WO2021008600A1

WO2021008600A1 - Cationic polymer nanoparticle/hil-22bp gene compound for preventing and treating cancer and preparation method therefor and application thereof

Info

Publication number: WO2021008600A1
Application number: PCT/CN2020/102600
Authority: WO
Inventors: 门可; 魏于全
Original assignee: 四川大学
Priority date: 2019-07-18
Filing date: 2020-07-17
Publication date: 2021-01-21
Also published as: CN110327299B; CN110327299A

Abstract

The present invention belongs to the field of biopharmaceuticals, and relates to a cationic polymer nanoparticle/hIL-22BP gene compound for preventing and treating cancer, a preparation method therefor and an application thereof. Provided in the present invention is a cationic polymer nanoparticle/hIL-22BP gene compound for preventing and treating cancer, which is prepared by compounding cationic polymer nanoparticles and a recombinant vector containing hIL-22BP-encoding gene by means of charge adsorption. The compound of the present invention may directly induce the apoptosis of tumor cells and block IL-22/IL-22R1 transmembrane signals to weaken growth and proliferation signals of the tumor cells, thereby achieving an inhibition effect on the proliferation of tumor cells and achieving the effect of treating tumors. According to animal experiments, the compound of the present invention has the effects of inhibiting the growth of various tumors such as lung cancer, ovarian cancer, liver cancer and colon cancer, the average tumor inhibition rate is higher than 60%, and the compound is a cancer treatment drug that has broad application prospects.

Description

Cationic polymer nanoparticle/hIL-22BP gene complex for preventing and treating cancer, and preparation method and application thereof

Technical field

The invention relates to a cationic polymer nanoparticle/hIL-22BP gene complex for preventing and treating cancer, a preparation method and application thereof, and belongs to the field of biopharmaceuticals.

Background technique

Gene therapy is one of the important methods of tumor biotherapy, and multiple gene therapy products have played a role in the clinical treatment of tumors. As an important part of gene therapy drugs, therapeutic genes used for tumor treatment achieve anti-tumor therapeutic effects through mechanisms such as inhibiting tumor cell proliferation, promoting tumor cell apoptosis, and regulating anti-tumor immunity. Further searching for and developing genes with good therapeutic effects is an important issue that still needs to be solved in tumor gene therapy.

Interleukin is recognized as one of the most important regulators of innate immunity and adaptive immunity related diseases. Interleukin-22 (Interleukin-22, IL-22) is mainly secreted by adaptive immune cells (CD4 positive T cells, CD8 positive T cells, etc.) and innate immune cells (LTi cells, NK cells, etc.), and belongs to IL-10 A member of the cytokine family. Since the discovery of IL-22 in 2000, it has been proven to play important roles including anti-inflammatory and immune regulation in endothelial cells in many tissues. On the one hand, IL-22 can promote tissue proliferation and regeneration and regulate the host's mucosal barrier defense, on the other hand, it may also cause histopathological inflammation. As one of the inflammatory cytokines in the tumor microenvironment, IL-22 plays an important role in the occurrence, development, proliferation, and apoptosis of malignant tumors including colon cancer, liver cancer, gastric cancer, lung cancer, pancreatic cancer, oral squamous cell carcinoma, and skin malignancies. It plays an important role in death and invasion and transfer. For example, studies have found that IL-22 and its transmembrane receptor IL-22R1 are highly expressed in colon cancer tissues, and the expression is positively correlated with tumor size; IL-22/IL-22R1 can inhibit tumors by activating the STAT3 signaling pathway Cell apoptosis can promote tumor growth and metastasis; in addition, IL-22 can also promote the occurrence and development of colon cancer through the expression of innate lymphocytes. Therefore, IL-22 is one of the important molecular targets for tumor therapy.

IL-22 binding protein (IL-22BP) is another receptor for IL-22 (IL-22R2) besides IL-22R1. The receptor exists outside the cell in the form of a soluble protein. It can compete with IL-22R1 to bind free IL-22 factors and block the related biological activities of IL-22, thereby preventing the downstream signal transduction of cytokines. Soluble antagonist of IL-22. Because the binding of IL-22BP and IL-22 has high specificity and high affinity, IL-22BP can effectively regulate the biological activity of IL-22 in vivo. In addition, the expression of IL-22BP in vivo is highly correlated with the function of IL-22/IL-22R1, and when the latter is out of balance, it will be expressed in large quantities to play a control and balance role. Therefore, IL-22BP plays an important role in regulating the inflammation and immune response induced by IL-22. Because IL-22/IL-22R1 can inhibit the apoptosis of tumor cells and promote the occurrence, growth and metastasis of tumors by activating the STAT3 signaling pathway, and promote the occurrence and development of malignant tumors. In addition, the expression of IL-22BP in cells can also directly induce tumor cell apoptosis, thereby inhibiting tumor cell proliferation. Therefore, by promoting the secretion and expression of soluble IL-22BP receptors by tumor cells, the neutralization of free extracellular IL-22 factors can be improved, and IL-22 and IL-22R1 transmembrane receptors on tumor cell membranes can be blocked or reduced. In combination, it weakens the growth and proliferation signal of tumor cells regulated by IL-22, and at the same time induces tumor cell apoptosis and killing, which can realize the hindering effect on tumor cell proliferation and achieve the effect of treating tumors.

Summary of the invention

The purpose of the present invention is to provide a cationic polymer nanoparticle/hIL-22BP gene complex for preventing and treating cancer and its preparation method and application.

The present invention provides a cationic polymer nanoparticle/hIL-22BP gene complex for preventing and treating cancer, which is a composite of cationic polymer nanoparticle and a recombinant vector containing hIL-22BP encoding gene through charge adsorption, wherein the cationic polymer Nanoparticles: The mass ratio of the recombinant vector containing hIL-22BP coding gene is (3-30):1, the nucleotide sequence of the hIL-22BP coding gene is shown in SEQ ID NO.1, the cationic polymer Nanoparticles are prepared from cationic lipids and polymer materials.

Further, the compounding method is to disperse the cationic polymer nanoparticles and the recombinant vector containing the hIL-22BP encoding gene in an aqueous solution and mix them thoroughly to obtain a compound.

Preferably, the solution is allowed to stand for 5-30 minutes after mixing.

Preferably, the solution is allowed to stand for 15 minutes after mixing.

Preferably, it is allowed to stand at room temperature.

Further, the aqueous solution is selected from one or more of 5% glucose solution, physiological saline, ultrapure water or deionized water.

Further, the mass ratio of the cationic polymer nanoparticles: the recombinant vector containing the gene encoding hIL-22BP is 10:1 or 30:1.

Further, the cationic polymer nanoparticles are prepared by a thin film hydration method.

Preferably, the cationic polymer nanoparticles are prepared by the following method: dissolving cationic lipids and polymer materials in a solvent together, removing the solvent and forming a film, taking the film out to dissolve in an aqueous solution, and shaking it sufficiently to obtain .

Preferably, the solvent is dichloromethane.

Preferably, the solvent is removed by rotary evaporation.

Preferably, the aqueous solution is selected from one or two or more of 5% glucose solution, physiological saline, ultrapure water or deionized water.

Further, the cationic lipid is DOTAP, DC-Cholesteral or a mixture thereof.

Preferably, the cationic lipid is DOTAP.

Further, the polymer material is selected from one or more of mPEG-PCL, mPEG-PLA, and mPEG-PLGA.

Preferably, the polymer material is mPEG-PCL.

Preferably, the molecular weight of mPEG-PCL is 4000 to 10000 Da.

Preferably, the molecular weight of mPEG-PCL is 4000 Da, wherein the mPEG fragment is 2000 Da and the PCL fragment is 2000 Da.

Further, the cationic polymer nanoparticles are prepared using DOTAP and mPEG-PCL as raw materials, wherein the mass ratio of DOTAP:mPEG-PCL is 1: (1-20).

Preferably, the mass ratio of DOTAP:mPEG-PCL is 1:9.

Further, the vector is a plasmid vector.

Preferably, the vector is pVAX1.

Further, the recombinant vector containing the gene encoding hIL-22BP has a nucleotide sequence as shown in SEQ ID NO.4.

Further, the recombinant vector containing the gene encoding hIL-22BP is prepared by the following method: cutting the vector with Hind III and Xbal I restriction enzymes, recovering vector fragments, and cutting with the same restriction enzymes. The processed and recovered hIL-22BP coding gene fragment is subjected to ligation reaction to obtain.

The present invention provides a method for preparing the composite: the cationic polymer nanoparticle and the recombinant vector containing the hIL-22BP encoding gene are composited through charge adsorption to obtain the composite.

The invention provides the use of the complex in the preparation of anticancer drugs.

Preferably, the drug is an anti-colon cancer, ovarian cancer, lung cancer and/or liver cancer drug.

The present invention provides an anti-cancer pharmaceutical composition, which is a preparation prepared by adding the compound as an active ingredient and adding pharmaceutically acceptable auxiliary materials or auxiliary ingredients.

Further, the preparation is an injection preparation or an oral preparation.

Further, the preparation is an anti-colon cancer, ovarian cancer, lung cancer and/or liver cancer preparation.

In the present invention, the amino acid sequence (SEQ ID No. 5, 263aa) of the protein encoded by the hIL-22BP encoding gene is:

The present invention provides a novel cationic nanoparticle/hIL-22BP gene complex, which can directly induce tumor cell apoptosis and block IL-22/IL-22R1 transmembrane signal to weaken the growth of tumor cells Proliferation signal realizes the hindering effect on tumor cell proliferation and achieves the effect of treating tumor. Animal experiments have proved that the compound of the invention has the effect of inhibiting the growth of various tumors such as lung cancer, ovarian cancer, liver cancer, colon cancer, etc., with an average tumor inhibition rate exceeding 60%, and is a cancer treatment drug with broad application prospects.

Description of the drawings

Figure 1 is a schematic diagram of the pVAX1 plasmid vector structure in Example 1;

Figure 2 is a schematic diagram of the pVAX1-hIL-22BP plasmid structure in Example 1;

Figure 3 is an agarose gel electrophoresis spectrum of the pVAX1-hIL-22BP plasmid in Example 1 identified by Hind III and XbaI double enzyme digestion;

Figure 4 is a diagram of the therapeutic effect of the colon cancer animal model in Test Example 1;

Figure 5 is a treatment effect diagram of the ovarian cancer animal model in Test Example 2;

Fig. 6 is a treatment effect diagram of a lung cancer animal model in Test Example 3;

Fig. 7 is a graph showing the therapeutic effect of a liver cancer animal model in Test Example 4.

Detailed ways

The present invention was completed based on the inventor's following findings:

Based on the ability of IL-22BP to compete with IL-22R1 to bind free IL-22 factors, thereby blocking the relevant biological activities of IL-22 to promote tumor growth, and the expression of IL-22BP in cells can directly induce tumor cell apoptosis The property of inhibiting tumor cell proliferation, the present invention attempts to construct a gene drug capable of high expression of IL-22BP in tumor tissues for cancer treatment. For this reason, the inventor chose cationic polymer nanoparticles as the gene delivery system, which has the advantages of low toxicity, large loading capacity, and convenient preparation.

The difficulty in preparing cationic polymer nanoparticles/hIL-22BP gene complex is to improve its transduction efficiency. Through repeated investigations, the inventor found that the feeding ratio of nanoparticles and genes is very critical for the expression of hIL-22BP in tumor tissues. Specifically, the mass ratio of nanoparticle: recombinant vector containing hIL-22BP encoding gene is within the range of (3-30):1, and the resulting complex can effectively introduce the hIL-22BP gene vector into the cell. Ideal hIL-22BP gene transcription level to ensure that the expressed human IL-22BP protein exerts a significant therapeutic effect on tumors. The results of the above investigation are detailed as follows:

Table 1 The influence of nanoparticle and gene feeding ratio on transduction efficiency

Note: The recombinant vectors and nanoparticles mentioned in the above table are prepared by referring to the methods of Examples 1 and 2 below, and the difference lies in the different feeding ratios.

The method for detecting gene transfer efficiency is as follows: pipette the transfected cells from the wells and collect them in the flow tube; re-wash them with 1ml PBS solution, and collect the remaining cells in the flow tube. Centrifuge at 1500 rpm for 3 min, discard the supernatant, and resuspend in 1 ml PBS. Repeat the washing twice, and finally add an appropriate amount of PBS to resuspend the cell pellet according to the amount of the cell pellet, and then use a flow cytometer to detect the transfection efficiency.

The method for detecting the mRNA level of IL-22BP in the cell is as follows: 48 hours after transfection, the total cell RNA is extracted by the Trizol method, and the obtained RNA is reverse transcribed into cDNA. Then the mRNA level was analyzed by qPCR method.

The present invention may also have the following additional technical features:

According to some embodiments of the present invention, the compounding method is to disperse the cationic polymer nanoparticles and the recombinant vector containing the hIL-22BP encoding gene in an aqueous solution and mix them thoroughly to obtain a compound. Among them, the aqueous solution is preferably one or more of 5% glucose solution, physiological saline, ultrapure water or deionized water, and most preferably ultrapure water or deionized water. Compared with other kinds of solvent environments, the compound prepared by ultrapure water or deionized water has the best stability and the best efficiency for the introduction of hIL-22BP gene vector. The results of the above investigation are detailed as follows:

Table 2 The effect of solvent types on the stability of the complex transduction efficiency

Note: The recombinant vectors and nanoparticles mentioned in the above table are prepared by referring to the methods in Examples 1 and 2 below, and the difference lies in the type of solvent.

Malvern particle size analyzer was used to analyze the particle size dispersion and PDI value of the composite.

According to some embodiments of the present invention, the cationic polymer nanoparticles are prepared using DOTAP and mPEG-PCL as raw materials. Compared with other types of cationic lipids such as DC-cholesterol and PEI, or other polymer materials such as mPEG-PLA, mPEG-PLGA, the preferred cationic polymer nanoparticles have high stability, uniform particle size distribution and particle size The advantage of being small and conducive to gene delivery and introduction.

According to some embodiments of the present invention, the mass ratio of DOTAP:mPEG-PCL is 1: (1-20). Thus, cationic polymer nanoparticles with good stability and low toxicity are prepared.

According to some embodiments of the present invention, the molecular weight of mPEG-PCL is 4000-10000 Da, most preferably 4000 Da, wherein the mPEG fragment is 2000 Da and the PCL fragment is 2000 Da. The prepared cationic polymer nanoparticles have a small particle size distribution.

The solution of the present invention will be explained below in conjunction with examples. Those skilled in the art will understand that the following embodiments are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the examples, the procedures shall be carried out in accordance with the techniques or conditions described in the literature in the field or in accordance with the product specification. The reagents or instruments used without the manufacturer's indication are all conventional products that are commercially available.

Example 1 Construction of hIL-22BP gene expression plasmid

1. Acquisition of hIL-22BP gene

1) According to the coding sequence of hIL-22BP (Gene ID: 116379, NM_052962) in the NCBI (National Center for Biotechnology Information) database of the National Center for Biotechnology Information, a cDNA plasmid containing the full-length hIL-22BP gene sequence was synthesized. The coding sequence of hIL-22BP (SEQ ID NO.1, 792bp) is:

2) Design and synthesize corresponding primers

Upstream primer: 5'-GGGAAAGCTTATGATGCCTAAACATTGCTTTC-3' (SEQ ID NO. 2), designed to introduce Hind III restriction site;

The downstream primer 5’-GGGATCTAGATCATGGAATTTCCACACATCTC-3’ (SEQ ID NO.3) is designed to introduce the Xbal I restriction site;

The DNA fragment of hIL-22BP was amplified by PCR, and the fragment was recovered by agarose gel electrophoresis to obtain the hIL-22BP gene fragment.

2. Cloning and construction of pVAX1-hIL-22BP plasmid

1) Using Hind III and Xbal I restriction enzymes, double digestion of the eukaryotic expression vector pVAX1 at 37°, agarose gel electrophoresis to recover the linearized pVAX1 vector fragment of the corresponding size, and the digestion treatment with the same conditions The hIL-22BP gene fragment recovered by agarose gel electrophoresis was subjected to a ligation reaction in a 37°C water bath for 2 hours, and the ligation product was transformed into DH5a E. coli competent cells and spread on a kanamycin-resistant LB plate. After 24 hours, the clones were picked and cultured overnight in 3 mL of LB medium containing kanamycin. The pVAX1-hIL-22BP plasmid recombinant plasmid was extracted the next day. The structure of the pVAX1 plasmid vector is shown in Figure 1, and the structure of the pVAX1-hIL-22BP plasmid is shown in Figure 2.

2) The pVAX1-hIL-22BP plasmid was digested with Hind III and Xbal I restriction enzymes, respectively. The agarose gel electrophoresis pattern is shown in Figure 3, where M represents DNAMarker and 1 represents pVAX1-hIL-22BP plasmid. 2 represents the result of double restriction digestion of pVAX1-hIL-22BP. Figure 3 shows that the connection is successful. The constructed pVAX1-hIL-22BP vector has a full length of 3717bp, and the full sequence (SEQ ID NO.4, 3717bp) is:

Example 2 Preparation of DMP cationic nanoparticles/hIL-22BP gene complex of the present invention

1. Preparation of DOTAP-mPEG-PCL polymer nanoparticle solution

Combine cationic lipid (2,3-dioleoyl-propyl)-trimethylamine (1,2-dioleoyl-3-trimethylammonium-propane, DOTAP) with methyl polyethylene glycol-polycaprolactone (mPEG-PCL) ) High molecular polymers (molecular weight 4000Da, PEG-PCL=2000Da-2000Da) are mixed in a mass ratio of 1:9, and the mixture is dissolved in dichloromethane, and placed on a rotary evaporator at 60°C Film formation after 30min. The formed film was taken out and dissolved in deionized water, and then shaken for 5 minutes under a water bath at 60° C. to obtain a DMP cationic polymer nanoparticle solution with a specific concentration.

2. Preparation of hIL-22BP gene expression plasmid

The pVAX1-hIL-22BP plasmid was transformed into DH5α E. coli competent cells and spread on LB plates containing kanamycin resistance. After 24 hours, the clones were picked and cultured overnight in 3 mL of LB medium containing kanamycin. The pVAX1-hIL-22BP plasmid recombinant plasmid was extracted the next day. The purified plasmid after testing can meet the requirements of in vivo and in vitro experiments.

3. Preparation of DMP cationic nanoparticles/hIL-22BP plasmid DNA gene complex

Add the nanoparticle solution (dispersed in deionized water at a concentration of 5mg/mL) to the pVAX1-IL-22BP solution (dispersed in deionized water) at a ratio of 10:1 (w/w) to DMP cationic nanoparticles: DNA The concentration is 1mg/mL), then immediately mix it with a pipette, and then stand for 15 minutes at room temperature to obtain DMP cationic nanoparticle plasmid DNA complex.

Example 3 Preparation of DCMP cationic nanoparticles/hIL-22BP gene complex of the present invention

1. Preparation of DC-Cholesterol-mPEG-PCL polymer nanoparticle solution

The cationic lipid 3β-[N-(N',N'-dimethylaminoethyl)carbamoyl]cholesterol (3β-[N-(N,N-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride, DC- Cholesteral) and methyl polyethylene glycol-polycaprolactone (mPEG-PCL) high molecular polymer (molecular weight 4000Da, PEG-PCL=2000Da-2000Da) are mixed at a mass ratio of 1:9, and the mixture is used The dichloromethane was dissolved and placed on a rotary evaporator at 60°C for 30 minutes to form a film. The formed film is taken out and dissolved in a deionized aqueous solution, and then shaken for 5 minutes under a water bath at 60° C. to obtain a DCMP cationic polymer nanoparticle solution with a specific concentration.

2. Preparation of hIL-22BP gene expression plasmid

3. Preparation of DCMP cationic nanoparticles/hIL-22BP plasmid DNA gene complex

Add the nanoparticle solution (dispersed in deionized water with a concentration of 5mg/mL) to the pVAX1-hIL-22BP solution (dispersed in deionized water) at a ratio of 10:1 (w/w) to DCMP cationic nanoparticles: DNA. The concentration is 1 mg/mL), immediately after mixing with a pipette, and then standing at room temperature for 15 minutes to obtain the DCMP cationic nanoparticle plasmid DNA complex of the present invention.

Example 4 Preparation of DPA cationic nanoparticles/hIL-22BP gene complex of the present invention

1. Preparation of DOTAP-mPEG-PLA polymer nanoparticle solution

The cationic lipid (2,3-dioleoyl-propyl)-trimethylamine (1,2-dioleoyl-3-trimethylammonium-propane, DOTAP) and α-methyl poly(oxyethylene)-poly(D,L) -Lactide) (mPEG-PLA) high molecular polymer (molecular weight 4000Da, PEG-PLA=2000Da-2000Da) is mixed at a mass ratio of 1:9, and the mixture is dissolved in dichloromethane and placed The film was formed after rotary evaporation at 60°C for 30 minutes on a rotary evaporator. The formed film is taken out and dissolved in a deionized water solution, and then shaken for 5 minutes under a water bath at 60° C. to obtain a DPA cationic polymer nanoparticle solution with a specific concentration.

2. Preparation of hIL-22BP gene expression plasmid

3. Preparation of DPA cationic nanoparticles/hIL-22BP plasmid DNA gene complex

Add the nanoparticle solution (dispersed in deionized water at a concentration of 5mg/mL) to the pVAX1-hIL-22BP solution (dispersed in deionized water) at a ratio of 10:1 (w/w) to DPA cationic nanoparticles: DNA The concentration is 1 mg/mL), then immediately mix it with a pipette, and then stand at room temperature for 15 minutes to obtain the DPA cationic nanoparticle plasmid DNA complex of the present invention.

Example 5 Preparation of DPGA cationic nanoparticles/hIL-22BP gene complex of the present invention

1. Preparation of DOTAP-mPEG-PLGA polymer nanoparticle solution

The cationic lipid (2,3-dioleoyl-propyl)-trimethylamine (1,2-dioleoyl-3-trimethylammonium-propane, DOTAP) was combined with methoxy polyethylene glycol-polyglycolide (mPEG) -PLGA) high molecular polymers (molecular weight 4000Da, PEG-PLGA=2000Da-2000Da) were mixed in a mass ratio of 1:9, and the mixture was dissolved in dichloromethane and placed on a rotary evaporator at 60°C Film is formed after 30 minutes of rotary steaming. The formed film was taken out and dissolved in a deionized aqueous solution, and then shaken for 5 minutes under a water bath at 60° C. to obtain a specific concentration of DPGA cationic polymer nanoparticle solution.

2. Preparation of hIL-22BP gene expression plasmid

3. Preparation of DPGA cationic nanoparticles/hIL-22BP plasmid DNA gene complex

Add the nanoparticle solution (dispersed in deionized water at a concentration of 5mg/mL) to the pVAX1-hIL-22BP solution (dispersed in deionized water) at a ratio of 10:1 (w/w) to DPGA cationic nanoparticles: DNA The concentration is 1 mg/mL), then immediately mix it with a pipette, and then stand at room temperature for 15 minutes to obtain the DPGA cationic nanoparticle plasmid DNA complex of the present invention.

The following test examples demonstrate the beneficial effects of the present invention.

Test Example 1 Anti-colon cancer test of cationic nanoparticle/hIL-22BP gene complex of the present invention

In order to study the anti-tumor effect of cationic nanoparticles/hIL-22BP gene complex in vivo, a human colon cancer heterotopic xenograft tumor model was established subcutaneously in BalB/c-nu mice (6-8 weeks old, female). The HCT116 human colon cancer cells cultured in vitro were digested with trypsin and fixed in serum-free and antibiotic-free 1640 medium. Each mouse was inoculated with 1×10 ⁷ cells subcutaneously. After 7 days of cell inoculation, start Randomized treatment according to the following (5 in each group):

A) Blank control group: 5% glucose solution;

B) Empty plasmid control group: the cationic nanoparticle/pVAX gene complex is placed in 5% glucose solution;

C) IL-22BP treatment group: DMP cationic nanoparticles/hIL-22BP gene complex was placed in a 5% glucose solution.

Intratumor injection was used for treatment. DMP cationic nanoparticles/DNA complexes were prepared according to the methods of Examples 1 and 2, and the proportions were as follows: Plasmid DNA: cationic nanoparticles = 1:10 (W/W), and the cationic nanoparticles The pellet/DNA complex is diluted in glucose solution and adjusted so that the final concentration of glucose is 5%. The injection volume of each mouse is 100 μL each time, which contains 5 μg plasmid and 50 μg cationic nanoparticles. It is administered once a day for a total of 7 treatments. The tumor volume was measured every day after the start of treatment. On the 9th day after the treatment, the animals were sacrificed and dissected, and the subcutaneous tumor tissue was separated and weighed. Tumor growth inhibition was analyzed by variance analysis, and P<0.05 was considered statistically significant. The tumor weights and tumor growth curves of the above groups of animals are shown in Figure 4, where Figure 4a shows the tumor growth curve, and Figure 4b shows the average tumor weight.

It can be seen from Figure 4 that the cationic nanoparticle/hIL-22BP gene complex treatment group has slow tumor growth, while the control group has a faster tumor growth. The cationic nanoparticle/hIL-22BP gene complex shows a strong inhibitory effect on tumor growth. Compared with the blank control group, the tumor inhibition rate reached 74.8%.

The above experimental results show that the cationic nanoparticle/hIL-22BP gene complex has a significant effect on human colon cancer.

Test Example 2 Anti-ovarian cancer test of the cationic nanoparticle/hIL-22BP gene complex of the present invention

In order to study the anti-tumor effect of cationic nanoparticles/hIL-22BP gene complex in vivo, a human ovarian cancer abdominal metastasis model was established in the abdominal cavity of BalB/c-nu mice (6-8 weeks old, female). The SKOV3 human ovarian cancer cells cultured in vitro were digested with trypsin, and the volume was fixed in serum-free and antibiotic-free DMEM medium, and 1×10 ⁷ cells were inoculated into the abdominal cavity of each mouse. After 7 days of cell inoculation, start Randomized treatment according to the following (5 in each group):

A) Blank control group: 5% glucose solution;

The treatment was performed by intraperitoneal injection. The DMP cationic nanoparticle/DNA complex was prepared according to the methods in Examples 1 and 2, and the formulation was as follows: Plasmid DNA: cationic nanoparticle = 1:10 (W/W), and the cationic nanoparticle/DNA complex The pellet/DNA complex is diluted in glucose solution and adjusted so that the final concentration of glucose is 5%. The injection volume of each mouse is 100 μL each time, which contains 5 μg plasmid and 50 μg cationic nanoparticles. It is administered once a day for a total of 7 treatments. On the 7th day after the treatment, the animals were sacrificed and dissected, the abdominal tumor tissues were separated, weighed and tumor nodules counted. Tumor growth inhibition was analyzed by variance analysis, and P<0.05 was considered statistically significant. The tumor weight and the number of tumor nodules of the above groups of animals are shown in Figure 5, where Figure 5a represents the average tumor weight, and Figure 5b represents the average number of tumor nodules.

It can be seen from Figure 5 that the cationic nanoparticle/hIL-22BP gene complex treatment group tumors grow slowly, while the control group tumors grow faster. The cationic nanoparticle/hIL-22BP gene complex exhibits a strong inhibitory effect on tumor growth. Compared with the blank control group, the tumor inhibition rate reached 71.1%.

The above experimental results show that the cationic nanoparticle/hIL-22BP gene complex has a significant effect on human ovarian cancer.

Test Example 3 Anti-lung cancer test of the cationic nanoparticle/hIL-22BP gene complex of the present invention

In order to study the anti-tumor effect of cationic nanoparticles/hIL-22BP gene complex in vivo, a human lung cancer heterotopic xenograft tumor model was established subcutaneously in BalB/c-nu mice (6-8 weeks old, female). The A549 human lung cancer cells cultured in vitro were digested with trypsin, and the volume was fixed in serum-free and antibiotic-free DMEM medium. Each mouse was subcutaneously inoculated with 1×10 ⁷ cells. After 7 days of cell inoculation, start pressing The following randomized treatments (5 in each group):

A) Blank control group: 5% glucose solution;

Intratumor injection was used for treatment. DMP cationic nanoparticles/DNA complexes were prepared according to the methods of Examples 1 and 2, and the proportions were as follows: Plasmid DNA: cationic nanoparticles = 1:10 (W/W), and the cationic nanoparticles The pellet/DNA complex is diluted in a glucose solution and adjusted so that the final concentration of glucose is 5%. The injection volume of each mouse is 100 μL each time, which contains 5 μg plasmid and 50 μg cationic nanoparticles. It is administered once a day for a total of 7 treatments. The tumor volume was measured every day after the start of treatment. On the 9th day after the treatment, the animals were sacrificed and dissected, and the subcutaneous tumor tissue was separated and weighed. Tumor growth inhibition was analyzed by variance analysis, and P<0.05 was considered statistically significant. The tumor weights and tumor growth curves of the above groups of animals are shown in Figure 6, where Figure 6a shows the tumor growth curve, and Figure 6b shows the average tumor weight.

It can be seen from Figure 6 that the cationic nanoparticle/hIL-22BP gene complex treatment group tumors grow slowly, while the control group tumors grow faster. The cationic nanoparticle/hIL-22BP gene complex exhibits a strong inhibitory effect on tumor growth. Compared with the blank control group, the tumor inhibition rate reached 66.87%.

The above experimental results show that the cationic nanoparticle/hIL-22BP gene complex has a significant effect on human lung cancer.

Test Example 4 Anti-liver cancer test of the cationic nanoparticle/hIL-22BP gene complex of the present invention

In order to study the anti-tumor effect of cationic nanoparticles/hIL-22BP gene complex in vivo, a human hepatocarcinoma xenograft tumor model was established subcutaneously in BalB/c-nu mice (6-8 weeks old, female). The HepG2 human liver cancer cells cultured in vitro were digested with trypsin, and the volume was fixed in serum-free and antibiotic-free DMEM medium. Each mouse was subcutaneously inoculated with 1×10 ⁷ cells. After 7 days of cell inoculation, start pressing The following randomized treatments (5 in each group):

A) Blank control group: 5% glucose solution;

Intratumor injection was used for treatment. DMP cationic nanoparticles/DNA complexes were prepared according to the methods of Examples 1 and 2, and the proportions were as follows: Plasmid DNA: cationic nanoparticles = 1:10 (W/W), and the cationic nanoparticles The pellet/DNA complex is diluted in glucose solution and adjusted so that the final concentration of glucose is 5%. The injection volume of each mouse is 100 μL each time, which contains 5 μg plasmid and 50 μg cationic nanoparticles. It is administered once a day for a total of 7 treatments. The tumor volume was measured every day after the start of treatment. On the 9th day after the treatment, the animals were sacrificed and dissected, and the subcutaneous tumor tissue was separated and weighed. Tumor growth inhibition was analyzed by variance analysis, and P<0.05 was considered statistically significant. The tumor weights and tumor growth curves of the above groups of animals are shown in Figure 7, where Figure 7a shows the tumor growth curve, and Figure 7b shows the average tumor weight.

It can be seen from Figure 7 that the cationic nanoparticle/hIL-22BP gene complex treatment group has slow tumor growth, while the control group has a faster tumor growth. The cationic nanoparticle/hIL-22BP gene complex shows a strong inhibitory effect on tumor growth. Effect, compared with the blank control group, the tumor inhibition rate reached 63%.

The above experimental results show that the cationic nanoparticle/hIL-22BP gene complex has a significant effect on human liver cancer.

It should be noted that the specific features, structures, materials, or characteristics described in this specification can be combined in any one or more embodiments in a suitable manner. In addition, those skilled in the art can combine and combine the different embodiments and the features of the different embodiments described in this specification without contradicting each other.

Claims

The cationic polymer nanoparticle/hIL-22BP gene complex for cancer prevention and treatment is characterized in that it is a composite of cationic polymer nanoparticles and a recombinant vector containing hIL-22BP encoding gene through charge adsorption. Among them, cationic polymer Nanoparticles: The mass ratio of the recombinant vector containing hIL-22BP coding gene is (3-30):1, the nucleotide sequence of the hIL-22BP coding gene is shown in SEQ ID NO.1, the cationic polymer Nanoparticles are prepared from cationic lipids and polymer materials.
The compound according to claim 1, wherein the compounding method is to disperse the cationic polymer nanoparticles and the recombinant vector containing hIL-22BP coding gene in an aqueous solution and mix them thoroughly to obtain the compound; Preferably, the solution is allowed to stand for 5-30 minutes after mixing; preferably, the solution is allowed to stand for 15 minutes after mixing; preferably, it is allowed to stand at room temperature.
The complex according to claim 2, wherein the aqueous solution is selected from one or more of 5% glucose solution, physiological saline, ultrapure water or deionized water.
The complex according to claim 1, wherein the mass ratio of the cationic polymer nanoparticles: the recombinant vector containing the hIL-22BP encoding gene is 10:1 or 30:1.
The composite of claim 1, wherein the cationic polymer nanoparticles are prepared by a thin film hydration method; preferably, the cationic polymer nanoparticles are prepared by the following method: Dissolve it together with the polymer material in a solvent, remove the solvent and form a film, take the film out and dissolve it in an aqueous solution, and shake it sufficiently to obtain; preferably, the solvent is dichloromethane; preferably, the solvent is removed by rotary evaporation; Preferably, the aqueous solution is selected from one or more of 5% glucose solution, physiological saline, ultrapure water or deionized water.
The complex according to claim 1 or 5, wherein the cationic lipid is DOTAP, DC-Cholesteral or a mixture thereof; preferably, the cationic lipid is DOTAP.
The composite of claim 1 or 5, wherein the polymer material is selected from

One or more than two of mPEG-PCL, mPEG-PLA, and mPEG-PLGA; preferably, the polymer material is mPEG-PCL; preferably, the molecular weight of mPEG-PCL is 4000～10000Da; preferably, mPEG- The molecular weight of PCL is 4000 Da, of which mPEG fragment is 2000 Da and PCL fragment is 2000 Da.
The composite according to any one of claims 1, 5 to 7, characterized in that the cationic polymer nanoparticles are prepared from DOTAP and mPEG-PCL as raw materials, wherein DOTAP: the quality of mPEG-PCL The ratio is 1: (1-20); preferably, the mass ratio of DOTAP:mPEG-PCL is 1:9.
The complex according to claim 1, wherein the vector is a plasmid vector; preferably, the vector is pVAX1.
The complex according to claim 1 or 9, wherein the recombinant vector containing the gene encoding hIL-22BP has a nucleotide sequence as shown in SEQ ID NO.4.
The complex according to claim 1 or 9, characterized in that: the recombinant vector containing the gene encoding hIL-22BP is prepared by the following method: cutting the vector with Hind III and Xbal I restriction enzymes, and recovering The vector fragment is subjected to ligation reaction with the hIL-22BP encoding gene fragment which is digested with the same restriction enzymes and recovered.
The method for preparing the complex according to any one of claims 1 to 11, characterized in that: the cationic polymer nanoparticles and the recombinant vector containing the hIL-22BP encoding gene are compounded by charge adsorption.
Use of the complex according to any one of claims 1 to 11 in the preparation of an anti-cancer drug; preferably, the drug is an anti-colon cancer, ovarian cancer, lung cancer and/or liver cancer drug.
The anti-cancer pharmaceutical composition is characterized in that it is a preparation prepared from the complex described in any one of claims 1-11 as an active ingredient and adding pharmaceutically acceptable excipients or auxiliary ingredients.
The pharmaceutical composition of claim 14, wherein the preparation is an injection preparation or an oral preparation.
The pharmaceutical composition according to claim 14 or 15, wherein the preparation is an anti-colon cancer, ovarian cancer, lung cancer and/or liver cancer preparation.