Membrane fusion liposome delivery system and preparation method and application thereof

Abstract

The invention relates to a membrane fusion liposome delivery system and a preparation method and application thereof, and belongs to the technical field of liposome preparation. The invention provides a membrane fusion liposome delivery system. The system takes lipidosome as a carrier, the surface of the lipidosome is modified with cRGD targeting molecules, and the interior of the lipidosome is loaded with a TPP-modified antitumor drug lonidamine; a DNA device CLTD is anchored on a liposome membrane through cholesterol, and the device is formed by a short-chain CAP containing an APE1 restriction enzyme cutting site and a long-chain LTD containing a CD63 aptamer and a RelA aptamer through base complementary pairing. The APE1 enzyme highly expressed in a tumor microenvironment is used as a specific trigger signal, and precise activated release of the drug in tumor cells and space-time controllable degradation of RelA protein are achieved. According to the system, chemical drug inhibition and nucleic acid aptamer mediated protein degradation are synergistically linked, off-target toxicity is effectively overcome, and a new strategy is provided for precise treatment of cancers.

Description

Technical Field

[0001] This invention belongs to the field of liposome preparation technology, and relates to membrane fusion liposome delivery system, its preparation method and application. Background Technology

[0002] The global incidence and mortality rates of cancer continue to rise, and breast cancer, as the most common malignant tumor in women, has become a major public health problem threatening women's health. With the development of precision medicine, targeted therapy has become a core direction in the field of cancer treatment. However, traditional small molecule targeted drugs face the bottleneck of a high proportion of "undruggable" targets—approximately 80% of human proteins are difficult to effectively regulate with traditional drugs due to the lack of a clear small molecule binding pocket and insufficient affinity for small molecules. Against this backdrop, targeted protein degradation (TPD) technology has emerged, providing a new approach to the regulation of "undruggable" targets and cancer treatment. The core mechanism of TPD technology is to utilize natural intracellular protein hydrolysis pathways (such as the ubiquitin-proteasome system and lysosomal degradation pathway) to target and deliver the target protein (POI) to the degradation system through synthetic degrading agents for clearance, rather than the "inhibition" effect of traditional drugs. Therefore, it has advantages such as high efficacy, low risk of drug resistance, and a wide range of tunable targets.

[0003] Currently developed TPD degraders mainly fall into four categories: proteolysis-targeting chimeras (PROTACs), lysosome-targeting chimeras (LYTACs), autophagy-targeting chimeras (AUTACs), and molecular glues. PROTACs use a dual-function linker to simultaneously connect POIs to E3 ubiquitin ligases, guiding ubiquitination of POIs and their degradation via proteasomes. LYTACs utilize lysosome-targeting receptors (such as CI-M6PR) to mediate the lysosomal degradation of POIs. AUTACs activate the autophagy pathway, encapsulating POIs into autophagosomes and fusing them with lysosomes for degradation. Molecular glues induce the interaction between E3 ubiquitin ligases and POIs, triggering POI ubiquitination and degradation. These technologies have shown potential in regulating various tumor-related targets (such as BTK, AR, and EGFR), but a key technological bottleneck remains: insufficient spatiotemporal control. Existing degradative agents mostly function non-specifically throughout the body, making it difficult to precisely target tumor tissues and specific subcellular structures within tumor cells. This can easily lead to the misdegradation of essential proteins in normal cells, causing toxic side effects and limiting their clinical translation. Therefore, constructing a TPD system with precise spatiotemporal control capabilities has become a core requirement for overcoming current technological bottlenecks and improving the safety and efficacy of tumor treatment.

[0004] Meanwhile, the rapid development of DNA nanotechnology has provided new tools for intelligent regulation in the biomedical field. DNA molecules possess unique programmable base-pairing properties, allowing for the precise design and formation of nanodevices with specific structures and functions (such as DNA origami, DNA logic gates, and DNA aptamers). Furthermore, DNA exhibits excellent biocompatibility, low immunogenicity, and ease of chemical modification, enabling precise regulation of intermolecular interactions within biological systems. Currently, DNA nanodevices are widely used in tumor diagnosis and treatment. For example, DNA catalytic amplifiers enable highly sensitive detection of tumor markers; DNA logic gates initiate therapeutic functions in response to tumor microenvironment signals (such as pH, enzymes, and small molecules); and DNA aptamers achieve targeted recognition of specific cells or proteins. The core advantage of DNA logic gates lies in their "response-output" intelligent decision-making capability. The logic gate only activates its downstream function when it receives specific tumor microenvironment signals (such as enzymes highly expressed by tumor cells or abnormal pH values), thereby achieving spatiotemporal control of the treatment process. This characteristic perfectly aligns with the spatiotemporal control requirements of TPD technology, providing crucial technical support for constructing a precisely regulated target protein degradation system. In the tumor pathological microenvironment, the expression level of apurinic / apyrimidinic endonuclease 1 (APE1) is significantly higher than in normal breast tissue and other breast cancer subtypes. APE1 is a key enzyme in the base excision repair pathway and also participates in regulating oxidative stress tolerance and radiotherapy / chemotherapy resistance in tumor cells. Its high expression is closely related to the malignant proliferation, invasion, metastasis, and poor prognosis of TNBC, making it an ideal "signaling molecule" for TNBC-specific targeting. Furthermore, tumor cells exhibit high expression of integrin αvβ3 receptors, and the cyclic peptide cRGD (cyclicArg-Gly-Asp) can specifically bind to integrin αvβ3. It has been widely used as a guide molecule in tumor-targeted delivery systems, significantly improving the enrichment efficiency of carriers in tumor tissues. In the molecular pathological mechanisms of tumors, abnormal activation of the nuclear factor κB (NF-κB) signaling pathway is one of the core pathways driving tumor development and progression. RelA (p65), as a key transcription factor in the NF-κB pathway, is continuously activated in tumor cells and regulates the expression of downstream pro-proliferation, anti-apoptotic, and pro-metastatic genes, making it an important target protein for TNBC treatment. Lysosomes, as important intracellular degradative organelles, have CD63 protein on their membrane surface as an ideal anchoring site for lysosomal targeted degradation. Targeting POIs to CD63 can achieve lysosomal-mediated specific degradation. Summary of the Invention

[0005] In view of this, one objective of the present invention is to provide an APE1 enzyme-responsive DNA-modified membrane fusion liposome delivery system, which has the function of spatiotemporally controlled lysosomal degradation of RelA protein; a second objective of the present invention is to provide a method for preparing an APE1 enzyme-responsive DNA-modified membrane fusion liposome delivery system, wherein the method constructs DNA-modified membrane fusion liposomes by embedding cholesterol into complementary DNA double strands containing APE1 restriction sites; a third objective of the present invention is to provide an application strategy for an APE1 enzyme-responsive DNA-modified membrane fusion liposome delivery system loaded with mitochondrial-targeting chlordamine in precision targeted tumor therapy. To achieve the above objectives, the present invention provides the following technical solutions: This invention provides a membrane fusion liposome delivery system, the membrane fusion liposome delivery system comprising the following components: Liposome carrier Lip-cRGD@TPP-LND: A liposome carrier is prepared from DMPC, DSPE-PEG2000-cRGD, and DOTAP, wherein the hydrophilic cavity of the liposome is loaded with an antitumor drug. CLTD: Composed of a short DNA CAP and a long DNA LTD; the short DNA CAP is 11-15 nt in length and is doped with an APE1 enzyme response site; the long DNA LTD contains an AP site sequence, a CD63 aptamer sequence, and a RelA aptamer sequence. The membrane-fused liposome delivery system Lip-cRGD@CTLD@TPP-LND was prepared by combining a cholesterol-hydrophobic terminal CLTD with a liposome carrier. Preferably, the antitumor drug is chlordamine; Preferably, the molar ratio of the short DNA chain CAP to the long DNA chain LTD is 1:1; Preferably, the nucleic acid sequence of the CAP is shown in SEQ ID NO:1, and the nucleic acid sequence of the LTD is shown in SEQ ID NO:2; Furthermore, the preparation method of the membrane-fused liposome delivery system is as follows: S1: Synthesis of Lip-cRGD@TPP-LND: 7.255 mg of DMPC, 1.516 mg of DSPE-PEG2000-cRGD, 1.963 mg of DOTAP, and TPP-TPP-LND were weighed and dissolved in 10 mL of chloroform. The mixture was then rotary evaporated under reduced pressure at 70 rpm and a water bath at 45 °C. The dissolved chloroform was then dissolved in 5 mL of phosphate buffer at pH 7.4. The mixture was sonicated for 30 minutes and centrifuged at 3500 rpm for 10 minutes. After filtration through a 200 nm filter, the mixture was repeatedly extruded using a microliposome extruder to obtain Lip-cRGD@TPP-LND. S2: Synthesis of CLTD: The lyophilized powders of CAP and LTD were dissolved in DEPC water to a final concentration of 100 nM. The two DNA strands were thoroughly mixed in a 1:1 ratio, heated at 95°C for 5 minutes, and then rapidly cooled to room temperature to prepare CLTD. S3: Add CLTD with cholesterol hydrophobic ends to Lip-cRGD@TPP-LND, incubate in PBS buffer at 37°C for 4 hours, and pass the thoroughly mixed micelles through a 200nm pore size polycarbonate membrane. Use a micro liposome extruder to extrude back and forth to obtain the membrane fusion liposome delivery system Lip-cRGD@CTLD@TPP-LND. Preferably, the Lip-cRGD@TPP-LND is obtained by dialysis for 2 days using a dialysis bag with a MWCO of 1000 Da; Furthermore, the membrane-fused liposome delivery system is used in the preparation of antitumor drugs; Preferably, the tumor is breast cancer.

[0006] The beneficial effects of this invention are as follows: This invention discloses an APE1 enzyme-responsive DNA-modified membrane fusion liposome delivery system. Using cRGD as the target molecule, cRGD is released into tumor cells to inhibit hexokinase 2, thereby preventing RelA from entering the nucleus. Simultaneously, a CTLD is embedded in the liposome membrane via cholesterol. The CAP chain is doped with an APE1 enzyme-responsive site, which can be specifically cleaved by the highly expressed APE1 enzyme in tumor cells, releasing the LTD. The two ends of this chain are a CD63 aptamer and a RelA aptamer. When the RelA aptamer has not yet bound to the RelA protein, a portion of its sequence is complementary to an insertion sequence of the CD63 aptamer. However, when the RelA aptamer collides with the RelA protein, the affinity for the protein is greater than the base complementarity, thus the RelA aptamer tends to bind to the RelA protein. Furthermore, after the RelA aptamer dissociates from the CD63 aptamer insertion sequence, the tertiary structure of the CD63 aptamer changes, thereby restoring its original conformation, allowing it to bind to the CD63 protein and pull RelA into the lysosome for degradation. Clonidamine LTD then exerts its antitumor effect in combination.

[0007] This invention achieves spatial control of "intracellular activation in tumor cells" through APE1 enzyme response and temporal control of "degradation initiated in the presence of target proteins" through RelA protein-mediated aptamer conformational changes. It also combines the delivery advantages of liposomes with the targeting specificity of DNA aptamers, effectively solving the problems of insufficient spatiotemporal control and high off-target toxicity in existing TPD technologies. This provides a new technical strategy and research foundation for precision cancer treatment and has significant scientific and clinical translational value.

[0008] Other advantages, objectives, and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination, or may be learned from practice of the invention. The objectives and other advantages of the invention can be realized and obtained through the following description. Attached Figure Description

[0009] To make the objectives, technical solutions, and advantages of the present invention clearer, the preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings, wherein: Figure 1 A schematic diagram of liposome synthesis for DNA strand assembly and DNA modification; Figure 2 A schematic diagram illustrating the mechanism of action of an APE1 enzyme-responsive DNA-modified membrane fusion liposome; Figure 3 To verify the APE1 enzyme-specific cleavage ability of the designed DNA strand; Among them, (a) the complementary length screening of AP-LTD and its complementary short chain; (bc) Fluorescence detection analysis of (b) AP-LTD with fluorescent groups and quenching groups and their complementary chains under the action of different types of enzymes and their statistical results (c); (de) Analysis of (d) the cleavage ability of different concentrations of APE1 enzyme on AP-LTD with fluorescent and quenching groups and their complementary strands and its statistics using fluorescence detection (e). (f) AP-LTD was treated with different methods to verify its APE1 enzyme-responsive cleavage ability; (g) Nucleic acid gel electrophoresis analysis of the cleavage ability of different concentrations of APE1 enzyme AP-LTD and its complementary strand; (h) Nucleic acid gel electrophoresis analysis of AP-LTD and its complementary strand under the action of different types of enzymes to determine AP site breakage; (i) Schematic diagram of APE1 enzyme-responsive cleavage of AP-LTD and its complementary strand; Figure 4 To characterize the morphology and perform a series of physicochemical characterizations of an APE1 enzyme-responsive DNA-modified membrane fusion liposome; Among them, (ab) the morphology of the synthesized APE1 enzyme-responsive DNA-modified membrane fusion liposomes was observed under transmission electron microscopy (a) and atomic force microscopy (b); (c) Particle size analysis of the synthesized liposomes containing different components: I: Lip-cRGD, II: Lip-cRGD@TPP-LND, III: Lip-cRGD@CLTD@TPP-LND; (d) Potential changes of the synthesized liposomes containing different components: I: Lip-cRGD, II: Lip-cRGD@TPP-LND, III: Lip-cRGD@CLTD@TPP-LND; (e) Flow cytometry detection of the tumor-specific targeting ability of liposomes; Figure 5 Different groups of fluorescent DNA strands were co-incubated with cells (a) to observe the localization of the DNA fluorescent strands (red fluorescence) and cell lysosomes (green fluorescence); (b) The fluorescence intensity of the DNA strands was analyzed by co-incubating the purified protein with DNA strands of different groups: I: Q-LTD, II: C-aptRelA+Q-LTD, III: Q-LTD+random protein, IV: Q-LTD+RelA; (c) The fluorescence intensity of the DNA strands was analyzed by co-incubating them with cell lysis buffer and different groups of DNA strands. I: Q-LTD, II: C-aptRelA+Q-LTD, III: Q-LTD+cell lysis buffer (RelA knockout cells), IV: Q-LTD+cell lysis buffer; (d) Western blot assay was used to detect the degradation capacity of different groups for RelA protein. I: blank control; I: LTD + APE1 enzyme inhibitor; III: C-aptRelA + LTD; IV: aptCD63 / RelA; V: LTD; Figure 6 A schematic diagram of the treatment regimen for 4T1 tumor-bearing mice (a): I: blank control, II: Lip-cRGD, III: Lip-cRGD@CLTD, IV: Lip-cRGD@TPP-LND, V: Lip-cRGD@CLTD@TPP-LND; (b) Bioluminescent images of 4T1-luc tumor growth in mice after different treatments: I: blank control, II: Lip-cRGD, III: Lip-cRGD@CLTD, IV: Lip-cRGD@TPP-LND, V: Lip-cRGD@CLTD@TPP-LND; (c) Changes in the volume of 4T1 tumors in different groups of mice during treatment, I: blank control, II: Lip-cRGD, III: Lip-cRGD@CLTD, IV: Lip-cRGD@TPP-LND, V: Lip-cRGD@CLTD@TPP-LND; (d) Tumor weight analysis in different groups of mice after treatment: I: blank control, II: Lip-cRGD, III: Lip-cRGD@CLTD, IV: Lip-cRGD@TPP-LND, V: Lip-cRGD@CLTD@TPP-LND; (e) Survival analysis of mice after different treatments: I: blank control, II: Lip-cRGD, III: Lip-cRGD@CLTD, IV: Lip-cRGD@TPP-LND, V: Lip-cRGD@CLTD@TPP-LND; (f) Changes in body weight of mice in different treatment groups: I: blank control, II: Lip-cRGD, III: Lip-cRGD@CLTD, IV: Lip-cRGD@TPP-LND, V: Lip-cRGD@CLTD@TPP-LND. Detailed Implementation

[0010] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the illustrations provided in the following embodiments are only schematic representations of the basic concept of the present invention. Unless otherwise specified, the following embodiments and features can be combined with each other.

[0011] The accompanying drawings are for illustrative purposes only and are schematic diagrams, not actual pictures. They should not be construed as limiting the invention. To better illustrate the embodiments of the invention, some parts in the drawings may be omitted, enlarged, or reduced, and do not represent the actual product dimensions. It is understandable to those skilled in the art that some well-known structures and their descriptions may be omitted in the drawings.

[0012] In the accompanying drawings of the embodiments of the present invention, the same or similar reference numerals correspond to the same or similar components. In the description of the present invention, it should be understood that if terms such as "upper," "lower," "left," "right," "front," and "rear" indicate the orientation or positional relationship based on the orientation or positional relationship shown in the drawings, they are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, the terms used to describe positional relationships in the drawings are only for illustrative purposes and should not be construed as limiting the present invention. For those skilled in the art, the specific meaning of the above terms can be understood according to the specific circumstances.

[0013] Example 1 Prepare a 500 mL round-bottom single-necked flask. Weigh out 7.255 mg of DMPC, 1.516 mg of DSPE-PEG2000-cRGD, and 1.963 mg of DOTAP using an electronic balance. Dissolve all three components (TPP-TPP-LND) in 10 mL of chloroform. Evaporate under reduced pressure overnight at 70 rpm in a 45°C water bath to obtain a homogeneous film. Then, dissolve the lipid film in 5 mL of phosphate-buffered saline (PBS) at pH 7. After complete dissolution, sonicate for 30 minutes. After sonication, centrifuge at 3500 rpm for 10 minutes to remove impurities or aggregates. Finally, filter through a 0.22 μm filter membrane, extrude repeatedly approximately 10 times using a microliposome extruder, and dialyze for 2 days using a dialysis bag with a MWCO of 1000 Da to obtain Lip-cRGD@TPP-LND.

[0014] First, the lyophilized powders of CAP and LTD were dissolved in DEPC water to a final concentration of 100 nM. The two DNA strands were then thoroughly mixed in a 1:1 ratio, heated at 95°C for 5 minutes, and then rapidly cooled to room temperature to complete the annealing process.

[0015] Table 1. DNA sequences used for CLTD synthesis

[0016] Note: / idSp / represents the AP site. DNA sequences with cholesterol-hydrophobic ends were added to the lipid mixture and incubated for 4 hours in 1×PBS buffer (pH 7.4) under mild conditions (37°C). The well-mixed and incubated lipid-DNA micelles were then passed through a 200 nm pore size polycarbonate membrane (Whatman; Diegem, Belgium) and extruded back and forth 10 times using a microliposome extruder. Uniformly sized DNA membrane fusion liposomes, Lip-cRGD@CTLD@TPP-LND, were obtained.

[0017] Example 2 To determine the complementary number of LTD and CAP chains, we synthesized CAP chains of different lengths and incubated them with LTD chains for complementarity. Nucleic acid gel electrophoresis results showed that a CAP chain of 14 nt length could successfully complement the LTD chain, forming a CTLD ( ). Figure 3 a). Co-incubation of CTLD with different cleavage enzymes revealed that the fluorescence intensity was highest in the presence of APE1 enzyme, demonstrating the APE1 enzyme cleavage specificity of this DNA. Figure 3 bc), and the gel electrophoresis experiment also yielded the same conclusion ( Figure 3 d). Figure 3 For example, the concentration of APE1 enzyme used was determined. Figure 3 h indicates that CTLD can only be effectively cleaved if the APE1 enzyme remains active.

[0018] Example 3 The morphology and physicochemical characterization of the Lip-cRGD@CTLD@TPP-LND synthesized in Example 1 above are as follows: Figure 4 As shown. Among them. Figure 4 ab represent the morphological displays of Lip-cRGD@CTLD@TPP-LND under transmission electron microscopy and atomic force microscopy, respectively. Figure 4 c is the liposome particle size distribution measured using a dynamic light scattering instrument. Figure 4 d represents the surface charge characteristics of liposomes as measured by a Zeta potentiometer. Figure 4The study demonstrated that the synthesized Lip-cRGD@CTLD@TPP-LND liposomes could target tumor cells without targeting immune cells.

[0019] Example 4 The function of LTD was validated at the cellular level, as follows: like Figure 5 As shown in Figure a, different groups of fluorescent DNA strands were co-incubated with 4T1 cells. Observation of the localization results of the DNA fluorescent strands (red fluorescence) and lysosomes (green fluorescence) confirmed that TLD could only release the DNA sequence targeting CD63 protein after binding to the RelA protein, and then co-localize with the lysosomes. Figures b and c also used purified protein or cell lysis buffer to co-incubate different groups of DNA strands, and the fluorescence intensity of the DNA strands was analyzed to further verify the above conclusions. Figure d confirmed through Western blotting that LTD can degrade the RelA protein.

[0020] Example 5 The antitumor activity of APE1 enzyme-responsive DNA-modified membrane fusion liposomes is studied, as detailed below: Balb / c mice (female, 6 weeks old) were housed at 25°C under a 12-hour dark / light cycle. 4T1-luc cells were subcutaneously injected into the Balb / c mice. The tumor-bearing mice were randomly divided into 5 groups of 5 mice each: I: blank control; II: Lip-cRGD; III: Lip-cRGD@CLTD; IV: Lip-cRGD@TPP-LND; V: Lip-cRGD@CLTD@TPP-LND. Tumor volume was measured every 3 days and calculated using the formula: Vtumor = L × W. 2 / 2 (L: longitudinal diameter of the tumor, W: transverse diameter of the tumor). Mice were euthanized 21 days after treatment.

[0021] Test results as follows Figure 6 As shown, a) is a schematic diagram of the treatment regimen for 4T1 tumor-bearing mice; b) is a bioluminescent image of 4T1-luc tumor growth in mice after different treatments; c) is the volume change of 4T1 tumors in different groups of mice during treatment; d) is the tumor weight analysis of different groups of mice after treatment; e) is the survival rate analysis of mice after different treatments; f) is the weight change of mice in different treatment groups. Figure 5 It can be seen that the APE1 enzyme-responsive DNA-modified membrane fusion liposomes prepared in this invention have anti-tumor effects.

[0022] In summary, this invention discloses an APE1 enzyme-responsive DNA-modified membrane fusion liposome delivery system, its preparation method, and its applications. The APE1 enzyme-responsive DNA-modified membrane fusion liposome delivery system disclosed in this invention mainly includes a cRGD targeting molecule, a DNA device CLTD anchored to the liposome membrane—containing a short DNA chain CAP complementary to the AP site sequence, and a long DNA chain LTD consisting of the AP site sequence, CD63 aptamer, and RelA aptamer sequences in sequence, and the antitumor drug chlordamine loaded in the hydrophilic cavity of the liposome. The DNA anchors the complementary short DNA chain CAP containing the APE1 cleavage site to both sides of the liposome membrane via cholesterol intercalation. The cRGD targeting molecule is modified on the liposome surface to achieve tumor cell targeting. On one hand, chlordamine is released into the tumor cells to inhibit hexokinase 2, thereby preventing RelA from entering the nucleus. On the other hand, the CLTD releases the LTD through specific cleavage of the AP site by the APE1 enzyme, thereby degrading the RelA protein. The delivery system uses biocompatible liposomes as its carriers, which can be used to precisely deliver the anti-tumor drug chlordamine and the spatiotemporally controlled degradation of RelA proteolysosomes to inhibit the occurrence and development of tumor cells, providing a new approach for precision cancer treatment.

[0023] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A membrane-fused liposome delivery system, characterized in that: The membrane-fused liposome delivery system comprises the following components: Liposome carrier Lip-cRGD@TPP-TPP-LND: A liposome carrier is prepared from DMPC, DSPE-PEG2000-cRGD, and DOTAP, wherein the hydrophilic cavity of the liposome is loaded with an antitumor drug. CLTD: Composed of a short DNA CAP and a long DNA LTD; the short DNA CAP is 11-15 nt in length and is doped with an APE1 enzyme response site; the long DNA LTD contains an AP site sequence, a CD63 aptamer sequence, and a RelA aptamer sequence. The membrane-fused liposome delivery system Lip-cRGD@CTLD@TPP-TPP-LND was prepared by combining a cholesterol hydrophobic terminal CLTD with a liposome carrier.

2. The membrane-fused liposome delivery system according to claim 1, characterized in that: The antitumor drug in question is chlordamine.

3. The membrane-fused liposome delivery system according to claim 1, characterized in that: The molar ratio of the short DNA chain CAP to the long DNA chain LTD is 1:

1.

4. The membrane-fused liposome delivery system according to claim 1, characterized in that: The nucleic acid sequence of the CAP is shown in SEQ ID NO:1, and the nucleic acid sequence of the LTD is shown in SEQ ID NO:

2.

5. The method for preparing the membrane-fused liposome delivery system according to any one of claims 1-4, characterized in that, Its preparation method is as follows: S1: Synthesis of Lip-cRGD@TPP-LND: DMPC 7.255 mg, DSPE-PEG2000-cRGD 1.516 mg, DOTAP 1.963 mg, and TPP-TPP-LND were weighed and dissolved together in 10 mL of chloroform. The mixture was then rotary evaporated under reduced pressure at 70 rpm and 45 °C in a water bath. The dissolved chloroform was then dissolved in 5 mL of phosphate buffer (pH 7.4). The mixture was sonicated for 30 minutes, centrifuged at 3500 rpm for 10 minutes, filtered through a 200 nm filter, and repeatedly extruded using a microliposome extruder to obtain Lip-cRGD@TPP-LND. S2: Synthesis of CLTD: The lyophilized powders of CAP and LTD were dissolved in DEPC water to a final concentration of 100 nM. The two DNA strands were thoroughly mixed in a 1:1 ratio, heated at 95°C for 5 minutes, and then rapidly cooled to room temperature to prepare CLTD. S3: Add CLTD with cholesterol hydrophobic ends to Lip-cRGD@TPP-LND, incubate in PBS buffer at 37°C for 4 hours, and pass the thoroughly mixed micelles through a 200nm pore size polycarbonate membrane. Use a micro liposome extruder to extrude back and forth to obtain the membrane fusion liposome delivery system Lip-cRGD@CTLD@TPP-LND.

6. The method for preparing the membrane-fused liposome delivery system according to claim 5, characterized in that: The Lip-cRGD@TPP-LND was obtained and dialysis was performed for 2 days using a dialysis bag with a MWCO of 1000 Da.

7. Use in the preparation of antitumor drugs using the membrane fusion liposome delivery system according to any one of claims 1-4.

8. The application according to claim 7, characterized in that: The tumor is breast cancer.

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