CN115850380A - Targeting cell-penetrating peptide for triple negative breast cancer and application thereof - Google Patents
Targeting cell-penetrating peptide for triple negative breast cancer and application thereof Download PDFInfo
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- CN115850380A CN115850380A CN202211514184.4A CN202211514184A CN115850380A CN 115850380 A CN115850380 A CN 115850380A CN 202211514184 A CN202211514184 A CN 202211514184A CN 115850380 A CN115850380 A CN 115850380A
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Abstract
The invention discloses a membrane-penetrating peptide combined with triple negative breast cancer (MDA-MB-231) in a targeted mode and application thereof. The triple negative breast cancer targeting cell-penetrating peptide is a polypeptide, and the amino acid sequences are respectively as follows: FKQDAWEAVDIR, APTTWFNSDSIT, FNSDSRSTHQED, IPLENQHKIYST, GSGFNESVARP, DGSMLNRMRGFS; also comprises a biological active substance of the triple negative breast cancer targeting cell-penetrating peptide formed on the surface of the microorganism and the polymer mixture. The invention can efficiently target triple-negative breast cancer tissues in a mouse body, pass through cell membranes of triple-negative breast cancer cells, and enter the triple-negative breast cancer cells, so that a cancer treatment medicament can be guided to effectively reach a focus part, and the targeted treatment of triple-negative breast cancer is realized.
Description
Technical Field
The invention belongs to a polypeptide in the field of biomedicine and application thereof, and particularly relates to a polypeptide capable of targeting a tumor tissue of triple negative breast cancer (MDA-MB-231) and entering a tumor cell and application thereof.
Background
Triple negative breast cancer refers to breast cancer in which estrogen receptor, progesterone receptor, and protooncogene expression are all negative. Due to the absence of multiple receptors, targeted delivery of drugs during treatment of triple negative breast cancer is difficult to achieve. Furthermore, the targets of most tumor targeting molecules are located on the surface of tumor tissue or tumor cells, making it difficult for targeted delivery of drug molecules into the interior of tumor cells.
Therefore, constructing a biomolecule which can target the tumor tissue of triple negative breast cancer and can enter the interior of tumor cells is a scientific problem to be solved urgently in the biomedical field at present.
Disclosure of Invention
In order to solve the problems in the background art, the invention aims to provide a targeting cell-penetrating polypeptide for triple negative breast cancer. The polypeptide can be targeted and gathered at a tumor tissue part of triple negative breast cancer in an organism, and can penetrate through a cell membrane of a tumor cell to enter the interior of the tumor cell. Provides possibility for realizing efficient and accurate treatment for the triple negative breast cancer.
In order to achieve the purpose, the invention adopts the following technical scheme.
1. Targeting cell-penetrating peptide for triple negative breast cancer
The targeting cell-penetrating peptide is polypeptide, and the amino acid sequence is selected from one of SEQ ID No. 1-SEQ ID No. 6.
The amino acid sequences of the targeting cell-penetrating peptide are respectively as follows:
FKQDAWEAVDIR、APTTWFNSDSIT、FNSDSRSTHQED、IPLENQHKIYST、GSGFNESSVARP、DGSMLNRMRGFS
table 1 amino acid sequence listing
| (Code) | Amino acid sequence | (Code) | Amino acid sequence |
| No.1 | FKQDAWEAVDIR | No.4 | IPLENQHKIYST |
| No.2 | APTTWFNSDSIT | No.5 | GSGFNESSVARP |
| No.3 | FNSDSRSTHQED | No.6 | DGSMLNRMRGFS |
2. A biologically active substance:
comprises a targeting cell-penetrating peptide aiming at triple negative breast cancer;
comprises microorganisms and polymer mixtures, and the targeting cell-penetrating peptide is formed on the surface of the mixture.
The polymer mixture includes covalently linked compounds or nanoparticles, and the microorganism includes engineered phage.
If the surface of the nano-particle is modified with targeting cell-penetrating peptide aiming at triple negative breast cancer;
if the compound is a compound connected by a covalent bond, the surface of the compound is modified with a targeting cell-penetrating peptide aiming at triple negative breast cancer;
if the target is the engineered phage, the targeting cell-penetrating peptide aiming at the triple negative breast cancer is displayed on the surface of the phage through genetic engineering.
The bioactive substance formed in the way has the same biomedical function as the targeting cell-penetrating peptide aiming at the triple negative breast cancer, namely, the bioactive substance is enriched at the tumor tissue part of the triple negative breast cancer in a targeted manner and enters the cell interior of the triple negative breast cancer.
The invention aims at the application of targeting cell-penetrating peptide or bioactive substance of triple negative breast cancer in the preparation of medicines.
3. A polynucleotide sequence capable of encoding the targeting cell-penetrating peptide against triple negative breast cancer.
4. A polynucleotide sequence capable of encoding said biologically active substance.
5. A polypeptide drug for treating diseases comprises the targeting cell-penetrating peptide aiming at triple negative breast cancer.
The medicine targets tumor tissues of triple negative breast cancer, enters tumor cells, and can realize diagnosis and treatment of triple negative breast cancer.
Targeting cell-penetrating peptide, bioactive substance or polypeptide medicine for triple negative breast cancer is enriched in tumor tissue of triple negative breast cancer in a targeted manner and enters into tumor cells. In particular to the application in efficient and accurate cancer targeting diagnosis and treatment.
The targeting cell-penetrating peptide for triple negative breast cancer, which is obtained by the invention, can be directionally enriched in tumor tissue parts of triple negative breast cancer in organisms and enter tumor cells. Is beneficial to realizing the targeted diagnosis and treatment of the triple negative breast cancer, and has wide application prospect in the field of biomedicine.
The inventor utilizes a phage 12 peptide library and carries out 5 rounds of screening aiming at triple negative breast cancer (MDA-MB-231) by a method of alternating in-vivo screening and in-vitro screening of tumor cells of a mouse tumor model. The targeting cell-penetrating polypeptide for triple negative breast cancer is finally obtained through monoclonal sequencing, exploration and verification.
Compared with other tumor targeting molecules, the targeting cell-penetrating polypeptide has the advantages that:
(1) The polypeptide sequence is short and small, and the synthesis production cost is low;
(2) The polypeptide fragment has stable structure, and the cost and difficulty of transportation and storage are reduced;
(3) Administration can be by a variety of means;
(4) Specific for triple negative breast cancer (MDA-MB-231);
(5) Can penetrate through tumor cell membranes to enter the interior of tumor cells while being targeted and enriched to tumor tissue sites.
The invention has the beneficial effects that:
the polypeptide can be targeted and enriched at the tumor tissue part of the triple negative breast cancer in an organism, and simultaneously passes through the cell membrane of the tumor cell to enter the cell.
The polypeptide provided by the invention provides a new idea for clinically aiming at the targeted diagnosis and treatment of triple-negative breast cancer, aiming at the targeting ability and the cell membrane penetrating ability of triple-negative breast cancer.
Drawings
FIG. 1 is a graph showing the output/input ratio of each round in the combined screening process of triple negative breast cancer tumor tissues and tumor cells using the phage 12 peptide library in example 1.
FIG. 2 is a graph showing 10 polypeptide sequences with higher frequency and their corresponding frequencies after the sequencing results were analyzed by DNAStar software in example 1.
FIG. 3 is a photograph of the fluorescence images of the mice in vivo obtained by injecting the fluorescence-labeled polypeptide molecules into tumor-bearing mice via tail vein in example 2.
FIG. 4 is an in vitro image of the major organs and tumor tissues (i: spleen; ii: kidney; iii: heart; iv: lung; v: liver; vi: tumor) of the mice injected with the fluorescently labeled polypeptide molecules into the tumor-bearing mice via tail vein in example 2.
Detailed Description
The present invention is further illustrated by the following examples and the accompanying drawings, wherein the following examples are only preferred embodiments of the present invention, and are not intended to limit the present invention, and various changes and modifications may be made therein by those skilled in the art without departing from the spirit and the principle of the present invention, and any modifications, equivalents, improvements, etc. made within the scope of the present invention are intended to be included in the present invention.
The examples of the invention are as follows:
example 1
5 rounds of phage in vivo and in vitro alternative screening were performed on the mouse tumor model and tumor cells of triple negative breast cancer using phage 12 peptide library constructed by phage display technology. Because the abundance of the phage 12 peptide library is large, 10 mu L of the phage 12 peptide library is selected for screening in the screening process, and the targeted cell-penetrating polypeptide for triple negative breast cancer is successfully obtained through the following operation steps in the experiment.
1.1 construction of nude mouse triple negative breast cancer model
1x10 5 The individual MDA-MB-231 cells were suspended in PBS solution and injected into the underarm area of nude mice by syringe. And when the diameter of the tumor tissue reaches 10mm, completing the construction of the model.
1.2, recovering and culturing the host bacterium E.coil.ER2738
LB solid plates were prepared and placed in a 37 ℃ incubator for 1 hour pre-heating. Dipping the melted E.coil.ER2738 bacterial solution by using a bacteria picking rod, coating the bacterial solution on the surface of an LB flat plate in a Z shape, and culturing the bacterial solution in an incubator at 37 ℃ for 12 hours. Add 5mL liquid LB medium containing tetracycline to the shake tube, pick up the monoclonal colonies in the plate using a sterile tip, and place the tip in the medium. The tubes were placed in a 37 ℃ shaker and incubated with shaking at 220rpm until the bacteria were in mid-log phase.
1.3, bonding
Sterile EP tubes were taken, 100 μ L PBS solution and 10 μ L phage 12 peptide library were added, and mixed by vortexing. The phage was injected into the nude mice via the tail vein, allowing the phage to reach the tumor site through blood circulation.
1.4, washing
After 1 hour, the nude mice were anesthetized. The nude mice were heart perfused with sterile physiological saline to wash out the phage that were not or weakly bound to the tumor site.
1.5, elution
Tumor tissue was dissected and added to the tissue grinder. 1mL of non-deformed tissue lysate was added to fully lyse the tumor tissue. Sucking the tissue homogenate into a sterile EP tube, centrifuging at 10000rpm for 5min, collecting supernatant, and performing phage titer determination and phage amplification and purification.
1.6 determination of phage titer
LB/IPTG/Xgal plates were placed in a 37 ℃ incubator and pre-warmed for 1 hour. The supernatant of the homogenate was diluted in a gradient, and 10. Mu.L of the diluted phage solution was added to 200. Mu.L of E.coil.ER2738 bacterial solution in the logarithmic phase. Standing for 15min. The infected phage were applied to the surface of LB/IPTG/Xgal plates and spread evenly using a sterile spreading bar. The coated plate was placed upside down in an incubator at 37 ℃ and incubated overnight. The next day the plates were examined for phage blue spots and counted.
1.7 preparation of bacterial liquid
20mL of LB medium was placed in a 250mL sterile conical flask, and 1mL of the host bacterium E.coil.ER2738, which was in the middle of logarithmic growth, was added. The Erlenmeyer flask was placed in a shaker at 37 ℃ and incubated with shaking at 220rpm until the bacteria were in the mid-log phase of growth.
1.8 amplification and purification of phages
The homogenate supernatant was added to 20mL of e.coil.er2738 bacterial solution in logarithmic growth phase. After standing at room temperature for 20min, the bacterial solution was placed in a shaker at 37 ℃ and cultured with shaking at 220rpm for 4.5 hours. Then, the bacterial solution was poured into a 50mL centrifuge tube and centrifuged at 12000 Xg for 20min. The supernatant was poured into a centrifuge tube containing 4mL of PEG/NaCl solution, placed in a 4 ℃ refrigerator and allowed to settle overnight. The next day, the tube was centrifuged at 12000 Xg for 20min and the supernatant discarded. Add 1mL of PBS solution to the centrifuge tube, redissolve the pellet, and transfer the resuspension to a 1.5mL EP tube, shake the tube for 1 hour at 37 ℃. The EP tube was then centrifuged at 12000 Xg for 10min and the supernatant was transferred to an EP tube containing 200mL of PEG/NaCl solution. The EP tube was placed at 4 ℃ and left for 1 hour to settle the phages. Then, the EP tube was centrifuged at 12000 Xg for 20min, the supernatant was discarded, and 100. Mu.L of PBS was added to resuspend the pellet.
1.9 second round tumor cell screening
MDA-MB-231 cells were seeded in 6-well plates, and the amplified phage solution was diluted with serum-free medium and added to the cell-well plates. And the cell well plate was placed in a cell incubator for 1 hour. Washing the cells for multiple times by using PBST buffer solution, and removing the unbound phage on the cell surface; removing the phage bound to the cell surface using an eluent; and finally, collecting the cells by using a cell scraper, adding a lysate to crack the cells, and collecting the phage inside the cells. The phage titer test and the amplification of the phage solution were then performed according to the procedures of 1.6 and 1.8.
1.10 third, fourth and fifth rounds of selection
And carrying out the screening of the tumor tissues in the nude mice of the third round, the screening of the tumor cells in vitro of the fourth round and the screening of the tumor tissues in the nude mice of the fifth round according to the same steps. The phage solution after each round of amplification and purification is used as the secondary phage peptide library for the next round of screening. The input amount of the phage for each round of screening is kept consistent, the titer of the elution product for each round of screening is determined as the output amount of the phage for each round, and the output/input ratio of the phage for each round is calculated.
The results of the output/input ratios are shown in fig. 1, and fig. 1 shows the output/input ratios of the respective rounds of in vivo tumor screening and in vitro tumor cell screening for triple negative breast cancer using the phage 12 peptide library. Wherein the output/input ratio of the first round of screening is 1.65 multiplied by 10 -4 (ii) a The output/input ratio of the second round of screening was 1.40X 10 -5 (ii) a The third output/input ratio is 1.25X 10 -3 (ii) a The fourth wheel output/input ratio is 3.2 x10 -4 (ii) a The output/input ratio of the fifth wheel is 5.63 multiplied by 10 -3 . The root experiment result shows that the output/input ratio is alternately increased round by round, so that the targeting cell-penetrating peptide for the triple negative breast cancer is effectively enriched.
1.11 phage-positive monoclonal
The eluted products of the third, fourth and fifth rounds were subjected to titer determination. Selecting a plate with no more than 100 plaques, randomly picking 100 phage blue plaques, adding the phage blue plaques into 100 shake culture tubes containing 5mL LB culture medium, and carrying out shake culture at 37 ℃ and 220rpm for 24 hours. mu.L of each culture was added to an EP tube containing 300. Mu.L of glycerol (50%) solution, mixed well and stored in a freezer at-80 ℃. And sequencing the residual bacteria liquid.
Analysis of DNA sequences of phage monoclonals
According to the sequencing result, finding the inserted exogenous DNA sequence and translating the corresponding amino acid sequence according to the principle of the triplet codon.
The results are shown in FIG. 2. FIG. 2 shows the polypeptide sequences with higher frequency of positive monoclonal phage and their repeats. The phage displaying the FKQDAWEAVDIR polypeptide sequence appeared 40 times, and the phage appeared most frequently.
The following examples are combined to verify the targeting effect of the polypeptide molecules in mice.
Example 2
2.1 construction of nude mouse triple negative Breast cancer model
Will be 1x10 5 The individual MDA-MB-231 cells were suspended in PBS solution and injected into the underarm area of nude mice by syringe. And when the diameter of the tumor tissue reaches 10mm, completing the construction of the model.
2.2 chemical Synthesis of Polypeptides
Chemically synthesizing six polypeptide molecules No. 1-No. 6, and connecting biotin to carboxyl terminal of the polypeptide. In addition, a polypeptide molecule with the sequence of KGYGVGLRFPAW is selected as a control group (CP), and biotin is connected to the carboxyl terminal of the polypeptide molecule.
2.3 labeling of fluorescein
The polypeptide molecule to which biotin is linked and the Cy 5-labeled streptavidin were dissolved in physiological saline and stirred at room temperature for 1 hour. Thereafter, excess Cy 5-streptavidin was removed by dialysis to obtain polypeptide molecules labeled with Cy5 fluorescein.
2.4 in vivo fluorescence imaging of mice
The polypeptide molecule marked with Cy5 fluorescein is injected into tumor-bearing mice through tail vein. And acquiring a live fluorescence image of the mouse and an in-vitro fluorescence image of a main tissue organ by a live fluorescence imager after 24 hours. According to the result of the fluorescence image, six different polypeptide molecules No. 1-No. 6 can be in the tumor-bearing mouse body, and effective targeting and enrichment aiming at triple negative breast cancer tissues can be realized (figure 3).
Therefore, the invention can efficiently target triple-negative breast cancer tissues in a mouse body, pass through cell membranes of triple-negative breast cancer cells, and enter the triple-negative breast cancer cells, so that a cancer treatment drug can be guided to effectively reach a focus part, and the targeted treatment of the triple-negative breast cancer is realized.
The gene and protein sequences related to the invention are as follows:
SEQ ID No.1；
name: amino acid sequence of targeting cell-penetrating peptide for triple negative breast cancer
Type of DNA: other DNA
The source is as follows: artificial Sequence (Artificial Sequence)
FKQDAWEAVDIR
SEQ ID No.2；
Name: amino acid sequence of targeting cell-penetrating peptide for triple negative breast cancer
Type of DNA: other DNA
The source is as follows: artificial Sequence (Artificial Sequence)
APTTWFNSDSIT
SEQ ID No.3；
The name is as follows: amino acid sequence DNA types of targeting cell-penetrating peptides for triple negative breast cancer: other DNA
The source is as follows: artificial Sequence (Artificial Sequence)
FNSDSRSTHQED
SEQ ID No.4；
Name: amino acid sequence DNA types of targeting cell-penetrating peptides for triple negative breast cancer: other DNA
The source is as follows: artificial Sequence (Artificial Sequence)
IPLENQHKIYST
SEQ ID No.5；
Name: amino acid sequence DNA types of targeting cell-penetrating peptides for triple negative breast cancer: other DNA
The source is as follows: artificial Sequence (Artificial Sequence)
GSGFNESSVARP
SEQ ID No.6；
Name: amino acid sequence DNA types of targeting cell-penetrating peptides for triple negative breast cancer: other DNA
The source is as follows: artificial Sequence (Artificial Sequence)
DGSMLNRMRGFS。
Claims (9)
1. A targeting cell-penetrating peptide for triple negative breast cancer MDA-MB-231, which is characterized in that: the targeting cell-penetrating peptide is polypeptide, and the amino acid sequence is selected from one of SEQ ID No. 1-SEQ ID No. 6.
2. A biologically active substance, characterized by:
the targeting cell-penetrating peptide, microorganism and polymer mixture containing the targeting cell-penetrating peptide, microorganism and polymer mixture according to claim 1, wherein the targeting cell-penetrating peptide for the triple negative breast cancer is formed on the surface.
3. A biologically active material according to claim 2, wherein:
the polymer mixture comprises covalently linked compounds or nanoparticles.
4. A biologically active material according to claim 2, wherein:
the microorganism comprises an engineered bacteriophage.
5. A polynucleotide sequence, wherein: can encode the triple negative breast cancer targeting cell-penetrating peptide of claim 1.
6. A polynucleotide sequence, wherein: is capable of encoding a biologically active substance according to claim 2.
7. A polypeptide drug, which comprises the triple negative breast cancer targeting cell-penetrating peptide of claim 1.
8. The use of the triple negative breast cancer targeting cell-penetrating peptide of claim 1, the bioactive substance of claim 2 or the polypeptide drug of claim 7, wherein: application in preparing triple negative breast cancer medicine.
9. Use according to claim 8, characterized in that:
the medicine is a medicine for targeted therapy of triple negative breast cancer.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116675739A (en) * | 2023-05-29 | 2023-09-01 | 浙江大学 | Colon cancer cell targeting membrane penetrating peptide and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116675739A (en) * | 2023-05-29 | 2023-09-01 | 浙江大学 | Colon cancer cell targeting membrane penetrating peptide and application thereof |
| CN116675739B (en) * | 2023-05-29 | 2024-04-09 | 浙江大学 | Colon cancer cell targeting membrane penetrating peptide and application thereof |
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