PBAE-DOPE-PEG2000 polymer-lipid nanocomposite as well as preparation method and application thereof

Abstract

The invention belongs to the field of biomedical materials, and particularly relates to a PBAE-DOPE-PEG2000 polymer-lipid nanocomposite and a preparation method and application thereof.The preparation method comprises the steps that a dimethyl sulfoxide-ethanol solution formed by mixing DOPE-PEG2000 lipid and PBAE serves as an oil phase, a sodium acetate / acetic acid buffer solution serves as a water phase, and the PBAE-DOPE-PEG2000 polymer-lipid nanocomposite is prepared through a microfluidic synthesis mode; the PBAE-DOPE-PEG2000 polymer-lipid nano composite material is prepared by the steps of carrying out ultrasonic treatment on the PBAE-DOPE-PEG The polymer-lipid nano composite material can efficiently load exogenous genes, the surface performance of the polymer-lipid nano composite material is changed and the stability of nano particles is improved by introducing DOPE-PEG2000 lipid, membrane fusion can be promoted, the risk of lysosome degradation is reduced, the transfection process of the exogenous genes in recipient cells is accelerated, and efficient expression in 293FT cells is realized. The efficient transfection of the polymer-lipid nanocomposite material is expected to show important application prospects in immune cell therapy or tumor cell therapy.

Description

Technical Field

[0001] The invention belongs to the field of biomedical materials, and specifically relates to a PBAE-DOPE-PEG2000 polymer-lipid nanocomposite material and a preparation method and application thereof. Background Art

[0002] The information disclosed in the background of the invention is only intended to enhance the understanding of the overall background of the invention and should not be necessarily regarded as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Gene therapy is the introduction of exogenous normal genes into target cells to correct or compensate for diseases caused by defective and abnormal genes, thereby achieving the purpose of treatment. The safe, efficient and stable expression of exogenous genes in target cells is the key to the success of gene therapy. However, naked DNA / RNA is easily hydrolyzed by nucleases in the extracellular environment and is easily degraded by lysosomes after entering the cell. Therefore, genes are often loaded on nanocarriers for delivery. At present, gene delivery vectors are divided into two categories: viral vectors and non-viral vectors. Although the transfection effect of viral vectors is better than that of non-viral vectors, the biosafety issues they bring cannot be ignored.

[0004] Cationic polymer carriers are one of the most representative non-viral carriers, mainly including polyethyleneimine (PEI), poly(β-amino ester) (PBAE), polyamidoamine (PAMAM), polylactic acid (PLA), etc., which have attracted extensive attention from researchers due to their wide sources of raw materials, simple synthesis methods, low immunogenicity and good biocompatibility. Although cationic polymer carriers have shown certain application potential, their cytotoxicity caused by their difficulty in degradation and low transfection efficiency also limit their clinical application.

[0005] Poly(β-amino ester) (PBAE) has diverse synthetic raw materials, simple preparation process, flexible chemical structure, adjustable particle size, pH sensitivity and good biodegradability. The ester bonds on the main chain can be hydrolyzed into small molecules and then excreted from the body, avoiding the high cytotoxicity caused by other cationic polymer carriers due to their difficulty in degradation. It has shown significant advantages in the field of gene delivery, but the problem of low transfection efficiency still exists. Therefore, it is necessary to find a new material system and its preparation method that can stably exist and improve the transfection efficiency of poly(β-amino ester) (PBAE) materials. Summary of the invention

[0006] In view of the needs of the prior art, the purpose of the present invention is to provide a PBAE-DOPE-PEG2000 polymer-lipid nanocomposite material and its preparation method and application. The present invention improves the stability of nanoparticles by modifying PBAE with functionalized PEG lipids (DOPE-PEG2000) to prevent the rapid degradation of the payload when entering the systemic blood. At the same time, DOPE-PEG2000 lipid (dioleoylphosphatidylethanolamine polyethylene glycol) modification can promote membrane fusion, achieve lysosomal escape, effectively release genes in cells, promote efficient gene transfection and expression, and further improve the transfection efficiency of PBAE-based nanomaterials.

[0007] Specifically, the present invention provides the following technical solutions:

[0008] In a first aspect of the present invention, a PBAE-DOPE-PEG2000 polymer-lipid nanocomposite material is provided, wherein the polymer-lipid nanocomposite material comprises a PBAE macromolecular material and a functionalized PEG lipid with surface modified PBAE; the particle size of the nanocomposite material is 50 to 200 nm;

[0009] Wherein, the PBAE polymer material is selected from one or more of PBAE447, PBAE446, and PBAE536; and the functionalized PEG lipid is DOPE-PEG2000.

[0010] The second aspect of the present invention provides a method for preparing a PBAE-DOPE-PEG2000 polymer-lipid nanocomposite material, comprising the following steps:

[0011] S1, adding amino alcohol to diacrylate, dissolving the obtained intermediate product in an organic solvent, and adding a capping group to cap it, to obtain a PBAE material;

[0012] S2, adding DOPE-PEG2000 dimethyl sulfoxide:ethanol solution to poly(β-amino lipid) dimethyl sulfoxide:ethanol solution, and the mixed solution after incubation is used as the oil phase component; sodium acetate / acetic acid buffer solution is used as the water phase component;

[0013] S3. Using a microfluidic chip, the oil phase and the water phase components are mixed to obtain a PBAE-DOPE-PEG2000 polymer-lipid nanocomposite material.

[0014] Preferably, in step S1, the diacrylate is selected from one of 1,4-butanediol diacrylate and 1,5-pentanediol diacrylate; the amino alcohol is selected from one of 4-amino-1-butanol and 3-amino-1-propanol; and the molar ratio of the diacrylate to the amino alcohol is 1:1 to 1.2:1.

[0015] Preferably, in step S1, the intermediate product is obtained by heating a diacrylate and an amino alcohol; further preferably, the temperature of the heating reaction is 70 to 90° C. and the time is 20 to 26 hours.

[0016] Preferably, in step S1, the organic solvent is selected from tetrahydrofuran and dimethyl sulfoxide.

[0017] Preferably, in step S1, the capping group is selected from one of 1-(3-aminopropyl)-4-methylpiperazine and 2-[(3-aminopropyl)amine]ethanol.

[0018] Preferably, in step S2, the poly(β-amino lipid) dimethyl sulfoxide:ethanol solution is prepared by mixing a poly(β-amino lipid) dimethyl sulfoxide solution with an ethanol solution, wherein the volume mass concentration of the poly(β-amino lipid) dimethyl sulfoxide solution is 100 to 200 mg / mL;

[0019] The poly(β-amino lipid) dimethyl sulfoxide:ethanol solution has a volume mass concentration of 6 to 24 mg / mL, and is specifically obtained by diluting the poly(β-amino lipid) dimethyl sulfoxide solution with a dimethyl sulfoxide:ethanol solution; the dimethyl sulfoxide:ethanol solution refers to a dimethyl sulfoxide to ethanol volume ratio of 1 to 2:1;

[0020] The DOPE-PEG2000 dimethyl sulfoxide:ethanol solution is prepared by mixing a DOPE-PEG2000 dimethyl sulfoxide solution with an ethanol solution. The volume mass concentration of the DOPE-PEG2000 dimethyl sulfoxide:ethanol solution is 3-5 mg / mL.

[0021] Preferably, in step S2, the mass ratio of DOPE-PEG2000 to poly(β-amino lipid) is 0.03:1 to 0.2:1.

[0022] Preferably, in step S2, the incubation temperature is 25-30°C and the time is 20-40 minutes.

[0023] Preferably, in step S2, the sodium acetate / acetic acid buffer solution is prepared from a sodium acetate buffer solution and an acetate buffer solution in a volume ratio of 2 to 4:1, the concentration of the sodium acetate / acetic acid buffer solution is 25 mM, and the pH value is 5.2; the concentrations of the sodium acetate buffer solution and the acetate buffer solution are both 25 mM.

[0024] Preferably, in step S3, the flow rate ratio of the water phase to the oil phase is 1 to 4:1, and then the obtained PBAE-DOPE-PEG2000 polymer-lipid nanocomposite particles are dialyzed twice with ammonia water and PBS respectively.

[0025] The third aspect of the present invention provides a use of the PBAE-DOPE-PEG2000 polymer-lipid nanocomposite material described in the first aspect in gene delivery.

[0026] The fourth aspect of the present invention provides a use of the PBAE-DOPE-PEG2000 polymer-lipid nanocomposite material described in the first aspect in the preparation of a therapeutic gene delivery preparation.

[0027] The fifth aspect of the present invention provides a PBAE-DOPE-PEG2000 polymer-lipid nanocomposite material loaded with exogenous genes, comprising the PBAE-DOPE-PEG2000 polymer-lipid nanocomposite material prepared in the second aspect and also comprising exogenous genes.

[0028] Preferably, the exogenous gene is selected from a green fluorescent protein (GFP) plasmid and a luciferase reporter gene (Luc) plasmid.

[0029] Preferably, the loading of exogenous genes is specifically performed by encapsulating the exogenous genes using a PBAE-DOPE-PEG2000 polymer-lipid nanocomposite material.

[0030] Further preferably, the specific operation of encapsulating the exogenous gene is: mixing the exogenous gene plasmid and the nanocomposite material, and uniformly dispersing the plasmid to obtain polymer-lipid nanocomposite particles loaded with the exogenous gene plasmid.

[0031] Further preferably, the mass ratio of the exogenous gene-loaded plasmid to PBAE is 1 / 90 to 1 / 30.

[0032] The beneficial effects achieved by one or more of the above technical solutions of the present invention are as follows:

[0033] (1) The PBAE-DOPE-PEG2000 polymer-lipid nanocomposite prepared by the present invention can be used to load exogenous genes. The introduction of functionalized PEG lipids changes its surface properties and improves the stability of nanoparticles, promotes membrane fusion, reduces the risk of lysosomal degradation, and accelerates the transfection process of exogenous genes in recipient cells, thereby achieving efficient expression.

[0034] (2) The transfection effect of the PBAE447-DOPE-PEG2000 polymer-lipid nanocomposite particles prepared by the present invention is significantly improved compared with that of PBAE447. When the amount of fluorescent protein particles added is 2.0 μg, the transfection efficiency of PBAE447-5% DOPE-PEG2000 and PBAE447-7% DOPE-PEG2000 polymer-lipid nanocomposite particles is increased by about 50% compared with that of PBAE447, and there is no significant change in cell viability; and fluorescence microscopy images show that the cell fluorescent protein expression of PBAE447-DOPE-PEG2000 polymer-lipid nanocomposite particles is more obvious. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0036] Figure 1 Provide a preparation roadmap for PBAE polymer materials;

[0037] Figure 2 is the nuclear magnetic resonance spectrum (NMR) of PBAE447;

[0038] Figure 3 The particle size distribution diagram and microscope image of the PBAE447-3% DOPE-PEG2000 polymer-lipid nanocomposite prepared in Example 1, wherein a is the particle size distribution diagram and b is the transmission electron microscope image;

[0039] Figure 4 The particle size distribution diagram and microscope image of the PBAE447-5% DOPE-PEG2000 polymer-lipid nanocomposite prepared in Example 2, wherein a is the particle size distribution diagram and b is the transmission electron microscope image;

[0040] Figure 5 The particle size distribution diagram and microscope image of the PBAE447-7% DOPE-PEG2000 polymer-lipid nanocomposite prepared in Example 3, wherein a is the particle size distribution diagram and b is the transmission electron microscope image;

[0041] Figure 6 The particle size distribution diagram and microscope image of the PBAE447-10% DOPE-PEG2000 polymer-lipid nanocomposite prepared in Example 4, wherein a is the particle size distribution diagram and b is the transmission electron microscope image;

[0042] Figure 7 The figure is a transfection efficiency diagram of in vitro cell culture of PBAE447-DOPE-PEG2000 nanoparticles loaded with fluorescent protein plasmids;

[0043] Figure 8 Fluorescence microscopy images of in vitro cell culture of PBAE447-DOPE-PEG2000 nanoparticles loaded with fluorescent protein particles;

[0044] Fig. 9 This is a comparative test chart of the transfection efficiency and cell viability of PBAE447, PBAE447-5% DOPE-PEG2000 and PBAE447-5% PEG2000 polymer-lipid nanocomposite particles. DETAILED DESCRIPTION

[0045] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0046] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0047] Example 1 :This embodiment provides a PBAE447-3% DOPE-PEG2000 polymer-lipid nanocomposite material, and its preparation method comprises the following steps:

[0048] (1) Synthesis of poly(β-amino ester) (PBAE447) polymer raw materials

[0049] ① Accurately weigh 1.223 g of 1,4-butanediol diacrylate and 0.5 g of 4-amino-1-butanol, and react in an oil bath at 70°C for 24 h to obtain an intermediate reactant;

[0050] ② The intermediate reactant obtained in step ① was dissolved in 5 mL of tetrahydrofuran (THF), and 6 mL of 1-(3-aminopropyl)-4-methylpiperazine tetrahydrofuran solution with a volume mass concentration of 90 mg / mL was prepared, and the intermediate reactant was added dropwise for end-capping, the speed was kept constant at 500 r / min, and the reaction was carried out at room temperature for 2 h;

[0051] ③ Wash twice with 5 volumes of ether, centrifuge, and vacuum dry to obtain the PBAE447 polymer material; then prepare a PBAE447 dimethyl sulfoxide (DMSO) solution with a volume mass concentration of 200 mg / mL and store it at -20°C.

[0052] (2) Preparation of PBAE447-3% DOPE-PEG2000 nanocomposites using a microfluidic device

[0053] ① Pipette 50 μL of PABE447 dimethyl sulfoxide (DMSO) solution with a volume mass concentration of 200 mg / mL, add an equal volume of ethanol solution, and dilute with a 1:1 volume ratio of DMSO:ethanol solution to obtain a PABE447 dimethyl sulfoxide:ethanol solution with a volume mass concentration of 18 mg / mL;

[0054] ② Prepare 56 μL of DOPE-PEG2000 dimethyl sulfoxide:ethanol solution with a volume mass concentration of 5 mg / mL, add 0.5 mL of PABE447 dimethyl sulfoxide:ethanol solution with a volume mass concentration of 18 mg / mL in step ①, add dimethyl sulfoxide:ethanol solution to a volume of 1 mL, and incubate at room temperature for 0.5 h;

[0055] ③Prepare 25mM sodium acetate / acetic acid buffer solution with a pH of 5.2: accurately weigh 1.701g sodium acetate trihydrate and dissolve it in 500mL pure water to obtain a 25mM sodium acetate buffer solution; pipette 0.72mL of acetic acid solution and dissolve it in 500mL pure water to obtain a 25mM acetate buffer solution; prepare a 25mM sodium acetate / acetic acid buffer solution with a pH of 5.2 according to a certain volume ratio;

[0056] ④ Using a microfluidic device, 25 mM sodium acetate / acetic acid buffer solution with pH = 5.2 as the aqueous phase, the dimethyl sulfoxide:ethanol solution in step ② as the oil phase, and a total flow rate of 3 mL / min to prepare a PBAE447-3% DOPE-PEG2000 polymer-lipid nanocomposite material; wherein the aqueous phase flow rate is 2 mL / min, and the oil phase flow rate is 1 mL / min;

[0057] ⑤ Using an activated dialysis bag with a molecular weight of 8-14K, dialyze the nanoparticles obtained in step ④ in a 0.5% ammonia solution for 1 hour, and then dialyze in a PBS solution for 1 hour.

[0058] like Figures 1-2 As shown, the nuclear magnetic resonance spectrum shows that the poly (β-amino lipid) (PBAE447) polymer material was successfully prepared. When the substituent groups of the raw materials used are changed, other types of PBAE polymer materials can be prepared accordingly.

[0059] like Figure 3 As shown, the PBAE447-3% DOPE-PEG2000 polymer-lipid nanocomposite (such as Figure 3 The transmission electron microscope image shows that the average particle size is about 50nm, the prepared nanoparticles are spherical particles with uniform particle size distribution, and the average hydrated particle size obtained by dynamic light scattering test is about 220nm (as shown in Figure 3 (shown in a).

[0060] Example 2 :This embodiment provides a PBAE447-5% DOPE-PEG2000 polymer-lipid nanocomposite material, and its preparation method comprises the following steps:

[0061] (1) Synthesizing poly(β-amino ester) (PBAE447) polymer raw material in the same manner as in Example 1;

[0062] (2) Preparation of PBAE447-5% DOPE-PEG2000 nanocomposites using a microfluidic device

[0063] ① Pipette 50 μL of PABE447 dimethyl sulfoxide (DMSO) solution with a volume mass concentration of 200 mg / mL, add an equal volume of ethanol solution, and dilute with a 1:1 volume ratio of DMSO:ethanol solution to obtain a PABE447 dimethyl sulfoxide:ethanol solution with a volume mass concentration of 18 mg / mL;

[0064] ② Prepare 95 μL of DOPE-PEG2000 dimethyl sulfoxide:ethanol solution with a volume mass concentration of 5 mg / mL, add 0.5 mL of PABE447 dimethyl sulfoxide:ethanol solution with a volume mass concentration of 18 mg / mL in step ①, add dimethyl sulfoxide:ethanol solution to a volume of 1 mL, and incubate at room temperature for 0.5 h;

[0065] ③Prepare 25mM sodium acetate / acetic acid buffer solution with a pH of 5.2: accurately weigh 1.701g sodium acetate trihydrate and dissolve it in 500mL pure water to obtain a 25mM sodium acetate buffer solution; pipette 0.72mL of acetic acid solution and dissolve it in 500mL pure water to obtain a 25mM acetate buffer solution; prepare a 25mM sodium acetate / acetic acid buffer solution with a pH of 5.2 according to a certain volume ratio;

[0066] ④ Using a microfluidic device, 25 mM, pH = 5.2 sodium acetate / acetic acid buffer solution is used as the aqueous phase, and the dimethyl sulfoxide:ethanol solution in step ② is used as the oil phase, and the PBAE447-5% DOPE-PEG2000 polymer-lipid nanocomposite material is prepared at a total flow rate of 3 mL / min; wherein the aqueous phase flow rate is 2 mL / min, and the oil phase flow rate is 1 mL / min;

[0067] ⑤ Using an activated dialysis bag with a molecular weight of 8-14K, dialyze the nanoparticles obtained in step ④ in a 0.5% ammonia solution for 1 hour, and then dialyze in a PBS solution for 1 hour.

[0068] like Figure 4 As shown, PBAE-5% DOPE-PEG2000 polymer-lipid nanocomposites (such as Figure 4 The transmission electron microscope image shows that the average particle size is about 40nm, the prepared nanoparticles are spherical particles with uniform particle size distribution, and the average hydrated particle size obtained by dynamic light scattering test is about 170nm (as shown in Figure 4 (shown in a).

[0069] Example 3 :This embodiment provides a PBAE447-7% DOPE-PEG2000 polymer-lipid nanocomposite material, and its preparation method comprises the following steps:

[0070] (1) Synthesizing poly(β-amino ester) (PBAE447) polymer raw material in the same manner as in Example 1;

[0071] (2) Preparation of PBAE447-7% DOPE-PEG2000 nanocomposites using a microfluidic device

[0072] ① Pipette 50 μL of PABE447 dimethyl sulfoxide (DMSO) solution with a volume mass concentration of 200 mg / mL, add an equal volume of ethanol solution, and dilute with a 1:1 volume ratio of DMSO:ethanol solution to obtain a PABE447 dimethyl sulfoxide:ethanol solution with a volume mass concentration of 18 mg / mL;

[0073] ② Prepare 136 μL of 5 mg / mL DOPE-PEG2000 dimethyl sulfoxide:ethanol solution, add 0.5 mL of 18 mg / mL PABE447 dimethyl sulfoxide:ethanol solution prepared in step ①, add dimethyl sulfoxide:ethanol solution to a volume of 1 mL, and incubate at room temperature for 0.5 h;

[0074] ③Prepare 25mM sodium acetate / acetic acid buffer solution with a pH of 5.2: accurately weigh 1.701g sodium acetate trihydrate and dissolve it in 500mL pure water to obtain a 25mM sodium acetate buffer solution; pipette 0.72mL of acetic acid solution and dissolve it in 500mL pure water to obtain a 25mM acetate buffer solution; prepare a 25mM sodium acetate / acetic acid buffer solution with a pH of 5.2 according to a certain volume ratio;

[0075] ④ Using a microfluidic device, 25 mM, pH = 5.2 sodium acetate / acetic acid buffer solution is used as the aqueous phase, and the dimethyl sulfoxide:ethanol solution in step ② is used as the oil phase, and the PBAE447-7% DOPE-PEG2000 polymer-lipid nanocomposite material is prepared at a total flow rate of 3 mL / min; wherein the aqueous phase flow rate is 2 mL / min, and the oil phase flow rate is 1 mL / min;

[0076] ⑤ Using an activated dialysis bag with a molecular weight of 8-14K, dialyze the nanoparticles obtained in step ④ in a 0.5% ammonia solution for 1 hour, and then dialyze in a PBS solution for 1 hour.

[0077] like Figure 5 As shown, PBAE-7% DOPE-PEG2000 polymer-lipid nanocomposites (such as Figure 5 The transmission electron microscope image shows that the average particle size is about 40nm, the prepared nanoparticles are spherical particles with uniform particle size distribution, and the average hydrated particle size obtained by dynamic light scattering test is about 180nm (as shown in Figure 5 (shown in a).

[0078] Example 4 :This embodiment provides a PBAE447-10% DOPE-PEG2000 polymer-lipid nanocomposite material, and its preparation method comprises the following steps:

[0079] (1) Synthesizing poly(β-amino ester) (PBAE447) polymer raw material in the same manner as in Example 1;

[0080] (2) Preparation of PBAE447-10% DOPE-PEG2000 nanocomposites using a microfluidic device

[0081] ① Pipette 50 μL of PABE447 dimethyl sulfoxide (DMSO) solution with a volume mass concentration of 200 mg / mL, add an equal volume of ethanol solution, and dilute with a 1:1 volume ratio of DMSO:ethanol solution to obtain a PABE447 dimethyl sulfoxide:ethanol solution with a volume mass concentration of 18 mg / mL;

[0082] ② Prepare 200 μL of DOPE-PEG2000 dimethyl sulfoxide:ethanol solution with a volume mass concentration of 5 mg / mL, add 0.5 mL of PABE447 dimethyl sulfoxide:ethanol solution with a volume mass concentration of 18 mg / mL in step ①, add dimethyl sulfoxide:ethanol solution to a volume of 1 mL, and incubate at room temperature for 0.5 h;

[0083] ③Prepare 25mM sodium acetate / acetic acid buffer solution with a pH of 5.2: accurately weigh 1.701g sodium acetate trihydrate and dissolve it in 500mL pure water to obtain a 25mM sodium acetate buffer solution; pipette 0.72mL of acetic acid solution and dissolve it in 500mL pure water to obtain a 25mM acetate buffer solution; prepare a 25mM sodium acetate / acetic acid buffer solution with a pH of 5.2 according to a certain volume ratio;

[0084] ④ Using a microfluidic device, 25 mM sodium acetate / acetic acid buffer solution with pH = 5.2 as the aqueous phase, the dimethyl sulfoxide:ethanol solution in step ② as the oil phase, and a total flow rate of 3 mL / min to prepare a PBAE447-10% DOPE-PEG2000 polymer-lipid nanocomposite material; wherein the aqueous phase flow rate is 2 mL / min, and the oil phase flow rate is 1 mL / min;

[0085] ⑤ Using an activated dialysis bag with a molecular weight of 8-14K, dialyze the nanoparticles obtained in step ④ in a 0.5% ammonia solution for 1 hour, and then dialyze in a PBS solution for 1 hour.

[0086] like Figure 6 As shown, PBAE-10% DOPE-PEG2000 polymer-lipid nanocomposites (such as Figure 6 The transmission electron microscope image shows that the average particle size is about 40nm, the prepared nanoparticles are spherical particles with uniform particle size distribution, and the average hydrated particle size obtained by dynamic light scattering test is about 180nm (as shown in Figure 6 (shown in a).

[0087] It can be seen from Examples 1 to 4 that as the DOPE-PEG2000 lipid content in the polymer-lipid nanocomposite material gradually increases, the PDI gradually decreases and the particle size becomes more uniform.

[0088] Comparative Example 1 :This comparative example provides a PBAE447-5% PEG2000 polymer nanocomposite material, and its preparation method comprises the following steps:

[0089] (1) Synthesizing poly(β-amino ester) (PBAE447) polymer raw material in the same manner as in Example 1;

[0090] (2) Preparation of PBAE447-5% PEG2000 nanocomposites using a microfluidic device

[0091] ① Pipette 50 μL of PABE447 dimethyl sulfoxide (DMSO) solution with a volume mass concentration of 200 mg / mL, add an equal volume of ethanol solution, and dilute with a 1:1 volume ratio of DMSO:ethanol solution to obtain a PABE447 dimethyl sulfoxide:ethanol solution with a volume mass concentration of 18 mg / mL;

[0092] ② Prepare 95 μL of PEG2000 dimethyl sulfoxide:ethanol solution with a volume mass concentration of 5 mg / mL, add 0.5 mL of PABE447 dimethyl sulfoxide:ethanol solution with a volume mass concentration of 18 mg / mL in step ①, add dimethyl sulfoxide:ethanol solution to a volume of 1 mL, and incubate at room temperature for 0.5 h;

[0093] ③Prepare 25mM sodium acetate / acetic acid buffer solution with a pH of 5.2: accurately weigh 1.701g sodium acetate trihydrate and dissolve it in 500mL pure water to obtain a 25mM sodium acetate buffer solution; pipette 0.72mL of acetic acid solution and dissolve it in 500mL pure water to obtain a 25mM acetate buffer solution; prepare a 25mM sodium acetate / acetic acid buffer solution with a pH of 5.2 according to a certain volume ratio;

[0094] ④ Using a microfluidic device, 25 mM sodium acetate / acetic acid buffer solution with pH = 5.2 as the aqueous phase, the dimethyl sulfoxide:ethanol solution in step ② as the oil phase, and a total flow rate of 3 mL / min to prepare a PBAE447-5% PEG2000 polymer-lipid nanocomposite material; wherein the aqueous phase flow rate is 2 mL / min, and the oil phase flow rate is 1 mL / min;

[0095] ⑤ Using an activated dialysis bag with a molecular weight of 8-14K, dialyze the nanoparticles obtained in step ④ in a 0.5% ammonia solution for 1 hour, and then dialyze in a PBS solution for 1 hour.

[0096] Test Example 1 :This application example provides a PBAE-3% DOPE-PEG2000 polymer-lipid nanocomposite material loaded with fluorescent protein particles, comprising the following steps:

[0097] (1) Synthesizing poly(β-amino ester) (PBAE447) polymer raw material in the same manner as in Example 1;

[0098] (2) Preparation of PBAE-3% DOPE-PEG2000 polymer-lipid nanocomposites loaded with fluorescent protein particles using a microfluidic device

[0099] ① Take 2 mL of PABE447 dimethyl sulfoxide (DMSO) solution with a volume mass concentration of 200 mg / mL, add an equal volume of ethanol solution, and dilute it with a 1:1 volume ratio of DMSO:ethanol solution to obtain a PABE447 dimethyl sulfoxide:ethanol solution with a volume mass concentration of 18 mg / mL;

[0100] ② Prepare 56 μL of DOPE-PEG2000 dimethyl sulfoxide:ethanol solution with a volume mass concentration of 5 mg / mL, add 0.5 mL of PABE447 dimethyl sulfoxide:ethanol solution with a volume mass concentration of 18 mg / mL in step (1), add dimethyl sulfoxide:ethanol solution to a total volume of 1 mL, and incubate at room temperature for 0.5 h;

[0101] ③Pipette 10 mL of sodium acetate / acetic acid buffer solution with a molar concentration of 25 mM and pH = 5.2;

[0102] ④ Using a microfluidic device, the dimethyl sulfoxide:ethanol solution in step ② is used as the oil phase, the sodium acetate / acetic acid buffer solution in step ③ is used as the aqueous phase, and PBAE-3% DOPE-PEG2000 polymer-lipid nanocomposite particles (1 ml) are prepared at a total flow rate of 3 mL / min, and the nanoparticles are collected in a centrifuge tube containing 0.05 mg of fluorescent protein plasmids, and the plasmids are evenly dispersed by a vortex mixer to obtain PBAE-3% DOPE-PEG2000 polymer-lipid nanocomposite particles loaded with fluorescent protein plasmids; wherein the aqueous phase flow rate is 2 mL / min, and the oil phase flow rate is 1 mL / min;

[0103] ⑤ Using an activated dialysis bag with a molecular weight of 8-14K, dialyze the nanoparticles obtained in step ④ in a 0.5% ammonia solution for 1 hour, and then dialyze in a PBS solution for 1 hour;

[0104] ⑥ Add 0.5μg, 1.0μg, 1.5μg, 2.0μg, 2.5μg, and 3.0μg of nanoparticles loaded with fluorescent protein (corresponding to nanoparticle solution volumes of 10μL, 20μL, 30μL, 40μL, 50μL, and 60μL) into the in vitro cultured 293FT cells and culture for 48h.

[0105] Test Example 2 :This application example provides a PBAE-5% DOPE-PEG2000 polymer-lipid nanocomposite material loaded with fluorescent protein particles, comprising the following steps:

[0106] (1) Synthesizing poly(β-amino ester) (PBAE447) polymer raw material in the same manner as in Example 1;

[0107] (2) Preparation of PBAE-5% DOPE-PEG2000 polymer-lipid nanocomposites loaded with fluorescent protein particles using a microfluidic device

[0108] ① Take 2 mL of PABE447 dimethyl sulfoxide (DMSO) solution with a volume mass concentration of 200 mg / mL, add an equal volume of ethanol solution, and dilute it with a 1:1 volume ratio of DMSO:ethanol solution to obtain a PABE447 dimethyl sulfoxide:ethanol solution with a volume mass concentration of 18 mg / mL;

[0109] ② Prepare 95 μL of DOPE-PEG2000 dimethyl sulfoxide:ethanol solution with a volume mass concentration of 5 mg / mL, add 0.5 mL of PABE447 dimethyl sulfoxide:ethanol solution with a volume mass concentration of 18 mg / mL in step (1), add dimethyl sulfoxide:ethanol solution to a total volume of 1 mL, and incubate at room temperature for 0.5 h;

[0110] ③Pipette 10 mL of sodium acetate / acetic acid buffer solution with a molar concentration of 25 mM and pH = 5.2;

[0111] ④ Using a microfluidic device, the dimethyl sulfoxide: ethanol solution in step ② is used as the oil phase, the sodium acetate / acetic acid buffer solution in step ③ is used as the aqueous phase, and the PBAE-5% DOPE-PEG2000 polymer-lipid nanocomposite particles (1 ml) are prepared at a total flow rate of 3 mL / min, and the nanoparticles are collected in a centrifuge tube containing 0.05 mg of fluorescent protein plasmids, and the plasmids are evenly dispersed by a vortex mixer to obtain PBAE-5% DOPE-PEG2000 polymer-lipid nanocomposite particles loaded with fluorescent protein plasmids; wherein the aqueous phase flow rate is 2 mL / min, and the oil phase flow rate is 1 mL / min;

[0112] ⑤ Using an activated dialysis bag with a molecular weight of 8-14K, dialyze the nanoparticles obtained in step ④ in a 0.5% ammonia solution for 1 hour, and then dialyze in a PBS solution for 1 hour;

[0113] ⑥ Add 0.5μg, 1.0μg, 1.5μg, 2.0μg, 2.5μg, and 3.0μg of nanoparticles loaded with fluorescent protein (corresponding to nanoparticle solution volumes of 10μL, 20μL, 30μL, 40μL, 50μL, and 60μL) into the in vitro cultured 293FT cells and culture for 48h.

[0114] Test Example 3 :This application example provides a PBAE-7% DOPE-PEG2000 polymer-lipid nanocomposite material loaded with fluorescent protein particles, comprising the following steps:

[0115] (1) Synthesizing poly(β-amino ester) (PBAE447) polymer raw material in the same manner as in Example 1;

[0116] (2) Preparation of PBAE-7% DOPE-PEG2000 polymer-lipid nanocomposites loaded with fluorescent protein particles using a microfluidic device

[0117] ① Take 2 mL of PABE447 dimethyl sulfoxide (DMSO) solution with a volume mass concentration of 200 mg / mL, add an equal volume of ethanol solution, and dilute it with a 1:1 volume ratio of DMSO:ethanol solution to obtain a PABE447 dimethyl sulfoxide:ethanol solution with a volume mass concentration of 18 mg / mL;

[0118] ② Prepare 136 μL of DOPE-PEG2000 dimethyl sulfoxide:ethanol solution with a volume mass concentration of 5 mg / mL, add 0.5 mL of PABE447 dimethyl sulfoxide:ethanol solution with a volume mass concentration of 18 mg / mL in step (1), add dimethyl sulfoxide:ethanol solution to a total volume of 1 mL, and incubate at room temperature for 0.5 h;

[0119] ③Pipette 10 mL of sodium acetate / acetic acid buffer solution with a molar concentration of 25 mM and pH = 5.2;

[0120] ④ Using a microfluidic device, the dimethyl sulfoxide: ethanol solution in step ② is used as the oil phase, the sodium acetate / acetic acid buffer solution in step ③ is used as the aqueous phase, and the PBAE-7% DOPE-PEG2000 polymer-lipid nanocomposite particles (1 ml) are prepared at a total flow rate of 3 mL / min, and the nanoparticles are collected in a centrifuge tube containing 0.05 mg of fluorescent protein plasmids, and the plasmids are evenly dispersed by a vortex mixer to obtain PBAE-7% DOPE-PEG2000 polymer-lipid nanocomposite particles loaded with fluorescent protein plasmids; wherein the aqueous phase flow rate is 2 mL / min, and the oil phase flow rate is 1 mL / min;

[0121] ⑤ Using an activated dialysis bag with a molecular weight of 8-14K, dialyze the nanoparticles obtained in step ④ in a 0.5% ammonia solution for 1 hour, and then dialyze in a PBS solution for 1 hour;

[0122] ⑥ Add 0.5μg, 1.0μg, 1.5μg, 2.0μg, 2.5μg, and 3.0μg of nanoparticles loaded with fluorescent protein (corresponding to nanoparticle solution volumes of 10μL, 20μL, 30μL, 40μL, 50μL, and 60μL) into the in vitro cultured 293FT cells and culture for 48h.

[0123] Test Example 4 :This application example provides a PBAE-10% DOPE-PEG2000 polymer-lipid nanocomposite material loaded with fluorescent protein particles, comprising the following steps:

[0124] (1) Synthesizing poly(β-amino ester) (PBAE447) polymer raw material in the same manner as in Example 1;

[0125] (2) Preparation of PBAE-10% DOPE-PEG2000 polymer-lipid nanocomposites loaded with fluorescent protein particles using a microfluidic device

[0126] ① Take 2 mL of PABE447 dimethyl sulfoxide (DMSO) solution with a volume mass concentration of 200 mg / mL, add an equal volume of ethanol solution, and dilute it with a 1:1 volume ratio of DMSO:ethanol solution to obtain a PABE447 dimethyl sulfoxide:ethanol solution with a volume mass concentration of 18 mg / mL;

[0127] ② Prepare 200 μL of 5 mg / mL DOPE-PEG2000 dimethyl sulfoxide:ethanol solution, add 0.5 mL of 18 mg / mL PABE447 dimethyl sulfoxide:ethanol solution in step (1), add dimethyl sulfoxide:ethanol solution to a total volume of 1 mL, and incubate at room temperature for 0.5 h;

[0128] ③Pipette 10 mL of sodium acetate / acetic acid buffer solution with a molar concentration of 25 mM and pH = 5.2;

[0129] ④ Using a microfluidic device, the dimethyl sulfoxide:ethanol solution in step ② is used as the oil phase, the sodium acetate / acetic acid buffer solution in step ③ is used as the aqueous phase, and the PBAE-10% DOPE-PEG2000 polymer-lipid nanocomposite particles (1 ml) are prepared at a total flow rate of 3 mL / min, and the nanoparticles are collected in a centrifuge tube containing 0.05 mg of fluorescent protein plasmids, and the plasmids are evenly dispersed by a vortex mixer to obtain PBAE-10% DOPE-PEG2000 polymer-lipid nanocomposite particles loaded with fluorescent protein plasmids; wherein the aqueous phase flow rate is 2 mL / min, and the oil phase flow rate is 1 mL / min;

[0130] ⑤ Using an activated dialysis bag with a molecular weight of 8-14K, dialyze the nanoparticles obtained in step ④ in a 0.5% ammonia solution for 1 hour, and then dialyze in a PBS solution for 1 hour;

[0131] ⑥ Add 0.5μg, 1.0μg, 1.5μg, 2.0μg, 2.5μg, and 3.0μg of nanoparticles loaded with fluorescent protein (corresponding to nanoparticle solution volumes of 10μL, 20μL, 30μL, 40μL, 50μL, and 60μL) into the in vitro cultured 293FT cells and culture for 48h.

[0132] Test Example 5 :This test example provides a PBAE447-5% PEG2000 polymer nanocomposite material loaded with fluorescent protein particles, and its preparation method comprises the following steps:

[0133] (1) Synthesizing poly(β-amino ester) (PBAE447) polymer raw material in the same manner as in Example 1;

[0134] (2) Preparation of PBAE447-5% PEG2000 nanocomposites using a microfluidic device

[0135] ① Pipette 50 μL of PABE447 dimethyl sulfoxide (DMSO) solution with a volume mass concentration of 200 mg / mL, add an equal volume of ethanol solution, and dilute with a 1:1 volume ratio of DMSO:ethanol solution to obtain a PABE447 dimethyl sulfoxide:ethanol solution with a volume mass concentration of 18 mg / mL;

[0136] ② Prepare 95 μL of PEG2000 dimethyl sulfoxide:ethanol solution with a volume mass concentration of 5 mg / mL, add 0.5 mL of PABE447 dimethyl sulfoxide:ethanol solution with a volume mass concentration of 18 mg / mL in step ①, add dimethyl sulfoxide:ethanol solution to a volume of 1 mL, and incubate at room temperature for 0.5 h;

[0137] ③Prepare 25mM sodium acetate / acetic acid buffer solution with a pH of 5.2: accurately weigh 1.701g sodium acetate trihydrate and dissolve it in 500mL pure water to obtain a 25mM sodium acetate buffer solution; pipette 0.72mL of acetic acid solution and dissolve it in 500mL pure water to obtain a 25mM acetate buffer solution; prepare a 25mM sodium acetate / acetic acid buffer solution with a pH of 5.2 according to a certain volume ratio;

[0138] ④ Using a microfluidic device, 25 mM sodium acetate / acetic acid buffer solution with pH = 5.2 as the aqueous phase, the dimethyl sulfoxide:ethanol solution in step ② as the oil phase, and a total flow rate of 3 mL / min to prepare a PBAE447-5% PEG2000 polymer-lipid nanocomposite material; wherein the aqueous phase flow rate is 2 mL / min, and the oil phase flow rate is 1 mL / min;

[0139] ⑤ Using an activated dialysis bag with a molecular weight of 8-14K, dialyze the nanoparticles obtained in step ④ in a 0.5% ammonia solution for 1 hour, and then dialyze in a PBS solution for 1 hour;

[0140] ⑥ Add 0.5μg, 1.0μg, 1.5μg, 2.0μg, 2.5μg, and 3.0μg of nanoparticles loaded with fluorescent protein (corresponding to nanoparticle solution volumes of 10μL, 20μL, 30μL, 40μL, 50μL, and 60μL) into the in vitro cultured 293FT cells and culture for 48h.

[0141] Application Example 1 :

[0142] Experimental Examples 1 to 4 of the present invention successfully synthesized PBAE447-DOPE-PEG2000 polymer-lipid nanocomposite particles loaded with fluorescent protein plasmids of various concentration gradients. The PBAE447-DOPE-PEG2000 polymer-lipid nanocomposite particles loaded with fluorescent protein plasmids were co-cultured with 293FT cells by setting different mass gradients. This application example used a flow cytometer to test its transfection efficiency, and measured the cell viability of each sample using a cell counting plate.

[0143] like Figure 7 As shown in the data, the transfection effect of PBAE447-DOPE-PEG2000 polymer-lipid nanocomposite particles was significantly improved compared with PBAE447. When the amount of fluorescent protein particles added was 2.0 μg, the transfection efficiency of PBAE447-5% DOPE-PEG2000 and PBAE447-7% DOPE-PEG2000 polymer-lipid nanocomposite particles was increased by about 50% compared with that of PBAE447, and there was no significant change in cell viability.

[0144] like Figure 8 As shown, fluorescence microscopic images were taken using a fluorescence microscope, and the results showed that the cell fluorescent protein expression of PBAE447-DOPE-PEG2000 polymer-lipid nanocomposite particles was more obvious.

[0145] Experimental Example 5 of the present invention successfully synthesized PBAE447-5% PEG2000 polymer-lipid nanocomposite particles loaded with fluorescent protein at different concentration gradients, and co-cultured the PBAE447-5% PEG2000 polymer-lipid nanocomposite particles with 293FT cells, tested the transfection efficiency by flow cytometry, and measured the cell viability between samples by cell counting plate.

[0146] Based on the above experimental results, the transfection efficiency and cell viability of PBAE447, PBAE447-5% DOPE-PEG2000 and PBAE447-5% PEG2000 nanoparticles were compared. Fig. 9 As shown, it can be seen that when the cell viability remains basically unchanged, the transfection efficiency of PBAE447-5% PEG2000 is not significantly improved compared with PABE447, but PBAE447-5% DOPE-PEG2000 has obvious advantages.

[0147] The DOPE-PEG2000 used in the present invention is an active phospholipid PEG derivative with good hydrophilicity and hydrophobicity. DOPE is an 18-carbon unsaturated phospholipid with high hydrophobicity. The unsaturated tail structure can form a more fluid lipid layer, and can also form a hexagonal phase (HII) organizational form. The HII phase can promote the fusion of lipid membrane and endosomal membrane, and promote the cytoplasmic release of nucleic acids. The PEG2000 skeleton provides good hydrophilicity, reduces particle dialysis aggregation, and enhances nanoparticle stability. DOPE-PEG2000 molecules combine the biocompatibility and stability of DOPE and the water solubility of PEG2000 chains at the same time, so that the PBAE polymer-lipid nanoparticles loaded with plasmids and modified by DOPE-PEG2000 can be quickly released intracellularly, and enhance lysosomal escape, further improving transfection performance.

[0148] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A PBAE-DOPE-PEG2000 polymer-lipid nanocomposite material, characterized in that: The polymer-lipid nanocomposite material comprises a PBAE macromolecular material and a functionalized PEG lipid with surface modified PBAE; the particle size of the nanocomposite material is 50 to 200 nm; Wherein, the PBAE polymer material is selected from one or more of PBAE447, PBAE446, and PBAE536; and the functionalized PEG lipid is DOPE-PEG2000.

2. A method for preparing the PBAE-DOPE-PEG2000 polymer-lipid nanocomposite material according to claim 1, characterized in that: The following steps are involved: S1, adding amino alcohol to diacrylate, dissolving the obtained intermediate product in an organic solvent, and adding a capping group to cap it, to obtain a PBAE material; S2, adding DOPE-PEG2000 dimethyl sulfoxide:ethanol solution to poly(β-amino lipid) dimethyl sulfoxide:ethanol solution, and the mixed solution after incubation is used as the oil phase component; sodium acetate / acetic acid buffer solution is used as the water phase component; S3. Using a microfluidic chip, the oil phase and the water phase components are mixed to obtain a PBAE-DOPE-PEG2000 polymer-lipid nanocomposite material.

3. The preparation method according to claim 2, characterized in that: In step S1, the diacrylate is selected from one of 1,4-butanediol diacrylate and 1,5-pentanediol diacrylate; the amino alcohol is selected from one of 4-amino-1-butanol and 3-amino-1-propanol; the molar ratio of the diacrylate to the amino alcohol is 1:1 to 1.2:1; The intermediate product is obtained by heating a diacrylate and an amino alcohol; preferably, the heating reaction temperature is 70 to 90° C. and the time is 20 to 26 hours.

4. The preparation method according to claim 2, characterized in that: In step S1, the organic solvent is selected from tetrahydrofuran and dimethyl sulfoxide; the end-capping group is selected from 1-(3-aminopropyl)-4-methylpiperazine and 2-[(3-aminopropyl)amine]ethanol.

5. The preparation method according to claim 2, characterized in that: In step S2, the poly(β-amino lipid) dimethyl sulfoxide:ethanol solution is prepared by mixing a poly(β-amino lipid) dimethyl sulfoxide solution with an ethanol solution, wherein the volume mass concentration of the poly(β-amino lipid) dimethyl sulfoxide solution is 100 to 200 mg / mL; The poly(β-amino lipid) dimethyl sulfoxide:ethanol solution has a volume mass concentration of 6 to 24 mg / mL, and is specifically obtained by diluting the poly(β-amino lipid) dimethyl sulfoxide solution with a dimethyl sulfoxide:ethanol solution; the dimethyl sulfoxide:ethanol solution refers to a dimethyl sulfoxide to ethanol volume ratio of 1 to 2:1; The DOPE-PEG2000 dimethyl sulfoxide:ethanol solution is prepared by mixing a DOPE-PEG2000 dimethyl sulfoxide solution with an ethanol solution. The volume mass concentration of the DOPE-PEG2000 dimethyl sulfoxide:ethanol solution is 3-5 mg / mL.

6. The preparation method according to claim 2, characterized in that: In step S2, the mass ratio of DOPE-PEG2000 to poly(β-amino lipid) is 0.03:1 to 0.2:1; Preferably, the incubation temperature is 25-30°C and the time is 20-40 minutes; The sodium acetate / acetic acid buffer solution is prepared from a sodium acetate buffer solution and an acetate buffer solution in a volume ratio of 2 to 4:

1. The concentration of the sodium acetate / acetic acid buffer solution is 25 mM and the pH value is 5.

2. The concentrations of the sodium acetate buffer solution and the acetate buffer solution are both 25 mM.

7. The preparation method according to claim 2, characterized in that: In step S3, the flow rate ratio of the water phase to the oil phase is 1 to 4:1, and then the obtained PBAE-DOPE-PEG2000 polymer-lipid nanocomposite particles are dialyzed twice with ammonia water and PBS respectively.

8. Use of the PBAE-DOPE-PEG2000 polymer-lipid nanocomposite material according to claim 1 in gene delivery.

9. Use of the PBAE-DOPE-PEG2000 polymer-lipid nanocomposite material according to claim 1 in preparing a therapeutic gene delivery preparation.

10. A PBAE-DOPE-PEG2000 polymer-lipid nanocomposite material loaded with exogenous genes, characterized in that: A PBAE-DOPE-PEG2000 polymer-lipid nanocomposite prepared by the preparation method according to any one of claims 2 to 8, and further comprising an exogenous gene; Preferably, the exogenous gene is selected from a green fluorescent protein plasmid and a luciferase reporter gene plasmid; Preferably, the loading of exogenous genes is specifically performed by encapsulating exogenous genes using PBAE-DOPE-PEG2000 polymer-lipid nanocomposite materials; Further preferably, the specific operation of encapsulating the exogenous gene is: mixing the exogenous gene plasmid and the nanocomposite material, and uniformly dispersing the plasmid to obtain polymer-lipid nanocomposite particles loaded with the exogenous gene plasmid; Further preferably, the mass ratio of the exogenous gene-loaded plasmid to PBAE is 1 / 90 to 1 / 30.

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