Multi-gene nanocarrier, preparation method therefor, application thereof, and Anti-cancer drug

Abstract

Provided are a multi-gene nanocarrier, a preparation method therefor, an application thereof, and an anti-cancer drug. The multi-gene nanocarrier comprises a three-branch central ligand and gene combination sequences covalently connected on each branch of the three-branch central ligand, the gene combination sequences on each branch all being the same; in the direction from the three-branch central ligand outward, the gene combination sequences contain at least two tumor suppressor genes selected in order from among TP53, BIM, and PTEN. The provided multi-gene nanocarrier has excellent anti-cancer activity and biological safety.

Description

A multi-gene nanocarrier, its preparation method and application, and an anticancer drug

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of Chinese patent application 202311497169.8 filed on November 10, 2023, the contents of which are incorporated herein by reference. Technical Field

[0003] The present invention relates to the field of biomedicine, and in particular to a multi-gene nanocarrier, a preparation method and application thereof, and an anticancer drug. Background Art

[0004] Extensive omics data from databases such as The Cancer Genome Atlas (TCGA), the International Cancer Genome Consortium (ICGC), and the Pan-Cancer Analysis of Whole Genomes (PCAWG) demonstrate that oncogenic driver genes are prone to mutations across all known cancer types, with a particularly pronounced prevalence in tumor suppressor genes and oncogenes, such as TP53, c-Myc, PIK3CA, KRAS, PTEN, CDKN2A, EGFR, and BIM. Notably, these genes often exhibit single-gene mutations or multi-gene combined mutations during tumor development, posing a significant challenge to current cancer treatment technologies based on single-gene targets.

[0005] Gene therapy is an emerging and highly effective treatment for complex diseases such as cancer caused by multiple gene mutations. For example, it can silence activated oncogenes or supplement tumor cells with normally functioning tumor suppressor genes to achieve functional cure. The effect of gene therapy is closely related to its delivery vector. Currently, the commonly used adeno-associated viruses (AAVs) and lentiviral vectors have shown great application potential in gene delivery, but are limited by the gene capacity of AAV vectors (~4.5kb) and lentiviral vectors (~8kb), and can usually only deliver 1-2 genes for treatment. In addition, the increase in the gene load of viral vectors may increase the structural instability of the virus and reduce the efficiency of multi-gene co-expression. More importantly, the safety issues of the viral vectors themselves cannot be ignored.

[0006] Summary of the Invention

[0007] The purpose of the present invention is to provide a multi-gene nanocarrier that has the potential to become a more efficient and safer new anticancer drug.

[0008] To achieve the above objectives, the first aspect of the present invention provides a multi-gene nanocarrier, which comprises a three-branched central ligand and a gene combination sequence covalently linked to each branch of the three-branched central ligand, wherein the gene combination sequence on each branch is the same;

[0009] In the direction of extending outward from the three-branched central ligand, the gene combination sequence contains at least two tumor suppressor genes selected from TP53, BIM, and PTEN in sequence.

[0010] A second aspect of the present invention provides a multi-gene nanocarrier comprising a three-branched central ligand and a gene combination sequence covalently linked to each branch of the three-branched central ligand, wherein the gene combination sequence on each branch is identical;

[0011] In the direction of extending outward from the three-branched central ligand, the gene combination sequence contains tumor suppressor genes and proto-oncogenes in sequence, the tumor suppressor gene is selected from one of TP53, BIM, and PTEN, and the proto-oncogene is MYC.

[0012] The third aspect of the present invention provides a method for preparing the multi-gene nanocarrier according to the first aspect and / or the second aspect, comprising the following steps:

[0013] (1) performing a click cross-linking reaction between the three-branched central compound represented by formula (I) and a single-stranded primer nucleic acid with an azide modification at the 5' end to obtain a branched primer triplet containing three primers;

[0014] (2) performing a branched PCR reaction in a PCR reaction system using the branched primer triplets as primers and the linear DNA as a template to obtain the multi-gene nanocarrier; the sequence information of the linear DNA is consistent with the gene combination sequence in the multi-gene nanocarrier described in the first aspect and / or the second aspect;

[0015] The fourth aspect of the present invention provides use of the multi-gene nanocarrier described in the first aspect and / or the second aspect in the preparation of anticancer drugs.

[0016] The fifth aspect of the present invention provides an anticancer drug, which is composed of an active ingredient and excipients, wherein the active ingredient contains the multi-gene nanocarrier described in the first aspect and / or the second aspect.

[0017] The technical solution provided by the present invention has at least the following advantages:

[0018] (1) The multi-gene nanocarrier provided by the present invention integrates various emerging gene regulation tools at different levels into a comprehensive toolbox and loads it into a chromatin-like payload. Guided by the central dogma, it simulates the chromosome-mediated gene decoding process at different levels and multiple spatiotemporal dimensions, realizing chromosome-mediated multi-level and multi-dimensional gene network regulation, thereby achieving the purpose of disease treatment. The strategy of simulating chromatin DNA to precisely regulate gene expression is expected to combat complex diseases in a manner close to chromosome regulation.

[0019] (2) The multi-gene nanocarrier provided by the present invention simultaneously loads the expression frames of multiple gene regulation into one body and maintains the scale of cell delivery, thereby achieving a balance in gene capacity, carrier scale, multi-gene co-expression and safety of the multi-gene delivery carrier.

[0020] (3) The technical solution provided by the present invention uses branched-chain PCR technology to construct a DNA multi-gene nanocarrier containing multi-component gene units (therapeutic gene targets such as tumor suppressor genes and oncogenes) with different gene regulatory functions (overexpression, RNA interference and other gene regulatory technologies), and verifies the multi-gene co-expression or regulation ability, cell apoptosis induction ability and cell proliferation inhibition ability of this type of vector in different cancer cell types. Finally, using mice with different cancer cell xenografts as a model, the therapeutic efficacy and safety of this type of vector in xenograft tumors are further verified, thereby verifying from both in vitro activity and in vivo activity that this type of multi-gene loaded multi-gene nanocarrier can achieve directional regulation of cancer cell apoptosis by balancing the expression level differences of tumor suppressor genes and oncogenes. The technical solution provided by the present invention helps to synchronously balance the regulation of genes related to tumor occurrence and development, further reduce potential side effects, toxicity and tolerance on the basis of improving anti-cancer activity, and provide technical support for the development of more efficient and safe pan-cancer genomic treatment methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1. Schematic diagram of branched primer triplet synthesis;

[0022] Figure 2. Schematic diagram of the NP-TP53-BIM-PTEN vector sequence structure;

[0023] Figure 3. Schematic diagram of the NP-TP53-shMYC vector sequence structure;

[0024] Figure 4. 1% agarose gel electrophoresis analysis of branched-chain PCR self-assembly products;

[0025] Figure 5. AFM characterization of NP-TP53-BIM-PTEN;

[0026] Figure 6. AFM characterization of NP-TP53-shMYC;

[0027] Figure 7. DLS characterization of NP-TP53-BIM-PTEN;

[0028] Figure 8. DLS characterization of NP-TP53-shMYC;

[0029] Figure 9. Serum stability analysis of NP-TP53-BIM-PTEN;

[0030] Figure 10. Serum stability analysis of NP-TP53-shMYC;

[0031] Figure 11. Expression analysis of the three genes NP-TP53-BIM-PTEN in NCI-H1299 cells;

[0032] Figure 12. Expression analysis of the NP-TP53-BIM-PTEN triple gene protein in NCI-H1299 cells;

[0033] Figure 13. Expression analysis of two gene mRNAs of NP-TP53-shMYC in MDA-MB-231 cells;

[0034] Figure 14. Expression analysis of two gene proteins of NP-TP53-shMYC in MDA-MB-231 cells;

[0035] Figure 15. Comparative analysis of the apoptosis-inducing activity of multi-gene nanocarriers, linear DNA, and plasmids on NCI-H1299 cells;

[0036] Figure 16. Comparative analysis of the proliferation inhibitory activity of multi-gene nanocarriers, linear DNA, and plasmids on NCI-H1299 cells;

[0037] FIG17 . Comparative analysis of the apoptosis-inducing activity of NP-TP53-shMYC, NP-TP53, and NP-shMYC on MDA-MB-231 cells;

[0038] Figure 18. Evaluation of the in vivo anti-tumor therapeutic effect of multi-gene nanocarriers;

[0039] Figure 19. Analysis of TP53, BIM, and PTEN gene expression levels in tumor tissues;

[0040] Figure 20. Quantitative analysis of apoptosis markers and cell proliferation marker Ki67 in tumor tissues;

[0041] Figure 21. Evaluation of the in vivo anti-tumor therapeutic effect of NP-TP53-shMYC multi-gene nanocarrier;

[0042] Figure 22. Analysis of TP53 and MYC gene expression levels in tumor tissues;

[0043] Figure 23. Analysis of TP53 and MYC expression levels in tumor tissues;

[0044] FIG24 . HE staining of different organs of MDA-MB-231 transplanted tumor-bearing mice;

[0045] Figure 25. Detection of immune inflammatory factors in the blood of MDA-MB-231 transplanted tumor-bearing mice;

[0046] Figure 26. HE staining of different organs of NCI-H1299 tumor-bearing mice;

[0047] Figure 27. Detection of immune inflammatory factors in the blood of NCI-H1299 tumor-bearing mice;

[0048] Figure 28. Expression analysis of three NP-PTB genes mRNA in NCI-H1299 cells;

[0049] Figure 29. Expression analysis of three NP-TBP-V gene mRNAs in NCI-H1299 cells. DETAILED DESCRIPTION

[0050] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0051] In the present invention, the phrase "selected in sequence" in "according to the direction extending outward from the three-branched central ligand, the gene combination sequence contains at least two tumor suppressor genes selected in sequence from TP53, BIM, and PTEN" means: selecting two or three tumor suppressor genes from "TP53, BIM, and PTEN" without changing the order. Exemplarily, according to the direction extending outward from the three-branched central ligand, the gene combination sequence is TP53-BIM-PTEN, TP53-BIM, TP53-PTEN, and BIM-PTEN.

[0052] In the present invention, TP53, BIM, and PTEN are all names of tumor suppressor genes, and MYC is the name of a proto-oncogene. p53 is the name of the protein expressed by TP53, c-Myc is the name of the protein expressed by MYC, and BIM protein and PTEN protein are proteins expressed by BIM and PTEN, respectively.

[0053] In the present invention, PCR refers to polymerase chain reaction.

[0054] Part 1

[0055] As mentioned above, the first aspect of the present invention provides a multi-gene nanocarrier, which comprises a three-branched central ligand and a gene combination sequence covalently linked to each branch of the three-branched central ligand, wherein the gene combination sequence on each branch is the same;

[0056] In the direction of extending outward from the three-branched central ligand, the gene combination sequence contains at least two tumor suppressor genes selected from TP53, BIM, and PTEN in sequence.

[0057] Preferably, in the direction of extending outward from the three-branched central ligand, the end of the gene combination sequence also contains the proto-oncogene MYC.

[0058] The inventors of the present invention discovered in their research that in the multi-gene nanocarrier, the arrangement order of the various tumor suppressor genes and oncogenes in the direction extending outward from the three-branched central ligand has an important influence on the overexpression and anti-cancer activity of the multi-gene nanocarrier. Under the arrangement order specifically required by the present invention, the prepared multi-gene nanocarrier has excellent overexpression, anti-cancer activity and biosafety.

[0059] Preferably, the gene combination sequence contains three tumor suppressor genes, TP53, BIM, and PTEN, in the direction extending outward from the three-branched central ligand. The inventors of the present invention have found in their research that under this preferred condition, the multi-gene nanocarrier provided by the present invention has better anti-cancer activity and higher biosafety. In addition, the inventors of the present invention have also found in their research that the TP53, BIM, and PTEN expression elements require the use of different types of promoters and transcription terminators to ensure the co-expression ability of the three genes.

[0060] Preferably, the three-branched central ligand is provided by a three-branched central compound represented by formula (I);

[0061] The present invention has no particular limitation on the specific method for preparing the three-branched central compound represented by formula (I). Those skilled in the art can make a selection based on technical means known in the art. However, in order to obtain the target compound with a higher yield, the present invention provides a preferred specific embodiment hereinafter, which will not be described in detail herein, and those skilled in the art should not interpret it as a limitation of the present invention.

[0062] Part 2

[0063] As mentioned above, the second aspect of the present invention provides a multi-gene nanocarrier, which comprises a three-branched central ligand and a gene combination sequence covalently linked to each branch of the three-branched central ligand, wherein the gene combination sequence on each branch is the same;

[0064] In the direction of extending outward from the three-branched central ligand, the gene combination sequence contains tumor suppressor genes and proto-oncogenes in sequence, the tumor suppressor gene is selected from one of TP53, BIM, and PTEN, and the proto-oncogene is MYC.

[0065] Preferably, the gene combination sequence includes TP53 and MYC in the direction extending outward from the three-branched central ligand. The inventors of the present invention have found that under this preferred condition, the multi-gene nanocarrier provided by the present invention has better anti-cancer activity and higher biosafety.

[0066] Preferably, the three-branched central ligand is provided by a three-branched central compound represented by formula (I).

[0067] The present invention has no particular limitation on the specific method for preparing the three-branched central compound represented by formula (I). Those skilled in the art can make a selection based on technical means known in the art. However, in order to obtain the target compound with a higher yield, the present invention provides a preferred specific embodiment hereinafter, which will not be described in detail herein, and those skilled in the art should not interpret it as a limitation of the present invention.

[0068] Part 3

[0069] As mentioned above, the third aspect of the present invention provides a method for preparing the multi-gene nanocarrier according to the first aspect and / or the second aspect, comprising the following steps:

[0070] (1) performing a click cross-linking reaction between the three-branched central compound represented by formula (I) and a single-stranded primer nucleic acid with an azide modification at the 5' end to obtain a branched primer triplet containing three primers;

[0071] (2) In a PCR reaction system, a branched PCR reaction is performed using the branched primer triplets as primers and the linear DNA as a template to obtain the multi-gene nanocarrier; the sequence information of the linear DNA is consistent with the gene combination sequence in the multi-gene nanocarrier described in the first aspect and / or the second aspect.

[0072] The "under the PCR reaction system" mentioned in the present invention means that under the conditions of having the necessary raw materials required for the PCR reaction, in addition to using the branched primer triplets as primers and the linear DNA as a template, it also contains necessary raw materials such as nucleotides, DNA polymerase and buffer, so that the branched PCR reaction can be successfully carried out.

[0073] The present invention has no particular limitation on the programmed reaction conditions for performing the branched PCR reaction. Those skilled in the art can make routine designs based on technical means known in the art. The present invention hereinafter exemplifies a preferred specific embodiment, which should not be construed as limiting the present invention by those skilled in the art.

[0074] The preparation method of the multi-gene nanocarrier of the present invention also includes post-processing means known in the art such as purification and recovery, which will not be described in detail herein and should not be construed as limiting the present invention by those skilled in the art.

[0075] Part 4

[0076] As mentioned above, the fourth aspect of the present invention provides the use of the multi-gene nanocarrier described in the first aspect and / or the second aspect in the preparation of anticancer drugs.

[0077] Preferably, the anticancer drug is selected from at least one of anti-breast cancer drugs, anti-lung cancer drugs, anti-liver cancer drugs, anti-colorectal cancer drugs, and anti-prostate cancer drugs.

[0078] More preferably, the anticancer drug is an anti-breast cancer drug and / or an anti-lung cancer drug.

[0079] Preferably, the anticancer drug is administered by in situ injection and / or intravenous injection.

[0080] Part 5

[0081] As mentioned above, the fifth aspect of the present invention provides an anticancer drug, which is composed of an active ingredient and excipients, and the active ingredient contains the multi-gene nanocarrier described in the first aspect and / or the second aspect.

[0082] Preferably, the content of the active ingredient is 0.01-99.99wt%, more preferably 0.1-99.9wt%, for example, it can be 0.01wt%, 0.1wt%, 1wt%, 3wt%, 5wt%, 10wt%, 15wt%, 20wt%, 25wt%, 30wt%, 35wt%, 40wt%, 45wt%, 50wt%, 55wt%, 60wt%, 65wt%, 70wt%, 75wt%, 80wt%, 85wt%, 90wt%, 95wt%, 99wt%.

[0083] Preferably, the excipient contains Liposome 2000 and / or Liposome 3000. The inventors of the present invention have found that when anticancer drugs contain Liposome 2000 and / or Liposome 3000 as excipients, the active ingredient multi-gene nanocarrier can have more efficient anticancer activity.

[0084] The present invention will be described in detail below through examples. In the following examples, unless otherwise specified, the raw materials are all commercially available. In the present invention, unless otherwise specified, the room temperature refers to 25±2°C.

[0085] Preparation Example 1: Preparation of branched primer triplets

[0086] 1) Synthesis of the three-branched central compound represented by formula (I)

[0087] Synthesis of Compound 1: Dissolve 10.0 mmol of triethanolamine (TEOA) in 50 mL of anhydrous chloroform, add 135 mmol of thionyl chloride dropwise, and reflux for 3 h. Remove the solvent by vacuum distillation, add 50 mL of dichloromethane, and filter to obtain 2.21 g of a white needle-like solid (92% yield). 1 H NMR (400MHz, d6-acetone): δ (ppm) 4.23 (t, J = 7.2 Hz, 6H), 3.73 (t, J = 7.2 Hz, 6H). 13 C NMR (100MHz, d6-acetone): δ (ppm) 56.01, 38.57.

[0088] Synthesis of Compound 2: Dissolve 5.00 mmol of Compound 1 in 25 mL of N,N-dimethylformamide (DMF), add 30.0 mmol of sodium azide, and stir at 60°C for 3 h. Add 50 mL of ethyl acetate, wash eight times with saturated sodium bicarbonate, water, and saturated brine, dry over anhydrous sodium sulfate, filter, and concentrate to obtain an oil. Add 4 M HCl in ethyl acetate to obtain 1.08 g of a white needle-like solid (83% yield). 1 H NMR (400MHz, CDCl3): δ (ppm) 4.06 (t, J = 5.2 Hz, 6H), 3.41 (t, J = 5.2 Hz, 6H). 13 C NMR (100MHz, CDCl3): δ (ppm) 52.89, 45.84.

[0089] Synthesis of Compound 3: Dissolve 4.15 mmol of Compound 2 in 15 mL of 1,4-dioxane, add 44.2 mg of palladium on carbon (10 wt%), then add 5 mL of aqueous ammonia (30 wt%). Vacuum H2 into the mixture, and stir at room temperature for 3 h. After the reaction, filter through Celite. Then, add ethyl acetate (3 × 10 mL) and water for extraction, then wash with saturated NaHCO3 solution. After separation, the organic phase is dried over anhydrous MgSO4 and filtered. The filtrate is evaporated to dryness and drained to obtain a yellow oily product, which is directly carried to the next step.

[0090] The synthesis of the compound represented by formula (I) follows: Under argon, compound 3 (0.42 mmol) was dissolved in 10 mL of dry DMF in two 50 mL round-bottom flasks. Diphenylcyclooctyne-activated ester (DBCO-NHS Ester, 1.38 mmol) was then added. The mixture was cooled to 0°C and then triethylamine (TEA, 1.26 mmol) was added in an ice-water bath (0°C). The mixture was allowed to react for 1 hour in an ice-water bath (0°C) before being warmed to room temperature and allowed to react overnight. After spot-blot detection, the reaction was complete. Extraction was performed by adding 10 mL of water and ethyl acetate (4 × 10 mL). After separation, the organic phases were combined, dried over anhydrous Na2SO4, and filtered. The filtrate was subjected to reduced pressure distillation on a rotary evaporator to remove the solvent. The filtrate was then directly separated by column chromatography (dichloromethane / methanol = 30:1, v / v) to obtain 296.1 mg of the product compound as a pale yellow viscous substance in a 70% yield. 1 H NMR (400MHz, CDCl3): δ (ppm) 7.64 (m, 12H): 7.35 (m, 12H), 3.34 (s, 6H), 2.76 (s, 12H), 2.60 (s, 6H), 2.51 (s, 6H). 13 C NMR (100MHz, CDCl3): δ (ppm) 172.52, 151.12, 148.26, 131.96, 129.50, 128.69, 128.15, 127.92, 127.60, 126.95, 125 .44,123.20,123.08,122.36,114.51,107.77,77.47,77.36,77.16,76.84,55.57,53.50,45.86,29.79,29.64,8.65. HRMS:calculated for[M+Na] + 1030.4268,found 1030.4265.

[0091] 2) Preparation of branched primer triplets

[0092] After correct mass spectrometric characterization, a click crosslinking reaction was performed using the cyclooctane group on the three-branched central compound represented by Formula (I) and an oligodeoxyribonucleic acid molecule with an azide modification at the 5' end, thereby obtaining a branched primer triplet containing three primers. The schematic diagram of the branched primer triplet synthesis is shown in Figure 1. The sequence information of the branched primer triplet is shown in Table 1.

[0093] Table 1 Sequence information of branched primer triplets

[0094] The specific process of branched primer triplet synthesis is as follows: customize primer F according to the primer sequence in Table 1 N3 and R N3 (Shanghai Sangon Biotech Co., Ltd.) Single-stranded nucleic acids (10 OD) with a 5'-end azide modification were first dissolved in 200 μL of double-distilled water (ddH2O). A small molecule triple-branched core compound dissolved in DMF (nucleic acid: triple-branched core compound = 3.3:1) was then added to the centrifuge tube. The mixture was shaken in a 37°C metal bath at a final concentration of 20 mM in PBS buffer overnight (10 hours). After the reaction, the fragments were purified using an 8% denaturing polyacrylamide gel (PAGE).

[0095] The branched primer triplets were purified as follows: the target band was excised under UV light, minced into fine pellets, and transferred to a 15 mL centrifuge tube. 0.3 M NaOAc was then added to the tube to a volume approximately twice that of the pellets, and the tube was rotated overnight. After extraction, the tube was centrifuged, and the supernatant was collected and transferred to a new 15 mL centrifuge tube. Three volumes of -20°C pre-cooled anhydrous ethanol were added to the tube containing the supernatant, mixed by inversion, and then precipitated at -20°C for 2 hours. After precipitation, the tube was centrifuged at 4°C for 30 minutes, and the supernatant was discarded. 2 mL of -20°C pre-cooled 75% ethanol was then added to the tube containing the pellet. The tube was then centrifuged at 4°C for 20 minutes, and the supernatant was discarded. The remaining ethanol in the tube was then vacuum dried. After drying, add 200 μL of ddH2O to the centrifuge tube, vortex to dissolve, measure the concentration, and store in a -20°C refrigerator.

[0096] The purified branched primer triplets were characterized by HRMS mass spectrometry, as shown in Table 2. 3 and R 3 The mass spectrometry results and the theoretical values ​​were within the allowable error range, indicating that the two branched primer triplets were synthesized correctly and could be used for subsequent branched PCR reactions to construct multi-gene nanocarriers.

[0097] Table 2 Mass spectrometry characterization of branched primers

[0098] Preparation Example 2: Design and Construction of Vectors for the Linear DNA Template L-TP53-BIM-PTEN

[0099] The simultaneous expression strategy for multi-component tumor suppressor genes utilizes a multi-gene nanoparticle vector element designed to overexpress three tumor suppressor genes (TP53, PTEN, and BIM). This vector is named NP-TP53-BIM-PTEN, and its linear DNA template is named L-TP53-BIM-PTEN. The sequence of L-TP53-BIM-PTEN includes the open reading frames of the TP53, BIM, and PTEN genes, as well as their respective promoters and terminators. From the 5' end to the 3' end, the following sequence is present: F1 box, CMV promoter, HA tag, TP53 ORF, BGH terminator, SV40 promoter, BIM ORF, SV40 pA terminator, EF1α promoter, FLAG tag, PTEN ORF, WPRE, hGH pA terminator, and R1 box element, totaling 7000 base pairs. A schematic diagram of the NP-TP53-BIM-PTEN vector sequence is shown in Figure 2.

[0100] The L-TP53-BIM-PTEN template was constructed from the plasmid template P-TP53-BIM-PTEN. The plasmid template was constructed using the Golden Gate assembly method. The specific process is as follows:

[0101] 1) PCR amplification and gel recovery to obtain linearized vector and insert fragment

[0102] According to Table 3, DNA fragments of varying lengths were amplified by PCR using different plasmids as templates and primers. The PCR reaction system consisted of 50 ng of plasmid template, 2.5 μM of upstream and downstream primers, and 1× Q5 Hot Start High-Fidelity Master Mix (New England Biolabs). The PCR reaction program was 98°C for 30 s (98°C, 10 s to 60°C, 10 s to 72°C, 1 min) followed by 30 cycles (72°C, 2 min). The resulting PCR products were named L-HA-TP53-BGH, L-SV40, L-BIM, L-SV40 polyA, L-EF1a, L-fPTEN, and L-WPRE-hGHpA. These PCR products were purified using a Gel DNA recovery kit (Zymo) for subsequent gene assembly reactions.

[0103] Table 3 Primer sequences for constructing P-TP53-BIM-PTEN

[0104] *Bold font indicates PaqCI recognition site.

[0105] 2) Construction of plasmid template P-TP53-BIM-PTEN by Golden Gate ligation

[0106] Purified DNA fragments of various lengths were prepared according to Table 4 and assembled using the following temperature program: (37°C, 5 min - 16°C, 5 min) × 30 cycles - 37°C, 5 min, 60°C, 5 min, 16°C, 30 min. After the reaction, 50 μL of Table 4 Chemically Competent Cells (ZC1015) were removed from -80°C and thawed in an ice bath. 5 μL of the ligation product was added to the 50 μL competent cells and mixed rapidly and gently. The mixture was then placed in an ice bath for 30 min, followed by a heat shock at 42°C for 90 s. The centrifuge tube was then quickly transferred to an ice bath and the cells were allowed to cool for 2-3 min. Finally, 100 μL of sterile LB medium (without antibiotics) was added and mixed thoroughly. The mixture was then added directly to Luria-Bertani (LB) solid medium containing the corresponding antibiotic, ampicillin. The mixture was evenly spread using sterile plate-spreading glass beads and incubated inverted at 37°C for 16 h. After colonies were formed, single colonies were selected and placed in 10 mL of sterile LB liquid medium. 10 μL of ampicillin (final concentration 100 μg / mL) was added and cultured at 37°C for 16 h. The bacterial solution was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing and analysis.

[0107] Table 4 Golden Gate Assembly ligation reaction system

[0108] 3) Amplification and recovery of the linear DNA template L-TP53-BIM-PTEN

[0109] After sequencing verification, the plasmid P-TP53-BIM-PTEN was expanded and extracted. Specifically, a 1 mL glycerol tube was added to 50 mL of LB liquid medium and shaken at 37°C at 200 rpm for 16-18 hours. The plasmid was extracted using a high-purity, endotoxin-free plasmid extraction kit (Cornwell), and the concentration was determined using a NanoDrop 2000. PCR amplification was performed using the primers listed in Table 5 (L-V2-F and L-V2-R) using 50 ng of plasmid template, 2.5 μM of upstream and downstream primers, and 1× Q5 Hot Start High-Fidelity Master Mix (New England Biolabs). The PCR reaction program was 98°C for 30 s (98°C, 10 s to 60°C, 10 s to 72°C, 1 min) × 30 cycles to 72°C for 2 min. The PCR products were separated by 1% agarose gel electrophoresis and purified using a gel purification kit (QIAGEN).

[0110] Table 5 Primers for linear DNA template amplification

[0111] Preparation Example 3: Design and Construction of Vectors for Linear DNA Template TP53-shMYC

[0112] Bidirectional regulation of tumor suppressor gene overexpression and oncogene downregulation utilizes a fusion vector that overexpresses the tumor suppressor gene TP53 and downregulates MYC with a shRNA. This vector, named NP-TP53-shMYC, uses a linear DNA template named L-TP53-shMYC. The L-TP53-shMYC vector sequence includes the promoters and terminators for both the TP53 and MYC shRNAs. From the 5' end to the 3' end, it contains the following sequence: F1 box, CMV promoter, TP53 ORF, BGH terminator, hU6 promoter, MYC shRNA, poly-T terminator, and R1 box, totaling 2932 bp. A schematic diagram of the NP-TP53-shMYC vector sequence is shown in Figure 3.

[0113] The construction template of L-TP53-shMYC is derived from the plasmid template P-TP53-shMYC. The construction of the plasmid template adopts the seamless cloning assembly ligation method. The specific process is as follows:

[0114] 1) PCR amplification and gel recovery to obtain linearized vector and insert fragment

[0115] According to Table 6, DNA fragments of varying lengths were amplified by PCR using different plasmids as templates and primers. The PCR reaction system consisted of 50 ng of plasmid template, 2.5 μM of upstream and downstream primers, and 1× Q5 Hot Start High-Fidelity Master Mix (New England Biolabs). The PCR reaction program was 98°C for 30 s (98°C, 10 s to 60°C, 10 s to 72°C, 1 min) followed by 30 cycles (98°C, 10 s to 60°C, 10 s to 72°C, 1 min) followed by 72°C for 2 min. The resulting PCR products were named L-TP53 and L-shMYC. These PCR products were purified using a Gel DNA recovery kit (ZYMO) for subsequent gene assembly reactions.

[0116] Table 6 Primer sequences for constructing P-TP53-shMYC

[0117] 2) Construction of plasmid template P-TP53-shMYC by seamless cloning, assembly and ligation

[0118] According to Table 7, purified DNA fragments of different lengths were prepared and assembled according to the following temperature program: 37°C, 15 min-16°C, 30 min. After the reaction was completed, 50 μL of Stabl4 Chemically Competent Cells (ZC1015) were removed from -80°C and thawed in an ice bath. 5 μL of the ligation product was added to the 50 μL competent cells and quickly and gently pipetted evenly. The cells were placed in an ice bath for 30 min, followed by a heat shock at 42°C for 90 s. The centrifuge tube was then quickly transferred to an ice bath and the cells were allowed to cool for 2-3 min. Finally, 100 μL of sterile LB medium (without antibiotics) was added and mixed. After mixing, the mixture was directly added to Luria-Bertani (LB) solid medium containing the corresponding antibiotic ampicillin. The mixture was evenly spread with sterile plate-spreading glass beads and incubated inverted at 37°C for 16 h. After colonies were formed, single colonies were selected and placed in 10 mL of sterile LB liquid medium. 10 μL of ampicillin (final concentration 100 μg / mL) was added and cultured at 37°C for 16 h. The bacterial solution was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing and analysis.

[0119] Table 7 Seamless cloning assembly ligation reaction system

[0120] 3) Amplification and recovery of the linear DNA template L-TP53-shMYC

[0121] After sequencing verification, the plasmid P-TP53-shMYC was expanded and extracted. The specific process was as follows: 1 mL of glycerol was added to 50 mL of LB liquid medium and shaken at 37°C at 200 rpm for 16-18 hours. The plasmid was extracted using a high-purity, endotoxin-free plasmid extraction kit (Kangwei Century), and the concentration was determined using a NanoDrop 2000 spectrophotometer. PCR amplification was performed using the primers (L-TM-F and L-TM-R) listed in Table 8 using 50 ng of plasmid template, 2.5 μM of upstream and downstream primers, and 1× Q5 Hot Start High-Fidelity Master Mix (New England Biolabs). The PCR reaction program was 98°C for 30 seconds (98°C, 10 seconds to 60°C, 10 seconds to 72°C, 1 minute) × 30 cycles to 72°C, 2 minutes. The PCR products were separated by 1% agarose gel electrophoresis and purified using a gel purification kit (QIAGEN).

[0122] Table 8 Primers for linear DNA template amplification

[0123] Example 1: Construction of multi-gene nanocarriers NP-TP53-BIM-PTEN and NP-TP53-shMYC

[0124] Prepare branched PCR reaction system according to Table 9. The linear DNA templates are L-TP53-BIM-PTEN and L-TP53-shMYC, and the primers are branched primer triplet F 3 and R 3 The final concentration was 0.25 μM. The branched-chain PCR reaction protocol was 95°C for 3 min, followed by 35 cycles of 95°C for 30 s to 58°C for 30 s to 72°C for 4 min, followed by 72°C for 5 min to 4°C for 8 h. PCR products were recovered using a PCR purification kit (Thermo Fisher Scientific). After elution, the concentration was determined and the purity of the products was analyzed by 1% agarose gel electrophoresis.

[0125] The results are shown in Figure 4, where A represents NP-TP53-BIM-PTEN and B represents NP-TP53-shMYC. As can be seen, NP-TP53-BIM-PTEN and NP-TP53-shMYC are completely localized in the gel wells and cannot move downward, and there are no bands corresponding to the linear DNA templates L-TP53-BIM-PTEN and L-TP53-shMYC, indicating that the multi-gene nanocarrier constructed by branched PCR was successfully synthesized.

[0126] Table 9 Branched PCR reaction system

[0127] Test Example 1: Characterization of the physicochemical properties of multi-gene nanocarriers NP-TP53-BIM-PTEN and NP-TP53-shMYC

[0128] 1) Atomic force microscopy (AFM) characterization

[0129] Take 5 μL of multi-gene nanocarrier with a concentration of 20 ng / μL and drop it onto a new clean mica sheet, place it in a clean box and wait for it to dry naturally, and perform scanning observation under AFM tap mode.

[0130] The results are shown in Figures 5 and 6 , from which it can be seen that the AFM results show that the average particle size of the branched PCR product NP-TP53-BIM-PTEN nanoparticles is 104.8±24.2 nm, and the average particle size of NP-TP53-shMYC is 350±30 nm.

[0131] 2) Dynamic light scattering (DLS) characterization

[0132] After dust removal through a 0.22 μm filter membrane, 80 μL of the multi-gene nanocarrier with a concentration of approximately 200 ng / μL was added to a micro-cuvette and subjected to DLS detection at 25°C.

[0133] The results are shown in Figures 7 and 8 , from which it can be seen that the DLS results show that in the aqueous solution state, the hydrodynamic diameter of NP-TP53-BIM-PTEN is 139.1±25.8 nm, and the hydrodynamic diameter of NP-TP53-shMYC is 300-450 nm.

[0134] 3) Serum stability characterization

[0135] The stabilities of multigene nanocarriers, linear DNA, and plasmids were compared in 30% fetal bovine serum (FBS) and analyzed by agarose gel electrophoresis.

[0136] Linear DNA incubation system: 2 μg linear DNA, 6 μL 100% FBS, 2 μL 100 mM PBS, ddH2O to 20 μL (8 copies), incubate at 37°C for 0 h, 2 h, 4 h, 8 h, 12 h, 24 h, 48 h, 72 h, respectively.

[0137] Plasmid incubation system: 2 μg plasmid, 6 μL 100% FBS, 2 μL 100 mM PBS, ddH2O to 20 μL (8 portions), incubate at 37°C for 0 h, 2 h, 4 h, 8 h, 12 h, 24 h, 48 h, 72 h, respectively.

[0138] Multigene nanocarrier incubation system: 2 μg multigene nanocarrier, 6 μL 100% FBS, 2 μL 100 mM PBS, ddH2O to 20 μL (8 portions), incubate at 37°C for 0 h, 2 h, 4 h, 8 h, 12 h, 24 h, 48 h, 72 h, respectively.

[0139] At each independent time point, 2.2 μL 1% SDS was added and the cells were incubated at 37°C for 5 min. The cells were then centrifuged at 13,000 rpm for 15 min at 4°C. The supernatant was loaded with 6× DNA loading buffer and analyzed by agarose gel electrophoresis.

[0140] The results are shown in Figures 9 and 10. As can be seen from Figure 9, the multi-gene nanocarrier has higher serum stability compared to linear DNA and plasmids. For example, L-TP53-BIM-PTEN and P-TP53-BIM-PTEN were completely degraded at 24 hours, while NP-TP53-BIM-PTEN was completely degraded as early as 72 hours. As can be seen from Figure 10, for NP-TP53-shMYC, the multi-gene nanocarrier has higher serum stability compared to linear DNA and plasmids. For example, L-TP53-shMYC and P-TP53-shMYC were completely degraded at 2 hours, while NP-TP53-BIM-PTEN was completely degraded as early as 96 hours.

[0141] Test Example 2: Multi-gene nanocarrier NP-TP53-BIM-PTEN simultaneously overexpresses three tumor suppressor genes in lung cancer cells

[0142] 1) Cell transfection

[0143] The type of cell culture medium is directly related to the cell type. This paper uses NCI-H1299 non-small cell lung cancer cells as an example to illustrate the cell expression and activity analysis process of multi-gene nanocarriers in NCI-1299 cells. The cell transfection process is specifically as follows:

[0144] a) During cell passage, add 2 mL of cell suspension to 10 mL of complete RPMI 1640 medium, mix thoroughly, and add 2 mL / well of cell suspension to a 6-well plate, ensuring even cell distribution and a cell abundance of approximately 60-70%. After incubation at 37°C, 5% CO₂ for 12-16 hours, transfect the multi-gene nanovectors into NCI-H1299 cells using the transfection reagent Lipofectamine 3000 (purchased from Invitrogen).

[0145] b) Dilute Lipofectamine 3000 in Opti-MEM: Dilute 5 μL of Lipofectamine 3000 with 195 μL of serum-free Opti-MEM, mix gently with a pipette, and let stand at room temperature for 5 minutes;

[0146] c) Diluting the gene vector with Opti-MEM: Based on a transfection concentration of 1.6 μg / mL, take 1.6 μg of the multi-gene nanocarrier, 5 μL of the P3000 auxiliary packaging component, and Opti-MEM, totaling 200 μL, and add them to a 1.5 mL centrifuge tube. Gently mix with a pipette. Repeat this process for other transfection methods.

[0147] d) Mix equal volumes of Lipofectamine 3000 liposomes diluted in Opti-MEM and multi-gene nanocarriers diluted in Opti-MEM and let stand at room temperature for 15 minutes;

[0148] e) Discard the original RPMI 1640 complete medium in the 6-well plate, add 600 μL of Opti-MEM medium, and evenly add 400 μL of the mixture from step d) dropwise to the corresponding wells. Gently shake the 6-well plate to evenly distribute the cells. Return the cells to a cell culture incubator at 37°C, 5% CO₂ for 4 hours. Then, replace with 2 mL / well of RPMI 1640 complete medium. Continue culturing for another 48 hours, then extract total RNA and total protein for subsequent fluorescence quantification and western blot analysis.

[0149] 2) Fluorescence quantitative PCR

[0150] 48 hours after transfection, cells were harvested and total RNA was extracted using an animal tissue / cell RNA extraction kit (Cornwell). RNA was used as a template to synthesize the first strand of cDNA using an RNA reverse transcription kit (Quanshijin). The reverse transcription reaction system was: 1 μg RNA, 1 μL gDNA Remover, 4 μL All-in-One SuperMix for qPCR, add nuclease-free ultrapure water (Nuclease-free H2O) to 1μL. The reverse transcription reaction program is: 42℃, 15min-85℃, 5s. Fluorescence quantitative PCR reaction system preparation: 1μL cDNA, 0.5μL quantitative primer (10μM), 10μL Green qPCR SuperMix (Full Gold) was supplemented with Nuclease-free H2O to 20 μL. The fluorescence quantitative PCR program was 94°C, 5 min-(94°C, 5 s-58°C, 20 s-72°C, 5 min) × 40 cycles. The relative expression of different genes was analyzed using 2 -ΔΔCq Method to calculate.

[0151] 3) Western blot analysis

[0152] 48 hours after transfection, cells were harvested and the supernatant discarded. An appropriate volume of RIPA lysis buffer was added to the cell pellet. 6Each cell requires approximately 100 μL of RIPA lysis buffer. Use a micropipette to repeatedly pipette several times to thoroughly loosen the cell pellet, then place on ice for 0.5 h. Once fully lysed, place the 1.5 mL centrifuge tube in a 4°C centrifuge and centrifuge at 12,000 rpm for 10 min. Transfer the supernatant to a fresh 1.5 mL centrifuge tube; this represents total protein. Total protein is quantified using the Thermo Fisher BCA assay kit. Based on the measured protein concentration, add appropriate amounts of 5× SDS loading buffer and ddH₂O to the protein stock solution, reducing the 5× SDS loading buffer to 1× SDS loading buffer. After thorough mixing, place the 1.5 mL EP tube containing the protein in a metal bath and heat at 100°C for 10 min. Remove the protein and load it for electrophoresis. Load the sample (15 μL) onto a 4-20 wt% precast gel and electrophorese at 140 V for 40 min. After electrophoresis, prepare a 1× transfer buffer (containing 20% ​​anhydrous methanol). Activate a PVDF membrane by soaking it in anhydrous methanol for 15 seconds. Remove the precast gel from the electrophoresis tank, attach the precast gel to the filter paper, and finally place the transfer rack into the transfer tank. Transfer the membrane at 90V for 120 minutes. After transfer, remove the PVDF membrane and wash it twice with TBST for 5 minutes each. Block the PVDF membrane with TBST containing 5% skim milk for 1 hour, then wash it three times with TBST for 5 minutes each. Apply primary antibody at various dilutions, completely immersing the PVDF membrane in the primary antibody dilution buffer and shaking overnight at 4°C. Before incubation with secondary antibody dilution, wash the membrane five times with TBST for 10 minutes each. Dilute the secondary antibody at a 1:5000 dilution, incubate the membrane in the secondary antibody dilution buffer for 1 hour, and then wash it three times with TBST for 10 minutes each. Finally, the luminescent substrate ECL was prepared in a 1:1 ratio of Solution A:Solution B in the dark. The PVDF membrane was soaked in ECL for approximately 5 minutes and placed in the darkroom of an exposure instrument. The exposure time was determined based on the intensity of the protein bands. The intensity of the protein bands was analyzed using Image J software for grayscale analysis.

[0153] The present invention uses the NCI-H1299 non-small cell lung cancer cell line to test the feasibility of simultaneously overexpressing three tumor suppressor genes in cancer cells using NP-TP53-BIM-PTEN. Using the transfection reagent Lipofectamine 3000, NP-TP53-BIM-PTEN was transfected into NCI-H1299 at different concentrations (0.2-3.2 μg / mL). As a negative control, the multi-gene nanocarrier NP-EGFP carrying the EGFP gene expression cassette was transfected into NCI-H1299 at a concentration of 2.0 μg / mL. After 48 hours of transfection, the mRNA expression levels and protein expression levels of TP53, BIM and PTEN in the cells were detected by qPCR and western blotting, respectively.

[0154] The results are shown in Figures 11 and 12, where in Figure 11, A is a graph showing the mRNA expression level of TP53, B is a graph showing the mRNA expression level of BIM, and C is a graph showing the mRNA expression level of PTEN; in Figure 12, A is a western blotting imaging graph; B is a statistical graph showing the expression level of TP53 protein; C is a statistical graph showing the expression level of BIM protein; and D is a statistical graph showing the expression level of PTEN protein.

[0155] Results showed that NP-EGFP was unable to induce the expression of p53, BIM, and PTEN proteins. In cells transfected with NP-TP53-BIM-PTEN, the mRNA and protein expression levels of TP53, BIM, and PTEN increased in a dose-dependent manner. As shown in Figure 11, when the NP-TP53-BIM-PTEN transfection concentration reached 3.2 μg / mL, TP53, BIM, and PTEN mRNA expression increased by 82639-fold, 685-fold, and 10-fold, respectively, compared to the untreated group. Figure 12 shows that compared to the untreated group, in NCI-H1299 cells transfected with 3.2 μg / mL NP-TP53-BIM-PTEN, the protein levels of p53, BIM, and PTEN increased by 75-fold, 57-fold, and 5-fold, respectively. These results demonstrate that NP-TP53-BIM-PTEN can simultaneously overexpress three tumor suppressor genes in the NCI-H1299 non-small cell lung cancer cell line.

[0156] Test Example 3: Multi-gene nanocarrier NP-TP53-shMYC synchronously overexpresses tumor suppressor genes and silences proto-oncogenes in breast cancer cells

[0157] The methods of cell transfection, fluorescent quantitative PCR, and western blot analysis are similar to those in Test Example 2, except that:

[0158] The present invention uses breast cancer cells MDA-MB-231 to test the feasibility of NP-TP53-shMYC in synchronously overexpressing tumor suppressor genes and silencing proto-oncogenes in MDA-MB-231 cancer cells. Using the transfection reagent Lipofectamine 3000, NP-TP53-shMYC was transfected into MDA-MB-231 at different concentrations (0.1 to 1.6 μg / mL). As a negative control, the multi-gene nanocarrier NP-EGFP carrying the EGFP gene expression cassette was transfected into MDA-MB-231 at a concentration of 2.0 μg / mL. After 48 hours of transfection, qPCR was used to detect the gene expression levels of TP53 and MYC in the cells. Western blotting was used to detect the protein expression levels of TP53 and MYC in the cells.

[0159] The results are shown in FIG13 and FIG14 . In FIG13 , A is a statistical graph showing the expression level of TP53 mRNA, and B is a statistical graph showing the expression level of MYC mRNA.

[0160] The results showed that NP-EGFP did not induce p53 expression or silence MYC protein. As shown in Figure 13, in MDA-MB-231 cells, TP53 mRNA transcription levels increased with increasing transfection doses of NP-TP53-shMYC. Specifically, at a dose of 1.2 μg / mL, the TP53 mRNA transcriptional fold increased by approximately 55-fold compared to the negative control group, indicating that NP-TP53-shMYC can overexpress the TP53 gene in breast cancer cells. Compared to the blank group and NP-EGFP, MYC mRNA transcription levels decreased with increasing transfection doses of NP-TP53-shMYC. Specifically, at a dose of 1.6 μg / mL, the MYC mRNA transcriptional fold decreased by 80% compared to the negative control group, indicating that NP-TP53-shMYC can downregulate MYC gene expression in breast cancer cells through RNA interference.

[0161] Western blotting was then used to detect the expression abundance of the corresponding proteins, and the results were consistent with the qPCR results. As can be seen in Figure 14, compared with the Blank group and NP-EGFP, as the transfection dose of NP-TP53-shMYC increased, the expression of p53 protein increased in a dose-dependent manner, while the expression of c-Myc protein decreased in a dose-dependent manner.

[0162] These results indicate that NP-TP53-shMYC can not only overexpress the tumor suppressor gene TP53 in cancer cells, but also downregulate the abnormally overexpressed proto-oncogene MYC in cancer cells through RNA interference, thus realizing a combined regulatory model of different gene regulation technologies.

[0163] Test Example 4: The multi-gene nanocarrier NP-TP53-BIM-PTEN, which simultaneously overexpresses three tumor suppressor genes, significantly increases cell apoptosis and proliferation inhibition efficiency

[0164] 1) Cell viability assay

[0165] The cell viability was detected using the CCK8 kit. The specific process is as follows:

[0166] a) Plating: NCI-H1299 cells were plated in 96-well plates at 1×10 cells per well. 4 cells (125 μL) and vortex thoroughly to keep the cells evenly distributed.

[0167] Four parallel groups were set up for each experimental group;

[0168] b) Transfection: 12-16 hours after plating cells, prepare a transfection mix in 5 replicates per group (×5) and transfer to 4 wells. Specifically, dilute 1.5 μL of Lipofectamine 3000 with 60.9 μL of serum-free Opti-MEM medium, mix gently with a pipette, and incubate at room temperature for 5 minutes. Based on a transfection concentration of 1.6 μg / mL, add 1.6 μg of multigene nanocarrier, 1.5 μL of P3000, and Opti-MEM (a total volume of 62.5 μL) to a 1.5 mL centrifuge tube and mix gently with a pipette. Repeat for other transfection methods. Mix equal volumes of Lipofectamine 3000 diluted in Opti-MEM and multigene nanocarrier diluted in Opti-MEM and incubate at room temperature for 15 minutes. Remove 87.5 μL of the original RPMI 1640 complete medium from the 96-well plate and add 25 μL of the transfection mix dropwise to the corresponding wells. The cells were returned to a cell culture incubator at 37°C and 5% CO2 and cultured for 48 h. 37.5 μL of RPMI 1640 complete medium was added to each well of the 96-well plate, maintaining a volume of 100 μL per well.

[0169] c) CCK8 treatment: After 48 h of culture, 10 μL of CCK8 solution (MedChemExpress) was added to each well. The culture plate was placed in an incubator and incubated for 2 h. The absorbance at 450 nm was measured using a microplate reader. After measuring the absorbance at 450 nm, the cell viability was calculated according to the formula: [(As-Ab) / (Ac-Ab)] × 100%, where As: absorbance of the experimental well (containing cells, culture medium, CCK-8 solution, and drug solution); Ac: absorbance of the control well (containing cells, culture medium, CCK-8 solution, but no drug solution); Ab: absorbance of the blank well (containing culture medium and CCK-8 solution, but no cells or drug solution).

[0170] 2) Cell apoptosis detection

[0171] Cell apoptosis was detected using the Annexin V-FITC / PI double staining cell detection kit and flow cytometry. The specific process is as follows:

[0172] a) Plating: NCI-H1299 cells were plated in a 12-well plate and the cells were evenly distributed.

[0173] b) Transfection: 12-16 hours after cell plating, the multi-gene nanocarriers were transfected into NCI-H1299 cells using the transfection reagent Lipofectamine 3000. The specific operation is as follows: dilute 2.5μL Lipofectamine 3000 with 97.5μL serum-free culture medium Opti-MEM, mix gently with a pipette, and let it stand at room temperature for 5 minutes; according to the transfection concentration of 4μg / mL, take 4μg multi-gene nanocarrier, 2.5μL P3000 and Opti-MEM, a total volume of 100μL, add them to a 1.5mL centrifuge tube, and mix gently with a pipette. The same applies to other different transfection masses; mix equal volumes of Lipofectamine3000 diluted with Opti-MEM and multi-gene nanocarrier diluted with Opti-MEM, and let it stand at room temperature for 15 minutes; discard the original RPMI 1640 complete culture medium in the 12-well plate, add 300μL Opti-MEM culture medium, and evenly add 200μL of the mixture to the corresponding wells, and gently shake the 12-well plate to make it evenly distributed. The cells were returned to a cell culture incubator at 37° C. and 5% CO 2 and cultured for 4 h. Then, RPMI 1640 complete medium was replaced at a volume of 1 mL / well and cultured for a further 48 h.

[0174] c) Cell collection: Use a pipette to collect the supernatant in a 15 mL centrifuge tube. After centrifugation, wash the cells once with PBS. Add 200 μL of trypsin to each well to digest the adherent cells. Digest for 2 minutes, then add 1 mL of RPMI 1640 complete medium. Combine the adherent digested cell suspension and the supernatant collected from the PBS-resuspended cell suspension. Centrifuge at 2,000 rpm for 3-5 minutes at room temperature. Discard the supernatant and collected cells. Resuspend the cells once in pre-chilled 1× PBS, centrifuge at 2,000 rpm for 3-5 minutes, and collect the cell pellet.

[0175] d) Dye incubation: Add 300 μL of 1× Binding Buffer, gently mix to suspend the cells, add 5 μL of Annexin V-FITC dye, and incubate at room temperature in the dark for 15 minutes. 5 minutes before loading, add 5 μL of PI dye and add 200 μL of 1× Binding Buffer.

[0176] e) Fluorescence signal collection: Cell apoptosis was detected using a flow cytometer (LSRFortessa, BD), and the software FlowJo-V10 was used to analyze the cell apoptosis.

[0177] The present invention used the NCI-H1299 non-small cell lung cancer cell line to test whether simultaneous overexpression of three tumor suppressor genes, NP-TP53-BIM-PTEN, in cancer cells increases cell apoptosis. Flow cytometry was performed 48 hours after transfection using FITC-labeled Annexin V and PI to detect cell apoptosis.

[0178] The results are shown in Figures 15 and 16. In Figure 15, A is a statistical graph showing the apoptosis rate of NCI-H1299 cancer cells at different concentrations of NP-TP53-BIM-PTEN; B is a statistical graph showing the apoptosis rate of NCI-H1299 cancer cells at different concentrations of L-TP53-BIM-PTEN; and C is a statistical graph showing the apoptosis rate of NCI-H1299 cancer cells at different concentrations of P-TP53-BIM-PTEN. In Figure 16, A is a statistical graph showing the relative cell viability of NCI-H1299 cancer cells at different concentrations of NP-TP53-BIM-PTEN; B is a statistical graph showing the relative cell viability of NCI-H1299 cancer cells at different concentrations of L-TP53-BIM-PTEN; and C is a statistical graph showing the relative cell viability of NCI-H1299 cancer cells at different concentrations of P-TP53-BIM-PTEN.

[0179] The results showed that NP-TP53-BIM-PTEN induced significant apoptosis in NCI-H1299 cells, and the apoptosis induction rate was dose-dependent. As can be seen in Figure 15A, within the transfection concentration range (0.1-4.0 μg / mL), the apoptosis rate induced by NP-TP53-BIM-PTEN was 13.55% to 94.90%, of which the average apoptosis induction rate of cells treated with 4.0 μg / mL NP-TP53-BIM-PTEN was close to 95%. As can be seen in Figure 15B and C, the apoptosis rates induced by L-TP53-BIM-PTEN and P-TP53-BIM-PTEN were relatively weak, with apoptosis induction rates maintained at 14.88% to 32.90% and 3.49% to 24.87%, respectively. At a transfection concentration of 4.0 μg / mL, the cell apoptosis rate induced by NP-TP53-BIM-PTEN was 2.9-fold and 3.7-fold higher than that of L-TP53-BIM-PTEN and P-TP53-BIM-PTEN, respectively.

[0180] 48 hours after transfection, cell proliferation inhibitory activity was further determined using a cell counting kit 8 (CCK8). The results showed that, as can be seen in Figure 16A, NP-TP53-BIM-PTEN can effectively inhibit the proliferation of NCI-H1299 cells. At a transfection concentration of 3.2 μg / mL, the inhibition rate of NP-TP53-BIM-PTEN on NCI-H1299 cell proliferation reached 42.00% ± 4.40%. As can be seen in Figure 16B and C, the inhibition rates of L-TP53-BIM-PTEN and P-TP53-BIM-PTEN on NCI-H1299 cell proliferation were 40.69% ± 3.77% and 32.92% ± 1.65%, respectively, which were significantly lower than the cell proliferation inhibition rate of NP-TP53-BIM-PTEN.

[0181] These results show that three tumor suppressor genes (TP53, BIM, PTEN) delivered by multi-gene nanocarriers can be expressed simultaneously in NCI-H1299 and significantly enhance the cell apoptosis induction rate, while also greatly reducing cell proliferation activity, indicating that restoring the expression capacity of multiple tumor suppressor genes in cancer cells will help improve the toxicity of gene therapy to cancer cells.

[0182] Test Example 5: Multi-gene nanocarrier NP-TP53-shMYC that simultaneously overexpresses tumor suppressor genes and silences proto-oncogenes significantly increases cell apoptosis activity

[0183] The method for detecting cell apoptosis is similar to that of Test Example 4, except that:

[0184] The present invention used breast cancer cells (MDA-MB-231) to test whether NP-TP53-shMYC, when used to simultaneously overexpress a tumor suppressor gene (TP53) and silence a proto-oncogene (MYC) via shRNA, increases cell apoptosis. MDA-MB-231 cells were transfected with varying concentrations of NP-TP53-shMYC (0 to 2.0 μg / mL). 48 hours after transfection, apoptosis was assessed by flow cytometry using FITC-labeled Annexin V and PI. NP-EGFP served as a negative control, while NP-TP53 and NP-shMYC served as single-gene controls for comparative analysis.

[0185] The results are shown in Figure 17. Flow cytometry analysis results showed that NP-EGFP could not induce apoptosis in MDA-MB-231 cells, while NP-TP53-shMYC could significantly induce apoptosis in MDA-MB-231 cells, and the apoptosis induction rate was dose-dependent.

[0186] As can be seen from Figure 17, when the transfection concentration of NP-TP53-shMYC is 2.0 μg / mL, the average apoptosis rate of MDA-MB-231 cells is close to 77.4%, while the average apoptosis rates of MDA-MB-231 cells induced by NP-TP53 and NP-shMYC are 54.1% and 56.3%, respectively. This indicates that the simultaneous targeting of TP53 and MYC genes has stronger apoptosis activity in cancer cells than that induced by single gene targeting. The bidirectional gene regulation of TP53 overexpression and MYC downregulation by RNA technology with the help of multi-gene nanocarriers helps to enhance the sensitivity of cancer cell apoptosis signaling pathways, thereby showing stronger apoptosis inducing activity.

[0187] Test Example 6: Multigene Nanocarrier NP-TP53-BIM-PTEN Synchronously Overexpressing Three Tumor Suppressor Genes Enhances Anti-tumor Effects in Xenograft Mice

[0188] 1) Construction of xenograft tumor mouse model

[0189] The animal model used to construct xenograft tumors is 6-8 week old BALB / c nude male mice. The specific operation is as follows: After purchasing mice from Beijing Weitonglihua Company, they are first cultured for one week under sterile conditions. The mouse culture conditions need to meet the following requirements: strictly follow the principle of sterile operation, the mice are caged in a laminar flow chamber, all the instruments used are sterilized at high temperature, the bedding, feed and drinking water are sterilized, the laboratory environment temperature is 18℃-28℃, the relative humidity is 40%-60%, the mice are free to eat and drink, the lighting is alternating for 12 hours (8:00-20:00), the bedding is kept dry, and the bedding is changed twice a week. When the mice are in good condition, NCI-H1299 cells (3×10 6 ) were injected subcutaneously in the right lower abdomen. 15 days after the injection of cells, a 100mm 3 The tumor mass indicated that the mouse model was successfully established.

[0190] 2) Multi-gene nanocarrier delivery

[0191] The mice with successful modeling were randomly divided into 6 groups, with 4 mice in each group, and treated by intratumoral administration. The multi-gene nanocarrier was delivered using Lipofectamine 2000, with each 100 μL injection volume containing 45 μL Lipofectamine 2000. The dosage of the multi-gene nanocarrier was determined according to the weight of the mouse, and was intratumorally administered at 2.25 mg / kg, once every two days, for a total of 3 doses. The specific grouping and dosing schedule are as follows: negative control group (100 μL PBS), multi-gene nanocarrier test group (100 μL multi-gene nanocarrier, 45 μg).

[0192] 3) Observation and recording of tumor growth status

[0193] After administration, the tumor volume, weight, and body weight of the mice were measured and recorded daily until the mice were processed. The tumor volume and body weight change trends were plotted based on the changes in weight, volume, and body weight over different days.

[0194] 4) Molecular marker tracing related to tumor growth and apoptosis

[0195] After the multi-gene nanocarrier enters the tumor tissue, whether it can effectively regulate the expression of related genes and induce apoptosis and inhibit cell proliferation in the tumor tissue requires further testing of changes in molecular markers related to tumor growth and apoptosis. Methods for detecting molecular markers related to tumor growth and apoptosis mainly include fluorescent quantitative PCR, Western blotting, TUNEL apoptosis analysis, and immunohistochemistry. The specific procedures for each method are as follows:

[0196] a) Fluorescence quantitative PCR

[0197] After tumor tissue was removed, the tissue was ground using a high-throughput tissue grinder, and total RNA was extracted using an Animal Tissue / Cell RNA Extraction Kit (Kangwei Century). The first strand of cDNA was synthesized using RNA as a template using an RNA Reverse Transcription Kit (Quanshijin). The reverse transcription reaction system was: 1 μg RNA, 1 μL gDNA Remover, 4 μL All-in-One SuperMix for qPCR, supplemented with Nuclease-free H2O to 1 μL. Reverse transcription reaction program: 42℃, 15min-85℃, 5s. Fluorescence quantitative PCR reaction system preparation: 1 μL cDNA, 0.5 μL quantitative primer (10 μM), 10 μL Green qPCR SuperMix (Full Gold) was supplemented with Nuclease-free H2O to 20 μL. The fluorescence quantitative PCR program was 94°C, 5 min-(94°C, 5 s-58°C, 20 s-72°C, 5 min) × 40 cycles. The relative expression of different genes was analyzed using 2 -ΔΔCq Method to calculate.

[0198] b) Western blot analysis

[0199] After tumor tissue removal, grind the tissue using a high-throughput tissue grinder and add an appropriate volume of RIPA lysis buffer. Once the tumor tissue is fully lysed, transfer the tissue solution to a 1.5 mL centrifuge tube and centrifuge at 12,000 rpm for 10 minutes at 4°C. Transfer the supernatant to a fresh 1.5 mL centrifuge tube, which represents total protein. Total protein is quantified using the Thermo Fisher BCA assay kit. Based on the measured protein concentration, add appropriate amounts of 5× SDS loading buffer and ddH₂O to the protein stock solution, reducing the 5× SDS loading buffer to 1× SDS loading buffer. After thorough mixing, place the 1.5 mL EP tube containing the protein in a metal bath and heat at 100°C for 10 minutes. The protein is then removed and loaded for electrophoresis. The sample (15 μL) is loaded onto a 4-20% precast gel and electrophoresed at 140 V for 40 minutes. After electrophoresis, prepare a 1x transfer buffer (containing 20% ​​anhydrous methanol). Activate a PVDF membrane by soaking it in anhydrous methanol for 15 seconds. Remove the precast gel from the electrophoresis tank, attach the precast gel to the filter paper, and finally place the transfer rack into the transfer tank. Transfer the membrane at 90V for 120 minutes. After transfer, remove the PVDF membrane and wash it twice with TBST for 5 minutes each. Block the PVDF membrane with TBST containing 5% skim milk for 1 hour and then wash it three times with TBST for 5 minutes each. Apply primary antibody at various dilutions, completely immersing the PVDF membrane in the primary antibody dilution solution and shaking overnight at 4°C. Before incubation with secondary antibody dilution, wash the membrane five times with TBST for 10 minutes each. Dilute the secondary antibody at a 1:5000 dilution, incubate the membrane in the secondary antibody dilution solution for 1 hour, and then wash it three times with TBST for 10 minutes each. Finally, the luminescent substrate ECL was prepared in a 1:1 ratio of Solution A:Solution B in the dark. The PVDF membrane was soaked in ECL for approximately 5 minutes and placed in the darkroom of an exposure instrument. The exposure time was determined based on the intensity of the protein bands. The intensity of the protein bands was analyzed using Image J software for grayscale analysis.

[0200] c) TUNEL apoptosis analysis

[0201] Tumor tissue was collected and placed in a paraffin tissue embedding cassette. It was then fixed in 4% paraformaldehyde for 48 hours and dehydrated in a stepwise manner using different ethanol gradients: 70% ethanol for 1 hour, 70% ethanol for 2 hours, 80% ethanol for 30 minutes, 90% ethanol for 30 minutes, 95% ethanol (I) for 30 minutes, 95% ethanol (II) for 1 hour, 100% ethanol (I) for 30 minutes, 100% ethanol (II) for 40 minutes, xylene (I) for 20 minutes, xylene (II) for 35 minutes, paraffin bath (I) for 1 hour, and paraffin bath (II) for 1 hour. The dehydrated tumor tissue was embedded in a paraffin embedding machine to prepare paraffin tissue blocks. The paraffin tissue blocks were sectioned using a paraffin microtome, and 4 μm thick tumor tissue sections were cut and stored in a sectioning cassette at room temperature. The sections were then placed in xylene (I) for 15 minutes, xylene (II) for 15 minutes, anhydrous ethanol (I) for 5 minutes, anhydrous ethanol (II) for 5 minutes, 85% alcohol for 5 minutes, 75% alcohol for 5 minutes, and finally washed with distilled water. Finally, the paraffin sections were dewaxed to water. After the sections were slightly shaken dry, a circle was drawn around the tissue using a histochemical pen (to prevent liquid from escaping). Proteinase K working solution was added to the circle to cover the tissue and incubated in a 37°C incubator for 15-30 minutes. The slides were washed three times in PBS (pH 7.4) on a decolorizing shaker, each for 5 minutes. After the sections were slightly shaken dry, permeabilization working solution was added to the circle to cover the tissue and incubated at room temperature for 20 minutes. The slides were washed three times in PBS (pH 7.4) on a decolorizing shaker, each for 5 minutes. Based on the number of slides and tissue size, a 1:9 mixture of Reagent 1 (TdT) and Reagent 2 (dUTP) from the TUNEL kit was prepared and kept away from light. Prepared TUNEL was added to the circle to cover the tissue. The sections were placed flat in a humidified chamber and incubated at 37°C for 2 hours. A small amount of water was added to maintain humidity. The sections were washed three times with PBS (pH 7.4), each for 5 minutes. After removing the PBS, DAPI stain was added dropwise to the circle and incubated at room temperature in the dark for 10 minutes. The slides were washed three times with PBS (pH 7.4) on a decolorizing shaker, each for 5 minutes. The sections were briefly dried and mounted with anti-fading mounting media. The sections were observed and images were acquired under a fluorescence microscope. DAPI excitation wavelength was 330-380 nm, emission wavelength 420 nm, emitting blue light; FITC excitation wavelength was 465-495 nm, emission wavelength 515-555 nm, emitting green light; CY3 excitation wavelength was 510-560 nm, emission wavelength 590 nm, emitting red light.

[0202] d) Immunohistochemical analysis of the proliferation marker Ki67

[0203] Paraffin sections were sequentially placed in xylene (I) for 20 minutes, xylene (II) for 20 minutes, xylene (III) for 15 minutes, anhydrous ethanol (I) for 5 minutes, anhydrous ethanol (II) for 5 minutes, 85% ethanol for 5 minutes, and 75% ethanol for 5 minutes. Wash with distilled water. Antigen retrieval was performed in a microwave oven filled with citric acid antigen retrieval buffer (pH 6.0) or EDTA antigen retrieval buffer (pH 9.0) for 5 minutes each on medium, medium-high, and high heat. After cooling naturally, the slides were washed three times with shaking in PBS (pH 7.4) for 5 minutes each.

[0204] Place the slides in 3% hydrogen peroxide solution and incubate at room temperature in the dark for 25 minutes. Wash and soak the slides three times in PBS (pH 7.4) with shaking, 5 minutes each time. After the slides are slightly dried, use a histochemical pen to draw a circle around the tissue. Add goat serum to the circle to evenly cover the tissue and block at room temperature for 30 minutes. Gently shake off the blocking solution and apply a 1:200 dilution of primary antibody to the slides. Incubate the slides flat in a humidified chamber at 4°C overnight. Wash the slides three times in PBS (pH 7.4) with shaking, 5 minutes each time. After the slides are slightly dried, add a secondary antibody (HRP-conjugated) of the same species as the primary antibody to the circle to cover the tissue and incubate at room temperature for 1 hour. Wash and soak the slides three times in PBS (pH 7.4) with shaking, 5 minutes each time. After the slides are slightly dried, add freshly prepared DAB colorimetric solution to the circle. Color development is monitored under a microscope. A positive color is brownish-yellow. Rinse the slides with tap water to terminate color development. Counterstain with hematoxylin for approximately 5-10 minutes, rinse with tap water, and then incubate with 1% hydrochloric acid alcohol for several seconds, rinse with tap water, then debluing with 1% ammonia solution for 1 minute, and rinse with tap water. Sections were then dehydrated and transparentized in 75% alcohol for 5 minutes, 85% alcohol for 5 minutes, anhydrous ethanol (I) for 5 minutes, anhydrous ethanol (II) for 5 minutes, and xylene (I) for 5 minutes. Sections were removed from the xylene, allowed to dry briefly, and mounted with neutral gum. After mounting, microscopic photographs were taken. Hematoxylin stains nuclei blue, while DAB stains positive cells brownish-yellow. Statistical analysis was performed using a semiquantitative method, with a staining score (scale 0-12) established: 1. Score based on the intensity of positive staining (weak: 1; mild: 2; strong: 3); 2. Score based on the proportion of positive cells of interest (0%, 0 points; <25%, 1 point; 26-50%, 2 points; 51-75%, 3 points; >76%, 4 points). Statistics were performed according to the established scoring system, with 4 mice in each group and 2 visual fields for each mouse.

[0205] The present invention uses a tumor-bearing mouse model transplanted with the NCI-H1299 non-small cell lung cancer cell line and uses an intratumoral injection method to test the anti-tumor therapeutic effect of NP-TP53-BIM-PTEN.

[0206] The in vivo anti-tumor therapeutic effect evaluation of the multi-gene nanocarrier is shown in Figure 18, wherein A is the treatment regimen; B is the tumor image of the mouse 12 days after administration, PBS: mice were administered with PBS containing Lipofectamine 2000; NP-TP53: mice were administered with NP-TP53 with the help of Lipofectamine 2000; NP-BIM: mice were administered with NP-BIM with the help of Lipofectamine 2000; NP-PTEN: mice were administered with NP-PTEN with the help of Lipofectamine 2000; NP-TP53-PTEN: mice were administered with NP-TP53-PTEN with the help of Lipofectamine 2000; NP-TP53-BIM-PTEN: mice were administered with the help of Lipofectamine 2000; C is the tumor growth curve within 12 days of administration. The experimental group was administered with a dose of 2.25 mg / kg every 2 days (days 1, 3, and 5) for a total of 3 treatments. The arrows indicate the time of administration; D is a statistical chart of tumor weights in different treatment groups on day 12.

[0207] As can be seen from the treatment scheme of Figure 18A, the successfully modeled mice were randomly divided into 6 groups, 4 mice per group, and treated by intratumoral administration. The multi-gene nanocarrier was delivered using Lipofectamine 2000, with 45 μL Lipofectamine 2000 contained in each 100 μL injection volume. The dosage of the multi-gene nanocarrier was determined according to the weight of the mouse, and was intratumorally administered at 2.25 mg / kg, once every two days, for a total of 3 doses. The specific grouping and dosing regimen are as follows: negative control group (100 μL PBS), multi-gene nanocarrier test group (100 μL multi-gene nanocarrier, 45 μg). The anti-tumor treatment test was set up as follows: PBS (negative control group), NP-TP53, NP-BIM, NP-PTEN, NP-TP53-PTEN, NP-TP53-BIM-PTEN.

[0208] As can be seen from Figure 18B, compared with the negative control in vivo, NP-TP53-PTEN and NP-TP53-BIM-PTEN showed significant anti-tumor activity, which was consistent with the anti-tumor effect in vitro.

[0209] As shown in Figures 18C and D, NP-TP53, NP-BIM, and NP-PTEN also exhibited antitumor activity compared to the negative control group, but their antitumor activity was lower than that of the NP-TP53-PTEN and NP-TP53-BIM-PTEN treatment groups. On day 12 of dosing, the average tumor volume and tumor weight of mice in the NP-TP53-BIM-PTEN group were reduced by 84.7% and 85.2%, respectively, compared to those in the PBS group. Similarly, on day 12 of dosing, the average tumor volume and tumor weight of mice in the NP-TP53-PTEN group were reduced by 80.8% and 81.6%, respectively, compared to those in the PBS group. The average tumor volume and tumor weight of mice in the NP-TP53 group were reduced by 76.9% and 76.0%, respectively, compared to those in the PBS group. The average tumor volume and tumor weight of mice in the NP-BIM group were reduced by 71.4% and 66.6%, respectively, compared to those in the PBS group. The average tumor volume and tumor weight of mice in the NP-PTEN group were reduced by 64.7% and 62.0%, respectively, compared to those in the PBS group. The mean tumor volume and weight of the multigene nanocarriers carrying two or three gene expression cassettes were significantly lower than those carrying a single gene expression cassette. Compared with NP-TP53, NP-BIM, and NP-PTEN, the mean tumor volume of NP-TP53-BIM-PTEN was reduced by 33.7%, 46.5%, and 56.6%, respectively. Tumor weights of mice were reduced by 38.5%, 55.9%, and 61.2%, respectively, compared with NP-TP53, NP-BIM, and NP-PTEN. Compared with NP-TP53 and NP-PTEN, the mean tumor volume of NP-TP53-PTEN was reduced by 16.7% and 45.4%, respectively. Tumor weights of mice were reduced by 23.2% and 51.6%, respectively, compared with NP-TP53 and NP-PTEN. The mean tumor volume and weight of mice in the NP-TP53-BIM-PTEN group were reduced by 20.4% and 19.9%, respectively, compared with the NP-TP53-PTEN group.

[0210] These results indicate that the three tumor suppressor genes (TP53, BIM, and PTEN) delivered by multi-gene nanocarriers can also be simultaneously expressed in NCI-H1299 xenograft tumors and exhibit anti-tumor therapeutic effects mediated by single gene overexpression.

[0211] The present invention further utilizes qPCR and Western blotting techniques to quantitatively analyze the expression levels of three tumor suppressor genes to verify whether the inhibition of tumor growth is associated with the restoration of TP53, BIM and PTEN gene expression in tumors.

[0212] The results are shown in Figure 19, where A is the qPCR detection of TP53 mRNA level in tumor tissue on day 12; B is the qPCR detection of BIM mRNA level in tumor tissue on day 12; C is the qPCR detection of PTEN mRNA level in tumor tissue on day 12; D and E are Western blotting detections of TP53, BIM, and PTEN protein levels in tumor tissue on day 12.

[0213] As shown in Figure 19A, qPCR analysis on day 12 after administration showed that the expression of these genes was upregulated in both the NP-TP53-BIM-PTEN and NP-TP53-PTEN groups compared to the PBS group. TP53 mRNA expression levels were increased by 16.9-fold and 5.5-fold in the NP-TP53-PTEN and NP-TP53-BIM-PTEN groups, respectively.

[0214] As can be seen from FIG19B , BIM mRNA expression in the NP-TP53-BIM-PTEN group was upregulated by 7.0-fold.

[0215] As can be seen from FIG19C , the PTEN mRNA expression levels in the NP-TP53-PTEN group and the NP-TP53-BIM-PTEN group were increased by 3.5 times and 1.6 times, respectively.

[0216] As can be seen in Figures 19D and E, consistent with the mRNA levels on day 12 after administration, p53 protein expression levels in the NP-TP53-PTEN and NP-TP53-BIM-PTEN groups were increased by 13.7-fold and 11.2-fold, respectively. BIM protein expression levels in the NP-TP53-BIM-PTEN group were increased by 4.3-fold. PTEN protein expression levels in the NP-TP53-PTEN and NP-TP53-BIM-PTEN groups were increased by 10.2-fold and 7.7-fold, respectively.

[0217] These results demonstrate that NP-TP53-BIM-PTEN can simultaneously produce the three designed proteins in a xenograft tumor model.

[0218] The present invention further utilizes TUNEL and immunohistochemistry techniques to quantitatively analyze apoptosis markers and cell proliferation marker Ki67 to verify whether tumor growth inhibition is directly correlated with increases in apoptosis signaling markers and cell proliferation markers in tumor tissue. The results are shown in Figure 20, where A is the TUNEL assay for apoptosis rates in NCI-H1299 xenograft tumor cells on day 12 after administration, using DAPI and DeadEnd™ fluorescent TUNEL system confocal microscopy for fluorescence imaging; B is the immunohistochemistry assay for expression of Ki67, a proliferation marker in NCI-H1299 xenograft tumor cells; C is the statistical analysis of apoptosis rates in tumor tissues treated with different drugs; and D is the semi-quantitative statistical analysis of Ki67 abundance in tumor tissues treated with different drugs.

[0219] As shown in Figure 20, A and C, on day 12 after administration, TUNEL assay results showed that the NP-TP53-BIM-PTEN group had the highest apoptosis rate, approximately 19.4 times that of the PBS group, slightly higher than the NP-TP53-PTEN group. The apoptosis rate in the NP-TP53-PTEN group was approximately 15.9 times that of the PBS group. For multi-gene nanocarriers carrying single gene expression cassettes, apoptosis rates in the NP-TP53, NP-PTEN, and NP-BIM groups were 8.9-fold, 4.4-fold, and 5.5-fold higher than those in the PBS group, respectively.

[0220] As can be seen from Figure 20 B and D, on day 12 after administration, Ki67 antibody immunohistochemistry was used to detect cell proliferation in different treatments. According to the staining score (0-12) standard of 4 mice per group and 2 fields per mouse, the results showed that compared with the PBS group, the expression of Ki67 protein in the NP-TP53, NP-BIM, NP-PTEN, NP-TP53-PTEN, and NP-TP53-BIM-PTEN groups was downregulated by 86.4%, 67.7%, 78.1%, 90.6%, and 97.9%, respectively.

[0221] The above results indicate that the synchronous overexpression of three tumor suppressor genes mediated by multigene nanocarriers (NP-TP53, NP-BIM, and NP-PTEN) showed better effects on tumor cell proliferation inhibition in xenograft tumors compared with multigene nanocarriers carrying single genes.

[0222] Test Example 7: Multigene Nanocarrier NP-TP53-shMYC Overexpressing Tumor Suppressor Genes and Silencing Proto-oncogenes Enhances Anti-tumor Effects in Mice Transplanted with Tumors

[0223] The methods for constructing a xenograft tumor mouse model, delivering multi-gene nanocarriers, observing and recording tumor growth, and evaluating tumor growth and apoptosis-related molecular marker tracing are similar to those in Test Example 6, except that:

[0224] The present invention uses an MDA-MB-231 transplanted tumor-bearing mouse model (5-week-old female BALB / c nude mice) and uses an intratumoral injection method to test the anti-tumor therapeutic effect of NP-TP53-shMYC. The successfully modeled mice were randomly divided into 7 groups, with 4 mice in each group, and treated by intratumoral administration. The multi-gene nanocarrier was delivered using Lipofectamine 2000, with 45 μL Lipofectamine 2000 contained in each 100 μL injection volume. The dosage of the multi-gene nanocarrier was determined according to the weight of the mouse, and was intratumorally administered at 2.25 mg / kg, once every two days, for a total of 3 doses. The specific grouping and dosing regimen are as follows: negative control group (100 μL PBS), multi-gene nanocarrier test group (100 μL multi-gene nanocarrier, 45 μg). The anti-tumor treatment test was set up with the following groups: PBS (negative control group), L-TP53-shMYC (added with Lipofectamine 2000), L-TP53-shMYC(-) (without Lipofectamine 2000), NP-TP53 (added with Lipofectamine 2000), NP-shMYC (added with Lipofectamine 2000), NP-TP53-shMYC (added with Lipofectamine 2000) and NP-TP53-shMYC(-) (without Lipofectamine 2000).

[0225] The results are shown in Figure 21 , where A is a photograph of the tumor excised and treated mice on day 16 after administration; B is a record of the weight changes of MDA-MB-231 tumor-bearing mice in each group within 16 days after administration (black arrows indicate each treatment time point); C is a record of the changes in relative tumor volume in each group of mice within 16 days after administration (black arrows indicate each treatment time point); and D is the relative tumor weight of the different treatment groups on day 16 after administration. The intratumoral injection dose for each mouse was 2.25 mg / kg, administered once every two days, for a total of three treatments (days 1, 3, and 5).

[0226] As can be seen from Figure 21A and B, consistent with the anti-tumor effect in vitro, NP-TP53-shMYC exhibited better anti-tumor effect in vivo, with anti-tumor activity higher than all other test groups, and its mice did not experience a significant decrease in body weight.

[0227] As shown in Figures 21C and 21D, tumor growth in mice treated with NP-TP53-shMYC essentially stagnated, with minimal change in tumor volume over 16 days. Compared to the untreated group, tumor volume decreased by 85.2% and tumor weight decreased by 82.9% on day 16 after dosing. This treatment significantly outperformed all other treatments, particularly the single-gene therapy groups NP-TP53 and NP-shMYC. In NP-TP53-treated mice, tumor volume and weight decreased by 47.9% and 48.2%, respectively, compared to the untreated group on day 16, while tumor volume and weight decreased by 57.6% and 56.7%, respectively, in NP-shMYC-treated mice compared to the untreated group. In contrast, L-TP53-shMYC(-) had no effect on tumor volume or weight. The antitumor activity of L-TP53-shMYC was enhanced only after the addition of Lipofectamine 2000, but remained significantly lower than that of NP-TP53-shMYC.

[0228] This result indicates that linear DNA-based in vivo therapy relies heavily on transfection agents to reduce nuclease resistance, but the serum stability advantage of multi-gene nanocarriers is well reflected in in vivo therapy. For example, in the case of transfection-assisted delivery with Lipofectamine 2000, NP-TP53-shMYC(-) has higher anti-tumor activity than NP-TP53 and NP-shMYC, but its anti-tumor activity is still lower than that of NP-TP53-shMYC delivered with the assistance of Lipofectamine 2000. This reveals that multi-gene nanocarriers have a certain degree of tumor tissue penetration ability and can autonomously enter cells to regulate related gene expression and cell physiological activity without relying on liposomes. Especially under the premise of synchronous regulation of the two key genes TP53 and MYC, multi-gene nanocarriers can exert therapeutic effects beyond single gene regulation, providing a reference basis for the subsequent optimization of different gene combinations.

[0229] The present invention further utilized qPCR and Western blotting techniques to quantitatively analyze the expression levels of TP53 and MYC in tumor tissues to verify whether the inhibition of tumor growth was directly correlated with the restoration of TP53 expression and the decrease in MYC expression in the tumors. Tumors were removed from mice 48 hours after the third administration, fragmented, and lysed for qPCR analysis. Tumors were removed from mice 48 hours after the third administration, fragmented, and lysed for Western blotting analysis.

[0230] The results are shown in Figures 22 and 23, wherein in Figure 22, A is the relative expression analysis of the TP53 gene; B is the relative expression analysis of the MYC gene.

[0231] As can be seen in Figure 22A, 48 hours after the third administration, the tumors in the mice were removed, broken and lysed for qPCR and western blotting analysis. Regarding the TP53 gene, compared to the negative control, the NP-shMYC multi-gene nanocarrier had no effect on TP53 expression, while NP-TP53 was able to increase the expression of the TP53 gene in tumor cells. L-TP53-shMYC could only increase the expression level of the TP53 gene in tumor tissues when delivered with the assistance of Lipofectamine 2000 transfection, while NP-TP53-shMYC could increase the expression level of the TP53 gene in tumor tissues regardless of whether or not Lipofectamine 2000 transfection was assisted. With the assistance of Lipofectamine 2000 transfection, it could increase the expression level of the TP53 gene by 900 times relative to the negative control group. The multiple increase in TP53 expression induced by it was much higher than that of NP-TP53 regulated by a single gene.

[0232] As shown in Figure 22B, for the MYC gene, the NP-shMYC multi-gene nanocarrier reduced MYC gene expression in tumor tissue by nearly 80% compared to the negative control, while NP-TP53 had no significant effect on MYC gene expression in tumor tissue. L-TP53-shMYC reduced MYC gene expression in tumor tissue only when delivered with the assistance of Lipofectamine 2000 transfection, while NP-TP53-shMYC reduced MYC gene expression in tumor tissue regardless of whether or not Lipofectamine 2000 transfection was used, achieving a MYC gene silencing efficiency of 40% with the assistance of Lipofectamine 2000 transfection.

[0233] As can be seen from Figure 23, the Western blotting analysis results are consistent with the qPCR results, both indicating that the bidirectional gene regulation strategy of overexpressing tumor suppressor genes and silencing oncogenes mediated by multi-gene nanocarriers can greatly enhance the anti-tumor effect of tumor-transplanted mice, and is far higher than the therapeutic effect of single gene regulation.

[0234] Test Example 8: Biosafety Evaluation of Multi-gene Nanocarriers NP-TP53-BIM-PTEN and NP-TP53-shMYC

[0235] The safety evaluation of multi-gene nanocarriers mainly includes organ tissue toxicity testing and immunotoxicity testing, which are detected by HE staining and ELISA detection of different inflammation-related factors. The specific operation process is as follows:

[0236] a) HE staining

[0237] After mice were sacrificed, heart, liver, spleen, lung, kidney, and tumor tissues were obtained and embedded. Tissues were then dehydrated in a series of ethanol solutions (75%, 85%, 95%, 100% (I), and 100% (II), with each level of ethanol for 40 minutes. Tissues were then immersed in three xylene baths for 30 minutes each. Tissues were then immersed in three paraffin baths for 1 hour in the first bath, 1.5 hours in the second bath, and 2 hours in the third bath. Liquid paraffin was poured into a mold box, and the wax-soaked tissue block was placed flat on the bottom, with the section facing downward. After the paraffin solidified, the embedding frame was removed. After the block had completely cooled and hardened, the block was trimmed, leaving a moderate amount of paraffin around the tissue for sectioning. The pre-chilled paraffin block was mounted on a paraffin microtome, with the section parallel to the blade. The blade was typically tilted 15 degrees. The rotary propeller was rotated to adjust the section thickness to 3-4 μm, resulting in uniform sections. Hold the writing brush in your left hand and rotate the handle of the microtome with your right hand. After the slice is brought out, lift it gently with the writing brush, and then use tweezers to gently tweeze the wax slice and place it in the slide box with the front side facing up. The water temperature is about 40℃. After it is flattened, pick up the slice. Attach the slice. Hold one end of the slide in your left hand and vertically immerse it in water to attach the slice. Use tweezers in your right hand to help push it and attach it to two-thirds of the slide. After the slice is attached, let it dry slightly in the air, bake it on a 60℃ slide baker for 1 hour, and then bake it in an oven for 2 hours. Place the paraffin slices in xylene (Ⅰ) for 15 minutes, xylene (Ⅱ) for 15 minutes, anhydrous ethanol (Ⅰ) for 10 minutes, anhydrous ethanol (Ⅱ) for 10 minutes, 95% alcohol for 10 minutes, and 85% alcohol for 10 minutes in sequence for gradient dewaxing. Paraffin sections were stained with hematoxylin for 5-10 minutes, rinsed with tap water, differentiated with 1% hydrochloric acid and alcohol for several seconds, rinsed with tap water, then blued with a saturated aqueous lithium carbonate solution for 1 minute, rinsed with running water for several seconds, stained with eosin solution for several seconds, and rinsed with running water. Dehydrated and mounted paraffin sections were sequentially placed in 75% ethanol for 2 minutes, 85% ethanol for 2 minutes, anhydrous ethanol (I) for 2 minutes, anhydrous ethanol (II) for 2 minutes, and xylene for 2 minutes to clear. Sections were removed from the xylene and mounted with neutral gum. Images of hematoxylin and eosin staining were obtained using a light microscope; nuclei appeared blue and cytoplasm red.

[0238] b) ELISA detection of inflammation-related factors

[0239] Before mice were sacrificed by hamstring rupture, whole blood was collected from the retroorbital vein in an anticoagulant EP tube and refrigerated at 4°C overnight. The next day, the blood was centrifuged at 4°C for approximately 30 minutes (2,000 rpm). The supernatant was collected for ELISA analysis of serum levels of interleukin-6 (IL-6), tumor necrosis factor (TNF-α), and interferons (IFN-α, IFN-β, and IFN-γ). Using mouse interleukin-6 (IL-6) as an example, the procedures for other cytokines are the same: the kit should be equilibrated at room temperature for 15-30 minutes after removal from refrigeration before use, and the sample should also be equilibrated at room temperature for 60 minutes before use. Standard and sample wells were set up, with 50 μL of the standard at different concentrations added to each well. Blank wells (blank control wells without sample or enzyme-linked reagent, all other steps remain the same) and test sample wells were set up. First, 40 μL of sample diluent was added to the test sample wells on the enzyme-linked plate, followed by 10 μL of the test sample (final sample dilution was 5-fold). When adding samples, add the sample to the bottom of the ELISA plate wells and gently shake to mix. Add 100μL of the enzyme-labeled reagent to each well, excluding the blank well. Seal the plate with a sealing film and incubate at 37°C for 60 minutes. Dilute the 20x concentrated wash buffer 20x with distilled water and set aside. Carefully remove the sealing film, discard the liquid, and spin dry. Fill each well with wash buffer, let it sit for 30 seconds, then discard. Repeat five times and pat dry. First, add 50μL of chromogen A to each well, then add 50μL of chromogen B. Gently shake to mix, and develop at 37°C in the dark for 15 minutes. Add 50μL of stop solution to each well to terminate the reaction (the blue color will immediately turn yellow). Use the blank well as the zero value and measure the absorbance (OD value) of each well in sequence at a wavelength of 450nm. The measurement should be performed within 15 minutes after adding the stop solution. With the concentration of the standard as the horizontal axis and the OD value as the vertical axis, a standard curve was drawn on the coordinate paper to detect the curve equation (four-parameter logistic fitting) and correlation coefficient R of each cytokine. 2 According to the OD value of the sample, the corresponding concentration is found from the standard curve, and then multiplied by the dilution multiple (5 times) to obtain the actual concentration of the sample.

[0240] Based on the verification of the anti-tumor effectiveness of the multi-gene nanocarrier, the present invention further evaluated the biosafety of two tumor-bearing mouse models. The two tumor-bearing mouse models are respectively MDA-MB-231 transplanted tumor-bearing mouse model (5-week-old female BALB / c nude mice) and NCI-H1299 transplanted tumor-bearing mouse model (6-8-week-old male BALB / c nude mice). The multi-gene nanocarrier is delivered by intratumoral injection. The morphology of NP-TP53-shMYC and NP-TP53-BIM-PTEN in different organs of the mouse (heart, liver, spleen, lung, kidney, tumor) is examined based on the HE staining of tissue sections. The ELISA method is used to detect whether the immune inflammatory factors (IL-6, TNF-α, IFN-α, IFN-β and IFN-γ) in the blood are abnormal, thereby evaluating the systemic organ toxicity and immunotoxicity risks of the multi-gene nanocarrier in mice.

[0241] The results are shown in Figures 24, 25, 26 and 27.

[0242] As shown in Figure 24, for the NP-TP53-shMYC multigene nanocarrier, on day 16 of dosing, mice were sacrificed and heart, liver, spleen, lung, kidney, and tumor tissues were harvested. Microscopic HE staining was performed to identify abnormal morphological changes based on the color (nuclei in blue, cytoplasm in red). The results showed that all organs (heart, liver, spleen, lung, and kidney) in the treated groups were consistent with those in the control group, with no significant abnormalities or lesions.

[0243] As shown in Figure 25, 16 days after administration and before the mice were sacrificed by hamstring fracture, whole blood was collected from the retroorbital vein of the mice and the levels of three immune and cytokines (TNF-α, IFN-α, and IL-6) in the blood were measured using ELISA kits. The levels of TNF-α, IFN-α, and IL-6 in all treated groups were close to those in the control group and remained within the normal range.

[0244] These results indicate that the NP-TP53-shMYC multi-gene nanocarrier has good biosafety.

[0245] As shown in Figure 26, for the NP-TP53-BIM-PTEN multigene nanocarrier, on day 12 of administration, mice were sacrificed and heart, liver, spleen, lung, kidney, and tumor tissues were harvested. Microscopic HE staining was performed to identify abnormal morphological changes based on color (nuclei are blue, cytoplasm is red). The results showed that all organs (heart, liver, spleen, lung, and kidney) in the treated groups were consistent with those in the control group, with no significant abnormalities or lesions.

[0246] As shown in Figure 27, 12 days after administration and before the mice were sacrificed by hamstring fracture, whole blood was collected from the retroorbital vein of the mice, and the levels of three immune and cytokines (IL-6, TNF-α, IFN-α, IFN-β, and IFN-γ) in the mice's blood were measured using ELISA kits. The levels of IL-6, TNF-α, IFN-α, IFN-β, and IFN-γ in all treated groups were close to those in the control group and remained within the normal range.

[0247] These results indicate that the NP-TP53-BIM-PTEN multi-gene nanocarrier has good biosafety.

[0248] Test Example 9: Effects of Different Gene Locations on Multi-Gene Co-expression in Multi-Gene Nanocarriers

[0249] To investigate the effect of the vector location of the expression elements for the three genes TP53, BIM, and PTEN on their co-expression, the NCI-H1299 non-small cell lung cancer cell line was used to test the feasibility of simultaneously overexpressing three tumor suppressor genes in cancer cells using the NP-PTEN-TP53-BIM construct. The co-expression of the plasmids P-PTEN-TP53-BIM (P-PTB) and NP-PTEN-TP53-BIM (NP-PTB) in NCI-H1299 cells was tested using the transfection reagent Lipofectamine 3000.

[0250] The results are shown in Figure 28 , where A is a schematic diagram of the PTEN-TP53-BIM (PTB) sequence; B is the qPCR detection of PTEN mRNA expression levels in P-PTB-transfected cells; C is the qPCR detection of TP53 mRNA expression levels in P-PTB-transfected cells; D is the qPCR detection of BIM mRNA expression levels in P-PTB-transfected cells; E is the western blotting detection of PTEN, TP53, and BIM protein expression levels in P-PTB-transfected cells; F is the NP-PTB dose-dependent expression analysis, which uses qPCR to detect TP53 mRNA expression levels in cells transfected with different doses of NP-PTB; G is the NP-PTB dose-dependent expression analysis, which uses qPCR to detect BIM mRNA expression levels in cells transfected with different doses of NP-PTB. H is the NP-PTB dose-dependent expression analysis, which uses qPCR to detect PTEN mRNA expression levels in cells transfected with different doses of NP-PTB.

[0251] As can be seen from Figure 28, qPCR and western blotting confirmed that P-PTB can simultaneously express TP53 and BIM genes, but cannot express PTEN gene. After preparing NP-PTB vector, NCI-H1299 was transfected with different doses (0-0.8μg). As a negative control, the multi-gene nanocarrier NP-EGFP carrying the EGFP gene expression cassette could not induce the expression of TP53, BIM and PTEN 48h after transfection. In cells transfected with NP-PTB, the mRNA levels of TP53 and BIM increased in a dose-dependent manner, but there was no significant increase in the induced expression of PTEN compared with the control group.

[0252] These results indicate that the NP-PTEN-TP53-BIM vector does not have the ability to express three genes synchronously, and the positions of the three gene expression elements are key considerations in vector design.

[0253] Test Example 10: Effects of Repetitive Promoters and Transcription Terminators on Multi-Gene Co-expression in Multi-Gene Nanocarriers

[0254] In order to verify the effect of using the same promoter and transcription terminator for the three gene expression elements of TP53, BIM and PTEN on the co-expression of the three genes, the NCI-H1299 non-small cell lung cancer cell line was selected to test the feasibility of NP-TP53-BIM-PTEN-V (NP-TBP-V) in simultaneously overexpressing three tumor suppressor genes in cancer cells.

[0255] The results are shown in Figure 29 , where A is a schematic diagram of the TP53-BIM-PTEN-V (TBP-V) sequence; B is the qPCR detection of TP53 mRNA expression levels in cells transfected with NP-TBP-V. C is the qPCR detection of BIM mRNA expression levels in cells transfected with NP-TBP-V. D is the qPCR detection of PTEN mRNA expression levels in cells transfected with NP-TBP-V.

[0256] As can be seen in Figure 29, qPCR confirmed that NP-TBP-V can express TP53 and BIM genes at the same time, but still expresses PTEN at a low level, although the PTEN expression level of NP-TBP-V exceeds that of NP-PTB.

[0257] These results indicate that the NP-TBP-V vector also does not have the ability to express three genes synchronously, and different types of promoters and transcription terminators are needed to ensure the co-expression ability of the three genes.

[0258] Vector sequence

[0259] Linear DNA template L-TP53-BIM-PTEN

[0260] F1-[CMV-HA-TP53-BGH]-[SV40P-BIM-SV40 pA]-[EF1a-FLAG-PTEN-WPRE-hGH pA]-R1 (SEQ ID NO: 25)

[0261] Linear DNA template L-TP53-shMYC

[0262] F1-[CMV-TP53-BGH]-[hU6-shMYC-PolyT]-R1 (SEQ ID NO: 26)

[0263] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, various simple variations of the technical solution of the present invention may be made, including combining the various technical features in any other appropriate manner. These simple variations and combinations should also be regarded as disclosed in the present invention and fall within the scope of protection of the present invention.

Claims

1. A multi-gene nanocarrier, characterized in that: The multi-gene nanocarrier comprises a three-branched central ligand and a gene combination sequence covalently connected to each branch of the three-branched central ligand, wherein the gene combination sequence on each branch is the same; In the direction extending outward from the three-branched central ligand, the gene combination sequence contains at least two tumor suppressor genes selected from TP53, BIM, and PTEN in sequence.

2. The multi-gene nanocarrier according to claim 1, characterized in that: In the direction extending outward from the three-branched central ligand, the end of the gene combination sequence also contains the proto-oncogene MYC.

3. The multi-gene nanocarrier according to claim 1, characterized in that: According to the direction extending outward from the three-branched central ligand, the gene combination sequence contains three tumor suppressor genes, TP53, BIM, and PTEN, in sequence.

4. A multi-gene nanocarrier, characterized in that: The multi-gene nanocarrier comprises a three-branched central ligand and a gene combination sequence covalently connected to each branch of the three-branched central ligand, and the gene combination sequence on each branch is the same; According to the direction extending outward from the three-branched central ligand, the gene combination sequence contains tumor suppressor genes and proto-oncogenes in sequence, the tumor suppressor gene is selected from one of TP53, BIM, and PTEN, and the proto-oncogene is MYC.

5. The multi-gene nanocarrier according to claim 4, characterized in that: In the direction extending outward from the three-branched central ligand, the gene combination sequence contains TP53 and MYC in sequence.

6. The multi-gene nanocarrier according to any one of claims 1 to 5, characterized in that: The three-branched central ligand is provided by a three-branched central compound represented by formula (I); 7. A method for preparing the multi-gene nanocarrier according to any one of claims 1 to 6, characterized in that: The following steps are involved: (1) performing a click cross-linking reaction on the three-branched central compound represented by formula (I) and a single-stranded primer nucleic acid with an azide modification at the 5' end to obtain a branched primer triplet containing three primers; (2) in a PCR reaction system, using the branched primer triplet as a primer and the linear DNA as a template to perform a branched PCR reaction to obtain the multi-gene nanocarrier; the sequence information of the linear DNA is consistent with the gene combination sequence in the multi-gene nanocarrier according to any one of claims 1 to 5; 8. Use of the multi-gene nanocarrier according to any one of claims 1 to 6 in the preparation of anticancer drugs.

9. The use according to claim 8, characterized in that: The anticancer drug is an anti-breast cancer drug and / or an anti-lung cancer drug.

10. An anticancer drug, characterized in that: The anticancer drug consists of active ingredients and excipients, and the active ingredients contain the multi-gene nanocarrier according to any one of claims 1 to 6.

11. The anticancer drug according to claim 10, characterized in that: The content of the active ingredient is 0.01-99.99 wt %.

12. The anticancer drug according to claim 10 or 11, characterized in that: The auxiliary material contains liposome 2000 and / or liposome 3000.

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