Lung targeting peptide and application thereof in preparation of extracellular vesicle drug delivery system

Abstract

The invention relates to the technical field of biological medicine, in particular to a lung targeting peptide and application thereof in preparation of an extracellular vesicle drug delivery system. The amino acid sequence of the lung targeting peptide provided by the invention is as shown in SEQ ID NO. 2. The lung targeting peptide with alveolar epithelium targeting property is obtained through a phage display technology, the lung targeting peptide is combined to the surface of mesenchymal stem cell-derived extracellular vesicles (EVs), and the prepared targeting delivery system can act on lung epithelial cells more accurately and more long-acting, so that effective repair and protection of a lung barrier are realized. The invention provides an effective targeted delivery strategy for precise treatment of lung diseases, and has a good application prospect.

Description

A lung-targeting peptide and its application in the preparation of extracellular vesicle drug delivery systems Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a lung-targeting peptide and its application in the preparation of extracellular vesicle drug delivery systems. Background Technology

[0002] The pulmonary microvascular-alveolar epithelial barrier is a highly selective structure composed of alveolar epithelial cells, pulmonary microvascular endothelial cells, and their intercalating basement membrane. Its main function is to maintain gas exchange and alveolar fluid homeostasis. This barrier effectively prevents plasma components and inflammatory cells from infiltrating into the alveolar cavity, while ensuring efficient diffusion exchange of oxygen and carbon dioxide. When damaged by factors such as infection, toxic substances, mechanical ventilation, or ischemia-reperfusion, the barrier structure and function are disrupted, leading to increased barrier permeability and alveolar edema, thereby triggering or aggravating diseases such as acute lung injury and acute respiratory distress syndrome. Impaired barrier integrity not only causes gas exchange disorders but also initiates inflammatory and oxidative stress responses, further amplifying tissue damage and functional imbalance. Therefore, how to protect or repair the pulmonary microvascular-alveolar epithelial barrier has become a key issue and an important breakthrough in the research and prevention of lung diseases.

[0003] Pulmonary surfactant proteins (SPs) are a class of substances located on the surface of alveolar epithelial cells, playing roles in immune defense and maintaining alveolar surface homeostasis. Based on their size, function, and hydrophilic or hydrophobic properties, they can be divided into two categories. Larger hydrophilic surfactant proteins A (SPA) and D (SPD) belong to the aggregatein protein family and participate in maintaining pulmonary immune homeostasis. They are associated with macrophage polarization and pathogen recognition, and can regulate the inflammatory microenvironment and promote repair. Relatively smaller and hydrophobic surfactant proteins B (SPB) and C (SP-C) are embedded in the lipid layer associated with pulmonary surfactant substances, playing a crucial role in the interfacial adsorption of pulmonary surfactant substances and protecting their stability during continuous respiratory cycles. In previous studies, Gu et al. fused pulmonary microvascular endothelial targeting peptide (LET) into LAMP-2B protein, and the resulting modified EVs had a significantly prolonged retention time in lung tissue, directly acting on damaged vascular endothelial cells and promoting barrier repair [Gu Z, Sun M, Liu J, et al. Endothelium-Derived Engineered Extracellular Vehicles Protect the Pulmonary Endothelial Barrier in Acute Lung Injury. AdvSci (Weinh). 2024;11(6): e2306156.]; Salazar-Puerta et al. co-transfected SP-A protein and the genes of anti-inflammatory cytokines IL-4 and IL-10 into adult mouse dermal fibroblasts, causing them to release engineered EVs with both SP-A surface modification and high concentrations of anti-inflammatory factors. The specific binding of SP-A to lung tissue receptors significantly enhanced lung targeting and anti-inflammatory efficacy [Salazar-Puerta, Ana I et al. "Engineered Extracellular [Vesicles Derived from Dermal Fibroblasts Attenuate Inflammation in a Murine Model of Acute Lung Injury.” Advanced materials (Deerfield Beach, Fla.) vol. 35,28(2023): e2210579.] Studies have found that the SP-C precursor protein (proSP-C) routinely and transiently appears on the cell membrane during transport from the endoplasmic reticulum to multivesicular bodies, but abnormally accumulates in the cell membrane and circulating endosomes under pathological conditions, providing opportunities for disease-specific targeting.

[0004] Phage display is a method for displaying exogenous peptides or protein fragments on the surface of bacteriophages. This technology involves fusing exogenous sequences with phage coat proteins and then performing high-throughput screening in vitro to obtain ligands with high affinity, specificity, and targeting for specific target molecules, thus playing a crucial role in targeted drug delivery. First established by Smith in 1985, this technology allows the insertion of target sequences into the pIII or pVIII gene of filamentous phages such as M13, enabling the phage to display the corresponding peptide when infecting the host bacteria. After multiple rounds of biological screening, specific binding molecules can be enriched from billions of candidate sequences. Due to its advantages of high sensitivity, broad screening range, and ease of molecular modification, phage display has been widely applied in antibody drug development, receptor recognition mechanism research, and disease-targeting peptide screening. Phage display technology also shows unique potential in targeted therapy for lung diseases. Short peptide sequences are screened for alveolar epithelial cells, lung microvascular endothelial cells, or inflammatory lesion tissues to modify nanoparticles such as liposomes and extracellular vesicles, enabling efficient lung delivery. However, a lung-targeting peptide that can specifically recognize alveolar epithelial cells is currently lacking. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a lung-targeting peptide and its application in an extracellular vesicle drug delivery system. The lung-targeting peptide provided by this invention can specifically recognize alveolar epithelial cells. After chemically coupling and modifying it onto the surface of extracellular vesicles, it significantly enhances the uptake of alveolar epithelial cells in vitro and exhibits good lung tissue enrichment capacity in vivo. This provides an effective targeted delivery strategy for the precision treatment of lung diseases and has promising application prospects.

[0006] To achieve the above objectives, the present invention provides the following technical solution: The present invention provides a lung-targeting peptide, the amino acid sequence of which is shown in SEQ ID NO.2.

[0007] This invention provides the application of the lung-targeting peptide described in the above-mentioned technical solution in the preparation of extracellular vesicle drug delivery systems.

[0008] This invention provides an extracellular vesicle drug delivery system, comprising: bis(stearoylphosphatidylethanolamine)-polyethylene glycol 2000-maleimide (DSPE-PEG2000-VPE), extracellular vesicles, and the lung-targeting peptide described above; the lung-targeting peptide is linked to the maleimide group on the DSPE-PEG2000-VPE via a thioether bond, and the DSPE-PEG2000-VPE is embedded in the phospholipid bilayer of the extracellular vesicles through the hydrophobic effect of DSPE.

[0009] Preferably, the extracellular vesicles include extracellular vesicles derived from mesenchymal stem cells.

[0010] This invention provides a method for preparing the extracellular vesicle drug delivery system described in the above technical solution, comprising the following steps: introducing a cysteine ​​residue at the C-terminus of the lung-targeting peptide and then performing pre-reduction to obtain a lung-targeting peptide with thiol activity; mixing the lung-targeting peptide with thiol activity with DSPE-PEG2000-VPE to obtain a lipopeptide conjugate (DSPE-PEG2000-Mal-Cys-VPE); and co-incubating the lipopeptide conjugate with extracellular vesicles to obtain the extracellular vesicle drug delivery system.

[0011] Preferably, the lung-targeting peptide with a C-terminus introduced into a cysteine ​​residue is pre-reduced using tris(2-carboxyethyl)phosphonic acid hydrochloride.

[0012] Preferably, the molar ratio of the lung-targeting peptide with thiol activity to the DSPE-PEG2000-VPE is 1:1 to 1.5.

[0013] Preferably, the mixing reaction time is 1.5~2.5 h; the co-incubation time is 0.8~1.5 h.

[0014] Preferably, the lung-targeting peptide with thiol activity is mixed and reacted with the DSPE-PEG2000-VPE and then dialyzed through a dialysis membrane with a molecular weight cutoff of 3.5 kDa, and the macromolecular fraction is collected as a purified lipopeptide conjugate.

[0015] This invention provides the application of the extracellular vesicle drug delivery system described above in the preparation of a drug delivery system for treating lung diseases.

[0016] Beneficial effects: This invention provides a lung-targeting peptide, the amino acid sequence of which is shown in SEQ ID NO.2. This invention obtains a lung-targeting peptide with alveolar epithelial targeting properties using phage display technology. This lung-targeting peptide is then bound to the surface of extracellular vesicles (EVs) derived from mesenchymal stem cells. The resulting targeted delivery system can act more precisely and for a longer duration on lung epithelial cells, thereby achieving effective repair and protection of the lung barrier. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0018] Figure 1 shows the affinity assay results of the targeting peptide VPEYWNAKRHYV with SP-C; where A is the BLI assay result of the binding affinity between the targeting peptide and SPC; B is the corresponding binding fitting curve. Figure 2 shows the molecular docking simulation results of the targeting peptide VPEYWNAKRHYV and SP-C. Figure 3 shows the characterization results of EVs and VPE-EVs; where A is the particle size distribution and particle size determination results by nanoparticle tracking analysis (NTA); B is the detection of positive (CD63, CD81, and TSG101) and negative (Calnexin) markers by Western blotting (WB); C is the morphology detected by transmission electron microscopy (TEM), showing a typical concave cup structure (scale: 200 nm). Figure 4 shows the in vitro uptake of VPE-EVs by alveolar epithelial cells. Figure 5 shows the in vivo imaging results of VPE-EVs in mice. Figure 6 shows the imaging results of ex vivo organs of mice treated with VPE-EVs; where A is the imaging results of the two groups of mouse tissues at 6 h and 72 h after administration (EV, VPE-EV). Figure 7 shows the fluorescence imaging results after h; B is the quantitative analysis results of the average fluorescence intensity of different tissues after drug administration; C is the clearance rate of EV in lung tissue calculated by dividing the average signal of the lung by the average signal of the liver; Figure 7 shows the co-localization of VPE-EVs with alveolar epithelial cells. Detailed Implementation

[0019] This invention provides a lung-targeting peptide, the amino acid sequence of which is shown in SEQ ID NO.2. The lung-targeting peptide provided by this invention is a novel, high-affinity alveolar epithelial cell-targeting peptide obtained through phage display technology. Using this lung-targeting peptide to functionalize EVs, a lung-targeting drug delivery system was constructed, exhibiting excellent targeting to lung tissue and specific alveolar epithelial cells both in vitro and in vivo, and demonstrating prolonged retention time at the target site. This system provides a novel carrier platform for developing efficient and precise treatment strategies for lung diseases.

[0020] Based on the above advantages, the present invention provides the application of the lung-targeting peptide described in the above technical solution in the preparation of extracellular vesicle drug delivery systems.

[0021] This invention provides an extracellular vesicle drug delivery system, comprising: DSPE-PEG2000-VPE, extracellular vesicles, and the lung-targeting peptide described above; the lung-targeting peptide is connected to the maleimide group on the DSPE-PEG2000-VPE via a thioether bond, and the DSPE-PEG2000-VPE is embedded in the phospholipid bilayer of the extracellular vesicles through the hydrophobic effect of DSPE.

[0022] In one embodiment, the extracellular vesicles include extracellular vesicles derived from mesenchymal stem cells.

[0023] The extracellular vesicle drug delivery system provided by the present invention has the following advantages: (1) Strong targeting: the lung-targeting peptide can specifically recognize SP-C, thereby significantly improving the binding and uptake efficiency of vesicles in alveolar epithelial cells; (2) Good enrichment: the modified vesicles have a significantly longer retention time in lung tissue, reducing non-specific distribution in clearing organs such as the liver and spleen; (3) Wide application: the lung-targeting peptide and its modified vesicles provided by the present invention can be used as a universal platform and are suitable for the construction of delivery systems for various lung diseases.

[0024] This invention provides a method for preparing the extracellular vesicle drug delivery system described in the above technical solution, comprising the following steps: introducing a cysteine ​​residue at the C-terminus of the lung-targeting peptide and then performing pre-reduction to obtain a lung-targeting peptide with thiol activity; mixing the lung-targeting peptide with thiol activity with DSPE-PEG2000-VPE to react, so that the lung-targeting peptide is covalently linked to DSPE-PEG2000-Mal through a thiol-maleimide click chemistry reaction to obtain a lipo-peptide conjugate; co-incubating the lipo-peptide conjugate with extracellular vesicles, and utilizing the characteristic of the hydrophobic tail of DSPE to spontaneously insert into the phospholipid bilayer of EVs, anchoring the targeting peptide to the surface of EVs to obtain the extracellular vesicle drug delivery system.

[0025] As one implementation method, lung-targeting peptides with C-terminal cysteine ​​residues introduced are pre-reduced using tris(2-carboxyethyl)phosphonic acid hydrochloride.

[0026] In one embodiment, the molar ratio of the lung-targeting peptide with thiol activity to the DSPE-PEG2000-VPE is 1:1 to 1.5.

[0027] In one implementation, the mixing reaction time is 1.5 to 2.5 h; the co-incubation time is 0.8 to 1.5 h.

[0028] As one implementation method, the lung-targeting peptide with thiol activity is mixed and reacted with the DSPE-PEG2000-VPE and then dialyzed through a dialysis membrane with a molecular weight cutoff of 3.5 kDa. The macromolecular fraction is collected as a purified lipopeptide conjugate.

[0029] Based on the above advantages, this invention provides the application of the extracellular vesicle drug delivery system described in the above technical solution in the preparation of a drug delivery system for treating lung diseases. The extracellular vesicle drug delivery system provided by this invention, while leveraging the low toxicity and low immunogenicity of extracellular vesicles, can precisely target drugs to the lungs, providing more alternative approaches for the treatment of lung diseases.

[0030] To further illustrate the present invention, the following detailed description, in conjunction with embodiments and accompanying drawings, describes a lung-targeting peptide provided by the present invention and its application in the preparation of extracellular vesicle drug delivery systems, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0031] Example 1: Lung-Targeting Peptide Screening Based on Phage Display Technology S1. Target Protein Immobilization: The target protein used in this invention for screening SP-C targeting peptides is commercially prepared recombinant human SP-C (SFTPC) protein, derived from a transient expression system of HEK293 mammalian cells, and purified using Anti-HA affinity chromatography. SDS-PAGE and Western Blot validation confirmed that the obtained protein has the expected molecular weight and good purity, meeting the requirements for use as a solid-phase antigen in phage display screening. All SP-C proteins used in the screening experiments of this invention were derived from the same batch of preparations mentioned above. SP-C (0.05 mg / mL, 150 μL per well) was incubated overnight at 4 ℃, and blocked with 5% BSA for 2 h the following day.

[0032] S2. Phage panning: The M13 phage display cyclic heptapeptide library (Ph.D.-C7C library) and linear dodecapeptide library (Ph.D.-12 library), purchased from New England Biolabs, were used. The amplified phage libraries were added to the coated wells and incubated at room temperature for 1 h. The cells were repeatedly washed with TBST, and the Tween-20 concentration was gradually increased in each round of panning to enhance the screening intensity.

[0033] S3, Elution and Amplification: The bound phages were eluted with 0.2 M Glycine-HCl (pH 2.2) and immediately neutralized with Tris-HCl; the eluted phages were then used to infect ER2738 E. coli for amplification and proceed to the next round of screening. After three rounds of screening, specifically bound phages were significantly enriched.

[0034] S4. Positive clone identification and sequence analysis: The phage supernatant and coated SFTPC protein of positive clones were detected by ELISA. Partial clone OD... 450The values ​​were significantly elevated, showing a marked difference compared to the control phage (original phage library), indicating that these clones have a strong binding ability to SP-C. One positive clone from the Ph.D.-C7C library and eight positive clones from the Ph.D.-12 library were selected for single-stranded DNA extraction and sequencing. Two clones showed obvious double peaks in the insert region, indicating template heterogeneity or incomplete clone purification, making it impossible to obtain a single, reliable insert sequence; therefore, they were discarded. The remaining seven clones were successfully sequenced. The successfully sequenced insert fragments were screened according to library construction requirements, and five insert sequences met the library structure (correct length, no stop codon, and normal translation). Further sequence alignment revealed that two clones had completely identical coding sequences, belonging to the same enriched clone. Ultimately, four polypeptide sequences were obtained, as follows: P1 (SEQ ID NO.1): WVSRPFQYQSLH; P2 (SEQ ID NO.2): VPEYWNAKRHYV; P3 (SEQ ID NO.3): HDASFIGHLPKH; P4 (SEQ ID NO.4): GVYRMTAASDHF.

[0035] Phylogenetic and hydrophilic analyses were performed on candidate peptide sequences to comprehensively evaluate their sequence enrichment characteristics, structural similarity, and physicochemical properties. Phylogenetic analysis was used to identify dominant sequences with high enrichment levels and stable structural features; hydrophilic analysis was used to screen peptides with good solubility and suitability for subsequent conjugation and biological applications. Ultimately, the target peptide P2 (SEQ ID NO.2) with the best binding potential and application feasibility was determined for subsequent affinity testing.

[0036] S5. Affinity determination and interaction analysis: Affinity constant (KD) determination: The affinity constant was determined and data fitted using biomembrane interferometry (BLI) on the Octet RED96 system. The dissociation constant KD between the target peptide P2 and SP-C protein was calculated to be 10.6 nM (Figure 1).

[0037] The three-dimensional structure of the peptide was predicted using AlphaFold2 software. The three-dimensional structure of the SP-C protein was obtained from the PDB database or through homology modeling. Molecular docking was performed using AutoDock Vina 1.5.6 software, and the docking results were visualized and analyzed using PyMOL software. The results showed that the targeting peptide P2 can embed in a binding pocket on the surface of the SP-C protein, and its Tyr4, Trp5, Arg8, His9 residues form hydrogen bonds and hydrophobic interactions with SP-C residues such as Gln107, Gln108, Leu109, Ile126, Ser130, Phe180, Lys140, Cys189, Gly190, Tyr196, and Ile197 (Figure 2).

[0038] Example 2 Preparation of DSPE-PEG2000-peptide-extracellular vesicles (VPE-EVs) S1. Preparation of DSPE-PEG2000-VPE: VPEYWNAKRHYV with a C-terminal cysteine ​​residue introduced was dissolved in PBS (pH 7.0) (1 mg / mL), and pre-reduced with 5 mM tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP) to maintain thiol activity. It was then mixed with DSPE-PEG2000-Mal at a molar ratio of 1:1.2 and reacted gently at room temperature for 2 h in the dark to allow the thiol groups on the peptide to undergo a specific addition reaction with the maleimide groups, forming stable thioether bonds. After the reaction was complete, L-cysteine ​​was added to terminate the reaction and block the residual maleimide, preventing subsequent non-specific cross-linking. The product was dialyzed through a dialysis membrane with a molecular weight cutoff (MWCO) of 3.5 kDa to remove free peptides and small molecule impurities. The macromolecular fraction collected was the purified DSPE-PEG2000-VPE.

[0039] S2. Isolation of extracellular vesicles: Collect the culture supernatant of human umbilical cord mesenchymal stem cells, and remove cells and impurities by sequential low-speed centrifugation (2000g, 10min; 10000g, 30min). Then, perform ultracentrifugation (100,000g, 70min, 4℃), collect the precipitate and resuspend it in PBS to obtain purified extracellular vesicles (EVs).

[0040] S3. Assembly and Characterization: The above-mentioned DSPE-PEG2000-VPE (1 mg / mL) was combined with purified EVs (10... 9 (A mixture of vesicles) was incubated at 37 °C for 1 h, allowing DSPE to embed into the vesicle phospholipid bilayer, forming surface-modified VPE-EVs. Unbound conjugates were removed by ultrafiltration (100 kD, 3000 rpm, 10 min, 2-3 times). Nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and Western blotting (WB) were used to verify the vesicle morphology, particle size, and marker proteins. The results showed that VPE-EVs maintained a typical cup-shaped morphology with a particle size of approximately 130 nm (Figure 3).

[0041] Example 3: In vitro and in vivo targeting evaluation of VPE-EVs. S1: In vitro uptake: VPE-EVs were labeled with the membrane dye DID and co-incubated with the alveolar epithelial cell line A549. A blank control group (Free-DID) was set up by co-incubating A549 cells with PBS. Fluorescence microscopy observation showed that, compared with the blank control group, red fluorescence was significantly aggregated in the A549 cells of the targeting peptide group, indicating that VPE-EVs were effectively taken up by the alveolar epithelial cells (Figure 4).

[0042] S2. In vivo distribution: DIR-labeled VPE-EVs were injected into the tail vein of mice, and in vivo fluorescence imaging was performed at time points of 1 h, 12 h, 24 h, and 72 h. DIL- or DIR-labeled EVs were set as control groups. The results showed that the fluorescence signal of VPE-EVs in the lung region was significantly stronger than that of unmodified EVs, and the retention time was longer. At 72 h, VPE-EVs still had a significant signal in the lungs, while ordinary EVs had been basically cleared. Further in vitro imaging of major organs such as the heart, liver, spleen, lungs, and kidneys revealed that VPE-EVs were mainly distributed in the lungs, while unmodified EVs were more distributed in the liver and spleen. (Figures 5 and 6) S3. Co-localization of alveolar epithelium: DID-labeled VPE-EVs were delivered into the lungs of mice via tracheal nebulization, and the lung tissue was immunofluorescence stained, with E-cadherin used to label alveolar epithelial cells. PBS was delivered into the lungs of mice via tracheal nebulization as a control group (Free-DID). The results showed that the red signal (VPE-EVs) and the green signal (E-cadherin) highly overlapped, further demonstrating that VPE-EVs have a clear alveolar epithelial cell targeting ability (Figure 7).

[0043] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A lung-targeting peptide, characterized in that, The amino acid sequence is shown in SEQ ID NO.

2.

2. The application of the lung-targeting peptide according to claim 1 in the preparation of an extracellular vesicle drug delivery system.

3. An extracellular vesicle drug delivery system, characterized in that, include: DSPE-PEG2000-VPE, extracellular vesicles, and the lung-targeting peptide of claim 1; the lung-targeting peptide is linked to the maleimide group on DSPE-PEG2000-VPE via a thioether bond, and DSPE-PEG2000-VPE is embedded in the phospholipid bilayer of the extracellular vesicles through the hydrophobic effect of DSPE.

4. The extracellular vesicle drug delivery system according to claim 3, characterized in that, The extracellular vesicles include extracellular vesicles derived from mesenchymal stem cells.

5. A method for preparing the extracellular vesicle drug delivery system according to claim 3 or 4, characterized in that, Includes the following steps: After introducing a cysteine ​​residue at the C-terminus of the lung-targeting peptide, it is pre-reduced to obtain a lung-targeting peptide with thiol activity; the lung-targeting peptide with thiol activity is mixed and reacted with DSPE-PEG2000-VPE to obtain a lipopeptide conjugate; the lipopeptide conjugate is co-incubated with extracellular vesicles to obtain the extracellular vesicle drug delivery system.

6. The preparation method according to claim 5, characterized in that, The lung-targeting peptide with a C-terminus introduced into a cysteine ​​residue was pre-reduced using tris(2-carboxyethyl)phosphonic acid hydrochloride.

7. The preparation method according to claim 5, characterized in that, The molar ratio of the lung-targeting peptide with thiol activity to the DSPE-PEG2000-VPE is 1:1 to 1.

5.

8. The preparation method according to claim 5, characterized in that, The mixing reaction time is 1.5~2.5 h; the co-incubation time is 0.8~1.5 h.

9. The preparation method according to claim 5 or 8, characterized in that, The lung-targeting peptide with thiol activity was mixed and reacted with the DSPE-PEG2000-VPE and then dialyzed through a dialysis membrane with a molecular weight cutoff of 3.5 kDa. The macromolecular fraction was collected as a purified lipopeptide conjugate.

10. The use of the extracellular vesicle drug delivery system of claim 3 or 4, or the extracellular vesicle drug delivery system prepared by the preparation method of any one of claims 5 to 9, in the preparation of a drug delivery system for treating lung diseases.

Images