Nano-carrier and preparation method and application thereof

Abstract

The invention belongs to the field of biomedical materials, and particularly relates to a nano-carrier and a preparation method and application thereof.The nano-carrier is prepared from PBAE and DMG-PEG2000, PBAE polymer materials form spherical particles, and the surfaces of the spherical particles are modified with DMG-PEG2000. The preparation method comprises the following steps: preparing a first solution obtained by dissolving PBAE in an organic solvent; preparing a second solution obtained by dissolving DMG-PEG2000 in an organic solvent; mixing and incubating the first solution and the second solution to obtain an oil phase; a micro-fluidic chip is used, and a water phase and an oil phase with the flow rate ratio of (1-4): 1 are mixed to prepare the nano-carrier. The stability of the nanoparticles is improved, aggregation among the nanoparticles is prevented, the DMG-PEG2000 shields the interaction between the nanoparticles and cell membranes through the steric hindrance effect, rapid dissociation can be achieved while in-vivo escape is achieved, and long circulation and passive targeting are achieved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical materials, and in particular relates to a nanocarrier 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] In recent years, the incidence of tumors has increased year by year. As a new type of therapy, immunotherapy has attracted widespread attention by enhancing or restoring the immune system's ability to identify, kill and eliminate tumor cells, so that the body's own immune system can find tumor cells hidden in the body and specifically kill and eliminate them. The key to immunotherapy is to introduce normal exogenous genes into target cells and achieve safe, efficient and stable expression in target cells. However, naked DNA / RNA is easily degraded by nucleases in the extracellular environment and is easily decomposed by lysosomes after entering cells. Therefore, nanocarriers are usually used to deliver genes. At present, gene delivery vectors are mainly divided into two categories: viral vectors and non-viral vectors. Based on biosafety considerations, although viral vectors have better transfection effects, the integration risks and immune responses they bring cannot be ignored.

[0004] Non-viral vectors are mainly divided into: polymer nanoparticles, polymer micelles, liposomes, polymer lipid composite nanoparticles, inorganic nanoparticles, etc. Among them, cationic polymer vectors are one of the most representative non-viral vectors. Due to their wide sources of raw materials, simple synthesis methods, low immunogenicity and good biocompatibility, they have attracted extensive attention from researchers. Poly(β-amino lipids) (PBAE) has a variety of synthetic raw materials, simple preparation processes, flexible chemical structures, adjustable particle sizes and good biodegradability. The ester bonds on its main chain can be hydrolyzed into small molecules and excreted from the body, thus avoiding the high cytotoxicity caused by other cationic polymer vectors due to their difficulty in degradation, and showing significant advantages in the field of gene delivery.

[0005] Although poly(β-amino ester) materials have shown certain application potential, how to prepare nanomaterials with high uniformity, good stability and long-term circulation in the body is truly related to their subsequent clinical application. Therefore, it is particularly important to find a new material system and preparation method that can improve the stability and circulation time of poly(β-amino ester) (PBAE). Summary of the invention

[0006] In view of the needs of the prior art, the purpose of the present invention is to provide a nanocarrier and a preparation method and application thereof. By changing the size and uniformity of the nanoparticles, the stability of the nanoparticles is further improved, and aggregation between particles is prevented. At the same time, DMG-PEG2000 lipids (dimyristoylglycerol-polyethylene glycol 2000) shield the interaction between the nanoparticles and the cell membrane through steric hindrance. The use of polyethylene glycol lipids containing short alkyl chains can achieve rapid dissociation while achieving in vivo escape, reducing the risk of "lysosomal escape" caused by PEGylation, thereby achieving long circulation and passive targeting.

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

[0008] In a first aspect of the present invention, a nanocarrier is provided, wherein the raw materials for preparation include: PBAE and DMG-PEG2000 in a mass ratio of 1: (0.03-0.2), wherein the PBAE polymer material constitutes spherical particles, and the surface of the spherical particles is modified with DMG-PEG2000.

[0009] Optionally, the particle size of the nanocarrier is 50 to 170 nm.

[0010] Optionally, the PBAE polymer material is selected from one or more of PBAE447, PBAE446, and PBAE536.

[0011] The second aspect of the present invention provides a method for preparing the above-mentioned nanocarrier, comprising the following steps:

[0012] S1, preparing a first solution in which PBAE is dissolved in an organic solvent; preparing a second solution in which DMG-PEG2000 is dissolved in an organic solvent;

[0013] S2, mixing and incubating the first solution and the second solution to obtain an oil phase, and using a sodium acetate / acetic acid buffer solution as an aqueous phase;

[0014] S3. Using a microfluidic chip, the water phase and the oil phase are mixed at a flow rate ratio of 1 to 4:1 to prepare a nanocarrier.

[0015] Optionally, in S1, the mass volume concentration of the first solution is 6 to 24 mg / mL, and the mass volume concentration of the second solution is 3 to 5 mg / mL.

[0016] Optionally, in S1, the organic solvent includes dimethyl sulfoxide and ethanol in a volume ratio of (1-2):1.

[0017] Optionally, in S1, the method for preparing the first solution includes: adding amino alcohol to diacrylate, dissolving the intermediate product obtained by the reaction in an organic component, then adding a capping group for end-capping, obtaining a solution in which PBAE is dissolved in the organic component, washing with excess ether and removing the supernatant, vacuum drying, and diluting with an organic solvent to obtain a first solution; the organic component is one or more of dimethyl sulfoxide and tetrahydrofuran.

[0018] Optionally, in 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 to 1.2):1.

[0019] Optionally, in S1, the reaction temperature for generating the intermediate product is 70 to 90° C. and the reaction time is 20 to 26 hours.

[0020] Optionally, in S1, the capping group is selected from one of 1-(3-aminopropyl)-4-methylpiperazine and 2-[(3-aminopropyl)amine]ethanol.

[0021] Optionally, in S2, the incubation temperature is 25-30° C. and the time is 20-40 min.

[0022] Optionally, in S2, the sodium acetate / acetic acid buffer solution is obtained by mixing a sodium acetate buffer solution with a concentration of 5 to 30 mM and an acetate buffer solution with a concentration of 25 to 30 mM in a volume ratio of (2 to 4):1.

[0023] Optionally, in S3, after the polymer-lipid nanocomposite particles are obtained by mixing, they are dialyzed with ammonia water and PBS, respectively.

[0024] In a third aspect, a nanocarrier loaded with an exogenous gene comprises the exogenous gene and the above-mentioned nanocarrier.

[0025] Optionally, the exogenous gene is a green fluorescent protein (GFP) plasmid or a luciferase reporter gene (Luc) plasmid.

[0026] In a fourth aspect, the method for preparing the above-mentioned nanocarrier loaded with exogenous genes comprises the following steps:

[0027] The exogenous gene plasmid is mixed with the nanocarrier.

[0028] Optionally, the mixing method is: dilute the exogenous gene plasmid in a buffer solution of acetic acid and sodium acetate at a plasmid mass concentration of 0.05 to 0.3 mg / mL, and use a vortex mixer to evenly disperse the plasmid.

[0029] Optionally, the mixing method is: directly mixing a set amount of the exogenous gene plasmid with the prepared nanoparticles by means of a vortex mixer to make the concentration of the exogenous gene plasmid be 0.03-0.1 mg / mL.

[0030] Optionally, the mass ratio of the exogenous gene-loaded plasmid to PBAE is 1 / 90 to 1 / 30.

[0031] The beneficial effects achieved by the present invention are as follows:

[0032] (1) The present invention modifies PBAE by functionalized PEG lipid (DMG-PEG2000), changes the size and uniformity of nanoparticles, further improves the stability of nanoparticles, and prevents aggregation between particles. At the same time, DMG-PEG2000 lipid (dimyristoylglycerol-polyethylene glycol 2000) shields the interaction between nanoparticles and cell membranes through steric hindrance. The use of polyethylene glycol lipids containing short alkyl chains can achieve rapid dissociation while achieving in vivo escape, reducing the risk of "lysosomal escape" caused by PEGylation, thereby achieving long circulation and passive targeting.

[0033] (2) The present invention uses microfluidic technology to achieve high-efficiency mixing of PBAE, DMG-PEG2000 dimethyl sulfoxide:ethanol solution and sodium acetate / acetic acid buffer solution, and obtains polymer-lipid composite nanoparticles with high uniformity and a size of 50 to 170 nm. In addition, the combination of plasmids and PBAE-DMG-PEG2000 polymer-lipid nanocomposites can be achieved through different loading methods.

[0034] (3) When the mass of the loaded plasmid is high or low, the transfection effect of PBAE447-DMG-PEG2000 polymer-lipid nanocomposite particles is slightly improved compared with PBAE447, and there is no significant change in cell viability, which has a certain reference value for trace or large-dose medication. 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 Flow chart of the microfluidic method for synthesizing nanocarriers in Example 1.

[0037] Figure 2 This is the synthetic route for the PBAE polymer material in Preparation Example 1.

[0038] Figure 3 This is the nuclear magnetic resonance spectrum (NMR) in Preparation Example 1.

[0039] Figure 4 The particle size distribution diagram and microscope image of the 3% DMG-PEG2000 nanocarrier prepared in Example 1, 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 5% DMG-PEG2000 nanocarrier prepared in Example 2, 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 7% DMG-PEG2000 nanocarrier prepared in Example 3, wherein a is the particle size distribution diagram and b is the transmission electron microscope image;

[0042] Figure 7 The particle size distribution diagram and microscope image of the 10% DMG-PEG2000 nanocarrier prepared in Example 4, wherein a is the particle size distribution diagram and b is the transmission electron microscope image;

[0043] Figure 8 The particle size distribution diagram and microscope image of the nanocarrier of PBAE447 prepared in Comparative Example 2, wherein a is the particle size distribution diagram and b is the transmission electron microscope image;

[0044] Fig. 9 The particle size fitting distribution diagram and microscope image of the nanocarrier containing 5% PEG2000 prepared in Comparative Example 1, wherein a is the particle size fitting distribution diagram drawn by Nano Measurer, and b is a transmission electron microscope image;

[0045] Fig.10 It is a graph of transfection efficiency of in vitro cell culture of nanocarriers loaded with fluorescent protein particles of Comparative Example 4 and Examples 5 to 8;

[0046] Fig.11 These are fluorescence microscopic images of in vitro cell culture of nanocarriers loaded with fluorescent protein particles of Comparative Example 4 and Examples 5 to 8.

[0047] Fig.12 The graphs are for the transfection efficiency and cell viability of the nanocarriers loaded with fluorescent protein particles of Comparative Example 3, Comparative Example 4 and Example 6. DETAILED DESCRIPTION

[0048] 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.

[0049] 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.

[0050] A nanocarrier, the preparation raw materials include: PBAE and DMG-PEG2000 in a mass ratio of 1: (0.03-0.2), the PBAE polymer material constitutes spherical particles, and the surface of the spherical particles is modified with DMG-PEG2000.

[0051] Optionally, the particle size of the nanocarrier is 50 to 170 nm.

[0052] Optionally, the PBAE polymer material is selected from one or more of PBAE447, PBAE446, and PBAE536.

[0053] A method for preparing the above-mentioned nanocarrier comprises the following steps:

[0054] S1, preparing a first solution of a set concentration in which PBAE is dissolved in an organic solvent, and preparing a second solution of a set concentration in which DMG-PEG2000 is dissolved in an organic solvent;

[0055] S2, mixing and incubating the first solution and the second solution as the oil phase, and using the sodium acetate / acetic acid buffer solution as the aqueous phase;

[0056] S3. Using a microfluidic chip, the water phase and the oil phase are mixed at a flow rate ratio of 1 to 4:1 to prepare a nanocarrier.

[0057] Optionally, in S1, the organic solvent includes dimethyl sulfoxide and ethanol in a volume ratio of (1-2):1.

[0058] Optionally, in S1, the mass volume concentration of the first solution is 6 to 24 mg / mL, and the mass volume concentration of the second solution is 3 to 5 mg / mL.

[0059] Optionally, in S1, the method for preparing the first solution includes: adding amino alcohol to diacrylate, dissolving the intermediate product obtained by the reaction in an organic component, then adding a capping group for end-capping, obtaining a solution in which PBAE is dissolved in the organic component, washing with excess ether and removing the supernatant, vacuum drying, and diluting with an organic solvent to obtain a first solution; the organic component is one or more of dimethyl sulfoxide and tetrahydrofuran.

[0060] Optionally, in 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 to 1.2):1.

[0061] Optionally, the reaction temperature for generating the intermediate product is 70 to 90° C. and the reaction time is 20 to 26 hours.

[0062] Optionally, the end-capping group is selected from one of 1-(3-aminopropyl)-4-methylpiperazine and 2-[(3-aminopropyl)amine]ethanol.

[0063] Optionally, in S2, the incubation temperature is 25-30° C. and the time is 20-40 min.

[0064] Optionally, in S2, the sodium acetate / acetic acid buffer solution is obtained by mixing a sodium acetate buffer solution with a concentration of 5 to 30 mM and an acetate buffer solution with a concentration of 25 to 30 mM in a volume ratio of (2 to 4):1.

[0065] Optionally, in S3, after mixing to obtain polymer-lipid nanocomposite particles, the particles are dialyzed with ammonia water and then with PBS.

[0066] A nanocarrier loaded with exogenous genes comprises the exogenous genes and the above nanocarrier.

[0067] Optionally, the exogenous gene is a green fluorescent protein (GFP) plasmid or a luciferase reporter gene (Luc) plasmid.

[0068] A method for preparing the above-mentioned nanocarrier loaded with exogenous genes comprises the following steps:

[0069] There are two methods for mixing the exogenous gene plasmid with the nanocarrier. The first method is to dilute the exogenous gene plasmid in a buffer solution of acetic acid and sodium acetate, use a vortex mixer to evenly disperse the plasmid in the buffer, and incubate at room temperature for about 30 minutes. The mass concentration of the diluted plasmid in the buffer is 0.05-0.3 mg / mL; the second method is to directly mix a certain amount of exogenous gene plasmid with the prepared nanoparticles with the help of a vortex mixer, and the final exogenous gene plasmid concentration is 0.03-0.1 mg / mL.

[0070] Optionally, the mass ratio of the exogenous gene-loaded plasmid to PBAE is 1 / 90 to 1 / 30.

[0071] Preparation Example 1

[0072] The preparation method of the PBAE447 polymer raw material in poly(β-amino ester) (PBAE) comprises the following steps:

[0073] S11. Accurately weigh 1.223 g of 1,4-butanediol diacrylate (CAS No.: 1070-70-8) and 0.5 g of 4-amino-1-butanol (CAS No.: 13325-10-5), react in an oil bath at 70°C for 24 h to obtain an intermediate reactant. Dissolve the intermediate product obtained in S11 in 5 mL of tetrahydrofuran (THF) to obtain an intermediate product solution in which the intermediate product is dissolved in an organic component.

[0074] S12, preparing a tetrahydrofuran solution of 1-(3-aminopropyl)-4-methylpiperazine (CAS No.: 4572-03-6) with a concentration of 90 mg / mL to obtain a blocking solution for providing a blocking group;

[0075] S13, adding the end-capping solution dropwise to the intermediate product solution while maintaining a rotation speed of 500 r / min, and reacting at room temperature for 2 h to obtain a reaction product;

[0076] S14. Wash the reaction product twice with 5 volumes of ether, centrifuge, and vacuum dry to obtain PBAE447 polymer material, dissolve it in dimethyl sulfoxide (DMSO) solution to a volume mass concentration of 200 mg / mL, and store it at -20°C.

[0077] The synthetic route in this example is as follows Figure 2 As shown, in Figure 2 In the process, the red group is provided by raw material B4 (1,4-butanediol diacrylate), the green group is provided by raw material S4 (4-amino-1-butanol), and the blue group is provided by raw material E7 (1-(3-aminopropyl)-4-methylpiperazine). The prepared PBAE447 is as follows: Figure 2 As shown in PBAE447 (B4-S4-E7) in the lower middle, when the substituent groups of the raw materials used are changed, other types of PBAE polymer materials can be prepared accordingly.

[0078] The PBAE447 prepared in this preparation example was subjected to nuclear magnetic resonance detection. Figure 3 As shown, the spectrum shows that the poly (β-amino ester) (PBAE447) polymer material was successfully prepared. When the substituent group of the raw material used is changed, other types of PBAE polymer materials can be prepared accordingly, and the corresponding test results also change accordingly.

[0079] Preparation Example 2

[0080] The preparation method of sodium acetate / acetic acid buffer solution comprises:

[0081] Accurately weigh 1.701 g of sodium acetate trihydrate and dissolve it in 500 mL of pure water to obtain a 25 mM sodium acetate buffer solution; also transfer 0.72 mL of acetic acid solution and dissolve it in 500 mL of pure water to obtain a 25 mM acetate buffer solution; according to a certain volume ratio, a 25 mM sodium acetate / acetic acid buffer solution with a pH of 5.2 is obtained.

[0082] Example 1

[0083] A method for preparing a nanocarrier, such as Figure 1 As shown, including:

[0084] S1. Pipette the dimethyl sulfoxide (DMSO) solution of PBAE447 obtained in Preparation Example 1, add an equal volume of ethanol solution, and then dilute it with a dimethyl sulfoxide:ethanol solution in a volume ratio of 1:1 to obtain a dimethyl sulfoxide:ethanol solution of PABE447 with a volume mass concentration of 18 mg / mL, which is the first solution; use a dimethyl sulfoxide:ethanol solution in a volume ratio of 1:1 to prepare a solution of DMG-PEG2000 (CAS No.: 160743-62-4) with a volume mass concentration of 5 mg / mL, which is the second solution; wherein, "dimethyl sulfoxide:ethanol solution" refers to a mixed solution of dimethyl sulfoxide and ethanol solution in a set volume ratio.

[0085] S2. Add 56 μL of the second solution to 0.5 mL of the first solution, then add dimethyl sulfoxide:ethanol solution to 1 mL, incubate at room temperature for 0.5 h to obtain an oil phase; use the sodium acetate / acetic acid buffer solution obtained in Preparation Example 2 as the aqueous phase.

[0086] S3. Prepare nanocarriers using a microfluidic chip, wherein the water phase flow rate is 2 mL / min, and the oil phase flow rate is 1 mL / min, to obtain a dispersion of polymer-lipid nanocomposite particles. Use an activated dialysis bag with a molecular weight of 8-14K ​​to dialyze the polymer-lipid nanocomposite particles in a 0.5wt% ammonia solution for 1 hour, and then dialyze in a PBS solution for 1 hour to obtain a dispersion of 3% DMG-PEG2000 nanocarriers, wherein 3% means that the mass of DMG-PEG2000 accounts for 3% of the total mass of all solutes in the solution.

[0087] Example 2

[0088] A method for preparing a nanocarrier, which differs from Example 1 in that:

[0089] In S1, 95 μL of the second solution was prepared;

[0090] In S2, 95 μL of the second solution was added to 0.5 mL of the first solution.

[0091] A dispersion of nanocarriers containing 5% DMG-PEG was prepared, wherein 5% means that the mass of DMG-PEG2000 accounts for 5% of the total mass of all solutes in the solution.

[0092] Example 3

[0093] A method for preparing a nanocarrier, which differs from Example 1 in that:

[0094] In S1, 136 μL of the second solution was prepared;

[0095] In S2, 136 μL of the second solution was added to 0.5 mL of the first solution.

[0096] A dispersion of nanocarriers containing 7% DMG-PEG was prepared, wherein 7% means that the mass of DMG-PEG2000 accounts for 7% of the total mass of all solutes in the solution.

[0097] Example 4

[0098] A method for preparing a nanocarrier, which differs from Example 1 in that:

[0099] In S1, 200 μL of the second solution was prepared;

[0100] In S2, 200 μL of the second solution was added to 0.5 mL of the first solution.

[0101] A dispersion of nanocarriers containing 10% DMG-PEG2000 was prepared, wherein 10% means that the mass of DMG-PEG2000 accounts for 10% of the total mass of all solutes in the solution.

[0102] Comparative Example 1

[0103] A method for preparing a nanocarrier, which differs from Example 1 in that:

[0104] In S1, the second solution was not used, but 95 μL of a dimethyl sulfoxide:ethanol solution of PEG2000 (CAS No.: 25322-68-3) with a volume mass concentration of 5 mg / mL was prepared as the third solution.

[0105] In S2, 95 μL of the third solution was added to 0.5 mL of the first solution.

[0106] A dispersion of nanocarriers containing 5% PEG2000 was prepared after dialysis, wherein 5% means that the mass of PEG2000 accounts for 5% of the total mass of all solutes in the solution.

[0107] Comparative Example 2

[0108] A method for preparing a nanocarrier, which differs from Example 1 in that:

[0109] The oil phase was prepared without using the second solution, and only the first solution containing PBAE447 was used as a raw material. The sodium acetate / acetic acid buffer solution obtained in Preparation Example 2 was used as an aqueous phase to prepare a dispersion of PBAE nanocarriers obtained after dialysis.

[0110] Example 5

[0111] In vitro cell transfection experiment of nanocarriers loaded with fluorescent protein particles.

[0112] Take 1 mL of the dispersion of the polymer-lipid nanocomposite particles prepared in S3 of Example 1, collect the polymer-lipid nanocomposite particles with a centrifuge tube containing 0.05 mg of fluorescent protein plasmids, and evenly disperse the plasmids with the help of a vortex mixer. The speed of the vortex mixer is 300-800 r / min, and the mixing time is 30-90 s. Then, use an activated dialysis bag with a molecular weight of 8-14K ​​to dialyze the nanocarrier loaded with fluorescent protein plasmids in a 0.5% ammonia solution for 1 h, and then put it into a PBS solution for dialyzation for 1 h to obtain a dispersion of the nanocarrier loaded with fluorescent protein plasmids.

[0113] Since the ratio of nanocarriers to fluorescent protein particles is fixed, 10 μL, 20 μL, 30 μL, 40 μL, 50 μL and 60 μL of the dispersion of nanocarriers loaded with fluorescent protein particles (the corresponding fluorescent protein particle masses are 0.5 μg, 1.0 μg, 1.5 μg, 2.0 μg, 2.5 μg and 3.0 μg, respectively) were added to the 293FT cells cultured in vitro and cultured for 48 h.

[0114] Example 6

[0115] In vitro cell transfection experiment of nanocarriers loaded with fluorescent protein particles.

[0116] The difference between this embodiment and embodiment 5 is that the dispersion of polymer-lipid nanocomposite particles prepared in S3 of embodiment 2 is used as a raw material to prepare a nanocarrier loaded with fluorescent protein particles for transfection.

[0117] Example 7

[0118] In vitro cell transfection experiment of nanocarriers loaded with fluorescent protein particles.

[0119] The difference between this embodiment and embodiment 5 is that the dispersion of polymer-lipid nanocomposite particles prepared in S3 of embodiment 3 is used as a raw material to prepare a nanocarrier loaded with fluorescent protein particles for transfection.

[0120] Example 8

[0121] In vitro cell transfection experiment of nanocarriers loaded with fluorescent protein particles.

[0122] The difference between this embodiment and embodiment 5 is that the dispersion of polymer-lipid nanocomposite particles prepared in S3 of embodiment 4 is used as a raw material to prepare a nanocarrier loaded with fluorescent protein particles for transfection.

[0123] Comparative Example 3

[0124] In vitro cell transfection experiment of nanocarriers loaded with fluorescent protein particles.

[0125] The difference between this example and Example 5 is that the nanocarrier containing 5% PEG2000 prepared in Comparative Example 1 is used as a raw material to prepare a nanocarrier loaded with fluorescent protein particles for transfection.

[0126] Comparative Example 4

[0127] In vitro cell transfection experiment of nanocarriers loaded with fluorescent protein particles.

[0128] The difference between this embodiment and embodiment 5 is that the PBAE nanocarrier prepared in comparative example 2 is used to prepare a nanocarrier loaded with fluorescent protein particles for transfection.

[0129] Test content

[0130] The 3% DMG-PEG2000 nanocarrier obtained in Example 1 was observed using a transmission electron microscope. Figure 4 As shown in (b), the average particle size is about 70nm, the prepared nanoparticles are spherical particles, and the particle size distribution is uniform; the dynamic light scattering test results are as follows Figure 4 As shown in (a) in the figure: the average hydrated particle size is about 170 nm.

[0131] The 5% DMG-PEG2000 nanocarrier obtained in Example 2 was observed using a transmission electron microscope. Figure 5 As shown in (b), the average particle size is about 60nm, the prepared nanoparticles are spherical particles, and the particle size distribution is uniform; the dynamic light scattering test results are as follows Figure 5 As shown in (a): the average hydrated particle size is about 150nm.

[0132] The 7% DMG-PEG2000 nanocarrier obtained in Example 3 was observed using a transmission electron microscope. Figure 6 As shown in (b), the average particle size is about 60nm, the prepared nanoparticles are spherical particles, and the particle size distribution is uniform; the dynamic light scattering test results are as follows Figure 6 As shown in (a): the average hydrated particle size is about 140nm.

[0133] The nanocarriers with 10% DMG-PEG2000 obtained in Example 4 were observed using a transmission electron microscope. Figure 7 As shown in (b), the average particle size is about 50 nm, the prepared nanoparticles are spherical particles, and the particle size distribution is uniform; the dynamic light scattering test results are as follows Figure 7 As shown in (a): the average hydrated particle size is about 110nm.

[0134] The nanocarrier of PBAE447 obtained in Comparative Example 2 was observed using a transmission electron microscope, and the image is as follows: Figure 8 As shown in (b), the average particle size is about 80nm, the prepared nanoparticles are spherical particles, and the particle size distribution is relatively uniform; the dynamic light scattering test results are as follows Figure 8 As shown in (a): the average hydrated particle size is about 200nm.

[0135] The nanocarriers containing 5% PEG2000 obtained in Comparative Example 1 were observed using a transmission electron microscope. Fig. 9 As shown in (b), there are two sizes of nanoparticles, one is about 100nm or larger, and the other is about 30nm or smaller. The Nano Measurer is used to perform particle size statistics on Figure (b). The average particle size of the larger one is 136.8nm, and the variance is 26.8nm. The prepared nanoparticles are spherical particles with a relatively dispersed particle size distribution.

[0136] From Comparative Example 2, it is known that the average hydrated particle size of PBAE447 nanoparticles is 200nm, the PDI is above 0.2, and the particle size uniformity is poor. From Examples 1 to 4, it can be seen that in each group of nanocarriers with different DMG-PEG2000 lipid contents, the PDI is below 0.1, and the uniformity of the nanoparticles is excellent. Compared with the PBAE447 nanoparticles in Comparative Example 2, the presence of DMG-PEG2000 has a greater effect on the uniformity of the PBAE nanoparticles, making the distribution of PBAE nanoparticles tend to be uniform, while PEG2000 does not have the above function.

[0137] The transfection efficiency in Comparative Example 2, Example 5, Example 6, Example 7 and Example 8 was tested by flow cytometry, and the cell viability of each sample was measured by a cell counting plate. Fig.10 and Fig.11 shown.

[0138] like Fig.10 As shown, when the mass of the loaded plasmid is high or low, the transfection effect of the DMG-PEG2000 nanocarrier of each embodiment is improved compared with the PBAE nanocarrier of comparative example 4, which has a certain reference role for trace or large-dose medication, and there is no obvious change in cell viability.

[0139] like Fig.11 As shown, fluorescence microscopic pictures were taken with a fluorescence microscope, and the results showed that when the amount of plasmid added was too low or too high, the cell fluorescent protein expression corresponding to the nanocarrier of Example 5 was more obvious, which has obvious advantages for trace or large-dose medication.

[0140] Combined with the above experimental results, the transfection efficiency and cell viability of the PBAE447 nanocarrier, the nanocarrier loaded with fluorescent protein particles prepared in Example 6 (nanocarrier with 5% DMG-PEG2000) and the nanocarrier loaded with fluorescent protein particles prepared in Comparative Example 3 (nanocarrier with 5% PEG2000) were compared. Fig.12 As shown, it can be seen that when the cell viability remains basically unchanged, the transfection efficiency of Comparative Example 3 is not significantly improved compared with Comparative Example 4, and the transfection efficiency of Example 6 is slightly improved.

[0141] The addition of functional lipid DMG-PEG2000 changes the particle size and uniformity of PBAE nanoparticles alone. The PDI of nanocarriers with different concentration gradients of DMG-PEG2000 in the above examples is all below 0.1, which well proves that the addition of DMG-PEG2000 improves the stability of nanoparticles and prevents aggregation between particles. At the same time, DMG-PEG2000 lipids (dimyristoylglycerol-polyethylene glycol 2000) reduce the interaction between nanoparticles and cell membranes through steric hindrance effects. The use of polyethylene glycol lipids containing short alkyl chains can not only achieve in vivo escape, but also quickly dissociate, reduce the adverse effects of PEGylation on "lysosomal escape", thereby achieving long circulation and passive targeting. Moreover, DMG-PEG2000 has sufficient advantages over DOPE-PEG2000 in terms of stability and immunogenicity. DMG-PEG2000 can stabilize the surface structure of nanoparticles, prevent them from aggregating in the blood circulation, cover up potential immunogenic sites on the surface, and reduce the activation of the immune system. Many liposome nanoparticles use DMG-PEG2000 as one of the liposome components because the addition of DMG-PEG2000 makes the liposomes more difficult to be recognized and cleared by the mononuclear phagocytic system (MPS), so the circulation life in the blood is longer. At the same time, the high water solubility and non-ionicity of DMG-PEG2000 enhance the compatibility of liposomes with biological membranes. Modifying the surface of PBAE nanoparticles with DMG-PEG2000 lipids may have significant advantages in the subsequent delivery and transfection of RNA.

[0142] 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 nanocarrier, characterized in that: The preparation raw materials include: PBAE and DMG-PEG2000 in a mass ratio of 1: (0.03-0.2); the PBAE polymer material constitutes spherical particles, and the surface of the spherical particles is modified with DMG-PEG2000.

2. The nanocarrier according to claim 1, wherein The particle size of the nanocarrier is 50 to 170 nm; Optionally, the PBAE polymer material is selected from one or more of PBAE447, PBAE446, and PBAE536.

3. A method for preparing a nanocarrier according to any one of claims 1 to 2, characterized in that: The following steps are involved: S1, preparing a first solution in which PBAE is dissolved in an organic solvent; preparing a second solution in which DMG-PEG2000 is dissolved in an organic solvent; S2, mixing and incubating the first solution and the second solution to obtain an oil phase, and using a sodium acetate / acetic acid buffer solution as an aqueous phase; S3. Using a microfluidic chip, the water phase and the oil phase are mixed at a flow rate ratio of 1 to 4:1 to prepare a nanocarrier.

4. The preparation method according to claim 3, characterized in that: In S1, the method for preparing the first solution comprises: adding amino alcohol to diacrylate, dissolving the intermediate product obtained by the reaction in an organic component, then adding a capping group to cap the solution, obtaining a solution in which PBAE is dissolved in the organic component, washing with excess ether and removing the supernatant, vacuum drying, and diluting with an organic solvent to obtain a first solution; the organic component is one or more of dimethyl sulfoxide and tetrahydrofuran; Optionally, 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 (1-1.2):1; Optionally, the reaction temperature for generating the intermediate product is 70 to 90° C. and the reaction time is 20 to 26 hours; Optionally, the end-capping group is selected from one of 1-(3-aminopropyl)-4-methylpiperazine and 2-[(3-aminopropyl)amine]ethanol.

5. The preparation method according to claim 3, characterized in that: In S1, the mass volume concentration of the first solution is 6 to 24 mg / mL, and the mass volume concentration of the second solution is 3 to 5 mg / mL; Optionally, in S1, the organic solvent includes dimethyl sulfoxide and ethanol in a volume ratio of (1-2):

1.

6. The preparation method according to claim 3, characterized in that: In S2, the sodium acetate / acetic acid buffer solution is obtained by mixing a sodium acetate buffer solution with a concentration of 5 to 30 mM and an acetate buffer solution with a concentration of 25 to 30 mM in a volume ratio of (2 to 4):1; Optionally, in S2, the incubation temperature is 25-30° C. and the time is 20-40 min.

7. The preparation method according to claim 3, characterized in that: In S3, after the polymer-lipid nanocomposite particles are obtained by mixing, they are dialyzed with ammonia water and PBS, respectively.

8. A nanocarrier loaded with exogenous genes, characterized in that: It comprises an exogenous gene and a nanocarrier as described in any one of claims 1-2.

9. The nanocarrier loaded with exogenous genes according to claim 8, characterized in that: The exogenous gene is a green fluorescent protein plasmid or a luciferase reporter gene plasmid.

10. A method for preparing a nanocarrier loaded with exogenous genes as claimed in any one of claims 8 to 9, characterized in that: The method comprises the following steps: mixing the exogenous gene plasmid with the nanocarrier; Optionally, the mixing method is: diluting the exogenous gene plasmid in a buffer solution of acetic acid and sodium acetate at a plasmid mass concentration of 0.05 to 0.3 mg / mL, and using a vortex mixer to evenly disperse the plasmid; Optionally, the mixing method is: directly mixing a set amount of the exogenous gene plasmid with the prepared nanoparticles by means of a vortex mixer to make the concentration of the exogenous gene plasmid be 0.03 to 0.1 mg / mL; Optionally, the mass ratio of the exogenous gene-loaded plasmid to PBAE is 1 / 90 to 1 / 30.

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