Branched poly (beta-amino ester) and preparation method and application thereof

Abstract

The invention relates to functionalized targeted branched poly (beta-amino ester), nanoparticles based on the functionalized targeted branched poly (beta-amino ester), and a preparation method and application of the functionalized targeted branched poly (beta-amino ester). In particular, the functionalized targeted branched poly (beta-amino ester) provided by the present disclosure can effectively deliver DNA, particularly functional DNA for treating and / or preventing various diseases and disorders.

Description

Technical Field

[0001] The present disclosure relates to functionalized targeted branched poly(β-amino esters), nanoparticles based thereon, methods for preparing the same, and uses thereof. Background Art

[0002] Gene therapy refers to the use of a certain method to introduce genetic material with specific functions into targeted tissues or cells to replace their diseased genes or inhibit the expression of diseased genes. Compared with emerging protein therapy and cell therapy, gene therapy can correct gene defects or mutations from the root, and has become one of the most promising treatments for various genetic diseases (such as RDEB, ichthyosis gene, cataract, hair loss, oral mucosal diseases and psoriasis-like syndrome (CAOP) syndrome, etc.) or acquired diseases (such as Alzheimer's disease, Parkinson's disease, rheumatoid arthritis, etc.).

[0003] However, the lack of safe and efficient vectors for cell and functional gene delivery has severely limited the clinical application of gene therapy. At present, gene vectors are mainly divided into viral vectors and non-viral vectors. Compared with viral vectors, non-viral vectors, especially cationic polymers, have the following advantages: 1) they can deliver larger gene fragments and have a wider range of applications; 2) they have low immunogenicity and higher safety; 3) they are flexible in design, easy to modify, and have tissue specificity; 4) they have good biodegradability and little cell damage; 5) they are simple to prepare, low in cost, and easy to mass produce. The current mainstream cationic polymers include polyethyleneimine (PEI), polydimethylaminoethyl methacrylate (PDMAEMA), polyamidoamine (PAMAM), etc., but these cationic polymers often have poor degradation properties, are prone to cause high post-transfection cytotoxicity, and the monomer types are often relatively single, resulting in limited clinical applications.

[0004] Due to the customizable composition, structure, relative molecular weight, biodegradability, high stability and good biocompatibility, linear and branched poly (β-amino esters) have shown certain application potential in clinical gene therapy for hereditary skin diseases, cancer, and gene delivery in various types of cell lines. However, the composite nanoparticles formed by poly (β-amino ester) cationic polymers and DNA are often retained in lysosomes after entering cells, and a small number of composite nanoparticles escape from lysosomes through the proton sponge effect, which can also cause certain cytotoxicity. In addition, the particle size of the composite nanoparticles is too large, which makes nuclear internalization difficult and the in vivo delivery efficiency is limited, which seriously hinders subsequent gene transfection and greatly limits its application research in clinical transformation. Summary of the invention

[0005] In order to overcome the shortcomings of the above-mentioned prior art, the present disclosure provides a branched poly (β-amino ester), nanoparticles based thereon, preparation methods thereof and uses thereof. In particular, the branched poly (β-amino ester) provided by the present disclosure can effectively deliver polynucleotides, in particular functional DNA for treating and / or preventing various diseases and disorders.

[0006] In one aspect, the present disclosure provides a branched poly(β-amino ester) having a structure shown in Formula (I), Formula (II), Formula (III), Formula (IV) or Formula (V):

[0007]

[0008]

[0009] in,

[0010] W1, W2, W3, W4 and W5 are each independently selected from alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkylcycloalkylalkyl, alkylheterocyclylalkyl, alkylarylalkyl or alkylheteroarylalkyl, wherein said alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkylcycloalkylalkyl, alkylheterocyclylalkyl, alkylarylalkyl and alkylheteroarylalkyl are optionally replaced by one or more R W replace;

[0011] L is selected from alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkylcycloalkyl, alkylheterocyclyl, alkylcycloalkylalkyl, alkylheterocyclylalkyl, alkylarylalkyl, alkylheteroarylalkyl, or heteroalkylarylalkylarylheteroalkyl, wherein said alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkylcycloalkylalkyl, alkylheterocyclylalkyl, alkylarylalkyl, alkylheteroarylalkyl, and heteroalkylarylalkylarylheteroalkyl are optionally replaced by one or more R L replace;

[0012] Each R1 is independently selected from R1 N or T;

[0013] R1 N is selected from alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkylcycloalkyl, alkylheterocyclyl, alkylaryl, alkylheteroaryl or nitrogen protecting group, wherein the alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkylcycloalkyl, alkylheterocyclyl, alkylaryl and alkylheteroaryl are optionally substituted by one or more R N replace;

[0014] Each R2 is independently selected from R2 N or T;

[0015] R2 Nis selected from alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkylcycloalkyl, alkylheterocyclyl, alkylaryl, alkylheteroaryl or a nitrogen protecting group, wherein the alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkylcycloalkyl, alkylheterocyclyl, alkylaryl, alkylheteroaryl, alkylcycloalkylalkyl, alkylheterocyclylalkyl, alkylarylalkyl and alkylheteroarylalkyl are optionally substituted with one or more R N replace;

[0016] Each T is independently an organelle targeting moiety;

[0017] Each R W , R L , R L and R N Each independently selected from the group consisting of halogen, cyano, nitro, oxy, -OR a 、-SR a 、-N(R a )2. -C(O)OR a 、-C(O)N(R a )2、-N(R a )C(O)OR a 、-OC(O)OR a 、-OC(O)N(R a )2、-N(R a )C(O)N(R a )2. -S(O)R a 、-S(O)OR a 、-S(O)N(R a )2. -OS(O)R a 、-N(R a )S(O)R a 、-S(O)2R a 、-S(O)2OR a 、-S(O)2N(R a )2, -OS(O)2R a 、-N(R a )S(O)2R a , alkyl, heteroalkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl;

[0018] Each R a are each independently selected from hydrogen or alkyl;

[0019] m1, m2, m3, m4, m5, m6, m7, m8, m9, m10, m11 and m12 are each independently in the range of 1 to 100.

[0020] In another aspect, the present disclosure provides a method for preparing a branched poly(β-amino ester) of formula (I), formula (II), formula (III), formula (IV), or formula (V), comprising:

[0021] 1-1) a multi-acrylate monomer having formula (I-1), formula (II-1), formula (III-1) or formula (IV-1):

[0022]

[0023] A branched poly(β-amino ester) P1 having a terminal double bond is obtained by reacting a diacrylate monomer having formula (2) and one or more amine monomers having formula (3) through Michael addition reaction:

[0024]

[0025] 1-2) subjecting the branched poly(β-amino ester) P1 obtained in step 1-1) to a Michael addition reaction with one or more end-capping amine monomers having the formula (4) to obtain a branched poly(β-amino ester) having a structure represented by the formula (I), formula (II), formula (III) or formula (IV):

[0026] R2-NH2

[0027] Formula (4)

[0028] or,

[0029] 2-1) A diamine monomer having the formula (V-1) is reacted with a diacrylate monomer having the formula (2) and one or more amine monomers having the formula (3) by Michael addition reaction to obtain a branched poly(β-amino ester) P2 having a double bond at the end:

[0030]

[0031] 2-2) The branched poly(β-amino ester) P2 prepared in step 2-1) is subjected to a Michael addition reaction with one or more end-capping amine monomers having the formula (4) to prepare a branched poly(β-amino ester) having a structure represented by the formula (V):

[0032] R2-NH2

[0033] Formula (4).

[0034] In another aspect, the present disclosure provides a composition comprising a branched poly(β-amino ester) of Formula (I), Formula (II), Formula (III), Formula (IV), or Formula (V) and a polynucleotide.

[0035] In another aspect, the present disclosure provides a method of delivering a polynucleotide to a subject, comprising administering to the subject a composition of the present disclosure.

[0036] In another aspect, the present disclosure provides a method of treating a disease in a subject, comprising administering to the subject a composition of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] FIG1 shows a schematic diagram of the synthesis of branched poly(β-amino ester)s of Examples 1-4.

[0038] Figure 2 It is the GPC curve of the branched poly (β-amino ester) of Example 1-4.

[0039] Figure 3 The branched poly (β-amino ester) of Examples 1-4 1 HNMR spectrum.

[0040] Figure 4 The PicoGreen results of the affinity performance test of the branched poly (β-amino ester) of Examples 1-4 to TGM1 DNA at different mass ratios.

[0041] Figure 5 The DLS characterization results of the particle size of the composite nanoparticles formed by the branched poly (β-amino ester) and TGM1 DNA in Examples 1-4 at different mass ratios.

[0042] Figure 6 The DLS characterization results of the Zeta potential of the composite nanoparticles formed by the branched poly (β-amino ester) and TGM1 DNA in Examples 1-4 at different mass ratios.

[0043] Figure 7 TEM microscopic morphology of the nanoparticles formed by the branched poly (β-amino ester) and TGM1 DNA of Examples 1-4.

[0044] Figure 8 The results are the characterization results of the cellular endocytosis efficiency of the composite nanoparticles formed by the branched poly (β-amino ester) of Examples 1 and 2 and DNA labeled with AF647 fluorescent dye.

[0045] Fig. 9 The organelle targeting properties of the composite nanoparticles formed by the branched poly (β-amino ester) of Examples 1 and 2 and DNA labeled with AF647 fluorescent dye were characterized.

[0046] Fig.10 The organelle targeting properties of the composite nanoparticles formed by the branched poly (β-amino ester) of Examples 1 and 2 and DNA labeled with AF647 fluorescent dye were characterized.

[0047] Fig.11The results are the transfection efficiency test results of the composite nanoparticles formed by the branched poly (β-amino ester) of Examples 1-4 and GFP DNA at different mass ratios in mouse embryonic fibroblast (3T3) cells.

[0048] Fig.12 The results of flow cytometry detection of the transfection efficiency of composite nanoparticles formed by branched poly (β-amino ester) of Examples 1-4 and GFP DNA in 3T3 cells.

[0049] Fig.13 The alarmBlue detection results of the viability of suspended cells after transfection of the composite nanoparticles formed by branched poly (β-amino ester) and GFP DNA in five different tissue cells (A2780 cells, 293T cells, 3T3 cells, SW1353 cells, Vero cells) in Examples 1 and 2.

[0050] Fig.14 The fluorescence detection results of the transfection efficiency of composite nanoparticles formed by the branched poly (β-amino ester) of Examples 1-4 and GFP DNA at different mass ratios in suspended cells (293F).

[0051] Fig.15 The results of cell flow cytometry detection of the transfection efficiency of composite nanoparticles formed by the branched poly (β-amino ester) of Example 1-4 and GFP DNA at a mass ratio of 20:1 in suspended cells (293F).

[0052] Fig.16 (a), 16(b), 16(c) and 16(d) are the alarmBlue detection results of the survival rate of suspended cells after transfection of composite nanoparticles formed by branched poly (β-amino ester) and GFP DNA in Examples 1-4 at mass ratios of 120:1, 100:1, 80:1, 60:1, 40:1, 20:1, 10:1 and 5:1, respectively, wherein the mass ratios are 120:1, 100:1, 80:1, 60:1, 40:1, 20:1, 10:1 and 5:1, respectively.

[0053] Fig.17 The qPCR detection results of the transfection efficiency of the composite nanoparticles formed by the branched poly (β-amino ester) of Examples 1-2 and COL7A1 DNA, TGM1 DNA and MBTPS1 DNA in HaCaT cells, respectively.

[0054] Fig.18The qPCR detection results of the transfection efficiency of the composite nanoparticles formed by the branched poly (β-amino ester) of Examples 1-2 and COL7A1 DNA, TGM1 DNA and MBTPS1 DNA in human primary fibroblast (NHF) cells, respectively.

[0055] Fig.19 The Western Blot test results show the transfection efficiency of the composite nanoparticles formed by the branched poly (β-amino ester) of Examples 1-2 and COL7A1 DNA, TGM1 DNA and MBTPS1 DNA in NHF cells and HaCaT cells, respectively.

[0056] Fig. 20 The Western Blot test results show the transfection efficiency of the composite nanoparticles formed by the branched poly (β-amino ester) of Example 1-2 and MBTPS1 DNA in C57 mice.

[0057] Fig.21 The Western Blot test results show the transfection efficiency of the composite nanoparticles formed by the branched poly (β-amino ester) of Example 1-2 and COL7A1 DNA in C57 mice.

[0058] Fig. 22 The HE staining results of the in vivo toxicity of the composite nanoparticles formed by the branched poly (β-amino ester) of Example 1-2 and MBTPS1 DNA after transfection in C57 mice.

[0059] Fig.23 The HE staining results of the in vivo toxicity of the composite nanoparticles formed by the branched poly (β-amino ester) of Example 1-2 and COL7A1 DNA after transfection in C57 mice.

[0060] Fig.24 The fluorescence results of the transfection efficiency of the composite nanoparticles formed by the branched poly (β-amino ester) and luciferase DNA of Example 1-2 administered by tail vein injection in C57 mice.

[0061] Fig.25 The results of the section are the transdermal efficiency of the composite nanoparticles formed by the branched poly (β-amino ester) and Cy3-labeled DNA in Example 1-2 administered by transdermal application in SD rats.

[0062] Fig.26 The results of Western Blot detection of the transfection efficiency of composite nanoparticles formed by the branched poly (β-amino ester) of Example 1-2 and COL1A1 DNA, CLO3A1 DNA or silk fibroin DNA administered transdermally in SD rats. DETAILED DESCRIPTION

[0063] Reference will now be made in detail to certain embodiments of the present disclosure, examples of which are shown in the attached structures and formulas. Although the present disclosure will be described in conjunction with the enumerated embodiments, it should be understood that the embodiments are not intended to limit the present disclosure to those embodiments. On the contrary, the present disclosure is intended to cover all alternatives, modifications and equivalents, which may be included within the scope of the present disclosure as defined in the claims. Those skilled in the art will recognize that many methods and materials similar to or equivalent to the methods and materials described herein may be used in practicing the present disclosure. The present disclosure is by no means limited to the methods and materials described. In the event that one or more of the incorporated references and similar materials (including but not limited to defined terms, term usage, described technology, etc.) are different from or contradictory to the present application, the present disclosure shall prevail. All references, patents, and patent applications cited in the present disclosure are hereby incorporated by reference in their entirety.

[0064] It should be understood that certain features of the disclosure described in the context of separate embodiments for the sake of clarity may also be provided in combination in a single embodiment. Conversely, various features of the disclosure described in the context of a single embodiment for the sake of brevity may also be provided separately or in any suitable sub-combination. It must be noted that, as used in the specification and the appended claims, the singular forms "a", "an", and "the" include their plural forms unless the context clearly indicates otherwise.

[0065] definition

[0066] Definitions of specific functional groups and chemical terms are described in more detail below. For purposes of this disclosure, chemical elements are identified according to the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th ed., inside cover, and specific functional groups are generally defined as described therein. In addition, general principles of organic chemistry as well as specific functional moieties and reactivities are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March, March's Advanced Organic Chemistry, 5th ed., John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; Carruthers, Some Modern Methods of Organic Synthesis, 3rd ed., Cambridge University Press, 1996. Press, Cambridge, 1987; the entire contents of each of which are incorporated herein by reference.

[0067] In various places of this disclosure, linking substituents are described. Where a structure clearly requires a linking group, the Markush variables listed for that group are to be understood as linking groups. For example, if a structure requires a linking group and the Markush group definition for that variable lists "alkyl", it is to be understood that the "alkyl" means a linking alkylene group.

[0068] As used herein, the term "substituted", whether or not there is the term "optionally" in front, means that one or more hydrogens of the specified part are replaced by suitable substituents. It should be understood that "replacement" or "substituted" includes the following implicit restrictions: the substitution is consistent with the allowed valence of the substituted atom, and the substitution produces a stable or chemically feasible compound, such as a compound that will not spontaneously transform by rearrangement, cyclization, elimination, etc. Unless otherwise indicated, the "optionally substituted" group can have a suitable substituent at each substitutable position of the group, and when one or more positions in any given structure can be substituted by more than one substituent selected from a specified group, the substituent can be the same or different at each position. It should be understood by those skilled in the art that, when appropriate, the substituent itself can be substituted. Unless specifically stated as "unsubstituted", reference to the chemical part herein should be understood to include substituted variables. For example, reference to "aryl" groups or parts implicitly includes both substituted and unsubstituted variables.

[0069] As used herein, when the term "about" is used before a given range or value (eg, temperature, time, amount, and concentration), it indicates an approximation that can vary by ±10%, ±5%, or ±1% of the given range or value.

[0070] When any variable (such as R i ) occurs more than one time in any constituent or formula for a compound, its definition at each occurrence is independent of its definition at every other occurrence. Thus, for example, if a group is shown as being comprised of 0 to 2 R i If the group is partially substituted, then the group may be optionally substituted with up to two R i Partially substituted, and R i In each occurrence, independently selected from R i Also, combinations of substituents and / or variables are permissible, but only if such combinations result in stable compounds.

[0071] As used herein, the term “C i-j " indicates a range of carbon atoms, wherein i and j are integers, and the range of carbon atoms includes the endpoints (i.e., i and j) and every integer point therebetween, and wherein j is greater than i. For example, C 1-6 Indicates a range of one to six carbon atoms, including one carbon atom, two carbon atoms, three carbon atoms, four carbon atoms, five carbon atoms, and six carbon atoms. In some embodiments, the term “C 1-12 ” indicates 1 to 12, especially 1 to 10, especially 1 to 8, especially 1 to 6, especially 1 to 5, especially 1 to 4, especially 1 to 3 or especially 1 to 2 carbon atoms.

[0072] As used herein, the term "alkyl" refers to a saturated straight or branched chain hydrocarbon group, whether used as part of another term or independently. i-j "Alkyl" refers to an alkyl group having i to j carbon atoms. In some embodiments, the alkyl group contains 1 to 12 carbon atoms. In some embodiments, the alkyl group contains 1 to 11 carbon atoms. In some embodiments, the alkyl group contains 1 to 11 carbon atoms, 1 to 10 carbon atoms, 1 to 9 carbon atoms, 1 to 8 carbon atoms, 1 to 7 carbon atoms, 1 to 6 carbon atoms, 1 to 5 carbon atoms, 1 to 4 carbon atoms, 1 to 3 carbon atoms, or 1 to 2 carbon atoms. Examples of alkyl groups include, but are not limited to, methyl, ethyl, 1-propyl (n-propyl), 2-propyl (isopropyl), 1-butyl (n-butyl), 2 -methyl-1-propyl (isobutyl), 2-butyl (sec-butyl), 2-methyl-2-propyl (tert-butyl), 1-pentyl (n-pentyl), 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl, 3,3-dimethyl-2-butyl, 1-heptyl, 1-octyl, etc. "C 1-12 Examples of "alkyl" include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, and dodecyl. 1-6 Examples of "alkyl" are methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl, 3,3-dimethyl-2-butyl, and the like.

[0073] The alkyl group may be optionally substituted with a substituent that independently replaces one or more hydrogen atoms on one or more carbons of the alkyl group. Examples of such substituents may include, but are not limited to, halogen, hydroxy, cyano, nitro, azido, acyl, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, alkoxy, haloalkyl, haloalkoxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylaryl, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, phosphate, phosphonic acid, The alkylamino groups of the present invention include alkylamino, ...

[0074] Whether used as part of another term or independently, as used herein, the term "aryl" refers to monocyclic and polycyclic systems having a total of 5 to 20 ring members, which may be optionally substituted independently by one or more substituents described herein, wherein at least one ring in the system is aromatic and wherein each ring in the system contains 3 to 12 ring members. Examples of "aryl" include, but are not limited to, phenyl, biphenyl, naphthyl, anthracenyl, etc., which may carry one or more substituents. As used herein, groups in which an aromatic ring is fused to one or more additional rings are also included within the scope of the term "aryl". In the case of a polycyclic system, only one ring is required to be aromatic (e.g., 2,3-dihydroindole), but all rings may be aromatic (e.g., quinoline). The second ring may also be a fused ring, a bridged ring, or a spiro ring. Examples of polycyclic aryls include, but are not limited to, benzofuranyl, indanyl, phthalimide, naphthimide, phenanthracenyl, or tetrahydronaphthyl, etc. Aryl may be optionally substituted by one or more substituents as described herein at one or more ring positions.

[0075] As used herein, the terms "cycloalkyl", "carbocyclyl" and "carbocycle" are interchangeable and, whether used as part of another term or independently, refer to monovalent, saturated or partially unsaturated or fully unsaturated monocyclic and polycyclic systems, which may be optionally independently substituted with one or more substituents described herein, wherein all ring atoms are carbon and contain at least three ring-forming carbon atoms. In some embodiments, the cycloalkyl may contain 3 to 12 ring-forming carbon atoms, 3 to 10 ring-forming carbon atoms, 3 to 9 ring-forming carbon atoms, 3 to 8 ring-forming carbon atoms, 3 to 7 ring-forming carbon atoms, 3 to 6 ring-forming carbon atoms, 3 to 5 ring-forming carbon atoms, 4 to 12 ring-forming carbon atoms, 4 to 10 ring-forming carbon atoms, 4 to 9 ring-forming carbon atoms, 4 to 8 ring-forming carbon atoms, 4 to 7 ring-forming carbon atoms, 4 to 6 ring-forming carbon atoms, 4 to 5 ring-forming carbon atoms. Cycloalkyl may be saturated or partially unsaturated. Cycloalkyl groups may be optionally substituted independently with one or more substituents described herein. In some embodiments, cycloalkyl groups may be saturated cyclic alkyl groups. In some embodiments, cycloalkyl groups may be unsaturated cyclic alkyl groups containing at least one double bond or triple bond in their ring system.

[0076] In some embodiments, the cycloalkyl group may be a saturated or unsaturated monocyclic carbocyclic ring system, examples of which include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl, cyclohexadienyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl, and cyclododecyl.

[0077] In some embodiments, cycloalkyl can be a saturated or unsaturated polycyclic (e.g., bicyclic and tricyclic) carbocyclic ring system, which can be a condensed ring, a spirocyclic ring or a bridged ring system. As used herein, the term "condensed ring" refers to a ring system with two rings sharing two adjacent atoms, the term "spirocyclic ring" refers to a ring system with two rings connected by a single common atom, and the term "bridged ring" refers to a ring system with two rings sharing three or more atoms. Examples of fused carbocyclic groups include but are not limited to naphthyl, benzopyrenyl, anthracenyl, acenaphthenyl, fluorenyl, etc. Examples of spiro carbocyclic groups include but are not limited to spiro [5.5] undecyl, spiropentadienyl, spiro [3.6] decyl, etc. Examples of bridged carbocyclic groups include, but are not limited to, bicyclo[1,1,1]pentenyl, bicyclo[2,2,1]heptenyl, bicyclo[2.2.1]heptyl, bicyclo[2.2.2]octyl, bicyclo[3.3.1]nonyl, bicyclo[3.3.3]undecyl, and the like.

[0078] As used herein, the term "haloalkyl" refers to an alkyl group substituted with one or more halogen atoms which independently replace one or more hydrogen atoms on one or more carbons of the alkyl group.

[0079] As used herein, the term "heteroalkyl" refers to an alkyl group, at least one of whose carbon atoms is replaced by a heteroatom selected from N, O, S or P. A heteroalkyl group can be a carbon group or a heteroatom group (i.e., a heteroatom can appear in the middle or at a terminal of the group), and can be optionally substituted independently with one or more substituents described herein. The term "heteroalkyl" encompasses alkoxy and heteroalkoxy.

[0080] As used herein, the term "heteroatom" refers to nitrogen, oxygen, sulfur, or phosphorus, and includes any oxidized form of nitrogen or sulfur and any quaternized form of basic nitrogen.

[0081] Whether used as part of another term or independently, as used herein, the term "heteroaryl" refers to an aryl group having one or more heteroatoms in addition to carbon atoms and optionally independently substituted by one or more substituents described herein. Examples of heteroaryl groups include, but are not limited to, thienyl, furyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, benzofuranyl, and pteridinyl. Heteroaryl also includes a group in which a heteroaromatic ring is fused to one or more aryl, cycloaliphatic, or heterocyclic rings, wherein an attachment group or point of attachment is located on the heteroaromatic ring. Non-limiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzothiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenathiazinyl, phenoxazinyl, tetrahydroquinolyl, tetrahydroisoquinolyl and pyrido[2,3-b]-1,4-oxazin-3(4H)-one. In some embodiments, the term "5- to 10-membered heteroaryl" refers to a 5- to 6-membered heteroaryl ring having 1 to 3 heteroatoms independently selected from nitrogen, oxygen, sulfur or phosphorus, or an 8- to 10-membered bicyclic heteroaryl ring having 1 to 4 heteroatoms independently selected from nitrogen, oxygen, sulfur or phosphorus. In certain embodiments, the term "5- to 12-membered heteroaryl" refers to a 5- to 6-membered heteroaryl ring having 1 to 3 heteroatoms independently selected from nitrogen, oxygen, sulfur, or phosphorus, or an 8- to 12-membered bicyclic heteroaryl ring having 1 to 4 heteroatoms independently selected from nitrogen, oxygen, sulfur, or phosphorus.

[0082] As used herein, the term "heterocycle" or "heterocyclyl" refers to a saturated, partially unsaturated or completely unsaturated carbocyclic group, wherein one or more ring atoms are independently selected from oxygen, sulfur, nitrogen, phosphorus, etc., and the remaining ring atoms are carbon, wherein one or more ring atoms may be optionally independently substituted by one or more substituents. In some embodiments, the heterocyclic group is a saturated heterocyclic group. In some embodiments, the heterocyclic group is an unsaturated heterocyclic group having one or more double bonds in its ring system. In some embodiments, the heterocyclic group may contain any oxidized form of carbon, nitrogen, sulfur or phosphorus and any quaternized form of basic nitrogen. "Heterocyclic group" also includes groups in which the heterocyclic group is fused to a saturated, partially unsaturated or completely unsaturated (i.e., aromatic) carbocyclic or heterocyclic ring. Where possible, the heterocyclic group may be carbon-connected or nitrogen-connected. In some embodiments, the heterocyclic ring is carbon-connected. In some embodiments, the heterocyclic ring is nitrogen-connected. For example, a group derived from pyrrole may be pyrrole-1-yl (nitrogen-connected) or pyrrole-3-yl (carbon-connected). Furthermore, a group derived from imidazole may be imidazol-1-yl (nitrogen attached) or imidazol-3-yl (carbon attached).

[0083] In some embodiments, the term "3 to 12 membered heterocyclic radical" refers to a 3 to 12 membered saturated or partially unsaturated monocyclic or polycyclic heterocyclic ring system having 1 to 3 heteroatoms independently selected from nitrogen, oxygen or sulfur. Condensed rings, spirocyclic rings and bridged ring systems are also included within the scope of this definition. Examples of monocyclic heterocyclic radicals include, but are not limited to, oxetane, 1,1-dioxothietane pyrrolidinyl, tetrahydrofuranyl, tetrahydrothienyl, pyrrolyl, furanyl, thienyl, pyrazolyl, imidazolyl, triazolyl, oxazolyl, thiazolyl, piperidinyl, piperazinyl, morpholinyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazinyl, pyridonyl, pyrimidonyl, pyrazinonyl, pyrimidonyl, pyridazinonyl, pyrrolidinyl, triazinonyl, etc. Examples of fused heterocyclic groups include, but are not limited to, phenyl fused rings or pyridyl fused rings, such as quinolyl, isoquinolyl, quinoxalinyl, quinolizinyl, quinazolinyl, azaindolizinyl, pteridinyl, chromenyl, isochromenyl, indolyl, isoindolyl, indolizinyl, indazolyl, purinyl, benzofuranyl, isobenzofuranyl, benzimidazolyl, benzothiophenyl, benzothiazolyl, carbazolyl, phenazinyl, phenothiazinyl, phenanthridinyl, imidazo[1,2-a]pyridinyl, [1,2,4]triazolo[4,3-a]pyridinyl, [1,2,3]triazolo[4,3-a]pyridinyl, etc. Examples of spiro heterocyclic groups include, but are not limited to, spiropyranyl, spirooxazinyl, etc. Examples of bridged heterocyclic groups include, but are not limited to, morphanyl, hexamethylenetetramine, 3-aza-bicyclo[3.1.0]hexane, 8-aza-bicyclo[3.2.1]octane, 1-aza-bicyclo[2.2.2]octane, 1,4-diazabicyclo[2.2.2]octane (DABCO), and the like.

[0084] As used herein, the term "alkylcycloalkyl" refers to a monovalent or divalent alkyl-cycloalkyl group. When the alkylcycloalkyl group is monovalent, it may be -alkyl-cycloalkyl or alkyl-cycloalkyl-. When the alkylcycloalkyl group is divalent, it may be -alkyl-cycloalkyl- or -cycloalkyl-alkyl-.

[0085] As used herein, the term "alkylheterocyclyl" refers to a monovalent or divalent alkyl-heterocyclyl. When the alkylheterocyclyl is monovalent, it may be -alkyl-heterocyclyl or alkyl-heterocyclyl-. When the alkylheterocyclyl is divalent, it may be -alkyl-heterocyclyl- or -heterocyclyl-alkyl-.

[0086] As used herein, the term "alkylaryl" refers to a monovalent or divalent alkyl-aryl group. When the alkylaryl group is monovalent, it may be -alkyl-aryl or alkyl-aryl-. When the alkylaryl group is divalent, it may be -alkyl-aryl- or -aryl-alkyl-.

[0087] As used herein, the term "alkylheteroaryl" refers to a monovalent or divalent alkyl-heteroaryl. When the alkylheteroaryl is monovalent, it may be -alkyl-heteroaryl or alkyl-heteroaryl-. When the alkylheteroaryl is divalent, it may be -alkyl-heteroaryl- or -heteroaryl-alkyl-.

[0088] As used herein, the term "alkylcycloalkylalkyl" refers to a monovalent -alkyl-cycloalkyl-alkyl or a divalent -alkyl-cycloalkyl-alkyl-.

[0089] As used herein, the term "alkylheterocyclylalkyl" refers to a monovalent -alkyl-heterocyclyl-alkyl or a divalent -alkyl-heterocyclyl-alkyl-.

[0090] As used herein, the term "alkylarylalkyl" refers to a monovalent -alkyl-aryl-alkyl or a divalent -alkyl-aryl-alkyl-.

[0091] As used herein, the term "alkylheteroarylalkyl" refers to a monovalent -alkyl-heteroaryl-alkyl or a divalent -alkyl-heteroaryl-alkyl-.

[0092] As used herein, the term "heteroalkylarylalkylarylheteroalkyl" refers to a monovalent -heteroalkyl-aryl-alkyl-aryl-heteroalkyl or a divalent -heteroalkyl-aryl-alkyl-aryl-heteroalkyl-.

[0093] As used herein, the term "nitrogen protecting group" together with the nitrogen atom to which the nitrogen protecting group is attached includes, but is not limited to, the following groups: methylcarbamate, ethylcarbamate, 9-fluorenylmethylcarbamate (Fmoc), 9-(2-sulfo)fluorenylmethylcarbamate, 9-(2,7-dibromo)fluoroenylmethylcarbamate, 2,7-di-tert-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthenyl)]methylcarbamate (DBD-Tmoc), 4-methoxybenzamidocarbamate (Phenoc), 2,2,2-trichloroethylcarbamate (Tr oc), 2-trimethylsilylethylcarbamate (Teoc), 2-phenylethylcarbamate (hZ), 1-(1-adamantyl)-1-methylethylcarbamate (Adpoc), 1,1-dimethyl-2-haloethylcarbamate, 1,1-dimethyl-2,2-dibromoethylcarbamate (DB-t-BOC), 1,1-dimethyl-2,2,2-trichloroethylcarbamate (TCBOC), 1-methyl-1-(4-biphenylyl)ethylcarbamate (Bpoc), 1-(3,5-di-tert-butylphenyl)-1-methylethylcarbamate (t-Bu meoc), 2-(2'- and 4'-pyridyl)ethylcarbamate (Pyoc), 2-(N,N-dicyclohexylamido)ethylcarbamate, tert-butylcarbamate (BOC or Boc), 1-adamantylcarbamate (Adoc), vinylcarbamate (Voc), allylcarbamate (Alloc), 1-isopropylallylcarbamate (Ipaoc), cinnamylcarbamate (Coc), 4-nitrocinnamylcarbamate (Noc), 8-quinolylcarbamate, N-hydroxypiperidinylcarbamate, alkyldithiocarbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz), p-nitrobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzyl carbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzyl carbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate, 2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate, 2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3-dithianyl)]methyl carbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc), 2,4-dimethylthiophenylcarbamate (Bmpc), 2-phosphinoethylcarbamate (Peoc), 2-triphenylphosphinoisopropylcarbamate (Ppoc), 1,1-dimethyl-2-cyanoethylcarbamate, m-chloro-p-acyloxybenzylcarbamate, p-(dihydroxyboryl)benzylcarbamate, 5-benzisoxazolylmethylcarbamate, 2-(trifluoromethyl)-6-chromonylmethylcarbamate (Tcroc), m-nitrophenylcarbamate, 3,5-dimethoxy benzyl carbamate, o-nitrobenzyl carbamate, 3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl (o-nitrophenyl) methyl carbamate, tert-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzyl carbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentyl carbamate, cyclopropyl methyl carbamate, p-decyloxybenzyl carbamate, 2,2-dimethoxyacyl vinyl carbamate, o-(N,N-dimethylformamido)benzyl 1,1-dimethyl-3-(N,N-dimethylformamido)propylcarbamate, 1,1-dimethylpropargylcarbamate, di(2-pyridyl)methylcarbamate, 2-furylmethylcarbamate, 2-iodoethylcarbamate, isobornylcarbamate, isobutylcarbamate, isonicotinoylcarbamate, p-(p-methoxyphenylazo)benzylcarbamate, 1-methylcyclobutylcarbamate, 1-methylcyclohexylcarbamate, 1-methyl-1- cyclopropylmethylcarbamate, 1-methyl-1-(3,5-dimethoxyphenyl)ethylcarbamate, 1-methyl-1-(p-phenylazophenyl)ethylcarbamate, 1-methyl-1-phenylethylcarbamate, 1-methyl-1-(4-pyridyl)ethylcarbamate, phenylcarbamate, p-(phenylazo)benzylcarbamate, 2,4,6-tri-tert-butylphenylcarbamate, 4-(trimethylammonium)benzylcarbamate, and 2,4,6-trimethylbenzylcarbamate. In some embodiments, the nitrogen protecting group is Boc, Cbz, Fmoc, trifluoroacetyl, trityl, or acetyl,

[0094] As used herein, the term "halo" or "halogen" refers to an atom selected from fluorine, chlorine, bromine and iodine.

[0095] As used herein, the term "haloalkyl" refers to an alkyl group substituted with one or more halogen atoms which independently replace one or more hydrogen atoms on one or more carbons of the alkyl group.

[0096] As used herein, the term "nitro" refers to a -NO2 group.

[0097] As used herein, the term "oxy" refers to a =0 group.

[0098] As used herein, the term "cyano" refers to a group of the formula "-C≡N".

[0099] As used herein, the term "organelle targeting moiety" refers to a moiety that targets an organelle.

[0100] As used herein, the term "endoplasmic reticulum targeting moiety" refers to a moiety that targets the endoplasmic reticulum.

[0101] As used herein, the term "molecular weight" is the weight average molecular weight (Mw).

[0102] Branched poly(β-amino ester)

[0103] In one aspect, the present disclosure provides a branched poly(β-amino ester) having a structure shown in Formula (I), Formula (II), Formula (III), Formula (IV) or Formula (V):

[0104]

[0105] in,

[0106] W1, W2, W3, W4 and W5 are each independently selected from alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkylcycloalkylalkyl, alkylheterocyclylalkyl, alkylarylalkyl or alkylheteroarylalkyl, wherein said alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkylcycloalkylalkyl, alkylheterocyclylalkyl, alkylarylalkyl and alkylheteroarylalkyl are optionally replaced by one or more R W replace;

[0107] L is selected from alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkylcycloalkyl, alkylheterocyclyl, alkylcycloalkylalkyl, alkylheterocyclylalkyl, alkylarylalkyl, alkylheteroarylalkyl, or heteroalkylarylalkylarylheteroalkyl, wherein said alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkylcycloalkylalkyl, alkylheterocyclylalkyl, alkylarylalkyl, alkylheteroarylalkyl, and heteroalkylarylalkylarylheteroalkyl are optionally replaced by one or more R L replace;

[0108] Each R1 is independently selected from R1 N or T;

[0109] R1 Nis selected from alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkylcycloalkyl, alkylheterocyclyl, alkylaryl, alkylheteroaryl or nitrogen protecting group, wherein the alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkylcycloalkyl, alkylheterocyclyl, alkylaryl and alkylheteroaryl are optionally substituted by one or more R N replace;

[0110] Each R2 is independently selected from R2 N or T;

[0111] R2 N is selected from alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkylcycloalkyl, alkylheterocyclyl, alkylaryl, alkylheteroaryl or a nitrogen protecting group, wherein the alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkylcycloalkyl, alkylheterocyclyl, alkylaryl, alkylheteroaryl, alkylcycloalkylalkyl, alkylheterocyclylalkyl, alkylarylalkyl and alkylheteroarylalkyl are optionally substituted with one or more R N replace;

[0112] Each T is independently an organelle targeting moiety;

[0113] Each R W , R L , R L and R N Each independently selected from the group consisting of halogen, cyano, nitro, oxy, -OR a 、-SR a 、-N(R a )2. -C(O)OR a 、-C(O)N(R a )2、-N(R a )C(O)OR a 、-OC(O)OR a 、-OC(O)N(R a )2、-N(R a )C(O)N(R a )2. -S(O)R a 、-S(O)OR a 、-S(O)N(R a )2. -OS(O)R a 、-N(R a )S(O)R a 、-S(O)2R a 、-S(O)2OR a 、-S(O)2N(R a )2, -OS(O)2R a 、-N(R a )S(O)2R a, alkyl, heteroalkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl;

[0114] Each R a are each independently selected from hydrogen or alkyl;

[0115] m1, m2, m3, m4, m5, m6, m7, m8, m9, m10, m11 and m12 are each independently in the range of 1 to 100.

[0116] In some embodiments, each R1 is independently R1 N , and at least one R2 is T.

[0117] In some embodiments, each R1 N independently selected from alkyl, heteroalkyl, alkylcycloalkyl, alkylheterocyclyl, alkylaryl or alkylheteroaryl, wherein the alkyl, heteroalkyl, alkylcycloalkyl, alkylheterocyclyl, alkylaryl and alkylheteroaryl are optionally substituted by one or more R N Replace, and each R N Independently -OR a or -N(R a )2.

[0118] In some embodiments, each R1 N independently selected from alkyl, heteroalkyl, alkylcycloalkyl, alkylheterocyclyl, alkylaryl or alkylheteroaryl, wherein the alkyl, heteroalkyl, alkylcycloalkyl, alkylheterocyclyl, alkylaryl and alkylheteroaryl are optionally substituted by one or more R N Replace, and each R N Independently -OR a or -N(R a )2. In some embodiments, each R1 N Independently selected from C 1-30 Alkyl, C 1-25 Alkyl, C 1-20 Alkyl, C 1-19 Alkyl, C 1-18 Alkyl, C 1-17 Alkyl, C 1-16 Alkyl, C 1-15 Alkyl, C 1-14 Alkyl, C 1-13 Alkyl, C 1-12 Alkyl, C 1-11 Alkyl, or C 1-10 In some embodiments, each R1 N Independently selected from C 1-30 Heteroalkyl, C 1-25 Heteroalkyl, C 1-20 Heteroalkyl, C 1-19 Heteroalkyl, C1-18 Heteroalkyl, C 1-17 Heteroalkyl, C 1-16 Heteroalkyl, C 1-15 Heteroalkyl, C 1-14 Heteroalkyl, C 1-13 Heteroalkyl, C 1-12 Heteroalkyl, C 1-11 Heteroalkyl, C 1-10 Heteroalkyl, C 1-9 Heteroalkyl, C 1-8 Heteroalkyl, C 1-7 Heteroalkyl, C 1-6 Heteroalkyl, C 1-5 Heteroalkyl, C 1-4 Heteroalkyl, C 1-3 Heteroalkyl, C 1-2 heteroalkyl, wherein the heteroalkyl contains 1-5 heteroatoms selected from N, O, and S. In some embodiments, each R1 N Independently selected from C 1-6 Alkyl C 3-12 Cycloalkyl, C 1-6 Alkyl C 3-9 Cycloalkyl or C 1-6 Alkyl C 5-9 In some embodiments, each R1 N Independently selected from C 1-6 Alkyl C 3-12 Heterocyclic group, C 1-6 Alkyl C 3-9 Heterocyclic or C 1-6 Alkyl C 5-9 A heterocyclic group containing 1-3 heteroatoms selected from N, O, and S. In some embodiments, each R1 N Independently selected from C 1-6 Alkyl C 6-12 Aryl or C 1-6 Alkyl C 6-9 In some embodiments, each R1 N Independently selected from C 1-6 Alkyl C 6-12 Heteroaryl or C 1-6 Alkyl C 6-9 Heteroaryl.

[0119] In some embodiments, each R1 is independently selected from the group consisting of:

[0120]

[0121] In some embodiments, each R1 is independently selected from the group consisting of:

[0122]

[0123] In some embodiments, each R2 N is independently selected from alkyl, heteroalkyl, alkylcycloalkyl, alkylheterocyclyl or alkylheterocyclylalkyl, wherein the alkyl, heteroalkyl, alkylcycloalkyl, alkylheterocyclyl and alkylheterocyclylalkyl are optionally substituted by one or more R N Replace, and each R N Independently -OR a or -N(R a )2.

[0124] In some embodiments, each R2 N is independently selected from alkyl, heteroalkyl, alkylcycloalkyl, alkylheterocyclyl or alkylheterocyclylalkyl, wherein the alkyl, heteroalkyl, alkylcycloalkyl, alkylheterocyclyl and alkylheterocyclylalkyl are optionally substituted by one or more R N Replace, and each R N Independently -OR a or -N(R a )2. In some embodiments, each R2 N Independently selected from C 1-30 Alkyl, C 1-25 Alkyl, C 1-20 Alkyl, C 1-19 Alkyl, C 1-18 Alkyl, C 1-17 Alkyl, C 1-16 Alkyl, C 1-15 Alkyl, C 1-14 Alkyl, C 1-13 Alkyl, C 1-12 Alkyl, C 1-11 Alkyl, or C 1-10 In some embodiments, each R2 N Independently selected from C 1-30 Heteroalkyl, C 1-25 Heteroalkyl, C 1-20 Heteroalkyl, C 1-19 Heteroalkyl, C 1-18 Heteroalkyl, C 1-17 Heteroalkyl, C 1-16 Heteroalkyl, C 1-15 Heteroalkyl, C 1-14 Heteroalkyl, C 1-13 Heteroalkyl, C 1-12 Heteroalkyl, C 1-11 Heteroalkyl, C 1-10 Heteroalkyl, C 1-9 Heteroalkyl, C 1-8 Heteroalkyl, C 1-7 Heteroalkyl, C 1-6 Heteroalkyl, C 1-5 Heteroalkyl, C 1-4Heteroalkyl, C 1-3 Heteroalkyl, C 1-2 heteroalkyl, wherein the heteroalkyl contains 1-5 heteroatoms selected from N, O, and S. In some embodiments, each R2 N Independently selected from C 1-6 Alkyl C 3-12 Cycloalkyl, C 1-6 Alkyl C 3-9 Cycloalkyl or C 1-6 Alkyl C 5-9 In some embodiments, each R2 N Independently selected from C 1-6 Alkyl C 3-12 Heterocyclic group, C 1-6 Alkyl C 3-9 Heterocyclic or C 1-6 Alkyl C 5-9 A heterocyclic group containing 1-3 heteroatoms selected from N, O, and S. In some embodiments, each R2 N Independently selected from C 1-6 Alkyl C 3-12 Heterocyclyl C 1-6 Alkyl, C 1-6 Alkyl C 3-9 Heterocyclyl C 1-6 Alkyl or C 1-6 Alkyl C 5-9 Heterocyclyl C 1-6 The heterocyclic group contains 1-3 heteroatoms selected from N, O and S.

[0125] In some embodiments, each R2 is independently selected from the group consisting of:

[0126]

[0127]

[0128] In some embodiments, each R2 is independently selected from the group consisting of:

[0129]

[0130] and T.

[0131] In some embodiments, the branched poly (β-amino ester) provided herein has a structure shown in formula (I), wherein In the repeating unit, at least 1, 2, 3, 4 or 5 R1 are T, and each R2 is independently R2 N .

[0132] In some embodiments, the branched poly (β-amino ester) has a structure as shown in formula (I), wherein W1 is an alkyl group or a heteroalkyl group, and the alkyl group and the heteroalkyl group are optionally replaced by one or more R W In some embodiments, W1 is C 1-30 Alkyl, C 1-25 Alkyl, C 1-20 Alkyl, C 1-19 Alkyl, C 1-18 Alkyl, C 1-17 Alkyl, C 1-16 Alkyl, C 1-15 Alkyl, C 1-14 Alkyl, C 1-13 Alkyl, C 1-12 Alkyl, C 1-11 Alkyl, C 1-10 Alkyl, C 1-9 Alkyl, C 1-8 Alkyl, C 1-7 Alkyl or C 1-6 Alkyl, the alkyl group is optionally substituted by one or more R W In some embodiments, each W1 is independently selected from C 1-30 Heteroalkyl, C 1-25 Heteroalkyl, C 1-20 Heteroalkyl, C 1-19 Heteroalkyl, C 1-18 Heteroalkyl, C 1-17 Heteroalkyl, C 1-16 Heteroalkyl, C 1-15 Heteroalkyl, C 1-14 Heteroalkyl, C 1-13 Heteroalkyl, C 1-12 Heteroalkyl, C 1-11 Heteroalkyl, C 1-10 Heteroalkyl, C 1-9 Heteroalkyl, C 1-8 Heteroalkyl, C 1-7 Heteroalkyl, C 1-6 Heteroalkyl, C 1-5 Heteroalkyl, C 1-4 Heteroalkyl, C 1-3 Heteroalkyl or C 1-2 A heteroalkyl group, wherein the heteroalkyl group contains 1-3 heteroatoms selected from N, O, S, and the heteroalkyl group is optionally substituted by one or more R W replace.

[0133] In some embodiments, W1 is

[0134] In some embodiments, the branched poly (β-amino ester) provided herein has a structure shown in formula (II), wherein In the repeating unit, at least 1, 2, 3, 4, 5 or 6 R1 are T, and each R2 is independently R2 N .

[0135] In some embodiments, the branched poly (β-amino ester) has a structure shown in formula (II), wherein W2 is an alkyl group or a heteroalkyl group, and the alkyl group and the heteroalkyl group are optionally replaced by one or more R W In some embodiments, W2 is C 1-30 Alkyl, C 1-25 Alkyl, C 1-20 Alkyl, C 1-19 Alkyl, C 1-18 Alkyl, C 1-17 Alkyl, C 1-16 Alkyl, C 1-15 Alkyl, C 1-14 Alkyl, C 1-13 Alkyl, C 1-12 Alkyl, C 1-11 Alkyl, C 1-10 Alkyl, C 1-9 Alkyl, C 1-8 Alkyl, C 1-7 Alkyl or C 1-6 Alkyl, the alkyl group is optionally substituted by one or more R W In some embodiments, each W2 is independently selected from C 1-30 Heteroalkyl, C 1-25 Heteroalkyl, C 1-20 Heteroalkyl, C 1-19 Heteroalkyl, C 1-18 Heteroalkyl, C 1-17 Heteroalkyl, C 1-16 Heteroalkyl, C 1-15 Heteroalkyl, C 1-14 Heteroalkyl, C 1-13 Heteroalkyl, C 1-12 Heteroalkyl, C 1-11 Heteroalkyl, C 1-10 Heteroalkyl, C 1-9 Heteroalkyl, C 1-8 Heteroalkyl, C 1-7 Heteroalkyl, C 1-6 Heteroalkyl, C 1-5 Heteroalkyl, C 1-4 Heteroalkyl, C 1-3 Heteroalkyl or C 1-2 A heteroalkyl group, wherein the heteroalkyl group contains 1-3 heteroatoms selected from N, O, S, and the heteroalkyl group is optionally substituted by one or more R W replace.

[0136] In some embodiments, W2 is

[0137] In some embodiments, the branched poly (β-amino ester) provided herein has a structure shown in formula (III), wherein In the repeating unit, at least 1, 2, 3, 4, 5, 6, 7 or 8 R1 are T, and each R2 is independently R2 N .

[0138] In some embodiments, the branched poly (β-amino ester) has a structure shown in formula (III), wherein W3 is an alkyl group or a heteroalkyl group, and the alkyl group and the heteroalkyl group are optionally replaced by one or more R W In some embodiments, W3 is C 1-30 Alkyl, C 1-25 Alkyl, C 1-20 Alkyl, C 1-19 Alkyl, C 1-18 Alkyl, C 1-17 Alkyl, C 1-16 Alkyl, C 1-15 Alkyl, C 1-14 Alkyl, C 1-13 Alkyl, C 1-12 Alkyl, C 1-11 Alkyl, C 1-10 Alkyl, C 1-9 Alkyl, C 1-8 Alkyl, C 1-7 Alkyl or C 1-6 Alkyl, the alkyl group is optionally substituted by one or more R W In some embodiments, each W3 is independently selected from C 1-30 Heteroalkyl, C 1-25 Heteroalkyl, C 1-20 Heteroalkyl, C 1-19 Heteroalkyl, C 1-18 Heteroalkyl, C 1-17 Heteroalkyl, C 1-16 Heteroalkyl, C 1-15 Heteroalkyl, C 1-14 Heteroalkyl, C 1-13 Heteroalkyl, C 1-12 Heteroalkyl, C 1-11 Heteroalkyl or C 1-10 A heteroalkyl group, wherein the heteroalkyl group contains 1-3 heteroatoms selected from N, O, S, and the heteroalkyl group is optionally substituted by one or more R W replace.

[0139] In some embodiments, W3 is

[0140] In some embodiments, the branched poly (β-amino ester) provided herein has a structure shown in formula (IV), wherein In the repeating unit, at least 1, 2, 3, 4, 5, 6, 7, 8 or 9 R1 are T, and each R2 is independently R2 N .

[0141] In some embodiments, the branched poly (β-amino ester) has a structure shown in formula (IV), wherein W4 is an alkyl group or a heteroalkyl group, and the alkyl group and the heteroalkyl group are optionally replaced by one or more R W In some embodiments, W4 is C 1-30 Alkyl, C 1-25 Alkyl, C 1-20 Alkyl, C 1-19 Alkyl, C 1-18 Alkyl, C 1-17 Alkyl, C 1-16 Alkyl, C 1-15 Alkyl, C 1-14 Alkyl, C 1-13 Alkyl, C 1-12 Alkyl, C 1-11 Alkyl, C 1-10 Alkyl, C 1-9 Alkyl, C 1-8 Alkyl, C 1-7 Alkyl or C 1-6 Alkyl, the alkyl group is optionally substituted by one or more R W In some embodiments, each W4 is independently selected from C 1-30 Heteroalkyl, C 1-25 Heteroalkyl, C 1-20 Heteroalkyl, C 1-19 Heteroalkyl, C 1-18 Heteroalkyl, C 1-17 Heteroalkyl, C 1-16 Heteroalkyl, C 1-15 Heteroalkyl, C 1-14 Heteroalkyl, C 1-13 Heteroalkyl, C 1-12 Heteroalkyl, C 1-11 Heteroalkyl or C 1-10 A heteroalkyl group, wherein the heteroalkyl group contains 1-3 heteroatoms selected from N, O, S, and the heteroalkyl group is optionally substituted by one or more R W replace.

[0142] In some embodiments, W4 is

[0143] In some embodiments, the branched poly (β-amino ester) provided herein has a structure shown in Formula (V), wherein In the repeating unit, at least 1 or 2 R1 are T, and each R2 is independently R2 N .

[0144] In some embodiments, the branched poly (β-amino ester) has a structure shown in formula (V), wherein W5 is an alkyl group, a heteroalkyl group or an alkyl heterocyclyl alkyl group, and the alkyl group, the heteroalkyl group and the alkyl heterocyclyl alkyl group are optionally replaced by one or more R W In some embodiments, W5 is C 1-30 Alkyl, C 1-20 Alkyl, C 1-18 Alkyl, C 1-16 Alkyl, C 1-14 Alkyl, C 1-12 Alkyl, C 1-10 Alkyl or C 1-6 Alkyl, the alkyl group is optionally substituted by one or more R W In some embodiments, each W5 is independently selected from C 1-30 Heteroalkyl, C 1-20 Heteroalkyl, C 1-18 Heteroalkyl, C 1-16 Heteroalkyl, C 1-14 Heteroalkyl, C 1-12 Heteroalkyl or C 1-10 A heteroalkyl group, wherein the heteroalkyl group contains 1-3 heteroatoms selected from N, O, S, and the heteroalkyl group is optionally substituted by one or more R W In some embodiments, W5 is C 1-6 Alkyl C 3-12 Heterocyclyl C 1-6 Alkyl, C 1-6 Alkyl C 3-9 Heterocyclyl C 1-6 Alkyl or C 1-6 Alkyl C 5-9 Heterocyclyl C 1-6 The heterocyclic group contains 1-3 heteroatoms selected from N, O, S, and the alkylheterocyclic group is optionally substituted by one or more R W replace.

[0145] In some embodiments, W5 is selected from:

[0146]

[0147] In some embodiments, each R1 N independently selected from alkyl, heteroalkyl, alkylcycloalkyl, alkylheterocyclyl, alkylaryl or alkylheteroaryl, wherein the alkyl, heteroalkyl, alkylcycloalkyl, alkylheterocyclyl, alkylaryl and alkylheteroaryl are optionally substituted by one or more R N Replace, and each R N Independently -OR a or -N(R a )2. In some embodiments, each R1N Independently selected from C 1-30 Alkyl, C 1-25 Alkyl, C 1-20 Alkyl, C 1-19 Alkyl, C 1-18 Alkyl, C 1-17 Alkyl, C 1-16 Alkyl, C 1-15 Alkyl, C 1-14 Alkyl, C 1-13 Alkyl, C 1-12 Alkyl, C 1-11 Alkyl, or C 1-10 In some embodiments, each R1 N Independently selected from C 1-30 Heteroalkyl, C 1-25 Heteroalkyl, C 1-20 Heteroalkyl, C 1-19 Heteroalkyl, C 1-18 Heteroalkyl, C 1-17 Heteroalkyl, C 1-16 Heteroalkyl, C 1-15 Heteroalkyl, C 1-14 Heteroalkyl, C 1-13 Heteroalkyl, C 1-12 Heteroalkyl, C 1-11 Heteroalkyl, C 1-10 Heteroalkyl, C 1-9 Heteroalkyl, C 1-8 Heteroalkyl, C 1-7 Heteroalkyl, C 1-6 Heteroalkyl, C 1-5 Heteroalkyl, C 1-4 Heteroalkyl, C 1-3 Heteroalkyl, C 1-2 heteroalkyl, wherein the heteroalkyl contains 1-5 heteroatoms selected from N, O, and S. In some embodiments, each R1 N Independently selected from C 1-6 Alkyl C 3-12 Cycloalkyl, C 1-6 Alkyl C 3-9 Cycloalkyl or C 1-6 Alkyl C 5-9 In some embodiments, each R1 N Independently selected from C 1-6 Alkyl C 3-12 Heterocyclic group, C 1-6 Alkyl C 3-9 Heterocyclic or C 1-6 Alkyl C 5-9 A heterocyclic group containing 1-3 heteroatoms selected from N, O, and S. In some embodiments, each R1 N Independently selected from C1-6 Alkyl C 6-12 Aryl or C 1-6 Alkyl C 6-9 In some embodiments, each R1 N Independently selected from C 1-6 Alkyl C 6-12 Heteroaryl or C 1-6 Alkyl C 6-9 Heteroaryl.

[0148] In some embodiments, each R1 is independently selected from the group consisting of:

[0149]

[0150] and T.

[0151] In some embodiments, each R1 is independently selected from the group consisting of:

[0152]

[0153] and T.

[0154] In some embodiments, each R2 N is independently selected from alkyl, heteroalkyl, alkylcycloalkyl, alkylheterocyclyl or alkylheterocyclylalkyl, wherein the alkyl, heteroalkyl, alkylcycloalkyl, alkylheterocyclyl and alkylheterocyclylalkyl are optionally substituted by one or more R N Replace, and each R N Independently -OR a or -N(R a )2. In some embodiments, each R2 N Independently selected from C 1-30 Alkyl, C 1-25 Alkyl, C 1-20 Alkyl, C 1-19 Alkyl, C 1-18 Alkyl, C 1-17 Alkyl, C 1-16 Alkyl, C 1-15 Alkyl, C 1-14 Alkyl, C 1-13 Alkyl, C 1-12 Alkyl, C 1-11 Alkyl, or C 1-10 In some embodiments, each R2 N Independently selected from C 1-30 Heteroalkyl, C 1-25 Heteroalkyl, C 1-20 Heteroalkyl, C 1-19 Heteroalkyl, C 1-18 Heteroalkyl, C 1-17Heteroalkyl, C 1-16 Heteroalkyl, C 1-15 Heteroalkyl, C 1-14 Heteroalkyl, C 1-13 Heteroalkyl, C 1-12 Heteroalkyl, C 1-11 Heteroalkyl, C 1-10 Heteroalkyl, C 1-9 Heteroalkyl, C 1-8 Heteroalkyl, C 1-7 Heteroalkyl, C 1-6 Heteroalkyl, C 1-5 Heteroalkyl, C 1-4 Heteroalkyl, C 1-3 Heteroalkyl, C 1-2 heteroalkyl, wherein the heteroalkyl contains 1-5 heteroatoms selected from N, O, and S. In some embodiments, each R2 N Independently selected from C 1-6 Alkyl C 3-12 Cycloalkyl, C 1-6 Alkyl C 3-9 Cycloalkyl or C 1-6 Alkyl C 5-9 In some embodiments, each R2 N Independently selected from C 1-6 Alkyl C 3-12 Heterocyclic group, C 1-6 Alkyl C 3-9 Heterocyclic or C 1-6 Alkyl C 5-9 A heterocyclic group containing 1-3 heteroatoms selected from N, O, and S. In some embodiments, each R2 N Independently selected from C 1-6 Alkyl C 3-12 Heterocyclyl C 1-6 Alkyl, C 1-6 Alkyl C 3-9 Heterocyclyl C 1-6 Alkyl or C 1-6 Alkyl C 5-9 Heterocyclyl C 1-6 The heterocyclic group contains 1-3 heteroatoms selected from N, O and S.

[0155] In some embodiments, each R2 is independently selected from the group consisting of:

[0156]

[0157]

[0158] In some embodiments, each R2 is independently selected from the group consisting of:

[0159]

[0160] In some embodiments, T is an endoplasmic reticulum targeting moiety.

[0161] In some embodiments, T is selected from:

[0162] In some embodiments, L is selected from alkyl, heteroalkyl, heterocyclyl, alkylheterocyclyl, alkylcycloalkylalkyl, alkylheterocyclylalkyl, or heteroalkylarylalkylarylheteroalkyl, wherein the alkyl, heteroalkyl, alkylcycloalkylalkyl, alkylheterocyclylalkyl, and heteroalkylarylalkylarylheteroalkyl are optionally replaced by one or more R L Replace, and each R L Independently -OR a 、-N(R a )2 or alkyl. In some embodiments, L is C 1-30 Alkyl, C 1-25 Alkyl, C 1-20 Alkyl, C 1-19 Alkyl, C 1-18 Alkyl, C 1-17 Alkyl, C 1-16 Alkyl, C 1-15 Alkyl, C 1-14 Alkyl, C 1-13 Alkyl, C 1-12 Alkyl, C 1-11 Alkyl, or C 1-10 In some embodiments, L is C 1-30 Heteroalkyl, C 1-25 Heteroalkyl, C 1-20 Heteroalkyl, C 1-19 Heteroalkyl, C 1-18 Heteroalkyl, C 1-17 Heteroalkyl, C 1-16 Heteroalkyl, C 1-15 Heteroalkyl, C 1-14 Heteroalkyl, C 1-13 Heteroalkyl, C 1-12 Heteroalkyl, C 1-11 Heteroalkyl or C 1-10 wherein the heteroalkyl group contains 1-5 heteroatoms selected from N, O, and S. In some embodiments, L is C 3-12 Heterocyclic group, C 3-11 Heterocyclic group, C 3-10 Heterocyclic group, C 3-9 Heterocyclic group, C 3-8 Heterocyclic group, C 3-7 Heterocyclic group, C 3-6 Heterocyclic group, C 4-9 Heterocyclic or C 5-9A heterocyclic group containing 1-3 heteroatoms selected from N, O, and S. In some embodiments, L is C 1-6 Alkyl C 3-12 Heterocyclic group, C 1-6 Alkyl C 3-9 Heterocyclic or C 1-6 Alkyl C 5-9 A heterocyclic group containing 1-3 heteroatoms selected from N, O, and S. In some embodiments, L is C 1-6 Alkyl C 3-12 Cycloalkyl C 1-6 Alkyl, C 1-6 Alkyl C 3-9 Cycloalkyl C 1-6 Alkyl or C 1-6 Alkyl C 5-9 Cycloalkyl C 1-6 In some embodiments, L is C 1-6 Alkyl C 3-12 Heterocyclyl C 1-6 Alkyl, C 1-6 Alkyl C 3-9 Heterocyclyl C 1-6 Alkyl or C 1-6 Alkyl C 5-9 Heterocyclyl C 1-6 wherein L is heteroalkyl, wherein each heteroalkyl group is independently C 1-30 Heteroalkyl, C 1-20 Heteroalkyl, C 1-18 Heteroalkyl, C 1-16 Heteroalkyl, C 1-14 Heteroalkyl, C 1-12 Heteroalkyl or C 1-10 heteroalkyl, the aryl groups are each independently C 6-12 Aryl, C 6-11 Aryl, C 6-10 Aryl, C 6-9 Aryl or C 6-8 Aryl, the alkyl groups are each independently C 1-30 Alkyl, C 1-20 Alkyl, C 1-18 Alkyl, C 1-16 Alkyl, C 1-14 Alkyl, C 1-12 Alkyl or C 1-10 alkyl.

[0163] In some embodiments, L is selected from the group consisting of:

[0164]

[0165]

[0166] In some embodiments, L is selected from the group consisting of:

[0167]

[0168] In some embodiments, ml, m2, m3, m4, m5, m6, m7, m8, m9, mlO, ml 1 and m12 are each independently in the range of 5 to 75.

[0169] In some embodiments, the molecular weight of the branched poly (β-amino ester) is in the range of 5.0 kDa to 40.0 kDa, in the range of 5.0 kDa to 50 kDa, in the range of 5.0 kDa to 40.0 kDa, in the range of 5.0 kDa to 30.0 kDa, in the range of 5.0 kDa to 20.0 kDa, in the range of 5.0 kDa to 10.0 kDa. In some embodiments, the molecular weight of the branched poly (β-amino ester) is about 5.0 kDa, 6.0 kDa, 7.0 kDa, or 8.0 kDa.

[0170] Synthesis method

[0171] In one aspect, the present disclosure provides a method for preparing a branched poly(β-amino ester) described herein, comprising:

[0172] 1-1) a multi-acrylate monomer having formula (I-1), formula (II-1), formula (III-1) or formula (IV-1):

[0173]

[0174]

[0175] A branched poly(β-amino ester) P1 having a terminal double bond is obtained by reacting a diacrylate monomer having formula (2) and one or more amine monomers having formula (3) through Michael addition reaction:

[0176]

[0177] 1-2) subjecting the branched poly(β-amino ester) P1 obtained in step 1-1) to a Michael addition reaction with one or more end-capping amine monomers having the formula (4) to obtain a branched poly(β-amino ester) having a structure represented by the formula (I), formula (II), formula (III) or formula (IV):

[0178] R2-NH2

[0179]

[0180] or,

[0181] 2-1) A diamine monomer having the formula (V-1) is reacted with a diacrylate monomer having the formula (2) and one or more amine monomers having the formula (3) by Michael addition reaction to obtain a branched poly(β-amino ester) P2 having a double bond at the end:

[0182]

[0183] 2-2) The branched poly(β-amino ester) P2 prepared in step 2-1) is subjected to a Michael addition reaction with one or more end-capping amine monomers having the formula (4) to prepare a branched poly(β-amino ester) having a structure represented by the formula (V):

[0184] R2-NH2.

[0185] Formula (4)

[0186] In some embodiments, the multi-acrylate monomer having formula (I-1) is

[0187] In some embodiments, the multi-acrylate monomer having formula (II-1) is

[0188]

[0189] In some embodiments, the multi-acrylate monomer having formula (III-1) is

[0190]

[0191] In some embodiments, the multiacrylate monomer having formula (IV-1) is

[0192]

[0193] In some embodiments, the diamine monomer having formula (V-1) is selected from the group consisting of:

[0194]

[0195] In some embodiments, the diacrylate monomer having formula (2) is selected from the group consisting of:

[0196]

[0197] In some embodiments, the one or more amine monomers having formula (3) are R1 N -NH2, the one or more end-capping amine monomers having formula (4) are selected from R2 N-NH2 or T-NH2, and at least one of the blocked amine monomers having formula (4) is T-NH2.

[0198] In some embodiments, the R1 N -NH2 is selected from the group consisting of:

[0199]

[0200] The R2 N -NH2 is selected from the group consisting of:

[0201]

[0202] The T-NH2 is selected from the following group:

[0203]

[0204] In some embodiments, the diacrylate monomer having formula (2), R2 N -NH2, T-NH2 and R1 N -NH2 reaction feed molar ratio is (1-3): (0.1-4): (0.02-2): (0.5-6); in step 1-1), the diacrylate monomer having formula (2), R1 N -NH2 and the multiacrylate monomer of formula (I-1), formula (II-1), formula (III-1) or formula (IV-1) are in a molar ratio of (1 to 3): (0.1 to 4): (0.5 to 3); in step 2-1), the diacrylate monomer of formula (2), R1 N The molar ratio of -NH2 and the diamine monomer having the formula (V-1) is (1 to 3):(0.1 to 4):(0.5 to 3).

[0205] In some embodiments, the one or more amine monomers having formula (3) are selected from R1 N -NH2 or T-NH2, at least one amine monomer having formula (3) is T-NH2, and the one or more end-capping amine monomers having formula (4) are R2 N -NH2.

[0206] In some embodiments, the R1 N -NH2 is selected from the group consisting of:

[0207]

[0208] The T-NH2 is selected from the following group:

[0209]

[0210] The R2 N-NH2 is selected from the group consisting of:

[0211]

[0212] In some embodiments, the diacrylate monomer having formula (2), R2 N -OH, T-OH and R1 N -NH2 reaction feed molar ratio is (1-3): (0.1-4): (0.02-2): (0.5-6); in step 1-1), the diacrylate monomer having formula (2), R1 N -NH2, a multiacrylate monomer of formula (I-1), formula (II-1), formula (III-1) or formula (IV-1) and T-NH2 in a molar ratio of (1 to 3): (0.1 to 4): (0.5 to 3): (0.02 to 2); in step 2-1), the diacrylate monomer of formula (2), R1 N The molar ratio of -NH2, the diamine monomer having the formula (V-1) and T-NH2 is (1 to 3):(0.1 to 4):(0.5 to 3):(0.02 to 2).

[0213] In some embodiments, step 1-1) and step 2-1) are performed at a temperature of 15 to 100° C. In some embodiments, step 1-1) and step 2-1) are performed at a temperature of 60 to 100° C., 70 to 100° C., 80 to 100° C., 90 to 100° C., or 90 to 95° C.

[0214] In some embodiments, step 1-1) and step 2-1) are performed at a temperature of about 90°C.

[0215] In some embodiments, the molecular weight of the branched poly(β-amino ester) P1 having a terminal double bond obtained in step 1-1) is in the range of 5 kDa to 40 kDa.

[0216] In some embodiments, the molecular weight of the branched poly(β-amino ester) P2 having a terminal double bond obtained in step 2-1) is in the range of 5 kDa to 40 kDa.

[0217] In some embodiments, step 1-2) and step 2-2) are performed at a temperature of 20-30° C. In some embodiments, step 1-2) and step 2-2) are performed at a temperature of 20-30° C., 22-30° C., 24-30° C., 24-28° C., or 24-26° C.

[0218] In some embodiments, step 1-2) and step 2-2) are performed at a temperature of about 25°C.

[0219] Compounds provided herein, including the synthesis of their salts, are described in the synthetic schemes in the examples. Compounds provided herein can be prepared using any known organic synthesis techniques and can be synthesized according to any of a variety of possible synthetic pathways, and therefore, these schemes are only illustrative and are not intended to limit other possible methods that can be used to prepare compounds provided herein. In addition, the steps in the schemes are for better illustration and can be changed when appropriate.

[0220] The reaction for preparing the disclosed compound can be carried out in a suitable solvent, which can be easily selected by the technician in the field of organic synthesis. Suitable solvents can be at a temperature where the reaction is carried out, such as at a temperature from the freezing temperature of the solvent to the boiling temperature of the solvent, generally not reacting with the starting material (reactant), intermediate or product. A given reaction can be carried out in a solvent or in a mixture of more than one solvent. Depending on the specific reaction step, the suitable solvent for the specific reaction step can be selected by a skilled person.

[0221] The preparation of the compounds of the present disclosure may involve the protection and deprotection of various chemical groups. The need for protection and deprotection and the selection of appropriate protecting groups can be easily determined by those skilled in the art. The chemical properties of protecting groups can be found in, for example, TW Greene and PGM Wuts, Protective Groups in Organic Synthesis, 3rd edition, Wiley & Sons, Inc., New York (1999), which is incorporated herein by reference in its entirety.

[0222] The reaction can be monitored by any suitable method known in the art. For example, the reaction can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g., 1 H or 13C), infrared spectroscopy (IR), spectrophotometry (e.g., UV-visible light), mass spectrometry (MS), or by chromatography, such as high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LCMS) or thin layer chromatography (TLC) to monitor product formation. Those skilled in the art can purify compounds by a variety of methods, including high performance liquid chromatography (HPLC) ("Preparative LC-MS Purification: Improved Compound Specific Method Optimization", Karl F. Blom, Brian Glass, Richard Sparks, Andrew P. Combs, "Journal of Combinatorial Chemistry (J. Combi. Chem.)", 2004, 6 (6), 874-883, incorporated herein by reference in its entirety) and normal phase silica chromatography.

[0223] Abbreviations used herein are defined as follows: “1×” or “×1” for once, “2×” or “×2” for twice, “3×” or “×3” for three times, “4×” or “×4” for four times, “5×” or “×5” for five times, “°C” for degrees Celsius, “eq” or “eq.” for equivalent (equivalent / equivalents), “g” for gram (gram / grams), “mg” for milligram (milligram / milligrams), “L” for liter (liter / liters), and “mL” or “ml” for milliliter (milliliter / mil). liters), "μL" for microliters (microliter / microliters), "N" for normal, "M" for mole, "mmol" for millimole / millimoles, "min" for minute / minutes, "h" or "hr" for hour / hour, "rt" or "rt" for room temperature, "atm" for atmospheric pressure, "psi" for pounds per square inch, "conc." for concentration, "sat" or "sat'd" for saturated, "MS" or "Mass Spec" for mass spectrometry, "ESI" for electrospray ionization mass spectrometry, "LCMS" for liquid chromatography mass spectrometry, "HPLC" for high performance liquid chromatography, "RP" for reverse phase, "TLC" or "tlc" for thin layer chromatography, "SM" for starting material, "NMR" for nuclear magnetic resonance spectroscopy, 1H" is a proton, "δ" is delta, "s" is a singlet, "d" is a doublet, "t" is a triplet, "q" is a quartet, "m" is a multiplet, "br" is broad, and "Hz" is Hertz. "α", "β", "R", "S", "E" and "Z" are stereochemical designations familiar to those skilled in the art.

[0224] Composition

[0225] In one aspect, the present disclosure provides a composition comprising a branched poly(β-amino ester) described herein and a polynucleotide.

[0226] In some embodiments, the polynucleotide is DNA or RNA.

[0227] In some embodiments, the DNA encodes C7 protein, TGM1 protein, MBTPS1 protein, COL1A1 protein, COL3A1 protein, silk protein, Cas9 protein, ADAM17 protein, TGF-β1 protein, ABCD1 protein, P53 protein, RPE65 protein, SMN1 protein, AADC protein, FVIII protein, VEGF protein or HGF protein.

[0228] In some embodiments, the mass ratio of the branched poly(β-amino ester) to the polynucleotide is in the range of 1:1 to 200:1. In some embodiments, the mass ratio of the branched poly(β-amino ester) to the polynucleotide is in the range of 5:1 to 120:1. In some embodiments, the mass ratio of the branched poly(β-amino ester) to the polynucleotide is in the range of 5:1 to 100:1. In some embodiments, the mass ratio of the branched poly(β-amino ester) to the polynucleotide is 120:1, 110:1, 100:1, 90:1, 80:1, 70:1, 60:1, 50:1, 40:1, 30:1, 20:1, 10:1, or 5:1.

[0229] In some embodiments, the composition is in the form of nanoparticles.

[0230] In some embodiments, the particle size of the nanoparticles is in the range of 50 nm to 500 nm. In some embodiments, the particle size of the nanoparticles is in the range of 150 nm to 400 nm. In some embodiments, the particle size of the nanoparticles is 150 nm, 200 nm, 250 nm, 300 nm, 350 nm, or 400 nm.

[0231] Treatment

[0232] In one aspect, the present disclosure provides a method of delivering a polynucleotide to a subject, comprising administering to the subject a composition described herein.

[0233] As used herein, the term "treatment" refers to reversing, alleviating a disease or condition as described herein or one or more symptoms thereof, delaying its onset or inhibiting its progression. In some embodiments, treatment may be administered after one or more symptoms have occurred. In other embodiments, treatment may be administered in the absence of symptoms. For example, treatment may be administered to susceptible individuals before the onset of symptoms (e.g., based on symptom history and / or based on genetic or other susceptibility factors). Treatment may also continue after symptoms subside, for example to prevent or delay their recurrence.

[0234] The compositions provided herein can be administered in pure form, in combination with other active ingredients, or in the form of pharmaceutical compositions disclosed herein. In some embodiments, the compositions provided herein can be administered to a subject in need simultaneously or sequentially in combination with one or more other active agents known in the art. The individual agents in such combinations are administered sequentially or simultaneously in separate or combined pharmaceutical compositions. Preferably, the individual agents will be administered simultaneously in the combined pharmaceutical compositions. Those skilled in the art will appreciate the appropriate dosage of known therapeutic agents.

[0235] In some embodiments, the administration is performed once a day, twice a day, three times a day, or once every two days, once every three days, once every four days, once every five days, once every six days, or once a week.

[0236] In some embodiments, the administration is by injection, but the compositions of the present disclosure can be administered by any medically acceptable route of administration deemed safe and appropriate by a physician of ordinary skill. Exemplary routes of administration include transdermal, subcutaneous injection, or tail vein injection.

[0237] In another aspect, the present disclosure provides a method of treating a disease in a subject, comprising administering to the subject a composition described herein.

[0238] In some embodiments, the compositions described herein are administered to a subject transdermally, subcutaneously, or by tail vein injection.

[0239] Reference will now be made in detail to certain embodiments of the present disclosure, examples of which are described in the accompanying structures and chemical formulas. Although the present disclosure will be described in conjunction with the enumerated embodiments, it should be understood that it is not intended to limit the present disclosure to those embodiments. On the contrary, the present disclosure is intended to cover all alternatives, modifications and equivalents that may be included within the scope of the present disclosure as defined by the claims. Those skilled in the art will recognize that many methods and materials are similar or equivalent to the methods or materials described herein, and the methods and materials may be used to practice the present disclosure. The present disclosure is in no way limited to the methods and materials described. If one or more of the combined documents and similar materials are different or contradictory from the present disclosure, including but not limited to defined terms, term usage, described technology, etc., then the present disclosure shall prevail.

[0240] It should be appreciated that certain features of the present disclosure described in the context of separate embodiments for clarity may also be provided in combination in a single embodiment. Conversely, various features of the present disclosure described in the context of a single embodiment for brevity may also be provided separately or in any suitable sub-combination.

[0241] Example

[0242] The general method of the present disclosure is further explained below. The compounds of the present disclosure can be prepared by methods known in the art. The detailed preparation methods of the preferred compounds of the present disclosure are described below. However, these are by no means limiting the preparation methods of the compounds of the present disclosure.

[0243] The synthesis of the compound (including its pharmaceutically acceptable salt) provided herein is described in the synthesis process in the example. The compound provided herein can be prepared using any known organic synthesis technology and can be synthesized according to any of a variety of possible synthetic pathways, and therefore, these processes are only illustrative and are not intended to limit other possible methods that can be used to prepare the compound provided herein. In addition, the steps in the process are to better illustrate and can be changed when appropriate. For the purpose of research and possible submission to the regulatory agency, the embodiment of the compound in the example is synthesized.

[0244] The reaction for preparing the compound of the present disclosure can be carried out in a suitable solvent, which can be easily selected by a technician in the field of organic synthesis. Suitable solvents can be at a temperature for reacting, for example, at a temperature between the freezing temperature of the solvent and the boiling temperature of the solvent, generally not reacting with starting materials (reactants), intermediates or products. A given reaction can be carried out in a solvent or in a mixture of more than one solvent. Depending on the specific reaction step, the suitable solvent for the specific reaction step can be selected by a skilled person.

[0245] The preparation of the compounds of the present disclosure may involve the protection and deprotection of various chemical groups. The need for protection and deprotection and the selection of appropriate protecting groups can be easily determined by those skilled in the art. The chemical properties of protecting groups can be found in, for example, TW Greene and PGM Wuts, Protective Groups in Organic Synthesis, 3rd Edition, Wiley & Sons, New York (1999), which is incorporated herein by reference in its entirety.

[0246] The reaction can be monitored according to any suitable method known in the art. For example, the reaction can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g., 1 H or 13 C), infrared spectroscopy, spectrophotometry (e.g., UV-visible light), mass spectrometry, or by chromatography, such as high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LCMS) or thin layer chromatography (TLC) to monitor product formation. Those skilled in the art can purify compounds by a variety of methods, including high performance liquid chromatography (HPLC) ("Preparative LC-MS Purification: Improved Compound-Specific Method Optimization", Karl F. Blom, Brian Glass, Richard Sparks, Andrew P. Combs, "Journal of Combinatorial Chemistry", 2004, 6 (6), 874-883, which is incorporated herein by reference in its entirety) and normal phase silica chromatography.

[0247] In the following examples, unless otherwise specified, TGM1 DNA, COL7A1 DNA and MBTPS1 DNA used in the present disclosure were purchased from Hanbio Biotech (Shanghai).

[0248] General method 1

[0249] The present disclosure provides methods for preparing the branched poly(β-amino esters) described herein, the general synthetic routes of which are shown in Figure 1(a)-1(b) , including the following steps:

[0250] 1) a diamine monomer (such as EDA) or a multi-acrylate monomer (such as PET4A, PETA, DPTH or TMPTA), a diacrylate monomer and an amine monomer (R1 N -NH2) through Michael addition reaction to obtain a branched poly (β-amino ester) P1 with a double bond at the end;

[0251] 2) reacting the branched poly(β-amino ester) P1 having a double bond at the end obtained in step 1) with a capping amine monomer (R2 N -NH2 and T-NH2) through Michael addition reaction to prepare the branched poly (β-amino ester) of the present invention.

[0252] General Method 2

[0253] The present disclosure provides methods for preparing the branched poly(β-amino esters) described herein, the general synthetic routes of which are shown in Figure 1(c)-1(d) , including the following steps:

[0254] 1) a diamine monomer (such as EDA) or a polyacrylate monomer (such as PET4A, PETA, DPTH or TMPTA), a diacrylate monomer, an amine monomer (R1 N -NH2 and T-NH2) through Michael addition reaction to obtain branched poly (β-amino ester) P1 with a double bond at the end;

[0255] 2) reacting the branched poly(β-amino ester) P1 having a double bond at the end obtained in step 1) with a capping amine monomer (R2 N -NH2) through a Michael addition reaction to prepare the branched poly (β-amino ester) of the present invention.

[0256] General method 3

[0257] The present disclosure provides a method for preparing a composition (e.g., composite nanoparticles) comprising the branched poly (β-amino ester) described herein and a polynucleotide, the method comprising forming uniform and stable composite nanoparticles by reacting the branched poly (β-amino ester) prepared in the present disclosure with DNA of different sizes through charge interaction.

[0258] The affinity of branched poly(β-amino ester) to DNA was tested, and the particle size, Zeta potential and microscopic morphology of the composite nanoparticles formed by branched poly(β-amino ester) and DNA were tested. The method is as follows:

[0259] A certain amount of branched poly (β-amino ester) solution was quickly added to the DNA solution, vortexed at high speed for 15 to 60 seconds, and then allowed to stand for 5 to 30 minutes, wherein the mass ratio of branched poly (β-amino ester) to DNA was (5 to 100): 1. Then PicoGreen working solution was added to test the fluorescence intensity, and then the DNA affinity efficiency was calculated.

[0260] A similar composite nanoparticle preparation method was used. After the nanoparticles were formed, 1 mL of deionized water was added and then tested using dynamic light scattering (DLS).

[0261] A similar method for preparing composite nanoparticles was used, in which the inorganic salt ions were washed with deionized water, centrifuged at high speed for 5 minutes, and then freeze-dried. Finally, the microscopic morphology of the composite nanoparticles was observed using a transmission electron microscope (TEM).

[0262] General Method 4

[0263] The present disclosure provides for transfection of DNA and cells of various sizes using the branched poly(β-amino esters) described herein, while testing for cytotoxicity, as follows:

[0264] 1) Cell culture: 293F cells, A2780 cells, 293T cells, 3T3 cells, SW1353 cells, Vero cells, HaCaT cells and NHF cells were cultured at 1.0×10 4 ~4.0×10 4 Cells were seeded at a density of 10 cells / well in a 96-well plate and cultured at 37°C overnight.

[0265] 2) A certain amount of the branched poly (β-amino ester) described herein is mixed with DNA (e.g., GFP DNA, TGM1 DNA, COL7A1 DNA, MBTPS1 DNA, COL1A1 DNA, COL3A1 DNA, fibroin DNA, Cas9 DNA, ADAM17 DNA, TGF-β1 DNA, ABCD1 DNA, P53 DNA, RPE65 DNA, SMN1 DNA, AADC DNA, FVIII DNA, VEGF DNA, and HGF DNA), allowed to stand for 5 to 40 minutes, and then added to the cells. 3) 48 hours after transfection, the cells transfected with GFP DNA were observed and photographed under a fluorescence microscope.

[0266] 4) 48 h after transfection, the efficiency of GFP DNA transfection was tested using flow cytometry.

[0267] 5) 48 h to 72 h after transfection, the cell supernatant was removed, and then the protein (C7 protein, TGM1 protein or MBTPS1 protein) expression efficiency was detected by western blotting and qPCR.

[0268] 6) 48h-72h after transfection, use alarmBlue to detect the viability of transfected cells. Specific embodiment:

[0270] Example 1 Synthesis of ER-HPAE-1

[0271] 1,4-Butanediol diacrylate, ethylenediamine and 5-amino-1-pentanol in a feed ratio of 2:0.2:1.5 were reacted at 90°C for 5 hours; a branched poly(β-amino ester) with a double bond at the end was obtained. Then 1,11-diamino-3,6,9-trioxaundecane and 4-(2-aminoethyl)benzenesulfonamide were added to achieve a final feed ratio of 1,4-butanediol diacrylate, ethylenediamine, 5-amino-1-pentanol, 1,11-diamino-3,6,9-trioxaundecane and 4-(2-aminoethyl)benzenesulfonamide of 2:0.2:1.5:0.5:0.1. The reaction was carried out at 25°C for 48 hours. The reaction solution was precipitated three times in excess ether to obtain a branched poly(β-amino ester) ER-HPAE-1 with a molecular weight of 7.5 kDa.

[0272] FIG. 1( a ) shows a schematic diagram of the synthesis of branched poly(β-amino ester) ER-HPAE-1 of this example. Figure 2 The GPC curve of the synthesis process of the branched poly (β-amino ester) ER-HPAE-1 in this example is shown. It can be found that as time goes by, the longer the retention time of the polymer in the GPC, the larger its molecular weight, which confirms that the polymer is successfully synthesized. Figure 3 of 1 As shown in the HNMR spectrum, the characteristic peaks of benzenesulfonamide appeared at 7.5-8.0, indicating that the branched poly (β-amino ester) ER-HPAE-1 was successfully synthesized.

[0273] Example 2 Synthesis of ER-HPAE-2

[0274] 1,4-Butanediol diacrylate, pentaerythritol tetraacrylate and 5-amino-1-pentanol in a feed ratio of 2:0.2:2.0 were reacted at 90°C for 5 hours; a branched poly(β-amino ester) with a double bond at the end was obtained. Then 1,11-diamino-3,6,9-trioxaundecane and N-(2-aminoethyl)-4-methylbenzenesulfonamide were added to achieve a final feed ratio of 1,4-butanediol diacrylate, pentaerythritol tetraacrylate, 5-amino-1-pentanol, 1,11-diamino-3,6,9-trioxaundecane and N-(2-aminoethyl)-4-methylbenzenesulfonamide of 2:0.2:2.0:0.6:0.1. The reaction was carried out at 25°C for 48 hours. The reaction solution was precipitated three times in excess ether to obtain a branched poly(β-amino ester) ER-HPAE-2 with a molecular weight of 7.1 kDa.

[0275] FIG1( b ) shows a schematic diagram of the synthesis of branched poly(β-amino ester) ER-HPAE-2 of this example. Figure 2The GPC curve of the synthesis process of the branched poly (β-amino ester) ER-HPAE-2 in this example is shown. It can be found that as time goes by, the longer the retention time of the polymer in the GPC, the larger its molecular weight, which confirms that the polymer is successfully synthesized. Figure 3 of 1 As shown in the HNMR spectrum, the characteristic peaks of benzenesulfonamide appeared at 7.5-8.0, indicating that the branched poly (β-amino ester) ER-HPAE-2 was successfully synthesized.

[0276] Example 3 Synthesis of ER-HPAE-3

[0277] 1,4-Butanediol diacrylate, polydipentaerythritol hexaacrylate, 5-amino-1-pentanol, dodecylamine and N-(2-aminoethyl)-4-methylbenzenesulfonamide in a feed ratio of 2:0.1:1.5:0.2:0.05 were reacted at 90°C for 5 hours to obtain a branched poly(β-amino ester) with a double bond at the end. Then 1,11-diamino-3,6,9-trioxaundecane was added to achieve a final feed ratio of 1,4-Butanediol diacrylate, polydipentaerythritol hexaacrylate, 5-amino-1-pentanol, dodecylamine, N-(2-aminoethyl)-4-methylbenzenesulfonamide and 1,11-diamino-3,6,9-trioxaundecane of 2:0.1:1.5:0.2:0.05:1. The reaction was carried out at 25°C for 48 hours. The reaction solution was precipitated in excess ether three times to obtain branched poly(β-amino ester) ER-HPAE-3 with a molecular weight of 7.4 kDa.

[0278] FIG1( c ) shows a schematic diagram of the synthesis of branched poly(β-amino ester) ER-HPAE-3 of this example. Figure 2 The GPC curve of the synthesis process of the branched poly (β-amino ester) ER-HPAE-3 in this example is shown. It can be found that as time goes by, the longer the retention time of the polymer in the GPC, the larger its molecular weight, which confirms that the polymer is successfully synthesized. Figure 3 of 1 As shown in the HNMR spectrum, the characteristic peaks of benzenesulfonamide appeared at 7.5-8.0, indicating that the branched poly (β-amino ester) ER-HPAE-3 was successfully synthesized.

[0279] Example 4 Synthesis of ER-HPAE-4

[0280] 1,4-Butanediol diacrylate, trimethylolpropane ethoxylate triacrylate, 4-amino-1-butanol and N-(2-aminoethyl)-4-methylbenzenesulfonamide in a feed ratio of 2:0.1:1.5:0.05 were reacted at 90°C for 8 hours to obtain a branched poly(β-amino ester) with a double bond at the end. Then 1,4-bis(3-aminopropyl)piperazine was added to achieve a final feed ratio of 1,4-butanediol diacrylate, trimethylolpropane ethoxylate triacrylate, 4-amino-1-butanol, N-(2-aminoethyl)-4-methylbenzenesulfonamide and 1,4-bis(3-aminopropyl)piperazine of 2:0.1:1.5:0.05:0.7. The reaction was carried out at 25°C for 48 hours. The reaction solution was precipitated three times in excess ether to obtain a branched poly(β-amino ester) ER-HPAE-4 with a molecular weight of 8.0 kDa.

[0281] FIG1( d ) shows a schematic diagram of the synthesis of branched poly(β-amino ester) ER-HPAE-4 of this example. Figure 2 The GPC curve of the synthesis process of the branched poly (β-amino ester) ER-HPAE-4 in this example is shown. It can be found that as time goes by, the longer the retention time of the polymer in the GPC, the larger its molecular weight, which confirms that the polymer is successfully synthesized. Figure 3 of 1 As shown in the HNMR spectrum, the characteristic peaks of benzenesulfonamide appeared at 7.5-8.0, indicating that the branched poly (β-amino ester) ER-HPAE-4 was successfully synthesized.

[0282] Example 5

[0283] PicoGreen was used to test the affinity of branched poly(β-amino ester) ER-HPAE-1 to ER-HPAE-4 prepared in Examples 1-4 for TGM1 DNA, wherein the mass ratios of branched poly(β-amino ester) to DNA were 30:1 and 60:1, respectively, and the amount of DNA used was 1 μg.

[0284] The branched poly (β-amino ester) solution was added to the DNA solution encoding green fluorescent protein, vortexed at high speed for 30 seconds, and then allowed to stand for 20 minutes to form composite nanoparticles. Then, the composite nanoparticle solution was diluted to 100 μL using TE buffer solution, and then 100 μL PicoGreen working solution was added, and the excited fluorescence intensity was detected under the conditions of an excitation wavelength of 480 nm and an emission wavelength of 520 nm.

[0285] Composite nanoparticles with a mass ratio of poly(β-amino ester) to TGM1 DNA of 30:1 and 60:1 were prepared by a method similar to the above, and the composite nanoparticles were diluted to 1 mL with deionized water, and then the particle size and surface potential were tested using dynamic light scattering (DLS). The composite nanoparticles were washed and freeze-dried, and their microscopic morphology was characterized using TEM.

[0286] Figure 4 The results of the affinity test of ER-HPAE-1 to ER-HPAE-4 to TGM1 DNA are shown. It can be seen that when the mass ratio of poly(β-amino ester) to TGM1 DNA is 30:1 and 60:1, ER-HPAE-1 to ER-HPAE-4 all show excellent DNA affinity, and the DNA affinity efficiency exceeds 95%.

[0287] Figure 5 The particle size test results of the obtained composite nanoparticles are shown. The results show that when the mass ratio of poly(β-amino ester) to TGM1 DNA is 30:1 and 60:1, the particle size of the obtained composite nanoparticles is 193nm to 380nm, which meets the conditions for efficient cell uptake.

[0288] Figure 6 The surface potential test results of the obtained composite nanoparticles are shown. The results show that when the mass ratio of poly(β-amino ester) to TGM1 DNA is 30:1 and 60:1, the Zeta potential range of the obtained composite nanoparticles is 18.0+mV to 27.0+mV, which can effectively shield the negative potential of DNA itself.

[0289] Figure 7 The microscopic morphology characterization results of the obtained composite nanoparticles are shown. The results show that when the mass ratio of poly(β-amino ester) to TGM1 DNA is 30:1 and 60:1, the obtained nanoparticles have uniform particle size distribution, compact spherical shape and are relatively stable, confirming their excellent stability and DNA protection efficiency.

[0290] Example 6

[0291] Skin effector 3T3 cells were cultured at 2.0×10 3The density of cells / well was inoculated in a 96-well plate, and the cells were cultured at 37°C overnight. The branched poly (β-amino ester) ER-HPAE-1 and ER-HPAE-2 of Examples 1 and 2 were combined with luciferase-encoding DNA labeled with AF647 fluorescent dye at a mass ratio of 30:1 to form composite nanoparticles ER-HPAE-1 / AF647-DNA and ER-HPAE-2 / AF647-DNA, wherein the amount of luciferase-encoding DNA labeled with AF647 fluorescent dye was 1.0 μg. The composite nanoparticles were then mixed with the culture medium and added to the cells. After incubation for 4 hours, Lyso-Tracker Green and Hoechst 33342 were used to stain the organelles and nuclei of the cells at 37°C for 10 minutes, and then the intracellular DNA distribution and organelle targeting efficiency were observed using a fluorescence microscope.

[0292] Figure 8 The results of characterizing the endocytosis efficiency of composite nanoparticles ER-HPAE-1 / AF647-DNA and ER-HPAE-2 / AF647-DNA are shown. From the fluorescence microscope photos, it can be found that a large number of red DNA molecules are surrounded by the blue cell nucleus. Without wishing to be limited by any particular theory, this may be due to the higher surface potential of the composite nanoparticles promoting their interaction with cells. These results confirm that the composite nanoparticles disclosed in the present invention have excellent cell uptake efficiency and can promote subsequent gene transfection.

[0293] Fig. 9 The organelle targeting characterization results of composite nanoparticles ER-HPAE-1 / AF647-DNA and ER-HPAE-2 / AF647-DNA are shown. It can be seen that the DNA labeled with AF647 fluorescent dye and the endoplasmic reticulum labeled with fluorescent dye ER-tracker Green have obvious overlap, indicating that the DNA molecules are clearly co-localized with the endoplasmic reticulum in the cell.

[0294] Fig.10 The organelle targeting characterization results of composite nanoparticles ER-HPAE-1 / AF647-DNA and ER-HPAE-2 / AF647-DNA are shown. According to the results of the magnified fluorescence photographs and PCC, not only was it observed that the DNA molecules were clearly co-localized with the endoplasmic reticulum in the cell, but also a large number of composite nanoparticles were located between the endoplasmic reticulum and the nucleus, providing conditions for further nuclear internalization. These results all confirm that the poly (β-amino ester)-based nanoparticles disclosed in the present invention have a good endoplasmic reticulum targeting effect, can significantly overcome the retention of composite nanoparticles in lysosomes, and help increase the entry of exogenous genes into the nucleus, thereby improving the gene transfection efficiency of nanoparticles.

[0295] Example 7

[0296] Skin effector 3T3 cells were cultured at 2.0×10 4 The cells were seeded in a 96-well plate at a density of 10 cells / well, and the cells were cultured at 37°C overnight. The composite nanoparticles formed by the branched poly (β-amino ester) ER-HPAE-1 to ER-HPAE-4 of Examples 1-4 and GFP DNA at a mass ratio of 30:1 and 60:1 were added to the serum-containing culture medium, mixed thoroughly, and then slowly added to the cells. The cells were then cultured for 48 hours, and the efficiency of GFP DNA transfection in 3T3 cells was tested using a fluorescence microscope and a flow cytometer.

[0297] Fig.11 The results of qualitative evaluation of the gene transfection performance of composite nanoparticles are shown. Fig.11 It can be found that the composite nanoparticles formed at mass ratios of 30:1 and 60:1 are both able to efficiently mediate efficient gene transfection, which indicates that the branched poly (β-amino ester)-based nanoparticles disclosed in the present invention have excellent biophysical properties and excellent cell targeting capabilities.

[0298] Fig.12 The results of quantitative evaluation of the transfection efficiency of composite nanoparticles in 3T3 cells are shown. The results of cell flow cytometry after transfection show that the composite nanoparticles formed at a mass ratio of 30:1 can efficiently mediate efficient gene transfection, with a transfection efficiency of 59.3% and a relative fluorescence intensity of 2.9*10 5 RLU, which indicates that the branched poly (β-amino ester)-based nanoparticles disclosed in the present invention have very excellent transfection performance and can meet the requirements of intracellular gene delivery in skin effector cells.

[0299] Fig.13 The results of the viability of suspended cells after transfection of the composite nanoparticles in five different tissue cells (A2780 cells, 293T cells, 3T3 cells, SW1353 cells, Vero cells) are shown. The alarmBlue results show that the viability of cells after transfection of the branched poly (β-amino ester)-based nanoparticles disclosed in the present invention exceeds 96%, and the highest reaches 100%, which is mainly due to the excellent biodegradability and biosafety of the composite nanoparticles disclosed in the present invention.

[0300] Example 8

[0301] The suspension cells (293F cells) were cultured at 4.0×10 4The density of cells / well and the composite nanoparticles formed by the branched poly (β-amino ester) ER-HPAE-1 to ER-HPAE-4 and GFP DNA of Examples 1-4 were fully mixed and directly inoculated into a 96-well plate, wherein the mass ratio of branched poly (β-amino ester) to GFP DNA was 10: 1, 20: 1, 30: 1, and 40: 1, respectively. The composite nanoparticles and 293F cells were cultured for 48 hours, and the cells transfected with GFP DNA were observed under a fluorescence microscope, and the gene transfection efficiency in the suspended cell species was evaluated by flow cytometry. The viability of the transfected cells was tested using alarmBlue.

[0302] Fig.14 The results of evaluating the transfection performance of composite nanoparticles in suspended cells are shown. Fig.14 It can be found that in 293F cells, due to the advantages of endoplasmic reticulum targeting in overcoming intracellular barriers such as low gene lysosomal escape efficiency and nuclear internalization restrictions, as well as the multiple terminal structures and 3D topological structure, the composite nanoparticles disclosed in the present invention exhibit extremely high GFP DNA transfection efficiency.

[0303] Fig.15 The quantitative results of cell flow cytometry after composite nanoparticle transfection are shown. The results show that the composite nanoparticles formed at a mass ratio of 20:1 can efficiently mediate gene transfection, with a transfection efficiency of 74% and a relative fluorescence intensity of more than 6.0*10 5 RLU, the performance is significantly better than the commercial transfection reagent Lipo 3000. This shows that the branched poly (β-amino ester)-based nanoparticles disclosed in the present invention can meet the requirements of intracellular gene delivery in suspended cells.

[0304] Fig.16 (a)-(d) are the survival rates of suspended cells after transfection of composite nanoparticles formed by branched poly(β-amino ester) ER-HPAE-1 to ER-HPAE-4 and GFP DNA at mass ratios of 120:1, 100:1, 80:1, 60:1, 40:1, 20:1, 10:1 and 5:1 in suspended cells. The alarmBlue results show that the survival rate of cells transfected with these composite nanoparticles exceeds 96%, with the highest reaching 100%, and with the increase of mass ratio, the activity of 293F cells after transfection with composite nanoparticles decreases slightly, but the extremely high cell survival rate can still be retained at high mass and low mass ratios, which is mainly due to the excellent biodegradability, biosafety and targeting of the composite nanoparticles disclosed in the present invention.

[0305] Example 9

[0306] In skin-related effector cells, the transfection efficiency of composite nanoparticles formed by branched poly(β-amino ester) ER-HPAE-1 and ER-HPAE-2 of Examples 1 and 2 and functional DNAs of different sizes (TGM1 DNA, COL7A1 DNA and MBTPS1 DNA) was tested.

[0307] HaCaT cells and primary NHF cells were cultured at 2.0*10 4 The cells were seeded in a 96-well plate at a density of 10 cells / well and cultured at 37°C overnight. The branched poly (β-amino ester) ER-HPAE-1 and ER-HPAE-2 of Examples 1 and 2 were mixed with TGM1DNA, COL7A1 DNA or MBTPS1 DNA at a mass ratio of 30:1 to form composite nanoparticles. The obtained composite nanoparticles were used to transfect HaCaT cells and primary NHF cells, respectively. After 36 hours of transfection, the transfected cells were digested, and the transfection efficiency of TGM1 mRNA, COL7A1 mRNA and MBTPS1 mediated by the composite nanoparticles was tested using qPCR. After 72 hours of transfection, the expression of TGM1 protein and COL7A1 protein by the composite nanoparticles was tested using a protein blotting test method. Cells were transfected with TGM1DNA, COL7A1 DNA and MBTPS1DNA solutions as blank controls.

[0308] Fig.17 The results of evaluating the expression efficiency of composite nanoparticles in HaCaT cells are shown. The qPCR results show that in HaCaT cells, the composite nanoparticles formed by the branched poly (β-amino ester) of Examples 1 and 2 expressed COL7A1 mRNA transcription levels that were increased by more than 3500 times, TGM1 mRNA transcription levels that were increased by more than 200 times, and MBTPS1 mRNA transcription levels that were increased by more than 350 times. This indicates that the composite nanoparticles disclosed herein have excellent functional DNA transfection efficiency in skin effector cells.

[0309] Fig.18 The results of evaluating the expression efficiency of composite nanoparticles in NHF cells are shown. The qPCR results show that in NHF cells, the composite nanoparticles formed by the branched poly (β-amino ester) of Examples 1 and 2 increased the transcription level of COL7A1 mRNA by more than 2400 times, the transcription level of TGM1 mRNA by more than 180 times, and the transcription level of MBTPS1 mRNA by more than 1100 times. This shows that the composite nanoparticles disclosed in the present invention have excellent functional DNA transfection efficiency in skin effector cells. .

[0310] Fig.19The results of the evaluation of the expression efficiency of the composite nanoparticles in NHF cells are shown. The results of protein blotting show that, compared with untransfected cells and cells transfected with the commercial transfection reagent jetPEI, in NHF cells, the composite nanoparticles formed by the branched poly (β-amino ester) of Example 1 and Example 2 present more obvious TGM1 protein bands, MBTPS1 protein bands and COL7A1 protein bands. This is mainly due to the excellent DNA affinity, compressibility, stable and compact microstructure and excellent endoplasmic reticulum targeting of the branched poly (β-amino ester)-based nanoparticles disclosed in the present invention. Therefore, the branched poly (β-amino ester)-based nanoparticles disclosed in the present invention can efficiently mediate the transfection of three functional DNAs (TGM1DNA, COL7A1 DNA and MBTPS1 DNA) of different sizes in skin effector cells, and meet the efficient gene delivery of suspended cells. These results preliminarily demonstrate that the composite nanoparticles disclosed herein have important application potential in the treatment of RDEB, ichthyosis gene, cataract, alopecia, oral mucosal diseases and psoriasis-like syndrome (CAOP) syndrome.

[0311] Example 10

[0312] The transfection efficiency of functional DNA (COL7A1 DNA or MBTPS1 DNA) of different sizes mediated by branched poly (β-amino ester) ER-HPAE-1 and ER-HPAE-2 of Examples 1 and 2 was tested in C57 mice (approved by the Animal Ethics Committee of the Children's Hospital Affiliated to Fudan University, No.: 2023220).

[0313] C57 mice were divided into 6 groups and injected subcutaneously twice (injection was performed every 48 hours). The branched poly (β-amino ester) ER-HPAE-1 and ER-HPAE-2 of Examples 1 and 2 were combined with COL7A1 DNA or MBTPS1 DNA to form composite nanoparticles (containing 7% F127) in an ethanol system, wherein the amount of COL7A1 DNA or MBTPS1 DNA was 15 μg, and the PBS group was used as a negative control. Finally, 72 hours after the second injection, the mice were killed by spinal dislocation, and the skin tissues were harvested for Western blotting and HE staining tests.

[0314] Fig. 20The results of evaluating the transfection efficiency of functional MBTPS1 DNA of different sizes mediated by branched poly (β-amino ester) ER-HPAE-1 and ER-HPAE-2 of Examples 1 and 2 in C57 mice are shown. The results of West Blot show that the two composite nanoparticles can efficiently express MBTPS1 protein in three transfected mice, further confirming that the composite nanoparticles disclosed in the present invention have important application potential in gene therapy of cataract, hair loss, oral mucosal diseases and psoriasis-like syndrome (CAOP) syndrome.

[0315] Fig.21 The results of evaluating the transfection efficiency of functional COL7A1 DNA of different sizes mediated by branched poly (β-amino ester) ER-HPAE-1 and ER-HPAE-2 of Examples 1 and 2 in C57 mice are shown. The results of Western Blot showed that the two composite nanoparticles were able to efficiently express C7 protein in the three transfected mice, further confirming that the nanoparticles have important application potential in RDEB gene therapy.

[0316] Fig. 22 The results of evaluating the in vivo toxicity of branched poly (β-amino ester) ER-HPAE-1 and ER-HPAE-2 of Examples 1 and 2 after transfection of functional MBTPS1 DNA of different sizes in C57 mice are shown. The results of HE staining show that the tissue structure of the skin of mice injected with two composite nanoparticles containing MBTPS1 DNA at the subcutaneous point 120 hours after injection is not significantly different from the normal tissue of non-transfected mice, so no obvious inflammation occurs after subcutaneous injection of composite nanoparticles, which confirms that the branched poly (β-amino ester) disclosed in the present invention has excellent safety and clinical application potential in in vivo gene delivery.

[0317] Fig.23 The results of evaluating the in vivo toxicity of branched poly (β-amino ester) ER-HPAE-1 and ER-HPAE-2 of Examples 1 and 2 after transfection of functional COL7A1 DNA of different sizes in C57 mice are shown. The results of HE staining show that the tissue structure of the skin of mice injected with two composite nanoparticles containing COL7A1 DNA at the subcutaneous point 120 hours after injection is not significantly different from the normal tissue of non-transfected mice, so no obvious inflammation occurs after subcutaneous injection of composite nanoparticles, which confirms that the branched poly (β-amino ester) disclosed in the present invention has excellent safety and clinical application potential in in vivo gene delivery.

[0318] Embodiment 11

[0319] The transfection efficiency of luciferase DNA mediated by branched poly (β-amino ester) ER-HPAE-1 and ER-HPAE-2 of Examples 1 and 2 was tested in C57 mice.

[0320] C57 mice were divided into three groups and injected subcutaneously once. The branched poly (β-amino ester) ER-HPAE-1 and ER-HPAE-2 obtained in Examples 1 and 2 were combined with DNA encoding luciferase to form composite nanoparticles (containing 7% F127) in an ethanol system, wherein the amount of DNA encoding luciferase was 20 μg. HPAE (HPAE without ABF, other monomer compositions were the same as those of the functionalized targeted branched poly (β-amino ester) in Example 1) / COL7A1 DNA or MBTPS1DNA nanoparticle group was used as a control, and in vivo imaging was performed 24 hours after in vivo transfection.

[0321] Fig.24 The results of evaluating the efficiency of luciferase DNA transfection mediated by branched poly (β-amino ester) ER-HPAE-1 and ER-HPAE-2 of Examples 1 and 2 in C57 mice by tail vein injection are shown. The in vivo luminescence results show that the branched poly (β-amino ester) ER-HPAE-1 and ER-HPAE-2 of Examples 1 and 2 can efficiently mediate DNA transfection by tail vein injection, wherein the highest luminescence intensity of luciferase mediated by branched poly (β-amino ester)-based nanoparticles of Example 1 exceeds 2.3*10 6 p / sec / cm2 / sr, far exceeding the luminescence intensity of luciferase mediated by HPAE (HPAE without ABF, other monomer compositions are the same as those of the branched poly (β-amino ester) in Example 1) based nanoparticles (no luminescence). These results show that the branched poly (β-amino ester) based nanoparticles disclosed in the present invention can overcome the complex physiological environment in the body (biomacromolecules such as proteins and nucleases) and efficiently mediate gene delivery, which also provides important support for further clinical applications.

[0322] Example 12

[0323] Female SD rats aged 6 to 8 weeks were selected, and the backs of the rats were shaved. Then, 300 μg of branched poly (β-amino ester) and 10 μg of Cy3-labeled DNA and laurocapram penetration enhancer were prepared into a mixed solution, and the final concentration of laurocapram was 1%. The mixed solution was then evenly applied to the shaved area on the back of the rats, and then the surface was covered with a medical dressing. After 4 hours, the rats were killed by cervical dislocation (approved by the Animal Ethics Committee of the Children's Hospital Affiliated to Fudan University, No.: 2023220), and the skin areas that had been applied were sampled, fixed in a paraformaldehyde solution, and then frozen sections were made. The prepared frozen sections were stained with nuclei and sealed, and finally fluorescence imaging was performed using a fluorescence microscope to observe the fluorescence distribution of cy3-labeled plasmids in skin tissue.

[0324] Fig.25 The results of evaluating the transdermal efficiency of Cy3-labeled DNA mediated by branched poly (β-amino ester) ER-HPAE-1 and ER-HPAE-2 of Examples 1 and 2, which were applied transdermally in female SD rats, are shown. The slice results show that after 4 hours of external application of composite nanoparticles to the skin, branched poly (β-amino ester) ER-HPAE-1 and ER-HPAE-2 of Examples 1 and 2 can efficiently deliver DNA to the dermis of the skin. The results confirm that the transdermal effect of branched poly (β-amino ester)-based nanoparticles of Examples 1 and 2 is significantly better than that of HPAE (HPAE without ABF, and the other monomer compositions are the same as those of the functionalized targeted branched poly (β-amino ester) in Example 1)-based nanoparticles and the commercial transfection reagent PEIpro (almost no luminescence is observed). These results indicate that the branched poly (β-amino ester)-based nanoparticles disclosed in the present invention have important clinical application potential in transdermal drug delivery.

[0325] Embodiment 13

[0326] Female SD rats aged 6 to 8 weeks were selected, and the backs of the rats were shaved. Then, 300 μg of branched poly (β-amino ester) and 10 μg of COL1A1, COL3A1 or silk protein plasmid were prepared into a mixed solution, and the mixed solution was evenly applied to the shaved area of ​​the rats' backs, and then the surface was covered with a medical dressing. After 48 hours, the rats were killed by cervical dislocation (approved by the Animal Ethics Committee of the Children's Hospital Affiliated to Fudan University, No.: 2023220), and the skin areas that had been applied were sampled, proteins were extracted, and the expression of COL1A1, COL3A1 or silk protein in the skin tissue was detected using Western Blot technology.

[0327] Fig.26The results of evaluating the transdermal efficiency of COL1A1, CLO3A1 or silk protein DNA mediated by branched poly (β-amino ester) ER-HPAE-1 and ER-HPAE-2 of Examples 1 and 2 applied transdermally on female SD rats are shown. Western Blot results show that after 48 hours of external application of composite nanoparticles to the skin, branched poly (β-amino ester) ER-HPAE-1 and ER-HPAE-2 of Examples 1 and 2 can efficiently deliver COL1A1, CLO3A1 or silk protein DNA to skin cells, and DNA successfully expresses corresponding proteins in cells. The results confirm that the transdermal effect of branched poly (β-amino ester)-based nanoparticles of Examples 1 and 2 is significantly better than that of HPAE (HPAE without ABF, and the other monomer compositions are the same as those of the functionalized targeted branched poly (β-amino ester) in Example 1)-based nanoparticles. These results indicate that the branched poly (β-amino ester)-based nanoparticles disclosed in the present invention have important application potential in medical beauty.

[0328] While the disclosure has been particularly shown and described with reference to particular embodiments, some of which are preferred embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the disclosure as herein disclosed.

Claims

1. A branched poly(β-amino ester) having a structure represented by formula (I), formula (II), formula (III), formula (IV) or formula (V): in, W 1 , W 2 , W 3 , W 4 and W 5 are each independently selected from alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkylcycloalkylalkyl, alkylheterocyclylalkyl, alkylarylalkyl, or alkylheteroarylalkyl, wherein the alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkylcycloalkylalkyl, alkylheterocyclylalkyl, alkylarylalkyl, and alkylheteroarylalkyl are optionally substituted by one or more R W replace; L is selected from alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkylcycloalkyl, alkylheterocyclyl, alkylcycloalkylalkyl, alkylheterocyclylalkyl, alkylarylalkyl, alkylheteroarylalkyl, or heteroalkylarylalkylarylheteroalkyl, wherein said alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkylcycloalkylalkyl, alkylheterocyclylalkyl, alkylarylalkyl, alkylheteroarylalkyl, and heteroalkylarylalkylarylheteroalkyl are optionally replaced by one or more R L replace; Each R 1 Independently selected from R 1 N or T; R 1 N is selected from alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkylcycloalkyl, alkylheterocyclyl, alkylaryl, alkylheteroaryl or nitrogen protecting group, wherein the alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkylcycloalkyl, alkylheterocyclyl, alkylaryl and alkylheteroaryl are optionally substituted by one or more R N replace; Each R 2 Independently selected from R 2 N or T; R 2 N is selected from alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkylcycloalkyl, alkylheterocyclyl, alkylaryl, alkylheteroaryl or a nitrogen protecting group, wherein the alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkylcycloalkyl, alkylheterocyclyl, alkylaryl, alkylheteroaryl, alkylcycloalkylalkyl, alkylheterocyclylalkyl, alkylarylalkyl and alkylheteroarylalkyl are optionally substituted with one or more R N replace; Each T is independently an organelle targeting moiety; Each R W , R L , R L and R N Each independently selected from the group consisting of halogen, cyano, nitro, oxy, -OR a 、-SR a 、-N(R a ) 2 、-C(O)OR a 、-C(O)N(R a ) 2 、-N(R a )C(O)OR a 、-OC(O)OR a 、-OC(O)N(R a ) 2 、-N(R a )C(O)N(R a ) 2 、-S(O)R a 、-S(O)OR a 、-S(O)N(R a ) 2 、-OS(O)R a 、-N(R a )S(O)R a 、-S(O) 2 R a 、-S(O) 2 OR a 、-S(O) 2 N(R a ) 2 、-OS(O) 2 R a 、-N(R a )S(O) 2 R a , alkyl, heteroalkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl; Each R a are each independently selected from hydrogen or alkyl; m1, m2, m3, m4, m5, m6, m7, m8, m9, m10, m11 and m12 are each independently in the range of 1 to 100.

2. The branched poly(β-amino ester) according to claim 1, in, Each R 1 Independently for R 1 N , and at least one R 2 is T.

3. The branched poly(β-amino ester) of claim 2, wherein each R 1 N independently selected from alkyl, heteroalkyl, alkylcycloalkyl, alkylheterocyclyl, alkylaryl or alkylheteroaryl, wherein the alkyl, heteroalkyl, alkylcycloalkyl, alkylheterocyclyl, alkylaryl and alkylheteroaryl are optionally substituted by one or more R N Replace, and each R N Independently -OR a or -N(R a ) 2 .

4. The branched poly(β-amino ester) of claim 3, wherein each R 1 Independently selected from the following group:

5. The branched poly(β-amino ester) of claim 4, wherein each R 1 Independently selected from the group consisting of:

6. The branched poly(β-amino ester) according to any one of claims 2 to 5, wherein each R 2 N is independently selected from alkyl, heteroalkyl, alkylcycloalkyl, alkylheterocyclyl or alkylheterocyclylalkyl, wherein the alkyl, heteroalkyl, alkylcycloalkyl, alkylheterocyclyl and alkylheterocyclylalkyl are optionally substituted by one or more R N Replace, and each R N Independently -OR a or -N(R a ) 2 .

7. The branched poly(β-amino ester) of claim 6, wherein each R 2 Independently selected from the group consisting of: and T.

8. The branched poly(β-amino ester) of claim 7, wherein each R 2 Independently selected from the group consisting of: and T.

9. The branched poly(β-amino ester) according to claim 1, having a structure shown in formula (I), in, exist In the repeating unit, at least 1, 2, 3, 4 or 5 R 1 is T, and Each R 2 Independently for R 2 N .

10. The branched poly(β-amino ester) of claim 9, wherein W 1 is alkyl or heteroalkyl, the alkyl and heteroalkyl are optionally substituted by one or more R W replace.

11. The branched poly(β-amino ester) of claim 10, wherein W 1 for 12. The branched poly(β-amino ester) according to claim 1, having a structure shown in formula (II), in, exist In the repeating unit, at least 1, 2, 3, 4, 5 or 6 R 1 is T, and Each R 2 Independently for R 2 N .

13. The branched poly(β-amino ester) of claim 12, wherein W 2 is alkyl or heteroalkyl, the alkyl and heteroalkyl are optionally substituted by one or more R W replace.

14. The branched poly(β-amino ester) of claim 13, wherein W 2 for 15. The branched poly(β-amino ester) according to claim 1, having a structure shown in formula (III), in, exist In the repeating unit, at least 1, 2, 3, 4, 5, 6, 7 or 8 R 1 is T, and Each R 2 Independently for R 2 N .

16. The branched poly(β-amino ester) of claim 15, wherein W 3 is alkyl or heteroalkyl, the alkyl and heteroalkyl are optionally substituted by one or more R W replace.

17. The branched poly(β-amino ester) of claim 16, wherein W 3 for 18. The branched poly(β-amino ester) according to claim 1, having a structure shown in formula (IV), in, exist In the repeating unit, at least 1, 2, 3, 4, 5, 6, 7, 8 or 9 R 1 is T, and Each R 2 Independently for R 2 N .

19. The branched poly(β-amino ester) of claim 18, wherein W 4 is alkyl or heteroalkyl, the alkyl and heteroalkyl are optionally substituted by one or more R W replace.

20. The branched poly(β-amino ester) of claim 19, wherein W 4 for 21. The branched poly(β-amino ester) according to claim 1, having a structure shown in formula (V), in, exist In the repeating unit, at least 1 or 2 R 1 is T, and Each R 2 Independently for R 2 N .

22. The branched poly(β-amino ester) of claim 21, wherein W 5 is alkyl, heteroalkyl or alkylheterocyclylalkyl, wherein the alkyl, heteroalkyl and alkylheterocyclylalkyl are optionally substituted by one or more R W replace.

23. The branched poly(β-amino ester) of claim 22, wherein W 5 Selected from:

24. The branched poly(β-amino ester) of any one of claims 9 to 23, wherein each R 1 N independently selected from alkyl, heteroalkyl, alkylcycloalkyl, alkylheterocyclyl, alkylaryl or alkylheteroaryl, wherein the alkyl, heteroalkyl, alkylcycloalkyl, alkylheterocyclyl, alkylaryl and alkylheteroaryl are optionally substituted by one or more R N Replace, and each R N Independently -OR a or -N(R a ) 2 .

25. The branched poly(β-amino ester) of claim 24, wherein each R 1 Independently selected from the following group: and T.

26. The branched poly(β-amino ester) of claim 25, wherein each R 1 Independently selected from the following group: and T.

27. The branched poly(β-amino ester) of any one of claims 9 to 26, wherein each R 2 N independently selected from alkyl, heteroalkyl, alkylcycloalkyl, alkylheterocyclyl, alkylheterocyclylalkyl, wherein the alkyl, heteroalkyl, alkylcycloalkyl, alkylheterocyclyl and alkylheterocyclylalkyl are optionally substituted by one or more R N Replace, and each R N Independently -OR a or -N(R a ) 2 .

28. The branched poly(β-amino ester) of claim 27, wherein each R 2 Independently selected from the group consisting of:

29. The branched poly(β-amino ester) of claim 28, wherein each R 2 Independently selected from the group consisting of:

30. The branched poly(β-amino ester) of any one of claims 1-29, wherein T is an endoplasmic reticulum targeting moiety.

31. The branched poly(β-amino ester) of claim 30, wherein T is selected from the group consisting of:

32. The branched poly(β-amino ester) of any one of claims 1 to 31, wherein L is selected from alkyl, heteroalkyl, heterocyclyl, alkylheterocyclyl, alkylcycloalkylalkyl, alkylheterocyclylalkyl, or heteroalkylarylalkylarylheteroalkyl, wherein the alkyl, heteroalkyl, alkylcycloalkylalkyl, alkylheterocyclylalkyl, and heteroalkylarylalkylarylheteroalkyl are optionally substituted with one or more R L Replace, and each R L Independently -OR a 、-N(R a ) 2 or alkyl.

33. The branched poly(β-amino ester) of claim 32, wherein L is selected from the group consisting of:

34. The branched poly(β-amino ester) of claim 33, wherein L is selected from the group consisting of:

35. The branched poly(β-amino ester) of any one of claims 1-34, wherein ml, m2, m3, m4, m5, m6, m7, m8, m9, mlO, ml 1 and m12 are each independently in the range of 5 to 75.

36. The branched poly(β-amino ester) of any one of claims 1-35, having a molecular weight in the range of 5.0 kDa to 40.0 kDa, in the range of 5.0 kDa to 50 kDa, or in the range of 5.0 kDa to 40.0 kDa.

37. The branched poly(β-amino ester) of claim 36, having a molecular weight of about 5.0 kDa, 6.0 kDa, 7.0 kDa, or 8.0 kDa.

38. A method for preparing a branched poly(β-amino ester) as claimed in any one of claims 1 to 37, wherein include: 1-1) a multi-acrylate monomer having formula (I-1), formula (II-1), formula (III-1) or formula (IV-1): A branched poly(β-amino ester) P1 having a terminal double bond is obtained by a Michael addition reaction with a diacrylate monomer having formula (2) and one or more amine monomers having formula (3): 1-2) subjecting the branched poly(β-amino ester) P1 obtained in step 1-1) to a Michael addition reaction with one or more end-capping amine monomers having the formula (4) to obtain a branched poly(β-amino ester) having a structure represented by the formula (I), formula (II), formula (III) or formula (IV): R 2 -NH 2 Formula (4) or, 2-1) A diamine monomer having the formula (V-1) is reacted with a diacrylate monomer having the formula (2) and one or more amine monomers having the formula (3) by Michael addition reaction to obtain a branched poly(β-amino ester) P2 having a double bond at the end: 2-2) The branched poly(β-amino ester) P2 prepared in step 2-1) is subjected to a Michael addition reaction with one or more end-capping amine monomers having the formula (4) to prepare a branched poly(β-amino ester) having a structure represented by the formula (V): R 2 -NH 2 Formula (4).

39. The method according to claim 38, in, The multi-acrylate monomer having formula (I-1) is The multi-acrylate monomer having formula (II-1) is The multi-acrylate monomer having formula (III-1) is The multi-acrylate monomer having formula (IV-1) is The diamine monomer having formula (V-1) is selected from the following group: The diacrylate monomer having formula (2) is selected from the following group:

40. The method according to claim 38 or 39, wherein the one or more amine monomers having formula (3) are R 1 N -NH 2 , and the one or more end-capping amine monomers having formula (4) are selected from R 2 N -NH 2 or T-NH 2 , and at least one of the end-capping amine monomers of formula (4) is T-NH 2 .

41. The method according to claim 40, in, R 1 N -NH 2 Select from the following group: R 2 N -NH 2 Select from the following group: T-NH 2 Select from the following group:

42. The method according to claim 40 or 41, in, A diacrylate monomer having the formula (2), R 2 N -NH 2 、T-NH 2 and R 1 N -NH 2 The reaction feed molar ratio is (1-3):(0.1-4):(0.02-2):(0.5-6); In step 1-1), the diacrylate monomer having formula (2), R 1 N -NH 2 and a multi-acrylate monomer having formula (I-1), formula (II-1), formula (III-1) or formula (IV-1) in a molar ratio of (1 to 3):(0.1 to 4):(0.5 to 3); In step 2-1), the diacrylate monomer having formula (2), R 1 N -NH 2 The molar ratio of the diamine monomer having the formula (V-1) is (1 to 3):(0.1 to 4):(0.5 to 3).

43. The method according to claim 38 or 39, wherein the one or more amine monomers having formula (3) are selected from R 1 N -NH 2 or T-NH 2 , and the one or more end-capping amine monomers having formula (4) are R 2 N -NH 2 .

44. The method according to claim 43, in, R 1 N -NH 2 Select from the following group: R 2 N -NH 2 Select from the following group: T-NH 2 Select from the following group:

45. The method according to claim 43 or 44, in, A diacrylate monomer having the formula (2), R 2 N -NH 2 、T-NH 2 and R 1 N -NH 2 The reaction feed molar ratio is (1-3):(0.1-4):(0.02-2):(0.5-6); In step 1-1), the diacrylate monomer having formula (2), R 1 N -NH 2 , a multi-acrylate monomer having formula (I-1), formula (II-1), formula (III-1) or formula (IV-1) and T-NH 2 The molar ratio is (1-3):(0.1-4):(0.5-3):(0.02-2); In step 2-1), the diacrylate monomer having formula (2), R 1 N -NH 2 , a diamine monomer having formula (V-1) and T-NH 2 The molar ratio is (1~3):(0.1~4):(0.5~3):(0.02~2).

46. ​​The method according to any one of claims 38 to 45, wherein step 1-1) and step 2-1) are performed at a temperature of 15 to 100°C.

47. The method according to claim 46, wherein step 1-1) and step 2-1) are performed at a temperature of about 90°C.

48. The method according to any one of claims 38 to 47, wherein the molecular weight of the branched poly(β-amino ester) P1 having a terminal double bond obtained in step 1-1) is in the range of 5 kDa to 40 kDa.

49. The method according to any one of claims 38 to 47, wherein the molecular weight of the branched poly(β-amino ester) P2 having a terminal double bond obtained in step 2-1) is in the range of 5 kDa to 40 kDa.

50. The method according to any one of claims 38 to 49, wherein step 1-2) and step 2-2) are performed at a temperature of 15 to 60°C.

51. The method of claim 50, wherein step 1-2) and step 2-2) are performed at a temperature of about 25°C.

52. A composition comprising the branched poly(β-amino ester) of any one of claims 1-37 and a polynucleotide.

53. The composition of claim 52, wherein the polynucleotide is DNA or RNA.

54. The composition of claim 53, wherein the DNA encodes C7 protein, TGM1 protein, MBTPS1 protein, COL1A1 protein, COL3A1 protein, silk protein, Cas9 protein, ADAM17 protein, TGF-β1 protein, ABCD1 protein, P53 protein, RPE65 protein, SMN1 protein, AADC protein, FVIII protein, VEGF protein, and HGF protein.

55. The composition of any one of claims 52-54, wherein the mass ratio of the branched poly(β-amino ester) to polynucleotide is in the range of 1:1 to 200:

1.

56. The composition of claim 55, wherein the mass ratio of the branched poly(β-amino ester) to polynucleotide is in the range of 5:1 to 120:

1.

57. A composition according to any one of claims 52-56, which is in the form of nanoparticles.

58. The composition of claim 57, wherein the nanoparticles have a particle size ranging from 50 nm to 500 nm.

59. The composition of claim 58, wherein the nanoparticles have a particle size ranging from 150 nm to 400 nm.

60. A method of delivering a polynucleotide to a subject, comprising administering to the subject the composition of any one of claims 52-59.

61. A method of treating a disease in a subject, comprising administering to the subject a composition as described in any one of claims 52-59.

62. The method of claim 60 or 61, wherein the composition is administered to the subject transdermally, subcutaneously, or intravenously.

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