Use of integrin-targeting peptide-collagenase drug combination and pharmaceutical composition constructed thereby

Abstract

The application discloses an integrin-targeting peptide-collagenase drug combination and a drug composition constructed by the integrin-targeting peptide-collagenase drug combination. The drug combination can effectively block mechanical signals and biochemical signals mediating fibrosis, efficiently inhibit fibroblast activation, reduce the secretion of extracellular matrix proteins, and reshape the fibrotic microenvironment. Efficient reduction of fibrosis can promote the penetration of subsequent therapeutic drugs, and has a cascade penetration effect. The constructed drug composition can synergistically deliver integrin-targeting peptides and collagenase, and after release, the integrin-targeting peptides and collagenase can synergistically act in the form of free molecules in fibrotic lesions, thereby providing a new strategy for efficient delivery of fibrosis treatment drugs.

Description

Technical Field

[0001] This invention relates to the application of an integrin-targeting peptide-collagenase drug combination and the pharmaceutical composition thereof, and more particularly to the application of an integrin-targeting peptide-collagenase drug combination with synergistic therapeutic effect on fibrosis and the pharmaceutical composition thereof. Background Technology

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

[0003] The signaling pathways leading to persistent fibroblast activation are mainly divided into two categories: the mechano-signaling pathway mediated by integrin transmission from the cell surface β1 subunit, and the biochemical signaling pathway mediated by factors such as TGF-β. Currently, anti-fibrotic strategies targeting fibroblast activation inhibition primarily focus on blocking either single biochemical or mechano-signaling pathways. Examples include 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, and integrin inhibitors.

[0004] Furthermore, CN118217246 modifies secondary nanoparticles with RGD peptides for co-delivery with collagenase. The RGD peptides primarily act as a target, specifically targeting and damaging type II alveolar epithelial cells, promoting effective internalization of the formulation. US20210059936 modifies the surface of liposomes with RGD peptides as a target to deliver collagenase, increasing its targeting specificity. All of these utilize integrin-targeting peptides (such as RGD cyclic peptides) to modify microparticle formulations for targeted delivery of collagenase to fibrotic lesions, achieving stronger lesion accumulation or cellular uptake. However, their mechanism of action remains limited to the local degradation of collagenase. Summary of the Invention

[0005] Purpose of the invention: The first purpose of the invention is to provide an application of an integrin-targeting peptide-collagenase drug combination with synergistic effect in the preparation of a drug for the treatment of fibrosis with dual pathways; the second purpose is to provide a pharmaceutical composition constructed from the drug combination.

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

[0007] The drug combination designed in this invention provides a dual-pathway synergistic inhibition antifibrotic strategy. On the one hand, collagenase is used to cleave type I and type III collagen into short peptides, weakening ECM rigidity. On the other hand, collagenase is also used to cleave the recognition sequences of integrins (α1β1, α2β1, α10β1, and α11β1) on type I and type III collagen, further reducing the mechanosensing between integrins and collagen, and blocking the mechanotransduction between collagen (type I and type III) and integrins.

[0008] On the other hand, the integrin-targeting peptide is released in single-molecule form and binds to the integrin receptor via the RGD sequence. It competitively binds to integrins (α5β1, α8β1, αvβ1, and αIIbβ1) with fibronectin, blocking the mechanotransduction between fibronectin and integrins. Furthermore, the released integrin-targeting peptide can also bind to αvβ6 and αvβ8 integrins on the surface of fibroblasts and tumor cells via the RGD sequence, reducing the release of active TGF-β and thus reducing fibroblast activation mediated by biochemical signaling pathways.

[0009] The RGD sequence is a core motif for integrin recognition and binding, capable of specific interactions with integrin ligand binding sites (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 and reduce ECM protein secretion by blocking the mechanotransduction and biochemical signal transduction mediating fibrosis, thereby disrupting the positive feedback loop between ECM rigidity and fibroblast activation and reshaping the fibrotic microenvironment.

[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 a type I collagenase.

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

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

[0016] Preferably, the drug is administered via local administration, intravenous injection, subcutaneous injection, or intramuscular injection.

[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 according to 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 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] Further 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 pharmaceutically acceptable carrier.

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

[0025] Preferably, the pharmaceutically acceptable carrier is selected from solvents, stabilizers, buffers, cosolvents, antioxidants, preservatives, osmotic pressure regulators, lipid materials, polymeric materials, or fillers.

[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 phospholipids (DPPD, DOPS, DEPE, DMPE, DSPE, DPPE, DOPE, DOPG, EPG, POPG, DPPG, DSPG, DMPG, DPPA, DEPC, DOPC, DMPC, POPC, DSPC, DPPC), X-PEG-Y (where 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-weight cholesterol, and other excipients. Molecular polymers and their derivatives (amylose, amylopectin, oxidized starch, hydroxyethyl starch, cross-linked starch, microcrystalline cellulose, cellulose acetate, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose carbonate, hydroxypropyl methylcellulose acetate succinate, gum arabic, chitosan, dextran, chitosan, hyaluronic acid, alginate, heparin, albumin), synthetic polymers and their derivatives (polyacrylic acid, polyhydroxyethyl methacrylate, carbomer, polyvinyl alcohol, polyvinyl acetate phthalate, polyvinylpyrrolidone, polyethylene glycol, polyethylene glycol-polylactic acid, polyethylene glycol-polycaprolactone, polyoxyethylene ether...) Sesame oil, polyoxyethylene fatty acid esters, poloxamer, polyglycolic acid, polylactic acid, polycaprolactone, glycolic acid-caprolactone copolymer, polysorbate, polyvinylpyridinium, poly(ethyleneimine), d-α-tocopherol polyethylene glycol 1000 succinate, amino acid polymers, poly(ethylene oxide)-poly(propylene oxide) block copolymers, polyphenolic materials (quercetin, kaempferol, myricetin, anthocyanins, luteolin, catechin, gallocatechin, gallocatechin gallate, epigallocatechin gallate, digaloyl-D-glucose, trigaloylglucose, tetragaloylglucose, pentagaloylglucose, gallic acid, digalic acid) Tannins, ellagitannins, ellagic acid, hydrolyzed tannins, polydopamine), emulsifiers (polyvinyl alcohol, polysorbate 20, polysorbate 80, sodium dodecyl sulfate, poloxamer 188, poloxamer 407, polyethylene glycol monostearate, polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil, octanoic acid caprylic acid glycerol), 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), sodium microcrystalline cellulose, sodium carboxymethyl cellulose, hydroxypropyl methylcellulose, magnesium stearate.

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

[0028] (1) Prepare solutions of integrin-targeting peptides and collagenase;

[0029] (2) Preparation of prefabricated system

[0030] Method 1: Mix the solution of integrin targeting peptide and collagenase prepared in step (1) with buffer solution and adjust the pH to 6.5-7.5; the mixing process is specifically microfluidic rapid mixing or simple mixing followed by ultrasonic, homogenization, slurrying, vortexing or stirring operations.

[0031] Method 2: Mix the solution of integrin targeting peptide and collagenase prepared in step (1) with buffer, then mix with stabilizer, and adjust the pH to 6.5-7.5; the mixing procedure is the same as in Method 1.

[0032] (3) Preparation of pharmaceutical formulations

[0033] Method 1: Mix the pre-prepared system prepared in step (2) with a solvent to obtain a solution containing integrin-targeting peptides and collagenase; the mixing process is specifically microfluidic technology for rapid mixing or simple mixing followed by ultrasonication, homogenization, slurrying, vortexing or stirring.

[0034] Method 2: Mix the pre-prepared system prepared in step (2) with polymer materials, freeze-dry and micronize the particles, and disperse them in an oil phase solution to obtain a suspension containing integrin targeting peptides and collagenase; the micronization process specifically includes ultrasonic treatment, high-pressure homogenization, ball milling, and air jet milling; the mixing process is the same as in Method 1.

[0035] Method 3: Mix the pre-prepared system prepared in step (2) with lipid or polymer materials, and prepare microspheres containing integrin targeting peptides and collagenase by emulsification-solvent evaporation, spray drying or phase separation. The emulsification-solvent evaporation method is specifically to dissolve the pre-prepared system prepared in step (2) and polymer materials in an organic solvent to form an oil phase. The oil phase is slowly added to an aqueous phase containing an emulsifier, and emulsified at high speed to form an emulsion. The organic solvent is then stirred to evaporate, and the microspheres are collected by centrifugation. The spray drying method is specifically to mix the pre-prepared system prepared in step (2) with polymer materials, and atomize and dry the mixture in a high-temperature airflow using a spray drying device to form microspheres. The phase separation method is specifically to dissolve the pre-prepared system prepared in step (2) and polymer materials in an organic solvent, and add a non-solvent to induce phase separation to form microspheres. The mixing process is the same as in Method 1.

[0036] Method 4: Mix the pre-prepared system prepared in step (2) with lipid or polymer materials, and prepare microcapsules containing integrin targeting peptides and collagenase by using the double emulsion method, interfacial polymerization method or coagulation method; The double emulsion method is specifically to use the pre-prepared system prepared in step (2) as the inner aqueous phase and mix it with the oil phase formed by the polymer material to form a primary emulsion (W / O); Add the primary emulsion to the outer aqueous phase (an aqueous solution containing an emulsifier), emulsify at high speed to form a double emulsion (W / O / W), stir to evaporate the organic solvent, and collect the microcapsules by centrifugation; The interfacial polymerization method is specifically to mix the pre-prepared system prepared in step (2) with a monomer as the aqueous phase; Dissolve another monomer in an organic solvent as the oil phase; Mix the two phases and polymerize at the interface to form microcapsules; The coagulation method is specifically to mix the pre-prepared system prepared in step (2) with the polymer material, and induce coagulation by adjusting the pH or adding a coagulant to form microcapsules; The mixing process is the same as in Method 1.

[0037] Method 5: Mix the pre-prepared system in step (2) with natural or synthetic polymer materials and prepare nanoparticles containing integrin targeting peptides and collagenase by ultrasonic or high-pressure homogenization. The mixing process is the same as in Method 1.

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

[0039] Method 7: Mix the pre-prepared system prepared in step (2) with lipid or polymer materials, and prepare liposomes containing integrin targeting peptides and collagenase by thin film dispersion, reverse evaporation or injection method; the thin film dispersion method specifically involves dissolving lipid materials such as phospholipids and cholesterol in an organic solvent, evaporating under reduced pressure to form a thin film, adding the pre-prepared system prepared in step (2) for hydration, and homogenizing by ultrasound or high pressure to obtain liposomes; the reverse evaporation method specifically involves dissolving lipid materials such as phospholipids and cholesterol in an organic solvent, mixing with the pre-prepared system prepared in step (2) to form an emulsion, removing the organic solvent to obtain liposomes; the injection method specifically involves slowly injecting the organic solution of lipid materials such as phospholipids and cholesterol into the pre-prepared system prepared in step (2), stirring and removing the organic solvent to obtain liposomes; the mixing process is the same as in Method 1; the organic solvent is removed by dialysis, ultrafiltration or evaporation.

[0040] More specifically, the ultrasonic frequency is 100–1000 W, and the ultrasonic time is 1–30 min; the centrifugation speed is 1000–10000 rpm, and the centrifugation time is 1–30 min; the homogenization is carried out by a high-pressure homogenizer or a micro-jet homogenizer, with a homogenization pressure of 300–1500 Pa and a homogenization number of 1–30 times; the slurry is achieved by a homogenizer, with a speed of 1000–10000 rpm and a homogenization time of 5–120 s; the vortex or stirring is achieved by a vortex mixer or a stirrer, with a vortex or stirring speed generally of 500–3000 rpm and a time of 1–30 min; the ball milling is achieved by a ball mill, with a speed of 50–400 rpm and a grinding time of 1–2 h; the air jet milling is achieved by an air jet mill, with an air jet pressure of 0.5–1.2 MPa; and the atomization pressure of the spray dryer is 0.1–0.5 MPa.

[0041] The pharmaceutically acceptable solvent is one or more of the following: water, buffer solution, glycerol, propylene glycol, diethyl 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 in this invention exhibits synergistic inhibitory effects through both mechanotransduction and biochemical signal transduction pathways. It can block the activation and proliferation of fibrotic cells, inhibit the initiation and maintenance of fibrosis, and promote matrix degradation. Simultaneously, by degrading the collagen matrix in fibrotic tissue, it reduces the volume and stiffness of the fibrotic tissue, restoring tissue elasticity. This efficient reduction of fibrosis also promotes the penetration of subsequent therapeutic drugs, exhibiting a cascade penetration-enhancing effect. The constructed drug composition can synergistically deliver integrin-targeting peptides and collagenases, which, upon release, exert a synergistic effect in the fibrotic lesions in the form of free molecules, providing a new strategy for the efficient delivery of fibrosis treatment drugs. Attached Figure Description

[0044] Figure 1 The release curves of RGD peptides and collagenase in the microcapsules prepared in Example 3 are shown.

[0045] Figure 2 The responsive release curves of RGD peptides and collagenase in the microspheres prepared in Example 4 are shown.

[0046] Figure 3 The responsive release curves of RGD peptide and collagenase in the nanoparticles prepared in Example 5 are shown.

[0047] Figure 4The responsive release curves of RGD peptides and collagenase in the gel prepared in Example 6 are shown.

[0048] Figure 5 The responsive release curves of RGD peptide and collagenase in the liposomes prepared in Example 7 are shown.

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

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

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

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

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

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

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

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

[0057] Figure 14 The results show the pharmacodynamic evaluation of the liposomes prepared in Example 7 for the treatment of pancreatic cancer. Detailed Implementation

[0058] The technical solution of the present invention will be further described below with reference to the embodiments.

[0059] The integrin-targeting peptide containing the RGD sequence is from Jier Biochemical Co., Ltd.

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

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

[0062] First, prepare a 0.01M phosphate-buffered saline (PBS) solution at pH 7.4. Then, dissolve 5 mg of CGPLGVRGD 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. Stir for 10 minutes. Subsequently, sonicate the mixture 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, aliquot into sterile tubes, and store in sealed containers at -80°C or 4°C.

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

[0064] First, prepare a 0.01M phosphate-buffered saline (PBS) solution at pH 7.4. Then, dissolve 5 mg of GRGDSP 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. Stir for 10 minutes. Subsequently, sonicate the mixture 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, aliquot into sterile tubes, and store in sealed containers at -80°C or 4°C.

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

[0066] First, prepare a 0.01M phosphate-buffered saline (PBS) solution at pH 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. Stir for 10 minutes. Subsequently, sonicate the mixture 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, aliquot into sterile test tubes, and store in sealed containers at -80°C or 4°C.

[0067] (4) Preparation of a solution containing RGDS, an integrin targeting peptide with RGD sequence, and collagenase.

[0068] First, prepare a 0.01M phosphate-buffered saline (PBS) solution at pH 7.4. Then, dissolve 5 mg of RGDS 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. Stir for 10 minutes. Subsequently, sonicate the mixture 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, aliquot into sterile test tubes, and store in sealed containers at -80°C or 4°C.

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

[0070] First, prepare a 0.01M phosphate-buffered saline (PBS) solution at pH 7.4. Then, dissolve 5 mg of RGDV 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. Stir for 10 minutes. Subsequently, sonicate the mixture 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, aliquot into sterile tubes, and store in sealed containers at -80°C or 4°C.

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

[0072] First, prepare a 0.01M phosphate-buffered saline (PBS) solution at pH 7.4. Then, dissolve 5 mg c(RGDfK) in 5 mL of PBS to prepare a 1 mg / mL RGD peptide solution, and dissolve 1 mg collagenase in 5 mL of PBS to prepare a 200 μg / mL collagenase solution. Stir for 10 minutes. Subsequently, sonicate the mixture 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, aliquot into sterile tubes, and store in sealed containers at -80°C or 4°C.

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

[0074] First, prepare a 0.01M phosphate-buffered saline (PBS) solution at pH 7.4. Then, dissolve 5 mg c(RGDyK) in 5 mL of PBS to prepare a 1 mg / mL RGD peptide solution, and dissolve 1 mg collagenase in 5 mL of PBS to prepare a 200 μg / mL collagenase solution. Stir for 10 minutes. Subsequently, sonicate the mixture 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, aliquot into sterile tubes, and store in sealed containers at -80°C or 4°C.

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

[0076] First, prepare a 0.01M phosphate-buffered saline (PBS) solution at pH 7.4. Then, dissolve 5 mg c(RGDfV) in 5 mL of PBS to prepare a 1 mg / mL RGD peptide solution, and dissolve 1 mg collagenase in 5 mL of PBS to prepare a 200 μg / mL collagenase solution. Stir for 10 minutes. Subsequently, sonicate the mixture 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, aliquot into sterile tubes, and store in sealed containers at -80°C or 4°C.

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

[0078] First, prepare a 0.01M phosphate-buffered saline (PBS) solution at pH 7.4. Then, dissolve 5 mg c(RGDfE) in 5 mL of PBS to prepare a 1 mg / mL RGD peptide solution, and dissolve 1 mg collagenase in 5 mL of PBS to prepare a 200 μg / mL collagenase solution. Stir for 10 minutes. Subsequently, sonicate the mixture 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, aliquot into sterile tubes, and store in sealed containers at -80°C or 4°C.

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

[0080] First, prepare a 0.01M phosphate-buffered saline (PBS) solution at pH 7.4. Then, dissolve 5 mg c (RGDfC) in 5 mL of PBS to prepare a 1 mg / mL RGD peptide solution, and dissolve 1 mg collagenase in 5 mL of PBS to prepare a 200 μg / mL collagenase solution. Stir for 10 minutes. Subsequently, sonicate the mixture 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, aliquot into sterile tubes, and store in sealed containers at -80°C or 4°C.

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

[0082] First, prepare a 0.01M phosphate-buffered saline (PBS) solution at pH 7.4. Then, dissolve 5 mg c(RGDf[N-Methyl]V) in 5 mL of PBS to prepare a 1 mg / mL RGD peptide solution, and dissolve 1 mg collagenase in 5 mL of PBS to prepare a 200 μg / mL collagenase solution. Stir for 10 minutes. Subsequently, sonicate the mixture 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, aliquot into sterile tubes, and store in sealed containers 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 a 0.01M phosphate-buffered saline (PBS) solution at pH 7.4. Then, dissolve 5 mg of c(RGDyC) 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. Stir for 10 minutes. Subsequently, sonicate the mixture 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, aliquot into sterile tubes, and store in sealed containers at -80°C or 4°C.

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

[0086] First, prepare a 0.01M phosphate-buffered saline (PBS) solution at pH 7.4. Then, dissolve 5 mg of RGD-PEG 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. Stir for 10 minutes. Subsequently, sonicate the mixture 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, aliquot into sterile tubes, and store in sealed containers at -80°C or 4°C.

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

[0088] First, prepare 0.01M phosphate-buffered saline (PBS) at pH 7.4; then dissolve 5 mg iRGD 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 mixture 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, aliquot into sterile test tubes, and seal and store at -80℃ or 4℃. (15) Pharmaceutical characterization of a solution containing RGD sequence integrin targeting peptide CGPLGVRGD and collagenase

[0089] Using the above preparation method, solutions containing RGD sequence integrin targeting peptide and collagenase (RGD / Col), solutions containing RGD sequence integrin targeting peptide (RGD), and solutions containing collagenase (Col) were prepared. The above formulations were subjected to pharmaceutical characterization, and the results are shown in Table 1. The prepared solutions were clear liquids with good stability and biological activity.

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

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

[0092] First, prepare a 0.01M phosphate-buffered saline (PBS) solution at pH 7.4. Then, dissolve 5 mg of CGPLGVRGD in 5 mL of PBS to prepare a 1 mg / mL RGD-containing peptide solution, dissolve 1 mg of collagenase in 5 mL of PBS to prepare a 200 μg / mL collagenase solution, and dissolve 20 mg of polymer (e.g., PVP) in 5 mL of PBS to prepare a 4 mg / mL solution. Stir for 10 minutes. Thoroughly mix the RGD-containing peptide solution, collagenase solution, and polymer solution. Aliquot the mixture into lyophilization vials and freeze-dry to obtain lyophilized particles. Add a stabilizer to the lyophilized particles and grind them until homogeneous. Simultaneously, prepare an oil phase by dissolving 10 mg of suspending agent (e.g., Tween-80) in 1 mL of solvent (e.g., medium-chain triglycerides) and stirring thoroughly until homogeneous. Disperse the lyophilized particles into the oil phase and mix thoroughly to form a stable microparticle suspension. Finally, the sample was filtered through a 0.22μm sterile filter, dispensed into sterile test tubes, and sealed for storage at -80℃ or 4℃.

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

[0094] Using the above preparation method, suspensions containing RGD sequence integrin targeting peptides and collagenase (RGD / Col), suspensions containing RGD sequence integrin targeting peptides (RGD), and suspensions containing collagenase (Col) were prepared. The above formulations were subjected to pharmaceutical characterization, and the results are shown in Table 1. The prepared suspensions exhibited 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 a 0.01M phosphate-buffered saline (PBS) solution at pH 7.4. Then, dissolve 5 mg of RGDSP 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. Add these solutions at a mass ratio of RGD / Col of 6.5:1 and stir until homogeneous to form a drug mixture. Simultaneously, prepare the oil phase by adding an appropriate amount of material (such as PLGA, PCL) to an appropriate amount of organic solvent (such as dichloromethane), heating to 60°C, and stirring to dissolve. 7.4 A surfactant (such as PVA) was added to 0.01M phosphate-buffered saline (PBS) to prepare an aqueous phase; then, the drug mixture was slowly added to the oil phase and ultrasonically emulsified (30% power, 3 min); the emulsion was treated 2-3 times with a high-pressure homogenizer at 1000-1500 psi to further refine the emulsion droplets; the promulgated emulsion was dropped into a large amount of the aqueous phase, and stirring was continued. The organic solvent was evaporated by rotary evaporation, and the microcapsules were separated by centrifugation. The microcapsules were washed 3 times with PBS to remove unreacted substances; they were aliquoted into sterile test tubes and sealed for storage at 4°C.

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

[0099] Using the above preparation method, microcapsules containing RGD sequence integrin targeting peptides and collagenase with acid-responsive release properties (RGD / Col), microcapsules containing RGD sequence integrin targeting peptides and collagenase without acid-responsive release properties (RGD / Col-NR (Non-Responsive)), microcapsules containing RGD sequence integrin targeting peptides with acid-responsive release properties (RGD), microcapsules containing collagenase with acid-responsive release properties (Col), and a blank microcapsule formulation were prepared. 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 types of microcapsules ranged from 0.6 to 0.7 μm, with a narrow particle size distribution and a PDI (polydispersity index) of less than 0.3, indicating that the prepared microcapsules had uniform particle size and good dispersibility, which is beneficial for the distribution and penetration of the microcapsules in vivo, thereby improving the bioavailability of the drug.

[0100] (3) In vitro release of RGD sequence integrin-targeting peptides and collagenases

[0101] Each formulation was mixed with PBS buffer at pH 6.5 and pH 7.4, 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 then replenished to the original volume using 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 formulation was mixed with PBS buffer at pH 6.5 and pH 7.4, respectively, and incubated at 37°C and 100 rpm for 48 hours. Ultrafiltration (MWCO = 300 kDa) was performed at 0, 1, 2, 3, 6, 9, 12, 24, and 48 hours to remove free C18-CD9 and VRGD cleaved by enzymes. The lipid concentrate was then replenished to the original volume using the corresponding PBS buffer. The dispersed RGD was quantified using the BCA method.

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

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

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

[0107] First, prepare a 0.01M phosphate-buffered saline (PBS) solution at pH 7.4. Then, dissolve 5 mg of RGD 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. Add these solutions at a mass ratio of RGD / Col of 7.5:1 and stir well to form a drug mixture. Dissolve 100 mg of chitosan in 2% acetic acid solution to form a chitosan solution. Mix the drug mixture (RGD peptide + collagenase) with the chitosan solution and stir well. Add 2% glutaraldehyde solution (at a 9:1 volume ratio) to crosslink the mixture and react at room temperature for 30 minutes. Collect the microspheres by centrifugation (10,000 × g, 10 min) and wash three times with PBS to remove unreacted glutaraldehyde and other impurities. Aliquot the microspheres into sterile tubes and store them at 4°C.

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

[0109] Using the above preparation method, microspheres containing RGD sequence integrin targeting peptides and collagenase with acid-responsive release characteristics (RGD / Col), microspheres containing RGD sequence integrin targeting peptides and collagenase without acid-responsive release characteristics (RGD / Col-NR), microspheres containing RGD sequence integrin targeting peptides with acid-responsive release characteristics (RGD), microspheres containing collagenase with acid-responsive release characteristics (Col), and a blank microsphere formulation were prepared. The particle size of the microspheres was determined by dynamic light scattering (DLS). The results are shown in Table 1. The particle size of the five types of microspheres ranged from 0.4 to 0.5 μm, with a narrow particle size distribution and a PDI (polydispersity index) of less than 0.3, indicating that the prepared microspheres had uniform particle size and good dispersibility, which is beneficial for the distribution and penetration of the microspheres in vivo, thereby improving the bioavailability of the drug.

[0110] (3) In vitro release of RGD sequence integrin-targeting peptides and collagenases

[0111] Each formulation was mixed with PBS buffer at pH 6.5 and pH 7.4, 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 then replenished to the original volume using 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 formulation was mixed with PBS buffer at pH 6.5 and pH 7.4, respectively, and incubated at 37°C and 100 rpm for 48 hours. Ultrafiltration (MWCO = 300 kDa) was performed at 0, 1, 2, 3, 6, 9, 12, 24, and 48 hours to remove free C18-CD9 and VRGD cleaved by enzymes. The lipid concentrate was then replenished to the original volume using the corresponding PBS buffer. The dispersed RGD was quantified using the BCA method.

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

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

[0116] (1) Preparation of nanoparticles containing RGD sequence integrin targeting peptides and collagenase

[0117] First, a 0.01M phosphate-buffered saline (PBS) solution at pH 7.4 was prepared. Then, 5 mg of c(RGDfK) was dissolved in 5 mL of PBS to prepare a 1 mg / mL RGD-containing peptide solution, and 1 mg of collagenase was dissolved in 5 mL of PBS to prepare a 200 μg / mL collagenase solution. These solutions were added at a mass ratio of RGD / Col of 6.0:1 and stirred thoroughly to form a drug mixture. Next, the mixture was added to 5 mL of a 2% polylactic-co-glycolic acid copolymer (PLGA) solution in dimethyl sulfoxide (DMSO) and stirred at high speed (10,000 rpm) for 30 min to form a proemulsion. Then, the proemulsion was slowly added to 50 mL of a 1% polyvinyl alcohol (PVA) aqueous solution and stirred for 1 h to form a double emulsion. Subsequently, the nanoparticles were collected by centrifugation at 4,000 × g for 15 min and washed three 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 peptides and collagenase

[0119] Using the above preparation method, nanoparticles containing RGD sequence integrin targeting peptides and collagenase with acid-responsive release characteristics (RGD / Col), nanoparticles containing RGD sequence integrin targeting peptides and collagenase without acid-responsive release characteristics (RGD / Col-NR), nanoparticles containing RGD sequence integrin targeting peptides with acid-responsive release characteristics (RGD), nanoparticles containing collagenase with acid-responsive release characteristics (Col), and a blank nanoparticle formulation were prepared. 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 types of nanoparticles was between 100 and 120 nm, with a narrow particle size distribution and a PDI (polydispersity index) of less than 0.3, indicating that the prepared nanoparticles had uniform particle size and good dispersibility, which is beneficial for the distribution and penetration of nanoparticles in vivo and improves the bioavailability of the drug.

[0120] (3) In vitro release of RGD sequence integrin-targeting peptides and collagenases

[0121] Each formulation was mixed with PBS buffer at pH 6.5 and pH 7.4, 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 then replenished to the original volume using 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 formulation was mixed with PBS buffer at pH 6.5 and pH 7.4, respectively, and incubated at 37°C and 100 rpm for 48 hours. Ultrafiltration (MWCO = 300 kDa) was performed at 0, 1, 2, 3, 6, 9, 12, 24, and 48 hours to remove free C18-CD9 and VRGD cleaved by enzymes. The lipid concentrate was then replenished to the original volume using the corresponding PBS buffer. The dispersed RGD was quantified using the BCA method.

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

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

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

[0127] First, prepare a 0.01M phosphate-buffered saline (PBS) solution at pH 7.4. Then, dissolve 5 mg of RGD-PEG 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. Add these solutions at a mass ratio of RGD / Col of 8.5:1 and stir well to form a drug mixture. Dissolve 100 mg of chitosan in 2% acetic acid solution and stir well to prepare a chitosan solution. Dissolve 100 mg of hyaluronic acid in 10 mL of ultrapure water and stir well to prepare a hyaluronic acid solution. Mix the chitosan solution and hyaluronic acid solution at a 1:1 volume ratio, add the RGD peptide and collagenase mixture, and stir well. Add 2% glutaraldehyde solution (at a 9:1 volume ratio) and crosslink at room temperature for 30 minutes. Place the mixture in an ultrasonicator and sonicate at 200W for 5 minutes to form a nanogel. The nanogel was collected by centrifugation (10,000×g, 10 min), washed three times with PBS, aliquoted into sterile test tubes, and sealed and stored at 4°C.

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

[0129] Using the above preparation method, nanogels containing RGD-sequence integrin-targeting peptides and collagenase with acid-responsive release properties (RGD / Col), nanogels containing RGD-sequence integrin-targeting peptides and collagenase without acid-responsive release properties (RGD / Col-NR), nanogels containing RGD-sequence integrin-targeting peptides with acid-responsive release properties (RGD), and nanogels containing collagenase with acid-responsive release properties (Col) were prepared, along with a blank nanogel formulation. The particle size of the nanogels was determined by dynamic light scattering (DLS) of the above formulations. The results are shown in Table 1. The formulations have good particle size, ranging from 100 to 120 nm, and exhibit good stability and biological activity.

[0130] (3) In vitro release of RGD sequence integrin-targeting peptides and collagenases

[0131] Each formulation was mixed with PBS buffer at pH 6.5 and pH 7.4, 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 then replenished to the original volume using 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 formulation was mixed with PBS buffer at pH 6.5 and pH 7.4, respectively, and incubated at 37°C and 100 rpm for 48 hours. Ultrafiltration (MWCO = 300 kDa) was performed at 0, 1, 2, 3, 6, 9, 12, 24, and 48 hours to remove free C18-CD9 and enzyme-cleaved VRGD. The lipid concentrate was then replenished to the original volume using the corresponding PBS buffer. The dispersed RGD was quantified using the BCA method.

[0134] The results are as follows Figure 4 As shown, the release of RGD peptides in RGD / Col is acid-responsive, while the RGD / Col-NR group basically does not release RGD peptides 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] Nanoparticles loaded with type I collagenase and RGD peptide can be prepared using thin-film dispersion, injection, or reverse solvent methods. 10 mg of soybean lecithin, 1.25 mg of cholesterol, 1.25 mg of DSPE-PEG-CREKA, and 4.836 mg of RGD-sequence-containing integrin-targeting peptide C18-CGPLGVRGD (from Jier Biochemical) were weighed into a 250 mL round-bottom flask. 1 mL of chloroform and 1 mL of methanol were added to dissolve the components. The mixture was then rotary evaporated at 37°C (120 rpm, 20 min) to form a lipid film, and vacuum dried 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). The TA HEPES solution was slowly added dropwise to the Col I HEPES solution under vortex. The above lipid membrane was hydrated using the TA and Col I HEPES solution (37℃, 150 rpm, 30 min). The solution was then sonicated with a probe (30% ultrasonic power, 2 min) to form a pale yellow, opalescent solution.

[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 formulation and incubated at room temperature for 10 min to break the hydrogen bonds 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] Using the above preparation method, liposomes Clip (RGD / Col) containing RGD sequence integrin targeting peptide and collagenase with acid-responsive and enzyme-responsive 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 acid-responsive and enzyme-responsive release characteristics, liposome Clip (Col) containing collagenase with acid-responsive and enzyme-responsive release characteristics, and a blank liposome preparation Clip were prepared. The particle size of the liposomes was measured by dynamic light scattering (DLS) of the above preparations. The results are shown in Table 1. The preparations have good particle size, between 60 and 80 nm, and exhibit good stability in PBS solution, physiological buffer solution, and DMEM medium containing 10% serum.

[0142] (3) In vitro release of RGD sequence integrin-targeting peptides and collagenases

[0143] Each formulation was mixed with PBS buffer (pH 6.5 containing 2 mg / mL type IV collagenase), PBS buffer (pH 7.4 containing 2 mg / mL type IV collagenase), and PBS solution (pH 7.4) at a ratio of 1:1, v / v. The mixtures were 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 ultrafiltration (MWCO = 300 kDa) was performed to remove the released Col I-FITC. The fluorescence intensity of the filtrate was measured, and the amount of Col I-FITC released was calculated. The concentrate was then replenished to the original volume using the corresponding PBS solution.

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

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

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

[0147] Table 1. Pharmacological parameters of the formulations prepared in Examples 1 to 7

[0148]

[0149]

[0150] Example 8: Investigation into the mechanism by which RGD sequence-containing integrin-targeting peptides and collagenase inhibit fibroblast activation

[0151] Using the integrin targeting peptide CGPLGVRGD containing the RGD sequence, RGD / Col group solution, Col group solution, RGD group solution and blank solution were prepared according to the method of Example 1. The mechanism by which the solution containing the integrin targeting peptide containing the RGD sequence and collagenase inhibits fibroblast activation was investigated.

[0152] Uniform tumor spheres were selected and transferred to 96-well plates using a sterile pipette tip. The spheres were then co-incubated with each formulation for 48 hours. The changes in the levels of mechanotransduction pathway protein (YAP) and TGF-β / Smad pathway protein (P-Smad2) were analyzed by Western blotting, and quantitative analysis was performed using ImageJ software.

[0153] The results are as follows Figure 6As shown, the collagenase released by RGD / Col hydrolyzes collagen and cleaves the recognition sequences of integrins (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 mechanosensing and inhibition of mechanotransmission, manifested as decreased YAP expression. In addition, the RGD peptide released from the formulation competes with LAP for binding to αvβ6 integrin on NIH 3T3 cells and αvβ6 and αvβ8 integrin on Panc-02 cells, resulting in reduced secretion of active TGF-β and inhibition of TGF-β-mediated biochemical signaling pathways, manifested as decreased P-Smad2 expression. In other words, RGD / Col inhibits fibroblast activation from both mechanotransmission and biochemical signal transduction aspects.

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

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

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

[0157] After preparing Panc-02 / NIH3T3 mixed tumor spheres using existing methods within the group, the spheres were transferred to 96-well plates using sterile pipette tips. The formulation was administered once every 24 hours, 100 μL each time, for a total of two administrations. After administration, the spheres were transferred to 1.5 mL centrifuge tubes, centrifuged at 2000 rpm for 5 min, the sphere pellet was collected, and 200 μL of blank DMEM medium was added for resuspending. The supernatant was collected again by centrifugation at 14000 rpm for 4 min, and cell debris was discarded.

[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) to neutralize and mix well, and measure the total amount of TGF-β.

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

[0161] (3) Use ELISA Kit to measure TGF-β content

[0162] Add the sample to be tested (50 μL) and enzyme-labeled reagent (100 μL) to the enzyme-labeled plate. Seal the plate with the sealing film and incubate at 37°C for 60 min. Remove the sealing film and discard the liquid. Fill each well with washing buffer (300 μL), let stand for 30 s and then discard. Repeat 5 times. Add chromogenic reagent A (50 μL) to each well first, then add chromogenic reagent B (50 μL), gently shake to mix, and develop color at 37°C in the dark for 15 min. Add 50 μL of stop solution to each well to stop the reaction. Measure the absorbance of each well sequentially 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, with the inhibitory effects increasing from smallest to largest as follows: (CLip-(RGD / Col) > CLIp-(Col) > CLIp-(RGD). Furthermore, the combination group showed a synergistic effect between the RGD peptide and Col I. In contrast, the non-releasing control group liposomes (DSPE-PEG-RGD) failed to release RGD peptide molecules in the microenvironment and could not compete with the latent TGF-β complex for binding to integrins, thus failing to reduce the production of active TGF-β.

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

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

[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), and 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. Cells in the logarithmic growth phase were used for experiments. HSC-T6 cells were seeded into 6-well plates. After cell attachment, the medium was replaced with blank medium and treated for 12 hours. Then, the respective drugs were added, and the cells were cultured for another 24 hours.

[0168] Western blot detection of type I collagen expression: 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 a PVDF membrane; the membrane was blocked with 5% skim milk for 1 hour; type I collagen primary antibody was added and incubated overnight at 4°C; the membrane was washed three times with TBST for 10 minutes each time; HRP-labeled secondary antibody was added and incubated at room temperature for 1 hour; the membrane was washed three times with TBST for 10 minutes each time. The membrane was developed with ECL chemiluminescence buffer, and the band gray values ​​were analyzed using ImageJ software.

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

[0170] The results are as follows Figure 8 As shown, compared with the control group, the expression of type I collagen in both the CGPLGVRGD group and the collagenase group decreased, but the decrease was small, approximately 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 that in the single drug group (P < 0.05).

[0171] Experimental results showed that a solution containing RGD-sequenced integrin-targeting peptides and collagenase significantly inhibited type I collagen secretion from HSC-T6 cells. The mechanism of action may include: the integrin-targeting peptides competitively bind to integrin receptors, blocking integrin-mediated signaling pathways, inhibiting HSC (Hepatic Stellate Cells) activation, and reducing ECM synthesis. Collagenase directly degrades existing collagen fibers, promoting ECM degradation. The combination of these two drugs simultaneously blocks both mechano- and biochemical signals mediating fibrosis, effectively inhibiting fibroblast activation, reducing extracellular matrix (ECM) protein secretion, and disrupting the positive feedback loop between ECM rigidity and fibroblast activation. The combined treatment group showed a synergistic effect, with significantly better inhibition of type I collagen expression than the single-drug group, suggesting that the combined use of these two drugs has better anti-hepatic fibrosis potential.

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

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

[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 DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO2. Cells in the logarithmic growth phase were used for experiments. Lung fibroblasts were seeded into 6-well plates. After cell attachment, the medium was replaced with blank medium and treated for 12 hours. Then, the drugs for each group were added and cultured for another 24 hours.

[0176] Western blot detection of type I collagen expression: 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 a PVDF membrane; the membrane was blocked with 5% skim milk for 1 hour; type I collagen primary antibody was added and incubated overnight at 4°C; the membrane was washed three times with TBST for 10 minutes each time; HRP-labeled secondary antibody was added and incubated at room temperature for 1 hour; the membrane was washed three times with TBST for 10 minutes each time. The membrane was developed with ECL chemiluminescence buffer, and the band gray values ​​were analyzed using ImageJ software.

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

[0178] Experimental results showed that the suspension containing RGD sequence integrin targeting peptide and collagenase could significantly inhibit the secretion of type I collagen by pulmonary 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 peptides and collagenases

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

[0181] Microcapsules were prepared according to the method in Example 3 and applied to the study of liver fibrosis treatment. The effect 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 was evaluated, and its anti-liver fibrosis potential was explored.

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

[0183] Liver fibrosis-related cell lines (such as hepatic stellate cells LX-2 or HepG2) were cultured in low-adsorption culture dishes to form homogeneous spheroids, simulating the liver fibrosis microenvironment. The morphology and size of the spheroids were observed under a microscope to ensure uniform diameter (approximately 100–200 μm) for experimental reproducibility. The spheroids were transferred to 96-well plates using sterile pipette tips and co-incubated with the respective formulations at 37°C and 5% CO2 for 48 hours to simulate the in vivo environment. After 48 hours, the spheroids were collected, digested, and analyzed by Western blotting (WB) to determine changes in α-SMA content in each group of spheroids.

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

[0185] Experimental results show that the simultaneous release of free RGD peptides and collagenases in the fibrotic microenvironment has a synergistic effect, effectively inhibiting fibroblast activation. The RGD / Col combined treatment group exhibited a synergistic effect, with significantly better fibroblast activation than the single treatment group, suggesting that the combined use 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 in Example 3 and applied to the study of liver fibrosis treatment. The effect of microcapsules containing RGD sequence integrin targeting peptide and collagenase on type I collagen expression in liver tissue of liver fibrosis model mice was evaluated, and its anti-liver fibrosis potential was explored.

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

[0189] Drug administration began on the second day after model establishment. Each group received the corresponding drug, while the normal control group received an equal volume of physiological saline. Administration method: tail vein injection, twice a week for 8 consecutive weeks. Twenty-four hours after the last administration, mice were sacrificed, and liver tissue was collected for Western blot analysis.

[0190] The results are as follows Figure 10 As shown, 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 levels of type I collagen in the integrin-targeting peptide group and the collagenase group decreased, by 65% ​​and 67%, respectively, with a relatively small decrease. The expression level of type I collagen in the RGD / Col combination therapy group was significantly reduced, with a reduction rate of approximately 75%, which was statistically different from the single therapy groups (P < 0.05).

[0191] Experimental results showed that microcapsules containing RGD sequence integrin targeting peptides and collagenase released free RGD sequence integrin targeting peptides and collagenase in the microenvironment, which could significantly inhibit the expression of type I collagen in the liver tissue of mice with liver fibrosis. 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-liver fibrosis potential.

[0192] Example 13: Pharmacodynamic evaluation of microspheres containing RGD sequence integrin targeting peptides 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 in Example 4 and applied to the study of pulmonary fibrosis treatment. The effect of microspheres containing RGD sequence integrin targeting peptide and collagenase on the α-SMA content in the pulmonary fibrosis microenvironment was evaluated, and their anti-pulmonary fibrosis potential was explored.

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

[0196] Cell lines associated with pulmonary fibrosis (such as MRC-5 and IMR-90) were cultured in low-adsorption culture dishes to form homogeneous spheroids, simulating the microenvironment of pulmonary fibrosis. The morphology and size of the spheroids were observed under a microscope to ensure uniform diameter (approximately 100-200 μm) for experimental reproducibility. The spheroids were transferred to 96-well plates using sterile pipette tips and co-incubated with the respective formulations at 37°C and 5% CO2 for 48 hours to simulate the in vivo environment. After 48 hours, the spheroids were collected, digested, and analyzed by Western blotting (WB) to determine changes in α-SMA content in each group of spheroids.

[0197] The results are as follows Figure 11 As shown, compared with the control group, the α-SMA expression in the RGD / Col-NR group remained at a high level, close to that of the control group; the α-SMA expression 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 α-SMA expression in the RGD / Col group was significantly decreased (P<0.05).

[0198] Experimental results showed that the synergistic effect of free RGD peptides and collagenase released in the fibrotic microenvironment effectively inhibited fibroblast activation. The RGD / Col combination therapy group exhibited a synergistic effect, with significantly better fibroblast activation than the single-drug group, suggesting that the combined use 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 in Example 4 and applied to the study of pulmonary fibrosis treatment. The effect of microspheres containing RGD sequence integrin targeting peptide and collagenase on type I collagen expression in the lung tissue of pulmonary fibrosis model mice was evaluated, and their anti-pulmonary fibrosis potential was explored.

[0201] Establishment of pulmonary fibrosis model: C57BL / 6 mice were randomly divided into 5 groups: 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 mice in the other groups were instilled with BLM (5 mg / kg) via tracheal infusion to establish a pulmonary fibrosis model.

[0202] Drug administration began on the second day after model establishment. Each group received the corresponding drug, while the normal control group received an equal volume of physiological saline. Administration method: tail vein injection, once daily for 14 consecutive days. Mice in the normal control group received an equal volume of physiological saline via tracheal infusion. Twenty-four hours after the last administration, mice were sacrificed, and lung tissue was collected for Western blot analysis.

[0203] The results are as follows Figure 11As shown, 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 levels of type I collagen in the integrin-targeting peptide group and the collagenase group decreased, by 67% and 70%, respectively, with a relatively small decrease. The expression level of type I collagen in the RGD / Col combination therapy group was significantly reduced, with a reduction rate of approximately 85%, which was statistically different from the single therapy groups (P < 0.05).

[0204] Experimental results showed that microspheres containing RGD sequence integrin targeting peptides and collagenase released free RGD sequence integrin targeting peptides and collagenase in the microenvironment, which could significantly inhibit the expression of type I collagen in the lung tissue of mice with pulmonary fibrosis. 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.

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

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

[0207] Nanoparticles were prepared according to the method in Example 5 and applied to the study of pancreatic fibrosis treatment. The inhibitory effect of nanoparticles containing RGD sequence integrin targeting peptides and collagenase on pancreatic stellate cells (PSCs) was evaluated, changes in α-SMA content were detected, and their 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] Pancreatic fibrosis model mice isolated PSCs and cultured them in DMEM medium containing 10% fetal bovine serum in an incubator at 37°C and 5% CO2. PSCs were seeded into 6-well plates, and when the cell density reached 80%, nanoparticles from different treatment groups were added, and the plates were cultured for another 48 hours.

[0210] Western blot analysis was performed to detect changes in α-SMA content. α-SMA is a marker of PSC activation, and its content changes directly reflect the degree of PSC activation. Cells were collected, and total protein was extracted. Protein concentration was determined using the BCA method. Proteins were separated by SDS-PAGE electrophoresis and transferred to a PVDF membrane. The membrane was blocked with 5% skim milk for 1 hour. α-SMA primary antibody was added, and the membrane was incubated overnight at 4°C. The membrane was washed three times with TBST for 10 minutes each time. HRP-labeled secondary antibody was added, and the membrane was incubated at room temperature for 1 hour. The membrane was washed three times with TBST for 10 minutes each time. The membrane was developed using ECL chemiluminescence buffer, and the band gray values ​​were analyzed using ImageJ software.

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

[0212] Experimental results showed that the free RGD peptides and collagenases released in the microenvironment had a good synergistic effect and could effectively inhibit fibroblast activation. The RGD / Col combined treatment group showed a synergistic effect, and its effect on fibroblast activation was significantly better than that of the single treatment group, suggesting that the combined use of the two drugs has better anti-pancreatic fibrosis potential.

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

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

[0215] Establishment of a pancreatic fibrosis model: C57BL / 6 mice were randomly divided into 5 groups: 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 other groups of mice were injected intratracheally with BLM (5 mg / kg) to establish a pancreatic fibrosis model. Pancreatic fibrosis in mice was induced by intraperitoneal injection of diethyldithiocarbamate (DDC) for 4 weeks.

[0216] Drug administration began on day 2 after model establishment. Each group received the corresponding drug, while the normal control group received an equal volume of physiological saline. Administration method: tail vein injection, once daily for 14 consecutive days. Mice in the normal control group received an equal volume of physiological saline via tracheal infusion. Twenty-four hours after the last administration, mice were sacrificed, and pancreatic tissue was collected for Western blot analysis.

[0217] The results are as follows Figure 12 As shown, 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 levels of type I collagen in the integrin-targeting peptide group and the collagenase group decreased, by 70% and 73%, respectively, with a relatively small decrease. The expression level of type I collagen in the RGD / Col combination therapy group was significantly reduced, with a reduction rate of approximately 90%, which was statistically different from the single therapy groups (P < 0.05).

[0218] Experimental results show that nanoparticles containing RGD sequence integrin targeting peptides and collagenase release free RGD sequence integrin targeting peptides and collagenase in the microenvironment, which can significantly inhibit the expression of type I collagen in pancreatic tissue of mice with pancreatic fibrosis. 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 fibrosis potential.

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

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

[0221] Nanogels were prepared according to the method in Example 6 and applied to the study of bladder cancer treatment. The effect 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 was evaluated, and its potential for treating bladder cancer was explored.

[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 homogeneous spheroids, simulating the fibrotic microenvironment of bladder cancer. The morphology and size of the spheroids were observed under a microscope to ensure uniform diameter (approximately 100–200 μm) for experimental reproducibility. The spheroids were transferred to 96-well plates using sterile pipette tips and co-incubated with the respective formulations at 37°C and 5% CO2 for 48 hours to simulate the in vivo environment. After 48 hours, the spheroids were collected, digested, and analyzed by Western blotting (WB) to determine changes in α-SMA content in each group of spheroids.

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

[0225] Experimental results showed that free RGD peptides and collagenases released in the microenvironment had a good synergistic effect and could effectively inhibit fibroblast activation. The RGD / Col combination therapy group showed a synergistic effect, and its effect on fibroblast activation was significantly better than that of the single therapy 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 a gel containing RGD sequence integrin targeting peptides and collagenase

[0227] Nanogels were prepared according to the method in Example 6 and applied to the treatment of bladder cancer fibrosis. The effects of a gel formulation containing RGD sequence integrin targeting peptides and collagenase on type I collagen expression in the bladder tissue of nude mice with bladder cancer xenografts were evaluated, and its anti-bladder cancer fibrosis potential was explored.

[0228] Establishment of a bladder cancer xenograft model: Human bladder cancer cells in the logarithmic growth phase were subcutaneously inoculated into the right back of nude mice, with 1 × 10⁶ cells per mouse. 6 One. Regularly monitor tumor growth; wait until the tumor volume reaches 100mm. 3 When left or right, group randomly.

[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 administered the corresponding drug, while the normal control group received physiological saline. Administration method: tail vein injection, twice a week for 4 consecutive weeks. Twenty-four hours after the last administration, nude mice were sacrificed, and bladder tissue was collected for Western blot analysis.

[0231] The results are as follows Figure 13 As shown, 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 levels of type I collagen in the integrin-targeting peptide group and the collagenase group decreased, by 65% ​​and 70%, respectively, with a relatively small decrease. The expression level of type I collagen in the RGD / Col combination therapy group was significantly reduced, with a reduction rate of approximately 80%, which was statistically different from the single therapy groups (P < 0.05).

[0232] Experimental results showed that the combination of RGD sequence integrin targeting peptide and collagenase in nanogels 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. 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-bladder cancer fibrosis potential.

[0233] Example 16: Pharmacodynamic evaluation of liposomes containing RGD sequence integrin targeting peptides 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 formulation to enhance the penetration of chemotherapeutic drugs in tumor spheroids was investigated.

[0236] Select uniform granules and transfer them to 96-well plates using a sterile pipette tip. Incubate the granules with each group of formulations for 48 hours, collect the granules, digest them, and analyze the changes in α-SMA content in each group of granules using Western blotting.

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

[0238] Experimental results showed that free RGD peptides and collagenases released in the microenvironment had a good synergistic effect and could effectively inhibit fibroblast activation. The RGD / Col combination therapy group showed a synergistic effect, and its effect on fibroblast activation was significantly better than that of the single therapy 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 in Example 7 and applied to the study of pancreatic cancer treatment. The effect of liposome formulation containing RGD sequence integrin targeting peptide and collagenase on type I collagen expression in pancreatic tissue of nude mice with pancreatic cancer xenograft was evaluated, and its anti-pancreatic cancer fibrosis potential was explored.

[0241] Establishment of a pancreatic cancer xenograft model: Human pancreatic cancer cells in the logarithmic growth phase were subcutaneously inoculated into the right back of nude mice, with 1 × 10⁶ cells per mouse. 6 One. Regularly monitor tumor growth; wait until the tumor volume reaches 100mm. 3 When left or right, group randomly.

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

[0243] Each group was administered the corresponding drug, while the normal control group received physiological saline. Administration method: tail vein injection, twice a week for 4 consecutive weeks. Twenty-four hours after the last administration, nude mice were sacrificed, and pancreatic tissue was collected for Western blot analysis.

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

[0245] Experimental results showed that liposomes containing RGD sequence integrin targeting peptides and collagenase could significantly inhibit the expression of type I collagen in pancreatic tissue of nude mice with pancreatic cancer xenografts. The combined drug group showed a synergistic effect, and its effect in 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. The application of an integrin-targeting peptide-collagenase drug combination in the preparation of a drug for treating fibrosis, wherein the drug combination consists of an integrin-targeting peptide and a collagenase; wherein the integrin-targeting peptide is selected from CGPLGVRGD, and the collagenase is type I collagenase; wherein the drug is a drug for treating liver fibrosis and pulmonary fibrosis.

2. The application according to claim 1, characterized in that, The drug can be administered via local administration, intravenous injection, subcutaneous injection, or intramuscular injection.

3. The application according to claim 1, characterized in that, The drug 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 pharmaceutically acceptable carrier.

4. The application according to claim 3, characterized in that, The mass ratio of the integrin-targeting peptide to collagenase is (5~10):

1.

5. The application according to claim 3, characterized in that, The drug comprises, by weight, 5 to 8.5 parts of integrin-targeting peptide, 1 part of collagenase, and 1 to 50 parts of pharmaceutically acceptable carrier.

6. The application according to claim 1, characterized in that, The dosage form of the drug is selected from solutions, suspensions, liposomes, microspheres, microcapsules, nanoparticles, and gels.

7. The application according to claim 3, characterized in that, The pharmaceutically acceptable carrier is selected from solvents, stabilizers, buffers, cosolvents, osmotic pressure regulators, lipid materials, or fillers.

8. In the application according to claim 7, the stabilizer is selected from antioxidants and preservatives.

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