IRGD-targeted and anti-fibrosis micromolecule dual-modified exosome as well as construction method and application of iRGD-targeted and anti-fibrosis micromolecule dual-modified exosome

Abstract

The invention relates to the technical field of biological medicines, in particular to an iRGD targeted and anti-fibrosis small molecule dual-modified exosome as well as a construction method and application thereof. The exosome utilizes the specific binding penetration enhancement effect of iRGD (the amino acid sequence is CRGDKGPDC) and a fibroblast high-expression integrin receptor, so that the enrichment efficiency of the exosome at a focus part is improved; the anti-fibrosis micromolecule Let-7a-5p can inhibit a TGF-beta signal channel by blocking Smad2 / 3 phosphorylation, and the anti-fibrosis micromolecule Let-7a-5p and the anti-fibrosis microRNA carried by the exosome generate a synergistic anti-fibrosis effect; the exosome phospholipid bilayer can also protect Let-7a-5p from enzymolysis and prolong the half-life period, and iRGD modification can reduce the non-targeted uptake rate. An animal model verifies that the double-modified exosome has a good skin fibrosis resisting effect, and side effects such as liver and kidney function damage are not found. The invention provides an effective and safe treatment scheme for resisting skin fibrosis.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and specifically to an iRGD-targeted and anti-fibrosis small molecule dual-modified exosome, and a construction method and application thereof. Background Art

[0002] Systemic sclerosis (SSc) is an autoimmune disease characterized by vascular abnormalities, immune dysregulation, and fibrosis. The disease is a serious threat to patients' lives, and its prognosis has therefore received widespread attention. Currently, the main clinical treatment is a combination of glucocorticoids and immunosuppressants, but remission rates are low, and safety and drug resistance are uncertain.

[0003] Stem cells are cells with the potential for self-renewal and multidirectional differentiation, and can autonomously repair damaged tissues. Human umbilical cord mesenchymal stem cells (HUMSCs) have the characteristics of lower immunogenicity, stronger differentiation ability and easier access. Stem cells mainly function through paracrine secretion, and human umbilical cord mesenchymal stem cell exosomes (HUMSCs-Ex) are a type of paracrine vesicle. Chinese patent application CN114042087A discloses a scheme for preparing human umbilical cord mesenchymal stem cell exosomes for the treatment of scleroderma. However, although exosomes have the advantage of natural delivery, unmodified exosomes are easily taken up by non-target cells. The existing technology has confirmed that anti-fibrotic small molecules have anti-fibrotic effects, but they have a short half-life in the body and poor targeting, which can easily lead to systemic toxic side effects. Summary of the Invention

[0004] (1) Technical issues to be resolved

[0005] In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides an iRGD-targeted and anti-fibrosis small molecule dual-modified exosome, as well as a construction method and application thereof, which solves the technical problems such as poor targeting of unmodified exosomes and the short in vivo half-life and poor targeting of the anti-fibrosis small molecule Let-7a-5p, thereby effectively improving the drug enrichment efficiency at the lesion site and prolonging the half-life of the anti-fibrosis small molecule Let-7a-5p, thereby achieving better anti-skin fibrosis treatment effects and reducing toxic side effects.

[0006] (2) Technical solution

[0007] In a first aspect, the present invention provides an exosome doubly modified with iRGD targeting and anti-fibrosis small molecules, comprising exosomes, wherein the exosome membrane is modified with iRGD peptide, and the exosome cavity encapsulates the anti-fibrosis small molecule Let-7a-5p, wherein the amino acid sequence of the iRGD peptide is CRGDKGPDC.

[0008] Among them, iRGD peptide was modified into the exosome membrane, and the specific binding penetration enhancement effect of iRGD with integrin receptors (αvβ3 / β5) highly expressed on fibroblasts was utilized to improve the enrichment efficiency of exosomes in the lesion site; Let-7a-5p was encapsulated into the exosome cavity, inhibiting the TGF-β signaling pathway by blocking Smad2 / 3 phosphorylation, and producing a synergistic anti-fibrotic effect with the anti-fibrotic microRNA carried by the exosomes themselves; the exosomal phospholipid bilayer protected Let-7a-5p from enzymatic hydrolysis, extending the drug half-life, and iRGD modification could reduce the non-target uptake rate, reducing the uptake of exosomes by non-target cells, thereby reducing the systemic toxic side effects caused by the targeting of the anti-fibrotic small molecule Let-7a-5p in vivo.

[0009] Preferably, the exosomes are derived from adipose-derived stem cells (ADSCs). ADSC-derived exosomes express immunomodulatory proteins (such as CD47 and PD-L1), naturally inhibit macrophage phagocytosis and T cell activation, and therefore have naturally low immunogenicity.

[0010] In a second aspect, the present invention further provides a method for constructing exosomes dual-modified with iRGD targeting and anti-fibrosis small molecules, comprising:

[0011] S1. Modifying the iRGD peptide to the exosome membrane by click chemistry, wherein the amino acid sequence of the iRGD peptide is CRGDKGPDC;

[0012] S2. The anti-fibrotic small molecule Let-7a-5p was encapsulated into the exosome cavity using ultrasonic drug loading method.

[0013] According to a preferred embodiment of the present invention, S1 includes the following steps:

[0014] S11. Preparation of iRGD-PEG-Chol Complex

[0015] iRGD peptide and PEG-2000-cholesterol Chol-PEG were mixed at a molar ratio of 1:3-1:5 in the dark, dissolved in serum-free PBS buffer, and incubated at 36.5-37.5°C with shaking to allow the thiol group of iRGD to covalently bind to the maleimide end of Chol-PEG via click chemistry to obtain an iRGD-PEG-Chol complex;

[0016] S12. Preparation of iRGD-modified exosomes

[0017] Purified exosomes were mixed with iRGD-PEG-Chol complexes in PBS at a particle molar ratio of 1:500-1:1000. The mixture was then centrifuged at 200-500 × g and 2-6°C to allow initial adsorption of the iRGD-PEG-Chol complex to the exosome membrane. The mixture was then transferred to a light-proof container and incubated at 2-6°C for 20-28 hours. During the incubation period, the mixture was gently mixed at predetermined intervals to allow the cholesterol hydrophobic end of the iRGD-PEG-Chol complex to insert into the exosome phospholipid bilayer.

[0018] S13, purification

[0019] Use a 100 kDa ultrafiltration tube and centrifuge to remove free iRGD peptide; finally, resuspend in cold PBS containing 0.1% BSA and sterilize through a microfiltration membrane. The filtrate is the iRGD peptide-modified exosomes.

[0020] According to a preferred embodiment of the present invention, S2 includes the following steps:

[0021] S21, the iRGD peptide-modified exosomes prepared in S1 were mixed with the anti-fibrotic small molecule Let-7a-5p at a molar ratio of 1:50-1:200 in PBS buffer;

[0022] S22. Immerse the probe sonicator 0.8-1.3 cm below the liquid surface and perform pulse sonication at 20-100 W for 1-5 min in an ice bath. Immediately cool the sample on ice after sonication.

[0023] S23. Separate the drug-loaded exosomes from the unencapsulated Let-7a-5p molecules by ultracentrifugation at (100,000-150,000) × g for 1-2 h or by size exclusion chromatography. Wash the precipitate 2-3 times with cold PBS to remove residual impurities to obtain exosomes dually modified with iRGD-targeted and anti-fibrosis small molecules.

[0024] Preferably, in S22, pulse ultrasonic treatment is performed at 20-40 W, with a pulse frequency of on for 2-5 seconds and off for 2-5 seconds, for intermittent heat dissipation.

[0025] According to a preferred embodiment of the present invention, the exosomes are exosomes derived from adipose-derived stem cells (ADSCs).

[0026] According to a preferred embodiment of the present invention, the exosome preparation method is as follows: adipose-derived stem cells are cultured in a serum-free medium for 48-72 hours, the culture supernatant is collected, and then centrifuged sequentially: 300×g for 10 minutes to remove cells → 2000×g for 20 minutes to remove dead cell debris → 10,000×g for 30 minutes to remove large vesicles and apoptotic bodies → finally ultracentrifuged at 100,000×g for 70 minutes, the precipitate is harvested at 2-6°C, and resuspended in pre-chilled PBS; the resuspension is sterilized by microfiltration membrane, host nucleic acid impurities are removed by sucrose density gradient centrifugation, and exosomes with a density range of 1.10-1.18 g / mL are collected and stored at -80°C in PBS containing 10% cryoprotectant for later use.

[0027] The present invention also relates to the use of the iRGD-targeted and anti-fibrosis small molecule dual-modified exosomes in the preparation of anti-skin fibrosis drugs.

[0028] (3) Beneficial effects

[0029] The present invention performs bifunctional modification on exosomes. On the one hand, iRGD peptide is introduced into the exosome membrane, and the specific binding and penetration enhancement effect of iRGD and the integrin receptor (αvβ3 / β5) highly expressed in fibroblasts is utilized to improve the enrichment efficiency of exosomes in the lesion site; on the other hand, the TGF-β pathway inhibitor Let-7a-5p is encapsulated in the exosome cavity, and Let-7a-5p is used to block Smad2 / 3 phosphorylation to inhibit the TGF-β signaling pathway, and amination realizes the anti-fibrosis effect. At the same time, this anti-fibrosis effect produces a synergistic effect with the anti-fibrosis microRNA carried by the exosomes themselves, further enhancing the anti-fibrosis function of the bifunctional modified exosomes of the present invention.

[0030] Among them, the TGF-β pathway inhibitor Let-7a-5p was encapsulated in the exosome cavity, and the exosome phospholipid bilayer was used to protect Let-7a-5p from enzymatic hydrolysis, thereby extending the drug half-life. At the same time, iRGD modification can reduce the non-target uptake rate, reduce the uptake of exosomes by non-target cells, and reduce the poor targeting of Let-7a-5p leading to systemic toxic side effects.

[0031] The constructed double-modified exosomes of the present invention were applied to a bleomycin-induced mouse skin fibrosis model. It was found that the skin volume fraction of the double-modified exosome-treated group was significantly reduced compared with the unmodified exosome group (p < 0.01), and no side effects such as liver and kidney damage were found. This shows that the constructed double-modified exosomes of the present invention have a better anti-skin fibrosis treatment effect and good drug safety.

[0032] The dual-functionalized exosomes of the present invention are the first fibrosis treatment system that combines targeted peptide modification with exosome-small molecule drugs, and have good clinical translation value. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Fluorescence images of FAM-labeled iRGD-modified exosome complexes detected by flow cytometry.

[0034] Figure 2 The drug loading rate of exosomes of the mixture of iRGD@ADSC-exo and Let-7a-5p before and after ultrasound, and the anti-RNase degradation effect of exosomes after encapsulation of Let-7a-5p.

[0035] Figure 3 The uptake rates of exosomes Exos, drug-loaded exosomes Let@Exos alone, targeted modified exosomes iRGD-Exos alone, and drug-loaded targeted modified exosomes Let@iRGD-Exos were compared in vitro by LoS primary fibroblasts and keratinocytes.

[0036] Figure 4 Tissue sections of the model group and control group of the skin fibrosis animal model.

[0037] Figure 5 The enrichment of exosomes and drugs after injection of exosomes Exos, drug-loaded exosomes Let@Exos alone, targeted modified exosomes iRGD-Exos alone, and drug-loaded targeted modified exosomes Let@iRGD-Exos into animal models of skin fibrosis.

[0038] Figure 6 These are tissue sections of the lesion site after injection of PBS, exosomes Exos, drug-loaded exosomes Let@Exos alone, targeted modified exosomes iRGD-Exos alone, and drug-loaded targeted modified exosomes Let@iRGD-Exos into the animal model of skin fibrosis.

[0039] Figure 7 The downregulation of α-SMA, COl1, and p-Smad2 / 3 in the model was observed after injection of exosomes Exos, drug-loaded exosomes Let@Exos alone, targeted modified exosomes iRGD-Exos alone, and drug-loaded targeted modified exosomes Let@iRGD-Exos into the skin fibrosis animal model. DETAILED DESCRIPTION

[0040] In order to better explain the present invention and facilitate understanding, the present invention is described in detail below through specific implementation methods in conjunction with the accompanying drawings.

[0041] The sources of the biological materials used in the present invention are described as follows:

[0042] 1. Adipose-derived mesenchymal stem cells (ADSC): self-isolation.

[0043] Tissue source: Adipose tissue (approximately 5 g) was obtained from healthy volunteers by abdominal liposuction, with approval from the ethics committee and informed consent from the patients. The isolation method was as follows:

[0044] (1) Enzymatic digestion: Adipose tissue was digested with 0.1% collagenase I (Gibco) at 37°C for 30 min and centrifuged (1200 rpm, 10 min) to remove the oil layer and bottom liquid;

[0045] (2) Red blood cell lysis: treated with ACK lysis buffer (Thermo,) for 5 min and washed with PBS;

[0046] (3) Primary culture: Resuspend the cells in ADSC complete medium (α-MEM + 10% FBS + 1% penicillin / streptomycin), culture at 37°C, 5% CO2, and passage to P3 for experiments.

[0047] (4) Identification criteria: Flow cytometry detection of CD73 / CD90 / CD105 positivity ≥ 95% (BD Biosciences antibodies), and verification of osteogenic / adipogenic / chondrogenic differentiation ability.

[0048] 2. The anti-fibrotic small molecule Let-7a-5p was purchased commercially from Sangon Biotech (Shanghai) Co., Ltd. (Sangon Biotech); catalog number: miRBase, number MIMAT0000062.

[0049] The anti-fibrotic small molecule Let-7a-5p is a microRNA with anti-fibrotic effects. It is chemically synthesized into a double-stranded form with a base sequence of 5′-UGAGGUAGUAGGUUGUAUAGUU-3′, purified by HPLC (purity ≥ 95%), and freeze-dried to store at -80°C. It is diluted to the working concentration with DEPC water before use.

[0050] 3. iRGD peptide (amino acid sequence CRGDKGPDC), commercially purchased from Peptides International, USA, with a purity of >98%.

[0051] Example 1

[0052] This example is to isolate and prepare exosomes ADSC-exo from adipose-derived mesenchymal stem cells (ADSCs); the isolation and culture methods of adipose-derived mesenchymal stem cells (ADSCs) (passages P3-P5) are described above. The culture conditions for ADSCs are α-MEM + 10% exosome-depleted FBS (to avoid serum exosome interference and foreign protein contamination). The detailed isolation and preparation process of exosomes ADSC-exo is as follows:

[0053] (1) ADSC cell pre-culture

[0054] When the ADSC cells reached 80% confluence, they were replaced with serum-free medium (containing 1% BSA) and cultured for 48 h to enrich exosomes; culture in a hypoxic environment (5% CO2, 3% O2) could increase exosome production.

[0055] (2) Separation method

[0056] Adipose-derived stem cells (ADSCs) were cultured in serum-free medium for 72 hours. The culture supernatant was collected and centrifuged sequentially: 300 × g (10 min) at 4°C to remove cells, 2000 × g (20 min) to remove dead cell debris, 10,000 × g (30 min) to remove large vesicles and apoptotic bodies, and finally, ultracentrifugation at 100,000 × g (Beckman Optima XPN-80 Ultracentrifuge, Type 70Ti rotor) for 70 min at 4°C to pellet exosomes. The pellets were then resuspended in pre-chilled PBS. Finally, the pellets were sterilized by filtration through a 0.22 μm filter and subjected to sucrose density gradient centrifugation to remove host nucleic acid impurities. Exosomes with a density range of 1.10-1.18 g / mL were collected to further minimize immunological risk. The exosomes were immediately aliquoted and stored at -80°C in PBS containing 10% glycerol (cryoprotectant) until further use.

[0057] (3) Key quality control indicators

[0058] Purity: Western blot verification of exosome markers (CD9 / CD63 / TSG101 positive), Calnexin negative.

[0059] Immunogenicity verification: Co-culture with PBMC in vitro, detect the release of inflammatory factors (TNF-α, IL-6) ≤ 10% of the untreated group and the test is qualified.

[0060] Sterility: The test results of Limulus amebocyte lysate (LIAL) were negative for endotoxin (≤0.25EU / mL) and mycoplasma.

[0061] The exosomes ADSC-exo prepared in this embodiment have low immunogenicity and high safety. ADSC-derived exosomes express immunomodulatory proteins (such as CD47, PD-L1), naturally inhibit macrophage phagocytosis and T cell activation, and have natural low immunogenicity. Exosome-free serum culture medium is used throughout the separation process to avoid contamination by heterologous proteins, and residual host cell DNA / RNA impurities are removed by 0.22μm filtration and sucrose density gradient centrifugation to further reduce the immune risk; stored at -80°C in PBS containing 10% glycerol (cryoprotectant) to avoid membrane rupture or increased immunogenicity caused by repeated freezing and thawing. This separation scheme takes into account both high efficiency and low immune risk, providing a safe carrier for subsequent targeted modification.

[0062] Example 2

[0063] In this example, the membrane of the exosome ADSC-exo prepared in Example 1 was modified with iRGD peptide, and Let-7a-5p was encapsulated into the exosome cavity to produce exosomes dually modified with iRGD targeting and anti-fibrosis small molecules. The preparation process is as follows:

[0064] (1) Preparation of iRGD-PEG-Chol complex

[0065] A FAM-fluorescently labeled iRGD peptide (amino acid sequence CRGDKGPDC) (facilitating screening and verification of the positive rate of modified products) was mixed with PEG-2000-cholesterol (Chol-PEG) at a molar ratio of 1:4 in the dark and dissolved in serum-free PBS buffer (pH 7.4). The mixture was incubated with shaking at 37°C for 2 hours to allow the thiol groups of iRGD to covalently bind to the maleimide ends of Chol-PEG via click chemistry, forming a stable iRGD-PEG-Chol complex. The complex synthesis efficiency was verified to be >90% by HPLC.

[0066] (2) Modification of the complex by co-incubation with ADSC-exo

[0067] Adipose-derived stem cell-derived exosomes (ADSC-exo, concentration ≥ 1 × 10 11 / mL) and mixed with iRGD-PEG-Chol complexes in PBS at a particle molar ratio of 1:600 ​​to a final volume of 2mL. First, centrifugation was performed at 350×g (4°C) for 3 hours to promote initial adsorption of the complex to the exosome membrane. After centrifugation, the complex was transferred to a light-proof container and incubated at 4°C for 24 hours. During this period, gentle mixing for 10 seconds every 6 hours was performed to ensure that the hydrophobic end of cholesterol was fully inserted into the exosome phospholipid bilayer.

[0068] (3) Purification and quality control

[0069] After incubation, use a 100 kDa ultrafiltration tube and centrifuge at 4°C and 4000×g for 10 minutes, repeating three times to remove free iRGD peptide; finally, resuspend in cold PBS containing 0.1% BSA, sterilize through a 0.22 μm filter membrane, and the filtrate is the target product.

[0070] like Figure 1 As shown, flow cytometry detection of FAM showed that the fluorescence peak of the iRGD-Exos group shifted to the right, the fluorescence positivity rate was ≥80%, and the negative rate of the unmodified group was <5%; NTA analysis of particle size (maintained 90±20nm) and TEM verification of membrane integrity confirmed that iRGD@ADSC-exo was obtained.

[0071] During the iRGD peptide modification process for ADSC-exo, it is important to maintain low temperature, avoid light, and maintain appropriate osmotic pressure. Finally, purification is required. Low temperature: Operate strictly at approximately 4°C throughout the process to prevent increased membrane fluidity that may cause the modification to dislodge. Protect from light: Use brown tubes or tin foil to protect the stability of FAM fluorescence. Buffer: Maintain an osmotic pressure of 280-320 mOsm / kg (PBS pH 7.4). Purification: Ultrafiltration is used to remove unbound complexes to ensure target specificity.

[0072] (4) iRGD@ADSC-exo purified in step (3) was mixed with Let-7a-5p (anti-fibrotic microRNA) at a molar ratio (exosomes: Let-7a-5p = 1:100-200) in PBS buffer (pH 7.4, to maintain physiological osmotic pressure and stability). The total volume of the mixture was controlled to 0.1-1 mL. A small volume facilitates the concentration of ultrasonic energy.

[0073] (5) Use a probe sonicator (Branson type) immersed approximately 1 cm below the liquid surface (to avoid bubbles or splashing) and perform pulsed sonication in an ice-water bath (0-4°C) at a power of 20-100 W (preferably controlled at 20-40 W, the power should not be too high to avoid damage to the exosome structure) for 1-5 min (preferably 2-3 min to avoid excessive cavitation and damage to the exosome membrane). The pulsed sonication frequency is on for 2-5 seconds / off for 2-5 seconds, with intermittent heat dissipation. After the sonication is completed, the sample is immediately placed on ice to cool.

[0074] The probe diameter of the probe ultrasound instrument is selected according to the sample size (3mm or 6mm).

[0075] (6) Purification

[0076] Separate drug-loaded exosomes from unencapsulated Let-7a-5p by ultracentrifugation (100,000-150,000 × g, 1-2 h) or size exclusion chromatography (SEC). Wash the pellet 2-3 times with cold PBS to remove residual impurities.

[0077] (7) Quality Inspection

[0078] Drug loading efficiency detection: The Let-7a-5p content in exosomes was measured by qPCR, and the drug loading capacity (e.g., number of molecules / exosome) was calculated.

[0079] like Figure 2 Figures A and B show the drug loading efficiency of Let-7a-5p in the exosomes of a mixture of iRGD@ADSC-exo and Let-7a-5p before and after sonication. Before sonication, the drug loading efficiency of the exosomes was 11.05%; after sonication, the drug loading efficiency of the exosomes was 47.64%.

[0080] like Figure 2 Figure C shows a comparison of the RNase-resistant effects of Let-7a-5p before and after encapsulation in the exosome cavity. The RNase group represents the free Let-7a-5p drug, without encapsulation within the exosome cavity, while the exo+RNase group represents the encapsulated Let-7a-5p drug. When equal amounts of RNase were added to both drug groups, the free Let-7a-5p molecules were severely degraded, with a retention rate of less than 5% compared to the untreated (no RNase) group. However, the retention rate of the encapsulated Let-7a-5p molecules exceeded 90%. This indicates that encapsulation of Let-7a-5p within the exosome cavity effectively protects Let-7a-5p from enzymatic degradation, extending its half-life.

[0081] Exosome integrity detection: Transmission electron microscopy (TEM) was used to observe the morphology, nanoparticle tracking analysis (NTA) was used to detect the particle size distribution (should be maintained at 50-150 nm), and Western blot was used to detect the presence of exosome markers (such as CD63 and TSG101) and modified iRGD.

[0082] Example 3

[0083] This example examines the in vitro uptake of exosomes (Exos), drug-loaded exosomes (Let@Exos), targeted modified exosomes (iRGD-Exos), and drug-loaded targeted modified exosomes (Let@iRGD-Exos) by target cells. The target cells in this example are LoS primary fibroblasts and keratinocytes, and the drug is Let-7a-5p (abbreviated as Let).

[0084] First, the dye PKH67 was used to stain exosomes Exos, drug-loaded exosomes Let@Exos, targeted modified exosomes iRGD-Exos, and drug-loaded targeted modified exosomes Let@iRGD-Exos, respectively. The stained exosomes were co-incubated with fibroblasts at 37°C for 24 hours. The relative fluorescence intensity of PKH67 in fibroblasts was detected to obtain the uptake rate of exosomes by fibroblasts.

[0085] The experimental results are as follows Figure 3 As shown. Figure 3As shown in A and B, LoS primary fibroblasts were co-cultured in vitro with exosomes Exos, drug-loaded exosomes Let@Exos alone, targeted modified exosomes iRGD-Exos alone, and drug-loaded targeted modified exosomes Let@iRGD-Exos. After the culture, the experimental results showed that the relative uptake rates of Los primary fibroblasts for exosomes Exos and drug-loaded exosomes Let@Exos were basically the same, both around 22%. The relative uptake rates of LoS primary fibroblasts for targeted modified exosomes iRGD-Exos alone and drug-loaded targeted modified exosomes Let@iRGD-Exos were also basically the same, both 34%.

[0086] like Figure 3 As shown in Figures C and D, the mixture of LoS primary fibroblasts and keratinocytes was co-cultured in vitro with exosomes Exos, drug-loaded exosomes Let@Exos alone, targeted modified exosomes iRGD-Exos alone, and drug-loaded targeted modified exosomes Let@iRGD-Exos, respectively. After the culture, the experimental results showed that the relative uptake rate of exosomes Exos and drug-loaded exosomes Let@Exos by the mixture of LoS primary fibroblasts and keratinocytes was low, approximately 21% and 17%; while the relative uptake rate of exosomes iRGD-Exos and drug-loaded targeted modified exosomes Let@iRGD-Exos by the mixture of LoS primary fibroblasts and keratinocytes was high, reaching 79% and 80%, respectively.

[0087] In summary, iRGD-modified exosomes can significantly increase the uptake rate of exosomes and their loaded drugs by target cells such as LoS primary fibroblasts, thereby reducing the uptake of exosomes by non-target cells and reducing the systemic toxic side effects caused by the poor targeting of Let-7a-5p.

[0088] Example 4

[0089] In this example, an animal model of skin fibrosis was constructed to verify the efficacy of drug-loaded targeted modified exosomes Let@iRGD-Exos in treating skin fibrosis. The experimental method is as follows:

[0090] 1. Establishment of mouse skin fibrosis model (bleomycin-induced method)

[0091] Purchase several C57BL / 6 mice (6-8 weeks old, male, weighing 20-25 g) and perform modeling as follows:

[0092] (1) Back hair removal

[0093] The hair on the back of the mouse was shaved with an electric shaver, and the remaining hair was treated with a depilatory cream (containing calcium thioacetate) to avoid skin damage.

[0094] (2) Bleomycin injection

[0095] Dissolve bleomycin (Sigma, B5507) in PBS to a concentration of 100 μg / mL (prepare immediately before use). Inject subcutaneously, 100 μL (containing 10 μg bleomycin) daily for 28 consecutive days (at the same site). The control group received an equal amount of PBS.

[0096] (3) Observation indicators:

[0097] Histological examination (gold standard): 48 hours after the last injection, skin tissue (including the injection center) was collected, fixed with 4% paraformaldehyde, embedded in paraffin and sliced. H&E staining was performed to observe epidermal thickening and inflammatory cell infiltration. Figure 4 The figure shows a comparison of tissue sections between the modeling group and the control group (H&E staining). As can be seen from the figure, the thickness of the skin epidermis (blue-purple nuclei) of the control group is normal, and the cells are arranged neatly; the collagen fibers (pink) in the dermis are evenly distributed, and there are no significant signs of inflammation or fibrosis. The layers are clear, and the interstitial cells are sparse, which is consistent with the morphological characteristics of healthy skin. In the modeling group, the skin epidermis is thickened: keratinocytes proliferate (densely stained nuclei); collagen fibers are coarse and disordered (increased and disordered pink areas); inflammatory infiltration and aggregation of lymphocytes / macrophages (dark-stained dots) can also be seen. The tissue sections of the modeling group are consistent with the typical manifestations of bleomycin-induced skin fibrosis, confirming that the model was successfully constructed.

[0098] Masson's trichrome staining was performed: hematoxylin-eosin (H&E), nuclei (blue-purple), and collagen / cytoplasm (pink). Collagen volume fraction (CVF) was calculated (using ImageJ to analyze the percentage of pink collagen area). A successful model was established when CVF increased by ≥50% compared to the control group.

[0099] 2. The mice with successful modeling were divided into 4 groups, with 6 mice in each group. Each group was subcutaneously injected with exosomes Exos, drug-loaded exosomes Let@Exos alone, targeted modified exosomes iRGD-Exos alone, and drug-loaded targeted modified exosomes Let@iRGD-Exos at the lesion site. The injection dose was calculated based on the number of exosomes (Exos particles). The number of exosomes was determined by NTA (nanoparticle tracking analysis), which was usually adjusted to 1×10 10 particles / mL (PBS was used as the dispersion solvent), and the single injection volume was 100 μL (containing 1×10 9 Exosomes were injected subcutaneously four times on days 1, 7, 14, and 21 after modeling. Multiple injections were performed around the fibrotic area to avoid direct damage to the lesion. Healthy mice served as a control group and received an equal amount of PBS.

[0100] 3. Efficacy testing: 24 hours after the last injection, the model mice were sacrificed, and the full-thickness skin was removed. Skin fluorescence (exosomes labeled with FAM fluorescent group) and tissue HE staining were performed, and skin fibrosis indicators were detected by qPCR.

[0101] Comparison of the four groups of processed fluorescence microscope images Figure 5 As shown. The four treatment groups include: Exos (unmodified exosomes); Let@Exos (exosomes loaded with Let-7a-5p); iRGD-Exos (exosomes modified with iRGD peptide); Let@iRGD-Exos (bifunctional exosomes modified with iRGD and loaded with Let-7a-5p). In the fluorescence images, red indicates exosome markers (such as CD63); green indicates targeting molecules (such as iRGD) or drugs (Let-7a-5p); and blue indicates cell nuclei (DAPI staining). Figure 5 It can be seen that in the Exos group, the red signal is evenly distributed and the green signal is weak (no targeting or drug loading); in the Let@Exos group, the red and green signals partially overlap (successful drug loading), but there is no targeted enrichment; in the iRGD-Exos group, the green signal (iRGD) accumulates around the cell membrane, showing enhanced targeting; in the Let@iRGD-Exos group, the red and green signals are highly co-localized, and the fluorescence intensity is highest around the blue nucleus, which confirms that the targeted delivery of exosomes enables the effective enrichment of drugs around the cells of the skin lesions.

[0102] The tissue sections of the five groups were treated as shown in Figure 6 , This figure shows the comparison of the effects of five treatment groups. The five treatment groups include: PBS group (negative control); Exos group (unmodified exosomes); Let@Exos group; iRGD-Exos group; Let@iRGD-Exos group (treatment group). Tissue section staining method: collagen fibers: pink purple (H&E); cell nuclei: dark purple. Figure 6 The tissue sections shown were analyzed for pathological characteristics. The results showed that the PBS group had loose collagen arrangement and normal epidermal thickness. The Exos group had slight collagen hyperplasia, but the fibrotic structure was still obvious. The Let@Exos group had reduced collagen deposition, but local disorder was still present. The iRGD-Exos group had more orderly collagen arrangement and reduced inflammatory infiltration. The Let@iRGD-Exos group had collagen close to normal tissue morphology, restored epidermal thickness, and significantly reduced fibrotic lesions. Compared with the other three exosome-injected groups, the Let@iRGD-Exos group had the best therapeutic effect.

[0103] In summary, targeted modification (iRGD) enhances the retention of exosomes in fibrotic areas; drug loading (Let-7a-5p) enhances the anti-fibrotic effect, and the dual-functional group (Let@iRGD-Exos) has the most significant synergistic effect.

[0104] See also Figure 7 As shown, by measuring the expression levels of skin fibrosis-related proteins in different treatment groups (quantitative PCR to detect mRNA levels), injection of drug-loaded, targeted exosomes, Let@iRGD-Exos, significantly reduced the expression levels of α-SMA, Col1, and p-Smad2 / 3 in mice. α-SMA, Col1, and p-Smad2 / 3 are key molecular markers of skin fibrosis. Anti-skin fibrosis treatments inhibit the TGF-β / Smad pathway, which in turn reduces p-Smad2 / 3 and downregulates α-SMA and Col1 synthesis, reversing the fibrotic process. Decreased levels of these markers indicate a significant anti-fibrosis treatment effect.

[0105] The present invention first modifies the exosome membrane (e.g., by attaching the target molecule, iRGD peptide). This modification process involves some potentially damaging treatments (e.g., click chemistry reaction conditions) that could damage the exosome's internal contents. To avoid damaging the potentially fragile encapsulated therapeutic molecule, Let-7a-5p, during the membrane modification process, it is preferred to load the drug molecule into the exosome lumen after the membrane modification is complete and the structure is stabilized.

[0106] Click chemistry is used to modify the exosome membrane surface with iRGD peptides. This method boasts high specificity, controllable targeting, and mild conditions, resulting in high yields (>90%) while avoiding the membrane disruption caused by traditional chemical cross-linking. iRGD fully integrates into the exosome phospholipid bilayer via its cholesterol hydrophobic end, ensuring that the targeting peptide faces outward, maintaining target binding activity. Ultrasound-assisted encapsulation of Let-7a-5p into the exosome cavity is used. Ultrasound cavitation temporarily opens the exosome membrane to trap the Let-7a-5p small molecule, resulting in high encapsulation efficiency, superior to electroporation or freeze-thaw methods. The exosome phospholipid bilayer protects Let-7a-5p from degradation by serum RNases, effectively extending its half-life. Ultrasound-assisted encapsulation also preserves exosome-derived natural anti-fibrotic miRNAs (such as miR-29b), which, along with Let-7a-5p, effectively inhibit skin fibrosis. The exosome dual modification method provided by the present invention takes into account both efficiency and safety, and achieves the synergistic functions of iRGD targeting, exosomes and Let-7a-5p.

[0107] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or make equivalent replacements for some or all of the technical features therein. These modifications or replacements, or when the technical features in the above embodiments do not conflict with each other, can be combined in the manner described in the embodiments, and these modifications, replacements or combinations do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An exosome dually modified with iRGD targeting and anti-fibrosis small molecules, characterized in that: The invention comprises exosomes, wherein the exosome membrane is modified with iRGD peptide, the exosome cavity encapsulates the anti-fibrosis small molecule Let-7a-5p, and the amino acid sequence of the iRGD peptide is CRGDKGPDC.

2. The exosome according to claim 1, wherein The exosomes are exosomes derived from adipose-derived stem cells (ADSCs).

3. A method for constructing exosomes dually modified with iRGD targeting and anti-fibrosis small molecules, characterized in that: include: S1. Modifying the iRGD peptide to the exosome membrane by click chemistry, wherein the amino acid sequence of the iRGD peptide is CRGDKGPDC; S2. The anti-fibrotic small molecule Let-7a-5p was encapsulated into the exosome cavity using ultrasonic drug loading method.

4. The construction method according to claim 3, characterized in that S1 includes: S11. Preparation of iRGD-PEG-Chol Complex iRGD peptide and PEG-2000-cholesterol Chol-PEG were mixed at a molar ratio of 1:3-1:5 in the dark, dissolved in serum-free PBS buffer, and incubated at 36.5-37.5°C with shaking to allow the thiol group of iRGD to covalently bind to the maleimide end of Chol-PEG via click chemistry to obtain an iRGD-PEG-Chol complex; S12. Preparation of iRGD-modified exosomes Purified exosomes were mixed with iRGD-PEG-Chol complexes in PBS at a particle molar ratio of 1:500-1:1000. The mixture was then centrifuged at 200-500 × g and 2-6°C to allow initial adsorption of the iRGD-PEG-Chol complex to the exosome membrane. The mixture was then transferred to a light-proof container and incubated at 2-6°C for 20-28 hours. During the incubation period, the mixture was gently mixed at predetermined intervals to allow the cholesterol hydrophobic end of the iRGD-PEG-Chol complex to insert into the exosome phospholipid bilayer. S13, purification Use a 100 kDa ultrafiltration tube and centrifuge to remove free iRGD peptide; finally, resuspend in cold PBS containing 0.1% BSA and sterilize through a microfiltration membrane. The filtrate is the iRGD peptide-modified exosomes.

5. The construction method according to claim 3 or 4, wherein S2 include: S21, the iRGD peptide-modified exosomes prepared in S1 were mixed with the anti-fibrotic small molecule Let-7a-5p at a molar ratio of 1:50-1:200 in PBS buffer; S22. Immerse the probe sonicator 0.8-1.3 cm below the liquid surface and perform pulse sonication at 20-100 W for 1-5 min in an ice bath. Immediately cool the sample on ice after sonication. S23. Separate the drug-loaded exosomes from the unencapsulated Let-7a-5p molecules by ultracentrifugation at (100,000-150,000) × g for 1-2 h or by size exclusion chromatography. Wash the precipitate 2-3 times with cold PBS to remove residual impurities to obtain exosomes dually modified with iRGD-targeted and anti-fibrosis small molecules.

6. The construction method according to claim 5, characterized in that: In S22, pulsed ultrasonic treatment is performed at 20-40 W with a pulse frequency of on for 2-5 seconds and off for 2-5 seconds, with intermittent heat dissipation.

7. The construction method according to claim 3, characterized in that: The exosomes are exosomes derived from adipose-derived stem cells (ADSCs).

8. The construction method according to claim 7, characterized in that: The exosome preparation method comprises culturing adipose-derived stem cells in a serum-free medium for 48-72 hours, collecting the culture supernatant, and then centrifuging in sequence: 300×g for 10 minutes to remove cells → 2000×g for 20 minutes to remove dead cell debris → 10,000×g for 30 minutes to remove large vesicles and apoptotic bodies → finally ultracentrifuging at 100,000×g for 70 minutes, harvesting the precipitate at 2-6°C, and resuspending it in pre-chilled PBS; the resuspension is sterilized by microfiltration membrane, and host nucleic acid impurities are removed by sucrose density gradient centrifugation. Exosomes with a density range of 1.10-1.18 g / mL are collected and stored at -80°C in PBS containing 10% cryoprotectant for future use.

9. Use of the iRGD-targeted and anti-fibrosis small molecule dual-modified exosomes according to claim 1 or 2 in the preparation of anti-skin fibrosis drugs.

10. Use of the exosomes constructed by the construction method according to any one of claims 3 to 8 in the preparation of an anti-skin fibrosis drug.

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