CN112870373A - Polypeptide polymer composite nanoparticle for mRNA delivery and preparation method and application thereof - Google Patents
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Abstract
The invention belongs to the technical field of biological medicines, and particularly relates to a polypeptide polymer composite nanoparticle for mRNA delivery, and a preparation method and application thereof. Aiming at the lack of the related research on the preparation of a nano compound by compounding cell-penetrating peptides and polymer nanoparticles DMP and the application thereof, the invention provides an mRNA delivery carrier consisting of polypeptides and polymer nanoparticles, and the polypeptide polymer composite nanoparticles for mRNA delivery are prepared from cell-penetrating peptides cRGD-R9 modified DMP-Mal nanoparticles. The nano-particles can be combined with mRNA through electrostatic interaction, can effectively introduce a target gene into cells, have the characteristics of low cytotoxicity, high transfection rate and the like, and have good application prospects in gene function research, gene therapy research and clinical application based on mRNA.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a polypeptide polymer composite nanoparticle for mRNA delivery, and a preparation method and application thereof.
Background
In recent years, rapid advances in the field of gene therapy have brought cancer patients with a number of innovative therapeutic approaches, with emphasis on the efficient delivery of nucleic acid drugs to target cells of patients and therapeutic expression to intervene in or treat cancer. Thus, there is a need for gene vectors that can efficiently and safely deliver nucleic acid drugs to patients. Genetic vectors are generally classified into: viral and non-viral, non-viral gene delivery has the advantages of greater loading capacity, lower immunogenicity, and fewer side effects than most viral vectors. In gene therapy, a good gene vector has the advantages of targeting property, effectiveness, safety, easiness in preparation and the like, so that the nucleic acid medicament has an anti-tumor effect. Therefore, we have focused on developing a safe and effective non-viral gene vector to deliver mRNA, which can be stably expressed in vivo, and can suppress the proliferation of tumor cells and achieve the purpose of gene therapy.
Methoxy polyethylene glycol-polycaprolactone (monomethoxyl poly (ethylene glycol) -b-poly (epsilon-caprolactone), mPEG-PCL for short, is a two-block copolymer, one end of which is hydrophilic chain segment methoxy polyethylene glycol and the other end of which is lipophilic chain segment poly (epsilon-caprolactone), and has good biocompatibility and safety, both mPEG and PCL are polymers approved by the United states Food and Drug Administration (FDA) for use in vivo, and have good application prospects in drug and gene introduction systems, DOTAP is amphiphilic cationic phospholipid which is used as a donor of positive charges and also widely applied to the preparation of gene delivery carriers, and the two can form cationic nano-particles DMP in aqueous solution through self-assembly to become a potential carrier for nucleic acid delivery, however, due to the special properties of mRNA molecules, the delivery capacity of the DMP nano-carrier to the mRNA molecules is limited, further improvement in its delivery ability is required to serve mRNA-based gene therapy research and drug development.
Cell Penetrating Peptides (CPPs) are a class of short peptides that are capable of crossing the cell membrane or tissue barrier. The CPPs can carry carried biological macromolecules such as protein, nucleic acid and nano particles into cells to play an effect function thereof through mechanisms such as endocytosis and direct penetration, thereby becoming an important technical tool for improving the drug delivery efficiency and having good application prospect in the field of biomedical research. The CPPs can realize the improvement of the cell delivery capacity of the nanoparticles through covalent coupling with the nano-carrier.
At present, there is no report on the preparation of a nano-composite by compounding cell-penetrating peptide and polymer nanoparticles DMP, and further there is no report on whether the obtained nano-composite has mRNA delivery.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: an mRNA delivery carrier consisting of polypeptide and polymer nanoparticles is developed to realize high-efficiency transmission of mRNA molecules in vivo.
The technical scheme for solving the technical problems comprises the following steps: polypeptide polymer composite nanoparticles for mRNA delivery are provided. The polypeptide polymer composite nanoparticle for mRNA delivery is prepared by modifying a DMP-Mal nanoparticle with cell-penetrating peptide cRGD-R9.
In the polypeptide polymer composite nanoparticle for mRNA delivery, the average particle size of the polypeptide polymer composite nanoparticle is 250nm, and the average potential is +20 mv.
In the polypeptide polymer composite nanoparticle for mRNA delivery, the mass ratio of the cell-penetrating peptide cRGD-R9 to the DMP-Mal nanoparticle is as follows: 1-5: 20 to 60.
In the polypeptide polymer composite nanoparticle for mRNA delivery, the DMP-Mal nanoparticle is prepared by cationic phospholipid DOTAP, mPEG-PCL polymer and Mal-PEG-PCl polymer, and the mass ratio of the DOTAP, mPEG-PCL polymer to the Mal-PEG-PCL polymer is as follows: 5: 45: 1.
the invention also provides a preparation method of the polypeptide polymer composite nanoparticle for mRNA delivery, which comprises the following steps:
weighing 45 parts of mPEG-PCL polymer, 5 parts of DOTAP and 1 part of Mal-PEG-PCL polymer, respectively dissolving the mPEG-PCL polymer, evaporating and volatilizing the solvent into a film by using a rotary evaporator, carrying out rotary evaporation on the film for 30min at 60 ℃, adding a proper amount of hydration solution to hydrate until the film is completely dissolved to obtain a DMP-Mal nanoparticle solution, mixing and incubating a proper amount of DMP-Mal nanoparticles and cRGD-R9 polypeptide, and dialyzing the reaction solution to obtain the polypeptide polymer composite nanoparticles for mRNA delivery.
In the preparation method of the polypeptide polymer composite nanoparticle for mRNA delivery, the solvent is at least one of dichloromethane, trichloromethane, acetone, tetrachloromethane, ethanol, methanol, diethyl ether, pentane, ethyl acetate, or cyclohexane.
In the preparation method of the polypeptide polymer composite nanoparticle for mRNA delivery, the hydration solution is at least one of double distilled water, deionized water, pure water or physiological saline.
The invention also provides application of the polypeptide polymer composite nanoparticle for mRNA delivery in delivery of mRNA molecules.
Wherein, in the above application, the mRNA molecule is mRNA of Bim, mRNA of IL-22BP or mRNA of IL-15.
The invention also provides a compound obtained by loading mRNA molecules on the polypeptide polymer composite nano-particles for mRNA delivery.
Furthermore, the invention also provides application of the compound in preparing a medicament for preventing or treating tumor diseases.
Further, in the above use, the tumor is colon cancer, lung cancer or breast cancer.
The invention has the beneficial effects that:
the polypeptide polymer composite nanoparticle DMP-039 for mRNA delivery provided by the invention can be combined with mRNA through electrostatic interaction, can effectively introduce a target gene into cells, has the characteristics of low cytotoxicity, high transfection rate and the like, and has good application prospects in gene function research, gene therapy research and clinical application based on mRNA. The DMP-039 nanoparticles can mediate different genes to exert curative effects, for example, mRNA molecules of genes such as IL-22BP, Bim or IL-15 delivered by the DMP-039 nanoparticles can effectively inhibit the growth of tumor tissues in vivo. The DM-039P nanoparticle is a relatively safe and efficient mRNA molecule non-viral gene delivery vector, and the prepared mRNA/DMP-039 compound provides a new strategy for treating diseases.
Drawings
FIG. 1 shows the chemical structure of DOTAP;
FIG. 2 shows the chemical structural formula of mPEG-PCL;
FIG. 3 shows the chemical structural formula of Mal-PEG-PCL;
FIG. 4 shows the structural formula of cRGD-R9 polypeptide;
FIG. 5 is a graph showing the effect of colon cancer animal model treatment in test example 1; a represents the mean tumor weight, b represents the mean tumor nodule number;
FIG. 6 is a graph showing the effect of the treatment of the breast cancer animal model in test example 2; a represents the tumor growth curve, b represents the average tumor weight;
FIG. 7 is a graph showing the therapeutic effect of the colon cancer pulmonary metastasis animal model in test example 3; a represents lung weight and b represents mean lung tumor nodule number.
Detailed Description
The invention provides a cell-penetrating peptide modified functional cationic polymer carrier, which is characterized in that firstly cationic phospholipid DOTAP, amphiphilic copolymer mPEG-PCL and Mal-PEG-PCL are self-assembled into DMP-Mal cationic nanoparticles, and then are subjected to addition reaction and covalent coupling with cRGD-R9 cell-penetrating peptide to prepare a cell-penetrating peptide modified mRNA delivery carrier, namely the DMP-039 nanoparticles. The DMP-039 nanoparticle has the advantages of high mRNA delivery efficiency, low cytotoxicity and the like.
The cationic phospholipid DOTAP adopted in the invention is chemically named as (2, 3-dioleoxypropyl) trimethyl ammonium chloride, is called DOTAP for short, and has a chemical structural formula shown in figure 1. According to the invention, the DOTAP is adopted to prepare the nanoparticles, on one hand, the DOTAP phospholipid is also amphiphilic and can be self-assembled into the nanoparticles together with the mPEG-PCL polymer which is amphiphilic; on the other hand, the DOTAP phospholipid has positive points, can generate electrostatic adsorption with mRNA molecules with negative electricity, and realizes the delivery of the mRNA; furthermore, since the cell membrane is also composed of lipids, the presence of DOTAP can facilitate transmembrane uptake of the nanoparticles.
One of the amphiphilic copolymers adopted in the invention is methoxy polyethylene glycol-polycaprolactone, which is called mPEG-PCL for short, and the chemical structural formula is shown in figure 2. The mPEG-PCL has biodegradability, biocompatibility and amphipathy, can be self-contained in aqueous solution to form nanoparticles, and is an ideal nano-drug carrier framework material.
One of the amphiphilic copolymers adopted in the invention is maleimide-polyethylene glycol-polycaprolactone, called Mal-PEG-PCL for short, and the chemical structural formula is shown in figure 3. Mal-PEG-PCL not only has biodegradability, biocompatibility and amphipathy, but also can be self-assembled into nanoparticles in aqueous solution, and can be covalently coupled with polypeptide through maleimide group, so that the Mal-PEG-PCL is an ideal nano-drug functionalized carrier framework material.
The cell-penetrating peptide cRGD-R9 adopted in the invention has a chemical structural formula shown in figure 4, is a short peptide with a cell penetrating function, and has a function of promoting the absorption of cells through membranes.
The invention determines the weight ratio of mPEG-PCL to DOTAP in the DMP-039 nanoparticles through a screening test, wherein the weight ratio of mPEG-PCL to DOTAP is adopted as follows: the DMP-039 nanoparticles are prepared at a ratio of 1: 1-20: 1, cell transfection experiments are carried out on the nanoparticles, and experiments show that the mPEG-PCL polymer has better transfection efficiency when 6-12 parts and 1 part of DOTAP are used, and 9 parts and 1 part of DOTAP are preferable.
The invention determines the weight ratio of Mal-PEG-PCL to mPEG-PCL in the DMP-039 nanoparticles through a screening test, wherein the weight ratio of Mal-PEG-PCL to mPEG-PCL is as follows: 1: 100-1: 20, preparing DMP-039 nanoparticles, and performing cell transfection experiments on the nanoparticles, wherein the experiments show that the transfection efficiency is better when 1 part of Mal-PEG-PCL polymer and 30-50 parts of mPEG-PCL polymer are used, and 1 part of Mal-PEG-PCL polymer and 45 parts of mPEG-PCL copolymer are preferred.
In the preparation process of the early-stage nano gene medicine, the mass ratio range of mRNA to DMP nano in the mRNA/DMP compound is set to be 1: 1-1: 50. Gel retardation analysis and cell transfection experiments show that the DMP-039 nanoparticles can effectively combine mRNA molecules in the proportion range and have better transfection efficiency. On this basis, the ratio is further reduced to 1: 10-1: 30, gel block analysis and cell transfection experiments were again performed to find that DMP-039 delivered the mRNA molecules optimally in this ratio range.
In the mPEG-PCL copolymer required by the preparation of the DMP-039 nanoparticles, the molecular weight ratio of mPEG and PCL is 1:1, and the total molecular weight range of mPEG-PCL is 4000 Da-8000 Da.
In the Mal-PEG-PCL copolymer required by the preparation of the DMP-039 nanoparticles, the molecular weight ratio of PEG and PCL is 1:1, and the total molecular weight range of Mal-PEG-PCL is 4000 Da-8000 Da.
In the cell-penetrating peptide required by the preparation of the DMP-039 nanoparticles, the molar ratio of the DMP nanoparticles to the cell-penetrating peptide is 30: 1.
The DMP-039 nanoparticles obtained by the invention have the average particle size of 250nm and the average potential of +20mv, have good mRNA binding capacity, can protect mRNA from being degraded by RNase enzyme, and have higher mRNA transfection capacity and lower cytotoxicity compared with a carrier material DMP which is not modified by cell-penetrating peptide and a gold-labeled transfection material PEI 25K.
The DMP-039 nanoparticle prepared by the invention belongs to biodegradable cationic nanoparticles, and is a novel non-viral vector of a gene transfer system. The nano-particle can be combined with mRNA through electrostatic interaction, can effectively introduce a target gene in the form of mRNA into tumor cells, and has the characteristics of low cytotoxicity, high transfection rate and the like.
The following examples are intended to illustrate specific embodiments of the present invention without limiting the scope of the invention to the examples.
The reagents used in the examples are all conventional commercial products.
Example 1 preparation of DMP-039 cationic nanoparticles
1. Preparation of DMP-Mal nanoparticles
Cationic lipid DOTAP, mPEG-PCL polymer (molecular weight 4000Da, PEG-PCL ═ 2000Da-2000Da) and Mal-PEG-PCL polymer (molecular weight 4000Da, PEG-PCL ═ 2000Da-2000Da) were mixed as 5: 45: 1, dissolving the mixture with dichloromethane, placing the mixture on a rotary evaporator, performing rotary evaporation at 60 ℃ for 45min, and forming a film. Taking out the formed membrane, dissolving the membrane in deionized water, oscillating the membrane for 5min under the condition of water bath at 60 ℃ to obtain DMP-Mal cationic polymer nanoparticle solution with specific concentration, and storing the DMP-Mal cationic polymer nanoparticle solution in a refrigerator at 4 ℃ for later use.
2. Preparation of DMP-039 nanoparticles
Dissolving the prepared DMP-Mal nano-particle aqueous solution with the concentration of 10mg/mL into Hepes buffer solution according to the mass ratio of the DMP-Mal nano-particle aqueous solution to the cRGD-R9 polypeptide of 30:1, uniformly mixing, and reacting at 4 ℃ overnight. After the reaction is finished, the mixed solution is put into a dialysis bag with the molecular weight of 2000Da, and the dialysis solution is distilled water and dialyzed overnight at the temperature of 4 ℃. The solution obtained after dialysis is the aqueous solution of DMP-039 nanoparticles, and is stored in a refrigerator at 4 ℃ for later use.
Example 2 preparation of IL-22BP mRNA/DMP-039 nanoparticle Gene complexes
1. Preparation of IL-22BP mRNA solution
The DNA fragment of IL-22BP gene was obtained by PCR amplification using pVAX1-IL-22BP plasmid as a template. Cutting off a fragment region gel with the same size as the target gene after agarose gel electrophoresis, and recovering the fragment by using a gel recovery kit to obtain a purified IL-22BP PCR product. Then, the T7 in vitro transcription kit is adopted, 0.5 mu g of IL-22BP gene PCR product is taken as a template, overnight transcription is carried out under the constant temperature condition of 37 ℃, and then 5 'end capping and 3' end tailing modification are carried out under the constant temperature condition of 37 ℃. And purifying the modified mRNA by using a transcription purification kit, and finally eluting by using deionized water to obtain an IL-22BP mRNA solution.
2. Preparation of IL-22BP mRNA/DMP-039 Complex
According to DMP-039 cationic nanoparticles: IL-22BP mRNA was expressed at 25: 1(w/w) adding the nanoparticle solution (dispersed in deionized water and having a concentration of 5mg/mL) into an mRNA solution (dispersed in deionized water and having a concentration of 1mg/mL), immediately blowing and uniformly mixing by using a pipette gun, and standing at room temperature for 15 minutes to obtain the DMP-039 cationic nanoparticle plasmid IL-22BP mRNA compound.
Example 3 preparation of Bim mRNA/DMP-039 nanoparticle Gene complexes
1. Preparation of Bim mRNA solution
The DNA fragment of the Bim gene is obtained by PCR amplification using pVAX1-Bim plasmid as a template. Cutting off a fragment region gel with the size consistent with that of the target gene after agarose gel electrophoresis, and recovering the fragment by using a gel recovery kit to obtain a purified Bim PCR product. Then, using T7 in vitro transcription kit, using 0.5. mu.g of Bim gene PCR product as template, carrying out overnight transcription at 37 ℃ constant temperature, and then carrying out 5 'end capping and 3' end tailing modification at 37 ℃. And purifying the modified mRNA by using a transcription purification kit, and finally eluting by using deionized water to obtain a Bim mRNA solution.
2. Preparation of Bim mRNA/DMP-039 Complex
According to DMP-039 cationic nanoparticles: bim mRNA was expressed at 25: 1(w/w) adding the nanoparticle solution (dispersed in deionized water and having a concentration of 5mg/mL) into an mRNA solution (dispersed in deionized water and having a concentration of 1mg/mL), immediately blowing and uniformly mixing by using a pipette gun, and standing for 15 minutes at room temperature to obtain the DMP-039 cationic nanoparticle plasmid Bim mRNA compound.
Example 4 preparation of IL-15mRNA/DMP-039 nanoparticle Gene complexes
1. Preparation of IL-15mRNA solution
The DNA fragment of IL-15 gene was obtained by PCR amplification using pVAX1-IL-15 plasmid as a template. Cutting off a fragment region gel with the same size as the target gene after agarose gel electrophoresis, and recovering the fragment by using a gel recovery kit to obtain a purified IL-15PCR product. Then, the in vitro transcription kit of T7 is adopted, 0.5 mu g of IL-15 gene PCR product is taken as a template, overnight transcription is carried out under the constant temperature condition of 37 ℃, and then 5 'end capping and 3' end tailing modification are carried out under the constant temperature condition of 37 ℃. And purifying the modified mRNA by using a transcription purification kit, and finally eluting by using deionized water to obtain an IL-15mRNA solution.
2. Preparation of IL-15mRNA/DMP-039 Complex
According to DMP-039 cationic nanoparticles: IL-15mRNA was expressed at 25: 1(w/w) adding the nanoparticle solution (dispersed in deionized water and having a concentration of 5mg/mL) into an mRNA solution (dispersed in deionized water and having a concentration of 1mg/mL), immediately blowing and uniformly mixing by using a pipette gun, and standing for 15 minutes at room temperature to obtain the DMP-039 cationic nanoparticle plasmid IL-15mRNA compound.
Test example 1 anti-colon cancer test of DMP-039 cationic nanoparticle/IL-22 BP mRNA gene complex of the present invention
In order to research the anti-tumor effect of the DMP-039 cationic nanoparticle/IL-22 BP mRNA gene complex in vivo, a colon cancer abdominal cavity metastasis tumor model is established in the abdominal cavity of a BalB/c mouse (female in 6-8 weeks old). C26 colon cancer cells cultured in vitro were trypsinized and aliquoted in serum-free, antibiotic-free DMEM medium, and the abdominal cavity of each mouse was inoculated with 2X 10 cells5Individual cells, 3 days after cell inoculation, randomized cohort treatment (5 per group) was started as follows:
A) blank control group: a 5% glucose solution;
B) blank vehicle control group: placing DMP-039 cationic nanoparticles in 5% glucose solution;
C) IL-22BP treatment group: the DMP-039 cationic nanoparticle/IL-22 BP mRNA gene complex was placed in a 5% glucose solution.
An intraperitoneal injection mode is adopted for treatment, and the DMP-039 cationic nanoparticle/IL-22 BP mRNA gene compound is prepared by the method of the embodiment 3, and the mixture ratio is as follows: mRNA: cationic nanoparticles ═ 1: 25(W/W), the cationic nanoparticle/mRNA complex was diluted in the glucose solution and adjusted so that the final glucose concentration was 5%. The injection volume of each mouse is 100 μ L, which contains 10 μ g of mRNA and 250 μ g of cationic nanoparticles. The administration is 1 time per day, and 16 times total treatment. After treatment, animals were sacrificed and dissected every other day, abdominal tumor tissue was isolated and weighed and tumor nodules counted. Tumor growth inhibition was analyzed by anova, and P <0.05 was considered statistically significant. The tumor weight and tumor nodule number of the above groups of animals are shown in FIG. 5, in which FIG. 5a shows the average tumor weight and FIG. 5b shows the average tumor nodule number.
As can be seen from FIG. 5, the DMP-039 cationic nanoparticle/IL-22 BP mRNA gene complex has slow tumor growth in the treatment group, but has faster tumor growth in the control group, and the DMP-039 cationic nanoparticle/IL-22 BP mRNA gene complex has extremely strong tumor growth inhibition effect, and the tumor inhibition rate reaches 77.5% compared with the blank control group.
Test example 2 anti-Breast cancer test of DMP-039 cationic nanoparticle/Bim mRNA Gene Complex of the present invention
In order to research the anti-tumor effect of the DMP-039 cationic nanoparticle/Bim mRNA gene complex in vivo, a breast cancer ectopic transplantation tumor model was established subcutaneously in BalB/c mice (6-8 weeks old, female). In vitro cultured 4T1 breast cancer cells were trypsinized and aliquoted in serum-free, antibiotic-free DMEM medium, and inoculated subcutaneously at 1.5X 10 per mouse6Individual cells, 5 days after cell inoculation, randomized cohort treatment (5 per group) was started as follows:
A) blank control group: a 5% glucose solution;
B) blank vehicle control group: placing DMP-039 cationic nanoparticles in 5% glucose solution;
C) bim treatment group: the DMP-039 cationic nanoparticle/Bim mRNA gene complex is placed in a 5% glucose solution.
An intratumoral injection mode is adopted for treatment, and the DMP-039 cationic nanoparticle/Bim mRNA gene compound is prepared by the method of the embodiment 3, and the mixture ratio is as follows: mRNA: cationic nanoparticles ═ 1: 25(W/W), the cationic nanoparticle/mRNA complex was diluted in the glucose solution and adjusted so that the final glucose concentration was 5%. The injection volume of each mouse is 100 μ L, which contains 10 μ g of mRNA and 250 μ g of cationic nanoparticles. The medicine is administered 1 time per day for 15 times. Tumor volume size was measured daily after treatment initiation. After treatment, animals were sacrificed and dissected every other day, subcutaneous tumor tissue was isolated and weighed. Tumor growth inhibition was analyzed by anova, and P <0.05 was considered statistically significant. The tumor weight and tumor growth curves of the above groups of animals are shown in FIG. 6, in which FIG. 6a shows the tumor growth curve and FIG. 6b shows the average tumor weight.
As can be seen from FIG. 6, the DMP-039 cationic nanoparticle/Bim mRNA gene complex treatment group has slow tumor growth, the control group has fast tumor growth, the DMP-039 cationic nanoparticle/Bim mRNA gene complex has extremely strong tumor growth inhibition effect, and the tumor inhibition rate reaches 69.9% compared with the blank control group.
Test example 3 anti-colon cancer lung metastasis tumor test of DMP-039 cationic nanoparticle/IL-15 mRNA gene complex of the present invention
In order to research the anti-tumor effect of the DMP-039 cationic nanoparticle IL-15mRNA gene complex in vivo, a lung metastasis tumor model was established in the lung of BalB/c mice (6-8 weeks old, female). C26 colon cancer cells cultured in vitro were trypsinized and aliquoted in serum-free, antibiotic-free DMEM medium, inoculated intravenously at 3X 10 per mouse5Individual cells, 1 day after cell inoculation, randomized cohort treatment (5 per group) was started as follows:
A) blank control group: a 5% glucose solution;
B) blank vehicle control group: placing DMP-039 cationic nanoparticles in 5% glucose solution;
C) IL-15 treatment group: the DMP-039 cationic nanoparticle/IL-15 mRNA gene complex was placed in a 5% glucose solution.
The treatment is carried out by tail vein injection, and the DMP-039 cationic nanoparticle/IL-15 mRNA gene compound is prepared by the method of the embodiment 3, and the mixture ratio is as follows: mRNA: cationic nanoparticles ═ 1: 25(W/W), the cationic nanoparticle/mRNA complex was diluted in the glucose solution and adjusted so that the final glucose concentration was 5%. The injection volume of each mouse is 100 μ L, which contains 10 μ g of mRNA and 250 μ g of cationic nanoparticles. The administration is 1 time per day for 13 times. Animals were sacrificed and dissected on day 3 after the end of treatment, lung tissue was isolated and weighed. Tumor growth inhibition was analyzed by anova, and P <0.05 was considered statistically significant. The lung weights and lung nodule counts for the above groups of animals are shown in FIG. 7, where FIG. 7a shows lung weights and FIG. 7b shows mean lung tumor nodule counts.
As can be seen from FIG. 7, the DMP-039 cationic nanoparticle/IL-15 mRNA gene complex treatment group has slow tumor growth, the control group has faster tumor growth, the DMP-039 cationic nanoparticle/IL-15 mRNA gene complex has extremely strong tumor growth inhibition effect, and the tumor inhibition rate reaches 67.1% compared with the blank control group.
The experimental results show that the DMP-039 cationic nanoparticle/IL-15 mRNA gene complex has a remarkable effect of resisting colon cancer lung metastasis tumors.
Claims (10)
1. A polypeptide polymer composite nanoparticle for mRNA delivery, characterized by: is prepared by modifying DMP-Mal nano-particles with cell-penetrating peptide cRGD-R9.
2. The polypeptide polymer composite nanoparticle for mRNA delivery according to claim 1, wherein: the average particle size of the polypeptide polymer composite nano-particles is 250nm, and the average potential is +20 mv.
3. The polypeptide polymer composite nanoparticle for mRNA delivery according to claim 1, wherein: the mass ratio of the cell-penetrating peptide cRGD-R9 to the DMP-Mal nanoparticles is as follows: 1-5: 20 to 60.
4. The polypeptide polymer composite nanoparticle for mRNA delivery according to claim 1, wherein: the DMP-Mal nanoparticle is prepared by cationic phospholipid DOTAP, mPEG-PCL polymer and Mal-PEG-PCl polymer together, wherein the mass ratio of the DOTAP and mPEG-PCL polymer to the Mal-PEG-PCL polymer is as follows: 5: 45: 1.
5. the method for preparing polypeptide polymer composite nanoparticles for mRNA delivery according to any one of claims 1 to 4, comprising the steps of:
weighing 45 parts of mPEG-PCL polymer, 5 parts of DOTAP and 1 part of Mal-PEG-PCL polymer, respectively dissolving the mPEG-PCL polymer, evaporating and volatilizing the solvent into a film by using a rotary evaporator, carrying out rotary evaporation on the film for 30min at 60 ℃, adding a proper amount of hydration solution to hydrate until the film is completely dissolved to obtain a DMP-Mal nanoparticle solution, mixing and incubating a proper amount of DMP-Mal nanoparticles and cRGD-R9 polypeptide, and dialyzing the reaction solution to obtain the polypeptide polymer composite nanoparticles for mRNA delivery.
6. Use of the polypeptide polymer composite nanoparticle for mRNA delivery according to any one of claims 1 to 4 for delivering mRNA molecules.
7. Use of the polypeptide polymer composite nanoparticle for mRNA delivery according to claim 6 in the delivery of mRNA molecules, characterized in that: the mRNA molecule is mRNA of Bim, mRNA of IL-22BP or mRNA of IL-15.
8. The polypeptide polymer composite nanoparticle for mRNA delivery according to any one of claims 1 to 4, wherein the polypeptide polymer composite nanoparticle is loaded with an mRNA molecule to obtain a composite.
9. Use of the complex of claim 8 for the preparation of a medicament for the prevention or treatment of a neoplastic disease.
10. Use according to claim 9, characterized in that: the tumor is colon cancer, lung cancer or breast cancer.
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