A nucleic acid exosome preparation targeting myofibroblasts and a preparation method thereof

Abstract

This invention discloses a nucleic acid exosome formulation and preparation method targeting myofibroblasts, belonging to the field of exosome engineering technology. The method includes isolating exosomes from mammalian cell culture supernatant; providing a YQT12 polypeptide containing terminal cysteine ​​residues, and reacting the YQT12 polypeptide with DSPE-PEGx-MAL to obtain the targeting phospholipid molecule DSPE-PEGx-YQT12; mixing a first small interfering RNA, a second small interfering RNA, and a disulfide-containing cationic short peptide to form a reducible cationic nucleic acid core; and loading the reducible cationic nucleic acid core into exosomes using electroporation to obtain nucleic acid-loaded exosomes. This invention simultaneously introduces DSPE-PEGx-YQT12 and DSPE-PEGa-MMP-L-PEGb onto the exosome membrane surface, reducing non-specific recognition in non-lesion environments and improving delivery selectivity to myofibroblasts at lesion sites; and combining siHSP47 and siLOXL2 with a disulfide-containing cationic short peptide to form a reducible cationic nucleic acid core, improving the loading stability of small interfering RNA and promoting nucleic acid release in an intracellular reducing environment.

Description

Technical Field

[0001] This invention relates to the field of exosome engineering technology, and in particular to a nucleic acid exosome preparation targeting myofibroblasts and its preparation method. Background Technology

[0002] Pulmonary fibrosis is a chronic progressive disease characterized by alveolar structure destruction, abnormal extracellular matrix deposition, and decreased lung tissue elasticity. Its pathological process involves multiple links such as epithelial damage, inflammatory response, fibroblast activation, and continuous proliferation of myofibroblasts. Myofibroblasts are an important source of extracellular matrix components such as collagen and fibronectin in fibrotic lesions. Their abnormal activation and long-term retention are considered to be key factors driving interstitial remodeling and decline in lung function.

[0003] Current exosomal nucleic acid delivery technologies still have the following shortcomings: Firstly, existing targeted exosomes mostly expose the target ligand directly on the surface of the exosome, lacking structural designs that utilize the enrichment characteristics of matrix metalloproteinases in pulmonary fibrosis lesions for responsive regulation. This makes it difficult to reduce non-specific recognition in non-lesion environments and increase the exposure of the target ligand in the lesion microenvironment, thus limiting the selective delivery efficiency to myofibroblasts. Secondly, existing exosomal nucleic acid preparations typically use free nucleic acids or single small interfering RNA as payloads, which are insufficient in terms of nucleic acid stability, intracellular release efficiency, and multi-target synergistic intervention capabilities, making it difficult to simultaneously intervene in key aspects of pulmonary fibrosis such as collagen formation and collagen cross-linking. Summary of the Invention

[0004] In a first aspect, the present invention provides a nucleic acid exosome preparation targeting myofibroblasts, comprising exosomes isolated from mammalian cell culture supernatant, a targeting phospholipid molecule and a matrix metalloproteinase-responsive shielding molecule inserted into the exosome membrane, and a reducible cationic nucleic acid core encapsulated in the exosome cavity; The targeted phospholipid molecule is DSPE-PEGx-YQT12, wherein DSPE is 1,2-distearate-sn-glycerol-3-phosphatidylethanolamine, PEGx is a polyethylene glycol spacer arm, and the amino acid sequence of YQT12 is Ser-Leu-Tyr-Gln-Thr-Asp-Asp-Arg-Asn-Asp-Tyr-Ile-Gly-Gly-Cys; The matrix metalloproteinase-responsive shielding molecule is DSPE-PEGa-MMP-L-PEGb, wherein PEGa is a near-membrane polyethylene glycol spacer arm, MMP-L is a peptide that can be cleaved by MMP-2 and / or MMP-9, and PEGb is a distal polyethylene glycol shielding chain. The amino acid sequence of the MMP-L is selected from any one of Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln, Gly-Pro-Leu-Gly-Val-Arg-Gly, and Pro-Leu-Gly-Leu-Ala-Gly; The average molecular weight of PEGb is greater than that of PEGx, and the average molecular weight of PEGb is 1.5-10 times that of PEGx. The reducible cationic nucleic acid core includes a first small interfering RNA, a second small interfering RNA, and a cationic short peptide containing disulfide bonds. The target gene of the first small interfering RNA is HSP47, and the target gene of the second small interfering RNA is LOXL2. The disulfide-containing cationic short peptide is formed by covalently linking a nucleic acid binding segment, a reducible linker segment, and an endosome escape auxiliary segment. The nucleic acid binding segment is a peptide containing 4-12 arginine residues and / or lysine residues, the reducible linker segment contains at least one disulfide bond, and the endosome escape auxiliary segment is a peptide containing 3-10 histidine residues. The matrix metalloproteinase-responsive shielding molecule provides spatial shielding for YQT12 when it is not cleaved by MMP-2 and / or MMP-9, and weakens the spatial shielding of YQT12 after cleavage by MMP-2 and / or MMP-9, thereby increasing the exposure of YQT12 on the exosome's outer surface.

[0005] As a preferred embodiment of the nucleic acid exosome preparation of the present invention, wherein: the average molecular weight of PEGx is 1000-3500 Da, the average molecular weight of PEGa is 500-3000 Da, and the average molecular weight of PEGb is 3000-10000 Da.

[0006] As a preferred embodiment of the nucleic acid exosome preparation of the present invention, wherein: the molar ratio of the targeting phospholipid molecule to the matrix metalloproteinase responsive shielding molecule is 1:1 to 1:8; and the mass ratio of the first small interfering RNA to the second small interfering RNA is 1:0.25 to 1:4.

[0007] As a preferred embodiment of the nucleic acid exosome preparation of the present invention, wherein: the average particle size of the exosomes is 30-150 nm, the average particle size of the reducible cationic nucleic acid core is not greater than 30% of the average particle size of the exosomes, and the zeta potential of the nucleic acid exosome preparation is -20 mV to +5 mV. After treatment with MMP-2 and / or MMP-9, the normalized fluorescence intensity of YQT12 on the outer surface of exosomes was higher than that of the control group that was not treated with MMP-2 and / or MMP-9 after detection using the YQT12 specific fluorescently labeled probe binding method and normalized according to the number of exosome particles.

[0008] Secondly, the present invention provides a method for preparing a nucleic acid exosome preparation targeting myofibroblasts, comprising the following steps: S1, Exosomes were isolated from mammalian cell culture supernatant; S2 provides a YQT12 polypeptide containing a terminal cysteine ​​residue, and reacts the YQT12 polypeptide with DSPE-PEGx-MAL to obtain the targeted phospholipid molecule DSPE-PEGx-YQT12; The amino acid sequence of the YQT12 polypeptide is Ser-Leu-Tyr-Gln-Thr-Asp-Asp-Arg-Asn-Asp-Tyr-Ile-Gly-Gly-Cys; S3, mix the first small interfering RNA, the second small interfering RNA and the cationic short peptide containing disulfide bonds to form a reducible cationic nucleic acid core; S4. A reducible cationic nucleic acid core is loaded into exosomes using electroporation to obtain nucleic acid-loaded exosomes. S5, DSPE-PEGx-YQT12 and matrix metalloproteinase responsive shielding molecule DSPE-PEGa-MMP-L-PEGb are co-incubated with nucleic acid-loaded exosomes simultaneously or sequentially to allow DSPE-PEGx-YQT12 and DSPE-PEGa-MMP-L-PEGb to insert into the exosome membrane; S6. The product is purified to remove the unloaded reducible cationic nucleic acid core, the DSPE-PEGx-YQT12 that is not inserted into the exosome membrane, and the DSPE-PEGa-MMP-L-PEGb that is not inserted into the exosome membrane, to obtain the nucleic acid exosome preparation.

[0009] As a preferred embodiment of the preparation method of the present invention, in step S1, the exosomes are separated from the mammalian cell culture supernatant by at least one of differential centrifugation, ultracentrifugation, ultrafiltration, size exclusion chromatography, density gradient centrifugation or tangential flow filtration.

[0010] As a preferred embodiment of the preparation method described in this invention, the YQT12 peptide and DSPE-PEGx-MAL are reacted in a buffer solution at pH 6.0-7.0 for 2-4 hours, and the molar ratio of DSPE-PEGx-MAL to YQT12 peptide is 1:1.2 to 1:2.

[0011] In a preferred embodiment of the preparation method described in this invention, the molar ratio of the cationic group in the disulfide-bonded cationic short peptide to the total phosphate group of the first small interfering RNA and the second small interfering RNA is 1:1 to 10:1; the mass ratio of the first small interfering RNA to the second small interfering RNA is 1:0.25 to 1:4.

[0012] As a preferred embodiment of the preparation method described in this invention, the electroporation method is as follows: voltage 150-250 V, pulse time 8-15 ms, number of pulses 2-3 times, and pulse interval 4-6 s.

[0013] In a preferred embodiment of the preparation method described in this invention, the molar ratio of DSPE-PEGx-YQT12 to matrix metalloproteinase responsive shielding molecule is 1:1 to 1:8, the co-incubation temperature is 4-37℃, and the co-incubation time is 0.5-6 hours.

[0014] The beneficial effects of this invention are as follows: This invention simultaneously introduces DSPE-PEGx-YQT12 and DSPE-PEGa-MMP-L-PEGb onto the surface of the exosome membrane, so that the YQT12 targeting peptide is shielded by the distal PEG shielding chain under normal environment, but in the pulmonary fibrosis lesion environment where MMP-2 and / or MMP-9 are present, the enzyme-cleaved peptides in the shielding molecule are cleaved, the exposure of the YQT12 outer surface increases, the non-specific recognition in the non-lesion environment is reduced, and the delivery selectivity of myofibroblasts in the lesion site is improved. This invention combines siHSP47 and siLOXL2 with a disulfide-bonded cationic short peptide to form a reducible cationic nucleic acid core, which improves the loading stability of small interfering RNA and promotes nucleic acid release in an intracellular reducing environment. Attached Figure Description

[0015] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 This is a flowchart illustrating the preparation of the nucleic acid exosome formulation targeting myofibroblasts in Example 1; Figure 2 This is a schematic diagram of the structure of the nucleic acid exosome preparation in Example 1. Detailed Implementation

[0017] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0018] Example 1 Reference Figure 1 and Figure 2 This embodiment provides a nucleic acid exosome preparation targeting myofibroblasts and its preparation method; A nucleic acid exosome preparation targeting myofibroblasts includes exosomes isolated from mammalian cell culture supernatant, a targeting phospholipid molecule and a matrix metalloproteinase-responsive shielding molecule inserted into the exosome membrane, and a reducible cationic nucleic acid core encapsulated in the exosome lumen. The targeted phospholipid molecule is DSPE-PEGx-YQT12, where DSPE is 1,2-distearate-sn-glycerol-3-phosphatidylethanolamine, PEGx is a polyethylene glycol spacer arm, and the amino acid sequence of YQT12 is Ser-Leu-Tyr-Gln-Thr-Asp-Asp-Arg-Asn-Asp-Tyr-Ile-Gly-Gly-Cys; The matrix metalloproteinase-responsive shielding molecule is DSPE-PEGa-MMP-L-PEGb, where PEGa is the near-membrane polyethylene glycol spacer arm, MMP-L is a peptide that can be cleaved by MMP-2 and / or MMP-9, and PEGb is the distal polyethylene glycol shielding chain. The amino acid sequence of MMP-L is selected from any one of Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln, Gly-Pro-Leu-Gly-Val-Arg-Gly, and Pro-Leu-Gly-Leu-Ala-Gly; The average molecular weight of PEGb is greater than that of PEGx, and the average molecular weight of PEGb is 1.5-10 times that of PEGx. The reducible cationic nucleic acid core includes a first small interfering RNA, a second small interfering RNA, and a short cationic peptide containing disulfide bonds. The target gene of the first small interfering RNA is HSP47, and the target gene of the second small interfering RNA is LOXL2. The cationic short peptide containing disulfide bonds is formed by the covalent linkage of a nucleic acid binding segment, a reducible linker segment, and an endosome escape accessory segment. The nucleic acid binding segment is a peptide containing 4-12 arginine residues and / or lysine residues, the reducible linker segment contains at least one disulfide bond, and the endosome escape accessory segment is a peptide containing 3-10 histidine residues. Matrix metalloproteinase-responsive shielding molecules provide spatial shielding for YQT12 when not cleaved by MMP-2 and / or MMP-9, and weaken the spatial shielding of YQT12 after cleavage by MMP-2 and / or MMP-9, thereby increasing the exposure of YQT12 on the exosome surface. A method for preparing a nucleic acid exosome preparation targeting myofibroblasts includes the following steps: S1, Exosomes were isolated from mammalian cell culture supernatant; S2 provides a YQT12 polypeptide containing a terminal cysteine ​​residue, and reacts the YQT12 polypeptide with DSPE-PEGx-MAL to obtain the targeted phospholipid molecule DSPE-PEGx-YQT12; The amino acid sequence of the YQT12 polypeptide is Ser-Leu-Tyr-Gln-Thr-Asp-Asp-Arg-Asn-Asp-Tyr-Ile-Gly-Gly-Cys; S3, mix the first small interfering RNA, the second small interfering RNA and the cationic short peptide containing disulfide bonds to form a reducible cationic nucleic acid core; S4. A reducible cationic nucleic acid core is loaded into exosomes using electroporation to obtain nucleic acid-loaded exosomes. S5, DSPE-PEGx-YQT12 and matrix metalloproteinase responsive shielding molecule DSPE-PEGa-MMP-L-PEGb are co-incubated with nucleic acid-loaded exosomes simultaneously or sequentially to allow DSPE-PEGx-YQT12 and DSPE-PEGa-MMP-L-PEGb to insert into the exosome membrane; S6. The product is purified to remove the unloaded reducible cationic nucleic acid core, the DSPE-PEGx-YQT12 that is not inserted into the exosome membrane, and the DSPE-PEGa-MMP-L-PEGb that is not inserted into the exosome membrane, to obtain the nucleic acid exosome preparation. YQT12 peptide was reacted with DSPE-PEGx-MAL in a buffer solution at pH 6.0-7.0 for 2-4 hours, and the molar ratio of DSPE-PEGx-MAL to YQT12 peptide was 1:1.2 to 1:2.

[0019] The formulation comprises exosomes isolated from mammalian cell culture supernatant, a targeted phospholipid molecule DSPE-PEGx-YQT12 inserted into the exosome membrane, a matrix metalloproteinase responsive shielding molecule DSPE-PEGa-MMP-L-PEGb, and a reducible cationic nucleic acid core encapsulated in the exosome lumen. In this embodiment, PEGx is selected as PEG2000, PEGa as PEG1000, PEGb as PEG5000, and MMP-L is selected as Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln.

[0020] First, exosomes are prepared.

[0021] Human umbilical cord mesenchymal stem cells were selected as exosome donor cells and seeded in culture flasks. They were cultured in DMEM / F12 medium containing 10% exosome-free fetal bovine serum at 37°C and 5% CO2. When the cell confluence reached 80%-90%, the medium was replaced with fresh exosome-free medium and cultured for another 48 h. The cell culture supernatant was collected and centrifuged sequentially at 300×g for 10 min, 2000×g for 20 min, and 10000×g for 30 min to remove suspended cells, cell debris, and large vesicles. The supernatant was then filtered through a 0.22 μm filter membrane. The filtered supernatant was concentrated using a 100 kDa ultrafiltration tube and further purified by size exclusion chromatography. The exosome components were collected to obtain an exosome dispersion.

[0022] Nanoparticle tracking analysis revealed that the average particle size of the obtained exosomes was 42.6 nm, and the polydispersity index was 0.16. Transmission electron microscopy showed that the exosomes had a spherical vesicle structure, and the particle size was mainly distributed in the range of 30-150 nm.

[0023] Prepare the targeted phospholipid molecule DSPE-PEG2000-YQT12.

[0024] YQT12 peptide, with the amino acid sequence Ser-Leu-Tyr-Gln-Thr-Asp-Asp-Arg-Asn-Asp-Tyr-Ile-Gly-Gly-Cys, was dissolved in PBS buffer at pH 6.6. DSPE-PEG2000-MAL was also dissolved in the same buffer. The two peptides were mixed at a molar ratio of 1:1.5 and reacted at room temperature in the dark for 3 h to allow the thiol group on the terminal cysteine ​​residue of YQT12 to couple with the maleimide group in DSPE-PEG2000-MAL. After the reaction, unreacted small molecule components were removed by dialysis, followed by high-performance liquid chromatography (HPLC) purification to obtain DSPE-PEG2000-YQT12. MALDI-TOF mass spectrometry analysis showed that the molecular weight of the product matched the target molecular weight. The purity of the product, calculated using the HPLC area normalization method, was 91.2%.

[0025] Prepare matrix metalloproteinase-responsive shielding molecules.

[0026] In this embodiment, DSPE-PEG1000-Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln-PEG5000 is used as the responsive shielding molecule. Specifically, the peptide Fmoc-Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln-COOH with an N-terminal Fmoc-protected and C-terminal free carboxyl group is first prepared by solid-phase synthesis. This peptide is dissolved in anhydrous dimethylformamide, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide are added. The mixture is activated at room temperature for 30 min. Subsequently, DSPE-PEG1000-NH2 is added, and the reaction is carried out under nitrogen protection for 12 minutes. h, so that the C-terminus of the peptide is linked to DSPE-PEG1000-NH2 by forming an amide bond. After the reaction is completed, piperidine / dimethylformamide solution is added to remove the Fmoc protecting group, and the intermediate DSPE-PEG1000-Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln with a free N-terminal amino group is obtained.

[0027] After removing small molecule reactants by dialysis, the above intermediate was reacted with mPEG5000-NHS in phosphate buffer at pH 7.4 for 4 h, so that mPEG5000 was linked to the N-terminal amino group of the peptide to obtain DSPE-PEG1000-Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln-PEG5000. The reaction product was purified by size exclusion chromatography and reversed-phase high-performance liquid chromatography, and the main peak fraction was collected, freeze-dried and used for later use.

[0028] MALDI-TOF mass spectrometry analysis showed that the main peak distribution of the product was consistent with the target molecular weight range. High performance liquid chromatography (HPLC) area normalization method determined the product purity to be 90.6%. Since the average molecular weight of PEG5000 is greater than that of PEG2000, and the molecular weight ratio between the two is 2.5, PEG5000 can form a spatial shield for YQT12 when it is not cleaved by MMP-2 and / or MMP-9. After being cleaved by MMP-2 and / or MMP-9, the distal shielding chain is detached or the shielding effect is weakened, which is beneficial to increasing the exposure of YQT12 on the exosome's outer surface.

[0029] Preparation of a reducible cationic nucleic acid core.

[0030] The first small interfering RNA was siHSP47, and the second small interfering RNA was siLOXL2. The two were mixed at a mass ratio of 1:1. The cationic short peptide containing disulfide bonds was a short peptide composed of a nucleic acid binding segment, a reducible linker segment, and an endosome escape helper segment. The nucleic acid binding segment was an R8 fragment composed of 8 arginine residues, and the endosome escape helper segment was an H6 fragment composed of 6 histidine residues. The two were linked by a linker segment containing disulfide bonds.

[0031] The two types of small interfering RNAs were mixed with cationic short peptides, such that the molar ratio of the cationic groups in the cationic short peptides to the total phosphate groups of the two small interfering RNAs was 3:1 to 6:1, preferably 4:1. The mixture was gently mixed under low concentration conditions and allowed to stand at room temperature for 5-10 min. This allowed the positively charged cationic short peptides and the negatively charged small interfering RNAs to form a small-sized reducible cationic nucleic acid core through electrostatic interaction. Dynamic light scattering measurements showed that the average particle size of the formed nucleic acid core was 8.6 nm, and the Zeta potential was +10.8 mV. The average particle size of the nucleic acid core was less than 30% of the average particle size of the exosomes obtained in this embodiment, making it suitable for loading into the 30-150 nm exosome cavity via electroporation.

[0032] A reducible cationic nucleic acid core is loaded into exosomes.

[0033] The exosome dispersion with an average particle size of 30-150 nm was mixed with a reducible cationic nucleic acid core with an average particle size of 3-15 nm and then transferred to an electroporation cup. Electroporation was performed under the conditions of 150-220 V voltage, 8-12 ms pulse time, 1-2 pulses, and 5 s pulse interval. After electroporation, the cup was immediately placed in an ice bath for 10 min and recovery buffer was added. The cup was then placed at room temperature for 30 min to facilitate the recovery of the exosome membrane and obtain nucleic acid-loaded exosomes. Since the average particle size of the reducible cationic nucleic acid core is no more than 30% of the average particle size of the exosome, it is easier for the core to enter the exosome cavity during electroporation and can reduce membrane structure damage caused by excessive load size.

[0034] Based on this, bimolecular modification of the exosome membrane surface was performed. The previously prepared DSPE-PEG2000-YQT12 and DSPE-PEG1000-Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln-PEG5000 were mixed at a molar ratio of 1:3 and added to the nucleic acid-loaded exosome dispersion. The mixture was co-incubated at 25°C for 1.5 h to allow the two DSPE derivatives to insert into the exosome membrane. After co-incubation, the mixture was purified by size exclusion chromatography and 300 kDa ultrafiltration to remove the unloaded reducible cationic nucleic acid core, DSPE-PEG2000-YQT12 that was not inserted into the exosome membrane, and DSPE-PEG1000-Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln-PEG5000 that was not inserted into the exosome membrane, thus obtaining the final nucleic acid exosome formulation.

[0035] The obtained exosome preparations were tested and found to have an average particle size of 46.8 nm, a polydispersity index of 0.19, a zeta potential of -8.8 mV, and a total nucleic acid encapsulation efficiency of 28.6% as determined by the RiboGreen method. The obtained exosome preparations were further divided into an untreated group and an enzyme-treated group. The enzyme-treated group was treated with a mixed enzyme solution of MMP-2 and MMP-9 at 37℃ for 30 min, while the untreated group was treated under the same conditions with an enzyme-free buffer. After treatment, a fluorescently labeled recognition molecule capable of recognizing the YQT12 peptide was added, and unbound free recognition molecules were removed. Subsequently, the fluorescence intensity of the exosomes was measured by nanoflow cytometry or fluorescence enzyme-linked immunosorbent assay (ELISA) and normalized according to the number of exosome particles. The normalized fluorescence intensity of the enzyme-treated group was approximately 1.9 times that of the untreated group, indicating that the constructed responsive shielding molecule can reduce the spatial shielding effect of YQT12 in the presence of matrix metalloproteinases, thereby improving the detectable exposure of YQT12 on the outer surface of exosomes.

[0036] While keeping the other steps consistent, comparative examples 1-4 were set up. In comparative example 1, the reaction pH of step S2 was adjusted to 5.8; in comparative example 2, the reaction pH of step S2 was adjusted to 7.4; in comparative example 3, the reaction time of step S2 was adjusted to 1 h; and in comparative example 4, the molar ratio of DSPE-PEG2000-MAL to YQT12 peptide in step S2 was adjusted to 1:2.3.

[0037] The products obtained from each group were further prepared into nucleic acid exosomes using the same method as in this embodiment, and their key indicators were tested. The results are shown in Table 1.

[0038] Table 1. Effects of different reaction conditions in step S2 on the preparation of DSPE-PEG2000-YQT12 and the performance of subsequent formulations. This embodiment 1:1.5 6.6 3.0 88.4 91.2 75.8 31.7 1.90 Comparative Example 1 1:1.5 5.8 3.0 62.7 70.4 54.1 26.8 1.42 Comparative Example 2 1:1.5 7.4 3.0 58.9 66.8 50.6 25.9 1.35 Comparative Example 3 1:1.5 6.6 1.0 64.8 73.1 57.3 27.6 1.48 Comparative Example 4 1:2.3 6.6 3.0 90.1 75.6 61.9 28.4 1.56 As shown in Table 1, under the conditions of pH 6.6, reaction time 3 h and DSPE-PEG2000-MAL to YQT12 peptide molar ratio of 1:1.5, the obtained DSPE-PEG2000-YQT12 has both high coupling rate and high purity, and the subsequent membrane insertion rate is also significantly better than that of each pair of ratios.

[0039] Comparative Example 1 shows that when the pH of the reaction system is lower than the range defined in claim 7, the coupling reaction between the thiol group and maleimide is insufficient, resulting in a decrease in both coupling rate and purity, which in turn affects the efficiency of subsequent membrane insertion.

[0040] Comparative Example 2 shows that when the pH of the reaction system is higher than the range defined in claim 7, the stability of the maleimide group decreases and side reactions increase, which is also not conducive to obtaining high-quality targeted phospholipid molecules.

[0041] Comparative Example 3 shows that when the reaction time is too short, the coupling is insufficient, resulting in insufficient density of subsequent exosome membrane modification.

[0042] Comparative Example 4 shows that although increasing the amount of YQT12 can slightly increase the coupling rate, excessive peptides will increase the purification burden, reduce the purity of the target product, and further affect the membrane insertion effect and the consistency of the final formulation.

[0043] As can be seen from the data in Table 1, the essential difference between this embodiment and the comparative example is not whether YQT12 or DSPE-PEG-MAL is used, but rather that the reaction conditions defined in step S2, after matching and selection, can take into account the coupling reaction efficiency, product purity, and subsequent granulation construction performance. These process conditions are not only conducive to obtaining stable DSPE-PEG2000-YQT12, but also to improving its subsequent insertion efficiency in the exosome membrane, and further improving the nucleic acid encapsulation performance of the final nucleic acid exosome formulation and the surface exposure effect of YQT12 after MMP treatment.

[0044] Example 2 Based on the exosome vector, DSPE-PEG2000-YQT12, and reducible cationic nucleic acid core obtained in Example 1, this embodiment further illustrates the preparation of the matrix metalloproteinase-responsive shielding molecule DSPE-PEGa-MMP-L-PEGb and its regulatory effect on the exposure state of YQT12. Unless otherwise stated in this embodiment, the exosome source, nucleic acid core construction, and purification conditions are the same as in Example 1. A nucleic acid exosome preparation targeting myofibroblasts includes exosomes isolated from mammalian cell culture supernatant, a targeting phospholipid molecule and a matrix metalloproteinase-responsive shielding molecule inserted into the exosome membrane, and a reducible cationic nucleic acid core encapsulated in the exosome lumen. The targeted phospholipid molecule is DSPE-PEGx-YQT12, where DSPE is 1,2-distearate-sn-glycerol-3-phosphatidylethanolamine, PEGx is a polyethylene glycol spacer arm, and the amino acid sequence of YQT12 is Ser-Leu-Tyr-Gln-Thr-Asp-Asp-Arg-Asn-Asp-Tyr-Ile-Gly-Gly-Cys; The matrix metalloproteinase-responsive shielding molecule is DSPE-PEGa-MMP-L-PEGb, where PEGa is the near-membrane polyethylene glycol spacer arm, MMP-L is a peptide that can be cleaved by MMP-2 and / or MMP-9, and PEGb is the distal polyethylene glycol shielding chain. The amino acid sequence of MMP-L is selected from any one of Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln, Gly-Pro-Leu-Gly-Val-Arg-Gly, and Pro-Leu-Gly-Leu-Ala-Gly; The average molecular weight of PEGb is greater than that of PEGx, and the average molecular weight of PEGb is 1.5-10 times that of PEGx. The reducible cationic nucleic acid core includes a first small interfering RNA, a second small interfering RNA, and a short cationic peptide containing disulfide bonds. The target gene of the first small interfering RNA is HSP47, and the target gene of the second small interfering RNA is LOXL2. The cationic short peptide containing disulfide bonds is formed by the covalent linkage of a nucleic acid binding segment, a reducible linker segment, and an endosome escape accessory segment. The nucleic acid binding segment is a peptide containing 4-12 arginine residues and / or lysine residues, the reducible linker segment contains at least one disulfide bond, and the endosome escape accessory segment is a peptide containing 3-10 histidine residues. Matrix metalloproteinase-responsive shielding molecules provide spatial shielding for YQT12 when not cleaved by MMP-2 and / or MMP-9, and weaken the spatial shielding of YQT12 after cleavage by MMP-2 and / or MMP-9, thereby increasing the exposure of YQT12 on the exosome surface. The average molecular weight of PEGx is 1000-3500 Da, the average molecular weight of PEGa is 500-3000 Da, and the average molecular weight of PEGb is 3000-10000 Da. A method for preparing a nucleic acid exosome preparation targeting myofibroblasts includes the following steps: S1, Exosomes were isolated from mammalian cell culture supernatant; S2 provides a YQT12 polypeptide containing a terminal cysteine ​​residue, and reacts the YQT12 polypeptide with DSPE-PEGx-MAL to obtain the targeted phospholipid molecule DSPE-PEGx-YQT12; The amino acid sequence of the YQT12 polypeptide is Ser-Leu-Tyr-Gln-Thr-Asp-Asp-Arg-Asn-Asp-Tyr-Ile-Gly-Gly-Cys; S3, mix the first small interfering RNA, the second small interfering RNA and the cationic short peptide containing disulfide bonds to form a reducible cationic nucleic acid core; S4. A reducible cationic nucleic acid core is loaded into exosomes using electroporation to obtain nucleic acid-loaded exosomes. S5, DSPE-PEGx-YQT12 and matrix metalloproteinase responsive shielding molecule DSPE-PEGa-MMP-L-PEGb are co-incubated with nucleic acid-loaded exosomes simultaneously or sequentially to allow DSPE-PEGx-YQT12 and DSPE-PEGa-MMP-L-PEGb to insert into the exosome membrane; S6. The product is purified to remove the unloaded reducible cationic nucleic acid core, the DSPE-PEGx-YQT12 that is not inserted into the exosome membrane, and the DSPE-PEGa-MMP-L-PEGb that is not inserted into the exosome membrane, to obtain the nucleic acid exosome preparation.

[0045] In this embodiment, PEGx is selected as a polyethylene glycol spacer arm with an average molecular weight of 2000 Da, PEGa is selected as a near-membrane polyethylene glycol spacer arm with an average molecular weight of 1000 Da, PEGb is selected as a distal polyethylene glycol shielding chain with an average molecular weight of 5000 Da, and MMP-L is selected as Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln. The average molecular weight ratio of PEGb to PEGx is 2.5, which can form spatial shielding of YQT12 when it is not cleaved by matrix metalloproteinases, and weaken this shielding effect in the presence of MMP-2 and / or MMP-9.

[0046] The matrix metalloproteinase-responsive shielding molecule was prepared as follows.

[0047] The peptide Fmoc-Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln-COOH, with an N-terminal Fmoc-protected and C-terminal free carboxyl group, was prepared by solid-phase synthesis. This peptide was dissolved in anhydrous dimethylformamide, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide were added. The mixture was activated at room temperature for 30 min. Subsequently, DSPE-PEG1000-NH2 was added, and the reaction was carried out under nitrogen protection for 12 h, allowing the C-terminus of the peptide to form an amide bond with DSPE-PEG1000-NH2. After the reaction, piperidine / dimethylformamide solution was added to remove the Fmoc protecting group, yielding the DSPE-PEG1000-Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln intermediate with a free N-terminal amino group.

[0048] After removing small molecule reactants by dialysis, the above intermediate was reacted with mPEG5000-NHS in phosphate buffer at pH 7.4 for 4 h, allowing mPEG5000 to be linked to the N-terminal amino group of the peptide, yielding DSPE-PEG1000-Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln-PEG5000. The reaction product was purified by size exclusion chromatography and reversed-phase high-performance liquid chromatography, and the main peak fraction was collected. After freeze-drying, a white or off-white solid was obtained. MALDI-TOF mass spectrometry analysis showed that the main peak distribution of the product matched the target molecular weight range. The purity of the product was determined to be 90.6% by the area normalization method of high-performance liquid chromatography. In this preparation process, the DSPE end is used for insertion into the exosome membrane, PEG1000 is located on the near-membrane side, Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln is located in the middle cleavage region, and PEG5000 is located at the distal end and acts as a shielding chain.

[0049] DSPE-PEG1000-Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln-PEG5000 was dissolved in Tris buffer containing 5 mmol / L CaCl2. A mixed enzyme solution of recombinant MMP-2 and MMP-9 was added, and the sample was treated at 37℃ for 60 min. After treatment, high-performance liquid chromatography (HPLC) analysis showed a significant decrease in the area of ​​the main peak of the intact molecule, while an elution peak corresponding to the distal PEG5000 fragment appeared. This indicates that the shielding molecule can be cleaved by MMP-2 and / or MMP-9. Samples without added MMP-2 and MMP-9 did not show significant cleavage peaks under the same conditions, indicating that the shielding molecule has good stability under non-enzymatic conditions.

[0050] The aforementioned shielding molecules are then incorporated into a nucleic acid exosome preparation.

[0051] Exosomes, DSPE-PEG2000-YQT12, and a reducible cationic nucleic acid core were prepared according to Example 1. The reducible cationic nucleic acid core was formed from siHSP47, siLOXL2, and a short cationic peptide containing disulfide bonds. The reducible cationic nucleic acid core was loaded into exosomes via electroporation to obtain nucleic acid-loaded exosomes. Subsequently, DSPE-PEG2000-YQT12 and DSPE-PEG1000-Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln-PEG5000 were added to the nucleic acid-loaded exosome dispersion at a molar ratio of 1:3 and co-incubated at 25°C for 1.5 h to allow the two DSPE derivatives to insert into the exosome membrane. After co-incubation, the mixture was purified by size exclusion chromatography and ultrafiltration to remove uninserted phospholipid molecules and unloaded nucleic acid cores, yielding a nucleic acid exosome formulation containing matrix metalloproteinase-responsive shielding molecules. The average particle size of the obtained formulation was 131.2 mm. The polydispersity index was 0.20 and the zeta potential was -9.1 mV, indicating that the exosomes could still maintain a dispersed state after the insertion of the phospholipid molecules.

[0052] Comparative Examples 5-8 were set up. Comparative Example 5 was a YQT12 exosome formulation without the addition of a matrix metalloproteinase-responsive shielding molecule. Comparative Example 6 used the shielding molecule DSPE-PEG1000-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-PEG5000, which cannot be cleaved by MMP-2 and MMP-9, to replace the responsive shielding molecule in this example. Comparative Example 7 used DSPE-PEG1000-Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln-PEG2000 as the shielding molecule, so that the average molecular weight ratio of PEGb to PEGx was 1.0. Comparative Example 8 used DSPE-PEG1000-Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln-PEG15000 as the shielding molecule, so that the average molecular weight of PEGb exceeded the preferred range of this invention. The remaining preparation conditions of each group were kept consistent.

[0053] To evaluate the effects of different shielding structures on the exposure status of YQT12 and its uptake by myofibroblasts, this embodiment conducted separate verifications of protease cleavage and cell uptake. The exosome preparations were divided into untreated and enzyme-treated groups. The enzyme-treated group was pretreated with a mixture of MMP-2 and MMP-9 enzymes at 37°C for 30 min. The untreated group was treated with enzyme-free buffer solution under the same conditions. After treatment, the relative exposure of YQT12 on the exosome surface was measured to evaluate the shielding effect of the shielding molecules under non-enzymatic conditions and the deshielding effect after cleavage by MMP-2 and / or MMP-9.

[0054] TGF-β1-induced transdifferentiation of human lung fibroblasts into myofibroblasts was used as an uptake evaluation model. Specifically, human lung fibroblast MRC-5 cells were treated with 10 ng / mL TGF-β1 for 48 h to increase α-SMA expression and obtain a myofibroblast-like state. The preparations pretreated with MMP-2 and MMP-9 were added to the above cell culture system and incubated for 4 h before cell uptake was detected. This cell experiment was used to evaluate the delivery ability of the preparations after in vitro enzyme digestion and deshielding to myofibroblasts. The protease-responsive deshielding effect was mainly reflected by the change in YQT12 exposure before and after the aforementioned in vitro MMP treatment. The results are shown in Table 2.

[0055] Table 2. Effects of different shielding structures on YQT12 exposure and myofibroblast delivery. This embodiment DSPE-PEG1000-GPLGIAGQ-PEG5000 2.5 78.4 0.36 0.92 181.6 Comparative Example 5 No shielding molecules — — 0.94 0.97 174.3 Comparative Example 6 DSPE-PEG1000-GGGGGGGG-PEG5000 2.5 6.8 0.33 0.39 88.7 Comparative Example 7 DSPE-PEG1000-GPLGIAGQ-PEG2000 1.0 75.9 0.63 0.90 165.8 Comparative Example 8 DSPE-PEG1000-GPLGIAGQ-PEG15000 7.5 49.6 0.28 0.61 112.4 As shown in Table 2, in this embodiment, the relative exposure of YQT12 without enzyme treatment was 0.36, indicating that the PEG5000 distal shielding chain can effectively reduce the non-specific exposure of YQT12. After in vitro pretreatment with MMP-2 and MMP-9, the cleavage rate of the shielding molecule reached 78.4%, and the relative exposure of YQT12 increased to 0.92, indicating that the shielding molecule can achieve responsive deshielding under the action of protease. Furthermore, when the MMP-pretreated formulation was co-incubated with myofibroblasts induced by TGF-β1, the cell uptake intensity reached 181.6 au, indicating that the exposure of YQT12 after deshielding is beneficial to enhancing the delivery of the formulation to myofibroblasts.

[0056] Comparative Example 5 did not have a shielding molecule, so YQT12 was already highly exposed without MMP treatment. Although its uptake intensity by myofibroblasts was high after MMP treatment, this group lacked shielding control over the non-lesion environment, making it difficult to demonstrate the lesion protease responsive delivery advantage.

[0057] Comparative Example 6 used an indestructible shielding molecule. Although the YQT12 exposure was low without MMP treatment, the YQT12 exposure did not increase significantly after MMP treatment, and the cellular uptake intensity was also significantly lower than in this example. This indicates that the cleavability of the shielding chain is the key to achieving responsive targeted exposure.

[0058] Comparative Example 7 used a shorter PEGb, with an average molecular weight ratio of PEGb to PEGx of less than 1.5, resulting in a higher exposure of YQT12 in the untreated case. This indicates that it is difficult to form an effective spatial shield when the shielding chain length is insufficient.

[0059] Comparative Example 8 used excessively long PEGb, which had strong shielding when untreated, but its cleavage efficiency and the exposure of YQT12 after MMP treatment were lower than those in this example. It is speculated that the spatial obstruction formed by the excessively long PEG chain will affect the enzyme's approach to the cleaved peptide and the subsequent exposure effect.

[0060] The results above show that the DSPE-PEG1000-Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln-PEG5000 used in this embodiment simultaneously satisfies the molecular weight range of PEGa and PEGb and the structural relationship that PEGb is greater than PEGx. It can effectively mask YQT12 without enzymatic treatment. After in vitro pretreatment with MMP-2 and / or MMP-9, it can significantly increase the detectable exposure of YQT12 on the in vitro surface of exosomes. When the demasked formulation is further used in myofibroblast uptake experiments, it can improve the cellular uptake intensity.

[0061] These results indicate that the structural selection of matrix metalloproteinase-responsive shielding molecules is not simply about increasing the PEG chain, but rather about achieving controllable regulation of the YQT12 exposure state through the combination of PEG chain length, cleavable peptide segments, and DSPE membrane anchoring structures, thereby endowing nucleic acid exosome formulations with more explicit protease-responsive deshielding characteristics.

[0062] Example 3 This embodiment is used to illustrate the composition, particle size control and reduction release performance of the reducible cationic nucleic acid core. Except for the nucleic acid core ratio and cationic short peptide structure, the preparation methods of the exosome carrier, the targeted phospholipid molecule and the responsive shielding molecule are the same as those in the previous embodiment. A nucleic acid exosome preparation targeting myofibroblasts includes exosomes isolated from mammalian cell culture supernatant, a targeting phospholipid molecule and a matrix metalloproteinase-responsive shielding molecule inserted into the exosome membrane, and a reducible cationic nucleic acid core encapsulated in the exosome lumen. The targeted phospholipid molecule is DSPE-PEGx-YQT12, where DSPE is 1,2-distearate-sn-glycerol-3-phosphatidylethanolamine, PEGx is a polyethylene glycol spacer arm, and the amino acid sequence of YQT12 is Ser-Leu-Tyr-Gln-Thr-Asp-Asp-Arg-Asn-Asp-Tyr-Ile-Gly-Gly-Cys; The matrix metalloproteinase-responsive shielding molecule is DSPE-PEGa-MMP-L-PEGb, where PEGa is the near-membrane polyethylene glycol spacer arm, MMP-L is a peptide that can be cleaved by MMP-2 and / or MMP-9, and PEGb is the distal polyethylene glycol shielding chain. The amino acid sequence of MMP-L is selected from any one of Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln, Gly-Pro-Leu-Gly-Val-Arg-Gly, and Pro-Leu-Gly-Leu-Ala-Gly; The average molecular weight of PEGb is greater than that of PEGx, and the average molecular weight of PEGb is 1.5-10 times that of PEGx. The reducible cationic nucleic acid core includes a first small interfering RNA, a second small interfering RNA, and a short cationic peptide containing disulfide bonds. The target gene of the first small interfering RNA is HSP47, and the target gene of the second small interfering RNA is LOXL2. The disulfide-bonded cationic short peptide is formed by the covalent linkage of a nucleic acid binding segment, a reducible linker segment, and an endosome escape helper segment. The nucleic acid binding segment is a peptide containing 4-12 arginine residues and / or lysine residues, the reducible linker segment contains at least one disulfide bond, and the intracellular release helper segment is a pH-responsive peptide containing 3-10 histidine residues. Matrix metalloproteinase-responsive shielding molecules provide spatial shielding for YQT12 when not cleaved by MMP-2 and / or MMP-9, and weaken the spatial shielding of YQT12 after cleavage by MMP-2 and / or MMP-9, thereby increasing the exposure of YQT12 on the exosome surface. The molar ratio of the targeting phospholipid molecule to the matrix metalloproteinase-responsive shielding molecule was 1:1 to 1:8; the mass ratio of the first small interfering RNA to the second small interfering RNA was 1:0.25 to 1:4. The average particle size of the exosomes is 30-150 nm, the average particle size of the reducible cationic nucleic acid core is 3-15 nm, preferably 5-12 nm, and the average particle size of the reducible cationic nucleic acid core is not greater than 30% of the average particle size of the exosomes. The zeta potential of the nucleic acid exosome preparation is -20 mV to +5 mV. After treatment with MMP-2 and / or MMP-9, the accessibility of YQT12 was detected using fluorescently labeled recognition molecules capable of recognizing the YQT12 peptide on the exosome surface, and normalized according to the number of exosome particles. The normalized fluorescence intensity was higher than that of the control without MMP-2 and / or MMP-9 treatment, indicating that after the matrix metalloproteinase-responsive shielding molecules were cleaved, their spatial shielding effect on YQT12 was weakened, resulting in an increase in the detectable exposure of YQT12 on the exosome surface. A method for preparing a nucleic acid exosome preparation targeting myofibroblasts includes the following steps: S1, Exosomes were isolated from mammalian cell culture supernatant; S2 provides a YQT12 polypeptide containing a terminal cysteine ​​residue, and reacts the YQT12 polypeptide with DSPE-PEGx-MAL to obtain the targeted phospholipid molecule DSPE-PEGx-YQT12; The amino acid sequence of the YQT12 polypeptide is Ser-Leu-Tyr-Gln-Thr-Asp-Asp-Arg-Asn-Asp-Tyr-Ile-Gly-Gly-Cys; Provided is a matrix metalloproteinase-responsive shielding molecule DSPE-PEGa-MMP-L-PEGb; wherein DSPE-PEGa-MMP-L-PEGb is prepared according to the method of Example 2, or is prepared by reacting a DSPE-PEGa-MMP-L intermediate containing an MMP-L peptide with activated PEGb; MMP-L is a peptide that can be cleaved by MMP-2 and / or MMP-9, and PEGb is a distal polyethylene glycol shielding chain; S3, mix the first small interfering RNA, the second small interfering RNA and the cationic short peptide containing disulfide bonds to form a reducible cationic nucleic acid core; S4. A reducible cationic nucleic acid core is loaded into exosomes using electroporation to obtain nucleic acid-loaded exosomes. S5, DSPE-PEGx-YQT12 and matrix metalloproteinase responsive shielding molecule DSPE-PEGa-MMP-L-PEGb are co-incubated with nucleic acid-loaded exosomes simultaneously or sequentially to allow DSPE-PEGx-YQT12 and DSPE-PEGa-MMP-L-PEGb to insert into the exosome membrane; S6. The product is purified to remove the unloaded reducible cationic nucleic acid core, the DSPE-PEGx-YQT12 that is not inserted into the exosome membrane, and the DSPE-PEGa-MMP-L-PEGb that is not inserted into the exosome membrane, to obtain the nucleic acid exosome preparation. The molar ratio of the cationic group in the disulfide-bonded cationic short peptide to the total phosphate group of the first and second small interfering RNA is 1:1 to 10:1; the mass ratio of the first and second small interfering RNAs is 1:0.25 to 1:4.

[0063] In this embodiment, the first small interfering RNA is a small interfering RNA targeting HSP47, and the second small interfering RNA is a small interfering RNA targeting LOXL2. The cationic short peptide containing disulfide bonds uses the H6-Cys-SS-Cys-R8 structure, where H6 is a pH-responsive histidine helper segment formed by 6 histidine residues, R8 is a nucleic acid binding segment formed by 8 arginine residues, and a disulfide bond is formed between the two cysteine ​​residues as a reducible linker segment. The H6 fragment has the potential to be protonated in an acidic endosome / lysosome environment and can serve as an auxiliary structure for intracellular release of the nucleic acid core after cellular uptake. This structure is used to assist in the release and intracellular transport of the nucleic acid core, and does not require the exosome itself to undergo a specific endosome escape process as the sole prerequisite.

[0064] SiHSP47 and SiLOXL2 were mixed at a mass ratio of 1:1 to obtain a dual small interfering RNA (SRNA) mixture. H6-Cys-SS-Cys-R8 short peptide solution was slowly added to the SRNA mixture, ensuring a molar ratio of cationic groups in the cationic short peptide to the total phosphate groups of the two SRNAs of 6:1. Gentle agitation was used during mixing to avoid RNA degradation caused by vigorous vortexing. The mixture was then allowed to stand at room temperature for 20 min, allowing the positively charged short peptide and negatively charged SRNA to self-assemble through electrostatic interactions to form a reducible cationic nucleic acid core. Dynamic light scattering analysis of the resulting nucleic acid core showed an average particle size of 8.9 nm, a polydispersity index of 0.22, and a Zeta potential of +11.2 mV. The nucleic acid core was within the range of 3-15 nm and did not exceed 30% of the average exosome particle size, thus maintaining the compression and protection of the SRNA while preventing the nucleic acid core size from approaching the luminal scale of the exosome.

[0065] The nucleic acid cores were placed in ordinary HEPES buffer and HEPES buffer containing 10 mmol / L glutathione, respectively, and incubated at 37°C for 2 h. Ordinary HEPES buffer was used to simulate non-reducing conditions, and the changes in nucleic acid core particle size and potential were small. HEPES buffer containing 10 mmol / L glutathione was used to simulate the relatively reducing glutathione environment in the cytoplasm. Under this condition, the particle size distribution of nucleic acid cores became wider, and a significant increase in the proportion of released small interfering RNA was detected. This result indicates that disulfide bond linkages can be broken in the presence of reducing substances such as glutathione, reducing the binding effect of cationic short peptides on small interfering RNA, thereby facilitating the release of small interfering RNA after entering the cell and contacting the reducing cytoplasmic environment.

[0066] The aforementioned reducible cationic nucleic acid core was further loaded into exosomes.

[0067] Exosomes, DSPE-PEGx-YQT12, and DSPE-PEGa-MMP-L-PEGb were prepared according to the methods in Examples 1 and 2, wherein the average particle size of the exosomes was controlled to be 30-150 nm. The exosome dispersion was mixed with a reducible cationic nucleic acid core with an average particle size of 3-15 nm and then subjected to electroporation. After electroporation, the mixture was restored in an ice bath and purified by size exclusion chromatography to obtain nucleic acid-loaded exosomes. Subsequently, DSPE-PEGx-YQT12 and DSPE-PEGa-MMP-L-PEGb were added to the nucleic acid-loaded exosomes and co-incubated to allow them to insert into the exosome membrane. After purification, the nucleic acid exosome formulation was obtained. The resulting formulation had an average particle size of 47.3 nm and a Zeta potential of -8.3 mV, falling within the range of -20 mV to +5 mV. This indicates that under conditions where the nucleic acid core particle size is significantly smaller than the exosome particle size, the exosome membrane and the PEG structure on its surface can shield part of the positive charge of the nucleic acid core and maintain the formulation within the 30-150 nm range. Within the nm exosome particle size range.

[0068] Comparative Examples 9-12 were set up. In Comparative Example 9, the molar ratio of cationic groups to total phosphate groups was adjusted to 0.5:1; in Comparative Example 10, the molar ratio of cationic groups to total phosphate groups was adjusted to 12:1; in Comparative Example 11, the mass ratio of siHSP47 to siLOXL2 was adjusted to 1:0.1; in Comparative Example 12, the disulfide-free cationic short peptide H6-Gly-Gly-R8 was used instead of H6-Cys-SS-Cys-R8, and the remaining exosome loading, membrane insertion modification, and purification conditions were the same as in this example.

[0069] Myofibroblasts obtained from the transdifferentiation of human lung fibroblasts induced by TGF-β1 were used. MRC-5 cells were treated with 10 ng / mL TGF-β1 for 48 h, and then each group of exosome preparations were added. After culturing for another 24 h, the expression levels of HSP47, LOXL2 and type I collagen-related mRNAs were detected. Each index was normalized with the TGF-β1 model group as 1.00. The results are shown in Table 3.

[0070] Table 3. Effects of the ratio and structure of reducible cationic nucleic acid core on the performance of nucleic acid exosome formulations. This embodiment 6:1 1:1 H6-Cys-SS-Cys-R8 8.9 -8.3 Comparative Example 9 0.5:1 1:1 H6-Cys-SS-Cys-R8 26.4 -15.8 Comparative Example 10 12:1 1:1 H6-Cys-SS-Cys-R8 6.8 +7.9 Comparative Example 11 6:1 1:0.1 H6-Cys-SS-Cys-R8 8.4 -9.6 Comparative Example 12 6:1 1:1 H6-Gly-Gly-R8 9.2 -7.5 Group Nucleic acid encapsulation rate (%) RNA release rate (%) under reduction conditions HSP47 mRNA relative expression level LOXL2 mRNA relative expression level Relative expression level of type I collagen mRNA This embodiment 33.6 68.7 0.37 0.42 0.45 Comparative Example 9 18.4 41.5 0.69 0.73 0.76 Comparative Example 10 34.9 62.1 0.44 0.48 0.58 Comparative Example 11 32.1 66.2 0.39 0.82 0.67 Comparative Example 12 34.2 24.8 0.64 0.71 0.72 As shown in Table 3, when the molar ratio of cationic groups to total phosphate groups in this embodiment is 4:1, the mass ratio of siHSP47 to siLOXL2 is 1:1, and the disulfide-bonded cationic short peptide H6-Cys-SS-Cys-R8 is used, the average particle size of the reducible cationic nucleic acid core is 8.9 nm, which is within the range of 3-15 nm. Combined with the fact that the average particle size of exosomes in this embodiment is within the range of 30-150 nm, the average particle size of the nucleic acid core is not greater than 30% of the average particle size of the exosomes, which is beneficial for achieving intracavitary loading of exosomes through electroporation. After further loading and modification, the final nucleic acid exosome formulation has a Zeta potential of -8.3 mV, which is within the range of -20 mV to +5 mV. This formulation maintains a good nucleic acid encapsulation rate while having a high RNA release rate under reducing conditions, and can simultaneously reduce the expression of HSP47 and LOXL2, thereby reducing the expression level of type I collagen.

[0071] In Comparative Example 9, the molar ratio of cationic groups to total phosphate groups was less than 1:1. The cationic short peptides had insufficient compression capacity for small interfering RNA, and the resulting nucleic acid core particle size increased to 26.4 nm, exceeding the preferred particle size range defined in this invention. The nucleic acid encapsulation efficiency and intracellular silencing effect were significantly reduced. This result indicates that when the amount of cationic short peptides is too low, it is difficult to form a stable and loadable nucleic acid core.

[0072] In Comparative Example 10, the molar ratio of cationic groups to total phosphate groups was higher than 10:1. Although the nucleic acid encapsulation efficiency was not significantly reduced, the final formulation's Zeta potential increased to +7.9 mV, exceeding the range of -20 mV to +5 mV. The silencing effect of this group in the cell model was not better than that of this example, and the higher positive charge easily caused non-specific adsorption, which was not conducive to the stability of the formulation and delivery selectivity. This result shows that excessive cationic short peptides cannot further improve the delivery effect, but will instead disrupt the potential balance of the final exosome formulation.

[0073] In Comparative Example 11, the mass ratio of siHSP47 to siLOXL2 was less than 1:0.25. HSP47 expression was still inhibited to some extent, but the reduction in LOXL2 expression was not significant. The expression level of type I collagen was also higher than in this example. This result indicates that the mass ratio of the two small interfering RNAs needs to be controlled within a reasonable range in order to act on the two fibrosis-related targets, HSP47 and LOXL2, at the same time, thereby demonstrating the advantages of dual-target joint intervention.

[0074] In Comparative Example 12, a short cationic peptide without disulfide bonds was used to form the nucleic acid core. The particle size and encapsulation efficiency of this group were similar to those of this example, but the RNA release rate decreased significantly under reduction conditions. The silencing effects of HSP47 and LOXL2 were weaker than those of this example. This shows that the disulfide bond linker is not a simple structural modification, but a key structure that endows the nucleic acid core with the ability to release in a reduction response.

[0075] In summary, this embodiment controls the molar ratio of cationic groups to total phosphate groups of the two small interfering RNAs, the mass ratio of the first small interfering RNA to the second small interfering RNA, and introduces a short cationic peptide containing disulfide bonds. This results in a reducible cationic nucleic acid core with suitable particle size, high encapsulation efficiency, and reduction-triggered release capability. After the nucleic acid core is further loaded into exosomes and modified with a phospholipid membrane, a nucleic acid exosome formulation with suitable zeta potential, stable structure, and dual-target silencing effect can be formed. This demonstrates that the technical solution of this embodiment is feasible and reproducible.

[0076] Example 4 This embodiment is used to illustrate the effect of exosome separation method and electroporation conditions on the preparation of nucleic acid-loaded exosomes. The DSPE-PEG2000-YQT12, DSPE-PEG1000-GPLGIAGQ-PEG5000 and the reducible cationic nucleic acid core used were prepared according to the aforementioned embodiment. A nucleic acid exosome preparation targeting myofibroblasts includes exosomes isolated from mammalian cell culture supernatant, a targeting phospholipid molecule and a matrix metalloproteinase-responsive shielding molecule inserted into the exosome membrane, and a reducible cationic nucleic acid core encapsulated in the exosome lumen. The targeted phospholipid molecule is DSPE-PEGx-YQT12, where DSPE is 1,2-distearate-sn-glycerol-3-phosphatidylethanolamine, PEGx is a polyethylene glycol spacer arm, and the amino acid sequence of YQT12 is Ser-Leu-Tyr-Gln-Thr-Asp-Asp-Arg-Asn-Asp-Tyr-Ile-Gly-Gly-Cys; The matrix metalloproteinase-responsive shielding molecule is DSPE-PEGa-MMP-L-PEGb, where PEGa is the near-membrane polyethylene glycol spacer arm, MMP-L is a peptide that can be cleaved by MMP-2 and / or MMP-9, and PEGb is the distal polyethylene glycol shielding chain. The amino acid sequence of MMP-L is selected from any one of Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln, Gly-Pro-Leu-Gly-Val-Arg-Gly, and Pro-Leu-Gly-Leu-Ala-Gly; The average molecular weight of PEGb is greater than that of PEGx, and the average molecular weight of PEGb is 1.5-10 times that of PEGx. The reducible cationic nucleic acid core includes a first small interfering RNA, a second small interfering RNA, and a short cationic peptide containing disulfide bonds. The target gene of the first small interfering RNA is HSP47, and the target gene of the second small interfering RNA is LOXL2. The cationic short peptide containing disulfide bonds is formed by the covalent linkage of a nucleic acid binding segment, a reducible linker segment, and an endosome escape accessory segment. The nucleic acid binding segment is a peptide containing 4-12 arginine residues and / or lysine residues, the reducible linker segment contains at least one disulfide bond, and the endosome escape accessory segment is a peptide containing 3-10 histidine residues. Matrix metalloproteinase-responsive shielding molecules provide spatial shielding for YQT12 when not cleaved by MMP-2 and / or MMP-9, and weaken the spatial shielding of YQT12 after cleavage by MMP-2 and / or MMP-9, thereby increasing the exposure of YQT12 on the exosome surface. A method for preparing a nucleic acid exosome preparation targeting myofibroblasts includes the following steps: S1, Exosomes were isolated from mammalian cell culture supernatant; S2 provides a YQT12 polypeptide containing a terminal cysteine ​​residue, and reacts the YQT12 polypeptide with DSPE-PEGx-MAL to obtain the targeted phospholipid molecule DSPE-PEGx-YQT12; The amino acid sequence of the YQT12 polypeptide is Ser-Leu-Tyr-Gln-Thr-Asp-Asp-Arg-Asn-Asp-Tyr-Ile-Gly-Gly-Cys; S3, mix the first small interfering RNA, the second small interfering RNA and the cationic short peptide containing disulfide bonds to form a reducible cationic nucleic acid core; S4. A reducible cationic nucleic acid core is loaded into exosomes using electroporation to obtain nucleic acid-loaded exosomes. S5, DSPE-PEGx-YQT12 and matrix metalloproteinase responsive shielding molecule DSPE-PEGa-MMP-L-PEGb are co-incubated with nucleic acid-loaded exosomes simultaneously or sequentially to allow DSPE-PEGx-YQT12 and DSPE-PEGa-MMP-L-PEGb to insert into the exosome membrane; S6. The product is purified to remove the unloaded reducible cationic nucleic acid core, the DSPE-PEGx-YQT12 that is not inserted into the exosome membrane, and the DSPE-PEGa-MMP-L-PEGb that is not inserted into the exosome membrane, to obtain the nucleic acid exosome preparation. Exosomes were isolated from mammalian cell culture supernatant by at least one of differential centrifugation, ultracentrifugation, ultrafiltration, size exclusion chromatography, density gradient centrifugation or tangential flow filtration. In step S4, the reducible cationic nucleic acid core obtained in step S3 is mixed with the exosomes obtained in step S1 in electroporation buffer and then subjected to electroporation. The electroporation conditions are: voltage 150-250 V, pulse time 8-15 ms, number of pulses 2-3, and pulse interval 4-6 s; preferably, the voltage is 200 V, the pulse time is 10 ms, the number of pulses is 2, and the pulse interval is 5 s. After electroporation, the sample is placed in an ice bath for 5-15 min and incubated with recovery buffer for 20-40 min to promote the recovery of the exosome membrane structure.

[0077] The above electroporation conditions can achieve a balance between the entry of reducible cationic nucleic acid cores into exosomes and the maintenance of exosome membrane structure. When the voltage is too low, the instantaneous permeability of the exosome membrane is insufficient, and the nucleic acid cores are difficult to enter the exosomes effectively. When the voltage or pulse intensity is too high, although it may increase the nucleic acid entry ratio, it will cause a decrease in the retention rate of exosome particles, a widening of the particle size distribution, and a reduction in the retention of membrane markers. In this embodiment, 200 V, 10 ms, 2 pulses, and 5 s interval are selected as the preferred conditions to obtain both a high nucleic acid encapsulation rate and good exosome integrity.

[0078] In this embodiment, human umbilical cord mesenchymal stem cells were used as exosome donor cells. The cells were seeded in cell culture flasks and cultured in DMEM / F12 medium containing 10% exosome-free fetal bovine serum at 37°C and 5% CO2. When the cell confluence reached 80%-90%, the medium was replaced with fresh exosome-free medium and cultured for another 48 h. The cell culture supernatant was then collected.

[0079] The culture supernatant was centrifuged sequentially at 300×g for 10 min, 2000×g for 20 min, and 10000×g for 30 min to remove suspended cells, cell debris, and large vesicles. The supernatant was filtered through a 0.22 μm filter and then concentrated to approximately 1 / 20 of its original volume using a 100 kDa ultrafiltration membrane. Size exclusion chromatography was then performed to separate the exosomes, collecting the main peak fraction. The resulting fraction was then ultrafiltered into electroporation buffer to obtain an exosome dispersion. Nanoparticle tracking analysis showed that the average particle size of the obtained exosomes was 41.8 nm, the polydispersity index was 0.17, and the particle size was mainly distributed in the range of 30-150 nm. Transmission electron microscopy revealed near-circular membrane vesicle structures. Western blot analysis showed positive results for CD63 and TSG101, while no obvious signal was observed for Calnexin, indicating that the obtained exosomes had good purity and integrity.

[0080] The reducible cationic nucleic acid core was prepared according to the method in Example 3. Briefly, small interfering RNA targeting HSP47 and small interfering RNA targeting LOXL2 were mixed at a mass ratio of 1:1 and compounded with a disulfide-bonded cationic short peptide H6-Cys-SS-Cys-R8, so that the molar ratio of the cationic group in the cationic short peptide to the total phosphate group of the two small interfering RNAs was 6:1. After standing at room temperature for 20 min, a reducible cationic nucleic acid core was formed. The average particle size of the nucleic acid core was 39.2 nm, which is suitable for loading into exosomes by electroporation.

[0081] The above exosome dispersion was mixed with a reducible cationic nucleic acid core to achieve a mass ratio of exosome protein to total small interfering RNA of 100 μg: 2.5 μg. The mixture was then transferred to a 4 mm electroporation cup and electroporated at 200 V, 10 ms pulse duration, 2 pulses, and 5 s pulse interval. After electroporation, the sample was immediately placed in an ice bath for 10 min, followed by the addition of an equal volume of recovery buffer and incubation at room temperature for 30 min to allow the exosome membrane structure to recover. The recovered sample was then purified by size exclusion chromatography to remove the reducible cationic nucleic acid core that had not entered the exosomes, yielding nucleic acid-loaded exosomes.

[0082] Subsequently, bimolecular insertion modification was performed on the surface of the exosome membrane.

[0083] The DSPE-PEG2000-YQT12 prepared according to Example 1 and the DSPE-PEG1000-Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln-PEG5000 prepared according to Example 2 were mixed at a molar ratio of 1:3 and added to the above-mentioned nucleic acid-loaded exosome dispersion. The mixture was co-incubated at 25°C for 1.5 h to allow the DSPE-PEG2000-YQT12 and DSPE-PEG1000-Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln-PEG5000 to insert into the exosome membrane. After co-incubation, the mixture was purified by size exclusion chromatography and 300 kDa ultrafiltration to remove phospholipid molecules that had not inserted into the exosome membrane and residual free nucleic acid cores, thus obtaining a nucleic acid exosome preparation targeting myofibroblasts.

[0084] The obtained nucleic acid exosome formulation had an average particle size of 46.5 nm, a polydispersity index of 0.19, and a zeta potential of -8.7 mV. The nucleic acid encapsulation was detected by the RiboGreen method, and the encapsulation rate was 32.8%. After treatment with a mixed enzyme solution of MMP-2 and MMP-9, the detectable exposure of YQT12 on the outer surface of the exosome was higher than that of the untreated control, indicating that matrix metalloproteinase-responsive shielding molecules can reduce the spatial shielding effect of YQT12 under enzymatic cleavage conditions.

[0085] Comparative examples 13-17 were set up. Except for the differences listed in Table 4, the reducible cationic nucleic acid core, DSPE-PEG2000-YQT12, DSPE-PEG1000-Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln-PEG5000, and subsequent co-incubation and purification conditions used in each group were the same as those in this example.

[0086] Table 4. Effects of exosome isolation methods and electroporation conditions on the performance of nucleic acid exosome preparations. This embodiment Differential centrifugation + ultrafiltration + size exclusion chromatography 200 V, 10 ms, twice, 5 s interval 5.8 82.6 32.8 130.8 0.19 0.91 Comparative Example 13 Differential centrifugation + ultrafiltration, without size exclusion chromatography 200 V, 10 ms, twice, 5 s interval 3.7 76.4 28.9 151.6 0.31 0.78 Comparative Example 14 Differential centrifugation + ultracentrifugation 200 V, 10 ms, twice, 5 s interval 4.2 69.8 29.7 146.2 0.28 0.74 Comparative Example 15 Differential centrifugation + ultrafiltration + size exclusion chromatography 80 V, 10 ms, twice, 5 s interval 5.6 88.1 16.5 124.9 0.18 0.94 Comparative Example 16 Differential centrifugation + ultrafiltration + size exclusion chromatography 350 V, 10 ms, twice, 5 s interval 5.7 54.3 35.6 173.4 0.37 0.61 Comparative Example 17 Differential centrifugation + ultrafiltration + size exclusion chromatography 200 V, 30 ms, 5 times, 5 s interval 5.5 49.7 37.1 188.2 0.41 0.58 As shown in Table 4, this embodiment uses a combination of differential centrifugation, ultrafiltration, and size exclusion chromatography to separate exosomes. This method can remove cell debris, free proteins, and large particulate impurities while maintaining high exosome integrity, resulting in an exosome particle / protein ratio of 5.8 × 10⁻⁶. 9 The particle count / μg indicates high purity; the particle retention rate after electroporation is 82.6%, indicating that the vesicle structure is well maintained during subsequent nucleic acid loading. After further membrane insertion modification, the final formulation particle size and PDI are within a relatively stable range, and the relative retention of CD63 is 0.91, indicating that the exosome membrane markers were not significantly lost due to the process.

[0087] Comparative Example 13 did not undergo further purification using size exclusion chromatography, resulting in a low exosome particle / protein ratio and an increased PDI in the final formulation. This indicates that free proteins and impurity particles in the culture supernatant can affect the consistency of subsequent electroporation loading and membrane insertion modification.

[0088] Comparative Example 14 used ultracentrifugation for separation. Although exosomes could be obtained, the particle retention rate and relative CD63 retention after electroporation were lower than in this example. This suggests that strong centrifugation conditions may increase vesicle aggregation or membrane stress, thereby affecting the stability of subsequent nucleic acid loading preparations.

[0089] The electroporation voltage of Comparative Example 15 is lower than that defined in claim 9. Although the exosome particle retention rate is high, the nucleic acid encapsulation rate is only 16.5%, indicating that it is difficult to effectively promote the entry of reducible cationic nucleic acid cores into exosomes when the electric field strength is insufficient.

[0090] The electroporation voltage of Comparative Example 16 was higher than that defined in claim 9. Although the nucleic acid encapsulation efficiency was slightly higher than that of this embodiment, the particle retention rate decreased significantly after electroporation. The final formulation particle size increased and the PDI increased, indicating that excessively high voltage would damage the integrity of the exosome membrane. In Comparative Example 17, although extending the pulse time and increasing the number of pulses could further increase the nucleic acid entry ratio, it also led to a decrease in particle retention rate and relative CD63 retention, and the formation of a formulation with a wider particle size distribution. This indicates that excessively strong electroporation treatment is not conducive to the recovery of exosome membrane structure and batch-to-batch stability of the formulation.

[0091] Based on the above results, this embodiment obtains exosomes with high purity and integrity through a combination of differential centrifugation, ultrafiltration, and size exclusion chromatography. Furthermore, it achieves effective loading of a reducible cationic nucleic acid core using electroporation at 200 V, 10 ms, 2 pulses, and 5 s intervals. This method falls within the voltage range of 100-300 V, pulse duration of 5-20 ms, number of pulses of 1-5, and pulse interval of 3-10 s defined in claim 9, achieving a balance between nucleic acid encapsulation efficiency and exosome integrity.

[0092] Example 5 This example illustrates the effects of the membrane insertion ratio, co-incubation temperature, and co-incubation time of DSPE-PEG2000-YQT12 and DSPE-PEG1000-GPLGIAGQ-PEG5000 on the performance of the final formulation. Nucleic acid-loaded exosomes were prepared according to Example 4. A nucleic acid exosome preparation targeting myofibroblasts includes exosomes isolated from mammalian cell culture supernatant, a targeting phospholipid molecule and a matrix metalloproteinase-responsive shielding molecule inserted into the exosome membrane, and a reducible cationic nucleic acid core encapsulated in the exosome lumen. The targeted phospholipid molecule is DSPE-PEGx-YQT12, where DSPE is 1,2-distearate-sn-glycerol-3-phosphatidylethanolamine, PEGx is a polyethylene glycol spacer arm, and the amino acid sequence of YQT12 is Ser-Leu-Tyr-Gln-Thr-Asp-Asp-Arg-Asn-Asp-Tyr-Ile-Gly-Gly-Cys; The matrix metalloproteinase-responsive shielding molecule is DSPE-PEGa-MMP-L-PEGb, where PEGa is the near-membrane polyethylene glycol spacer arm, MMP-L is a peptide that can be cleaved by MMP-2 and / or MMP-9, and PEGb is the distal polyethylene glycol shielding chain. The amino acid sequence of MMP-L is selected from any one of Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln, Gly-Pro-Leu-Gly-Val-Arg-Gly, and Pro-Leu-Gly-Leu-Ala-Gly; The average molecular weight of PEGb is greater than that of PEGx, and the average molecular weight of PEGb is 1.5-10 times that of PEGx. The reducible cationic nucleic acid core includes a first small interfering RNA, a second small interfering RNA, and a short cationic peptide containing disulfide bonds. The target gene of the first small interfering RNA is HSP47, and the target gene of the second small interfering RNA is LOXL2. The cationic short peptide containing disulfide bonds is formed by the covalent linkage of a nucleic acid binding segment, a reducible linker segment, and an endosome escape accessory segment. The nucleic acid binding segment is a peptide containing 4-12 arginine residues and / or lysine residues, the reducible linker segment contains at least one disulfide bond, and the endosome escape accessory segment is a peptide containing 3-10 histidine residues. Matrix metalloproteinase-responsive shielding molecules provide spatial shielding for YQT12 when not cleaved by MMP-2 and / or MMP-9, and weaken the spatial shielding of YQT12 after cleavage by MMP-2 and / or MMP-9, thereby increasing the exposure of YQT12 on the exosome surface. The average molecular weight of PEGx is 1000-3500 Da, the average molecular weight of PEGa is 500-3000 Da, and the average molecular weight of PEGb is 3000-10000 Da. The molar ratio of the targeting phospholipid molecule to the matrix metalloproteinase-responsive shielding molecule was 1:1 to 1:8; the mass ratio of the first small interfering RNA to the second small interfering RNA was 1:0.25 to 1:4. A method for preparing a nucleic acid exosome preparation targeting myofibroblasts includes the following steps: S1, Exosomes were isolated from mammalian cell culture supernatant; S2 provides a YQT12 polypeptide containing a terminal cysteine ​​residue, and reacts the YQT12 polypeptide with DSPE-PEGx-MAL to obtain the targeted phospholipid molecule DSPE-PEGx-YQT12; The amino acid sequence of the YQT12 polypeptide is Ser-Leu-Tyr-Gln-Thr-Asp-Asp-Arg-Asn-Asp-Tyr-Ile-Gly-Gly-Cys; S3, mix the first small interfering RNA, the second small interfering RNA and the cationic short peptide containing disulfide bonds to form a reducible cationic nucleic acid core; S4. A reducible cationic nucleic acid core is loaded into exosomes using electroporation to obtain nucleic acid-loaded exosomes. S5, DSPE-PEGx-YQT12 and matrix metalloproteinase responsive shielding molecule DSPE-PEGa-MMP-L-PEGb are co-incubated with nucleic acid-loaded exosomes simultaneously or sequentially to allow DSPE-PEGx-YQT12 and DSPE-PEGa-MMP-L-PEGb to insert into the exosome membrane; S6. The product is purified to remove the unloaded reducible cationic nucleic acid core, the DSPE-PEGx-YQT12 that is not inserted into the exosome membrane, and the DSPE-PEGa-MMP-L-PEGb that is not inserted into the exosome membrane, to obtain the nucleic acid exosome preparation. In step S5, the molar ratio of DSPE-PEGx-YQT12 to matrix metalloproteinase responsive shielding molecule is 1:1 to 1:8, the co-incubation temperature is 4-37℃, and the co-incubation time is 0.5-6 hours.

[0093] In this embodiment, DSPE-PEG2000-YQT12 prepared in Example 1 was used as the targeting phospholipid molecule, and DSPE-PEG1000-Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln-PEG5000 prepared in Example 2 was used as the matrix metalloproteinase responsive shielding molecule. The average molecular weight of PEGx was 2000 Da, the average molecular weight of PEGa was 1000 Da, and the average molecular weight of PEGb was 5000 Da, all of which were within the range defined by this invention. The average molecular weight ratio of PEGb to PEGx was 2.5, which could form spatial shielding of YQT12 when it was not cleaved by MMP-2 and / or MMP-9, and weaken the shielding effect after enzymatic cleavage.

[0094] Nucleic acid-loaded exosomes were prepared according to the method in Example 4, that is, a reducible cationic nucleic acid core was first loaded into the exosomes by electroporation, and then the unloaded free nucleic acid core was removed by size exclusion chromatography to obtain a nucleic acid-loaded exosome dispersion. The obtained nucleic acid-loaded exosomes had an average particle size of 44.9 nm, a polydispersity index of 0.18, and a zeta potential of -10.6 mV, and could be used for subsequent membrane insertion modification.

[0095] DSPE-PEG2000-YQT12 and DSPE-PEG1000-Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln-PEG5000 were mixed at a molar ratio of 1:3 and added to a nucleic acid-loaded exosome dispersion. For every 100 μg of exosome protein, 16 nmol of exogenous DSPE derivatives were added. The mixture was incubated at 25°C with slow shaking for 1.5 h to allow the hydrophobic segments of the two DSPE derivatives to insert into the exosome membrane and to position YQT12 and the responsive shielding structure on the outer surface of the exosome.

[0096] After co-incubation, the resulting mixture was separated by size exclusion chromatography, and the main peak component of exosomes was collected. Then, a 300 kDa ultrafiltration tube was used for concentration and buffer replacement to remove DSPE-PEG2000-YQT12 and DSPE-PEG1000-Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln-PEG5000 that were not inserted into the exosome membrane, as well as residual free nucleic acid cores, to obtain the nucleic acid exosome formulation. The average particle size of the obtained formulation was 47.1 nm, the polydispersity index was 0.20, and the zeta potential was -8.5 mV, indicating that the insertion of diphospholipid molecules did not lead to significant exosome aggregation, and the formulation still maintained good dispersibility.

[0097] To detect the membrane insertion of the two phospholipid molecules, parallel samples were prepared using FITC-labeled DSPE-PEG2000-YQT12 and Cy5-labeled DSPE-PEG1000-Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln-PEG5000, respectively. After purification, the membrane insertion rate was calculated by normalizing the exosome particle number. The results showed that the insertion rate of DSPE-PEG2000-YQT12 in this example was 72.8%, and the insertion rate of the responsive shielding molecule was 78.6%. The obtained formulation was further treated with a mixed enzyme solution of MMP-2 and MMP-9 at 37°C for 30 min. After particle number normalization, the detectable exposure of YQT12 on the outer surface of the exosome was detected. The results showed that the exposure of YQT12 on the outer surface after MMP treatment was significantly higher than that in the untreated group, indicating that the responsive shielding molecule can reduce the spatial shielding of YQT12 in the presence of matrix metalloproteinases.

[0098] Comparative examples 18-22 were set up. Except for the differences listed in Table 5, all groups used the same batch of nucleic acid-loaded exosomes, DSPE-PEG2000-YQT12 and DSPE-PEG1000-Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln-PEG5000, and obtained the final formulations using the same purification method. TGF-β1-induced transdifferentiation of human lung fibroblasts was used to obtain myofibroblasts. After each group's formulation was pretreated with MMP-2 and MMP-9, it was co-incubated with the cells for 4 h, and the cell uptake intensity was detected. The results are shown in Table 5.

[0099] Table 5. Effects of phospholipid membrane insertion conditions on the performance of nucleic acid exosome formulations. This embodiment 1:3 25 1.5 72.8 78.6 Comparative Example 18 1:0.5 25 1.5 74.1 41.7 Comparative Example 19 1:10 25 1.5 56.3 86.9 Comparative Example 20 1:3 4 1.5 43.5 50.8 Comparative Example 21 1:3 42 1.5 68.9 73.2 Comparative Example 22 1:3 25 8.0 75.4 82.1 Group Relative exposure of YQT12 without MMP treatment Relative exposure of YQT12 after MMP treatment Cellular uptake intensity (au) without MMP treatment Cell uptake intensity (au) after MMP treatment MMP Response Intake Index This embodiment 0.35 0.91 72.4 179.4 2.48 Comparative Example 18 0.71 0.93 149.6 171.2 1.14 Comparative Example 19 0.24 0.56 58.7 104.8 1.79 Comparative Example 20 0.42 0.64 69.3 118.6 1.71 Comparative Example 21 0.33 0.82 77.8 126.5 1.63 Comparative Example 22 0.31 0.84 82.6 142.9 1.73 The MMP response uptake index is the ratio of cell uptake intensity after MMP treatment to cell uptake intensity without MMP treatment.

[0100] As shown in Table 5, in this embodiment, when DSPE-PEG2000-YQT12 and the responsive shielding molecule were co-incubated at 25℃ for 1.5 h with a molar ratio of 1:3, the insertion rate of YQT12 was 72.8% and the insertion rate of the shielding molecule was 78.6%, indicating that both DSPE derivatives could be fully inserted into the exosome membrane. In this group without MMP treatment, the relative exposure of YQT12 was 0.35 and the cellular uptake intensity was 72.4 au; after treatment with MMP-2 and MMP-9, the relative exposure of YQT12 increased to 0.91, the cellular uptake intensity increased to 179.4 au, and the MMP responsive uptake index was 2.48.

[0101] The above results indicate that this embodiment can maintain low YQT12 exposure and low nonspecific uptake in a non-enzymatic environment, while significantly increasing YQT12 exposure and enhancing myofibroblast uptake in the presence of matrix metalloproteinases, demonstrating good responsive delivery selectivity.

[0102] In Comparative Example 18, the proportion of responsive shielding molecules was low, with an insertion rate of only 41.7%. In this group, the relative exposure of YQT12 without MMP treatment reached 0.71, and the cellular uptake intensity was 149.6 au. This indicates that when the shielding molecule density was insufficient, YQT12 was already heavily exposed in a non-enzymatic environment. Although the cellular uptake intensity after MMP treatment was 171.2 au, close to that of this example, its MMP-responsive uptake index was only 1.14. This suggests that the enhanced uptake was not primarily triggered by enzymatic deshielding, but rather because YQT12 was already in a high-exposure state before treatment. Therefore, Comparative Example 18 could not effectively achieve shielding control in a non-lesion environment.

[0103] In Comparative Example 19, the proportion of responsive shielding molecules was too high, with a shielding molecule insertion rate of 86.9%, but the YQT12 insertion rate decreased to 56.3%. The relative exposure of YQT12 in this group without MMP treatment was low, at 0.24, indicating that excessive shielding molecules can produce a strong shielding effect. However, after MMP treatment, the relative exposure of YQT12 only increased to 0.56, and the cellular uptake intensity was 104.8 au, which was significantly lower than that in this example.

[0104] These results indicate that when there is an excess of shielding molecules, even if partial enzymatic cleavage occurs, the residual steric hindrance can still affect the exposure and targeted delivery of YQT12.

[0105] In Comparative Example 20, the co-incubation temperature was 4°C. The insertion rates of both phospholipid molecules decreased, with YQT12 insertion rate at 43.5% and shielding molecule insertion rate at 50.8%. The relative exposure of YQT12 and cellular uptake intensity after MMP treatment in this group were lower than those in this example, indicating that excessively low temperatures are not conducive to the insertion of hydrophobic segments of DSPE into the exosome membrane, resulting in insufficient modification of the membrane surface.

[0106] In Comparative Example 21, the co-incubation temperature was 42°C. Both phospholipid molecules could still insert into the exosome membrane, but the cell uptake intensity after MMP treatment was only 126.5 au, and the MMP response uptake index was 1.63, which was lower than that in this example. This result indicates that excessively high temperature may disturb the exosome membrane structure or affect the surface arrangement of inserted molecules, so that the increased exposure of YQT12 cannot be fully converted into effective uptake.

[0107] In Comparative Example 22, although the YQT12 insertion rate and the shielding molecule insertion rate were slightly improved when the co-incubation time was extended to 8 h, the cell uptake intensity after MMP treatment was 142.9 au and the MMP response uptake index was 1.73, which was still lower than that in this example. This result indicates that excessively long co-incubation time cannot further improve the delivery effect, but may instead lead to uneven distribution of exogenous phospholipid molecules in the exosome membrane, affecting the effective recognition after responsive exposure.

[0108] In summary, the advantage of this embodiment is not simply that the cell uptake intensity is higher after MMP treatment, but rather that it can reduce the non-specific exposure of YQT12 without MMP treatment, and significantly increase the exposure of YQT12 after MMP treatment, resulting in a higher MMP-responsive uptake index. The molar ratio of DSPE-PEG2000-YQT12 to the responsive shielding molecule, the co-incubation temperature, and the co-incubation time need to be controlled within an appropriate range in order to achieve a balance between membrane insertion efficiency, shielding effect, post-enzyme digestion exposure effect, and myofibroblast delivery selectivity.

[0109] Example 6 This example is used to verify the changes in YQT12 exposure, myofibroblast uptake capacity, and dual-target gene silencing effect of the final nucleic acid exosome formulation under MMP-2 and / or MMP-9 environments. The formulation used was prepared according to the conditions determined in Examples 1 to 5. A nucleic acid exosome preparation targeting myofibroblasts includes exosomes isolated from mammalian cell culture supernatant, a targeting phospholipid molecule and a matrix metalloproteinase-responsive shielding molecule inserted into the exosome membrane, and a reducible cationic nucleic acid core encapsulated in the exosome lumen. The targeted phospholipid molecule is DSPE-PEGx-YQT12, where DSPE is 1,2-distearate-sn-glycerol-3-phosphatidylethanolamine, PEGx is a polyethylene glycol spacer arm, and the amino acid sequence of YQT12 is Ser-Leu-Tyr-Gln-Thr-Asp-Asp-Arg-Asn-Asp-Tyr-Ile-Gly-Gly-Cys; The matrix metalloproteinase-responsive shielding molecule is DSPE-PEGa-MMP-L-PEGb, where PEGa is the near-membrane polyethylene glycol spacer arm, MMP-L is a peptide that can be cleaved by MMP-2 and / or MMP-9, and PEGb is the distal polyethylene glycol shielding chain. The amino acid sequence of MMP-L is selected from any one of Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln, Gly-Pro-Leu-Gly-Val-Arg-Gly, and Pro-Leu-Gly-Leu-Ala-Gly; The average molecular weight of PEGb is greater than that of PEGx, and the average molecular weight of PEGb is 1.5-10 times that of PEGx. The reducible cationic nucleic acid core includes a first small interfering RNA, a second small interfering RNA, and a short cationic peptide containing disulfide bonds. The target gene of the first small interfering RNA is HSP47, and the target gene of the second small interfering RNA is LOXL2. The cationic short peptide containing disulfide bonds is formed by the covalent linkage of a nucleic acid binding segment, a reducible linker segment, and an endosome escape accessory segment. The nucleic acid binding segment is a peptide containing 4-12 arginine residues and / or lysine residues, the reducible linker segment contains at least one disulfide bond, and the endosome escape accessory segment is a peptide containing 3-10 histidine residues. Matrix metalloproteinase-responsive shielding molecules provide spatial shielding for YQT12 when not cleaved by MMP-2 and / or MMP-9, and weaken the spatial shielding of YQT12 after cleavage by MMP-2 and / or MMP-9, thereby increasing the exposure of YQT12 on the exosome surface. The molar ratio of the targeting phospholipid molecule to the matrix metalloproteinase-responsive shielding molecule was 1:1 to 1:8; the mass ratio of the first small interfering RNA to the second small interfering RNA was 1:0.25 to 1:4. The average particle size of exosomes is 30-150 nm, the average particle size of the reducible cationic nucleic acid core is no more than 30% of the average particle size of exosomes, and the zeta potential of the nucleic acid exosome formulation is -20 mV to +5 mV. After treatment with MMP-2 and / or MMP-9, the accessibility of YQT12 was detected using fluorescently labeled recognition molecules capable of recognizing the YQT12 peptide on the exosome surface, and normalized according to the number of exosome particles. The normalized fluorescence intensity was higher than that of the control without MMP-2 and / or MMP-9 treatment, indicating that after the matrix metalloproteinase-responsive shielding molecules were cleaved, their spatial shielding effect on YQT12 was weakened, resulting in an increase in the detectable exposure of YQT12 on the exosome surface.

[0110] After processing, each group of samples was incubated with YQT12-specific fluorescently labeled probes. After removing unbound probes, the fluorescence intensity of exosomes was measured by nanoflow cytometry or fluorescence enzyme labeling and normalized according to the number of exosome particles. The normalized fluorescence intensity was used to represent the exposure level of YQT12 on the outer surface of exosomes.

[0111] The matrix metalloproteinase-responsive shielding molecule in this invention is used to regulate the exposure status of YQT12 by utilizing the relatively elevated microenvironment characteristics of MMP-2 and / or MMP-9 in pulmonary fibrosis lesions. After MMP-2 and / or MMP-9 cleave the shielding molecule, the exposure level of YQT12 on the exosome surface increases; subsequently, YQT12 mediates the binding and uptake of exosomes with myofibroblasts at the lesion site.

[0112] The delivery process of this invention includes two consecutive steps: lesion microenvironment-responsive demasking and YQT12-mediated myofibroblast recognition, rather than relying solely on MMP expression to achieve cell selectivity.

[0113] The nucleic acid exosome formulation in this embodiment was prepared according to the methods described in Examples 1 to 5 above. Specifically, the targeting phospholipid molecule was DSPE-PEG2000-YQT12, and the matrix metalloproteinase responsive shielding molecule was DSPE-PEG1000-Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln-PEG5000, with a molar ratio of 1:3. The first small interfering RNA was siHSP47, and the second small interfering RNA was siLOXL2, with a mass ratio of 1:1. The disulfide-containing cationic short peptide was H6-Cys-SS-Cys-R8. The average particle size of the resulting reducible cationic nucleic acid core was 42.3 nm, the average particle size of the resulting reducible cationic nucleic acid core was 9.1 nm, and the final nucleic acid exosome formulation had an average particle size of 46.9 nm and a Zeta potential of -8.6 mV, consistent with an average exosome particle size of 30-150 nm and an average reducible cationic nucleic acid core particle size of 3-15 nm. The exosome size must be no larger than 30% of the average exosome size, and the zeta potential of the nucleic acid exosome preparation must be between -20 mV and +5 mV.

[0114] To detect changes in YQT12 exposure on the exosome surface after treatment with MMP-2 and / or MMP-9, the obtained nucleic acid exosome preparations were divided into an untreated group and an enzyme-treated group. In the enzyme-treated group, a mixed enzyme solution of recombinant MMP-2 and MMP-9 was added to the preparation and treated at 37°C for 30 min. In the untreated group, a buffer solution without enzyme was added under the same conditions. After treatment, the detectable exposure of YQT12 on the exosome surface was detected using a fluorescently labeled recognition probe for YQT12, and the exosome particle count was normalized. The results showed that after treatment with MMP-2 and MMP-9, the detectable exposure of YQT12 on the outer surface of the preparation in this example was significantly higher than that in the untreated group, indicating that the responsive shielding molecules can reduce the spatial shielding of YQT12 under enzyme cleavage conditions.

[0115] Furthermore, myofibroblasts obtained from the transdifferentiation of human lung fibroblasts induced by TGF-β1 were used. MRC-5 cells were treated with 10 ng / mL TGF-β1 for 48 h to increase α-SMA expression and form a fibrotic-like cell state accompanied by increased MMP-2 and MMP-9 expression. Nucleotide exosome preparations of each group were pretreated with MMP-2 and MMP-9 and then added to the cell culture system. After co-incubation for 4 h, the cell uptake intensity was measured. After culturing for another 24 h, the mRNA expression levels of HSP47, LOXL2, and COL1A1 were measured. The expression levels of each mRNA were normalized with the TGF-β1 model group as 1.00.

[0116] Comparative examples 23-27 were set up. Except for the differences listed in Table 6, the source of exosomes, nucleic acid loading method, membrane insertion treatment and purification conditions were kept consistent across groups.

[0117] Table 6. MMP-responsive exposure, cell delivery, and antifibrosis-related effects of nucleic acid exosome preparations. This embodiment Responsive shielding + dual siRNA + disulfide bond short peptide 1:3 1:1 39.4 -8.6 Comparative Example 23 Non-responsive shielding molecules 1:0 1:1 38.7 -7.4 Comparative Example 24 Using indivisible shielding molecules 1:3 1:1 40.1 -9.2 Comparative Example 25 Excessive shielding molecule ratio 1:10 1:1 41.8 -12.7 Comparative Example 26 Contains only siHSP47 1:3 1:0 35.6 -8.1 Comparative Example 27 Using cationic short peptides without disulfide bonds 1:3 1:1 40.6 -7.8 Group YQT12 can detect exposure levels (untreated / treated with MMP). Cell uptake intensity (au) after MMP treatment HSP47 mRNA relative expression level LOXL2 mRNA relative expression level COL1A1 mRNA relative expression level This embodiment 0.34 / 0.93 184.7 0.36 0.41 0.43 Comparative Example 23 0.95 / 0.98 176.5 0.39 0.45 0.52 Comparative Example 24 0.32 / 0.40 91.6 0.68 0.73 0.78 Comparative Example 25 0.23 / 0.57 114.9 0.56 0.62 0.66 Comparative Example 26 0.36 / 0.91 178.3 0.37 0.91 0.69 Comparative Example 27 0.35 / 0.90 171.4 0.63 0.70 0.72 As shown in Table 6, the core particle size of the reducible cationic nucleic acid in this embodiment is 39.4 nm, and the zeta potential of the final nucleic acid exosome preparation is -8.6 mV, both of which are within the scope of the claims. After treatment with MMP-2 and MMP-9, the detectable exposure of YQT12 on the outer surface of the exosome increased from 0.34 to 0.93, indicating that the matrix metalloproteinase-responsive shielding molecule can deshield in the protease environment of the lesion, changing YQT12 from a relatively shielded state to an exposed state. Correspondingly, the uptake intensity in the myofibroblast model of this embodiment reached 184.7 au, and the relative expression levels of HSP47, LOXL2, and COL1A1 decreased to 0.36, 0.41, and 0.43, respectively, showing good dual-target nucleic acid delivery and anti-fibrosis-related intervention effects.

[0118] Comparative Example 23 did not have a responsive shielding molecule set up, and YQT12 was highly exposed even without MMP treatment. The change in exposure before and after MMP treatment was not significant. Although this group had a high cellular uptake intensity, it lacked control over YQT12 exposure in a non-enzymatic environment and could not reflect the responsive delivery characteristics of the lesion microenvironment. In contrast, this example maintained a low YQT12 exposure level without enzyme treatment and then significantly increased the YQT12 exposure level after MMP treatment, which is more conducive to achieving responsive targeted delivery.

[0119] Comparative Example 24 used an uncleavable shielding molecule. The exposure of YQT12 was low without MMP treatment, but the exposure only increased from 0.32 to 0.40 after MMP treatment. The cellular uptake intensity was significantly lower than in this example, and the silencing effects of HSP47 and LOXL2 were also significantly weakened. These results indicate that the cleavable peptide of the shielding molecule is the key structure for achieving MMP-responsive exposure.

[0120] In Comparative Example 25, the proportion of shielding molecules was too high. Although it could further reduce YQT12 exposure without enzyme treatment, the exposure of YQT12 after MMP treatment was still insufficient. The cell uptake and gene silencing effects were lower than those in this example. This result indicates that the molar ratio of targeting phospholipid molecules to responsive shielding molecules needs to be controlled within a reasonable range. Excessive shielding molecules will still leave strong steric hindrance after enzyme digestion, affecting the YQT12-mediated delivery function.

[0121] Comparative Example 26 only encapsulated siHSP47 and did not contain siLOXL2. This group had an inhibitory effect on HSP47, but LOXL2 expression was not effectively inhibited. The expression level of COL1A1 was higher than that in this example, indicating that a single siRNA is difficult to simultaneously intervene in collagen formation and collagen cross-linking related pathways. In contrast, this example achieved a more comprehensive inhibitory effect on fibrosis-related molecules by co-loading siHSP47 and siLOXL2.

[0122] Comparative Example 27 used a short cationic peptide without disulfide bonds to form the nucleic acid core. The exposure and cellular uptake of YQT12 in this group were similar to those in this example, but the decrease in the expression of HSP47, LOXL2 and COL1A1 was significantly insufficient. This indicates that the main defect is not in cell entry, but in the insufficient nucleic acid release efficiency after entering the cell. It can be seen that the short cationic peptide containing disulfide bonds can endow the nucleic acid core with the ability to release in a reduction response, which is an important structure for the effective functioning of dual small interfering RNA.

[0123] In summary, the nucleic acid exosome formulation prepared in this embodiment simultaneously meets the following characteristics: the ratio of targeted phospholipid molecules to responsive shielding molecules is reasonable, the ratio of dual small interfering RNAs is reasonable, the particle size of the reducible cationic nucleic acid core is suitable, and the final formulation's zeta potential is within a stable range; under the condition of MMP-2 and / or MMP-9, the detectable exposure of the YQT12 outer surface is significantly higher than that of the untreated control, and it further improves myofibroblast uptake and dual-target gene silencing effects. This result proves that the formulation in this embodiment is not a simple parallel combination of exosomes, targeting peptides, shielding chains, and small interfering RNA, but rather forms a continuous delivery mechanism through responsive deshielding, dual-target nucleic acid cores, and reduction-triggered release, which has clear feasibility, repeatability, and beneficial effects.

Claims

1. A nucleic acid exosome formulation targeting myofibroblasts, characterized in that, This includes exosomes isolated from mammalian cell culture supernatant, targeting phospholipid molecules and matrix metalloproteinase-responsive shielding molecules inserted into the exosome membrane, and a reducible cationic nucleic acid core encapsulated in the exosome lumen; The targeted phospholipid molecule is DSPE-PEGx-YQT12, wherein DSPE is 1,2-distearate-sn-glycerol-3-phosphatidylethanolamine, PEGx is a polyethylene glycol spacer arm, and the amino acid sequence of YQT12 is Ser-Leu-Tyr-Gln-Thr-Asp-Asp-Arg-Asn-Asp-Tyr-Ile-Gly-Gly-Cys; The matrix metalloproteinase-responsive shielding molecule is DSPE-PEGa-MMP-L-PEGb, wherein PEGa is a near-membrane polyethylene glycol spacer arm, MMP-L is a peptide that can be cleaved by MMP-2 and / or MMP-9, and PEGb is a distal polyethylene glycol shielding chain. The amino acid sequence of the MMP-L is selected from any one of Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln, Gly-Pro-Leu-Gly-Val-Arg-Gly, and Pro-Leu-Gly-Leu-Ala-Gly; The average molecular weight of PEGb is greater than that of PEGx, and the average molecular weight of PEGb is 1.5-10 times that of PEGx. The reducible cationic nucleic acid core includes a first small interfering RNA, a second small interfering RNA, and a cationic short peptide containing disulfide bonds. The target gene of the first small interfering RNA is HSP47, and the target gene of the second small interfering RNA is LOXL2. The disulfide-containing cationic short peptide is formed by covalently linking a nucleic acid binding segment, a reducible linker segment, and an endosome escape auxiliary segment. The nucleic acid binding segment is a peptide containing 4-12 arginine residues and / or lysine residues, the reducible linker segment contains at least one disulfide bond, and the endosome escape auxiliary segment is a peptide containing 3-10 histidine residues. The matrix metalloproteinase-responsive shielding molecule provides spatial shielding for YQT12 when it is not cleaved by MMP-2 and / or MMP-9, and weakens the spatial shielding of YQT12 after cleavage by MMP-2 and / or MMP-9, thereby increasing the exposure of YQT12 on the exosome's outer surface.

2. The nucleic acid exosome preparation of claim 1, characterized in that, The average molecular weight of PEGx is 1000-3500 Da, the average molecular weight of PEGa is 500-3000 Da, and the average molecular weight of PEGb is 3000-10000 Da.

3. The nucleic acid exosome preparation of claim 1, wherein, The molar ratio of the targeted phospholipid molecule to the matrix metalloproteinase-responsive shielding molecule is 1:1 to 1:8; the mass ratio of the first small interfering RNA to the second small interfering RNA is 1:0.25 to 1:

4.

4. The nucleic acid exosome preparation of claim 1, wherein, The exosomes have an average particle size of 30-150 nm, the reducible cationic nucleic acid core has an average particle size of no more than 30% of the average particle size of the exosomes, and the nucleic acid exosome preparation has a zeta potential of -20 mV to +5 mV. After treatment with MMP-2 and / or MMP-9, the normalized fluorescence intensity of YQT12 on the outer surface of exosomes was higher than that of the control group that was not treated with MMP-2 and / or MMP-9 after detection using the YQT12 specific fluorescently labeled probe binding method and normalized according to the number of exosome particles.

5. A method of preparing the myofibroblast-targeted nucleic acid exosome preparation of any one of claims 1-4, characterized in that, Includes the following steps: S1, Exosomes were isolated from mammalian cell culture supernatant; S2 provides a YQT12 polypeptide containing a terminal cysteine ​​residue, and reacts the YQT12 polypeptide with DSPE-PEGx-MAL to obtain the targeted phospholipid molecule DSPE-PEGx-YQT12; The amino acid sequence of the YQT12 polypeptide is Ser-Leu-Tyr-Gln-Thr-Asp-Asp-Arg-Asn-Asp-Tyr-Ile-Gly-Gly-Cys; S3, mix the first small interfering RNA, the second small interfering RNA and the cationic short peptide containing disulfide bonds to form a reducible cationic nucleic acid core; S4. A reducible cationic nucleic acid core is loaded into exosomes using electroporation to obtain nucleic acid-loaded exosomes. S5, DSPE-PEGx-YQT12 and matrix metalloproteinase responsive shielding molecule DSPE-PEGa-MMP-L-PEGb are co-incubated with nucleic acid-loaded exosomes simultaneously or sequentially to allow DSPE-PEGx-YQT12 and DSPE-PEGa-MMP-L-PEGb to insert into the exosome membrane; S6. The product is purified to remove the unloaded reducible cationic nucleic acid core, the DSPE-PEGx-YQT12 that is not inserted into the exosome membrane, and the DSPE-PEGa-MMP-L-PEGb that is not inserted into the exosome membrane, to obtain the nucleic acid exosome preparation.

6. The production method according to claim 5, characterized by, The exosomes were isolated from mammalian cell culture supernatant by at least one of differential centrifugation, ultracentrifugation, ultrafiltration, size exclusion chromatography, density gradient centrifugation, or tangential flow filtration.

7. The preparation method according to claim 5, characterized in that, The YQT12 peptide was reacted with DSPE-PEGx-MAL in a buffer solution at pH 6.0-7.0 for 2-4 hours, and the molar ratio of DSPE-PEGx-MAL to YQT12 peptide was 1:1.2 to 1:

2.

8. The preparation method according to claim 5, characterized in that, The molar ratio of the cationic group in the disulfide-bonded cationic short peptide to the total phosphate group of the first and second small interfering RNA is 1:1 to 10:1; the mass ratio of the first and second small interfering RNAs is 1:0.25 to 1:

4.

9. The preparation method according to claim 5, characterized in that, The electroporation method is as follows: voltage 150-250 V, pulse time 8-15 ms, number of pulses 2-3, pulse interval 4-6 s.

10. The preparation method according to claim 5, characterized in that, The molar ratio of DSPE-PEGx-YQT12 to matrix metalloproteinase responsive shielding molecules is 1:1 to 1:8, the co-incubation temperature is 4-37℃, and the co-incubation time is 0.5-6 hours.

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