CN113105625A

CN113105625A - Succinic acid vitamin E modified polyethyleneimine derivative, and preparation method and application thereof

Info

Publication number: CN113105625A
Application number: CN202110400857.2A
Authority: CN
Inventors: 王升启; 杨静; 任晋; 曹艺明; 王鑫; 李蕾
Original assignee: Institute of Pharmacology and Toxicology of AMMS
Current assignee: Institute of Pharmacology and Toxicology of AMMS
Priority date: 2021-04-14
Filing date: 2021-04-14
Publication date: 2021-07-13
Anticipated expiration: 2041-04-14
Also published as: CN113105625B

Abstract

The invention provides a succinic acid vitamin E modified polyethyleneimine derivative, a preparation method and application thereof, and relates to the field of biomedical materials. In vitro experiments show that the prepared vitamin E succinate modified derivative carrier can effectively protect mRNA, carry the mRNA to enter cells and efficiently express the mRNA in the cells; in vivo experiments show that the derivative can successfully deliver and express mRNA into a body and has good safety. The polyethyleneimine derivative as a nucleic acid delivery vector has the advantages of low toxicity and high transfection efficiency.

Description

Succinic acid vitamin E modified polyethyleneimine derivative, and preparation method and application thereof

Technical Field

The invention relates to the technical field of biomedical materials, in particular to a succinic acid vitamin E modified polyethyleneimine derivative, a preparation method and application thereof.

Background

mRNA-based drugs, particularly mRNA vaccines, have been widely demonstrated to be a promising strategy for immunotherapy. The unique advantages of mRNA vaccines, including their high efficacy, relatively low side effects, and low cost, have led to their widespread use in preclinical and clinical trials for a variety of infectious diseases and cancers. With the outbreak of new coronary epidemic, the advantages of mRNA vaccine in dealing with emergent infectious diseases are exerted, and currently, mRNA vaccine developed by fevere in combination with BioNTech and mRNA vaccine developed by modern company are approved by the Food and Drug Administration (FDA) of the united states for emergency use.

mRNA drugs have a broad prospect, but because of their size, charge, and susceptibility to degradation, naked mRNA cannot readily cross cell membranes into the cytoplasm. Thus, to exert a therapeutic effect on mRNA, the aid of a delivery vehicle is required. At present, the delivery vectors for mRNA mainly include liposomes, lipid nanoparticles, polymers, and the like. Among them, lipid nanoparticles are the most commonly used carriers, but their potential toxicity has been the focus of debate.

Polyethyleneimine (PEI) is a cationic polymer that has been used in gene vector research in recent years, and binds nucleic acids by electrostatic interaction, compressing them into nanoscale complexes, thereby facilitating entry into cells and escape from lysosomes. The delivery efficiency of PEI is related to molecular weight, wherein large molecular weight PEI with a molecular weight of 25kDa is called "gold standard" for transfection due to its higher transfection efficiency, but also causes strong cytotoxicity due to its strong electrical charge and non-degradability. In contrast, small molecular weight PEI is easily degraded, but the transfection efficiency is low. Therefore, efforts have been made to structurally modify PEI to increase transfection capacity while reducing cytotoxicity.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a succinic acid vitamin E modified polyethyleneimine derivative, and a preparation method and application thereof.

The technical scheme provided by the invention is as follows:

in one aspect, a vitamin E succinate modified polyethyleneimine derivative, wherein the polyethyleneimine derivative is obtained by conjugating vitamin E succinate to polyethyleneimine.

Further, the polyethyleneimine is preferably branched polyethyleneimine; more preferably, the branched polyethylenimine has a molecular weight of 1.8 kDa.

In one embodiment, the vitamin E succinate is in an amount anywhere from 1 to 10.

In particular embodiments, the amount of vitamin E succinate can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; in a preferred embodiment, the amount of vitamin E succinate is 2.

The invention modifies the succinic acid vitamin E on the polyethyleneimine with low molecular weight to obtain the succinic acid vitamin E modified polyethyleneimine derivative carrier. The invention selects the polyethyleneimine in a branched chain form; preferably a branched polyethyleneimine having a small molecular weight, such as a branched polyethyleneimine having a molecular weight of 10kDa or less; more preferably 5kDa or less.

In another aspect, the present invention provides a method for preparing the foregoing polyethyleneimine derivative, the method comprising:

(a) activating succinic acid vitamin E;

(b) and (b) reacting the activated product obtained in the step (a) with branched polyethyleneimine with the molecular weight of 1.8kDa, and separating and purifying the product to obtain the target product.

In one embodiment, the step (a) is vitamin E succinate activation with EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, i.e. carbodiimide).

In a preferred embodiment, the vitamin E succinate is dissolved in chloroform, EDC is added to be completely dissolved, and the mixture is stirred and reacted at room temperature to obtain the vitamin E succinate.

In one embodiment, the amount of modified tocopheryl succinate on each polyethyleneimine is controlled in step (b) by controlling the feed ratio of activated tocopheryl succinate to polyethyleneimine during synthesis.

In another aspect, the present invention provides a complex obtained by entrapping a nucleic acid by the polyethyleneimine derivative.

In one embodiment, the nucleic acid comprises a plasmid, mRNA, DNA vaccine, or RNA vaccine; preferably, the RNA vaccine is a COVID-19mRNA vaccine.

The nucleic acid vaccine is prepared through introducing exogenous gene (DNA or RNA)) encoding certain antigen protein directly into animal cell, synthesizing antigen protein via the expression system of host cell, and inducing host to produce immune response to the antigen protein to reach the aim of preventing and treating diseases. In the present invention, in a broad sense, the nucleic acid includes a nucleic acid vaccine including a DNA vaccine or an RNA vaccine.

The invention provides an application of a succinic acid vitamin E modified polyethyleneimine derivative as an in vitro mRNA transfection reagent or an in vivo mRNA delivery vector.

In one embodiment, the complex is a complex of a polyethyleneimine derivative carrier and mRNA;

preferably, the binding ratio of the polyethyleneimine derivative carrier to mRNA is (8-48) to 1; preferably (16-48) 1; more preferably (32-40): 1.

In another aspect, the invention also provides the use of the polyethyleneimine derivative in nucleic acid transfection or delivery vectors.

In one embodiment, the nucleic acid transfection comprises in vivo transfection or in vitro transfection.

The invention also provides application of the polyethyleneimine derivative in delivery of COVID-19mRNA vaccine. The delivery is for non-disease treatment purposes.

In one embodiment, the transfected subject comprises a cell or a mammal; preferably, the cell is any one of a HEK 293T cell, a HeLa cell, a Vero cell or a DC2.4 cell.

Has the advantages that:

the invention modifies and conjugates the vitamin E succinate onto the polyethyleneimine with low molecular weight, and the obtained polyethyleneimine derivative can be used as a carrier for delivering mRNA, can effectively protect the mRNA, carries the mRNA to enter cells, and efficiently expresses the mRNA.

The succinic acid vitamin E modified polyethyleneimine derivative has lower cytotoxicity and better safety. The succinic acid vitamin E modified polyethyleneimine derivative is high in efficiency of transfecting cells with mRNA carried by the carrier, and can also be used for in vivo delivery.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 shows the synthetic route and nuclear magnetism of the PVES vector of the present invention¹H NMR characterization;

FIG. 2 is a morphological characterization of a PVES vector of the present invention;

FIG. 3 is a graph showing the particle size and potential of PVES carriers of the present invention;

FIG. 4 shows the results of gel blocking experiments for the PVES/mRNA complexes of the present invention;

FIG. 5 shows the results of the ribozyme stability test of the PVES/mRNA complex of the present invention;

FIG. 6 is a morphological characterization of the PVES/mRNA complexes of the present invention;

FIG. 7 is a graph showing the particle size and potential characterization of the PVES/mRNA complexes of the present invention;

FIG. 8 shows the expression of fluorescent proteins of the present invention after transfection of 293T cells with PVES/EGFP mRNA under different N/P conditions (wherein PEI1.8 kDa and PEI25 kDa are negative and positive controls);

FIG. 9 shows the fluorescent protein expression of PVES/EGFP mRNA transfected with HeLa, Vero and DC2.4 cells under the condition of 32N/P ═ N (PEI 1.8kDa and PEI25 kDa as negative and positive controls);

FIG. 10 shows the in vitro cytotoxic activity of the PVES vectors of the present invention (the viability of 293T cells was determined at different PVES vector concentrations, and PEI1.8 k and PEI25 k were used as negative and positive controls);

FIG. 11 shows PVES vector-wrapped Luciferase mRNA in vivo imaging results;

FIG. 12 is a graph showing the change in body weight of mice injected intramuscularly with PVES vector;

FIG. 13 is a pathological diagram of liver and kidney tissues of a mouse injected intramuscularly with PVES vector;

FIG. 14 shows the fluorescent protein expression of different PVES/EGFP mRNAs after transfection into 293T cells;

FIG. 15 is a graph showing the results of evaluating the expression of GFP mRNA by detecting the number of GFP-positive cells by flow cytometry;

FIG. 16 shows the expression of fluorescent protein after transfection of HeLa cells with PVES/EGFP plasmids;

FIG. 17 is a schematic representation of COVID-19mRNA vaccine mouse immunization, sample collection;

FIG. 18 shows the results of ELISA assay of antibody levels after intramuscular injection of PVES/COVID-19mRNA vaccine into mice;

FIG. 19 shows the results of the serum antibody titer of PVES/COVID-19mRNA vaccine administered intramuscularly to mice;

FIG. 20 is a graph showing the change in body weight of PVES/COVID-19mRNA vaccine after intramuscular injection to mice.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1 Synthesis and characterization of PVES vectors

1.1 Synthesis of PVES vectors

PVES is a condensation product of branched 1.8kDa PEI and Vitamin E Succinate (VES).

The specific operation is as follows: 100mg of Vitamin E Succinate was weighed out and dissolved in 8mL of CHCl at room temperature₃In (5), carbodiimide (EDC) (2.0eq.) was added and dissolved completely, and the reaction was stirred for 1 h. And then adding PEI1.8 kDa (0.5eq) to dissolve completely, continuing to react for 8h at room temperature, carrying out reduced pressure rotary evaporation to remove the solvent, adding a proper amount of pure water to dissolve, transferring to a dialysis bag (with the molecular weight cut-off of 1000Da) for dialysis overnight, collecting dialysate, and carrying out freeze drying to obtain the PVES product. Further through¹The final product was confirmed by H NMR.

FIG. 1 shows the synthetic route and nuclear magnetism of PVES vector¹And H NMR characterization results.

1.2 structural characterization of PVES vectors

¹H NMR(400MHz,CD₃OD)δ_H:3.16-2.73(CH₂CH₂NH)，2.49-2.29(COCH₂CH₂)，2.12-1.07(H of VES)，0.87(CH₃)。

Example 2 morphology and particle size and potential characterization of PVES vectors

Morphological characterization of PVES vectors

The morphological characteristics of the PVES carrier were observed using a Transmission Electron Microscope (TEM). Preparing the PVES carrier into a solution with the concentration of 1 mug/microliter, then dripping 10 microliter of the solution on a carbon copper net, standing for 3min, and then sucking the solution by using absorbent paper. Then 0.5% phosphotungstic acid is dripped for negative dyeing, after 30s, the dye is sucked dry and observed under an electron microscope. FIG. 2 shows the morphological characterization of PVES vectors. Relatively uniform nanoparticles can be observed under an electron microscope, and the size of the nanoparticles is about 200 nm.

Characterization of particle size and potential of PVES support

The particle size and zeta potential of the PVES nano micelle are detected by a Dynamic Light Scattering (DLS) nano laser particle sizer. At a concentration of 1. mu.g/. mu.L, the particle diameter of PVES was 185.6nm, and the zeta potential was 69.8 mV. Figure 3 is a particle size and potential characterization of PVES supports.

Example 3 preparation of PVES/mRNA complexes

The preparation of PVES/mRNA complexes is carried out by mixing mRNA solutions with PVES solutions. Specifically, mRNA stock solution (0.1. mu.g/. mu.L) was mixed with an equal volume of PVES solution, vortexed gently, and incubated at room temperature for 30 min. The PVES/mRNA ratio was calculated based on the nitrogen/phosphorus (N/P) ratio.

Similarly, 1.8kDa and 25kDa PEI/mRNA complexes were prepared as controls.

EXAMPLE 4 agarose gel electrophoresis retardation experiment

Agarose gel electrophoresis can be used to study the binding capacity of PVES to mRNA.

In the experiment, PVES/mRNA complexes with N/P from 1 to 32 and containing 1. mu.g of mRNA were first prepared, with a final volume of 5. mu.L. Vortex mixing, standing for 10min, loading on pre-prepared 1% agarose gel, and performing electrophoresis at 100V for 45 min. The run gel was observed for banding in a chemiluminescent gel imaging system and photographed. FIG. 4 shows gel blocking experiments for PVES/mRNA complexes.

The results show that: PVES can effectively combine with mRNA to form a complex, and completely block the migration of mRNA when N/P is more than or equal to 4.

Example 5 ribozyme stability experiment

Agarose gel electrophoresis was also used to study the tolerance of the PVES/mRNA complex to RNase A.

In the experiment, PVES/mRNA complexes with N/P of 16 and 32 and containing 1. mu.g of mRNA were first prepared in a final volume of 5. mu.L. Vortex, mix and stand for 10 min. Then RNase A solution was added to react for 10min, and the mixture was loaded on 1% agarose gel and electrophoresed at 100V for 45 min. The run gel was observed for banding in a chemiluminescent gel imaging system and photographed. FIG. 5 shows the results of the ribozyme stability experiment of the PVES/mRNA complex. The results show that: after PVES encapsulated mRNA, RNase A was added to the PVES/mRNA complexes to show good RNase A tolerance compared to mRNA samples alone.

Example 6 morphology and particle size and potential characterization of PVES/mRNA complexes

Morphological characterization of PVES/mRNA complexes

In order to understand the morphology of the PVES/mRNA complex, it was also characterized using TEM. In the experiment, PVES/mRNA complex with N/P of 32 was prepared, then 10. mu.L of complex liquid was dropped on a carbon film copper net, after a few minutes the excess solution was blotted off with absorbent paper, then a drop of 0.5% phosphotungstic acid was dropped on the copper net for negative staining, blotted off with absorbent paper for 30 seconds, followed by observation under an electron microscope. FIG. 6 is a morphological characterization of PVES/mRNA complexes. It was shown that PVES can compress mRNA into spherical nanoparticles with a diameter of about 100-200 nm.

Measurement of particle size and potential of PVES/mRNA Complex

In this experiment, the particle size and potential of the PVES/mRNA complex were measured under different N/P conditions.

When the N/P is 8 or more, PVES can effectively compress mRNA into nanoparticles. The particle size of PVES/mRNA tended to be constant with increasing N/P. Zeta potential measurements show that the surface charge of PVES/mRNA is relatively constant with increasing N/P. FIG. 7 is a graph showing the particle size and potential of the PVES/mRNA complex.

Example 7 cell transfection and Condition selection

1.293T cell transfection

HEK-293T cells in good growth state were seeded into 24-well plates at a concentration of 2X 10⁵And/well, placing in an incubator at 37 ℃, and standing and culturing until the cell fusion degree is about 70-90%. In this experiment, mRNA encoding Green Fluorescent Protein (GFP) was used as a reporter gene.

To examine the transfection effect of PVES on EGFP mRNA, different N/P PVES/mRNA complexes were prepared and incubated for 4h in FBS-free and double-antibody-free medium, followed by incubation for 24h with 10% FBS. Meanwhile, PEI1.8 kDa/EGFP mRNA and PEI25 kDa/EGFP mRNA complex transfected cells were prepared in the same manner as a control group. And observing the expression effect of the EGFP protein in the cells through a fluorescence microscope after the culture is finished, and photographing. The percentage of EGFP positive cells was also quantified by flow cytometry and transfection efficiency was calculated.

FIG. 8 shows the expression of fluorescent protein after transfection of 293T cells with PVES/EGFP mRNA under different N/P conditions, and PEI1.8 kDa and PEI25 kDa are negative and positive controls. The transfection efficiency of the PVES/mRNA complex is increased sequentially with the increase of N/P, and the transfection efficiency reaches the highest when the N/P is 32, and after that, the increase of the N/P transfection efficiency is not obvious. Therefore, N/P-32 was considered the optimal N/P for transfection.

HeLa, Vero and DC2.4 cell transfection

Similarly, other three different cell lines, namely HeLa cells, Vero cells and DC2.4 cells, are selected for qualitative and quantitative evaluation of the cell transfection efficiency of PVES.

FIG. 9 shows the fluorescent protein expression of PVES/EGFP mRNA transfected HeLa, Vero and DC2.4 cells under N/P32 condition, and PEI1.8 kDa and PEI25 kDa as negative and positive controls. Under the conditions of 32 and 40N/P, PVES can successfully transfect EGFP mRNA into three cell lines, and the transfection efficiency is higher than that of a positive control PEI25 kDa group.

Example 8 evaluation of in vitro cytotoxic Activity of PVES vectors

The influence of PVES vectors on the cell survival rate is evaluated by a CCK8 method, different concentrations of PVES vectors are prepared, the influence of PVES vectors on the cell survival rate of 293T cells is evaluated, and therefore in vitro cytotoxic activity of the PVES vectors is determined, and PEI1.8 kDa and PEI25 kDa are used as negative and positive controls.

FIG. 10 shows the in vitro cytotoxic activity of PVES vectors. The viability of 293T cells was determined at different PVES vector concentrations, with PEI1.8 k and PEI25 k as negative and positive controls.

The experimental result shows that PEI25 kDa shows higher toxicity in 293T cells, the cell survival rate is only about 20% at the concentration of 60 mu g/mL, and the survival rate of the PVES vector is still about 80%, which indicates that the PVES vector has no cytotoxic activity basically.

Example 9 evaluation of delivery Effect in mice

1. Evaluation of in vivo delivery Effect in mice

To investigate the ability of PVES vectors to carry mRNA for expression in vivo, studies were performed using mRNA encoding luciferase protein. This experiment was done using female BALB/mouse (6-8 weeks) and mice were raised under SPF conditions. The PVES/Luciferase mRNA complex was injected intramuscularly at a mRNA dose of 10. mu.g. And performing living body imaging 24h after injection, and observing the expression condition of the luciferase protein.

The result of in vivo imaging shows that after PVES/Luciferase mRNA is injected into a mouse intramuscularly, a stronger signal of Luciferase protein can be detected, which indicates that the PVES vector can carry the mRNA to enter the mouse for expression, and shows that the PVES vector can be used as a better in vivo delivery vector. FIG. 11 shows PVES vector-encapsulated Luciferase mRNA in vivo imaging results.

Evaluation of in vivo toxicity of PVES vectors

After the PVES vector is injected into a mouse intramuscularly, the injection part, the weight change and the pathological changes of liver and kidney of the mouse are continuously monitored, and the results show that the injection part is not inflamed, the mouse has no obvious weight loss and liver and kidney pathological changes, which indicates that the vector is used for in vivo mRNA delivery and has better safety.

Fig. 12 is a graph of the change of body weight of the mice, and fig. 13 is a graph of liver and kidney histopathology of the mice.

Example 10. effect of PVES/mRNA complexes with varying amounts of vitamin E succinate on mRNA delivery.

PVES was prepared by condensing vitamin E succinate with varying numbers (2, 4 and 6) with branched 1.8kDa PEI. The mRNA solution was mixed with the prepared PVES solution to form a PVES/mRNA complex. The effect of PVES transfection on EGFP mRNA (N/P. cndot.32) was examined, and FIG. 14 shows the fluorescent protein expression of 293T cells transfected with different PVES/EGFP mRNAs. As can be seen from the figure, the transfection efficiency was the best when the number of tocopheryl succinates was 2.

Fig. 15 shows the results of evaluating GFP mRNA expression by flow cytometry, and the mRNA transfection experiment was performed with N/P32.

Example 11 delivery of PVES vectors to plasmids.

The effect of PVES vector delivery to plasmid was examined, and fig. 16 shows the expression of fluorescent protein after transfection of Hela cells with PVES/EGFP plasmid (N/P ═ 32). The plasmid used was a self-constructed pCAG-eGFP plasmid. As can be seen from the figure, the PVES vector can successfully transfect the EGFP plasmid into HeLa cells, and the 48h protein expression amount is higher than 24 h.

Example 12 delivery effect of PVES vector for mRNA vaccine.

To investigate the delivery effect of PVES vectors for mRNA vaccines, in vivo immunization experiments with COVID-19mRNA vaccine were performed in mice. Female BALB/c mice at 6-8 weeks were randomized into 3 groups of 5 mice each, including: blank group, PVES/mRNA group. Intramuscular injection was used with an mRNA dose of 30. mu.g. The blank group was given corresponding sterile water. FIG. 17 shows a schematic of COVID-19mRNA vaccine mice immunization, sample collection. And then antibody detection (ELISA determination) is carried out, as shown in figure 18, a PVES vector is adopted to deliver the new crown mRNA vaccine, after three times of immunization, the antibody level is gradually increased and is obviously higher than that of a blank group and a vector group, and the fact that the vector can carry the new crown mRNA vaccine into a body and successfully induce humoral immune response is shown. Furthermore, evaluation of serum antibody titer showed that after three immunizations, the antibody titer of the vaccine group was significantly higher than that of the blank group and the vehicle group (as shown in fig. 19).

After the mice are immunized by intramuscular injection of PVES/mRNA, the injection parts, the weight change and the pathological changes of liver and kidney of the mice are continuously monitored, and the weights of the mice in a vaccine group and a vector group are not obviously reduced in the whole mRNA vaccine immunization period (as shown in figure 20), which indicates that the vaccine and the vector have no obvious toxicity and better safety.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A vitamin E succinate modified polyethyleneimine derivative, wherein the polyethyleneimine derivative is obtained by conjugating vitamin E succinate to polyethyleneimine.

2. Polyethyleneimine derivative according to claim 1, wherein the amount of vitamin E succinate is any number from 1 to 10.

3. A method for preparing the polyethyleneimine derivative according to claim 1 or 2, wherein the method comprises:

(a) activating succinic acid vitamin E;

(b) reacting the activated product of step (a) with branched polyethyleneimine, separating and purifying the product to obtain the target product.

4. The method of claim 3, wherein step (a) is activating vitamin E succinate with EDC;

preferably, the vitamin E succinate is dissolved in the trichloromethane, EDC is added to be completely dissolved, and the mixture is stirred and reacts at room temperature to obtain the vitamin E succinate.

5. A complex obtained by encapsulating a nucleic acid with the polyethyleneimine derivative according to claim 1 or claim 2.

6. The complex of claim 5, wherein the nucleic acid comprises a plasmid, mRNA, DNA vaccine, or RNA vaccine; preferably, the RNA vaccine is a COVID-19mRNA vaccine.

7. The complex of claim 5, wherein the complex is a complex of the polyethyleneimine derivative carrier and mRNA;

8. Use of a polyethyleneimine derivative according to claim 1 or 2 in a nucleic acid transfection or delivery vector.

9. The use of claim 8, wherein the nucleic acid transfection comprises in vivo transfection or in vitro transfection.

10. The use of claim 8, wherein the transfected subject comprises a cell or mammal; preferably, the cell is any one of a HEK 293T cell, a HeLa cell, a Vero cell or a DC2.4 cell.