Phosphatidyl polymer, preparation method, mRNA phosphatidyl polymer delivery system and application

Abstract

The invention relates to the technical field of biological medicine, in particular to a phosphatidyl polymer, a preparation method, an mRNA phosphatidyl polymer delivery system and application. The phosphatidyl polymer has a structure as shown in a formula I. The phosphatidyl polymer can be self-assembled with mRNA and modified amphiphilic molecules to form an mRNA delivery system. According to the invention, the organ targeting characteristic of the compound in vivo can be changed by regulating and controlling the mass ratio of the phosphatidyl polymer to the mRNA during preparation of the compound. Along with the increase of the proportion of the phosphatidyl polymer in the mass ratio formula, the target organ of the compound changes from spleen to liver to lung. Compared with a traditional mRNA delivery system, the organ targeting characteristics of the delivery system can be regulated and controlled only by changing the mass ratio formula, efficient in-vivo and in-vitro mRNA delivery efficiency is achieved, the large-scale production difficulty and cost of raw materials are reduced, and a new platform is provided for efficient, accurate and universal delivery of mRNA.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a phosphatidyl polymer, a preparation method, an mRNA phosphatidyl polymer delivery system and applications. Background Art

[0002] Messenger RNA (mRNA) has significant potential in innovating treatments for challenging diseases. In addition to COVID-19, mRNA vaccines, therapeutics, or gene editing tools are actively being developed for influenza virus, cytomegalovirus, tumors, etc. Recently, the U.S. Food and Drug Administration (FDA) approved a non-COVID-19 mRNA vaccine, mRNA-1345, marking an important milestone in the advancement of mRNA technology.

[0003] The effectiveness of mRNA technology depends on the creation of safe, efficient and highly selective delivery systems that can target specific tissues and cell types. Lipid nanoparticles (LNPs) are considered one of the most successful non-viral synthetic polymers and have been shown to be able to deliver mRNA specifically to the liver. However, the challenge lies in effectively and selectively delivering mRNA to other organs, such as the lungs and spleen. Summary of the invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a phosphatidyl polymer, a preparation method, an mRNA phosphatidyl polymer delivery system and application, so as to achieve multi-organ targeting of mRNA.

[0005] To achieve the above object, the present invention provides a phosphatidyl polymer having a structure shown in Formula I:

[0006]

[0007] in,

[0008] x, y, z, a are the degree of polymerization, 10≤x+y+z+a≤600, x≥0, y>0, z>0, a>0;

[0009] m and n are the lengths of the lipid chains, respectively.

[0010] Preferably, 10≤x+y+z+a≤600, more preferably 30≤x+y+z+a≤100.

[0011] m is the length of the lipid chain, preferably, 4≤m≤16, more preferably, 5≤m≤9.

[0012] n is the length of the lipid chain, preferably, 4≤n≤16, more preferably, 4≤n≤7.

[0013] The present invention provides a method for preparing the above-mentioned phosphatidyl polymer, comprising the following steps:

[0014] The PEI polymer represented by formula Ia, the phospholipid compound represented by formula Ib and the epoxy compound represented by formula Ic are reacted to obtain the phosphatidyl polymer represented by formula I;

[0015]

[0016] in,

[0017] x, y, z, a are the degree of polymerization, 10≤x+y+z+a≤600, x≥0, y>0, z>0, a>0;

[0018] m and n are the lengths of the lipid chains, respectively.

[0019] The ranges of x, y, z, a, m, and n are as described in the full text.

[0020] The reaction temperature is preferably 45 to 85° C., and the reaction time is preferably 12 to 72 hours.

[0021] Preferably, the phospholipid compound represented by formula Ib is prepared according to the following method:

[0022] The dioxaphosphocycloalkane compound represented by formula Ib-1 and the alkyl alcohol represented by formula Ib-2 are reacted to obtain the phospholipid compound represented by formula Ib;

[0023]

[0024] m is the length of the lipid chain.

[0025] The range of m is the same as described above.

[0026] Preferably, the above reaction is carried out in the presence of an acid binding agent. The acid binding agent may be an acid binding agent well known to those skilled in the art, including but not limited to triethylamine.

[0027] The phosphatidyl polymer provided by the present invention carries cations and has electrostatic interaction with the mRNA carrying negative charges.

[0028] Based on this, the present invention provides an mRNA phosphatidyl polymer delivery system, comprising: mRNA, the above-mentioned phosphatidyl polymer and a modified amphiphilic molecule;

[0029] The phosphatidyl polymer and mRNA form a core structure through electrostatic interaction, and the modified amphiphilic molecule is anchored on the surface of the core structure through hydrophobic interaction to form a spherical core-shell structure.

[0030] The core-shell structure has high stability and can be used for mRNA transfection.

[0031] The present invention has no particular limitation on the type of the mRNA, which may be a common mRNA well known to those skilled in the art.

[0032] Preferably, the mRNA includes at least one of: self-reporter gene mRNA, genome editing mRNA, antigen mRNA, tumor suppressor gene mRNA, interleukin mRNA or cell chimeric antigen receptor mRNA or other functional mRNA.

[0033] Optionally, the self-reporter gene mRNA is selected from one or more of GFP, eGFP, Luc, mCherry, betagal or renillaLuc; the genome editing mRNA is selected from one or more of Cas9, Cas9Nickase or Cre recombinase; the antigen mRNA is selected from OVA; the tumor suppressor gene mRNA is selected from one or more of p53 or PTEN; the interleukin mRNA is selected from one or more of IL-10, IL-12 or IL-6; the cell chimeric antigen receptor mRNA is selected from anti-human CD19-specific chimeric antigen receptor; the other functional mRNA is selected from HAase.

[0034] Optionally, the modified amphiphilic molecule includes at least one of a pegylated amphiphilic molecule, an amphiphilic surfactant, an amphiphilic protein or other modified molecules.

[0035] Optionally, the PEGylated amphiphilic molecule is selected from one or more of DSPE-PEG and DMG-PEG.

[0036] Optionally, the amphiphilic surfactant is CO-520, F127.

[0037] Optionally, the amphipathic protein is albumin.

[0038] Optionally, the other modifying molecules are selected from one or more of mannitol, modified chitosan, dextran, carboxydextran, liposomes, tetraethyl orthosilicate, polyacrylic acid, KH560, KH550, diethylenetriaminepentaacetic acid, meglumine, arginine, polyglutamic acid or polypeptides.

[0039] Preferably, the mass ratio of the mRNA to the phosphatidyl polymer is 1:(0.1-100), more preferably 1:(2-50).

[0040] Preferably, the ratio of the mass of the modified amphiphilic molecule to the total mass of the mRNA and the phosphatidyl polymer is 1:(0.1-100), more preferably 1:(20-40).

[0041] The composite nanoparticles prepared by the present invention through different mass ratio formulas have differentiated surface potentials, pK a These parameters enable the composite nanoparticles to have the ability to specifically target and transfect different organs after intravenous infusion.

[0042] In some specific embodiments of the present invention, the mass ratio of the phosphatidyl polymer, mRNA, and modified amphiphilic molecule is 2:1:0.075, 10:1:0.275, or 50:1:1.275.

[0043] The present invention provides a method for preparing the mRNA phosphatidyl polymer delivery system, comprising the following steps:

[0044] The acidic buffer solution containing the phosphatidyl polymer and the buffer solution containing mRNA are mixed to assemble the phosphatidyl polymer and mRNA complex particles through electrostatic interaction;

[0045] The above-mentioned phosphatidyl polymer and mRNA complex particles are modified with biocompatible modification molecules to obtain the mRNA phosphatidyl polymer delivery system.

[0046] The buffer system in the acidic buffer solution of the phosphatidyl polymer is preferably a sodium citrate buffer solution, and the pH value of the sodium citrate buffer solution is preferably 4.4.

[0047] The buffer system in the mRNA buffer solution is preferably a sodium citrate buffer solution or OPTI-MEM, and the pH value of the sodium citrate buffer solution is preferably 4.4.

[0048] In some specific embodiments of the present invention, the phosphatidyl polymer is firstly dissolved in a solvent to prepare a solution containing the phosphatidyl polymer with a concentration of 1 to 20 mg / mL.

[0049] The solvent is preferably one or more of methanol, formaldehyde, ethanol, acetaldehyde, ethylene glycol, diethylene glycol, DMSO, DMF, benzyl alcohol, hydrazine hydrate, sodium borohydride, hydroiodic acid, acetone, dichloromethane, and chloroform.

[0050] Then, the solution containing the phosphatidyl polymer is uniformly mixed with the acidic buffer solution to obtain the acidic buffer solution containing the phosphatidyl polymer. The pH range of the acidic buffer solution containing the phosphatidyl polymer is preferably 1 to 6; the reaction time is preferably 30 seconds to 24 hours; the reaction temperature is preferably 0 to 37°C; the acidic buffer solution is preferably one or more of citric acid, citrate, acetic acid, acetate, hydrochloric acid, hydrochloride, sulfuric acid, sulfate, nitric acid, and nitrate; the pH range of the acidic buffer solution is preferably 1 to 6.

[0051] In the acidic buffer solution containing the phosphatidyl polymer, the concentration of the phosphatidyl polymer is preferably 0.1 to 1000 mg / mL, more preferably 1 to 20 mg / mL. In some specific embodiments of the present invention, the concentration of the phosphatidyl polymer is 10 mg / mL.

[0052] While preparing the acidic buffer solution containing the phosphatidyl polymer, a buffer solution containing mRNA can be prepared. Specifically, the mRNA mother solution is diluted with a buffer solution to prepare a buffer solution containing mRNA at a concentration of 0.01 to 2 mg / mL. The buffer solution is preferably an acidic buffer solution or a serum-free cell culture medium. The acidic buffer solution is preferably one or more of citric acid, citrate, acetic acid, acetate, hydrochloric acid, hydrochloride, sulfuric acid, sulfate, nitric acid, and nitrate, and the pH range of the acidic buffer solution is preferably 1 to 6; the serum-free cell culture medium is preferably one of OPTI-MEM culture medium, DMEM culture medium, and RPMI 1640 culture medium.

[0053] In the mRNA-containing buffer solution, the concentration of the mRNA is preferably 0.1 to 1000 g / L, more preferably 0.01 to 2 mg / mL. In some specific embodiments of the present invention, the concentration of the mRNA is 0.1 mg / mL.

[0054] Then, the buffer solution containing mRNA and the acidic buffer solution containing phosphatidyl polymer are mixed evenly, and after standing for a period of time, complex particles composed of mRNA and phosphatidyl polymer are obtained; the standing time is preferably 30 seconds to 24 hours; the reaction temperature is preferably 0 to 37°C.

[0055] Finally, a solution containing biocompatible modification molecules is added to the solution containing the mRNA-phosphatidyl polymer composite nanoparticles, and after standing for a period of time, the mRNA phosphatidyl polymer delivery system nanoparticles are obtained.

[0056] In the solution containing the biocompatibility modifying molecule, the concentration of the biocompatibility modifying molecule is preferably 0.1 to 1000 mg / mL, more preferably 0.1 to 10 mg / mL. In some specific embodiments of the present invention, the concentration of the biocompatibility modifying molecule is 0.1 mg / mL.

[0057] In the above preparation process, the solution mixing method includes but is not limited to: at least one of dripping, stirring, standing, microfluidic equipment, peristaltic pump, microinjection pump, etc.

[0058] In some specific embodiments of the present invention, the above preparation method comprises the following steps:

[0059] (1) dispersing the phosphatidyl polymer in an acidic buffer solution, and diluting the mRNA mother solution with the buffer solution;

[0060] (2) mixing the acidic buffer solution of the phosphatidyl polymer and the buffer solution of the mRNA by pipetting or microfluidics and allowing to stand, preferably for 15 minutes, to obtain a core dispersion solution;

[0061] (3) Adding the biocompatible modified molecule to the above acidic buffer solution by pipetting or microfluidics, and letting it stand, preferably for 15 minutes, to obtain composite nanoparticles.

[0062] The test results show that the cell transfection efficiency of mRNA in the above-mentioned mRNA phosphatidyl polymer delivery system provided by the present invention is ≥50%, preferably ≥60%, more preferably ≥70%, more preferably ≥80%, more preferably ≥90%, and optimally ≥95%.

[0063] The types of mRNA delivered by the delivery system provided by the present invention are not limited, and combined with its multi-organ targeting ability, it has broad application prospects.

[0064] Based on this, the present invention provides the use of the above-mentioned mRNA phosphatidyl polymer delivery system or the mRNA phosphatidyl polymer delivery system prepared by the above-mentioned preparation method in the preparation of tumor therapeutic drugs, tumor vaccines, tumor targeted drugs, in vivo and in vitro tumor diagnostic materials, inflammation-related disease therapeutic drugs, infectious disease therapeutic drugs, infectious disease prevention drugs, wound healing therapeutic drugs, cardiovascular and cerebrovascular chronic disease therapeutic drugs, organ fibrosis therapeutic drugs, viral infection therapeutic drugs, viral infection prevention drugs, in vivo and in vitro gene editing therapeutic drugs, non-alcoholic fatty liver therapeutic drugs, intestinal disease therapeutic drugs, new antigen vaccines, cell induced differentiation drugs, protein beauty agents, protein defect disease therapeutic drugs, aging repair products, anti-aging products, etc. or as a carrier of the above-mentioned drugs.

[0065] The present invention also relates to downstream products prepared by the above-mentioned mRNA phosphatidyl polymer delivery system, including but not limited to cell mRNA transfection kits, in vivo mRNA transfection kits, mRNA vaccines, etc.

[0066] Compared with the prior art, the present invention provides a phosphatidyl polymer having a structure shown in Formula I. The phosphatidyl polymer can self-assemble with mRNA and modified amphiphilic molecules to form an mRNA delivery system.

[0067] The present invention has the following advantages: the delivery system can achieve an in vivo and in vitro mRNA delivery efficiency comparable to that of LNP, and can achieve specific targeted transfection on various organs including the spleen, liver and lungs. This is achieved by adjusting the mass ratio formula, which is not only universal and efficient, but also simple and controllable in the regulation method. The organ targeting properties of the complex in vivo can be changed by only regulating the mass ratio formula of the phosphatidyl polymer and mRNA when preparing the complex. As the proportion of the phosphatidyl polymer in the mass ratio formula increases, the target organ of the complex changes from spleen to liver and then to lung.

[0068] Compared with traditional mRNA delivery systems, the organ targeting characteristics of the delivery system can be regulated by simply changing the mass ratio formula, with high efficiency of in vivo and in vitro mRNA delivery, reducing the difficulty and cost of large-scale production of raw materials, and providing a new platform for efficient, accurate and universal delivery of mRNA. The preparation process is simple and the cost is low, which is very suitable for industrial promotion.

[0069] The phosphatidyl polymer used has the advantages of being environmentally friendly, safe and reliable, simple in process, low in cost and high in yield, easy to control in quality, and easy to produce on a large scale. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 is the PLm prepared in Example 1 1 HNMR;

[0071] Figure 2 The PEI-x%PL10 prepared in Example 2 1 HNMR;

[0072] Figure 3 PEI-25%PL10 / y%E8 prepared in Example 3 1 HNMR;

[0073] Figure 4 The dynamic light scattering results of the composite nanoparticles prepared in Example 4 at a concentration of 0.2 mg / mL in water;

[0074] Figure 5TEM image of the composite nanoparticles prepared in Example 5;

[0075] Figure 6 The apparent pK of the composite nanoparticles prepared in Example 4-5 is a Test results;

[0076] Figure 7 The test results of the mRNA delivery efficiency of the composite nanoparticles prepared in Example 4-5 to 293T cells in vitro;

[0077] Figure 8 The results of the test of the cellular endocytosis and endosomal escape ability of the composite nanoparticles prepared in Example 4-5;

[0078] Fig. 9 The test results of the ability of the composite nanoparticles prepared in Example 4-5 to selectively deliver mRNA to organs after intravenous reinfusion into mice;

[0079] Fig.10 This is the organ enrichment of the composite nanoparticles prepared in Example 4-5 after intravenous reinfusion into mice. DETAILED DESCRIPTION

[0080] In order to further illustrate the present invention, the following is a detailed description in conjunction with the embodiments. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, rather than limiting the claims of the invention.

[0081] All raw materials of the present invention have no particular limitation on their sources, and can be purchased from the market or prepared according to conventional methods known to those skilled in the art.

[0082] Example 1: Preparation of phospholipid molecules (PLm)

[0083] Alkyl diphosphinate molecules (PLm) were synthesized using 2-chloro-2-oxo-1,3,2-dioxaphospholane (COP) and the corresponding alkylated alcohol as raw materials. Alkylated alcohol (1.00 mmol) and triethylamine (106 mg, 1.05 mmol) were dissolved in anhydrous tetrahydrofuran (THF) (20 mL). Subsequently, 2-chloro-2-oxo-1,3,2-dioxaphospholane (COP, 150 mg, 1.05 mmol) was dissolved in THF (10 mL). The solution was then dropped into the above mixture under an ice-water bath, and the reaction was allowed to proceed overnight at 25 °C. Triethylamine hydrochloride was then removed by filtration, and the resulting filtrate was evaporated to produce PLm molecules. The yield of all PLm molecules exceeded 85%.

[0084] The prepared PLm was subjected to NMR analysis, and the results are shown in Figure 1, using deuterated chloroform as the deuterated reagent, the results showed that the chemical shift of 1.68ppm should be the methylene signal peak of the phosphatidyl group, and the appearance of this peak indicated the successful formation of the phosphatidyl group. The chemical shifts of 0.89ppm and 1.27ppm were the signal peaks of the methyl and methylene of the phospholipid tail, respectively, and the appearance of these peaks indicated that the lipid tail was successfully modified on the COP molecule.

[0085] Example 2: Preparation of PEI polymer modified with phospholipid molecules (PEI-x% PLm)

[0086] Branched PEI 1800 (100 mg, 2.33 mmol) and PLm prepared in Example 1 (0.23 mmol, x% = 10%; 0.58 mmol, x% = 25%; 1.16 mmol, x% = 50%; 1.74 mmol, x% = 75%) were added to a dry reaction bottle and dissolved in anhydrous DMSO. The reaction was sealed and stirred at 85°C for 24 hours. The solvent was then removed by vacuum drying to obtain a crude product of PEI-x% PLm. The crude product was directly used for subsequent screening.

[0087] The prepared PEI-x%PLm was analyzed by nuclear magnetic resonance, and the results are shown in Figure 2 , using deuterated DMSO as the deuterated reagent, the results showed that the chemical shift of 4.50ppm should be the methylene signal peak on the dioxaphospholane ring, and the disappearance of this peak indicated the successful ring opening of the dioxaphospholane ring. The chemical shifts of 0.89ppm and 1.27ppm were the signal peaks of the methyl and methylene on the phospholipid tail, respectively, and the appearance of these peaks indicated that the phospholipid tail was successfully modified on PEI.

[0088] Example 3: Preparation of phosphatidyl polymer modified with epoxy and phospholipid molecules (PEI-25% PL10 / y% En)

[0089] Add branched PEI 1800 (10 mg, 0.0056 mmol), PL10 (15 mg, 0.057 mmol) and octyl oxide (epoxide of C8, E8) (the modification rate y% calculated by the feed ratio is 25%) to a dry reaction bottle and dissolve them together in DMSO. Then stir the reaction system at a temperature of 80°C for 24 hours. The crude product is then obtained by vacuum drying and used for preliminary screening. For the refined product, the crude product is placed in a dialysis bag and dialyzed in pure water for two days. The liquid in the dialysis bag is collected and freeze-dried to produce a white or light yellow viscous solid.

[0090] The prepared PEI-25% PL10 / y% En was analyzed by nuclear magnetic resonance. The results are shown in Figure 3, using deuterated DMSO as the deuterated reagent, the results showed that the chemical shift of 4.43ppm should be the methylene signal peak on the epoxy group, and the disappearance of this peak indicated the successful ring opening of the epoxy group. The chemical shifts of 0.89ppm and 1.27ppm were the signal peaks of methyl and methylene on the phospholipid tail, respectively, and the enhancement of the peak integration indicated the successful modification of octyl oxide on the PEI backbone.

[0091] Example 4: Preparation of mRNA-loaded complex nanoparticles (MACO)

[0092] The mRNA was diluted in sterile citrate buffer (pH 4.4, 10 mM). The ethanol solution containing the PEI-based polymer prepared in Example 3 was quickly mixed with the mRNA aqueous solution in the following weight ratios: 1:1, 2:1, 5:1, 10:1, 20:1, 30:1, 50:1 and 100:1 (wt / wt), and the volume ratio was 1:3. After incubation for 15 minutes, Pluronic F127 (concentration of 2.5% wt) was introduced and incubated for another 15 minutes. MACO was then diluted three times using 1× PBS buffer for in vitro mRNA delivery.

[0093] Example 5: Particle size and zeta potential test of MACO

[0094] To further reduce the size, MACO was synthesized using a microfluidic mixing system (Nanowetech). The mRNA was diluted in citrate buffer (pH 4.4, 10 mM), while the PEI-based polymer prepared in Example 3 was dissolved in ethanol. In the OBMPO test chip, the two phases were mixed rapidly at a flow rate of 12 ml / min with a water-ethanol volume ratio of 3:1. The particle size and TEM images of MACO were determined using dynamic light scattering (DLS) technology with a Zetasizer Nano-ZS (Malvern, UK). The results are shown in Figure 4 and Figure 5 The MACOs prepared with all the mass ratios had good assembly.

[0095] Example 6: Apparent pKa Test of MACO

[0096] The pKa of MACOs was determined by dissolving MACOs (10 μg Luc mRNA) in 5 mL NaCl-saturated ethanol. After that, diluted 0.01 M HCl aqueous solution was added to the ethanol solution in 10 μL increments, and the pH value at each stage was recorded. The pKa of MACOs was then calculated using the Henderson-Hasselbalch equation. The results are shown in Figure 6 The pKa of all MACOs is in the range of 6-7, which is beneficial for the delivery of mRNA.

[0097] Example 7: In vitro mRNA delivery test of MACO to 293T cells

[0098] 293T cells The cells were cultured in Dulbecco's modified Eagle's medium at 37°C and 5% CO2. One day before the experiment, 1 × 10 5 Cells were seeded into 24-well plates (Costa). Nanoparticles (NPs) containing 100 ng of Luc mRNA (MACO) were added to each well and incubated for 24 h. The cells were then lysed and luciferase substrate was added. Luminescence was quantified by 20 / 20 luminometer. Cell viability of 293T cells treated with MACO was assessed by CCK8 assay. Figure 7 Under the appropriate mass ratio formulation, MACO exhibited strong in vitro mRNA delivery capabilities.

[0099] Example 8: Testing of the intracellular endocytosis and endosomal escape ability of MACO

[0100] To evaluate the endocytosis and endosomal escape of MACO, 293T cells were seeded onto Lab-Tek chambered coverslips at a density of 1.5 × 10 per well. 5 cells. After 12 hours, the medium containing metabolic waste was removed and replaced with 1 ml of fresh medium containing 10% FBS. MACO was prepared as described above (polymer / mRNA weight ratios were 2 / 1, 10 / 1, 50 / 1, and 500 ngLuc mRNA per well). After incubation for 3 hours, staining was performed with Lysotracker Green DND26 (1 / 5000 dilution) and Hoechst 33342 (0.1 mg / mL) at 37°C for 15 minutes and washed 3 times. Slides were imaged using a confocal microscope (Zeiss LSM 700) and analyzed using ZEN 2010 software v.6.0.62 (Carl Zeiss MicroImaging). The results are shown in Figure 8 . From the significant intracellular red fluorescence (representing MACO complex nanoparticles), it can be analyzed that all tested MACOs showed excellent cellular endocytosis ability. The white arrows indicate the areas where red fluorescence and green fluorescence (representing endosomes) are not co-localized. This indicates that MACO nanoparticles have successfully escaped from endosomes.

[0101] Example 9: In vivo transfection and in vivo organ enrichment test of MACO complex nanoparticles

[0102] For in vivo mRNA transfection experiments, MACO complexes were synthesized as described above. The complexes were intravenously (iv) administered to 6-8 week old BALB / c mice at a Luc mRNA dose of 0.25 mg / kg. After 24 hours, 100 μL of D-luciferin potassium salt solution (30 mg / mL) was administered intraperitoneally. Ten minutes later, mice were euthanized, and their organs (heart, lungs, liver, spleen, kidneys, and lymph nodes) were collected and collected at PerkinElmer The images were analyzed using Living Image analysis software (Perkin Elmer). Fig. 9 As the mass ratio formula of MACO was adjusted from 2 / 1 to 10 / 1 to 50 / 1 (polymer / mRNA), its target organ changed from spleen to liver to lung.

[0103] For in vivo biodistribution studies, MACO complexes were synthesized as described above. The complexes were administered intravenously (iv) to 6-8 week old BALB / c mice at a Luc mRNA dose of 0.25 mg / kg. After 6 hours, the mice were euthanized and their organs (heart, lungs, liver, spleen, kidneys and lymph nodes) were harvested and analyzed at PerkinElmer The images were analyzed using Living Image analysis software (Perkin Elmer). Fig.10 Regardless of how the mass ratio formulation of MACO was changed, all tested MACOs were mainly enriched in the liver.

[0104] The above embodiments are only used to help understand the method and core idea of ​​the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

1. A phosphatidyl polymer having a structure shown in Formula I: in, x, y, z, a are the degree of polymerization, 10≤x+y+z+a≤600, x≥0, y>0, z>0, a>0; m and n are the lengths of the lipid chains, respectively.

2. The phosphatidyl polymer according to claim 1, characterized in that The 4≤n≤16, the 4≤m≤16.

3. The method for preparing the phosphatidyl polymer according to any one of claims 1 to 2, comprising the following steps: The PEI polymer represented by formula Ia, the phospholipid compound represented by formula Ib and the epoxy compound represented by formula Ic are reacted to obtain the phosphatidyl polymer represented by formula I; in, x, y, z, a are the degree of polymerization, 10≤x+y+z+a≤600, x≥0, y>0, z>0, a>0; m and n are the lengths of the lipid chains, respectively.

4. The preparation method according to claim 3, characterized in that: The phospholipid compound represented by formula Ib is prepared according to the following method: The dioxaphosphocycloalkane compound represented by formula Ib-1 and the alkyl alcohol represented by formula Ib-2 are reacted to obtain the phospholipid compound represented by formula Ib; m is the length of the lipid chain.

5. An mRNA phosphatidyl polymer delivery system, comprising: mRNA, the phosphatidyl polymer and the modified amphiphilic molecule according to any one of claims 1 to 2; The phosphatidyl polymer and mRNA form a core structure through electrostatic interaction, and the modified amphiphilic molecule is anchored on the surface of the core structure through hydrophobic interaction to form a spherical core-shell structure.

6. The mRNA phosphatidyl polymer delivery system according to claim 5, characterized in that: The mRNA includes: at least one of self-reporter gene mRNA, genome editing mRNA, antigen mRNA, tumor suppressor gene mRNA, interleukin mRNA or cell chimeric antigen receptor mRNA or other functional mRNA.

7. The mRNA phosphatidyl polymer delivery system according to claim 5, characterized in that: The modified amphiphilic molecules include: at least one of pegylated amphiphilic molecules, amphiphilic surfactants, amphiphilic proteins or other modified molecules.

8. The mRNA phosphatidyl polymer delivery system according to claim 5, characterized in that: The mass ratio of the mRNA to the phosphatidyl polymer is 1:(0.1-100); the ratio of the mass of the modified amphiphilic molecule to the total mass of the mRNA and the phosphatidyl polymer is 1:(0.1-100).

9. The method for preparing the mRNA phosphatidyl polymer delivery system according to any one of claims 5 to 8, comprising the following steps: Mixing an acidic buffer solution containing the phosphatidyl polymer according to any one of claims 1 to 2 and a buffer solution containing mRNA, and assembling through electrostatic interaction to obtain complex particles of the phosphatidyl polymer and mRNA; The above-mentioned phosphatidyl polymer and mRNA complex particles are modified with biocompatible molecules to obtain the mRNA phosphatidyl polymer delivery system.

10. Use of the mRNA phosphatidyl polymer delivery system according to any one of claims 5 to 8 or the mRNA phosphatidyl polymer delivery system prepared by the preparation method according to claim 9 in the preparation of tumor therapeutic drugs, tumor vaccines, tumor targeted drugs, in vivo and in vitro tumor diagnostic materials, inflammation-related disease therapeutic drugs, infectious disease therapeutic drugs, infectious disease prevention drugs, wound healing therapeutic drugs, cardiovascular and cerebrovascular chronic disease therapeutic drugs, organ fibrosis therapeutic drugs, viral infection therapeutic drugs, viral infection prevention drugs, in vivo and in vitro gene editing therapeutic drugs, non-alcoholic fatty liver therapeutic drugs, intestinal disease therapeutic drugs, new antigen vaccines, cell-induced differentiation drugs, protein beauty agents, protein defect disease therapeutic drugs, aging repair products, anti-aging products, or as a carrier of the above drugs.

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