An outer membrane vesicle surface modification system with a nucleic acid support layer, a construction method and application thereof

Abstract

The application discloses an outer membrane vesicle surface modification system with a nucleic acid support layer, a construction method and application thereof. The system comprises an outer membrane vesicle carrier layer, a connecting interface layer, a nucleic acid support layer and a functional nucleic acid layer, and forms a layered structure of "outer membrane of outer membrane vesicle-connecting interface-nucleic acid support layer-functional nucleic acid molecule". The nucleic acid support layer is formed by pre-assembly of multiple single-stranded nucleic acids through base complementary pairing, is located between the outer membrane of the outer membrane vesicle and the functional nucleic acid molecule, and serves as an independent space support structure to indirectly connect and outwardly display the functional nucleic acid. The application further discloses a construction method of the system and application of the system in preparation of tumor-targeted drug delivery. By introducing the independent nucleic acid support layer between the functional nucleic acid and the surface of the outer membrane vesicle, the programmable loading and density regulation of the functional nucleic acid are realized, the cell binding capacity, uptake efficiency, serum stability and anti-tumor efficacy output of the functional nucleic acid are significantly enhanced, and the application has platform application potential.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine, nucleic acid drug delivery and bacterial outer membrane vesicle engineering modification technology, specifically to an outer membrane vesicle surface modification system with a nucleic acid support layer, its construction method and application. Background Technology

[0002] Outer membrane vesicles (OMVs) are nanoscale membrane vesicles naturally secreted by Gram-negative bacteria during their growth, typically ranging from 20 to 300 nm in diameter. OMVs possess a natural lipid bilayer structure, excellent cellular uptake capacity, and intrinsic immunostimulatory properties, and have been studied and used as drug delivery carriers in recent years, showing particular potential in tumor immunotherapy and targeted drug delivery.

[0003] In existing technologies, nucleic acid modification of the OMV surface mainly includes methods such as direct insertion of single-stranded nucleic acids into the lipid membrane, lipid tail anchoring, or chemical coupling (e.g., maleimide-thiol, NHS ester-amino coupling), which directly immobilize functional nucleic acids with targeting or therapeutic functions onto the outer membrane surface of the OMV. However, these monolayer direct connection methods still have room for improvement in the following aspects: the distance between the functional nucleic acid and the OMV membrane surface is relatively short, and its spatial conformation and binding activity may be affected by the steric hindrance of membrane surface components; the arrangement of functional nucleic acids on the OMV surface is relatively random, and the ability to achieve high-density, programmable loading needs to be further improved; in addition, existing modification methods lack an independent spatial support structure between the OMV outer membrane and the functional nucleic acid, limiting the ability to regulate the topological display, density adjustment, and modular replacement of functional nucleic acids.

[0004] Therefore, there is an urgent need for an OMV surface nucleic acid modification system that differs from traditional monolayer direct modification. This system introduces an independent nucleic acid support layer between the OMV outer membrane and the functional nucleic acid, enabling indirect connection, spatial display, density regulation, and functional enhancement of functional nucleic acid molecules. This system has research value and application prospects. Summary of the Invention

[0005] To address the aforementioned issues, this invention provides an outer membrane vesicle surface modification system with a nucleic acid support layer, its construction method, and its applications, aiming to solve the following technical problems: Existing functional nucleic acids on the OMV surface are mostly directly fixed structures, lacking an independent support layer, resulting in insufficient controllability of the spatial arrangement of functional nucleic acids; the ability of existing OMV surface nucleic acid modification systems to achieve high-density, programmable, and modular loading of functional nucleic acids needs further improvement; when functional nucleic acids are directly attached to the OMV outer membrane surface, they may be affected by steric hindrance on the membrane surface, leading to issues that cell binding, uptake, and drug delivery need improvement; and existing OMV nucleic acid modification systems still have room for improvement in balancing structural stability, loading adjustability, and functional nucleic acid delivery efficiency.

[0006] The technical solution of this invention is: To achieve the above objectives, the present invention provides an outer membrane vesicle surface modification system with a nucleic acid support layer, the system comprising: 1. an OMV carrier layer; 2. a connection interface layer; 3. a nucleic acid support layer; and 4. a functional nucleic acid layer. Its structure is as follows: OMV outer membrane — junction interface — nucleic acid support layer — functional nucleic acid molecules The nucleic acid support layer is located between the OMV outer membrane and the functional nucleic acid molecules. As an independent spatial support structure, it allows the functional nucleic acids to be indirectly connected and displayed to the outside through the nucleic acid support layer.

[0007] 2.1 OMV Carrier Layer The OMV is derived from the outer membrane vesicles of Gram-negative bacteria (such as Escherichia coli), retaining the lipid bilayer, membrane proteins, and membrane-related components. The OMV serves as a basic carrier, supporting the nucleic acid support layer and acting as a delivery platform for functional nucleic acid molecules.

[0008] In one embodiment of the present invention, the extracted OMV was detected by dynamic light scattering (DLS), and its main particle size was approximately 206 nm. After modification with a nucleic acid support layer and functional nucleic acid molecules, an OMV@Y@Aptamer-MMAE system was formed, with a main particle size of approximately 302 nm. The increase in particle size from 206 nm to 302 nm, and the intact vesicle morphology shown by transmission electron microscopy (TEM), indicate that a new outer layer structure was formed on the surface of the OMV.

[0009] 2.2 Connection Interface Layer The interface layer is used to fix the nucleic acid support layer to the surface of the OMV outer membrane. The connection methods include hydrophobic lipid anchoring, bioorthogonal click chemistry, maleimide-thiol coupling, NHS ester-amino coupling, or biotin-avidin linkage.

[0010] 2.3 Nucleic Acid Support Layer The nucleic acid support layer is the core structure of this invention. The nucleic acid support layer is formed by the pre-assembly of multiple single-stranded nucleic acids through complementary base pairing, and has already formed a stable spatial configuration before being connected to the OMV.

[0011] In one embodiment, the nucleic acid support layer is a Y-shaped DNA framework structure, comprising: (1) a central hybridization region; (2) an anchoring arm connected to the OMV; (3) a functional arm connected to a functional nucleic acid; and (4) optional fluorescent labeling, reactive groups, or drug-linking groups.

[0012] 2.4 Functional Nucleic Acid Layer Functional nucleic acid molecules are attached to the outer side of the nucleic acid support layer to achieve targeted recognition, drug delivery, gene regulation, or therapeutic functions. These functional nucleic acids include nucleic acid aptamers, aptamer-drug conjugates (ApDCs), siRNA, antisense oligonucleotides, miRNA mimics or inhibitors, and CpG immunomodulatory nucleic acids.

[0013] Those skilled in the art will understand that, since the nucleic acid support layer is linked to the functional nucleic acid through complementary base pairing, any nucleic acid molecule with a known sequence can be loaded onto the support layer by designing complementary sequences. Therefore, the technical solution of the present invention is universal and can be extended to various types of functional nucleic acids.

[0014] In embodiments of the present invention, the functional nucleic acid is Aptamer or Aptamer-MMAE.

[0015] This invention provides a method for constructing an outer membrane vesicle functional nucleic acid delivery system with a nucleic acid support layer, comprising the following steps: Step 1: Preparation of OMV Gram-negative bacteria were cultured, and the culture supernatant was collected. OMV was obtained by centrifugation, filtration, and ultracentrifugation. The obtained OMV was characterized by TEM and DLS.

[0016] Step 2: Pre-assembling the nucleic acid support layer Multiple single-stranded nucleic acids were mixed in a specific ratio and annealed in a suitable buffer solution to form a Y-shaped nucleic acid support layer through base pairing. The assembly efficiency of the Y-shaped structure and functional nucleic acids was verified by nucleic acid gel electrophoresis.

[0017] Step 3: Attach the nucleic acid support layer to the OMV surface The pre-assembled Y-shaped nucleic acid support layer was incubated with OMV under specific conditions, allowing the nucleic acid support layer to be fixed to the outer membrane surface of the OMV via anchoring arms or linker groups. The optimal linker concentration of the nucleic acid support layer was screened using flow cytometry.

[0018] Step 4: Loading functional nucleic acid molecules Functional nucleic acid molecules were incubated with an OMV (Omnivirus Vaccine) layer already connected to a nucleic acid support layer, allowing the functional nucleic acids to bind to the outer side of the support layer through complementary pairing. The optimal loading concentration of the functional nucleic acids was screened using flow cytometry. These results demonstrate that the system of this invention can achieve adjustable loading density of functional nucleic acids on the OMV surface by regulating the concentration of the nucleic acid support layer and the functional nucleic acid.

[0019] The beneficial effects of this invention are: 1. Formation of an independent nucleic acid support layer: A pre-assembled nucleic acid support layer is introduced between the outer membrane of the OMV and the functional nucleic acid, so that the functional nucleic acid is indirectly displayed on the outside of the OMV, reducing the influence of steric hindrance on the membrane surface.

[0020] 2. Achieve a layered connection structure: Form a layered structure of "OMV outer membrane - connection interface - nucleic acid support layer - functional nucleic acid", which is different from the existing single-layer structure of direct fixation of single-stranded nucleic acid.

[0021] 3. Programmable loading and density control: By adjusting the concentration of the nucleic acid support layer and functional nucleic acid, the loading density of functional nucleic acid on the OMV surface can be controlled (e.g., 250 nM for Y-frames, 500 nM for Aptamer), with a parameterized control window.

[0022] 4. Enhanced cell binding and uptake: Compared with OMV@Aptamer or Y@Aptamer, OMV@Y@Aptamer showed stronger cell surface binding and intracellular uptake in a variety of tumor cells (KPC1199, BxPC-3, Capan-1).

[0023] 5. Enhanced drug delivery: CCK8 experiments showed that OMV@Y@Aptamer-MMAE enhanced the in vitro killing activity against tumor cells compared to OMV@Aptamer-MMAE and Y@Aptamer-MMAE, proving that this delivery system can improve the delivery efficiency of functional nucleic acid drugs.

[0024] 6. Improved nucleic acid stability: Serum stability experiments showed that after incubation in 10% FBS for 48 hours, the fluorescence retention rate of the system of the present invention was still close to 60%, indicating that the nucleic acid support layer and OMV carrier can improve the stability of functional nucleic acids. Attached Figure Description

[0025] Figure 1 This is a schematic diagram of the overall structure of the OMV outer membrane, connection interface, nucleic acid support layer, and functional nucleic acid of the present invention; Figure 2 This is a schematic diagram of the nucleic acid backbone of the present invention; Figure 3 This is a schematic diagram of the nucleic acid molecule of the present invention; Figure 4These are OMV@Y@Aptamer-MMAE and OMV projection electron microscope images of the present invention; Figure 5 This is a DLS curve diagram of the present invention; Figure 6 This is a nucleic acid gel electrophoresis image of the present invention; Figure 7 This is the concentration screening chart of the present invention; Figure 8 This is a confocal co-location diagram of the OMV@Y@Aptamer of the present invention; Figure 9 This is a graph showing the screening results of particle size, potential, and polydispersity index of different OMV@Y@Aptamer formulations of this invention; Figure 10 These are confocal images of OMV@Y@Aptamer combined with KPC1199 cells, and flow cytometry quantitative results of OMV@Y@Aptamer combined with KPC1199 cells; Figure 11 These are confocal images of three samples; Figure 12 These are the results of toxicity tests on three samples; Figure 13 This is the result of the serum stability experiment of this invention.

[0026] Figure label: 1-OMV outer membrane, 2-first reactive group, 3-nucleic acid backbone, 4-functional nucleic acid molecule, 5-T-type structure, 6-X-type four-arm structure, 7-Y-type three-pronged structure, 8-DNA nanoframework structure, 9-siRNA, 10-antisense oligonucleotide, 11-ApDC molecule. Detailed Implementation

[0027] The embodiments of the present invention will be further described below with reference to the accompanying drawings.

[0028] Example 1: Preparation and Characterization of OMV Gram-negative bacteria (such as Escherichia coli) were cultured to the stationary phase. The culture supernatant was collected and centrifuged at low speed (10,000 g, 30 min) to remove bacterial cells. The supernatant was filtered through a 0.22 μm filter membrane and then ultracentrifuged (100,000 g, 1.5 h) to precipitate OMV. The precipitate was resuspended in PBS, and the protein concentration was determined by the BCA method.

[0029] Experimental results: Transmission electron microscopy revealed typical vesicle morphology, such as... Figure 4 a is a transmission electron microscope image of OMV@Y@Aptamer-MMAE. Figure 4 b is a transmission electron microscope image of OMV; dynamic light scattering analysis shows that the average particle size of OMV is 206 nm. Figure 5 a), the average particle size of OMV@Y@Aptamer-MMAE is 302 nm ( Figure 5 b).

[0030] Example 2: Assembly verification of Y-shaped nucleic acid support layer and Aptamer In this embodiment, the nucleic acid support layer adopts a Y-shaped triangular structure (7) (as shown in the figure). Figure 2 As shown), the functional nucleic acid uses Aptamer (nucleic acid aptamer).

[0031] Three DNA single strands were designed and synthesized. In this embodiment, the Y-shaped nucleic acid framework was named comp3-Y-C6-PolyT-Chol, and the sequence of one representative single strand is as follows (taking one anchoring arm strand as an example, the other two strands are designed to complement it to form a Y-shaped trifurcation structure): 5-AAAACCCAGGTTCTCTTTT-(iSymmetric)-TTTTT-(iSp6)-TTTTTTTTTTTTTTTTTTTTTT-Chol-3' Among them: AAAACCCAGGTTCTCTTTT is the functional arm sequence of the Y-type nucleic acid framework (complementary to Aptamer); (iSymmetric) is the symmetric spacer arm, used to increase spatial flexibility; TTTTT is the flexible linker sequence; (iSp6) is the six-carbon spacer arm; TTTTTTTTTTTTTTTTTTTTTTT is the PolyT flexible linker arm; Chol is the cholesterol modification group located at the 3' end, used to insert into the OMV lipid bilayer through hydrophobic interaction, so as to realize the anchoring of the Y-type nucleic acid framework on the OMV surface.

[0032] The design principles of the three single strands are as follows: the above sequence is an anchoring arm of the Y-shaped framework; the other two strands (functional arm and central strand) are designed according to the principle of complementary base pairing, so that the complementary regions shared by the three strands form a stable Y-shaped tripartite central hybridization region, and each arm end retains a specific sequence for connection with OMV or hybridization with functional nucleic acids; the Y-shaped nucleic acid framework (Y) is formed through complementary base pairing. Aptamer, the Y framework, and the complex of the two after annealing (Y@Aptamer) are subjected to non-denaturing nucleic acid gel electrophoresis.

[0033] Experimental results: such as Figure 6 As shown in figure a, the Y@Aptamer complex exhibits a different migration location than Aptamer alone or the Y framework alone. Further fluorescence gel electrophoresis using FITC-labeled Y framework and Cy5-labeled Aptamer showed that both co-localized at the Y@Aptamer complex band. Figure 6 (b) This proves that the Y-shaped frame was successfully assembled with Aptamer.

[0034] Example 3: Optimization of Y-shaped frame and Aptamer loading concentration In this embodiment, the Y-framework is attached to the surface of the OMV outer membrane (1) via a first reactive group (2) (such as cholesterol modification). In this embodiment, the Y-framework is attached to the OMV surface via cholesterol anchoring. Specifically, a cholesterol group is modified at the 3' end of one of the DNA strands constituting the Y-framework. This cholesterol group inserts into the lipid bilayer of the OMV outer membrane through hydrophobic interactions, thereby anchoring the pre-assembled Y-framework to the OMV surface.

[0035] Annealing assembly of the Y@Aptamer complex and its connection with OMV: (1) Mix the Y-type nucleic acid framework with Aptamer at a molar ratio of 1:2, wherein the final concentration of the Y-type nucleic acid framework is 250 nM and the final concentration of Aptamer is 500 nM. The mixing system includes 5 μL of Y-type nucleic acid framework, 10 μL of Aptamer and 5 μL of ddH2O.

[0036] (2) Heat the mixture to 95°C and hold for 10 min, then cool it down to 4°C to allow the Y-type nucleic acid framework and Aptamer to form the Y@Aptamer complex through complementary sequences.

[0037] (3) The Y@Aptamer formed by annealing was added to 30 μL of OMV solution (protein concentration 200 μg / mL) and mixed well. It was then incubated at 37℃ and 220 rpm for 4 h. During the incubation, the cholesterol groups on the Y-type nucleic acid framework were inserted into the lipid bilayer of the OMV outer membrane, which anchored the Y@Aptamer to the surface of the OMV as a whole, forming OMV@Y@Aptamer.

[0038] This method does not rely on OMV genetic modification or damage the main OMV membrane structure. It can form a layered connection structure between the OMV outer membrane and the functional Aptamer, consisting of "OMV outer membrane—cholesterol anchoring interface—Y-shaped nucleic acid support layer—Aptamer". The overall structure is as follows: Figure 1 As shown. With a fixed OMV concentration, Y-frames of different concentrations (0-500 nM) were added for incubation, and the FITC fluorescence intensity was detected by flow cytometry.

[0039] Experimental results: such as Figure 7 As shown in Figure a, the Y-framework was modified on the OMV surface in a concentration-dependent manner, and the preferred concentration of the Y-framework was 250 nM.

[0040] Based on OMV modified with a 250 nM Y framework, different concentrations (0-1000 nM) of Cy5-labeled Aptamer were added for incubation. Flow cytometry results showed that Aptamer loading was concentration-dependent.

[0041] Experimental results: such as Figure 7 As shown in b, the optimal concentration of Aptamer was found to be 500 nM.

[0042] Besides cholesterol insertion, Y-shaped nucleic acid frameworks can also be attached to the OMV surface via covalent chemical reactions. For example, reactive groups such as amino, carboxyl, thiol, azide, or alkynyl groups can be introduced at the ends of the Y-shaped nucleic acid framework, and the Y-shaped nucleic acid framework can be fixed to the OMV surface using amino, carboxyl, or modified reactive groups through amidation, maleimide-thiol coupling, or click chemistry reactions.

[0043] Taking amidation as an example, EDC / NHS can be used to activate the carboxyl groups on the OMV surface, causing them to couple with the amino-modified Y-type nucleic acid framework to form stable amide bonds. This method can also achieve the immobilization of the Y-type nucleic acid framework on the OMV surface.

[0044] Example 4: Construction and co-localization verification of OMV@Y@Aptamer The OMV@Y@Aptamer system constructed in this embodiment fully presents... Figure 1 The layered structure shown is: OMV outer membrane (1) — first reactive group (2) — nucleic acid backbone (3) (Y-type trifurcation structure (7)) — functional nucleic acid molecule (4) (Aptamer).

[0045] The OMV obtained in Example 1 was incubated with the preferred concentration of Y@Aptamer (Y frame 250 nM, Aptamer 500 nM) selected in Example 3 for 2 hours at room temperature, and washed twice with PBS to obtain OMV@Y@Aptamer.

[0046] Experimental results: Confocal results show that Y-FITC and Aptamer-Cy5 are co-localized on the OMV surface. Figure 8 This demonstrates that the Y-shaped nucleic acid framework and Aptamer are co-located on the OMV surface, forming a complex structure with a spatial hierarchy.

[0047] Example 5: Structural characterization of OMV@Y@Aptamer The OMV@Y@Aptamer prepared in Example 4 was observed by transmission electron microscopy and dynamic light scattering detection.

[0048] Experimental results: Transmission electron microscopy showed that the modified vesicles retained their intact morphology. Figure 4a); Dynamic light scattering detection showed an average particle size of 302 nm ( Figure 5 b). Compared with the 206 nm of unmodified OMV, the particle size is significantly increased, indicating that the nucleic acid support layer and functional nucleic acid molecules were successfully constructed on the OMV surface.

[0049] Example 6: OMV@Y@Aptamer prescription screening Multiple OMV@Y@Aptamer formulations were prepared using different combinations of Aptamer sequences, OMV concentrations, and Y-frame concentrations, and their particle size, zeta potential, and polydispersity index (PDI) were measured.

[0050] Experimental results: as shown in Table 1 and Figure 9 As shown, the particle size range is 200-310 nm, the potential is -18 to -52 mV, and the PDI is 0.115-0.181. Two preferred formulations, OMV@Y@apt6 and OMV@Y@apt3, were obtained through screening.

[0051] Table 1. Specific data on prescription screening

[0052] Example 7: Evaluation of the binding ability of OMV@Y@Aptamer to tumor cells In this embodiment, the OMV@Aptamer group was set as a control. In this group, Aptamer was directly attached to the surface of the OMV outer membrane (1) through the first reactive group (2) (such as cholesterol modification) (without nucleic acid backbone (3)).

[0053] OMV@Aptamer-MMAE, Y@Aptamer-MMAE, and OMV@Y@Aptamer-MMAE (all Aptamer labeled with Cy5) were prepared and co-incubated with KPC1199 cells. The cell binding ability was detected by flow cytometry and confocal microscopy.

[0054] Experimental results: such as Figure 10 a (confocal) and Figure 10 As shown in flow cytometry (b), compared to the Y@Aptamer group and the OMV@Aptamer group, the OMV@Y@Aptamer group showed a better binding signal with cells, indicating that the system of the present invention can enhance the binding ability of functional nucleic acids to tumor cells.

[0055] Example 8: Evaluation of OMV@Y@Aptamer cell uptake capacity Aptamer, Y@Aptamer, and OMV@Y@Aptamer (Cy5 labeled) were prepared and co-incubated with BxPC-3, KPC1199, and Capan-1 cells, respectively. Cell uptake was observed using a confocal microscope.

[0056] Experimental results: In BxPC-3 cells ( Figure 11 a) Compared to Aptamer alone, both Y@Aptamer and OMV@Y@Aptamer showed better cellular uptake, with the OMV@Y@Aptamer group exhibiting the strongest uptake signal.

[0057] In KPC1199 cells ( Figure 11 (b) Similarly, the OMV@Y@Aptamer group was observed to have the strongest intracellular fluorescence signal.

[0058] In Capan-1 cells ( Figure 11 c), the results were consistent, with the OMV@Y@Aptamer group showing the best cell uptake effect.

[0059] The above results indicate that the Y-shaped nucleic acid support layer can enhance the cellular uptake of Aptamer, and the effect is further enhanced when the OMV carrier is used in combination with the nucleic acid support layer.

[0060] Example 9: Efficacy evaluation of OMV@Y@Aptamer-MMAE Y@Aptamer-MMAE, OMV@Aptamer-MMAE, and OMV@Y@Aptamer-MMAE were prepared and co-incubated with BxPC-3, Capan-1, and KPC1199 cells for 72 hours. Cell viability was detected by CCK8 assay.

[0061] Experimental results: In BxPC-3 cells ( Figure 12 a) OMV@Aptamer-MMAE showed stronger cytotoxicity than Y@Aptamer-MMAE; OMV@Y@Aptamer-MMAE showed further enhanced cytotoxicity.

[0062] In Capan-1 cells ( Figure 12 (b) The results were consistent, with OMV@Y@Aptamer-MMAE showing the highest cell killing efficiency.

[0063] In KPC1199 cells ( Figure 12 c), OMV@Y@Aptamer-MMAE also showed the strongest cytotoxicity.

[0064] The above results indicate that OMV@Y@Aptamer-MMAE can further enhance cytotoxicity compared to OMV@Aptamer-MMAE and Y@Aptamer-MMAE, suggesting that the system of the present invention can enhance the delivery and efficacy of Aptamer-MMAE.

[0065] Example 10: OMV@Y@Aptamer-MMAE serum stability assay OMV@Y@Aptamer-MMAE (Aptamer-Cy5) was added to a culture medium containing 10% fetal bovine serum, and the fluorescence intensity was measured at 0, 2, 4, 8, 24, and 48 hours, respectively, and the relative fluorescence retention rate was calculated (with 0 hours as 100%).

[0066] Experimental results: such as Figure 13 As shown, after 48 hours in 10% FBS, the fluorescence retention rate was still close to 60%. This result indicates that the Y-shaped nucleic acid support layer and OMV can improve nucleic acid stability.

[0067] The embodiments described above are merely illustrative of specific implementations of the present invention, and while the descriptions are detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A surface modification system for outer membrane vesicles with a nucleic acid support layer, characterized in that, The system includes: Outer membrane vesicle carrier layer; Connect the interface layer; Nucleic acid support layer; Functional nucleic acid layer; The nucleic acid support layer is located between the outer membrane of the outer membrane vesicle and the functional nucleic acid molecule. As an independent spatial support structure, the functional nucleic acid molecule is indirectly connected through the nucleic acid support layer and exposed to the outside, forming a layered structure of "outer membrane vesicle outer membrane - connection interface - nucleic acid support layer - functional nucleic acid molecule".

2. The system according to claim 1, characterized in that, The nucleic acid support layer is formed by pre-assembling multiple single-stranded nucleic acids through complementary base pairing. The nucleic acid support layer is selected from at least one of Y-type DNA framework, T-type nucleic acid structure, X-type four-arm nucleic acid structure, tetrahedral DNA structure, DNA nanoframework or DNA origami structure.

3. The system according to claim 2, characterized in that, The nucleic acid support layer is a Y-shaped DNA framework, which includes a central hybridization region, anchoring arms connected to outer membrane vesicles, and functional arms connected to functional nucleic acids.

4. The system according to claim 1, characterized in that, The connection interface layer fixes the nucleic acid support layer to the outer membrane surface of the outer membrane vesicles through at least one of the following methods: hydrophobic lipid anchoring, bioorthogonal click chemistry, maleimide-thiol coupling, NHS ester-amino coupling, or biotin-avidin linkage.

5. The system according to claim 1, characterized in that, The functional nucleic acid is selected from at least one of nucleic acid aptamers, aptamer-drug conjugates, siRNA, antisense oligonucleotides, miRNA mimics or inhibitors, and CpG.

6. A method for constructing an outer membrane vesicle surface modification system with a nucleic acid support layer according to any one of claims 1-5, characterized in that, Includes the following steps: (1) Preparation of outer membrane vesicles; (2) Multiple single-stranded nucleic acids are pre-assembled into a nucleic acid support layer through complementary base pairing; (3) Fix the pre-assembled nucleic acid support layer in step (2) onto the surface of the outer membrane vesicles obtained in step (1) through the connection interface; (4) Load functional nucleic acid molecules onto the outside of the nucleic acid support layer described in step (3).

7. The construction method according to claim 6, characterized in that, The nucleic acid support layer described in step (2) is formed by annealing multiple single-stranded nucleic acids in a magnesium-containing buffer system; the concentration of the nucleic acid support layer incubated with the outer membrane vesicles in step (3) is 100-500 nM; the loading concentration of the functional nucleic acid in step (4) is 100-1000 nM.

8. Use of the outer membrane vesicle surface modification system with a nucleic acid support layer prepared by the system according to any one of claims 1-5 or the construction method according to any one of claims 6-7 in the preparation of drugs for tumor-targeted delivery.

9. The use according to claim 8, characterized in that, The system enhances the cell binding, cell uptake, serum stability, and antitumor drug delivery of functional nucleic acids through a nucleic acid support layer.

10. A pharmaceutical composition for targeted tumor therapy, characterized in that, The system comprises the system according to any one of claims 1-5 or the system prepared by the method according to any one of claims 6-7, and a pharmaceutically acceptable carrier or excipient.

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