Preparation method and application of amino acid polymer nucleic acid carrier

Abstract

This invention provides an amino acid-based polymer with the structure shown in Formula I, relating to the field of biomedical technology. The polymer provided by this invention is prepared by introducing amino acid monomers into the side chains of linear polyethyleneimine (LPEI). Using the polymer of this invention as a nucleic acid drug delivery carrier, its simple composition allows for efficient nucleic acid delivery as a single-component carrier, exhibiting excellent transfection efficiency and biocompatibility.

Description

Technical Field

[0001] This invention relates to the field of nucleic acid delivery technology, specifically to a method for preparing an amino acid polymer and its application in nucleic acid delivery. Background Technology

[0002] Gene therapy marks a revolutionary leap in biomedical research, demonstrating immense clinical potential in various applications, including preventative and therapeutic vaccines. These therapies encompass protein replacement therapy, cancer immunotherapy, cell reprogramming, and gene editing. While gene therapy opens a promising new path, its clinical application still faces numerous challenges. This is primarily due to the susceptibility of nucleic acids to degradation when administered in their raw form, and their relatively limited localization within the body. For nucleic acid drugs to function effectively in vivo, safe, efficient, and stable delivery systems are required to protect nucleic acids from degradation and allow for cellular uptake and release. To date, various nucleic acid delivery systems have been developed, including lipids, lipid-like materials, polymers, inorganic materials, and cell membranes. Lipids, lipid-like materials, and polymers are the most commonly used. Although lipid nanoparticle-based nucleic acid drug delivery systems are currently at the forefront of clinical translation, several limitations remain, such as complex fabrication processes, thermal stability issues, and liver aggregation problems.

[0003] Polymer carriers have attracted much attention due to their multifunctional structure, ease of functionalization, and robust stability. Common polymer carriers include polyethyleneimine, polyamides, dendritic polymers, and poly-β-amino esters. These polymers interact with the negative charges of nucleic acids through their positive charges, forming stable complexes that protect nucleic acids from degradation and facilitate their entry into cells. Polyethyleneimine (PEI) is one of the most widely used cationic polymers for nucleic acid delivery. PEI provides a high density of positive charges for nucleic acid complexation and endosome escape. Studies have shown that PEI promotes endosome escape through a "proton sponge" effect. However, a problem is that PEI has poor biocompatibility due to its excessive cationic charge and lack of biodegradable bonds. The biocompatibility and in vivo stability of polymer carriers are crucial for in vivo nucleic acid delivery.

[0004] To address this issue, it is essential to reduce cytotoxicity while maintaining nucleic acid transfection efficiency. Therefore, designing an amino acid-based polymer library for nucleic acid delivery is crucial. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing and applying an amino acid polymer. The amino acid polymer provided by this invention has low toxicity and side effects as a nucleic acid drug delivery carrier and has a high efficiency in nucleic acid delivery.

[0006] This invention designs an amino acid-based polymer library for nucleic acid delivery. First, a biocompatible amino acid monomer is attached with a double bond via esterification. Then, the double bond reacts with the amino group in the LPEI (Liquid Polymer Extraction Intake), introducing the amino acid into the LPEI to obtain the final polyamino acid polymer library.

[0007] This invention provides an amino acid polymer with the structure shown in Formula I.

[0008]

[0009] Wherein, 300≤n≤500000, and n can be 300, 500, 1000, 5000, 10000, 50000, 100000, 50000, or any value between the two above; or any value between the two above.

[0010] The molar ratio of x / n is 1:(1 to 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 to 10000; or any value between the two mentioned above.

[0011] The R is selected from -H, -(CH2). m R1; m is selected from integers 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 this invention, the polymer has any of the following structures:

[0014]

[0015] This 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, and then reacted with di-tert-butyl dicarbonate and triethylamine. After the reaction, the mixture is extracted and purified to obtain intermediate a.

[0017] B) Intermediate a and 1,3-propanediol were dissolved in an organic solvent, and EDC and DMAP were added to react. After the reaction, the mixture was purified to obtain intermediate b.

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

[0019] D) React intermediate c and linear PEI of formula (III) in an organic solvent. Dialyze and freeze-dry the reaction product to obtain a freeze-dried product. Then react the freeze-dried product with trifluoroacetic acid to obtain the final product.

[0020]

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

[0022] The molar ratio of x / n is 1:(1 to 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 to 10000; or any value between the two mentioned above.

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

[0024] The preparation equation for the polymer of this invention is as follows:

[0025]

[0026] The preparation method of the amino acid polymer of the present invention firstly dissolves the amino acid described in formula (II) in an organic solvent, adds ditert-butyl dicarbonate and triethylamine to react, and then extracts and purifies the mixture to obtain intermediate a.

[0027] Preferably, the amino acid monomer represented by Formula II of the present invention 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 25-100°C, more preferably 25-50°C, and even more preferably 25°C; the reaction time is 2-24 h, more preferably 5 h.

[0030] Specifically, the molar ratio of ditert-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, ditert-butyl dicarbonate and triethylamine described in formula (II) is 1:(1-1.5):1.2.

[0032] Following the reaction, extraction and purification processes yield intermediate a. This invention does not limit the specific extraction steps described, as those well-known to those skilled in the art are acceptable.

[0033] The purification described above is performed using silica gel column chromatography. The mobile phase typically used is 100% DCM to 80%:30% DCM / MeOH. This involves first using the fluidity of 100% DCM to flush down the more polar compounds, and then gradually increasing the amount of methanol to separate products with similar polarities.

[0034] Intermediate a and 1,3-propanediol were dissolved in an organic solvent, and EDC and DMAP were added to react. After the reaction, the mixture was purified to obtain the intermediate.

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

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

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

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

[0039] Intermediate b and acrylic acid were added to an organic solvent, and EDC and DMAP were added to react. After the reaction, the mixture was purified to obtain intermediate c.

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

[0041] The concentrations of intermediate b and acrylic acid with organic solvents are 5–20 M.

[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] Intermediate c and linear PEI of formula (III) were reacted in an organic solvent to obtain the reaction product.

[0044] The molar ratio of intermediate c to linear PEI of formula (III) is 1:(1 to 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 to 10000; or any value between the above two.

[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 even more 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 lyophilized to obtain a lyophilized product; the dialysis is performed using a dialysis bag with a molecular weight cutoff of 500–10000 Da. This invention does not limit the specific parameters of the lyophilization process described above, as those well known to those skilled in the art are acceptable.

[0048] The lyophilized product is then reacted with trifluoroacetic acid to obtain the final product.

[0049] The molar ratio of the lyophilized product to trifluoroacetic acid was 1:(1-5), the reaction temperature was 0-35℃, and the reaction time was 4-24h.

[0050] The application of the amino acid polymers described in the above technical solutions of this invention or the amino acid polymers prepared by the above preparation methods as nucleic acid delivery carriers in the preparation of drugs.

[0051] The drugs described in this invention include nucleic acid drugs.

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

[0053] The above polymer is loaded onto the surface of nucleic acids.

[0054] The present invention also provides a method for preparing a drug, wherein an amino acid polymer and a nucleic acid are separately prepared into aqueous solutions; the two are then mixed and incubated to obtain the drug.

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

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

[0057] The in vitro delivery is preferably performed in cells such as 293, CHO, HeLa, MCF-7, DC2.4, HepG2, B16F10, A549, L20, and BMDCs to deliver nucleic acid drugs.

[0058] The amino acid polymer described in this invention, as a nucleic acid drug delivery carrier, exhibits good in vitro transfection effects when used in the aforementioned cells for nucleic acid drug delivery.

[0059] This invention provides an amino acid-based polymer with the structure shown in Formula I, relating to the field of biomedical technology. The polymer provided by this invention is prepared by introducing amino acid monomers into the side chains of linear polyethyleneimine (LPEI), resulting in an amino acid-modified polymer. Using the polymer of this invention as a nucleic acid drug delivery carrier, its simple composition allows for efficient nucleic acid delivery as a single-component carrier, exhibiting excellent transfection efficiency and biocompatibility. Attached Figure Description

[0060] Figure 1 The NMR spectrum of the amino acid polymer prepared in Example 1;

[0061] Figure 2 The image shows the in vitro transfection effect of the amino acid polymer prepared in Example 3. Detailed Implementation

[0062] This 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 this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and fall within the scope of this invention. The method and application of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the method and application described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0063] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

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

[0065] In this application, the term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. A and B can be singular or plural.

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

[0067] It should be understood that the order of the steps or the order in which certain actions are performed is not important as long as the invention remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0068] The use of any and all instances or exemplary language such as “e.g.” or “including” in this document is merely intended to better illustrate the invention and is not intended to limit the scope of the invention unless the claims are made. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0069] Furthermore, 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 precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately." Here, "approximately" generally means an actual value within plus or minus 10%, 5%, 1%, or 0.5% of a particular value or range.

[0070] It should be understood that in the various embodiments of this application, the order of the above processes does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. 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 this application.

[0071] The embodiments and comparative examples of this invention describe some examples, in which the embodiments illustrate certain implementations of the invention. However, this does not mean that the effects of the invention can only be achieved in these examples.

[0072] To further illustrate the present invention, the following describes in detail, with reference to embodiments, a method for preparing an amino acid polymer and its application in nucleic acid delivery.

[0073] Example 1

[0074] The amino acid polymer was prepared according to the following method:

[0075]

[0076] Step 1:

[0077] Alanine (1.0 g) was dissolved in THF (10 mL), and a THF solution of triethylamine (2.14 mL) and Boc₂O (2.4 g) (10 mL) was added under ice bath conditions. The mixture was stirred at room temperature for 5 hours. After concentration under reduced pressure, hexane was added for recrystallization. The product a was obtained by filtration.

[0078] Step 2:

[0079] Compound a (1 g) and 1,3-propanediol (1.18 g) were dissolved in DCM (20 mL) and stirred until dissolved. EDC (0.98 g) and DMAP (0.18 g) were added to the reaction solution. After the reaction was complete, the mixture was washed with 1 M hydrochloric acid solution and sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and 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) and stirred until dissolved. EDC (0.75 g) and DMAP (0.15 g) were added to the reaction solution, and the mixture was stirred at room temperature for 24 hours. After the reaction was complete, the mixture was washed with 1 M hydrochloric acid solution and sodium chloride, dried over anhydrous sodium sulfate, filtered, and 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 and set aside. 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 thoroughly. The mixture was reacted at 80 °C for 24 h. After the reaction was completed, the lyophilized product was dialyzed through a 3500 Da dialysis bag. The lyophilized product was then added to 30 mL of TFA and reacted at room temperature for 4 h. After sedimentation and filtration with diethyl ether, the final product was obtained by lyophilization through a 3500 Da dialysis bag.

[0084] Comparative Example 2

[0085]

[0086] Dissolve 1.0 g of LPEI-25K in 10 mL of water and set aside. Dissolve 0.45 g of n-octyl acrylate in 50 mL of DMF. Pour the LPEI aqueous solution into the DMF solution and mix well. React at 80 °C for 24 h. After the reaction is complete, dialyze the lyophilized product LPEI-OA using a 3500 Da dialysis bag.

[0087] Example 3

[0088] The LPEI-Ala was analyzed by proton NMR spectroscopy, and the resulting NMR spectrum is shown below. Figure 1 As shown, the strong signal peak at a chemical shift of around 3.0 ppm is the characteristic absorption peak of -CH2- on the LPEI chain, the peak at 2.31 ppm is the characteristic absorption peak of -CH3- on the amino acid side chain, and the peak at 3.0-4.0 ppm is the characteristic absorption peak of -OCH2-.

[0089] Example 4

[0090] The LPEI-Ala / mRNA complex is prepared as follows:

[0091] LPEI-Ala (the amino acid polymer obtained in Example 1) was dissolved in enzyme-free water to obtain a carrier solution; the carrier solution and mRNA solution were mixed evenly in a certain proportion to obtain an mRNA delivery system. The molar ratio of LPEI grafted with 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 in the delivery system prepared in Example 4

[0097] 1. In vitro nucleic acid delivery

[0098] (1) Cell Culture

[0099] Continuous cell culture was performed using a 5% fetal bovine serum incubator at 37°C. Cells stored in liquid nitrogen were removed and placed in a 37°C water bath until the solution was completely dissolved. The cells were then quickly transferred to a centrifuge tube containing 5 mL of the above culture medium, mixed, and centrifuged at 1000 rpm for 5 min. The cell pellet in the centrifuge tube was retained, and 10 mL of culture medium was added. The pellet was then transferred to a culture dish and placed in the above-mentioned incubator for further culture. 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 cultured cells were divided into groups of 1 × 10⁶ cells per well. 4 The cells were seeded at a density of 200 μL in 96-well plates and cultured overnight. The polymer nucleic acid drug delivery carrier and luciferase substrate prepared in Example 1 were used to detect the transfection efficiency of the complex in cells using a microplate reader. Table 2 shows the transfection efficiency of the complex.

[0102] Table 2

[0103]

[0104] Figure 2 The image shows 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 carrying green fluorescent protein mRNA (GFP-mRNA) showed significant green fluorescent protein expression in HeLa, DC2.4 and L20 cells.

[0105] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An amino acid polymer with the structure shown in Formula I, Equation I in, 300≤n≤500000, the molar ratio of x / n is 1∶(1~10000); The R is selected from -H, -(CH2). m R1; m is selected from integers between 1 and 8; 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, Includes the following steps: A) The amino acid described in formula (II) is dissolved in an organic solvent, and then reacted with di-tert-butyl dicarbonate and triethylamine. After the reaction, the mixture is extracted and purified to obtain intermediate a. B) Intermediate a and 1,3-propanediol were dissolved in an organic solvent, and EDC and DMAP were added to react. After the reaction, the mixture was purified to obtain intermediate b. C) Intermediate b and acrylic acid were added to an organic solvent, and EDC and DMAP were added to react. After the reaction, the mixture was purified to obtain intermediate c. D) React intermediate c and linear PEI of formula (III) in an organic solvent. Dialyze and freeze-dry the reaction product to obtain a freeze-dried product. Then react the freeze-dried product with trifluoroacetic acid to obtain the final product. Formula (II); Equation (III).

4. The preparation method according to claim 3, characterized in that, In step A), the organic solvent is one of THF, DMF, or DCM, the reaction temperature is 25~100℃, and the reaction time is 2~24h.

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

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

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

8. The use of an amino acid polymer according to any one of claims 1 to 2 or an amino acid polymer prepared by any one of claims 3 to 7 as a nucleic acid delivery carrier in the preparation of a drug.

9. A drug, characterized in that, The raw materials include amino acid polymers and nucleic acids; the amino acid polymer is 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.

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

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