A terminal-modified cationic polyester, a living body delivery system and its application

Abstract

The present application discloses a terminal-modified cationic polyester, a live delivery system and application. The cationic polyester of the present application is a hyperbranched polymer formed by polymerization of three monomers, monomer P, monomer S and monomer M, or two monomers, monomer P and monomer S, with monomer T, and the terminal carboxyl group of the hyperbranched polymer is modified with polyethylene glycol; monomer P is a cyclic lactone; monomer S is an organic acid with two or more carboxyl groups at the terminal; monomer M is a compound containing two hydroxyl groups and one secondary amine or tertiary amine; monomer T is a compound containing three or more hydroxyl groups; the number average molecular weight of polyethylene glycol is 0.4 to 20k. The cationic polyester of the present application, when the terminal carboxyl group of the hyperbranched polymer is modified with polyethylene glycol, can improve the stability of the complex when it is delivered in vivo together with the cationic polyester modified with the terminal group E, thereby significantly improving the efficiency of live delivery. In addition, the preparation cost of the cationic polyester of the present application is low and environmentally friendly and pollution-free.

Description

Technical Field

[0001] The present application relates to the technical field of nucleic acid delivery materials, and in particular to a terminal-modified cationic polyester, a living body delivery system and applications. Background Art

[0002] Compared with other technologies such as DNA or viral vectors, mRNA technology has the advantages of high efficiency, high safety, shorter production cycle and lower production cost. However, due to the instability of mRNA molecules and low in vivo delivery efficiency, the application of mRNA technology has been limited. To achieve the widespread application of mRNA technology, a suitable delivery vector is needed to deliver it into the body in order to have a practical effect. At present, the commercially successful delivery technology is mainly lipid nanoparticle (LNP) technology based on ionizable cationic lipids used by companies such as Moderna and Biotech. Although LNP has been widely used in new crown vaccines, its relatively low efficiency and significant side effects greatly limit its scope of application. Therefore, the development of an efficient and non-toxic in vivo delivery system is still a bottleneck problem that inhibits the development of mRNA technology.

[0003] Baida Liankang Biotechnology (Shenzhen) Co., Ltd. has developed a new biodegradable hyperbranched cationic polyester that can be used for DNA and mRNA delivery based on a biodegradable cationic polyester that can be used for DNA and mRNA delivery reported by Yale University in the United States. The efficiency of gene delivery using this hyperbranched cationic polyester is significantly better than that of linear polymers and the best commercial mRNA transfection reagents currently available, and its cytotoxicity is lower. In addition, the hyperbranched cationic polyester is soluble in ethanol and has potential clinical application potential. However, in-depth studies have found that the complex formed by hyperbranched cationic polyester and gene drugs has a high efficiency of gene delivery at the cell level in vitro, but the efficiency of this complex is significantly reduced when delivered in vivo.

[0004] Therefore, how to improve the in vivo delivery efficiency of the complex formed by hyperbranched cationic polyester and gene drug is an important technical problem that needs to be solved urgently. Summary of the invention

[0005] The purpose of the present application is to provide an improved end-group-modified cationic polyester, a living body delivery system and applications.

[0006] In order to achieve the above objectives, this application adopts the following technical solutions:

[0007] The first aspect of the present application discloses a terminal-modified cationic polyester, which is a hyperbranched polymer (abbreviated as HBPA) formed by polymerizing three monomers, namely, monomer P, monomer S and monomer M, or two monomers, namely, monomer P and monomer S, with monomer T, and the terminal carboxyl group of the hyperbranched polymer is modified with polyethylene glycol (abbreviated as PEG); wherein the monomer P is a cyclic lactone; the monomer S is an organic acid having two or more carboxyl groups at the terminal; the monomer M is a compound containing two hydroxyl groups and one secondary amine or tertiary amine; the monomer T is a compound containing three or more hydroxyl groups; and the number average molecular weight of the polyethylene glycol is 0.4 to 20k.

[0008] It should be noted that the applicant has previously developed a hyperbranched polymer end group with a cationic polyester modified with a group E (abbreviated as HBPA-E), and the end group modified compound E provided by the group E is a compound containing at least one primary amine, secondary amine or tertiary amine group. Although HBPA-E has a high in vitro transfection efficiency; however, the efficiency is significantly reduced during in vivo delivery. Analysis shows that the complex formed by HBPA-E and nucleic acid drugs has a significantly positive charge on its surface, which leads to a significant reduction in its efficiency during in vivo delivery. Therefore, the present application has developed a new end group modified cationic polyester, namely a cationic polyester modified with a carboxyl end group PEG (abbreviated as HBPA-PEG). The HBPA-PEG of the present application, on the one hand, can consume part of the carboxyl groups through PEG modification, making the electrical properties of the hyperbranched polymer more neutral; on the other hand, PEG modification has better stability, and PEG is hydrophilic. When used in combination with HBPA-E for in vivo delivery, PEG forms a stable structure on the surface of the complex formed with the nucleic acid drug, enhancing the stability of the in vivo delivery system, thereby significantly improving the in vivo delivery efficiency of the nucleic acid drug, and solving the problem that the efficiency of the cationic polyester modified with the terminal group E is significantly reduced during in vivo delivery.

[0009] It should also be noted that the key to the present application is to use PEG modification to improve the stability of the in vivo delivery system composed of HBPA-PEG and HBPA-PEG and HBPA-E; however, the nucleic acid delivery efficiency of HBPA-PEG modified with PEG alone is not good, especially with the increase of PEG molecular weight, although the stability will be improved, it will affect the delivery efficiency; therefore, the present application preferably uses 0.4~20k PEG, and more preferably, for example, 1~5k PEG is used.

[0010] In one implementation of the present application, the monomer P is a cyclic lactone with a fatty chain length of 6 to 35.

[0011] In one implementation of the present application, the monomer P is selected from but not limited to at least one of cyclohexyl lactone, cyclododecanolide, cyclopentadecanolide and cyclohexadecanolide.

[0012] In one implementation of the present application, the monomer S is an organic acid with a carbon chain length of 3 to 18.

[0013] In one implementation of the present application, the monomer S is selected from but not limited to at least one of adipic acid, sebacic acid and 1,2,3-propanetricarboxylic acid.

[0014] In one implementation of the present application, the monomer M is a compound having a carbon chain length of 4 to 36 and containing two hydroxyl groups and a secondary amine or a tertiary amine.

[0015] In one implementation of the present application, the monomer M is selected from but not limited to at least one of diethanolamine, methyldiethanolamine and ethyldiethanolamine.

[0016] In one implementation of the present application, monomer T is a compound having a carbon chain length of 4 to 54 and containing three or more hydroxyl groups.

[0017] In one implementation of the present application, the monomer T is selected from but not limited to at least one of trimethylolpropane, 3-(hydroxymethyl)-1,5-pentanediol, triethanolamine, and N,N,N',N'-tetrahydroxyethylethylenediamine.

[0018] In one implementation of the present application, the molar ratio of polyethylene glycol to hyperbranched polymer is 5:1 to 1:10.

[0019] It should be noted that the molar ratio of polyethylene glycol to hyperbranched polymer actually reflects the number or degree of PEG modification of the terminal carboxyl groups of the hyperbranched polymer. It can be understood that HBPA has a hyperbranched structure and multiple terminal carboxyl groups. When the molecular weight of PEG is the same, the more terminal carboxyl groups are modified by PEG, the higher the stability of HBPA-PEG, but the lower the delivery efficiency; in this case, the corresponding HBPA-PEG needs to reduce the proportion in the in vivo delivery system composed of HBPA-PEG and HBPA-E. Therefore, the molar ratio of polyethylene glycol to hyperbranched polymer can be a larger range, such as 5:1 to 1:10. In general, whether it is the molecular weight of PEG or the number of terminal carboxyl modifications of PEG, the efficiency of the in vivo delivery system composed of HBPA-PEG and HBPA-E during in vivo delivery shall prevail; in principle, if the molecular weight of PEG in HBPA-PEG is larger, and / or the number of terminal carboxyl modifications of PEG is greater, the amount of HBPA-PEG used in the in vivo delivery system will be relatively smaller, so as to ensure that the composition formed by HBPA-PEG, HBPA-E and nucleic acid drugs has better stability and good delivery efficiency, thereby having a higher in vivo delivery efficiency.

[0020] In one implementation of the present application, the number average molecular weight of the cationic polyester is 1-30k, preferably 2-20k.

[0021] In one implementation of the present application, the terminal carboxyl group of the hyperbranched polymer is also capped with a neutral compound and / or modified with a group E, wherein the terminal group-modified compound E providing the group E modification is a compound containing at least one primary amine, secondary amine or tertiary amine group.

[0022] It should be noted that PEG modification can only end-cap part of the carboxyl groups of the hyperbranched polymer, and the remaining uncapped carboxyl groups make the charge of HBPA-PEG still negative. In the process of forming a complex with the nucleic acid drug, the charge of the nucleic acid drug is negative, and it attracts the positively charged HBPA-E to form a complex; therefore, if the charge of HBPA-PEG is negative, it will cause unnecessary losses to HBPA-E or other positively charged lipids. Based on such considerations, in a further improvement scheme of the present application, the remaining carboxyl groups other than the terminal carboxyl groups modified with PEG are all end-capped with neutral compounds and / or modified with group E.

[0023] In the present application, the small molecule polymers conventionally used in the art can be used for the end-capping of the remaining carboxyl groups, such as polymers with a molecular weight of less than 1000, including but not limited to primary amine compounds, alcohol compounds and epoxy compounds. These small molecule polymers only end-cap the carboxyl groups, so that the electrical properties of HBPA-PEG change from negative to neutral; therefore, the present application uniformly refers to them as neutral compounds or neutral compound end-capping. Group E modification is actually the end group modification method in HBPA-E; therefore, group E modification can make HBPA-PEG positively charged. In this case, the positively charged HBPA-PEG can not only have better stability, but also attract each other with nucleic acid drugs, play a part of the function of HBPA-E, so that the in vivo delivery system has a higher delivery efficiency. The present application study confirms that whether it is neutral compound end-capping or group E modification, as long as the remaining carboxyl groups are end-capped, the in vivo delivery efficiency is better than that of HBPA-PEG without end-capping.

[0024] In one implementation of the present application, the neutral compound includes at least one of a primary amine compound, an alcohol compound and an epoxy compound.

[0025] In one implementation of the present application, the primary amine compound is a small molecule compound containing one primary amine and having a molecular weight less than 1000.

[0026] In one implementation of the present application, the primary amine compound is at least one of ethylamine, propylamine, butylamine, ethanolamine and n-propanolamine.

[0027] In one implementation of the present application, the alcohol compound is at least one of ethanol, propanol and butanol.

[0028] In one implementation of the present application, the terminal group-modified compound E for modifying the remaining carboxyl groups of HBPA-PEG is at least one of E1 to E26; preferably, the terminal group-modified compound E is at least one of E1 to E10, E12, E14 to E21, E25, and E26; more preferably, the terminal group-modified compound E is at least one of E2, E4, E9, E10, E12, E14, E15, E25, and E26.

[0029] In one implementation of the present application, the cationic polyester is a hyperbranched polymer having a structure shown in Formula 1;

[0030] Formula 1

[0031]

[0032] Wherein, x, y, and z are independent integers ranging from 1 to 200.

[0033] n is an integer from 0 to 200,

[0034] j and k are integers from 0 to 30,

[0035] l, m, o, p, q are independent integers from 1 to 20,

[0036] R x is hydrogen, or a substituted or unsubstituted alkyl group containing 1 to 18 carbon atoms, or a substituted or unsubstituted aromatic group containing at least one benzene ring, or a substituted or unsubstituted heterocyclic group containing at least one heterocyclic ring, or a substituted or unsubstituted alkoxy group containing 1 to 18 carbon atoms and at least one oxygen atom;

[0037] J is hydrogen, at least one of R1 is a group modified by polyethylene glycol, the others are groups formed by end-capping of neutral compounds or groups E provided by end-group modification compound E, in which case there is no R2; or, J is a carbonyl group, at least one of R1 and R2 is a group modified by polyethylene glycol, the others are groups formed by end-capping of neutral compounds or groups E provided by end-group modification compound E;

[0038] In Formula 1, the truncation line represents a branch structure, the first monomer connected to the branch structure is P or S, and other monomers are subsequently connected to form a hyperbranched structure.

[0039] Herein, connecting other monomers means, for example, subsequently connecting monomers in the order of formula 1 to form a branched structure based on the branched structure, and so on, to form a dendrite-like hyperbranched structure.

[0040] It should be noted that in the present application, when only monomers P and S are polymerized with monomer T to form a hyperbranched polymer, that is, n is 0, if J is a carbonyl group, one end of the carbonyl group is connected to R2, and the other end is connected to the hydroxyl group of monomer T. It can be understood that in this case, the structure of the general formula 1 will be adaptively adjusted, that is, monomer T is connected to the carbonyl group J at the end.

[0041] The second aspect of the present application discloses a living body delivery system, which includes a first polymer and a second polymer; the first polymer is the cationic polyester of the present application; the second polymer is a polymer whose end group of the hyperbranched polymer is modified with a group E, that is, a cationic polyester modified with an end group group E, wherein the end group modified compound E providing the group E modification is a compound containing at least one primary amine, secondary amine or tertiary amine group.

[0042] It should be noted that the in vivo delivery system of the present application, i.e., the gene delivery material for in vivo delivery, is mainly composed of the HBPA-PEG of the present application and the previously developed HBPA-E. The combination of the two can significantly improve the efficiency of in vivo delivery, solving the problem that the efficiency of cationic polyester modified with the terminal group E alone is significantly reduced when performing in vivo delivery. It is analyzed that when the in vivo delivery system of the present application forms a complex with the nucleic acid drug, the PEG in the HBPA-PEG is hydrophilic and will form a stable structure on the surface of the complex, thereby improving the overall stability of the complex during the in vivo delivery process, thereby improving the efficiency of in vivo delivery.

[0043] In the present application, the terminal group modified compound E of the cationic polyester modified by the terminal group E of the second polymer HBPA-E is at least one of E1 to E26.

[0044] Preferably, the terminal group modified compound E is at least one of E1 to E10, E12, E14 to E21, E25, and E26.

[0045] More preferably, the terminal group modified compound E is at least one of E2, E4, E9, E10, E12, E14, E15, E25, and E26.

[0046] It should be noted that the terminal group E modified cationic polyesters obtained by terminal group modification using terminal group modified compounds E1 to E26 in the present application all have good transfection efficiency and low cytotoxicity; in particular, the terminal group E modified cationic polyesters obtained by terminal group modification of E2, E4, E9, E10, E12, E14, E15, E25, and E26 have a transfection efficiency that is significantly better than other terminal group modified cationic polyesters, and is also significantly better than the currently most efficient commercial transfection reagent LipoMM.

[0047] In one implementation of the present application, the second polymer, i.e., the cationic polyester modified with the terminal group E, is a hyperbranched polymer having a structure shown in Formula 2;

[0048] Formula 2

[0049]

[0050] Wherein, x, y, and z are independent integers ranging from 1 to 200.

[0051] n is an integer from 0 to 200,

[0052] j and k are integers from 0 to 30,

[0053] l, m, o, p, q are independent integers from 1 to 20,

[0054] R x is hydrogen, or a substituted or unsubstituted alkyl group containing 1 to 18 carbon atoms, or a substituted or unsubstituted aromatic group containing at least one benzene ring, or a substituted or unsubstituted heterocyclic group containing at least one heterocyclic ring, or a substituted or unsubstituted alkoxy group containing 1 to 18 carbon atoms and at least one oxygen atom;

[0055] J is hydrogen, R1 is modified by the group E provided by the terminal group modification compound E, and there is no R2; or, J is a carbonyl group, and both R1 and R2 are modified by the group E provided by the terminal group modification compound E;

[0056] In Formula 2, the truncation line represents a branch structure, the first monomer connected to the branch structure is P or S, and other monomers are subsequently connected to form a hyperbranched structure.

[0057] It should be noted that in the present application, the main chains of HBPA-PEG and HBPA-E, i.e., hyperbranched polymers, have the same structural principle, but the end groups are modified differently. Of course, in the in vivo delivery system, the specific number average molecular weights of HBPA-PEG and HBPA-E, or the arrangement order of the monomers, may be different.

[0058] In one implementation of the present application, the number average molecular weight of the second polymer is 1-30k, preferably 2-20k.

[0059] In one implementation of the present application, the mass ratio of the first polymer to the second polymer is 0.01:9.99 to 2.5:7.5.

[0060] It should be noted that the key to this application is the combined use of HBPA-PEG and HBPA-E; it is understandable that, in principle, as long as HBPA-PEG is added to HBPA-E, it can improve the efficiency of in vivo delivery to varying degrees; however, this application has found that the in vivo delivery system formed by the mass ratio of the first polymer to the second polymer is 0.01:9.99 to 2.5:7.5 has a relatively high in vivo delivery efficiency. Among them, if the amount of the first polymer HBPA-PEG is too small, the delivery system is not stable enough in the body, resulting in a decrease in delivery efficiency; conversely, if the amount of the first polymer HBPA-PEG is too large, its cell delivery efficiency is extremely low, resulting in almost no in vivo delivery efficiency.

[0061] The third aspect of the present application discloses the use of the cationic polyester of the present application or the in vivo delivery system of the present application in the delivery of nucleic acid drugs.

[0062] In one implementation of the present application, nucleic acid drugs include but are not limited to mRNA, circular RNA, siRNA, microRNA, saRNA and DNA.

[0063] It should be noted that the key to this application is that a new nucleic acid drug delivery material, namely the cationic polyester HBPA-PEG of this application, is improved, which can solve the problem that the efficiency of in vivo delivery using the terminal group E to modify the cationic polyester is significantly reduced. The in vivo delivery system of this application can also be used for cell transfection, for example, the in vivo delivery system of this application is used to encapsulate nucleic acid drugs and deliver them into cells. The types of these cells include but are not limited to HEK293T, A549, HeLa, U87, HUVEC, Jurkat, RAW264.7, iPSC and MSC.

[0064] The fourth aspect of the present application discloses a nucleic acid delivery particle, comprising a wrapping material and a nucleic acid wrapped in the wrapping material; the wrapping material is the cationic polyester of the present application or the wrapping material at least comprises the cationic polyester of the present application, or the wrapping material is the living body delivery system of the present application or the wrapping material at least comprises the living body delivery system of the present application; the nucleic acid is at least one of mRNA, circular RNA, siRNA, microRNA, saRNA and DNA.

[0065] It should be noted that the nucleic acid delivery particles using the in vivo delivery system of the present application not only have the advantages of low cytotoxicity and better meeting clinical use requirements, but also can significantly improve the efficiency of in vivo delivery, solving the problem of significantly reduced efficiency when using cationic polyester modified with terminal group E for in vivo delivery alone. It can be understood that the key to the nucleic acid delivery particles of the present application is to use the cationic polyester of the present application or the in vivo delivery system of the present application, and the specific embedding method and nucleic acid delivery method can refer to the prior art.

[0066] It should also be noted that the wrapping material of the present application may also include other materials besides the cationic polyester of the present application or the living body delivery system of the present application according to different designs or requirements of the wrapping layer, which are not specifically limited here.

[0067] In one implementation of the present application, the particle size of the nucleic acid delivery particle is 30-500 nm.

[0068] The fifth aspect of the present application discloses a kit for nucleic acid drug delivery, comprising at least one of the following components:

[0069] (a) the cationic polyester of the present application;

[0070] (b) the in vivo delivery system of the present application;

[0071] (b) The nucleic acid delivery particle of the present application.

[0072] It should be noted that the kit for nucleic acid drug delivery of the present application can be a kit for a certain specific nucleic acid drug that has been embedded, or it can be the cationic polyester of the present application or the in vivo delivery system of the present application. The user can design and embed the corresponding nucleic acid drug according to the needs. It can be understood that the key to the kit of the present application is that it contains the cationic polyester, in vivo delivery system or nucleic acid delivery particles of the present application. As for other conventional reagents required for nucleic acid delivery, such as some lipids or polymer materials, etc., they can be obtained by referring to the prior art or commercially purchased. Of course, for ease of use, some reagents can also be combined into the kit of the present application, which is not specifically limited here.

[0073] The sixth aspect of the present application discloses the use of the cationic polyester of the present application, or the in vivo delivery system of the present application, or the nucleic acid delivery particle of the present application, or the kit of the present application in the treatment of diseases based on nucleic acid drug delivery.

[0074] The seventh aspect of the present application discloses a method for administering a nucleic acid drug in vivo, comprising using the in vivo delivery system of the present application or a packaging material containing the in vivo delivery system of the present application to package the nucleic acid drug to form nucleic acid delivery particles, and using the nucleic acid delivery particles to deliver the nucleic acid drug.

[0075] It should be noted that the key to the administration method of the present application is to use the in vivo delivery system of the present application to deliver nucleic acid drugs. As for the specific operation of in vivo delivery and other auxiliary materials required, reference can be made to the existing technology and no specific limitation is made here.

[0076] Due to the adoption of the above technical solution, the beneficial effects of this application are:

[0077] The cationic polyester of the present application modifies the terminal carboxyl group of the hyperbranched polymer with polyethylene glycol, and when it is delivered in vivo together with the cationic polyester modified with the terminal group E, the stability of the complex can be improved, thereby significantly improving the efficiency of in vivo delivery. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] Figure 1 is a synthetic route for the hyperbranched polymer and the end group E and PEG modification in the examples of the present application;

[0079] Figure 2 is the 1H-NMR spectrum of HBPA-E14-3 before and after end group modification in the embodiment of the present application;

[0080] Figure 3 is the GPC spectrum of HBPA-E14-1 and PACA-E14 in the examples of the present application;

[0081] Figure 4 is a quantitative graph of the end group modified E14 of HBPA-E14-1 and PACA-E14 in the examples of the present application;

[0082] Figure 5 is the 1H-NMR spectrum of HBPA-PEG before and after PEG modification in the examples of the present application;

[0083] Figure 6 is the GPC spectrum of HBPA-PEG before and after PEG modification in the examples of the present application;

[0084] Figure 7 is the particle size test result of the complex of HBPA-E14-2 and mRNA in the example of the present application;

[0085] Figure 8 These are the test results of mRNA transfection efficiency of different HBPA-E in A549 and HEK 293T cells in the examples of this application;

[0086] Fig. 9 It is the test result of DNA transfection efficiency of different HBPA-E in HEK 293T cells in the examples of this application;

[0087] Fig.10The test results of mRNA transfection efficiency of HBPA-E modified with 26 different terminal groups E in A549 cells in the examples of the present application are as follows;

[0088] Fig.11 This is the in vitro transfection efficiency result of the complex of HBPA-E14 / HBPA-PEG / mRNA in the example of the present application;

[0089] Fig.12 This is the in vivo lung delivery effect of the HBPA-E14 / HBPA-PEG / mRNA complex in the examples of this application. DETAILED DESCRIPTION

[0090] The existing commercial gene delivery materials have low transfection efficiency and high toxicity, which greatly limits the widespread application of gene therapy based on nucleic acid drug delivery. Although studies have reported that the biodegradable linear polymer (PACA) obtained by polymerization of three monomers P, S, and M catalyzed by immobilized lipase based on in vitro synthetic biology technology has high transfection efficiency and lower cytotoxicity; however, the transfection efficiency of the linear polymer is still not ideal, and the prepared gene delivery material is difficult to use in actual clinical practice because it needs to be dissolved in toxic organic solvents.

[0091] The applicant creatively adds a new monomer T on the basis of the polymerization of the original three monomers, or replaces the monomer M with the monomer T, thereby preparing a new type of hyperbranched polymer with a hyperbranched structure. Specifically, a hyperbranched polymer is formed by polymerization of three monomers, monomer P, monomer S and monomer M, or two monomers, monomer P and monomer S, with monomer T, and the end group of the hyperbranched polymer is modified with a group E, thereby forming a cationic polyester modified with an end group group E. Among them, monomer P is a cyclic lactone; monomer S is an organic acid with two or more carboxyl groups at the end group; monomer M is a compound with two hydroxyl groups and one secondary amine or tertiary amine at the end group; monomer T is a compound containing three or more hydroxyl groups; and the end group modified compound E provided with the group E modification is a compound containing at least one primary amine, secondary amine or tertiary amine group.

[0092] The cationic polyester modified with terminal group E developed by the applicant has significantly improved gene transfection efficiency, which is significantly better than the original linear polymer and the best existing commercial mRNA transfection reagent, and has lower cytotoxicity; more importantly, it is soluble in ethanol and does not need to be dissolved in toxic organic solvents, is easy to scale up industrially and can better meet clinical use needs, and has great practical application prospects.

[0093] However, in-depth research has found that the efficiency of in vivo delivery of cationic polyesters modified with terminal group E alone is significantly reduced. In order to solve this problem, the present application creatively proposes to modify the terminal carboxyl groups of hyperbranched polymers with polyethylene glycol, and the polymer thus formed is the HBPA-PEG of the present application.

[0094] The HBPA-PEG of the present application also has the advantages of low cytotoxicity and better suitability for clinical use; moreover, when combined with a cationic polyester modified with an end group E, a new delivery system is formed, which can significantly improve the efficiency of in vivo delivery and solve the problem that the efficiency of cationic polyester modified with an end group E is significantly reduced during in vivo delivery.

[0095] In the present application, the preparation method of the cationic polyester modified with the terminal group E comprises: adding each monomer and the catalyst to the solvent, sequentially carrying out the first stage polymerization reaction and the second stage polymerization reaction in an inert atmosphere, after the reaction is completed, removing the catalyst to obtain a hyperbranched polymer; then, under the action of a coupling agent, the hyperbranched polymer is end-modified with the terminal group modification compound E to obtain a cationic polyester with the terminal group modified with the group E; wherein the conditions of the first stage polymerization reaction are: temperature 85-95°C, reaction vacuum degree 50-1000mbar, reaction time 12-24h; the conditions of the second stage polymerization reaction are: temperature 85-95°C, reaction vacuum degree 1-30mbar, reaction time 12-72h. The molar ratio of monomer P to monomer S is 0.1:10-4:1, the molar ratio of monomer M to monomer S is 0:10-20:10, and the molar ratio of monomer T to monomer S is 0.1:10-20:10. The catalyst is an immobilized lipase, and the amount of the immobilized lipase is 3-50wt% of the total mass of each monomer. The solvent is at least one of diphenyl ether, n-dodecane, 1-butyl-3-methylimidazolium hexafluorophosphate, dimethylacetamide and o-phenylenedimethyl ether, and the amount of the solvent is 100-500wt% of the total mass of each monomer. Wherein, removing the catalyst specifically includes filtering the reaction solution with a filtering device after the reaction is completed and collecting the filtrate; obtaining the hyperbranched polymer specifically includes adding n-hexane to the filtrate to precipitate the hyperbranched polymer, centrifuging, removing the supernatant, adding dichloromethane to the precipitate to dissolve, then adding n-hexane to precipitate the hyperbranched polymer, centrifuging, removing the supernatant, repeating the dissolution with dichloromethane, precipitation with n-hexane, and centrifugation at least twice; finally, drying the precipitate to obtain the hyperbranched polymer. The coupling agent is at least one of N,N'-carbonyldiimidazole, carbodiimide, phosphorus cation and urea cation. Carbodiimides include but are not limited to diisopropylcarbodiimide, dicyclohexylcarbodiimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide. Phosphorus cations include but are not limited to benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate and benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate; urea cations include but are not limited to 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate and O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate. The molar ratio of the coupling agent to the hyperbranched polymer is 2:1 to 50:1; preferably, the molar ratio of the coupling agent to the hyperbranched polymer is 4:1 to 15:1; in addition, the molar ratio of the coupling agent to the end group modified compound E is 5:1 to 1:10.The end group modification of the hyperbranched polymer is carried out by using the end group modification compound E, specifically comprising: dissolving the hyperbranched polymer in dichloromethane, adding a coupling agent, stirring at room temperature for at least 10 hours in an inert atmosphere, concentrating the reaction solution, adding at least 3 volumes of ether, centrifuging, removing the precipitate, and obtaining a supernatant; removing the solvent in the supernatant, adding the dried product to dichloromethane to dissolve, adding the end group modification compound E under stirring, reacting at room temperature for at least 10 hours, and obtaining a hyperbranched polymer with a modified end group having a group E, that is, the cationic polyester of the present application. Further, after the end group modification compound E is added to the reaction solution at room temperature, an equal volume of deionized water is added to the reaction solution, vortexing, centrifuging and layering, and removing the upper aqueous phase; repeating the operation of adding an equal volume of deionized water, vortexing, centrifuging and layering, and removing the upper aqueous phase at least 3 times; finally, adding at least 3 volumes of n-hexane, vortexing, centrifuging, removing the supernatant, and drying the precipitate, and obtaining a cationic polyester modified with the end group E.

[0096] In the present application, the preparation method of HBPA-PEG includes adding each monomer and a catalyst to a solvent, sequentially carrying out a first stage polymerization reaction and a second stage polymerization reaction in an inert atmosphere, and after the reaction is completed, removing the catalyst to obtain a hyperbranched polymer; then, under the action of a coupling agent, polyethylene glycol is first added to carry out polyethylene glycol modification of the terminal carboxyl group, and if the remaining carboxyl groups need to be capped, a neutral compound capped compound or a terminal modified compound E modified with a group E is added to cap the remaining carboxyl groups to obtain HBPA-PEG; wherein the conditions for the first stage polymerization reaction are a temperature of 85 to 95°C, a reaction vacuum of 50 to 1000 mbar, and a reaction time of 12 to 24 hours; the conditions for the second stage polymerization reaction are a temperature of 85 to 95°C, a reaction vacuum of 1-30 mbar, and a reaction time of 12 to 72 hours. The molar ratio of monomer P to monomer S is 0.1:10-4:1, the molar ratio of monomer M to monomer S is 0:10-20:10, and the molar ratio of monomer T to monomer S is 0.1:10-20:10. The catalyst, solvent, coupling agent and their dosage are the same as those in the preparation method of cationic polyester modified with terminal group E. The molar ratio of coupling agent to hyperbranched polymer is 2:1-50:1; preferably, the molar ratio of coupling agent to hyperbranched polymer is 4:1-15:1; the molar ratio of polyethylene glycol to coupling agent is 1:5-10:1, and the molar ratio of neutral compound-terminated compound or terminal group modified compound E to coupling agent is 1:1-50:1. In the present application, polyethylene glycol is first added to modify the terminal carboxyl group with polyethylene glycol, and then a compound for capping the remaining carboxyl groups is added, specifically comprising: dissolving the hyperbranched polymer in dichloromethane, adding a coupling agent, stirring at room temperature for at least 3 hours in an inert atmosphere, adding polyethylene glycol, and reacting at room temperature for at least 48 hours; then, adding a coupling agent to the reaction solution, stirring at room temperature for at least 3 hours, adding a neutral compound capped compound or a terminal group modified compound E modified with group E, and reacting at room temperature for at least 10 hours to obtain HBPA-PEG. Further, after adding a neutral compound capped compound or a terminal group modified compound E modified with group E, reacting at room temperature for at least 10 hours, adding an equal volume of deionized water to the reaction solution, vortexing, centrifuging and layering, and removing the upper aqueous phase; repeating the operation of adding an equal volume of deionized water, vortexing, centrifuging and layering, and removing the upper aqueous phase for at least 5 times; finally, adding at least 3 times the volume of n-hexane, vortexing, centrifuging, removing the supernatant, and drying the precipitate to obtain the HBPA-PEG of the present application.

[0097] The present application is further described in detail below through specific examples. The following examples are only used to further illustrate the present application and should not be construed as limiting the present application.

[0098] Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0099] Example

[0100] 1. Synthesis and end group modification of hyperbranched polymer (HBPA)

[0101] (1)HBPA synthesis

[0102] In this example, a hyperbranched polymer (HBPA) is first formed by polymerizing four monomers P, S, M and T, and then the terminal group E is modified on HBPA to obtain HBPA-E. Specifically, the monomer P in this example is cyclopentadecanolide (PDL), the monomer S is sebacic acid (SA), the monomer M is methyldiethanolamine (MDEA), and the monomer T is triethanolamine (TEA). The synthetic route of HBPA is as follows: Figure 1 As shown in Figure (a), seven tests and two controls were designed in this example. The raw materials were added to a round-bottom flask according to the molar ratio of the four monomers P(PDL) / S(SA) / M(MDEA) / T(TEA) in Table 1, and immobilized lipase (CALB) was added at 10Wt% of the total raw material mass, and finally 200Wt% of diphenyl ether was added. After the reaction system was replaced with argon three times, it was stirred and heated to 90°C at 60mbar for 24 hours, and then the reaction was continued at 2.1mbar pressure for 48 hours. After the reaction was completed, the reaction solution was filtered to remove the lipase. The filtrate was added with n-hexane, vortexed and centrifuged, and the supernatant was poured out. The precipitate was dissolved with dichloromethane, and then n-hexane was added to precipitate it, centrifuged, and the supernatant was poured out. The above operation was repeated 3 times. The precipitate was vacuum dried for one day to obtain HBPA. Among them, the polymer synthesized by the control is a linear polymer PACA, refer to US20200399424 A1.

[0103] Table 1 Characterization of polymers synthesized with different feed ratios and their modified E14 products

[0104]

[0105] In Table 1, "a" is calculated based on 1H-NMR results, and "b" is calculated based on GPC results.

[0106] (2) Modification of terminal group E

[0107] In this example, as shown in Table 1, terminal group modified compound E14 was used. In this example, N,N'-carbonyldiimidazole (CDI) was used as a coupling agent to modify the terminal group of the polymer.

[0108] like Figure 1As shown in Figure (b), CDI was used as a coupling agent, 250 mg of HBPA was dissolved in 5 mL of ultra-dry dichloromethane (DCM), and 40 eq of CDI was added under stirring. After the reaction system was replaced with argon three times, it was stirred at room temperature overnight. The reaction solution was concentrated to 3 mL, 3 volumes of ether were added, vortexed, centrifuged, and the precipitate was removed. The supernatant was decompressed and dried, ultra-dry DCM was added to dissolve, and 40 eq of E14 was added under stirring, and the reaction was allowed to react at room temperature for 24 hours. After the reaction was completed, an equal volume of deionized water was added to the reaction solution, and after vortexing and centrifugation, the upper aqueous phase was removed. Another equal volume of deionized water was added, and the above operation was repeated 5 times. 3 volumes of n-hexane were added to the lower layer of dichloromethane solution, vortexed, and centrifuged. The precipitate was vacuum dried for one day to obtain HBPA-E14. For control 1, PACA-E14 was obtained.

[0109] Using a method similar to that of Experiment 2, this example further used 25 terminal group modification compounds from E1 to E26, except E14, to modify the terminal groups of HBPA to synthesize other HBPA-E.

[0110] The 26 terminal group modified compounds from E1 to E26 are as follows:

[0111]

[0112] (3) PEG modification

[0113] The synthetic route of the cationic polyester modified with carboxyl PEG and terminated with primary amine compounds is as follows: Figure 1 As shown in Figure (c), HBPA was dissolved in ultra-dry dichloromethane (DCM), and CDI (0.5 eq-COOH) was added under stirring. The reaction system was replaced with argon three times, stirred at room temperature for 3 hours, and then mPEG was added. 2K -NH2 (0.1eq-COOH), continue the reaction at room temperature for 48 hours. If other carboxyl groups need to be capped, continue the capping treatment, otherwise skip this step and proceed to the subsequent reaction. Capping treatment: add CDI (1eq-COOH) to the reaction solution, stir at room temperature for 3 hours, add ethanolamine (5eq-COOH), and continue the reaction at room temperature overnight.

[0114] After the reaction is completed, add an equal volume of deionized water to the reaction solution, vortex and centrifuge to separate the layers, and then remove the upper aqueous phase. Add another equal volume of deionized water and repeat the above operation 5 times. Add 3 times the volume of n-hexane to the dichloromethane solution, vortex and centrifuge. The precipitate was vacuum dried for one day to obtain a cationic polyester modified with terminal carboxyl PEG and capped with a primary amine compound, labeled as HBPA-PEG. Using a similar method, PEGs of different molecular weights, specifically PEGs with molecular weights of 1k, 5k, 10k and 20k, were used to modify HBPA to obtain different HBPA-PEGs.

[0115] In this example, HBPA-PEG with and without carboxyl groups were prepared for subsequent experiments. The "HBPA-PEG" mentioned in the following experiments is normal HBPA-PEG with carboxyl groups terminated, unless otherwise specified.

[0116] 2. Structural Characterization of HBPA, HBPA-PEG and HBPA-E

[0117] (1) Structural characterization of HBPA and HBPA-E

[0118] The molecular structures of HBPA and HBPA-E can be characterized by 1H-NMR. The results are shown in Table 1. Some of the results are shown in Table 1. Figure 2 shown. Figure 2 The spectra of HBPA in Experiment 3 before and after end group modification, namely, 1H-NMR spectra of HBPA-3 and HBPA-E14-3, with the solvent being CDCl3. In the spectra, the characteristic peak of the PDL unit is f (δ≈4.08ppm), the characteristic peak of the SA unit is b (δ≈1.60ppm), the characteristic peak of the MDEA unit is g (δ≈4.20ppm), and the characteristic peak of the TEA unit is h (δ≈2.85ppm). The presence of the TEA unit indicates that the polymer structure is in line with the expected assumptions and may have a hyperbranched structure, while the characteristic peak k (δ≈2.22ppm) can prove that the end group has successfully modified the group E. The ratio of each repeating unit of the polymer can be converted by calculating the integrated area of ​​each characteristic peak. The results show that the molar ratio of the P / S / M / T repeating units of HBPA-E14-3 obtained by CDI modification is 1:9:4.5:1.9, which is very close to the feed ratio of 1:9:6:2. This indicates that the reaction of each monomer raw material in the reaction is basically complete and the reaction efficiency is very high. Other 1H-NMR results are shown in Table 1. The results show that even with the continuous increase of T feed, the enzyme-catalyzed reaction is close to complete, and the reaction can be very controllable to obtain hyperbranched polymers without crosslinking.

[0119] The experiment showed that the reduction of monomer M had little effect on the hyperbranched polymer; therefore, this example further studied the case where the proportion of monomer M was reduced to 0, that is, the polymerization reaction was directly carried out using monomers P, S and T. Specifically, the molar ratio of the feed was P:S:T = 1:9:6, and the remaining steps and parameters were the same as "(1) HBPA synthesis". The results showed that the three monomers P, S and T can also obtain a polymer with a hyperbranched structure without crosslinking. The hyperbranched polymer obtained from the three monomers P, S and T has similar physical and chemical properties to the hyperbranched polymer obtained from the four monomers P, S, M and T.

[0120] At the same time, GPC was used to characterize the molecular weight and distribution of HBPA-E. GPC measurements were performed using a Waters 1515 column and a 2414 refractive index (RI) detector at 35°C. The mobile phase was DMF containing 0.1% LiBr at a flow rate of 1 mL / min, and a linear polymethyl methacrylate standard was used as the standard. The results are shown in Table 1, and some of the results are shown in Table 2. Figure 3 shown. Figure 3 These are the GPC spectra of HBPA-E14-1 of Experiment 1 and PACA-E14 of Control 1, respectively. Figure 3 The results showed that the GPC spectra of hyperbranched HBPA-E14-1 and linear PACA-E14 were both single-peak, with a number average molecular weight of M n The molecular weights of the two are similar, but the PDIs are significantly different. The PDIs of other HBPA-E14s are shown in Table 1, which are similar to those of HBPA-E14-1.

[0121] In addition, in this example, HBPA-E14 and PACA-E14 prepared in Table 1 were dissolved in ethanol to test their solubility. The results are shown in Table 1. The results in Table 1 show that the branched HBPA-E in this example is easily soluble in ethanol, with a solubility greater than 25 mg / mL, while the linear PACA-E is poorly soluble in ethanol, with a solubility less than 10 mg / mL.

[0122] Finally, in this example, the ninhydrin method was used to quantify the end-group modified E14 content of HBPA-E14 (Mn=8769) and PACA-E14 (Mn=8296) with similar number average molecular weights. The specific method is as follows: 1. Prepare ninhydrin colorimetric agent: weigh 200 mg of ninhydrin, dissolve it in 9 mL of ethanol, add ethanol to make up to 10 mL, and prepare a 20 mg / mL ninhydrin ethanol solution. 2. Sample determination: Take about 10 mg of each HBPA-E14 and PACA-E14 sample, prepare a 5 mg / mL ethanol solution, take 100 μL of each and mix them evenly with 200 μL of ninhydrin ethanol solution, place on a shaker and heat and oscillate at 60°C for 10 minutes, cool to room temperature, take 200 μL of each to measure the absorbance at 570 nm, and convert the absorbance value into the relative concentration of the end group E14. The results are as follows. Figure 4 As shown in the figure, the relative concentration of end group E14 in PACA-E14 is 1, while at the same material concentration, the relative concentration of end group E14 in HBPA-E14-1 is 3.01, indicating that its end group E14 content is nearly 3 times that of PACA-E14. Considering that the molecular weights of the two are very close, the significantly higher end group content of HBPA-E14-1 than that of PACA-E14 should be due to its branched structure, which once again verifies the branched structure of HBPA.

[0123] (2) Structural characterization of HBPA and HBPA-PEG

[0124] The molecular structures of HBPA and HBPA-PEG were characterized by 1H-NMR. Figure 5 The spectra of HBPA in experiment 2 before and after end group modification, namely HBPA-2 and HBPA-PEG 2k 1H-NMR spectrum, the solvent is CDCl3. In the spectrum, the characteristic peak of the PDL unit is f (δ≈4.05ppm), the characteristic peak of the SA unit is b (δ≈1.60ppm), the characteristic peak of the MDEA unit is g (δ≈4.16ppm), the characteristic peak of the TEA unit is h (δ≈2.83ppm), the characteristic peak of the PEG unit is p (δ≈3.65ppm), and the characteristic peaks of the ETA (ethanolamine) unit are r (δ≈3.71ppm) and q (δ≈3.42ppm). The presence of the TEA unit proves that the polymer structure is in line with the expected assumption and is a hyperbranched structure, while the characteristic peaks k (δ≈2.19ppm) and p, r, q can prove that the end groups have been successfully modified with ETA and PEG. The ratio of each repeating unit of the polymer can be converted by calculating the integrated area of ​​each characteristic peak. The results show that the molar ratio of P / S / M / T repeating units of HBPA-PEG modified by CDI is 1:9:5.8:1.1, which is very close to the feed ratio of 1:9:6.2:1. 2k After modification, its molecular structure has not changed. At the same time, PEG can be calculated 2k The molar ratio of HBPA to PEG is 1:1, which means that on average one HBPA molecule modifies one PEG. 2k .

[0125] In addition, GPC can also be used to characterize the molecular weight and distribution of HBPA-PEG. GPC measurements were performed using a Waters 1515 column and a 2414 refractive index (RI) detector at 35°C. The mobile phase was DMF containing 0.1% LiBr at a flow rate of 1 mL / min, and polystyrene standards were used as standards. Figure 6 As shown in the figure, the GPC spectra of hyperbranched HBPA-PEG and HBPA are both single peaks, with a number average molecular weight of M n The molecular weights of HBPA-PEG and HBPA-PEG are 6948 and 3791Da, respectively, and the PDIs are 1.85 and 1.98, respectively. The PDIs of the two are close, but the molecular weights are significantly different. This indicates that compared with HBPA, the end group modification of PEG and ETA leads to a significant increase in molecular weight.

[0126] In addition, PEG 2k The modified HBPA-PEG is easily soluble in ethanol (>25 mg / mL), which is very different from the properties of unmodified HBPA.

[0127] 3. Characterization of Materials and mRNA Complexes

[0128] After the polymer material HBPA-E14-2 was prepared, it was mixed with Firefly Luciferase mRNA (N1-Me-Pseudo UTP, Nanjing Novozyme Biotechnology Co., Ltd.) at different mass ratios and the diameter of the complex was measured. The complex of PACA-E14 and mRNA was also used as a control. Specifically, the polymer materials HBPA-E14-2 and PACA-E14 were dissolved in ethanol and DMSO to prepare a 25 mg / mL polymer solution, and then different amounts of the polymer solution were added to a certain volume of NaAc buffer at pH 4.9 and vortexed. At the same time, a NaAc solution at pH 4.9 containing 20 μg / mL mRNA was prepared, and an equal volume of the polymer solution was added to the mRNA solution and vortexed to finally obtain a complex solution with a polymer and mRNA mass ratio of 25:1, 50:1, 75:1, 100:1, 150:1, and 200:1. HBPA-E14-2 and PACA-E14 were subjected to the above mass ratio control test. The dosage of HBPA-E14-2 and PACA-E14 is calculated based on the mass ratio. For example, if the mass ratio of polymer to mRNA is 25:1, 20 μL of 25 mg / mL polymer solution, i.e., 500 μg of polymer, is added to 980 μL of pH 4.9 NaAc buffer and vortexed. At the same time, a pH 4.9 NaAc solution containing 20 μg / mL mRNA is prepared, and an equal volume of the polymer solution is added to the mRNA solution and vortexed to obtain a mass ratio of polymer to mRNA of 25:1.

[0129] At the same time, the obtained polymer materials HBPA-E14-2 and HBPA-PEG were mixed according to the mass ratio in Table 2 to prepare a living body delivery system.

[0130] Table 2 Characterization of the physicochemical properties of the complexes of HBPA-E14 / HBPA-PEG / mRNA at different ratios

[0131]

[0132] Different in vivo delivery systems and Firefly Luciferase mRNA were mixed at a mass ratio of 75:1 and the diameter of the complex was measured. At the same time, according to the control example 1 in Table 2, the complex of HBPA-E14 and mRNA was used as a control. Specifically, the polymer materials HBPA-E14-2 and HBPA-PEG were dissolved in ethanol to prepare a 25 mg / mL polymer solution, and then the polymer mixed solution with different HBPA-PEG contents, HBPA-PEG mass content of 0, 0.1, 1, 5, 10, 25, 50 and 100%, was added to a pH 4.9 NaAc buffer and vortexed. At the same time, a pH 4.9 NaAc solution containing 20 μg / mL mRNA was prepared, and an equal volume of the polymer solution was added to the mRNA solution and vortexed to finally obtain a in vivo delivery system.

[0133] The particle size of the HBPA-E14 / mRNA complex was measured by a particle size analyzer (Malvern Panalytical Zetasizer Pro) at 25°C. Figure 7 As shown, Figure 7 The horizontal axis is the mass ratio of polymer to mRNA, and the vertical axis is the particle size of the complex, in nm.

[0134] Figure 7 The results showed that the diameter of the HBPA-E14-2 / mRNA complex exceeded 300nm when the mass ratio was 25:1, while the diameter decreased significantly and gradually stabilized at 100-200nm when the mass ratio was 50:1-200:1. This shows that after the mass ratio is greater than 50:1, the complex of HBPA-E14-2 and mRNA tends to be stable. PACA-E14 and mRNA also showed similar results.

[0135] At the same time, this example also tested the in vivo delivery system with different HBPA-PEG contents in Table 2. The mass content of HBPA-PEG was 0, 0.1, 1, 5, 10, 25, 50 and 100%, respectively, and the particle size of the mRNA complex was listed in Table 2. The mass ratio of HBPA-E14 to mRNA was 75:1. From the results in Table 2, it can be seen that the particle size of the complex changes significantly with the change of HBPA-PEG content. When the HBPA-PEG content is less than 50%, as the HBPA-PEG in the complex continues to increase, the particle size of the complex continues to decrease and gradually stabilizes. When HBPA-PEG increases to 100%, its particle size increases compared to 50%. These results show that an appropriate amount of PEG will make the particle size of the complex smaller.

[0136] IV. Test of mRNA transfection efficiency of HBPA-E14 / HBPA-PEG in vitro cells

[0137] After obtaining the complex of HBPA-PEG and mRNA or DNA expressing luciferase (FireflyLuciferase DNA, Guangzhou Aiki Biotechnology Co., Ltd.), this example tested its transfection efficiency in different cell models. The specific method of cell transfection is as follows: A549 and HEK293T cells (ATCC) were inoculated in a 48-well cell culture plate (2.5×10 4 / well), the cells were cultured in DMEM at 37°C and 5% CO2 for 12 hours to adhere to the wall, and then the DMEM medium (250 μL / well) was replaced, and the test samples and positive controls were added and cultured for 24 hours (mRNA) or 48 hours (DNA), and the samples were added according to the final mRNA of 2 μg / mL per well. The preparation method of the complex of the test material and mRNA or DNA is the same as "III. Characterization of the complex of materials and mRNA". The positive control of mRNA transfection uses Lipofectamine MessengerMAX (LipoMM) from Thermo Fisher, and the positive control of DNA transfection uses Lipofectamine2000 (Lipo2k).

[0138] The specific method of testing the lysis and luminescence effect after cell transfection is as follows: 24 hours (mRNA) or 48 hours (DNA) after cell transfection, remove the culture medium in the well plate and place it in a -80℃ refrigerator for 10 minutes. Take out the well plate and place it at 4℃, take 40μL of lysis solution to the 48-well plate, cover the bottom surface, let it stand for 5 minutes, then take 280μL of test solution to the 48-well plate, then take 160μL of the mixture to the black microplate reader plate, and finally use a spray gun to take 40μL of D-Luciferin and add it to each well. After reacting at room temperature for 2 minutes, put it into the microplate reader to test the 560nm luminescence.

[0139] The results of mRNA and DNA transfection efficiency tests of different HBPA-E in A549 and HEK 293T cells are shown in Figures 8 to 10 The specific experimental designs are as follows:

[0140] In this example, the mRNA transfection efficiency of the polymers of control 1, test 1, test 2, test 3, test 5, test 6 and test 7 in A549 and HEK293T cells was tested after they were respectively complexed with mRNA at a polymer:mRNA mass ratio of 75:1. The results are shown in the figure. Figure 8 shown.

[0141] Figure 8The results showed that in A549 cells, all hyperbranched materials showed significantly higher mRNA transfection effects than the commercial control LipoMM, and much higher than the transfection effect of linear PACA-E14. Among them, HBPA-E14-3 had the highest transfection efficiency, nearly 4 times that of LipoMM. The linear PACA-E14 was weaker than LipoMM. The transfection results of HEK293T cells were consistent with those of A549. This shows that the cell transfection effect of hyperbranched polymers on mRNA is significantly improved compared with linear polymers, and is better than the most efficient commercial transfection reagents currently available. This effect has also been verified on different cells. Considering that this hyperbranched polymer can also be dissolved in ethanol, it has great application potential in both in vitro and in vivo mRNA transfection.

[0142] In addition, this example also tested the DNA transfection efficiency of different HBPA-E in HEK 293T cells. Specifically, the DNA transfection efficiency of the polymers of control 1, test 1, test 2, test 3, test 5, test 6 and test 7 after being complexed with DNA at a polymer:DNA mass ratio of 75:1 in HEK 293T cells was tested. The results are shown in FIG. Fig. 9 shown.

[0143] Fig. 9 The results showed that all hyperbranched materials showed significantly higher DNA transfection effects than the commercial control Lipo2k, and much higher than the transfection effect of linear PACA-E14, among which HBPA-E14-2 had the highest transfection efficiency. This shows that hyperbranched polymer materials can also be used for DNA transfection.

[0144] This example also tests the mRNA transfection efficiency of HBPA-E modified with 26 different terminal group modification compounds with group E in A549 cells. Specifically, the mRNA transfection efficiency of 26 HBPA-E modified with 26 terminal group modification compounds E1 to E26 and mRNA at a polymer:mRNA mass ratio of 75:1 in A549 cells was tested. The results are as follows: Fig.10 shown.

[0145] Fig.10The results showed that different end group modifications had an effect on the mRNA transfection efficiency, among which HBPA-E with various end group modifications showed significantly higher transfection efficiency than LipoMM, such as E2, E4, E9, E10, E12, E14, E15, E25 and E26. Except that the polymers modified with E11, E13, E22, E23 and E24 had significantly lower mRNA transfection efficiency than LipoMM, the polymers modified with E8, E17, E18 and E21 had the same mRNA transfection efficiency as LipoMM, and the other end group modified polymers had higher mRNA transfection efficiency than LipoMM.

[0146] Finally, this example tested the mRNA transfection efficiency of the in vivo delivery system with different HBPA-PEG contents in Table 2 (HBPA-PEG mass contents were 0, 0.1, 1, 5, 10, 25, 50 and 100%) in A549 cells. The results are shown in Fig.11 shown.

[0147] Fig.11 The results show that when the HPBA-PEG content is 0.1%, the mRNA delivery efficiency in the cells is only slightly lower than when the content is 0. As the HPBA-PEG content in the delivery system increases, when the content is 1-10%, the mRNA delivery efficiency is partially reduced. For example, when the HPBA-PEG content is 10%, the transfection efficiency is 70% of when the HPBA-PEG content is 0. When HPBA-PEG is greater than 25%, its cell delivery efficiency is greatly reduced, almost to 0. This also shows that although PEG is widely reported to have the effect of improving the stability of the delivery system in vivo, excessive PEG content will significantly inhibit the system's delivery efficiency to mRNA. Therefore, the HPBA-PEG content in the in vivo delivery system needs to be appropriate.

[0148] 5. Test of mRNA transfection effect of HBPA-E14 / HBPA-PEG in vivo

[0149] In this case, the mRNA transfection effect of HBPA-E14 / HBPA-PEG-2 in vivo was tested. The specific method is as follows: 4-6 week old C57 mice (weighing about 20g) were purchased from Guangdong Medical Experimental Animal Center and monitored and raised at the SPF (specific pathogen-free) environmental level. HBPA-E14 was mixed with HBPA-PEG2k and mRNA at a mass ratio of 75:8.3:1 to form HBPA-E14 / HBPA-PEG2k / mRNA complexes, where HBPA-PEG2k-1 was HBPA-PEG2k that was only PEG-modified but the remaining carboxyl groups were not capped, and HBPA-PEG2k-2 was HBPA-PEG2k that was PEG-modified and the remaining carboxyl groups were all capped. At the same time, HBPA-E14 was mixed with other HBPA-PEGs of different molecular weights (1k, 5k, 10k and 20k, all capped) according to the same PEG content, and then formed different HBPA-E14 / HBPA-PEG / mRNA complexes with mRNA. Then, HBPA-E14 / mRNA and different HBPA-E14 / HBPA-PEG / mRNA complexes were injected into mice through pulmonary administration (administered by inserting a pulmonary spray needle into the trachea, 5 μg dose) to observe their luciferase expression effects in vivo, with normal saline and simple mRNA as controls. Fig.12 As shown, Fig.12 The luciferase expression intensity of the HBPA-E14 / mRNA complex administered to the lung was taken as 1, and the intensity of the remaining groups relative to the HBPA-E14 / mRNA group was calculated.

[0150] Fig.12 In the figure, the abscissa "Control" represents the blank group, "mRNA" represents direct pulmonary administration of mRNA, "HBPA-E / mRNA" represents pulmonary administration of HBPA-E14 / mRNA complex, and "HBPA-E / HBPA-PEG / mRNA" represents pulmonary administration of HBPA-E14 / HBPA-PEG / mRNA complex (PEG1k-20k represent PEGs of different molecular weights, respectively).

[0151] Fig.12The results showed that no fluorescence was detected in the direct mRNA pulmonary administration and blank groups, while the HBPA-E / mRNA and HBPA-E / HBPA-PEG / mRNA groups showed significant luciferase expression effects, and all HBPA-E / HBPA-PEG / mRNA groups showed higher pulmonary delivery efficiency than the HBPA-E / mRNA group. Among them, the delivery efficiency of HBPA-E / HBPA-PEG2k-2 / mRNA was the highest, more than twice that of the HBPA-E / mRNA group. These results show that the in vivo delivery system composed of HBPA-E / HBPA-PEG can improve the efficiency of in vivo mRNA delivery compared with a single HBPA-E, and PEG2k is more effective than other molecular weight PEG modifications. In addition, after PEG modification of HBPA, the effect of capping the remaining carboxyl groups is better than that of uncapped groups.

[0152] According to the above experiments, HBPA-PEG modified with PEG molecular weight of 1k-20k can improve the efficiency of in vivo mRNA delivery to varying degrees. For PEG with a molecular weight less than 1k, such as PEG of 0.4-1k, these small molecular weight PEGs are widely used for drug modification and have been proven to have good safety (Phlebology. 2010; 25(3): 124-1311; https: / / asclera.com;); therefore, it can be reasonably expected that these molecular weight PEGs or PEGs with smaller molecular weights can also be safely used for in vivo delivery of the present application and can improve the efficiency of in vivo delivery to varying degrees.

[0153] The above contents are further detailed descriptions of the present application in combination with specific implementation methods, and it cannot be determined that the specific implementation of the present application is limited to these descriptions. For ordinary technicians in the technical field to which the present application belongs, several simple deductions or substitutions can be made without departing from the concept of the present application.

Claims

1. A terminal group-modified cationic polyester, characterized in that: The cationic polyester is a hyperbranched polymer formed by polymerization of three monomers, monomer P, monomer S and monomer M, or two monomers, monomer P and monomer S, with monomer T, and the terminal carboxyl group of the hyperbranched polymer is modified with polyethylene glycol; Wherein, monomer P is a cyclic lactone; Monomer S is an organic acid having two or more carboxyl groups at the terminal end; The monomer M is a compound containing two hydroxyl groups and a secondary or tertiary amine; Monomer T is a compound containing three or more hydroxyl groups; The number average molecular weight of polyethylene glycol is 0.4 to 20k.

2. The cationic polyester according to claim 1, characterized in that: Monomer P is a cyclic lactone with a fatty chain length of 6 to 35.

3. The cationic polyester according to claim 2, characterized in that: The monomer P is at least one of cyclohexyl lactone, cyclododecanolide, cyclopentadecanolide and cyclohexadecanolide.

4. The cationic polyester according to claim 1, characterized in that: The monomer S is an organic acid having a carbon chain length of 3 to 18.

5. The cationic polyester according to claim 4, characterized in that: The monomer S is at least one of adipic acid, sebacic acid and 1,2,3-propanetricarboxylic acid.

6. The cationic polyester according to claim 1, characterized in that: The monomer M is a compound having a carbon chain length of 4 to 36.

7. The cationic polyester according to claim 6, characterized in that: The monomer M is at least one of diethanolamine, methyldiethanolamine and ethyldiethanolamine.

8. The cationic polyester according to claim 1, characterized in that: The monomer T is a compound having a carbon chain length of 4 to 54.

9. The cationic polyester according to claim 8, characterized in that: The monomer T is at least one of trimethylolpropane, 3-(hydroxymethyl)-1,5-pentanediol, triethanolamine, and N,N,N',N'-tetrahydroxyethylethylenediamine.

10. The cationic polyester according to any one of claims 1 to 5, characterized in that: The molar ratio of polyethylene glycol to the hyperbranched polymer is 5:1 to 1:

10.

11. The cationic polyester according to claim 10, characterized in that: The number average molecular weight of the cationic polyester is 1-30k.

12. The cationic polyester according to claim 11, characterized in that: The carboxyl groups of the hyperbranched polymer are also capped with neutral compounds and / or modified with groups E, wherein the end group modified compound E providing the group E modification is a compound containing at least one primary amine, secondary amine or tertiary amine group.

13. The cationic polyester according to claim 12, characterized in that: The neutral compound includes at least one of a primary amine compound, an alcohol compound and an epoxy compound.

14. The cationic polyester according to claim 12, characterized in that: The primary amine compound is a small molecule compound containing one primary amine and having a molecular weight less than 1000.

15. The cationic polyester according to claim 12, characterized in that: The primary amine compound is at least one of ethylamine, propylamine, butylamine, ethanolamine and n-propanolamine.

16. The cationic polyester according to claim 12, characterized in that: The alcohol compound is at least one of ethanol, propanol and butanol.

17. The cationic polyester according to claim 12, characterized in that: The terminal group modified compound E is at least one of E1 to E26; 18. The cationic polyester according to claim 12, characterized in that: The cationic polyester is a hyperbranched polymer with a structure shown in Formula 1; Formula 1 Wherein, x, y, and z are independent integers ranging from 1 to 200. n is an integer from 0 to 200, j and k are integers from 0 to 30, l, m, o, p, q are independent integers from 1 to 20, R x is hydrogen, or a substituted or unsubstituted alkyl group containing 1 to 18 carbon atoms, or a substituted or unsubstituted aromatic group containing at least one benzene ring, or a substituted or unsubstituted heterocyclic group containing at least one heterocyclic ring, or a substituted or unsubstituted alkoxy group containing 1 to 18 carbon atoms and at least one oxygen atom; J is hydrogen, at least one of R1 is a group modified by polyethylene glycol, and the others are groups formed by end-capping of neutral compounds and / or groups E provided by end-group modification compound E, and in this case there is no R2; or, J is a carbonyl group, in this case at least one of R1 and R2 is a group modified by polyethylene glycol, and the others are groups formed by end-capping of neutral compounds and / or groups E provided by end-group modification compound E; In Formula 1, the truncation line represents a branch structure, the first monomer connected to the branch structure is P or S, and other monomers are subsequently connected to form a hyperbranched structure.

19. A living body delivery system, characterized in that: including a first polymer and a second polymer; The first polymer is the cationic polyester according to any one of claims 1 to 18; The second polymer is a polymer whose end group of the hyperbranched polymer in the cationic polyester according to any one of claims 1 to 18 is modified with a group E, wherein the end group modification compound E providing the group E modification is a compound containing at least one primary amine, secondary amine or tertiary amine group.

20. The living body delivery system according to claim 19, characterized in that: The terminal group modified compound E is at least one of E1 to E26; 21. The living body delivery system according to claim 19 or 20, characterized in that: The second polymer is a hyperbranched polymer having a structure shown in Formula 2; Formula 2 Wherein, x, y, and z are independent integers ranging from 1 to 200. n is an integer from 0 to 200, j and k are integers from 0 to 30, l, m, o, p, q are independent integers from 1 to 20, R x is hydrogen, or a substituted or unsubstituted alkyl group containing 1 to 18 carbon atoms, or a substituted or unsubstituted aromatic group containing at least one benzene ring, or a substituted or unsubstituted heterocyclic group containing at least one heterocyclic ring, or a substituted or unsubstituted alkoxy group containing 1 to 18 carbon atoms and at least one oxygen atom; J is hydrogen, R1 is modified by the group E provided by the terminal group modification compound E, and there is no R2; or, J is a carbonyl group, and both R1 and R2 are modified by the group E provided by the terminal group modification compound E; In Formula 2, the truncation line represents a branch structure, the first monomer connected to the branch structure is P or S, and other monomers are subsequently connected to form a hyperbranched structure.

22. The living body delivery system according to claim 19 or 20, characterized in that: The number average molecular weight of the second polymer is 1-30k.

23. The living body delivery system according to claim 19 or 20, characterized in that: The mass ratio of the first polymer to the second polymer is 0.01:9.99 to 2.5:7.

5.

24. Use of the cationic polyester according to any one of claims 1 to 18, or the in vivo delivery system according to any one of claims 19 to 23 in nucleic acid drug delivery.

25. The use according to claim 24, characterized in that: The nucleic acid drugs include but are not limited to mRNA, circular RNA, siRNA, microRNA, saRNA and DNA.

26. A nucleic acid delivery particle, characterized in that: including a packaging material and a nucleic acid encapsulated in the packaging material; The packaging material comprises the cationic polyester according to any one of claims 1 to 18, or the in vivo delivery system according to any one of claims 19 to 23; The nucleic acid is at least one of mRNA, circular RNA, siRNA, microRNA, saRNA and DNA.

27. The nucleic acid delivery particle according to claim 26, characterized in that: The particle size of the nucleic acid delivery particle is 30-500 nm.

28. A kit for nucleic acid drug delivery, characterized in that: It comprises at least one of the following components, (a) the cationic polyester according to any one of claims 1 to 18; (b) the living body delivery system according to any one of claims 19 to 23; (c) The nucleic acid delivery particle according to claim 26 or 27.

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