CN113925975A

CN113925975A - preparation method of p53 and UTX messenger RNA nanoparticles and application of messenger RNA

Info

Publication number: CN113925975A
Application number: CN202111011631.XA
Authority: CN
Inventors: 孔娜; 谢恬
Original assignee: Hangzhou Normal University
Current assignee: Hangzhou Normal University
Priority date: 2021-08-31
Filing date: 2021-08-31
Publication date: 2022-01-14

Abstract

The invention discloses a preparation method of p53 and UTX messenger RNA nanoparticles and application thereof in bladder cancer treatment, belonging to the technical field of genetic engineering. The tumor cancer suppressor gene messenger RNA nano-particle is prepared by the following method: 1) in vitro synthesis of chemically modified p53-mRNA and UTX-mRNA, 2) preparation of lipid polymer composite messenger RNA nanoparticles. In the invention, p53 and UTX messenger RNA are successfully delivered into bladder cancer cells through a nano system, and efficiently and rapidly induce apoptosis and inhibit the expression of transfer-promoting factors in vivo and in vitro, thereby obviously inhibiting the growth and the transfer of tumor cells; the RNA nanoparticle delivery system with the bladder mucosa adhesion function can increase the retention time of p53-mRNA and UTX-mRNA medicaments in the bladder, so that messenger RNA enters tumor cells to play an anti-tumor role.

Description

preparation method of p53 and UTX messenger RNA nanoparticles and application of messenger RNA

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a preparation method of p53 and UTX messenger RNA nanoparticles and application of messenger RNA.

Background

Genetic analysis of bladder cancer (BCa) revealed that tumor suppressor genes play an important role in the development of bladder cancer. Among these inactivated genes, the p53 gene, which has the ability to inhibit tumor growth, is deleted by up to 50%, accompanied by the deletion of another tumor suppressor gene, UTX, which encodes a histone demethylase named lysine-specific demethylase 6A (Kdm6A), an important epigenetic regulator, functioning as histone H3K27 demethylase. The mutation rate of UTX of bladder cancer is about 20% -29%, therefore, p53 and UTX gene and protein coded by the gene have important significance in BCa.

Non-viral vectors, liposomal reagents and viral vectors are currently the most commonly used tools for DNA transfection. However, (i) non-viral lipofectamine reagents can only be used in vitro (due to poor in vivo stability), and (ii) viral vector-based technologies may raise concerns such as low packaging capacity, high production costs, high immunogenicity, and potential environmental spread. In addition, DNA transfection methods may result in the risk of genomic integration and mutation. It would not be an easy task to design an alternative method that would effectively recover p53, UTX functionality, but avoid the above problems.

Here we provide an mRNA Nanoparticle (NP) -mediated strategy to target up-regulation of p53, UTX levels in BCa. This approach avoids the potential risk of deleterious integration into the host genome through insertional mutagenesis by viral vector or plasmid DNA, and mRNA can still express proteins rapidly and stably even in cells that are difficult to transfect or do not divide. Nanoparticles for delivery of mRNA were constructed from the FDA-approved polymer poly (lactic-co-glycolic) acid (PLGA) for protection and loading of mRNA/lipid complexes. A layer of lipid-polyethylene glycol (PEG) was used to further protect the NP core and stabilize the mRNA NP system under physiological conditions. There are no current studies reported using NPs to intravesically deliver mRNA to specific orthotopic bladder tumors. Here, we imparted our mRNA NPs with mucoadhesive properties through surface modification to deliver p53, UTX-mRNA efficiently within the bladder. Our results from different levels (including protein level, cell level, experimental animals) prove that p53 and UTX messenger RNA are successfully delivered into bladder cancer cells through a nano system, and cell apoptosis is efficiently and rapidly induced in vivo and in vitro and the expression of a transfer-promoting factor is inhibited, so that the growth and the transfer of tumor cells are obviously inhibited; and the RNA nanoparticle delivery system with the bladder mucosa adhesion function can increase the retention time of p53-mRNA and UTX-mRNA medicaments in the bladder, so that messenger RNA enters tumor cells to play an anti-tumor role.

Disclosure of Invention

Aiming at the bottleneck of traditional RNA delivery in the prior art, such as short in vivo circulation time, easy degradation by RNA enzyme, poor endosome escape capability and the like, the application aims to provide a preparation method of p53 and UTX messenger RNA nanoparticles and a technical scheme of application of the messenger RNA, the invention combines the advantages of polymer biomaterials (chemical composition, molecular structure and physical and chemical properties are easy to regulate) and multiple biological functions of polypeptide materials (tissue penetrability, organ and cell targeting, membrane penetrability and other functions), prepares a novel polymer adhesion property nanoparticle carrier for intravesical delivery of cancer suppressor genes p53-mRNA and UTX-mRNA, realizes first mRNA-based repair of cancer suppressor factors p53-mRNA and UTX-mRNA in bladder cancer tissues, and realizes obvious cancer proliferation and metastasis inhibition effects after the repair of p53-mRNA and UTX-mRNA in preclinical animal models of bladder cancer in situ .

The technical scheme effectively solves the potential gene mutation risk existing in the DNA treatment mode; and two international problems that small molecule drugs are ineffective under the condition of cancer suppressor gene deletion, can overcome the limitations of bladder cancer perfusion treatment (the problems of short retention time, drug adhesion failure, failure of sustained release and the like), and has huge clinical transformation potential.

The invention aims to disclose p53 and UTX messenger RNA nanoparticles.

The second purpose of the invention is to disclose the preparation method of the p53 and UTX messenger RNA nano-particle.

The third purpose of the invention is to disclose the application of the p53 and UTX messenger RNA nanoparticles in the treatment of bladder cancer.

The purpose of the invention is realized by the following technical scheme:

the preparation method of the p53-mRNA and UTX-mRNA nano-particle comprises the following steps:

1) in vitro synthesis of chemically modified p53-mRNA and UTX-mRNA

Human p53 carrying a T7 promoter and UTX gene open reading frame plasmid form linearized DNA, then a P53 and UTX open reading frame containing a T7 promoter are amplified by adopting PCR, and PCR products are purified to obtain purified polymerase chain reaction products;

reacting MEGAscript T7 transcription kit with purified polymerase chain reaction products, 3 ' -O-Me-m7G (5 ') ppp (5 ') G cap structure analogues, guanosine triphosphate, 5-methyl-cytidine triphosphate, adenosine triphosphate and pseudouridine-5 ' -triphosphate, carrying out deoxyribonuclease treatment after the reaction is finished, and adding a 3 ' poly (A) kit into an IVT RNA transcript by using a poly (A) kit to obtain p53/UTX messenger RNA;

p53/UTX messenger RNA is purified by MEGAclear kit, treated with thermosensitive phosphatase and further purified to obtain the required mRNA;

2) preparation of lipid polymer composite messenger RNA nanoparticles

Synthesizing cationic lipid G0-C14 by using PAMAM dendrimer G0 and 1, 2-epoxy tetradecane;

mixing the mRNA obtained in the step 1) with G0-C14 DMF by adopting an optimized self-assembly strategy to form an mRNA/G0-C14 complex, then quickly adding 250G of PLGA into 5mg/ml of DMF, and mixing with the complex to obtain a uniform mixed solution;

under magnetic stirring, dripping the mixed solution into 10 ml of nucleic acid-free HyPure water, wherein 1mg of DSPE-PEG-NH2, or 1mg of DSPE-PEG-NH2/DSPE-PEG-SH mixture, or 1mg of DSPE-PEG-SH is constructed to obtain a lipid PEG outer layer of NPs;

after self-assembly and stabilization, the formed NPs are washed by precooled enzyme-free pure water by an Amicon filter tube to remove free compounds and organic solvents, and the washed nanoparticle precipitate is resuspended into adhesive mRNA NPs with different concentrations by PBS to obtain p53-mRNA and UTX-mRNA nanoparticles.

Further, the chemical modification reaction system in the step 1) specifically comprises: the amount of the purified PCR product added was 1-2. mu.g, the amount of 3 '-O-Me-m 7G (5') ppp (5 ') G cap structural analog added was 6mM, the amount of guanosine triphosphate added was 1.5mM, the amount of 5-methylcytosine triphosphate added was 7.5mM, the amount of adenosine triphosphate added was 7.5mM, the amount of pseudouridine-5' -triphosphate added was 7.5mM, the reaction temperature was 37 ℃ and the reaction time was 4 hours.

Further, the temperature of the heat-sensitive phosphatase treatment in the step 1) is 37 ℃ and the treatment time is 30 minutes.

Further, in the step 2) self-assembly strategy, 16 μ G of mRNA and G0-C14250 μ G are mixed for 15s, wherein the concentration of the mRNA is 1mg/ml, and the concentration of G0-C14 is 2.5 mg/ml.

Further, the weight ratio of the DSPE-PEG-NH2/DSPE-PEG-SH mixture in the step 2) is 1: 1.

the p53-mRNA and UTX-mRNA nano-particles obtained by any method are applied to preparing the medicines for treating tumors.

Furthermore, the tumor treatment drug is a drug for recovering the cancer inhibition function of p53 and UTX.

Furthermore, the medicine for recovering the cancer inhibition function of p53 and UTX is specifically a medicine for recovering the cancer inhibition function of p53 and UTX in bladder cancer.

Furthermore, the medicine for treating tumor increases the retention time of p53-mRNA and UTX-mRNA medicine in bladder through RNA nano-particles with bladder mucosa adhesion function, so that messenger RNA enters tumor cells to play the role of anti-tumor.

The invention has the following beneficial effects:

1) in the invention, the p53 and UTX messenger RNA nanoparticles are successfully delivered to bladder cancer cells KU19-19 through a nano system, and efficiently and rapidly induce apoptosis and inhibit cancer cell metastasis, thereby obviously inhibiting the growth of bladder tumor cells;

2) the p53 and UTX messenger RNA nanoparticles have the advantages of overcoming the limitations of bladder cancer perfusion treatment (the problems of short retention time, incapability of adhering and sustained release of medicaments and the like), and efficiently recovering the cancer inhibition functions of p53 and UTX, thereby generating the effects of inhibiting tumor growth and inhibiting metastasis in vivo and in vitro experiments.

Drawings

FIG. 1 is a schematic diagram of the preparation process of messenger RNA nanoparticles and the intracellular biological process of the present invention;

FIG. 2 is a representation of messenger RNA nanoparticles with mucosal adhesion properties;

wherein, A: observing the forms of non-adhesive mRNA NPs and adhesive mRNA NPs (NPs-NH 2, NPs-NH2/SH and NPs-SH) by transmission electron microscope imaging, wherein the scale bar is 200 nanometers; b: confocal microscopy pictures of bladder walls of mice after 2-hour incubation of non-adhered mRNA NPs, adhered mRNA NPs-NH2, adhered mRNA NPs-NH2/SH or adhered mRNA NPs-SH, wherein mRNA is marked with Cy5 (red fluorescence), NPs are marked with FITC (green fluorescence), and the scale bar is 400 microns; c: confocal microscopy of the bladder wall of mice after 2h incubation with different mRNANPs followed by 3 h incubation in urine, mRNA labeled with Cy5 (red fluorescence), NPs labeled with FITC (green fluorescence) at scale bar: 400 μm;

FIG. 3 shows the biodistribution of mRNA NPs in xenograft tumor nude mice;

a: biodistribution image display of different organs (Lu: lung; K: kidney; H: heart; L: liver; T: tumor; S: spleen), B: quantitatively analyzing the biodistribution of free Cy5-mRNA and Cy5-mRNA NPs in mice from panel A;

FIG. 4 is a delivery efficiency test of a nanocarrier delivery system;

wherein, A: after incubation with Cy5-mRNA NPs (red) for various periods of time (1,3,6 hours), the bladder cancer KU19-19 cells were imaged by Confocal Laser Scanning Microscopy (CLSM). Endosome staining with LysoTracker Green (Green), nuclear staining with Hoechst 33342 (blue); b: flow Cytometry (FCM) analysis of the in vitro transfection efficiency of KU19-19 cells after treatment of different concentrations of EGFP-positive cells (% EGFP-positive cells) with unloaded NPs, free EGFP-mRNA or EGFP-mRNA NPs; C-D: bioluminescence imaging of luciferase protein expression following treatment of KU19-19 cells with different concentrations of empty NPs or Luc-mRNA NPs;

FIG. 5 shows cell proliferation assays before and after treatment of bladder cancer cells with p53/UTX-mRNA NPs, KU 19-19;

a relative proliferation of KU19-19 cells after treatment with unloaded NPs, EGFP-mRNA NPs or p53/UTX-mRNA NPs. Untreated cells were not in control; b: colony formation assay after treatment of KU19-19 cells with different concentrations of unloaded NPs, EGFP-mRNA NPs or p53/UTX-mRNA NPs in 6-well plates. Untreated cells were not in control; c: quantitative analysis of the colony formation experiment;

FIG. 6 shows the effect of bladder perfusion treatment of tumor by p53/UTX-mRNA NPs and its mechanism;

wherein, A: immunohistochemical IHC detection of tumor apoptosis marker (activated caspase3 protein, namely CC 3) and proliferation inhibition (Ki67 and PCNA proliferation marker); b: collecting metastatic lymph nodes in the mice after different treatments; c: counting the mouse metastatic lymph nodes;

FIG. 7 is inhibition of metastasis following bladder perfusion treatment with p53/UTX-mRNA NPs;

wherein, A: detecting the expression of an apoptosis factor CC3 and a metastasis related protein N-cadherin of the mouse orthotopic bladder cancer after the treatment of unloaded NPs by in vivo immunofluorescence, wherein the KU19-19 mouse orthotopic bladder cancer is subjected to in vivo immunofluorescence; b: in vivo immunofluorescence assay KU19-19 mouse in situ bladder cancer model expression of apoptosis factor CC3 and metastasis associated protein N-cadherin after treatment with p53/UTX-mRNA NPs.

Detailed Description

In order to facilitate the understanding of the technical scheme of the invention, the preparation method of p53 and UTX messenger RNA nanoparticles and the application thereof in the treatment of bladder cancer are further described in the following by combining specific examples and experimental examples.

Materials:

l-cystine dimethyl ester dihydrochloride ((H-cys-ome) 2.2 HCl), trimethylamine, cationic ethylenediamine Polyamidoamine (PAMAM) generation 0 dendrimer (G0), and fatty acid dichloride were from Sigma-Aldrich.

Dimyristoylphosphatidylethanolamine-ethylene glycol having a polyethylene glycol Molecular Weight (MW) of 2000 and distearoylphosphatidylethanolamine-ethylene glycol having a polyethylene glycol Molecular Weight (MW) of 5000 are available from Avanti Polar Lipids.

Lipofectamine 2000 (Lip 2 k) was purchased from Invitrogen, USA, (Calsbad, Calif.).

TriLink Biotechnology, Inc. (TriLink Biotechnologies) purchased enhanced Green fluorescent protein messenger RNAEGFPmRNA (modified with 5-methylcytidine and pseudouridine) and cy5-Fluc messenger RNA (control cy5 labeled messenger RNA) from CleanCap ™.

The main antibodies used in immunohistochemical experiments included: p53 antibody (Santa Cruz, sc-126; 1:500 dilution), UTX (GeneTex Cat # GTX121246, RRID: AB _10722382, 1:200 dilution), Cleaved-Caspase3 antibody (Cell Signaling Technology, # 9661; 1:500 dilution). GAPDH antibody was obtained from Cell Signaling Technology (Cell Signaling Technology, 5174; 1:2000 dilution), beta-Actin antibody (Cell Signaling Technology; 1:2000 dilution), anti-rabbit and anti-mouse horseradish peroxidase (HRP) conjugated secondary antibodies. The second antibody used for confocal laser scanning microscope experiments includes: alexa Fluor 488 labeled goat anti-rabbit IgG (Life technologies, A-11034) and Alexa Fluor 647 labeled goat anti-mouse IgG (Life technologies, A-28181). The cationic lipid compound G0-C14 was prepared by ring-opening reaction of 1, 2-epoxytetradecane with G0 according to the above method (38). As described in our previous studies, hydrophobic dithioamide polymers were synthesized by a one-step polycondensation of (H-Cys-OMe) 2.2 HCl and fatty acid dichloride and characterized by NMR with mercury VX-300 spectrometer at 400 MHz (Wailan, USA).

Cell line:

human bladder cancer cell line KU19-19(DSMZ Cat # ACC-395, RRID: CVCL _ 1344)) was purchased from DSMZ cell bank and RPMI-1640 medium was used to culture KU19-19 cells. 1% penicillin/streptomycin antibiotic (Thermo-Fisher Scientific) and 10% fetal bovine serum (FBS; Gibco @) were added to the cell culture medium.

Example 1: preparation of p53 and UTX messenger RNA nano-particle

In vitro synthesis of chemically modified p53-mRNA and UTX-mRNA:

enhanced Green Fluorescent Protein (EGFP) and p53/UTX messenger RNA (mRNA) were synthesized using in vitro transcription technology (IVT). An untranslated region (UTR) is designed at the 5' end of the RNA to enhance translation initiation of the mRNA. To improve its stability and translation efficiency, an inverted cap Analog (ARCR) was further applied to the 5' end of the mRNA. To avoid immune stimulation by mRNA, conventional cytidine triphosphate and uridine triphosphate are replaced with 5-Methyl-cytidine triphosphate (5 '-Methyl-CTP) and pseudouridine-5' -triphosphate (Pseudo-UTP). The specific process is as follows:

the human p53/UTX gene Open Reading Frame (ORF) plasmid carrying the T7 promoter was purchased from Addgene. The p53/UTX open reading frame containing the T7 promoter was amplified by Polymerase Chain Reaction (PCR) and PCR product purification was performed according to the purification kit. For In Vitro Transcription (IVT), MEGAscript T7 transcription kit (Ambion) was used with 1-2 μ G of purified polymerase chain reaction product (i.e., template), 6mM 3 ' -O-Me-m7G (5 ') ppp (5 ') G cap analog (anti-inverted cap analog, ARCA), 1.5mM guanosine triphosphate, 7.5mM 5-Methyl-cytidine triphosphate (5 ' -Methyl-CTP), 7.5mM adenosine triphosphate and 7.5mM pseudouridine-5 ' -triphosphate (Pseudo-UTP) (TriLink Biotech, Inc.). The reaction was carried out at 37 ℃ for 4 hours, followed by DNase treatment. Subsequently, a 3' poly (a) kit was added to the IVT RNA transcripts using the poly (a) kit (Ambion). Messenger RNA from p53/UTX was purified using the MEGAclear kit (ambion) and then treated with a heat-sensitive phosphatase (New England Biolabs, USA) at 37 ℃ for 30 minutes, followed by further purification to give the desired mRNA product.

Electrostatic complexation of G0-C14 with messenger RNA:

to evaluate the complexation of cationic compound G0-C14 with messenger RNA, electrophoretic studies were performed on naked p53/UTX messenger RNA or p53/UTX messenger RNA with G0-C14 (weight ratio of G0-C14/messenger RNA: 0.1, 1, 5, 10, 15 and 20) using E-Gel prefabricated agarose gels (Invitrogen). To assess the stability of messenger RNA in organic solvent (DMF), naked messenger RNA was incubated with organic solvent for 30 minutes, then dropped into agarose gel, the gel was imaged under UV light, and bands from each set were analyzed.

preparation of p53/UTX messenger RNA nanoparticles, i.e., lipopolymer-complexed messenger RNA nanoparticles:

PAMAM dendrimer G0 and 1, 2-epoxy tetradecane synthesize cationic lipid G0-C14 with a molar ratio of 1:7, and 16 mu G of mRNA (1 mg/ml) and 250 mu G G0-C14 (2.5mg/ml) DMF are lightly mixed for 15s by adopting an optimized self-assembly strategy to be completely electron-adsorbed to form mRNA/G0-C14 complex. Subsequently, 250. mu.g PLGA was rapidly added to DMF (5mg/ml) and mixed with the complex to give a homogeneous solution; under the magnetic stirring of 1000 rpm, the mixed solution is finally dropped into 10 ml of nucleic acid-free Hypure water, wherein 1mg of DSPE-PEG-NH2, or 1mg of DSPE-PEG-NH2/DSPE-PEG-SH mixture (weight ratio is 1:1), or 1mg of DSPE-PEG-SH is used for constructing the lipid PEG outer layer of NPs, and the general preparation process is shown in FIG. 1.

The method specifically comprises the following steps:

1) synthesizing cationic lipid G0-C14 by using PAMAM dendrimer G0 and 1, 2-epoxy tetradecane, wherein the molar ratio is 1: 7;

2) using an optimized self-assembly strategy, 16. mu.g of mRNA (1 mg/ml) was gently mixed with 250. mu. G G0-C14 (2.5mg/ml) DMF for 15s for complete electron adsorption to form the mRNA/G0-C14 complex. Subsequently, 250. mu.g PLGA was rapidly added to DMF (5mg/ml) and mixed with the complex to give a homogeneous solution;

3) under the magnetic stirring of 1000 r/min, finally dropping the mixed solution into 10 ml of nucleic acid-free HyPure water, wherein 1mg of DSPE-PEG-NH2, or 1mg of DSPE-PEG-NH2/DSPE-PEG-SH mixture (weight ratio is 1:1), or 1mg of DSPE-PEG-SH is used for constructing a lipid PEG outer layer of NPs;

4) after 30 minutes of self-assembly stabilization, the formed NPs were washed with pre-cooled, enzyme-free pure water using an Amicon filter tube to remove free compounds and organic solvents. The washed nanoparticle precipitate was resuspended in PBS to form adherent mRNA NPs of different concentrations for in vitro and in vivo experiments.

Characterization of synthetic messenger RNA nanoparticles:

experimental materials and corresponding reagents: l-cystine dimethyl ester dihydrochloride ((H-cys-ome) 2.2 HCl), trimethylamine, cationic ethylenediamine Polyamidoamine (PAMAM) generation 0 dendrimer (G0), and fatty acid dichloride were from Sigma-Aldrich. Dimyristoylphosphatidylethanolamine-ethylene glycol having a polyethylene glycol Molecular Weight (MW) of 2000 and distearoylphosphatidylethanolamine-ethylene glycol having a polyethylene glycol Molecular Weight (MW) of 5000 are available from Avanti Polar Lipids. TriLink Biotechnology, Inc. (TriLink Biotechnologies) purchased enhanced green fluorescent protein messenger RNA, EGFPMRNA (modified with 5-methylcytidine and pseudouridine) and cy5-Fluc messenger RNA (control cy5 labeled messenger RNA) from CleanCap ™.

The experimental procedure was as described above for the specific preparation.

The experimental results are as follows: we measured the size and stability of phosphate buffer at 37 ℃ in 24 hours and messenger RNA nanoparticles after urine wash using dynamic light scattering (DLS, Bruk Haiwen instruments Inc., USA) (as shown in FIGS. 2A, B, C, where A: Transmission Electron microscopy image for morphology of non-adherent mRNA NPs and adherent mRNA NPs (NPs-NH 2, NPs-NH2/SH, NPs-SH) B: non-adherent mRNA NPs, adherent mRNA NPs-NH2, adherent mRNA NPs-NH2/SH or adherent mRNA NPs-SH 2 hours after incubation.B: mouse bladder wall confocal microscopy images after incubation.Cy 5 for mRNA (red fluorescence), FITC for NPs (green fluorescence). C: mouse bladder wall confocal microscopy images 2 hours after incubation with different mRNA NPs for 2 hours, followed by Cy5 for mRNA 3 hours in urine solution (red fluorescence), NPs were labeled with FITC (green fluorescence). The synthesized adhesive nanoparticles can effectively overcome the problems of limitations (short retention time, incapability of adhering and releasing medicaments) of bladder cancer perfusion treatment, so that mRNA medicaments can effectively act on tumor parts. The invention discloses that the functional NPs are adhesive NPs which can be adhered in the bladder and continuously transmit p53/UTX-mRNA NPs at the tumor site. The abundant functional groups (such as amine and sulfydryl) on the surface of the drug delivery system can effectively promote the adhesion to the surface of bladder mucosa and realize the delivery in the bladder. Therefore, in the present invention, instead of using DSPE-PEG-nh 8926 (100%) to achieve systemic delivery of long circulating mRNA NPs, instead of using DSPE-PEG-nh2(100%), we used DSPE-PEG-nh2(100%), DSPE-PEG-nh2/DSPE-PEG-sh hybrid (50% vs 50%) or DSPE-PEG-sh (100%) to coat NPs to make them adhesive. As can be seen from the TEM image (FIG. 2A), as the SH-terminated surface modification ratio increases, the obtained adherent mRNA NPs have a tendency to aggregate (i.e., the degree of aggregation: mRNA NPs-SH > messenger rn a NPs-NH2/SH ratio; mRNA NPs-NH 2), as compared with non-adherent mRNA NPs. To further assess the adhesion of different surface functionalized mRNA NPs, we performed double staining experiments on mouse bladder tissue after different treatments of these mRNA NPs. Fluorescein (FITC) -labeled PLGA and cy 5-labeled mRNA were used to generate these different NPs, so we could track both NPs simultaneously with the green signal and mRNA simultaneously with the red signal. PBS containing Dual-labelled NPs (i.e., non-mucosal mRNA NPs, mucosal mRNA NPs-NH2, mucosal mRNA NPs-NH2/SH or mucosal mRNA NPs-SH) were used to incubate the mouse at 37 ℃ for 2 h. After simple PBS washing, mRNA NPs with different signals were observed for sample morphology in the bladder wall (fig. 2B). The signals of the three adhesive mRNA NPs on the bladder wall are higher than those of the non-adhesive mRNA NPs. Meanwhile, the bladder wall treated with mRNA NPs-sh showed the highest signal among the three adhering mRNA NPs. To better mimic the activity of the bladder after treatment, we incubated the bladder with urine for another 3 hours at 37 ℃ and then washed with simple PBS, respectively, after the same treatment procedure with different NPs (fig. 2C). We can observe that non-adherent mRNA NPs are easily washed by urine, since almost all signals are cleared from the bladder wall. However, all three adherent mRNA NPs retain detectable signal on the bladder wall. The mRNA NPs-sh showed the highest adhesion and uptake ability among 3 kinds of adhesion mRNA NPs. These results can be explained by the fact that thionps are capable of forming-s-s-bonds with cysteine-rich domains in mucus glycoproteins, which covalent interactions are much stronger than non-covalent ones (e.g. van der waals forces, hydrogen bonding, ionic interactions with mucus layer anionic substructures). Since mRNA NPs-SH showed the best adhesion properties on the bladder, we used these mRNA NPs-SH in subsequent studies.

Experimental example 1: nanoparticle Biodistribution (BD) study and quantitative detection of delivery efficiency

Nanoparticle Biodistribution (BD) study:

the experimental method and the steps are as follows: in vivo pharmacokinetic studies, healthy BALB/c mice (6 weeks old, n =3 per group) were injected with free Cy5-mRNA and Cy5-mRNA NPs. Blood was taken via retro-orbital venous blood at various predetermined time intervals (0, 0.5, 1,2, 4, 8, 12 and 24 hours). The wound was gently pressed for one minute to stop bleeding. The fluorescence intensity of Cy5-mRNA was measured with a microplate reader. After background removal, pharmacokinetics were assessed by measuring the percentage of cy5 mRNA in blood at these time points, after normalization with the initial time points (0 hours). In the biodistribution study, KU19-19 tumorigenic nude mice (n = 3/group) were injected with free Cy5-mRNA and Cy5-mRNA NPs (mRNA dose at 750 μ g/kg animal body weight). After 24 hours, all mice were sacrificed and organ and tumor distribution was observed using Syngene multiple imaging system pxi (synoptics ltd), england.

The experimental results are as follows: the depegylation mediated by serum albumin plays a key role in the cellular uptake, Pharmacokinetics (PK) and tumor accumulation of the mixed lipid polymer nanoparticles. We examined the biodistribution (BioD) and tumor accumulation of these nanoparticles, and FIG. 3 Biodistribution (BD) study results, A: different organ biodistribution image display (Lu: lung; K: kidney; H: heart; L: liver; T: tumor; S: spleen). B: the biodistribution of free Cy5-mRNA and Cy5-mRNA NPs in mice was quantitatively analyzed from panel A. The fluorescent signal of naked Cy5-mRNA was barely detectable in the tumors 24 hours after injection.

Quantitative detection of delivery efficiency of green fluorescent EGFP messenger RNA nanoparticles in cytoplasm:

experimental materials and corresponding reagents TriLink Biotechnology, Inc. (TriLink Biotechnologies) purchased enhanced Green fluorescent protein messenger RNA EGFPMRNA (modified with 5-methyl-cytidine triphosphate and pseudouridine-5' -triphosphate) and cy5-Fluc messenger RNA (control cy5 labeled messenger RNA) from CleanCap ™. Human bladder cancer cell line KU19-19(DSMZ Cat # ACC-395, RRID: CVCL _ 1344)) was purchased from DSMZ cell bank and RPMI-1640 medium was used to culture KU19-19 cells. 1% penicillin/streptomycin antibiotic (Thermo-Fisher Scientific) and 10% fetal bovine serum (FBS; Gibco @) were added to the cell culture medium.

The experimental steps are as follows: KU19-19 cells were plated evenly in 96-well plates at a density of 5000 cells per well. After 24 hours of cell attachment, cells were transfected with different concentrations (0.102, 0.207, 0.415, or 0.830 μ g/ml) of enhanced green fluorescent protein messenger RNA for 24 hours, then 0.1ml of fresh complete medium was added and further cultured for 24 hours to assess cell viability and transfection efficiency. Lip2k was used as a positive control for transfection efficiency compared to nanocarriers. Cell viability was tested by AlarmaBlue, a non-toxic cell viability assay that can examine real-time cell proliferation by a microplate reader (TECAN, Infinite M200 Pro). Absorbance was measured at 545 nm and 590 nm using a 96-well Spectramax plate reader (molecular devices, Senyvale, Calif.). To determine the transfection efficiency of cells, cells were treated with nanoparticles or enhanced green fluorescent protein messenger RNA of Lip2k for 24 hours, detached with 2.5% ethylenediaminetetraacetic acid (EDTA) trypsin and collected in phosphate buffer solution, and then Evaluated for Green Fluorescent Protein (EGFP) expression using a flow cytometer (haddock Biosystems, germany). The percentage of cells transfected with enhanced green fluorescent protein positive was calculated using Flowjo software.

The experimental results are as follows: to further validate the effectiveness of in vitro transfection, we selected enhanced green fluorescent protein mRNA (EGFP-mRNA) as the model mRNA. Wherein FIG. 4B is the enhancement of the transfection efficiency of the Green fluorescent protein messenger RNA (EGFPmRNA) nanoparticles, FIG. 4A is the nanoparticles' effective delivery of cy 5-labeled mRNA into the cytoplasm and is time-dependent, and most of the mRNA nanoparticles first co-localize with lysoTracker Green (LysoTracker Green) 1 hour after entering the cell (see FIG. 4A, wherein A: confocal microscopy observes that the nanoparticles deliver cy 5-labeled mRNA into the cytoplasm). After 3 hours of culture, a part of the cy 5-labeled mRNA entered the cytoplasm, and after 6 hours of culture, a large amount of cy 5-labeled mRNA escaped from the lysosome and diffused into the cytoplasm. In contrast, naked mRNA was unable to enter cells after 6 hours of culture. Wherein 4B: the intracellular delivery efficiency of the composite nanoparticles to EGFPMRNA was detected in KU19-19 cells by flow cytometry.

The p53/UTX messenger RNA nanoparticle and the application thereof are explained in detail by specific experimental examples below.

Experimental example 2: nano-carrier delivery of p53/UTX-mRNA in bladder cancer cell KU19-19 for recovering p53 and UTX cancer inhibition function

The experimental method and the steps are as follows:

1) and (3) detecting cell proliferation: KU19-19 cells were plated evenly in 96-well plates at a density of 2000 cells/well. PBS, unloaded nanoparticles, EGFP-mRNA NPs, p53/UTX-mRNANPs were added 24 hours after cell adherence. After further culturing for 1,2, 3, 4, 5 and 6 days, the cell proliferation capacity was continuously measured by Alarma Blue.

2) Cell colony formation assay: the proliferation potency of the cells was determined by soft agar colony formation. Cells were treated with PBS, unloaded nanoparticles, EGFP-mRNA NPs, p53/UTX-mRNANPs, respectively, for 48 hours. Cells were then suspended in 0.36% agarose (Invitrogen), diluted in complete medium, and then re-seeded at low density (about 1000 cells per well) into 6-well plates containing 0.75% agarose pre-formed layers for 2 weeks of culture. The plates were then washed with phosphate buffer, fixed in 4% paraformaldehyde for 20 minutes, and then stained with 0.005% crystal violet. The image is scanned and analyzed.

The experimental results are as follows: in order to research the function that the nano-carrier mediated p53/UTX-mRNA can effectively recover tumor suppressor factors p53 and UTX, a cell proliferation Alarma Blue method is adopted to detect the proliferation capacity of bladder cancer KU19-19 cells after the p53/UTX-mRNA nanoparticles are treated (see figure 5A), and at the same time, whether the p53/UTX-mRNA nanoparticles have the clone formation inhibition effect on tumor cells KU19-19 is detected, after the p53/UTX-mRNA nanoparticles are used for treatment, compared with an empty nanoparticle and a non-functional EGFP-mRNA nanoparticle treatment group, the cell colony formation of the p53/UTX-mRNA nanoparticles is also obviously inhibited (see figure 4B, C, wherein B, the inhibition of the p53/UTX-mRNA on the clone formation of the KU19-19 is observed in a cell colony formation test, C, the inhibition efficiency of the clone formation after the p53/UTX-mRNA nanoparticles with different concentrations are treated is counted), further proves that the p53/UTX gene complementation mediates a remarkable tumor proliferation inhibition function, in KU9-19 cells, the cell proliferation of p53/UTX-mRNA nanoparticles is remarkably inhibited under the action of the concentration of 0.8 mu g/ml, and the proliferation inhibition effect is not caused by the empty nanoparticles and the EGFP-mRNA.

Experimental example 3: the bladder perfusion mucosa adhesion type p53/UTX-mRNA NPs remarkably inhibit the tumor growth of mouse orthotopic bladder cancer and inhibit adjacent lymphatic metastasis

The experimental method and the steps are as follows:

1. constructing a mouse orthotopic bladder cancer model: 8-week-old female BALB/c nude mice are used for establishing nude mice in-situ human bladder cancer models. KU19-19 cells were cultured to logarithmic phase, digested with 0.25% trypsin and washed 3 times with PBS, and the cell concentration was adjusted to 1 x 107/ml with 1% FBS-containing RPMI1640 medium for use. Female BALB/c nude mice were anesthetized with 0.6% sodium pentobarbital intraperitoneal injection at a dose of 60mg/kg and then catheterized with a venous indwelling needle under aseptic procedure. The specific method comprises the following steps: (1) disinfecting the urethral orifice and the surrounding skin of the mouse by iodophor, clamping the soft tube at the side of the venous indwelling needle, and withdrawing the needle core by about 3mm outwards; (2) the needle tube is coated with sterile paraffin oil and then slowly inserted into the urethral orifice of a mouse, if resistance is met, the direction is adjusted and then the needle tube enters, and if resistance-free propulsion is about 1.2cm, the needle tube can enter the bladder cavity; (3) after the mice are successfully intubated, the needle core is pulled out by fixing the needle tube, a 1ml syringe is connected, and then the bladder perfusion operation of the mice can be carried out, all the mice are firstly irrigated for 1 time by 0.1ml PBS, the urine is washed away, then the bladder mucosa is pretreated, 0.1ml0.1mol/L HCl is irrigated to act for 20s and then is extracted, then 0.1ml0.1mol/L NaOH is irrigated into the bladder, about 5s later all the liquid is extracted, 0.1ml KU19-19 cells are irrigated into the bladder to carry out the tumor cell planting, and the vein indwelling needle is left for about 2 h.

2. Bladder perfusion treatment of p53/UTX-mRNA NPs: bladder perfusion therapy with p53/UTX-mRNA NPs was initiated on day 3 after the infusion of KU19-19 bladder cancer cells, once every three days for 5 consecutive times. Carrying out intraperitoneal injection of 0.6% sodium pentobarbital at the dose of 60mg/kg for anesthesia, then carrying out urethral catheterization by using a venous indwelling needle under the aseptic operation (the method is the same as the above), irrigating for 1 time by using 0.1ml PBS, washing the urine, infusing 0.1ml p53/UTX-mRNA NPs into the bladder, and indwelling the venous indwelling needle for about 2 hours, once every three days and continuously for 5 times.

3. Preparation of HE stained paraffin sections: after the treatment of different groups, dead mice or euthanized mice were dissected in time and bladder tumor tissues, kidney, heart, liver, spleen, lung and lymph node tissues of systemic metastases were retained. Fixing: generally, the tissue thickness is not more than 0.5 cm, and the tissue and cell protein is denatured and coagulated in a prepared fixative (10% formalin, Bouin's fixative) to prevent autolysis or bacterial decomposition after cell death, thereby maintaining the original morphological structure of the cell; and (3) decoloring: gradually removing water from the tissue block by using low-concentration to high-concentration alcohol as a dehydrating agent. Placing the tissue block in a transparent agent xylene which is soluble in alcohol and paraffin for transparency, replacing the alcohol in the tissue block with xylene, and performing later-stage wax dipping and embedding; wax dipping and embedding: placing the transparent tissue block into melted paraffin, placing the tissue block into a paraffin dissolving box for heat preservation, and embedding after the paraffin is completely immersed into the tissue block: preparing a container, pouring melted paraffin, quickly clamping tissue blocks soaked with paraffin, putting the tissue blocks into the container, cooling and solidifying the tissue blocks into blocks, and hardening the embedded tissue blocks; slicing: the embedded wax blocks are fixed on a microtome and cut into thin sections, typically 5-8 microns thick. The cut slices are often folded, put into heated water for ironing, then pasted on a glass slide, put into a thermostat at 45 ℃ for drying, dewaxed and dyed in the later stage, and then HE pathological sections are made.

4. Immunohistochemical IHC staining: preparation of tissue sections IHC staining was performed as described above, with the following staining procedure: (1) the sections were washed twice with 5 minutes each time with rinse solution; (2) soaking in 3% H202/methanol at room temperature, and incubating for 10 min; (3) washing with rinsing solution twice, each time for 5 minutes; (4) dropwise adding 1% FBS into the section, and sealing for 1 hour at room temperature; (5) diluting antibodies CC3, Ki67 and PCNA in a blocking solution according to the ratio of 1 to 200; (6) removing the blocking solution from the sections, adding 100-400 ul of diluted primary antibody to each section, and incubating overnight at 4 ℃; (7) removing antibody, washing with rinsing solution for 5 min for 3 times; (8) washing with rinsing solution for 5 min for 3 times; (9) adding a secondary antibody dropwise at 37 ℃, incubating for 10-30 minutes, and washing for 5 minutes x3 times by PBS; (10) the developer develops color for 3-15 minutes (DAB or NBT/BCIP). To test the therapeutic effect of p53/UTX-mRNA NPs, IHC used the apoptosis marker CC3, as well as the tumor proliferation markers Ki-67 and PCNA.

The experimental results are as follows: in this section, the messenger RNA NPs complement p53/UTX, so that the mouse orthotopic bladder cancer tumor can be effectively inhibited in proliferation and metastasis. We established an orthotopic bladder tumor model in BALB/c nude mice and used mRNA NPs with mucoadhesive properties for intravesical acid infusion therapy, which is the most common administration route for current clinical treatment BCa (i.e. drug injection into the bladder through a catheter). However, (i) almost all of the drug in the bladder is usually washed away quickly on the first urination, and (ii) BCa tumors have a low chance of exposure to the drug, which greatly limits the effectiveness of this treatment. Therefore, it is important to prolong the retention time of the therapeutic NPs in the bladder and improve the intake of the therapeutic NPs into BCa tissues, and the invention invents the functional NPs as adhesive NPs which can be adhered in the bladder and continuously deliver p53/UTX-mRNA NPs at the tumor site. The abundant functional groups (such as amine and sulfydryl) on the surface of the drug delivery system can effectively promote the adhesion to the surface of bladder mucosa and realize the delivery in the bladder. Therefore, in the present invention, instead of using DSPE-PEG-nh 8926 (100%) to achieve systemic delivery of long circulating mRNA NPs, instead of using DSPE-PEG-nh2(100%), we used DSPE-PEG-nh2(100%), DSPE-PEG-nh2/DSPE-PEG-sh hybrid (50% vs 50%) or DSPE-PEG-sh (100%) to coat NPs to make them adhesive. As can be seen from the TEM image (FIG. 2A), as the SH-terminated surface modification ratio increases, the obtained adherent mRNA NPs have a tendency to aggregate (i.e., the degree of aggregation: mRNA NPs-SH > messenger rn a NPs-NH2/SH ratio; mRNA NPs-NH 2), as compared with non-adherent mRNA NPs. To further assess the adhesion of different surface functionalized mRNA NPs, we performed double staining experiments on mouse bladder tissue after different treatments of these mRNA NPs. Fluorescein (FITC) -labeled PLGA and cy 5-labeled mRNA were used to generate these different NPs, so we could track both NPs simultaneously with the green signal and mRNA simultaneously with the red signal. PBS containing Dual-labelled NPs (i.e., non-mucosal mRNA NPs, mucosal mRNA NPs-NH2, mucosal mRNA NPs-NH2/SH or mucosal mRNA NPs-SH) were used to incubate the mice at 37 ℃ for 2h of bladder. After simple PBS washing, mRNA NPs with different signals were observed for sample morphology in the bladder wall (fig. 2B). The signals of the three adhesive mRNA NPs on the bladder wall are higher than those of the non-adhesive mRNA NPs. Meanwhile, the bladder wall treated with mRNA NPs-sh showed the highest signal among the three adhering mRNA NPs. To better mimic the activity of the bladder after treatment, we incubated the bladder with urine for another 3 hours at 37 ℃ and then washed with simple PBS, respectively, after the same treatment procedure with different NPs (fig. 2C). We can observe that non-adherent mRNA NPs are easily washed by urine, since almost all signals are cleared from the bladder wall. However, all three adherent mRNA NPs retain detectable signal on the bladder wall. The mRNA NPs-sh showed the highest adhesion and uptake ability among 3 kinds of adhesion mRNA NPs. These results can be explained by the fact that thionps are capable of forming-s-s-bonds with cysteine-rich domains in mucus glycoproteins, which covalent interactions are much stronger than non-covalent ones (e.g. van der waals forces, hydrogen bonding, ionic interactions with mucus layer anionic substructures). Since mRNA NPs-SH exhibit the best adhesion properties on the bladder, these mRNA NPs-SH are used in vivo animal models.

KU19-19 bladder cancer cells are injected into the bladder of an immunodeficient nude mouse through the urethra to establish an orthotopic bladder tumor model. Tumor growth and adjacent tissue metastasis were monitored by assessing the mean brightness of the tumor site by bioluminescence imaging. After 3d, mice were randomized into 3 groups, and each 3d bladder was injected with PBS, empty NPs-SH or p53/UTX-mRNA NPs-SH. Compared with the control group (PBS) and the control group NP (empty NPs-SH), the p53/UTX-mRNA NPs-SH intravesical treatment can effectively reduce the burden of in situ tumors, obviously induce the expression of a tumor apoptosis factor CC3, reduce the levels of tumor proliferation factors Ki67 and PCNA (figure 6A) and reduce the systemic lymph node metastasis (figure 6B, C). The p53/UTX-mRNA NPs-SH treatment was further verified to inhibit tumor metastasis in situ by counting the number of macroscopic lymph node metastatic nodes for each group of mice. Mice intravesically injected with p53/UTX-mRNA NPs-SH exhibited a reduced number of macroscopic perilymphatic metastases compared to the control (PBS) and control NP (empty NPs-SH) groups (FIG. 6B, C). Therefore, the experimental result shows that the p53/UTX protein level can be effectively inhibited by adhering mRNA NPs to the tumor proliferation and lymphatic metastasis of the tumor-bearing mice.

Experimental example 4: mechanism research of bladder perfusion type p53/UTX-mRNA nanoparticle for inhibiting proliferation and metastasis of bladder cancer

The experimental method and the steps are as follows:

3. Immunofluorescence (IF) staining: tissues were fixed with 4% paraformaldehyde (Electron Microcopy Sciences) for 15 min at room temperature and then infiltrated in 0.2% Polyethyleneglycol octylphenyl ether (Triton X-100) -phosphate buffer for 10 min. The samples were further incubated with phosphate-blocked buffer (containing 2% bovine serum albumin, 2% normal goat serum and 0.2% gel) for 30 minutes at room temperature. Subsequently, fixation was performed with 4% paraformaldehyde (Electron Microcopy Sciences) for 15 minutes. The primary antibody was incubated overnight at 4 ℃, washed with phosphate buffer, and then incubated with Alexa Fluor 647-labeled goat anti-mouse IgG (molecular probes) in blocking buffer (1: 1000 dilution) for 60 minutes at room temperature. Finally, the stained sample was washed with phosphate buffer, and the cell nucleus was stained with Hoechst 33342 (molecular probes-Invitrogen, H1399, 1:2000 phosphate buffer dilution) and treated with an anti-quenching reagent (Life Technologies). The slides were imaged on a laser confocal microscope (lycra, germany).

The experimental results are as follows: to elucidate the potential mechanism by which p53/UTX-mRNA inhibits BCa metastasis in vivo, we first analyzed in situ BCa tumors in mice after different treatments. Since N-Cadherin generally exerts a positive effect of promoting tumor migration by EMT, we first examined the expression of N-Cadherin in different groups. Compared to PBS-treated mice, N-Cadherin (green) was overexpressed in BCa tumors in situ (FIG. 7A), while p53/UTX-mRNA NPs-SH treatment effectively complemented p53/UTX, inhibiting N-Cadherin levels in BCa tumors in situ (FIG. 7B). Furthermore, co-localization between BCa cells (DAPI staining; blue) and apoptosis factors (CC3 staining; a distinct red colour visible in tumors of the p53/UTX-mRNA NPs treatment group (FIG. 7B), indicating significant apoptosis of cancer cells however, in mouse BCa tumors in situ receiving unloaded NP treatment, this co-localization was not observed (FIG. 7A), indicating that restoration of p53/UTX by p53/UTX-mRNA NPs-SH can successfully prevent the tumor migration process by down-regulating expression of N-Cadherin. consistent with previous experimental data in vitro, we also observed that restoration of the p53/UTX pathway in situ UT BCa tumors by p53/UTX-mRNA NPs-SH treatment can up-regulate expression of CC3 (FIG. 7B), p53/UTX as a putative tumor suppressor, and that the p 53/NAA proliferation markers (PCNA 67-Ki) can be significantly down-regulated by p53/UTX-mRNA NPs-SH-restoration of the p53/UTX pathway in situ UT BCa tumors And simultaneously inhibits a transfer related factor N-cadherin.

While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims; meanwhile, any equivalent changes, modifications and variations of the above embodiments according to the essential technology of the present invention are within the scope of the technical solution of the present invention.

Claims

The preparation method of p53-mRNA and UTX-mRNA nano-particles is characterized by comprising the following steps:

1) in vitro synthesis of chemically modified p53-mRNA and UTX-mRNA

Human p53 carrying a T7 promoter and UTX gene open reading frame plasmid form linearized DNA, then a P53 and UTX open reading frame containing a T7 promoter are amplified by adopting PCR, and PCR products are purified to obtain purified polymerase chain reaction products;

reacting MEGAscript T7 transcription kit with purified polymerase chain reaction products, 3 ' -O-Me-m7G (5 ') ppp (5 ') G cap structure analogues, guanosine triphosphate, 5-methyl-cytidine triphosphate, adenosine triphosphate and pseudouridine-5 ' -triphosphate, carrying out deoxyribonuclease treatment after the reaction is finished, and adding a 3 ' poly (A) kit into an IVT RNA transcript by using a poly (A) kit to obtain p53/UTX messenger RNA;

p53/UTX messenger RNA is purified by MEGAclear kit, treated with thermosensitive phosphatase and further purified to obtain the required mRNA;

2) preparation of lipid polymer composite messenger RNA nanoparticles

Synthesizing cationic lipid G0-C14 by using PAMAM dendrimer G0 and 1, 2-epoxy tetradecane;

mixing the mRNA obtained in the step 1) with G0-C14 DMF by adopting an optimized self-assembly strategy to form an mRNA/G0-C14 complex, then quickly adding 250G of PLGA into 5mg/ml of DMF, and mixing with the complex to obtain a uniform mixed solution;

under magnetic stirring, dripping the mixed solution into 10 ml of nucleic acid-free HyPure water, wherein 1mg of DSPE-PEG-NH2, or 1mg of DSPE-PEG-NH2/DSPE-PEG-SH mixture, or 1mg of DSPE-PEG-SH is constructed to obtain a lipid PEG outer layer of NPs;

after self-assembly and stabilization, the formed NPs are washed by precooled enzyme-free pure water by an Amicon filter tube to remove free compounds and organic solvents, and the washed nanoparticle precipitate is resuspended into adhesive mRNA NPs with different concentrations by PBS to obtain p53-mRNA and UTX-mRNA nanoparticles.
2. The method for preparing p53-mRNA and UTX-mRNA nanoparticles as claimed in claim 1, wherein the chemical modification reaction system in step 1) is specifically: the amount of the purified PCR product added was 1-2. mu.g, the amount of 3 '-O-Me-m 7G (5') ppp (5 ') G cap structural analog added was 6mM, the amount of guanosine triphosphate added was 1.5mM, the amount of 5-methylcytosine triphosphate added was 7.5mM, the amount of adenosine triphosphate added was 7.5mM, the amount of pseudouridine-5' -triphosphate added was 7.5mM, the reaction temperature was 37 ℃ and the reaction time was 4 hours.
3. The method for preparing p53-mRNA and UTX-mRNA nanoparticles according to claim 1, wherein the temperature of the heat-sensitive phosphatase treatment in step 1) is 37 ℃ and the treatment time is 30 minutes.
4. The method for preparing p53-mRNA and UTX-mRNA nanoparticles as claimed in claim 1, wherein in the self-assembly strategy of step 2), mRNA 16 μ G is mixed with G0-C14250 μ G for 15s, wherein the concentration of mRNA is 1mg/ml and the concentration of G0-C14 is 2.5 mg/ml.
5. The method for preparing nanoparticles of p53-mRNA and UTX-mRNA as claimed in claim 1, wherein the weight ratio of the mixture DSPE-PEG-NH2/DSPE-PEG-SH in step 2) is 1: 1.
6. the application of the p53-mRNA and UTX-mRNA nano-particle obtained by the method of any one of claims 1 to 5 in preparing a medicament for treating tumors.
7. The use according to claim 6, wherein the tumor treatment drug is a drug that restores the anti-cancer functions of p53 and UTX.
8. Use according to claim 7, characterized in that the agent for restoring the cancer-suppressing function of p53 and UTX is in particular an agent for restoring the cancer-suppressing function of p53 and UTX in bladder cancer.
9. The use as claimed in claim 6, wherein the medicament for treating tumor is prepared by increasing the retention time of p53-mRNA and UTX-mRNA in bladder by using RNA nanoparticles with bladder mucosa adhesion function, so that messenger RNA enters tumor cells to exert anti-tumor effect.