Preparation method and application of amino acid polymer nucleic acid carrier

Abstract

The invention provides an amino acid polymer with a structure as shown in a formula I, and relates to the technical field of biological medicines. The polymer provided by the invention is an amino acid modified polymer prepared by introducing an amino acid monomer into a linear polyethyleneimine (LPEI) side chain. The polymer is used as a nucleic acid drug delivery carrier, the components of the polymer are simple, efficient nucleic acid delivery can be achieved through a single-component carrier, and excellent transfection efficiency and biocompatibility are shown; # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the technical field of nucleic acid delivery, and in particular to a preparation method of an amino acid polymer and application of the amino acid polymer in nucleic acid delivery. Background Art

[0002] Gene therapy marks a revolutionary leap in biomedical research, and it has shown great clinical application potential in a variety of applications such as preventive and therapeutic vaccines. Such therapies include protein replacement therapy, cancer immunotherapy, cell reprogramming, and gene editing. Although gene therapy has opened up a promising new path for us, its clinical application still faces many challenges. This is mainly due to the fact that nucleic acids are easily degraded when administered in naked form and their localization in the body is relatively limited. In order to work in vivo, nucleic acid drugs require safe, effective and stable delivery systems to protect nucleic acids from degradation and allow cellular uptake and nucleic acid release. To date, a variety of nucleic acid delivery systems have been developed, including lipids, lipid-like materials, polymers, inorganic materials and cell membranes. Among them, lipids, lipid-like materials and polymers are the most commonly used. Although the nucleic acid drug delivery system of lipid nanoparticles is at the forefront of current clinical transformation, there are still many limitations, such as cumbersome preparation process, thermal stability problems, liver aggregation problems, etc.

[0003] Polymer carriers have attracted much attention due to their multifunctional structure, easy functionalization and robust stability. Common polymer carriers include polyethyleneimine, polyamide, dendrimers and poly-β-amino esters. These polymers interact with the negative charges of nucleic acids through their positive charges to form stable complexes, thereby protecting nucleic acids from degradation and promoting their entry into cells. Polyethyleneimine (PEI) is one of the most widely used cationic polymers for nucleic acid delivery. PEI can provide a high density of positive charges for nucleic acid complexation and endosomal escape. Studies have shown that PEI promotes endosomal escape through the "proton sponge" effect. But the problem is that PEI has poor biocompatibility due to excessive cationic charges and lack of biodegradable bonds. The biocompatibility and in vivo stability of polymer carriers are crucial for in vivo nucleic acid delivery.

[0004] To solve this problem, it is necessary to design an amino acid-based polymer library for nucleic acid delivery while maintaining nucleic acid transfection efficiency and reducing cytotoxicity. Summary of the invention

[0005] The purpose of the present invention is to provide a preparation method and application of an amino acid polymer. The amino acid polymer provided by the present invention has low toxic and side effects as a nucleic acid drug delivery carrier and has efficient nucleic acid delivery capability.

[0006] The present invention designs an amino acid-based polymer library for nucleic acid delivery. First, amino acid monomers with good biocompatibility are connected with double bonds through esterification reaction, and then the double bonds are used to react with amino groups in LPEI to introduce amino acids into LPEI to obtain the final polyamino acid polymer library.

[0007] The present invention provides an amino acid polymer having a structure shown in formula I,

[0008]

[0009] Among them, 300≤n≤500000, and n can specifically be 300, 500, 1000, 5000, 10000, 50000, 100000, 500000; or a range value between any two of the above.

[0010] The molar ratio of x / n is 1:(1-10000); specifically, it can be 1:1, 1:3, 1:5, 1:8, 1:10, 1:15, 1:20, 1:25, 1:30, 1:50, 1:100, 1:500, 1:1000, 1:5000, 1-10000; or a range between any two of the above.

[0011] The R is selected from -H, -(CH2) m R1; the m is selected from an integer between 1 and 8; specifically, it can be 1, 2, 3, 4, 5, 6, 7, or 8.

[0012] The R1 is selected from one or more of the following structures: -H, -CH3, -COOH, -OH, -NH2, -SH, -(C=O)-NH2,

[0013] In the present invention, the polymer has any of the following structures:

[0014]

[0015] The present invention provides a method for preparing the amino acid polymer described in the above technical solution, comprising the following steps:

[0016] A) the amino acid described in formula (II) is dissolved in an organic solvent, di-tert-butyl dicarbonate and triethylamine are added for reaction, and then extracted and purified to obtain intermediate a;

[0017] B) dissolving intermediate a and 1,3-propylene glycol in an organic solvent, adding EDC and DMAP for reaction, and purifying to obtain intermediate b;

[0018] C) adding intermediate b and acrylic acid to an organic solvent, adding EDC and DMAP to react, and after the reaction, purifying to obtain intermediate c;

[0019] D) reacting the intermediate c and the linear PEI of formula (III) in an organic solvent, dialyzing and freeze-drying the reaction product to obtain a freeze-dried product; and then reacting the freeze-dried product with trifluoroacetic acid to obtain;

[0020]

[0021] 300≤n≤500000, where n can specifically be 300, 500, 1000, 5000, 10000, 50000, 100000, 500000, or a range value between any two of the above.

[0022] The molar ratio of x / n is 1:(1-10000); specifically, it can be 1:1, 1:3, 1:5, 1:8, 1:10, 1:15, 1:20, 1:25, 1:30, 1:50, 1:100, 1:500, 1:1000, 1:5000, 1-10000; or a range between any two of the above.

[0023] The R is selected from -H, -(CH2) m R1; the m is selected from an integer between 1 and 8; specifically, it can be 1, 2, 3, 4, 5, 6, 7, or 8.

[0024] The preparation formula of the polymer of the present invention is as follows:

[0025]

[0026] The preparation method of the amino acid polymer of the present invention comprises the following steps: firstly dissolving the amino acid described in formula (II) in an organic solvent, adding di-tert-butyl dicarbonate and triethylamine to react, and then extracting and purifying the reaction to obtain an intermediate a.

[0027] Preferably, the amino acid monomer represented by formula II has any of the following structures:

[0028]

[0029] In some specific embodiments of the present invention, in step A), the organic solvent is one of THF, DMF, and DCM, more preferably THF; the reaction temperature is 25 to 100° C., more preferably 25 to 50° C., and further preferably 25° C.; the reaction time is 2 to 24 h, more preferably 5 h;

[0030] Specifically, the molar ratio of di-tert-butyl dicarbonate to triethylamine is 1:(1-5), more preferably 1:(1-2), and even more preferably 1:1.2.

[0031] The molar ratio of the amino acid described in formula (II), di-tert-butyl dicarbonate and triethylamine is 1:(1-1.5):1.2.

[0032] After the reaction, the intermediate a is obtained by extraction and purification. The present invention does not limit the specific extraction steps, which are well known to those skilled in the art.

[0033] The above purification is silica gel column chromatography, and the mobile phase used is generally 100% DCM to 80%: 30% DCM / MeOH, that is, first use 100% DCM to wash down the more polar compounds, and then gradually increase the amount of methanol to separate products with similar polarity.

[0034] The intermediate a and 1,3-propylene glycol are dissolved in an organic solvent, EDC and DMAP are added for reaction, and after the reaction, the intermediate is purified to obtain the intermediate.

[0035] According to the present invention, the molar ratio of the intermediate a to 1,3-propylene glycol is 1:(1-20), more preferably 1:(1-10), and further preferably 1:3.

[0036] The organic solvent is one or more of THF, DMF, DCM, DMSO, and CH3OH, and DMF or DCM is more preferred; the concentration of the intermediate a and 1,3-propylene glycol dissolved in the organic solvent is 5 to 20M.

[0037] The molar ratio of intermediate a, EDC and DMAP is 1:(1-2):(0.1-1).

[0038] The reaction temperature of the present invention is 25 to 100° C., more preferably 25 to 50° C., and further preferably 25° C.; the reaction time is 2 to 72 h, and more preferably 24 h.

[0039] The intermediate b and acrylic acid are dissolved in an organic solvent, and EDC and DMAP are added for reaction. After the reaction, the intermediate c is obtained by purification.

[0040] The molar ratio of intermediate b to acrylic acid is 1:(1-10), more preferably 1:(1-5), and further preferably 1:1.2. The crude product obtained by the reaction is subjected to silica gel column chromatography to obtain intermediate b.

[0041] The concentration of the intermediate b, acrylic acid and the organic solvent is 5-20M.

[0042] In some specific embodiments, the molar ratio of intermediate b, acrylic acid, EDC and DMAP is 1:(1-2):(1-2):(0.1-1).

[0043] The intermediate c and the linear PEI of the formula (III) are reacted in an organic solvent to obtain a reaction product.

[0044] The molar ratio of intermediate c and linear PEI of formula (III) is 1:(1-10000); 1:1, 1:3, 1:5, 1:8, 1:10, 1:15, 1:20, 1:25, 1:30, 1:50, 1:100, 1:500, 1:1000, 1:5000, 1-10000; or a range between any two of the above.

[0045] In some specific embodiments, the organic solvent is one or more of THF, DMF, DMSO and deionized water, more preferably DMF, DMSO and deionized water, and further preferably a mixed solution of DMF and deionized water.

[0046] In some specific embodiments, the reaction temperature is 25-100° C., more preferably 25° C.; the reaction time is 2-72 h, more preferably 72 h.

[0047] The reaction product is dialyzed and freeze-dried to obtain a freeze-dried product; the dialysis is performed using a dialysis bag with a molecular weight cutoff of 500 to 10,000 Da. The present invention does not limit the specific parameters of the freeze-drying, which are well known to those skilled in the art.

[0048] The freeze-dried product is then reacted with trifluoroacetic acid to obtain;

[0049] The molar ratio of the freeze-dried product to trifluoroacetic acid is 1:(1-5), the reaction temperature is 0-35° C., and the reaction time is 4-24 h.

[0050] The amino acid polymer described in the above technical solution of the present invention or the amino acid polymer prepared by the preparation method described in the above technical solution is used as a nucleic acid delivery carrier in the preparation of drugs.

[0051] The drugs of the present invention include nucleic acid drugs.

[0052] The present invention also provides a drug, the raw materials of which include an amino acid polymer and a nucleic acid; the amino acid polymer is an amino acid polymer described in any one of the above technical solutions or an amino acid polymer prepared by the preparation method described in any one of the above technical solutions.

[0053] The above polymer is loaded on the surface of nucleic acid.

[0054] The present invention also provides a method for preparing the medicine, which comprises preparing the amino acid polymer and the nucleic acid into aqueous solutions respectively; and mixing and incubating the two to obtain the medicine.

[0055] In some embodiments of the present invention, the mass ratio of the amino acid polymer to the nucleic acid is 2.5 to 5:1; specifically, it can be 2.5:1, 3:1, 4:1, 5:1; or any range value therebetween.

[0056] Preferably, the nucleic acid drug is selected from one or more of DNA, mRNA aptamer, and siRNA.

[0057] The in vitro delivery is preferably carried out by delivering nucleic acid drugs in 293, CHO, HeLa, MCF-7, DC2.4, HepG2, B16F10, A549, L20, BMDCs and other cells.

[0058] The amino acid polymer of the present invention has a good in vitro transfection effect when used as a nucleic acid drug delivery carrier to deliver nucleic acid drugs in the above cells.

[0059] The present invention provides an amino acid polymer having a structure shown in formula I, and relates to the field of biomedicine technology. The polymer provided by the present invention is prepared by introducing amino acid monomers into the side chain of linear polyethyleneimine (LPEI) to obtain an amino acid modified polymer. The polymer of the present invention is used as a nucleic acid drug delivery carrier, and its components are simple, and a single-component carrier can achieve efficient nucleic acid delivery and show excellent transfection efficiency and biocompatibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 This is the NMR image of the amino acid polymer prepared in Example 1;

[0061] Figure 2 This is a diagram showing the in vitro transfection effect of the amino acid polymer prepared in Example 3. DETAILED DESCRIPTION

[0062] The present invention provides a method for preparing an amino acid polymer and its application in nucleic acid delivery. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they all fall within the scope of protection of the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0063] It should be understood that the expression "one or more of..." includes each of the items recited after the expression individually and in various different combinations of two or more of the recited items, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited items should be understood to have the same meaning, unless otherwise understood from the context.

[0064] The use of the terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, for example not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.

[0065] In this application, the term "and / or" describes the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can represent: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural.

[0066] In this application, "at least one" means one or more, "more than one" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single or plural items.

[0067] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the present invention remains operable. In addition, two or more steps or actions may be performed simultaneously.

[0068] The use of any and all examples or exemplary language, such as "for example" or "including", herein is intended only to better illustrate the invention and does not limit the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating that any non-claimed element is essential to the practice of the invention.

[0069] In addition, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.

[0070] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution, some or all of the steps can be executed in parallel or sequentially, and the execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0071] Some cases are recorded in the embodiments and comparative examples of the present invention, wherein the embodiments show certain implementation modes of the present invention. However, this does not mean that the effects of the present invention can only be achieved in these cases.

[0072] To further illustrate the present invention, the preparation method of an amino acid polymer provided by the present invention and its application in nucleic acid delivery are described in detail below in conjunction with examples.

[0073] Example 1

[0074] The amino acid polymers were prepared as follows:

[0075]

[0076] Step 1:

[0077] Alanine (1.0 g) was dissolved in THF (10 mL), triethylamine (2.14 mL) and Boc2O (2.4 g) in THF (10 mL) were added under ice bath, and stirred at room temperature for 5 hours. After concentrating under reduced pressure, n-hexane was added for recrystallization. Product a was obtained by suction filtration.

[0078] Step 2:

[0079] Compound a (1 g) and 1,3-propylene glycol (1.18 g) were dissolved in DCM (20 mL), stirred to dissolve, and EDC (0.98 g) and DMAP (0.18 g) were added to the reaction solution. After the reaction was complete, the separated liquid was washed with 1M hydrochloric acid solution and sodium chloride solution, dried with anhydrous sodium sulfate, filtered, and the excess solvent was removed by rotary evaporation. The residue was purified by silica gel chromatography (100% DCM-80%: 20% DCM / MeOH) to obtain product b.

[0080] Step 3:

[0081] Compound b (1 g) and acrylic acid (0.3 g) were dissolved in DCM (20 mL), stirred to dissolve, EDC (0.75 g) and DMAP (0.15 g) were added to the reaction solution, and stirred at room temperature for 24 hours. After the reaction was complete, the separated liquid was washed with 1M hydrochloric acid solution and sodium chloride, dried over anhydrous sodium sulfate, filtered, and the excess solvent was removed by rotary evaporation. The residue was purified by silica gel chromatography (100% DCM-80%: 20% DCM / MeOH) to obtain product c.

[0082] Step 4:

[0083] LPEI-25K (1.0 g) was dissolved in 10 ml of water for later use, and the intermediate product c (0.88 g) was dissolved in 50 mL of DMF. The LPEI aqueous solution was poured into the DMF solution and mixed evenly, and reacted at 80°C for 24 h. After the reaction was completed, the freeze-dried product was dialyzed with a 3500 Da dialysis bag, and the freeze-dried product was added with 30 ml of TFA and reacted at room temperature for 4 h, then ether was precipitated and filtered, and the final product was dialyzed and freeze-dried with a 3500 Da dialysis bag.

[0084] Comparative Example 2

[0085]

[0086] LPEI-25K (1.0 g) was dissolved in 10 ml of water for later use, n-octyl acrylate (0.45 g) was dissolved in 50 mL of DMF, the LPEI aqueous solution was poured into the DMF solution and mixed evenly, and reacted at 80° C. for 24 h. After the reaction was completed, the freeze-dried product LPEI-OA was dialyzed with a 3500 Da dialysis bag.

[0087] Example 3

[0088] The hydrogen spectrum analysis of LPEI-Ala gave the following NMR image: Figure 1 As shown in the figure, the strong signal peak at about 3.0ppm chemical shift is the characteristic absorption peak of -CH2- on the LPEI chain, 2.31ppm is attributed to the characteristic absorption peak of -CH3- on the amino acid side chain, and 3.0-4.0ppm is attributed to the characteristic absorption peak of -OCH2-.

[0089] Example 4

[0090] The preparation method of LPEI-Ala / mRNA complex is as follows:

[0091] LPEI-Ala (amino acid polymer obtained in Example 1) was dissolved in enzyme-free water to obtain a carrier solution; the carrier solution and the mRNA solution were mixed evenly in a certain ratio to obtain an mRNA delivery system. The molar ratio of LPEI grafted alanine and the mass ratio of LPEI-Ala to mRNA are shown in Table 1.

[0092] Table 1

[0093]

[0094]

[0095] Example 5

[0096] Evaluation of the in vivo delivery performance of luciferase mRNA of the delivery system prepared in Example 4

[0097] 1. In vitro nucleic acid delivery

[0098] (1) Cell culture

[0099] Continuous cell culture was performed in an incubator containing 10% fetal bovine serum and 5% volume fraction of carbon dioxide at a culture temperature of 37°C; the cells stored in liquid nitrogen were taken out and placed in a 37°C constant temperature water bath, and after the solution was completely dissolved, they were quickly transferred to a centrifuge tube containing 5 mL of the above culture medium, mixed and centrifuged at 1000 rpm for 5 minutes; the cell pellet in the centrifuge tube was retained, 10 mL of culture medium was added, transferred to a culture dish, and placed in the above constant temperature incubator for culture, and the culture medium was changed every other day.

[0100] (2) Cell transfection

[0101] B16F10 cells and luciferase mRNA were used to evaluate the transfection ability of the material: the above cultured cells were cultured at 1×10 4 The cells were planted in a 96-well plate at a density of 100 μL and cultured overnight in 200 μL culture medium; the polymer nucleic acid drug delivery vector and luciferase substrate prepared in Example 1 were used to detect the transfection efficiency of the complex in the cells by using an ELISA instrument. Table 2 shows the transfection efficiency of the complex.

[0102] Table 2

[0103]

[0104] Figure 2 This is a diagram of the in vitro transfection effect of the nucleic acid drug carrier prepared in Example 5. The results show that the amino acid polymer carrier of Example 1 loaded with mRNA expressing green fluorescent protein (GFP-mRNA) delivered to HeLa, DC2.4 and L20 cells all showed obvious expression of green fluorescent protein.

[0105] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An amino acid polymer having a structure shown in formula I, in, 300≤n≤500000, the molar ratio of x / n is 1:(1-10000); The R is selected from -H, -(CH2) m R1; said m is selected from an integer between 1 and 8; The R1 is selected from one or more of the following structures: -H, -CH3, -COOH, -OH, -NH2, -SH, -(C=O)-NH2, 2. The amino acid polymer according to claim 1, characterized in that The polymer has any of the following structures:

3. A method for preparing the amino acid polymer according to claim 1 or 2, characterized in that: The steps include: A) the amino acid described in formula (II) is dissolved in an organic solvent, di-tert-butyl dicarbonate and triethylamine are added for reaction, and then extracted and purified to obtain intermediate a; B) dissolving intermediate a and 1,3-propylene glycol in an organic solvent, adding EDC and DMAP to react, and after the reaction, purifying to obtain intermediate b; C) adding intermediate b and acrylic acid to an organic solvent, adding EDC and DMAP to react, and after the reaction, purifying to obtain intermediate c; D) reacting the intermediate c and the linear PEI of formula (III) in an organic solvent, dialyzing and freeze-drying the reaction product to obtain a freeze-dried product; and then reacting the freeze-dried product with trifluoroacetic acid to obtain; 4. The preparation method according to claim 3, characterized in that: The organic solvent in step A) is one of THF, DMF and DCM, the reaction temperature is 25-100° C., and the reaction time is 2-24 h.

5. The preparation method according to claim 3, characterized in that: The organic solvent in step B) is one or more of THF, DMF, DCM, DMSO or CH3OH, the reaction time is 2 to 72 hours, and the reaction temperature is 25 to 100°C; the purification is performed by silica gel column chromatography.

6. The preparation method according to claim 3, characterized in that: The organic solvent in step C) is one or more of THF, DMF, DCM, DMSO or CH3OH, the reaction time is 2 to 72 hours, and the reaction temperature is 25 to 100°C; the purification is performed by silica gel column chromatography.

7. The preparation method according to claim 3, characterized in that: The organic solvent in step D) is one or more of THF, DMF, DMSO and deionized water, the reaction temperature is 25-100° C., and the reaction time is 2-72 hours; The molar ratio of the linear PEI of formula (III) to the intermediate c is 1:(1-10000); The dialysis is performed using a dialysis bag with a molecular weight cutoff of 500 to 10,000 Da.

8. Use of the amino acid polymer according to any one of claims 1 to 2 or the amino acid polymer prepared by the preparation method according to any one of claims 3 to 7 as a nucleic acid delivery carrier in the preparation of drugs.

9. A drug, characterized in that The raw materials include amino acid polymers and nucleic acids; the amino acid polymers are the amino acid polymers described in any one of claims 1 to 2 or the amino acid polymers prepared by the preparation method described in any one of claims 3 to 7.

10. The drug according to claim 9, characterized in that The nucleic acid is selected from one or more of DNA, mRNA aptamer or siRNA.

Images