A 293t exosome for targeted delivery of pirfenidone with membrane-mounted fap, and a preparation method and application thereof

Abstract

The application belongs to the technical field of targeted drugs, and particularly relates to 293T exosome for targeted delivery of pirfenidone with FAP on the membrane as well as a preparation method and application thereof. The application uses exosome secreted by 293T cells as a natural nanocarrier, modifies FAP antibody with fibroblast targeting property on the membrane surface of the exosome through copper-free click chemistry, and uses an ultrasonic method to load anti-fibrosis drug PFD into the exosome, so as to construct an engineered exosome system (293T exo antiFAP+PFD ) with functions of targeted recognition and drug delivery. The system is delivered to the lung through a nebulization inhalation mode, can realize accurate enrichment and stable release of PFD in a pulmonary fibrosis lesion, and thus effectively relieves damage to the lung tissue structure and collagen deposition, and significantly improves the anti-fibrosis effect. The technology solves the problems of low drug delivery efficiency, large side effects and poor targeting in traditional treatment, and provides a safe, efficient and accurate treatment strategy for pulmonary fibrosis.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of targeted drugs, and particularly relates to 293T exosomes for targeted delivery of pirfenidone with membrane-carrying FAP as well as a preparation method and application thereof. BACKGROUND

[0002] Pulmonary fibrosis (PF) is a chronic and progressive interstitial lung disease caused by various pathogenic factors, characterized by destruction of alveolar structure, abnormal deposition of extracellular matrix and irreversible decline of lung function. Although pirfenidone (PFD) and nintedanib and other anti-fibrosis drugs have been used in clinical treatment, the therapeutic effect of pulmonary fibrosis is still not ideal due to low bioavailability, significant side effects and insufficient targeting. Therefore, developing a new type of efficient, safe and targeted drug delivery system has become an urgent direction for breakthrough in the field of pulmonary fibrosis treatment. SUMMARY

[0003] The application aims to provide 293T exosomes for targeted delivery of pirfenidone with membrane-carrying FAP as well as a preparation method and application thereof, which are based on "239T exosomes for targeted delivery of PFD with membrane-carrying FAP" and used for treating pulmonary fibrosis through the atomization pathway, so as to solve the technical bottlenecks of low delivery efficiency, poor targeting and large side effects in the current treatment of pulmonary fibrosis and promote the clinical conversion application of exosome engineering in lung diseases.

[0004] To achieve the above-mentioned purpose, the technical scheme adopted by the application is as follows:

[0005] A preparation method of 293T exosomes for targeted delivery of pirfenidone with membrane-carrying FAP, comprising the following steps:

[0006] S1. Extraction of 293T cell-derived exosomes: 293T cells are inoculated in a T175 cell bottle and cultured in DMEM high-glucose medium to 90% confluence, then cultured in FBS-free medium for 48 hours, the supernatant is collected, and exosomes are extracted from the 293T cell culture supernatant by ultrahigh-speed centrifugation, and particle size and morphology analysis is performed by NTA and TEM, and exosome-specific membrane proteins (CD63, TSG101) are detected by Western blot to confirm the purity and source, which ensures that high-quality 293Texo with complete structure and clear source is obtained, laying a foundation for subsequent modification;

[0007] S2. Modification of exosome membrane with FAP targeting molecules: antiFAP antibody was conjugated to the surface of exosome membrane by copper-free click chemistry, the modified exosomes were verified by immunolabeling, Western blot and TEM, which maintained the original structure and targeting ability;

[0008] S3. Packaging PFD into the lumen of the modified exosomes: PFD was dissolved in DMSO, and PFD was mixed with 293T exo antiFAP exosome suspension at a ratio of 2:1, treated with 20% power ultrasound, cycled for 30 seconds work / 30 seconds interval, a total of 3 minutes, repeated six times, the drug encapsulation rate and release behavior were detected by high performance liquid chromatography (HPLC), and the delivery performance was evaluated, and finally the engineered exosomes 293T with dual functions of targeting and drug loading were obtained. exoantiFAP+PFD .

[0009] Further, the specific operation of step S2 is: adding DBCO reagent in DMSO into the exosome solution resuspended in PBS, stirring at 4℃ for 1 hour; adding Azide modified antiFAP antibody, continuing to stir at 4℃ for 24 hours; washing with 100 kDa ultrafiltration tube for 3 times to remove unbound antibody.

[0010] Another object of the present application is to provide a 293T exosome with FAP membrane carrying targeted delivery of pirfenidone prepared by the above method, and a C57BL / 6 mouse lung fibrosis model is established by giving bleomycin through nasal drop method to simulate the pathological process of the disease. The destruction of alveolar structure and collagen deposition are confirmed by HE and Masson staining to verify the success of modeling. The model is used for subsequent evaluation of the therapeutic effect and mechanism of the delivery system in vivo.

[0011] Still another object of the present application is to provide a use of 293T exosome with FAP membrane carrying targeted delivery of pirfenidone, and the 293T exosome with FAP membrane carrying targeted delivery of pirfenidone is used for preparing a drug for treating pulmonary fibrosis disease by inhalation.

[0012] The present application uses 293T cell secreted exosomes as natural nanocarriers, modifies FAP antibody with fibroblast targeting function on the surface of exosome membrane by copper-free click chemistry method, and uses ultrasonic method to load anti-fibrosis drug PFD into the inside of exosome, and constructs an engineered exosome system (293T exo antiFAP+PFDThis system delivers PFD to the lungs via nebulized inhalation, enabling precise enrichment and stable release of PFD in pulmonary fibrosis lesions. This effectively alleviates lung tissue structural damage and collagen deposition, significantly improving anti-fibrotic efficacy. This technology addresses the problems of low drug delivery efficiency, significant side effects, and poor targeting in traditional treatments, providing a safe, efficient, and precise treatment strategy for pulmonary fibrosis.

[0013] The advantages of this invention are:

[0014] 1. This invention modifies the membrane surface of exosomes derived from 293T cells with FAP antibodies, enabling the exosomes to target and recognize fibroblasts that highly express FAP in pulmonary fibrosis lesions. This significantly improves the enrichment of exosomes in the lesion area and stably loads PFDs without affecting their morphology and structure, effectively avoiding the problems of drug diffusion, degradation or systemic toxicity that occur in traditional delivery methods.

[0015] 2. This invention achieves high-concentration enrichment and sustained release of PFD in pulmonary fibrosis areas, significantly inhibiting collagen deposition and fibroblast activation, while avoiding adverse reactions such as gastrointestinal discomfort and liver function damage caused by oral or intravenous administration, thus enhancing the effectiveness of local treatment and patient tolerance.

[0016] 3. This invention delivers engineered exosomes via nebulized inhalation, offering advantages such as non-invasiveness, ease of operation, and high patient compliance. Furthermore, by combining a targeting mechanism with a drug loading system, it enables precise intervention and structural repair of pulmonary fibrosis without increasing the treatment burden, providing an innovative paradigm for the future clinical translation of exosomes in lung diseases. Attached Figure Description

[0017] Figure 1 Transmission electron microscopy (TEM) observations, NTA particle size analysis, and Western blot results of exosomes treated with different methods (A: TEM observations of exosomes treated with different methods; B: NTA particle size analysis of exosomes treated with different methods; C: Western blot results of marker proteins of exosomes treated with different methods).

[0018] Figure 2 HLPC results after exosomes loaded with PFD (A: HLPC results of PFD; B: HLPC results of exosomes loaded with PFD).

[0019] Figure 3 Establishment of a mouse model of pulmonary fibrosis (A: Lung tissues of normal control mice (NC) and pulmonary fibrosis mice (PF); B: HE staining results of lung tissues of normal control mice (NC) and pulmonary fibrosis mice (PF)).

[0020] Figure 4 Condition of lung tissue in mice after nebulization treatment;

[0021] Figure 5 HE staining and Masson staining results of mouse lung tissue after nebulization treatment (A: HE staining results of mouse lung tissue after nebulization treatment; B: Masson staining results of mouse lung tissue after nebulization treatment).

[0022] Figure 6 In vivo imaging in mice to monitor the fluorescence distribution of exosomes;

[0023] Figure 7 Expression of p-Smad3 and TGF-β1 proteins in different mouse lung tissues. Detailed Implementation

[0024] The 293T cells used in this invention were purchased from Jiangsu Kaiji Biotechnology Co., Ltd., catalog number: KGG3101-1; the T175 cells and DMEM high glucose culture medium were also obtained through conventional commercial channels. Example

[0025] As shown in the figure, a method for preparing 293T exosomes for targeted delivery of pirfenidone via a membrane-loaded FAP includes the following steps:

[0026] I. 293T cell-derived exosomes (293T) exo Extraction and identification of ) Figure 1 ).

[0027] (1) 293T cells were seeded into T175 cell culture flasks and cultured in DMEM high-glucose medium (FBS without exosomes) to 90% confluence. The cells were then cultured for another 48 hours in FBS-free medium, and the supernatant was collected. The following centrifugation steps were performed sequentially to remove impurities: centrifugation at 2000 g for 20 minutes to remove dead cells and debris; filtration through a 0.22 μm filter; ultracentrifugation at 110000 g for 90 minutes to precipitate exosomes; resuspending the exosome precipitate in 1 PBS; and ultracentrifugation at 110000 g to obtain approximately 200 μL of exosome solution. All steps were performed at 4 °C.

[0028] (2) 10 μL of exosome suspension was added to a copper grid, dried at room temperature and stained, and its morphology was observed by transmission electron microscopy. In addition, the particle size and concentration were detected by a nanoparticle tracking analyzer. The expression levels of exosome membrane marker proteins CD63 and TSG101 were detected by Western blot.

[0029] II. FAP-targeted molecule modification of exosome membranes (293T) exo antiFAP () Figure 1 ).

[0030] (1) Coupling antiFAP antibody (Abcam, ab53066) to the exosome membrane surface using copper-free click chemistry: DBCO reagent in DMSO was added to the exosome-containing solution in PBS and stirred at 4°C for 1 hour; Azide-modified antiFAP antibody was added and stirred at 4°C for 24 hours; and the solution was washed three times with a 100 kDa ultrafiltration tube to remove unbound antibody.

[0031] (2) Continue to observe the "cup-shaped" structure of exosomes using transmission electron microscopy, indicating that the exosome results are complete. Figure 1 A); The size of exosomes was detected by NTA method, and the results showed that the particle size of exosomes was mainly distributed between 50 nm and 150 nm. Figure 1 B); Western blot analysis of the retention of exosomal membrane proteins CD63 and TSG101 showed that the marker proteins were significantly expressed ( Figure 1 C).

[0032] III. PFD loading and HPLC validation (293T) exo antiFAP+PFD () Figure 2 ).

[0033] (1) PFD (MCE, HY-B0673) is dissolved in DMSO, and PFD is mixed with 293T in a 2:1 ratio. exo antiFAP Exosome suspension. The exosomes were sonicated at 20% power for 30 seconds on, 30 seconds off, for a total of 3 minutes, repeated six times. They were then incubated at 37°C for 1 hour to restore membrane structure. Loaded exosomes were collected using a 100 kDa ultrafiltration tube.

[0034] (2) Drug loading was determined using an HPLC system (Waters 2695): C18 column, mobile phase methanol / water (60 / 40), flow rate 1 mL / min; detection wavelength 310 nm, PFD showed a significant peak at approximately 10.4-10.5 minutes. Figure 2 A), the same exosomes loaded with PFD showed a significant peak at approximately 10.4–10.5 minutes. Figure 2 B) indicates that the PFD has been successfully encapsulated by the exosomes. Encapsulation efficiency (EE%) and drug loading content (DL%) are calculated using the following formulas: EE% = (mass of PFD in drug-loaded exosomes / initial mass of added PFD) × 100%;

[0035] DL% = (PFD mass in drug-loaded exosomes / total exosome mass) × 100%.

[0036] In this experiment, EE% was approximately 72%, and DL% was approximately 24.4%.

[0037] IV. Establishment of a mouse pulmonary fibrosis model using nasal drops ( Figure 3 ).

[0038] Six- to eight-week-old male C57BL / 6 mice were selected and acclimatized for one week before modeling. Bleomycin (Bleomycin, MCE, HY-108345) 5 mg / kg was administered intranasally in 50 μL of physiological saline. Body weight and clinical status were recorded every two days. Mice were sacrificed on day 10, and lung tissue was obtained through necropsy. Macroscopic observation was performed first, and changes in lung volume, color, and texture were recorded. Figure 3 A); Subsequently, histological analysis was performed, and changes in alveolar structure were assessed by HE staining; the results showed that the lung tissue of the model group (PF) exhibited obvious fibrotic characteristics, including alveolar structural collapse, interstitial thickening, and inflammatory cell infiltration; while the lung tissue of the control group (NC) had a relatively intact structure, thinner alveolar walls, and significantly reduced inflammatory response. Figure 3 B) indicates that the fibrosis model has been successfully constructed and will proceed to the next stage of treatment experiments.

[0039] V. Atomized delivery 293T exo antiFAP+PFD Treatment of pulmonary fibrosis and evaluation of its effectiveness Figure 4 ).

[0040] Using a CALVIN ultrasonic nebulizer (model: KW-DM-YWH), the nebulization dose was set to a total exosome particle count of 1×10⁻⁶ per nebulization session. 10 / kg, nebulized liquid volume 0.5 - 1 mL. The nebulization process lasts 10–15 minutes. Mice are placed in the inhalation chamber and inhale without pressure. The treatment cycle is once daily for 7 days.

[0041] VI. Evaluation of the efficacy of exosome nebulization therapy for pulmonary fibrosis ( Figure 5 , Figure 6 and Figure 7 ).

[0042] Lung tissue structural recovery and collagen deposition were assessed using HE and Masson staining; exosome distribution was tracked in vivo in real time using a fluorescence imaging system; and the expression levels of p-Smad3 and TGF-β1 in lung tissue were analyzed by Western blot. HE staining results indicated that 293T... exo The alveolar structure was disordered, the interstitium was significantly thickened, and there was extensive infiltration of inflammatory cells, suggesting severe pulmonary fibrosis. 293T exo+PFD The group showed moderate lung tissue damage, with partial alveolar collapse and interstitial fibrosis still visible, but the inflammatory infiltration was slightly reduced. In contrast, the 293T group...exo antiFAP+PFD The alveolar structure of the group was relatively intact, the interstitium was significantly thinned, and the number of inflammatory cells was significantly reduced, suggesting that it has a better therapeutic effect in improving lung tissue structure. Figure 5 A).

[0043] Masson staining was used to further assess the degree of collagen deposition. 293T exo The group showed extensive collagen fiber (blue) deposition, distributed in the alveolar septa and around blood vessels, suggesting high-level fibrosis. 293T exo+PFD The degree of collagen deposition decreased in this group, but large areas of blue fibers still remained. A significant difference was observed in group 293T. exo antiFAP+PFD Almost no obvious collagen deposition was observed in the group, and the lung tissue structure was close to normal. Figure 5 B) indicates that this group had the strongest intervention effect in anti-fibrosis.

[0044] Results from fluorescence imaging tracking of exosome distribution showed that 293T exo The 293T group rapidly diffused throughout the body after injection, indicating weak targeting and tissue retention capabilities. exo+PFD The 293T group diffused to other tissues 8 hours after injection, with limited enrichment in the lungs. In contrast, the 293T group... exo antiFAP+PFD The sample remained primarily concentrated in the lung region after 8 and 24 hours, demonstrating excellent tissue targeting and retention capabilities. Figure 6 This result further validates that antiFAP modification helps improve the lung targeting efficiency of exosome delivery systems.

[0045] Western blot results showed that at 293T exo In this group, p-Smad3 and TGF-β1 expression levels were high, suggesting that this signaling pathway is highly activated in the context of pulmonary fibrosis. 293T exo+PFD The expression of p-Smad3 and TGF-β1 in the group was slightly decreased, indicating that PFD has a certain anti-fibrotic effect, but the inhibitory effect is limited. In contrast, the 293T group showed a slight decrease. exo antiFAP+PFD The expression levels of p-Smad3 and TGF-β1 in the group were significantly downregulated, suggesting that antiFAP modification enhanced the antifibrotic effect of exosome-delivered PFD. Figure 7 ), effectively inhibiting the activation of the TGF-β / Smad signaling pathway ( Figure 7 ).

Claims

1. A method for preparing 293T exosomes for targeted delivery of pirfenidone via a membrane-loaded FAP, characterized in that, Includes the following steps: Extraction of exosomes from S1.293T cells: 293T cells were seeded in T175 cell flasks and cultured in DMEM high glucose medium to 90% confluence. They were then cultured in FBS-free medium for 48 hours. The supernatant was collected and exosomes were extracted from the 293T cell culture supernatant by ultracentrifugation. S2. FAP-targeting molecule modification of exosome membranes: Anti-FAP antibodies were coupled to the surface of exosome membranes using copper-free click chemistry to prepare 293T. exo antiFAP exosomes; S3. Encapsulate PFD within the modified exosome cavity: Dissolve PFD in DMSO, and mix PFD with 293T at a 2:1 ratio. exo antiFAP The exosome suspension, wherein the PFD is pirfenidone, is subjected to ultrasonic treatment at 20% power, with a cycle of 30 seconds working and 30 seconds rest, for a total of 3 minutes, and repeated six times to obtain 293T exosomes with membrane-loaded FAP for targeted delivery of pirfenidone.

2. The method as described in claim 1, characterized in that: The specific operation of step S2 is as follows: add the DBCO reagent in DMSO to the exosome solution resuspended in PBS, and stir at 4°C for 1 hour; add Azide-modified antiFAP antibody, and continue stirring at 4°C for 24 hours; wash three times with a 100 kDa ultrafiltration tube to remove unbound antibodies.

3. The membrane prepared by the method of any one of claims 1-2 carries FAP-targeted delivery of 293T exosomes of pirfenidone.

4. The application of the membrane-loaded FAP-based targeted delivery of pirfenidone 293T exosomes as described in claim 3, characterized in that: The membrane-loaded FAP-targeted delivery pirfenidone 293T exosomes are used in the preparation of drugs for the treatment of pulmonary fibrosis, administered via nebulized inhalation.

Images