Plasmid-loaded targeted liposome compound as well as preparation method and application thereof

Abstract

The invention relates to a plasmid-loaded targeted liposome compound, and a preparation method and application thereof. The liposome compound comprises liposome and plasmid pPEA entrapped by the liposome, and the plasmid pPEA comprises an expression vector pGAVPO containing a blue light induced transcription factor GAVPO protein and pU5-PEA of a light-operated gene expression system capable of expressing PEA. The targeting biological material for modifying the liposome is fused on the liposome to form a liposome compound pPEA (at) GPH-L entrapping the pseudomonas aeruginosa exotoxin light-operated gene and the liposome targeting material. The liposome disclosed by the invention can target tumor cells, has great lethality on the tumor cells, is safe and effective, and has important significance in preparation of anti-tumor medicines, especially medicines for treating breast cancer.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a plasmid-targeted liposome complex, a preparation method and use thereof. Background Art

[0002] Breast cancer is now the most common cancer in the world, second only to lung cancer, and is the leading cause of cancer death in women worldwide, accounting for approximately one quarter of all new cancer cases worldwide and 13-15% of all cancer-related deaths. The incidence of breast cancer is closely related to economic development, and it is expected that the incidence of breast cancer in the current economic transition regions of the world will increase significantly. Breast cancer is classified by molecular subtypes into Luminal A, Luminal B, HER2-OE and triple negative. Triple negative breast cancer (TNBC) is the most aggressive breast cancer subtype, with a poor prognosis and the highest recurrence rate within 5 years.

[0003] The treatment of breast cancer is mostly surgery, radiotherapy combined with chemotherapy, and neoadjuvant therapy. In terms of chemotherapy, the chemotherapy drugs approved in my country are: platinum; anthracyclines, taxanes, antimetabolites, and cytoskeletal inhibitors. Chemotherapy drugs were originally an effective treatment method due to rapidly dividing cells such as tumors. However, even if these drugs have a certain efficacy in cancer treatment, their application is limited. The main disadvantage is that the division rate of tumor cells is not necessarily related to the proliferation of cancer cells, so the effect of chemotherapy drugs is poor due to the enrichment of cancer stem cells, which is closely related to disease recurrence and systemic side effects on living organs. Chemotherapy drugs also exhibit low water solubility, nonspecific tumor targeting, rapid elimination in the body, and severe side effects. Chemotherapy resistance and enrichment of tumor stem cells after chemotherapy are very common manifestations in breast cancer patients, increasing the possibility of metastasis and tumor formation.

[0004] For neoadjuvant therapy, there are anti-HER2 therapy, other targeted therapy, endocrine therapy, and bisphosphonate therapy. Within each type, there are several treatment options. Most options are recommended in the relevant guidelines of official medical authorities around the world. For other targeted therapies, the recommended dosing schedule is specified as neoadjuvant for pembrolizumab and adjuvant for abemaciclib. Endocrine therapy for estrogen receptor (ER)-positive disease - tamoxifen and aromatase inhibitors (AI). For premenopausal women, ovarian suppression or ablative therapy is used. Endocrine therapy for ER-positive breast cancer is usually recommended after surgery, but neoadjuvant delivery can be performed for women with a low risk of recurrence. Bisphosphonates are recommended for adjuvant therapy in postmenopausal women. For most women after breast-conserving surgery, adjuvant radiation therapy is recommended. This includes whole-breast radiation therapy, sometimes with additional radiation enhancement to the tumor bed, or partial breast radiation therapy. Recommended radiation therapy after mastectomy includes the chest wall. Radiation therapy to regional lymph nodes is recommended after breast-conserving surgery or mastectomy. Most breast cancer patients have limited treatment options. Therefore, there is an urgent need to develop new and effective therapies with high targeting specificity and low tumor toxicity to meet the medical needs of breast cancer patients. Traditional chemotherapy treatment methods have insufficient specificity, high risk of drug resistance, and serious side effects. Therefore, it is extremely necessary to develop new strategies for breast cancer treatment. Summary of the invention

[0005] One of the purposes of the present invention is to provide a plasmid-targeted liposome complex.

[0006] The second object of the present invention is to provide a method for preparing a plasmid-targeted liposome complex.

[0007] The third object of the present invention is to provide a plasmid-targeted liposome complex or a use of the plasmid-targeted liposome complex prepared by the method described above in the preparation of tumor drugs.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] A plasmid-targeted liposome complex is characterized in that it includes liposomes and plasmid pPEA entrapped by the liposomes, wherein the plasmid pPEA includes an expression vector pGAVPO containing a blue light-induced transcription factor GAVPO protein and pU5-PEA, a light-controlled gene expression system capable of expressing PEA.

[0010] Preferably, the liposome is fused with a targeting biological material for modifying the liposome.

[0011] Preferably, the ratio of pGAVPO to pU5-PEA is one of 1:10, 1:5, 1:1, 5:1 and 10:1.

[0012] Preferably, the pPEA is coated with a cationic material.

[0013] Preferably, the targeting biomaterial comprises 1,3-glycerol distearate (GDS), thiolated hyaluronic acid (HA) and bifunctional polyethylene glycol (MAL-PEG 2000 -NHS) and synthesized by substitution reaction.

[0014] Preferably, the particle size of the liposome complex is 121.6±3.6 nm, and the concentration is 0.01-0.5 mg / mL.

[0015] In another aspect, the present invention provides a method for preparing a plasmid-targeted liposome complex, comprising the following steps:

[0016] (a) Preparation of targeted biomaterial GDS-PEG-HA;

[0017] (b) constructing the light-controlled gene pPEA and extracting and purifying the light-controlled gene;

[0018] (c) preparing pPEA-cation complex;

[0019] (d) Preparation of liposome complex pPEA@GPH-L, first preparing blank liposome L, fusing GDS-PEG-HA on the blank liposome L, and then encapsulating the pPEA-cationic complex in the blank liposome L with targeting material; or first encapsulating the pPEA-cationic complex in the blank liposome L to obtain pPEA@L, then fusing GDS-PEG-HA on pPEA@L, and finally extruding with an extruder to obtain the liposome complex pPEA@GPH-L encapsulating the Pseudomonas aeruginosa exotoxin light-controlled gene and liposome targeting material.

[0020] Preferably, the GDS-PEG-HA is prepared by the following steps:

[0021] (e) First use GDS and Mal-PEG 2000 -NHS reacts with the active ester group on NHS to obtain GDS-PEG;

[0022] (f) HA is added again, and the carboxyl groups on HA are thiolated with cysteamine to obtain HA-SH;

[0023] (g) Finally, add Mal-PEG 2000 -NHS and HA-SH, HA-SH and Mal-PEG 2000 -The maleimide group on NHS was reacted, the pH value was adjusted, and GDS-PEG-HA was obtained after dialysis in a dialysis bag.

[0024] Preferably, in step (e), EDC and DMAP are further added, and ammonia gas is introduced to mix the mixture, and then the mixture is separated by silica gel column chromatography, and the solvent is rotary evaporated.

[0025] Preferably, in step (f), equal amounts of cysteamine are added twice, and EDC and NHS are added respectively during the two additions, nitrogen is continuously introduced, the pH value is adjusted, and then the mixture is placed in a dialysis bag for dialysis to prepare a lyophilized powder for later use.

[0026] Preferably, the developing solvent for chromatography is DCM / CH3OH / triethylamine in a volume ratio of 90:10:1.

[0027] Preferably, the pH value in step (g) is 7, and the pH value in step (f) is 5.0-5.5.

[0028] Preferably, the pPEA-cationic complex is obtained by adding pPEA and a cationic material into a HEPES buffer, stirring vigorously, and incubating at room temperature for 25 minutes, and the cationic material is a chitosan solution.

[0029] Preferably, the N / P of the pPEA and chitosan solution is one of 0, 2, 4, 6, 8, 10, 12, and 14.

[0030] On the other hand, the present invention also provides the use of the plasmid-targeted liposome complex or the plasmid-targeted liposome complex prepared by the method described above in the preparation of tumor drugs.

[0031] Preferably, the tumor is gastric cancer, colorectal cancer, liver cancer or breast cancer.

[0032] Beneficial effects:

[0033] The present invention utilizes a light-controlled gene of Pseudomonas aeruginosa exotoxin that overcomes tumor drug resistance, and uses nanotechnology to encapsulate the gene, supplemented by a biomaterial with both targeting and sustained-release effects, and expresses Pseudomonas aeruginosa exotoxin to kill cancer cells through local light irradiation, prevent cancer cell metastasis, and overcome multiple drug resistance. Utilizing the significant advantages of the nano drug delivery system in structural design and modifiability, a drug-loaded liposome complex with CD44 targeting ability is prepared. The carrier also contains a PEG fragment, which can prolong the in vivo circulation time and target the tumor microenvironment to reduce the off-target effect of Pseudomonas aeruginosa exotoxin gene drug delivery in cancer cells. Overcome the drug resistance problem in the treatment of breast cancer, increase the half-life of the drug in the body, exert long-term drug efficacy, and reduce the difficulty of treating breast cancer. This new treatment strategy breaks through the existing treatment bottleneck, can reduce the patient's systemic side effects, improve the clinical treatment effect of breast cancer, and achieve safe, effective, and stable effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The specific implementation modes of the present invention will be further described in detail below in conjunction with the accompanying drawings.

[0035] Figure 1 This is the H-NMR spectrum of GDS-PEG-HA.

[0036] Figure 2 Fourier-infrared spectra of GDS-PEG, HA, HA-SH and GDS-PEG-HA.

[0037] Figure 3 This is the sequence diagram of pGAVPO.

[0038] Figure 4 This is the sequence diagram of pU5-PEA.

[0039] Figure 5 This is a diagram of the composition and mechanism of action of the light-controlled gene expression system.

[0040] Figure 6 Download the gene compression capacity graph of gene liposomes for different N / P.

[0041] Figure 7 Schematic diagram of the preparation of pPEA@GPH-L.

[0042] Figure 8 TEM results of pPEA@GPH-L.

[0043] Fig. 9 This is the particle size potential change diagram of pPEA@GPH-L.

[0044] Fig.10 The graph shows the hemolysis results of pPEA@GPH-L at different concentrations and the quantitative analysis of hemolysis rate (n=3).

[0045] Fig.11 The results of gel electrophoresis bands after different gene-carrying liposomes were treated with DNase I enzyme.

[0046] Fig.12 Figure 3 is a pPEA standard curve and a pPEA cumulative release curve in PBS at pH 7.4.

[0047] Fig.13 The graph shows the uptake of Free C6, C6@L, C6@GPH-L and C6@GPH-L+HA by 4T1 cells.

[0048] Fig.14 The figure shows the cytotoxicity of the blank vector to 4T1 cells.

[0049] Fig.15 This is a picture of liver tissue pathological section after using siNOX1-2.

[0050] Fig.16 The cytotoxicity of pPEA@Lipo 2000 at different powers with a pGAVPO to pU5-PEA ratio of 1:1 and the toxicity of the light-controlled Pseudomonas aeruginosa exotoxin gene on 4T1 cells.

[0051] Fig.17 This is a diagram showing the expression of PEA inside and outside cells after blue light irradiation.

[0052] Fig.18 These are the Transwell results of Saline (Light Off), Saline (Light On), pPEA@GPH-L (Light Off), pPEA@L (Light On) and pPEA@GPH-L (Light On) on 4T1 cells at 24 hours.

[0053] Fig.19 The migration effects of Saline (Light Off), Saline (Light On), pPEA@GPH-L (Light Off), pPEA@L (Light On) and pPEA@GPH-L (Light On) on 4T1 cells at 0h or 24h and the quantitative calculation of relative healing area.

[0054] Fig. 20 The graph shows the inhibitory effects of Saline (Light Off), Saline (Light On), pPEA@GPH-L (Light Off), pPEA@L (Light On) and pPEA@GPH-L (Light On) on 4T1 tumor spheres.

[0055] Fig.21 This is a diagram of the in vivo biodistribution study of free IR775, IR775@L, and IR775@GPH-L.

[0056] Fig. 22 The in vitro imaging and quantitative results of important organs and tumor tissues of each group of mice 24 hours after administration.

[0057] Fig.23 Figure 1 shows the BALB / c mouse model bearing 4T1 breast cancer.

[0058] Fig.24 The figure shows the in vivo anti-tumor effect.

[0059] Fig.25 These are the results of H&E, TUNEL, Ki67 and CD31 staining of tumor tissues in each treatment group.

[0060] Fig.26 These are H&E section staining images of the heart, liver, spleen, lung, and kidney of mice in each group after treatment.

[0061] Fig. 27 The figure shows the changes in mouse body weight during the treatment period.

[0062] Fig.28 This is the result graph of mouse serum AST, ALT, CRE, and BUN values ​​after administration. DETAILED DESCRIPTION

[0063] Example 1.

[0064] 1. Synthesis and characterization of GDS-PEG-HA.

[0065] 1. Synthesis of GDS-PEG.

[0066] Weigh 0.650-0.755 g of GDS (CAS: 1323-83-7) (molecular weight 625.05, 0.0012 mol), 0.200-0.230 g of EDC (molecular weight 191.70, 0.0012 mol), and 0.125-0.154 g of 4-dimethylaminopyridine (DMAP, 0.0012 mol) in an eggplant-shaped flask, and slowly drop 10 mL of deionized water. Pass ammonia gas, shake and mix thoroughly for 1 hour in the dark, and then add 0.40-0.55 g of Mal-PEG at room temperature. 2000 -NHS (molecular weight about 2000) in 10ml deionized water solution was slowly added dropwise to the reaction system. After the addition was completed, stirring was continued at room temperature for 12h. After the reaction was completed, the mixture was separated by silica gel column chromatography with DCM / CH3OH / triethylamine (90:10:1, v:v:v) as the developing solvent, and the product GDS-PEG was obtained by rotary evaporation of the solvent. GDS provides fatty chains to form a liposome bilayer structure.

[0067]

[0068] 2. Synthesis of intermediate HA-SH

[0069] Weigh 90-100 mg HA (hyaluronic acid) in 10 mL of deionized water and stir overnight to allow for complete swelling. Weigh 3.750-3.950 g EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide) and 0.55-0.10 g cysteamine ( ) is added to the reaction solution, the pH value is adjusted to 5.0-5.5, and then nitrogen is continuously introduced into the mixed system. The system is magnetically stirred for 2 hours, and 2.150-2.450 g NHS and 0.55-0.10 g cysteamine are added. The cysteamine added twice is equal. The pH value is adjusted to 5.0-5.5, and the reaction is continued. The reaction is allowed to react at room temperature for 2 hours. The reaction solution is placed in a 8000-14000 Da dialysis bag for dialyzation against deionized water for 3 days (nitrogen protection, and 0.025 mM protective agent tri(2-carboxyethyl)phosphine, i.e. TCEP, is added to the dialyzate). The solution is freeze-dried to obtain freeze-dried powder for later use.

[0070]

[0071] 3. Synthesis of GDS-PEG-HA

[0072] Take 120-180 mg Mal-PEG 2000 -GDS was stirred in 10 mL of deionized water overnight to allow it to fully swell, and 10 mL of HA-SH aqueous solution was slowly added dropwise under constant stirring, the pH value was adjusted, and the reaction was allowed to proceed for 12 h. After the synthesis was completed, the reaction solution was placed in a dialysis bag and dialyzed for 72 hours, then placed in a vial and freeze-dried, and the product was confirmed by proton magnetic resonance spectroscopy.

[0073] pass Figure 1 From the H-NMR spectrum of GDS-PEG-HA, it can be seen that the peaks of GDS-PEG and HA-SH are retained, while the peak of MAL disappears, proving that the final product is successfully synthesized.

[0074]

[0075] Infrared spectra of GDS-PEG (A), HA (B), HA-SH (C) and GDS-PEG-HA (D) Figure 2 shown. Figure 2 Figure B shows the characteristic peaks of HA. The stretching vibration of OH is at 3400 cm -1 Nearby, the stretching vibration of CH is at 2920 cm -1 Nearby, the stretching vibration of amide C=O is at 1640 cm -1 Nearby, the bending vibration of CH is at 1343 cm -1 Nearby, and 1107cm -1 It is the stretching vibration of COC. A and D show that by comparing the infrared analysis results of GDS-PEG-HA and GDS-PEG, -CH2- is at 945 cm -1 The stretching vibration at 1029 cm -1 The stretching vibration at 1602 cm-1 The stretching vibration at the position is significantly enhanced, which proves that HA is successfully connected to PEG. It proves that the synthesis of GDS-PEG-HA is successful, which is abbreviated as GPH.

[0076] 2. Construction, extraction and purification of light-controlled gene expression system

[0077] 1. Construction of light-controlled genes

[0078] (1) Construct an expression vector pGAVPO containing the blue light-induced transcription factor GAVPO protein, such as Figure 3 As shown; the CMV promoter in pCDNA3.1 (produced by Invitrogen) was recombined with the GAVPO fragment to form pGAVPO, the specific sequence of GAVPO is shown in SEQ ID NO: 1:

[0079] MKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTKRSPLTRAHLTEVESRLERLERSIAATRSHTLYAPGGYDIMGYLIQIMKRPNPQVELGPVDTSVALILCDLKQKDTPIVYASEA FLYMTGYSNAEVLGRNCRFLQSPDGMVKPKSTRKYVDSNTINTMRKAIDRNAEVQVVVNFKKNGQRFVNFLTMIPVRDETGEYRYSMGFQCETELQYPYDVPDYAEFQYLPDTDDRHRIEEKRKRT YETFKSIMKKSPFSGPTDPRPPPRRIAVPSRSSASVPKPAPQPYPFTSSLSTINYDEFPTMVFPSGQISQASALAPAPPQVLPQAPAPAPAMVSALAQAPAPVPVLAPGPPQAVAPPAPKPTQAG EGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNSEFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGDEDFSSIADMDFSALLSQISSDYKDDDDK.

[0080] (2) The reporter plasmid pU5-pPEA containing the light-controlled fragment 5×UASG capable of expressing PEA (Pseudomonas aeruginosa exotoxin A fragment) was constructed by site-directed mutagenesis. Figure 4 The specific sequence of PEA is shown in SEQ ID NO: 2:

[0081] .

[0082] The light-controlled gene consists of a light-sensitive transcription factor GAVPO and a transcription unit that expresses the target gene (pU5-GOI). Under blue light irradiation, the transcription factor can homodimerize and bind to its specific promoter, thereby expressing the target gene. Figure 5 As shown. The GAVPO protein expressed by the pGAVPO gene transcription will form a dimer after blue light irradiation, and then bind to the 5×UASG fragment on the pU5-GOI plasmid to trigger the transcription and expression of the target gene. When there is no blue light irradiation, the GAVPO protein will not undergo dimerization, avoiding binding to the 5×UASG fragment and interrupting the expression process. The light-controlled gene expression system achieves precise and controllable expression of the target gene by regulating light.

[0083] The blue light-induced transcription factor (pGAVPO) and the transcription unit of the target gene (pU5-PEA) together constitute the light-controlled gene expression system pPEA. After pGAVPO enters the cell, it first expresses the GAVPO protein through the CMV promoter. Under the stimulation of blue light irradiation, the GAVPO protein forms a reversible dimer, merges the 5×UASG (U5) fragment located in the pU5-PEA unit, and then transcribes the gene fragment. Under the condition of blue light irradiation, the system can construct the Pseudomonas aeruginosa exotoxin light-controlled gene pPEA (pGAVPO and pU5-PEA) by combining the encoded PEA gene (pU5-PEA) with the blue light-induced transcription factor (pGAVPO).

[0084] 2. Extraction and purification

[0085] Take 1 μL of the plasmid to be extracted (pPEA) and transfer it into 100 μL of E. coli competent cells, incubate it at 37 °C for 2h on LB medium, and then screen the cells on the corresponding resistance plate. Isolate the positive colonies by overnight culture and continue to incubate in LB medium conical flasks. After 24 hours, centrifuge the bacteria and extract and purify the plasmid according to the instructions of the high-purity plasmid extraction kit. The cells were lysed by the modified SDS-alkaline lysis method. The silica matrix membrane in the centrifugal adsorption column selectively binds the plasmid DNA in the solution under high salt and low pH conditions, thereby adsorbing impurities, while other bacterial components are removed by protein removal solution and rinsing solution. Finally, a low salt and high pH elution buffer was used to elute the purified plasmid DNA from the silica-based membrane. The extracted plasmid was stored in a -20 °C refrigerator for a long time.

[0086] 3. Agarose gel electrophoresis experiment

[0087] First, gene-carrying liposomes with different nitrogen-phosphorus ratios were prepared. According to the planned N / P ratio, appropriate amounts of pPEA and cationic materials were added to the HEPES buffer. The cationic materials included protamine, polyethyleneimine, ε-polylysine or chitosan, preferably chitosan. The pPEA and chitosan solutions were stirred vigorously and incubated at room temperature for 20 minutes to obtain pPEA-chitosan complexes. 9 mg of egg yolk phosphatidylcholine (PC) and 1 mg of cholesterol (Chol) were placed in a 50 mL 21-mouth round-bottom (or eggplant-shaped) flask filled with chloroform at 55°C and 80 rpm, and the chloroform was removed by rotary evaporation to obtain a transparent and uniform liposome membrane. 2 mL of chitosan-gene complex PBS hydrated liposome membrane solution containing a NP ratio of 0 (i.e., chitosan solution without plasmid) was added to the round-bottom flask containing the liposome membrane, and incubated uniformly in a 50°C water bath for 30 minutes. Then the lipid mixture was placed in a water bath of an ultrasonic cleaner for 30 minutes, and finally the liposomes were placed in a probe ultrasonicator for 5 minutes (10%, 10 minutes, 1 second working, 1 second interval) under ice bath conditions. The gene-loaded liposomes pPEA@L were obtained for the first time, and the preparation method of the gene-loaded liposomes with NP ratios of 0, 2, 4, 6, 8, 10, 12, and 14 was the same as above.

[0088] Agarose (2% w / v) solution was dissolved in a 1L volume conical flask containing 200mL TBE buffer to prepare agarose gel, and the mixture was heated in a microwave for 3 minutes. After boiling, it was cooled briefly, and the conical flask was carefully shaken with insulated gloves to fully dissolve the agarose, and then continued to microwave. Repeat the above steps to make the agarose particles disappear completely and form a transparent and uniform hot agarose TBE solution. When the agarose solution is cooled to about 50°C, the nucleic acid dye DuRed (20μL) is added, and the mixture is poured into the gel plate, covered with a dustproof plate, and cooled at room temperature for 20 minutes to wait for the agarose gel to solidify.

[0089] Carefully place the solidified agarose gel into a gel electrophoresis instrument filled with TBE buffer. The mass of the gene added to each sample well is 2 μg. The samples are divided into 8 groups, and the NPs are 0, 2, 4, 6, 8, 10, 12, and 14. Add 5 μL of loading buffer to each sample, mix well, and add to the sample well. Connect the electrophoresis instrument to the electrophoresis tank, set the electrophoresis voltage to 110V, and ensure that the current is above 40 mA during electrophoresis. When the band is about 2 cm away from the front end of the gel, stop the power supply, develop the DNA band fragments of each group under ultraviolet light, observe the development results with an ultraviolet imager, and take photos to record.

[0090] Agarose gel electrophoresis can be used to observe the compression of genes, such as Figure 6As shown in the figure, as the nitrogen-phosphorus ratio increases, the brightness of the gene running out of the sample well becomes lower and lower, and gradually only exists in the sample well. When the nitrogen-phosphorus ratio exceeds 4, pPEA is completely compressed. Therefore, N / P=4 was used in subsequent experiments to prepare gene-carrying liposomes.

[0091] Different nitrogen-phosphorus ratios will affect the compression capacity of genes. When the amount of genes remains unchanged, when cationic materials are added, the N / P ratio increases, resulting in an enhanced compression capacity of genes. If the N / P value is low, the genes cannot be effectively compressed, resulting in insufficient transfection efficiency; if the N / P value is too large, the gene drug may not be released and the transfection efficiency is not high. At the same time, the physical and chemical properties of gene-carrying materials are significantly affected by the nitrogen-phosphorus ratio (N / P). Generally, a higher nitrogen-phosphorus ratio can improve gene transfection efficiency and vector stability, but it will also increase cytotoxicity. Therefore, correctly calculating the ratio of positive and negative charges of the gene complex and selecting an appropriate N / P ratio are two particularly critical factors in gene drug delivery.

[0092] 4. Preparation and Validation of Liposomes

[0093] 1. Preparation of plasmid-targeted liposomes

[0094] Dissolve 9 mg of egg yolk phosphatidylcholine (PC) and 1 mg of cholesterol (Chol) in chloroform, evaporate under reduced pressure at 50°C and 80 rpm to remove chloroform, and obtain liposome membrane. Add 2 mL of PBS to hydrate the film, and incubate in a 60°C water bath for 30 min. The incubated liposomes are ultrasonicated in an ultrasonic cleaner semi-immersed in a water bath for 30 min, and finally ultrasonicated in an ice bath with a probe ultrasonicator for 5 min (10%, 10 min, working 1 s, interval 1 s). After the ultrasonication, squeeze through a 220 nm membrane, and repeat 12 times to obtain the control liposome L.

[0095] According to the best NP ratio obtained, pPEA plasmid and chitosan solution according to the best NP ratio were added to HEPES buffer (1X) and incubated at room temperature for 25 minutes after vigorous stirring to obtain pPEA-chitosan complex. The liposome membrane was prepared by thin film dispersion method. 22.5 mg of egg yolk phosphatidylcholine (PC) and 2.5 mg of cholesterol (Chol) were weighed in a 50 mL eggplant-shaped bottle for making blank liposome L, and 10 mL of chloroform (as a solvent, used to dissolve the liposomes, and then evaporated to dryness by rotary evaporation to form a liposome film on the flask) and GDS-PEG-HA (5 mg / ml) were added. After complete dissolution, CHCl3 was removed on a rotary evaporator at 50-55 ° C until a uniform lipid film was formed. The prepared pPEA-chitosan complex was added and shaken evenly in a 55 ° C water bath until the lipid film was evenly detached, and then hydrated for 60 minutes under rotary evaporation. Alternatively, the pPEA-cation complex can be first encapsulated in a blank liposome L to obtain pPEA@L, and then GDS-PEG-HA is fused to pPEA@L. The cell is treated with an ultrasonic cell disruptor in an ice bath (10%, 20 min, 1 second on, 1 second off), and then the extruder passes through a 220 nm filter membrane 10 to 20 times, preferably 13 times, to form pPEA-loaded lipid pPEA@GPH-L (liposomes encapsulating Pseudomonas aeruginosa exotoxin light-controlled gene and liposome targeting material) with tumor angiogenesis targeting function. The pPEA@GPH-L production process is as follows: Figure 7 shown.

[0096] HA binds to CD44 receptors highly expressed on the cell surface of various tumors (gastric cancer, colorectal cancer, liver cancer, breast cancer, etc.), increasing the ability to target tumor cells. By combining HA with cationic nanostructures through electrostatic interaction, the cytotoxicity of gene-loaded liposome complexes can be reduced. HA will be degraded by hyaluronidase highly expressed in tumor tissues, releasing the gene complex from the liposomes. Modifying HA with polyethylene glycol (PEG) can prevent it from being captured and cleared by macrophages, increase the residence time of liposomes in the body, and play a role in long circulation in the body.

[0097] Reference preparation: pPEA-loaded liposomes (pPEA@L), prepared as described in the preparation of plasmid-loaded liposomes, without adding GDS-PEG-HA during film formation.

[0098] 2. Particle size and Zeta potential characterization

[0099] This patent uses Malvern dynamic light scattering particle size analyzer and Zetasizer Software to measure the particle size, polydispersity index (PI) and Zeta potential of the prepared liposomes. The detection of Malvern dynamic light scattering particle size analyzer is based on the principle of dynamic light scattering. Simply put, the detection of particle size is achieved by measuring the fluctuation of particle scattered light caused by the Brownian motion of nanoparticles. The basic principle is that the rapid Brownian motion of small particles causes the scattered light to fluctuate rapidly, while the slow Brownian motion of large particles causes the scattered light to fluctuate slowly. The model of the particle size pool is Disposable folded capillary (DTS1070). The Zetasizer Software is set to an aqueous phase system, the equilibrium temperature is 25°C, and the equilibrium time is 120 seconds, and finally Table 1 is obtained.

[0100] Table 1: Physicochemical properties of pPEA@GPH-L

[0101] Nanocarrier Particle size (nm) Zeta potential (mV) PI L 105.5±2.1 -19.9±0.5 0.253 GPH-L 109.1±1.8 -20.1±1.2 0.288 pPEA@GPH-L 121.6±3.6 -18.9±2.7 0.216 PI: polydispersity index. Data are presented as mean±SD (n=3)

[0102] As shown in Table 1, the obtained control liposome particle size was 105.5±2.1nm, PI was 0.253, and Zeta Potential was -19.9±0.5mV. After inserting the targeting material GPH, the particle size of GPH-L (liposome containing liposome targeting material) increased slightly to 109.1±1.8nm. After loading the gene particles, the particle size of pPEA@GPH-L increased by 11.5% compared with GPH-L, while the potential only increased by 1.2mV, proving that the core of the chitosan gene complex was completely encapsulated.

[0103] 3. Morphological characterization (TEM)

[0104] A drop of 50 μL of pPEA@GPH-L sample solution (1 mg / mL) was carefully dripped onto the center of a TEM grid with carbon-coated copper and incubated at 37°C for 20 minutes in a closed environment of a humidified container. Excess liquid was carefully removed from the edge of the copper grid with a clean qualitative filter paper, and then freshly prepared 1% phosphotungstic acid was dripped onto the center of the copper grid for 5 minutes. Excess liquid was then carefully removed from the edge of the copper grid with a clean qualitative filter paper. Observe with a transmission electron microscope (TEM), select a suitable field of view and take a picture.

[0105] TEM results are as follows Figure 8As shown. pPEA@GPH-L is a regular sphere with a distinct core-shell bilayer structure and is shiny overall. In the TEM image, the particle size of pPEA@GPH-L is about 80 nm. This is because the Malvern particle sizer measures the polymerized hydration radius of the liposomes, while TEM measures the slightly compressed morphology of the liposomes in a dehydrated state. At the same time, the Malvern particle sizer is a macroscopic statistical result, calculated based on Stokes-Einstein, while the TEM measurement is a microscopic result.

[0106] 4. Storage stability test

[0107] pPEA@GPH-L was stored in a refrigerator at 4°C for 4 weeks, and the physical storage stability of pPEA@GPH-L was characterized on days 1, 2, 3, 5, 7, 14, and 28, and then the particle size and Zeta potential were measured. The storage stability of the liposome complex was evaluated by measuring the changes in particle size and Zeta potential during storage.

[0108] Fig. 9 The graph of the particle size potential change of the gene-carrying membrane fusion liposome complex pPEA@GPH-L shows that the particle size of pPEA@GPH-L is relatively stable when stored at 4°C, with an average particle size of about 129.3±7.2nm and an average potential of about -20.0±1.3mV, which remains stable within 28 days.

[0109] 5. Biocompatibility test

[0110] Mouse blood was obtained by removing the eyeballs. 6-8 week old Balb / c female mice were taken, and the beards of the experimental animals were cut off with surgical scissors in advance to prevent blood from accidentally contaminating the beards and causing hemolysis during the blood collection process. During the blood collection process, the scalp of the mouse neck was grasped with the thumb, index finger, and middle finger of the left hand, and the tail was fixed with the little finger and ring finger. Gently press the skin around the eyeball to be removed to make the eyeball congested and protruding. Use tweezers to deeply clamp the eyeball, quickly pull out the eyeball, and let the blood flow from the eye socket into the anticoagulant EDTA tube containing sodium heparin prepared in advance. When the mouse's blood dripping speed slows down, the mouse heart can be gently pressed to speed up the heart's pumping speed to obtain more blood, and the mouse was killed by dislocating the neck. When squeezing the heart, the force should be moderate. Excessive force will not only cause the animal to die midway, making the blood collection process incomplete, but also cause hemolysis, affecting the experimental results.

[0111] The mouse blood was centrifuged at 900xg (4°C) for 10 minutes, the supernatant was discarded, the pellet obtained by centrifugation was washed twice, and carefully resuspended with 0.9% NaCl (w / v). A 4% (w / v) red blood cell (RBC) solution was prepared with 0.9% NaCl in a collection tube (2.5 mL volume), and 50 μL of pPEA@GPH-L samples with concentrations of 0.01, 0.1, 0.5, and 1 mg / mL were added to the red blood cell (RBC) solution, followed by incubation at 37°C for 30 minutes, centrifugation at 800xg for 10 minutes, and 100 μL of the supernatant was transferred to a 96-well plate, set at a UV absorption wavelength of 540 nm, and measure the absorbance of the supernatant of each group. A negative control (0.9% (w / v) NaCl) (0% hemolysis) and a positive control (ultrapure water) (100% hemolysis) were included to calculate the relative hemolysis rate.

[0112] like Fig.10 As shown in A, it can be observed that when the concentration of pPEA@GPH-L is less than or equal to 0.5 mg / mL, no serious hemolysis occurs; then the relative hemolysis rate at different concentrations is calculated, as shown in Fig.10 The results of B show that when the concentration of pPEA@GPH-L increases, the hemolysis rate gradually increases. At a concentration of 1 mg / mL, the hemolysis rate of pPEA@GPH-L is 16.62±0.19%, indicating that its biocompatibility is poor. Therefore, the upper limit of the concentration of pPEA@GPH-L in subsequent experiments is 0.5 mg / mL.

[0113] 6. Enzyme stability test of pPEA@GPH-L

[0114] Protecting pDNA in liposome complexes from nuclease degradation is one of the important conditions for the success of gene delivery.

[0115] Therefore, the nuclease resistance of pPEA@L and pPEA@GPH-L liposomes was studied. 2 μL of DNase I buffer (containing 50 mM Tris-HCl buffer system at pH 7.5 and 10 mM MgCl2) was added to the nucleic acid-loaded liposomes (containing 10 μg pPEA) prepared above and mixed well, and then 10 U / μL of DNase I buffer and 1 μL of 0.9% NaCl solution were added to make a DNase I (1 U / μg pDNA) solution and mixed well. In a parallel operation of the control group, 10 μL of pPEA was taken, and 19 μL of DNase I buffer and 1 μL of 10 U / μL DNase I were added to the 0.9% NaCl solution (1 U / μg pDNA) and mixed well. The above mixed system was incubated on a 37°C water bath constant temperature oscillator at a frequency of 100 rpm for 30 min. After the reaction was completed, 5 μL of ethylenediaminetetraacetic acid (0.5 M, pH 8.0) was added and incubated for 10 min to terminate the reaction. Heparin was added to the liposomes to make the final concentration of heparin in the mixed system reach 1%, and the mixture was shaken at 37°C for 3 hours. The plasmid pPEA in the liposomes all entered the free state solution, and then electrophoresed on agarose gel.

[0116] like Fig.11 As shown, 1 is pPEA + DNase I; 2 is pPEA; 3 is pPEA@L + DNase I; 4 is pPEA@GPH-L + DNase I.

[0117] In addition to the stability of the vector itself, the most important prerequisite for liposome-based gene delivery is to protect the gene from being decomposed by gene metabolic enzymes in the metabolic system. Fig.11 According to the experimental results, there is no significant difference between the complete gene band and the control group that was not treated with DNase I. After the free group gene was damaged by DNase I, no complete band was found during gel electrophoresis. Further analysis of each gene vector group showed that the electrophoresis results of the pPEA@GPH-L group were brighter. This is because the targeted polymer material GPH was fused to the liposome, blocking the exposure of the gene and improving the stability of the vector. Therefore, it can be concluded that the surface-modified gene-carrying liposomes can better protect genes from DNaseI degradation in vivo and improve the stability of genes in vivo.

[0118] 7. In vitro gene release experiment

[0119] First, draw a standard curve, take Hochest 33258 solution (0.5 mg / mL, 30 μL) and place it in a brown opaque 100 mL volumetric flask, then add TNE buffer (1X, pH7.4) to obtain Hochest diluent (0.15 μg / mL). Add 20 μg pPEA gene to 2 mL Hochest diluent, vortex to mix, and measure its maximum excitation and emission wavelengths respectively. Take pPEA gene (250 μg) in a 10 mL volumetric flask, add Hochest 33258 diluent to make up to volume, and obtain pPEA gene solution (25 μg / mL), then take 0.02, 0.1, 0.2, 0.4, 0.8, 1.2, 1.6 and 2 mL of pPEA gene solution and place it in a 5 mL volumetric flask, and add TNE buffer to make up to volume. Measure the fluorescence intensity by a fluorescence spectrophotometer (excitation wavelength: 365 nm, emission wavelength: 460 nm) to obtain a standard curve.

[0120] An appropriate amount of pPEA@L and pPEA@GPH-L was placed in a PBS (pH 7.4) release medium to make the pPEA concentration 150 μg / mL. The mixture was shaken at 300 rpm in a water bath at 37°C. Then, at different time points (1 h, 2 h, 4 h, 8 h, 24 h, 48 h and 72 h), 200 μL of PBS release solution was taken out from the release medium and refrigerated. After centrifugation at 14,000 x g for 25 min at 4°C, 100 μL of supernatant was added to Hochest 33258 diluent to make the volume 2 mL. The fluorescence intensity was detected by a fluorescence spectrophotometer. The concentration was calculated according to the gene concentration standard curve fitting formula. The remaining 100 μL PBS release solution and the centrifugal precipitate were returned to the release container of each group. 100 μL PBS (pH 7.4) buffer was added. The cumulative release rate was obtained according to the calculation result of the gene concentration, and a cumulative release rate line graph was prepared.

[0121] like Fig.12 As shown in A, the standard curve of pPEA was first measured to be y=171.7x–2.6443, R2=0.9984. The in vitro release curve of pPEA@GPH-L is shown in Fig.12 As shown in Figure B, pPEA@L has a faster release rate, with a cumulative release rate of 63.65±3.30% at 72h, while the cumulative release rate of pPEA@GPH-L at 72h is 43.56±3.0%. This is because the GDS-PEG-HA fused to the surface of the liposome is not easy to leak out of the liposome under the electrostatic effect of the negative charge of the HA carboxyl group. At this time, the biological material on the surface of the macromolecular lipid material makes it more difficult to separate from the carrier, thereby reducing its release rate. Therefore, pPEA@GPH-L has good sustained-release properties.

[0122] All data were analyzed using Prism 9.0 one-way ANOVA, and differences between multiple groups were analyzed using one-way ANOVA and Tukey's multiple comparison analysis. Unless otherwise specified, data are mean standard deviation. Test values ​​*p < 0.05, **p < 0.01 and ***p < 0.001 indicate significant differences.

[0123] Example 2

[0124] In vitro biological evaluation of pPEA@GPH-L

[0125] 1. Culture of 4T1 breast cancer cells

[0126] Preparation of RPMI-1640 complete medium: Under sterile conditions, add 50 ml of thawed fetal bovine serum (FBS) and 5 ml of penicillin / streptomycin double antibody to 500 mL of RPMI-1640 medium, mix well and store in a 4°C refrigerator away from light to avoid repeated freezing and thawing.

[0127] Recovery of 4T1 cells: Gently shake the cryovial in a 37°C water bath. To reduce the possibility of contamination, do not immerse the O-ring and cryovial cap in water. To prevent ice crystals from damaging the cells during thawing, the thawing process should be rapid (approximately 2 minutes). Remove the vial from the water bath once the contents are completely thawed and decontaminate by dipping or spraying with 70% ethanol. Start aseptic operation from then on. Transfer the contents of the cryovial to a centrifuge tube containing 9.0 mL of complete medium and centrifuge at approximately 125xg for 5 to 10 minutes. Resuspend the cell pellet in complete medium (pH 7.0 to 7.6) that has been pre-incubated in the incubator for at least 15 minutes and then dispense into 25 cm 2 or 75cm 2 The purpose of this step is to prevent the medium from being too alkaline. The incubator should be a sterile incubator with 5% CO2 and 37°C.

[0128] Medium changes for 4T1 cells: Replace complete medium every 2 to 3 days.

[0129] Passaging of 4T1 cells: When 4T1 cells grow to 80% of the density of the culture medium, they should be subcultured. Remove the culture medium and wash with 0.25% trypsin-0.53mM EDTA solution. Remove the solution and add another 1-2mL of trypsin-EDTA solution. Incubate the culture flask at room temperature (or 37°C) until the cells are detached from the bottom of the culture flask. Add fresh culture medium, blow dry cells, and centrifuge the cell suspension at approximately 125xg for 5 to 10 minutes. Discard the supernatant in the centrifuge, gently blow the cell pellet with 9.0mL of complete culture medium, make it completely resuspended, and then aliquot into new culture flasks.

[0130] Cryopreservation of 4T1 cells: Store in liquid nitrogen vapor phase, not submerged in liquid nitrogen at -70°C. Storage in liquid nitrogen at -70°C will result in loss of viability.

[0131] 2. Construction of light-controlled lighting device

[0132] Use blue LED light (460nm) to build an irradiation light plate of the same size as the 96-well plate, and use electromagnetic relays to switch and control blue light irradiation to achieve different light intensities.

[0133] 3. Cellular uptake experiment

[0134] Fluorescent nanoparticle preparation: Coumarin 6-encapsulated liposomes (C6@L), that is, 10 μg / mL of C6 was added to CHCl3 in which lipids were dissolved, and the rest of the operation was completed in the dark to obtain C6@L and C6@GPH-L (liposomes encapsulating coumarin 6 and targeting materials). The operation of encapsulating IR775 nanoparticles was the same as that of C6, to obtain IR775@L and IR775@GPH-L (liposomes encapsulating IR775 and targeting materials).

[0135] Breast cancer 4T1 cells in the logarithmic growth phase were evenly plated in a 24-well cell culture plate at a density of 1×105 cells / well and cultured overnight. After overnight culture, the culture medium was discarded, the cells were washed 3 times with pre-cooled PBS, Free C6, C6@L liposomes and C6@GPH-L liposomes were added, and the C6 concentration was 10μg / mL. The cells were incubated for 4 hours, washed several times with pre-cooled sterile PBS at about 4°C, and the nuclear dye Hoechst 33258 (nuclei Dye) was added at 10 μg / mL. The cells were incubated at 37°C for 10 minutes, washed twice with ice-cold PBS, and 500μL of paraformaldehyde (4%, w / v) was added to each well. The cells were fixed for 10 minutes, and then the paraformaldehyde was washed with PBS. The anti-fluorescence quencher was dripped to just cover the bottom surface of the center of each sample well. Cell uptake was observed and photographed using a fluorescence microscope. In addition, a control well was set up, 1 mg / mL HA was added in advance and incubated for 30 minutes before adding C6@GPH-L liposomes, and subsequent operations were referred to the above.

[0136] Free C6, C6@L, and C6@GPH-L were used to study cellular uptake. Fig.13As shown. After staining, the cell nucleus is blue, and green is the fluorescence of C6. It is easy to find that Free C6 tumor cells have almost no uptake, and the C6@L group shows a certain amount of fluorescence, which is because liposomes can promote cell uptake. The green fluorescence intensity of the C6@GPH-L group is higher than that of the C6@L group, because HA can increase the amount of protein entering the cell through the CD44 receptor on the surface of the tumor cell membrane, thereby enhancing the ability of cell uptake. To further verify the mechanism, the cells were incubated with 1 mg / mL HA for 30 minutes in advance, and it was found that the treated cells were basically unable to uptake C6, indicating that C6 was taken up into the cell-mediated endocytosis through the CD44 receptor.

[0137] 4. CCK-8 cell survival rate experiment

[0138] Cell Counting Reagent-8 (CCK-8 for short) is a rapid, highly sensitive, non-radioactive colorimetric detection kit based on (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonylbenzene)-2H-tetrazolyl monosodium salt) (WST-8), which is widely used for cell proliferation, counting and toxicity detection. CCK-8 reagent can be added directly to the cell sample without pre-mixing the various components. In the presence of electron coupling agents, WST-8 can be reduced by some dehydrogenases in the mitochondria to produce orange-yellow formazan. The more the cells proliferate, the faster the color develops and the darker the final color result. The greater the cell toxicity, the lower the absorbance. For the same cell, the color depth (formazan production) is linearly related to the number of cells. CCK-8 is used to study the toxicity of breast cancer cells under different conditions. Take 4T1 cells in the logarithmic growth phase and digest them with trypsin. 200μL / well (10,000 cells / well) were inoculated in a 96-well plate with an inoculation density of about 10,000 4T1 cells / well and cultured overnight. The culture medium was discarded and the samples to be tested were added respectively. Three parallel experimental wells were set for each sample, and RPMI-1640 culture medium was used as a blank control, and the cells were incubated in the incubator for 24 h. After the culture was completed, the culture medium was discarded, and 10μL of the newly prepared CCK-8 toxicity detection solution was added respectively, and the cells were incubated in the incubator for another 4 hours. After incubation, the cells were shaken for 1 minute with an ELISA reader, and the absorbance was measured at a wavelength of 450nm to calculate the cell specific activity.

[0139] To investigate the toxicity of blank liposomes Lipo and liposomes containing liposome targeting materials (GPH-L) on 4T1 tumor cells, the final concentrations of blank liposomes Lipo and GPH-L were 10, 50, 125, 250, 500, and 1000 μg / mL, respectively, and then administered and incubated for 48 hours (n=3).

[0140] like Fig.14As shown, blank liposomes (Lipo) and GPH-L were less toxic to cells, and at high concentration (1000 μg / mL), the cell viability of blank liposomes (Lipo) was 79.6±2.9%, while that of GPH-L was 83.4±2.2%, indicating that the carrier had good biocompatibility.

[0141] To investigate the toxicity of blue light power to 4T1 cells, serum-free RPMI-1640 culture medium was added to each well, and then the well plate was placed under LED gradient blue light intensity (0.3, 0.6, 1, 1.3, 1.6, 2 W / m 2 ) were incubated in an incubator for 48 h (n=3).

[0142] To study the toxicity of pGAVPO and pU5-PEA at different ratios on cells, Lipo 2000 was used to load pGAVPO and pU5-PEA at different ratios at 1.3 W / m 2 The cells were cultured under blue light for 48 h (n=3).

[0143] To study the toxicity of different light intensities on cells under the optimal ratio of pGAVPO to pU5-PEA, Lipo 2000 was used to carry the optimal ratio of pGAVPO to pU5-PEA and the cells were exposed to gradient blue light (0.3, 0.6, 1, 1.3, 1.6, 2 W / m 2 ) for 48 h (n=3).

[0144] To investigate the cytotoxicity of pPEA, pPEA@L, and pPEA@GPH-L to 4T1 cells, pPEA, pPEA@L, and pPEA@GPH-L were diluted with serum-free RPMI-1640 medium and mixed with 1 μg of pGAVPO and 1 μg of pU5-PEA in each well. The cells were then incubated in a dark room or under blue light irradiation (1.3 W / m 2 ) conditions for 48 h (n=3).

[0145] The light-controlled gene expression system designed in this patent is triggered by blue light induction, and blue light irradiation must be performed during the treatment process. Therefore, the effect of blue light on the viability of 4T1 cells is also a factor that we must examine.

[0146] like Fig.15 As shown in A, the effect of different blue light powers on the viability of 4T1 cells was studied. The power range selected in this experiment was 0.3W / m 2 Up to 2W / m 2 The results show that the highest blue light power (2W / m 2 ), the cell survival rate was 89.7%, indicating that within the irradiation power range of this experiment, blue light had no significant effect on the survival rate of 4T1 cells.

[0147] Then, the toxicity of the mixed ratio of pGAVPO and pU5-PEA to 4T1 cells was studied. pPEA and pPEA embedded in Lipo 2000 were selected, and the mixing ratio of pGAVPO and pU5-PEA was set to 10:1, 5:1, 1:1, 5:1 and 10:1. Fig.15 From the results of B, we can see that when the mixing ratio of pGAVPO and pU5-PEA is 1:1, the cell survival rates of free genes and commercially available cationic liposomes Lipo 2000 are 40.3±4.4% and 23.5±1.2%, respectively, which are the lowest among all experimental groups, indicating that the mixing ratio of pGAVPO and pU5-PEA is 1:1, which has the best cell killing effect. Subsequent experiments all use a 1:1 dosing ratio.

[0148] In order to further determine the blue light irradiation power that can produce the best drug delivery effect, under the condition of a pGAVPO and pU5-PEA ratio of 1:1, commercially available cationic liposome Lipo 2000 was used to carry the pPEA gene - its cytotoxicity was studied under different blue light powers. Fig.16 As shown in A, the toxicity of Pseudomonas aeruginosa exotoxin to 4T1 cells increases with the increase of blue light irradiation power, and ... 2 The increase is more obvious in the range, and the toxicity is when the power exceeds 1.3W / m 2 When the blue light power is 2W / m 2 The survival rate of 4T1 cells treated for 24 hours was 27.6%. 2 The irradiation power is carried out.

[0149] The non-light-exposed group was used as a control to investigate whether the cytotoxicity was induced by blue light (1.3 W / m 2 ) is caused by the activation of the light-controlled gene expression system after irradiation. Fig.16 The results of B showed that in the absence of blue light irradiation, there was no obvious cell killing effect in each group; after blue light irradiation, the cell survival rate of the pPEA@GPH-L group was 21.9%, which was not statistically different from the ability of the pPEA@L group to kill tumor cells.

[0150] 5. Determination of PEA expression in and outside 4T1 cells

[0151] In order to study the expression of PEA content inside and outside 4T1 cells after blue light irradiation, this patent detected the PEA content inside and outside 4T1 cells after using different groups of preparations. 4T1 cells were cultured at 5×10 5The cells were seeded at a density of 10 cells / well in a 6-well plate. After 12 hours, the medium was diluted with serum-free RPMI-1640 medium to dilute pPEA@L and pPEA@GPH-L. The dose was 10 μg pPEA (5 μg pGAVPO and 5 μg pU5-PEA) per well. 460 nm blue light (power 1.3 W / m 2 ) After 48 hours of treatment, the cell culture supernatant was first taken to obtain the extracellular sample, and then the lower layer of adherent cells was digested, and the intracellular sample was obtained by cell ultrasonic disruptor at a constant temperature of 0°C. Finally, the PEA content ELISA kit was used to detect the intracellular and extracellular PEA content (n=3).

[0152] The double antibody sandwich method Pseudomonas aeruginosa exotoxin A (PEA) enzyme-linked immunosorbent assay kit was used to study the expression of PEA content inside and outside cells after blue light treatment. The PEA content outside the cells (supernatant) and inside the cells (cell rupture) was detected respectively. This patent detected the expression of PEA inside and outside 4T1 cells after treatment with different groups of preparations. Fig.17 As shown, after blue light irradiation, PEA was detected inside and outside the cells of the drug-treated group compared with the Saline group, indicating that the light-controlled gene expression system can successfully express PEA in cells.

[0153] 6. Transwell experiment

[0154] The chamber of the Transwell chamber device is called the upper chamber, and the culture plate is called the lower chamber. The upper chamber is filled with the upper medium, and the lower chamber is filled with the lower medium. The upper and lower layers of the culture medium are separated by a polycarbonate membrane. Cells are seeded in the upper chamber. Due to the permeability of the polycarbonate membrane, the components in the lower medium will affect the cells in the upper chamber, so that the effects of the components of the lower medium on cell growth and movement can be studied. Using different polycarbonate membranes, co-culture, cell chemotaxis, cell migration, cell invasion, etc. can be studied.

[0155] Five groups were set up, namely the pPEA@GPH-L group (without blue light irradiation), pPEA@L group (blue light irradiation), pPEA@GPH-L group (blue light irradiation) nanoparticle solution containing 1μg pGAVPO and 1μg pU5-PEA plasmid, and two control groups without adding plasmids. The cells were irradiated under blue light for 24 hours, and a control group without blue light control was set up in the group without adding plasmids. That is, the final groups were: Saline (Light Off) group, Saline (Light On) group, pPEA@GPH-L (Light Off) group, pPEA@L (Light On) group, and pPEA@GPH-L (Light On) group. Fig.18 shown.

[0156] 4T1 cells in the logarithmic growth phase were plated in 6-well plates, and drugs were added after they adhered to the plate for 24 hours. Cell suspension was prepared, washed with PBS, digested with trypsin, and resuspended in serum-free medium. The number of 4T1 cells in each well was 1×10 5 . Culture medium containing 15% serum was added to the lower chamber of the 6-well plate. After all preparations were completed, the transwell chamber was placed in the 6-well plate, 300 μL of the prepared 4T1 cell suspension (serum-free culture medium) was added to the upper chamber, and it was placed in the culture medium. Incubate in the incubator for 48 hours under light. After incubation, the chamber was removed, the culture medium was aspirated, and 600 μL of paraformaldehyde (4%, w / v) fixative was added to the 6-well plate and fixed for 20-30 minutes. After that, the fixative was discarded, washed 2-3 times with PBS, and 600 μL of 0.5% ready-to-use crystal violet was added to each well of the 6-well plate and stained at room temperature for 20 minutes. Then, the excess crystal violet was washed 3 times with PBS for 5 minutes each time. The upper side of the chamber was gently wiped with qualitative filter paper and allowed to dry for 10-15 minutes. The photos were taken under an inverted microscope, with appropriate exposure time adjusted, and the background was yellow. In the non-illumination group, no blue light device was used during the 48-h culture, and other operations were the same as above.

[0157] 7. 4T1 cell scratch test

[0158] Take 4T1 cells and 24-well plates in the logarithmic growth phase, digest them with trypsin, and inoculate them at a density of about 1×106 4T1 cells / well. The amount of liquid in each well is about 1 mL, and culture them overnight. When the cells grow to a density of 90-100%, discard the culture medium, draw a straight line in the middle of the well plate with a 10μL pipette tip, wash twice with PBS, and record the scratch image under a microscope. Add nanoparticle solutions containing 1μg pGAVPO and 1μg pU5-PEA plasmid to the pPEA@GPH-L group (no blue light illumination), pPEA@L group (blue light illumination), and pPEA@GPH-L group (blue light illumination), respectively, and set up two control groups without adding plasmids. Irradiate the cells under blue light for 24 h, and set up a control group without blue light control in the group without adding plasmids. After irradiation, use a microscope to record the scratch image, such as Fig.19 shown.

[0159] The results are as follows Fig.18 and Fig.19As shown, the crystal violet absorbance of the Saline group, the light group, and the pPEA@GPH-L group without blue light irradiation is large, and the scratches have different degrees of healing. This shows that without the activation of blue light (460nm) irradiation, the light-controlled Pseudomonas aeruginosa exotoxin gene cannot function, and thus has no effect on cell migration. After blue light (460nm) irradiation treatment, the scratches in the pPEA@GPH-L treatment group not only did not heal at 24h compared to 0h, but the scratches expanded. At the same time, the crystal violet absorbance of the pPEA@GPH-L treatment group in the transwell experiment was relatively small, indicating that the expression of Pseudomonas aeruginosa exotoxin effectively inhibited the growth and migration of 4T1 cells. This shows that pPEA@GPH-L has the potential to treat breast cancer.

[0160] 8. 3D tumor sphere growth inhibition experiment

[0161] In vitro 3D tumor spheres were used to predict the inhibitory effects of pPEA@L and pPEA@GPH-L on solid breast tumors. 4T1 tumor sphere model was established: 200 mg of low melting point agarose was weighed and added to 10 mL of RPMI-1640 medium without FBS, heated at 90 °C until the liquid became clear, and the gel was added to a 96-well plate (60 μL per well) with a 300 μL dispenser while hot. The agarose gel was cooled and solidified at the bottom of the well plate. 10,000 4T1 cells in the logarithmic growth phase were added to each well. After 4 days of conventional culture, 4T1 tumor spheres were formed. The formation of spheres was recorded with an inverted microscope (recorded as day 1). pPEA@L and two groups of pPEA@GPH-L were diluted with RPMI-1640 medium without FBS, with a dose of 4 μg pPEA (2 μg pGAVPO and 2 μg pPEA) per well, and the interval was 12 hours, and the maximum time was 12 hours with blue light (1.3 W / m 2 ), and an inverted microscope was used to take photos and record the changes of tumor spheroids on the 3rd and 5th days. In addition, a group of only light (1.3W / m 2 ) control group and a blank control group without light.

[0162] like Fig. 20As shown, under Light Off, the structure of the tumor spheres in the Saline group became more and more dense as the number of days increased; a similar situation can be observed in the Control group under blue light irradiation, indicating that blue light irradiation itself does not inhibit the growth of 4T1 tumor spheres. In addition, it can be proved that pPEA@GPH-L does not inhibit the growth of 4T1 tumor spheres without blue light irradiation. After adding pPEA@L and pPEA@GPH-L, accompanied by blue light irradiation, it can be observed that the structure of the tumor spheres becomes loose on the 3rd day; on the 5th day, it can be found that the tumor spheres have been completely lysed. It is speculated that this phenomenon is because pPEA@L and pPEA@GPH-L can specifically express PEA in tumor sphere cells under Light On. The above results also prove that this treatment regimen has a certain killing effect on solid breast tumors.

[0163] All data were analyzed using Prism 9.0 one-way ANOVA, and differences between multiple groups were analyzed using one-way ANOVA and Tukey's multiple comparison analysis. Unless otherwise specified, data are mean standard deviation. Test values ​​*p < 0.05, **p < 0.01 and ***p < 0.001 indicate significant differences.

[0164] Example 3

[0165] In vivo antitumor study of pPEA@GPH-L

[0166] 1. Establishment of tumor-bearing mouse model

[0167] Establishment of mouse breast cancer orthotopic tumor model: When the 4T1 cells in the logarithmic growth phase were cultured to a density of about 80%, the cells were digested with trypsin, washed 3 times with PBS, and resuspended in RPMI-1640 complete medium. 100 μL of the cell (1×106 cells) resuspension was subcutaneously inoculated into the right breast of 10 female BALB / C mice (6-8 weeks old, Shanghai Jiesijie Experimental Animal Co., Ltd.) until the tumor grew to 100 mm. 3 The tumor was removed and minced in RPMI-1640 complete medium. Female BALB / c mice (6-8 weeks old) for tumor implantation were shaved on the right side of the subcutaneous breast of the mice, and then the minced tumor solution was injected subcutaneously at the shaved area. After 15 days, when the tumor size reached 100mm 3 Subsequent experiments were conducted.

[0168] 2. Liposome complex targeted distribution experiment

[0169] When the tumor volume reaches about 200 mm 3Afterwards, 9 tumor-bearing BALB / c mice were randomly divided into 3 groups (n=3) and injected with 0.9% (w / v) saline, IR775@L, and IR775@GPH-L (IR775 dose was 10μg / mouse) via the tail vein. Mice were anesthetized by intraperitoneal injection of sodium pentobarbital saline solution (20mg / mL, 50μL) and imaged at 4h, 8h, and 24h using a small animal in vivo imager (Kodak, USA, excitation: 750nm, emission: 780nm). All mice were killed 24h after administration and the heart, liver, spleen, lung, kidney, and tumor were removed. The removed organs were imaged using a small animal imaging system under the same conditions.

[0170] like Fig.21 As shown, the accumulation of IR775@L and IR775@GPH-L in tumors at 2 hours, 8 hours and 24 hours after administration was studied by in vivo imaging of small animals. The accumulation of IR775@L and IR775@GPH-L at more sites in the tumor may be due to the good protection of liposomes on genes. The fluorescence intensity of the tumor site in the IR775@GPH-L group was stronger than that of IR775@L at 8 hours, indicating that IR775@GPH-L can accumulate in the tumor site in advance, reflecting the targeting effect of HA. After 24 hours of administration, the fluorescence of the tumor site in the IR775@GPH-L group did not weaken, which was due to the long circulation effect of PEG.

[0171] Fig. 22 The in vitro distribution of IR775, IR775@L, and IR775@GPH-L after tail vein injection in mice. 24 hours after administration, the fluorescence intensity of IR775 in the tumor was the highest in the IR775@GPH-L group, indicating that IR775@GPH-L had the best tumor aggregation effect 24 hours after administration; the tumor accumulation of IR775@L was second only to IR775-Tig@CML, and the accumulation of IR775@L was relatively low, indicating that the liposome had a protective gene effect, which may be due to the targeting effect of HA and the long circulation of PEG. At 24h, the content of IR775@GPH-L liposomes in the liver was higher than that of the other two groups, and the fluorescence intensity was relatively high. The accumulation of IR775@GPH-L liposomes in the tumor site was much higher than that in the liver. The above results show that IR775@GPH-L has a good tumor targeting effect and accumulates in the tumor site.

[0172] 3. In vivo pharmacodynamics tumor inhibition experiment

[0173] Tumor volume reaches 100mm 3After about 12 hours, 48 ​​tumor-bearing BALB / c female mice were randomly divided into 5 groups (n=12). Each group of mice was injected with two groups of 0.9% (w / v) saline through the tail vein, pPEA@L, pPEA@GPH-L (50μg pGAVPO, 50μg pU5-PEA), and the administration was once every 3 days for a total of 3 administrations. The time of the first administration was recorded as the first day. A total of 4 treatment groups were set up, namely Saline (Light Off), pPEA@GPH-L (Light Off), pPEA@L (Light On) and pPEA@GPH-L (Light On). For the Saline (Light off), pPEA@L (Light On) and pPEA@GPH-L (Light On) treatment groups, 24 hours after administration, the mice were wrapped with black opaque strips, and only the tumor site was exposed, and then fixed on a foam board and irradiated with blue light for 12 hours. The control group was set up with an equal volume of saline injected into the tail vein without blue light irradiation. Treatment ended on day 9.

[0174] Two mice were randomly selected from each group and killed, and the tumors were removed for immunofluorescence staining. The tumor size (V = (tumor long diameter) × (tumor short diameter) 2 / 2) and mouse weight were recorded every 3 days with a vernier caliper. At the end of the 22-day observation, 5 mice in each group were sacrificed for scientific research. The removed tumors were photographed and recorded in size to directly show the treatment effect of each group. The remaining mice were used to study the survival rate of each treatment group (n = 5). Out of humanitarian considerations, mice with tumors larger than 1500mm 3 The mice were euthanized. The survival rate of mice in each group was calculated and the survival rate curve was drawn.

[0175] In order to study the anti-tumor effect of pPEA@GPH-L in vivo, this patent established a BALB / c mouse model bearing 4T1 breast cancer (such as Fig.23 As shown), Saline, pPEA@L and pPEA@GPH-L were injected into the tail vein of each group of mice. The treatment group given the gene drug was given blue light irradiation treatment. Previous in vivo distribution imaging results showed that pPEA@GPH-L accumulated most in the tumor 24 hours after administration. Considering the therapeutic effect, this patent started blue light irradiation treatment of the treatment group 24 hours after administration. A negative control group was set up with saline (0.9% w / v) without blue light irradiation and saline (0.9% w / v) with blue light irradiation in the tail vein.

[0176] After a 22-day observation period, the size of all tumors was Fig.24 As shown in A, the tumor size was measured every 3 days with a vernier caliper. Fig.24As shown in B. It can be found that the tumor volume of the control group increased rapidly, while the pPEA@GPH-L (Light off) group had almost no tumor inhibition effect on breast cancer. Compared with the first two groups, both the pPEA@L (Light On) and pPEA@GPH-L (Light On) treatment groups had certain tumor inhibition effects, among which the pPEA@GPH-L (Light On) group had the strongest tumor inhibition effect, and the average tumor volume at 22 days was the smallest.

[0177] The therapeutic effect of the pPEA@GPH-L group was better than that of the pPEA@L group, which proved the excellent efficacy of light-controlled gene targeted therapy. At the same time, pPEA@GPH-L has a stronger anti-tumor effect, probably due to the targeting effect of HA and the long circulation effect of PEG.

[0178] Mouse survival rate chart ( Fig.24 C) showed that 60% of the mice in the pPEA@GPH-L (Light On) group survived for 45 days, while the survival rates of mice in the pPEA@GPH-L (Light off) and pPEA@L (Light On) groups at 45 days were 0% and 40%, respectively, and all mice in the control group given saline (0.9% w / v) died on day 33. The above results show that the pPEA@GPH-L (Light On) group can significantly improve the survival rate of tumor-bearing mice.

[0179] 4. Immunofluorescence staining of tumor tissue

[0180] The resected tumor was first washed with 0.9% (w / v) saline, placed in a small EP tube, and immersed in 4% (w / v) paraformaldehyde solution for tissue fixation. The fixed tumor was embedded in paraffin, and the tumor tissue was observed by H&E staining, TUNEL pathological staining was used to observe tumor tissue cell apoptosis, Ki67 and CD31 double staining was used to observe tumor tissue pathology, DAPI (20 μg / mL) was added to counterstain the cell nucleus, and photos were taken under a fluorescence microscope.

[0181] like Fig.25As shown in the figure, the results of H&E staining show that compared with the Saline group, the cells in the tumor tissues after pPEA@L (Light On) and pPEA@GPH-L (Light On) treatment shrank, among which the pPEA@GPH-L treatment group had the most severe shrinkage. Regardless of whether there was light exposure or not, the H&E results of the tumor tissues of the two control groups showed that the tumor cells were relatively dense and there was no cell damage. In the TUNEL staining results of the tumor tissues, green marks apoptotic cells, and the stronger the green fluorescence, the more apoptotic cells there are; blue marks the cell nucleus to mark the cell position. It can be observed that the apoptosis signal in the pPEA@GPH-L (Light On) treatment group is the strongest, indicating that the most tumor cells have apoptosis after pPEA@GPH-L (Light On) treatment, and has the best treatment effect.

[0182] Tumor tissue was double-stained with red markers Ki67 and CD31 to further characterize the proliferation in tumor tissue. Ki67 is an antigen associated with proliferating cells, and its function is closely related to mitosis. Ki67 index detection is indispensable in the process of cell proliferation. The higher the Ki67, the higher the level of malignant transformation of the tumor. CD31 can mark tumor angiogenesis. It can be seen that the proliferating cells and tumor angiogenesis in the pPEA@GPH-L (Light On) treatment group were significantly reduced compared with the control group. In summary, it shows that pPEA@GPH-L (Light On) has the strongest tumor inhibition effect and the best tumor killing effect after treatment.

[0183] 5. Preliminary in vivo safety testing

[0184] From the start of administration, tumor size and mouse weight were recorded every 3 days until the end of observation on the 21st day. Plasma samples were collected from mice sacrificed under conditions that avoided hemolysis. Whole blood samples were placed at room temperature for 2 hours or at 4°C overnight, then centrifuged at 3000rpm for 15 minutes at 2-8°C, and dispensed into EP tubes containing anticoagulant heparin. The samples were stored at -20°C or -80°C to avoid repeated freezing and thawing. Thawed samples should be centrifuged again and then tested. The samples should be clear and transparent, without bubbles, and suspended matter should be removed by centrifugation. Sample hemolysis affects the experiment. Subsequently, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured to evaluate liver function, and blood urea nitrogen (BUN) and creatinine (CREA) were used to measure renal function. Important organs (heart, liver, spleen, lung, and kidney) of the sacrificed mice were collected, fixed with 4% (w / v) paraformaldehyde, embedded in paraffin, sliced, and stained with H&E. The stained slices were observed and photographed for determination of their histopathological changes.

[0185] The important organs (heart, liver, spleen, lung, and kidney) of euthanized mice were collected for H&E staining, and the staining sections were observed and photographed to determine the histopathological changes. Fig.26 As shown, the results of H&E staining showed that no obvious pathological changes were observed in important organs such as the heart, liver, spleen, lung, and kidney in each group, indicating that the Pseudomonas aeruginosa exotoxin A gene has a safe anti-tumor effect.

[0186] In addition, the change in mouse body weight during the in vivo antitumor experiment is also an indicator of systemic toxicity. Fig. 27 It can be seen that the body weight of each group of mice did not change significantly during the in vivo tumor inhibition experiment.

[0187] Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured to assess liver function, and blood urea nitrogen (BUN) and creatinine (CREA) to determine renal function. Fig.28 It was shown that after the treatment, the blood biochemical indicators of each group did not change much compared with the normal saline control group and were all within the normal range, indicating that the toxicity of the patented pPEA@GPH-L to the liver and kidneys was negligible, and preliminarily showed that the patented vector had good safety.

[0188] All data were analyzed using Prism 9.0 one-way ANOVA, and differences between multiple groups were analyzed using one-way ANOVA and Tukey's multiple comparison analysis. Unless otherwise specified, data are mean standard deviation. Test values ​​*p < 0.05, **p < 0.01 and ***p < 0.001 indicate significant differences.

[0189] In summary, the liposome-targeted biomaterial GDS-PEG-HA was successfully synthesized; the gene-loaded liposome complex pPEA@GPH-L was prepared, with a particle size of 121.6±3.6nm, a potential of -18.9±2.7mV, an obvious shell-core structure, and a regular spherical shape. When the nitrogen-phosphorus ratio (N / P) was 4, it had a good gene compression ability and good storage stability. The cumulative release experiment of pPEA@GPH-L showed that the cumulative gene release rate at 72h was 63.65±3.30%, with a certain sustained release result. In vitro cell experiments showed that pPEA@GPH-L could enhance the uptake of 4T1 cells; in vivo distribution experiments showed that pPEA@GPH-L could effectively target tumor tissues.

[0190] In vitro pharmacodynamics experiments showed that pPEA@GPH-L could inhibit the growth of tumor spheroids, inhibit wound healing, and prevent migration; under blue light irradiation, pPEA@GPH-L could express PEA in 4T1 cells and kill cells, resulting in reduced activity of 4T1 cells.

[0191] In vivo pharmacodynamic experiments showed that under local blue light irradiation, the pPEA@GPH-L treatment group significantly inhibited tumor growth, induced tumor cell apoptosis, prolonged tumor-bearing tumor growth, increased mouse survival rate, and increased tumor cell apoptosis and necrosis compared with the pPEA@L treatment group.

[0192] CCK-8 and hemolysis experiments showed that pPEA@GPH-L had good biocompatibility. In the in vivo experiment, the liver and kidney function indicators of mice in each group were normal at the end of treatment, and H&E staining of important organ tissues showed no obvious lesions, which preliminarily showed that pPEA@GPH-L had good biosafety.

[0193] The series of detailed descriptions listed above are only specific descriptions of feasible implementation methods of the present invention. They are not intended to limit the scope of protection of the present invention. Those skilled in the art can design many other modifications and implementation methods, which will fall within the scope and spirit of the principles disclosed in this application. More specifically, within the scope of the disclosure, drawings and claims of this application, various modifications and improvements can be made to the components and / or layout of the subject combination layout. In addition to the modifications and improvements made to the components and / or layout, other uses will also be obvious to those skilled in the art.

Claims

1. A plasmid-targeted liposome complex, characterized in that: The invention comprises liposome and plasmid pPEA contained by the liposome. The plasmid pPEA comprises an expression vector pGAVPO containing transcription factor GAVPO protein induced by blue light and pU5-PEA of a light-controlled gene expression system capable of expressing PEA.

2. The liposome complex according to claim 1, characterized in that The liposome is fused with a targeting biological material for modifying the liposome.

3. The liposome complex according to claim 1, characterized in that The ratio of pGAVPO to pU5-PEA is one of 1:10, 1:5, 1:1, 5:1 and 10:

1.

4. The liposome complex according to claim 1, characterized in that The pPEA is coated with cationic materials.

5. The liposome complex according to claim 2, characterized in that The targeting biomaterials include 1,3-glycerol distearate (GDS), thiolated hyaluronic acid (HA) and bifunctional polyethylene glycol (MAL-PEG 2000 -NHS) and synthesized by substitution reaction.

6. The liposome complex according to any one of claims 2 to 5, characterized in that: The particle size of the liposome complex is 121.6±3.6 nm, and the concentration is 0.01-0.5 mg / mL.

7. A method for preparing a plasmid-targeted liposome complex, characterized in that: The steps include: (a) Preparation of targeted biomaterial GDS-PEG-HA; (b) constructing the light-controlled gene pPEA and extracting and purifying the light-controlled gene; (c) preparing pPEA-cation complex; (d) Preparation of liposome complex pPEA@GPH-L, first preparing blank liposome L, fusing GDS-PEG-HA on the blank liposome L, and then encapsulating the pPEA-cationic complex in the blank liposome L with targeting material; or first encapsulating the pPEA-cationic complex in the blank liposome L to obtain pPEA@L, then fusing GDS-PEG-HA on pPEA@L, and finally extruding with an extruder to obtain the liposome complex pPEA@GPH-L encapsulating the Pseudomonas aeruginosa exotoxin light-controlled gene and liposome targeting material.

8. The method according to claim 7, characterized in that The GDS-PEG-HA is prepared by the following steps: (e) First use GDS and Mal-PEG 2000 -NHS reacts with the active ester group on NHS to obtain GDS-PEG; (f) HA is added again, and the carboxyl groups on HA are thiolated with cysteamine to obtain HA-SH; (g) Finally, add Mal-PEG 2000 -NHS and HA-SH, HA-SH and Mal-PEG 2000 -The maleimide group on NHS was reacted, the pH value was adjusted, and GDS-PEG-HA was obtained after dialysis in a dialysis bag.

9. The method according to claim 8, characterized in that In step (e), EDC and DMAP are further added, and ammonia gas is introduced to mix well. The mixture is then separated by silica gel column chromatography, and the solvent is rotary evaporated.

10. The method according to claim 8, characterized in that In step (f), equal amounts of cysteamine are added twice, and EDC and NHS are added respectively during the two additions. Nitrogen is continuously introduced, the pH value is adjusted, and then the mixture is placed in a dialysis bag for dialysis to prepare a lyophilized powder for later use.

11. The method according to claim 9, characterized in that The developing solvent for chromatography was DCM / CH3OH / triethylamine in a volume ratio of 90:10:

1.

12. The method according to claim 10, characterized in that The pH value in step (g) is 7, and the pH value in step (f) is 5.0-5.

5.

13. The method according to claim 7, characterized in that The pPEA-cationic complex is obtained by adding pPEA and a cationic material into a HEPES buffer, stirring vigorously, and incubating at room temperature for 25 minutes. The cationic material is a chitosan solution.

14. The method according to claim 13, characterized in that The N / P of the pPEA and chitosan solution is one of 0, 2, 4, 6, 8, 10, 12, and 14.

15. Use of the plasmid-targeted liposome complex according to any one of claims 1 to 6 or the plasmid-targeted liposome complex prepared by the method according to any one of claims 7 to 14 in the preparation of tumor drugs.

16. The use according to claim 15, characterized in that The tumor is gastric cancer, colorectal cancer, liver cancer or breast cancer.

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