaluates the targeting ability of the nanodrug before and after vasoactive intestinal peptide modification to pathological FLS through in vivo experiments. Using the same immunofluorescence method as in Example 3, vimentin and VPAC1 were used to co-localize pathological FLS in the synovial tissue of OA mice, and the targeting ability of the nanodrug before and after vasoactive intestinal peptide modification to pathological FLS in osteoarthritis mice was evaluated by immunofluorescence staining.

[0073] The results show that ( Figure 4 ), compared with the control group without vasoactive intestinal peptide modification, the nanodrugs modified with vasoactive intestinal peptide showed Cy5 fluorescence reaction on the 14th day, and the fluorescence signal of Cy5 was highly consistent with Vimentin (green) and VPAC1 (red); then, the fluorescence intensity of different delivery platforms was statistically analyzed by Image J software, and the statistical results are shown in the figure. Figure 5 As shown, compared with the unmodified vasoactive intestinal peptide delivery platform, the fluorescence intensity (synovial tissue) of the vasoactive intestinal peptide-modified delivery platform on the 14th day was significantly improved, indicating that the vasoactive intestinal peptide modification can effectively achieve the targeted accumulation of nanodrug S / C@CP in pathological fibroblast-like synoviocytes.

[0074] Example 6: Screening of nanomedicine targeting pathological FLS

[0075] In the drug delivery system, the targeting mode of the carrier loaded with nano drugs will affect the targeting ability of the drug, the therapeutic effect, etc. In order to further verify the necessity of the large-pore calcium ion-doped silica (Ca-MSN-PDA) drug carrier wrapped in VIP peptide modification dopamine, this example compares the method of charge modification carrier (unmodified VIP peptide). Since negatively charged carriers are generally difficult to bind to cell membranes, positively charged carriers can more easily bind to cell membranes through electrostatic attraction, promoting cell uptake. Therefore, this example uses polyethyleneimine (PEI) to positively charge the Ca-MSN-PDA carrier to further construct the drug delivery system Ca-MSN-PDA-PEI, and the remaining methods and steps are the same as in Example 1. Afterwards, the two nano delivery platforms were labeled with the fluorescent group Cy5-NHS according to the method of Example 3, and the joint tissue was collected 14 days after joint injection for section staining. By comparing the fluorescence signals of different tissue parts, the targeting ability of nano drugs in osteoarthritis with two targeting methods was compared.

[0076] The experimental results are as follows Figure 6 As shown, the nano-delivery platform using PEI positive charge mainly accumulates in cartilage tissue. This is because cartilage tissue is a highly negatively charged tissue in the joints. Therefore, through the interaction between positive and negative charges, Ca-MSN-PDA-PEI accumulates in cartilage tissue. When Ca-MSN-PDA is modified with VIP peptide, the nano-drug is mainly targeted and accumulated in synovial cells. Therefore, the modification method using VIP peptide can effectively improve the targeting ability of nano-drugs to synovial cells.

[0077] Example 7: Targeted therapeutic effect of vasoactive intestinal peptide-modified nanomedicine on osteoarthritis

[0078] Due to the coating of polydopamine, the nanodrug has good photothermal therapy (NIR). Therefore, the present invention further evaluates the therapeutic effect of vasoactive intestinal peptide-modified nanodrug combined with photothermal therapy on osteoarthritis. The drug targeted delivery system S / C@CPV prepared in Example 1 was used to treat osteoarthritis mice by photothermal therapy. Then, the joint tissue was stained with histological safranin fast green ( Figure 7 a in the figure), Osteoarthritis Score International (OARSI) and Synovial Score ( Figure 7 b). The results showed that the combination therapy based on VIP peptide, SOD / CAT and photothermal therapy effectively achieved the treatment of early synovial inflammation ( Figure 7 a and b in the figure); and, the secretion of MMP13 and ADAMTS5 in the synovium was evaluated by immunohistochemical staining of MMP13 and ADAMTS5 in joint tissues. The results showed that after S / C@CPV combined with photothermal therapy was used to treat mice, MMP13 in the tissues ( Figure 7 c and d), ADAMTS5 ( Figure 7 e and f) secretion was inhibited, indicating that based on the targeting effect of vasoactive intestinal peptide, early precise targeting of SOD / CAT and combined photothermal therapy for the treatment of early osteoarthritis were achieved.

[0079] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A drug targeted delivery system targeting pathological fibroblast-like synoviocytes, characterized in that: The invention comprises vasoactive intestinal peptide and a drug delivery carrier, wherein the vasoactive intestinal peptide specifically targets pathological fibroblast-like synoviocytes.

2. The drug targeted delivery system according to claim 1, characterized in that The drug delivery carrier includes any one or more of an organic nanocarrier and an inorganic nanocarrier.

3. The drug targeted delivery system according to claim 2, characterized in that: The drug delivery carrier is an inorganic nanocarrier, and the vasoactive intestinal peptide is modified on the surface of the inorganic nanocarrier.

4. The drug targeted delivery system according to claim 1, characterized in that It also includes drugs for treating osteoarthritis, which include any one or more of small molecule compounds, nucleic acid drugs, and macromolecular proteins.

5. The drug targeted delivery system according to claim 4, characterized in that: The mass ratio of the vasoactive intestinal peptide to the drug delivery carrier is 0.5:

1.

6. A drug for targeted treatment of osteoarthritis, characterized in that: The invention comprises a drug targeted delivery system as described in any one of claims 1 to 5.

7. The method for preparing the drug targeted delivery system according to any one of claims 1 to 5, characterized in that: Vasoactive intestinal peptide was modified onto the surface of drug delivery carrier.

8. The preparation method according to claim 7, characterized in that: The following steps are involved: S1. Drug loading on silica carrier; S2. Polydopamine encapsulated silica; S3. Vasoactive intestinal peptide modified drug delivery vector.

9. The preparation method according to claim 8, characterized in that: The drug in step (1) includes any one or more of small molecule compounds, nucleic acid drugs, and macromolecular proteins.

10. Use of vasoactive intestinal peptide for preparing a targeting ligand of a drug targeting delivery system specifically targeting pathological fibroblast-like synoviocytes, characterized in that: The drug targeted delivery system is as described in any one of claims 1-5.

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