Application of integrin targeting peptide-collagenase medicine composition and medicine composition constructed by integrin targeting peptide-collagenase medicine composition

Abstract

The invention discloses application of an integrin targeting peptide-collagenase medicine composition and a medicine composition constructed by the integrin targeting peptide-collagenase medicine composition. The pharmaceutical composition can effectively block mechanical signals and biochemical signals mediating fibrosis, efficiently inhibit fibroblast activation, reduce secretion of extracellular matrix protein and remodel a fibrosis microenvironment. The fibrosis can be efficiently relieved, the permeation of subsequent treatment drugs can be promoted, and the cascade permeation promotion effect is achieved. The constructed pharmaceutical composition can synergistically deliver integrin targeting peptide and collagenase, and after being released, the integrin targeting peptide and collagenase play a synergistic role in fibrosis lesions in a free molecule form, and a new strategy is provided for efficient delivery of fibrosis treatment drugs.

Description

Technical Field

[0001] The present invention relates to an application of an integrin targeting peptide-collagenase drug combination and a drug composition constructed therefrom, and in particular to an application of an integrin targeting peptide-collagenase drug combination with synergistic therapeutic efficacy for fibrosis and a drug composition constructed therefrom. Background Art

[0002] Fibrosis diseases such as liver fibrosis and pulmonary fibrosis, as well as tumors with fibrotic symptoms such as pancreatic cancer and bladder cancer, are closely related to the continuous activation of fibroblasts. After fibroblasts are activated into myofibroblasts, they secrete a large amount of extracellular matrix (ECM) components mainly composed of collagen and fibronectin, which leads to excessive deposition and cross-linking of ECM, promoting the progression of fibrosis.

[0003] The signaling pathways that cause sustained activation of fibroblasts are mainly divided into two categories: mechanical signaling pathways mediated by ECM rigidity transmitted by cell surface β1 subunit integrin, and biochemical signaling pathways mediated by factors such as TGF-β. At present, anti-fibrosis strategies for inhibiting fibroblast activation mainly focus on single-blocking biochemical signaling pathways or single-blocking mechanical signaling pathways, such as TGF-β1 receptor inhibitors, PI3K / Akt signaling pathway inhibitors, Smad signaling pathway inhibitors, MAPK signaling pathway inhibitors, Rho kinase (ROCK) inhibitors, extracellular matrix (ECM) remodeling inhibitors, integrin inhibitors, etc.

[0004] In addition, CN118217246 modifies RGD peptide on secondary nanoparticles and delivers them together with collagenase. The RGD peptide mainly acts as a target head to specifically target damaged type II alveolar epithelial cells, promoting the effective internalization of the preparation. US20210059936 modifies the RGD peptide on the surface of liposomes as a target head for delivering collagenase and increasing the targeting of collagenase. All of the above use integrin targeting peptides (such as RGD cyclic peptides) to modify microparticle preparations to target the delivery of collagenase to fibrotic lesions, achieving stronger lesion accumulation or cellular uptake, and its mechanism of action is still limited to the local degradation of collagenase. Summary of the invention

[0005] Purpose of the invention: The first purpose of the present invention is to provide an integrin targeting peptide-collagenase drug combination with synergistic effect for use in the preparation of dual-pathway high-efficiency drugs for the treatment of fibrosis, and the second purpose is to provide a pharmaceutical composition constructed by the drug combination.

[0006] Technical solution: The use of the integrin targeting peptide-collagenase drug combination of the present invention in the preparation of a drug for treating fibrosis, wherein the molar ratio of the integrin targeting peptide to the collagenase is (500-1000):1.

[0007] The drug combination designed by the present invention provides an anti-fibrosis strategy of dual-pathway synergistic inhibition. On the one hand, collagenase is used to cut type I and type III collagen into short peptides to weaken the rigidity of ECM, and collagenase is also used to cut the recognition sequence of integrin (α1β1, α2β1, α10β1 and α11β1) on type I and III collagen, further reducing the mechanical sensing between integrin and collagen, and blocking the mechanical transduction between collagen (type I and type III) and integrin.

[0008] On the other hand, the integrin targeting peptide is released in the form of a single molecule, binds to the integrin receptor through the RGD sequence, and blocks the mechanical transduction between fibronectin and integrin by competing with fibronectin for integrin (α5β1, α8β1, αvβ1, and αIIbβ1). In addition, the released integrin targeting peptide can also bind to αvβ6 and αvβ8 integrins on the surface of fibroblasts and tumor cells through the RGD sequence, reducing the release of active TGF-β, thereby reducing the activation of fibroblasts mediated by biochemical signaling pathways.

[0009] The RGD sequence is the core motif for integrin recognition and binding, and can specifically interact with the ligand binding sites of integrins (such as αvβ3, αvβ5, α5β1, etc.). Therefore, peptides containing the RGD sequence have the potential to bind to integrins. Overall, the collagenase and integrin targeting peptides in the drug combination effectively inhibit fibroblast activation, reduce ECM protein secretion, block the positive feedback loop between ECM rigidity and fibroblast activation, and reshape the fibrotic microenvironment by blocking the mechanical and biochemical signal transduction that mediates fibrosis.

[0010] Preferably, the molar ratio of the integrin targeting peptide to collagenase is (500-850):1.

[0011] Preferably, the integrin targeting peptide contains an RGD sequence.

[0012] Further preferably, the integrin targeting peptide is selected from GRGDSP, C18-CGPLGVRGD, GRGDS, RGDS, RGDV, c(RGDfK), c(RGDyK), c(RGDfV), c(RGDfE), c(RGDfC), c(RGDf[N-Methyl]V), c(RGDyC), RGD-PEG or iRGD.

[0013] Preferably, the collagenase is type I collagenase.

[0014] Preferably, the drug is a drug for treating liver fibrosis, lung fibrosis, pancreatic fibrosis or fibrotic tumors.

[0015] More preferably, the fibrotic tumor is selected from triple-negative breast cancer, pancreatic cancer or bladder cancer with fibrotic symptoms.

[0016] Preferably, the drug is administered topically, intravenously, subcutaneously or intramuscularly.

[0017] Preferably, the drug is used in combination with radiotherapy, chemotherapy, immunotherapy or biological therapy to treat fibrosis.

[0018] The pharmaceutical composition for treating fibrosis of the present invention comprises, by weight, 0.5 to 50 parts of integrin targeting peptide, 0.5 to 50 parts of collagenase and 1 to 100 parts of a pharmaceutically acceptable carrier.

[0019] Preferably, the integrin targeting peptide is CGPLGVRGD, C18-CGPLGVRGD, GRGDS, RGDS, RGDV, c(RGDfK), c(RGDyK), c(RGDfV), c(RGDfE), c(RGDfC), c(RGDf[N-Methyl]V), c(RGDyC), RGD-PEG or iRGD, and the collagenase is type I collagenase.

[0020] Preferably, the mass ratio of the integrin targeting peptide to collagenase is (5-10):1.

[0021] More preferably, the mass ratio of the integrin targeting peptide to collagenase is (5-8.5):1.

[0022] More preferably, the mass ratio of the integrin targeting peptide to collagenase is selected from 5:1, 6:1, 6.5:1, 7.5:1 or 8.5:1.

[0023] Preferably, the pharmaceutical composition comprises, by weight, 5 to 8.5 parts of integrin targeting peptide, 1 part of collagenase and 1 to 50 parts of a pharmaceutically acceptable carrier.

[0024] Preferably, the dosage form of the pharmaceutical composition is selected from the group consisting of solutions, suspensions, liposomes, microspheres, microcapsules, nanoparticles, and gels.

[0025] Preferably, the pharmaceutically acceptable carrier is selected from a solvent, a stabilizer, a buffer, a cosolvent, an antioxidant, a preservative, an osmotic pressure regulator, a lipid material, a polymer material or a filler.

[0026] Specifically, the pharmaceutically acceptable carrier is selected from one or more of the following excipients: egg yolk lecithin, soybean lecithin, hydrogenated soybean lecithin or synthetic lecithin (DPPD, DOPS, DEPE, DMPE, DSPE, DPPE, DOPE, DOPG, EPG, POPG, DPPG, DSPG, DMPG, DPPA, DEPC, DOPC, DMPC, POPC, DSPC, DPPC), X-PEG-Y (wherein X is selected from DSPE, DPPE, DMPE or DOPE; Y is selected from 0 to 10 amino acid sequences; the molecular weight of PEG is 1000 to 5000), cholesterol, natural high Molecular polymers and their derivatives (amylose, amylopectin, oxidized starch, hydroxyethyl starch, cross-linked starch, microcrystalline cellulose, cellulose acetate, methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, hydroxypropyl methyl cellulose carbonate, hydroxypropyl methyl cellulose acetate succinate, gum arabic, chitin, dextran, chitosan, hyaluronic acid, alginate, heparin, albumin), synthetic polymers and their derivatives (polyacrylic acid, polyhydroxyethyl methacrylate, carbomer, polyvinyl alcohol, polyvinyl acetate phthalate, polyvinyl pyrrolidone, polyethylene glycol, polyethylene glycol-polylactic acid, polyethylene glycol-polycaprolactone, polyoxyethylene castor Sesame oil, polyoxyethylene fatty acid esters, poloxamer, polyglycolic acid, polylactic acid, polycaprolactone, glycolic acid-caprolactone copolymer, polysorbate, polyvinyl pyridone, poly(ethylene imine), d-α-tocopheryl polyethylene glycol 1000 succinate, amino acid polymer, poly(ethylene oxide)-poly(propylene oxide) block copolymer), polyphenol materials (quercetin, kaempferol, myricetin, anthocyanin, oleander, catechol, gallocatechin gallate, epigallocatechin gallate, digalloyl-D-glucose, trigalloyl glucose, tetragalloyl glucose, pentagalloyl glucose, gallic acid, digallic acid , tannic acid, ellagitannin, ellagic acid, hydrolyzed tannin, polydopamine), emulsifiers (polyvinyl alcohol, polysorbate 20, polysorbate 80, sodium lauryl sulfate, poloxamer 188, poloxamer 407, polyethylene glycol monostearate, polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil, caprylic capric macrogol glyceride), crosslinking agents (glutaraldehyde, genipin, formaldehyde, epichlorohydrin, carbodiimide, N-hydroxysuccinimide, divinyl sulfone, bisacrylamide, diisocyanate, melamine formaldehyde resin, calcium chloride, sodium tripolyphosphate, sodium sulfate, barium chloride, sodium citrate), microcrystalline cellulose sodium, carboxymethyl cellulose sodium, hypromellose, magnesium stearate.

[0027] The method for preparing the pharmaceutical composition of the present invention comprises the following steps:

[0028] (1) preparing a solution of integrin targeting peptide and collagenase;

[0029] (2) Preparation of prefabricated system

[0030] Method 1: The integrin targeting peptide and collagenase solution prepared in step (1) are mixed with a buffer solution and the pH is adjusted to 6.5-7.5; the mixing step is specifically a microfluidic technology rapid mixing or a simple mixing and an ultrasonic, homogenizing, slurrying, vortexing or stirring operation.

[0031] Method 2: The integrin targeting peptide and collagenase solution prepared in step (1) are mixed with a buffer solution, and then mixed with a stabilizer, and the pH is adjusted to 6.5-7.5; the mixing process is the same as that of method 1.

[0032] (3) Preparation of pharmaceutical preparations

[0033] Method 1: The prefabricated system prepared in step (2) is mixed with a solvent to obtain a solution containing integrin targeting peptide and collagenase; the mixing step is specifically a rapid mixing by microfluidic technology or a simple mixing and an ultrasonic, homogenizing, slurrying, vortexing or stirring operation.

[0034] Method 2: The prefabricated system prepared in step (2) is mixed with a polymer material, freeze-dried and the particles are micronized and dispersed in an oil phase solution to obtain a suspension containing integrin targeting peptide and collagenase; the micronization process is specifically ultrasonic treatment, high-pressure homogenization treatment, ball milling treatment, and air flow crushing treatment; the mixing process is the same as method 1.

[0035] Method three: The prefabricated system prepared in step (2) is mixed with lipid or polymer materials, and prepared by emulsification-solvent evaporation method, spray drying method or phase separation method to obtain microspheres containing integrin targeting peptide and collagenase; the emulsification-solvent evaporation method is specifically to dissolve the prefabricated system prepared in step (2) and the polymer material in an organic solvent to form an oil phase; the oil phase is slowly added to the aqueous phase containing an emulsifier, emulsified at high speed to form an emulsion, stirred to evaporate the organic solvent, and centrifuged to collect the microspheres; the spray drying method is specifically to mix the prefabricated system prepared in step (2) with the polymer material, atomized and dried in a high-temperature air flow through a spray drying device to form microspheres; the phase separation method is specifically to dissolve the prefabricated system prepared in step (2) and the polymer material in an organic solvent, add a non-solvent to induce phase separation, and form microspheres; the mixing process is the same as method one.

[0036] Method 4: The prefabricated system prepared in step (2) is mixed with lipid or polymer materials, and prepared by double emulsion method, interfacial polymerization method or coacervation method to obtain microcapsules containing integrin targeting peptide and collagenase; the double emulsion method specifically comprises the following steps: the prefabricated system prepared in step (2) is used as the inner aqueous phase, and is mixed with the oil phase formed by the polymer material to form colostrum (W / O); the colostrum is added to the outer aqueous phase (aqueous solution containing an emulsifier), emulsified at high speed to form a double emulsion (W / O / W), the organic solvent is stirred to evaporate, and the microcapsules are collected by centrifugation; the interfacial polymerization method specifically comprises the following steps: the prefabricated system prepared in step (2) is mixed with a monomer as the aqueous phase; another monomer is dissolved in an organic solvent as the oil phase; the two phases are mixed, and a polymerization reaction occurs at the interface to form microcapsules; the coacervation method specifically comprises the following steps: the prefabricated system prepared in step (2) is mixed with the polymer material, and coacervation is induced by adjusting the pH or adding a coagulant to form microcapsules; the mixing process is the same as that of method 1.

[0037] Method 5: The prefabricated system prepared in step (2) is mixed with natural or synthetic polymer materials and prepared by ultrasonic or high-pressure homogenization to obtain nanoparticles containing integrin targeting peptide and collagenase; the mixing process is the same as method 1.

[0038] Method 6: The prefabricated system prepared in step (2) is mixed with natural or synthetic polymer materials, and cross-linked by ion cross-linking or adding a cross-linking agent to obtain a gel containing integrin targeting peptide and collagenase; the mixing process is the same as method 1.

[0039] Method seven: The prefabricated system prepared in step (2) is mixed with lipid or polymer materials, and prepared by thin film dispersion method, reverse evaporation method or injection method to obtain liposomes containing integrin targeting peptide and collagenase; the thin film dispersion method specifically comprises dissolving lipid materials such as phospholipids and cholesterol in an organic solvent, evaporating under reduced pressure to form a thin film, adding the prefabricated system prepared in step (2) to hydrate, and subjecting to ultrasonic or high-pressure homogenization treatment to obtain liposomes; the reverse evaporation method specifically comprises dissolving lipid materials such as phospholipids and cholesterol in an organic solvent, mixing with the prefabricated system prepared in step (2) to form an emulsion, removing the organic solvent, and obtaining liposomes; the injection method specifically comprises slowly injecting an organic solution of lipid materials such as phospholipids and cholesterol into the prefabricated system prepared in step (2), stirring and removing the organic solvent, and obtaining liposomes; the mixing process is the same as that of method one; and the organic solvent is removed by dialysis, ultrafiltration or evaporation.

[0040] More specifically, the ultrasonic frequency is 100-1000W, and the ultrasonic time is 1-30min; the centrifugal speed is 1000-10000rpm, and the centrifugal time is 1-30min; the homogenization is implemented by a high-pressure homogenizer or a microfluidizer, the homogenization pressure is 300-1500Pa, and the homogenization times are 1-30 times; the homogenization is achieved by a homogenizer, the speed is 1000-10000rpm, and the homogenization time is 5-120s; the vortex or stirring is achieved by a vortex instrument or a stirrer, and the vortex or stirring speed is generally 500-3000rpm, and the time is 1-30min; the ball milling treatment is achieved by a ball mill, the speed is 50-400rpm, and the grinding time is 1-2h; the air flow crushing treatment is achieved by an air flow crusher, and the air flow pressure is 0.5-1.2MPa; the atomization pressure of the spray dryer is 0.1-0.5MPa.

[0041] The pharmaceutically acceptable solvent is one or more of water, buffer, glycerol, propylene glycol, ether, dimethyl sulfoxide, N,N-dimethylformamide, formamide, ethanol, acetone, methanol, tetrahydrofuran, dichloromethane, chloroform, ethyl acetate, and isopropanol.

[0042] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0043] The drug combination designed by the present invention has a synergistic inhibitory effect of mechanical signal transduction and biochemical signal transduction dual pathways, can block the activation and proliferation of fibrotic cells, inhibit the initiation and maintenance of fibrosis, and promote matrix degradation; at the same time, by degrading the collagen matrix in the fibrotic tissue, the volume and hardness of the fibrotic tissue are reduced, and the elasticity of the tissue is restored. Efficient reduction of fibrosis can also promote the penetration of subsequent therapeutic drugs, and has a cascade penetration-promoting effect. The constructed drug composition can synergistically deliver integrin targeting peptides and collagenase, and after release, it plays a synergistic role in fibrotic lesions in the form of free molecules, providing a new strategy for the efficient delivery of fibrosis therapeutic drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 This is the responsive release curve of RGD peptide and collagenase in the microcapsule prepared in Example 3;

[0045] Figure 2 This is the responsive release curve of RGD peptide and collagenase in the microspheres prepared in Example 4;

[0046] Figure 3 This is the responsive release curve of RGD peptide and collagenase in the nanoparticles prepared in Example 5;

[0047] Figure 4This is the responsive release curve of RGD peptide and collagenase in the gel prepared in Example 6;

[0048] Figure 5 This is the responsive release curve of RGD peptide and collagenase in the liposome prepared in Example 7;

[0049] Figure 6 The results of the study on the mechanism of RGD peptide and collagenase combined to inhibit fibroblast activation;

[0050] Figure 7 The results show the effect of RGD peptide and collagenase combined on the secretion of active TGF-β.

[0051] Figure 8 The pharmacodynamic evaluation results of the solution prepared in Example 1 for the treatment of liver fibrosis;

[0052] Fig. 9 The pharmacodynamic evaluation results of the suspension prepared in Example 2 for the treatment of pulmonary fibrosis;

[0053] Fig.10 The pharmacodynamic evaluation results of the microcapsules prepared in Example 3 for the treatment of liver fibrosis;

[0054] Fig.11 The pharmacodynamic evaluation results of the microspheres prepared in Example 4 for the treatment of pulmonary fibrosis;

[0055] Fig.12 The pharmacodynamic evaluation results of the nanoparticles prepared in Example 5 for the treatment of pancreatic fibrosis;

[0056] Fig.13 The pharmacodynamic evaluation results of the gel prepared in Example 6 for the treatment of bladder cancer;

[0057] Fig.14 The pharmacodynamic evaluation results of the liposomes prepared in Example 7 for the treatment of pancreatic cancer. DETAILED DESCRIPTION

[0058] The technical solution of the present invention is further described below in conjunction with embodiments.

[0059] The integrin targeting peptide containing RGD sequence was from Gill Biochemical Co., Ltd.

[0060] Example 1: Preparation and formulation characterization of a solution containing an RGD sequence integrin targeting peptide and collagenase

[0061] (1) Preparation of a solution of the integrin targeting peptide CGPLGVRGD containing the RGD sequence and collagenase

[0062] First, prepare 0.01 M phosphate buffer (PBS) with a pH of 7.4; then dissolve 5 mg CGPLGVRGD in 5 mL PBS to prepare a 1 mg / mL RGD peptide solution, and dissolve 1 mg collagenase in 5 mL PBS to prepare a 200 μg / mL collagenase solution, and stir for 10 minutes; then, sonicate the mixed solution for 15 minutes (300 W, 40 kHz) to ensure uniform dispersion; centrifuge for 10 minutes (10,000 × g) to remove insoluble matter; finally, filter through a 0.22 μm sterile filter, dispense into sterile test tubes, and seal and store at -80°C or 4°C.

[0063] (2) Preparation of a solution containing the RGD sequence-containing integrin targeting peptide GRGDSP and collagenase

[0064] First, 0.01 M phosphate buffer (PBS) with a pH of 7.4 was prepared; then 5 mg of GRGDSP was dissolved in 5 mL of PBS to prepare a 1 mg / mL RGD peptide solution, and 1 mg of collagenase was dissolved in 5 mL of PBS to prepare a 200 μg / mL collagenase solution, which were stirred for 10 minutes; then, the mixed solution was sonicated for 15 minutes (300 W, 40 kHz) to ensure uniform dispersion; insoluble matter was removed by centrifugation for 10 minutes (10,000×g); finally, the mixture was filtered through a 0.22 μm sterile filter, dispensed into sterile test tubes, and sealed and stored at -80°C or 4°C.

[0065] (3) Preparation of a solution containing the RGD sequence-containing integrin targeting peptide GRGDS and collagenase

[0066] First, prepare 0.01 M phosphate buffer (PBS) with a pH of 7.4; then dissolve 5 mg of GRGDS in 5 mL of PBS to prepare a 1 mg / mL RGD peptide solution, and dissolve 1 mg of collagenase in 5 mL of PBS to prepare a 200 μg / mL collagenase solution, and stir for 10 minutes; then, sonicate the mixed solution for 15 minutes (300 W, 40 kHz) to ensure uniform dispersion; centrifuge for 10 minutes (10,000×g) to remove insoluble matter; finally, filter through a 0.22 μm sterile filter, dispense into sterile test tubes, and seal and store at -80°C or 4°C.

[0067] (4) Preparation of a solution containing the RGD sequence-containing integrin targeting peptide RGDS and collagenase

[0068] First, prepare 0.01 M phosphate buffer (PBS) with a pH of 7.4; then dissolve 5 mg RGDS in 5 mL PBS to prepare a 1 mg / mL RGD peptide solution, and dissolve 1 mg collagenase in 5 mL PBS to prepare a 200 μg / mL collagenase solution, and stir for 10 minutes; then, sonicate the mixed solution for 15 minutes (300 W, 40 kHz) to ensure uniform dispersion; centrifuge for 10 minutes (10,000×g) to remove insoluble matter; finally, filter through a 0.22 μm sterile filter, dispense into sterile test tubes, and seal and store at -80°C or 4°C.

[0069] (5) Preparation of a solution containing the RGD sequence integrin targeting peptide RGDV and collagenase

[0070] First, 0.01 M phosphate buffer (PBS) with a pH of 7.4 was prepared; then 5 mg RGDV was dissolved in 5 mL PBS to prepare a 1 mg / mL RGD peptide solution, and 1 mg collagenase was dissolved in 5 mL PBS to prepare a 200 μg / mL collagenase solution, and stirred for 10 minutes; then, the mixed solution was sonicated for 15 minutes (300 W, 40 kHz) to ensure uniform dispersion; centrifuged for 10 minutes (10,000×g) to remove insoluble matter; finally, filtered through a 0.22 μm sterile filter, dispensed into sterile test tubes, and sealed and stored at -80°C or 4°C.

[0071] (6) Preparation of a solution containing RGD sequence integrin targeting peptide c (RGDfK) and collagenase

[0072] First, 0.01 M phosphate buffer (PBS) with a pH of 7.4 was prepared; then 5 mg c(RGDfK) was dissolved in 5 mL PBS to prepare a 1 mg / mL RGD peptide solution, and 1 mg collagenase was dissolved in 5 mL PBS to prepare a 200 μg / mL collagenase solution, and stirred for 10 minutes; then, the mixed solution was sonicated for 15 minutes (300 W, 40 kHz) to ensure uniform dispersion; insoluble matter was removed by centrifugation for 10 minutes (10,000×g); finally, the mixture was filtered through a 0.22 μm sterile filter, dispensed into sterile test tubes, and sealed and stored at -80°C or 4°C.

[0073] (7) Preparation of a solution containing an RGD sequence integrin targeting peptide c (RGDyK) and collagenase

[0074] First, 0.01 M phosphate buffer (PBS) with a pH of 7.4 was prepared; then 5 mg c(RGDyK) was dissolved in 5 mL PBS to prepare a 1 mg / mL RGD peptide solution, and 1 mg collagenase was dissolved in 5 mL PBS to prepare a 200 μg / mL collagenase solution, and stirred for 10 minutes; then, the mixed solution was sonicated for 15 minutes (300 W, 40 kHz) to ensure uniform dispersion; insoluble matter was removed by centrifugation for 10 minutes (10,000 × g); finally, the mixture was filtered through a 0.22 μm sterile filter, dispensed into sterile test tubes, and sealed and stored at -80°C or 4°C.

[0075] (8) Preparation of a solution containing RGD sequence integrin targeting peptide c (RGDfV) and collagenase

[0076] First, 0.01 M phosphate buffer (PBS) with a pH of 7.4 was prepared; then 5 mg c(RGDfV) was dissolved in 5 mL PBS to prepare a 1 mg / mL RGD peptide solution, and 1 mg collagenase was dissolved in 5 mL PBS to prepare a 200 μg / mL collagenase solution, and stirred for 10 minutes; then, the mixed solution was sonicated for 15 minutes (300 W, 40 kHz) to ensure uniform dispersion; insoluble matter was removed by centrifugation for 10 minutes (10,000×g); finally, the mixture was filtered through a 0.22 μm sterile filter, dispensed into sterile test tubes, and sealed and stored at -80°C or 4°C.

[0077] (9) Preparation of a solution containing RGD sequence integrin targeting peptide c (RGDfE) and collagenase

[0078] First, 0.01 M phosphate buffer (PBS) with a pH of 7.4 was prepared; then 5 mg c(RGDfE) was dissolved in 5 mL PBS to prepare a 1 mg / mL RGD peptide solution, and 1 mg collagenase was dissolved in 5 mL PBS to prepare a 200 μg / mL collagenase solution, and stirred for 10 minutes; then, the mixed solution was sonicated for 15 minutes (300 W, 40 kHz) to ensure uniform dispersion; insoluble matter was removed by centrifugation for 10 minutes (10,000 × g); finally, the mixture was filtered through a 0.22 μm sterile filter, dispensed into sterile test tubes, and sealed and stored at -80°C or 4°C.

[0079] (10) Preparation of a solution containing RGD sequence integrin targeting peptide c (RGDfC) and collagenase

[0080] First, 0.01 M phosphate buffer (PBS) with a pH of 7.4 was prepared; then 5 mg c(RGDfC) was dissolved in 5 mL PBS to prepare a 1 mg / mL RGD peptide solution, and 1 mg collagenase was dissolved in 5 mL PBS to prepare a 200 μg / mL collagenase solution, and stirred for 10 minutes; then, the mixed solution was sonicated for 15 minutes (300 W, 40 kHz) to ensure uniform dispersion; centrifuged for 10 minutes (10,000×g) to remove insoluble matter; finally, filtered through a 0.22 μm sterile filter, dispensed into sterile test tubes, and sealed and stored at -80°C or 4°C.

[0081] (11) Preparation of a solution containing the RGD sequence integrin targeting peptide c(RGDf[N-Methyl]V) and collagenase

[0082] First, 0.01 M phosphate buffer (PBS) with a pH of 7.4 was prepared; then 5 mg c(RGDf[N-Methyl]V) was dissolved in 5 mL PBS to prepare a 1 mg / mL RGD peptide solution, and 1 mg collagenase was dissolved in 5 mL PBS to prepare a 200 μg / mL collagenase solution, and stirred for 10 minutes; then, the mixed solution was sonicated for 15 minutes (300 W, 40 kHz) to ensure uniform dispersion; insoluble matter was removed by centrifugation for 10 minutes (10,000×g); finally, the mixture was filtered through a 0.22 μm sterile filter, dispensed into sterile test tubes, and sealed and stored at -80°C or 4°C.

[0083] (12) Preparation of a solution containing RGD sequence integrin targeting peptide c (RGDyC) and collagenase

[0084] First, prepare 0.01 M phosphate buffer (PBS) with a pH of 7.4; then dissolve 5 mg c(RGDyC) in 5 mL PBS to prepare a 1 mg / mL RGD peptide solution, and dissolve 1 mg collagenase in 5 mL PBS to prepare a 200 μg / mL collagenase solution, and stir for 10 minutes; then, sonicate the mixed solution for 15 minutes (300 W, 40 kHz) to ensure uniform dispersion; centrifuge for 10 minutes (10,000 × g) to remove insoluble matter; finally, filter through a 0.22 μm sterile filter, dispense into sterile test tubes, and seal and store at -80°C or 4°C.

[0085] (13) Preparation of a solution containing the RGD sequence integrin targeting peptide RGD-PEG and collagenase

[0086] First, prepare 0.01 M phosphate buffer (PBS) with a pH of 7.4; then dissolve 5 mg RGD-PEG in 5 mL PBS to prepare a 1 mg / mL RGD peptide solution, and dissolve 1 mg collagenase in 5 mL PBS to prepare a 200 μg / mL collagenase solution, and stir for 10 minutes; then, sonicate the mixed solution for 15 minutes (300 W, 40 kHz) to ensure uniform dispersion; centrifuge for 10 minutes (10,000×g) to remove insoluble matter; finally, filter through a 0.22 μm sterile filter, dispense into sterile test tubes, and seal and store at -80°C or 4°C.

[0087] (14) Preparation of a solution containing the RGD sequence-containing integrin targeting peptide iRGD and collagenase

[0088] First, 0.01 M phosphate buffer (PBS) with a pH of 7.4 was prepared; then 5 mg of iRGD was dissolved in 5 mL of PBS to prepare a 1 mg / mL RGD peptide solution, and 1 mg of collagenase was dissolved in 5 mL of PBS to prepare a 200 μg / mL collagenase solution, and stirred for 10 minutes; then, the mixed solution was sonicated for 15 minutes (300 W, 40 kHz) to ensure uniform dispersion; insoluble matter was removed by centrifugation for 10 minutes (10,000 × g); finally, the mixture was filtered through a 0.22 μm sterile filter, dispensed into sterile test tubes, and sealed and stored at -80°C or 4°C. (15) Formulation characterization of the solution of the RGD sequence-containing integrin targeting peptide CGPLGVRGD and collagenase

[0089] Using the above preparation method, a solution containing RGD sequence integrin targeting peptide and collagenase (RGD / Col), a solution containing RGD sequence integrin targeting peptide (RGD), and a solution containing collagenase (Col) were prepared, and the above preparations were taken for pharmaceutical characterization, and the results are shown in Table 1. The prepared solution is a clear liquid with good stability and biological activity.

[0090] Example 2: Preparation and formulation characterization of a suspension containing an RGD sequence integrin targeting peptide and collagenase

[0091] (1) Preparation of a suspension containing an RGD sequence integrin targeting peptide and collagenase

[0092] First, prepare 0.01M phosphate buffer (PBS) with a pH of 7.4; then dissolve 5mg CGPLGVRGD in 5mL PBS to prepare a 1mg / mL RGD peptide solution, dissolve 1mg collagenase in 5mL PBS to prepare a 200μg / mL collagenase solution, dissolve 20mg polymer material (such as PVP) in 5mL PBS to prepare a 4mg / mL solution, and stir for 10 minutes; fully stir the RGD peptide solution, collagenase solution and polymer material solution to mix evenly. The mixed solution is divided into freeze-drying bottles for freeze drying to obtain freeze-dried particles. The freeze-dried particles are added with a stabilizer and ground evenly. At the same time, prepare the oil phase, dissolve 10mg suspending agent (such as Tween-80) in 1mL solvent (such as medium-chain triglycerides), and stir until uniform. Disperse the freeze-dried particles into the oil phase, mix evenly, and form a stable microparticle suspension. Finally, filter through a 0.22 μm sterile filter, dispense into sterile test tubes, seal and store at -80°C or 4°C.

[0093] (2) Pharmaceutical characterization of the suspension containing the RGD sequence integrin targeting peptide and collagenase

[0094] Using the above preparation method, suspensions containing RGD sequence integrin targeting peptide and collagenase (RGD / Col), suspensions containing RGD sequence integrin targeting peptide (RGD), and suspensions containing collagenase (Col) were prepared, and the above preparations were characterized by pharmaceutics, and the results are shown in Table 1. The prepared suspensions have good stability and biological activity.

[0095] Example 3: Preparation and formulation characterization of microcapsules containing RGD sequence integrin targeting peptide and collagenase

[0096] (1) Preparation of microcapsules containing RGD sequence integrin targeting peptide and collagenase

[0097] First, prepare 0.01M phosphate buffer (PBS) at pH 7.4; then dissolve 5mg GRGDSP in 5mL PBS to prepare a 1mg / mL RGD peptide solution, and dissolve 1mg collagenase in 5mL PBS to prepare a 200μg / mL collagenase solution, add them at a mass ratio of RGD / Col of 6.5:1, stir evenly, and use as a drug mixed solution; at the same time, prepare an oil phase, add an appropriate amount of material (such as PLGA, PCL) to an appropriate amount of organic solvent (such as dichloromethane), heat to 60°C, and stir to dissolve; at pH A surfactant (such as PVA) is added to 7.4 0.01M phosphate buffer (PBS) to prepare an aqueous phase; then, the drug mixture solution is slowly added to the oil phase, and probe ultrasonic emulsification is performed (30% power, 3 min); the emulsion is treated 2 to 3 times with a high-pressure homogenizer at 1000 to 1500 psi to further refine the droplets; the colostrum is dripped into a large amount of aqueous phase, stirring is continued, the organic solvent is rotary evaporated, the microcapsules are separated by centrifugation, and washed 3 times with PBS to remove unreacted products; the microcapsules are divided into sterile test tubes, sealed and stored at 4°C.

[0098] (2) Pharmaceutical characterization of microcapsules containing RGD sequence integrin targeting peptide and collagenase

[0099] Using the above preparation method, microcapsules containing RGD sequence integrin targeting peptide and collagenase with acid responsive release characteristics (RGD / Col), microcapsules containing RGD sequence integrin targeting peptide and collagenase without acid responsive release characteristics (RGD / Col-NR (Non-Responsive)), microcapsules containing RGD sequence integrin targeting peptide with acid responsive release characteristics (RGD), microcapsules containing collagenase with acid responsive release characteristics (Col), and blank microcapsule preparations were prepared respectively. The above preparations were taken and the particle size of the microcapsules was determined by dynamic light scattering (DLS). The results are shown in Table 1. The particle size of the five microcapsules is between 0.6 and 0.7 μm, and the particle size distribution is narrow. The PDI (polydispersity index) is less than 0.3, indicating that the prepared microcapsules have uniform particle size and good dispersibility, which is conducive to the distribution and penetration of the microcapsules in the body and improves the bioavailability of the drug.

[0100] (3) In vitro release of RGD sequence integrin targeting peptide and collagenase

[0101] Each group of preparations was mixed with pH 6.5 PBS buffer and pH 7.4 PBS buffer, respectively, and incubated at 37°C and 100 rpm for 48 hours. The released Col I-FITC was removed by ultrafiltration (MWCO = 300 kDa) at 0, 1, 2, 3, 6, 9, 12, 24, and 48 hours, the fluorescence intensity of the filtrate was measured, and the amount of Col I-FITC released was calculated. The concentrate was supplemented to the original volume with the corresponding PBS solution.

[0102] The results are as follows Figure 1 As shown, the release of Col I in RGD / Col is acid-responsive, while the RGD / Col-NR group basically does not release Col I under acidic conditions.

[0103] Each group of preparations was mixed with pH 6.5 PBS buffer and pH 7.4 PBS buffer, respectively, and incubated at 37°C and 100 rpm for 48 hours. Free C18-CD9 and VRGD cleaved by enzyme were removed by ultrafiltration (MWCO = 300 kDa) at 0, 1, 2, 3, 6, 9, 12, 24, and 48 hours, and the lipid concentrate was supplemented to the original volume with the corresponding PBS buffer, and the dispersed RGD was quantified using the BCA method.

[0104] The results are as follows Figure 1 As shown, the release of RGD peptide in RGD / Col is acid-responsive, while the RGD / Col-NR group basically does not release RGD peptide under acidic conditions.

[0105] Example 4: Preparation and formulation characterization of microspheres containing RGD sequence integrin targeting peptide and collagenase

[0106] (1) Preparation of microspheres containing RGD sequence integrin targeting peptide and collagenase

[0107] First, prepare 0.01M phosphate buffer (PBS) at pH 7.4; then dissolve 5mg GRGDS in 5mL PBS to prepare a 1mg / mL RGD peptide solution, and dissolve 1mg collagenase in 5mL PBS to prepare a 200μg / mL collagenase solution, add them at a mass ratio of RGD / Col of 7.5:1, stir evenly, and use them as a drug mixed solution; dissolve 100mg chitosan in 2% acetic acid solution to form a chitosan solution. The mixed solution (RGD peptide + collagenase) was mixed with the chitosan solution and stirred evenly. Add 2% glutaraldehyde solution (9:1 volume ratio) for cross-linking and react at room temperature for 30 minutes. Collect the microspheres by centrifugation (10,000×g, 10 minutes) and wash them 3 times with PBS. Remove unreacted glutaraldehyde and other impurities. The microspheres were divided into sterile tubes and sealed and stored at 4°C.

[0108] (2) Pharmaceutical characterization of microspheres containing RGD sequence integrin targeting peptide and collagenase

[0109] Using the above preparation method, microspheres (RGD / Col) containing RGD sequence integrin targeting peptide and collagenase with acid responsive release characteristics, microspheres (RGD / Col-NR) containing RGD sequence integrin targeting peptide and collagenase without acid responsive release characteristics, microspheres (RGD) containing RGD sequence integrin targeting peptide with acid responsive release characteristics, microspheres containing collagenase with acid responsive release characteristics (Col), and blank microsphere preparations. Take the above preparations and measure the particle size of the microspheres by dynamic light scattering (DLS). The results are shown in Table 1. The particle size of the five microspheres is between 0.4 and 0.5 μm, and the particle size distribution is narrow, and the PDI (polydispersity index) is less than 0.3, indicating that the prepared microspheres have uniform particle size and good dispersibility, which is conducive to the distribution and penetration of the microspheres in the body and improves the bioavailability of the drug.

[0110] (3) In vitro release of RGD sequence integrin targeting peptide and collagenase

[0111] Each group of preparations was mixed with pH 6.5 PBS buffer and pH 7.4 PBS buffer, respectively, and incubated at 37°C and 100 rpm for 48 hours. The released Col I-FITC was removed by ultrafiltration (MWCO = 300 kDa) at 0, 1, 2, 3, 6, 9, 12, 24, and 48 hours, the fluorescence intensity of the filtrate was measured, and the amount of Col I-FITC released was calculated. The concentrate was supplemented to the original volume with the corresponding PBS solution.

[0112] The results are as follows Figure 2 As shown, the release of Col I in RGD / Col is acid-responsive, while the RGD / Col-NR group basically does not release Col I under acidic conditions.

[0113] Each group of preparations was mixed with pH 6.5 PBS buffer and pH 7.4 PBS buffer, respectively, and incubated at 37°C and 100 rpm for 48 hours. Free C18-CD9 and VRGD cleaved by enzyme were removed by ultrafiltration (MWCO = 300 kDa) at 0, 1, 2, 3, 6, 9, 12, 24, and 48 hours, and the lipid concentrate was supplemented to the original volume with the corresponding PBS buffer, and the dispersed RGD was quantified using the BCA method.

[0114] The results are as follows Figure 2 As shown, the release of RGD peptide in RGD / Col is acid-responsive, while the RGD / Col-NR group basically does not release RGD peptide under acidic conditions.

[0115] Example 5: Preparation and formulation characterization of nanoparticles containing RGD sequence integrin targeting peptide and collagenase

[0116] (1) Preparation of Nanoparticles Containing RGD Sequence Integrin Targeting Peptide and Collagenase

[0117] First, 0.01M phosphate buffer (PBS) with a pH of 7.4 was prepared; then 5mg c(RGDfK) was dissolved in 5mL PBS to prepare a 1mg / mL RGD peptide solution, and 1mg collagenase was dissolved in 5mL PBS to prepare a 200μg / mL collagenase solution, which was added at a mass ratio of RGD / Col of 6.0:1 and stirred evenly to form a drug mixed solution; then, the mixed solution was added to 5mL 2% polylactic-co-glycolic acid (PLGA) dimethyl sulfoxide (DMSO) solution and stirred at high speed (10,000rpm) for 30min to form colostrum. Then, the colostrum was slowly added to 50mL 1% polyvinyl alcohol (PVA) aqueous solution and stirred for 1h to form a double emulsion. Subsequently, the nanoparticles were collected by centrifugation at 4,000×g for 15min and washed 3 times with deionized water to remove unencapsulated peptides and enzymes. Finally, the nanoparticles were dispersed in PBS, filtered through a 0.22 μm sterile filter, dispensed into sterile test tubes, and sealed and stored at 4 °C.

[0118] (2) Pharmaceutical characterization of nanoparticles containing RGD sequence integrin targeting peptide and collagenase

[0119] Using the above preparation method, nanoparticles (RGD / Col) containing RGD sequence integrin targeting peptide and collagenase with acid responsive release characteristics, nanoparticles (RGD / Col-NR) containing RGD sequence integrin targeting peptide and collagenase without acid responsive release characteristics, nanoparticles (RGD) containing RGD sequence integrin targeting peptide with acid responsive release characteristics, nanoparticles (Col) containing collagenase with acid responsive release characteristics, and blank nanoparticle preparations were prepared. The above preparations were taken and the particle size of the nanoparticles was determined by dynamic light scattering (DLS). The results are shown in Table 1. The particle size of the five nanoparticles is between 100 and 120 nm, and the particle size distribution is narrow, and the PDI (polydispersity index) is less than 0.3, indicating that the prepared nanoparticles have uniform particle size and good dispersibility, which is conducive to the distribution and penetration of the nanoparticles in the body and improves the bioavailability of the drug.

[0120] (3) In vitro release of RGD sequence integrin targeting peptide and collagenase

[0121] Each group of preparations was mixed with pH 6.5 PBS buffer and pH 7.4 PBS buffer, respectively, and incubated at 37°C and 100 rpm for 48 hours. The released Col I-FITC was removed by ultrafiltration (MWCO = 300 kDa) at 0, 1, 2, 3, 6, 9, 12, 24, and 48 hours, the fluorescence intensity of the filtrate was measured, and the amount of Col I-FITC released was calculated. The concentrate was supplemented to the original volume with the corresponding PBS solution.

[0122] The results are as follows Figure 3 As shown, the release of Col I in RGD / Col is acid-responsive, while the RGD / Col-NR group basically does not release Col I under acidic conditions.

[0123] Each group of preparations was mixed with pH 6.5 PBS buffer and pH 7.4 PBS buffer, respectively, and incubated at 37°C and 100 rpm for 48 hours. Free C18-CD9 and VRGD cleaved by enzyme were removed by ultrafiltration (MWCO = 300 kDa) at 0, 1, 2, 3, 6, 9, 12, 24, and 48 hours, and the lipid concentrate was supplemented to the original volume with the corresponding PBS buffer, and the dispersed RGD was quantified using the BCA method.

[0124] The results are as follows Figure 3 As shown, the release of RGD peptide in RGD / Col is acid-responsive, while the RGD / Col-NR group basically does not release RGD peptide under acidic conditions.

[0125] Example 6: Preparation and pharmaceutical characterization of gel containing RGD sequence integrin targeting peptide and collagenase

[0126] (1) Preparation of gel containing RGD sequence integrin targeting peptide and collagenase

[0127] First, prepare 0.01M phosphate buffer (PBS) with a pH of 7.4; then dissolve 5mg RGD-PEG in 5mL PBS to prepare a 1mg / mL RGD peptide solution, and dissolve 1mg collagenase in 5mL PBS to prepare a 200μg / mL collagenase solution, add it at a mass ratio of RGD / Col of 8.5:1, stir evenly, and use it as a drug mixed solution; dissolve 100mg chitosan in 2% acetic acid solution, stir evenly, and prepare chitosan solution. Dissolve 100mg hyaluronic acid in 10mL ultrapure water, stir evenly, and prepare hyaluronic acid solution. Mix the chitosan solution and the hyaluronic acid solution in a volume ratio of 1:1, add the mixed solution of RGD peptide and collagenase, and stir evenly. Add 2% glutaraldehyde solution (9:1 volume ratio) and crosslink at room temperature for 30 minutes. Place the mixed solution in an ultrasonicator and ultrasonicate it at 200W power for 5 minutes to form a nanogel. The nanogels were collected by centrifugation (10,000×g, 10 min), washed three times with PBS, dispensed into sterile tubes, and sealed and stored at 4°C.

[0128] (2) Pharmaceutical characterization of the gel containing the RGD sequence integrin targeting peptide and collagenase

[0129] Using the above preparation method, nanogels (RGD / Col) containing RGD sequence integrin targeting peptide and collagenase with acid responsive release characteristics, nanogels (RGD / Col-NR) containing RGD sequence integrin targeting peptide and collagenase without acid responsive release characteristics, nanogels (RGD) containing RGD sequence integrin targeting peptide with acid responsive release characteristics, nanogels (Col) containing collagenase with acid responsive release characteristics, and blank nanogel preparations were prepared respectively. The above preparations were taken and the particle size of the nanogel was measured by dynamic light scattering (DLS). The results are shown in Table 1. The preparations have a good particle size, the particle size is between 100 and 120 nm, and have good stability and biological activity.

[0130] (3) In vitro release of RGD sequence integrin targeting peptide and collagenase

[0131] Each group of preparations was mixed with pH 6.5 PBS buffer and pH 7.4 PBS buffer, respectively, and incubated at 37°C and 100 rpm for 48 hours. The released Col I-FITC was removed by ultrafiltration (MWCO = 300 kDa) at 0, 1, 2, 3, 6, 9, 12, 24, and 48 hours, the fluorescence intensity of the filtrate was measured, and the amount of Col I-FITC released was calculated. The concentrate was supplemented to the original volume with the corresponding PBS solution.

[0132] The results are as follows Figure 4As shown, the release of Col I in RGD / Col is acid-responsive, while the RGD / Col-NR group basically does not release Col I under acidic conditions.

[0133] Each group of preparations was mixed with pH 6.5 PBS buffer and pH 7.4 PBS buffer, respectively, and incubated at 37°C and 100 rpm for 48 hours. Free C18-CD9 and VRGD cleaved by enzyme were removed by ultrafiltration (MWCO = 300 kDa) at 0, 1, 2, 3, 6, 9, 12, 24, and 48 hours, and the lipid concentrate was supplemented to the original volume with the corresponding PBS buffer, and the dispersed RGD was quantified using the BCA method.

[0134] The results are as follows Figure 4 As shown, the release of RGD peptide in RGD / Col is acid-responsive, while the RGD / Col-NR group basically does not release RGD peptide under acidic conditions.

[0135] Example 7: Preparation and formulation characterization of liposomes containing RGD sequence integrin targeting peptide and collagenase

[0136] (1) Preparation of liposomes containing RGD sequence integrin targeting peptide and collagenase

[0137] Nanoformulations loaded with type I collagenase and RGD peptide can be prepared by thin film dispersion method, injection method or reverse solvent method. Weigh 10 mg soybean lecithin, 1.25 mg cholesterol, 1.25 mg DSPE-PEG-CREKA, and 4.836 mg integrin targeting peptide C18-CGPLGVRGD containing RGD sequence (from Gill Biochemical) in a 250 mL eggplant-shaped bottle, add 1 mL chloroform and 1 mL methanol to dissolve, and form a lipid film by rotary evaporation at 37 ° C (120 rpm, 20 min), and vacuum dry overnight.

[0138] Preparation of liposome nanoparticles by hydration: 0.802 mg of type I collagenase Col I and 1.12 mg of tannic acid TA were weighed and dissolved in 0.5 mL of HEPES solution (10 mM, pH = 7.4), and the TA HEPES solution was slowly added to the Col I HEPES solution under vortexing. The HEPES solution of TA and Col I was used to hydrate the lipid film (37°C, 150 rpm, 30 min), and probe ultrasound was used (ultrasonic power 30%, ultrasound time 2 min) to form a light yellow solution with opalescence.

[0139] Removal of free Col I-FITC and TA: After preparing liposomes using FITC-labeled Col I (Col I-FITC), Urea was added to the preparation and incubated at room temperature for 10 min to disrupt the hydrogen bond between TA and Col I. Free Col I-FITC and TA were removed by ultrafiltration (3000 rpm, 30 min, MWCO = 300 kDa).

[0140] (2) Pharmaceutical characterization of liposomes containing RGD sequence integrin targeting peptide and collagenase

[0141] By using the above preparation method, liposome Clip (RGD / Col) containing RGD sequence integrin targeting peptide and collagenase with acidic responsiveness and enzyme responsiveness release characteristics, liposome DEPE-PEG-RGD / Col containing RGD sequence integrin targeting peptide and collagenase without responsive release characteristics, liposome Clip (RGD) containing RGD sequence integrin targeting peptide with acidic responsiveness and enzyme responsiveness release characteristics, liposome Clip (Col) containing collagenase with acidic responsiveness and enzyme responsiveness release characteristics, and blank liposome preparation Clip were prepared respectively. The above preparations were taken and the particle size of the liposomes was measured by dynamic light scattering (DLS). The results are shown in Table 1. The preparations have a good particle size between 60 and 80 nm, and have good stability in PBS solution, physiological buffered saline solution, and DMEM culture medium containing 10% serum.

[0142] (3) In vitro release of RGD sequence integrin targeting peptide and collagenase

[0143] Each group of preparations was mixed with pH 6.5 PBS buffer (containing 2 mg / mL type IV collagenase), pH 7.4 PBS buffer (containing 2 mg / mL type IV collagenase), and pH 7.4 PBS solution (1:1, v / v), and incubated at 37°C and 100 rpm for 48 hours. Urea was added at 0, 1, 2, 3, 6, 9, 12, 24, and 48 hours, and the released Col I-FITC was removed by ultrafiltration (MWCO=300 kDa). The fluorescence intensity of the filtrate was measured and the amount of Col I-FITC released was calculated, and the concentrate was supplemented to the original volume with the corresponding PBS solution.

[0144] The results are as follows Figure 5 As shown in the figure, the release of Col I in CLIp-(RGD / Col) mainly depends on the cleavage of the collagenase IV responsive peptide, and the acidic environment will further promote the release of Col I. However, the liposomes prepared using DSPE-PEG-RGD basically do not release Col I in an acidic environment containing collagenase IV.

[0145] Each group of preparations was mixed with pH 6.5 PBS buffer (containing 2 mg / mL type IV collagenase) or pH 7.4 PBS solution (1:1, v / v), incubated at 37°C and 100 rpm for 48 hours, and ultrafiltration (MWCO=300 kDa) was used to remove free C18-CD9 and VRGD cleaved by the enzyme at 0, 1, 2, 3, 6, 9, 12, 24, and 48 hours. The lipid concentrate was supplemented to the original volume with the corresponding PBS buffer, and the dispersed RGD was quantified using the BCA method.

[0146] The results are as follows Figure 5 As shown, the release of RGD peptide in CLIp-(RGD / Col) is responsive to collagenase IV, while liposomes prepared using DSPE-PEG-RGD basically do not release RGD peptide in an acidic environment containing collagenase IV.

[0147] Table 1 Pharmaceutical parameters of the preparations prepared in Examples 1 to 7

[0148]

[0149]

[0150] Example 8: Mechanism of inhibition of fibroblast activation by combining integrin targeting peptide containing RGD sequence and collagenase

[0151] Using the RGD sequence-containing integrin targeting peptide CGPLGVRGD, RGD / Col group solution, Col group solution, RGD group solution and blank solution were prepared according to the method of Example 1, and the mechanism of the solution containing the RGD sequence-containing integrin targeting peptide and collagenase in inhibiting fibroblast activation was explored.

[0152] Uniform tumor spheres were selected and transferred to a 96-well plate using a sterile pipette tip. The tumor spheres were incubated with the preparations in each group for 48 h. The changes in the levels of mechanical transduction pathway protein (YAP) and TGF-β / Smad pathway protein (P-Smad2) were analyzed by WB, and quantitative analysis was performed using Image J software.

[0153] The results are as follows Figure 6As shown, the collagenase released by RGD / Col hydrolyzes collagen and cuts the recognition sequence of integrin (α1β1, α2β1, α10β1 and α11β1) on collagen. The RGD peptide competes with fibronectin for binding to integrins (α5β1, α8β1, αvβ1 and αIIbβ1) on the surface of fibroblasts, resulting in impaired mechanical sensing and inhibition of the mechanical pathway, which is manifested by decreased YAP expression. In addition, the RGD peptide released in the preparation competes with LAP for binding to αvβ6 integrin on NIH 3T3 cells and αvβ6 and αvβ8 integrins on Panc-02 cells, resulting in decreased secretion of active TGF-β and inhibition of the TGF-β-mediated biochemical signaling pathway, which is manifested by decreased P-Smad2 expression. That is, RGD / Col inhibits fibroblast activation from both mechanical signaling and biochemical signaling.

[0154] Example 9: Effect of the combined use of integrin targeting peptide containing RGD sequence and collagenase on the secretion of active TGF-β

[0155] After preparing CLIp-(RGD / Col), CLIp-(Col), CLIp-(RGD), CLIp and liposomes without responsive RGD peptide (non-releasing type) according to the method of Example 7, the inhibition of active TGF-β release by extracellular RGD molecules was verified.

[0156] (1) Preparation and collection of tumor spheres

[0157] After preparing Panc-02 / NIH3T3 mixed tumor spheres using the existing method in the group, the tumor spheres were transferred to a 96-well plate using a sterile pipette tip, and the preparation was given once every 24 hours, 100 μL each time, for a total of two doses; after the administration, the tumor spheres were transferred to a 1.5 mL centrifuge tube and centrifuged at 2000 rpm for 5 min. The tumor sphere pellet was collected and resuspended in 200 μL blank DMEM culture medium, and centrifuged again at 14000 rpm for 4 min to collect the supernatant and discard the cell debris.

[0158] (2) Activation and dilution of cell culture supernatant

[0159] Take 100 μL of supernatant, add 20 μL of HCL (1N), mix well and incubate at room temperature for 10 min, then add 20 μL of NaOH (1.2N) for neutralization and mix well, and measure the total amount of TGF-β.

[0160] Take 100 μL of cell supernatant and measure directly the active TGF-β content.

[0161] (3) Measurement of TGF-β content using ELISAKit

[0162] Add the sample to be tested (50 μL) to the enzyme-labeled plate, add the enzyme-labeled reagent (100 μL), seal the plate with a sealing film and incubate at 37°C for 60 min; remove the sealing film, discard the liquid, fill each well with washing solution (300 μL), let it stand for 30 seconds and then discard, repeat 5 times; first add color developer A (50 μL) to each well, then add color developer B (50 μL), gently shake and mix, and color at 37°C in the dark for 15 min; add 50 μL of stop solution to each well to terminate the reaction, measure the absorbance value of each well in sequence at a wavelength of 450 nm, and substitute it into the standard curve to calculate the TGF-β content.

[0163] The results are as follows Figure 7 As shown, CLIp-(RGD / Col), CLIp-(RGD), and CLIp-(Col) all significantly reduced the production of active TGF-β, and the inhibitory effects from small to large were: (CLip-(RGD / Col)>CLip-(Col)>CLip-(RGD), and there was a synergistic effect between the RGD peptide and Col I in the combination group; while the non-releasing control group liposomes (DSPE-PEG-RGD) failed to release RGD peptide molecules in the microenvironment, could not compete with the latent TGF-β complex for binding to integrin, and therefore failed to reduce the production of active TGF-β.

[0164] Example 10: Pharmacodynamic evaluation of a solution containing an RGD sequence integrin targeting peptide and collagenase

[0165] A solution was prepared according to the method of Example 1 and applied to the study of liver fibrosis treatment, to evaluate the effect of a solution containing an RGD sequence integrin targeting peptide and collagenase on the secretion of type I collagen in the extracellular matrix (ECM) by hepatic stellate cells (HSC-T6), and to explore its anti-liver fibrosis potential.

[0166] Experimental groups: blank control group, solution containing RGD sequence integrin targeting peptide and collagenase (RGD / Col), solution containing RGD sequence integrin targeting peptide (RGD), solution containing collagenase (Col).

[0167] HSC-T6 hepatic stellate cells were cultured in a 37°C, 5% CO2 incubator in DMEM medium containing 10% fetal bovine serum, and cells in the logarithmic growth phase were used for the experiment. HSC-T6 cells were seeded in a 6-well plate, and after the cells adhered to the wall, they were replaced with blank medium for 12 hours, and then each group of drugs was added and cultured for 24 hours.

[0168] Western blot was used to detect the expression of type I collagen: cells were collected and total protein was extracted; protein concentration was determined by BCA method; proteins were separated by SDS-PAGE electrophoresis and transferred to PVDF membrane; 5% skim milk was used for blocking for 1 hour; type I collagen primary antibody was added and incubated at 4°C overnight; the membrane was washed with TBST 3 times, 10 minutes each time; HRP-labeled secondary antibody was added and incubated at room temperature for 1 hour; the membrane was washed with TBST 3 times, 10 minutes each time. ECL luminescent solution was used for development, and the gray value of the bands was analyzed by Image J software.

[0169] The solution was prepared according to the method of Example 1. Compared with the control group, the expression of type I collagen in the GRGDSP group and the collagenase group decreased by about 75% and 79%, respectively. The expression of type I collagen in the GRGDSP combined with collagenase group was significantly reduced, with a reduction rate of 87%, which was statistically different from the group using the drug alone (P < 0.05); compared with the control group, the expression of type I collagen in the GRGDS group and the collagenase group was reduced by about 80% and 83%, respectively. The expression of type I collagen in the GRGDS combined with collagenase group was significantly reduced, with a reduction rate of 89%, which was statistically different from the group using the drug alone (P < 0.05); compared with the control group, the expression of type I collagen in the RGDS group and the collagenase group was reduced by about 78% and 76%, respectively. The expression of type I collagen in the RGDS combined with collagenase group was significantly reduced, with a reduction rate of 86%, which was statistically different from the group using the single drug (P < 0.05); compared with the control group, the expression of type I collagen in the RGDV group and the collagenase group was reduced, about 68% and 73%, respectively. The expression of type I collagen in the RGDV combined with collagenase group was significantly reduced, with a reduction rate of 84%, which was statistically different from the group using the single drug (P < 0.05); compared with the control group, the expression of type I collagen in the c(RGDfK) group and the collagenase group was reduced, about 69% and 73%, respectively. The expression of type I collagen in the c(RGDfK) combined with collagenase group was significantly reduced, with a reduction rate of 87%, which was statistically different from the group using the single drug (P < 0.05); compared with the control group, the expression of type I collagen in the c(RGDyK) group and the collagenase group was reduced, about 64% and 68%, respectively. The expression of type I collagen in the c(RGDyK) combined with collagenase group was significantly reduced, with a reduction rate of 80%, which was statistically different from the group using the drug alone (P<0.05); compared with the control group, the expression of type I collagen in the c(RGDfV) group and the collagenase group was reduced by about 75% and 77%, respectively. The expression of type I collagen in the c(RGDfV) combined with collagenase group was significantly reduced, with a reduction rate of 89%, which was statistically different from the group using the drug alone (P<0.05); compared with the control group, the expression of type I collagen in the c(RGDfE) group and the collagenase group was reduced by about 73% and 77%, respectively. The expression of type I collagen in the c(RGDfE) combined with collagenase group was significantly reduced, with a reduction rate of 87%, which was statistically different from the group using either drug alone (P < 0.05). Compared with the control group, the expression of type I collagen in the c(RGDfC) group and the collagenase group decreased by approximately 69% and 71%, respectively.The expression of type I collagen in the c(RGDfC) combined with collagenase group was significantly reduced, with a reduction rate of 85%, which was statistically different from the group using the single drug (P < 0.05); compared with the control group, the expression of type I collagen in the c(RGDf[N-Methyl]V) group and the collagenase group was reduced by about 73% and 76%, respectively. The expression of type I collagen in the c(RGDf[N-Methyl]V) combined with collagenase group was significantly reduced, with a reduction rate of 87%, which was statistically different from the group using the single drug (P < 0.05); compared with the control group, the expression of type I collagen in the c(RGDyC) group and the collagenase group was reduced by about 77% and 78%, respectively. The expression of type I collagen in the c(RGDyC) combined with collagenase group was significantly reduced, with a reduction rate of 90%, which was statistically different from the group using the drug alone (P < 0.05); compared with the control group, the expression of type I collagen in the RGD-PEG group and the collagenase group was reduced by about 79% and 81%, respectively. The expression of type I collagen in the RGD-PEG combined with collagenase group was significantly reduced, with a reduction rate of 94%, which was statistically different from the group using the drug alone (P < 0.05); compared with the control group, the expression of type I collagen in the iRGD group and the collagenase group was reduced by about 78% and 81%, respectively. The expression of type I collagen in the iRGD combined with collagenase group was significantly reduced, with a reduction rate of 95%, which was statistically different from the group using the drug alone (P < 0.05).

[0170] The results are as follows Figure 8 As shown in the figure, compared with the control group, the expression of type I collagen in the CGPLGVRGD group and the collagenase group decreased, but the decrease was small, about 80% and 85% respectively. The expression of type I collagen in the combined drug group was significantly reduced, with a reduction rate of 95%, which was statistically different from the single drug group (P < 0.05).

[0171] The experimental results showed that the combined solution of integrin targeting peptide containing RGD sequence and collagenase can significantly inhibit the secretion of type I collagen by HSC-T6 cells. Its mechanism of action may include: integrin targeting peptide competitively binds to integrin receptors, blocks integrin-mediated signaling pathways, inhibits HSCs (Hepatic Stellate Cells) activation, and reduces ECM synthesis. Collagenase directly degrades the formed collagen fibers and promotes ECM degradation. The combination of the two simultaneously blocks the mechanical and biochemical signals that mediate fibrosis, effectively inhibits the activation of fibroblasts, reduces the secretion of extracellular matrix (ECM) proteins, and blocks the positive feedback loop between ECM rigidity and fibroblast activation. The combined drug group showed a synergistic effect, and its effect of inhibiting the expression of type I collagen was significantly better than that of the single drug group, suggesting that the combined use of the two drugs has better anti-liver fibrosis potential.

[0172] Example 11: Pharmacodynamic evaluation of a suspension containing an RGD sequence integrin targeting peptide and collagenase

[0173] The suspension was prepared according to the method of Example 2 and applied to the study of pulmonary fibrosis treatment, to evaluate the effect of the suspension combined with RGD sequence integrin targeting peptide and collagenase on the expression of type I collagen in the treatment of pulmonary fibrosis, and to explore its anti-pulmonary fibrosis potential.

[0174] Experimental groups: blank control group, suspension containing RGD sequence integrin targeting peptide and collagenase (RGD / Col), suspension containing RGD sequence integrin targeting peptide (RGD), and suspension containing collagenase (Col).

[0175] Lung fibroblasts were cultured in a 37°C, 5% CO2 incubator in DMEM medium containing 10% fetal bovine serum, and cells in the logarithmic growth phase were used for the experiment. Lung fibroblasts were seeded in a 6-well plate, and after the cells adhered to the wall, they were replaced with blank medium for 12 hours, and then each group of drugs was added and cultured for another 24 hours.

[0176] Western blot was used to detect the expression of type I collagen: cells were collected and total protein was extracted; protein concentration was determined by BCA method; proteins were separated by SDS-PAGE electrophoresis and transferred to PVDF membrane; 5% skim milk was used for blocking for 1 hour; type I collagen primary antibody was added and incubated at 4°C overnight; the membrane was washed with TBST 3 times, 10 minutes each time; HRP-labeled secondary antibody was added and incubated at room temperature for 1 hour; the membrane was washed with TBST 3 times, 10 minutes each time. ECL luminescent solution was used for development, and the gray value of the bands was analyzed by Image J software.

[0177] The results are as follows Fig. 9 As shown in the figure, compared with the control group, the expression of type I collagen in the integrin targeting peptide group and the collagenase group decreased, but the decrease was small, about 60% and 65% respectively. The expression of type I collagen in the combined drug group was significantly reduced, with a reduction rate of 75%, which was statistically different from the single drug group (P < 0.05).

[0178] The experimental results showed that the combined suspension of integrin targeting peptide containing RGD sequence and collagenase can significantly inhibit the secretion of type I collagen by lung fibroblasts. The combined drug group showed a synergistic effect, and its effect of inhibiting type I collagen expression was significantly better than that of the single drug group, suggesting that the combined use of the two drugs has better anti-pulmonary fibrosis potential.

[0179] Example 12: Pharmacodynamic evaluation of microcapsules containing RGD sequence integrin targeting peptide and collagenase

[0180] (1) In vitro pharmacodynamic evaluation of microcapsules containing RGD sequence integrin targeting peptide and collagenase

[0181] Microcapsules were prepared according to the method of Example 3 and applied to the study of liver fibrosis treatment. The effects of microcapsules containing RGD sequence integrin targeting peptide and collagenase on the content of α-SMA (α-smooth muscle actin) in the liver fibrosis microenvironment were evaluated to explore its anti-liver fibrosis potential.

[0182] Experimental groups: blank control group, RGD / Col group, RGD / Col-NR group, RGD group, and Col group.

[0183] Cell lines related to liver fibrosis (such as hepatic stellate cells LX-2 or HepG2) were cultured in low-adsorption culture dishes to form uniform tumor spheres (Spheroids) to simulate the microenvironment of liver fibrosis. The morphology and size of the tumor spheres were observed under a microscope to ensure that the diameter of the tumor spheres was uniform (about 100-200 μm) to ensure the repeatability of the experiment. The tumor spheres were transferred to a 96-well plate using a sterile pipette tip, and the tumor spheres were co-incubated with each group of preparations and incubated in an incubator at 37°C and 5% CO2 for 48 hours to simulate the in vivo environment. After 48 hours, the tumor spheres were collected and digested, and the changes in the content of α-SMA in the tumor spheres of each group were analyzed by WB method.

[0184] The results are as follows Fig.10 As shown in the data, compared with the control group, the expression of α-SMA in the RGD / Col-NR group remained at a high level, close to that in the control group; the expression of α-SMA in the RGD group and collagenase group decreased, but the effect was weaker than that in the RGD / Col group (P < 0.05), and the expression of α-SMA in the RGD / Col group was significantly decreased (P < 0.05).

[0185] The experimental results showed that the simultaneous release of free RGD peptide and collagenase in the fibrotic microenvironment had a synergistic effect and could effectively inhibit the activation of fibroblasts. The RGD / Col combination group showed a synergistic effect, and its fibroblast activation effect was significantly better than that of the single drug group, suggesting that the combination of the two drugs has better anti-liver fibrosis potential.

[0186] (2) In vivo pharmacodynamic evaluation of microcapsules containing RGD sequence integrin targeting peptide and collagenase

[0187] Microcapsules were prepared according to the method of Example 3 and applied to the study of liver fibrosis treatment. The effects of microcapsules containing RGD sequence integrin targeting peptide and collagenase on the expression of type I collagen in liver tissue of liver fibrosis model mice were evaluated to explore their anti-liver fibrosis potential.

[0188] Establishment of liver fibrosis model: C57BL / 6 mice were randomly divided into 5 groups, namely normal control group, blank control group, RGD / Col group, RGD / Col-NR group, RGD group, and Col group. Except for the normal control group, mice in other groups were intraperitoneally injected with CCl4 (0.5mL / kg, 20% CCl4 olive oil solution, twice a week) to establish liver fibrosis model for 8 weeks. Mice in the normal control group were intraperitoneally injected with an equal amount of olive oil.

[0189] Drug administration began on the second day after modeling, with each group receiving the corresponding drug, and the normal control group receiving an equal amount of saline. Administration method: tail vein injection, twice a week, for 8 consecutive weeks. 24 hours after the last administration, the mice were killed and liver tissues were taken for Western blot detection.

[0190] The results are as follows Fig.10 As shown in the figure, compared with the normal control group, the expression level of type I collagen in the liver tissue of mice in the blank preparation control group was significantly increased. Compared with the blank preparation control group, the expression level of type I collagen in the RGD / Col-NR group was close to that in the control group; the expression level of type I collagen in the integrin targeting peptide group and collagenase group decreased by 65% ​​and 67%, respectively, with a small decrease. The expression level of type I collagen in the RGD / Col combined group was significantly reduced, with a reduction rate of about 75%, which was statistically different from the single drug group (P < 0.05).

[0191] The experimental results showed that the microcapsules containing RGD sequence integrin targeting peptide and collagenase released free RGD sequence integrin targeting peptide and collagenase in the microenvironment, which could significantly inhibit the expression of type I collagen in the liver tissue of mice with liver fibrosis model. The combination group showed a synergistic effect, and its effect of inhibiting type I collagen expression was significantly better than that of the single drug group, suggesting that the combined use of the two drugs has better anti-liver fibrosis potential.

[0192] Example 13: Pharmacodynamic evaluation of microspheres containing RGD sequence integrin targeting peptide and collagenase

[0193] (1) In vitro pharmacodynamic evaluation of microspheres containing RGD sequence integrin targeting peptide and collagenase

[0194] Microspheres were prepared according to the method of Example 4 and applied to the study of pulmonary fibrosis treatment. The effects of microspheres containing RGD sequence integrin targeting peptide and collagenase on the α-SMA content in the pulmonary fibrosis microenvironment were evaluated to explore their anti-pulmonary fibrosis potential.

[0195] Experimental groups: blank control group, RGD / Col group, RGD / Col-NR group, RGD group, and Col group.

[0196] Cell lines related to pulmonary fibrosis (such as MRC-5, IMR-90, etc.) were cultured in low-adsorption culture dishes to form uniform spheroids to simulate the pulmonary fibrosis microenvironment. The morphology and size of the spheroids were observed under a microscope to ensure that the diameter of the spheroids was uniform (about 100-200 μm) to ensure the repeatability of the experiment. The spheroids were transferred to a 96-well plate using a sterile pipette tip, and the spheroids were co-incubated with each group of preparations and incubated in an incubator at 37°C and 5% CO2 for 48 hours to simulate the in vivo environment. After 48 hours, the spheroids were collected, and after digestion, the WB method was used to analyze the changes in the content of α-SMA in the spheroids of each group.

[0197] The results are as follows Fig.11 As shown in the data, compared with the control group, the expression of α-SMA in the RGD / Col-NR group was maintained at a high level, close to that in the control group; the expression of α-SMA in the RGD group and collagenase group was reduced, but the effect was weaker than that in the RGD / Col group (P < 0.05), and the expression of α-SMA in the RGD / Col group was significantly reduced (P < 0.05).

[0198] The experimental results showed that the synergistic effect of free RGD peptide and collagenase released in the fibrotic microenvironment effectively inhibited the activation of fibroblasts. The RGD / Col combination group showed a synergistic effect, and its fibroblast activation effect was significantly better than that of the single drug group, suggesting that the combination of the two drugs has better anti-pulmonary fibrosis potential.

[0199] (2) In vivo pharmacodynamic evaluation of microspheres containing RGD sequence integrin targeting peptide and collagenase

[0200] Microspheres were prepared according to the method of Example 4 and applied to the study of pulmonary fibrosis treatment. The effects of microspheres containing RGD sequence integrin targeting peptide and collagenase on the expression of type I collagen in lung tissue of pulmonary fibrosis model mice were evaluated to explore their anti-pulmonary fibrosis potential.

[0201] Establishment of pulmonary fibrosis model: C57BL / 6 mice were randomly divided into 5 groups, namely normal control group, blank control group, RGD / Col group, RGD / Col-NR group, RGD group, and Col group. Except for the normal control group, BLM (5 mg / kg) was instilled into the trachea of ​​mice in the other groups to establish pulmonary fibrosis model.

[0202] The drug administration started on the second day after modeling, and each group was given the corresponding drug, and the normal control group was given an equal amount of normal saline. Administration method: tail vein injection, once a day, for 14 consecutive days. The mice in the normal control group were instilled with an equal amount of normal saline through the trachea. 24 hours after the last administration, the mice were killed and the lung tissues were taken for Western blot detection.

[0203] The results are as follows Fig.11As shown in the figure, compared with the normal control group, the expression level of type I collagen in the lung tissue of mice in the blank preparation control group was significantly increased. Compared with the blank preparation control group, the expression level of type I collagen in the RGD / Col-NR group was close to that in the control group; the expression level of type I collagen in the integrin targeting peptide group and collagenase group decreased by 67% and 70%, respectively, with a small decrease. The expression level of type I collagen in the RGD / Col combined group was significantly reduced, with a reduction rate of about 85%, which was statistically different from the single drug group (P < 0.05).

[0204] The experimental results showed that the microspheres containing RGD sequence integrin targeting peptide and collagenase released free RGD sequence integrin targeting peptide and collagenase in the microenvironment, which could significantly inhibit the expression of type I collagen in the lung tissue of mice with pulmonary fibrosis model. The combination group showed a synergistic effect, and its effect of inhibiting type I collagen expression was significantly better than that of the single drug group, suggesting that the combined use of the two drugs has better anti-pulmonary fibrosis potential.

[0205] Example 14: Pharmacodynamic evaluation of nanoparticles containing RGD sequence integrin targeting peptide and collagenase

[0206] (1) In vitro pharmacodynamic evaluation of nanoparticles containing RGD sequence integrin targeting peptide and collagenase

[0207] Nanoparticles were prepared according to the method of Example 5 and applied to the study of pancreatic fibrosis treatment. The inhibitory effect of nanoparticles combined with RGD sequence integrin targeting peptide and collagenase on pancreatic stellate cells (PSCs) was evaluated, the changes in α-SMA content were detected, and its anti-pancreatic fibrosis potential was explored.

[0208] Experimental groups: blank control group, RGD / Col group, RGD / Col-NR group, RGD group, and Col group.

[0209] PSCs were isolated from pancreatic fibrosis model mice and cultured in DMEM medium containing 10% fetal bovine serum in an incubator at 37°C and 5% CO2. PSCs were seeded in 6-well plates, and when the cell density reached 80%, nanoparticles from different treatment groups were added and cultured for 48 hours.

[0210] Western blot was used to detect the changes in α-SMA content. α-SMA is a marker of PSCs activation, and its content changes directly reflect the degree of PSCs activation. Cells were collected and total protein was extracted; protein concentration was determined by BCA method; protein was separated by SDS-PAGE electrophoresis and transferred to PVDF membrane; 5% skim milk was used for blocking for 1 hour; α-SMA primary antibody was added and incubated at 4°C overnight; TBST was used to wash the membrane 3 times, 10 minutes each time; HRP-labeled secondary antibody was added and incubated at room temperature for 1 hour; TBST was used to wash the membrane 3 times, 10 minutes each time. ECL luminescent solution was used for development, and the gray value of the bands was analyzed by Image J software.

[0211] The results are as follows Fig.12 As shown in the data, compared with the control group, the expression of α-SMA in the RGD / Col-NR group remained at a high level, close to that in the control group; the expression of α-SMA in the RGD group and collagenase group decreased, but the effect was weaker than that in the RGD / Col group (P < 0.05), and the expression of α-SMA in the RGD / Col group was significantly decreased (P < 0.05).

[0212] The experimental results showed that the free RGD peptide and collagenase released in the microenvironment had a good synergistic effect and could effectively inhibit the activation of fibroblasts. The RGD / Col combination group showed a synergistic effect, and its fibroblast activation effect was significantly better than that of the single drug group, suggesting that the combination of the two drugs has better anti-pancreatic fibrosis potential.

[0213] (2) In vivo pharmacodynamic evaluation of nanoparticles containing RGD sequence integrin targeting peptide and collagenase

[0214] Nanoparticles were prepared according to the method of Example 5 and applied to the study of pancreatic fibrosis treatment. The effect of nanoparticles containing RGD sequence integrin targeting peptide and collagenase on the expression of type I collagen in pancreatic tissue of pancreatic fibrosis model mice was evaluated to explore its anti-pancreatic fibrosis potential.

[0215] Establishment of pancreatic fibrosis model: C57BL / 6 mice were randomly divided into 5 groups, namely normal control group, blank control group, RGD / Col group, RGD / Col-NR group, RGD group, and Col group. Except for the normal control group, the pancreatic fibrosis model was established by intratracheal instillation of BLM (5 mg / kg) in the other groups of mice. Pancreatic fibrosis was induced in mice by intraperitoneal injection of diethyldithiocarbamate (DDC) for 4 weeks.

[0216] The drug administration started on the second day after the model was established. Each group was given the corresponding drug, and the normal control group was given an equal amount of normal saline. Administration method: tail vein injection, once a day, for 14 consecutive days. The mice in the normal control group were instilled with an equal amount of normal saline through the trachea. 24 hours after the last administration, the mice were killed and pancreatic tissues were collected for Western blot detection.

[0217] The results are as follows Fig.12 As shown in the figure, compared with the normal control group, the expression level of type I collagen in the pancreatic tissue of mice in the blank preparation control group was significantly increased. Compared with the blank preparation control group, the expression level of type I collagen in the RGD / Col-NR group was close to that in the control group; the expression level of type I collagen in the integrin targeting peptide group and collagenase group decreased by 70% and 73%, respectively, with a small decrease. The expression level of type I collagen in the RGD / Col combined group was significantly reduced, with a reduction rate of about 90%, which was statistically different from the single drug group (P < 0.05).

[0218] The experimental results showed that nanoparticles containing RGD sequence integrin targeting peptide and collagenase released free RGD sequence integrin targeting peptide and collagenase in the microenvironment, which could significantly inhibit the expression of type I collagen in the pancreatic tissue of pancreatic fibrosis model mice. The combination group showed a synergistic effect, and its effect of inhibiting type I collagen expression was significantly better than that of the single drug group, suggesting that the combined use of the two drugs has better anti-pancreatic fibrosis potential.

[0219] Example 15: Pharmacodynamic evaluation of gel containing RGD sequence integrin targeting peptide and collagenase

[0220] (1) In vitro pharmacodynamic evaluation of a gel containing an RGD sequence integrin targeting peptide and collagenase

[0221] Nanogels were prepared according to the method of Example 6 and applied to the study of bladder cancer treatment. The effects of nanogels containing RGD sequence integrin targeting peptides and collagenase on the content of α-SMA (α-smooth muscle actin) in the bladder cancer microenvironment were evaluated to explore their potential for treating bladder cancer.

[0222] Experimental groups: blank control group, RGD / Col group, RGD / Col-NR group, RGD group, and Col group.

[0223] Bladder cancer cells (such as T24 or 5637 cell lines) were cultured in low-adsorption culture dishes to form uniform spheroids to simulate the fibrotic microenvironment of bladder cancer. The morphology and size of the spheroids were observed under a microscope to ensure that the diameter of the spheroids was uniform (about 100-200 μm) to ensure the repeatability of the experiment. The spheroids were transferred to a 96-well plate using a sterile pipette tip, and the spheroids were co-incubated with each group of preparations and incubated in an incubator at 37°C and 5% CO2 for 48 hours to simulate the in vivo environment. After 48 hours, the spheroids were collected and digested, and the changes in the content of α-SMA in the spheroids of each group were analyzed by WB.

[0224] The results are as follows Fig.13 As shown in the data, compared with the control group, the expression of α-SMA in the RGD / Col-NR group remained at a high level, close to that in the control group; the expression of α-SMA in the RGD group and collagenase group decreased, but the effect was weaker than that in the RGD / Col group (P < 0.05), and the expression of α-SMA in the RGD / Col group was significantly decreased (P < 0.05).

[0225] The experimental results showed that the free RGD peptide and collagenase released in the microenvironment had a good synergistic effect and could effectively inhibit the activation of fibroblasts. The RGD / Col combination group showed a synergistic effect, and its fibroblast activation effect was significantly better than that of the single drug group, suggesting that the combination of the two drugs can better improve the level of fibrosis in bladder cancer.

[0226] (2) In vivo pharmacodynamic evaluation of the gel containing the RGD sequence integrin targeting peptide and collagenase

[0227] Nanogels were prepared according to the method of Example 6 and applied to the treatment of bladder cancer fibrosis. The effects of the RGD sequence-containing integrin targeting peptide and collagenase combined gel preparation on the expression of type I collagen in the bladder tissue of nude mice with bladder cancer transplanted tumor models were evaluated to explore its anti-bladder cancer fibrosis potential.

[0228] Establishment of bladder cancer transplant model: Human bladder cancer cells in the logarithmic growth phase were inoculated subcutaneously on the right back of nude mice. The number of cells inoculated per mouse was 1×10 6 Observe the tumor growth regularly and wait until the tumor volume reaches 100mm 3 When left and right, randomly group them.

[0229] Experimental groups: normal control group, blank control group, RGD / Col group, RGD / Col-NR group, RGD group, and Col group.

[0230] Each group was given corresponding drugs, and the normal control group was given normal saline. Administration method: tail vein injection, twice a week, for 4 consecutive weeks. 24 hours after the last administration, the nude mice were killed and the bladder tissue was taken for Western blot detection.

[0231] The results are as follows Fig.13 As shown in the figure, compared with the normal control group, the expression level of type I collagen in the bladder tissue of mice in the blank preparation control group was significantly increased. Compared with the blank preparation control group, the expression level of type I collagen in the RGD / Col-NR group was close to that in the control group; the expression level of type I collagen in the integrin targeting peptide group and collagenase group decreased by 65% ​​and 70%, respectively, with a small decrease. The expression level of type I collagen in the RGD / Col combined group was significantly reduced, with a reduction rate of about 80%, which was statistically different from the single drug group (P < 0.05).

[0232] The experimental results showed that the nanogel containing RGD sequence integrin targeting peptide and collagenase released free RGD sequence integrin targeting peptide and collagenase in the microenvironment, which could significantly inhibit the expression of type I collagen in the bladder tissue of mice with bladder cancer fibrosis model. The combination group showed a synergistic effect, and its effect of inhibiting type I collagen expression was significantly better than that of the single drug group, suggesting that the combined use of the two drugs has better anti-bladder cancer fibrosis potential.

[0233] Example 16: Pharmacodynamic evaluation of liposomes containing RGD sequence integrin targeting peptide and collagenase

[0234] (1) In vitro pharmacodynamic evaluation of liposomes containing RGD sequence integrin targeting peptide and collagenase

[0235] After preparing CLIp-(RGD / Col), CLIp-(Col), CLIp-(RGD), DEPE-PEG-RGD / Col and CLIp according to the method of Example 7, the ability of the preparations to enhance the penetration of chemotherapeutic drugs in tumor spheres was investigated.

[0236] Uniform tumor spheres were selected and transferred to a 96-well plate using a sterile pipette tip. The tumor spheres were incubated with the preparations in each group for 48 h, and the tumor spheres were collected. After digestion, the changes in the α-SMA content in the tumor spheres in each group were analyzed using the WB method.

[0237] The results are as follows Fig.14As shown in the data, compared with the control group, the expression of α-SMA in the DEPE-PEG-RGD / Col group remained at a high level, close to that of the control group; the expression of α-SMA in the CLIp-(RGD) group and the collagenase group decreased, but the effect was weaker than that in the CLIp-(RGD / Col) group (P < 0.05), and the expression of α-SMA in the CLIp-(RGD / Col) group was significantly decreased (P < 0.05).

[0238] The experimental results showed that the free RGD peptide and collagenase released in the microenvironment had a good synergistic effect and could effectively inhibit the activation of fibroblasts. The RGD / Col combination group showed a synergistic effect, and its fibroblast activation effect was significantly better than that of the single drug group, suggesting that the combination of the two drugs can better improve the level of fibrosis in pancreatic cancer.

[0239] (2) In vivo pharmacodynamic evaluation of liposomes containing RGD sequence integrin targeting peptide and collagenase

[0240] Liposomes were prepared according to the method of Example 7 and applied to the study of pancreatic cancer treatment. The effects of liposome preparations containing RGD sequence integrin targeting peptide and collagenase on the expression of type I collagen in pancreatic tissue of nude mice with pancreatic cancer transplanted tumor models were evaluated to explore its anti-fibrosis potential of pancreatic cancer.

[0241] Establishment of pancreatic cancer transplant model: Human pancreatic cancer cells in the logarithmic growth phase were inoculated subcutaneously on the right back of nude mice. The number of cells inoculated per mouse was 1×10 6 Observe the tumor growth regularly and wait until the tumor volume reaches 100mm 3 When left and right, randomly group them.

[0242] Experimental groups: normal control group, blank control group CLIp, CLIp-(RGD / Col) group, CLIp-(Col) group, CLIp-(RGD) group, and DEPE-PEG-RGD / Col group.

[0243] Each group was given corresponding drugs, and the normal control group was given normal saline. Administration method: tail vein injection, twice a week, for 4 consecutive weeks. 24 hours after the last administration, the nude mice were killed and the pancreatic tissue was obtained for Western blot detection.

[0244] The results are as follows Fig.14As shown in the figure, compared with the normal control group, the expression level of type I collagen in the pancreatic tissue of nude mice in the blank control group CLIp was significantly increased. Compared with the blank control group CLIp, the expression level of type I collagen in the integrin targeting peptide group and collagenase group decreased, but the decrease was small, 80% and 85%, respectively. The expression level of type I collagen in the combined drug group was significantly reduced, with a reduction rate of 90%, which was statistically different from the single drug group (P < 0.05).

[0245] The experimental results showed that RGD sequence-containing integrin targeting peptide and collagenase combined with liposomes could significantly inhibit the expression of type I collagen in the pancreatic tissue of nude mice with pancreatic cancer transplanted tumor models. The combined drug group showed a synergistic effect, and its effect of inhibiting type I collagen expression was significantly better than that of the single drug group, suggesting that the combined use of the two drugs has better anti-pancreatic cancer fibrosis potential.

Claims

1. Use of an integrin targeting peptide-collagenase drug combination in the preparation of a drug for treating fibrosis, wherein the molar ratio of the integrin targeting peptide to the collagenase is (500-1000):

1.

2. The use according to claim 1, characterized in that: The integrin targeting peptide is selected from CGPLGVRGD, C18-CGPLGVRGD, GRGDSP, GRGDS, RGDS, RGDV, c(RGDfK), c(RGDyK), c(RGDfV), c(RGDfE), c(RGDfC), c(RGDf[N-Methyl]V), c(RGDyC), RGD-PEG or iRGD, and the collagenase is type I collagenase.

3. The use according to claim 1, wherein the drug is a drug for treating liver fibrosis, lung fibrosis, pancreatic fibrosis or fibrotic tumors.

4. The use according to claim 1, characterized in that: The drug is administered by local administration, intravenous injection, subcutaneous injection or intramuscular injection.

5. A pharmaceutical composition for treating fibrosis, characterized in that: In parts by weight, it comprises 0.5 to 50 parts of integrin targeting peptide, 0.5 to 50 parts of collagenase and 1 to 100 parts of pharmaceutically acceptable carrier.

6. The pharmaceutical composition according to claim 5, characterized in that: The integrin targeting peptide is CGPLGVRGD, C18-CGPLGVRGD, GRGDSP, GRGDS, RGDS, RGDV, c(RGDfK), c(RGDyK), c(RGDfV), c(RGDfE), c(RGDfC), c(RGDf[N-Methyl]V), c(RGDyC), RGD-PEG or iRGD, and the collagenase is type I collagenase.

7. The pharmaceutical composition according to claim 5, characterized in that: The mass ratio of the integrin targeting peptide to collagenase is (5-10):

1.

8. The pharmaceutical composition according to claim 5, characterized in that: In parts by weight, it comprises 5 to 8.5 parts of integrin targeting peptide, 1 part of collagenase and 1 to 50 parts of pharmaceutically acceptable carriers.

9. The pharmaceutical composition according to claim 5, characterized in that: The dosage form is selected from the group consisting of solution, suspension, liposome, microsphere, microcapsule, nanoparticle and gel.

10. The pharmaceutical composition according to claim 5, wherein the pharmaceutically acceptable carrier is selected from a solvent, a stabilizer, a buffer, a cosolvent, an antioxidant, a preservative, an osmotic pressure regulator, a lipid material, a polymer material or a filler.

Images