Fap-targeted engineered exosomes for treating hypertrophic scars and preparation method and application thereof

Abstract

This invention provides a FAP-targeted engineered exosome for treating hypertrophic scars, its preparation method, and its application, belonging to the field of biopharmaceutical manufacturing technology. After co-incubation with an ferroptosis inducer, the FAP-targeting small molecule ligand SUC-Lys(Ac)-PEG3-UAMC1110 is covalently linked to the exosome membrane surface using an EDC / NH-mediated amide bond coupling strategy to obtain engineered exosomes. These engineered exosomes are then used to prepare microneedle patches. The microneedle patches allow the engineered exosomes to accumulate at the scar site and preferentially enter FAP-highly expressed scar fibroblasts, inhibiting their abnormal proliferation, migration, and pro-fibrotic phenotype, reducing collagen deposition, and promoting hypertrophic scar tissue remodeling. This invention combines the advantages of strong targeting, low off-target toxicity, minimally invasive drug delivery, sustained local retention, and stable efficacy, making it suitable for preparing drugs to treat or improve hypertrophic scars, and possessing good clinical translational potential.

Description

Technical Field

[0001] This application belongs to the field of biopharmaceutical manufacturing technology, specifically relating to a FAP-targeted engineered exosome for treating hypertrophic scars, its preparation method, and its application. Background Technology

[0002] Hypertrophic scars are a common adverse outcome of skin injury, characterized by persistent abnormal activation of scar fibroblasts, manifested as excessive proliferation, migration, and transformation into myofibroblast phenotypes. This leads to abnormal deposition of extracellular matrix such as collagen, thickening of scar tissue, and structural disorder. Currently used clinical treatments generally suffer from insufficient targeting, unstable efficacy, high recurrence rates, or easy damage to normal tissue.

[0003] In recent years, ferroptosis, as a novel programmed cell death mechanism driven by iron-dependent lipid peroxidation, has provided new insights for selectively inhibiting aberrantly activated fibroblasts. However, existing ferroptosis inducers (such as systemic xc)... - Inhibitors and other drugs are prone to causing non-specific cytotoxicity when applied topically, resulting in a narrow therapeutic window. Therefore, there is an urgent need for delivery systems that can precisely target and enrich fibroblasts in lesions and scars.

[0004] Exosomes (sEVs) are considered ideal drug carriers due to their good biocompatibility, low immunogenicity, and natural transcellular delivery capabilities. However, sEVs still face challenges in local applications, such as short retention time, impaired percutaneous penetration, and non-specific uptake. There is an urgent need to provide a delivery molecule that can specifically target pathogenic fibroblasts and form a stable link with exosomes. Summary of the Invention

[0005] The purpose of this invention is to provide a small molecule ligand for fibroblast activation protein (FAP) that has good delivery characteristics to pathogenic fibroblasts and is stably linked to exosomes.

[0006] This invention provides a small molecule ligand for targeting fibroblast activation proteins, having the following molecular structure: SUC-Lys(Ac)-PEG3-UAMC1110; The SUC is succinyl, Lys(Ac) is Nε-acetylated lysine, PEG3 is triethylene glycol, and - indicates covalent linkage.

[0007] The present invention provides an engineered exosome, comprising an exosome loaded with a scar treatment drug; the surface of the exosome is modified with a small molecule ligand targeting the fibroblast activation protein.

[0008] Preferably, the fibroblast activation protein targeting small molecule ligand is coupled via an amide bond formed between the activated carboxyl group in the succinyl group and the amino group on the surface of the exosome.

[0009] Preferably, the mass ratio of the protein content of the exosomes to the fibroblast activation protein targeting small molecule ligand is 5~15:0.5~2.

[0010] Preferably, the scar treatment drug includes a ferroptosis inducer; The loading concentration of the ferroptosis inducer is 85~95 μM; The types of ferroptosis inducers include erastin.

[0011] Preferably, the cell source of the exosomes includes at least one of the following: adipose-derived mesenchymal stem cells, fibroblasts, bone marrow mesenchymal stem cells, and umbilical cord mesenchymal stem cells.

[0012] This invention provides a method for preparing the engineered exosomes, comprising the following steps: Exosomes and scar treatment drugs were co-incubated to remove free scar treatment drugs, resulting in exosomes loaded with scar treatment drugs. Engineered exosomes were obtained by coupling exosomes loaded with scar treatment drugs with small molecule ligands targeting fibroblast activation proteins.

[0013] The present invention provides a microneedle patch, wherein the cavity of the microneedle contains the engineered exudate or the engineered exudate prepared by the preparation method.

[0014] The present invention provides a drug for treating scars, comprising the engineered exudate or the engineered exudate prepared by the preparation method.

[0015] This invention provides the application of the engineered exudate or the engineered exudate prepared by the preparation method in the preparation of drugs for hypertrophic scars.

[0016] This invention provides a small molecule ligand targeting fibroblast activation proteins, having the following molecular structure: SUC-Lys(Ac)-PEG3-UAMC1110; where SUC is succinyl, Lys(Ac) is Nε-acetylated lysine, PEG3 is triethylene glycol, and - indicates covalent linkage. UAMC1110 in this small molecule ligand is a drug that inhibits FAP enzyme activity, effectively blocking collagen metabolism, fibroblast activation, and inhibiting scar hyperplasia. The succinyl group enhances coupling efficiency, stability, and multivalent binding affinity. Simultaneously, Nε-acetylated lysine serves as the molecular backbone and linker site, reducing immunogenicity, while triethylene glycol acts as a flexible linker group, improving molecular water solubility and prolonging the in vivo half-life. Therefore, this small molecule ligand not only targets and inhibits pathogenic fibroblasts, exerting its therapeutic effect to reduce scar hyperplasia, but also provides an effective exosome binding site, facilitating the preparation of exosome delivery systems.

[0017] This invention provides an engineered exosome comprising an exosome loaded with a scar treatment drug; the surface of the exosome is modified with a small molecule ligand targeting the fibroblast activation protein. This invention improves delivery selectivity by covalently linking the FAP-targeting ligand to the exosome membrane surface, enabling it to preferentially recognize and enter scar fibroblasts with high FAP expression. Simultaneously, this invention loads a ferroptosis inducer into the engineered exosome with FAP targeting, which can trigger ferroptosis-related processes within the target cells, reducing the risk of off-target damage caused by free drug diffusion and expanding the therapeutic window. This invention uses a soluble microneedle patch for percutaneous minimally invasive delivery, allowing the engineered exosome to be continuously released locally in the dermis and prolonging its residence time, avoiding the pain and uneven distribution caused by multi-point injections, and improving local utilization and treatment stability. Attached Figure Description

[0018] Figure 1 A schematic diagram of the synthetic route for the FAP-targeted small molecule ligand SUC-Lys(Ac)-PEG3-UAMC1110; Figure 2 This is a schematic diagram of the preparation process of the engineered exosomes (A) and the soluble microneedle patch (B) loaded with engineered exosomes of the present invention; Figure 3 The figure shows the characterization results of exosomes and their different formulations (unloaded, loaded with ferroptosis inducers, and loaded with ferroptosis inducers with surface-coupled FAP targeting ligands). A is the morphology figure, B is the particle size / concentration analysis figure, and C is the marker protein detection figure. Figure 4 Internalization diagram of engineered exosomes in scar fibroblasts and human dermal fibroblasts (A) and quantitative comparison diagram (B and C). Figure 5The figure shows the validation results of ferroptosis-related changes induced by engineered exosomes. A is the protein immunoassay result, B is the mitochondrial membrane potential change graph, and C is the statistical bar graph. Figure 6 The images show the characterization and in vivo retention results of soluble microneedle patches loaded with engineered exosomes. In the images, A is the morphology of the microneedle patch, B is the in vivo fluorescence imaging image compared with direct injection, and C is the fluorescence signal statistics. Figure 7 The image shows the therapeutic effect of soluble microneedle patches in a rabbit ear hypertrophic scar model; A is the phenotype, B is the HE staining image, and C is the SEI statistical results for each group. Figure 8 The synthetic route for 3Mal-Lys(Ac)-PEG3-UAMC1110; Figure 9 The HPLC results for 3Mal-Lys(Ac)-PEG3-UAMC1110 are as follows: 1 is 6.481, 2 is 6.802, 3 is 6.988, 4 is 9.708, 5 is 9.936, 6 is 10.118, and 7 is 10.525. Figure 10 MS detection results for 3Mal-Lys(Ac)-PEG3-UAMC1110; Figure 11 Results for the connection efficiency of 3Mal-Lys(Ac)-PEG3-UAMC1110. Detailed Implementation

[0019] This invention provides a small molecule ligand for targeting fibroblast activation proteins, having the following molecular structure: SUC-Lys(Ac)-PEG3-UAMC1110; The SUC is succinyl, Lys(Ac) is Nε-acetylated lysine, PEG3 is triethylene glycol, and - indicates covalent linkage.

[0020] In this invention, the small molecule ligand can react with the amino group on the exosome surface via the activated carboxyl group in the succinyl group to form an amide bond. Simultaneously, UAMC1110 in the small molecule ligand targets and inhibits FAP enzyme activity in fibroblasts, thereby inhibiting fibroblast metastasis.

[0021] In this invention, the method for preparing the fibroblast activation protein-targeting small molecule ligand preferably includes the following steps: The N-Boc-PEG3-carboxylic acid solution was activated by mixing HATU and DIEA, and then reacted with a 4-quinolinecarboxamide derivative (UAMC1110) to obtain a Boc-protected intermediate. The Boc-protected intermediate is separated after de-Boc protection to obtain a Boc-free intermediate. Fmoc-Lys(Ac) was activated by EDC and HOBt, and then reacted with a Boc-free intermediate and N-methylmorpholine to obtain a second intermediate. The second intermediate was deprotected from Fmoc to obtain an intermediate containing a free amino group; The intermediate containing free amino groups was subjected to a third reaction with succinic anhydride and DIEA to introduce a succinyl-terminal carboxyl group, and then purified to obtain SUC-Lys(Ac)-PEG3-UAMC1110.

[0022] In this invention, the working concentration of the N-Boc-PEG3-carboxylic acid solution is preferably 10-50 mM, and can be 20 mM. The working concentration of HATU is preferably 0.5 mM-2 mM, and can be 1 mM. The working concentration of DIEA is preferably 2 mM-5 mM, and can be 3-4 mM. The working concentration of the 4-quinoline carboxamide derivative is preferably 0.3-0.8 mM, and can be 0.5 mM. During activation, the solvent is DMF. The activation time is preferably 3-7 min, and can be 5 min. The working concentration of the 4-quinoline carboxamide derivative is preferably 0.1-1 mM, and can be 0.5 mM. The first reaction time is preferably 1-2 h, and can be 1.5 h. The first reaction temperature is preferably 1-2 h, and can be 1.5 h.

[0023] In this invention, during the deprotection of the Boc-protected intermediate, the solvent is preferably removed under reduced pressure beforehand. The reagent used for deprotection is preferably trifluoroacetic acid (TFA). The separation is preferably performed by precipitating the deprotection reaction system with cold diethyl ether, followed by centrifugation, collection of the precipitate, and drying.

[0024] In this invention, the working concentration of Fmoc-Lys(Ac) is preferably 0.1~1 M, and can be 0.5 M. The working concentration of EDC is preferably 0.1~1 M, and can be 0.28 M. The working concentration of HOBt is preferably 0.1~1 M, and can be 0.23 M. The working concentration of the Boc-removing intermediate is preferably 10~50 mg / ml, and can be 30 mg / ml. The working concentration of N-methylmorpholine for Boc removal is preferably 0.1~0.3 M, and can be 0.2 M. The activation time of EDC and HOBt is preferably 8~12 min, and can be 10~12 min. The activation temperature of EDC and HOBt is preferably 0℃. The temperature of the second reaction is preferably 20~27℃, and can be 22~25℃, or 23~24℃. The reaction time of the second reaction is preferably 1.5~2.5 h, and can be 2 h.

[0025] In this invention, the second intermediate is used for deprotection, preferably by removing the solvent under reduced pressure. The solvent for removing Fmoc protection is preferably a DMF solution containing 20% ​​piperidine.

[0026] In this invention, the working concentration of the intermediate containing the free amino group is preferably 0.1~1 M, and can be 0.68 M. The working concentration of the succinic anhydride is preferably 0.1~0.5 M, and can be 0.2 M. The working concentration of the DIEA is preferably 0.1~0.5 M, and can be 0.2 M. The temperature of the third reaction is preferably 20~27℃, and can be 22~25℃, or 23~24℃. The time of the third reaction is preferably 1.5~2.5 h, and can be 2 h. The purification method is preferably reversed-phase high-performance liquid chromatography.

[0027] The present invention provides an engineered exosome, comprising an exosome loaded with a scar treatment drug; the surface of the exosome is modified with a small molecule ligand targeting the fibroblast activation protein.

[0028] In this invention, the cell source of the exosomes preferably includes at least one of the following: adipose-derived mesenchymal stem cells, fibroblasts, bone marrow mesenchymal stem cells, and umbilical cord mesenchymal stem cells. In this embodiment, the preparation method of exosomes is illustrated using adipose-derived mesenchymal stem cells as an example. This invention does not impose any particular limitation on the preparation method of the exosomes; any exosome collection method well-known in the art can be used.

[0029] In this invention, the fibroblast activation protein targeting small molecule ligand preferably couples with the amino group on the surface of the exosome via an amide bond formed by the activated carboxyl group in the succinyl group. The mass ratio of the protein in the exosome to the fibroblast activation protein targeting small molecule ligand is preferably 5-15:0.5-2, and can be 10:1. The function of the fibroblast activation protein targeting small molecule ligand is to preferentially recognize and enter scar fibroblasts with high FAP expression, thereby improving delivery selectivity.

[0030] In this invention, the scar treatment drug preferably includes a ferroptosis inducer. The type of ferroptosis inducer is preferably erastin. The loading concentration of the ferroptosis inducer is preferably 150-250 mM, and can be 200 mM. The ferroptosis inducer is loaded into engineered exosomes with FAP targeting, which can trigger ferroptosis-related processes within target cells, reducing the risk of off-target damage caused by free drug diffusion and expanding the therapeutic window.

[0031] This invention provides a method for preparing the engineered exosomes, comprising the following steps: Exosomes and scar treatment drugs were co-incubated to remove free scar treatment drugs, resulting in exosomes loaded with scar treatment drugs. Engineered exosomes were obtained by coupling exosomes loaded with scar treatment drugs with small molecule ligands targeting fibroblast activation proteins.

[0032] In this invention, the preferred number and molar ratio of the exosomes and the scar treatment drug is (1.0 ~ 10) × 10. 10 150~250 μmol, which can be 1.0 × 10 10 200 μmol. The co-incubation is preferably at 36-38°C, and can be 37°C. The co-incubation time is preferably 0.5-1.5 h, and can also be 1 h. The method for removing free scar treatment drug is preferably ultracentrifugation followed by washing and resuspension. The ultracentrifugation temperature is preferably 4°C. The ultracentrifugation speed is preferably 90,000×g-110,000×g, and can also be 100,000×g. The ultracentrifugation time is preferably 60-80 min, and can be 70 min. The resuspension method preferably involves washing twice with PBS.

[0033] In this invention, the coupling reaction preferably uses EDC:NHS to activate the fibroblast activation protein targeting small molecule ligand, and the activated small molecule ligand is then coupled to an exosome loaded with a ferroptosis inducer via an amide bond. The mass ratio of the fibroblast activation protein targeting small molecule ligand, EDC, and NHS is preferably 0.8~1.2:1~2:0.8~1.2, and can also be 1:1.5:1. The reaction solvent for activation is preferably 50 mM MES buffer (pH 5.5). The activation time is preferably 15~30 min, and can be 20~25 min. The activation temperature is preferably 20~27℃. The purpose of activation is to activate the carboxyl group in the small molecule ligand to an activated ester, preparing for subsequent exosome coupling. The amide bond coupling temperature is preferably 0~8℃, and can be 4℃. The amide bond coupling is preferably a shaking reaction. After the coupling reaction, purification is preferably also included. The preferred purification method involves ultracentrifugation at 4°C and 100,000×g for 70 min, discarding the supernatant; resuspending the precipitate in PBS and then ultracentrifuging again at 4°C and 100,000×g for 70 min to remove unreacted ligands.

[0034] The present invention provides a microneedle patch, wherein the cavity of the microneedle contains the engineered exudate or the engineered exudate prepared by the preparation method.

[0035] In this invention, the microneedle patch further includes a backing layer. The material of the backing layer is preferably polyvinyl alcohol (PVA).

[0036] In this invention, the preferred method for preparing the microneedle patch is to prepare a needle tip mixture containing engineered exosomes; The needle tip mixture is injected into the microneedle mold and degassed to obtain a mold containing the needle tip mixture. The backing material solution is injected into the microneedle mold, cross-linked and cured, and then dried to obtain a complete microneedle.

[0037] In this invention, the solvent for the needle tip mixture containing engineered exosomes is preferably a 20% (w / v) GelMA solution. The final concentration of the engineered exosomes is preferably (1~10)×10⁻⁶. 12 particles / mL, which can be 4 × 10 12 particles / mL. The microneedle mold is preferably a PDMS microneedle mold. The height of the conical needle tip cavity in the PDMS microneedle mold is preferably 500~900 μm, and can be 700 μm. The volume of the needle tip mixture injected into the PDMS microneedle mold is preferably 500~600 μL / microneedle. The degassing method is preferably vacuum degassing under vacuum conditions to remove air bubbles in the needle tip cavity and promote full filling of the needle tip cavity.

[0038] In this invention, the concentration of the backing material solution is preferably 20% (w / v) PVA solution. The curing method is preferably photocrosslinking. The light intensity for photocrosslinking is preferably 405 nm. The function of photocrosslinking is to shape the needle tip structure and achieve a stable bond with the backing layer. The drying method is preferably drying with silica gel desiccant, followed by drying at 4°C until fully formed, and then demolding from the mold to obtain a soluble microneedle patch loaded with engineered exosomes.

[0039] The present invention provides a drug for treating scars, comprising the engineered exudate or the engineered exudate prepared by the preparation method.

[0040] In this invention, the dosage form of the drug is preferably an injection solution or an injection powder. This invention does not impose any particular limitation on the preparation method of the injection solution or injection powder; any preparation method well-known in the art can be used. The mass percentage of engineered exosomes in the drug is preferably 10% to 90%, but can be 20% to 80%, 30% to 70%, or even 50%.

[0041] This invention provides the application of the engineered exosomes or engineered exosomes prepared by the preparation method in the preparation of drugs for hypertrophic scars.

[0042] The engineered exosomes described in this invention use exosomes as drug delivery carriers. First, exosomes are obtained by separating and purifying the supernatant from donor cell culture. Then, a ferroptosis inducer is co-incubated with the exosomes under predetermined conditions to achieve drug loading. Free drug is removed by ultracentrifugation, yielding exosomes loaded with erastin. Based on this, the FAP-targeting small molecule ligand SUC-Lys(Ac)-PEG3-UAMC1110 is synthesized and covalently linked to the exosome membrane surface via EDC / NHS-mediated amide bond coupling. This enables the exosomes to selectively recognize and enrich FAP-highly expressing scar fibroblasts, thereby constructing engineered exosomes with FAP targeting and ferroptosis-inducing activity. Further, the engineered exosomes are mixed with a soluble microneedle tip material and perfused into a mold, then combined with a backing material to obtain a soluble microneedle patch, achieving percutaneous minimally invasive delivery and localized sustained retention to scar tissue. Through the synergistic effect of FAP-targeted enrichment and microneedle percutaneous delivery, engineered exosomes can increase the local effective concentration at the lesion site, reduce non-specific diffusion, and trigger ferroptosis-related processes in scar fibroblasts, inhibiting their abnormal proliferation, migration, and pro-fibrotic phenotype, thereby reducing collagen deposition and promoting scar tissue remodeling. At the same time, it reduces the off-target toxicity risk of free drugs and improves the safety and stability of treatment.

[0043] In one embodiment of the invention, a rabbit ear hypertrophic scar model was established, and a mature hypertrophic scar was formed on postoperative day 28. Microneedle patches containing engineered exosomes (sEVs) were used. ErF -DMNP) administration resulted in more significant improvement in scar appearance, and histological evaluation showed decreased dermal thickness and more regular tissue structure, more closely resembling the blank control group (NS). The scar elevation index (SEI) statistical results indicated that sEVs ErF -The most significant decrease in DMNP levels was observed from day 28 post-surgery (before treatment) to day 49 post-surgery (after completing 3 treatments), with efficacy superior to other control groups.

[0044] The following detailed description, in conjunction with embodiments, illustrates a FAP-targeted engineered exosome for treating hypertrophic scars, its preparation method, and its application, but these should not be construed as limiting the scope of protection of this invention.

[0045] Example 1 The synthetic route of the FAP-targeting ligand SUC-Lys(Ac)-PEG3-UAMC1110 is as follows: Figure 1 As shown, the specific steps are as follows: Step 1) Construction of PEG3-UAMC1110 intermediate: N-Boc-PEG3-carboxylic acid was dissolved in 10 mL of anhydrous DMF to obtain a N-Boc-PEG3-carboxylic acid solution with a final concentration of 20 mM. HATU and DIEA with a final concentration of 1 mM and 4 mM were added and activated at room temperature for 5 min. 4-quinolinecarboxamide derivative with a final concentration of 0.5 mM was added and reacted for 2 h to obtain the Boc protected intermediate.

[0046] Step 2) Deprotection: After the reaction is complete, remove the solvent under reduced pressure, add 5 mL of TFA to remove the Boc protecting group, precipitate with cold diethyl ether, collect by centrifugation and dry to obtain the Boc-free intermediate.

[0047] Step 3) Connecting Fmoc-Lys(Ac): Dissolve 0.24 M Fmoc-Lys(Ac) in 15 mL DMF, add 0.28 M EDC and 0.23 M HOBt at 0 °C and activate for 10 min at 0 °C; add the intermediate obtained in step 2 and react at room temperature for 2 h.

[0048] Step 4) De-Fmoc: After desolventizing under reduced pressure, the Fmoc protecting group is removed with 20% piperidine / DMF, and the intermediate containing free amino groups is obtained by washing with diethyl ether.

[0049] Step 5) Introduction of succinyl group: The intermediate obtained in step 4 was reacted with 0.68M succinic anhydride and 0.5M DIEA in DMF at room temperature for 2 h to introduce a succinyl terminal carboxyl group and purified to obtain SUC-Lys(Ac)-PEG3-UAMC1110.

[0050] Example 2 Methods for extracting exosomes Step 1) Donor cell culture and supernatant collection: Adipose-derived mesenchymal stem cells (ADSCs) were selected as donor cells. After the cells were in good condition, they were replaced with a culture medium containing exosome-free serum and cultured continuously for 24 h. The cell culture supernatant was then collected.

[0051] Step 2) Supernatant pretreatment: Centrifuge the supernatant at 10,000×g for 10 min at 4°C to remove cell debris and large particulate impurities, and transfer the supernatant to a new centrifuge tube.

[0052] Step 3) Magnetic bead pretreatment: Vortex the bottled magnetic beads for 30 s, add 350 μL to a centrifuge tube, centrifuge at 4℃ and 3,000×g for 2 min, and discard the supernatant.

[0053] Step 4) Magnetic bead washing: Add 10 mL of PBS pre-cooled at 4℃ and filtered through 0.22 μm, vortex to mix for 30 s, centrifuge at 4℃ and 3,000×g for 5 min, and discard the supernatant.

[0054] Step 5) Exosome capture: Add 4 mL of Buffer EXA, 1 mL of Buffer EXB and 14.65 mL of the supernatant obtained in Step 2 to a centrifuge tube in sequence, and mix in a rotary mixer at 4°C for 40 min.

[0055] Step 6) Magnetic separation: Place the centrifuge tube on a magnetic rack and let it stand at 4°C for 10 minutes. After the magnetic beads have gathered, discard the supernatant.

[0056] Step 7) Residual liquid removal: Centrifuge at 4 ℃, 3,000×g for 1 min, and discard the residual liquid.

[0057] Step 8) Exosome elution: Add 0.5 mL Buffer EXE and mix well. Centrifuge at 4 °C and 7,000 × g for 2 min. Transfer the supernatant to an EP tube.

[0058] Step 9) Filtration and impurity removal: After rinsing needle filters A and B with EXE, filter the exosome solution separately to obtain a purified exosome (sEVs) suspension, and freeze for later use.

[0059] Example 3 Identification of exosomes (sEVs) prepared in Example 2 1. Electron microscopy (TEM) identification Add 10 μL of sEVs solution to a copper grid, let it stand at room temperature for 10 min, and then remove excess liquid. Negative stain with 2% uranium acetate for 1 min, dry, and then image under a transmission electron microscope (80 kV) to observe the morphology of exosomes.

[0060] 2. NTA (Nanoparticle Tracking Analysis) The nanoparticle suspension was irradiated with a laser light source, and the scattered light from the nanoparticles was detected. The concentration of the nanoparticles was calculated by counting the number of scattered particles. Specifically, the concentration of the separated sEVs was determined using a ZetaView PMX 110 particle matrix under 405 nm emission light, and the sEVs were diluted with PBS to 1x10⁻⁶. 7 particles / mL ~1x10 9 The particle count was measured per mL, and their size and mass were determined. Simultaneously, the particle trajectories of exosomes were analyzed.

[0061] 3. sEVs protein extraction (1) Take 50 μL of sEVs lysis buffer and 150 μL of sEVs solution at a ratio of 1:3 and mix well. Centrifuge 1 mL of cell suspension, add 1 mL of lysis buffer to the cell pellet, mix well, and boil at 100 °C for 5 min.

[0062] (2) Transfer to an ice-water mixture to cool for 5 min, control the centrifugal acceleration to 12,000×g, centrifuge at 4℃ for 5 min, and transfer the supernatant to a new EP tube.

[0063] 4. Western Blot Detection (1) Protein concentration determination.

[0064] The protein concentration of sEVs was determined according to the instructions of the BCA method protein concentration assay kit. The simplified procedure is as follows: Mix solution A and solution B from the kit at a volume ratio of 50:1 to prepare the working solution. Dilute the protein standard to 0.5 mg / mL using sterile ddH2O, and then serially dilute the standard using ddH2O again. Add the diluted standard and sample to a 96-well plate, then add 200 μL of the prepared working solution to each well. Incubate at 37 ℃ for 30 min, and detect the absorbance at 562 nm using a microplate reader. Plot a standard curve and calculate the protein concentration in the sample using a regression equation.

[0065] (2) Electrophoresis and membrane transfer.

[0066] Using a polyacrylamide gel preparation kit, SDS-PAGEGel of appropriate concentration was prepared according to the molecular weight of the target antibody. The required sample volume was calculated based on the protein concentration in the sample, added to the wells of the polyacrylamide gel, and transferred to an electrophoresis tank for electrophoresis. Electrophoresis was performed at a constant voltage of 80 V until the indicator (bromophenol blue) in the sample entered the separating gel, and then at a constant voltage of 120 V until the indicator reached the bottom of the gel. Electrophoresis was stopped, the polyacrylamide gel was peeled off, the stacking gel was removed, and the separating gel was retained. The gel was placed on a PVDF membrane, sandwiched with filter paper and a sponge to prepare a sandwich structure. The membrane was then inserted into the electroporation tank, electroporation buffer was added, and electroporation was performed at a constant current of 200 mA for 2 h. The PVDF membrane was then removed, washed three times with TBST for 5 min each time, and then placed in 5% skim milk powder (prepared with TBST) and blocked at room temperature for 1 h.

[0067] (3) Antibody incubation Primary antibody incubation: Remove the PVDF membrane from the blocking solution, wash it three times with TBST for 5 minutes each time, transfer it to the prepared primary antibody solution, and incubate overnight on a horizontal shaker at 4 ℃.

[0068] Secondary antibody incubation: Wash the membrane three times with TBST for 5 min each time, then transfer it to the prepared secondary antibody solution. The secondary antibody is diluted with 5% skim milk powder (prepared with TBST). Incubate at room temperature for 1.5 h. See Table 1 for details.

[0069] Table 1. Antibody types and dilution ratios

[0070] (4) Develop and photograph (a) Washing: After the secondary antibody incubation is completed, wash the PVDF membrane 3 times for 5 min each time using TBST.

[0071] (b) Lift the PVDF membrane and drain off any excess detergent.

[0072] (c) Lay the membrane flat on the developing plate, add the prepared ECL reaction solution, and place it in the chemiluminescence imaging instrument.

[0073] (d) Photography: Prepare the chemiluminescence solution, place the band into an exposure machine (GE), add the chemiluminescence solution, expose, and acquire images. If necessary, ImageJ software can be used to analyze the grayscale values ​​of the protein bands.

[0074] Example 4 sEVs loaded with ferroptosis inducers Er Preparation method of ) Take approximately 1.0 × 10 10 One exosome was mixed with ferroptosis inducer erastin at a final concentration of 200 μM and incubated at 37°C for 1 h to obtain a drug-containing incubation system; The drug-containing incubation system was centrifuged at 4°C and 100,000×g for 70 min, and the supernatant was discarded. The precipitate was resuspended in PBS and centrifuged again at 4°C and 100,000×g for 70 min. The PBS washing was repeated twice to obtain the loaded erastin exosomes after the removal of free erastin.

[0075] Example 5 FAP-targeted engineered exosomes (sEVs) ErF Preparation method of ) 1. Dissolve 1 mg of the SUC-Lys(Ac)-PEG3-UAMC1110 small molecule ligand prepared in Example 1 in 200 μL of 50 mM MES buffer (pH 5.5). Add EDC and NHS at a ratio of approximately 1:1.5:1 (mass ratio) of small molecule ligand: EDC: NHS. React at room temperature for 30 min to form an activated ester that can react with primary amines by the terminal carboxyl group of the ligand.

[0076] 2. The obtained activated ester was added to 1 mL of the exosome suspension (PBS, pH 7.4) loaded with ferroptosis inducer prepared in Example 3, and the mixture was gently shaken and reacted overnight at 4 °C to allow the ligand to undergo amide bond coupling with the amino groups on the exosome membrane surface, thereby obtaining engineered exosomes that present the FAP-targeting ligand on the surface.

[0077] 3. The reaction system was centrifuged at 4 ℃ and 100,000 × g for 70 min, and the supernatant was discarded. The precipitate was resuspended in PBS and centrifuged again at 4 ℃ and 100,000 × g for 70 min. Repeated washing was performed to remove unreacted ligands, and engineered exosomes were obtained. The morphology of the engineered exosomes was observed.

[0078] The results are as follows Figure 3 As shown, engineered exosomes (sEVs) ErF The exosomes retained their typical morphological characteristics, with no obvious aggregation or structural damage, suggesting that the drug loading and surface coupling processes did not adversely affect the integrity of the exosomes.

[0079] Particle size and concentration analysis showed that the particle size distribution and concentration of exosomes remained stable before and after engineering. Western blot results showed that the engineered exosomes still expressed typical exosome marker proteins (TSG101, CD9, CD63) and did not express endoplasmic reticulum-specific markers such as Calnexin, thus confirming that the obtained formulation was engineered exosomes with good purity and stability.

[0080] Example 6 Cell-targeted experiments Step 1) Cell preparation: Harvest scar fibroblasts (HSFs) and human dermal fibroblasts (HDFs), and seed them into 6-well plates, and culture them until the cell density is about 60%–80%.

[0081] Step 2) Exosome fluorescent labeling: Take the engineered exosomes (sEVs) prepared in Example 4 ErF The exosomes were labeled with membrane dyes (such as DiI or PKH26) according to the reagent instructions. After labeling, the free dye was removed by ultracentrifugation or ultrafiltration, and the suspension was resuspended in PBS to obtain a fluorescently labeled exosome suspension.

[0082] Step 3) Cell internalization: Add fluorescently labeled exosomes to cell culture medium and incubate at 37 °C for 2 h; after incubation, discard the supernatant and wash three times with PBS to remove uninternalized exosomes.

[0083] Step 4) Imaging observation: Cell fixation (4% paraformaldehyde), cell nuclear staining (DAPI), and observation of the fluorescence distribution and intensity of exosomes in the cells using confocal microscopy.

[0084] Test results as follows Figure 4 Compared with the human dermal fibroblast (HDF) group, the fluorescence intensity in scar fibroblasts (HSFs) was significantly increased, indicating that scar fibroblasts have good specific phagocytic activity towards engineered exosomes.

[0085] Example 5 1. Western blot detection of ferroptosis-related proteins was performed according to the method described in Example 3; the grayscale values ​​of the bands were quantified using ImageJ and normalized with internal reference proteins.

[0086] 2. Mitochondrial membrane potential (JC-1) detection Step 1) Cell seeding: Seed scar fibroblasts (HSFs) into 6-well plates and culture until the cell density is about 60%~80%.

[0087] Step 2) Treatment and Incubation: PBS (control), free ferroptosis inducer (Er), and ferroptosis inducer-loaded exosomes (sEVs) were added according to the experimental groups. Er ) and engineered exosomes (sEVs) with FAP targeting ErF The sample was processed and incubated at 37 °C.

[0088] Step 3) JC-1 staining: Discard the culture medium, wash the cells with PBS 1–2 times; add JC-1 working solution to cover the cells, and incubate at 37°C in the dark for about 20–30 min.

[0089] Step 4) Washing and detection: After incubation, discard the staining solution, wash 2–3 times with JC-1 staining buffer or PBS, and then use flow cytometry to detect the red / green fluorescence intensity.

[0090] Test results are shown Figure 5 Compared to the control, the free iron death inducer group and sEVs Er Groups, sEVs ErF Significant depolarization changes were observed in the mitochondrial membrane potential of the group's cells.

[0091] Example 6 Preparation method of soluble microneedle patches loaded with engineered exosomes The preparation process is shown in the diagram below. Figure 2 As shown in Figures A–B, the steps are as follows: Step 1) Needle tip mixture: Prepare a 20% (w / v) GelMA solution; add the engineered sEVs prepared in Example 4. ErF The mixture was then thoroughly mixed to obtain a needle tip mixture, wherein the final concentration of engineered exosomes was 4 × 10⁻⁶. 12 particles / mL.

[0092] Step 2) Mold Infusion: Provide a PDMS microneedle mold (needle tip cavity height approximately 700 μm, conical structure); add approximately 500–600 μL of needle tip mixture to the mold to cover it and enter the needle tip cavity.

[0093] Step 3) Vacuum degassing and filling: Place the mold under vacuum conditions to remove air bubbles from the needle tip cavity and promote full filling of the cavity.

[0094] Step 4) Backing layer: Prepare a 20% (w / v) PVA solution; add about 400–500 μL of PVA solution to the mold as a backing layer to cover and bond the needle tip layer.

[0095] Step 5) Photocuring: Photocrosslinking curing is performed using 405 nm light to form the needle tip structure and firmly bond it with the backing layer.

[0096] Step 6) Drying and demolding: Place the mold in a sealed container / drying tank, place silica gel desiccant inside the tank, and dry at 4°C until fully formed; then demold to obtain a soluble microneedle patch loaded with engineered sEVs.

[0097] The results are as follows Figure 6 As shown in Figure A, the microneedle array has a complete morphology and the needle tip structure is clear.

[0098] 2. In Vivo Intravascular Isolation (IVIS) Comparative Experiment Step 1) Fluorescent labeling of exosomes: Engineered exosomes (sEVs) ErFThe membrane was fluorescently labeled with 1,1′-octadecyl-3,3,3′,3′-tetramethylindocyanine perchlorate (DiI); after labeling, the free dye was removed by ultracentrifugation or ultrafiltration, and the membrane was resuspended in PBS to obtain a fluorescently labeled exosome suspension.

[0099] Step 2) DMNP loading: The fluorescently labeled exosomes obtained in Step 1 are loaded into a soluble microneedle patch according to the method of this embodiment.

[0100] Step 3) Drug administration and control: A DMNP delivery group and an intradermal injection control group were set up; the DMNP group was applied to the scar site for 10–15 minutes until the needle tip dissolved and then removed; the injection group was injected intradermally with an equal amount of exosome suspension at the same site.

[0101] Step 4) Dosage: Both groups were administered the drug at equal doses, with 2 × 10⁻⁶ doses at each administration site. 10 particles.

[0102] Step 5) In vivo imaging: The fluorescence signal at the treatment site was monitored using a small animal in vivo imaging system (IVIS, PerkinElmer, USA) for up to 7 days after administration.

[0103] Step 6) Statistics: Quantify the fluorescence intensity at each time point and plot the fluorescence intensity-time decay curve to compare the local retention and decay process of the two administration methods.

[0104] The results are as follows Figure 6 As shown in Figure B, the microneedle delivery system (sEVs) ErF DMNPs have significantly longer local retention time and sustained drug release characteristics compared to traditional injection.

[0105] Example 7 Establishment of a rabbit ear hypertrophic scar model and efficacy verification 1. To evaluate the transdermal ability, solubility, minimal invasiveness, sustained-release properties and biocompatibility of microneedle arrays, a rabbit ear hypertrophic scar model was established according to published methods.

[0106] (1) Husbandry environment: Female New Zealand white rabbits weighing about 2.5–3.0 kg were selected and kept in a standard animal house with an ambient temperature of about 25 ℃ and a 12-hour day-night cycle; they were kept in single cages for 1 week before the experiment.

[0107] (2) Surgical treatment: The rabbit ear hair was shaved one day before the operation. On the day of the operation, sodium pentobarbital (30mg / kg) was injected into the ear vein for anesthesia; under sterile conditions, four full-thickness wounds with a diameter of 10 mm were prepared on the proximal ventral surface of each rabbit ear using a corneal punch, the periosteum was removed but the cartilage was preserved, and blood vessels were avoided; hemostasis was achieved by compression and disinfection with povidone-iodine.

[0108] (3) Scar formation: After about 28 days, the wound will re-epithelialize and form a raised dark red scar with a scar thickness / surrounding normal skin thickness ratio >2; infected or necrotic wounds will be removed.

[0109] 2. Drug efficacy verification experiment After successfully establishing a rabbit ear hypertrophic scar model and forming a mature hypertrophic scar on the 28th day after surgery, this example was used to compare and evaluate the in vivo efficacy of different formulations / administration methods.

[0110] 1. Grouping and Dosing Regimen Step 1) Grouping: After confirming scar maturation on day 28 post-surgery, the animals were randomly divided into 7 groups (n = 3 per group): NS group (normal skin control), HS group (untreated scar control), Er group (free erastin), sEVs Er Group (exosomes loaded with erastin), sEVs ErF Group (FAP-targeted loaded erastin engineered exosomes), sEVs ErF -DMNP group (load sEVs) ErF The soluble microneedle patch and the TA group (triamcinolone, clinical control).

[0111] Step 2) Start time and frequency of drug administration: Start drug administration from the 28th day after surgery (W3, before the first treatment), once a week, for a total of 3 treatments (corresponding to "after 3 treatments" is W6 / 49th day after surgery).

[0112] Step 3) Administration method and dosage (equal amount principle): For Er, sEVs Er sEVs ErF Group: The formulation was injected intradermally into the scar tissue; for groups containing sEVs, the exosome dosage was 2.0 × 10⁻⁶ per administration site. 10 For particles, the erastin dose in group Er was consistent with that in the groups mentioned above.

[0113] For sEVs ErF-DMNP group: One DMNP patch (1.2 cm in diameter, approximately 300 needles / patch) was applied to each scar surface for 10–15 minutes, and removed after the needle tips had fully dissolved and released the sEVs; microneedle-delivered sEVs ErF The loading capacity was calculated based on the prepared perfusion volume to ensure that the delivered dose was equivalent to that of the injection group (approximately 2.0 × 10⁻⁶). 10 (particles / sites).

[0114] For the HS group: treat with an equal volume of carrier.

[0115] For the TA group: TA was injected into the scar, and the dosage was set according to the commonly used clinical protocol (as a positive control).

[0116] 2. Photographic record of scar appearance Photographic records: The appearance of each group of scars was photographed on the 28th day after surgery (before the first treatment) and the 49th day after surgery (after the completion of 3 treatments) to compare the changes in scar elevation, color and border after different treatments.

[0117] Result: As Figure 7 As shown in Figure A, all groups exhibited mature hypertrophic scar appearance on postoperative day 28; after completing 3 treatments, sEVs ErF -The DMNP group showed more significant improvement in the appearance of scars.

[0118] 3. Histological evaluation: H&E includes the following steps: Timing of tissue sampling: Scar tissue was taken on the 49th day after surgery (after the completion of 3 treatments) (adjacent normal dermis was also taken as a reference if necessary).

[0119] Staining: The tissue was fixed with 4% paraformaldehyde, dehydrated, and embedded in paraffin. The section thickness was about 4–5 μm. The slides were prepared and observed using the standard H&E staining procedure.

[0120] Result: As Figure 7 As shown in Figure B, after completing 3 treatments, sEVs ErF The DMNP group showed decreased dermal thickness and a more regular tissue structure, which was closer to that of the NS group.

[0121] 4. Calculation of Scar Elevation Index (SEI) Step 7) SEI Calculation Method: Measure the thickness of the scar tissue and the thickness of the adjacent normal dermis on the H&E section, and calculate the Scar Increase Index (SEI). The calculation formula I is: SEI = Scar thickness / Adjacent normal dermal thickness (Formula I)

[0122] Use ImageJ to perform thickness measurement and statistical analysis.

[0123] The results are as follows Figure 7 As shown in C, sEVs ErF - The SEI in the DMNP group decreased most significantly from day 28 post-surgery (before treatment) to day 49 post-surgery (after completing 3 treatments), and the efficacy was superior to other control groups (including the TA group).

[0124] Comparative Example 1 A method for synthesizing 3Mal-Lys(Ac)-PEG3-UAMC1110 according to Figure 8 3Mal-Lys(Ac)-PEG3-UAMC1110 was synthesized using the method described above, and the results were detected by HPLC-MS. (See attached table). Figure 9 and Figure 10 .

[0125] Comparative Example 2 The 3Mal-Lys(Ac)-PEG3-UAMC1110 and the ferroptosis-inducing exosomes (sEVs) prepared in Example 3 were used. Er Covalent coupling was performed by dissolving 3Mal-Lys(Ac)-PEG3-UAMC1110 in 200 μL of PBS buffer (pH 7.4) to obtain a 5 mM 3Mal-Lys(Ac)-PEG3-UAMC1110 solution. This solution was then mixed with 1 mL of exosome suspension loaded with ferroptosis inducer (approximately 1 mg of protein). The mixture was gently shaken at 4 °C for 12 h to form covalent bonds via a Michael addition reaction between the maleimide group and the thiol groups on the exosome membrane surface. After the reaction, the mixture was ultracentrifuged at 100,000 × g for 70 min at 4 °C. The supernatant was discarded, the precipitate was resuspended in PBS, and then washed again by ultracentrifugation to remove unreacted ligands, yielding engineered exosomes modified with 3Mal ligands.

[0126] See results Figure 11 Compared to SUC-Lys(Ac)-PEG3-UAMC1110, the coupling efficiency of 3Mal-Lys(Ac)-PEG3-UAMC1110 with exosomes is significantly reduced. This is because the carboxyl group at the succinyl (SUC) end, after EDC / NHS activation, can efficiently form stable amide bonds with the abundant amino groups on the exosome membrane surface, with mild reaction conditions and high coupling efficiency. In contrast, the maleimide (3Mal) group relies on the naturally occurring thiol groups on the exosome membrane surface for a Michael addition reaction, but the abundance of thiol groups on the exosome membrane surface is low, and the reaction process is easily affected by factors such as pH and redox environment, resulting in poor coupling efficiency. This comparative result indicates that the SUC-terminal structure used in this invention is more suitable for exosome surface modification, achieving higher ligand coupling efficiency and more stable engineered exosome construction.

[0127] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A small molecule ligand targeting fibroblast activation proteins, characterized in that, It has the following molecular structure: SUC-Lys(Ac)-PEG3-UAMC1110; The SUC is succinyl, Lys(Ac) is Nε-acetylated lysine, PEG3 is triethylene glycol, and - indicates covalent linkage.

2. An engineered exosome, characterized in that, The exosomes include those loaded with drugs for treating scars; the surface of the exosomes is modified with the fibroblast activation protein targeting small molecule ligand of claim 1.

3. The engineered exosomes according to claim 2, characterized in that, The fibroblast activation protein targeting small molecule ligand is coupled via an amide bond formed between the activated carboxyl group in the succinyl group and the amino group on the surface of the exosome.

4. The engineered exosome according to claim 2, characterized in that, The mass ratio of the protein content of the exosomes to the fibroblast activation protein targeting small molecule ligand is 5~15:0.5~2.

5. The engineered exosome according to claim 2, characterized in that, The medication for treating scars includes ferroptosis inducers; The loading concentration of the ferroptosis inducer is 85~95 μM; The types of ferroptosis inducers include erastin.

6. The engineered exosomes according to any one of claims 2 to 5, characterized in that, The cell sources of the exosomes include at least one of the following: adipose-derived mesenchymal stem cells, fibroblasts, bone marrow mesenchymal stem cells, and umbilical cord mesenchymal stem cells.

7. A method for preparing engineered exosomes according to any one of claims 2 to 6, characterized in that, Includes the following steps: Exosomes and scar treatment drugs were co-incubated to remove free scar treatment drugs, resulting in exosomes loaded with scar treatment drugs. Engineered exosomes were obtained by coupling exosomes loaded with scar treatment drugs with small molecule ligands targeting fibroblast activation proteins.

8. A microneedle patch, characterized in that, The cavity of the microneedle contains the engineered exudate as described in any one of claims 2 to 6 or the engineered exudate prepared by the preparation method described in claim 7.

9. A drug for treating scars, characterized in that, Includes the engineered exosomes described in any one of claims 2 to 6 or the engineered exosomes prepared by the preparation method described in claim 7.

10. The use of the engineered exudate as described in any one of claims 2 to 6 or the engineered exudate prepared by the preparation method described in claim 7 in the preparation of a drug for hypertrophic scars.

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