Highly branched poly(β-amino esters) containing hydrophobic alkyl chains, their preparation methods, and their applications in mRNA delivery.

Abstract

This invention belongs to the field of biomedical materials technology and discloses a highly branched poly(β-amino ester) containing hydrophobic alkyl chains, its preparation method, and its application in mRNA delivery. The highly branched poly(β-amino ester) is prepared by a Mike addition reaction of acrylate monomers and small molecule amine monomers. Using an amino monomer as a capping agent, the highly branched poly(β-amino ester) is functionalized and capped to obtain a highly branched poly(β-amino ester) containing hydrophobic alkyl chains, achieving efficient mRNA delivery in cells such as HeLa. The highly branched poly(β-amino ester) containing hydrophobic alkyl chains of this invention is inexpensive, easy to design and prepare, and exhibits superior transfection performance, showing significant clinical application potential in mRNA drug delivery.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to a highly branched poly(β-amino ester) containing hydrophobic alkyl chains, its preparation method, and its application in mRNA delivery. Background Technology

[0002] mRNA drugs have enormous application potential in infectious disease vaccines, oncology, protein replacement, and gene therapy. However, the lack of safe and efficient vectors limits the clinical application of mRNA gene therapy. Currently, over 70% of clinical trials still use viral vectors, but viral vectors have potential immunogenicity and teratogenicity, complex preparation procedures, and high costs. Among non-viral vectors, commercially available lipid nanoparticle delivery (LNP) technology is controlled by Arbutus in Canada, and breaking through related technology patents for LNPs in the short term seems relatively difficult. Furthermore, their poor low-temperature storage and stability greatly limit their commercial application. While the potential toxicity of inorganic nanoparticles can be modulated by controlling their size and surface modification, their poor biodegradability in vivo is a crucial factor to consider in practical applications.

[0003] Polymers have become one of the non-viral vectors with clinical translational potential due to their low immunogenicity, high safety, flexible design, and easier production and chemical modification processes. Poly(β-amino esters), with their wide availability of monomers, easily tunable structure and properties, and in vivo degradability, have shown great promise for drug, protein, and nucleic acid delivery. However, current research on the application of poly(β-amino esters) in mRNA delivery has only explored polymers prepared using polyamines as branching units; there are no reports on the construction of poly(β-amino esters) using tri(tetra)acrylate monomers as branching units for efficient mRNA delivery. Summary of the Invention

[0004] In order to overcome the shortcomings of the prior art, the present invention aims to provide a highly branched poly(β-amino ester) containing hydrophobic alkyl chains, its preparation method and its application in mRNA delivery. This solves the problem that in the prior art, the application of poly(β-amino ester) in mRNA delivery has only explored polymers prepared with polyamines as branching units, and there have been no reports on the construction of poly(β-amino ester) with tri(tetra) acrylate monomers as branching units for efficient mRNA delivery.

[0005] To achieve the above objectives, the present invention employs the following technical solution: a highly branched poly(β-amino ester) containing a hydrophobic alkyl chain, wherein the highly branched poly(β-amino ester) has the following structural formula:

[0006]

[0007] Wherein, R1 is one of the following structures:

[0008]

[0009] R2 is one of the following structures:

[0010]

[0011] Where n = 2-16, m = 3-50.

[0012] This invention also discloses a method for preparing highly branched poly(β-amino ester) containing hydrophobic alkyl chains, comprising the following steps:

[0013] Step 1: Highly branched poly(β-amino ester) is prepared by reacting acrylate monomers and small molecule amine monomers via a Mike addition reaction.

[0014] Step 2: Using amino monomers as end-capping agents, highly branched poly(β-amino ester) is functionalized and end-capped to obtain highly branched poly(β-amino ester) containing hydrophobic alkyl chains.

[0015] Preferably, the acrylate monomers include diacrylate monomers and tri(tetra)acrylate monomers; the small molecule amine monomers include hydrophilic amines and hydrophobic amines; the reaction molar ratio of acrylate monomers and small molecule amine monomers is (1-2):1; wherein the ratio of diacrylate monomers to tri(tetra)acrylate monomers is 9:1-1:9; and the ratio of hydrophilic amines to hydrophobic amines is 9:1-1:9.

[0016] Preferably, the Mike addition reaction in step one is carried out at 60-120°C for 12-48 hours, and the concentration of the reactants dissolved in dimethyl sulfoxide is 200-500 mg / mL.

[0017] Preferably, in step two, the molar ratio of the capping agent to the acrylate monomer is 0.5-3:1.

[0018] Preferably, the functionalization end-capping reaction temperature is 25°C and the reaction time is 48 hours.

[0019] Preferably, the diacrylate monomer has one of the following structures:

[0020]

[0021] Preferably, the tri(tetra)acrylate monomer has one of the following structures:

[0022]

[0023] Preferably, the small molecule hydrophilic amine monomer has one of the following structures:

[0024]

[0025] The small molecule hydrophobic amine monomer includes one of the following structures:

[0026]

[0027] The amino-containing capped monomer R2-NH2 has one of the following structures:

[0028]

[0029] This invention also discloses the application of highly branched poly(β-amino ester) containing hydrophobic alkyl chains in the preparation of mRNA drug delivery vectors.

[0030] Compared with existing technologies, this invention has the following advantages: This invention discloses highly branched poly(β-amino esters) containing hydrophobic alkyl chains, constructed using tri(tetra)acrylate monomers as branching units. By using different monomer combinations, regulating the branching structure, and using different end-capping monomers, a class of biodegradable highly branched polymers with tunable structural components, structure, and properties can be synthesized. Due to their special topological structure, they enhance the encapsulation and protection of mRNA, exhibiting high mRNA delivery efficiency and cell activity. This is in contrast to the commercially available mRNA delivery reagents currently used in this field. Compared to Lipofectamine MessengerMAX™, the highly branched poly(β-amino ester) containing hydrophobic alkyl chains disclosed in this invention has higher delivery efficiency, lower toxicity, and greater clinical potential.

[0031] This invention also discloses a method for preparing the aforementioned highly branched poly(β-amino ester) containing hydrophobic alkyl chains. Using commercially available acrylate monomers and small-molecule organic amines, highly branched poly(β-amino ester) is prepared via Michael addition. The molecular weight of the polymer is monitored by gel permeation chromatography, and the molecular weight is controlled within the range of 2000-40000 Da. Using an amino monomer as a capping agent, the highly branched poly(β-amino ester) is capped, and poly(β-amino ester) with different chemical compositions, branched structures, and terminal groups is prepared in a one-pot process, exhibiting good mRNA transfection performance. The preparation method disclosed in this invention successfully prepares highly branched poly(β-amino ester) containing hydrophobic alkyl chains with advantages of low cost, simple synthetic route, and easy control of structure and properties.

[0032] Furthermore, the molar ratio of diacrylate monomer to tri(tetra)acrylate monomer serves two purposes: firstly, it regulates the reaction rate, and secondly, it ensures that the resulting polymer exhibits good mRNA delivery efficiency and safety.

[0033] Furthermore, the reaction time and temperature range were well controlled, resulting in a product with a suitable molecular weight.

[0034] Furthermore, the molar ratio of the capping agent to the acrylate monomer ensures that the capping reaction proceeds completely without any side reactions.

[0035] This invention also discloses the use of highly branched poly(β-amino ester) containing hydrophobic alkyl chains in the preparation of mRNA drug delivery carriers. Due to its main chain peptide bond structure, it has good biodegradability; the ionizable or positively charged groups can effectively compress mRNA, thereby achieving delivery. The effectiveness, accuracy and wide applicability of this invention have been verified in in vitro cell experiments, and it has good prospects for clinical translation. Attached Figure Description

[0036] Figure 1 A schematic diagram illustrating the synthesis of highly branched poly(β-amino ester);

[0037] Figure 2 This is a schematic diagram of the physical phase state of the highly branched poly(β-amino ester) containing hydrophobic alkyl chains prepared in Example 1 at 25°C.

[0038] Figure 3 The highly branched poly(β-amino ester) containing hydrophobic alkyl chains prepared in Example 1 1 H NMR spectrum;

[0039] Figure 4 The image shows the gel permeation chromatography (GPC) curve of the highly branched poly(β-amino ester) containing hydrophobic alkyl chains obtained in Example 1 after purification.

[0040] Figure 5 The TEM microstructure of the complex formed by the highly branched poly(β-amino ester) containing hydrophobic alkyl chains and mRNA prepared in Example 1 is shown.

[0041] Figure 6 The potential of the complex formed by the highly branched poly(β-amino ester) containing hydrophobic alkyl chains and mRNA prepared in Example 1;

[0042] Figure 7a The image shows the fluorescence of HeLa cells after transfection with mRNA encoding green fluorescent protein using the highly branched poly(β-amino ester) containing hydrophobic alkyl chains prepared in Example 1;

[0043] Figure 7b This is a fluorescence image of HeLa cells after transfection with mRNA encoding green fluorescent protein using the commercial Lipo Messenger from Example 1.

[0044] Figure 8The cell viability of the highly branched poly(β-amino ester) containing hydrophobic alkyl chains prepared in Example 1 after transfection into HeLa cells;

[0045] Figure 9 This is a schematic diagram of the physical phase state of the highly branched poly(β-amino ester) containing hydrophobic alkyl chains prepared in Example 2 at 25°C.

[0046] Figure 10 The 1H NMR spectrum of the highly branched poly(β-amino ester) containing hydrophobic alkyl chains prepared in Example 2 is shown below.

[0047] Figure 11 The image shows the gel permeation chromatography (GPC) curve of the highly branched poly(β-amino ester) containing hydrophobic alkyl chains obtained in Example 2 after purification.

[0048] Figure 12 This is a schematic diagram of the physical phase state of the highly branched poly(β-amino ester) containing hydrophobic alkyl chains prepared in Example 3 at 25°C.

[0049] Figure 13 The highly branched poly(β-amino ester) containing hydrophobic alkyl chains prepared in Example 3 1 H NMR spectrum;

[0050] Figure 14 The image shows the gel permeation chromatography (GPC) curve of the highly branched poly(β-amino ester) containing hydrophobic alkyl chains obtained in Example 3 after purification. Detailed Implementation

[0051] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0052] It should be noted that the terms "first," "second," etc., in the specification, claims, and accompanying drawings of this invention are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate so that the embodiments of the invention described herein can be implemented in orders other than those illustrated or described herein. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover a non-exclusive inclusion; for example, a process, method, system, product, or apparatus that comprises a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such processes, methods, products, or apparatus.

[0053] The present invention will now be described in further detail with reference to the accompanying drawings:

[0054] For the preparation method and synthetic route of highly branched poly(β-amino esters) containing hydrophobic alkyl chains, please refer to [link / reference]. Figure 1 This includes the following steps:

[0055] Step 1: Add a certain amount of acrylate monomers (diacrylate monomers, tri(tetra)acrylate monomers, etc.) and small molecule organic amines (hydrophilic amines, hydrophobic amines) to a flask containing dimethyl sulfoxide. The molar ratio of acrylate monomers to small molecule organic amines is (1-2):1; wherein the ratio of diacrylate monomers to triacrylate monomers is 9:1-1:9; and the ratio of hydrophilic amines to hydrophobic amines is 9:1-1:9. React at 60-120℃ for 12-48 hours. During the reaction, use gel permeation chromatography to monitor the molecular weight of the polymer. When the molecular weight of the polymer reaches 2000-40000 Da, terminate the reaction.

[0056] Step 2: Add a certain amount of end-capping agent and dimethyl sulfoxide to the reaction flask and react at 25°C for 48 hours. The molar ratio of end-capping agent to raw acrylate monomer is 0.5-3:1. Purify the product using precipitation method and vacuum dry to obtain highly branched poly(β-amino ester) containing hydrophobic alkyl chains.

[0057] The functional, highly branched poly(β-amino ester) prepared in this invention is used for mRNA delivery, comprising the following steps:

[0058] 1) Cell culture: HeLa cells were cultured under standard culture conditions at a rate of 0.5*10⁻⁶. 4 -2.0*10 4 Cells were seeded at a density of 100 cells / well in 96-well plates and cultured for 24 hours.

[0059] 2) Mix the solution of highly branched poly(β-amino ester) containing hydrophobic alkyl chains with the mRNA solution encoding green fluorescent protein, incubate for 15-30 min, and then transfer to a 96-well plate for transfection. The polymer:mRNA mass ratio is 10-200:1, and the mRNA mass per well is 25-250 ng.

[0060] 3) 24-48 h after transfection, observe and photograph the transfected green fluorescent protein cells under a fluorescence microscope.

[0061] 5) 24-48 h after transfection, remove the cell supernatant and add Alamar Blue solution. Incubate in an incubator for 30 min and then perform cell viability testing.

[0062] Example 1

[0063] The molar ratio of acrylate monomers (bisphenol A ethoxylated diacrylate, EO / phenol = 1.5:trimethylolpropane triacrylate molar ratio = 5:1) and small organic amine monomers (5-amino-1-pentanol and n-octylamine molar ratio = 8:1) was 1.5:1, and the concentration of the reactants in dimethyl sulfoxide was 400 mg / mL. The reaction was carried out at 80℃ for 12 h; then, 0.6 times the molar amount of acrylate monomer 1-(3-aminopropyl)-4-methylpiperazine was added, and the reaction was carried out at 25℃ for 48 h. After purification and drying, the resulting branched poly(β-amino ester) had a molecular weight of 6000 Da, a molecular weight distribution of 2.3, and m = 10. The structural formula is as follows:

[0064]

[0065] The physical phase diagram of the highly branched poly(β-amino ester) containing hydrophobic alkyl chains obtained in this embodiment at 25°C is shown below. Figure 2 As shown; the highly branched poly(β-amino ester) containing hydrophobic alkyl chains prepared in this embodiment 1 H NMR spectrum as shown Figure 3 As shown; the GPC curve of the highly branched poly(β-amino ester) containing hydrophobic alkyl chains prepared in this embodiment is as follows. Figure 4 As shown.

[0066] The highly branched poly(β-amino ester) containing hydrophobic alkyl chains prepared in this embodiment was used for extracellular mRNA delivery experiments. HeLa cells were seeded at 10,000 cells per well in 96-well plates and cultured for 24 hours. The polymer was then uniformly mixed with mRNA at mass ratios of 10, 30, and 60:1, with 25 ng of mRNA per well. After static incubation for 20 minutes, composite nanoparticles were formed and then transferred to 96-well plates for transfection. The microstructure of the nanoparticles under the 30:1 condition is shown in the figure below. Figure 5 As shown, the nanoparticles are uniform in size, and their potentials are as follows: Figure 6 As the amount of polymer increases, the positive charge increases accordingly, which can effectively compress mRNA; the mRNA delivery efficiency of the polymer in HeLa cells is as follows: Figure 7a and Figure 7b Cell viability after transfection is as follows Figure 8 The polymer's mRNA transfection efficiency and safety are significantly better than those of commercial products.

[0067] Example 2

[0068] The molar ratio of acrylate monomers (bisphenol A glycerol diacrylate: pentaerythritol triacrylate in a molar ratio of 3:1) and small organic amine monomers (3-amino-1,2-propanediol: dodecaneamine in a molar ratio of 6:1) was 1.1:1, and the concentration of the reactants in dimethyl sulfoxide was 200 mg / mL. The reaction was carried out at 100℃ for 24 h; then, propylenediamine, at a molar ratio equal to that of the acrylate monomers, was added, and the reaction was carried out at 25℃ for 48 h. After purification and drying, the resulting branched poly(β-amino ester) had a molecular weight of 15000 Da, a molecular weight distribution of 3.4, and m = 20. The structural formula is as follows:

[0069]

[0070] The physical phase diagram of the highly branched poly(β-amino ester) containing hydrophobic alkyl chains obtained in this embodiment at 25°C is shown below. Figure 9 As shown; the highly branched poly(β-amino ester) containing hydrophobic alkyl chains prepared in this embodiment 1 H NMR spectrum as shown Figure 10 As shown, the chemical composition of the product is basically consistent with that of the raw materials; the GPC curve of the highly branched poly(β-amino ester) containing hydrophobic alkyl chains prepared in this example is shown in the figure. Figure 11 As shown.

[0071] Example 3

[0072] The molar ratio of acrylate monomers (bisphenol A ethoxylated diacrylate, EO / phenol = 2: pentaerythritol tetraacrylate molar ratio = 9:1) and small organic amine monomers (2-aminoethylmorpholine and tetradecylamine molar ratio = 4:1) was 1.3:1, and the concentration of the reactants in dimethyl sulfoxide was 350 mg / mL. The reaction was carried out at 80℃ for 36 h; then 1,11-diamino-3,6,9-trioxaundecane was added in 1.3 times the molar amount of the acrylate monomers, and the reaction was carried out at 25℃ for 48 h. After purification and drying, the resulting branched poly(β-amino ester) had a molecular weight of 22000 Da, a molecular weight distribution of 3.2, and m = 25. The structural formula is as follows:

[0073]

[0074] The physical phase diagram of the highly branched poly(β-amino ester) containing hydrophobic alkyl chains obtained in this embodiment at 25°C is shown below. Figure 12 As shown; the highly branched poly(β-amino ester) containing hydrophobic alkyl chains prepared in this embodiment 1 H NMR spectrum as shown Figure 13 As shown, the chemical composition of the product is consistent with that of the raw materials; the GPC curve of the highly branched poly(β-amino ester) containing hydrophobic alkyl chains prepared in this example is shown in the figure. Figure 14 As shown.

[0075] This invention discloses a method for preparing a class of biodegradable, highly branched poly(β-amino esters) containing hydrophobic alkyl chains and their application in mRNA delivery, belonging to the field of biomedical materials technology. Highly branched poly(β-amino esters) were prepared by selecting different acrylate monomers and small molecule amine monomers, achieving efficient mRNA delivery in typical HeLa cells. Compared with mainstream commercial transfection reagents (jetPEI, Lipofectamine), this type of polymer is cheaper, easier to design and prepare, and has superior transfection performance, showing significant clinical application potential in mRNA drug delivery. Compared to using small molecule amines as branching units, highly branched poly(β-amino esters) containing hydrophobic alkyl chains, using tri(tetra)acrylate monomers as branching units, can introduce more ester bonds, resulting in polymers that degrade faster, exhibit lower cytotoxicity, and have greater clinical application prospects. Effective encapsulation of mRNA protects it from degradation by nucleases in vivo before translation and expression of proteins. Meanwhile, current research indicates that the escape efficiency of mRNA in endosomes is only 1-4%, and improving endosome escape efficiency is a key step in achieving efficient mRNA transfection. Introducing hydrophobic alkyl chains increases the hydrophobicity of the polymer, enhancing its affinity for mRNA and thus enabling efficient mRNA encapsulation. Furthermore, the fusion of these hydrophobic alkyl chains with the endosome membrane effectively increases the escape efficiency of the mRNA within the endosome, thereby improving transfection efficiency. Compared to traditional synthetic methods, a multi-component synthetic strategy was proposed to rapidly and effectively evaluate and screen for branched poly(β-amino esters) with superior performance. These results further demonstrate that highly branched poly(β-amino esters) containing hydrophobic alkyl chains can effectively reduce biotoxicity, increase mRNA encapsulation efficiency and endosome escape efficiency, achieving efficient intracellular mRNA delivery.

[0076] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A highly branched poly(β-amino ester) containing a hydrophobic alkyl chain, characterized in that, The highly branched poly(beta-amino ester) has the following structural formula: R1 is one of the following structures: ； R2 is one of the following structures: ； n = 2-16, and m = 3-50. The preparation method of the highly branched poly(beta-amino ester) containing a hydrophobic alkyl chain comprises the following steps: Step one, a highly branched poly(beta-amino ester) is prepared by Michael addition reaction of an acrylic ester monomer and a small molecule amine monomer. The acrylic ester monomer is a diacrylic ester monomer and a triacrylic ester monomer; the small molecule amine monomer comprises a hydrophilic amine and a hydrophobic amine; the reaction molar ratio of the acrylic ester monomer and the small molecule amine monomer is (1-2):1; the diacrylic ester monomer:triacrylic ester monomer = 9:1-1:9; the hydrophilic amine:hydrophobic amine = 9:1-1:

9. Step two, the highly branched poly(beta-amino ester) is capped by using an amino monomer as a capping agent to obtain the highly branched poly(beta-amino ester) containing a hydrophobic alkyl chain.

2. Process for the preparation of a highly branched poly(β-amino ester) containing hydrophobic alkyl chains according to claim 1, characterized in that, The preparation method comprises the following steps: Step one, a highly branched poly(beta-amino ester) is prepared by Michael addition reaction of an acrylic ester monomer and a small molecule amine monomer. Step two, the highly branched poly(beta-amino ester) is capped by using an amino monomer as a capping agent to obtain the highly branched poly(beta-amino ester) containing a hydrophobic alkyl chain.

3. Process for the preparation of a highly branched poly(β-amino ester) containing hydrophobic alkyl chains according to claim 2, characterized in that, The acrylic ester monomer is a diacrylic ester monomer and a triacrylic ester monomer; the small molecule amine monomer comprises a hydrophilic amine and a hydrophobic amine; the reaction molar ratio of the acrylic ester monomer and the small molecule amine monomer is (1-2):1; the diacrylic ester monomer:triacrylic ester monomer = 9:1-1:9; the hydrophilic amine:hydrophobic amine = 9:1-1:

9.

4. Process for the preparation of a highly branched poly(β-amino ester) containing hydrophobic alkyl chains according to claim 2, characterized in that, In step one, the Michael addition reaction is performed at 60-120°C for 12-48 h, and the concentration of the reactants dissolved in dimethyl sulfoxide is 200-500 mg / mL.

5. The method for preparing highly branched poly(β-amino ester) containing hydrophobic alkyl chains according to claim 2, characterized in that, In step two, the molar ratio of the capping agent to the acrylic ester monomer is (0.5-3):

1.

6. The process for the preparation of highly branched poly(β-amino esters) containing hydrophobic alkyl chains according to claim 2, characterized in that, The functional capping reaction temperature is 25°C, and the reaction time is 48 h.

7. Process for the preparation of a highly branched poly(β-amino ester) containing hydrophobic alkyl chains according to any one of claims 2 to 6, characterized in that, The diacrylic ester monomer is one of the following structures: 。 8. Process for the preparation of a highly branched poly(β-amino ester) containing hydrophobic alkyl chains according to any one of claims 2 to 6, characterized in that, The triacrylate monomer is .

9. Process for the preparation of a highly branched poly(β-amino ester) containing hydrophobic alkyl chains according to any one of claims 2 to 6, characterized in that, The small molecule hydrophilic amine monomer is one of the following structures: ； The small molecule hydrophobic amine monomer is one of the following structures: ； The amino-containing capping monomer R2-NH2 is one of the following structures: 。 10. Use of the highly branched poly(beta-amino ester) containing a hydrophobic alkyl chain in claim 1 in the preparation of an mRNA drug delivery carrier.

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