General preparation method of DNA framework vesicle composite structure containing cell membrane components and its application in constructing virus-like nanocarriers

Abstract

The present invention discloses a general preparation method of a DNA framework vesicle composite structure containing cell membrane components and its application in constructing virus-mimicking nanocarriers. The present invention utilizes DNA frameworks to reconstruct liposomes containing cell membrane fragments inside the liposomes, making full use of the excellent performance of DNA frameworks and cell membrane fragments, and designs a composite nanocarrier that is highly biomimetic in structure and highly flexible in function; and the Spike and ORF3a proteins of the new coronavirus are modified in the DNA framework-liposome composite nanocarrier, imitating the cell infection mechanism of the new coronavirus, and utilizing the synergistic effect of the two proteins to enhance the endocytosis and lysosomal escape ability of the nanocarrier, significantly improving the intracellular delivery efficiency of siRNA, and having broad application prospects. The present invention can obtain cell membrane fragments containing any functional membrane protein, endow the DNA framework-liposome composite nanocarrier with specific functions, and has broad universality in the field of drug delivery.

Description

Technical Field

[0001] The invention relates to a general preparation method of a DNA framework vesicle composite structure containing cell membrane components and application in constructing virus-mimicking nano-carriers, belonging to the fields of biomedicine and DNA nano-technology. Background Art

[0002] Virus-mimicking nanocarriers are nanomaterials designed to improve the efficiency of drug delivery systems by mimicking the recognition, endocytosis and delivery mechanisms of viruses in host cells. Viruses have become the inspiration for nanocarrier design due to their efficient intracellular delivery and specific cell recognition capabilities. This type of nanocarrier strives to achieve efficient drug delivery by mimicking the characteristics of the virus's shell, surface proteins, nucleic acid packaging mechanism, etc., especially in the fields of gene therapy, vaccine delivery and cancer treatment.

[0003] The origin of the research on virus-like nanocarriers can be traced back to the end of the 20th century. Although early carriers have demonstrated their potential to a certain extent, they still have obvious limitations in terms of delivery efficiency and safety, such as non-specific toxicity and low delivery efficiency. With the deepening of research, the design of virus-like nanocarriers has gradually become more complex and sophisticated. For example, researchers have developed carriers that can responsively release drugs under specific conditions, or use specific enzymes in the tumor microenvironment to trigger drug release. It is also possible to design nanomaterials with highly biomimetic properties through the protein self-assembly properties of the virus shell. These new generation of virus-like carriers are not only closer to natural viruses in structure, but also more targeted and controllable in function.

[0004] However, viral nanocarriers still face many challenges. First, although viral-like vectors reduce the toxicity of traditional vectors to a certain extent, they may trigger immune responses and may even produce unpredictable side effects during metabolism in the body. Secondly, the physical properties of the vector, such as size and shape, will significantly affect its efficiency of internalization by cells. Overly large vectors may have difficulty entering cells through endocytosis, while overly small vectors may be quickly cleared in the body. The shape and size of the currently developed viral-like nanocarriers are difficult to precisely control. Low intracellular delivery efficiency is also one of the challenges faced by viral-like nanocarriers. Even if the viral-like nanocarriers successfully enter the cells, many vectors will enter the lysosomes. The viral-like vectors often lack an effective lysosomal escape mechanism, resulting in the degradation of the drug before reaching the target site, which in turn leads to low drug delivery efficiency. Therefore, it is of utmost importance to select viruses with strong lysosomal escape ability as the object of imitation. The novel coronavirus (SARS-CoV-2, referred to as the new coronavirus) has strong targeting and lysosomal escape capabilities. Its main structural proteins include Spike protein (spike), E protein (envelope), M protein (transmembrane) and N protein (nucleocapsid). The receptor binding domain (RBD) of the Spike protein is responsible for binding to the target cell receptor ACE2 and mediating the recognition of the virus. The latest studies have shown that in addition to mediating receptor binding, the Spike protein also has the function of mediating membrane fusion and lysosomal targeting. As people's understanding of the new coronavirus becomes more and more in-depth, the important roles of other new coronavirus viral proteins have gradually been revealed. The ORF3a protein encoded by the new coronavirus is a non-structural protein that can help the virus escape. On the one hand, it evades autophagy monitoring by blocking the fusion of autophagosomes and lysosomes. On the other hand, it destroys the functional integrity of lysosomes to achieve lysosomal escape of the virus.

[0005] It is very important to choose a suitable carrier for the design of virus-like nanostructures. DNA nanotechnology is an important milestone in the development of chemistry and biology in the 20th century. DNA nanotechnology was founded by Professor Nadrian Seeman in the 1980s. Through the principle of base complementary pairing, a series of complementary oligonucleotide single chains were designed and self-assembled into nanostructures of certain geometric shapes. The emergence of DNA origami in 2006 further expanded the application of DNA nanotechnology in chemistry, biology, and materials. DNA origami is a process of folding a long DNA chain as a template with hundreds of complementary staple chains into a nanostructure of a specific shape. DNA nanostructures are not only flexible and changeable in structure, but also can be used as a framework to guide the precise assembly of other materials. For example, the synthesis of liposome vesicles with controllable shape and size can be induced inside the DNA framework. This DNA framework-liposome composite nanocarrier has the advantages of low toxicity and modification diversity.

[0006] In recent years, the construction of nanocarriers using cell membrane fragments has become a hot research direction in the field of nanobiotechnology. Cell membrane fragments are considered to be ideal nanocarrier materials due to their natural biocompatibility and complex functionality. By combining these membrane fragments with nanoparticles, the external environment of biological cells can be effectively simulated, thereby achieving more precise drug delivery and immune escape. Its main advantages include: (1) High biocompatibility: Cell membrane fragments are derived from organisms and can significantly reduce immune responses and enhance the targeting ability of drugs. (2) Multifunctionality: The various proteins and receptor molecules carried on the cell membrane give nanocarriers specific biological functions, such as target recognition, endocytosis and transmembrane transport. (3) Good stability: Nanocarriers, coated with cell membranes, can effectively protect the contents from degradation by the external environment and prolong the circulation time of drugs. However, this technology also has certain limitations: (1) Complex preparation process: The separation and purification of cell membranes require strict condition control, and the combination process with nanoparticles is complex, which affects the feasibility of large-scale production. (2) Poor controllability: Due to the natural complexity of the cell membrane, it is difficult to achieve precise membrane size and functional consistency, which may lead to unstable drug delivery effects. In summary, cell membrane fragments have shown great potential as nanocarriers in the field of drug delivery, but their preparation process and functional controllability still need to be further optimized to achieve wider application.

[0007] In summary, it is of great significance to use DNA frameworks and cell membrane fragments to construct a new and universal virus-like nanocarrier and other functional nanocarriers for the delivery of various drugs. Summary of the invention

[0008] The purpose of the present invention is: in view of the shortcomings of the prior art, the present invention aims to provide a general preparation method of a DNA framework vesicle composite structure containing cell membrane components and apply it to the construction of virus-like nanocarriers, and then for drug (such as nucleic acid drugs) delivery. The present invention innovatively uses the DNA framework structure to guide internal reconstruction and form liposome vesicles containing cell membrane fragments, so that it has efficient homologous cell recognition and lysosomal escape capabilities, thereby enabling efficient drug delivery; the present invention imitates the infection mechanism of the new coronavirus, modifies the key functional proteins of the new coronavirus, and successfully develops a new drug delivery system, showing broad application potential in the direction of drug delivery.

[0009] In order to achieve the above object, the present invention provides the following technical solutions:

[0010] In a first aspect of the present invention, a general method for preparing a DNA framework vesicle composite nanocarrier containing cell membrane components is provided. The preparation method uses a DNA framework to guide the reconstruction of a liposome vesicle containing cell membrane fragments, and specifically comprises the following steps:

[0011] Step 1: Use DNA nanostructure design software to construct a soccer ball-shaped DNA framework structure;

[0012] Step 2: Use the long-chain ssDNA template p7560 in the M13 phage genome as the skeleton of the DNA framework structure, then design a complementary staple chain, the long-chain ssDNA template and the staple chain are annealed, base pairing self-assembles to obtain a football-shaped DNA framework, and then separates and purifies to obtain a purified DNA framework;

[0013] Step 3: The purified DNA framework is hybridized with the cholesterol-modified seed chain through the internal Handle chain, and mixed with phospholipids, cell membrane fragments and surfactant octyl glucoside. Under the guidance of the seed chain cholesterol, the cell membrane fragments, whose main components are lipid bilayers, fuse with the phospholipids, and then separated and purified by dialysis and isopycnic centrifugation to obtain a DNA framework vesicle composite nanocarrier containing cell membrane components; the composition of the phospholipids includes DOPC, DOPE, cholesterol, PEG2k-PE and Rho-PE. Octyl glucoside (n-Octylglucoside, OG) is a mild, non-denaturing surfactant. OG is added in this step to disperse phospholipids and cell membrane fragments, so that the phospholipids and cell membrane fragments are evenly mixed.

[0014] Furthermore, the soccer ball-shaped DNA framework structure is a truncated icosahedron structure.

[0015] Furthermore, the ratio of the phospholipids to the cell membrane fragments is 1 mM phospholipids: 0.2-0.4 mg / mL cell membrane fragments.

[0016] Furthermore, the composition of the phospholipids includes 34-38% DOPC, 48-52% DOPE, 8-12% cholesterol, 1-3% PEG2k-PE and 1-3% Rho-PE.

[0017] Furthermore, the ratio of the phospholipids to the cell membrane fragments is 1 mM phospholipids: 0.2 mg / mL cell membrane fragments.

[0018] Furthermore, the composition of the phospholipids includes 36% DOPC, 50% DOPE, 10% cholesterol, 2% PEG2k-PE and 2% Rho-PE. And / or, the concentration of the surfactant during the dialysis process is 0.6%.

[0019] Furthermore, the cell membrane fragments are cell membrane fragments modified with functional membrane proteins.

[0020] Furthermore, the functional membrane protein is firstly transfected into the tool cells via plasmid to construct cells overexpressing the functional membrane protein, and then the cell membrane fragments are extracted to obtain the cell membrane fragments modified with the functional membrane protein.

[0021] Among them, tool cells are selected according to the research object or actual application requirements.

[0022] The second aspect of the present invention provides the application of the general preparation method described in the first aspect in constructing a novel coronavirus-mimicking DNA framework-containing cell membrane fragment liposome composite nanocarrier, wherein the cell membrane fragments in step 3 are cell membrane fragments modified with novel coronavirus functional membrane proteins, and the novel coronavirus functional membrane proteins are ORF3a and / or Spike proteins.

[0023] The third aspect of the present invention provides a method for constructing a novel coronavirus-like DNA framework-containing cell membrane fragment liposome composite nanocarrier, which specifically comprises the following steps:

[0024] Transfecting an overexpression plasmid of ORF3a protein and / or Spike protein into a tool cell to construct an ORF3a protein and / or Spike protein overexpression cell, and extracting cell membrane fragments containing ORF3a protein and / or Spike protein from the overexpression cell;

[0025] The general preparation method described in the first aspect of the present invention is used to construct a novel coronavirus-mimicking DNA framework-cell membrane fragment-containing liposome composite nanocarrier; wherein the cell membrane fragments are the above-mentioned cell membrane fragments containing ORF3a protein and / or Spike protein.

[0026] Furthermore, the tool cells are selected according to the research object or actual application requirements. In some embodiments of the present invention, in order to study the regulation of cell function in colorectal cancer and cervical cancer, the tool cells selected are human colon cancer cell line HCT116 cells and / or human cervical cancer cell line Hela cells.

[0027] The fourth aspect of the present invention provides the use of a novel coronavirus-like DNA framework-cell membrane fragment-containing liposome composite nanocarrier constructed by the construction method described in the third aspect in delivering drugs in vitro or preparing a drug delivery system.

[0028] The fifth aspect of the present invention provides a method for preparing a drug delivery system, which carries drugs on the basis of the novel coronavirus-like DNA framework-containing cell membrane fragment liposome composite nanocarrier constructed by the construction method described in the third aspect of the present invention, that is, the preparation method includes: incubating and combining the cholesterol-modified drug with the novel coronavirus-like DNA framework-containing cell membrane fragment liposome composite nanocarrier, so that the drug is combined with the liposome inside the novel coronavirus-like DNA framework-containing cell membrane fragment liposome composite nanocarrier, thereby providing stable protection and preventing it from being rapidly degraded by nucleases in the cell.

[0029] Furthermore, the drugs include nucleic acid drugs and polypeptide and protein drugs.

[0030] Furthermore, the nucleic acid drugs include siRNA, mRNA, lncRNA, RNA aptamers, ASO (antisense oligonucleotide) and gRNA of the CRISPR / Cas system.

[0031] In some embodiments of the present invention, the carried drug is a nucleic acid drug siRNA. In order to carry the siRNA onto the liposome of the composite nanocarrier, the siRNA is conjugated with a cholesterol molecule through an organic chemical synthesis method to achieve connection, and then the conjugated cholesterol-siRNA is fused with a phospholipid to achieve uniform dispersion and fusion of the siRNA and the liposome inside the composite structure.

[0032] Among them, the method of connecting siRNA with cholesterol molecules through conjugation reaction is a method known in the art.

[0033] In some embodiments of the present invention, the siRNA is a siRNA targeting the anti-apoptotic gene Bcl2 (Sense: Cy5-GUGAAGUCAACAUGCCUGC, SEQ ID NO: 92; Anti-sense: Chol-GCAGGCAUGUUGACUUCAC, SEQ ID NO: 93).

[0034] The sixth aspect of the present invention provides use of the drug delivery system prepared by the preparation method described in the fifth aspect of the present invention in preparing drugs.

[0035] Furthermore, the drugs include anti-tumor drugs and vaccines.

[0036] The present invention uses DNA nanostructure design software to construct a football-shaped DNA framework structure, and realizes the self-assembly of the DNA framework by temperature annealing, purifies and evaluates its shape and integrity. Then, cell membrane fragments are obtained by cell membrane extraction technology. Then, the seed chain modified with cholesterol is mixed with phospholipids and cell membrane fragments, and a DNA framework-liposome composite nanostructure containing cell membrane fragments is constructed by dialysis and isopycnic centrifugation. On this basis, the ORF3a protein and Spike protein of the new coronavirus are modified into the composite nanostructure by transfection technology to realize the construction of the new coronavirus nanostructure. Further optimization steps include regulating the ratio of phospholipids to membrane fragments to maximize the carrying of membrane fragments and siRNA, and ensuring the best liposome reconstruction conditions containing cell membrane fragments. The experimental results show that the nanostructure containing the DNA framework is significantly better than the structure without the DNA framework in terms of siRNA delivery efficiency, reflecting that the target gene silencing effect is better, and the experimental results are more stable. In addition, the nanostructure modified with cell membrane fragments exhibits a significant homing effect, which can effectively deliver siRNA to cells with the same cell membrane source, and the delivery efficiency is higher. By destroying the integrity of the lysosomal membrane, ORF3a protein significantly improves the delivery efficiency of siRNA, and the synergistic effect of Spike protein and ORF3a protein further enhances the delivery effect of siRNA. Experiments with different delivery times show that under short-term delivery conditions, the DNA framework-liposome composite nanostructure exhibits the best delivery efficiency. As time goes on, the delivery efficiency of the novel coronavirus-like nanostructure modified with ORF3a protein is significantly improved. The present invention has important application prospects in drug delivery, especially in the field of gene therapy.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] (1) Controllability and stability of multifunctional composite nanostructures: The present invention combines three different types of nanocarrier materials: DNA framework, liposomes and cell membrane fragments. By complementing each other's advantages, a composite nanocarrier with high biomimetic structure and high functional flexibility is designed. The football-shaped DNA framework structure constructed by DNA origami provides a stable framework for the reconstruction of the internal liposome vesicles. At the same time, the optimized exploration of the liposome phospholipid components and the full fusion of cell membrane fragments ensures the successful assembly of the three nanomaterials of DNA framework, liposome and cell membrane fragments, and achieves the consistency of the size and shape of the nanocarrier, thereby improving the stability and repeatability of the delivery system.

[0039] (2) Targeting and safety: The composite nanocarrier of the present invention extracts cell membrane fragments through cell membrane extraction technology and guides the reconstruction of liposome vesicles containing cell membrane fragments within the DNA framework. The cell membrane fragments are successfully loaded into the composite nanocarrier through the fusion of the optimal ratio of phospholipid and membrane fragment concentrations. By utilizing the "homing effect" between homologous cell membranes and the repulsion between heterologous cell membranes, high targeting to specific cell types and efficient drug delivery are achieved. At the same time, the DNA nanocarrier and cell membrane fragments have extremely low toxicity and good biocompatibility, which further enhances its safety in clinical applications.

[0040] (3) Implementation of virus-like infection mechanism: The present invention modifies the Spike protein and ORF3a protein of the new coronavirus onto a DNA framework-liposome composite nanocarrier, mimics the cell infection mechanism of the new coronavirus, and utilizes the synergistic effect of the ORF3a protein and the Spike protein to enhance the lysosomal escape ability of the nanocarrier, significantly improving the intracellular delivery efficiency of siRNA, thereby achieving the regulation of cell function; providing a new strategy for the design of nanocarriers.

[0041] (4) Broad application potential and universality: The DNA framework-liposome composite nanocarrier in the present invention is not only suitable for the delivery of siRNA, but can also be extended to the delivery of other nucleic acid drugs (such as mRNA, ASO, CRISPR / Cas system) and peptide and protein drugs. It plays an important role in the development of anti-tumor drugs and vaccine delivery, and has broad application potential. At the same time, the three technologies of membrane protein overexpression technology, cell membrane fragment extraction and successful modification of cell membrane fragments are combined to obtain cell membrane fragments containing any functional membrane protein, breaking the functional limitations of cell membrane fragments and giving DNA framework-liposome composite nanocarriers specific functions. It has broad universality in the field of drug delivery. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 Schematic diagram of the construction of a virus-mimicking DNA framework-liposome composite nanocarrier containing cell membrane fragments; wherein: A is a schematic diagram of a truncated icosahedral (soccer ball-shaped) DNA framework structure; B is a schematic diagram of separating and collecting pure DNA football structures by agarose gel electrophoresis; C is an immunofluorescence localization map of ORF3a protein overexpressed in HCT116 cells; D is a schematic diagram of the principle and process of modifying cell membrane fragments containing the key functional proteins ORF3a and Spike protein of the new coronavirus and siRNA into a DNA framework-liposome composite nanostructure;

[0043] Figure 2In order to construct DNA framework-liposome composite nanocarriers containing cell membrane fragments, the optimal concentrations of phospholipid components and OG were determined by density gradient centrifugation to explore the optimal structural reconstruction conditions; among them: A was a phospholipid concentration of 1 mM, a cell membrane fragment concentration of 2 mg / mL, the phospholipid components were: 36% DOPC, 50% DOPE, 10% cholesterol, 2% PEG2k-PE and 2% Rho-PE, and the OG concentration was 0.3%; B was a phospholipid concentration of 1 mM, a cell membrane fragment concentration of 2 mg / mL, the phospholipid ratio was: 36% DOPC, 50% DOPE, 10% cholesterol, 2% PEG2k-PE and 2% Rho-PE, and the OG concentration was 0.6%; C was a phospholipid concentration of 1 mM, a cell membrane fragment concentration of 2 mg / mL, the phospholipid ratio was: 36% DOTAP, 50% DOPE, 10% cholesterol, 2% PEG2k-PE and 2% Rho-PE, OG concentration is 0.3%; D is phospholipid concentration of 1 mM, cell membrane fragment concentration is 2 mg / mL, phospholipid ratio is: 36% DOTAP, 50% DOPE, 10% cholesterol, 2% PEG2k-PE and 2% Rho-PE, OG concentration is 0.6%.

[0044] Figure 3 In the process of constructing DNA framework-liposome composite nanocarriers containing cell membrane fragments, the optimal ratio of phospholipids and cell membrane fragments was determined by density gradient centrifugation and Western Blot analysis, and the optimal structural reconstruction conditions were further explored based on the determination of phospholipid components and OG concentrations; among them: A is a phospholipid concentration of 1 mM and a cell membrane fragment concentration of 1 mg / mL; B is a phospholipid concentration of 1 mM and a cell membrane fragment concentration of 0.2 mg / mL; C is a phospholipid concentration of 0.2 mM and a cell membrane fragment concentration of 1 mg / mL; D is a phospholipid concentration of 0.2 mM and a cell membrane fragment concentration of 0.2 mg / mL.

[0045] Figure 4 The characterization results of the virus-mimicking DNA framework-liposome composite nanocarrier containing cell membrane fragments; wherein: A is the electron microscopy characterization result; B is the zeta potential measurement result; C is the nanoflow cytometry detection result; D is the Western Blot detection result; in the figure, the FL group is a liposome structure without an external DNA framework, which is divided into unmodified liposomes (FL), liposomes modified with membrane fragments (FL-CM) and liposomes modified with membrane fragments containing ORF3a protein (FL-CM3a); the SL group is a composite nanostructure with an external DNA framework, which is divided into a structure without a modified DNA framework (SL), a DNA framework structure modified with membrane fragments (SL-CM), and a DNA framework structure modified with membrane fragments containing ORF3a protein (SL-CM3a);

[0046] Figure 5 The results of the siRNA delivery efficiency test of the viral DNA framework-liposome composite nanocarrier containing cell membrane fragments; wherein: A is a flowchart of the experimental design for determining the homing effect of membrane fragments from different sources by homologous and heterologous stimulation of two cell lines using a structure modified with membrane fragments derived from HCT116 cells; B is a flowchart of the experimental design for determining the homing effect of membrane fragments from different sources by homologous stimulation of HCT116 cell lines using a structure modified with membrane fragments derived from HCT116 cells, detecting the expression level of Bcl2 protein by Western Blot after homologous stimulation of HCT116 cell lines to detect the siRNA delivery efficiency; C is a flowchart of the experimental design for determining the homing effect of membrane fragments from different sources by heterologous stimulation of Hela cell lines using a structure modified with membrane fragments derived from HCT116 cells, detecting the expression level of Bcl2 protein by Western Blot after heterologous stimulation of Hela cell lines using a structure modified with membrane fragments derived from HCT116 cells to detect the siRNA delivery efficiency; D is a flowchart of the experimental design for determining the homing effect of membrane fragments from different sources by homologous and heterologous stimulation of two cell lines using a structure modified with membrane fragments derived from Hela cells; E is a flowchart of the experimental design for determining the homing effect of membrane fragments from different sources by homologous stimulation of Hela cell lines using a structure modified with membrane fragments derived from Hela cells Blot was used to detect the expression level of Bcl2 protein to detect the delivery efficiency of siRNA; F is a structure modified by membrane fragments derived from Hela cells. After heterologous stimulation of HCT116 cell line, the expression level of Bcl2 protein was detected by Western Blot to detect the delivery efficiency of siRNA;

[0047] Figure 6 The confocal microscopy images of siRNA delivery by the virus-mimicking DNA framework-liposome composite nanocarrier containing cell membrane fragments under homologous and heterologous conditions, respectively; wherein: A is a structure modified by membrane fragments derived from HCT116 cells, and the distribution of siRNA-Cy5 was observed after homologous stimulation of HCT116 cells for 8 hours; B is a structure modified by membrane fragments derived from HCT116 cells, and the distribution of siRNA-Cy5 was observed after heterologous stimulation of Hela cells for 8 hours;

[0048] Figure 7The figure is a result diagram of the relationship between the delivery efficiency of siRNA of the virus-like DNA framework-liposome composite nanocarrier containing cell membrane fragments and the delivery time; wherein: A is the delivery efficiency test result of siRNA with different nanostructures within 1 hour of delivery; B is the delivery efficiency test result of siRNA with different nanostructures within 2 hours of delivery; C is the delivery efficiency test result of siRNA with different nanostructures within 4 hours of delivery; D is the delivery efficiency test result of siRNA with different nanostructures within 8 hours of delivery; E is the summary result of the delivery efficiency of siRNA with different nanostructures at different delivery times;

[0049] Figure 8 To test the toxicity of each structure and the commercial transfection reagent Lipo2000 on cells for CCK-8 experiments;

[0050] Fig. 9 The results are as follows: A is the immunofluorescence localization of Spike protein after overexpression of Spike protein in HCT116 cells; B is the cell membrane fragments extracted from HCT116 cells and Hela cells, and Western Blot was used to determine that the cell membrane fragments contained Spike protein; C is a schematic diagram of the structure of the holographic novel coronavirus-imitation DNA framework-liposome composite nanocarrier containing cell membrane fragments; D is the delivery efficiency test results of siRNA with different nanostructures in HCT116 cells when the delivery time is 8 hours; E is the delivery efficiency test results of siRNA with different nanostructures in Hela cells when the delivery time is 8 hours. DETAILED DESCRIPTION

[0051] In order to make the present invention more clearly understood, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0052] The present invention relates to the use of DNA frameworks to guide the reconstruction of liposome vesicles containing cell membrane fragments in the context of virus-mimicking nanocarriers, and overexpression of key functional proteins of the new coronavirus on the cell membrane fragments, simulating the mechanism of the new coronavirus infecting cells, and aims to deliver nucleic acid drugs, such as siRNA, through the virus-mimicking composite nanostructure of the present invention, for targeted silencing of the expression of specific genes, thereby regulating cell functions. The present invention mainly includes the following parts:

[0053] 1.1 DNA framework directs the reconstitution of liposome vesicles containing cell membrane fragments

[0054] The DNA framework structure adopts a truncated icosahedron design, which is based on the spherical morphology of the new coronavirus. The truncated icosahedron consists of 12 pentagons and 20 hexagons, with a total of 60 vertices and 90 edges and a diameter of about 74 nm. This geometric shape has good stability and modifiability, and can be multifunctionalized through surface modification. The DNA framework structure is realized by programmed DNA self-assembly technology. First, the long single-stranded DNA (ssDNA) from the M13 phage is folded into a three-dimensional structure of a truncated icosahedron using caDNAno and Tiamat software for modeling. Then, complementary staple chains are designed to self-assemble through base pairing. The assembled structure is preliminarily identified by agarose gel electrophoresis and purified by rate zone centrifugation. Finally, the shape and integrity of the structure are characterized by atomic force microscopy (AFM) and transmission electron microscopy (TEM) to ensure its stability and functionality in practical applications.

[0055] Inside the DNA framework, 90 Handle chains extend out from the DNA framework, and these Handle chains will become anchor points for liposome synthesis. Then, through the principle of base complementary pairing, cholesterol-modified anti-Handle chains, called Seed chains, are hybridized on the Handle chains to form hydrophobic anchor points, allowing phospholipid molecules to aggregate on these anchor points. Subsequently, the cell membrane fragments and phospholipid molecules obtained by cell membrane fragment extraction technology are mixed with the surfactant OG, and the surfactant is gradually removed by dialysis to induce the spontaneous formation of liposomes inside the DNA framework. The liposome vesicles containing cell membrane fragments formed can not only carry siRNA, but also modify viral functional proteins to achieve the design of functional bionic carriers that simulate the viral infection mechanism.

[0056] 1.2 Modification of ORF3a protein

[0057] In order to realize the lysosomal escape function of the composite nanocarrier, the present invention modifies the ORF3a protein of the new coronavirus on the surface of the liposome. ORF3a is an important non-structural protein that can help the new coronavirus escape lysosomal degradation. In order to modify the ORF3a protein to the surface of the liposome, the present invention utilizes the characteristics of the ORF3a protein being localized in the cell membrane, and obtains cell membrane fragments containing the ORF3a protein through cell membrane extraction technology (cell membrane extraction technology is a method known in the art). Because the main component of the cell membrane fragments is the lipid component, the extracted membrane fragments are mixed with the phospholipid component using the principle of similar compatibility, and the ORF3a protein can be evenly modified on the surface of the liposome, thereby enhancing the lysosomal escape ability of the nanocarrier. This modification method not only allows the ORF3a protein to be evenly distributed on the surface of the liposome, but also can further enhance the stability and functionality of the composite nanocarrier in the intracellular environment by increasing the fusion of the liposome with the cell membrane.

[0058] 1.3 Modification of Spike Protein

[0059] In order to further mimic the cell infection mechanism of the new coronavirus, the present invention modifies the Spike protein into a DNA framework-liposome composite nanostructure. Spike protein plays a key role in the invasion and infection of the virus. It is a large glycoprotein located on the surface of the virus and consists of S1 subunits and S2 subunits. The S1 subunit is responsible for the initial binding of the virus to the host cell. This subunit contains a receptor binding domain (RBD) that binds to the ACE2 receptor on the surface of the host cell. The binding of RBD is a key step in viral infection. This is the first step for the virus to invade the cell. This binding is highly specific, ensuring that the virus can effectively recognize and attach to the target cell. The S2 subunit is responsible for the fusion of the virus and the host cell membrane. After the virus binds to the cell membrane, the structure of the S2 subunit changes, prompting the fusion of the virus membrane and the host cell membrane, so that the genetic material of the virus can enter the host cell, thereby realizing the replication and spread of the virus. Not only that, the Spike protein can also be located in the lysosome, which plays an important role in the escape of the virus lysosome and cell infection. In the present invention, the characteristic of Spike protein that it can be expressed on the cell membrane surface was first determined. In order to modify the Spike protein into our composite nanostructure, the method of first overexpressing Spike, then extracting membrane fragments, and finally mixing the membrane fragments with phospholipids was adopted. The Spike protein can then be evenly modified on the liposome surface.

[0060] 1.4 siRNA Carrying and Protection

[0061] In order to carry nucleic acid drug siRNA onto the liposome of DNA framework-liposome composite nanocarrier, in the present invention, siRNA is carried by cholesterol-modified siRNA, which can be well embedded in the bilayer of liposome by taking advantage of the lipophilicity of cholesterol molecules, and cholesterol-modified siRNA (synthesized by Sangon Biotechnology (Shanghai) Co., Ltd.) is fused with phospholipids to achieve uniform dispersion and fusion of siRNA and liposomes inside the composite structure, thereby providing stable protection and preventing it from being rapidly degraded by nucleases in cells. The carried siRNA can effectively interfere with the expression of target genes and achieve regulation of cell functions.

[0062] 1.5 Optimization of phospholipid composition, OG concentration, and membrane fragment ratio

[0063] In order to successfully fuse membrane fragments, siRNA and phospholipids as much as possible, in the present invention, the components of phospholipids, the concentration of OG and the ratio of phospholipids to membrane fragments are explored during dialysis to ensure that the synthesized composite nanostructure can carry membrane fragments and siRNA to the maximum extent possible, thereby achieving the effect of improving drug delivery efficiency.

[0064] The present invention is further described in detail by the following examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product instructions; the materials, reagents, etc. used, unless otherwise specified, are conventional commercially available products.

[0065] Example 1: Construction of virus-like DNA framework-liposome composite nanocarrier containing cell membrane fragments

[0066] This example describes a method for constructing a DNA framework-liposome composite nanocarrier containing cell membrane fragments, wherein the outer structure is a truncated icosahedral (soccer ball-shaped) DNA framework structure with a diameter of about 74 nm, and the inner structure reconstructs a single and full liposome vesicle structure containing cell membrane fragments. The specific construction steps are as follows:

[0067] Step 1: Using DNA nanostructure design software caDNAno and Tiamat, the long ssDNA template p7560 in the M13 phage genome was used as a backbone to fold into a football-shaped structure ( Figure 1A in the middle). After inputting the template sequence, hundreds of complementary short staple chains (staple chains, five of which were modified with TAMRA fluorescent groups for experimental observation) were synthesized. The template chain and the staple chain were mixed in a 1×TE-Mg²⁺ buffer (25 mM Tris EDTA, 10mM MgCl2, pH 7.4) at a molar ratio of 1:10, and the self-assembly of the DNA framework structure was achieved by programmed temperature annealing (95°C, 5 minutes; 65°C to 4°C, 1°C decrease every 15 minutes). Subsequently, the product was subjected to rate zone centrifugation under a glycerol gradient (15%-45%, v / v) to separate the well-assembled structure from excess staple chains and aggregates. The pure DNA football structure ( Figure 1 The purified DNA soccer ball was then filtered and washed with 1×TE-Mg²⁺ buffer using an Amicon ultracentrifugal filter (30 kDa, 7500 rpm, 5 min, repeated three times). Finally, the concentration of the soccer ball DNA framework was determined using NanoDrop and adjusted to 50 nM. The shape and integrity of the purified soccer ball DNA framework were observed and evaluated using atomic force microscopy (AFM) and transmission electron microscopy (TEM).

[0068] Step 2: To construct a virus-like DNA framework-liposome composite nanocarrier containing cell membrane fragments, a soccer-shaped DNA framework was used as the shell to induce the formation of liposome vesicles containing cell membrane fragments with controllable shape and size inside, and nucleic acid drugs siRNA were carried to regulate the expression of target genes. First, the commercially available ORF3a protein overexpression plasmid (SARS-CoV-2 (2019-nCoV) ORF3A protein Gene ORF cDNA clone expression plasmid, SinoBiological) was transfected into cells. During the transfection process, 5 μL Lipo8000 and 5 μL 10 ng / μL overexpression plasmid were mixed in DMEM medium containing serum and added to 2×10 7 After 48 hours of transfection, the cells were collected and the successful overexpression of ORF3a protein was verified by Western blot and confocal microscopy ( Figure 1C). After successfully overexpressing ORF3a protein, the cell membrane fragments were extracted. The specific steps are as follows (for isolating cell membranes from HCT116 cells or HeLa cells): First, the cells were gently scraped from the culture dish, suspended in 1 mL PBS, centrifuged at 4°C, 1200 g for 10 minutes, and the supernatant was discarded. Subsequently, the pellet was washed with 1 mL PBS, centrifuged at 600 g for 5 minutes, and the supernatant was removed. The pellet was centrifuged at 600 g for 1 minute to further remove the residual PBS. Then, 1 mL of Regent A (Beyotime, P0033) in the Cell Membrane Protein and Cytoplasmic Protein Extraction Kit was added and the cells were gently resuspended. The suspended cell pellet was placed in an ice bath for 15 minutes. After the ice bath, ultrasonic treatment was performed using an ultrasonic disruptor. Subsequently, the cell fragments were centrifuged at 700 g at 4°C for 10 minutes, and the supernatant was collected. Next, the supernatant was centrifuged at 14000 g at 4°C for 30 minutes. After centrifugation, the supernatant was removed and the resulting precipitate was the cell membrane fragments.

[0069] Step 3: Reconstruction of liposome vesicles containing membrane fragments of ORF3a protein inside the DNA framework. First, the 90 Handle chains inside the DNA framework (see Table 1) were incubated with the cholesterol-modified Seed chain (5'-TATGAGAAGTTAGGAATGTTA-Chol-3', SEQ ID NO: 91) (containing 1% OG, w / v, Seed chain is also called displacement chain, which can form complementary pairing with Handle chain and participate in chain displacement reaction) at 37°C for 1 hour for complementary hybridization. Subsequently, the internally modified cholesterol DNA framework was mixed with the phospholipid molecules used to reconstitute liposomes (liposome 1: 36% DOPC, 50% DOPE, 10% cholesterol, 2% PEG2k-PE, 2% Rho-PE; liposome 2: 36% DOTAP, 50% DOPE, 10% cholesterol, 2% PEG2k-PE, 2% Rho-PE) in 1× hydration buffer (25 mM HEPES, 140 mM KCl, 10 mM MgCl2, pH 7.5). The final concentration of the DNA framework was 15 nM. The final concentrations of OG were set to 0.3% and 0.6%, respectively. Taking advantage of the similar compatibility between phospholipids and cell membrane fragments, cell membrane fragments containing ORF3a protein were directly mixed with DNA frameworks and phospholipids. To load siRNA into the composite structure, siRNA was modified with cholesterol for embedding into liposomes, and Cy5 labeling was used to monitor the loading efficiency and distribution of siRNA. The solution was dialyzed overnight in 2L of 1× hydration buffer to remove the surfactant, and then subjected to isopycnic centrifugation in an iodixanol gradient at 50 krpm for 5 hours. After ultracentrifugation, the fractions were collected from top to bottom into 96 wells, with 200 μL collected per well (200 μL / layer × 24 layers), and their composition and location were analyzed by SDS agarose gel (1.5% agarose containing 0.1% SDS) electrophoresis. To ensure the successful fusion of membrane fragments, siRNA and phospholipids, the components of phospholipids were first explored during the dialysis process, and two liposomes with different components, liposome 1 (36% DOPC, 50% DOPE, 10% cholesterol, 2% PEG2k-PE, 2% Rho-PE) and liposome 2 (36% DOTAP, 50% DOPE, 10% cholesterol, 2% PEG2k-PE, 2% Rho-PE) were designed. Then, the OG concentrations used to disperse phospholipids and cell membrane fragments were set to 0.3% and 0.6%, respectively. The fixed phospholipid concentration was 1 mM, and the concentration of membrane fragments was 2 mg / mL. The experiment set up four groups of different phospholipid components and OG concentrations, namely: liposome 1 / 0.3% OG ( Figure 2 Middle A), liposome 1 / 0.6% OG ( Figure 2Middle B), liposome 2 / 0.3% OG ( Figure 2 C), liposome 2 / 0.6% OG ( Figure 2 D), density gradient centrifugation and Western Blot analysis were used to determine the optimal structural reconstruction conditions. The phospholipid composition and OG concentration of liposomes 1 / 0.6% OG were finally selected based on the coincidence of phospholipid and siRNA fluorescence ( Figure 2 Middle B).

[0070] Next, the ratio of phospholipids to membrane fragments was optimized to ensure that the synthesized composite nanostructures could carry the membrane fragments and siRNA to the maximum extent. The input amount of siRNA was fixed at 160 pmol, and the concentrations of phospholipids and membrane fragments were adjusted based on Figure 2 The results of the experiment were analyzed to reduce the concentration of membrane fragments. Four groups of different phospholipid and membrane fragment ratios were set up: 1 mM phospholipid / 1 mg / mL containing ORF3a membrane fragments ( Figure 3 Middle A), 1 mM phospholipids / 0.2 mg / mL containing ORF3a membrane fragments ( Figure 3 Middle B), 0.2 mM phospholipids / 1 mg / mL containing ORF3a membrane fragments ( Figure 3 C), 0.2 mM phospholipids / 0.2 mg / mL containing ORF3a membrane fragments ( Figure 3 The optimal structural reconstruction conditions were determined by density gradient centrifugation and Western Blot analysis, and the final ratio was 1 mM phospholipid-0.2 mg / mL CM3a (containing ORF3a membrane fragments) ( Figure 3 Middle B).

[0071] Step 4: Characterize the performance of the viral DNA framework-containing cell membrane fragment liposome composite nanocarrier. First, characterize it by electron microscopy. The results show that the liposome structure without the DNA framework outside is uneven in size and easy to aggregate, while the soccer-lipo composite nanostructure with the DNA framework shows higher size uniformity and stability ( Figure 4 The zeta potential measurement showed that the electronegativity of the modified membrane fragments SL-CM and SL-CM3a increased significantly ( Figure 4 The results of nanoflow cytometry further confirmed the high stability of the DNA framework-liposome composite nanostructure ( Figure 4 Meanwhile, Western Blot analysis confirmed that the collected nanostructures were successfully modified with membrane fragments and ORF3a protein ( Figure 4 Middle D).

[0072] Table 1 Handle chain sequence list

[0073]

[0074] Example 2: Verification of the effects of various components of the virus-mimicking DNA framework-liposome composite nanocarrier containing cell membrane fragments on the siRNA delivery efficiency

[0075] This embodiment relates to the verification of the siRNA delivery efficiency of each component in the virus-like DNA framework-liposome composite nanocarrier containing cell membrane fragments. In order to clarify the role of different components in siRNA delivery, the present invention constructs six different structures, including Free-lipo (FL), Free-lipo-CM (FL-CM), Free-lipo-CM3a (FL-CM3a), Soccer-lipo (SL), Soccer-lipo-CM (SL-CM), Soccer-lipo-CM3a (SL-CM3a). Among them, the FL group is a liposome structure without an external DNA framework, which is divided into unmodified liposomes (FL), liposomes modified with membrane fragments (FL-CM) and liposomes modified with membrane fragments containing ORF3a protein (FL-CM3a). The SL group is a composite nanostructure with an external DNA framework, which is divided into a structure without a modified DNA framework (SL), a DNA framework structure modified with membrane fragments (SL-CM), and a DNA framework structure modified with membrane fragments containing ORF3a protein (SL-CM3a).

[0076] In order to further explore the homing effect of cell membrane fragments from different sources in siRNA delivery, in this example, ORF3a protein was overexpressed in human colon cancer cell line HCT116 and human cervical cancer cell line Hela, membrane fragments from these two tumor cell lines were prepared, and these membrane fragments were used to modify the above six nanostructures. Figure 5 Middle A and Figure 5 As shown in D, the structures modified with membrane fragments from HCT116 and Hela cells were used to stimulate the two cell lines homologously and heterologously to determine the homing effects of membrane fragments from different sources. Figure 5 The membrane fragments in A are from HCT116 cells. Figure 5The membrane fragments in D are derived from Hela cells. During the experiment, the expression level of the siRNA target gene Bcl2 was detected by Western Blot to evaluate the siRNA delivery efficiency of each structure. This experiment used siRNA targeting the anti-apoptotic gene Bcl2 (Sense: Cy5-GUGAAGUCAACAUGCCUGC, SEQ ID NO: 92; Anti-sense: Chol-GCAGGCAUGUUGACUUCAC, SEQ ID NO: 93), set the siRNA dosage to 20 pmol, and the stimulation time to 8 hours. After the experiment, the cells were washed and cultured for another 40 hours, and finally the expression level of Bcl2 protein was detected by Western Blot. The degree of reduction in Bcl2 expression level reflects the delivery efficiency of siRNA. The experimental results show that in the homologous system ( Figure 5 In B and E, all nanostructures modified with membrane fragments showed high siRNA delivery efficiency. In particular, in the SL-CM3a structure modified with ORF3a protein, the expression level of Bcl2 was significantly reduced, indicating that its siRNA delivery ability was better than that of other structures. Figure 5 In C and F, the structure modified with heterologous membrane fragments showed lower siRNA delivery efficiency and higher Bcl2 expression level. This indicates that the modification of heterologous membrane fragments has a certain inhibitory effect on siRNA delivery. In contrast, even in the heterologous system, the SL-CM3a structure still showed a high delivery efficiency, proving that the ORF3a protein plays an important role in the siRNA delivery process. In addition, in the SL group, the presence of the DNA framework significantly improved the delivery stability of the structure and reduced the volatility of the experimental results.

[0077] In order to further observe the effects of homologous and heterologous systems on siRNA delivery, the distribution of siRNA in cells was observed by confocal microscopy after the six structures were co-incubated with cells for 8 hours under homologous and heterologous systems. Figure 6 As shown in A, the membrane fragments are from HCT116 cells, and the stimulated cells are HCT116 cells. It can be seen that after 8 hours of incubation, the siRNA (red fluorescence) carried by the six structures were delivered to the cells. Figure 6Middle B), that is, the membrane fragments came from HCT116 cells, and the cells stimulated by the structure were Hela cells. The confocal microscopy results showed that in the FL and SL groups, siRNA was widely distributed in the cytoplasm, while in the FL-CM, FL-CM3a, SL-CM, and SL-CM3a groups modified with membrane fragments, a large amount of siRNA accumulated on the cell membrane surface and could not enter the cell, indicating that the repulsion effect between heterologous cell membranes hindered the delivery of siRNA carried by the structure into the cell.

[0078] The results of this example show that the siRNA delivery efficiency of the three structures containing DNA framework (SL, SL-CM, SL-CM3a) is significantly better than that of the three structures without DNA framework (FL, FL-CM, FL-CM3a), which is specifically reflected in the following aspects:

[0079] (1) Better silencing effect of target genes: The three groups of structures containing DNA frameworks showed stronger siRNA delivery ability in the silencing experiment of the target gene Bcl2. Compared with the negative control group, the expression level of Bcl2 was significantly reduced, indicating that its siRNA delivery efficiency and gene silencing effect were significantly better than those of the structure without DNA framework.

[0080] (2) The experimental results are more stable: The structure containing the DNA framework showed a more stable siRNA delivery effect under multiple experimental conditions. In contrast, the structure without the DNA framework showed large data fluctuations in the experiment, resulting in unstable siRNA delivery results. The presence of the DNA framework improves the overall stability of the nanostructure, making the silencing effect of the target gene more reproducible and reliable.

[0081] Therefore, through this optimized design, the nanostructure containing the DNA framework not only improves the efficiency of gene silencing, but also significantly improves the stability of the experimental results, ensuring the effectiveness and consistency of the nanocarrier in practical applications.

[0082] The results of this example also show that the nanostructures modified with cell membrane fragments exhibit a significant homing effect during siRNA delivery, which is specifically reflected in:

[0083] (1) Homologous enhancement of delivery efficiency: When the nanostructures modified with cell membrane fragments were used to stimulate cells with the same cell membrane origin, the siRNA delivery efficiency was significantly improved, which was manifested as a stronger silencing effect on the target gene Bcl2. Compared with the negative control group, the expression level of the target gene was significantly reduced, indicating that the modification of homologous cell membrane fragments can enhance the delivery efficiency of the nanostructure.

[0084] (2) Heterogeneous inhibition of delivery efficiency: When the nanostructures modified with cell membrane fragments are used to stimulate cells with different cell membrane origins, their siRNA delivery efficiency is significantly inhibited, which is manifested as a weakened target gene silencing effect, and even no significant difference compared with the control group. This inhibitory effect indicates that the nanostructures modified with heterologous cell membrane fragments have a tendency to decrease in efficacy during siRNA delivery.

[0085] (3) Homing effect: Nanostructures modified with cell membrane fragments exhibit a strong homing effect, that is, they tend to deliver siRNA more effectively to cells with the same cell membrane origin, while the delivery efficiency is significantly reduced when stimulating cells of different origins. This effect has been verified in multiple experiments, indicating that nanostructures modified with cell membrane fragments can improve the targeting and efficiency of siRNA delivery through the homing mechanism.

[0086] Example 3: Study on the effect of nanostructures with different modification methods on siRNA delivery time

[0087] This embodiment aims to explore the siRNA delivery efficiency of nanostructures with different modification modes at different delivery times. To this end, the present invention conducted a detailed time gradient experiment on six nanostructures (FL, FL-CM, FL-CM3a, SL, SL-CM, SL-CM3a), and set four different delivery time points of 1 hour, 2 hours, 4 hours and 8 hours to evaluate the siRNA delivery effect at different time points. The experimental design includes the following steps: First, the above six nanostructures are prepared, and Cy5-labeled siRNA is embedded in the structure. Then, these nanostructures are co-incubated with HCT116 cells, and the delivery process is terminated after 1 hour, 2 hours, 4 hours and 8 hours, respectively. After the delivery, the cells continue to be cultured in complete medium for 48 hours, and then the expression level of the siRNA target gene Bcl2 is detected by Western Blot. The experimental results show that different delivery times have a significant effect on the siRNA delivery efficiency. Within the 1-hour delivery time, the SL structure has the highest siRNA delivery efficiency, which is manifested by a significant decrease in the expression level of Bcl2; while the delivery efficiency of other structures is relatively low ( Figure 7 Middle A). Within 2 hours of delivery, the SL structure still performed well, while the delivery efficiency of other structures was improved. Figure 7 In the middle (B), the SL-CM3a structure showed the strongest siRNA delivery ability at 4 and 8 hours of delivery, especially at 8 hours, when the Bcl2 expression level was significantly reduced compared with the control group Lipo2000 ( Figure 7 This indicates that the SL structure has a higher delivery efficiency in a short time, while other modified structures require a longer time to exert their delivery effect.

[0088] Example 4: Cytotoxicity test of virus-like DNA framework-liposome composite nanocarrier containing cell membrane fragments

[0089] In this example, the toxicity of six nanostructures (FL, FL-CM, FL-CM3a, SL, SL-CM, SL-CM3a) to cells was detected by CCK-8 experiment, and compared with the commercial transfection reagent Lipo2000 to explore the biosafety of the viral DNA framework-liposome containing cell membrane fragments composite nanocarrier.

[0090] like Figure 8 The results showed that after 8 h of Lipo2000 stimulation, the cell viability decreased significantly, indicating that Lipo2000 has obvious toxicity to cells, and as the dose of Lipo2000 increases, the cytotoxicity increases. After the six nanostructures FL, FL-CM, FL-CM3a, SL, SL-CM, and SL-CM3a were incubated with cells for 8 h, the cell activity almost did not decrease, indicating that the structure had almost no toxicity to cells, and as the dose of the structure increased, the cytotoxicity did not change significantly. This shows that compared with the commercial transfection reagent Lipo2000, in the present invention, the virus-mimicking DNA framework-cell membrane fragment-containing liposome composite nanocarrier can achieve the huge advantage of having the same low toxicity as the liposome structure without a DNA framework.

[0091] Example 5: Further verification of the delivery efficiency of the novel coronavirus DNA framework-liposome composite nanocarrier containing cell membrane fragments for siRNA, and investigation of the synergistic delivery effect of ORF3a and Spike protein

[0092] In this example, on the basis of modifying ORF3a, the novel coronavirus Spike protein was further modified, a holographic novel coronavirus-mimicking DNA framework-liposome composite nanocarrier was constructed, and its synergistic effect and delivery efficiency in siRNA delivery were explored. First, HCT116 cells (or Hela cells) were transfected with ORF3a protein overexpression plasmid (SARS-CoV-2 (2019-nCoV) ORF3A protein Gene ORF cDNA clone expression plasmid, SinoBiological) and Spike protein overexpression plasmid (SARS-CoV-2 (2019-nCoV) Spike GeneORF cDNA clone expression plasmid, SinoBiological), and after overexpressing Spike protein, its expression localization was verified by immunofluorescence ( Fig. 9Then, membrane fragments containing both ORF3a protein and Spike protein were obtained by cell membrane extraction technology, and Western Blot was used to determine whether the membrane fragments extracted from HCT116 cells and Hela cells contained ORF3a protein and Spike protein ( Fig. 9 Next, a SL-CM3a-CMSp composite nanostructure was constructed, in which both the ORF3a protein and the Spike protein were modified ( Fig. 9 In C). To further explore the roles of ORF3a protein and Spike protein in siRNA delivery, we constructed four structures, SL, SL-CM3a, SL-CMSp, and SL-CM3a-CMSp, and tested the delivery efficiency in HCT116 and Hela cell lines. Western Blot results showed that in HCT116 cells, the SL-CM3a-CMSp structure had the strongest delivery efficiency and the lowest Bcl2 expression level when the delivery time was 8 hours ( Fig. 9 This indicates that the synergistic effect of ORF3a and Spike protein plays a significant role in promoting siRNA delivery.

[0093] The same experiment was verified in Hela cells, and the results showed that the SL-CM3a-CMSp structure performed best in terms of delivery efficiency, which was consistent with the conclusion in HCT116 cells ( Fig. 9 Middle E).

[0094] The above description is only a preferred embodiment of the present invention and is not any formal or substantial limitation of the present invention. It should be pointed out that a person skilled in the art can make several improvements and supplements without departing from the present invention, and these improvements and supplements should also be regarded as the protection scope of the present invention.

Claims

1. A general preparation method of a DNA framework vesicle composite nanocarrier containing cell membrane components, characterized in that: The following steps are involved: Step 1: Use DNA nanostructure design software to construct a soccer ball-shaped DNA framework structure; Step 2: using the long-chain ssDNA template p7560 in the M13 phage genome as the skeleton of the DNA framework structure, and then designing a staple chain complementary thereto, the long-chain ssDNA template and the staple chain are annealed, base-paired and self-assembled to obtain a soccer-shaped DNA framework, which is then separated and purified to obtain a purified DNA framework; Step 3: hybridizing the purified DNA framework with the cholesterol-modified seed chain through the internal Handle chain, and mixing with phospholipids, cell membrane fragments and surfactant octyl glucoside. Under the guidance of the seed chain cholesterol, the cell membrane fragments, whose main components are lipid bilayers, fuse with the phospholipids, and then separate and purify by dialysis and isopycnic centrifugation to obtain a DNA framework vesicle composite nanocarrier containing cell membrane components; the composition of the phospholipids includes DOPC, DOPE, cholesterol, PEG2k-PE and Rho-PE; The cell membrane fragments are cell membrane fragments modified with functional membrane proteins; The functional membrane protein is firstly transfected into the tool cells via plasmid to construct cells overexpressing the functional membrane protein, and then the cell membrane fragments are extracted to obtain the cell membrane fragments modified with the functional membrane protein.

2. The general preparation method according to claim 1, characterized in that: The soccer ball-shaped DNA framework structure is a truncated icosahedral structure; and / or, the ratio of the phospholipids to the cell membrane fragments is 1 mM phospholipids: 0.2-0.4 mg / mL cell membrane fragments; and / or, the composition of the phospholipids includes 34-38% DOPC, 48-52% DOPE, 8-12% cholesterol, 1-3% PEG2k-PE and 1-3% Rho-PE.

3. The general preparation method according to claim 1, characterized in that: The ratio of the phospholipids to the cell membrane fragments is 1 mM phospholipids: 0.2 mg / mL cell membrane fragments; and / or, the composition of the phospholipids includes 36% DOPC, 50% DOPE, 10% cholesterol, 2% PEG2k-PE and 2% Rho-PE; and / or, the concentration of the surfactant during the dialysis process is 0.6%.

4. The use of the general preparation method described in any one of claims 1 to 3 in constructing a novel coronavirus-like DNA framework-containing cell membrane fragment liposome composite nanocarrier, characterized in that: The cell membrane fragments in step 3 are cell membrane fragments modified with functional membrane proteins of the new coronavirus, and the functional membrane proteins of the new coronavirus are ORF3a and / or Spike proteins.

5. A method for constructing a novel coronavirus-like DNA framework-liposome composite nanocarrier containing cell membrane fragments, characterized in that: The specific steps include: Transfecting an overexpression plasmid of ORF3a protein and / or Spike protein into a tool cell to construct an ORF3a protein and / or Spike protein overexpression cell, and extracting cell membrane fragments containing ORF3a protein and / or Spike protein from the overexpression cell; The general preparation method described in claim 1 is used to construct a novel coronavirus-mimicking DNA framework-cell membrane fragment-containing liposome composite nanocarrier; wherein the cell membrane fragments are cell membrane fragments containing ORF3a protein and / or Spike protein.

6. Use of the novel coronavirus-like DNA framework-cell membrane fragment-containing liposome composite nanocarrier constructed by the construction method of claim 5 in in vitro drug delivery or preparation of a drug delivery system.

7. A method for preparing a drug delivery system, characterized in that: On the basis of the novel coronavirus-like DNA framework-containing cell membrane fragment liposome composite nanocarrier constructed by the method described in claim 5, a drug is loaded, that is, the preparation method comprises: incubating and combining the cholesterol-modified drug with the novel coronavirus-like DNA framework-containing cell membrane fragment liposome composite nanocarrier, so that the drug is combined with the liposome inside the novel coronavirus-like DNA framework-containing cell membrane fragment liposome composite nanocarrier, thereby providing stable protection and preventing it from being rapidly degraded by nucleases in the cell; the drug includes nucleic acid drugs as well as polypeptide and protein drugs.

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