CN114085815B - Engineered exosomes carrying nucleic acid type iron death inducer, construction method and application thereof - Google Patents
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Abstract
The invention relates to an engineering exosome carrying nucleic acid type iron death inducer, a construction method and application thereof. The existing iron death inducer has no targeting property and poor biocompatibility, so that the in-vivo application of the iron death inducer is greatly limited. The invention constructs an expression vector of SP94-lamp2b-RRM fusion protein, then constructs an exosome with the SP94-lamp2b-RRM fusion protein, and finally constructs an engineering exosome loaded with multi-siRNA, thus obtaining the engineering exosome loaded with nucleic acid iron death inducer. The engineering exosome contains RNA binding protein, and can wrap a large amount of RNA containing RNA binding protein binding site sequence into the exosome during the biosynthesis process, thereby achieving the effect of drug loading; the multi-siRNA with the GPX4 and the DHODH being simultaneously targeted is loaded, so that the expression of the GPX4 and the DHODH in the cell can be effectively reduced, and further the death of the cell iron is induced; can further induce the death of hepatoma cell iron by being combined with sorafenib, and overcome sorafenib drug resistance.
Description
Technical Field
The invention relates to the field of biological medicine, in particular to an engineering exosome carrying a nucleic acid type iron death inducer, and a construction method and application thereof.
Background
Hepatocellular carcinoma (HCC) is one of the most common malignant tumors, severely threatening the health and life of the patient. The early liver cancer can be effectively treated by methods such as tumor ablation, surgical excision and the like. However, most liver cancer patients are already in middle and late stages when diagnosis is confirmed, the optimal treatment period is missed, and no better treatment method exists at present. Sorafenib (sorafenib) is approved by the FDA for first-line treatment of advanced liver cancer and unresectable liver cancer, and can inhibit the activity of various kinases to further inhibit proliferation of tumor cells, promote apoptosis of tumor cells, and achieve the effect of inhibiting malignant progression of tumors. Although sorafenib showed a better therapeutic effect at the beginning of liver cancer treatment, sorafenib-treated tumors rarely resolved completely and most patients developed recurrent resistance. Thus, combination therapy may be a better treatment option in order to improve survival and quality of life for liver cancer patients.
Iron death (ferroptosis) is a newly described form of programmed cell death characterized by iron-dependent accumulation of lipid peroxides to a lethal dose. There is growing evidence that iron death can be induced by inhibiting cystine/glutamate transporter (system xc-) activity, down-regulating GPX4 or DHODH, and accumulating Reactive Oxygen Species (ROS). Recent reports suggest that sorafenib can induce iron death by inhibiting system xc-. Furthermore, numerous studies have shown that the anticancer activity of sorafenib is primarily dependent on induction of iron death by inhibition of the activity of system xc-. However, tumor cells can escape from the iron death process by means of high-expression iron death suppression genes (such as GPX4 and DHODH) or low-expression iron death induction genes (such as ACSL4 and TFRC) and the like, so that the survival and the growth of the tumor cells are improved. We found through public database analysis and in vitro cytology experiments that two key genes inhibiting iron death, GPX4 and DHODH, were enriched in sorafenib resistant patients and cells and correlated with poor prognosis for HCC patients receiving sorafenib treatment. Thus, further enhancement of the antitumor effect of sorafenib from the iron death point of view may be an effective strategy to increase the therapeutic effect of sorafenib and overcome sorafenib resistance.
Iron death is one of the important ways of sorafenib to exert anti-tumor effects, so the combination of sorafenib and an iron death inducer would be an effective way to overcome liver cancer sorafenib resistance and improve patient survival and quality of life. Based on the important role of iron death in tumorigenesis and development and treatment of drug resistance, the safe and effective iron death inducer has wide application prospect in tumor treatment. However, currently available iron death inhibitors are mostly poorly soluble and have strong systemic toxicity, and the above drawbacks are mainly due to the fact that currently available iron death inducers are not targeted and have poor biocompatibility, limiting the in vivo application of the iron death inducers.
Disclosure of Invention
The invention aims to provide an engineering exosome carrying a nucleic acid type iron death inducer, a construction method and application thereof, wherein the exosome can effectively carry multi-siRNA targeting GPX4 and DHODH simultaneously, carry the multi-siRNA into liver cancer cells, induce the iron death process of the liver cancer cells, enhance the treatment effect of sorafenib, overcome the limitation (poor biocompatibility and targeting property and great toxic and side effect) of the existing iron death inducer in vivo and enhance the sensitivity of liver cancer to sorafenib treatment.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
a method for constructing an engineered exosome carrying a nucleic acid-type iron death inducer, comprising the steps of:
the method comprises the following steps:
step 1: constructing an SP94-lamp2b-RRM fusion protein expression vector:
inserting CDS region sequences of three proteins of SP94, lamp2b and RRM into a pcDNA3.1 eukaryotic expression vector by a molecular cloning method to obtain an SP94-lamp2b-RRM fusion protein expression vector, and transfecting the fusion protein expression vector into 293T cells;
step 2: construction of exosomes harboring the SP94-lamp2b-RRM fusion protein:
culturing the transfected cells in the step 1, and collecting the supernatant to obtain exosomes with SP94-lamp2b-RRM fusion proteins;
step 3: construction of engineered exosomes loaded with multi-siRNA:
the SP94-lamp2b-RRM fusion protein expression vector and multi-RNA containing GPX4 and DHODH siRNA and RNA binding protein binding sequence are co-transfected into 293T cells, then culturing is carried out, and supernatant is collected to obtain the engineering exosomes loaded with multi-siRNA.
The CDS region sequence of the SP94 is:
TCTTTCTCTATTATTCATACTCCTATTCTTCCTCTT。
the CDS region sequence of lamp2b is:
ATGGTGTGCTTCCGCCTCTTCCCGGTTCCGGGCTCAGGGCTCGTTCTGGTCTGCCTAGTCCTGGGAGCTG
TGCGGTCTTATGCATTGGAACTTAATTTGACAGATTCAGAAAATGCCACTTGCCTTTATGCAAAATGGCA
GATGAATTTCACAGTACGCTATGAAACTACAAATAAAACTTATAAAACTGTAACCATTTCAGACCATGGC
ACTGTGACATATAATGGAAGCATTTGTGGGGATGATCAGAATGGTCCCAAAATAGCAGTGCAGTTCGGAC
CTGGCTTTTCCTGGATTGCGAATTTTACCAAGGCAGCATCTACTTATTCAATTGACAGCGTCTCATTTTC
CTACAACACTGGTGATAACACAACATTTCCTGATGCTGAAGATAAAGGAATTCTTACTGTTGATGAACTT
TTGGCCATCAGAATTCCATTGAATGACCTTTTTAGATGCAATAGTTTATCAACTTTGGAAAAGAATGATG
TTGTCCAACACTACTGGGATGTTCTTGTACAAGCTTTTGTCCAAAATGGCACAGTGAGCACAAATGAGTT
CCTGTGTGATAAAGACAAAACTTCAACAGTGGCACCCACCATACACACCACTGTGCCATCTCCTACTACA
ACACCTACTCCAAAGGAAAAACCAGAAGCTGGAACCTATTCAGTTAATAATGGCAATGATACTTGTCTGC
TGGCTACCATGGGGCTGCAGCTGAACATCACTCAGGATAAGGTTGCTTCAGTTATTAACATCAACCCCAA
TACAACTCACTCCACAGGCAGCTGCCGTTCTCACACTGCTCTACTTAGACTCAATAGCAGCACCATTAAG
TATCTAGACTTTGTCTTTGCTGTGAAAAATGAAAACCGATTTTATCTGAAGGAAGTGAACATCAGCATGT
ATTTGGTTAATGGCTCCGTTTTCAGCATTGCAAATAACAATCTCAGCTACTGGGATGCCCCCCTGGGAAG
TTCTTATATGTGCAACAAAGAGCAGACTGTTTCAGTGTCTGGAGCATTTCAGATAAATACCTTTGATCTA
AGGGTTCAGCCTTTCAATGTGACACAAGGAAAGTATTCTACAGCCCAAGAGTGTTCGCTGGATGATGACA
CCATTCTAATCCCAATTATAGTTGGTGCTGGTCTTTCAGGCTTGATTATCGTTATAGTGATTGCTTACGT
AATTGGCAGAAGAAAAAGTTATGCTGGATATCAGACTCTG。
the CDS region sequence of the RRM is as follows:
ATGGCAGTTCCCGAGACCCGCCCTAACCACACTATTTATATCAACAACCTCAATGAGAAGATCAAGAAGG
ATGAGCTAAAAAAGTCCCTGTACGCCATCTTCTCCCAGTTTGGCCAGATCCTGGATATCCTGGTATCACG
GAGCCTGAAGATGAGGGGCCAGGCCTTTGTCATCTTCAAGGAGGTCAGCAGCGCCACCAACGCCCTGCGC
TCCATGCAGGGTTTCCCTTTCTATGACAAACCTATGCGTATCCAGTATGCCAAGACCGACTCAGATATCA
TTGCCAAGATGAAAGGCACCTTCGTGGAGCGGGAC。
the sequence of the multi-RNA is:
sense(5'-3')：GGAGUAACGAAGAGAUCAACAAUGGGCCAUAAAUUCCGAAAU；
antisense(5'-3')：AUUUCGGAAUUUAUGGCCCAUUGCACUUGAUCUCUUCGUUACUCC。
the engineered exosomes carrying the nucleic acid-type iron death inducer obtained by the above-described construction method.
The application of the engineering exosome in preparing anti-liver cancer drugs.
The engineering exosomes can deliver multi-RNA to liver cancer cells and induce the death of liver cancer cell iron.
The application of the engineering exosome combined with sorafenib in preparing anti-liver cancer drugs is disclosed.
The engineering exosomes can synergistically induce hepatoma cell iron death with sorafenib.
Compared with the prior art, the invention has the following beneficial effects:
1. the invention provides a novel siRNA drug-loading mode-engineering exosomes: the engineering exosome membrane contains RNA binding protein, so that a large amount of RNA containing RNA binding protein binding site sequence can be wrapped into the exosome during the biosynthesis process, thereby achieving the effect of drug loading.
2. The invention effectively loads multi-siRNA targeting GPX4 and DHODH simultaneously: can effectively reduce the expression of GPX4 and DHODH in cells, thereby inducing the death of cell iron.
3. The present invention provides multi-siRNA comprising an RNA binding protein binding sequence: the multi-siRNA can be loaded into the engineered exosomes described above in large quantities and then delivered into the target cells for use.
4. The engineered exosome carries targeting peptide SP94 of liver cancer cell, which can be absorbed by liver cancer cell without affecting the function of other organs.
5. The engineering exosome can be combined with sorafenib to further induce the death of hepatoma cell iron, thereby overcoming sorafenib resistance.
6. The engineering exosome also has better biocompatibility, does not show obvious toxic or side effect in the body of a mouse, and has better safety and wide application prospect.
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In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other embodiments of the drawings can be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic structural diagram of an engineered exosome.
FIG. 2 is a schematic diagram of the delivery of loaded multi-siRNA by an engineered exosome to a hepatoma cell.
FIG. 3 is a western blot result of multi-siRNA loaded engineered exosomes inducing reduced expression of GPX4 and DHODH in hepatoma cells.
FIG. 4 is the CCK8 results of engineered exosomes loaded with multi-siRNA to increase sensitivity of hepatoma cells to sorafenib.
FIG. 5 is a schematic representation of the enrichment of engineered exosomes at the liver cancer site in mice.
FIG. 6 is a schematic of the in situ liver cancer growth of a multi-siRNA loaded engineered exosomes in combination with sorafenib in mice.
Fig. 7 is a graph of the statistical results of fig. 6.
FIG. 8 is a graphical representation of survival results of tumor-bearing mice after treatment with multi-siRNA loaded engineered exosomes in combination with sorafenib.
Detailed Description
In order that the invention may be readily understood, a more complete description of the invention will be rendered by reference to the appended drawings. The drawings illustrate preferred embodiments of the invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
The invention constructs an engineering exosome capable of targeting liver cancer cells, namely an engineering exosome carrying nucleic acid iron death inducer, which can effectively load multi-siRNA simultaneously targeting high-expression iron death suppression genes GPX4 and DHODH, carry the multi-siRNA into liver cancer cells and induce the iron death process of the liver cancer cells.
The method for constructing the engineering exosome specifically comprises the following steps:
step 1: constructing an SP94-lamp2b-RRM fusion protein expression vector:
the CDS region sequences of the SP94, lamp2b and RRM proteins are inserted into a pcDNA3.1 eukaryotic expression vector by a molecular cloning method to obtain an SP94-lamp2b-RRM fusion protein expression vector, and the fusion protein expression vector is transfected into 293T cells.
The CDS region sequence of SP94 is SEQ ID NO:1:
TCTTTCTCTATTATTCATACTCCTATTCTTCCTCTT。
the CDS region sequence of lamp2b is SEQ ID NO:2:
ATGGTGTGCTTCCGCCTCTTCCCGGTTCCGGGCTCAGGGCTCGTTCTGGTCTGCCTAGTCCTGGGAGCTG
TGCGGTCTTATGCATTGGAACTTAATTTGACAGATTCAGAAAATGCCACTTGCCTTTATGCAAAATGGCA
GATGAATTTCACAGTACGCTATGAAACTACAAATAAAACTTATAAAACTGTAACCATTTCAGACCATGGC
ACTGTGACATATAATGGAAGCATTTGTGGGGATGATCAGAATGGTCCCAAAATAGCAGTGCAGTTCGGAC
CTGGCTTTTCCTGGATTGCGAATTTTACCAAGGCAGCATCTACTTATTCAATTGACAGCGTCTCATTTTC
CTACAACACTGGTGATAACACAACATTTCCTGATGCTGAAGATAAAGGAATTCTTACTGTTGATGAACTT
TTGGCCATCAGAATTCCATTGAATGACCTTTTTAGATGCAATAGTTTATCAACTTTGGAAAAGAATGATG
TTGTCCAACACTACTGGGATGTTCTTGTACAAGCTTTTGTCCAAAATGGCACAGTGAGCACAAATGAGTT
CCTGTGTGATAAAGACAAAACTTCAACAGTGGCACCCACCATACACACCACTGTGCCATCTCCTACTACA
ACACCTACTCCAAAGGAAAAACCAGAAGCTGGAACCTATTCAGTTAATAATGGCAATGATACTTGTCTGC
TGGCTACCATGGGGCTGCAGCTGAACATCACTCAGGATAAGGTTGCTTCAGTTATTAACATCAACCCCAA
TACAACTCACTCCACAGGCAGCTGCCGTTCTCACACTGCTCTACTTAGACTCAATAGCAGCACCATTAAG
TATCTAGACTTTGTCTTTGCTGTGAAAAATGAAAACCGATTTTATCTGAAGGAAGTGAACATCAGCATGT
ATTTGGTTAATGGCTCCGTTTTCAGCATTGCAAATAACAATCTCAGCTACTGGGATGCCCCCCTGGGAAG
TTCTTATATGTGCAACAAAGAGCAGACTGTTTCAGTGTCTGGAGCATTTCAGATAAATACCTTTGATCTA
AGGGTTCAGCCTTTCAATGTGACACAAGGAAAGTATTCTACAGCCCAAGAGTGTTCGCTGGATGATGACA
CCATTCTAATCCCAATTATAGTTGGTGCTGGTCTTTCAGGCTTGATTATCGTTATAGTGATTGCTTACGT
AATTGGCAGAAGAAAAAGTTATGCTGGATATCAGACTCTG。
the CDS region sequence of RRM (which is the domain responsible for binding to RNA in the RNA binding protein SNRPA) is SEQ ID NO:3:
ATGGCAGTTCCCGAGACCCGCCCTAACCACACTATTTATATCAACAACCTCAATGAGAAGATCAAGAAGG
ATGAGCTAAAAAAGTCCCTGTACGCCATCTTCTCCCAGTTTGGCCAGATCCTGGATATCCTGGTATCACG
GAGCCTGAAGATGAGGGGCCAGGCCTTTGTCATCTTCAAGGAGGTCAGCAGCGCCACCAACGCCCTGCGC
TCCATGCAGGGTTTCCCTTTCTATGACAAACCTATGCGTATCCAGTATGCCAAGACCGACTCAGATATCA
TTGCCAAGATGAAAGGCACCTTCGTGGAGCGGGAC。
step 2: construction of exosomes harboring the SP94-lamp2b-RRM fusion protein:
culturing the cells transfected in the step 1 by using a culture medium without exosome serum, and collecting supernatant to obtain exosome with SP94-lamp2b-RRM fusion protein;
step 3: construction of engineered exosomes loaded with multi-siRNA:
the SP94-lamp2b-RRM fusion protein expression vector and multi-RNA containing GPX4 and DHODH siRNA and RNA binding protein binding sequence are co-transfected into cells, then culture is carried out by using a culture medium without exosome serum, and the supernatant is collected to obtain the engineered exosomes loaded with multi-siRNA.
The sequence of the multi-RNA is:
sense(5'-3')：GGAGUAACGAAGAGAUCAACAAUGGGCCAUAAAUUCCGAAAU(SEQ ID NO：4)；
antisense(5'-3')：AUUUCGGAAUUUAUGGCCCAUUGCACUUGAUCUCUUCGUUACUCC(SEQ ID NO：5)。
in the above steps, firstly, a fusion protein expression vector is transfected into a cell, and it is confirmed that a corresponding exosome containing the fusion protein can be obtained (step 2); the fusion protein expression vector and the multi-siRNA are then co-transfected into the cell (step 3), the purpose of which is to load the multi-siRNA into the exosomes.
The engineering exosome carrying the nucleic acid type iron death inducer is obtained through the construction method, the nucleic acid type iron death inducer specifically refers to multi-siRNA capable of simultaneously targeting GPX4 and DHODH, the GPX4 and the DHODH are key molecules for inhibiting iron death in tumor cells, and the reduction of the expression of the two molecules can effectively induce cell iron death. The engineering exosome carries targeting peptide SP94 of liver cancer cells, is loaded with GPX4 and DHODH siRNA, contains RNA binding protein, can be used for treating liver cancer, can deliver multi-RNA to liver cancer cells, effectively reduces the expression of GPX4 and DHODH in the cells, and induces iron death of the liver cancer cells; the engineering exosome can also be used together with sorafenib, can synergistically induce the death of hepatoma cell iron with sorafenib, can further enhance the treatment effect of sorafenib, and overcomes the drug resistance of hepatoma cells to sorafenib.
Example 1: co-culturing the engineering exosomes and liver cancer cells:
as shown in fig. 2, after co-culturing the obtained engineered exosomes with liver cancer cells, it was found by using a confocal experiment that the engineered exosomes were able to deliver the loaded multi-siRNA to the liver cancer cells.
Example 2: synergistic combination of engineered exosomes with sorafenib:
co-culturing the engineering exosomes and sorafenib-resistant HepG2 cells, and detecting the expression condition and the cell activity of GPX4 and DHODH in the cells by using real time PCR, western blot, clone formation experiments, CCK8 experiments and the like, wherein the results show that the expression level of GPX4 and DHODH in the cells treated by the engineering exosomes carrying multi-siRNA is obviously reduced, and the sensitivity of the cells to sorafenib is enhanced as shown in figures 3 and 4.
Example 3: enrichment of engineered exosomes at liver cancer sites in model mice:
the engineered exosomes were stained with DIR dye, then the engineered exosomes were injected into liver cancer model mice in a tail vein of 100 μg (protein level) per mouse, and the distribution of exosomes in mice was detected by using a small animal living body imaging technique, as can be seen from fig. 5, the engineered exosomes with targeting polypeptide sequences were enriched in liver cancer sites of mice.
Example 4: engineered exosomes in combination with sorafenib act on model mice:
in-situ liver cancer mice with sorafenib and engineering exosomes resistant to sorafenib are treated in a combined mode, and the results of detection on tumor growth conditions in the mice through a small animal living body imaging technology show that sorafenib cannot effectively inhibit tumor progression of the model mice, but tumors of mice in a sorafenib and engineering exosomes combined group are obviously reduced, and the survival time is obviously prolonged, as shown in fig. 6, 7 and 8.
The foregoing description of the invention has been presented for purposes of illustration and description, and is not intended to be limiting. Several simple deductions, modifications or substitutions may also be made by a person skilled in the art to which the invention pertains, based on the idea of the invention.
SEQUENCE LISTING
<110> Chinese people's air force medical university
<120> engineered exosomes carrying nucleic acid type iron death inducer, method for constructing same, and use thereof
<130> 20211015
<160> 5
<170> PatentIn version 3.5
<210> 1
<211> 36
<212> DNA
<213> CDS
<400> 1
tctttctcta ttattcatac tcctattctt cctctt 36
<210> 2
<211> 1230
<212> DNA
<213> CDS
<400> 2
atggtgtgct tccgcctctt cccggttccg ggctcagggc tcgttctggt ctgcctagtc 60
ctgggagctg tgcggtctta tgcattggaa cttaatttga cagattcaga aaatgccact 120
tgcctttatg caaaatggca gatgaatttc acagtacgct atgaaactac aaataaaact 180
tataaaactg taaccatttc agaccatggc actgtgacat ataatggaag catttgtggg 240
gatgatcaga atggtcccaa aatagcagtg cagttcggac ctggcttttc ctggattgcg 300
aattttacca aggcagcatc tacttattca attgacagcg tctcattttc ctacaacact 360
ggtgataaca caacatttcc tgatgctgaa gataaaggaa ttcttactgt tgatgaactt 420
ttggccatca gaattccatt gaatgacctt tttagatgca atagtttatc aactttggaa 480
aagaatgatg ttgtccaaca ctactgggat gttcttgtac aagcttttgt ccaaaatggc 540
acagtgagca caaatgagtt cctgtgtgat aaagacaaaa cttcaacagt ggcacccacc 600
atacacacca ctgtgccatc tcctactaca acacctactc caaaggaaaa accagaagct 660
ggaacctatt cagttaataa tggcaatgat acttgtctgc tggctaccat ggggctgcag 720
ctgaacatca ctcaggataa ggttgcttca gttattaaca tcaaccccaa tacaactcac 780
tccacaggca gctgccgttc tcacactgct ctacttagac tcaatagcag caccattaag 840
tatctagact ttgtctttgc tgtgaaaaat gaaaaccgat tttatctgaa ggaagtgaac 900
atcagcatgt atttggttaa tggctccgtt ttcagcattg caaataacaa tctcagctac 960
tgggatgccc ccctgggaag ttcttatatg tgcaacaaag agcagactgt ttcagtgtct 1020
ggagcatttc agataaatac ctttgatcta agggttcagc ctttcaatgt gacacaagga 1080
aagtattcta cagcccaaga gtgttcgctg gatgatgaca ccattctaat cccaattata 1140
gttggtgctg gtctttcagg cttgattatc gttatagtga ttgcttacgt aattggcaga 1200
agaaaaagtt atgctggata tcagactctg 1230
<210> 3
<211> 315
<212> DNA
<213> CDS
<400> 3
atggcagttc ccgagacccg ccctaaccac actatttata tcaacaacct caatgagaag 60
atcaagaagg atgagctaaa aaagtccctg tacgccatct tctcccagtt tggccagatc 120
ctggatatcc tggtatcacg gagcctgaag atgaggggcc aggcctttgt catcttcaag 180
gaggtcagca gcgccaccaa cgccctgcgc tccatgcagg gtttcccttt ctatgacaaa 240
cctatgcgta tccagtatgc caagaccgac tcagatatca ttgccaagat gaaaggcacc 300
ttcgtggagc gggac 315
<210> 4
<211> 42
<212> RNA
<213> Synthesis
<400> 4
ggaguaacga agagaucaac aaugggccau aaauuccgaa au 42
<210> 5
<211> 45
<212> RNA
<213> Synthesis
<400> 5
auuucggaau uuauggccca uugcacuuga ucucuucguu acucc 45
Claims (6)
1. A method for constructing an engineered exosome carrying a nucleic acid-type iron death inducer, comprising the steps of:
the method comprises the following steps:
step 1: constructing an SP94-lamp2b-RRM fusion protein expression vector:
inserting CDS region sequences of three proteins of SP94, lamp2b and RRM into a pcDNA3.1 eukaryotic expression vector by a molecular cloning method to obtain an SP94-lamp2b-RRM fusion protein expression vector, and transfecting the fusion protein expression vector into 293T cells;
step 2: construction of exosomes harboring the SP94-lamp2b-RRM fusion protein:
culturing the transfected cells in the step 1, and collecting the supernatant to obtain exosomes with SP94-lamp2b-RRM fusion proteins;
step 3: construction of engineered exosomes loaded with multi-siRNA:
co-transfecting SP94-lamp2b-RRM fusion protein expression vector and multi-RNA containing GPX4 and DHODH siRNA and RNA binding protein binding sequence into 293T cells, culturing, and collecting supernatant to obtain engineered exosomes loaded with multi-siRNA;
the CDS region sequence of the SP94 is:
TCTTTCTCTATTATTCATACTCCTATTCTTCCTCTT；
the CDS region sequence of lamp2b is:
ATGGTGTGCTTCCGCCTCTTCCCGGTTCCGGGCTCAGGGCTCGTTCTGGTCTGCCTAGTCCTGGGAGCTG
TGCGGTCTTATGCATTGGAACTTAATTTGACAGATTCAGAAAATGCCACTTGCCTTTATGCAAAATGGCA
GATGAATTTCACAGTACGCTATGAAACTACAAATAAAACTTATAAAACTGTAACCATTTCAGACCATGGC
ACTGTGACATATAATGGAAGCATTTGTGGGGATGATCAGAATGGTCCCAAAATAGCAGTGCAGTTCGGAC
CTGGCTTTTCCTGGATTGCGAATTTTACCAAGGCAGCATCTACTTATTCAATTGACAGCGTCTCATTTTC
CTACAACACTGGTGATAACACAACATTTCCTGATGCTGAAGATAAAGGAATTCTTACTGTTGATGAACTT
TTGGCCATCAGAATTCCATTGAATGACCTTTTTAGATGCAATAGTTTATCAACTTTGGAAAAGAATGATG
TTGTCCAACACTACTGGGATGTTCTTGTACAAGCTTTTGTCCAAAATGGCACAGTGAGCACAAATGAGTT
CCTGTGTGATAAAGACAAAACTTCAACAGTGGCACCCACCATACACACCACTGTGCCATCTCCTACTACA
ACACCTACTCCAAAGGAAAAACCAGAAGCTGGAACCTATTCAGTTAATAATGGCAATGATACTTGTCTGC
TGGCTACCATGGGGCTGCAGCTGAACATCACTCAGGATAAGGTTGCTTCAGTTATTAACATCAACCCCAA
TACAACTCACTCCACAGGCAGCTGCCGTTCTCACACTGCTCTACTTAGACTCAATAGCAGCACCATTAAG
TATCTAGACTTTGTCTTTGCTGTGAAAAATGAAAACCGATTTTATCTGAAGGAAGTGAACATCAGCATGT
ATTTGGTTAATGGCTCCGTTTTCAGCATTGCAAATAACAATCTCAGCTACTGGGATGCCCCCCTGGGAAG
TTCTTATATGTGCAACAAAGAGCAGACTGTTTCAGTGTCTGGAGCATTTCAGATAAATACCTTTGATCTA
AGGGTTCAGCCTTTCAATGTGACACAAGGAAAGTATTCTACAGCCCAAGAGTGTTCGCTGGATGATGACA
CCATTCTAATCCCAATTATAGTTGGTGCTGGTCTTTCAGGCTTGATTATCGTTATAGTGATTGCTTACGT
AATTGGCAGAAGAAAAAGTTATGCTGGATATCAGACTCTG；
the CDS region sequence of the RRM is as follows:
ATGGCAGTTCCCGAGACCCGCCCTAACCACACTATTTATATCAACAACCTCAATGAGAAGATCAAGAAGG
ATGAGCTAAAAAAGTCCCTGTACGCCATCTTCTCCCAGTTTGGCCAGATCCTGGATATCCTGGTATCACG
GAGCCTGAAGATGAGGGGCCAGGCCTTTGTCATCTTCAAGGAGGTCAGCAGCGCCACCAACGCCCTGCGC
TCCATGCAGGGTTTCCCTTTCTATGACAAACCTATGCGTATCCAGTATGCCAAGACCGACTCAGATATCA
TTGCCAAGATGAAAGGCACCTTCGTGGAGCGGGAC；
the sequence of the multi-RNA is:
sense(5'-3')：GGAGUAACGAAGAGAUCAACAAUGGGCCAUAAAUUCCGAAAU；
antisense(5'-3')：AUUUCGGAAUUUAUGGCCCAUUGCACUUGAUCUCUUCGUUACUCC。
2. the engineered exosome carrying a nucleic acid-type iron death inducer obtained by the construction method according to claim 1.
3. The use of the engineered exosome of claim 2 in the manufacture of an anti-hepatoma drug.
4. A use according to claim 3, characterized in that:
the engineering exosomes can deliver multi-RNA to liver cancer cells and induce the death of liver cancer cell iron.
5. The use of the engineered exosome combination sorafenib of claim 2 in the manufacture of an anti-liver cancer medicament.
6. The use according to claim 5, characterized in that:
the engineering exosomes can synergistically induce hepatoma cell iron death with sorafenib.
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