A lung-targeting peptide and application thereof in preparation of an extracellular vesicle drug delivery system

Abstract

The present application 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 present application is shown as SEQ ID NO.2.The lung targeting peptide with alveolar epithelial targeting property is obtained by phage display technology, and the lung targeting peptide is combined to the surface of extracellular vesicles (EVs) derived from mesenchymal stem cells, so that the prepared targeted delivery system can more accurately and more effectively act on lung epithelial cells, and then the effective repair and protection of the lung barrier are realized.The present application provides an effective targeted delivery strategy for the precise treatment of lung diseases, and has a good application prospect.

Description

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 can be 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: This 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-Mal), extracellular vesicles, and the lung-targeting peptide described in the above technical solution; the lung-targeting peptide is linked to the maleimide group on the DSPE-PEG2000-Mal via a thioether bond, and the DSPE-PEG2000-Mal 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 above, comprising 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 was mixed and reacted with DSPE-PEG2000-Mal to obtain a lipopeptide conjugate (DSPE-PEG2000-Mal-Cys-VPE). The lipopeptide conjugate was co-incubated 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-Mal 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-Mal 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 utilizes phage display technology to obtain a lung-targeting peptide with alveolar epithelial targeting properties. By binding this lung-targeting peptide to the surface of extracellular vesicles (EVs) derived from mesenchymal stem cells, a targeted delivery system is prepared that 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 1The results show the affinity assay for the target peptide VPEYWNAKRHYV and SP-C; where A represents the BLI assay result of the binding affinity between the target peptide and SPC; and B represents the corresponding binding curve. Figure 2 The results of molecular docking simulations between the target peptide VPEYWNAKRHYV and SP-C; Figure 3 Characterization results for EVs and VPE-EVs are shown below; A represents the particle size distribution and particle size determined by nanoparticle tracking analysis (NTA); B represents the positive (CD63, CD81, and TSG101) and negative (Calnexin) markers detected by Western blotting (WB); C represents the morphology detected by transmission electron microscopy (TEM), showing a typical concave cup structure (scale: 200 nm). Figure 4 Results of in vitro uptake of VPE-EVs by alveolar epithelial cells; Figure 5 These are in vivo imaging results from VPE-EVs mice; Figure 6 The images show the results of in vitro organ imaging in VPE-EV mice. Among them, A shows the fluorescence imaging results of the two groups of mouse tissues after 6 h and 72 h after administration (EV, VPE-EV); B shows the quantitative analysis results of the average fluorescence intensity of different tissues after administration; C shows the clearance rate of EV in lung tissue calculated by dividing the average lung signal by the average liver signal. Figure 7 VPE-EVs were co-localized 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-Mal, extracellular vesicles, and the lung-targeting peptide described above; the lung-targeting peptide is connected to the maleimide group on the DSPE-PEG2000-Mal via a thioether bond, and the DSPE-PEG2000-Mal 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 this 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 this invention can be used as a universal platform 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 above, comprising 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-Mal, so that the lung-targeting peptide is covalently linked to DSPE-PEG2000-Mal through a thiol-maleimide click chemistry reaction to obtain a lipopeptide conjugate. The lipopeptide conjugate was co-incubated with extracellular vesicles, and the target peptide was anchored to the surface of the EVs by utilizing the property that the hydrophobic tail of the DSPE spontaneously inserts into the phospholipid bilayer of EVs, thus obtaining 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-Mal 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] In one embodiment, the lung-targeting peptide with thiol activity is mixed and reacted with the DSPE-PEG2000-Mal, 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: Screening of lung-targeting peptides 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 in HEK293 mammalian cells, and purified using Anti-HA affinity chromatography. SDS-PAGE and Western Blot analysis 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 the above-mentioned preparation. SP-C (0.05 mg / mL, 150 μL per well) was incubated overnight at 4 ℃, followed by blocking with 5% BSA for 2 h the next 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... 450 The values ​​were significantly elevated, showing a marked difference compared to the control phage (original phage library), indicating that these clones had 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, normal translation). Further sequence alignment showed that two clones had completely identical coding sequences, belonging to the same enriched clone, ultimately yielding four polypeptide sequences, 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 Measurement and Interaction Analysis: Affinity Constant (KD) Measurement: Affinity constant measurement and data fitting were performed 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) Preparation of S1, 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( ) were mixed and incubated at 37 ℃ for 1 h. DSPE, through its hydrophobic interaction, embedded itself 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 vivo and in vitro 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 established by co-incubating A549 cells with PBS. Fluorescence microscopy results showed that, compared with the blank control group, red fluorescence was significantly aggregated in the A549 cells of the targeted 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 1 h, 12 h, 24 h, and 72 h. DIL- or DIR-labeled EVs were used as control groups. Results showed that VPE-EVs exhibited significantly stronger fluorescence signals in the lung region than unmodified EVs, and their retention time was longer. At 72 h, VPE-EVs still showed significant signals in the lungs, while ordinary EVs were largely cleared. Further in vivo 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 abundant in the liver and spleen. Figure 5 and Figure 6 ) S3. Alveolar Epithelial Co-localization: DID-labeled VPE-EVs were delivered to the lungs of mice via tracheal nebulization, and the lung tissue was immunofluorescence stained with E-cadherin to label alveolar epithelial cells. A control group (Free-DID) was set up by delivering PBS to the lungs of mice via tracheal nebulization. Results showed that the red signal (VPE-EVs) and the green signal (E-cadherin) highly overlapped, further demonstrating that VPE-EVs have a clear targeting ability to alveolar epithelial cells. 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: The invention comprises bisstearoylphosphatidylethanolamine-polyethylene glycol 2000-maleimide, extracellular vesicles, and the lung-targeting peptide of claim 1; wherein the lung-targeting peptide is connected to the maleimide group on the bisstearoylphosphatidylethanolamine-polyethylene glycol 2000-maleimide via a thioether bond, and the bisstearoylphosphatidylethanolamine-polyethylene glycol 2000-maleimide is embedded in the phospholipid bilayer of the extracellular vesicles through the hydrophobic interaction 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 was mixed and reacted with the bisstearoylphosphatidylethanolamine-polyethylene glycol 2000-maleimide to obtain a lipo-peptide conjugate. The lipopeptide conjugate was 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 bisstearoylphosphatidylethanolamine-polyethylene glycol 2000-maleimide 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 bisstearoylphosphatidylethanolamine-polyethylene glycol 2000-maleimide, 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-peptide 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.

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