Polymer with double pH response as well as preparation method and application thereof

Abstract

The invention provides a polymer with double pH response as well as a preparation method and application thereof. The polymer has a structure as shown in a formula I, the polymer can be well dispersed into nano particles with uniform particle sizes in a PBS buffer solution with the pH value of 7.4, and tertiary amine groups in the PBS buffer solution with the pH value of 5.0 can be protonized and can be hydrolyzed to release active pharmaceutical ingredients for in-vivo release under an acidic condition. Besides, according to the polymer with double pH response provided by the invention, a main chain segment is a polyethylene glycol-polycarbonate chain segment, polycarbonate can realize in-vivo degradation, and the existence of a polyethylene glycol (PEG) block also ensures low irritation of the polymer during in-vivo circulation, which is verified in a cytotoxicity experiment. Therefore, the polymer provided by the invention also has relatively high biological safety.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials, specifically relating to a polymer with dual pH response, its preparation method, and its applications. Background Technology

[0002] In many biological processes, various stimuli play a crucial role. These include temperature, specific enzymes, redox reactions, light, and magnetic fields. Biomaterials can utilize internal or external stimuli to treat pathological or injured sites. Currently developed materials that utilize internal or external stimuli include proton sponge polymers, cell-penetrating peptides, charge-reversible materials, and pore-forming materials.

[0003] Generally, pH is the most representative and commonly used internal stimulus in pathological sites, including endosome / lysosomal maturation, antigen processing, protein and lipid metabolism, and tumor pathophysiology. Researchers have developed various pH-sensitive polymers and nanoparticles for drug delivery and imaging applications in disease treatment. For many biological processes, pH changes are small compared to normal physiological pH (pH=7.4). The extracellular pH of tumors has been reported to be around 6.8, early lysosomal pH to 5-6, and late lysosomal pH to 4-5. However, the pH-sensitive systems of small molecules or oligomers are generally not very efficient at responding to these subtle pH differences. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a polymer with dual pH response, a method for preparing the polymer, and its application.

[0005] To achieve this objective, the present invention adopts the following technical solution:

[0006] In a first aspect, the present invention provides a polymer with dual pH response, having the structure of Formula I:

[0007] Formula I;

[0008] Where k = 20~230, k:q = (1~3):1, m:q = (0.2~0.8):1;

[0009] Q represents a residue of the active pharmaceutical ingredient after the loss of a hydroxyl group; the active pharmaceutical ingredient is a small molecule compound or a biological macromolecule fragment with physiological activity.

[0010] Preferably, the polymer, under acidic conditions, QO - It combines with a hydrogen atom to form Q-OH; wherein, Q-OH is the active pharmaceutical ingredient.

[0011] Preferably, under acidic conditions, the nitrogen-containing seven-membered ring in the polymer undergoes protonation by accepting a proton.

[0012] Preferably, the acidic condition is an aqueous solution environment with pH ≤ 6 or a physiologically acidic microenvironment.

[0013] Preferably, the structural formula of the active pharmaceutical ingredient is shown in formulas A-1 to A-6 below:

[0014] Formula A-1; Formula A-2; Formula A-3; Formula A-4; Formula A-5; Formula A-6.

[0015] Preferably, the polymer is selected from any one of the following formulas I-1 to I-6:

[0016] Formula I-1;

[0017] Formula I-2;

[0018] Formula I-3;

[0019] Formula I-4;

[0020] Formula I-5;

[0021] Formula I-6.

[0022] In a second aspect, the present invention provides a method for preparing the above-mentioned polymer with dual pH response, characterized by comprising the following steps:

[0023] S1: Provides the polyethylene glycol-polycarbonate segment shown in Formula 1;

[0024] S2: The polyethylene glycol-polycarbonate segment shown in Formula 1 is modified with a nitrogen-containing seven-membered ring to obtain the polymer shown in Formula 2. Then, the carboxyl group is modified to obtain the polymer shown in Formula 3. After the ethylene oxide group is modified, the polymer shown in Formula 4 is obtained. Finally, the polymer shown in Formula 4 is mixed with the active pharmaceutical ingredient and reacted to obtain the polymer with dual pH response shown in Formula I.

[0025] Formula 1; Formula 2; Formula 3; Formula 4.

[0026] Preferably, the method for nitrogen-containing seven-membered ring modification is as follows:

[0027] Protonated 2-(aza-1-yl)ethane-1-thiol was reacted with the polyethylene glycol-polycarbonate segment shown in Formula 1 under ultraviolet light in the presence of a photoinitiator, and deprotonated to obtain the polymer shown in Formula 2.

[0028] Preferably, the method for carboxyl modification is as follows:

[0029] The polymer shown in Formula 2 was reacted with a thiol-containing carboxyl compound under ultraviolet light in the presence of a photoinitiator to obtain the polymer shown in Formula 3.

[0030] Preferably, the method for ethylene oxide modification is as follows:

[0031] In an inert atmosphere, the polymer shown in Formula 3 and an ethylene-oxygenated alcohol are reacted in the presence of a catalyst and a carbodiimide condensing agent to obtain the polymer shown in Formula 4.

[0032] Preferably, the photoinitiator is selected from any one or more of benzoin dimethyl ether, 2-hydroxy-2-methylphenylacetone, 4'-aminophenylacetone, or benzophenone.

[0033] Preferably, the thiol-containing carboxyl compound is selected from mercaptopropionic acid.

[0034] Preferably, the ethylene-oxygenated alcohol is selected from 2-ethyleneoxyethanol.

[0035] Preferably, the catalyst is selected from any one or more of 4-dimethylaminopyridine, 4-pyrrolidinylpyridine, or 4-piperidinylpyridine.

[0036] Preferably, the carbodiimide condensing agent is selected from any one or more of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, dicyclohexylcarbodiimide, or diisopropylcarbodiimide.

[0037] Preferably, the molar ratio of the protonated 2-(aza-1-yl)ethane-1-thiol to the polyethylene glycol-polycarbonate segment shown in Formula 1 is (0.2q~0.8q):1.

[0038] Preferably, the molar ratio of the polymer shown in Formula 2 to the thiol-containing acidic substance is 1:(1~3)(qm).

[0039] Preferably, the molar ratio of the polymer shown in Formula 3 to the ethylene-oxygenated alcohol is 1:(1.5~3)(qm).

[0040] Preferably, the reaction between the polymer represented by Formula 4 and the active pharmaceutical ingredient is as follows:

[0041] In an inert atmosphere, the polymer and active pharmaceutical ingredient shown in Formula 4 undergo an electrophilic addition reaction in the presence of a strong acid catalyst.

[0042] Preferably, the strong acid catalyst is selected from p-toluenesulfonic acid.

[0043] Thirdly, the present invention provides the application of the above-mentioned dual-pH responsive polymer in drug delivery, vaccine adjuvants, or targeted therapy.

[0044] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0045] This invention provides a polymer with dual pH response, having the structure of Formula I. This polymer can be well dispersed into uniformly sized nanoparticles in a PBS buffer solution at pH 7.4. Under acidic conditions, such as in a PBS buffer solution at pH 5.0, the tertiary amine groups can be protonated and hydrolyzed to release the active pharmaceutical ingredient for in vivo release.

[0046] Furthermore, the polymer with dual pH response provided by this invention has a main chain segment of polyethylene glycol-polycarbonate, wherein the polycarbonate can be degraded in vivo, and the presence of the polyethylene glycol (PEG) block ensures low irritation during in vivo circulation, which has been verified in cytotoxicity experiments. Therefore, the polymer provided by this invention also has high biocompatibility. Attached Figure Description

[0047] Figure 1 This is a schematic diagram of the synthetic route for the polymer with dual pH response of the present invention;

[0048] Figure 2 The 1H NMR spectrum of 2-(aza-1-yl)ethane-1-thiol;

[0049] Figure 3 The hydrogen NMR spectrum of Ph-DA;

[0050] Figure 4 PEG 5000 -PAGEC's hydrogen NMR spectrum;

[0051] Figure 5 PEG 5000 -H NMR spectrum of PC7A-PAGEC;

[0052] Figure 6 PEG 5000 -1H NMR spectrum of the product after carboxyl modification with PC7A-PAGEC;

[0053] Figure 7 PEG 5000After modification with the carboxyl group of PC7A-PAGEC, the product was further modified with the ethylene oxy group to obtain the 1H NMR spectrum.

[0054] Figure 8 PEG 5000 -H NMR spectrum of PC7A-P (Ph-DA);

[0055] Figure 9 PEG 5000 - PC7A-P (Ph-DA) was assembled with rabies protein, and the titer of neutralizing antibodies in mice was obtained by injection (compared with conventional aluminum adjuvant and rabies protein assembly and unadjuvanted rabies protein assembly).

[0056] Figure 10 PEG 5000 Physiological indicators and blood routine evaluation results of mice after PC7A-P (Ph-DA) injection (compared with mice injected with conventional aluminum oleanol and PBS). Detailed Implementation

[0057] The technical solution of the present invention will be clearly and completely described below with reference to 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 of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0058] To better understand the technical solution of this invention, it is described in detail in the following parts.

[0059] Part One

[0060] This invention provides a polymer with dual pH response, having the structure of Formula I:

[0061] Formula I;

[0062] Where k = 20~230, such as 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, or 230, etc. It should be noted that k refers to the number of repeating units of the monomer in the polyethylene glycol segment, which is directly related to the molecular weight of polyethylene glycol. For example, when k = 23, the corresponding molecular weight of polyethylene glycol is 1000; when k = 113, the corresponding molecular weight of polyethylene glycol is 5000; when k = 227, the corresponding molecular weight of polyethylene glycol is 10000.

[0063] In this invention, the ratio of k:q = (1~3):1 can be 1:1, 1:2 or 1:3, etc. The ratio refers to the ratio of repeating units, which is known to those skilled in the art and can also be regarded as the molar ratio.

[0064] In this invention, m:q = (0.2~0.8):1, such as 0.2:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1, or 0.8:1, etc. Here, m refers to the number of repeating units in PC7A, and qm refers to the number of repeating units in PQ. The ratio between the two refers to the ratio of repeating units in PC7A to PQ, which is well known to those skilled in the art and can also be considered as a molar ratio.

[0065] In this invention, Q in the polymer structure represents a pharmaceutically active component that has lost a hydroxyl residue; the pharmaceutically active component is a small molecule compound or a biomolecular fragment with physiological activity. In this invention, under acidic conditions (an aqueous solution with pH ≤ 6 or a physiologically acidic microenvironment), QO... - It combines with a hydrogen atom to form Q-OH; where Q-OH is the active pharmaceutical ingredient.

[0066] In some embodiments of the present invention, the structural formula of the pharmaceutically active component is shown in formulas A-1 to A-6 as follows:

[0067] Formula A-1; Formula A-2; Formula A-3; Formula A-4; Formula A-5; Formula A-6.

[0068] Specifically, in some embodiments of the present invention, the polymer is selected from any one of the following formulas I-1 to I-6:

[0069] Formula I-1;

[0070] Formula I-2;

[0071] Formula I-3;

[0072] Formula I-4;

[0073] Formula I-5;

[0074] Formula I-6.

[0075] The polymer of Formula I provided by this invention uses polyethylene glycol and polycarbonate as the main chain segments, and introduces a nitrogen-containing seven-membered ring and residue Q of the pharmaceutically active component into the side chain of the polycarbonate. Specifically, under acidic conditions, the nitrogen-containing seven-membered ring can accept a proton and undergo protonation. In lysosomes, protonation can promote the release of more Cl... - Entering the lysosome increases the ion concentration within it. To balance osmotic pressure, more water enters the lysosome, facilitating lysosomal escape and drug release. Simultaneously, QO... - It can be released and combined with a hydrogen atom to transform into Q-OH (the Q-OH being the active pharmaceutical ingredient). Therefore, the polymer of Formula I provided by the present invention has a dual pH response; under acidic conditions, both the nitrogen-containing seven-membered ring and residue Q can respond, which is beneficial for drug delivery and immune response.

[0076] In this invention, the acidic conditions refer to an aqueous solution environment with pH ≤ 6 or a physiologically acidic microenvironment.

[0077] Part Two

[0078] The present invention also provides a method for preparing the above-mentioned polymer with dual pH response, comprising the following steps:

[0079] S1: Provides the polyethylene glycol-polycarbonate segment shown in Formula 1;

[0080] S2: The polyethylene glycol-polycarbonate segment shown in Formula 1 is modified with a nitrogen-containing seven-membered ring to obtain the polymer shown in Formula 2. Then, the carboxyl group is modified to obtain the polymer shown in Formula 3. After the ethylene oxide group is modified, the polymer shown in Formula 4 is obtained. Finally, the polymer shown in Formula 4 is mixed with the active pharmaceutical ingredient and reacted to obtain the polymer with dual pH response shown in Formula I.

[0081] Formula 1; Formula 2; Formula 3; Formula 4.

[0082] According to the present invention, a polyethylene glycol-polycarbonate segment of Formula 1 is first provided.

[0083] In this invention, the polyethylene glycol-polycarbonate segments can be prepared using methods well known to those skilled in the art.

[0084] For example, polyethylene glycol-polycarbonate segments are used as PEG. 5000 Taking the PAGEC chain segment as an example, PEG is synthesized. 5000 The methods for the -PAGEC chain segment are as follows:

[0085] (1) The catalysts trifluoroacetic acid [N,N'-bis(salicyl)ethylenediaminecobalt]Salen-Co-TFA (0.0001~0.1 eq, structure as shown in formula B) and co-catalyst trifluoroacetic acid [bis(triphenylphosphine)ammonium]PPN-TFA (0.0001~0.1 eq, structure as shown in formula C) were added to the reactor, and then the chain transfer agent polyethylene glycol monomethyl ether-5000 (PEG) was added sequentially. 5000 0.001~1 eq) and monomer allyl glycidyl ether (AGE, 1 eq), and a small amount of solvent, preferably dichloromethane (DCM), is added to dissolve the reactants;

[0086] (2) Charge with 2~5 MPa, preferably 3 MPa CO2 and react at room temperature for 2~3 days;

[0087] (3) The polymer is dissolved in a solvent, preferably DCM, and then precipitated in diethyl ether to remove byproducts and catalyst. After vacuum drying, PEG is obtained. 5000 -PAGEC.

[0088] Formula B; Formula C.

[0089] After obtaining the polyethylene glycol-polycarbonate segment shown in Formula 1, it is modified with a nitrogen-containing seven-membered ring according to the present invention.

[0090] In this invention, the method for modifying the nitrogen-containing seven-membered ring is as follows:

[0091] Protonated 2-(aza-1-yl)ethane-1-thiol was reacted with the polyethylene glycol-polycarbonate segment (PEG-PAGEC) shown in Formula 1 under ultraviolet light in the presence of a photoinitiator, and deprotonated to obtain the polymer shown in Formula 2.

[0092] 2-(aza-1-yl)ethane-1-thiol can be purchased directly as a finished product or prepared in-house.

[0093] In some embodiments of the present invention, cyclothioethane and cyclohexylimine are preferably dissolved separately in a solvent, preferably DCM, and then sequentially transferred to a flask. The mixture is stirred at room temperature for 1-3 h, and excess solvent and unreacted reactants are removed under reduced pressure to obtain 2-(aza-1-yl)ethane-1-thiol. The reaction formula is as follows:

[0094] .

[0095] In some embodiments of the present invention, it is preferred to protonate 2-(aza-1-yl)ethane-1-thiol by reacting it with hydrogen chloride; then, the protonated 2-(aza-1-yl)ethane-1-thiol ((0.2q~0.8q)eq) and the polyethylene glycol-polycarbonate segment (1 eq) shown in Formula 1 are added sequentially to a reaction flask, and a photoinitiator ((0.0001q~0.8q)eq) benzoin dimethyl ether (DMPA) is added. The reaction is carried out under ultraviolet light for 1~5 h, preferably 2~4 h; finally, the polymer is dissolved in a solvent, preferably DCM, and precipitated in ether to remove byproducts; the product is dissolved in PBS solution at pH=9, stirred for 2~8 h, preferably 4~6 h to remove protonation, dialyzed, and vacuum dried to obtain the polymer shown in Formula 2, abbreviated as: PEG-PC7A-PAGEC.

[0096] The molar ratio of the protonated 2-(aza-1-yl)ethane-1-thiol to the polyethylene glycol-polycarbonate segment shown in Formula 1 is (0.2q~0.8q):1; the reaction temperature is room temperature.

[0097] Then, according to the present invention, carboxyl modification is performed.

[0098] In this invention, the method for carboxyl group modification is as follows:

[0099] The polymer shown in Formula 2 is reacted with an acidic substance containing thiol groups under ultraviolet light in the presence of a photoinitiator to obtain the polymer shown in Formula 3.

[0100] Specifically, the polymer (1 eq) shown in Formula 2 and a thiol-containing carboxyl compound (such as mercaptopropionic acid, ((1~3)(qm) eq, where (1~3) and (qm) are multiplicative) are sequentially added to a reaction flask, and the photoinitiator benzoin dimethyl ether (DMPA, (0.0001~0.3)(qm) eq) is added. The reaction is carried out under ultraviolet light for 1~3 h, preferably 1.5~2 h. Finally, the polymer is dissolved with DCM, precipitated in diethyl ether to remove byproducts, and vacuum dried to obtain the polymer shown in Formula 3, abbreviated as: PEG-PC7A-P1.

[0101] Wherein, the molar ratio of the polymer shown in Formula 2 to the acidic substance containing thiol groups is 1:(1~3)(qm); the reaction temperature is room temperature.

[0102] Then, according to the present invention, ethylene oxide modification is performed.

[0103] In this invention, the method for ethylene oxide modification is as follows:

[0104] In an inert atmosphere, the polymer shown in Formula 3 and an alcohol containing ethylene oxide are reacted in the presence of a catalyst and a carboxyl activator to obtain the polymer shown in Formula 4.

[0105] Specifically, the polymer (1 eq) shown in Formula 3 is preferably added sequentially to a flask along with the catalyst 4-dimethylaminopyridine (DMAP, (0.1~1)(qm)eq) and an ethylene-oxygenated alcohol (such as 2-ethyleneoxyethanol, (1.5~3)(qm)eq), and a solvent, preferably DCM, is added to completely dissolve the polymer. The reaction is carried out under N2 protection for 10~60 min, preferably 30 min. Then, the carboxyl activator 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, (1.5~3)(qm)eq) is dissolved in a solvent, preferably DCM, and slowly added to the above reaction flask. The reaction is carried out for 10~24 h, preferably 15~20 h. After removing the solvent by rotary evaporation, the obtained product is transferred to a dialysis bag and dialyzed with deionized water for a period of time to remove unreacted monomers. The solution in the dialysis bag is collected, centrifuged, and freeze-dried to finally obtain the polymer shown in Formula 4, abbreviated as PEG-PC7A-P2.

[0106] The above dialysis procedure is as follows: the dialysis time is 48 hours. For the first 8 hours, the deionized water is changed every 2 hours. For the middle 16 hours, the deionized water is changed every 4 hours. For the last 24 hours, the deionized water is changed every 12 hours.

[0107] Wherein, the molar ratio of the polymer shown in Formula 3 to the ethylene-oxygenated alcohol is 1:(1.5~3) (qm); the reaction temperature is room temperature.

[0108] It should be noted that the photoinitiator involved in the above method is selected from any one or more of benzoin dimethyl ether, 2-hydroxy-2-methylphenylacetone, 4'-aminophenylacetone, or benzophenone; the thiol-containing acidic substance is selected from any one or more of mercaptoacetic acid, mercaptopropionic acid, or mercaptobutyric acid; the ethyleneoxy alcohol is selected from any one or more of 2-ethyleneoxyethanol, 3-ethyleneoxy-1-propanol, or 1-ethyleneoxy-2-propanol; the catalyst is selected from any one or more of 4-dimethylaminopyridine, 4-pyrrolidinylpyridine, or 4-piperidinylpyridine; and the carbodiimide condensing agent is selected from any one or more of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, dicyclohexylcarbodiimide, or diisopropylcarbodiimide.

[0109] Finally, according to the present invention, the polymer shown in Formula 4 is reacted with the active pharmaceutical ingredient.

[0110] In some embodiments of the present invention, the active pharmaceutical ingredient, Ph-DA, is first prepared. Specifically, this includes the following steps:

[0111] Preferredly, dopamine hydrochloride (1 eq) was dissolved in methanol (MeOH) and added to a reaction flask, followed by the addition of triethylamine (1 eq). Benzoyl chloride (1 eq) dissolved in tetrahydrofuran (THF) and triethylamine (1 eq) dissolved in MeOH were added dropwise to the reaction flask alternately. The reaction was carried out at -20°C for 2 h, diluted with distilled water, and quenched with concentrated hydrochloric acid. The mixture was extracted with ethyl acetate, concentrated by rotary evaporation, and a white solid precipitated, namely Ph-DA.

[0112] The parameters involved in the preparation of the active pharmaceutical ingredient can be adjusted by those skilled in the art according to actual needs, and will not be elaborated here.

[0113] In some embodiments of the present invention, the polymer shown in Formula 4 is preferably added to a reaction flask under an inert atmosphere, preferably N2 protection, and dissolved in a solvent, preferably DMSO. Then, at 0°C, Ph-DA and p-toluenesulfonic acid (PTSA) dissolved in a solvent, preferably DMSO, are slowly added to the above reaction flask; the reaction flask is moved to room temperature and the reaction is allowed to proceed for 10–24 h, preferably 15–20 h. After the reaction is complete, a saturated Na2CO3 solution is added to remove excess p-toluenesulfonic acid; the obtained product is then transferred to a dialysis bag and dialyzed with deionized water for a period of time to remove unreacted monomers and impurity ions such as Na2CO3; the solution in the dialysis bag is collected, centrifuged, and freeze-dried to obtain the final product.

[0114] The above dialysis procedure is as follows: the dialysis time is 24 hours, with the deionized water changed every 2 hours for the first 8 hours and every 4 hours for the next 16 hours.

[0115] In summary, the synthetic route for the polymers exhibiting dual pH responses can be represented as follows:

[0116]

[0117] The preparation method of the above-mentioned dual pH responsive polymer provided by the present invention is convenient, easy to implement, and conducive to industrial production.

[0118] Part Three

[0119] The present invention also provides the application of the above-mentioned dual pH responsive polymer in drug delivery, vaccine adjuvants, and targeted therapy.

[0120] The present invention provides a polymer with dual pH response, enabling intelligent lysosomal escape. Generally, a significant characteristic of the lysosomal pathway is its pH value being lower than the normal physiological pH of 7.4. Utilizing this characteristic, the present invention designs the aforementioned polymer with dual pH response, which remains stable at the physiological pH of 7.4, but undergoes a rapid change in physicochemical properties in an acidic environment, thus achieving intelligent lysosomal escape.

[0121] The polymer provided by this invention contains a nitrogen-containing seven-membered ring that can induce a proton sponge effect. The tertiary amine group can be protonated under suitable acidic pH conditions, causing the lysosome to absorb water and burst, precisely releasing the antigen into the cytoplasm. Furthermore, the acetal bond is acid-labile and can break under acidic conditions, leading to structural disintegration and rapid release of the attached drug-active component residues, forming the drug-active component.

[0122] This invention can assemble antigens and pH-responsive materials into nanovaccines. After endocytosis, the nanovaccines undergo rapid structural changes in acidic lysosomes, disrupting the membrane structure and enabling lysosomal escape. This allows for the efficient delivery of antigens and active drug components to the cytoplasm, greatly promoting the presentation of MHC class I antigens and the activation of cellular immunity.

[0123] To further illustrate the present invention, the following embodiments provide a detailed description. The experimental materials used in the following embodiments of the present invention are all commercially available products.

[0124] Preparation Example 1

[0125] This preparation example provides a C7ASH, and the preparation method is as follows:

[0126] Cyclothione (2 eq) and cyclohexylimine (1 eq) were dissolved separately in DCM and transferred sequentially to flasks. The mixtures were stirred at room temperature for 3 h, and excess solvent and unreacted raw materials were removed under reduced pressure.

[0127] The reaction formula is as follows, and the C7ASH 1H NMR spectrum is as follows. Figure 2 .

[0128] .

[0129] Preparation Example 2

[0130] This preparation example provides a Ph-DA, and the preparation method is as follows:

[0131] Dopamine hydrochloride (1 eq) was dissolved in methanol (MeOH) and added to a reaction flask. Triethylamine (1 eq) was then added to the flask. Benzoyl chloride (1 eq) was dissolved in tetrahydrofuran (THF) and triethylamine (1 eq) was dissolved in MeOH and added to the reaction flask dropwise alternately. The reaction was carried out at -20℃ for 2 h. The reaction was quenched with diluted concentrated hydrochloric acid. The mixture was extracted with ethyl acetate and concentrated by rotary evaporation. The white solid precipitated was Ph-DA.

[0132] The reaction formula is as follows, and the Ph-DA hydrogen NMR spectrum is as follows. Figure 3 .

[0133] .

[0134] Example 1

[0135] This embodiment provides a preparation method for dual pH-responsive polymerization, and its preparation flow chart is shown below. Figure 1 As shown, the specific experimental steps include:

[0136] Step 1: Synthesize PEG 5000 -PAGEC chain segment;

[0137] Step 2: The product obtained in Step 1 is modified with a nitrogen-containing seven-membered ring to obtain PEG. 5000 -PC7A-PAGEC;

[0138] Step 3: Modify the polymer obtained in Step 2 with carboxyl groups;

[0139] Step 4: Modify the polymer obtained in Step 3 with ethylene oxide groups;

[0140] Step 5: Use the polymer obtained in Step 4 to synthesize the final product, PEG. 5000 -PC7A-P (Ph-DA);

[0141] Among them, the above step one synthesizes PEG. 5000 The PAGEC chain segment, the specific steps are as follows:

[0142] Step 11: Add the catalyst Salen-Co-TFA (0.002 eq, structure as shown in Formula I) and the co-catalyst PPN-TFA (0.002 eq, structure as shown in Formula II) to the reactor, and then add the chain transfer agent polyethylene glycol monomethyl ether-5000 (PEG) in sequence. 5000 ,0.016 eq) and monomer allyl glycidyl ether (AGE, 1 eq), a small amount of dichloromethane (DCM) was added to dissolve the reactants;

[0143] Step 12: Charge with 3 MPa CO2 and react at room temperature for two days;

[0144] Step 13: Dissolve the polymer in DCM, precipitate it in diethyl ether to remove byproducts and catalyst, and vacuum dry to obtain PEG. 5000 -PAGEC, using 1 NMR was performed using 1H-NMR, and the 1H NMR spectrum is shown below. Figure 4 As shown.

[0145] Formula I Formula II.

[0146] In step two, PEG is synthesized. 5000 The PC7A-PAGEC method is as follows:

[0147] Step 21: Protonation is carried out by reacting 2-(aza-1-yl)ethane-1-thiol (1 eq) with hydrogen chloride (1 eq);

[0148] Step 22: Protonated 2-(aza-1-yl)ethane-1-thiol (20 eq) is reacted with the PEG obtained in Step 1. 5000 -PAGEC (1 eq) was added sequentially to the reaction flask, followed by benzoin dimethyl ether (DMPA) (2 eq), and the reaction was carried out under UV light at room temperature for 4 h.

[0149] Step 23: Dissolve the polymer in DCM, precipitate it in diethyl ether to remove byproducts, dissolve the product in PBS solution at pH 9, stir for 4 h to remove protonation, dialyze, and then vacuum dry to obtain PEG. 5000 -PC7A-PAGEC, using 1 NMR was performed using 1H-NMR, and the 1H NMR spectrum is shown below. Figure 5 As shown.

[0150] In step three, the carboxyl group modification method is as follows:

[0151] Step 31: Take the PEG obtained in Step 2 5000 -PC7A-PAGEC (1 eq) and mercaptopropionic acid (60 eq) were added sequentially to a reaction flask, followed by benzoin dimethyl ether (DMPA) (6 eq), and the mixture was reacted at room temperature under UV light for 2 h.

[0152] Step 32: Dissolve the polymer with DCM, precipitate it in diethyl ether to remove byproducts, and vacuum dry to obtain a polymer with carboxyl groups. 1 NMR was performed using 1H-NMR, and the 1H NMR spectrum is shown below. Figure 6 As shown.

[0153] In step four, the ethylene oxide modification method is as follows:

[0154] Step 41: Add the product obtained in step 3 (1 eq) to a flask along with 4-dimethylaminopyridine (DMAP) (6 eq) and 2-ethyleneoxyethanol (60 eq), add DCM to dissolve it completely, and react at room temperature for 30 min under N2 protection.

[0155] Step 42: Dissolve 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) (60 eq) in DCM and slowly add it to the reaction flask in step 42. React at room temperature for 20 h.

[0156] Step 43: Remove DCM by rotary evaporation;

[0157] Step 44: Transfer the product obtained in Step 43 to a dialysis bag and dialyze with deionized water for a period of time to remove unreacted monomers. The dialysis time in this step is 48 hours. For the first 8 hours, change the deionized water every 2 hours; for the middle 16 hours, change the deionized water every 4 hours; and for the last 24 hours, change the deionized water every 12 hours.

[0158] Step 45: Collect the solution from the dialysis bag, centrifuge and freeze-dry the solution to obtain the ethylene-oxygenated polymer. 1 NMR was performed using 1H-NMR, and the 1H NMR spectrum is shown below. Figure 7 As shown.

[0159] In step five, PEG 5000 The synthesis method of -PC7A-P (Ph-DA) is as follows:

[0160] Step 51: Add the product (1 eq) obtained in step 4 to a reaction flask under N2 protection, and dissolve it in DMSO;

[0161] Step 52: At 0°C, dissolve Ph-DA (40 eq) and p-toluenesulfonic acid (PTSA) (4 eq) in DMSO and slowly add them to the reaction flask from Step 51.

[0162] Step 53: Move the reaction flask from Step 51 to room temperature and react for 20 h;

[0163] Step 54: Add excess saturated Na2CO3 solution to remove excess p-toluenesulfonic acid;

[0164] Step 55: Transfer the product obtained in step 54 to a dialysis bag and dialyze it with deionized water for a period of time to remove unreacted monomers and impurity ions such as Na2CO3.

[0165] Step 56: Collect the solution from the dialysis bag, centrifuge and freeze-dry the solution to obtain the final product. 1 NMR was performed using 1H-NMR, and the 1H NMR spectrum is shown below. Figure 8 As shown;

[0166] In step 55, the dialysis time is 24 hours. For the first 8 hours, the deionized water is changed every 2 hours, and for the next 16 hours, the deionized water is changed every 4 hours.

[0167] Characterization and performance testing

[0168] I. Analysis of polymer molecular weight and distribution using gel permeation chromatography (GPC)

[0169] This invention uses GPC to detect the number-average molecular weight (Mn) and molecular weight distribution (Mw / Mn) of polymers. Six mg of the polymer to be tested was prepared into a 3 mg / ml solution using DCM. The column was calibrated with a monodisperse polystyrene standard. Chromatographically pure DCM was used as the mobile phase, the flow rate was 1.0 ml / min, and the temperature was 30℃. The results of the polymer molecular weight and distribution at different stages are shown in Table 1.

[0170] Table 1

[0171] Polymer (mg) Mn Mw PDI <![CDATA[PEG 5000 -PAGEC]]> 13299 18220 1.37 <![CDATA[PEG 5000 -PC7A-PAGEC]]> 17072 28510 1.67 <![CDATA[PEG 5000 -PC7A-P(Ph-DA)]]> 29994 39598 1.32

[0172] II. Analysis of polymer PEG using dynamic light scattering (DLS) 5000 -PC7A-PAGEC nanoparticle size dispersed in water

[0173] Take the polymer to be tested, PEG 5000 PC7A-PAGEC was dispersed in PBS buffer at pH 7.4 and pH 5.0 to prepare 0.1 mg / ml dispersions for testing at 4℃. The results are shown in Table 2.

[0174] Table 2

[0175]

[0176] As can be seen from Table 2, the size of nanoparticles decreases and hydrophilicity increases under acidic conditions, proving that tertiary amino groups in the polymer can be protonated under simulated lysosome pH=5.0 conditions.

[0177] III. Analysis of polymer PEG using dynamic light scattering (DLS) 5000 -PC7A-P (Ph-DA) nanoparticle size dispersed in water

[0178] Take the polymer to be tested, PEG 5000-PC7A-P (Ph-DA) was dispersed in PBS buffer at pH 5.0 to prepare a dispersion of 0.1 mg / ml. After standing for 48 h, the dispersion was tested at 4℃. Two peaks were observed in the nanoparticle size after standing: 108.9 nm and 199.8 nm. The 199.8 nm size is similar to the particle size of Ph-DA after self-assembly in water, confirming that PEG... 5000 The acetal bond in -PC7A-P (Ph-DA) can be hydrolyzed to release Ph-DA under simulated lysosomal pH=5.0 conditions.

[0179] IV. Neutralizing Antibody Titration

[0180] The purified RABV G protein was mixed with PEG. 5000 -PC7A-P (Ph-DA) was mixed in PBS buffer at pH 7.4, with an injection volume of 200 μL per mouse (containing PEG). 5000 Mice were immunized three times via intramuscular injection of PC7A-P (Ph-DA) 200 μg and RABV G 1 μg. The control group received Alum, with RABV G protein dissolved in pH 7.4 PBS buffer, and each mouse was injected with 200 μL of Alum (20 μg Alum, 1 μg RABV G). The blank control group received 200 μL of RABV G protein dissolved in pH 7.4 PBS buffer, with each mouse injected with 1 μg RABV G.

[0181] Injections were administered at 0, 2, and 4 weeks. Blood was collected via the infraorbital venous plexus at 5 and 6 weeks. Serum was separated and neutralizing antibody levels were measured.

[0182] Neutralizing antibody titration results are as follows Figure 9 As shown, PEG 5000 RABV neutralizing antibodies can be detected in the serum of mice immunized with PC7A-P (Ph-DA). The average neutralizing antibody titer is higher than the international standard of 0.5 IU / ml, which can produce 100% protection.

[0183] V. Safety Evaluation

[0184] PEG 5000 -PC7A-P (Ph-DA) was dispersed in PBS buffer at pH 7.4 at a concentration of 200 mg / mL and administered to mice via intramuscular injection three times, with each injection being 200 μL.

[0185] Mice were injected intramuscularly three times with PBS buffer at pH 7.4, with each injection consisting of 200 μL.

[0186] Commercial aluminum adjuvant (Alum) was dispersed in PBS buffer at pH 7.4 at a concentration of 20 mg / mL and immunized mice via intramuscular injection three times, with each injection being 200 μL.

[0187] Physiological indicators and blood routine tests were performed on the three groups of mice. The levels of ATS, ALT, BUN, TP, WBC, RBC, HGB, and PLT in their blood were measured. The results are as follows: Figure 10 As shown. Compared with aluminum adjuvant, 10 times PEG 5000 Mice immunized with -PC7A-P (Ph-DA) showed almost no liver or kidney damage. The results of ATS, ALT, and BUN were similar to those of mice that received only three intramuscular injections of PBS buffer. The increase in white blood cell (WBC) and platelet (PLT) counts was significantly lower than that induced by aluminum adjuvant, indicating a milder inflammatory response and good biosafety.

[0188] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A polymer with dual pH response, characterized in that, It has the structure of Formula I: Formula I; Where k = 20~230, k:q = (1~3):1, m:q = (0.2~0.8):1; Q represents a residue of the active pharmaceutical ingredient after the loss of a hydroxyl group; the active pharmaceutical ingredient is a small molecule compound or a biological macromolecule fragment with physiological activity.

2. The polymer with dual pH response according to claim 1, characterized in that, The polymer, under acidic conditions, QO - It combines with a hydrogen atom to form Q-OH; wherein, Q-OH is the active pharmaceutical ingredient; Under acidic conditions, the nitrogen-containing seven-membered ring in the polymer undergoes protonation by accepting a proton. The acidic conditions are an aqueous solution environment with pH ≤ 6 or a physiologically acidic microenvironment.

3. The polymer with dual pH response according to claim 1 or 2, characterized in that, The structural formulas of the active pharmaceutical ingredient are shown in formulas A-1 to A-6 below: Formula A-1; Formula A-2; Formula A-3; Formula A-4; Formula A-5; Formula A-6.

4. The polymer with dual pH response according to any one of claims 1 to 3, characterized in that, The polymer is selected from any one of the following formulas I-1 to I-6: Formula I-1; Formula I-2; Formula I-3; Formula I-4; Formula I-5; Formula I-6.

5. A method for preparing a polymer with dual pH response as described in any one of claims 1 to 4, characterized in that, Includes the following steps: S1: Provides the polyethylene glycol-polycarbonate segment shown in Formula 1; S2: The polyethylene glycol-polycarbonate segment shown in Formula 1 is modified with a nitrogen-containing seven-membered ring to obtain the polymer shown in Formula 2. Then, the carboxyl group is modified to obtain the polymer shown in Formula 3. After the ethylene oxide group is modified, the polymer shown in Formula 4 is obtained. Finally, the polymer shown in Formula 4 is mixed with the active pharmaceutical ingredient and reacted to obtain the polymer with dual pH response shown in Formula I. Formula 1; Formula 2; Formula 3; Formula 4.

6. The preparation method according to claim 5, characterized in that, The method for nitrogen-containing seven-membered ring modification is as follows: Protonated 2-(aza-1-yl)ethane-1-thiol and the polyethylene glycol-polycarbonate segment shown in Formula 1 were reacted under ultraviolet light in the presence of a photoinitiator, and deprotonated to obtain the polymer shown in Formula 2. The method for carboxyl group modification is as follows: The polymer shown in Formula 2 was reacted with a carboxyl compound containing a thiol group under ultraviolet light in the presence of a photoinitiator to obtain the polymer shown in Formula 3. The method for ethylene oxide modification is as follows: In an inert atmosphere, the polymer shown in Formula 3 and an ethylene-oxygenated alcohol are reacted in the presence of a catalyst and a carbodiimide condensing agent to obtain the polymer shown in Formula 4.

7. The preparation method according to claim 5 or 6, characterized in that, The photoinitiator is selected from any one or more of benzoin dimethyl ether, 2-hydroxy-2-methylphenylacetone, 4'-aminophenylacetone or benzophenone; The thiol-containing carboxyl compound is selected from mercaptopropionic acid; The alcohols containing ethylene oxide are selected from 2-ethyleneoxyethanol; The catalyst is selected from any one or more of 4-dimethylaminopyridine, 4-pyrrolidinylpyridine or 4-piperidinylpyridine; The carbodiimide condensing agent is selected from any one or more of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, dicyclohexylcarbodiimide, or diisopropylcarbodiimide.

8. The preparation method according to claim 6 or 7, characterized in that, The molar ratio of the protonated 2-(aza-1-yl)ethane-1-thiol to the polyethylene glycol-polycarbonate segment shown in Formula 1 is (0.2q~0.8q):1; The molar ratio of the polymer shown in Formula 2 to the thiol-containing acidic substance is 1:(1~3) (qm). The molar ratio of the polymer shown in Formula 3 to the ethylene-oxygenated alcohol is 1:(1.5~3) (qm).

9. The preparation method according to any one of claims 5 to 8, characterized in that, The reaction between the polymer shown in Formula 4 and the active pharmaceutical ingredient is as follows: In an inert atmosphere, the polymer and active pharmaceutical ingredient shown in Formula 4 undergo an electrophilic addition reaction in the presence of a strong acid catalyst.

10. The use of the dual-pH responsive polymer according to any one of claims 1 to 4, or the dual-pH responsive polymer prepared by the preparation method according to any one of claims 5 to 9, in drug delivery, vaccine adjuvants, or targeted therapy.

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