CN108250267B - Polypeptide, polypeptide-siRNA induction co-assembly and application thereof - Google Patents
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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- A—HUMAN NECESSITIES
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
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Abstract
本发明提供了一种多肽，其结构式如下所示，
The present invention provides a polypeptide whose structural formula is as follows,
Description
Technical Field
The invention belongs to the field of bioengineering, and relates to a polypeptide, in particular to a polypeptide, a polypeptide-siRNA induced co-assembly and application thereof.
Background
RNAi technology is one of the most widely used biological techniques due to its high specificity and efficient expression regulation of specific gene mrnas. Therefore, RNAi technology is widely used in many fields such as functional genomics, genetics, gene therapy, viral disease therapy, and the like. However, both in vivo and in vitro, siRNA is easily degraded by ubiquitous RNase and siRNA cannot induce RNAi effect through cell membrane alone, so RNAi technology cannot be aided by highly efficient, low-toxic siRNA carriers.
Commonly used siRNA transfection vectors include viral vectors, liposomes, high polymers, inorganic nanoparticles, polypeptide biological vectors, and the like. The virus vector is siRNA vector mainly comprising adenovirus, retrovirus and lentivirus. Viral vectors are one of the most efficient siRNA vectors known to be transfected, but because viruses may be uncontrollably inserted into the genome of a host, the use of viral vectors is greatly limited due to safety and operational complexity.
A chemical modified carrier represented by liposome and high polymer is a siRNA carrier which is most widely applied at present. The liposome (such as lipo-2000) is an amphiphilic molecule consisting of a hydrophilic head part and a hydrophobic tail part, the liposome can form a uniform and stable compound with siRNA in water, the siRNA is wrapped in a cavity consisting of liposome molecules and enters cells in an endocytosis or membrane fusion mode, and the siRNA molecules in the liposome are further released to trigger RNAi effect. However, due to the strong cytotoxicity of liposomes themselves towards cell membranes, liposome-mediated transfection of siRNA is generally performed at lower liposome concentrations and is mainly limited to transfection at the in vitro and cellular level.
The PEI molecule utilizes electrostatic action to load and wrap siRNA, and after entering cells through endocytosis, the PEI molecule utilizes proton sponge effect to break endocytosis bodies and promote the release of the siRNA. As the molecular weight of PEI increases, the more siRNA can be loaded, but the cytotoxicity is higher. Therefore, PEI molecules are generally used only for transfection at a low dose cellular level, before being further optimized.
The non-polar metal nano particle is a novel siRNA carrier represented by nano gold. It is mainly combined with siRNA through electrostatic interaction, enters cells through endocytosis and is successfully released to trigger RNAi effect. The transfection efficiency is high, and no acute toxic effect is generated on cells, but because the inorganic metal nanoparticles are too stable and are difficult to be eliminated by organisms, the metal nanoparticles are easy to accumulate in vivo, and a potential toxic reaction is caused, so that the application of the metal nanoparticles is greatly limited.
Although these vectors can achieve effective transfection of siRNA, they also have respective problems, and there are still many limitations and difficulties that cannot be achieved for siRNA delivery in a complex system. The siRNA carrying mode is convenient, simple, efficient and safe and still is the direction concerned by researchers.
The polypeptide is used as an endogenous molecule in an organism, and has very high biocompatibility and low cytotoxicity. At present, by reasonably designing the sequence and the structure of the polypeptide, the polypeptide siRNA vector with low toxicity, degradability and high transfection efficiency can be obtained. The appearance of polypeptide carriers provides new direction and activity for the development of siRNA technology. The polypeptide siRNA carrier mainly comprises two categories of a cell-penetrating peptide carrier and an amphiphilic polypeptide carrier, wherein the cell-penetrating peptide carrier loads negative siRNA in a covalent crosslinking or electrostatic adsorption mode by mainly utilizing the electropositivity and the cell-penetrating property of polypeptide, and then enters cells through endocytosis to trigger RNAi effect. Amphiphilic polypeptides are another widely used type of polypeptide siRNA carrier, and mainly utilize the sequence of RNA binding protein and the sequence of cell-penetrating peptide to realize the process of loading siRNA and carrying into cells. However, in order to increase the delivery efficiency of polypeptide carriers, polypeptide molecules are often modified with specific groups, such as: S-S bonds sensitive to reducing environment, PH sensitive trichloroquinolines, and the like. Although the modifications can improve the efficiency of polypeptide carrying siRNA, the inconvenience of preparation, synthesis, purification and separation is brought, and the length of the polypeptide carrier is generally more than 20 amino acids, which further increases the difficulty of synthesis and separation and limits the application of the polypeptide siRNA carrier. Therefore, the development of novel, simple, safe and efficient polypeptide siRNA vectors is always the direction of attention of researchers.
Disclosure of Invention
Aiming at the technical problems of the siRNA carrier in the prior art, the invention provides a polypeptide, a polypeptide-siRNA induction co-assembly and application thereof, and aims to solve the technical problems of complicated preparation and purification, high toxicity and poor effect of the siRNA carrier in the prior art.
The invention provides a polypeptide, the structural formula of which is shown as follows,
wherein R is arginine, M is methionine, E is glutamic acid, H is histidine, and W is tryptophan.
The invention also provides a polypeptide-siRNA induction co-assembly body, which consists of the polypeptide and siRNA.
The invention also provides application of the polypeptide-siRNA induced co-assembly in preparing a medicament for treating cervical cancer.
The invention provides a short peptide molecule based on nucleic acid induction co-assembly, which can be co-assembled with siRNA at normal temperature to form stable nanoparticles and realize intracellular delivery of siRNA molecules to cause RNAi effect, thereby achieving the purpose of down-regulating the mRNA expression level of a target gene. The short peptide molecule is simple and convenient to synthesize, only has 9 amino acids, is suitable for mass preparation, is beneficial to solving the problem of complicated preparation and purification of the polypeptide siRNA carrier, and provides a new direction for developing the polypeptide siRNA carrier which is simple, convenient, low in toxicity and high in efficiency.
The invention relates to a polypeptide molecule Wpc based on induced co-assembly in the presence of nucleic acid molecules (single-stranded RNA, double-stranded RNA, single-stranded DNA, double-stranded DNA, plasmids, single nucleotide molecules and the like). The polypeptide and siRNA form a co-assembled nano particle, and then the carrier siRNA is delivered to a target cell to trigger RNAi effect, so that the regulation and control effect on target gene mRNA is realized.
The polypeptide and the target siRNA are mixed and incubated for 5min at normal temperature to form stable and uniform nano particles, and the polypeptide can be obviously observed to carry the siRNA marked by fluorescence into cells efficiently through flow cytometry and laser confocal microscopy. RT-PCR analysis further verifies that the co-assembled nanoparticles can induce RNAi effect in cells, so that the content of target gene mRNA is reduced. The cytotoxicity experiment proves that the co-assembled nano particle has higher biocompatibility and very low cytotoxicity and hemolytic toxicity. The anti-cell proliferation experiment is a cell cycle experiment and further proves that the polypeptide-siRNA (Survivin gene) nano particle can effectively block the cervical cancer cell line HeLa in the G2 stage and block the growth of the cervical cancer cell line HeLa. And the human-derived heterogeneous cervical cancer tumor model proves that the polypeptide-siRNA (Survivin gene) can well inhibit the proliferation of the cervical cancer tumor body.
The invention proves that the polypeptide can form stable and uniform polypeptide-nucleic acid (such as siRNA) composite nanoparticles under the induction of nucleic acid molecules through experiments such as an Atomic Force Microscope (AFM), a scanning tunnel microscope (SEM), a dynamic light particle size scatterometer (DLS), flow cytometry, laser confocal microscopy, RT-PCR analysis, cytotoxicity experiments (MTT), a human source heterogeneous cervical cancer tumor transplantation model and the like, and the formed composite nanoparticles can deliver siRNA into target cells, release and further trigger RNAi effect. Can be used as a novel, simple, low-toxicity and high-efficiency polypeptide siRNA carrier. In addition, the polypeptide carrier carrying mRNA of a key gene survivin aiming at cervical cancer cells can inhibit the growth of mouse human-derived xenograft cervical cancer tumors.
The polypeptide molecule of the invention does not need to introduce any non-natural modification, and only two Met molecules existing in the polypeptide sequence react with a ring closing reagent (double halogenated hydrocarbon) in water (containing 1% formic acid) for 12h, so that the electropositivity and the stability of the polypeptide can be increased. And the modification on the side chain can be eliminated in a reducing environment (such as in a cell), so that the positive charge added on the polypeptide can enhance the combination of the polypeptide and the negative siRNA, be conductive to generating a polypeptide-siRNA compound and protect the siRNA from being degraded by RNase. Meanwhile, after the polypeptide-siRNA complex enters a cell, the reducing environment in the cell can eliminate the electropositive modification of the polypeptide and weaken the interaction between the polypeptide and the siRNA, thereby promoting the release of the siRNA in cytoplasm.
Compared with the prior art, the invention has remarkable technical progress. The invention is a simple, convenient, low-toxicity and high-efficiency polypeptide siRNA delivery system which delivers siRNA into target cells and triggers RNAi effect. The polypeptide-siRNA (survivin) co-assembled nanoparticles based on the polypeptide delivery system have obvious effect on the anti-proliferation of cervical cancer cell lines and human source xenograft cervical cancer models. The invention develops a simple, convenient, low-toxicity and high-efficiency polypeptide siRNA delivery system based on biological endogenous polypeptide molecules, thereby not only reducing the cytotoxicity of the siRNA delivery vehicle, but also avoiding fussy and complicated polypeptide preparation and purification steps. Provides a new direction for the development of polypeptide siRNA carrier.
Drawings
FIG. 1 is an agarose gel block electrophoresis of polypeptide Wpc; for 0.4. mu.g siRNA, 10. mu.g polypeptide can be completely loaded, and the loading degree is higher.
FIG. 2 is an Atomic Force Microscope (AFM) image of a polypeptide-siRNA co-assembled nanoparticle; after the polypeptide and the siRNA are incubated at normal temperature, the nano particles with uniform size are formed.
FIG. 3 is a Scanning Electron Microscope (SEM) image of polypeptide-siRNA co-assembled nanoparticles.
FIG. 4 is a graph of Dynamic Light Scattering (DLS) of polypeptide-siRNA co-assembled nanoparticles.
FIG. 5 is Zeta potential diagram of polypeptide-siRNA co-assembled nanoparticles.
FIG. 6 is a flow cytogram of polypeptide-siRNA co-assembled nanoparticles; a is the average fluorescence intensity value of HeLa cells after the nano particles are transfected; b is a transfection efficiency chart of polypeptide-siRNA nano particles to HeLa cells. The result shows that the polypeptide can carry siRNA into HeLa cells with high efficiency.
FIG. 7 is a real-time confocal microscopy imaging of polypeptide-siRNA co-assembled nanoparticles; the polypeptide can effectively disperse siRNA uniformly in the whole cell, and does not influence the shape of the cell.
FIG. 8 is a graph of the cytotoxicity (MTT) assay and hemolytic toxicity assay of the polypeptide-siRNA co-assembled nanoparticles on different cell lines; a is MTT toxicity determination diagram of polypeptide to QSG7701 cell; b is an MTT toxicity determination graph of the polypeptide on HeLa cells; MTT toxicity assay graph of C polypeptide to HEK-293T cell; d is a hemolytic toxicity assay graph of the polypeptide on fresh mouse blood cells; the results show that even at high concentrations, the polypeptide carrier has no significant cytotoxicity or hemolytic toxicity for different cell types.
FIG. 9 is a diagram of RT-PCR of polypeptide-siRNA co-assembled nanoparticles for inducing mRNA expression level change of related genes by siRNA transfection of different cell lines. A is an RT-PCR picture for reflecting mRNA expression level after transfecting Survivin genes in HeLa cells; b is a RT-PCR picture of the reaction mRNA expression level after transfection of K-ras gene in Miapaca-2 cells; c is an RT-PCR picture for reflecting the mRNA expression level after the LSD-1 gene is transfected in PA-1 cells; d is an RT-PCR picture for reflecting the mRNA expression level after Bcl-2 gene is transfected in HeLa cells; the results show that polypeptide-siRNA complexes are able to achieve down-regulation of the mRNA levels of the corresponding genes for different cell lines, especially for HeLa cells and Miapaca-2 cell lines. The polypeptide can be used as a simple, low-toxicity and high-efficiency siRNA carrier.
FIG. 10 is a bright field diagram of the antiproliferative inhibition effect of polypeptide-siRNA (survivin) nanoparticles on HeLa of cervical cancer cells; the growth of HeLa cells was significantly inhibited compared to the control group.
FIG. 11 shows that polypeptide-siRNA (survivin) nanoparticles block the cell cycle at the G2 stage after treating the cervical cancer cell HeLa.
FIG. 12 is a graph of tumor size versus time in nude mice after administration of polypeptide-siRNA (survivin) nanoparticles; the result shows that the polypeptide-siRNA (survivin) nano particle can effectively inhibit the growth of the nude mouse human source xenograft cervical cancer tumor.
FIG. 13 is a graph of weight versus time for nude mice during administration of polypeptide-siRNA (survivin) nanoparticles; body weight of the nude mice remained stable throughout the dosing period.
FIG. 14 is a HE stained section of each organ of a mouse collected after 10 times of administration of polypeptide-siRNA (survivin) nanoparticles; the results show that the polypeptide-siRNA (survivin) nano-particle has no teratogenic toxicity to each organ of the mouse.
FIG. 15 is an immunohistochemistry chart of tumor tissues collected from mice after 10 full doses of polypeptide-siRNA (survivin) nanoparticles; the results show that the survivin protein in the tumor tissues is down-regulated and Tunel and caspase-3 are up-regulated compared with the control after the polypeptide-siRNA (survivin) nano-particles are administrated.
FIG. 16 shows the working principle of the polypeptide-siRNA of the present invention.
Detailed Description
The invention is further described below with reference to the accompanying drawings.
Example 1 preparation and isolation and purification of the polypeptide:
the polypeptides were synthesized on resin using MBHA resin according to standard-Fmoc solid phase polypeptide synthesis techniques (taking polypeptide Wpc as an example):
the specific operation steps are as follows:
(1) polypeptide solid phase synthesis: weighing Rink amide MBHA resin in a peptide connecting tube, adding N, N-Dimethylformamide (DMF), and swelling for 40min with nitrogen. Adding a 50% (v/v) morpholine solution in N, N-Dimethylformamide (DMF), bubbling nitrogen for 40min, and removing the Fmoc protective group. The resin was washed with DMF and DCM alternately 3 times for 1min each time, and the prepared Fmoc-AA-OH (5eq, 0.4M, DMF) solution, 6-chlorobenzotriazole-1, 1, 3, 3-tetramethyluronium Hexafluorophosphate (HCTU) (4.9eq, 0.38M, DMF) solution, N, N-Diisopropylethylamine (DIPEA) (7.0eq) were mixed well and added to the resin under nitrogen bubbling for 2 h. The reaction was removed, the resin was washed alternately 3 times with DMF and DCM for 1min each, and deprotection was performed as described above, followed by the next amino acid.
(2) The following amino acids were the same as described above. After the last amino acid Arg, the resin was washed alternately 3 times with DMF and DCM for 1min each time, then with methanol, and the resin was drained and awaited for further processing.
(4) Intramolecular S salt ring closure: with shearing fluid (trifluoroacetic acid (TFA), Triisopropylsilane (TIPS) and H2O (v: v: v ═ 9.5:0.25:0.25)) the polypeptide was cut from the resin (1 ml of shear solution was added to 75mg of resin and sheared at room temperature for 3 hours), the shear solution was dried with dry nitrogen, and the resulting mixture was driedH2O and acetonitrile (v: v ═ 6:4) were dissolved, and a ring-closing reagent 1, 2-dibromomethylbenzene (5.0eq) and 1% formic acid were added to the solution, and the mixture was reacted for 12 hours with exclusion of light.
(5) Polypeptide purification: after the reaction solution after the reaction is filtered, water and acetonitrile can be used as mobile phases, and the high performance liquid chromatography is directly used for purification and separation.
Example 2 polypeptides under induction by siRNA (or other nucleic acid molecule), co-assembled polypeptide-siRNA nanoparticles are formed.
The loading degree of polypeptide to siRNA is judged by agarose gel blocking electrophoresis, the amount of siRNA is 0.4 mug, the aqueous solution of polypeptide and siRNA are incubated for 5min at normal temperature, so that stable polypeptide-siRNA co-assembled nano particles can be formed, and finally, 0.4 mug of siRNA can be completely loaded by 10 mug of polypeptide. The concentration of the polypeptide was determined from the absorption of Trp at 280nm (FIG. 1).
The co-assembled nanoparticles formed by the polypeptide and the siRNA were characterized by an Atomic Force Microscope (AFM), which demonstrated that the polypeptide-siRNA formed regular uniform nanoparticles (FIG. 2).
Characterization of the co-assembled nanoparticles formed by the polypeptide and siRNA was performed using Scanning Electron Microscopy (SEM), demonstrating that the polypeptide-siRNA forms regular uniform nanoparticles (fig. 3).
The co-assembled nanoparticles formed by the polypeptide-siRNA are further characterized by using a dynamic light scattering particle size analyzer, and the polypeptide-siRNA is proved to form regular and uniform nanoparticles (figure 4).
The polypeptide-siRNA formed co-assembly nanoparticles were further characterized by Zeta potential, and the results showed that the potential of siRNA was significantly changed after forming co-assembly nanoparticles with polypeptide, which was helpful for penetrating cell membrane (FIG. 5).
Example 3 polypeptide-siRNA co-assembled nanoparticles can be used as a simple, low-toxicity transfection reagent to effectively transfect siRNA into various cell lines and trigger RNAi effects.
In order to verify whether the polypeptide-siRNA co-assembled nanoparticles can be used as siRNA carriers to realize the effect of carrying siRNA, the most intuitive method is to utilize flow cytometry and laser confocal microscope imaging to determine and observe the fluorescence of transfected cells in real time. Using HeLa cells as a model, incubating polypeptide and fluorescence labeled (FAM) siRNA for 5min at normal temperature according to a proportion determined by agarose gel electrophoresis, adding the incubated polypeptide and fluorescence labeled (FAM) siRNA into the HeLa cells incubated by an OPTi culture medium in advance, continuously culturing for 4h in an incubator, washing, digesting the cells, and detecting the fluorescence intensity of the HeLa cells by a flow cytometer. It can be clearly seen that the fluorescence intensity value of the cells after the polypeptide-siRNA nanoparticle transfection is very high (obviously higher than that of the conventional commercial transfection reagents lipo-2000 and oligo), and the peak pattern of the cells is very normal without causing a great influence on the cell state (FIG. 6).
By observing the HeLa cells transfected for 4h in real time by using a confocal laser microscope, it can be clearly seen that the polypeptide-siRNA complexes labeled by fluorescence are uniformly distributed in the whole cells, and the transfection efficiency (fluorescence intensity) is obviously higher than that of the conventional commercial transfection reagents lipo-2000 and oligo (FIG. 7).
As a transfection reagent, it is necessary to have no toxic effect on normal cell lines, so we measured the cytotoxicity (MTT) of the polypeptide in 3 different cell lines (HeLa, HEK-293T and QSG7701), and the results showed that the polypeptide had no significant cytotoxicity even under 6-fold conditions (600. mu.g/ml) of the concentration used for transfection. Furthermore, we also utilized fresh mouse blood cells (10)8-109One), and different concentrations of the polypeptide were incubated at 37 deg.C (0.1% SDS as a positive control), and the hemolytic toxicity of the polypeptide was determined, which indicated that the polypeptide had little hemolytic toxicity compared to the positive control. The above results indicate that our polypeptide can be used as a simple, low-toxicity siRNA carrier (FIG. 8).
Subsequently, we tried the gene knockout efficiency of polypeptide-siRNA co-assembled nanoparticles in different cell lines (HeLa, Miapaca-2, PA-1 and A2780). After 2.0 mu g of siRNA and 50 mu g of polypeptide are incubated for 5min at normal temperature to form stable and uniform co-assembled nanoparticles, the co-assembled nanoparticles are added into HeLa (or other cell lines) which is incubated by an OPTi culture medium in advance, the co-assembled nanoparticles are placed in an incubator to be cultured for 48h continuously, RNA is extracted, reverse transcription is carried out, and the change of the mRNA level of the related gene is analyzed by RT-PCR. We found that polypeptide-siRNA complexes are able to achieve down-regulation of mRNA levels of the corresponding genes, especially against HeLa cells and Miapaca-2 cell lines. The above results indicate that our polypeptide can be used as a novel siRNA carrier with simplicity, low toxicity and effectiveness for transfection of various cell lines (FIG. 9).
Table 1 shows the siRNA sequences used during transfection in different cell lines, and Table 2 shows the primer sequences used after transfection when RT-PCR was used to analyze the changes in mRNA expression levels of the genes of interest.
Table one: siRNA sequences used
Table two: primer sequences used
Example 4 polypeptide-siRNA (survivin) nanoparticles anti-cervical cancer cell proliferation and anti-human xenograft cervical cancer model proliferation effect.
In order to evaluate whether the polypeptide vector can be applied to the field of cancer treatment based on RNAi effect, a cervical cancer cell HeLa is taken as a model, a cell proliferation inhibition experiment is utilized, and the proliferation condition of the HeLa cell is observed 48h after polypeptide-siRNA (survivin) nanoparticles are transfected. The result shows that the polypeptide-siRNA (survivin) nanoparticle can inhibit the proliferation of the cervical cancer cell HeLa. (fig. 10) then we further verified that polypeptide-sirna (survivin) nanoparticles can arrest the cell cycle of HeLa cells at the G2 phase using a cell cycle analysis kit, thereby inhibiting proliferation and spreading of HeLa cells (fig. 11).
In addition, we further verified the anti-tumor growth effect of the polypeptide-siRNA (survivin) nanoparticles in a human-derived xenograft cervical cancer model of nude mice. After the nude mice are inoculated with cervical cancer tumors, the nude mice are continuously cultured for 14 days and the like to grow, then, the administration of polypeptide-siRNA (Survivin) nanoparticles by intratumoral injection is started, the administration is carried out once every two days, 0.34OD siRNA (Survivin) is carried out every time, the weight and the tumor size of the mice are measured before the administration every time, the mice are killed after the 21 st day, the tumors and organs of the mice are taken out, the HE staining is utilized to analyze whether the organ tissues of the mice are normal, and the immunohistochemistry is utilized to analyze the expression conditions of corresponding Survivin the tumor tissues, caspase-3 and Tunel. The results show that the polypeptide-siRNA (survivin) nano-particle can effectively inhibit the growth of the human-derived cervical cancer tumor (figure 12), the body weight of the mouse does not change obviously in the whole administration process (figure 13), the HE staining result shows that various tissue organs of the mouse do not distort and are normal together compared with the control, (figure 14) the immunohistochemical analysis shows that survivin protein in the tumor tissue is down-regulated and Tunel and caspase-3 is up-regulated compared with the control (figure 15).
As shown in fig. 16, after siRNA is added, electrostatic adsorption induces polypeptide to self-assemble into nanospheres, which carry siRNA to penetrate membrane and release at target position, thus exerting the effect of siRNA to knock out target gene and inhibiting the growth of tumor cells.
The results show that the polypeptide-siRNA (survivin) nano-particle can effectively inhibit the growth of the human source cervical cancer tumor implanted in different ways, does not poison the normal tissues and organs of the mouse, and also verifies that the polypeptide carrier can be applied to the cancer treatment field based on the RNAi effect.
Sequence listing
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Claims (3)
1. A polypeptide, characterized by: the structural formula is shown as follows,
2. a polypeptide-siRNA inducing co-assembly, comprising: consists of the polypeptide of claim 1 and siRNA.
3. Use of the polypeptide-siRNA inducible co-assembly of claim 2 in the preparation of a medicament for treating cervical cancer.
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