CN114699420A - Composition for treating prostate cancer and preparation method and application thereof - Google Patents
Composition for treating prostate cancer and preparation method and application thereof Download PDFInfo
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- CN114699420A CN114699420A CN202210226933.7A CN202210226933A CN114699420A CN 114699420 A CN114699420 A CN 114699420A CN 202210226933 A CN202210226933 A CN 202210226933A CN 114699420 A CN114699420 A CN 114699420A
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Abstract
The invention discloses a composition for treating prostate cancer and a preparation method and application thereof. The composition for treating prostate cancer comprises a carrier and mRNA of KLF 5; the mass ratio of the vector to the mRNA of the KLF5 is 1-128: 1; the carrier is phospholipid, and the phospholipid comprises the following components in a molar ratio of 1: 0.5-2: 0.5-2: (2, 3-dioleoyl-propyl) -trimethylammonium chloride, 1, 2-dioleoyl-sn-glycero-3-phosphate, cholesterol, and distearoylphosphatidylethanolamine-polyethylene glycol 2000 at 0.1-0.5. The composition for treating the prostate cancer can inhibit the development, the deterioration and the metastasis of the prostate cancer by inhibiting the growth of tumor cells, inhibiting the generation of new vessels of tumor focuses and promoting the infiltration of immune cells at multiple angles by restoring the expression of key transcription factors of the prostate cancer. The material used in the composition for treating prostatic cancer has high biological safety and low toxic and side effects compared with chemoradiotherapy medicines.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a composition for treating prostate cancer, and a preparation method and application thereof.
Background
Prostate cancer refers to the pathological types of prostate cancer that occur in the epithelial malignancies of the prostate including adenocarcinoma (acinar adenocarcinoma), ductal adenocarcinoma, urothelial carcinoma, squamous cell carcinoma, adenosquamous carcinoma. Wherein the prostatic adenocarcinoma accounts for more than 95%. The occurrence of prostate cancer is related to genetic factors, and if the relative risk of a person without prostate cancer in the family is 1, the absolute risk is 8; the relative risk of the hereditary prostate cancer family member suffering from the prostate cancer is 5, and the absolute risk is 35-45. In addition, the onset of prostate cancer is related to sexual activity and eating habits. Greater sexual activity is at increased risk of prostate cancer. High fat diet also has a certain relationship with morbidity.
Existing prostate cancer treatments include surgical resection and chemotherapy. Among them, the operative treatment has a large trauma and a long recovery time after the operation, and even partial nerves, blood vessels and intestinal organs are damaged. In addition, serious complications such as urinary incontinence, bladder neck contracture, erectile dysfunction, etc. may occur after the operation, which has a great influence on the quality of life of the patient. Among them, in the general reaction of chemotherapy, patients may have symptoms such as nausea, vomiting, anorexia, diarrhea, and ulcer of oral mucosa. In addition, some chemotherapeutic drugs have toxic effects on the cardiovascular system and heart failure can occur severely. Some chemotherapy drugs can cause acute chemical pneumonia and chronic pulmonary fibrosis, and even respiratory failure. In addition, the single drug of radiotherapy and chemotherapy can only inhibit the growth of tumor from one side.
Therefore, there is an urgent need to find a safer and more effective composition for treating prostate cancer to replace surgical resection and chemotherapy.
Disclosure of Invention
The first technical problem to be solved by the invention is as follows:
a composition for treating prostate cancer is provided. The composition for treating prostate cancer can inhibit tumor cell growth by restoring the expression of the key transcription factor of prostate cancer.
The second technical problem to be solved by the invention is:
a preparation method of the composition for treating the prostatic cancer is provided.
The invention also provides an intravenous injection which comprises the composition for treating prostatic cancer and pharmaceutically acceptable auxiliary materials.
The invention also provides application of the composition for treating the prostate cancer in a prostate cancer medicament.
The invention also provides application of the composition for treating the prostatic cancer in an immunomodulator.
In order to solve the first technical problem, the invention adopts the technical scheme that:
a composition for treating prostate cancer comprising a carrier and mRNA of KLF 5; the mass ratio of the vector to the mRNA of the KLF5 is 1-128: 1;
the carrier comprises the following components in a molar ratio of 1: 0.5-2: 0.5-2: (2, 3-dioleoyl-propyl) -trimethylammonium chloride, 1, 2-dioleoyl-sn-glycero-3-phosphate, cholesterol, and distearoylphosphatidylethanolamine-polyethylene glycol 2000 at 0.1-0.5. Preferably, the carrier comprises a molar ratio of 1: 1: 1: 0.1 of (2, 3-dioleoyl-propyl) -trimethylammonium chloride, 1, 2-dioleoyl-sn-glycerol-3-phosphoric acid, cholesterol and distearoylphosphatidylethanolamine-polyethylene glycol 2000.
KLF5 is a zinc finger transcription factor, and KLF5 is a basic transcription factor involved in the regulation of various vital activities. On one hand, the KLF5 gene is a cancer suppressor gene which is high-frequency deleted in human prostate cancer, on the other hand, KLF5 protein plays a dual role in canceration, and is subjected to acetylation modification induced by TGF-beta signals, the acetylated KLF5 can reverse the proliferation promoting function of the protein in normal epithelial cells and inhibit nude mouse tumorigenesis of prostate cancer cell lines PC-3 and DU145, and deacetylation blocks the inhibiting function.
Messenger RNA (mrna) is a large class of RNA molecules that transfer genetic information from DNA to ribosomes where it serves as a template for protein synthesis and determines the amino acid sequence of the peptide chain of the protein product of gene expression. RNA polymerase transcribes the primary transcript mRNA (referred to as pre-mRNA) into processed mature mRNA, which is translated into protein. As in DNA, mRNA genetic information is also stored in nucleotide sequences that are arranged into codons consisting of every three base pairs. Each codon encodes a specific amino acid, with the exception of a stop codon, because it terminates protein synthesis. rRNA is a central component of ribosomal protein manufacturing machinery.
High levels of KLF5 mRNA are present in humans and mice in the digestive tract including small intestine, large intestine, stomach, pancreas, as well as placenta, testis and prostate, skeletal muscle and lung. KLF5 mRNA is also found in the bladder and uterus of humans and rabbits. Although KLF5 is expressed primarily in epithelial cells, it is also expressed in cardiovascular smooth muscle and cornea and in lymphoid cells, neuronal cells. Although most tissues express 3.3kb of transcription, testis tissue is approximately 1.5kb in expression. Furthermore, the expression of KLF5 was higher in cell proliferation than in differentiated cells.
If the molar ratio of the carrier is outside this range, the solution of the present invention cannot be achieved.
If the choice of carrier does not include (2, 3-dioleoyl-propyl) -trimethylammonium chloride, 1, 2-dioleoyl-sn-glycero-3-phosphate, cholesterol, and distearoylphosphatidylethanolamine-polyethylene glycol 2000, the effect of the composition of the present invention for treating prostate cancer will be greatly compromised.
According to one embodiment of the invention, the support is a nanocarrier.
The nano carrier is natural or synthetic polymer and inorganic nano material, and is a nano dispersion system formed in different forms. Including nanoparticles, nanocapsules, nanomicelles, nanoemulsions, and the like. The nano carrier is a submicron drug carrier conveying system belonging to the nanometer microscopic category. The drug is encapsulated in submicron particles, so that the release speed can be adjusted, the permeability of a biological membrane is increased, the distribution in a body is changed, the bioavailability is improved, and the like.
Wherein, Solid Lipid Nanoparticles (SLN) are a new generation submicron drug delivery system developed in the early 90 s of the 20 th century, and refer to a Solid colloidal particle drug delivery system which is prepared by wrapping or embedding a drug in a lipid core with a particle size of 10-1000nm and taking Solid natural or synthetic lipids such as lecithin, triacylglycerol and the like as carriers, wherein the Solid colloidal particle drug delivery system takes Solid natural or synthetic lipids with low toxicity, good biocompatibility and biodegradability as carriers.
According to one embodiment of the invention, the carrier is dissolved in an organic solvent comprising at least one of ethanol, diethyl ether, tetrahydrofuran, methanol and acetone.
According to one embodiment of the invention, the mRNA of KLF5 is dissolved in an inorganic solvent.
According to one embodiment of the invention, the volume ratio of organic solvent in the carrier to inorganic solvent in the mRNA of KLF5 is 1: 1-10, preferably 1: 3.
according to one embodiment of the invention, the mass ratio of said vector to said mRNA of KLF5 is between 32 and 64: 1.
according to one embodiment of the invention, in the composition for treating prostate cancer, the mRNA of KLF5 is dissolved in an inorganic solvent, and the mRNA concentration of KLF5 is 0.5-10 μ g/mL.
According to one embodiment of the invention, in the composition for treating prostate cancer, the mRNA of KLF5 is dissolved in an inorganic solvent, and the mRNA concentration of KLF5 is preferably 2 μ g/mL.
In order to solve the second technical problem, the technical scheme adopted by the invention is as follows:
a method of preparing the composition for treating prostate cancer, comprising the steps of:
mixing the carrier and mRNA of KLF5 to obtain the composition for treating prostate cancer.
In another aspect of the invention, the invention also relates to the application of the composition for treating prostate cancer in the preparation of medicaments for treating prostate cancer.
In a further aspect of the invention, there is also provided a use of a composition for the treatment of prostate cancer in an immunomodulatory agent.
One of the technical schemes at least has one of the following advantages or beneficial effects:
1. the composition for treating the prostate cancer can inhibit the development, the deterioration and the metastasis of the prostate cancer by inhibiting the growth of tumor cells, inhibiting the generation of new vessels of tumor focuses and promoting the infiltration of immune cells at multiple angles by restoring the expression of key transcription factors of the prostate cancer.
2. The material used in the composition for treating prostate cancer has high biocompatibility and low side effect, and the carrier and the KLF5 mRNA are biodegradable materials, so that the composition has low toxic and side effects compared with chemoradiotherapy medicines.
3. The carrier in the composition for treating the prostatic cancer can be used for treating the prostatic cancer by intravenous injection in vivo and inhibiting the development, the deterioration and the metastasis of the prostatic cancer.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.
FIG. 1 is a graph of transfection efficiency and mean fluorescence intensity measurements for different ratios of vector and EGFP mRNA delivery to LNCaP cell lines.
FIG. 2 shows the results of Western Blotting analysis of protein expression after delivery of the compositions for treating prostate cancer prepared in examples 1 to 3 and examples 8 to 12 to LNCaP cell line.
Fig. 3 is a graph showing the results of experimental tests on mice injected with the composition for treating prostate cancer prepared in example 13.
FIG. 4 shows the results of Western Blotting analysis of protein expression after delivery of the compositions prepared in examples 4-7 for treating prostate cancer to LNCaP cell line.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the scope of the present invention.
In the examples, the mRNA of KLF5 was purchased from yunzhi biotechnology (guangzhou) limited.
The mRNA sequence of KLF5 is detailed in SEQ ID NO. 1.
Example 1
A method for preparing the composition for treating prostate cancer comprises the following steps:
the carrier was dissolved in ethanol and mRNA of KLF5 was dissolved in water at a volume ratio of ethanol to water of 1: 3;
the mass ratio of the components is 32: 1 (excluding the mass of ethanol and water) the carrier was mixed with mRNA of KLF5, and ultrafiltration was performed using an ultrafiltration tube to remove ethanol, to obtain the above composition for the treatment of prostate cancer.
The carrier is prepared by the following steps of 1: 1: 1: 0.1 of (2, 3-dioleoyl-propyl) -trimethylammonium chloride (DOTAP), 1, 2-dioleoyl-sn-glycerol-3-phosphate (DOPE), cholesterol and distearoylphosphatidylethanolamine-polyethylene glycol 2000(DSPE-PEG 2000).
Example 2
A method for preparing the composition for treating prostate cancer comprises the following steps:
the carrier was dissolved in ethanol and mRNA of KLF5 was dissolved in water at a volume ratio of ethanol to water of 1: 3;
the mass ratio of 64: 1 (excluding the mass of ethanol and water) mixing the carrier with mRNA of KLF5, and ultrafiltering with an ultrafiltration tube to remove ethanol to obtain the above composition for treating prostate cancer.
The carrier is prepared by the following steps of 1: 1: 1: 0.1 of (2, 3-dioleoyl-propyl) -trimethylammonium chloride (DOTAP), 1, 2-dioleoyl-sn-glycerol-3-phosphate (DOPE), cholesterol and distearoylphosphatidylethanolamine-polyethylene glycol 2000(DSPE-PEG 2000).
Example 3
A method for preparing the composition for treating prostate cancer comprises the following steps:
the carrier was dissolved in ethanol and mRNA of KLF5 was dissolved in water at a volume ratio of ethanol to water of 1: 3;
the mass ratio of the components is 128: 1 (excluding the mass of ethanol and water) mixing the carrier with mRNA of KLF5, and ultrafiltering with an ultrafiltration tube to remove ethanol to obtain the above composition for treating prostate cancer.
The carrier is prepared by the following steps of 1: 1: 1: 0.1 of (2, 3-dioleoyl-propyl) -trimethylammonium chloride (DOTAP), 1, 2-dioleoyl-sn-glycerol-3-phosphate (DOPE), cholesterol and distearoylphosphatidylethanolamine-polyethylene glycol 2000(DSPE-PEG 2000).
Example 4
A method for preparing the composition for treating prostate cancer comprises the following steps:
the carrier was dissolved in ethanol and mRNA of KLF5 was dissolved in water at a volume ratio of ethanol to water of 1: 3;
the mass ratio of the components is 32: 1 (excluding the mass of ethanol and water) mixing the carrier with mRNA of KLF5, and ultrafiltering with an ultrafiltration tube to remove ethanol to obtain the above composition for treating prostate cancer.
The carrier is prepared by the following steps of 1: 0.5: 0.5: 0.1 of (2, 3-dioleoyl-propyl) -trimethylammonium chloride (DOTAP), 1, 2-dioleoyl-sn-glycerol-3-phosphate (DOPE), cholesterol and distearoylphosphatidylethanolamine-polyethylene glycol 2000(DSPE-PEG 2000).
The mass ratio of the mRNA of the KLF5 to the carrier is 32: 1.
example 5
A method for preparing the composition for treating prostate cancer comprises the following steps:
the carrier was dissolved in ethanol and mRNA of KLF5 was dissolved in water at a volume ratio of ethanol to water of 1: 3;
the mass ratio of the components is 32: 1 (excluding the mass of ethanol and water) the carrier was mixed with mRNA of KLF5, and ultrafiltration was performed using an ultrafiltration tube to remove ethanol, to obtain the above composition for the treatment of prostate cancer.
The carrier is prepared by sequentially mixing 1: 2: 2: 0.5 of (2, 3-dioleoyl-propyl) -trimethylammonium chloride (DOTAP), 1, 2-dioleoyl-sn-glycerol-3-phosphoric acid (DOPE), cholesterol and distearoylphosphatidylethanolamine-polyethylene glycol 2000(DSPE-PEG 2000).
The mass ratio of the mRNA of the KLF5 to the carrier is 32: 1.
example 6
A method for preparing the composition for treating prostate cancer comprises the following steps:
the carrier was dissolved in ethanol and mRNA of KLF5 was dissolved in water at a volume ratio of ethanol to water of 1: 1;
the mass ratio of the components is 32: 1 (excluding the mass of ethanol and water) mixing the carrier with mRNA of KLF5, and ultrafiltering with an ultrafiltration tube to remove ethanol to obtain the above composition for treating prostate cancer.
The carrier is prepared by sequentially mixing 1: 1: 1: 0.1 of (2, 3-dioleoyl-propyl) -trimethylammonium chloride (DOTAP), 1, 2-dioleoyl-sn-glycerol-3-phosphate (DOPE), cholesterol and distearoylphosphatidylethanolamine-polyethylene glycol 2000(DSPE-PEG 2000).
Example 7
A method for preparing the composition for treating prostate cancer comprises the following steps:
the carrier was dissolved in ethanol and mRNA of KLF5 was dissolved in water at a volume ratio of ethanol to water of 1: 10;
the mass ratio of the components is 32: 1 (excluding the mass of ethanol and water) the carrier was mixed with mRNA of KLF5, and ultrafiltration was performed using an ultrafiltration tube to remove ethanol, to obtain the above composition for the treatment of prostate cancer.
The carrier is prepared by the following steps of 1: 1: 1: 0.1 of (2, 3-dioleoyl-propyl) -trimethylammonium chloride (DOTAP), 1, 2-dioleoyl-sn-glycerol-3-phosphate (DOPE), cholesterol and distearoylphosphatidylethanolamine-polyethylene glycol 2000(DSPE-PEG 2000).
Example 8
A method for preparing the composition for treating prostate cancer comprises the following steps:
the carrier is dissolved in ethanol, and the mRNA of KLF5 is dissolved in water, wherein the volume ratio of the ethanol to the water is 1: 3;
the mass ratio of the components is 1: 1 (excluding the mass of ethanol and water) mixing the carrier with mRNA of KLF5, and ultrafiltering with an ultrafiltration tube to remove ethanol to obtain the above composition for treating prostate cancer.
The carrier is prepared by sequentially mixing 1: 1: 1: 0.1 of (2, 3-dioleoyl-propyl) -trimethylammonium chloride (DOTAP), 1, 2-dioleoyl-sn-glycerol-3-phosphate (DOPE), cholesterol and distearoylphosphatidylethanolamine-polyethylene glycol 2000(DSPE-PEG 2000).
Example 9
A method for preparing the composition for treating prostate cancer comprises the following steps:
the carrier was dissolved in ethanol and mRNA of KLF5 was dissolved in water at a volume ratio of ethanol to water of 1: 3;
the mass ratio of the components is 2: 1 (excluding the mass of ethanol and water) mixing the carrier with mRNA of KLF5, and ultrafiltering with an ultrafiltration tube to remove ethanol to obtain the above composition for treating prostate cancer.
The carrier is prepared by the following steps of 1: 1: 1: 0.1 of (2, 3-dioleoyl-propyl) -trimethylammonium chloride (DOTAP), 1, 2-dioleoyl-sn-glycerol-3-phosphate (DOPE), cholesterol and distearoylphosphatidylethanolamine-polyethylene glycol 2000(DSPE-PEG 2000).
Example 10
A method for preparing the composition for treating prostate cancer comprises the following steps:
the carrier was dissolved in ethanol and mRNA of KLF5 was dissolved in water at a volume ratio of ethanol to water of 1: 3;
the mass ratio of the components is 4: 1 (excluding the mass of ethanol and water) mixing the carrier with mRNA of KLF5, and ultrafiltering with an ultrafiltration tube to remove ethanol to obtain the above composition for treating prostate cancer.
The carrier is prepared by the following steps of 1: 1: 1: 0.1 of (2, 3-dioleoyl-propyl) -trimethylammonium chloride (DOTAP), 1, 2-dioleoyl-sn-glycerol-3-phosphate (DOPE), cholesterol and distearoylphosphatidylethanolamine-polyethylene glycol 2000(DSPE-PEG 2000).
Example 11
A method for preparing the composition for treating prostate cancer comprises the following steps:
the carrier is dissolved in ethanol, and the mRNA of KLF5 is dissolved in water, wherein the volume ratio of the ethanol to the water is 1: 3;
the mass ratio of the components is 8: 1 (excluding the mass of ethanol and water) mixing the carrier with mRNA of KLF5, and ultrafiltering with an ultrafiltration tube to remove ethanol to obtain the above composition for treating prostate cancer.
The carrier is prepared by the following steps of 1: 1: 1: 0.1 of (2, 3-dioleoyl-propyl) -trimethylammonium chloride (DOTAP), 1, 2-dioleoyl-sn-glycerol-3-phosphate (DOPE), cholesterol and distearoylphosphatidylethanolamine-polyethylene glycol 2000(DSPE-PEG 2000).
Example 12
A method for preparing the composition for treating prostate cancer comprises the following steps:
the carrier was dissolved in ethanol and mRNA of KLF5 was dissolved in water at a volume ratio of ethanol to water of 1: 3;
the mass ratio of the components is 16: 1 (excluding the mass of ethanol and water) mixing the carrier with mRNA of KLF5, and ultrafiltering with an ultrafiltration tube to remove ethanol to obtain the above composition for treating prostate cancer.
The carrier is prepared by the following steps of 1: 1: 1: 0.1 of (2, 3-dioleoyl-propyl) -trimethylammonium chloride (DOTAP), 1, 2-dioleoyl-sn-glycerol-3-phosphate (DOPE), cholesterol and distearoylphosphatidylethanolamine-polyethylene glycol 2000(DSPE-PEG 2000).
Example 13
A method for preparing the composition for treating prostate cancer comprises the following steps:
a composition for treating prostate cancer was obtained by mixing 20 micrograms of mRNA of KLF5 with 320 micrograms of vehicle in 100 microliters of PBS.
The carrier is prepared by the following steps of 1: 1: 1: 0.1 of (2, 3-dioleoyl-propyl) -trimethylammonium chloride (DOTAP), 1, 2-dioleoyl-sn-glycerol-3-phosphate (DOPE), cholesterol and distearoylphosphatidylethanolamine-polyethylene glycol 2000(DSPE-PEG 2000).
And (3) performance testing:
in the test example, RPMI1640 medium was used as the medium, and 10% fetal bovine serum was added to the medium.
In the test example, RPMI-1640 medium was purchased from Gibco (USA).
In the test example, the medium was exposed to 5% CO2And culturing in a constant temperature incubator at 37 ℃.
RPMI is an abbreviation for Roswell Park Memorial Institute, referred to the Roseviv Park Community of Memorial. RPMI is a type of cell culture medium developed by this institute, 1640 is the medium code.
In the test examples, BALB/c nude mice were from Experimental animals technology, Inc., Vitronlia, Beijing.
In the test example, LNCaP cells originated from the beijing counseling hospital.
Ingredient list of RPMI-1640 cell culture medium
In the performance test, the selected experimental group, the control group 1 and the control group 2 comprise the following medicaments:
experimental groups: a composition prepared for treating prostate cancer selected from the group consisting of examples 1-3 and examples 8-12.
Control group 1: 64. mu.g of the vector was selected and dissolved in 100. mu.L of PBS.
Wherein, the carrier is prepared by the following components in a molar ratio of 1: 1: 1: 0.1 of (2, 3-dioleoyl-propyl) -trimethylammonium chloride, 1, 2-dioleoyl-sn-glycerol-3-phosphoric acid, cholesterol and distearoylphosphatidylethanolamine-polyethylene glycol 2000.
Control group 2: 100 μ L of PBS was used.
Control group 3: mu.g of mRNA from KLF5 was selected and dissolved in 100. mu.L of PBS.
EGFP mRNA test group: the EGFP mRNA test group differed from the compositions prepared in examples 1 to 3 and examples 8 to 12 for treating prostate cancer only in that mRNA of KLF5 was replaced with EGFP mRNA. The method specifically comprises the following steps: dissolving the carrier in ethanol, and dissolving EGFP mRNA in water, wherein the volume ratio of ethanol to water is 1: 3; the mass ratio of the components is 1: 1,2: 1,4: 1,8: 1,16: 1,32: 1,64: 1,128: 1 (excluding the mass of ethanol and water) the vehicle was mixed with the mRNA of EGFP, and ultrafiltered using an ultrafiltration tube to remove ethanol, to give the composition of the EGFP mRNA test group. The carrier is prepared by the following steps of 1: 1: 1: 0.1 of (2, 3-dioleoyl-propyl) -trimethylammonium chloride (DOTAP), 1, 2-dioleoyl-sn-glycerol-3-phosphate (DOPE), cholesterol and distearoylphosphatidylethanolamine-polyethylene glycol 2000(DSPE-PEG 2000).
1. Since the mRNA of KLF5 has no fluorescent signal, the mass ratio between the vector and EGFP mRNA (mRNA enhancing green fluorescent protein) was tested to test the effect of different mass ratios of vector to EGFP mRNA on infection efficiency and average fluorescence intensity, after which the EGFP mRNA could be converted to mRNA of KLF 5. The test method is as follows: the EGFP mRNA test group of compositions, in which the concentration of EGFP mRNA was 2. mu.g/mL, was incubated with LNCaP cells, and after 24 hours the cells were suspended with trypsin and analyzed for fluorescence distribution using the GFP channel of a flow cytometer (BD FACSCCanto).
The test results are shown in fig. 1.
As can be appreciated from fig. 1: the transfection efficiency is best when the mass ratio of the carrier to the EGFP mRNA is 32-128, namely the transfection efficiency is best when the mass ratio of the carrier to the mRNA of KLF5 is 32-128.
2. The compositions of experimental, control 1, control 2 and control 3 were delivered to LNCaP cell line for protein expression and analyzed by Western Blotting as follows: the compositions of the experimental group, control group 1, control group 2 and control group 3 were added to 1mL of LNCaP cell culture medium, respectively, and after 24 hours, total cell protein was collected using RIPA cell lysate (shanghai bi yunnan biotechnology limited). After determining the total protein amount with BCA kit (Invitrogen), a sample of the same total protein amount was used for polyacrylamide gel electrophoresis. The protein bands on the polyacrylamide gel (Shanghai Biyun Biotechnology Co., Ltd.) were transferred to nitrocellulose membranes (Shanghai Biyun Biotechnology Co., Ltd.) and blocked with 5% milk (Shanghai Yunyan leaf Biotechnology Co., Ltd.). The primary antibodies of the ACTIN protein and the KLF5 protein are ab5694 and ab137676(abcam), respectively, the secondary antibody is ab205718(abcam), and the secondary antibody developing solution is TMB developing solution (Shanghai Bin Yuntan biotechnology Co., Ltd.). The electrophoresis system comprises a power supply, a vertical electrophoresis tank and a membrane rotating instrument which are purchased from Berle company. Gel imaging systems were purchased from ThermoFisher, Inc.
The test results are shown in FIG. 2.
As can be seen from fig. 2, the expression of KLF5 protein was hardly observed in the LNCaP cells treated by the control group 1-2, the expression of a small amount of KLF5 protein was observed in the LNCaP cells treated by the control group 3, and the expression of the internal reference actinin corresponding to the experimental group and the control group 1-3 was significantly increased in the LNCaP cells treated by the composition for treating prostate cancer of examples 1-3 and 8-12 used in the experimental group compared to the former KLF5 protein, indicating that the expression of the KLF5 protein in the LNCaP cell line could be significantly enhanced by the composition for treating prostate cancer of the experimental group (the expression of the KLF5 protein was the same in the LNCaP cells treated by the composition for treating prostate cancer of examples 1-3 and 8-12, and thus only limited enumeration was performed in fig. 2).
3. Testing the effect of modified LNCaP cells on inhibiting tumor cell growth:
12 BALB/c nude mice were injected subcutaneously with 1X 106And (4) establishing a prostate cancer xenograft subcutaneous tumor model by using the LNCaP cells and the average weight of the mice being 20 g. Subcutaneous tumor of mouse grows to 100mm3Then, the three groups of medicines are randomly and evenly divided, and three groups of medicines are respectively injected, wherein the first group of medicines is as follows: the composition prepared in example 13 for the treatment of prostate cancer; the second group of drugs is: 320 μ g of vehicle, 100 μ l PBS; the third group of drugs is: 100 microliters of PBS was administered repeatedly every 3 days. After 3 weeks, the test comparison was carried out, and the test results are shown in fig. 3, in which only one of the mice is shown in fig. 3, and the other mice in the same group exhibited substantially the same tumor size.
The carrier is prepared by the following steps of 1: 1: 1: 0.1 of (2, 3-dioleoyl-propyl) -trimethylammonium chloride (DOTAP), 1, 2-dioleoyl-sn-glycerol-3-phosphate (DOPE), cholesterol and distearoylphosphatidylethanolamine-polyethylene glycol 2000(DSPE-PEG 2000).
As can be seen from FIG. 3, after 3 weeks, the first phase groupThe subcutaneous tumors were significantly reduced in mice compared to the second and third groups, wherein the average tumor size of the second group was 1043mm3Third group mean tumor size 1156mm3First group mean tumor size 368mm3It can be seen that not only the first group of drugs can reduce mouse tumor size, but the second group of drugs can also reduce mouse tumor size.
The composition for treating prostate cancer prepared in examples 4 to 7 and the composition for control 2 were added to 1mL of LNCaP cell culture medium, respectively, and after 24 hours, total cellular protein was collected using RIPA cell lysate (shanghai bi yunshi biotechnology limited). After determination of the total protein amount with the BCA kit (Invitrogen), a sample of the same total protein amount was used for polyacrylamide gel electrophoresis. The protein bands on the polyacrylamide gel (Shanghai Biyun Biotechnology Co., Ltd.) were transferred to nitrocellulose membranes (Shanghai Biyun Biotechnology Co., Ltd.) and blocked with 5% milk (Shanghai Yunyan leaf Biotechnology Co., Ltd.). The primary antibodies of the ACTIN protein and the KLF5 protein are ab5694 and ab137676(abcam), respectively, the secondary antibody is ab205718(abcam), and the secondary antibody developing solution is TMB developing solution (Shanghai Bin Yuntan biotechnology Co., Ltd.). The electrophoresis system comprises a power supply, a vertical electrophoresis tank and a membrane rotating instrument which are purchased from Berle company. Gel imaging systems were purchased from ThermoFisher, Inc. The test results are shown in fig. 4.
As can be seen from fig. 4, KLF5 protein expression was sequentially enhanced in LNCaP cells treated with the compositions for treating prostate cancer prepared in examples 4-7.
The above description is only an example of the present invention and is not intended to limit the scope of the present invention, and all equivalent modifications made by the present invention as described in the specification of the present invention or directly or indirectly applied to the related technical fields are included in the scope of the present invention.
Claims (8)
1. A composition for treating prostate cancer, characterized by:
mRNA comprising a vector and KLF 5; the mass ratio of the vector to the mRNA of the KLF5 is 1-128: 1;
the carrier is phospholipid, and the phospholipid comprises the following components in a molar ratio of 1: 0.5-2: 0.5-2: (2, 3-dioleoyl-propyl) -trimethylammonium chloride, 1, 2-dioleoyl-sn-glycero-3-phosphate, cholesterol, and distearoylphosphatidylethanolamine-polyethylene glycol 2000 at 0.1-0.5.
2. A composition for use in the treatment of prostate cancer according to claim 1, wherein: the carrier is dissolved in an organic solvent, wherein the organic solvent comprises at least one of ethanol, diethyl ether, tetrahydrofuran, methanol and acetone.
3. A composition for use in the treatment of prostate cancer according to claim 1, wherein: the mass ratio of the carrier to the mRNA of the KLF5 is 32-64: 1.
4. a method of preparing a composition for treating prostate cancer according to claims 1-3, wherein: the method comprises the following steps: mixing the vector and mRNA of KLF5 to obtain the composition for treating prostate cancer.
5. The method of claim 4, wherein: the mRNA of KLF5 was added as a solution in which the mRNA concentration of KLF5 was 0.5-10. mu.g/mL.
6. An intravenous injection characterized by: a composition for treating prostate cancer comprising a compound according to any one of claims 1 to 3 and a pharmaceutically acceptable excipient.
7. Use of a composition according to any one of claims 1 to 3 for the treatment of prostate cancer in the preparation of an immunomodulator.
8. Use of a composition according to any one of claims 1 to 3 for the treatment of prostate cancer in the manufacture of a medicament for the treatment of prostate cancer.
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