Tumor stem cell targeting plasmid based on logic gating circuit, nano-carrier and preparation method and application of tumor stem cell targeting plasmid

Abstract

The invention belongs to the technical field of genetic engineering and biological medicine, and particularly discloses a tumor stem cell targeting plasmid based on a logic gating circuit, a nano-carrier and a preparation method and application of the tumor stem cell targeting plasmid and the nano-carrier. The tumor stem cell targeting plasmid provided by the invention comprises a first plasmid pGal4 and a second plasmid pUAS, a logic AND gate circuit based on protein degradation is designed by utilizing two transcription factors Sox4 and Maff specifically expressed by liver cancer stem cells, and only the liver cancer stem cells with Sox4 and Maff simultaneously highly expressed can express stable target protein GAL4; meanwhile, a target protein GAL4 of an AND gate circuit is set as a primary signal by utilizing a cascade amplification principle of signal transduction, and the signal is used for driving expression (secondary signal) of downstream GSDMD-N and immune activation factors and improving the expression quantity of GSDMD-N in liver cancer stem cells, so that specific pyroptosis of tumor stem cells is induced, and the effect of cancer immunotherapy is enhanced.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering and biomedicine technology, and specifically relates to a tumor stem cell targeting plasmid based on a logic gate circuit, a nanocarrier, and a preparation method and application thereof. Background Art

[0002] There are many different types of tumor cells, and the group of cells with the strongest tumorigenic ability is called tumor stem cells. Tumor stem cells have the ability to self-renew and differentiate, and play an important role in tumor occurrence, metastasis, drug resistance and recurrence. Recent studies have found that the self-renewal of tumor stem cells is precisely regulated by the Notch, Wnt / -catenin and Hedgehog signaling pathways. However, these pathways have limited effects in targeting liver cancer stem cells. The reasons are: 1) There are large individual differences among liver cancer patients, and some patients do not have these pathways activated; 2) These signaling pathways play a core role in the regulation of individual development, tissue stem cells and tissue homeostasis, and the related targeted therapies have large side effects; 3) The regulation of these pathways is complex, and there are multiple cross- and compensatory regulation methods, which makes intervention difficult.

[0003] To solve this problem, Chinese patent application CN107184992A (Taizhou People's Hospital) discloses a pHRE-Egr1-HSV-TK / PEI-PtFe-NPs complex for targeted killing of liver cancer stem cells. The tumor stem cell surface specific antigen CD133 is used as a target, and PtFe magnetic nanoparticles (PtFe-NPs) are used as hyperthermia magnetic media and gene carriers. The suicide gene recombinant plasmid combined with anti-CD133 McAb targeting nuclides are delivered to the tumor site. Under the action of an external alternating magnetic field, the nanomagnetic particles are heated and heated for hyperthermia, and the expression of the target gene is increased under the regulation of the hypoxia radiation dual sensitive promoter, thereby organically combining multiple treatment technologies of nuclides, genes, and hyperthermia to synergistically target and kill liver cancer stem cells. Therefore, exploring new intervention strategies for tumor stem cells is a difficult point in tumor research and tumor intervention, and has important clinical application potential. Summary of the invention

[0004] The main technical problem solved by the present invention is to provide a tumor stem cell targeting plasmid based on a logic gate circuit, and utilizes two transcription factors Sox4 and Maff specifically expressed by liver cancer stem cells to design a logic AND gate circuit based on protein degradation, wherein the AND gate circuit comprises TEV-degron and TEVP, and only liver cancer stem cells that simultaneously highly express Sox4 and Maff will express a stable target protein GAL4; at the same time, utilizing the cascade amplification principle of signal transduction, the target protein GAL4 of the AND gate circuit is set as a primary signal, and the signal is used to drive the expression of downstream GSDMD-N and immune activation factors (such as CCL5, IL-15) (secondary signal), and the two are connected through GAL4 / UAS that has no background activation in liver cancer stem cells, thereby increasing the expression levels of GSDMD-N and immune activation factors in liver cancer stem cells.

[0005] Secondly, the present invention provides a tumor stem cell targeted nanocarrier and a preparation method thereof, wherein the tumor stem cell targeted plasmid is firstly encapsulated with oligopeptide-terminated poly(β-amino ester), and then mixed with a targeting protein (such as CXCR4, acting as a targeting element) to further precisely target liver cancer stem cells, thereby inducing specific pyroptosis of tumor stem cells.

[0006] Again, the present invention provides an application of a tumor stem cell targeting plasmid and a nanocarrier in the preparation of anti-tumor drugs.

[0007] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0008] A cancer stem cell targeting plasmid based on a logic gate circuit, comprising a first plasmid and a second plasmid;

[0009] The first plasmid includes elements connected in the following order: Sox4 transcription factor binding site-GAL4 binding domain / VP16 activation domain fusion protein coding region-TEV cleavage site-dihydrofolate reductase Degron coding region;

[0010] The first plasmid also includes elements connected in the following order: Maff transcription factor binding site-TEV protease coding region;

[0011] The second plasmid includes elements connected in the following order: UAS site-GSDMD-N coding region and / or immune activation factor coding region.

[0012] As a preferred embodiment of the present invention, a TATA box and a Kozak sequence are sequentially connected between the Sox4 transcription factor binding site and the GAL4 binding domain / VP16 activation domain fusion protein coding region.

[0013] As a preferred embodiment of the present invention, the GAL4 binding domain / VP16 activation domain fusion protein coding region, the TEV cleavage site, and the dihydrofolate reductase Degron coding region are connected by a linker.

[0014] As a preferred embodiment of the present invention, the first plasmid comprises a nucleotide sequence as shown in SEQ ID NO: 1, corresponding to the elements connected in the following order: Sox4 transcription factor binding site-TATA box-Kozak sequence-GAL4 binding domain / VP16 activation domain fusion protein coding region-linker 1-TEV cleavage site-linker 2-dihydrofolate reductase Degron coding region.

[0015] As a preferred embodiment of the present invention, a TATA box and a Kozak sequence are sequentially connected between the Maff transcription factor binding site and the TEV protease coding region.

[0016] As a preferred embodiment of the present invention, the first plasmid further comprises a nucleotide sequence as shown in SEQ ID NO: 2, corresponding to the elements connected in the following order: Maff transcription factor binding site-TATA box-Kozak sequence-TEV protease coding region.

[0017] As a preferred embodiment of the present invention, the first plasmid comprises a nucleotide sequence as shown in SEQ ID NO: 3. In this sequence, the sequence shown in SEQ ID NO: 1 and the sequence shown in SEQ ID NO: 2 are connected via a 2×STOP sequence.

[0018] As a preferred embodiment of the present invention, the nucleotide sequence of the first plasmid is shown in SEQ ID NO:4.

[0019] As a preferred embodiment of the present invention, the immune activation factors include but are not limited to CCL5 and IL-15.

[0020] As a preferred embodiment of the present invention, the second plasmid comprises elements connected in the following order: UAS site-IL-15 coding region-CCL5 coding region-GSDMD-N coding region.

[0021] As a preferred embodiment of the present invention, a TATA box and a Kozak sequence are sequentially connected between the UAS site and the IL-15 coding region.

[0022] Specifically, the IL-15, CCL5, and GSDMD-N coding regions are connected by 2A linkers, including but not limited to P2A, T2A, E2A, and F2A.

[0023] As a preferred embodiment of the present invention, the second plasmid comprises a nucleotide sequence as shown in SEQ ID NO: 5, corresponding to the elements connected in the following order: UAS site-TATA box-Kozak sequence-IL-15 coding region-P2A linker-CCL5 coding region-T2A linker-GSDMD-N coding region.

[0024] As a preferred embodiment of the present invention, the nucleotide sequence of the second plasmid is shown as SEQ ID NO:6.

[0025] As a preferred embodiment of the present invention, the mass ratio of the first plasmid to the second plasmid in the tumor stem cell targeting plasmid is 1-1.2:1-1.2.

[0026] A tumor stem cell targeted nanocarrier comprises a tumor stem cell targeted plasmid wrapped by an oligopeptide-modified poly(β-amino ester) shell, on which a targeting protein is self-assembled.

[0027] As a preferred embodiment of the present invention, the oligopeptide-modified poly(β-amino ester) (PBAE) can be prepared by referring to the method disclosed in patent CN105324120B (Sarria CETS Private Foundation Chemical Research Institute).

[0028] As a preferred embodiment of the present invention, the targeting protein includes but is not limited to CXCR4.

[0029] As a preferred embodiment of the present invention, the size of the nanocarrier is 10-1000 nm.

[0030] A method for preparing a tumor stem cell-targeted nanocarrier comprises the following steps:

[0031] The tumor stem cell targeting plasmid is firstly wrapped with oligopeptide-modified poly(β-amino ester), and then mixed with the targeting protein for self-assembly to obtain a tumor stem cell targeting nanocarrier.

[0032] As a preferred embodiment of the present invention, the preparation of the oligopeptide-modified poly(β-amino ester) comprises the following steps:

[0033] (1) mixing 1-4-butanediol diacrylate, 5-amino-1-pentanol and dodecyl primary amine to obtain a polymer through reaction;

[0034] (2) The oligopeptide and the polymer are mixed in DMSO to prepare the oligopeptide-modified poly(β-amino ester) through reaction.

[0035] In step (1), the molar ratio of 1-4-butanediol diacrylate to 5-amino-1-pentanol is 1-3:1.

[0036] In step (1), the reaction conditions are: stirring the reaction at room temperature for at least 20 hours.

[0037] In step (1), the molar ratio of dodecyl primary amine to 5-amino-1-pentanol is 1-1.2:1-1.2.

[0038] In step (2), the molar ratio of the oligopeptide to the polymer is 1.5-3:1.

[0039] In step (2), the oligopeptide is oligopeptide R 6 , the amino acid sequence is: C-RRRRRR-NH2.

[0040] In step (2), the reaction conditions are: stirring the reaction at room temperature for at least 12 hours.

[0041] In step (2), after the reaction is completed, the product is precipitated with a mixed solution of ether and acetone, and then washed and purified with ether. The volume ratio of ether to acetone in the mixed solution is 6-8:3.

[0042] As a preferred embodiment of the present invention, the mass ratio of the oligopeptide-modified poly(β-amino ester) to the tumor stem cell targeting plasmid is 40-60:1.

[0043] As a preferred embodiment of the present invention, the targeting protein includes but is not limited to CXCR4.

[0044] As a preferred embodiment of the present invention, the mass ratio of the oligopeptide-modified poly(β-amino ester) to the targeting protein is 1-1.2:1.

[0045] A tumor stem cell targeting plasmid based on a logic gate circuit or a tumor stem cell targeting nanocarrier is used in the preparation of an anti-tumor drug.

[0046] As a preferred embodiment of the present invention, the drug further comprises pharmaceutically acceptable excipients, including but not limited to solvents, wetting agents, excipients, antioxidants, and preservatives.

[0047] As a preferred embodiment of the present invention, the dosage form of the drug is a pharmaceutically acceptable dosage form, including but not limited to injection, dispersion, suspension, and spray.

[0048] Beneficial effects of the present invention:

[0049] The tumor stem cell targeting plasmid based on logic gate circuit provided by the present invention comprises a first plasmid pGal4 (as shown in SEQ ID NO: 4) and a second plasmid pUAS (as shown in SEQ ID NO: 6). In order to control the precise expression of GSDMD-N and immune activation factors (such as CCL5, IL-15), the present invention utilizes two transcription factors Sox4 and Maff specifically expressed by liver cancer stem cells, designs a logic AND gate circuit based on protein degradation, and the AND gate circuit comprises TEV-degron and TEVP, and only liver cancer stem cells with high expression of Sox4 and Maff at the same time will express stable target protein GAL4. In terms of system sensitivity, the present invention utilizes the cascade amplification principle of signal transduction, sets the target protein GAL4 of the AND gate circuit as a primary signal, and the signal is used to drive the expression of downstream GSDMD-N and immune activation factors (secondary signal), and the two are connected through GAL4 / UAS without background activation in liver cancer stem cells, so as to further increase the expression amount of GSDMD-N and immune activation factors in liver cancer stem cells.

[0050] The tumor stem cell targeted nanocarrier provided by the present invention first encapsulates the tumor stem cell targeted plasmid with oligopeptide-terminated poly(β-amino ester), and then mixes with the targeting protein (such as CXCR4), thereby accurately targeting liver cancer stem cells. Nanocarrier PBAE-R regulated by transcription factors 6 / CXCR4@pGla4 / pUAS can achieve full expression of biomimetic GSDMD and induce specific pyroptosis of tumor stem cells, thereby enhancing the effect of cancer immunotherapy.

[0051] The present invention constructs transcription factor promoter plasmids pGal4 and pUAS encoding biomimetic GSDMD protein, and uses logic AND gate circuits and tumor stem cell targeting peptides to ensure that the killing element is accurately introduced into liver cancer stem cells and specifically expressed in liver cancer stem cells. The present invention transforms tumor stem cells (key cells for tumor immune escape) into activated cells of tumor immunity for the first time, providing new ideas for tumor stem cells and systemic anti-tumor. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 This is the design map of the first plasmid pGla4.

[0053] Figure 2 This is the design map of the second plasmid pUAS.

[0054] Figure 3 Functional verification of the pGla4 / pUAS plasmid system designed based on the concept of cascade amplification and logic AND gate circuits;

[0055] A: Schematic diagram of four different logic paths: [0, 0], [0, 1], [1, 0], [1, 1];

[0056] B: Schematic diagram of pGla4 expression;

[0057] C: Flow cytometry detection of Gla4 expression in different cells;

[0058] D: Schematic diagram of pmKate2 and pGla4 / pmKate plasmid expression;

[0059] E: Fluorescence image of Hepa1-6 cells after transfection of pmKate2 and pGla4 / pmKate plasmids;

[0060] F: Statistical chart;

[0061] G: Schematic diagram of pICG and pGla4 / pUAS plasmid expression;

[0062] H: Pyroptosis of Hepa1-6 after transfection with pGla4 / pUAS plasmid;

[0063] I: WB detection of GSDMD-N protein expression in Hepa1-6 cells transfected with pGla4 / pUAS plasmid and in HEK293T and CHO-K1 cells transfected with different pathways;

[0064] J: WB detection of the expression of IL15, CCL5, and GSDMD-N proteins in Hepa1-6 cells after transfection of pICG and pGla4 / pUAS plasmids;

[0065] K: qPCR detection of the expression of IL15 and CCL5 after pGla4 / pUAS plasmid transfected cells with different logic pathways;

[0066] L: qPCR detection of the expression of IL15, CCL5, and GSDMD-N in MC38 and Hepa1-6 cells after transfection of pICG and pGla4 / pUAS plasmids;

[0067] M: Flow cytometry detection of the expression of IL15, CCL5, and GSDMD-N proteins in MC38 and Hepa1-6 cells after transfection of pICG and pGla4 / pUAS plasmids.

[0068] Figure 4 For PBAE-R 6 Characterization of / CXCR4@pGla4 / pUAS nanocarriers;

[0069] A: PBAE-R 6 Schematic diagram of the construction process of / CXCR4@pGla4 / pUAS;

[0070] B: PBAE-R 6 / CXCR4@pGla4 / pUAS particle size distribution;

[0071] C: particle size;

[0072] D: PBAE-R 6 PBAE-R 6 / CXCR4 vector gel retardation of pDNA;

[0073] E: PBAE-R 6 PBAE-R 6 / GFP expression after CXCR4 and Lipo were transfected into Hepa1-6 cells with pEGFP;

[0074] F: PBAE-R 6 , R 6 , PBAE H NMR spectrum.

[0075] Figure 5 For PBAE-R 6 / CXCR4 tumor targeting in vivo and in vitro in mice;

[0076] A: Flow cytometry detection;

[0077] B: Statistical chart;

[0078] C: Confocal imaging of PBAE-R 6 @DIR and PBAE-R 6 / CXCR4@DIR phagocytosis of Hepa1-6 cells at different times;

[0079] D: PBAE-R 6 / lysosomal escape at different times after CXCR4@DIR treatment of Hepa1-6 and MC38 cells;

[0080] E: Small Animal In Vivo Imaging Detection PBAE-R 6 @DIR and PBAE-R 6 / Targeting of CXCR4@DIR in mice;

[0081] F: In vitro fluorescence of mouse heart, liver, spleen, lung, kidney, and tumor;

[0082] G: Statistical chart.

[0083] Figure 6 To verify the in vitro functionality of the nanocarrier;

[0084] A: Flow cytometry detection of PBAE-R 6 / CXCR4@pGla4 / pUAS、PBAE-R 6 @Expression of IL15, CCL5, and GSDMD proteins in Hepa1-6 cells after treatment with pGla4 / pUAS, pGla4 / pUAS, and PBS;

[0085] B: Statistical chart;

[0086] C: Flow cytometry detection of Annexin V / 7-AAD staining of PBS, pGla4 / pUAS, and PBAE-R 6 @pGla4 / pUAS、PBAE-R 6 / CXCR4@pGla4 / pUAS-treated Hepa1-6 cells;

[0087] D: Statistical chart;

[0088] E: T cell proliferation;

[0089] F: Statistical chart;

[0090] G:CD8 + INF-γT cells;

[0091] H: Statistical graph;

[0092] I: NK cell proliferation;

[0093] J: Statistical graph;

[0094] K: PBAE-R 6 / CXCR4@pGla4 / pUAS、PBAE-R 6 @Pyroptosis caused by treatment of Hepa1-6 cells with pGla4 / pUAS, pGla4 / pUAS, and PBS;

[0095] L: WB test PBAE-R 6 / CXCR4@pGla4 / pUAS、PBAE-R 6 @Expression of IL15, CCL5, and GSDMD proteins in Hepa1-6 cells after treatment with pGla4 / pUAS, pGla4 / pUAS, and PBS;

[0096] M: qPCR detection of PBAE-R 6 / CXCR4@pGla4 / pUAS、PBAE-R 6 @Expression of IL15, CCL5, and GSDMD in Hepa1-6 cells after treatment with pGla4 / pUAS, pGla4 / pUAS, and PBS.

[0097] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the above drawings obtained in the experimental examples are briefly introduced. It should be understood that the above drawings only show some experimental examples of the present invention and should not be regarded as any limitation on the scope of protection of the claims. For ordinary technicians in this field, other related drawings can also be obtained based on these drawings without creative work. DETAILED DESCRIPTION

[0098] The technical solution of the present invention will be clearly and completely described below in conjunction with specific embodiments and experimental examples. However, it should be understood by those skilled in the art that the embodiments are only used to illustrate the technical solution of the present invention and should not be regarded as limiting the scope of protection of the present invention. Based on the following embodiments, all other implementation schemes obtained by ordinary technicians in the field without making creative work, such as modification, deformation or simple replacement, should belong to the scope of protection of the present invention.

[0099] Unless otherwise specified, the experimental methods used in the following embodiments and experimental examples are all conventional methods; the raw materials (including biological materials), reagents, culture media, instruments, etc. used are all commonly used in the art, available to the public or commercially available unless otherwise specified; the terms and abbreviations involved have the conventional meanings in the art, such as PBS buffer is phosphate buffered saline.

[0100] Tumor-targeted gene circuits often require promoters with high cell state specificity as cancer sensors, which can be difficult to find. In addition, natural mammalian promoters can span several kb and are therefore difficult to adapt to the limited coding capacity of many viral vectors. Therefore, the present invention constructs compact de novo synthetic promoters that exhibit enhanced specificity for cancer stem cells compared to endogenous cancer stem cell-specific promoters.

[0101] Differentiation of tumor cells based on the integration of multiple tumor markers can significantly enhance targeting specificity. Therefore, in order to enhance the circuit selectivity for a single synthetic promoter, the present invention designed a synthetic RNA-based circuit that integrates the activity of two synthetic liver cancer stem cell-specific promoters and produces output proteins only when both promoters are significantly active, similar to an AND gate. Specifically, input promoter 1 (P1) regulates the autoinhibitory mRNA encoding the output protein, while input promoter 2 (P2) regulates the RNA molecule that relieves autoinhibition, thereby achieving output production. This RNA-based circuit design can reduce the off-target immunogenicity of the circuit by minimizing the expression of exogenous proteins in normal cells.

[0102] Example 1

[0103] This embodiment provides a tumor stem cell targeting plasmid based on a logic gate circuit, including a first plasmid and a second plasmid;

[0104] The first plasmid includes the nucleotide sequence shown in SEQ ID NO: 1, corresponding to the elements linked in the following order: Sox4 transcription factor binding site-TATA box-Kozak sequence-GAL4 binding domain / VP16 activation domain fusion protein coding region-Linker 1-TEV cleavage site-Linker 2-Dihydrofolate reductase Degron coding region;

[0105] The first plasmid also includes a nucleotide sequence as shown in SEQ ID NO: 2, corresponding to the elements linked in the following order: Maff transcription factor binding site-TATA box-Kozak sequence-TEV protease coding region;

[0106] The second plasmid includes the nucleotide sequence shown in SEQ ID NO: 5, corresponding to the elements linked in the following order: UAS site-TATA box-Kozak sequence-IL-15 coding region-P2A linker-CCL5 coding region-T2A linker-GSDMD-N coding region.

[0107] In other embodiments of the present invention, the first plasmid comprises a nucleotide sequence as shown in SEQ ID NO: 3, in which the sequence shown in SEQ ID NO: 1 and the sequence shown in SEQ ID NO: 2 are connected via a 2×STOP sequence.

[0108] Example 2

[0109] This embodiment provides a tumor stem cell targeting plasmid based on a logic gate circuit, including a first plasmid pGal4 and a second plasmid pUAS, both of which are commissioned to be synthesized by Sangon Biotech (Shanghai) Co., Ltd.;

[0110] The nucleotide sequence of the first plasmid pGal4 is shown in SEQ ID NO: 4, and the plasmid map is shown in Figure 1 As shown;

[0111] The nucleotide sequence of the second plasmid pUAS is shown in SEQ ID NO: 6, and the plasmid map is shown in Figure 2 shown.

[0112] This embodiment provides a method for preparing a tumor stem cell targeting plasmid based on a logic gate circuit, comprising:

[0113] (1) After the synthesis of the first plasmid pGal4 and the second plasmid pUAS, they were cloned into E. coli DH5α puncture bacteria to preserve the strains, and then amplified by shaking in LB liquid medium. The first plasmid pGal4 and the second plasmid pUAS were extracted according to the operation steps of the plasmid extraction kit. The extracted plasmids were measured for concentration and stored in a -20°C refrigerator. (2) The first plasmid pGal4 and the second plasmid pUAS were taken out and mixed evenly at a mass ratio of 1:1 to obtain a tumor stem cell targeting plasmid.

[0114] In other embodiments of the present invention, the mass ratio of the first plasmid to the second plasmid in the tumor stem cell targeting plasmid can be arbitrarily adjusted within a given range.

[0115] Example 3

[0116] This embodiment provides a tumor stem cell targeted nanocarrier, comprising an oligopeptide R 6 The tumor stem cell targeting plasmid (same as Example 2) is encapsulated by a modified poly(β-amino ester) shell, and the targeting protein CXCR4 is self-assembled on the shell; the size of the nanocarrier is 100-300 nm.

[0117] This embodiment provides a method for preparing a tumor stem cell-targeted nanocarrier, comprising the following steps:

[0118] (1) 5-amino-1-pentanol and dodecyl primary amine (the molar ratio of the three is 2:1:1) are added to 1-4-butanediol acrylate, and the mixture is magnetically stirred for 24 hours to obtain a polymer solution.

[0119] (2) oligopeptide R 6 Dissolved in DMSO, added to the polymer solution, stirred overnight at room temperature, the molar ratio of oligopeptide to polymer was 2.1:1.

[0120] (3) The oligopeptide-modified polymer solution was added to a mixed solution of ether and acetone (volume ratio of 7:3) to produce a precipitate, and the precipitate was washed twice with ether for purification. The purified precipitate was vacuum dried, dissolved in DMSO at a concentration of 100 mg / mL, and stored at -20°C with a desiccant.

[0121] (4) PBAE-R in DMSO storage solution 6 The PBAE-R and tumor stem cell targeting plasmids were dissolved in 25 mM MgAc2 (magnesium acetate), with a nucleic acid concentration of 0.83 ng / μL and a polymer concentration of 50 ng / μL, respectively, and mixed by pipetting at a volume ratio of 1:1, vortexed for about 10 seconds, and then self-assembled at room temperature for 20 minutes to obtain PBAE-R with plasmids. 6 .

[0122] (5) PBAE-R carrying plasmid 6 The target protein CXCR4 was dissolved in PBS buffer at a mass ratio of 1:1 and vortexed for 30 min to obtain the tumor stem cell targeting nanocarrier PBAE-R 6 / CXCR4@pGla4 / pUAS.

[0123] In other embodiments of the present invention, the raw materials and preparation parameters of the tumor stem cell-targeted nanocarriers can be adjusted arbitrarily within a given range without substantially affecting the physicochemical properties and biological functions of the nanocarriers.

[0124] Example 4

[0125] This embodiment provides an application of a tumor stem cell-targeted nanocarrier in the preparation of an anti-tumor drug, including:

[0126] The tumor stem cell targeting nanocarrier (same as in Example 3) is mixed with pharmaceutically acceptable excipients such as excipients and preservatives to prepare a medicinal injection for inducing tumor-specific cell pyroptosis and enhancing the effect of cancer immunotherapy.

[0127] In other embodiments of the present invention, a tumor stem cell targeting plasmid (same as in Example 2) is used to prepare an anti-tumor drug.

[0128] Experimental example

[0129] 1. Design, synthesis and verification of plasmids pGal4 and pUAS

[0130] 1.1. Complete plasmid design map and sequence

[0131] The plasmid map of the first plasmid pGal4 is as follows Figure 1 The complete plasmid sequence is shown in SEQ ID NO: 4;

[0132] The plasmid map of the second plasmid pUAS is as follows Figure 2 The complete sequence of the plasmid is shown in SEQ ID NO: 6.

[0133] 1.2 Synthesis and extraction of plasmid

[0134] The designed first plasmid pGal4 and the second plasmid pUAS were fully synthesized by Sangon Biotechnology (Shanghai) Co., Ltd. After synthesis, they were cloned into E. coli DH5α puncture bacteria to preserve the strains, and then amplified by shaking in LB liquid culture medium. The first plasmid pGal4 and the second plasmid pUAS were extracted respectively according to the operating procedures of a commercial plasmid extraction kit. The extracted plasmids were measured for concentration and stored in a -20°C refrigerator.

[0135] 1.3. Agarose gel electrophoresis to verify whether the plasmid is successfully constructed

[0136] Rinse the gel-making mold and comb with distilled water, place them on the gel-making plate, seal the edge of the mold, and insert the comb. Prepare agarose gel (1%) of appropriate concentration using gel buffer according to the size of the DNA fragment to be separated: accurately weigh agarose dry powder, add it to the conical flask used for gel preparation, quantitatively add 30mL of electrophoresis buffer, heat it in a microwave oven to melt, cool it for a while, add red fluorescent nucleic acid dye (EB), gently rotate to mix it thoroughly, quickly pour it into the electrophoresis tank, and wait for it to solidify to process the sample. Add 10× volume of loading buffer loading buffet to the DNA sample, and add the corresponding amount of triple distilled water to dilute and mix it. After 40 minutes at room temperature, the gel is completely condensed. Carefully pull out the comb, add the sample and marker to the gel hole, place the gel in the electrophoresis tank, pour the electrophoresis buffer into the electrophoresis tank (if there are bubbles in the sample hole, try to remove them), then turn on the power, red is the positive pole, black is the negative pole, the DNA sample moves from the negative pole to the positive pole (the end close to the sample hole is negative), set the electrophoresis conditions: 120V, 300mA, electrophoresis for 30 minutes. The electrophoresis results show that the fragment sizes of the first plasmid pGal4 and the second plasmid pUAS are consistent with expectations, indicating that both plasmids were successfully constructed.

[0137] 1.4. Plasmid expression verification

[0138] The expression of target protein in plasmid-transfected cells was detected by flow cytometry. The operation steps are as follows:

[0139] 1) Transfect hepa1-6 cell line (or other cell lines) using Lipofectamine 2000 transfection method.

[0140] 2) Hepa1-6 cells were plated one day in advance and transfection was performed when the cells grew to about 90%.

[0141] 3) 0.5 h before transfection, replace the culture medium (appropriate amount of Opti-MEM I culture medium without serum), 2 mL for 6-well plates.

[0142] 4) Prepare 1.5mL tubes, divided into A and B tubes:

[0143] Tube A: 200 μL Opti-MEM I medium, 2 μg target plasmid, pipette to mix;

[0144] Tube B: 200 μL Opti-MEM I medium, 10 μL Lipofectamine 2000.

[0145] 5) Vortex tubes A and B for 10 seconds each, and then incubate at room temperature for 5 minutes.

[0146] 6) Mix tubes A and B, shake on a vortexer to mix well, and then incubate at room temperature for 20 minutes.

[0147] 7) Add the mixed solution evenly to the Hepa1-6 cells and place them in an incubator for culture.

[0148] 8) After 6 hours of transfection, the culture medium (containing 10% FBS serum) was replaced.

[0149] 9) After 36 hours, the expression of target protein was detected by flow cytometry.

[0150] 1.5. Plasmid Function Verification

[0151] Verify whether the target plasmid can induce cell pyroptosis by transfection. The steps are as follows:

[0152] 1) An empty plasmid without the core target fragment of pyroptosis protein was used as a negative control, and the above-synthesized plasmid was used as the experimental group. The experimental groups were respectively subjected to AnnexinV-PE and 7-AAD single staining for adjustment and compensation.

[0153] 2) Collect adherent cells (hepa1-6 cells) and their supernatant, digest the cells with EDTA-free trypsin, collect the cells after terminating the digestion, centrifuge at 3500 rpm and 4°C for 5 min, and discard the supernatant.

[0154] 3) Washing cells: Wash the cells twice with pre-cooled PBS, centrifuging at 3500 rpm and 4°C for 5 min each time, and discard the supernatant.

[0155] 4) Resuspend cells: Add 100 μL 1× Binding Buffer and gently blow to obtain a single-cell suspension.

[0156] 5) Cell staining: Add 5 μL Annexin V-PE and 5 μL 7-AAD Staining Solution (both are commercial kits), blow gently to mix, incubate at room temperature for 10 min in the dark, then add 400 μL 1× Binding Buffer and mix gently.

[0157] 6) The stained samples were detected by flow cytometry within 1 hour.

[0158] 1.6. Verification of plasmid-induced tumor-specific pyroptosis

[0159] To construct MC38 cells with Sox4 and Maff knocked down individually or simultaneously, the steps are as follows:

[0160] 1) Take HEK-293T cells with good growth status and add 8×10 4The cells / well were plated in a 24-well plate and placed in an incubator overnight to allow the cells to adhere to the wall. Lentivirus was produced in HEK-293T cells.

[0161] 2) The next day, when the cells grew to 80-90%, the cells were transfected.

[0162] 3) 8 hours after virus packaging, replace with fresh complete medium.

[0163] 4) MC38 host cells are plated in advance, and it is best if the cells grow to about 30% during infection.

[0164] 5) After 48 hours of virus packaging, the virus liquid was collected and filtered with a 0.45 μm filter head, the original cell culture medium was discarded, the filtered virus liquid was added to the host cells, and 1‰ polybrene was added.

[0165] 6) Seal the culture plate with sealing film, centrifuge at 2000 rpm and 25°C for 30 min, remove the sealing film and place in an incubator for culture.

[0166] 7) After 6 hours of culture, the culture medium was replaced with new one. After two days of culture, 10% puromycin was added to screen the cells.

[0167] 1.7 Western blot (WB) experiment

[0168] (1) Sample preparation

[0169] 1) Collect cells and add cell lysis buffer; to prevent protein degradation, add protease inhibitors (such as PMSF) and phosphatase inhibitors; vortex repeatedly to lyse cells, then centrifuge at 12000 rpm and 4°C for 10 min, and collect the supernatant.

[0170] 2) Use the BCA protein quantification kit to quantify the protein concentration, set the standard curve, calculate the protein concentration by measuring the absorbance of the sample, and then dilute the protein sample according to the quantitative result to ensure that the amount of protein added to the electrophoresis gel in each sample is consistent.

[0171] (2) SDS-PAGE gel electrophoresis

[0172] 1) Prepare separation gel and stacking gel according to conventional methods, add appropriate amount of TEMED and APS, quickly pour into the gel plate, add stacking gel (usually 5%) after the gel solidifies, and wait for solidification.

[0173] 2) Add an appropriate amount of SDS sample buffer (to help protein decomposition and ensure that it is negatively charged) to the cell lysate, and heat the sample in a 95°C water bath for 5 minutes; load the same amount of protein sample (30 μg) into each well of the electrophoresis gel, and set a molecular weight marker (Protein ladder) control sample for comparison.

[0174] 3) Place the gel plate in the electrophoresis tank and add 1× electrophoresis buffer (Tris-Glycine-SDS); start electrophoresis, set the current to 80V at the beginning of the electrophoresis process, increase the current to 120V after the sample enters the separation gel, and perform electrophoresis until the molecular weight marker reaches the expected position.

[0175] 4) After electrophoresis, soak the gel and membrane (PVDF membrane is first soaked in methanol) in transfer buffer, and then clamp the gel and membrane between the transfer clamps to ensure that the membrane is in close contact with the gel.

[0176] 5) Place the fixture into the transfer tank, add transfer buffer (containing Tris-Glycine and methanol), transfer the membrane at a low voltage (30V) for 2 hours, and check the transfer of proteins on the membrane after completion.

[0177] 6) After transfer, wash the membrane with TBST (Tris-buffered saline with Tween) to remove excess transfer buffer, and then block the membrane with 5% skim milk powder at room temperature for 1 hour.

[0178] 7) After blocking, wash the membrane, add the diluted primary antibody of the target protein, gently shake the reaction plate, and incubate the membrane overnight at 4°C to ensure that the antibody and the target protein are fully bound.

[0179] 8) After the primary antibody incubation, remove the membrane and wash it three times with TBST, 5 min each time, to remove unbound antibodies.

[0180] 9) Dilute the secondary antibody and add it to the membrane, incubate overnight at 4°C.

[0181] 10) After incubation with the secondary antibody, remove the membrane and wash it three times with TBST, 5 minutes each time, to remove unbound secondary antibody.

[0182] 11) Prepare ECL developing reagent (composed of ECL-A and ECL-B), develop using chemiluminescence (ECL), mix the two reagents according to the instruction manual, apply them evenly on the membrane surface, then expose with a developer, observe the bright spots, and record the image.

[0183] 1.8. qRT-PCR detection of target gene expression in each cell line

[0184] (1) Extraction of total cellular RNA

[0185] HEK293T, CHO-K1, MC38, Hepa1-6 and other cells were cultured in advance and total cell RNA was extracted, and cDNA was obtained by reverse transcription.

[0186] Table 1 Pretreatment system

[0187] Components Dosage Template RNA 1 μg 4×gDNAwiperMix 4μL <![CDATA[ddH2O]]> Add to 16 μL

[0188] The above system was placed in a PCR instrument and the program was set to: 42°C for 2 min to obtain the pretreated product.

[0189] Table 2 Reverse transcription reaction system

[0190] Components Dosage The product after pretreatment 16μL 5×HiscriptⅢRT Super Mix for PCR 4μL

[0191] The above system was placed in a PCR instrument and the program was set as follows: 37°C for 15 min, 85°C for 5 s, after which the product concentration was adjusted to 4 ng / μL.

[0192] (2) qPCR detection of target gene expression

[0193] Table 3 qRT-PCR reaction system

[0194]

[0195]

[0196] Sox4:

[0197] Upstream primer F: 5′-GAAAGAAGGTGAAGTCGGGCAAC-3′;

[0198] Downstream primer R: 5′-AGAACGATGCAGCCGCCTT-3′.

[0199] Maff:

[0200] Upstream primer F: 5′-TATGCCTTAACCTTTGGCGTC-3′;

[0201] Downstream primer R: 5′-CGATGGCAAGCGTGATGACT-3′.

[0202] The detection primers for other genes are designed as commonly used primers and will not be described in detail here.

[0203] The above system was placed in an 8-tube tube and mixed evenly, and then placed in a qPCR tube to detect the expression level of the target gene in each cell line.

[0204] 2. Characterization of Nano-drug Delivery Systems

[0205] 2.1. Particle size and Zeta potential analysis

[0206] The Zeta potential and particle size distribution of the nanocarriers were detected. Different nanocarriers were ultrasonically dispersed in sterile water, and 1 mL of sample was taken into a potential cup and a particle size cup respectively. The Zeta potential and particle size distribution of the nanocarriers were detected using a Malvern particle size potential analyzer.

[0207] 2.2. Morphology test of nanocarriers

[0208] Transmission electron microscopy (TEM) is a commonly used intuitive qualitative analysis method in the characterization of nanomaterials. The specific method is: take an appropriate amount of nanocarriers and place them in sterile water for ultrasonic dispersion, then dilute them with sterile water to an appropriate concentration, and ultrasonicate for 15 minutes to evenly disperse the nanocarriers; carefully take the copper mesh out of the sample box, place it in a bacterial culture dish lined with filter paper, use a 20μL pipette to absorb the ultrasonically uniform sample and drop it onto the copper mesh, repeat the addition twice, then place it in a fume hood to dry, and observe and take pictures with a transmission electron microscope.

[0209] 2.3 Determination of encapsulation efficiency and drug loading of nanocarriers

[0210] Differential centrifugation was used to separate the free drugs in the nanocarriers and the supernatant. First, different drug loading groups were designed, and the mass ratios of PBAE-R6 / CXCR4 and pGal4 / pUAS were set to 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, and 80:1, respectively, and different nanocarriers were prepared; centrifugation was performed at 14000 rpm and 4°C for 30 min, the supernatant was collected and filtered with a 0.22 μm filter head, and the filtrate was operated according to the experimental steps of the AceQ Universal SYBR qPCR Master Mix kit. The content of pGal4 / pUAS in the supernatant was detected by Qubit, and the encapsulation efficiency and drug loading of the nanocomplex for pGal4 / pUAS were calculated using the following formula.

[0211]

[0212] 3. Study on the activity of nano drug delivery system at the cellular level

[0213] 3.1. Cellular uptake ability study

[0214] In order to observe the cellular uptake of nanocarriers and evaluate the uptake level of tumor cells for the nanodrug delivery system, Hepa1-6 cells were first inoculated in a special laser confocal dish, and then the fluorescently labeled nanocarriers were incubated with the cells for 1h, 2h and 4h, respectively; they were fixed with paraformaldehyde for 30min, and then the cells were washed with PBS; finally, DAPI was added to stain the cell nuclei, and after staining, the cells were washed with PBS, and anti-fluorescence quenchers were added, and the cells were observed and recorded under a confocal microscope.

[0215] 3.2 Lysosomal escape assay

[0216] (1) Hepa1-6 and MC38 cells were seeded in 6-well plates containing complete medium DMEM at a seeding density of 500,000 per well to ensure that the cells were fully attached and grew within 24 hours.

[0217] (2) Using PBAE-R 6 / CXCR4 was encapsulated with fluorescent dye DIR and added to the attached Hepa1-6 and MC38 cells at four time points: 2, 4, 6, and 8 hours.

[0218] (3) Prepare the Hoechst dye solution, and then dilute the Hoechst 33342 dye to 5 μg / mL with culture medium. After culturing the cells for 24 h, gently aspirate the old culture medium in the culture dish, add a sufficient amount of Hoechst dye solution to each well to ensure that the cells are completely immersed in the solution, and gently shake to mix. Place the culture dish back in a 37°C, 5% CO2 incubator and incubate for 15 min.

[0219] (4) After incubation, add PBS buffer for washing and gently shake the culture dish 3 times, each time for about 5 minutes, to remove unbound Hoechst dye.

[0220] (5) Use a fluorescence microscope to observe the staining of the cell nucleus. Hoechst dye has blue fluorescence under ultraviolet light. Set the excitation wavelength to 355 nm and the emission wavelength to 460 nm to observe the morphology of the cell nucleus.

[0221] 4. In vivo imaging of C57 mice

[0222] 4.1 Establishment of subcutaneous tumor model in C57BL / 6 mice

[0223] (1) Hepa1-6 cell culture

[0224] Hepa1-6 cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin at 37°C and 5% CO2. When the cells reached 85% confluence, they were digested with trypsin and then washed with PBS buffer. After washing, the cells were resuspended in PBS buffer and the concentration of injected tumor cells was adjusted to 5×10 6 cells / 200μL.

[0225] (2) Establishment of mouse subcutaneous tumor model

[0226] Use 6-8 week old female C57BL / 6 mice, place them in a standard laboratory environment, maintain a 12h light-dark cycle, allow the mice to adapt to the new environment2, and avoid stress; use a microsyringe to inject 100 μL of Hepa1-6 cells subcutaneously into the right abdomen of the mouse, gently tighten the skin during injection to ensure that the injection point is accurate and avoid cell leakage, and gently press the injection site after injection to help the cells distribute evenly; measure the size of the tumor with a vernier caliper every 2-3 days, and calculate the tumor volume. The formula is as follows:

[0227] Tumor volume (mm 3 ) = length × width 2 / 2; where length is the longest dimension and width is the vertical dimension.

[0228] (3) Fluorescent labeling and DIR injection

[0229] PBAE-R was prepared according to the method of Example 3 6 / CXCR4@DIR and PBAE-R 6 @DIR solution, first mix DIR with PBAE-R 6 / CXCR、PBAE-R 6 Dissolve in PBS, vortex for 30 seconds, and let stand for 30 minutes to obtain PBAE-R 6 / CXCR4@DIR and PBAE-R 6 @DIR; in mice, tumor volume reached 80mm 3 The above two solutions (0.5 mg / kg) were injected into the tail vein.

[0230] (4) In vivo imaging

[0231] Mice were anesthetized with isoflurane and placed on the imaging platform of the in vivo imager. The imager was turned on and an imaging mode suitable for DIR fluorescence was selected. The excitation wavelength was set to 740 nm, the emission wavelength was set to 790 nm, and the appropriate exposure time was set to ensure that the image was not overexposed or too dark. Photos were taken and observed at 1, 2, 4, 8, 12, 24, and 48 h.

[0232] 4.2 CD8 + Co-culture experiment of T cells and Hepa1-6 cell supernatant

[0233] (1) C57 mouse CD8 + T cell sorting

[0234] 1) Tissue collection: Mice were killed by cervical dislocation, and the spleen and lymph nodes were removed under sterile conditions and placed in a culture dish filled with PBS buffer (pH 7.2) for infiltration.

[0235] 2) Preparation of single-cell suspension: Grind the spleen and lymph nodes using sterilized glass slides, filter through a mesh, centrifuge at 2000 rpm and 4°C for 5 min, and collect lymphocyte single-cell suspension.

[0236] 3) Lysis of red blood cells and adjustment of cell density: Add 5 mL of 1× red blood cell lysis buffer, lyse at room temperature for 8 min, wash twice with PBS, and resuspend in PBS containing 1 mM EDTA + 2% FBS to adjust the cell density to 1×10 8 cell / mL.

[0237] 4) Blocking: Use a pipette to carefully transfer the cell suspension to a 5 mL cell sorting round-bottom test tube (12×75 mm), add 50 μL / mL rat serum, and block at room temperature for 30 min.

[0238] 5) Antibody incubation: After blocking, the antibody Mouse CD8 in the cell sorting kit was added + The T Cell Isolation Cocktail mixture (50 μL / mL) was added to the cell suspension, carefully pipetted to mix, and allowed to stand at room temperature for 10 min.

[0239] 6) Magnetic bead incubation: Take the streptavidin magnetic beads in the cell sorting kit, mix them on a vortexer, add the mixed magnetic beads to the cell suspension at a volume of 125 μL / mL, blow gently to mix, and incubate at room temperature for 5 minutes.

[0240] 7) Magnetic sorting: Add PBS containing 1mM EDTA + 2% FBS to the cell suspension with magnetic beads to a volume of 2.5mL, gently pipette to mix, and then carefully place the cell sorting round-bottom test tube in the EasySep magnetic pole and let it stand for 2.5min.

[0241] 8) CD8 + Obtaining T cells: Lift the magnetic pole and pour the liquid in the tube into a new centrifuge tube. The cells retained in the liquid are the CD8 obtained through negative screening. + T cells.

[0242] (2) CFSE staining to detect CD8 + Proliferation of T cells

[0243] 1) CFSE staining: Take the sorted CD8 + T cells were washed twice with PBS, then resuspended in 2 mL PBS and counted, and the cell density was adjusted to 7 × 10 6 cell / mL; add CFSE dye solution to make the final concentration of 2μM, incubate at room temperature in the dark for 10min; add pre-cooled serum to make the final concentration of 30%, place on ice for 10min, and stop staining; add 10% FBS 1640 medium to resuspend the cells, centrifuge at 3000rpm, 4℃ for 5min, wash twice, count, add 10% FBS 1640 medium to adjust the cell density to 1×10 6 cell / mL.

[0244] 2) Pretreatment of Hepa1-6 cells: Hepa1-6 cells in the logarithmic growth phase were cultured at 5×10 4 The cells were plated at a density of 100 cells / mL in a 6-well plate (2 mL per well), and the plate was placed in a 37°C, 5% CO2 cell culture incubator for culture. After 12 hours of stabilization of the cell state in each group, 8 replicate wells were set up in each group, and different drugs were added for 48 hours. The experimental groups were: PBS group, pGal4 / pUAS, PBAE-R 6 @pGal4 / pUAS、PBAE-R 6 / CXCR4@pGal4 / pUAS, the drug concentration was quantified by pGal4 / pUAS (2 μg / mL), and the amount of nanocarrier was substituted into the drug loading calculation formula; after 48 hours of drug treatment, the cell supernatant was collected in groups.

[0245] 3) Co-culture: Take 100 μL of CFSE from 1) + CD8 + T cell suspension, extract 100 μL of pretreated Hepa1-6 cells from 2) and add them to a round-bottom 96-well plate at a ratio of 10:1; add 0.5 μg / mL anti-mouse CD3, 0.5 μg / mL anti-mouse CD28 and 100 ng / mL mouse recombinant IL-2 to each well, and place the well plate in an incubator for co-culture for 72 hours.

[0246] 4) Flow cytometry antibody staining: After the co-culture, the cells were collected into a 1.5 mL Eppendorf tube, supplemented with PBS to 1.5 mL, centrifuged at 3500 rpm and 4°C for 5 min, 0.3 μL anti-mouse CD8α PE antibody was added, and incubated on ice in the dark for 30 min.

[0247] 5) Flow cytometry: After staining, add 1 mL of PBS and centrifuge, add 250 μL of PBS to resuspend, transfer the cell suspension to a flow cytometry tube, and wait for detection.

[0248] (3) Flow cytometry detection of IFN-γ secretion by CD8+ T cells

[0249] 1) Extract 100 μL CFSE + CD8 + T cell suspension and 100 μL pretreated Hepa1-6 cells were added to a round-bottom 96-well plate at a ratio of 10:1; 0.5 μg / mL anti-mouse CD3, 0.5 μg / mL anti-mouse CD28 and 100 ng / mL mouse recombinant IL-2 were added to each well (CD3 antibody is not required for co-culture of NK cells), and the well plate was placed in an incubator for co-culture for 72 h.

[0250] 2) Addition of protein transport inhibitors: 4 h before the end of co-culture, add 0.2 μL of protein transport inhibitor to each well to make the final concentration 1 μL / mL, and continue culturing in the incubator for 4 h.

[0251] 3) Surface antibody staining: Collect the cells into a 1.5 mL Eppendorf tube, fill the tube with PBS and centrifuge, discard the supernatant, resuspend with 50 μL PBS, add 0.3 μL anti-mouse CD45 FITC and 0.3 μL anti-mouse CD8α PE antibodies, add 0.3 μL anti-mouse CD3 FITC and 0.3 μL anti-mouse NK1.1 PE antibodies to the NK tube, and incubate on ice in the dark for 30 min.

[0252] 4) IFN-γ intracellular factor staining: After staining, add 1 mL of PBS to the tube for centrifugal washing, discard the supernatant (keep a portion of the liquid), place the cells on a vortex to mix thoroughly, then add 200 μL of 4% paraformaldehyde fixative, and fix at room temperature for 30 min; after fixation, add 800 μL of membrane permeabilization agent and centrifuge, discard the supernatant, resuspend the cells with 50 μL of membrane permeabilization agent, add 0.3 μL of anti-mouse IFN-γAPC antibody, and incubate on ice in the dark for 30 min.

[0253] 5) Flow cytometry: After staining, add 1 mL of PBS and centrifuge, resuspend with 250 μL of PBS, transfer the cell suspension to a flow cytometry tube, and wait for detection.

[0254] 5. Experimental results

[0255] 5.1. pGla4 / pUAS plasmid system based on cascade amplification and logic AND gate circuit concept design

[0256] Sox4 and Maff are transcription factors that are specifically and highly expressed in tumor stem cells and can regulate various biological characteristics of tumor cells, such as proliferation, survival and drug resistance. The present invention uses the transcription factors Sox4 and Maff that are specifically expressed by tumor stem cells to design a logic AND gate circuit pGla4 / pUAS based on protein degradation. The circuit is composed of TEV-degron and TEVP. Combined with the principle of signal transduction cascade amplification, the target protein GLA4 of the AND gate circuit is set as the primary signal to drive the expression of downstream GSDMD-N, CCL5 and IL-15 (secondary signal). The two are connected through the background activated GAL4 / UAS in liver cancer cells. Only when Sox4 and Maff are highly expressed at the same time, tumor stem cells will express stable target proteins.

[0257] To verify the function of tumor stem cell targeting plasmids, the present invention proposes four different logic pathway models: [0,0], [0,1], [1,0] and [1,1], where [0,0] represents cells with low expression of both Sox4 and Maff, [0,1] represents cells with high expression of only Maff, [1,0] represents cells with high expression of only Sox4, and [1,1] represents cells with high expression of both Sox4 and Maff. Combined with cell experiments, such as the construction of knockdown and overexpression cell lines, immunoblotting, flow cytometry, immunofluorescence, cell transfection and qRT-PCR, it can be seen that only when Sox4 and Maff are highly expressed at the same time, tumor cells transfected with pGla4 / pUAS plasmids will express stable GSDMD-N protein and induce tumor-specific cell pyroptosis. In addition, the release of IL-15 and CCL5 immune factors further enhances the specificity of tumor immune response. In terms of improving the sensitivity of the system, cell experiments also confirmed the successful construction of the GLA4 / UAS signal cascade amplification system, which can significantly enhance the expression of GSDMD-N, IL-15, and CCL5 in liver cancer stem cells (such as Figure 3 shown).

[0258] 5.2 Construction of tumor stem cell-targeted nanocarriers

[0259] The application of nanocarriers in gene delivery has become one of the core technologies of gene therapy and molecular targeted therapy. Nanocarriers can reduce toxicity to normal cells by modifying the surface with ligands that target tumor cells or tumor stem cells. Poly(β-amino ester) (PBAE) has an adjustable chemical structure and excellent biodegradability. It can effectively encapsulate nucleic acid molecules and promote the binding and endocytosis of gene carriers to cell membranes through its cationic properties. The present invention introduces specific targeting functional groups on the surface of PBAE through chemical modification, so that it can specifically recognize and enter tumor stem cells. In order to further improve the targeting and efficiency of PBAE in gene delivery, the present invention incorporates oligopeptide R 6 Copolymerized with PBAE to form PBAE-R 6 The oligopeptide complex was successfully synthesized and confirmed by nuclear magnetic resonance (NMR) analysis. Dynamic light scattering (DLS) characterization results showed that these nanoparticles exhibited a uniform spherical structure with a moderate particle size, which was conducive to intracellular uptake and gene release.

[0260] PBAE-R was shown by flow cytometry 6 @pGla4 / pUAS has the highest transfection efficiency. In order to better improve the delivery efficiency and targeting, the present invention further uses PBAE-R 6 Combined with CXCR4 receptor-specific peptide, a tumor cell-targeting nanocarrier system PBAE-R was constructed. 6 / CXCR4@pGla4 / pUAS. Flow cytometry and agarose gel blocking experiments revealed that when PBAE-R 6 When the binding ratio with CXCR4 is 1:1, the gene delivery efficiency and targeting effect of the nanocarrier system are optimal. It can not only efficiently deliver genes, but also achieve precise targeting of tumor cells, providing a new therapeutic strategy for gene therapy and immunotherapy of tumors (such as Figure 4 shown).

[0261] 5.3. Validation of the targeting properties of nanocarriers in vivo and in vitro

[0262] Whether the nanocarrier can deliver effective drugs to the tumor site and whether the drug can successfully escape from the lysosome is the key factor to improve the therapeutic effect of tumors. 6 The lysosomal escape ability of PBAE-R / CXCR4 nanocarriers in Hepa1-6 and MC38 cells. 6 / CXCR4 nanocarriers can effectively escape lysosomes. This finding suggests that PBAE-R 6 / CXCR4 not only effectively delivers drugs in tumor cells, but also successfully avoids premature degradation of drugs, thereby improving the intracellular activity of drugs. 6 In order to improve the targeting of CXCR4 nanocarrier, the present invention combines the nanocarrier with the fluorescent dye DIR and uses PBAE-R 6 and PBAE-R 6 Through various experimental methods such as flow cytometry, confocal imaging and small animal in vivo imaging, it was found that PBAE-R 6 / CXCR4 nanocarriers can significantly improve the ability to target tumor sites. Especially in small animal in vivo imaging experiments, PBAE-R 6 The fluorescence signal of the CXCR4 group at the tumor site was significantly stronger than that of the PBAE-R group. 6 group, further demonstrated that PBAE-R 6 In summary, the PBAE-R6 / CXCR4 nanocarrier prepared by the present invention not only has excellent lysosomal escape ability, but also can significantly improve the accumulation of drugs in tumor sites through CXCR4 targeting peptides (such as Figure 5 shown).

[0263] 5.4. In vitro functional verification of tumor cell-targeted nanocarriers

[0264] The present invention aims to evaluate the function of nanocarriers in in vitro tumor treatment, explore its expression ability at the tumor site, and examine the effect of its output signal on tumor immune response. The output of the nanocarrier includes: GSDMD-N, which can induce cell pyroptosis, immune activation factor CCL5 and IL-15. GSDMD-N is the activation domain of GSDMD, which can promote cell pyroptosis by lysing cell membranes, and enhance local immune response by releasing inflammatory mediators, thereby effectively promoting the activation of tumor immunity. As a chemokine, CCL5 can induce T cells to infiltrate the tumor microenvironment by binding to its receptor CXCR5, and at the same time promote the enrichment of natural killer cells (NK cells) in liver cancer sites through the CCR1 receptor, which helps to strengthen the reversal of tumor immune escape. IL-15 is a key cytokine for T cell activation, which plays an important role in promoting the proliferation and activation of effector T cells, and is also a potential therapeutic molecule in tumor immunotherapy. Since tumor stem cells specifically and highly express Sox4 and Maff genes in the tumor microenvironment, these genes can drive the expression of GSDMD-N, CCL5 and IL-15, ultimately inducing pyroptosis of tumor stem cells and promoting the activation of tumor immunity.

[0265] In order to evaluate the effects of different treatment strategies, the present invention set up four control groups: PBS group, pure plasmid pGla4 / pUAS group without nanocarrier, nanocarrier PBAE-R6@pGla4 / pUAS group without targeting peptide, and PBAE-R6@pGla4 / pUAS group containing targeting CXCR4 peptide. 6 / CXCR4@pGla4 / pUAS group. Flow cytometry results showed that PBAE-R 6 @pGla4 / pUAS and PBAE-R 6 / CXCR4@pGla4 / pUAS can successfully express GSDMD-N, CCL5 and IL-15, and the addition of targeting peptides significantly increased the expression levels of these immune factors. Through co-culture with spleen cells of homologous tumor-bearing mice, Western Blot, qRT-PCR and AnnexinV / 7-AAD staining analysis, the results showed that PBAE-R 6 / CXCR4@pGla4 / pUAS nanocarrier group can significantly promote the occurrence of cell pyroptosis, T cell maturation, and NK cell activation, which indicates that PBAE-R 6 Potential application value of / CXCR4@pGla4 / pUAS nanocarrier in tumor immunotherapy (such as Figure 6 shown).

[0266] Although the technical solution of the present invention has been described in detail above with general descriptions, specific implementation methods and test examples, it should be noted that the embodiments and test examples are only used to illustrate the technical solution and technical effects of the present invention, and should not be regarded as any limitation on the protection scope of the present invention. Simple deformation, modification or improvement based on the technical concept of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A tumor stem cell targeting plasmid based on a logic gated circuit, characterized in that: including a first plasmid and a second plasmid; The first plasmid includes elements connected in the following order: Sox4 transcription factor binding site-GAL4 binding domain / VP16 activation domain fusion protein coding region-TEV cleavage site-dihydrofolate reductase Degron coding region; The first plasmid also includes elements connected in the following order: Maff transcription factor binding site-TEV protease coding region; The second plasmid includes elements connected in the following order: UAS site-GSDMD-N coding region and / or immune activation factor coding region.

2. The tumor stem cell targeting plasmid according to claim 1, characterized in that: The first plasmid includes the nucleotide sequence shown in SEQ ID NO: 1, corresponding to the elements connected in the following order: Sox4 transcription factor binding site-TATA box-Kozak sequence-GAL4 binding domain / VP16 activation domain fusion protein coding region-linker 1-TEV cleavage site-linker 2-dihydrofolate reductase Degron coding region; And / or, the first plasmid further comprises a nucleotide sequence as shown in SEQ ID NO: 2, corresponding to the elements connected in the following order: Maff transcription factor binding site-TATA box-Kozak sequence-TEV protease coding region.

3. The tumor stem cell targeting plasmid according to claim 1, characterized in that: The first plasmid comprises the nucleotide sequence shown in SEQ ID NO: 3; And / or, the second plasmid comprises the nucleotide sequence shown in SEQ ID NO: 5, corresponding to the elements connected in the following order: UAS site-TATA box-Kozak sequence-IL-15 coding region-P2A linker-CCL5 coding region-T2A linker-GSDMD-N coding region.

4. The tumor stem cell targeting plasmid according to claim 1, characterized in that: The nucleotide sequence of the first plasmid is shown in SEQ ID NO: 4; and / or, the nucleotide sequence of the second plasmid is shown in SEQ ID NO: 6; And / or, the mass ratio of the first plasmid to the second plasmid in the tumor stem cell targeting plasmid is 1-1.2:1-1.

2.

5. A tumor stem cell targeting nanocarrier, characterized in that: The invention comprises the tumor stem cell targeting plasmid according to any one of claims 1 to 4, which is wrapped by a poly (β-amino ester) shell modified with oligopeptides, and a targeting protein is self-assembled on the shell.

6. The tumor stem cell targeting nanocarrier according to claim 5, characterized in that: The targeting protein includes but is not limited to CXCR4; And / or, the size of the nanocarrier is 10-1000 nm.

7. A method for preparing a tumor stem cell-targeting nanocarrier according to any one of claims 5 to 6, characterized in that: The following steps are involved: The tumor stem cell targeting plasmid is firstly wrapped with oligopeptide-modified poly(β-amino ester), and then mixed with the targeting protein for self-assembly to obtain a tumor stem cell targeting nanocarrier.

8. The preparation method according to claim 7, characterized in that: The mass ratio of the oligopeptide-modified poly(β-amino ester) to the tumor stem cell targeting plasmid is 40-60:1; And / or, the mass ratio of the oligopeptide-modified poly(β-amino ester) to the targeting protein is 1-1.2:

1.

9. Use of the tumor stem cell targeting plasmid according to any one of claims 1 to 4, or the tumor stem cell targeting nanocarrier according to any one of claims 5 to 6 in the preparation of an anti-tumor drug.

10. The use according to claim 9, characterized in that: The drug also includes pharmaceutically acceptable excipients, including but not limited to solvents, wetting agents, excipients, antioxidants, and preservatives; And / or, the dosage form of the drug is a pharmaceutically acceptable dosage form, including but not limited to injection, dispersion, suspension, and spray.

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