Phosphatidyl polymers, methods of preparation, mRNA phosphatidyl polymer delivery systems, and uses

Abstract

This invention relates to the field of biomedical technology, and more particularly to a phosphatidyl polymer, its preparation method, an mRNA phosphatidyl polymer delivery system, and its applications. The phosphatidyl polymer has the structure shown in Formula I. The phosphatidyl polymer can self-assemble with mRNA and modified amphiphilic molecules to form an mRNA delivery system. This invention, by adjusting the mass ratio of the phosphatidyl polymer to mRNA during complex preparation, can alter the organ-targeting characteristics of the complex in vivo. With increasing proportion of phosphatidyl polymer in the mass ratio formulation, the target organ of the complex changes from the spleen to the liver and then to the lung. Compared to traditional mRNA delivery systems, this invention allows for control of the organ-targeting characteristics of the delivery system simply by changing the mass ratio formulation, achieves high in vitro and in vivo mRNA delivery efficiency, reduces the difficulty and cost of large-scale raw material production, and provides a new platform for efficient, precise, and universal mRNA delivery.

Description

Technical Field

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

[0002] Messenger RNA (mRNA) holds significant potential for the treatment of innovative and challenging diseases. In addition to COVID-19, mRNA vaccines, therapeutics, and gene-editing tools are being actively developed for influenza viruses, cytomegaloviruses, tumors, and more. Recently, the U.S. Food and Drug Administration (FDA) approved the non-COVID-19 mRNA vaccine mRNA-1345, marking a significant 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 for specific tissues and cell types. Lipid nanoparticles (LNPs) are considered among the most successful non-viral synthetic polymers, demonstrating their ability to specifically deliver mRNA to the liver. However, the challenge lies in efficiently 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 an application, thereby achieving multi-organ targeting of mRNA.

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

[0006]

[0007] in,

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

[0009] m and n are the lengths of the lipid chain, 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] This invention provides a method for preparing the above-mentioned phosphatidyl polymer, comprising the following steps:

[0014] The PEI polymer shown in Formula Ia, the phospholipid compound shown in Formula Ib, and the epoxy compound shown in Formula Ic are reacted to obtain the phosphatidyl polymer shown in Formula I.

[0015]

[0016] in,

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

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

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

[0020] The preferred temperature for the above reaction is 45–85°C, and the preferred reaction time is 12–72 h.

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

[0022] The dioxophosphorus heterocyclic alkane compound shown in Formula Ib-1 and the alkyl alcohol shown in Formula Ib-2 are reacted to obtain the phospholipid compound shown in 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 can be any 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 has cations and electrostatic interactions with negatively charged mRNA.

[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 interactions, and the modified amphiphilic molecule is anchored to the surface of the core structure through hydrophobic interactions, forming a spherical core-shell structure.

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

[0031] The present invention does not specifically limit the type of mRNA mentioned above, and it can be any general mRNA well known to those skilled in the art.

[0032] Preferably, the mRNA includes at least one of the following: self-reporter gene mRNA, genome editing mRNA, antigen mRNA, tumor suppressor gene mRNA, interleukin mRNA, or 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 chimeric antigen receptor mRNA is selected from anti-human CD19-specific chimeric antigen receptor; and the other functional mRNAs are selected from HAase.

[0034] Optionally, the modified amphiphilic molecule includes at least one of: polyethylene glycol-modified amphiphilic molecules, amphiphilic surfactants, amphiphilic proteins, or other modified molecules.

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

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

[0037] Optionally, the amphiphilic protein is albumin.

[0038] Optionally, the other modifying molecules are selected from one or more of mannitol, modified chitosan, dextran, carboxyglucan, 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 phosphatidyl polymer is 1:(0.1-100), more preferably 1:(20-40).

[0041] The composite nanoparticles prepared by this invention through different mass ratio formulations exhibit differentiated surface potentials, pK. a And lipid tail density. These parameters enable the complex nanoparticles 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] This invention provides a method for preparing the above-mentioned mRNA phosphatidyl polymer delivery system, comprising the following steps:

[0044] An acidic buffer solution containing the above-mentioned phosphatidyl polymer and a buffer solution containing mRNA were mixed, and the complex particles of phosphatidyl polymer and mRNA were assembled by electrostatic interaction.

[0045] The complex particles of the above-mentioned phosphatidyl polymer and mRNA were modified with biocompatible modifying molecules to obtain an mRNA phosphatidyl polymer delivery system.

[0046] The buffer system in the acidic buffer solution of the above-mentioned 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 buffer solution for the above-mentioned mRNA is preferably 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 above-mentioned phosphatidyl polymer is first dissolved in a solvent to prepare a solution containing phosphatidyl polymer with a concentration of 1 to 20 mg / mL.

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

[0050] The solution containing the phosphatidyl polymer is then mixed thoroughly with an acidic buffer solution to obtain an acidic buffer solution containing the phosphatidyl polymer. The pH range of the acidic buffer solution containing the phosphatidyl polymer is preferably 1–6; the reaction time is preferably 30 seconds–24 hours; the reaction temperature is preferably 0–37°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 1–6.

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

[0052] Concurrently with the preparation of the acidic buffer solution containing the phosphatidyl polymer, a buffer solution containing mRNA can also be prepared. Specifically, the mRNA stock solution is diluted with the buffer solution to prepare a buffer solution containing mRNA with a concentration of 0.01–2 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 1–6; the serum-free cell culture medium is preferably one of OPTI-MEM medium, DMEM medium, and RPMI 1640 medium.

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

[0054] Then, the buffer solution containing mRNA is mixed evenly with the acidic buffer solution containing phosphatidyl polymer, and after standing for a period of time, a complex particle composed of mRNA and phosphatidyl polymer is 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 modifying molecules was added to the solution containing the mRNA-phosphatidyl polymer composite nanoparticles. After standing for a period of time, the mRNA-phosphatidyl polymer delivery system nanoparticles were obtained.

[0056] In the solution containing the biocompatible modified molecules, the concentration of the biocompatible modified molecules is preferably 0.1–1000 mg / mL, more preferably 0.1–10 mg / mL. In some specific embodiments of the present invention, the concentration of the biocompatible modified molecules 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 the following: dripping, stirring, standing, microfluidic equipment, peristaltic pump, microinjection pump, etc.

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

[0059] (1) Disperse the phosphatidyl polymer in an acidic buffer solution and dilute the mRNA stock solution with the buffer solution;

[0060] (2) The acidic buffer solution of phosphatidyl polymer and the buffer solution of mRNA are mixed by pipetting or microfluidic method and allowed to stand, preferably for 15 minutes, to obtain the kernel dispersion solution.

[0061] (3) Add the biocompatible modified molecules to the above acidic buffer solution by pipetting or microfluidic method, let stand, preferably for 15 minutes, to obtain composite nanoparticles.

[0062] The experimental results show that the cell transfection efficiency of the 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 most preferably ≥95%.

[0063] The delivery system provided by this invention can deliver an unlimited variety of mRNAs, and combined with its multi-organ targeting capability, it has broad application prospects.

[0064] Based on this, the present invention provides the application 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 diagnostic tumor materials, drugs for treating inflammation-related diseases, drugs for treating infectious diseases, drugs for preventing infectious diseases, drugs for wound healing, drugs for treating cardiovascular and cerebrovascular chronic diseases, drugs for treating organ fibrosis, drugs for treating viral infections, drugs for preventing viral infections, drugs for treating gene editing in vivo and in vitro, drugs for treating non-alcoholic fatty liver disease, drugs for treating intestinal diseases, neoantigen vaccines, cell differentiation-inducing drugs, protein cosmetics, drugs for treating protein deficiency diseases, aging repair products, anti-aging products, etc., or as a carrier for 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 the structure shown in Formula I. The above-mentioned phosphatidyl polymer can self-assemble with mRNA and modified amphiphilic molecules to form an mRNA delivery system.

[0067] This invention offers the following advantages: the delivery system achieves in vitro and in vivo mRNA delivery efficiency comparable to LNP, and enables specific targeted transfection in various organs, including the spleen, liver, and lungs. This is achieved by adjusting the mass ratio formula, which is not only versatile and efficient but also simple and controllable. The organ-targeting characteristics of the complex in vivo can be altered simply by adjusting the mass ratio of phosphatidyl polymer to mRNA during complex preparation. As the proportion of phosphatidyl polymer in the mass ratio formula increases, the target organ of the complex changes from the spleen to the liver and then to the lungs.

[0068] Compared to traditional mRNA delivery systems, this new system allows for control over organ-targeting characteristics simply by altering the mass ratio formulation. It boasts high in vitro and in vivo mRNA delivery efficiency, reduces the difficulty and cost of large-scale raw material production, and provides a new platform for efficient, precise, and universal mRNA delivery. The preparation process is simple and low-cost, making it highly suitable for industrial-scale application.

[0069] The phosphatidyl polymer used has advantages such as being environmentally friendly, safe and reliable, having a simple process, low cost and high yield, easy quality control, and easy large-scale production. Attached Figure Description

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

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

[0072] Figure 3 The 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 Examples 4-5 a Test results;

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

[0077] Figure 8 The results of the cell endocytosis and endosome escape ability tests of the composite nanoparticles prepared in Examples 4-5 are shown.

[0078] Figure 9 The results show the organ-selective mRNA delivery capability of the composite nanoparticles prepared in Examples 4-5 after intravenous reinfusion into mice;

[0079] Figure 10 The organ enrichment of the composite nanoparticles prepared in Examples 4-5 after intravenous reinfusion into mice. Detailed Implementation

[0080] To further illustrate the present invention, a detailed description is provided below with reference to embodiments. However, it should be understood that these descriptions are merely for further illustrating the features and advantages of the present invention, and not for limiting the scope of the claims.

[0081] There are no particular restrictions on the source of any raw materials used in this invention; they can be purchased from the market or prepared using conventional methods known to those skilled in the art.

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

[0083] Alkyl secondary phosphonate molecules (PLm) were synthesized from 2-chloro-2-oxo-1,3,2-dioxophosphazenecyclopentane (COP) and the corresponding alkylated alcohol. The 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-dioxophosphazenecyclopentane (COP, 150 mg, 1.05 mmol) was dissolved in THF (10 mL). This solution was then added dropwise to the mixture under an ice-water bath, and the reaction was carried out overnight at 25 °C. Triethylamine hydrochloride was then removed by filtration, and the resulting filtrate was evaporated to produce PLm molecules. The yields of all PLm molecules exceeded 85%.

[0084] Nuclear magnetic resonance analysis was performed on the prepared PLm, and the results are shown in the figure. Figure 1Using deuterated chloroform as the deuteration reagent, the results showed that the methylene signal peak at a chemical shift of 1.68 ppm should be the methylene signal peak at the edge of the phosphatidyl group, indicating the successful formation of the phosphatidyl group. The signal peaks at chemical shifts of 0.89 ppm and 1.27 ppm, respectively, represent the methyl and methylene signal peaks at the phospholipid tail, indicating successful modification of the lipid tail 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 flask and dissolved in anhydrous DMSO. The reaction was carried out under sealed stirring at 85 °C for 24 hours. The solvent was then removed by vacuum drying to obtain a crude product of PEI-x%PLm. This crude product was used directly for subsequent screening.

[0087] Nuclear magnetic resonance analysis was performed on the prepared PEI-x%PLm, and the results are shown in the figure. Figure 2 Using deuterated DMSO as the deuteration reagent, the results showed that the methylene signal peak at chemical shift 4.50 ppm should be the methylene signal peak on the dioxaphospentyl ring, and the disappearance of this peak indicates successful ring-opening of the dioxaphospentyl ring. The methyl and methylene signal peaks at chemical shifts of 0.89 ppm and 1.27 ppm, respectively, represent the methyl and methylene signal peaks on the phospholipid tail, indicating successful modification of the phospholipid tail on PEI.

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

[0089] Branched PEI 1800 (10 mg, 0.0056 mmol), PL10 (15 mg, 0.057 mmol), and epioctane oxide (C8 epoxide, E8) (the modification rate y% calculated from the feed ratio was 25%) were added to a dry reaction flask and dissolved together in DMSO. The reaction system was then stirred at 80 °C for 24 hours. The crude product was subsequently obtained by vacuum drying and used for preliminary screening. For the purified product, the crude product was placed in a dialysis bag and dialyzed in pure water for two days. The liquid in the dialysis bag was collected and freeze-dried, producing a white or pale yellow viscous solid.

[0090] Nuclear magnetic resonance analysis was performed on the prepared PEI-25%PL10 / y%En, and the results are shown in the figure. Figure 3Using deuterated DMSO as the deuteration reagent, the results showed that the methylene signal peak at chemical shift 4.43 ppm should be the methylene signal peak on the epoxy group, and the disappearance of this peak indicates successful ring opening of the epoxy group. The methyl and methylene signal peaks at chemical shifts of 0.89 ppm and 1.27 ppm are the methyl and methylene signal peaks on the phospholipid tail, respectively, and the enhancement of the integral of these peaks indicates successful modification of the PEI backbone with epoxy octane.

[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 rapidly mixed with the aqueous mRNA solution at the following weight ratios: 1:1, 2:1, 5:1, 10:1, 20:1, 30:1, 50:1, and 100:1 (wt / wt), with a volume ratio of 1:3. After incubation for 15 minutes, Pluronic F127 (2.5% wt) was introduced and incubated for another 15 minutes. The MACO was then diluted three-fold with 1×PBS buffer for in vitro mRNA delivery.

[0093] Example 5: Particle size and zeta potential testing 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-containing polymer prepared in Example 3 was dissolved in ethanol. The two phases were rapidly mixed at a flow rate of 12 ml / min in an OBMPO test chip, 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 (Malvin, UK). The results are shown in [Figure number missing]. Figure 4 and Figure 5 All MACOs prepared according to the specified mass ratios exhibited good assembly.

[0095] Example 6: Apparent pKa test of MACO

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

[0097] Example 7: Test of MACO's in vitro mRNA delivery capability in 293T cells

[0098] 293T cells Cultured in Dulbecco modified Eagle medium at 37°C and 5% CO2. One × 10⁶ cells per well were cultured the day before the experiment. 5 Cells were seeded into 24-well plates (Costa). Nanoparticles (NP) containing 100 ng of LucmRNA (MACO) were added to each well and incubated for 24 hours. Cells were then lysed and luciferase substrate was added. Quantitative chemiluminescence was performed using a 20 / 20 spectrophotometer. Cell viability of MACO-treated 293T cells was assessed using the CCK8 assay; results are shown below. Figure 7 When formulated with appropriate mass ratios, MACO exhibits strong in vitro mRNA delivery capabilities.

[0099] Example 8: Test of MACO's cellular endocytosis and endosome escape capabilities

[0100] To assess MACO endocytosis and endosome escape, 293T cells were seeded onto Lab-Tek chamber coverslips at a density of 1.5 × 10⁶ cells per well. 5 Cells were collected. After 12 hours, the culture 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 ratio of 2 / 1, 10 / 1, 50 / 1, and 500 ng Luc mRNA per well). After incubation for 3 hours, the cells were stained with Lysotracker Green DND26 (1 / 5000 dilution) and Hoechst 33342 (0.1 mg / mL) at 37°C for 15 minutes and washed 3 times. The 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 the figure. Figure 8 Analysis of the significant intracellular red fluorescence (representing MACO complex nanoparticles) indicates that all tested MACO nanoparticles exhibited excellent endocytic capacity. White arrows indicate regions where red and green fluorescence (representing endosomes) do not co-localize. This suggests that the MACO nanoparticles successfully escaped from the endosomes.

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

[0102] For in vivo mRNA transfection experiments, the MACO complex was synthesized as previously described. The complex was administered intravenously (iv) to 6-8 week old BALB / c mice at a dose of 0.25 mg / kg of Luc mRNA. Twenty-four hours later, 100 μL of LD-fluorescein potassium solution (30 mg / mL) was administered intraperitoneally. Ten minutes later, the mice were euthanized, and their organs (heart, lungs, liver, spleen, kidneys, and lymph nodes) were collected and processed by PerkinElmer. Imaging on a Lumina LT Series III. The generated images were analyzed using Living Image analysis software (Perkin Elmer), and the results are as follows: Figure 9 As the mass ratio formulation of MACO was adjusted from 2 / 1 to 10 / 1 to 50 / 1 (polymer / mRNA), its target organ changed from the spleen to the liver and then to the lungs.

[0103] For in vivo biodistribution studies, the MACO complex was synthesized as previously described. The complex was administered intravenously (iv) to 6-8 week old BALB / c mice at a dose of 0.25 mg / kg of Luc mRNA. Six hours later, the mice were euthanized, and their organs (heart, lungs, liver, spleen, kidneys, and lymph nodes) were harvested and processed in PerkinElmer. Imaging on a Lumina LT Series III. The generated images were analyzed using Living Image analysis software (Perkin Elmer), and the results are as follows: Figure 10 Regardless of variations in the mass ratio and formulation of MACO, all tested MACO samples were primarily enriched in the liver.

[0104] The above description of the embodiments is only for the purpose of helping to understand the method and core ideas of the present invention. It should be noted that those skilled in the art can make several improvements and modifications to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

1. A phosphatidyl polymer having the structure shown in Formula I: Equation I in, x, y, z, a are the degree of aggregation, 10≤x+y+z+a≤600, x≥0, y>0, z>0, a>0; 4≤n≤16, 4≤m≤16.

2. The method for preparing the phosphatidyl polymer according to claim 1, comprising the following steps: The PEI polymer shown in Formula Ia, the phospholipid compound shown in Formula Ib, and the epoxy compound shown in Formula Ic are reacted to obtain the phosphatidyl polymer shown in Formula I. Formula Ia; Formula Ib; Formula Ic; Equation I in, x, y, z, a are the degree of aggregation, 10≤x+y+z+a≤600, x≥0, y>0, z>0, a>0; 4≤n≤16, 4≤m≤16.

3. The preparation method according to claim 2, characterized in that, The phospholipid compound represented by formula Ib is prepared according to the following method: The dioxophosphorus heterocyclic alkane compound shown in Formula Ib-1 and the alkyl alcohol shown in Formula Ib-2 are reacted to obtain the phospholipid compound shown in Formula Ib. Formula Ib-1; ; 4≤m≤16。 4. An mRNA phosphatidyl polymer delivery system, comprising: mRNA, the phosphatidyl polymer and modified amphiphilic molecule as described in claim 1; The phosphatidyl polymer and mRNA form a core structure through electrostatic interactions, and the modified amphiphilic molecule is anchored to the surface of the core structure through hydrophobic interactions, forming a spherical core-shell structure.

5. The mRNA phosphatidyl polymer delivery system according to claim 4, characterized in that, The mRNA includes at least one of the following: self-reporter gene mRNA, genome editing mRNA, antigen mRNA, tumor suppressor gene mRNA, interleukin mRNA, or chimeric antigen receptor mRNA.

6. The mRNA phosphatidyl polymer delivery system according to claim 4, characterized in that, The modified amphiphilic molecule includes at least one of the following: polyethylene glycol-modified amphiphilic molecule, amphiphilic surfactant, and amphiphilic protein.

7. The mRNA phosphatidyl polymer delivery system according to claim 4, characterized in that, The mass ratio of the mRNA to the phosphatidyl polymer is 1:(0.1~100); the mass ratio of the modified amphiphilic molecule to the total mass of the mRNA and the phosphatidyl polymer is 1:(0.1~100).

8. A method for preparing the mRNA phosphatidyl polymer delivery system according to any one of claims 4 to 7, comprising the following steps: An acidic buffer solution containing the phosphatidyl polymer of claim 1 and a buffer solution containing mRNA are mixed, and the complex particles of phosphatidyl polymer and mRNA are assembled by electrostatic interaction. The complex particles of the above-mentioned phosphatidyl polymer and mRNA were modified with biocompatible molecules to obtain an mRNA phosphatidyl polymer delivery system.

9. The application of the mRNA phosphatidyl polymer delivery system according to any one of claims 4 to 7 or the mRNA phosphatidyl polymer delivery system prepared by the preparation method according to claim 8 as a drug carrier; The drugs include tumor treatment drugs, tumor vaccines, tumor-targeted drugs, in vivo and in vitro diagnostic tumor materials, drugs for treating inflammation-related diseases, drugs for treating infectious diseases, drugs for preventing infectious diseases, drugs for wound healing, drugs for treating cardiovascular and cerebrovascular chronic diseases, drugs for treating organ fibrosis, drugs for treating viral infections, drugs for preventing viral infections, drugs for treating gene editing in vivo and in vitro, drugs for treating non-alcoholic fatty liver disease, drugs for treating intestinal diseases, neoantigen vaccines, cell differentiation-inducing drugs, protein cosmetic agents, drugs for treating protein deficiency diseases, aging repair products, and anti-aging products.

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