CN117363652A - Dual-transcription factor regulated dual-start plasmid, nano material, preparation method and application thereof - Google Patents
Dual-transcription factor regulated dual-start plasmid, nano material, preparation method and application thereof Download PDFInfo
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- CN117363652A CN117363652A CN202311301531.XA CN202311301531A CN117363652A CN 117363652 A CN117363652 A CN 117363652A CN 202311301531 A CN202311301531 A CN 202311301531A CN 117363652 A CN117363652 A CN 117363652A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4702—Regulators; Modulating activity
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
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- C12N2800/00—Nucleic acids vectors
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- C12N2800/107—Plasmid DNA for vertebrates for mammalian
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2810/00—Vectors comprising a targeting moiety
- C12N2810/50—Vectors comprising as targeting moiety peptide derived from defined protein
- C12N2810/80—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates
- C12N2810/85—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates mammalian
Abstract
The invention belongs to the fields of genetic engineering and biological medicine, and particularly discloses a dual-promoter plasmid and a nano material regulated by dual transcription factors, and a preparation method and application thereof. According to the invention, a recombinant plasmid for specifically expressing the pyroprotein based on the regulation and control of Sox4 and Hif-1 alpha dual transcription factors is constructed for the first time, and the CDS region of GSDMD and clear-caspase-1 (P20-P10) genes and the combined promoter sequence of Sox4 and Hif-1 alpha are cloned to the plasmid by a genetic engineering means to obtain the dual-promoter plasmid regulated and controlled by the dual transcription factors, so that the GSDMD is specifically expressed in tumor cells and active fragments are released, and the aim of accurately treating tumors is fulfilled. Based on the recombinant plasmid, the invention also provides a nano material regulated by double transcription factors and a preparation method thereof, and the effect of cancer immunotherapy is enhanced by precisely inducing tumor cell apoptosis.
Description
Technical Field
The invention belongs to the fields of genetic engineering and biological medicine, and in particular relates to a double-start plasmid and a nano material regulated and controlled by double transcription factors, and a preparation method and application thereof.
Background
Pyroapoptosis is a programmed cell death pattern mediated by the Gasdermin (GSDM) protein family, which occurs in dependence on caspase 1/4/5/11 (caspase-1/4/5/11), which performs in dependence on the GSDM protein family, which is cleaved by caspase-1 into N-and C-terminal domains, the N-terminal domain being perforated in the cell membrane, resulting in cell swelling and cell rupture, forming a bubble-like structure of the plasma membrane pores, further resulting in release of pro-inflammatory substances such as interleukin 1 beta and 18 (interleukin 1 beta/18, IL-1 beta/18) to extracellular stimuli.
Recent studies have shown that pyrosis triggers the release of endogenous danger signals and tumor-associated antigens, can generate an effective and durable anti-tumor immune response, and suggest that induction of pyrosis has become a promising strategy for promoting cancer immunotherapy. For example, strategies for enhancing cancer immunotherapy using chemotherapeutic agents, as well as some inorganic nanomaterials to induce pyro-death, have been widely studied. However, these strategies still face challenges such as limited efficacy and systemic toxicity, mainly due to low bioavailability and non-specific biodistribution, thus limiting the immune response induced by pyrosis. Therefore, the GSDM protein is enhanced to specifically express at the tumor site, and the scorch of tumor cells is accurately activated, thereby providing a promising approach for improving tumor immunotherapy. Delivery of a plasmid encoding a GSDM protein, which enhances its expression in tumor cells, thereby inducing tumor cell apoptosis, may be a potential strategy for treating cancer. However, indiscriminate expression of GSDM proteins causes apoptosis in normal cells, which leads to systemic toxic side effects.
In order to realize tumor specific expression of target protein, chinese patent CN114657210B (Zhongshan Da Sun Yixian commemorative Hospital) discloses a nano material based on a GSDMD protein N-terminal peptide segment, wherein the material core is GSDMD-N plasmid and is loaded with GSDMD-N-terminal sequence (shown as SEQ ID NO: 2), and the coating material is nano carrier PDPA, in particular to weak acid response Meo-PEG-B-PDPA. The patent synthesizes bromopolyethylene glycol (Meo-PEG-Br) firstly, then synthesizes Meo-PEG-b-PDPA by utilizing an atom transfer radical polymerization method, then mixes GSDMD-N plasmid with G0-C14 solution, and then mixes with Meo-PEG-b-PDPA to obtain GSDMD-N-PDPA nano material. Because GSDMD-N has tumor killing function and potential inherent immune chemotactic function, the prepared nano material has the functions of tumor killing and local immune function enhancement, and can effectively promote the release of plasmids once entering an acidic microenvironment of tumors, so that a large amount of inherent immune cell chemotactic factors in tumor cells are obviously increased. In addition, the physiological characteristics of tumor endogenous including pH, certain enzymes and redox potential are well developed for plasmid-targeted delivery and protein-specific expression, and Mn, one of microelements essential for human body, also shows good effects in promoting antigen presentation, activating innate immune response. Therefore, the development of efficient and tumor-specific cell apoptosis strategies is critical to promote cancer immunotherapy.
Disclosure of Invention
The invention mainly solves the technical problem of providing a dual-start plasmid for expressing the pyroprotein GSDMD and clear-caspase-1 (P20-P10) based on the regulation and control of Sox4 and Hif-1 alpha dual transcription factors.
Secondly, the invention provides a nano material based on Sox4 and Hif-1 alpha dual transcription factor regulation and control and a preparation method thereof, so as to realize accurate induction of tumor cell apoptosis, thereby enhancing the effect of cancer immunotherapy.
The invention further provides application of the double-start plasmid and the nano material regulated by the double transcription factors in preparing medicaments for treating cancers.
In order to solve the technical problems, the invention provides the following technical scheme:
a dual transcription factor regulated dual promoter plasmid comprising elements linked in the following order: hif-1 alpha transcription factor binding site-clear-caspase-1 gene CDS region-Sox 4 transcription factor binding site-GSDMD gene CDS region.
As a preferred embodiment of the present invention, the plasmid comprises elements linked in the following order: hif-1. Alpha. Transcription factor binding site-clear-caspase-1 gene CDS region-SV 40Poly (A) termination signal-Sox 4 transcription factor binding site-GSDMD gene CDS region-SV 40Poly (A) termination signal.
As a preferred embodiment of the present invention, the NCBI accession number of the clear-caspase-1 gene is NM-009807.2.
As a preferred embodiment of the present invention, NCBI accession number of the GSDMD gene is NM-026960.4.
As a preferred embodiment of the invention, the plasmid comprises a sequence as set forth in SEQ ID NO:1, corresponding to elements linked in the following order: hif-1. Alpha. Transcription factor binding site-clear-caspase-1 gene CDS region-SV 40Poly (A) termination signal-Sox 4 transcription factor binding site-GSDMD gene CDS region-SV 40Poly (A) termination signal.
As a preferred embodiment of the present invention, the plasmid is a plasmid having a sequence as set forth in SEQ ID NO:1, and a nucleotide sequence shown in the formula 1.
As a preferred embodiment of the present invention, the nucleotide sequence of the plasmid is shown in SEQ ID NO: 2.
The nano material comprises an inner core, wherein the inner core comprises calcium carbonate nano particles and double-start plasmids regulated by double transcription factors, and the double-start plasmids regulated by the double transcription factors are uniformly distributed on the surfaces of the calcium carbonate nano particles.
As a preferred embodiment of the present invention, the calcium carbonate nanoparticle is loaded with manganese. During preparation, manganese is firstly loaded on the calcium carbonate nano-particles, and then the manganese is mixed with the double-start plasmid regulated by the double transcription factors, so that the double-start plasmid regulated by the double transcription factors is uniformly distributed on the surfaces of the calcium carbonate nano-particles loaded with manganese.
As a preferred embodiment of the present invention, the nanomaterial comprises an outer membrane layer that is wrapped around the outer surface of the inner core. Specifically, the outer membrane layer is composed of cell membrane vesicles. Sources of the cell membrane vesicles include, but are not limited to, MC38 cells, and also can be encapsulated using erythrocyte membranes and the like. Further, targeting substances, such as folic acid, can be added to the adventitia layer to increase the targeting of tumors.
As a preferred embodiment of the present invention, the nanomaterial is sized from 100 to 200nm.
A preparation method of a nano material regulated by double transcription factors comprises the following steps: mixing the calcium carbonate nano particles and the dual-start plasmids regulated by the dual transcription factors in the solution to obtain the inner core of the nano material.
As a preferred embodiment of the present invention, the method further comprises: manganese is firstly loaded on the calcium carbonate nano particles, and then the manganese is mixed with double-start plasmids regulated by double transcription factors.
As a preferred embodiment of the present invention, the method further comprises: mixing and extruding the inner core of the nano material with the cell membrane vesicle to obtain the nano material.
As a preferred embodiment of the present invention, the calcium carbonate nanoparticles are prepared using a gas diffusion process, or are commercially available. Specifically, the preparation method of the calcium carbonate nano-particles comprises the following steps: calcium salts (e.g. CaCl) 2 ) Ultrasonic dispersing in absolute ethanol, mixing with ammonium bicarbonate (NH) 4 HCO 3 ) And (3) reacting at room temperature to obtain the calcium carbonate nano particles.
As a preferred embodiment of the present inventionThe preparation method of the manganese-loaded calcium carbonate nano-particles comprises the following steps: manganese salts (e.g. MnCl 2 ) Dispersing in absolute ethanol, adding calcium carbonate nanoparticles, and mixing.
As a preferred embodiment of the invention, the preparation method of the inner core of the nano material comprises the following steps: mixing manganese-loaded calcium carbonate nanoparticles and dual-initiation plasmids regulated by dual transcription factors in a solution (such as PBS buffer solution).
As a preferred embodiment of the invention, the preparation method of the cell membrane vesicle comprises the following steps: and carrying out ultrasonic treatment on the extracted cell membrane fragments, and then extruding to obtain the cell membrane derivative vesicles, namely the cell membrane vesicles.
An application of dual-transcription factor regulated dual-start plasmid or dual-transcription factor regulated nano material in preparing the medicines for treating cancer.
As a preferred embodiment of the invention, the medicament is prepared into a pharmaceutically acceptable dosage form by adopting pharmaceutically acceptable auxiliary materials. Such adjuvants include, but are not limited to, wetting agents, excipients, antioxidants, preservatives, and the like. Such dosage forms include, but are not limited to, injections, tablets, dispersions, suspensions, and the like.
As a preferred embodiment of the invention, the medicament is used for inducing the scorch of tumor cells and enhancing the effect of cancer immunotherapy.
The invention has the beneficial effects that:
the invention constructs a recombinant plasmid for specifically expressing the pyroprotein based on the regulation of Sox4 and Hif-1 alpha dual transcription factors for the first time, clones the CDS region of GSDMD and clear-caspase-1 (P20-P10) genes and the combined start sequence of Sox4 and Hif-1 alpha on the plasmid through a genetic engineering means to obtain the dual start plasmid regulated by the dual transcription factors, so as to realize the specific expression of GSDMD in tumor cells and release of active fragments, thereby achieving the aim of accurately treating tumors.
The invention also provides a nano material based on Sox4 and Hif-1 alpha dual transcription factor regulation and control, and a preparation method and application thereof, so as to realize accurate induction of tumor cell apoptosis, thereby enhancing the effect of cancer immunotherapy.
Drawings
FIG. 1 is a map of plasmid pPHS design in experimental example.
FIG. 2 shows the size of plasmid pPHS verified by agarose gel electrophoresis in experimental example;
in the figure, the left lane: control plasmid (2300 bp), right lane: plasmid pPHS (6348 bp).
FIG. 3 shows the flow cytometry verification of pPHS-regulated P20-P10-His and GSDMD-Flag protein expression in experimental examples;
in the figure, a: P20-P10-His protein expression, B: GSDMD-Flag protein expression.
FIG. 4 shows the killing ability of pPHS to MC38 by flow cytometry in experimental examples.
FIG. 5 is a schematic diagram of the construction flow of the nanocarrier M-CNP/Mn@pPHS in experimental example.
FIG. 6 shows the particle size distribution and Zeta potential distribution of the nanocarriers in the experimental example;
in the figure, A-B: particle size distribution of CNP/MnNPs and nanocarriers, C: zeta potential profiles of different nano-formulations.
FIG. 7 is a transmission electron microscope image of the nanocarrier in the experimental example;
in the figure, a: transmission electron microscopy of CNP/MnNPs, B: M-CNP/Mn@pPHS transmission electron microscope image.
FIG. 8 is a PDI diagram of the nanocarrier in experimental example.
FIG. 9 is a graph showing the analysis of the uptake capacity of tumor cells on nanocarriers in experimental examples;
in the figure, a: flow chart after incubation of M-CNP/Mn@pPHS with MC38 cells, B: CLSM image.
FIG. 10 shows relative cell viability (n=3) of MC38 cells treated with different concentrations (0, 20, 40, 60 and 80. Mu.g/mL) of CNP/Mn, CNP/Mn@pPHS, MCNP@pPHS and M-CNP/Mn@pPHS in experimental examples.
FIG. 11 is a phase contrast microscope image of experimental examples after M-CNP/Mn@pPHS (20. Mu.g/mL) was incubated with MC38, MC38 shSox4 and MC38 shSox 4/shHif-1. Alpha. Cells for 24h, respectively;
in the figure, red arrows indicate scorched cells.
FIG. 12 shows the results of the detection of cytotoxicity and tumor cell apoptosis in experimental examples;
in the figure, A-B: detecting apoptosis by a flow cytometer based on annexin v-FITC/PI; c: morphological photographs after different drugs treatment of MC38 cells, wherein M-CNP/Mn@pPHS treatment group cells were swollen, large bubble-like structures (black arrows), scale: 25 μm; d: lactate dehydrogenase kit detects (LDH) release.
FIG. 13 shows the tumor volume and body weight of MC38 tumor-bearing mice in different treatment groups in the experimental example.
In order to more clearly illustrate the technical solution of the embodiments of the present invention, the above description of the drawings obtained in the experimental examples is briefly described. It is to be understood that the above-described drawings illustrate only some examples of the invention and are not to be considered limiting of the scope of the claims. Other relevant drawings may be made by those of ordinary skill in the art without undue burden from these drawings.
Detailed Description
The technical scheme of the present invention will be clearly and completely described in the following in connection with specific examples and experimental examples. It should be understood by those skilled in the art that the examples are only for illustrating the technical scheme of the present invention and should not be construed as limiting the scope of the present invention. All other embodiments, such as modified, modified or simply substituted embodiments, which would be apparent to one of ordinary skill in the art without undue effort based on the examples described below, are intended to be within the scope of the present invention.
The experimental methods used in the following examples and experimental examples are all conventional methods unless otherwise specified; the raw materials (including biological materials), reagents, media, instruments, etc. used, unless otherwise specified, are all commonly used in the art, publicly available or commercially available; the terms and abbreviations are all the conventional meanings in the art, such as PBS for phosphate buffer, and PBST for phosphate Tween buffer.
Example 1
The present example provides a dual promoter plasmid regulated by dual transcription factors, comprising elements linked in the following order: hif-1 alpha transcription factor binding site-clear-caspase-1 gene CDS region-SV 40Poly (A) termination signal-Sox 4 transcription factor binding site-GSDMD gene CDS region-SV 40Poly (A) termination signal (nucleotide sequence shown as SEQ ID NO: 1).
In other embodiments of the invention, the plasmid is a plasmid inserted between Pci I and Not I of the pEGFP-N1 vector as shown in SEQ ID NO:1, and a nucleotide sequence shown in the formula 1.
Example 2
The embodiment provides a dual-promoter plasmid regulated by dual transcription factors, the nucleotide sequence of which is shown in SEQ ID NO: 2.
Example 3
The embodiment provides a nano material regulated by double transcription factors, which comprises an inner core and an outer film layer, wherein the inner core comprises manganese-loaded calcium carbonate nano particles and double-start plasmids regulated by double transcription factors, and the double-start plasmids regulated by the double transcription factors are uniformly distributed on the surfaces of the manganese-loaded calcium carbonate nano particles; the outer membrane layer is wrapped on the outer surface of the inner core and is composed of cell membrane vesicles; the size of the obtained nano material is 100-200nm.
The embodiment provides a preparation method of a nano material regulated by double transcription factors, which comprises the following steps:
preparing calcium carbonate nano particles by adopting a gas diffusion method;
MnCl is added to 2 Dispersing in absolute ethanol, adding calcium carbonate nanoparticles, and mixing to load manganese thereon;
mixing manganese-loaded calcium carbonate nano particles and dual-start plasmids regulated by dual transcription factors in PBS buffer solution to obtain a nano material core;
mixing and extruding the cell membrane vesicle with the inner core to obtain the nano material.
In other embodiments of the invention, the nanomaterial comprises only an inner core comprising manganese-loaded calcium carbonate nanoparticles and dual transcription factor-regulated dual-start plasmids, wherein the dual transcription factor-regulated dual-start plasmids are uniformly distributed on the surface of the manganese-loaded calcium carbonate nanoparticles.
Example 4
The embodiment provides an application of a nano material regulated by double transcription factors in preparing a medicament for treating cancer, and in particular relates to the application of the nano material regulated by double transcription factors in preparing the medicament for treating cancer:
the nanometer material is mixed with excipient, preservative and other pharmaceutically acceptable supplementary material to prepare medicine injection for inducing tumor cell death and enhancing cancer immunological treatment effect.
In other embodiments of the invention, dual transcription factor regulated dual promoter plasmids are used to prepare medicaments for the treatment of cancer.
Experimental example
1. Design, synthesis and verification of plasmid pPHS
1.1, complete plasmid design map and sequence
The plasmid map is shown in FIG. 1, and the plasmid full sequence is shown as follows (or shown as SEQ ID NO: 2):
GGATCCGGACGTGGGGTAAGGACGTGGGGTAAGGACGTGGGGTAAGGACGTGGGGTAAGGACGTGGGGTAAGGACGTGGGGTAAGAGGGTATATAATGGAATTCATGAACAAAGAAGATGGCACATTTCCAGGACTGACTGGGACCCTCAAGTTTTGCCCTTTAGAAAAAGCCCAGAAGTTATGGAAAGAAAATCCTTCAGAGATTTATCCAATAATGAATACAACCACTCGTACACGTCTTGCCCTCATTATCTGCAACACAGAGTTTCAACATCTTTCTCCGAGGGTTGGAGCTCAAGTTGACCTCAGAGAAATGAAGTTGCTGCTGGAGGATCTGGGGTATACCGTGAAAGTGAAAGAAAATCTCACAGCTCTGGAGATGGTGAAAGAGGTGAAAGAATTTGCTGCCTGCCCAGAGCACAAGACTTCTGACAGTACTTTCCTTGTATTCATGTCTCATGGTATCCAGGAGGGAATATGTGGGACCACATACTCTAATGAAGTTTCAGATATTTTAAAGGTTGACACAATCTTTCAGATGATGAACACTTTGAAGTGCCCAAGCTTGAAAGACAAGCCCAAGGTGATCATTATTCAGGCATGCCGTGGAGAGAAACAAGGAGTGGTGTTGTTAAAAGATTCAGTAAGAGACTCTGAAGAGGATTTCTTAACGGATGCAATTTTTGAAGATGATGGCATTAAGAAGGCCCATATAGAGAAAGATTTTATTGCTTTCTGCTCTTCAACACCAGATAATGTGTCTTGGAGACATCCTGTCAGGGGCTCACTTTTCATTGAGTCACTCATCAAACACATGAAAGAATATGCCTGGTCTTGTGACTTGGAGGACATTTTCAGAAAGGTTCGATTTTCATTTGAACAACCAGAATTTAGGCTACAGATGCCCACTGCTGATAGGGTGACCCTGACAAAACGTTTCTACCTCTTCCCGGGACATCATCATCACCATCACCATCATCACCATCACTAACTCGAGAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTAAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCTAGAAGAAGAACAAAGGACTAGGGTAAAGAAGAACAAAGGACTAGGGTAAAGAAGAACAAAGGACTAGGGTAAAGAAGAACAAAGGACTAGGGTAAAGAAGAACAAAGGACTAGGGTAAAGAAGAACAAAGGACTAGGGTAAGAGGGTATATAATGAGATCTATGCCATCGGCCTTTGAGAAAGTGGTCAAGAATGTGATCAAGGAGGTAAGCGGCAGCAGAGGCGATCTCATTCCGGTGGACAGCCTGCGGAACTCCACCAGCTTCAGGCCCTACTGCCTTCTGAACAGGAAATTTTCAAGCTCAAGGTTCTGGAAACCCCGTTATTCATGTGTCAACCTGTCAATCAAGGACATCCTGGAGCCCAGTGCTCCAGAACCAGAACCGGAGTGTTTTGGCTCCTTCAAAGTCTCTGATGTCGTCGATGGGAACATTCAGGGCAGAGTGATGTTGTCAGGCATGGGAGAAGGGAAAATTTCTGGTGGGGCTGCAGTGTCTGACAGTTCCAGTGCCTCCATGAATGTGTGTATACTGCGTGTGACTCAGAAGACCTGGGAGACCATGCAGCATGAAAGGCACCTTCAGCAGCCTGAGAACAAAATCCTGCAACAGCTTCGGAGTCGTGGGGATGACCTGTTTGTGGTGACCGAGGTGCTGCAGACAAAGGAGGAAGTGCAGATCACTGAGGTCCACAGCCAAGAGGGCTCAGGCCAGTTTACGCTGCCTGGAGCTTTATGCTTGAAGGGTGAAGGCAAGGGCCACCAAAGCCGGAAGAAGATGGTGACCATTCCTGCAGGCAGCATCCTGGCATTCCGAGTGGCCCAACTGCTTATTGGCTCTAAATGGGATATCCTTCTCGTCTCAGATGAGAAACAGAGGACCTTTGAGCCCTCCTCAGGTGACAGAAAAGCAGTGGGCCAGAGGCACCATGGCCTCAATGTGCTTGCTGCGCTTTGTTCCATCGGAAAGCAGCTCAGTCTCCTGTCAGATGGGATTGATGAGGAGGAATTAATTGAGGCGGCAGACTTCCAGGGCCTGTATGCTGAGGTGAAGGCTTGCTCCTCAGAACTGGAGAGCTTGGAAATGGAGTTGAGACAACAGATACTGGTGAACATCGGAAAGATTTTACAGGACCAGCCCAGCATGGAAGCCTTAGAGGCCTCACTAGGGCAGGGCCTGTGCAGTGGCGGCCAGGTGGAGCCTCTGGACGGCCCAGCTGGCTGCATCCTTGAGTGTCTGGTGCTTGACTCTGGAGAACTGGTGCCGGAACTCGCAGCCCCTATCTTCTACCTGCTGGGAGCACTGGCTGTGCTGAGTGAAACCCAGCAGCAGCTGCTAGCTAAGGCTCTGGAGACAACGGTGCTGTCAAAGCAGCTGGAGTTGGTGAAGCACGTCTTGGAACAGAGCACCCCGTGGCAGGAGCAGAGTTCTGTGTCCCTGCCCACCGTGCTCCTTGGGGACTGCTGGGATGAAAAGAATCCCACCTGGGTCTTGCTAGAAGAATGTGGCCTAAGGCTGCAGGTAGAATCCCCCCAGGTGCACTGGGAACCAACGTCTCTGATCCCCACAAGTGCGCTCTATGCCTCCCTGTTCCTATTGTCAAGTCTAGGCCAGAAACCTTGTGATTACAAGGATGACGACGATAAGGACTATAAGGACGATGATGACAAGGACTACAAAGATGATGACGATAAATAAAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATAAGCGGCCGCGACTCTAGATCATAATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTAAGGCGTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTCCTGAGGCGGAAAGAACCAGCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAGATCGATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAAGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGAGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGCGGGACTCTGGGGTTCGAAATGACCGACCAAGCGACGCCCAACCTGCCATCACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCTAGGGGGAGGCTAACTGAAACACGGAAGGAGACAATACCGGAAGGAACCCGCGCTATGACGGCAATAAAAAGACAGAATAAAACGCACGGTGTTGGGTCGTTTGTTCATAAACGCGGGGTTCGGTCCCAGGGCTGGCACTCTGTCGATACCCCACCGAGACCCCATTGGGGCCAATACGCCCGCGTTTCTTCCTTTTCCCCACCCCACCCCCCAAGTTCGGGTGAAGGCCCAGGGCTCGCAGCCAACGTCGGGGCGGCAGGCCCTGCCATAGCCTCAGGTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGT。
1.2 Synthesis and extraction of plasmids
The binding promoter sequences of clear-caspase-1 (P20-P10) (NCBI accession number: NM-009807.2) and GSDMD (NCBI accession number: NM-026960.4) genes CDS region, and Sox4 and Hif-1α were cloned into two cleavage sites of Pci I and NotI of pEGFP-N1 vector, and complete gene synthesis and subcloning were completed by Hua Dada gene company. The plasmid pPHS is cloned into DH5 alpha colibacillus to preserve strain after synthesis, and then shake-bacteria amplification is carried out in LB liquid culture medium. The extraction of plasmid pPHS was carried out according to the experimental procedure of the commercial kit. The extracted plasmid is stored in a refrigerator at-20 ℃ after the concentration is measured.
1.3, agarose gel electrophoresis to verify if the plasmid construction was successful
Washing the glue making mold and the comb with distilled water, placing the glue making mold and the comb on a glue making flat plate, closing the edge of the mold, and inserting the comb. Agarose gel (1%) of appropriate concentration was prepared with gel buffer according to the size of the DNA fragment to be isolated: accurately weighing 0.30g of agarose dry powder, adding the powder into a triangular flask for preparing gel, and quantitatively adding 30mL of electrophoresis buffer. Heating and melting in a microwave oven, cooling for a while, adding red fluorescent nucleic acid dye (EB) 3 μl, lightly rotating to mix thoroughly, rapidly pouring into an electrophoresis tank, standing for solidifying sample, adding 10×volume of sample loading buffer loadingbuffet into DNA sample, and adding corresponding amount of triple distilled water for diluting and mixing. After 30 minutes (30-45 minutes) at room temperature, the gel was completely coagulated, the comb was carefully pulled out, the sample and Marker were added to the gel well, and the gel was placed in the electrophoresis tank. The electrophoresis buffer, such as bubbles in the sample well, is poured into the electrophoresis tank and the bubbles are removed. Then, the power supply is switched on, red is the positive electrode, black is the negative electrode, the DNA sample swims from the negative electrode to the positive electrode (one end close to the sample adding hole is negative), and electrophoresis conditions are set: 120V,300mA, for 30min, the results are shown in FIG. 2.
1.4, verification of plasmid expression
The procedure for plasmid expression verification was as follows:
1) The MC38 cell line was transfected by calcium phosphate transfection.
2) MC38 cells are spread one day in advance, and transfection is performed when the cells grow to about 90%.
3) Medium (DMEM medium with 10% FBS) was replaced 0.5h (0.5-1 h) before transfection, and 1.5mL in 6-well plates.
4) Prepare 1.5mL tube, split into A, B tubes:
i.A tube: 180 mu L of water, 20 mu L of calcium chloride solution and 2 mu g of skeleton plasmid are blown and evenly mixed;
tube b: 200. Mu. LHBS buffer.
5) The tube A was placed on an oscillator, the gun was adjusted to 70. Mu.L, HBS in the tube B was added dropwise to the tube A3 times, slowly in the dropwise addition, immediately after each addition, air was blown in and mixed thoroughly, and the mixture was left to stand for 5 minutes after all addition.
6) The mixture was added dropwise to MC38 cells and cultured in an incubator.
7) After 6h (all 6-8 h) of transfection the medium (containing 10% FBS serum) was replaced.
8) The expression of the target protein was detected in a post-flow manner for 36h (24-48 h is available), and the result is shown in FIG. 3.
1.5 functional verification of plasmid
The transfection is used for verifying whether the target plasmid can induce cell apoptosis, and the operation steps are as follows:
1) The empty plasmid without the target fragment of the core of the pyroptoprotein is used as a negative control, and the plasmid is designed as an experimental group. The experimental groups were taken for annexin V-PE and 7-AAD single staining, respectively, for adjustment of compensation.
2) The adherent cells and the supernatant thereof were collected, the cells were digested with pancreatin free of EDTA, the cells were collected after termination of the digestion, and centrifuged at 3500rpm at 4℃for 5min, and the supernatant was discarded.
3) Washing the cells: the cells were washed twice with pre-chilled PBS, centrifuged at 3500rpm at 4℃for 5min each, and the supernatant discarded. Cell resuspension: mu.L of 1 Xbinding Buffer was added and gently swirled until a single cell suspension was obtained.
4) Cell staining: add 5. Mu.L Annexin V-PE and 5. Mu.L 7-AAD Staining Solution (both commercial kits), gently blow well, incubate for 10min at room temperature (20-25 ℃) in the dark, then add 400. Mu.L 1 Xbinding Buffer, gently mix.
5) The stained sample was examined with a flow cytometer within 1 hour, and the results are shown in fig. 4.
2. Construction of M-CNP/Mn@pPHS nano-carrier
The M-CNP/Mn@pPHS nano-carrier is constructed by the following steps (the preparation flow is shown in figure 5):
1) Preparing a calcium carbonate nano carrier by using a gas diffusion method: accurately weigh CaCl 2 Adding into beaker containing absolute ethanol, and performing ultrasonic treatment to obtain CaCl 2 Uniformly dispersing in absolute ethanol, mixing beaker with other 4 materials containing NH 4 HCO 3 Placing the beaker in a vacuum dryer, reacting for 48 hours at room temperature, centrifuging to obtain CNP, and dispersing in a vacuum dryer containing MnCl 2 Repeatedly centrifuging and washing in the absolute ethanol solution of (2), and collecting the pure CNP/Mn.
2) Uniformly dispersing the obtained CNP/Mn into PBS, proportionally adding pPHS, stirring for 12h by a magnetic stirrer, and centrifugally collecting CNP/Mn@pPHS nano particles.
3) Extracting MC38 cell membrane with commercial cell membrane extraction kit, selecting MC38 cell in logarithmic growth phase, and extracting cell membrane from MC38 cell according to kit instruction to obtain cell membrane fragments.
4) Adding fragments of MC38 cell membranes to ddH 2 And (3) performing ultrasonic treatment in O, and then extruding by using an extruder to obtain the cell membrane vesicles. The cancer cell membrane vesicles were mixed with the above-collected nanoparticles, and simultaneously extruded multiple times with an extruder (pore size 200 nm) to obtain M-CNP/Mn@pPHS.
3. Characterization of nano drug delivery systems
3.1 analysis of Zeta potential
Detecting Zeta potential and particle size distribution of the nano carrier: suspending the sample in ddH 2 In O (1 mg/mL), the size distribution and Zeta potential of the nanoparticles were measured by Zetasizer Nano ZS (Malvern, nano ZS, U.K.) after 10min of sonication, and the results are shown in FIG. 6.
3.2 topography testing of nanocarriers
The Transmission Electron Microscope (TEM) is used for characterizing the nano material, and the specific method is as follows: the copper mesh was placed on a filter paper, 100. Mu.L of the nano-carrier prepared in advance was extracted with a pipette, dropped on the copper mesh, repeated 3 times of operation, and then dried in an oven, and the morphology of the nano-particles was observed by TEM (HT 7700, hitachi, japan), and the result is shown in FIG. 7.
3.3 analysis of nanocarrier stability
An appropriate amount of nanocarriers were weighed and ultrasonically dispersed in sterile water, and their hydrated particle size and PDI were measured daily with a Markov particle sizer and continuously examined for 7 days, the results of which are shown in FIG. 8.
3.4, encapsulation efficiency of nano-carrier and drug-loading rate measurement
Separating free medicine in nano carrier and supernatant by differential centrifugation, firstly setting different mass ratios of M-CNP/Mn and pPHS to prepare M-CNP/Mn@pPHS, and then centrifuging at 10000rpm and 4 ℃ for 60min to collect supernatant; will result inFor the supernatant of (a) using Equalbit 1X dsDNA HS detection kit and Qubit TM Flex fluorometer was used to measure double-stranded DNA concentration and the encapsulation and drug loading rates were calculated using the following formulas, and the results are shown in Table 1.
3.5 Activity study of nanodelivery System at cellular level
3.5.1 investigation of cell uptake Capacity
In order to observe the uptake condition of cells on nano materials, the uptake level of tumor cells on a nano drug-carrying system is estimated, firstly, a 5-FAM fluorescent marked M-CNP/Mn@5-FAM nano carrier is prepared, MC38 cells with good growth state are selected, and the density is adjusted to be 1 multiplied by 10 5 cells/wells were plated in confocal dishes and then placed in 5% CO 2 Culturing overnight in an incubator at 37 ℃. After the culture is finished, discarding the old culture medium at the upper layer, adding the prepared fluorescent nano carrier M-CNP/Mn@5-FAM and MC38 cells for incubation, discarding the supernatant after incubation for 1, 2 and 4 hours, washing 2 times by PBS, and adding PBS buffer solution containing DAPI for incubation for 30min. After the incubation, the dish bottom was washed 3 times with PBS, put into a light-shielding wet box, and taken to a laser confocal microscope for observation of the uptake, and the result is shown in FIG. 9.
3.5.2 MTT reduction method
Cytotoxicity was assessed using a typical MTT reduction assay: cells were first seeded into 96-well plates, dosed after cell attachment, and different treatment groups were set: CNP/Mn group, CNP/Mn@pPHS group, M-CNP@pPHS group, M-CNP/Mn@pPHS group, PBS group, and different concentrations were set: 0. 20, 40, 60 and 80. Mu.g/mL. At time points 24, 48 and 72 hours, 20 mu LMTT solution is added, after 4 hours of incubation in dark place, 150 mu L of dimethyl sulfoxide (DMSO) is added into each hole, the mixture is placed on an oscillator for oscillation in dark place, finally the absorbance of each hole is measured at the wavelength of 490nm through an enzyme-labeling instrument, the cell survival rate of each experimental group is calculated respectively, so as to evaluate the toxicity of the constructed nano-carrier to normal cells and TNBC cells, and the experiment is repeated for 3 times, and the result is shown in figure 10.
3.5.3 nanometer carrier M-CNP/Mn@pPHS induced tumor specific pyrosis verification
A. Construction of stable Sox4 and Hif-1 alpha MC38 cells knocked down singly or simultaneously
1) shSox4 and shHif-1 alpha synthesized by the company are subcloned between the Age I and EcoR I cleavage sites of the pLVX-puro vector.
2) Lentiviruses were generated in HEK-293T and host cell MC38 was infected with the virus.
3) After 48 hours incubation puromycin (0.1 mg/mL) was added and empty vector was transferred to cells as negative control.
4) After lentivirus transfection and puromycin screening, each group of cellular RNAs was extracted and the knock-out efficiency was detected by RT-qPCR.
B. Observing the influence of M-CNP/Mn@pPHS on normal and tumor cell lines by a phase contrast microscope
1) Selecting MC38, CHO-K1, RAW264.7, BMDC, MC38 shSox4, MC38 shHif-1α, MC38 shSox4/shHif-1α cell line with good growth state, and adjusting density to 2×10 5 cells/well were plated in 6-well plates, which were then placed in a chamber containing 5% CO 2 Is cultured overnight in an incubator at 37 ℃.
2) After the completion of the culture, the old medium was discarded, and M-CNP/Mn@pPHS (20. Mu.g/mL) prepared in advance was added to the medium to incubate MC38 cells, respectively.
3) After 24h incubation, the change in morphology of each group of cells was observed using a phase contrast microscope and stored with photographs, the results of which are shown in fig. 11.
C. M-CNP/Mn@pPHS induced cell apoptosis verification
Annexin-V/PI staining experiments, lactate Dehydrogenase (LDH) release experiments, and microscopic observation of tumor cell death morphology were analyzed by flow cytometry, and the results are shown in FIG. 12.
3.7, establishment of C57BL/6 mouse subcutaneous colon cancer tumor model
In vivo anticancer effects Using female C57BL/6 miceTo construct the MC38 subcutaneous tumor model, MC38 cells were resuspended in 200 μlpbs and injected subcutaneously on the right side of the back of the mice. After 7 days, the mice were randomly divided into 5 groups until the tumors grew to 40-80mm 3 PBS, CNP/Mn, CNP/Mn@pPHS, M-CNP@pPHS, and M-CNP/Mn@pPHS were intravenously injected to the tail of the mice. The medicines are injected again at intervals of 3 days, the administration is carried out 5 times, the tumor volume and the weight change of the mice are monitored every 1 day, and the in-vivo treatment effect of the medicines is observed through the monitoring of the indexes. After the treatment is finished, the mice are subjected to segment neck sacrifice, then tumor tissues are stripped for photographing, and organs such as tumors, hearts, livers, spleens, lungs, kidneys and the like of 1 mouse are taken for H in each group&E analysis, the results are shown in FIG. 13.
The tumor volume calculation formula is as follows:
tumor volume (mm) 3 ) =length×width 2 2; wherein the length is the longest dimension and the width is the vertical dimension.
4. Experimental results
4.1, particle size, zeta potential
The physicochemical properties of the prepared different nano-carriers are characterized by adopting a plurality of technical means, as shown in figures 6A-6B, dynamic Light Scattering (DLS) results show that the hydrodynamic diameter of CNP/MnNPs is 100.46+/-0.82 nm; TEM observes that M-CNP/Mn@pPHS wrapped by cell membrane has complete membrane structure with a diameter of about 150nm, and dynamic light scattering results show that the hydrodynamic diameter is 153.17 + -1.69 nm, which is about 50nm larger than that of CNP/Mn due to the wrapping by cell membrane.
Zeta potential (Zeta potential) is the potential of the Shear Plane, also called the Zeta potential or electrokinetic potential, and is an important indicator for the stability of colloidal dispersions. As shown in FIG. 6C, analysis of the Zeta potential of each nanoparticle involved in the preparation process revealed that the Zeta potentials of CNP/Mn@pPHS and M-CNP/Mn@pPHS were reduced from 7.83.+ -. 0.34mV to-24.33.+ -. 1.11mV and-32.63.+ -. 0.67mV, respectively, which may be related to the loading of pPHS and the coating of cell membranes, which are negatively charged.
4.2 topographical features of nanocarriers
From the transmission electron microscope images of FIGS. 7A-7B, it can be observed that CNP/MnNPs, M-CNP/Mn@pPHS all exhibit a monodispersed state and are spherical in size. Compared with CNP/MnNPs, the surface of M-CNP/Mn@pPHS is obviously coated with a layer of transparent substance, which proves that the coating of MC38 cell membranes is successful, and the loading rate of the nano drug delivery system is more stable.
4.3 stability analysis of nanocarriers
As shown in FIG. 8, the particle size and PDI value of M-CNP/Mn@pPHS remained relatively stable by PDI detection for 7 consecutive days, demonstrating that the nanocarriers remained well stable and dispersible in sterile water within 7 days.
4.4, encapsulation efficiency of nano-carrier and drug-loading rate measurement
In order to obtain better encapsulation efficiency and drug loading rate of the nano-carrier, different proportions are designed for measurement. As shown in Table 1, at a mass ratio of 10:1, the encapsulation efficiency of pPHS reached a maximum.
TABLE 1 encapsulation efficiency and drug loading of nanocarriers
4.5, analysis of cellular uptake Capacity
In order to evaluate the uptake of the nanocarriers by tumor cells, the tumor cells were analyzed by flow cytometry using 5-FAM labeled nanoparticles followed by incubation with MC38 tumor cells. As shown in FIG. 9, the degree of internalization of M-CNP/Mn@5-FAM by tumor cells increased with increasing culture time. Quantitative analysis shows that the uptake rate of M-CNP/Mn@5-FAM after 4h incubation is significantly higher than that of CNP/Mn@5-FAM, which is mainly attributed to the cell membrane layer mediated tumor targeting ability. Consistent with the flow cytometer results, the uptake results of confocal laser scanning microscopy evaluation showed that M-CNP/Mn@5-FAM showed brighter green fluorescence, representing an increase in cellular uptake.
4.6 MTT reduction assay to assess cytotoxicity
The result of evaluating the toxicity of the nanocarrier to normal cells is shown in fig. 10, and compared with the control group, the survival rate of the CNP/Mn to MC38 cells is not affected, which indicates that the nanoparticles have no effect on the activity of tumor cells, and meanwhile, the nanoparticles have no obvious proliferation inhibition to MC38 cells, which indicates that the CNP/Mn nanoparticles have good drug safety. The CNP/Mn@pPHS, M-CNP@pPHS and M-CNP/Mn@pPHS nano-carrier treatment groups containing pPHS gene medicaments have obvious influence on the cell survival rate, and particularly, the treatment of the M-CNP/Mn@pPHS leads to the cell activity to be obviously lower than that of other groups, and obviously inhibits the proliferation of tumor cells. The experimental result shows that the M-CNP/Mn@pPHS nano-carrier has obvious killing effect on tumor cells under the action of pPHS and has good medicinal safety.
4.7 verification of tumor-specific apoptosis induced by nanocarrier M-CNP/Mn@pPHS
In order to study the accurate regulation and control effect of Sox4 and Hif-1 alpha dual transcription factors on tumor cell specific killing, MC38 cells in which Sox4 and Hif-1 alpha are knocked down singly or simultaneously are constructed. Together with MC38 tumor cell line, CHO-K1, RAW264.7 and BMDC, the important effect of Sox4 and Hif-1 alpha double transcription factor on accurate killing of tumor cells in M-CNP/Mn@pPHS treatment is explored. As shown in FIG. 11, M-CNP/Mn@pPHS can not induce the scorch of cells knocked down with Sox4 and Hif-1 alpha, and further proves that the specific tumor cell scorch induced by double regulation of Sox4 and Hif-1 alpha. MC38 cells knocked out of Sox4 or Hif-1 alpha and MC38 cells knocked out of Sox4 and Hif-1 alpha simultaneously survive, so that the cell morphology is clear, and the specific tumor cell pyrosis induced by double regulation of Sox4 and Hif-1 alpha is further confirmed. Single modulation means killing as long as responding to a high expression, and is easy to bring toxic and side effects. And Sox4 and Hif-1 alpha are over-expressed in tumor tissues, so that double regulation and control are more accurate and safer, and toxic and side effects and accidental injury to normal cells can be reduced.
4.8, analysis of the cellular therapeutic Effect of the Nano drug delivery System
As shown in FIG. 9, after CNP/Mn@pPHS, M-CNP@pPHS, M-CNP/Mn@pPHS treatment, the coke death rate of tumor cells was significantly increased (FIG. 12A), and a large amount of LDH was released (FIG. 12B), while a bubble-like structure was present around MC38 cells (FIG. 12C). Compared with the CNP/Mn@pPHS group, the M-CNP/Mn@pPHS group has the further improved capability of inducing cell apoptosis, which indicates that the membrane coating can improve the phagocytosis of cells and enhance the capability of inducing cell apoptosis.
4.9, MC38 tumor volume Change in tumor-bearing mice
In order to explore the anti-tumor effect of M-CNP/Mn@pPHS in animals, a MC38 mouse subcutaneous transplantation tumor model is specifically constructed, and the experimental result is shown in FIG. 13. The results show that the tumor of the whole vector M-CNP/Mn@pPHS group is obviously smaller than that of other groups, the optimal anti-tumor effect is shown, and the weight of each group of mice is hardly changed during the treatment period, so that the normal life of the mice is not influenced by the nano vector.
In summary, the invention constructs a recombinant plasmid for specifically expressing the pyroprotein based on the regulation of Sox4 and Hif-1 alpha dual transcription factors for the first time, clones the CDS region of GSDMD and clear-caspase-1 (P20-P10) genes and the combined promoter sequence of Sox4 and Hif-1 alpha to the plasmid through a genetic engineering means to obtain the dual-promoter plasmid regulated by the dual transcription factors, so as to realize the specific expression of GSDMD in tumor cells and release of active fragments, thereby achieving the purpose of accurately treating tumors. The nanomaterial based on Sox4 and Hif-1 alpha dual transcription factor regulation provided by the invention can accurately induce tumor cell apoptosis, enhance the effect of cancer immunotherapy, and has no obvious toxic or side effect.
Although the technical solutions of the present invention have been described in detail in the foregoing general description, the specific embodiments and the test examples, it should be noted that the examples and the test examples are only for illustrating the technical solutions and the technical effects of the present invention, and should not be construed as limiting the scope of the present invention. Simple variations, modifications or improvements made on the basis of the technical idea of the invention fall within the scope of the invention as claimed.
Claims (10)
1. A dual promoter plasmid regulated by dual transcription factors, characterized in that: the plasmid comprises elements linked in the following order: hif-1 alpha transcription factor binding site-clear-caspase-1 gene CDS region-Sox 4 transcription factor binding site-GSDMD gene CDS region.
2. The dual promoter plasmid of claim 1, wherein: the plasmid comprises elements linked in the following order: hif-1. Alpha. Transcription factor binding site-clear-caspase-1 gene CDS region-SV 40Poly (A) termination signal-Sox 4 transcription factor binding site-GSDMD gene CDS region-SV 40Poly (A) termination signal.
3. The dual promoter plasmid of claim 1 or 2, wherein: the NCBI accession number of the clear-caspase-1 gene is NM_009807.2;
and/or, the NCBI accession number of the GSDMD gene is nm_026960.4.
4. The dual promoter plasmid of claim 2, wherein: the plasmid comprises the sequence as set forth in SEQ ID NO:1, and a nucleotide sequence shown in the specification;
and/or, the plasmid is a plasmid inserted between Pci I and Not I of pEGFP-N1 vector as shown in SEQ ID NO:1, and the corresponding nucleotide sequence is shown as SEQ ID NO: 2.
5. A nano material regulated by double transcription factors is characterized in that: the nanomaterial comprises an inner core, wherein the inner core comprises calcium carbonate nanoparticles and the dual-initiation plasmids regulated by the dual transcription factors according to any one of claims 1 to 4, and the dual-initiation plasmids regulated by the dual transcription factors are uniformly distributed on the surfaces of the calcium carbonate nanoparticles.
6. The nanomaterial of claim 5, characterized in that: and manganese is loaded on the calcium carbonate nano particles.
7. The nanomaterial of claim 5, characterized in that: the nanomaterial comprises an outer membrane layer which is wrapped on the outer surface of the inner core and is composed of cell membrane vesicles.
8. A method for preparing a dual transcription factor regulated nanomaterial according to any of claims 5 to 7, characterized in that: the method comprises the following steps: mixing the calcium carbonate nano particles and the dual-start plasmids regulated by the dual transcription factors in the solution to obtain the inner core of the nano material.
9. The method of manufacturing according to claim 8, wherein: firstly, loading manganese on the calcium carbonate nano particles, and then mixing the manganese with double-start plasmids regulated by double transcription factors;
and/or, the method further comprises: mixing and extruding the inner core of the nano material with the cell membrane vesicle to obtain the nano material.
10. Use of a dual transcription factor regulated dual priming plasmid according to any one of claims 1 to 4, or a dual transcription factor regulated nanomaterial according to any one of claims 5 to 7, for the preparation of a medicament for the treatment of cancer.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109136265A (en) * | 2018-09-25 | 2019-01-04 | 吉林大学 | GSDMD-N gene recombination plasmid and methods and applications in mammary carcinoma model |
| WO2021036793A1 (en) * | 2019-08-23 | 2021-03-04 | National Institute Of Biological Sciences, Beijing | Pyroptosis-induced immunotherapy |
| CN112939951A (en) * | 2021-01-29 | 2021-06-11 | 华南理工大学 | Cell apoptosis drug prodrug, preparation method thereof and cell apoptosis drug |
| CN114657210A (en) * | 2022-03-03 | 2022-06-24 | 中山大学孙逸仙纪念医院 | Nano material based on GSDMD protein N-terminal peptide fragment loading and application thereof |
-
2023
- 2023-10-10 CN CN202311301531.XA patent/CN117363652A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109136265A (en) * | 2018-09-25 | 2019-01-04 | 吉林大学 | GSDMD-N gene recombination plasmid and methods and applications in mammary carcinoma model |
| WO2021036793A1 (en) * | 2019-08-23 | 2021-03-04 | National Institute Of Biological Sciences, Beijing | Pyroptosis-induced immunotherapy |
| CN112939951A (en) * | 2021-01-29 | 2021-06-11 | 华南理工大学 | Cell apoptosis drug prodrug, preparation method thereof and cell apoptosis drug |
| CN114657210A (en) * | 2022-03-03 | 2022-06-24 | 中山大学孙逸仙纪念医院 | Nano material based on GSDMD protein N-terminal peptide fragment loading and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| XIAOXI WANG 等: ""Logic "AND Gate Circuit"-Based Gasdermin Protein Expressing Nanoplatform Induces Tumor-Specific Pyroptosis to Enhance Cancer Immunotherapy"", 《ACS NANO》, vol. 18, 20 February 2024 (2024-02-20), pages 6946 - 6962 * |
| 傅心雨 等: ""经典途径细胞焦亡模型的构建"", 《中国兽医学报》, vol. 41, no. 11, 30 November 2021 (2021-11-30), pages 2238 - 2242 * |
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