Polyhydroxy polyamino acid as well as preparation method and application thereof

Abstract

The invention discloses polyhydroxy polyurethane as well as a preparation method and application thereof, and the polyhydroxy polyurethane has a structure as shown in a formula 1. Polyamino acids with double bonds on side chains and different polymerization degrees are used, side chain modification is carried out through a click reaction to obtain aminated polyamino acid, polyhydroxy polyamino acid is prepared through a simple ring-opening reaction, the polyhydroxy polyamino acid is compounded with cholesterol, auxiliary lipid and DMG-PEG to be used for delivery of mRNA, spleen targeting is obvious, and good comprehensive performance is achieved.

Description

Technical Field

[0001] This application relates to the field of biomedical materials, and in particular to a polyhydroxy polyamino acid, its preparation method, and its application. Background Technology

[0002] Despite lipid nanoparticles (LNPs) being the current mainstream mRNA carriers, their core component—ionizable cationic lipids—has significant drawbacks: high cytotoxicity leading to inflammatory responses, questionable long-term in vivo safety, excessive liver targeting severely limiting their potential for treating extrahepatic diseases, and complex formulations resulting in stringent manufacturing processes and poor storage stability.

[0003] Therefore, developing a polyamino acid alternative to LNP for mRNA vectors that can improve formulation stability and relax storage conditions is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0004] In view of this, this application provides a polyhydroxy polyamino acid (PM-OH) that is combined with cholesterol, cofactor lipids, and DMG-PEG for mRNA delivery, exhibiting good overall performance.

[0005] This application provides a polyhydroxy polyurethane having the structure of Formula 1:

[0006] ;

[0007] Where n is an integer from 1 to 100.

[0008] In some specific implementations, n is between 5 and 12.

[0009] This application also provides a method for preparing a polyhydroxy polyurethane, comprising:

[0010] The γ-allyl-L-glutamic acid carboxylic anhydride was polymerized and then dialyzed for the first time to obtain poly(γ-allyl-L-glutamic acid ester).

[0011] Poly(γ-allyl-L-glutamate), cysteine ​​hydrochloride and photoinitiator were mixed and irradiated with ultraviolet light, followed by a second dialysis to obtain side-chain-amino acid-based polyamino acids.

[0012] Amminated polyamino acids with side chains and glycidyl ethers are mixed and reacted to obtain polyhydroxy polyurethane.

[0013] In some specific implementations, the polymerization reaction takes 40 to 50 hours and the polymerization temperature is 20°C to 30°C; the molecular weight of the first dialysis is 500 Da to 10000 Da and the first dialysis takes 48 to 96 hours.

[0014] In some specific implementations, the molar ratio of poly(γ-allyl-L-glutamate), cysteine ​​hydrochloride, and photoinitiator is 1:(4-6):(0.1-0.3), the UV irradiation time is 0.2h to 1h, and the UV irradiation temperature is 20℃ to 30℃; the molecular weight of the second dialysis is 500Da to 10000Da, and the second dialysis time is 48h to 96h.

[0015] In some specific implementations, the molar ratio of the side-chain-aminated polyamino acid to glycidyl is 1:(2-4); the amination reaction time is 2h to 6h; and the amination reaction temperature is 70℃ to 90℃.

[0016] The reaction with amino groups is followed by a third dialysis, wherein the molecular weight of the third dialysis is 500 Da to 10000 Da, and the duration of the third dialysis is 48 h to 96 h.

[0017] In some specific implementations, the preparation method of the γ-allyl-L-glutamic acid carboxylic anhydride includes:

[0018] L-glutamic acid, allyl alcohol, and trimethylchlorosilane were mixed and reacted to obtain γ-allyl-L-glutamic acid ester;

[0019] γ-Allyl-L-glutamic acid ester, ultra-dry tetrahydrofuran, and triphosgene were mixed and reacted at room temperature to obtain γ-Allyl-L-glutamic acid carboxylic anhydride.

[0020] This application also provides a composition comprising, by weight parts: 40 to 60 parts of polyhydroxy polyamino acid, 20 to 30 parts of cholesterol, 10 to 20 parts of auxiliary lipids, and 5 to 10 parts of DMG-PEG.

[0021] In some specific implementations, the auxiliary lipid includes one or more of 1,2-distearyl-sn-glycerol-3-phosphate choline, 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine, or 1,2-dioleoyl-sn-glycerol-3-phosphate choline.

[0022] This application also provides a method for delivering mRNA, comprising:

[0023] A composition was prepared by mixing polyhydroxy polyamino acids, cholesterol, auxiliary lipids, and DMG-PEG; mRNA was dissolved in an aqueous sodium citrate solution, and the composition was mixed with the aqueous sodium citrate solution of mRNA for intramuscular injection.

[0024] This application uses polyamino acids (PALG) with double bonds on their side chains of different degrees of polymerization. The side chains are modified by click reaction to obtain amino polyamino acids (PM). A polyhydroxy polyamino acid (PM-OH) is prepared by simple ring-opening reaction. This polyamino acid is then combined with cholesterol, auxiliary lipids, and DMG-PEG for mRNA delivery. It shows significant spleen targeting and good overall performance. Attached Figure Description

[0025] Figure 1 The 1H NMR spectrum of the monomer ALG-NCA provided in Example 1 of this application;

[0026] Figure 2 The 1H NMR spectrum of the polyamino acid PM provided in Example 1 of this application;

[0027] Figure 3 The 1H NMR spectrum of the polyhydroxy polyamino acid PM-OH provided in Example 1 of this application;

[0028] Figure 4 This is a diagram showing the mRNA muscle transfection of the compositions provided in Example 2 and Comparative Example 1 of this application in mice;

[0029] Figure 5 This is a GPC effluent curve of polyamino acid PM with different degrees of polymerization in Example 3 of this application;

[0030] Figure 6 This is a diagram showing the distribution of PM-OH in organs within the body.

[0031] Figure 7 Distribution map of SM102 in vivo;

[0032] Figure 8 The images show the transfection effects of PM-OH with different degrees of polymerization. Detailed Implementation

[0033] 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.

[0034] 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.

[0035] It should be understood that the order of steps or the sequence of actions is not important as long as this application remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0036] The use of any and all instances or exemplary language such as “e.g.” or “include” in this document is intended merely to better illustrate the application and does not constitute a limitation on the scope of the application. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of this application.

[0037] Furthermore, the numerical ranges and parameters used to define this application are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any numerical value inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise explicitly stated, 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 that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.

[0038] To overcome these bottlenecks, this application proposes to directly replace the cationic lipids in LNPs with polyhydroxy polyamino acids to construct a novel delivery system. The abundant hydroxyl groups (-OH) introduced through the side chains fundamentally solve the key problems of cationic lipids while retaining the advantages of LNPs' efficient encapsulation and endosome escape framework: the hydrophilicity and electroneutrality of the hydroxyl groups significantly neutralize the positive charge density, greatly reducing cytotoxicity and inflammatory responses; the hydrogen bond network they form enhances the stability of the mRNA complex and its anti-protein adsorption capacity, potentially reducing liver accumulation and expanding extrahepatic targeting; under acidic conditions, the hydroxyl groups can synergistically promote endosome escape with protonated amino groups; the natural amino acid-based backbone endows it with excellent biodegradability, ensuring safe and controllable metabolites and avoiding the long-term safety risks of synthetic lipids from the source. Furthermore, the well-defined structure and simplified component design are expected to improve formulation stability and relax storage conditions. Polyhydroxy polyamino acids, as a revolutionary alternative to cationic lipids, provide a core technological breakthrough for developing safer, more efficient, and more broadly targeted next-generation mRNA delivery systems.

[0039] This application provides a polyhydroxy polyurethane having the structure of Formula 1:

[0040] ;

[0041] Where n is an integer from 1 to 100, which can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100.

[0042] In some specific implementations, n is between 5 and 12.

[0043] In some specific implementations, the number-average molecular weight of the polyhydroxy polyurethane is between 1,000 and 20,000.

[0044] This application also provides a method for preparing a polyhydroxy polyurethane, comprising:

[0045] The γ-allyl-L-glutamic acid carboxylic anhydride was polymerized and then dialyzed for the first time to obtain poly(γ-allyl-L-glutamic acid ester).

[0046] Poly(γ-allyl-L-glutamate), cysteine ​​hydrochloride and photoinitiator were mixed and irradiated with ultraviolet light, followed by a second dialysis to obtain side-chain-amino acid-based polyamino acids.

[0047] Amminated polyamino acids with side chains and glycidyl ethers are mixed and reacted to obtain polyhydroxy polyurethane.

[0048] This application first reacts L-glutamic acid, allyl alcohol, and trimethylchlorosilane to obtain γ-allyl-L-glutamic acid ester (ALG); then, it reacts γ-allyl-L-glutamic acid ester, ultra-dry tetrahydrofuran, and triphosgene at room temperature to obtain γ-allyl-L-glutamic acid carboxylic anhydride. In some specific implementations, the molar ratio of L-glutamic acid, allyl alcohol, and trimethylchlorosilane is 1:(8-10):(2-4), preferably 1:10:3. In some specific implementations, the reaction time is 20 to 30 hours, preferably 24 hours, and the reaction temperature is 20°C to 30°C, preferably 25°C. The resulting solution is concentrated by rotary evaporation and precipitated using a large amount of diethyl ether to obtain γ-allyl-L-glutamic acid ester. In some specific implementations, the molar ratio of triphosgene to ALG is 1.5:1. In some specific implementations, the room temperature reaction time is 2 to 6 hours, preferably 4 hours.

[0049] This application then proceeds to polymerize γ-allyl-L-glutamic acid carboxylic anhydride and performs a first dialysis to obtain poly(γ-allyl-L-glutamic acid ester). In some specific implementations, the polymerization reaction time is 40 to 50 hours, preferably 48 hours, and the polymerization reaction temperature is 20°C to 30°C, preferably 25°C; the molecular weight of the first dialysis is 500 Da to 10000 Da, and the first dialysis time is 48 to 96 hours.

[0050] This application then mixes poly(γ-allyl-L-glutamate), cysteine ​​hydrochloride, and a photoinitiator, irradiates them under ultraviolet light, and performs a second dialysis to obtain a side-chain-amino acid with amino groups. In some specific implementations, the molar ratio of poly(γ-allyl-L-glutamate), cysteine ​​hydrochloride, and the photoinitiator is 1:(4-6):(0.1-0.3), preferably 1:5:0.2; the ultraviolet irradiation time is 0.2 h to 1 h, preferably 0.5 h; the ultraviolet irradiation temperature is 20 °C to 30 °C, preferably 25 °C; the molecular weight of the second dialysis is 500 Da to 10000 Da; and the second dialysis time is 48 h to 96 h. The wavelength of the ultraviolet irradiation is 365 nm.

[0051] This application then reacts a mixture of side-chain-aminated polyamino acids and glycidyl ether to obtain a polyhydroxy polyurethane. In some specific implementations, the molar ratio of the side-chain-aminated polyamino acids to glycidyl ether is 1:(2-4), preferably 1:3; the aminated reaction time is 2 h to 6 h, preferably 4 h; and the aminated reaction temperature is 70 °C to 90 °C, preferably 80 °C. The aminated reaction is followed by a third dialysis, wherein the molecular weight of the third dialysis is 500 Da to 10000 Da, and the third dialysis time is 48 h to 96 h.

[0052] In some specific implementations, the preparation method of the polyhydroxy polyurethane includes: A) mixing L-glutamic acid and allyl alcohol in a flask and adding trimethylchlorosilane. The solution is milky white in the initial stage of the reaction until the reaction solution becomes clear, at which point the reaction is stopped;

[0053] B) The solution from step A) was concentrated by rotary evaporation and then precipitated using a large amount of diethyl ether to obtain γ-allyl-L-glutamate (ALG).

[0054] C) Place the ALG obtained in step B) in a flask, add ultra-dry tetrahydrofuran and triphosgene, and react at room temperature until the solid is completely dissolved, then stop the reaction.

[0055] D) Pour the solution from step C) into a large amount of n-hexane, let it stand, collect the yellow oily substance at the bottom, dissolve it with ethyl acetate, wash it with saturated sodium carbonate until the organic phase is clear, dry it with anhydrous sodium sulfate, filter it, and dry it under vacuum to obtain the product γ-allyl-L-glutamic acid carboxylic anhydride (ALG-NCA).

[0056] E) Dissolve the polymer obtained in step D) with ultra-dry DMF, and initiate polymerization with n-hexylamine;

[0057] F) After polymerization, the solution from step E) is directly placed into a dialysis bag, filtered and freeze-dried after a period of time to obtain poly(γ-allyl-L-glutamate) (PALG).

[0058] G) Dissolve the polymer obtained in step F) with DMF, add cysteine ​​hydrochloride and photoinitiator, bubble with nitrogen for a period of time, and irradiate the reaction solution with a 365nm ultraviolet lamp for 30 minutes.

[0059] H) Dialyze and freeze-dry the solution from step G) to obtain side-chain aminated polyamino acids (PM).

[0060] I) Dissolve the polymer obtained in step H) with DMSO, add glycidyl ether to react with the amino groups on the polymer side chains to obtain polyhydroxy polyamino acid (PM-OH).

[0061] This application also provides a composition comprising, by weight parts: 40 to 60 parts of polyhydroxy polyamino acids (which may be 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, or 60 parts, preferably 57.6 parts), 20 to 30 parts of cholesterol (which may be 20, 22, 24, 26, 28, or 30 parts, preferably 23.2 parts), 10 to 20 parts of auxiliary lipids (which may be 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 parts, preferably 12.8 parts), and 5 to 10 parts of DMG-PEG (which may be 5, 5.5, 6, 7, 8, 9, 9.5, or 10 parts, preferably 6.4 parts).

[0062] In some specific implementations, the auxiliary lipid includes one or more of 1,2-distearyl-sn-glycerol-3-phosphate choline, 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine, or 1,2-dioleoyl-sn-glycerol-3-phosphate choline.

[0063] This application also provides a method for delivering mRNA, comprising:

[0064] A composition was prepared by mixing polyhydroxy polyamino acids, cholesterol, auxiliary lipids, and DMG-PEG; mRNA was dissolved in an aqueous sodium citrate solution, and the composition was mixed with the aqueous sodium citrate solution of mRNA for intramuscular injection.

[0065] The present application is further illustrated below with reference to embodiments. The scope of protection of the present application is not limited to the following embodiments.

[0066] Example 1

[0067] This embodiment provides a polyhydroxy polyamino acid, the preparation method of which includes:

[0068] 5 g of γ-allyl-L-glutamate (ALG) was placed in a 500 mL flask, 120 mL of ultra-dry tetrahydrofuran was added, followed by 3.5 g of triphosgene. The reaction was carried out at room temperature until the solution became clear. The solution was poured into 1000 mL of cold n-hexane to settle, and the yellow oily residue at the bottom was collected. This residue was dissolved in ethyl acetate and washed with saturated sodium bicarbonate until the organic phase transition became clear. The residue was then dried over anhydrous sodium sulfate, filtered, and concentrated by rotary evaporation to obtain the monomer ALG-NCA. The monomer ALG-NCA was subjected to 1H NMR spectroscopy, and the results are shown below. Figure 1 As shown.

[0069] 1.0 g of monomer ALG-NCA was dissolved in 20 mL of ultra-dry DMF, and 23.6 mg of n-hexylamine was added to initiate polymerization. The reaction was continued at room temperature for 48 h. After the reaction, the product was dialyzed and lyophilized to obtain polyamino acid PALG. Further, 230 mg of PALG was dissolved in DMF, and 700 mg of cysteine ​​hydrochloride and 50 mg of photoinitiator were added. After bubbling under nitrogen for 30 min, the product was irradiated with a 365 nm UV lamp for 30 min. After the reaction, the product was dialyzed and lyophilized to obtain side-chain-aminated polyamino acid PM. The polyamino acid PM was analyzed by 1H NMR spectroscopy, and the results are shown below. Figure 2 As shown.

[0070] 200 mg of product PM was dissolved in DMSO, 100 mg of glycidyl ether was added, and the mixture was reacted at 80 °C for 4 hours. After dialyzing and lyophilization, the polyhydroxy polyamino acid PM-OH was obtained. The polyhydroxy polyamino acid PM-OH was analyzed by 1H NMR spectroscopy, and the results are as follows: Figure 3 As shown.

[0071] Example 2

[0072] This embodiment provides a composition, the preparation method of which includes:

[0073] The polymer PM-OH was dissolved in ethanol to obtain a 10 mg / mL solution, and then mixed with cholesterol, cofactor lipids, and DMG-PEG (all three in 10 mg / mL ethanol solution) to obtain a composition. The mixing mass ratio was 57.6:23.2:12.8:6.4. This embodiment also provides a method for mRNA delivery, comprising: dissolving mRNA in an aqueous sodium citrate solution, vortexing the composition with nucleic acids, and finally administering the solution via intramuscular injection.

[0074] Example 3

[0075] To obtain PALG with different molecular weights, monomers ALG-NCA (0.75 / 3.75 / 7.5 g) were dissolved in ultra-dry DMF, and 35.4 mg of n-hexylamine was added to initiate polymerization. The reaction was continued at room temperature for 48 h. After the reaction, the mixture was dialyzed and lyophilized to obtain polyamino acid PALG with degrees of polymerization of 10 / 50 / 100. PALG was further dissolved in DMF, and 700 mg of cysteine ​​hydrochloride and 50 mg of photoinitiator were added. After bubbling with nitrogen for 30 min, the mixture was irradiated with a 365 nm UV lamp for 30 min. After the reaction, the mixture was dialyzed and lyophilized to obtain polyamino acid PM with side-chain amination. The GPC elution curves of polyamino acid PM with different degrees of polymerization are shown below. Figure 5 As shown.

[0076] Comparative Example 1

[0077] This comparative example provides a composition using conventional SM102 as a cationic lipid, with the composition having an SM102:cholesterol:coenzyme:DMG-PEG ratio of 57.6:23.2:12.8:6.4. Compared to formulations using conventional SM102 as a cationic lipid, the mRNA transfection images of the compositions provided in Example 2 and Comparative Example 1 in mice are shown below. Figure 4 As shown in the diagram (left SM102, right PM-OH), it can be seen that replacing SM102 with PM-OH improves muscle transfection performance, and PM-OH exhibits better spleen targeting. The organ distribution map of PM-OH in vivo is shown below. Figure 6 As shown, the distribution map of SM102 in vivo is as follows: Figure 7 As shown in the figure. The transfection effect of PM-OH with different degrees of polymerization is illustrated in the figure. Figure 8 As shown, the left leg represents PM-OH with a degree of polymerization of 10, and the right leg represents PM-OH with a degree of polymerization of 100. Figure 4 The right leg contains PM-OH with a degree of polymerization of 50, indicating that a moderate degree of polymerization yields the best transfection effect.

[0078] The above description is merely a preferred embodiment of this application, but the scope of protection of this application is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in this application, based on the technical solution and application concept of this application, should be included within the scope of protection of this application.

Claims

1. A polyhydroxy polyurethane, characterized in that, has a structure of formula 1: ； wherein n is an integer from 1 to 100.

2. The polyhydroxy polyurethane of claim 1, wherein, n is 5 to 12.

3. A process for the preparation of a polyhydroxy polyurethane, characterized in that, comprising: polymerizing a gamma-allyl-L glutamic acid carboxylic anhydride, first dialysis to obtain poly (gamma-allyl-L glutamate) ; mixing the poly (gamma-allyl-L glutamate), cysteamine hydrochloride and a photoinitiator, ultraviolet irradiation, second dialysis to obtain a side chain aminated polyamino acid; mixing the side chain aminated polyamino acid and glycidol, amino reaction to obtain a polyhydroxy polyurethane.

4. The production method according to claim 3, characterized by, The polymerization time is 40 to 50 hours, and the polymerization temperature is 20 to 30 degrees Celsius; the first dialysis molecular weight is 500 to 10,000 Da, and the first dialysis time is 48 to 96 hours.

5. The production method according to claim 3, wherein The molar ratio of the poly (gamma-allyl-L glutamate), cysteamine hydrochloride and photoinitiator is 1: (4-6) : (0.1-0.3), the ultraviolet irradiation time is 0.2 to 1 hour, and the ultraviolet irradiation temperature is 20 to 30 degrees Celsius; the second dialysis molecular weight is 500 to 10,000 Da, and the second dialysis time is 48 to 96 hours.

6. The preparation method according to claim 3, characterized in that, The molar ratio of the side chain aminated polyamino acid and glycidol is 1: (2-4) ; the amino reaction time is 2 to 6 hours, and the amino reaction temperature is 70 to 90 degrees Celsius; The amino reaction further comprises third dialysis, the third dialysis molecular weight is 500 to 10,000 Da, and the third dialysis time is 48 to 96 hours.

7. The preparation method according to claim 3, characterized in that, The preparation method of the gamma-allyl-L glutamic acid carboxylic anhydride comprises: mixing L-glutamic acid, propenol and trimethylsilyl chloride to obtain gamma-allyl-L glutamate; mixing the gamma-allyl-L glutamate, ultradry tetrahydrofuran and triphosgene at room temperature to obtain gamma-allyl-L glutamic acid carboxylic anhydride.

8. A composition characterized in that, The composition comprises, in mass parts: 40 to 60 parts of polyhydroxy polyamino acid, 20 to 30 parts of cholesterol, 10 to 20 parts of auxiliary lipid, and 5 to 10 parts of DMG-PEG.

9. The composition of claim 8, wherein, The auxiliary lipid comprises one or more of 1,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine or 1,2-dioleoyl-sn-glycero-3-phosphocholine.

10. A method of delivery of mRNA, characterized in that, comprising: mixing the polyhydroxy polyamino acid, cholesterol, auxiliary lipid and DMG-PEG to obtain a composition; dissolving the mRNA in a sodium citrate aqueous solution, mixing the composition and the sodium citrate aqueous solution of the mRNA, and performing intramuscular injection.

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