Branched poly(β-amino ester), and preparation method therefor and use thereof

Abstract

The present disclosure relates to a functionalized targeted branched poly(β-amino ester), a nanoparticle based thereon, a preparation method therefor, and the use thereof. Particularly, the functionalized targeted branched poly(β-amino ester) provided in the present disclosure is capable of effectively delivering biomolecules, particularly functional biomolecules for treating and / or preventing various diseases and conditions.

Description

Branched poly(beta-amino esters) and methods of making and uses thereof TECHNICAL FIELD

[0001] The present disclosure relates to functionalized targeted branched poly(beta-amino esters), nanoparticles based thereon, methods of making the same, and uses thereof. BACKGROUND

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

[0003] However, the lack of safe and efficient carriers for cell and functional gene delivery seriously limits the clinical application of gene therapy. At present, gene carriers 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) can deliver larger gene fragments, and have a wider range of applications; 2) have low immunogenicity and high safety; 3) are flexible in design, easy to modify, and have tissue specificity; 4) have good biodegradability and cause little cell damage; 5) are simple to prepare, have low cost, and are easy to mass-produce. The current mainstream cationic polymers include polyethyleneimine (PEI), poly (dimethylamino ethyl methacrylate) (PDMAEMA), polyamidoamine (PAMAM), etc. However, these cationic polymers often have poor degradation performance, easily cause high post-transfection cytotoxicity, and the monomer types are often single, which limits their clinical application.

[0004] Due to customizable composition, structure, relative molecular mass, biodegradability, high stability, and good biocompatibility, linear and branched poly(beta-amino esters) exhibit certain application potential in clinical gene therapy of genetic skin diseases, cancer, and gene delivery of various types of cell lines. However, the complex nanoparticles formed by poly(beta-amino esters) cationic polymers and DNA often remain in lysosomes after entering cells, and a small part of the complex nanoparticles that escape lysosomes through the proton sponge effect also cause certain cytotoxicity. In addition, the complex nanoparticles have a large particle size, causing difficulty in nuclear internalization, and limited delivery efficiency in vivo, which seriously hinders subsequent gene transfection and greatly limits the application research in clinical transformation. SUMMARY

[0005] To overcome the drawbacks of the above prior art, the present disclosure provides a branched poly(β-amino ester), nanoparticles based thereon, methods of making the same and uses thereof. In particular, the branched poly(β-amino ester) provided by the present disclosure is capable of efficiently delivering biomolecules, particularly functional biomolecules for the treatment and / or prevention of various diseases and disorders.

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

[0007] wherein,

[0008] W1, W2, W3, W4and W5are 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 with one or more R W ;

[0009] L is selected from alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkylcycloalkyl, alkylheterocyclyl, alkylcycloalkylalkyl, alkylheterocyclylalkyl, alkylarylalkyl, alkylheteroarylalkyl or heteroalkylarylalkylarylheteroalkyl, wherein the alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkylcycloalkylalkyl, alkylheterocyclylalkyl, alkylarylalkyl, alkylheteroarylalkyl and heteroalkylarylalkylarylheteroalkyl are optionally substituted with one or more R L ;

[0010] each R1is independently selected from R1 N or T;

[0011] R1 N is selected from alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkylcycloalkyl, alkylheterocyclyl, alkylaryl, alkylheteroaryl or a nitrogen protecting group, the alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkylcycloalkyl, alkylheterocyclyl, alkylaryl and alkylheteroaryl being optionally substituted with one or more R N ;

[0012] each R2is independently selected from R2 N or T;

[0013] R2 Nselected from alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkylcycloalkyl, alkylheterocyclyl, alkylaryl, alkylheteroaryl, or a nitrogen protecting group, said alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkylcycloalkyl, alkylheterocyclyl, alkylaryl, alkylheteroaryl, alkylcycloalkylalkyl, alkylheterocyclylalkyl, alkylarylalkyl, and alkylheteroarylalkyl being optionally substituted with one or more R N substituents;

[0014] each T is independently an organelle targeting moiety;

[0015] each R W , R L , and R N is each independently selected from the group consisting of halogen, cyano, nitro, oxo, -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;

[0016] each R a is each independently selected from hydrogen or alkyl;

[0017] m1, m2, m3, m4, m5, m6, m7, m8, m9, m10, m11, and m12 are each independently in the range of 1 to 100.

[0018] In another aspect, the present disclosure provides a method of preparing a branched poly(beta-amino ester) of Formula (I), Formula (II), Formula (III), Formula (IV), or Formula (V), comprising:

[0019] 1-1) subjecting a multi-acrylate monomer having Formula (I-1), Formula (II-1), Formula (III-1), or Formula (IV-1):

[0020] to a Michael addition reaction with a di-acrylate monomer having Formula (2) and one or more amine monomers having Formula (3) to obtain a branched poly(beta-amino ester) P1 having terminal double bonds:

[0021] 1-2) subjecting the branched poly(beta-amino ester) P1 obtained in step 1-1) to a Michael addition reaction with one or more capping amine monomers having Formula (4) to obtain a branched poly(beta-amino ester) having a structure represented by Formula (I), Formula (II), Formula (III), or Formula (IV):

[0022] or,

[0023] 2-1) subjecting a diamine monomer having Formula (V-1) to a Michael addition reaction with a di-acrylate monomer having Formula (2) and one or more amine monomers having Formula (3) to obtain a branched poly(beta-amino ester) P2 having terminal double bonds:

[0024] 2-2) subjecting the branched poly(beta-amino ester) P2 obtained in step 2-1) to a Michael addition reaction with one or more capping amine monomers having Formula (4) to obtain a branched poly(beta-amino ester) having a structure represented by Formula (V):

[0025] In another aspect, the present disclosure provides a composition comprising a branched poly(beta-amino ester) of Formula (I), Formula (II), Formula (III), Formula (IV), or Formula (V) and a biomolecule.

[0026] In another aspect, the present disclosure provides a method of delivering a biomolecule to a subject, comprising administering to the subject a composition of the present disclosure.

[0027] 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 DRAWINGS

[0028] FIG. 1 shows a schematic of the synthesis of branched poly(beta-amino esters) of Examples 1-4.

[0029] Figure 2 is a GPC curve of the branched poly( -amino ester) of Examples 1-4.

[0030] Figure 3 is a GPC curve of the branched poly( -amino ester) of Examples 1-4. 1 HNMR spectrum.

[0031] Figure 4 is PicoGreen results of the affinity performance test of the branched poly( -amino ester) of Examples 1-4 to TGM1 DNA at different mass ratios.

[0032] Figure 5 is DLS characterization results of the particle size of the complex nanoparticles formed by the branched poly( -amino ester) of Examples 1-4 and TGM1 DNA at different mass ratios.

[0033] Figure 6 is DLS characterization results of the Zeta potential of the complex nanoparticles formed by the branched poly( -amino ester) of Examples 1-4 and TGM1 DNA at different mass ratios.

[0034] Figure 7 is a TEM micro-morphology of the nanoparticles formed by the branched poly( -amino ester) of Examples 1-4 and TGM1 DNA.

[0035] Figure 8 is characterization results of the cellular endocytosis efficiency of the complex nanoparticles formed by the branched poly( -amino ester) of Examples 1 and 2 and AF647 fluorescent dye-labeled DNA.

[0036] Figure 9 is characterization of the organelle targeting of the complex nanoparticles formed by the branched poly( -amino ester) of Examples 1 and 2 and AF647 fluorescent dye-labeled DNA.

[0037] Figure 10 is characterization of the organelle targeting of the complex nanoparticles formed by the branched poly( -amino ester) of Examples 1 and 2 and AF647 fluorescent dye-labeled DNA.

[0038] Figure 11 is detection results of the transfection efficiency of the complex nanoparticles formed by the branched poly( -amino ester) of Examples 1-4 and GFP DNA at different mass ratios in mouse embryonic fibroblast (3T3) cells.

[0039] Figure 12 is cell flow detection results of the transfection efficiency of the complex nanoparticles formed by the branched poly( -amino ester) of Examples 1-4 and GFP DNA in 3T3 cells.

[0040] Figure 13 is alarmBlue detection results of the survival rate of suspended cells after transfection of the complex nanoparticles formed by the branched poly( -amino ester) of Examples 1 and 2 and GFP DNA in five different tissue cells (A2780 cells, 293T cells, 3T3 cells, SW1353 cells, Vero cells).

[0041] Figure 14 is a fluorescence detection result of the transfection efficiency of the complex nanoparticles formed by the branched poly( -amino ester)s of Examples 1-4 and GFP DNA at different mass ratios in suspension cells (293F).

[0042] Figure 15 is a cell flow detection result of the transfection efficiency of the complex nanoparticles formed by the branched poly( -amino ester)s of Examples 1-4 and GFP DNA at a mass ratio of 20:1 in suspension cells (293F).

[0043] Figures 16(a), 16(b), 16(c) and 16(d) are alarmBlue detection results of the survival rate of the suspension cells after transfection of the complex nanoparticles formed by the branched poly( -amino ester)s of Examples 1-4 and GFP DNA at mass ratios of 120:1, 100:1, 80:1, 60:1, 40:1, 20:1, 10:1 and 5:1, respectively, in suspension cells, wherein the mass ratios are 120:1, 100:1, 80:1, 60:1, 40:1, 20:1, 10:1 and 5:1, respectively.

[0044] Figure 17 is a qPCR detection result of the transfection efficiency of the complex nanoparticles formed by the branched poly( -amino ester)s of Examples 1-2 and COL7A1 DNA, TGM1 DNA and MBTPS1 DNA, respectively, in HaCaT cells.

[0045] Figure 18 is a qPCR detection result of the transfection efficiency of the complex nanoparticles formed by the branched poly( -amino ester)s of Examples 1-2 and COL7A1 DNA, TGM1 DNA and MBTPS1 DNA, respectively, in human primary fibroblast (NHF) cells.

[0046] Figure 19 is a Western Blot detection result of the transfection efficiency of the complex nanoparticles formed by the branched poly( -amino ester)s of Examples 1-2 and COL7A1 DNA, TGM1 DNA and MBTPS1 DNA, respectively, in NHF cells and HaCaT cells.

[0047] Figure 20 is a Western Blot detection result of the transfection efficiency of the complex nanoparticles formed by the branched poly( -amino ester)s of Examples 1-2 and MBTPS1 DNA in C57 mice.

[0048] Figure 21 is a Western Blot detection result of the transfection efficiency of the complex nanoparticles formed by the branched poly( -amino ester)s of Examples 1-2 and COL7A1 DNA in C57 mice.

[0049] Figure 22 is HE staining results of in vivo toxicity of the complex nanoparticles of branched poly(beta-amino ester)s of Examples 1-2 and MBTPS1 DNA in C57 mice after transfection.

[0050] Figure 23 is HE staining results of in vivo toxicity of the complex nanoparticles of branched poly(beta-amino ester)s of Examples 1-2 and COL7A1 DNA in C57 mice after transfection.

[0051] Figure 24 is fluorescence results of transfection efficiency of the complex nanoparticles of branched poly(beta-amino ester)s of Examples 1-2 and luciferase DNA in C57 mice administered by tail vein injection.

[0052] Figure 25 is section results of transdermal efficiency of the complex nanoparticles of branched poly(beta-amino ester)s of Examples 1-2 and Cy3 labeled DNA in SD rats administered by smearing transdermally.

[0053] Figure 26 is Western Blot detection results of transfection efficiency of the complex nanoparticles of branched poly(beta-amino ester)s of Examples 1-2 and COL1A1 DNA, CLO3A1 DNA or silk protein DNA in SD rats administered by smearing transdermally.

[0054] Figure 27 is detection results of transfection efficiency of the complex nanoparticles of branched poly(beta-amino ester)s of Examples 1-4 and GFP mRNA in SW1353 cells.

[0055] Figure 28 is detection results of transfection efficiency of the complex nanoparticles of branched poly(beta-amino ester)s of Examples 1-4 and GFP mRNA in HeLa cells.

[0056] Figure 29 is detection results of transfection efficiency of the complex nanoparticles of branched poly(beta-amino ester)s of Examples 1-4 and BSA-FITC in HeLa cells.

[0057] Figure 30 is detection results of intracellular delivery efficiency of the complex nanoparticles of branched poly(beta-amino ester)s of Examples 1-4 and BSA-FITC in HaCaT cells.

[0058] Figures 31 and 32 are detection results of transfection efficiency of the complex nanoparticles of branched poly(beta-amino ester)s of Examples 1-4 and GFP siRNA in 293T-GFP cells.

[0059] FIGS. 33 and 34 are results of transfection efficiency assays of complex nanoparticles formed by branched poly( -amino ester)s of Examples 1-4 and GFP siRNA in HeLa-GFP cells. DETAILED DESCRIPTION

[0060] Reference will now be made in detail to certain embodiments of the present disclosure, examples of which are illustrated in the accompanying structures and formulas. While the present disclosure will be described in conjunction with the enumerated embodiments, it will be understood that they 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 that can be included within the scope of the present disclosure as defined by the claims. One skilled in the art will recognize many methods and materials as being suitable for use in practicing the present disclosure. It is intended to include within the scope of the present disclosure, all such alternatives, modifications and equivalents as can be permitted by law. Nothing in the present disclosure is intended to be construed as limiting the present disclosure to one or more particular methods or materials. Rather, the present disclosure is to cover all statutory equivalents, as well as all equivalents of these methods and materials that are within the scope of the present disclosure, as defined by the claims. All references, patents and / or applications disclosed herein are hereby incorporated by reference in their entirety, as if each had been individually incorporated by reference.

[0061] It should be understood that certain features of the present disclosure, which are, for clarity, described in the context of separate embodiments, can also be provided in combination in a single embodiment. Conversely, various features of the present disclosure, which are, for brevity, described in the context of a single embodiment, can also be provided separately or in any suitable

[0062] DEFINITIONS

[0063] Definitions of specific functional groups and chemical terms are described in more detail below. For purposes of this disclosure, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75thEd., inside cover, and specific functional group terms are generally defined as described therein. Additionally, general principles of organic chemistry, and specific functional moieties and reactivity descriptions are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March, March’s Advanced Organic Chemistry, 5thEdition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; Carruthers, Some Modern Methods of Organic Synthesis, 3rdEdition, Cambridge University Press, Cambridge, 1987; each of which is incorporated herein by reference in its entirety.

[0064] Throughout this disclosure, connecting substituents are described. Where a structure explicitly requires a connecting group, the Markush variable recited in connection with that group should be understood to be a connecting group. For example, if a structure requires a connecting group and the Markush group definition recitation lists “alkyl,” then it should be understood that the “alkyl” represents a connecting alkylene group.

[0065] As used herein, the term "substituted," whether preceded by the term "optionally" or not, means that one or more hydrogens of the designated moiety is replaced by a suitable substituent. It is understood that "substitution" or "substituted" includes the implicit proviso that such substitution is in accordance with permitted valence of the atom to which the moiety is substituted, and the substitution results in a stable or chemically feasible compound, e.g., a compound that does not spontaneously undergo rearrangement, cyclization, eliminations or other conversion in which the stability of the compound is adversely affected. Unless otherwise indicated, an "optionally substituted" group can have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure can be substituted with more than one substituent selected from a specified group, the substituent may, at each position, be either the same or different. The skilled artisan will appreciate that the substituents themselves can be further substituted, if appropriate. Unless specifically identified as "unsubstituted," a reference to a chemical moiety herein is understood to include substituted variants. For example, a reference to an "aryl" group or moiety implicitly includes both substituted and unsubstituted variants.

[0066] As used herein, the term "about," when used in reference to a given range or value (e.g., a temperature, time, amount, and concentration), indicates approximations that can vary by ±10%, ±5%, or ±1% of the given range or value.

[0067] When any variable (e.g., 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 to be substituted with 0 to 2 R i groups, then at each occurrence the group can optionally be substituted with up to two R i groups, and the R i groups are at each occurrence independently selected from the definition of R i . Also, combinations of substituents and / or variables are permissible, but only if such combinations result in stable compounds.

[0068] As used herein, the term "C i-j " indicates a range of the number of carbon atoms, wherein i and j are integers, and the range of the number of carbon atoms includes the endpoints (i.e., i and j) and every integer point in between, 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.

[0069] Regardless of whether used as part of another term or independently, the term "alkyl" as used herein refers to a saturated straight-chain or branched-chain hydrocarbon group. The term "C 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 (i-propyl), 1 -butyl (n-butyl), 2-methyl-l -propyl (i-butyl), 2-butyl (sec-butyl), 2-methyl-2-propyl (t-butyl), 1-pentyl (n-pentyl), 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-l -butyl, 2-methyl-l -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, and the like. "C 1-12 Examples of "alkyl" include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl. "C 1-6 Examples of "alkyl" include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl. "C

[0070] The alkyl groups can be optionally substituted with a substituent that independently replaces one or more of the hydrogens on one or more carbons of the alkyl group. Examples of such substituents can include, but are not limited to, halogen, hydroxyl, 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, phosphonato, phosphinato, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, formylamino, and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonates, sulfamoyl, sulfamido, aryl, heteroaryl, saturated or partially unsaturated cyclic alkyl, or saturated or partially unsaturated heterocyclic radical. Alkenyl, alkynyl, aryl, heteroaryl, saturated or partially unsaturated cyclic alkyl, and saturated or partially unsaturated heterocyclic radicals as described below can also be similarly substituted. The term "alkyl" as used herein refers to a straight-chain or branched-chain monovalent alkyl radical having from one to twelve carbon atoms, preferably one to eight carbon atoms, and more preferably one to six carbon atoms. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, t-butyl, pentyl, isopentyl, hexyl, isohexyl, and the like. The alkyl groups can be optionally substituted with a substituent that independently replaces one or more of the hydrogens on one or more carbons of the alkyl group. Examples of such substituents can include, but are not limited to, halogen, hydroxyl, 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, phosphonato, phosphinato, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, formylamino, and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonates, sulfamoyl, sulfamido, aryl, heteroaryl, saturated or partially unsaturated cyclic alkyl, or saturated or partially unsaturated heterocyclic radical. Alkenyl, alkynyl, aryl, heteroaryl, saturated or partially unsaturated cyclic alkyl, and saturated or partially unsaturated heterocyclic radicals as described below can also be similarly substituted.

[0071] As used herein, the term "aryl" refers to monocyclic and polycyclic systems having from 5 to 20 ring members, which can be optionally substituted independently with one or more substituents described herein, either as part of another term or used independently, wherein at least one ring in the system is aromatic and wherein each ring in the system contains from 3 to 12 ring members. Examples of "aryl" include, but are not limited to, phenyl, biphenyl, naphthyl, anthryl, and the like, which can bear 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 polycyclic systems, only one ring need be aromatic (e.g., 2,3-dihydroindole), but all rings can be aromatic (e.g., quinoline). The second ring can also be a fused, bridged, or spiro ring. Examples of polycyclic aryl groups include, but are not limited to, benzofuryl, indanyl, phthalimidyl, naphthalimidyl, indolizinyl, or tetrahydronaphthyl, and the like. The aryl group can be optionally substituted at one or more ring positions with one or more substituents as described herein.

[0072] As used herein, the terms "cycloalkyl," "carbocyclic group," and "carbocycle" are interchangeable and, whether used alone or as part of another term, refer to monovalent, saturated or partially unsaturated or fully unsaturated monocyclic and polycyclic ring systems that can 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, a cycloalkyl group can 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. A cycloalkyl group can be saturated or partially unsaturated. A cycloalkyl group can be optionally independently substituted with one or more substituents described herein. In some embodiments, a cycloalkyl group can be a saturated cyclic alkyl group. In some embodiments, a cycloalkyl group can be an unsaturated cyclic alkyl group containing at least one double bond or a triple bond in its ring system.

[0073] In some embodiments, a cycloalkyl group can 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.

[0074] In some embodiments, a cycloalkyl group can be a saturated or unsaturated polycyclic (e.g., bicyclic and tricyclic) carbocyclic ring system, which can be a fused, spiro, or bridged ring system. As used herein, the term "fused" refers to a ring system having two rings that share two adjacent atoms, the term "spiro" refers to a ring system having two rings connected by a single common atom, and the term "bridged" refers to a ring system having two rings that share three or more atoms. Examples of fused carbocyclic groups include, but are not limited to, naphthyl, benzopyrenyl, anthryl, acenaphthyl, fluorenyl, and the like. Examples of spiro carbocyclic groups include, but are not limited to, spiro[5.5]undecyl, spiropentadienyl, spiro[3.6]decyl, and the like. Examples of bridged carbocyclic groups include, but are not limited to, bicyclo[l,l,l]pentenyl, bicyclo[2,2,l]heptenyl, bicyclo[2.2.1]heptyl, bicyclo[2.2.2]octyl, bicyclo[3.3.1]nonyl, bicyclo[3.3.3]undecyl, and the like.

[0075] As used herein, the term "haloalkyl" refers to an alkyl group substituted with one or more halogen atoms independently replacing one or more hydrogen atoms on one or more carbons of the alkyl group.

[0076] As used herein, the term "heteroalkyl" refers to an alkyl group in which at least one of the carbon atoms is replaced by a heteroatom selected from N, O, S, or P. Heteroalkyl groups can be carbon-based or heteroatom-based (i.e., the heteroatom can appear in the middle of the group or at the end), and can be optionally substituted independently with one or more substituents described herein. The term "heteroalkyl" encompasses alkoxy and heteroalkoxy groups.

[0077] 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 a basic nitrogen.

[0078] As used herein, the term "heteroaryl," whether used by itself, as part of a larger moiety, or in combination with another term, refers to an aryl group that has one or more heteroatoms in addition to carbon atoms and can be optionally substituted independently with one or more substituents described herein. Examples of heteroaryl groups include, but are not limited to, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, pteridinyl, naphthyridinyl, benzofuranyl, and pteridinyl. Heteroaryl also includes groups in which a heteroaromatic ring is fused with one or more aryl, cycloaliphatic, or heterocyclyl rings, where the radical or point of attachment is at the heteroaromatic ring. Non-limiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenanthridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and pyrido[2,3-b]-l,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.

[0079] As used herein, the term "heterocycle" or "heterocyclyl" refers to a saturated, partially unsaturated, or fully unsaturated carbocyclic group in which one or more ring atoms are heteroatoms independently selected from oxygen, sulfur, nitrogen, phosphorus, and the like, with the remaining ring atoms being carbon, wherein one or more ring atoms can be optionally independently substituted with one or more substituents. In some embodiments, the heterocyclyl group is a saturated heterocyclyl group. In some embodiments, the heterocyclyl group is an unsaturated heterocyclyl group having one or more double bonds in its ring system. In some embodiments, the heterocyclyl group can contain any oxidized form of carbon, nitrogen, sulfur, or phosphorus, and any quaternized form of basic nitrogen. "Heterocyclyl" also includes groups in which the heterocyclyl is fused or linked to a saturated, partially unsaturated, or fully unsaturated (i.e., aromatic) carbocyclic or heterocyclic ring. Where possible, the heterocyclyl group can be carbon-linked or nitrogen-linked. In some embodiments, the heterocycle is carbon-linked. In some embodiments, the heterocycle is nitrogen-linked. For example, a group derived from pyrrole can be a pyrrol-1-yl group (nitrogen-linked) or a pyrrol-3-yl group (carbon-linked). Furthermore, a group derived from imidazole can be an imidazol-1-yl group (nitrogen-linked) or an imidazol-3-yl group (carbon-linked).

[0080] In some embodiments, the term "3- to 12-membered heterocyclyl" 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. Fused, spiro, and bridged ring systems are also included within the scope of this definition. Examples of monocyclic heterocyclyl groups include, but are not limited to, oxetanyl, 1,1-dioxothietanyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydrothiophenyl, pyrrolyl, furanyl, thiophenyl, pyrazolyl, imidazolyl, triazolyl, oxazolyl, thiazolyl, piperidyl, piperazinyl, morpholinyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazinyl, pyridonyl, pyrimidonyl, pyrazinonyl, pyridazonyl, pyrrolidinyl, triazonyl, and the like. Examples of fused heterocyclyl groups include, but are not limited to, phenyl fused rings or pyridyl fused rings such as quinolyl, isoquinolyl, quinoxalyl, quinolizinyl, quinazolinyl, azaindolizinyl, cinnolinyl, chromenyl, isochromenyl, indolyl, isoindolyl, indolizinyl, indazolyl, purinyl, benzofuranyl, isobenzofuranyl, benzimidazolyl, benzothiophenyl, benzothiazolyl, carbazolyl, phenoxazinyl, phenothiazinyl, phenazinyl, imidazo[l,2-a]pyridinyl, [l,2,4]triazolo[4,3-a]pyridinyl, [l,2,3]triazolo[4,3-a]pyridinyl, and the like. Examples of spiro heterocyclyl groups include, but are not limited to, spirooxetanyl, spirooxazinyl, and the like. Examples of bridged heterocyclyl groups include, but are not limited to, morphanyl, hexamethylenetetraminyl, 3-aza-bicyclo[3.1.0]hexanyl, 8-aza-bicyclo[3.2.1]octanyl, 1-aza-bicyclo[2.2.2]octanyl, 1,4-diazabicyclo[2.2.2]octane (DABCO), and the like.

[0081] As used herein, the term "alkylcycloalkyl" refers to a monovalent or divalent alkyl-cycloalkyl group. When alkylcycloalkyl is monovalent, it can be -alkyl-cycloalkyl or alkyl-cycloalkyl-. When alkylcycloalkyl is divalent, it can be -alkyl-cycloalkyl- or -cycloalkyl-alkyl-.

[0082] As used herein, the term "alkylheterocyclyl" refers to a monovalent or divalent alkyl-heterocyclyl group. When alkylheterocyclyl is monovalent, it can be -alkyl-heterocyclyl or alkyl-heterocyclyl-. When alkylheterocyclyl is divalent, it can be -alkyl-heterocyclyl- or -heterocyclyl-alkyl-.

[0083] As used herein, the term "alkylaryl" refers to a monovalent or divalent alkyl-aryl group. When alkylaryl is monovalent, it can be -alkyl-aryl or alkyl-aryl-. When alkylaryl is divalent, it can be -alkyl-aryl- or -aryl-alkyl-.

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

[0085] As used herein, the term "alkylcycloalkylalkyl" refers to a monovalent -alkyl-cycloalkyl-alkyl or divalent -alkyl-cycloalkyl-alkyl-.

[0086] As used herein, the term "alkylheterocyclylalkyl" refers to a monovalent -alkyl-heterocyclyl-alkyl or divalent -alkyl-heterocyclyl-alkyl-.

[0087] As used herein, the term "alkylarylalkyl" refers to a monovalent -alkyl-aryl-alkyl or divalent -alkyl-aryl-alkyl-.

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

[0089] As used herein, the term "heteroalkylarylalkylheteroalkyl" refers to a monovalent -heteroalkyl-aryl-alkyl-aryl-heteroalkyl or divalent -heteroalkyl-aryl-alkyl-aryl-heteroalkyl-.

[0090] As used herein, the term "nitrogen protecting group" in conjunction with a nitrogen atom to which the nitrogen protecting group is attached includes, but is not limited to, the following groups: methylcarbamate, ethylcarbamate, 9-fluorenylmethyl carbamate (Fmoc), 9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluoroenylmethyl carbamate, 2,7-di-tert-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methyl carbamate (DBD-Tmoc), 4-methoxybenzylic carbamate (Phenoc), 2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate (Teoc), 2-phenylethyl carbamate (hZ), 1-(1- adamantyl)-1-methylethyl carbamate (Adpoc), 1,1-dimethyl-2-haloethyl carbamate, 1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC), 1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC), 1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc), 1-(3,5-di-tert-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 2-(2'- and 4'-pyridyl)ethyl carbamate (Pyoc), 2-(N,N-dicyclohexylamido)ethyl carbamate, t-butyl carbamate (BOC or Boc), 1-adamantyl carbamate (Adoc), vinyl carbamate (Voc), allyl carbamate (Alloc), 1-isopropylallyl carbamate (Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc), 8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyl dithio carbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz), p-nitrobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzyl carbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfanyl benzyl carbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate, 2-methylthioethyl carbamate, 2-methylsulfinylethyl carbamate, 2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3-dithianyl)]methyl carbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc), 2,4-dimethylsulfanylphenylcarbamate (Bmpc), 2-phosphinyl ethyl carbamate (Peoc), 2-triphenylphosphinopropyl carbamate (Ppoc), 1,1-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate, p-(dihydroxyboryl)benzyl carbamate, 5-benzisoxazolylmethyl carbamate, 2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc), m-nitrophenyl carbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate, 3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl (o-nitrophenyl)methyl carbamate, t- amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzyl carbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentyl carbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate, 2,2-dimethoxyacylvinyl carbamate, o-(N,N-dimethylcarboxamido)benzyl carbamate, 1,1-dimethyl-3-(N,N-dimethylcarboxamido)propyl carbamate, 1,1-dimethylpropargyl carbamate, di(2-pyridyl)methyl carbamate, 2-furylmethyl carbamate, 2-iodoethyl carbamate, isobornyl carbamate, isobutyl carbamate, isoxazyl carbamate, p-(p-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate, 1-methylcyclohexyl carbamate, 1-methyl-1-cyclopropylmethyl carbamate, 1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate, 1-methyl-1-(p-phenylazophenyl)ethyl carbamate, 1-methyl-1-phenylethyl carbamate, 1-methyl-1-(4-pyridyl)ethyl carbamate, phenyl carbamate, p-(phenylazo)benzyl carbamate, 2,4,6-tri-t-butylphenyl carbamate, 4-(trimethylammonio)benzyl carbamate, and 2,4,6-trimethylbenzyl carbamate. In some embodiments, the nitrogen protecting group is Boc, Cbz, Fmoc, trifluoroacetyl, trityl, or acetyl,

[0091] As used herein, the term "halo" or "halogen" refers to an atom selected from fluorine, chlorine, bromine, and iodine.

[0092] As used herein, the term "haloalkyl" refers to an alkyl group substituted with one or more halogen atoms independently replacing one or more hydrogen atoms on a carbon of the alkyl group.

[0093] As used herein, the term "nitro" refers to a -NO2 group.

[0094] As used herein, the term "oxo" refers to a =0 group.

[0095] As used herein, the term "cyano" refers to a group of formula "-CºN".

[0096] As used herein, the term "organelle targeting moiety" refers to a structural moiety that targets an organelle.

[0097] As used herein, the term "endoplasmic reticulum targeting moiety" refers to a structural moiety that targets the endoplasmic reticulum.

[0098] As used herein, the term "molecular weight" is weight average molecular weight (Mw).

[0099] As used herein, the term "biomolecule" refers to a protein, peptide, amino acid, glycoprotein, nucleic acid, nucleotide, nucleoside, oligonucleotide, sugar, oligosaccharide, lipid, hormone, proteoglycan, saccharide, polypeptide, polynucleotide, polysaccharide, drug, prodrug, and the like that can be found in a living organism, including isolated cells. A biomolecule need not be a naturally occurring molecule, but can be a molecule that has been introduced into a living organism or ancestor of a living organism, e.g., directly, by transgenic methods, or otherwise.

[0100] Branched poly(beta-amino ester)

[0101] In one aspect, the present disclosure provides a branched poly(beta-amino ester) having a structure according to Formula (I), Formula (II), Formula (III), Formula (IV), or Formula (V):

[0102] wherein,

[0103] W1, W2, W3, W4, and W5are 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 with one or more R W substituents;

[0104] L is selected from alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkylcycloalkyl, alkylheterocyclyl, alkylcycloalkylalkyl, alkylheterocyclylalkyl, alkylarylalkyl, alkylheteroarylalkyl, or heteroalkylarylalkylheteroarylalkyl, wherein the alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkylcycloalkylalkyl, alkylheterocyclylalkyl, alkylarylalkyl, alkylheteroarylalkyl, and heteroalkylarylalkylheteroarylalkyl are optionally substituted with one or more R L substituents;

[0105] Each R1 is independently selected from R1 N Or T;

[0106] R1 N The group is selected from alkyl, heteroalkyl, cycloalkyl, heterocyclic, aryl, heteroaryl, alkylcycloalkyl, alkylheterocyclic, alkylaryl, alkylheterocyclic, or nitrogen-protecting groups, wherein the alkyl, heteroalkyl, cycloalkyl, heterocyclic, aryl, heteroaryl, alkylcycloalkyl, alkylheterocyclic, alkylaryl, and alkylheterocyclic groups are optionally protected by one or more R groups. N replace;

[0107] Each R2 is independently selected from R2 N Or T;

[0108] R2 N The group is selected from alkyl, heteroalkyl, cycloalkyl, heterocyclic, aryl, heteroaryl, alkylcycloalkyl, alkylheterocyclic, alkylaryl, alkylheterocyclic, or nitrogen-protecting groups, wherein the alkyl, heteroalkyl, cycloalkyl, heterocyclic, aryl, heteroaryl, alkylcycloalkyl, alkylheterocyclic, alkylaryl, alkylheterocyclic, alkylcycloalkyl, alkylheterocyclic, alkylarylalkyl, and alkylheterocyclic are optionally protected by one or more R groups. N replace;

[0109] Each T cell independently targets specific organelles;

[0110] Each R W R L R L and R N Each is independently selected from the following groups: halogen, cyano, nitro, oxygen, -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, heterocyclic, aryl, and heteroaryl;

[0111] Each R a Each is independently selected from hydrogen or alkyl groups;

[0112] m1, m2, m3, m4, m5, m6, m7, m8, m9, m10, m11 and m12 are each independently in the range of 1 to 100.

[0113] In some implementations, each R1 is independent of R1. N And at least one R2 is T.

[0114] In some implementations, each R1 N Independently selected from alkyl, heteroalkyl, alkylcycloalkyl, alkylheterocyclic, alkylaryl, or alkylheteroaryl, wherein the alkyl, heteroalkyl, alkylcycloalkyl, alkylheterocyclic, alkylaryl, and alkylheteroaryl groups are optionally separated by one or more R groups. N Replace, and each R N Independently for -OR a or -N(R) a )2.

[0115] In some implementations, each R1 N Independently selected from alkyl, heteroalkyl, alkylcycloalkyl, alkylheterocyclic, alkylaryl, or alkylheteroaryl, wherein the alkyl, heteroalkyl, alkylcycloalkyl, alkylheterocyclic, alkylaryl, and alkylheteroaryl groups are optionally separated by one or more R groups. N Replace, and each R N Independently for -OR a or -N(R) a 2. In some implementations, each R1 N Selected independently 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 Alkyl group. In some embodiments, each R1 N Selected independently from C1-30 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 The heteroalkyl group contains 1-5 heteroatoms selected from N, O, and S. In some embodiments, each R1 N Selected independently 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 Cycloalkyl. In some embodiments, each R1 N Selected independently from C 1- 6-alkyl C 3-12 Heterocyclic group, C 1-6 Alkyl C 3-9 Heterocyclic group or C 1-6 Alkyl C 5-9 A heterocyclic group, wherein the heterocyclic group contains 1-3 heteroatoms selected from N, O, and S. In some embodiments, each R1 N Selected independently from C 1-6 Alkyl C 6-12 Aryl or C 1- 6-alkyl C 6-9 Aryl. In some implementations, each R1 N Selected independently from C 1-6 Alkyl C 6-12 heteroaryl or C 1-6 Alkyl C 6-9 Mixed aromatic compounds.

[0116] In some implementations, each R1 is independently selected from the following group:

[0117] In some implementations, each R1 is independently selected from the following group:

[0118] In some embodiments, each R2 N is independently selected from alkyl, heteroalkyl, alkylcycloalkyl, alkylheterocyclyl, or alkylheterocyclylalkyl, said alkyl, heteroalkyl, alkylcycloalkyl, alkylheterocyclyl, and alkylheterocyclylalkyl optionally substituted with one or more R N , and each R N is independently -OR a , or -N(R a )2.

[0119] In some embodiments, each R2 N is independently selected from alkyl, heteroalkyl, alkylcycloalkyl, alkylheterocyclyl, or alkylheterocyclylalkyl, said alkyl, heteroalkyl, alkylcycloalkyl, alkylheterocyclyl, and alkylheterocyclylalkyl optionally substituted with one or more R N , and each R N is independently -OR a , or -N(R a )2. In some embodiments, each R2 N is 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 alkyl. In some embodiments, each R2 N 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, C1- 5 heteroalkyl, C 1-4 Heteroalkyl, C 1-3 Heteroalkyl, C 1-2 The heteroalkyl group contains 1-5 heteroatoms selected from N, O, and S. In some embodiments, each R2 N Selected independently 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 Cycloalkyl. In some embodiments, each R2 N Selected independently from C 1-6 Alkyl C 3-12 Heterocyclic group, C 1-6 Alkyl C 3-9 Heterocyclic group 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 Selected independently from C 1-6 Alkyl C 3-12 Heterocyclic C 1-6 Alkyl, C 1-6 Alkyl C 3-9 Heterocyclic C 1-6 Alkyl or C 1-6 Alkyl C 5-9 Heterocyclic C 1-6 Alkyl group, wherein the heterocyclic group contains 1-3 heteroatoms selected from N, O, and S.

[0120] In some implementations, each R2 is independently selected from the following group:

[0121] And T.

[0122] In some implementations, each R2 is independently selected from the following group:

[0123] And T.

[0124] In some embodiments, the branched poly(β-amino ester) provided herein has the structure shown in formula (I), wherein, in In a repeating unit, at least 1, 2, 3, 4, or 5 R1s are T, and each R2 is independently R2. N .

[0125] In some embodiments, the branched poly(β-amino ester) has a structure according to Formula (I), wherein W1is an alkyl or heteroalkyl group, the alkyl and heteroalkyl groups being optionally substituted with one or more R W In some embodiments, W1is C 1-30 In some embodiments, W1is C 1-25 In some embodiments, W1is C 1-20 In some embodiments, W1is C 1-19 In some embodiments, W1is C 1-18 In some embodiments, W1is C 1-17 In some embodiments, W1is C 1-16 In some embodiments, W1is C 1-15 In some embodiments, W1is C 1-14 In some embodiments, W1is C 1-13 In some embodiments, W1is C 1-12 In some embodiments, W1is C 1-11 In some embodiments, W1is C 1-10 In some embodiments, W1is C 1-9 In some embodiments, W1is C 1-8 In some embodiments, W1is C 1-7 In some embodiments, W1is C 1-6 In some embodiments, W1is C W In some embodiments, each W1is independently selected from C 1-30 In some embodiments, W1is C 1-25 In some embodiments, W1is C 1-20 In some embodiments, W1is C 1-19 In some embodiments, W1is C 1-18 In some embodiments, W1is C 1-17 In some embodiments, W1is C 1-16 In some embodiments, W1is C 1-15 In some embodiments, W1is C 1-14 In some embodiments, W1is C 1-13 In some embodiments, W1is C 1-12 In some embodiments, W1is C 1-11 In some embodiments, W1is C 1-10 In some embodiments, W1is C 1-9 In some embodiments, W1is C 1-8 In some embodiments, W1is C 1-7 In some embodiments, W1is C 1-6 In some embodiments, W1is C 1-5 In some embodiments, W1is C 1-4 In some embodiments, W1is C 1-3 In some embodiments, W1is C 1-2 In some embodiments, W1is C W In some embodiments, W1is C

[0126] In some embodiments, W1is

[0127] In some embodiments, the branched poly(β-amino ester) provided herein has a structure according to Formula (II), wherein, in In some embodiments, at least 1, 2, 3, 4, 5, or 6 R1in the repeat unit is T, and each R2is independently R2 N .

[0128] In some embodiments, the branched poly(β-amino ester) has the structure of Formula (II), wherein W2is an alkyl or heteroalkyl, the alkyl and heteroalkyl being optionally substituted with one or more R W In some embodiments, W2is C 1-30 In some embodiments, W2is C 1-25 In some embodiments, W2is C 1-20 In some embodiments, W2is C 1-19 In some embodiments, W2is C 1-18 In some embodiments, W2is C 1-17 In some embodiments, W2is C 1-16 In some embodiments, W2is C 1-15 In some embodiments, W2is C 1-14 In some embodiments, W2is C 1-13 In some embodiments, W2is C 1-12 In some embodiments, W2is C 1-11 In some embodiments, W2is C 1-10 In some embodiments, W2is C 1-9 In some embodiments, W2is C 1-8 In some embodiments, W2is C 1-7 In some embodiments, W2is C 1-6 In some embodiments, W2is C W In some embodiments, each W2is independently selected from C 1-30 In some embodiments, W2is C 1-25 In some embodiments, W2is C 1-20 In some embodiments, W2is C 1-19 In some embodiments, W2is C 1-18 In some embodiments, W2is C 1-17 In some embodiments, W2is C 1-16 In some embodiments, W2is C 1-15 In some embodiments, W2is C 1-14 In some embodiments, W2is C 1-13 In some embodiments, W2is C 1-12 In some embodiments, W2is C 1-11 In some embodiments, W2is C 1-10 In some embodiments, W2is C 1-9 In some embodiments, W2is C 1-8 In some embodiments, W2is C 1-7 In some embodiments, W2is C 1-6 In some embodiments, W2is C 1-5 In some embodiments, W2is C 1-4 In some embodiments, W2is C 1-3 In some embodiments, W2is C 1-2 In some embodiments, W2is C W In some embodiments, W2is C

[0129] In some embodiments, W2is In some embodiments, W2is

[0130] In some embodiments, the branched poly(β-amino ester) provided herein has a structure according to Formula (III), wherein, in the repeat unit, at least 1, 2, 3, 4, 5, 6, 7, or 8 R1is T, and each R2is independently R2 In some embodiments, the branched poly(β-amino ester) provided herein has a structure according to Formula (III), wherein, in the repeat unit, at least 1, 2, 3, 4, 5, 6, 7, or 8 R1is T, and each R2is independently R2 N .

[0131] In some embodiments, the branched poly(β-amino ester) provided herein has a structure according to Formula (III), wherein W3is alkyl or heteroalkyl, said alkyl and heteroalkyl being optionally substituted with one or more R W In some embodiments, W3is 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, said alkyl being optionally substituted with one or more R W In some embodiments, each W3is 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 heteroalkyl, said heteroalkyl containing 1-3 heteroatoms selected from N, O, S, and said heteroalkyl being optionally substituted with one or more R W In some embodiments, W3is

[0132] In some embodiments, W3is

[0133] In some embodiments, the branched poly(β-amino ester) provided herein has a structure according to Formula (IV), wherein, in the repeat unit, at least 1, 2, 3, 4, 5, 6, 7, or 8 R1is T, and each R2is independently R2 In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, or 9 of R1in the repeat unit is T, and each R2is independently R2 N .

[0134] In some embodiments, the branched poly( -amino ester) has the structure of Formula (IV), wherein W4is an alkyl or heteroalkyl, the alkyl and heteroalkyl being optionally substituted with one or more R W In some embodiments, W4is C 1-30 In some embodiments, W4is C 1-25 In some embodiments, W4is C 1-20 In some embodiments, W4is C 1-19 In some embodiments, W4is C 1-18 In some embodiments, W4is C 1-17 In some embodiments, W4is C 1-16 In some embodiments, W4is C 1-15 In some embodiments, W4is C 1-14 In some embodiments, W4is C 1-13 In some embodiments, W4is C 1-12 In some embodiments, W4is C 1-11 In some embodiments, W4is C 1-10 In some embodiments, W4is C 1-9 In some embodiments, W4is C 1-8 In some embodiments, W4is C 1-7 In some embodiments, W4is C 1-6 In some embodiments, W4is C W In some embodiments, each W4is independently selected from C 1-30 In some embodiments, W4is C 1-25 In some embodiments, W4is C 1-20 In some embodiments, W4is C 1-19 In some embodiments, W4is C 1-18 In some embodiments, W4is C 1-17 In some embodiments, W4is C 1-16 In some embodiments, W4is C 1-15 In some embodiments, W4is C 1-14 In some embodiments, W4is C 1-13 In some embodiments, W4is C 1-12 In some embodiments, W4is C 1-11 In some embodiments, W4is C 1-10 In some embodiments, W4is C W In some embodiments, W4is C

[0135] In some embodiments, W4is

[0136] In some embodiments, the branched poly( -amino ester) provided herein has the structure of Formula (V), wherein, in the repeat unit, In some embodiments, at least 1 or 2 of R1in the repeat unit is T, and each R2is independently R2 N .

[0137] In some embodiments, the branched poly(β-amino ester) has a structure according to Formula (V), wherein W5is an alkyl, heteroalkyl, or alkylheterocyclylalkyl group, said alkyl, heteroalkyl, and alkylheterocyclylalkyl groups are optionally substituted with one or more R W In some embodiments, W5is 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, said alkyl is optionally substituted with one or more R W In some embodiments, each W5is 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 heteroalkyl, said heteroalkyl contains 1-3 heteroatoms selected from N, O, S, and said heteroalkyl is optionally substituted with one or more R W In some embodiments, W5is 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 alkyl, said heterocyclyl contains 1-3 heteroatoms selected from N, O, S, and said alkylheterocyclylalkyl is optionally substituted with one or more R W substituents.

[0138] In some embodiments, W5is selected from:

[0139] In some embodiments, each R1 N is independently selected from alkyl, heteroalkyl, alkylcycloalkyl, alkylheterocyclyl, alkylaryl, or alkylheteroaryl, said alkyl, heteroalkyl, alkylcycloalkyl, alkylheterocyclyl, alkylaryl, and alkylheteroaryl are optionally substituted with one or more R N substituents, and each R N is independently -OR a or -N(R a )2. In some embodiments, each R1 Nindependently 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 alkyl. 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 containing 1-5 heteroatoms selected from the group consisting of N, O, 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 cycloalkyl. In some embodiments, each R1 N independently selected from C 1- 6 alkyl C 3-12 heterocyclyl, C 1-6 alkyl C 3-9 heterocyclyl, or C 1-6 alkyl C 5-9 heterocyclyl containing 1-3 heteroatoms selected from the group consisting of N, O, S. In some embodiments, each R1 N independently selected from C 1-6 alkyl C6-12 aryl or C 1- 6alkyl C 6-9 aryl. In some embodiments, each R1 N is independently selected from C 1-6 alkyl C 6-12 heteroaryl or C 1-6 alkyl C 6-9 heteroaryl.

[0140] In some embodiments, each R1is independently selected from the group consisting of:

[0141] and T.

[0142] In some embodiments, each R1is independently selected from the group consisting of:

[0143] and T.

[0144] In some embodiments, each R2 N is independently selected from alkyl, heteroalkyl, alkylcycloalkyl, alkylheterocyclyl, or alkylheterocyclylalkyl, optionally substituted with one or more R N , and each R N is independently -OR a , or -N(R a )2. In some embodiments, each R2 N is 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 alkyl. In some embodiments, each R2 N 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, C1-13 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 The heteroalkyl group contains 1-5 heteroatoms selected from N, O, and S. In some embodiments, each R2 N Selected independently 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 Cycloalkyl. In some embodiments, each R2 N Selected independently from C 1-6 Alkyl C 3-12 Heterocyclic group, C 1-6 Alkyl C 3-9 Heterocyclic group 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 Selected independently from C 1-6 Alkyl C 3-12 Heterocyclic C 1-6 Alkyl, C 1-6 Alkyl C 3-9 Heterocyclic C 1-6 Alkyl or C 1-6 Alkyl C 5-9 Heterocyclic C 1-6 Alkyl group, wherein the heterocyclic group contains 1-3 heteroatoms selected from N, O, and S.

[0145] In some implementations, each R2 is independently selected from the following group:

[0146] In some implementations, each R2 is independently selected from the following group:

[0147] In some implementations, T stands for the endoplasmic reticulum targeting portion.

[0148] In some implementation schemes, T is selected from:

[0149] In some embodiments, L is selected from alkyl, heteroalkyl, heterocyclyl, alkylheterocyclyl, alkylcycloalkylalkyl, alkylheterocyclylalkyl, or heteroalkylarylalkylheteroalkyl, wherein the alkyl, heteroalkyl, alkylcycloalkylalkyl, alkylheterocyclylalkyl, and heteroalkylarylalkylheteroalkyl are optionally substituted with one or more R L , and each R L is 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 alkyl. 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 heteroalkyl, wherein the heteroalkyl contains 1-5 heteroatoms selected from N, O, S. In some embodiments, L is C 3-12 heterocyclyl, C 3-11 heterocyclyl, C 3-10 heterocyclyl, C 3-9 heterocyclyl, C 3-8 heterocyclyl, C 3-7 heterocyclyl, C 3-6 heterocyclyl, C 4-9 heterocyclyl, or C 5-9 heterocyclyl, which heterocyclyl contains 1-3 heteroatoms selected from N, O, S. In some embodiments, L is C 1-6 alkylC 3-12 heterocyclyl, C 1-6 alkylC 3-9 heterocyclyl, or C 1-6 alkylC 5-9heteroalkyl, C 1-6 alkyl C 3-12 cycloalkyl C 1-6 alkyl, C 1-6 alkyl C 3-9 cycloalkyl C 1- 6alkyl or C 1-6 alkyl C 5-9 cycloalkyl C 1-6 alkyl. In some embodiments, L is C 1-6 alkyl C 3-12 heterocyclyl C 1- 6alkyl, C 1-6 alkyl C 3-9 heterocyclyl C 1-6 alkyl or C 1-6 alkyl C 5-9 heterocyclyl C 1-6 alkyl, the heterocyclyl containing 1-3 heteroatoms selected from N, O, S. In some embodiments, L is heteroalkyl aryl alkyl aryl heteroalkyl, wherein each of the heteroalkyl 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, each of the aryl is independently C 6-12 aryl, C 6-11 aryl, C 6-10 aryl, C 6-9 aryl or C 6-8 aryl, each of the alkyl is 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.

[0150] In some embodiments, L is selected from the group consisting of:

[0151] In some embodiments, L is selected from the group consisting of:

[0152] In some embodiments, m1, m2, m3, m4, m5, m6, m7, m8, m9, m10, m11, and m12 are each independently in the range of 5 to 75.

[0153] In some embodiments, the branched poly(beta-amino ester) has a molecular weight in the range of 5.0 kDa to 40.0 kDa, 5.0 kDa to 50 kDa, 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 branched poly(beta-amino ester) has a molecular weight of about 5.0 kDa, 6.0 kDa, 7.0 kDa, or 8.0 kDa.

[0154] In some embodiments, the present application provides a branched poly(beta-amino ester) having a structure shown in ER-HPAE-1, ER-HPAE-2, ER-HPAE-3, or ER-HPAE-4:

[0155] In some embodiments, the branched poly(beta-amino ester) has a molecular weight of about 5.0 kDa, 6.0 kDa, 7.0 kDa, or 8.0 kDa.

[0156] Synthesis method

[0157] In one aspect, the present disclosure provides a method of preparing a branched poly(beta-amino ester) described herein, comprising:

[0158] 1-1) subjecting a multi-acrylate monomer having formula (I-1), formula (II-1), formula (III-1), or formula (IV-1):

[0159] to a Michael addition reaction with a di-acrylate monomer having formula (2) and one or more amine monomers having formula (3), to obtain a branched poly(beta-amino ester) P1 having a terminal double bond:

[0160] 1-2) subjecting the branched poly(beta-amino ester) P1 prepared in step 1-1) to a Michael addition reaction with one or more capping amine monomers having formula (4), to thereby prepare a branched poly(beta-amino ester) having a structure shown in formula (I), formula (II), formula (III), or formula (IV):

[0161] or,

[0162] 2-1) subjecting a diamine monomer of formula (V-1) to a Michael addition reaction with a diacrylate monomer of formula (2) and one or more amine monomers of formula (3) to obtain a branched poly(beta-amino ester) P2 terminated with a double bond:

[0163] 2-2) subjecting the branched poly(beta-amino ester) P2 produced in step 2-1) to a Michael addition reaction with one or more capping amine monomers of formula (4) to produce a branched poly(beta-amino ester) having the structure of formula (V):

[0164] In some embodiments, the multiacrylate monomer of formula (I-1) is

[0165] In some embodiments, the multiacrylate monomer of formula (II-1) is

[0166] In some embodiments, the multiacrylate monomer of formula (III-1) is

[0167] In some embodiments, the multiacrylate monomer of formula (IV-1) is

[0168] In some embodiments, the diamine monomer of formula (V-1) is selected from the group consisting of:

[0169] In some embodiments, the diacrylate monomer of formula (2) is selected from the group consisting of:

[0170] In some embodiments, the one or more amine monomers of formula (3) is R1 N -NH2, the one or more capping amine monomers of formula (4) is selected from R2 N -NH2or T-NH2, and at least one capping amine monomer of formula (4) is T-NH2.

[0171] In some embodiments, the R1 N -NH2is selected from the group consisting of:

[0172] The R2 N -NH2is selected from the group consisting of:

[0173] The T-NH2is selected from the group consisting of:

[0174] In some embodiments, the diacrylate monomer of formula (2), R2 N -NH2, T-NH2, and R1 N The molar ratio of the reaction feed of the diacrylate monomer of formula (2), R1 N -NH2, and the polyacrylate monomer of formula (I-1), formula (II-1), formula (III-1), or formula (IV-1) is (1-3):(0.1-4):(0.5-3); in step 2-1), the diacrylate monomer of formula (2), R1 N -NH2, and the diamine monomer of formula (V-1) is (1-3):(0.1-4):(0.5-3).

[0175] In some embodiments, the one or more amine monomers of formula (3) is selected from R1 N -NH2, or T-NH2, at least one amine monomer of formula (3) is T-NH2, and the one or more capped amine monomer of formula (4) is R2 N -NH2.

[0176] In some embodiments, the R1 N -NH2is selected from the group consisting of:

[0177] The T-NH2is selected from the group consisting of:

[0178] The R2 N -NH2is selected from the group consisting of:

[0179] In some embodiments, the diacrylate monomer of formula (2), R2 N -OH, T-OH, and R1 N The molar ratio of the reaction feed of the diacrylate monomer of formula (2), R1 N -NH2, the polyacrylate monomer of formula (I-1), formula (II-1), formula (III-1), or formula (IV-1), and T-NH2is (1-3):(0.1-4):(0.5-3):(0.02-2); in step 2-1), the diacrylate monomer of formula (2), R1 Nthe molar ratio of -NH2, the diamine monomer of Formula (V-1), and T-NH2 is (1-3):(0.1-4):(0.5-3):(0.02-2).

[0180] In some embodiments, steps 1-1) and 2-1) are performed at a temperature of 15-100 °C. In some embodiments, steps 1-1) and 2-1) are performed at a temperature of 60-100 °C, 70-100 °C, 80-100 °C, 90-100 °C, or 90-95 °C.

[0181] In some embodiments, steps 1-1) and 2-1) are performed at a temperature of about 90 °C.

[0182] In some embodiments, the branched poly(beta-amino ester) P1 resulting from step 1-1) has a molecular weight in the range of 5 kDa to 40 kDa.

[0183] In some embodiments, the branched poly(beta-amino ester) P2 resulting from step 2-1) has a molecular weight in the range of 5 kDa to 40 kDa.

[0184] In some embodiments, steps 1-2) and 2-2) are performed at a temperature of 20-30 °C. In some embodiments, steps 1-2) and 2-2) are performed at a temperature of 20-30 °C, 22-30 °C, 24-30 °C, 24-28 °C, or 24-26 °C.

[0185] In some embodiments, steps 1-2) and 2-2) are performed at a temperature of about 25 °C.

[0186] The synthesis of the compounds provided herein, including salts thereof, is illustrated in the synthetic schemes in the Examples. The 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 routes, and thus, the schemes are merely illustrative and not intended to limit other possible methods that can be used to prepare the compounds provided herein. Furthermore, the steps in the schemes are for better illustration and can be changed as appropriate.

[0187] The reactions for preparing the compounds of the present disclosure can be carried out in suitable solvents which can be readily selected by one of ordinary skill in the art of organic synthesis. Suitable solvents can be substantially non-reactive with the starting materials (reactants), intermediates, or products at the temperatures at which the reactions are carried out, e.g., temperatures between the solvent's freezing and boiling temperatures. A given reaction can be carried out in one solvent or a mixture of more than one solvent. The choice of a suitable solvent will depend on the specific reaction step

[0188] The preparation of compounds of the present disclosure can involve the protection and deprotection of various chemical groups. The need for protection and deprotection, and the selection of appropriate protecting groups can be readily determined by one skilled in the art. The chemical nature of the protecting groups can be found in, for example, T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 3rd edition, Wiley & Sons, Inc., New York (1999), which is incorporated herein by reference in its entirety.

[0189] The reaction can be monitored according to any suitable method known in the art. For example, product formation can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g., 1 H or 13 C), infrared spectroscopy (IR), spectrophotometry (e.g., UV-visible light), mass spectrometry (MS), or by chromatographic means, such as high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LCMS), or thin layer chromatography (TLC). Compounds can be purified 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, J. Combi. Chem., 2004, 6(6), 874-883, incorporated herein by reference in its entirety) and normal phase silica chromatography.

[0190] The abbreviation definitions as used herein are as follows: "1x" or "x1" is one time, "2x" or "x2" is two times, "3x" or "x3" is three times, "4x" or "x4" is four times, "5x" or "x5" is five times, "°C" is degrees Celsius, "eq" or "eq." is equivalent, "g" is gram(s), "mg" is milligram(s), "L" is liter(s), "mL" or "ml" is milliliter(s), "pL" is microliter(s), "N" is normal, "M" is molar, "mmol" is millimole(s), "min" is minute(s), "h" or "hr" is hour(s), "r.t." or "rt" is room temperature, "atm" is atmosphere, "psi" is pounds per square inch, "conc." is concentration, "sat" or "sat'd" is saturated, "MS" or "Mass Spec" is mass spectrometry, "ESI" is electrospray ionization mass spectrometry, "LCMS" is liquid chromatography mass spectrometry, "HPLC" is high performance liquid chromatography, "RP" is reverse phase, "TLC" or "tlc" is thin layer chromatography, "SM" is starting material, "NMR" is nuclear magnetic resonance spectroscopy, "1H" is proton, "δ" is delta, "s" is singlet, "d" is doublet, "t" is triplet, "q" is quartet, "m" is multiplet, "br" is broad, and "Hz" is hertz. "α", "β", "R", "S", "E", and "Z" are stereochemical designations familiar to one of ordinary skill in the art. 1 H" is proton, "δ" is delta, "s" is singlet, "d" is doublet, "t" is triplet, "q" is quartet, "m" is multiplet, "br" is broad, and "Hz" is hertz. "α", "β", "R", "S", "E", and "Z" are stereochemical designations familiar to one of ordinary skill in the art.

[0191] Composition

[0192] In one aspect, the present disclosure provides a composition comprising a branched poly(beta-amino ester) and a biomolecule described herein.

[0193] In some embodiments, the biomolecule is a polynucleotide, a protein, a peptide, a sugar, a polysaccharide, a glycoprotein, a lipid, a hormone, a drug, or a prodrug.

[0194] In some embodiments, the biomolecule is a polynucleotide. In some embodiments, the polynucleotide is DNA or RNA.

[0195] In some embodiments, the DNA encodes a C7 protein, a TGM1 protein, a MBTPS1 protein, a COL1A1 protein, a COL3A1 protein, a fibroin protein, a Cas9 protein, a ADAM17 protein, a TGF-β1 protein, a ABCD1 protein, a P53 protein, a RPE65 protein, a SMN1 protein, a AADC protein, a FVIII protein, a VEGF protein, or a HGF protein.

[0196] In some embodiments, the RNA is an mRNA or an siRNA.

[0197] In some embodiments, the RNA encodes a C7 protein, a TGM1 protein, a MBTPS1 protein, a COL1A1 protein, a COL3A1 protein, a fibroin protein, a Cas9 protein, a ADAM17 protein, a TGF-β1 protein, a ABCD1 protein, a P53 protein, a RPE65 protein, a SMN1 protein, a AADC protein, a FVIII protein, a VEGF protein, or a HGF protein.

[0198] In some embodiments, the biomolecule is a protein. In some embodiments, the protein is a serum protein. In some embodiments, the protein is a fluorescein isothiocyanate labeled bovine serum albumin (BSA-FITC).

[0199] In some embodiments, the mass ratio of the branched poly(beta-amino ester) to the biomolecule is in the range of 1:1 to 200:1. In some embodiments, the mass ratio of the branched poly(beta-amino ester) to the biomolecule is in the range of 5:1 to 120:1. In some embodiments, the mass ratio of the branched poly(beta-amino ester) to the biomolecule is in the range of 5:1 to 100:1. In some embodiments, the mass ratio of the branched poly(beta-amino ester) to the biomolecule 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.

[0200] In some embodiments, the biomolecule is a polynucleotide, and the branched poly(beta-amino ester) has a mass ratio to the polynucleotide in the range of 1 : 1 to 200: 1. In some embodiments, the branched poly(beta-amino ester) has a mass ratio to the polynucleotide in the range of 5: 1 to 120: 1. In some embodiments, the branched poly(beta-amino ester) has a mass ratio to the polynucleotide in the range of 5: 1 to 100: 1. In some embodiments, the branched poly(beta-amino ester) has a mass ratio to the polynucleotide of 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.

[0201] In some embodiments, the composition has the form of a nanoparticle.

[0202] In some embodiments, the nanoparticle has a particle size in the range of 50 nm to 500 nm. In some embodiments, the nanoparticle has a particle size in the range of 150 nm to 400 nm. In some embodiments, the nanoparticle has a particle size of 150 nm, 200 nm, 250 nm, 300 nm, 350 nm, or 400 nm.

[0203] Methods of treatment

[0204] In one aspect, the present disclosure provides a method of delivering a biomolecule to a subject, comprising administering to the subject a composition described herein.

[0205] As used herein, the term "treatment" refers to reversing, alleviating, delaying the onset of, or inhibiting the progress of a disease or disorder, or one or more symptoms thereof, as described herein. In some embodiments, treatment can be administered after one or more symptoms have developed. In other embodiments, treatment can be administered in the absence of symptoms. For example, treatment can be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a genetic or other susceptibility, or in light of a symptom history). Treatment can also continue after symptoms have resolved, for example to prevent or delay their recurrence.

[0206] The compositions provided herein can be administered in pure form, in combination with other active ingredients, or as pharmaceutical compositions of the present disclosure. In some embodiments, the compositions provided herein can be administered simultaneously or sequentially with one or more other active agents known in the art to the subject in need thereof. The individual agents in such combinations are administered in separate or unitary pharmaceutical compositions. Preferably, the individual agents will be administered concurrently in a single pharmaceutical composition. The skilled artisan will appreciate that appropriate dosages of the known therapeutic agents would be known.

[0207] In some embodiments, the administration is 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, once a week.

[0208] 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 transdermally, subcutaneously, or intravenously.

[0209] 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.

[0210] In some embodiments, the disease is selected from recessive dystrophic epidermolysis bullosa (RDEB), ichthyosis, cataracts, alopecia, oral mucosal disease, or pustular psoriasis syndrome (CAOP).

[0211] In some embodiments, the compositions described herein are administered to a subject by transdermally, subcutaneously, or intravenously.

[0212] Reference will now be made in detail to certain embodiments of the present disclosure, examples of which are illustrated in the accompanying structures and formulas. While the present disclosure will be described in conjunction with the enumerated embodiments, it will be understood that it is not intended to limit the present disclosure to those embodiments. On the contrary, it is intended to cover all alternatives, modifications, and equivalents that can be included within the scope of the present disclosure as defined by the claims. One skilled in the art will recognize many methods and materials as being suitable for the practice of this disclosure. No limitation on the scope of the disclosure is intended by indicating any of the following which can not be used in practicing the present disclosure. If one or more of the incorporated references and similar materials differ from or contradict this disclosure, including by way of definition, usage of terms, described techniques, etc., this disclosure controls.

[0213] It should be appreciated that certain features of the present disclosure, which are, for clarity, described in the context of separate embodiments, can also be provided in combination in a single embodiment. Conversely, various features of the present disclosure, which are, for brevity, described in the context of a single embodiment, can also be provided separately or in any suitable

[0214] EMBODIMENTS

[0215] The general methodology of the present disclosure is further illustrated below. The compounds of the present disclosure can be prepared by methods known in the art. Detailed methods of preparation of preferred compounds of the present disclosure are illustrated below. These are by no means limiting to the methods of preparation of the compounds of the present disclosure.

[0216] The synthetic procedures described herein illustrate the synthesis of the compounds (including their pharmaceutically acceptable salts). The compounds provided herein can be prepared using any known organic synthetic technique and can be synthesized according to any of a variety of possible synthetic routes; therefore, these procedures are merely illustrative and not intended to limit other possible methods that can be used to prepare the compounds provided herein. Furthermore, the steps in the described procedures are for better illustration and may be changed where appropriate. Embodiments for synthesizing the compounds in the examples are provided for research purposes and possible submission to regulatory authorities.

[0217] The reactions used to prepare the compounds of this disclosure can be carried out in suitable solvents that can be readily selected by those skilled in the art of organic synthesis. Suitable solvents are those that, at the temperature at which the reaction takes place, for example, between the freezing temperature and boiling temperature of the solvent, do not substantially react with the starting materials (reactants), intermediates, or products. A given reaction can be carried out in one solvent or a mixture of more than one solvent. Depending on the specific reaction step, a suitable solvent for that particular reaction step can be selected by those skilled in the art.

[0218] The preparation of the compounds disclosed herein 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 readily determined by those skilled in the art. The chemical properties of protecting groups can be found, for example, in TW Greene and PGM Uts, Protecting Groups in Organic Synthesis, 3rd Edition, Wiley & Co., New York (1999), which is incorporated herein by reference in its entirety.

[0219] The reaction can be monitored using any suitable method known in the field. For example, it can be monitored using spectroscopic techniques, such as nuclear magnetic resonance spectroscopy (e.g., 1 H or 13 C) Infrared spectroscopy, spectrophotometry (e.g., UV-Vis), mass spectrometry, or chromatographic methods such as high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LCMS), or thin-layer chromatography (TLC) can be used to monitor product formation. Those skilled in the art can purify compounds using a variety of methods, including high-performance liquid chromatography (HPLC) (“Preparative LC-MS Purification: Optimization of an Improved Compound-Specific Method”, 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.

[0220] In the following examples, unless otherwise specified, the TGM1 DNA, COL7A1 DNA and MBTPS1 DNA used in the present disclosure were purchased from Hanheng Biotechnology (Shanghai).

[0221] General Method 1

[0222] The present disclosure provides a method for preparing branched poly(beta-amino ester) described herein, the general synthetic route of the method is shown in FIG. 1(a)-1(b), comprising the following steps:

[0223] 1) Michael addition reaction of diamine monomer (e.g. EDA) or multi-acrylate monomer (e.g. PET4A, PETA, DPTH or TMPTA), di-acrylate monomer and amine monomer (R1 N -NH2) to obtain branched poly(beta-amino ester) P1 with terminal double bond;

[0224] 2) Michael addition reaction of branched poly(beta-amino ester) P1 with terminal double bond prepared in step 1) and capping amine monomer (R2 N -NH2) to obtain branched poly(beta-amino ester) of the present disclosure.

[0225] General Method 2

[0226] The present disclosure provides a method for preparing branched poly(beta-amino ester) described herein, the general synthetic route of the method is shown in FIG. 1(c)-1(d), comprising the following steps:

[0227] 1) Michael addition reaction of diamine monomer (e.g. EDA) or multi-acrylate monomer (e.g. PET4A, PETA, DPTH or TMPTA), di-acrylate monomer, amine monomer (R1 N -NH2) and T-NH2) to obtain branched poly(beta-amino ester) P1 with terminal double bond;

[0228] 2) Michael addition reaction of branched poly(beta-amino ester) P1 with terminal double bond prepared in step 1) and capping amine monomer (R2 N -NH2) to obtain branched poly(beta-amino ester) of the present disclosure.

[0229] General Method 3

[0230] The present disclosure provides a method for preparing a composition (e.g. complex nanoparticle) comprising branched poly(beta-amino ester) described herein and biomolecule, the method comprising forming uniform and stable complex nanoparticle by charge interaction of branched poly(beta-amino ester) prepared in the present disclosure and DNA of different sizes.

[0231] The branched poly(β-amino ester) is tested for its affinity to biomolecules (e.g., DNA), and the size, Zeta potential, and micro-morphology of the complex nanoparticles formed by the branched poly(β-amino ester) and biomolecules are tested. The method is as follows:

[0232] A certain amount of branched poly(β-amino ester) solution is quickly added to a biomolecule solution, vortexed at high speed for 15-60 s, and then left to stand for 5-30 min, where the mass ratio of branched poly(β-amino ester) to biomolecule is (5-100): 1. Then PicoGreen working solution is added to test the fluorescence intensity, and then the DNA affinity efficiency is calculated.

[0233] Using a similar method of preparing complex nanoparticles, after the nanoparticles are formed, 1 mL of deionized water is added, and then dynamic light scattering (DLS) is used for testing.

[0234] Using a similar method of preparing complex nanoparticles, inorganic salt ions are washed using deionized water, high-speed centrifugation is performed for 5 min, and then freeze-drying is performed, and finally transmission electron microscopy (TEM) is used to observe the micro-morphology of the complex nanoparticles.

[0235] General Method 4

[0236] The present disclosure provides transfection of DNA, RNA, proteins, and cells of various sizes using the branched poly(β-amino ester) described herein, while testing for cytotoxicity, as follows:

[0237] 1) Cell culture: 293F cells, A2780 cells, 293T cells, 3T3 cells, SW1353 cells, Vero cells, HaCaT cells, and NHF cells are seeded in a 96-well plate at a density of 1.0 x 10 4 ~ 4.0 x 10 4 cells / well, and cultured overnight at 37°C.

[0238] 2) Mix a certain amount of branched poly(β-amino ester) described herein with DNA (e.g., GFP DNA, TGM1 DNA, COL7A1 DNA, MBTPS1 DNA, COL1A1 DNA, COL3A1 DNA, silk fibroin DNA, Cas9 DNA, ADAM17 DNA, TGF-β1 DNA, ABCD1 DNA, P53 DNA, RPE65 DNA, SMN1 DNA, AADC DNA, FVII DNA, VEGF DNA, and HGF DNA), RNA (e.g., GFP mRNA, TGM1 mRNA, COL7A1 mRNA, and GFP siRNA), or protein (e.g., BSA-FITC) for 5-40 min, and then add to cells. 3) After 48 h of transfection, observe and take pictures of cells transfected with GFP DNA under a fluorescence microscope.

[0239] 4) After 48 h of transfection, use a flow cytometer to test the efficiency of transfection of GFP DNA, GFP RNA, or protein.

[0240] 5) After 48-72 h of transfection, remove the cell supernatant, and then use Western blotting and qPCR to detect the expression efficiency of protein (C7 protein, TGM1 protein, or MBTPS1 protein).

[0241] 6) After 48-72 h of transfection, use alarmBlue to detect the survival rate of cells after transfection.

[0242] Specific embodiments:

[0243] Example 1 synthesis of ER-HPAE-1

[0244] 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 h to obtain branched poly(β-amino ester) with a terminal double bond. 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 h. The reaction solution was precipitated in excess ether three times to obtain branched poly(β-amino ester) ER-HPAE-1 with a molecular weight of 7.5 kDa.

[0245] Figure 1(a) shows a schematic diagram of the synthesis of branched poly( -amino ester) ER-HPAE-1 of this example. Figure 2 shows the GPC curve during the synthesis of branched poly( -amino ester) ER-HPAE-1 of this example, it can be found that the longer the retention time of the polymer in GPC means the larger its molecular weight, which confirms that the polymer is successfully synthesized. As shown in Figure 3, the characteristic peak of benzene sulfonamide appears at 7.5-8.0, indicating that branched poly( -amino ester) ER-HPAE-1 is successfully synthesized. 1 As shown in the HNMR spectrum, the characteristic peak of benzene sulfonamide appears at 7.5-8.0, indicating that branched poly( -amino ester) ER-HPAE-1 is successfully synthesized.

[0246] Example 2 Synthesis of ER-HPAE-2

[0247] The raw material ratio of 1,4-butanediol diacrylate, pentaerythritol tetraacrylate and 5-amino-1-pentanol was 2:0.2:2.0, and the reaction was carried out at 90°C for 5h; branched poly( -amino ester) with double bond at the end was obtained. Then 1,11-diamino-3,6,9-trioxaundecane and N-(2-aminoethyl)-4-methylbenzenesulfonamide were added, so that the final raw material 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 was 2:0.2:2.0:0.6:0.1. The reaction was carried out at 25°C for 48h. The reaction solution was precipitated in excess ether for three times to obtain branched poly( -amino ester) ER-HPAE-2 with a molecular weight of 7.1kDa.

[0248] Figure 1(b) shows a schematic diagram of the synthesis of branched poly( -amino ester) ER-HPAE-2 of this example. Figure 2 shows the GPC curve during the synthesis of branched poly( -amino ester) ER-HPAE-2 of this example, it can be found that the longer the retention time of the polymer in GPC means the larger its molecular weight, which confirms that the polymer is successfully synthesized. As shown in Figure 3, the characteristic peak of benzene sulfonamide appears at 7.5-8.0, indicating that branched poly( -amino ester) ER-HPAE-2 is successfully synthesized. 1 As shown in the HNMR spectrum, the characteristic peak of benzene sulfonamide appears at 7.5-8.0, indicating that branched poly( -amino ester) ER-HPAE-2 is successfully synthesized.

[0249] Example 3 Synthesis of ER-HPAE-3

[0250] The reactants of 1,4-butanediol diacrylate, polydi-pentaerythritol hexaacrylate, 5-amino-1-pentanol, dodecylamine and N-(2-aminoethyl)-4-methylbenzenesulfonamide with a feed ratio of 2:0.1:1.5:0.2:0.05 were reacted at 90°C for 5h to obtain branched poly(β-amino ester) with double bond at the end. Then 1,11-diamino-3,6,9-trioxaundecane was added to reach a final feed ratio of 1,4-butanediol diacrylate, polydi-pentaerythritol 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 solution was reacted at 25°C for 48h. The reaction solution was precipitated in excess ether for three times to obtain branched poly(β-amino ester) ER-HPAE-3 with a molecular weight of 7.4kDa.

[0251] Figure 1(c) shows a schematic diagram of the synthesis of branched poly(β-amino ester) ER-HPAE-3 of the present example. Figure 2 shows the GPC curve during the synthesis of branched poly(β-amino ester) ER-HPAE-3 of the present example. It can be found that the longer the retention time of the polymer in GPC, the larger the molecular weight of the polymer, which confirms that the polymer is successfully synthesized. As shown in Figure 3, the characteristic peak of benzene sulfonamide appears at 7.5-8.0 in the HNMR spectrum, indicating that branched poly(β-amino ester) ER-HPAE-3 is successfully synthesized. 1 HNMR spectrum as shown in Figure 3, the characteristic peak of benzene sulfonamide appears at 7.5-8.0, indicating that branched poly(β-amino ester) ER-HPAE-3 is successfully synthesized.

[0252] Example 4 Synthesis of ER-HPAE-4

[0253] The reactants of 1,4-butanediol diacrylate, trimethylolpropane ethoxylate triacrylate, 4-amino-1-butanol and N-(2-aminoethyl)-4-methylbenzenesulfonamide with a feed ratio of 2:0.1:1.5:0.05 were reacted at 90°C for 8h to obtain branched poly(β-amino ester) with double bond at the end. Then 1,4-bis(3-aminopropyl)piperazine was added to reach 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 solution was reacted at 25°C for 48h. The reaction solution was precipitated in excess ether for three times to obtain branched poly(β-amino ester) ER-HPAE-4 with a molecular weight of 8.0kDa.

[0254] Figure 1(d) shows a schematic diagram of the synthesis of branched poly(β-amino ester) ER-HPAE-4 of the present example. Figure 2 shows the GPC curve during the synthesis of branched poly(β-amino ester) ER-HPAE-4 of the present example, it can be found that the longer the retention time of the polymer in GPC, the larger the molecular weight of the polymer, which confirms that the polymer is successfully synthesized. As shown in Figure 3, the characteristic peak of the benzene sulfonamide appears at 7.5-8.0, indicating that branched poly(β-amino ester) ER-HPAE-4 is successfully synthesized. 1 HNMR spectrum shows that the characteristic peak of benzene sulfonamide appears at 7.5-8.0, indicating that branched poly(β-amino ester) ER-HPAE-4 is successfully synthesized.

[0255] Example 5

[0256] The affinity of branched poly(β-amino ester) ER-HPAE-1 to ER-HPAE-4 prepared in Examples 1-4 to TGM1 DNA was tested by PicoGreen, wherein the mass ratio of branched poly(β-amino ester) to DNA was 30:1 and 60:1 respectively, and the amount of DNA was 1 μg.

[0257] The branched poly(β-amino ester) solution was added to the DNA solution encoding green fluorescent protein, and vortexed at high speed for 30 s, and then left to stand for 20 min, to form complex nanoparticles respectively. Then, the complex nanoparticle solution was diluted to 100 μL using TE buffer solution, and then 100 μL of PicoGreen working solution was added, and the excited fluorescence intensity was detected under the condition of excitation wavelength of 480 nm and emission wavelength of 520 nm.

[0258] Complex nanoparticles of poly(β-amino ester) and TGM1 DNA with a mass ratio of 30:1 and 60:1 respectively were prepared by a method similar to the above, and the complex nanoparticles were diluted to 1 mL using deionized water, and then the particle size and surface potential of the complex nanoparticles were tested by dynamic light scattering (DLS). The complex nanoparticles were washed and freeze-dried, and the micro-morphology thereof was characterized by TEM.

[0259] Figure 4 shows the results of the affinity test of ER-HPAE-1 to ER-HPAE-4 to TGM1 DNA. 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 is more than 95%.

[0260] Figure 5 shows the particle size test results of the obtained complex nanoparticles. 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 complex nanoparticles is 193 nm-380 nm, which meets the condition of efficient uptake by cells.

[0261] Figure 6 shows the surface potential test results of the obtained composite nanoparticles. 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 of the obtained composite nanoparticles ranges from 18.0 + mV to 27.0 + mV, which can effectively shield the negative potential of DNA itself.

[0262] Figure 7 shows the micro-morphology characterization results of the obtained composite nanoparticles. 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 distribution of the obtained nanoparticles is uniform, compact spherical and relatively stable, which confirms its excellent stability and DNA protection efficiency.

[0263] Example 6

[0264] Skin effect cells 3T3 cells were seeded in a 96-well plate at a density of 2.0 x 10 3 Example 1 and 2, branched poly(β-amino ester) ER-HPAE-1 and ER-HPAE-2, were formed into composite nanoparticles ER-HPAE-1 / AF647-DNA and ER-HPAE-2 / AF647-DNA with AF647 fluorescent dye-labeled luciferase-encoding DNA at a mass ratio of 30:1, wherein the amount of AF647 fluorescent dye-labeled luciferase-encoding DNA was 1.0 μg. Then the composite nanoparticles were mixed with the culture medium and added to the cells, and after incubation for 4 h, Lyso-Tracker Green and Hoechst 33342 were used to stain the organelles and nuclei of the cells at 37°C for 10 min, and then the distribution of DNA in the cells and the organelle targeting efficiency were observed using a fluorescence microscope.

[0265] Figure 8 shows the endocytosis efficiency characterization results of the composite nanoparticles ER-HPAE-1 / AF647-DNA and ER-HPAE-2 / AF647-DNA. From the fluorescence microscope photos, it can be found that a large number of red DNA molecules surround the blue cell nucleus, which may be due to the high surface potential of the composite nanoparticles promoting their interaction with cells, without wishing to be bound by any particular theory. These results confirm that the composite nanoparticles of the present disclosure have excellent cell uptake efficiency, which can promote subsequent gene transfection.

[0266] Figure 9 shows the results of organelle targeting characterization of the composite nanoparticles ER-HPAE-1 / AF647-DNA and ER-HPAE-2 / AF647-DNA. It can be seen that the AF647 fluorescent dye-labeled DNA has obvious overlap with the endoplasmic reticulum labeled by the fluorescent dye ER-tracker Green, which indicates that the DNA molecules have obvious colocalization with the endoplasmic reticulum in the cell.

[0267] Figure 10 shows the results of organelle targeting characterization of the composite nanoparticles ER-HPAE-1 / AF647-DNA and ER-HPAE-2 / AF647-DNA. According to the results of the enlarged fluorescence photos and PCC, not only is the DNA molecule observed to have obvious colocalization with the endoplasmic reticulum in the cell, but also a large number of composite nanoparticles are located between the endoplasmic reticulum and the nucleus, providing conditions for further nuclear internalization. These results all confirm that the poly( -amino ester)-based nanoparticles of the present disclosure have good endoplasmic reticulum targeting effect, can significantly overcome the retention of the composite nanoparticles in the lysosome, and help to increase the entry of the exogenous gene into the nucleus, thereby improving the gene transfection efficiency of the nanoparticles.

[0268] Example 7

[0269] Skin effect cells 3T3 cells were seeded in a 96-well plate at a density of 2.0 x 10 4 cells / well, and the cells were cultured overnight at 37°C. The branched poly( -amino ester) ER-HPAE-1 to ER-HPAE-4 of Examples 1-4 were added to the composite nanoparticles formed with GFP DNA at a mass ratio of 30:1 and 60:1 in the serum-containing culture medium, mixed thoroughly, and then slowly added to the cells. Then the cells were cultured for 48 h, and the efficiency of transfection of the 3T3 cells with the GFP DNA was tested using a fluorescence microscope and a flow cytometer.

[0270] Figure 11 shows the results of qualitative evaluation of the gene transfection performance of the composite nanoparticles. It can be seen from Figure 11 that the composite nanoparticles formed at a mass ratio of 30:1 and 60:1 can both efficiently mediate the efficient transfection of the gene, which indicates that the branched poly( -amino ester)-based nanoparticles of the present disclosure have excellent biophysical performance and excellent cell targeting ability.

[0271] Figure 12 shows the results of quantitative evaluation of the transfection efficiency of the composite nanoparticles in 3T3 cells. The results of the flow cytometry of the cells after transfection show that the composite nanoparticles formed at a mass ratio of 30:1 can both efficiently mediate the efficient transfection of the gene, with a transfection efficiency of 59.3% and a relative fluorescence intensity of 2.9 x 10 5RLU, which indicates that the branched poly(β-amino ester)-based nanoparticles of the present disclosure have very excellent transfection performance and can meet the intracellular gene delivery of skin effect cells.

[0272] Figure 13 shows the survival rate results of suspended cells after transfection of the composite nanoparticles in five different tissue cells (A2780 cells, 293T cells, 3T3 cells, SW1353 cells, and Vero cells). The alarmBlue results show that the survival rate of cells after transfection of the branched poly(β-amino ester)-based nanoparticles of the present disclosure is more than 96%, and the highest is 100%, which is mainly due to the excellent biodegradability and biosafety of the composite nanoparticles of the present disclosure.

[0273] Example 8

[0274] The suspended cells (293F cells) were mixed with 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 density of 4.0 x 10 4 cells / well, and 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 further cultured for 48 h, and the cells transfected with GFP DNA were observed under a fluorescence microscope, and the gene transfection efficiency in the suspended cells was evaluated by flow cytometry. The survival rate of the cells after transfection was tested using alarmBlue.

[0275] Figure 14 shows the evaluation results of the transfection performance of the composite nanoparticles in the suspended cells. As can be seen from Figure 14, in 293F cells, due to the advantages of endoplasmic reticulum targeting in overcoming the intracellular barriers such as low gene lysosome escape efficiency and nuclear internalization limitation, as well as the multiple terminal structure and 3-dimensional topological structure, the composite nanoparticles of the present disclosure exhibit extremely high GFP DNA transfection efficiency.

[0276] Figure 15 shows the quantitative results of flow cytometry of the cells after transfection of the composite nanoparticles. The results show that the composite nanoparticles formed at a mass ratio of 20:1 can efficiently mediate gene transfection, and the transfection efficiency reaches 74%, and the relative fluorescence intensity is more than 6.0 x 10 5 RLU, which is significantly better than the commercial transfection reagent Lipo3000. This indicates that the branched poly(β-amino ester)-based nanoparticles of the present disclosure can meet the intracellular gene delivery of the suspended cells.

[0277] Figure 16(a)-(d) show the cell viability of the suspended cells after transfection of the complex nanoparticles of branched poly( -amino ester) ER-HPAE-1 to ER-HPAE-4 of Examples 1-4 and GFP DNA at mass ratios of 120:1, 100:1, 80:1, 60:1, 40:1, 20:1, 10:1 and 5:1. The alarmBlue results show that the cell viability after transfection of these complex nanoparticles is over 96%, and the highest is 100%, and with the increase of the mass ratio, the activity of the 293F cells after transfection of the complex nanoparticles has a slight decrease, but at high and low mass ratios, it can still retain a very high cell viability, which is mainly due to the excellent biodegradability, biosafety and targeting of the complex nanoparticles of the present disclosure.

[0278] Example 9

[0279] The transfection efficiency of the complex nanoparticles of branched poly( -amino ester) ER-HPAE-1 and ER-HPAE-2 of Examples 1 and 2 and DNA of different sizes and functions (TGM1 DNA, COL7A1 DNA and MBTPS1 DNA) in skin-related effector cells was tested.

[0280] HaCaT cells and primary NHF cells were seeded in 96-well plates at a density of 2.0*10 4 cells / well and cultured overnight at 37°C. The branched poly( -amino ester) ER-HPAE-1 and ER-HPAE-2 of Examples 1 and 2 were mixed with TGM1 DNA, COL7A1 DNA or MBTPS1 DNA to form complex nanoparticles at a mass ratio of 30:1. The resulting complex nanoparticles were used to transfect HaCaT cells and primary NHF cells, respectively. After 36h of transfection, the transfected cells were digested, and qPCR was used to test the TGM1 mRNA, COL7A1 mRNA and MBTPS1 transfection efficiency mediated by the complex nanoparticles. After 72h of transfection, Western blotting was used to test the expression of TGM1 protein and COL7A1 protein mediated by the complex nanoparticles. The TGM1 DNA, COL7A1 DNA and MBTPS1 DNA solution was used to transfect the cells as a blank control.

[0281] Figure 17 shows the results of the evaluation of the expression efficiency of the composite nanoparticles in HaCaT cells. The qPCR results show that the composite nanoparticles formed by the branched poly(b-amino ester)s of Examples 1 and 2 in HaCaT cells increased the COL7A1 mRNA transcription level by more than 3500 times, the TGM1 mRNA transcription level by more than 200 times, and the MBTPS1 mRNA transcription level by more than 350 times. This indicates that the composite nanoparticles of the present disclosure have excellent functional DNA transfection efficiency in skin effector cells.

[0282] Figure 18 shows the results of the evaluation of the expression efficiency of the composite nanoparticles in NHF cells. The qPCR results show that the composite nanoparticles formed by the branched poly(b-amino ester)s of Examples 1 and 2 in NHF cells increased the COL7A1 mRNA transcription level by more than 2400 times, the TGM1 mRNA transcription level by more than 180 times, and the MBTPS1 mRNA transcription level by more than 1100 times. This indicates that the composite nanoparticles of the present disclosure have excellent functional DNA transfection efficiency in skin effector cells.

[0283] Figure 19 shows the results of the evaluation of the expression efficiency of the composite nanoparticles in NHF cells. The results of the Western blotting show that the composite nanoparticles formed by the branched poly(b-amino ester)s of Examples 1 and 2 in NHF cells exhibited more obvious TGM1 protein bands, MBTPS1 protein bands, and COL7A1 protein bands than the untransfected cells and the cells transfected by the commercial transfection reagent jetPEI. This is mainly due to the excellent DNA affinity, compressibility, stable compact microstructure, and excellent endoplasmic reticulum targeting of the branched poly(b-amino ester)-based nanoparticles of the present disclosure, so that the branched poly(b-amino ester)-based nanoparticles of the present disclosure can efficiently mediate the transfection of three different sizes of functional DNA (TGM1 DNA, COL7A1 DNA, and MBTPS1 DNA) in skin effector cells and meet the high-efficiency gene delivery of suspension cells. These results preliminarily confirm that the composite nanoparticles of the present disclosure have important application potential in the treatment of RDEB, ichthyosis genes, cataracts, alopecia, oral mucosal diseases, and CAOP syndrome.

[0284] Example 10

[0285] The branched poly(b-amino ester)s ER-HPAE-1 and ER-HPAE-2 of Examples 1 and 2 were tested for their transfection efficiency of different sizes of functional DNA (COL7A1 DNA or MBTPS1 DNA) in C57 mice (approved by the Animal Ethics Committee of the Pediatric Hospital Affiliated to Fudan University, number: 2023220).

[0286] C57 mice were divided into 6 groups and subcutaneously injected twice (one injection every 48 h). Branched poly(β-amino ester) ER-HPAE-1 and ER-HPAE-2 of Examples 1 and 2 were complexed with COL7A1 DNA or MBTPS1 DNA in an ethanol system (containing 7% F127) with the amount of COL7A1 DNA or MBTPS1 DNA being 15 μg, and PBS group as a negative control. Finally, 72 h after the second injection, the mice were sacrificed by the method of spinal dislocation, and the skin tissue was harvested for Western blot and HE staining tests.

[0287] Figure 20 shows the results of evaluating the transfection efficiency of branched poly(β-amino ester) ER-HPAE-1 and ER-HPAE-2 of Examples 1 and 2 mediated by functional MBTPS1 DNA of different sizes in C57 mice. The results of Western Blot show that both complex nanoparticles can efficiently express MBTPS1 protein in three transfected mice, further confirming that the complex nanoparticles of the present disclosure have important application potential in the gene therapy of cataract, alopecia, oral mucosa disease and CAOP syndrome.

[0288] Figure 21 shows the results of evaluating the transfection efficiency of branched poly(β-amino ester) ER-HPAE-1 and ER-HPAE-2 of Examples 1 and 2 mediated by functional COL7A1 DNA of different sizes in C57 mice. The results of Western Blot show that both complex nanoparticles can efficiently express C7 protein in three transfected mice, further confirming that the nanoparticles have important application potential in the gene therapy of RDEB.

[0289] Figure 22 shows the results of evaluating the in vivo toxicity after transfection of branched poly(β-amino ester) ER-HPAE-1 and ER-HPAE-2 of Examples 1 and 2 mediated by functional MBTPS1 DNA of different sizes in C57 mice. The results of HE staining show that there is no obvious difference between the tissue structure of the skin of the mice subcutaneously injected with two kinds of complex nanoparticles containing MBTPS1 DNA 120 hours later and the normal tissue of the untransfected mice, and thus no obvious inflammation occurs after subcutaneous injection of the complex nanoparticles, which confirms that the branched poly(β-amino ester) of the present disclosure has excellent safety and clinical application potential in in vivo gene delivery.

[0290] Figure 23 shows the results of evaluating the in vivo toxicity of branched poly( -amino ester) ER-HPAE-1 and ER-HPAE-2 of Examples 1 and 2 mediated transfection of functional COL7A1 DNA of different sizes in C57 mice. The results of HE staining show that there is no significant difference in the tissue structure of the skin of the mice subcutaneously injected with the two kinds of composite nanoparticles containing COL7A1 DNA 120 hours later and the normal tissue of the untransfected mice, and therefore there is no obvious inflammation after subcutaneous injection of the composite nanoparticles, which confirms that the branched poly( -amino ester) of the present disclosure has excellent safety and clinical application potential in in vivo gene delivery.

[0291] Example 11

[0292] The transfection efficiency of luciferase DNA mediated by branched poly( -amino ester) ER-HPAE-1 and ER-HPAE-2 of Examples 1 and 2 was tested in C57 mice.

[0293] The C57 mice were divided into 3 groups and subcutaneously injected once. The branched poly( -amino ester) ER-HPAE-1 and ER-HPAE-2 obtained in Examples 1 and 2 were formed into composite nanoparticles (containing 7% F127) with DNA encoding luciferase in an ethanol system, and the amount of DNA encoding luciferase was 20 pg. The HPAE (HPAE not containing ABF, other monomer composition being the same as the functionalized targeted branched poly( -amino ester) of Example 1) / COL7A1 DNA or MBTPS1 DNA nanoparticle group was used as a control, and in vivo imaging was performed 24 hours after in vivo transfection.

[0294] Figure 24 shows the results of evaluating the transfection efficiency of luciferase DNA mediated by branched poly( -amino ester) ER-HPAE-1 and ER-HPAE-2 of Examples 1 and 2 in C57 mice by tail vein injection. The results of in vivo luminescence show that the branched poly( -amino ester) ER-HPAE-1 and ER-HPAE-2 of Examples 1 and 2 can efficiently mediate DNA transfection by tail vein injection, and the highest luminescence intensity of luciferase mediated by the branched poly( -amino ester) based nanoparticles of Example 1 is more than 2.3*10 6 p / sec / cm2 / sr, which is much higher than the luminescence intensity of luciferase mediated by HPAE (HPAE not containing ABF, other monomer composition being the same as the branched poly( -amino ester) of Example 1) based nanoparticles (no luminescence is observed). These results show that the branched poly( -amino ester) based nanoparticles of the present disclosure can efficiently mediate gene delivery in the complex physiological environment (biological macromolecules such as proteins and nucleases) in vivo, which also provides important support for further clinical application.

[0295] Example 12

[0296] Female SD rats aged 6 to 8 weeks were selected, the back of the rats was shaved, and then 300 μg of branched poly(β-amino ester) and 10 μg of Cy3-labeled DNA, laurocapram penetration enhancer were configured into a mixed solution, and the final concentration of laurocapram was 1%. The mixed solution was evenly applied to the shaved area of the back of the rat, and then covered with a medical dressing. After 4 hours, the rats were sacrificed by cervical dislocation (approved by the Animal Ethics Committee of the Children's Hospital of Fudan University, number: 2023220), and the skin area applied was sampled, fixed in paraformaldehyde solution, and then frozen sectioned. The prepared frozen sections were stained, mounted, and finally imaged using a fluorescence microscope to observe the fluorescence distribution of cy3-labeled plasmid in the skin tissue.

[0297] Figure 25 shows the results of evaluating the transdermal efficiency of branched poly(β-amino ester) ER-HPAE-1 and ER-HPAE-2 of Examples 1 and 2 for mediating Cy3-labeled DNA in female SD rats by transdermal application. The results of the sectioning show that after 4 hours of external application of the 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 layer 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, with the same monomer composition as the functionalized targeting branched poly(β-amino ester) in Example 1) -based nanoparticles and commercial transfection reagent PEIpro (almost no luminescence). These results show that the branched poly(β-amino ester)-based nanoparticles of the present disclosure have important clinical application potential in transdermal drug delivery.

[0298] Example 13

[0299] Female SD rats aged 6 to 8 weeks were selected, the back of the rats was shaved, and then 300 μg of branched poly(β-amino ester) and 10 μg of plasmid of COL1A1, COL3A1 or silk protein were configured into a mixed solution, and then the mixed solution was evenly applied to the shaved area of the back of the rat, and then covered with a medical dressing. After 48 hours, the rats were sacrificed by cervical dislocation (approved by the Animal Ethics Committee of the Children's Hospital of Fudan University, number: 2023220), and the skin area applied was sampled, and the protein was extracted, and the expression of COL1A1, COL3A1 or silk protein in the skin tissue was detected using Western Blot technology.

[0300] Figure 26 shows the results of evaluating the transdermal efficiency of the branched poly(beta-amino ester) ER-HPAE-1 and ER-HPAE-2 of Examples 1 and 2 in mediating COL1A1, CLO3A1 or fibroin DNA transdermally administered by smearing on female SD rats. The results of Western Blot show that the branched poly(beta-amino ester) ER-HPAE-1 and ER-HPAE-2 of Examples 1 and 2 can efficiently deliver COL1A1, CLO3A1 or fibroin DNA to skin cells and the DNA is successfully expressed into corresponding proteins in cells after smearing the composite nanoparticles on the skin for 48 hours. The results confirm that the transdermal effect of the branched poly(beta-amino ester)-based nanoparticles of Examples 1 and 2 is significantly better than that of HPAE (HPAE without ABF, other monomer composition is the same as the functionalized targeting branched poly(beta-amino ester) in Example 1)-based nanoparticles. These results show that the branched poly(beta-amino ester)-based nanoparticles of the present disclosure have important application potential in medical aesthetics.

[0301] Example 14

[0302] SW1353 and HeLa cells were seeded in 96-well plates at a density of 1.0 x 10 4 cells / well and incubated at 37°C overnight. The branched poly(beta-amino ester) ER-HPAE-1 to ER-HPAE-4 of Examples 1-4 were added to the composite nanoparticles formed with GFP mRNA at a mass ratio of 60:1 in serum-containing medium, mixed thoroughly and then slowly added to the cells. The cells were then incubated for 48 h and tested for their efficiency in transfecting GFP mRNA in SW1353 and HeLa cells using fluorescence microscopy.

[0303] Figure 27 shows the results of evaluating the transfection performance of the composite nanoparticles formed by the branched poly(beta-amino ester) ER-HPAE-1 to ER-HPAE-4 of Examples 1-4 with GFP mRNA in SW1353 cells. It can be found that the composite nanoparticles of the present disclosure exhibit extremely high GFP mRNA transfection efficiency in SW1353 cells due to the advantages of endoplasmic reticulum targeting in overcoming intracellular barriers such as low efficiency of lysosomal escape of genes and nuclear internalization limitations, as well as the multiple terminal structure and three-dimensional topological structure.

[0304] Figure 28 shows the results of the evaluation of the transfection performance of the complex nanoparticles formed by the branched poly( -amino ester)s ER-HPAE-1 to ER-HPAE-4 of Examples 1-4 and GFP mRNA in HeLa cells. It can be found that, due to the advantages of endoplasmic reticulum targeting in overcoming the intracellular barriers such as low lysosome escape efficiency and nuclear internalization limitation, as well as the multi-terminal structure and three-dimensional topology, the complex nanoparticles of the present disclosure exhibit extremely high GFP mRNA transfection efficiency in HeLa cells.

[0305] Example 15

[0306] HeLa and HaCaT cells were seeded in 96-well plates at a density of 1.0 x 10 4 cells / well and incubated at 37 °C overnight. The complex nanoparticles formed by the branched poly( -amino ester)s ER-HPAE-1 to ER-HPAE-4 of Examples 1-4 and BSA-FITC at a mass ratio of 60:1, respectively, were added to the serum-containing medium, and after thorough mixing, they were slowly added to the cells. Then the cells were further incubated for 24 h, and the BSA-FITC intracellular delivery efficiency of the complex nanoparticles in HeLa and HaCaT cells was tested using a fluorescence microscope.

[0307] Figure 29 shows the results of the evaluation of the transfection performance of the complex nanoparticles formed by the branched poly( -amino ester)s ER-HPAE-1 to ER-HPAE-4 of Examples 1-4 and BSA-FITC in HeLa cells. It can be found that, due to the advantages of endoplasmic reticulum targeting in overcoming the intracellular barriers such as low lysosome escape efficiency and nuclear internalization limitation, as well as the multi-terminal structure and three-dimensional topology, the complex nanoparticles of the present disclosure exhibit extremely high BSA-FITC intracellular delivery efficiency in HeLa cells.

[0308] Figure 30 shows the results of the evaluation of the transfection performance of the complex nanoparticles formed by the branched poly( -amino ester)s ER-HPAE-1 to ER-HPAE-4 of Examples 1-4 and BSA-FITC in HaCaT cells. It can be found that, due to the advantages of endoplasmic reticulum targeting in overcoming the intracellular barriers such as low lysosome escape efficiency and nuclear internalization limitation, as well as the multi-terminal structure and three-dimensional topology, the complex nanoparticles of the present disclosure exhibit extremely high BSA-FITC intracellular delivery efficiency in HaCaT cells.

[0309] Example 16

[0310] 293T-GFP cells and HeLa-GFP cells were seeded in 96-well plates at a density of 1.0 x 10 4The branched poly( -amino ester)s ER-HPAE-1 to ER-HPAE-4 of Examples 1-4 were complexed with GFP siRNA at a mass ratio of 60:1 to form complex nanoparticles, which were added to the serum-containing medium and mixed well before being slowly added to the cells. The cells were then incubated for another 48 h, and the efficiency of the complex nanoparticles in transfecting GFP siRNA in 293T-GFP cells and HeLa-GFP cells was tested using a fluorescence microscope.

[0311] Figures 31 and 32 show the results of the evaluation of the transfection performance of the complex nanoparticles formed by the branched poly( -amino ester)s ER-HPAE-1 to ER-HPAE-4 of Examples 1-4 and GFP siRNA in 293T-GFP cells. It can be found that, in 293T-GFP cells, the complex nanoparticles of the present disclosure exhibit very high GFP siRNA transfection efficiency due to the advantages of endoplasmic reticulum targeting in overcoming intracellular barriers such as low lysosome escape efficiency and nuclear internalization limitation, as well as the multi-terminal structure and three-dimensional topological structure.

[0312] Figures 33 and 34 show the results of the evaluation of the transfection performance of the complex nanoparticles formed by the branched poly( -amino ester)s ER-HPAE-1 to ER-HPAE-4 of Examples 1-4 and GFP siRNA in HeLa-GFP cells. It can be found that, in HeLa-GFP cells, the complex nanoparticles of the present disclosure exhibit very high GFP siRNA transfection efficiency due to the advantages of endoplasmic reticulum targeting in overcoming intracellular barriers such as low lysosome escape efficiency and nuclear internalization limitation, as well as the multi-terminal structure and three-dimensional topological structure.

[0313] While the present disclosure has been particularly shown and described with reference to specific embodiments, in which a part is a preferred embodiment, it will be understood by those skilled in the art that various changes in form and details can be made therein without departing from the spirit and scope of the disclosure as disclosed herein.

Claims

1. A branched poly(β-amino ester) having the structure shown in formula (I), formula (II), formula (III), formula (IV) or formula (V): wherein, W1, W2, W3, W4, and W5are 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 with one or more R W substituents; L is selected from alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkylcycloalkyl, alkylheterocyclyl, alkylcycloalkylalkyl, alkylheterocyclylalkyl, alkylarylalkyl, alkylheteroarylalkyl, or heteroalkylarylalkylheteroalkyl, wherein the alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkylcycloalkylalkyl, alkylheterocyclylalkyl, alkylarylalkyl, alkylheteroarylalkyl, and heteroalkylarylalkylheteroalkyl is optionally substituted with one or more R L substituents; each R1is independently selected from R1 N or T; R1 N is selected from alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkylcycloalkyl, alkylheterocyclyl, alkylaryl, alkylheteroaryl, or a nitrogen protecting group, said alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkylcycloalkyl, alkylheterocyclyl, alkylaryl, and alkylheteroaryl being optionally substituted with one or more R N substituents; each R2is independently selected from R2 N or T; R2 N is selected from alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkylcycloalkyl, alkylheterocyclyl, alkylaryl, alkylheteroaryl, or a nitrogen protecting group, said alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkylcycloalkyl, alkylheterocyclyl, alkylaryl, alkylheteroaryl, alkylcycloalkylalkyl, alkylheterocyclylalkyl, alkylarylalkyl, and alkylheteroarylalkyl optionally substituted with one or more R N substituents; each T is independently an endoplasmic reticulum targeting moiety; each R W , R L , and R N is independently selected from the group consisting of halogen, cyano, nitro, oxo, -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; each R is independently selected from hydrogen or alkyl; a 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) of claim 1, wherein, each R1is independently R1 N , and at least one R2is T.

3. The branched poly(β-amino ester) of claim 2, wherein each R1 N is independently selected from alkyl, heteroalkyl, alkylcycloalkyl, alkylheterocyclyl, alkylaryl, or alkylheteroaryl, the alkyl, heteroalkyl, alkylcycloalkyl, alkylheterocyclyl, alkylaryl, and alkylheteroaryl optionally substituted with one or more R N , and each R N is independently -OR a , or -N(R a )2.

4. The branched poly(β-amino ester) of claim 3, wherein each R1is independently selected from the group consisting of:

5. The branched poly(β-amino ester) of claim 4, wherein each R1is independently selected from the group consisting of:

6. The branched poly( -amino ester) of any one of claims 2-5, wherein each R2 N is independently selected from alkyl, heteroalkyl, alkylcycloalkyl, alkylheterocyclyl, or alkylheterocyclylalkyl, the alkyl, heteroalkyl, alkylcycloalkyl, alkylheterocyclyl, and alkylheterocyclylalkyl optionally substituted with one or more R N , and each R N is independently -OR a , or -N(R a )2.

7. The branched poly(β-amino ester) of claim 6, wherein each R2is independently selected from the group consisting of:

8. The branched poly(β-amino ester) of claim 7, wherein each R2is independently selected from the group consisting of:

9. The branched poly(beta-amino ester) of claim 1, having a structure according to Formula (I), wherein, In of the repeating units are T, and each R2is independently R2 N .

10. The branched poly( -amino ester) of claim 9, wherein W1is an alkyl or heteroalkyl, the alkyl and heteroalkyl being optionally substituted with one or more R W substituents.

11. The branched poly(β-amino ester) of claim 10, wherein W1 is 12. The branched poly(beta-amino ester) of claim 1, having a structure according to Formula (II), wherein, In of the repeating units are T, and each R2is independently R2 N .

13. The branched poly( -amino ester) of claim 12, wherein W2 is an alkyl or heteroalkyl, the alkyl and heteroalkyl being optionally substituted with one or more R W substituents.

14. The branched poly(β-amino ester) of claim 13, wherein W2 is 15. The branched poly(beta-amino ester) of claim 1, having a structure according to Formula (III), wherein, In of the repeating units are T, and each R2is independently R2 N .

16. The branched poly(β-amino ester) of claim 15, wherein W3 is an alkyl or heteroalkyl, the alkyl and heteroalkyl being optionally substituted with one or more R W substituents.

17. The branched poly(β-amino ester) of claim 16, wherein W3 is 18. The branched poly(beta-amino ester) of claim 1, having a structure according to Formula (IV), wherein, In of the repeating units are T, and each R2is independently R2 N .

19. The branched poly(β-amino ester) of claim 18, wherein W4 is an alkyl or heteroalkyl, the alkyl and heteroalkyl being optionally substituted with one or more R W substituents.

20. The branched poly(β-amino ester) of claim 19, wherein W4 is 21. The branched poly(beta-amino ester) of claim 1, having a structure according to Formula (V), wherein, In of the repeating units are T, and each R2is independently R2 N .

22. The branched poly(β-amino ester) of claim 21, wherein W5 is alkyl, heteroalkyl, or alkylheterocyclylalkyl, the alkyl, heteroalkyl, and alkylheterocyclylalkyl groups being optionally substituted with one or more R W substituents.

23. The branched poly(β-amino ester) of claim 22, wherein W5 is selected from:

24. The branched poly(β-amino ester) of any one of claims 9-23, wherein each R1 N is independently selected from alkyl, heteroalkyl, alkylcycloalkyl, alkylheterocyclyl, alkylaryl, or alkylheteroaryl, the alkyl, heteroalkyl, alkylcycloalkyl, alkylheterocyclyl, alkylaryl, and alkylheteroaryl optionally substituted with one or more R N , and each R N is independently -OR a , or -N(R a )2.

25. The branched poly(β-amino ester) of claim 24, wherein each R1is independently selected from the group consisting of: and T.

26. The branched poly(β-amino ester) of claim 25, wherein each R1is independently selected from the group consisting of: and T.

27. The branched poly(β-amino ester) of any one of claims 9-26, wherein each R2 N is independently selected from alkyl, heteroalkyl, alkylcycloalkyl, alkylheterocyclyl, alkylheterocyclylalkyl, the alkyl, heteroalkyl, alkylcycloalkyl, alkylheterocyclyl, and alkylheterocyclylalkyl groups being optionally substituted with one or more R N , and each R N is independently -OR a , or -N(R a )2.

28. The branched poly(β-amino ester) of claim 27, wherein each R2is independently selected from the group consisting of:

29. The branched poly(β-amino ester) of claim 28, wherein each R2is independently selected from the group consisting of:

30. The branched poly(β-amino ester) of claim 29, wherein the T is selected from:

31. The branched poly(β-amino ester) of any one of claims 1-30, 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 , and each R L is independently -OR a , -N(R a )2, or alkyl.

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

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

34. The branched poly(beta-amino ester) of any one of claims 1-33, wherein m1, m2, m3, m4, m5, m6, m7, m8, m9, m10, m11, and m12 are each independently in the range of 5 to 75.

35. The branched poly(beta-amino ester) of any one of claims 1-34, having a molecular weight in the range of 5.0 kDa to 40.0 kDa, 5.0 kDa to 50 kDa, or 5.0 kDa to 40.0 kDa.

36. The branched poly(beta-amino ester) of claim 35, having a molecular weight of about 5.0 kDa, 6.0 kDa, 7.0 kDa, or 8.0 kDa.

37. The branched poly(β-amino ester) of claim 1 having the structure shown in ER-HPAE-1, ER-HPAE-2, ER-HPAE-3, or ER-HPAE-4: wherein ER-HPAE-1 has a molecular weight of 7.5 kDa, ER-HPAE-2 has a molecular weight of 7.1 kDa, ER-HPAE-3 has a molecular weight of 7.4 kDa, and ER-HPAE-4 has a molecular weight of 8.0 kDa.

38. A method of making the branched poly(beta-amino ester) of any one of claims 1-37, comprising: 1-1) polymerizing a multiacrylate monomer having the formula (I-1), the formula (II-1), the formula (III-1), or the formula (IV-1): with diacrylate monomers of formula (2) and one or more amine monomers of formula (3) via Michael addition reaction to give branched poly(beta-amino ester) P1 with terminal double bonds: 1-2) subjecting the branched poly(beta-amino ester) P1 produced in step 1-1) to one or more capping amine monomers having Formula (4) via a Michael addition reaction, thereby producing a branched poly(beta-amino ester) having a structure according to Formula (I), Formula (II), Formula (III), or Formula (IV): R2-NH2 Formula (4) or, 2-1) subjecting the diamine monomer of formula (V-1) to Michael addition reaction with the diacrylate monomer of formula (2) and one or more amine monomers of formula (3) to obtain branched poly(beta-amino ester) P2 with double bond at the end: 2-2) subjecting the branched poly(beta-amino ester) P2 produced in step 2-1) to one or more capping amine monomers having Formula (4) via a Michael addition reaction, thereby producing a branched poly(beta-amino ester) having a structure according to Formula (V): R2-NH2 Formula (4).

39. The method of claim 38, wherein, The multiacrylate monomer of formula (I-1) is The multiacrylate monomer of formula (II-1) is The multiacrylate monomer of formula (III-1) is The multiacrylate monomer of formula (IV-1) is The diamine monomer of formula (V-1) is selected from the group consisting of: The diacrylate monomer of formula (2) is selected from the group consisting of:

40. The method of claim 38 or 39, wherein the one or more amine monomers of Formula (3) are R1 N -NH2, and the one or more capped amine monomers of Formula (4) are selected from R2 N -NH2or T-NH2, and at least one capped amine monomer of Formula (4) is T-NH2.

41. The method of claim 40, wherein, R1 N -NH2 is selected from the group consisting of: R2 N -NH2 is selected from the group consisting of: T-NH2is selected from the group consisting of:

42. The method of claim 40 or 41, wherein, diacrylate monomer of formula (2), R2 N -NH2, T-NH2, and R1 N The reaction feed molar ratio of -NH2, T-NH2, and R1 is (1-3):(0.1-4):(0.02-2):(0.5-6); In step 1-1), the diacrylate monomer of formula (2), R1 N the molar ratio of the diacrylate monomer of formula (2), R1 the diacrylate monomer of formula (2), R1 the diacrylate monomer of formula (2), R1 the diacrylate monomer of formula (2), R1 the diacrylate monomer of formula (2), R1 the diacrylate monomer of formula (2), R1 the diacrylate monomer of formula (2), R1 the diacrylate monomer of formula (2), R1 the diacrylate monomer of formula (2), R1 the diacrylate monomer of formula (2), R1 the diacrylate monomer of formula (2), R1 the diacrylate In step 2-1), the diacrylate monomer of formula (2), R1 N The molar ratio of the diamine monomer of formula (V-1) is (1-3):(0.1-4):(0.5-3).

43. The method of claim 38 or 39, wherein the one or more amine monomers having formula (3) is selected from R1 N -NH2or T-NH2, and the one or more capped amine monomers having formula (4) is R2 N -NH2.

44. The method of claim 43, wherein, R1 N -NH2 is selected from the group consisting of: R2 N -NH2 is selected from the group consisting of: T-NH2is selected from the group consisting of:

45. The method of claim 43 or 44, wherein, diacrylate monomer of formula (2), R2 N -NH2, T-NH2, and R1 N The reaction feed molar ratio of -NH2, T-NH2, and R1 is (1-3):(0.1-4):(0.02-2):(0.5-6); In step 1-1), the diacrylate monomer of formula (2), R1 N the molar ratio of the diacrylate monomer of formula (2), R1 the molar ratio of the diacrylate monomer of formula (2), R1 the molar ratio of the diacrylate monomer of formula (2), R1 the molar ratio of the diacrylate monomer of formula (2), R1 the molar ratio of the diacrylate monomer of formula (2), R1 the molar ratio of the diacrylate monomer of formula (2), R1 the molar ratio of the diacrylate monomer of formula (2), R1 the molar ratio of the diacrylate monomer of formula (2), R1 the molar ratio of the diacrylate monomer of formula (2), R1 the molar ratio of the diacrylate monomer of formula (2), In step 2-1), the diacrylate monomer of formula (2), R1 N The molar ratio of the diamine monomer of formula (V-1) and T-NH2 is (1-3):(0.1-4):(0.5-3):(0.02-2).

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

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

48. The method of any one of claims 38-47, wherein the branched poly(beta-amino ester) PI resulting from step 1-1) has a molecular weight in the range of 5 kDa to 40 kDa.

49. The method of any one of claims 38-47, wherein the branched poly(beta-amino ester) P2 resulting from step 2-1) has a molecular weight in the range of 5 kDa to 40 kDa.

50. The method of any one of claims 38-49, wherein step 1-2) and step 2-2) are performed at a temperature of 15-60 °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(beta-amino ester) of any one of claims 1-37 and a biomolecule.

53. The composition of claim 52, wherein the biomolecule is a polynucleotide, a protein, a peptide, a sugar, a polysaccharide, a glycoprotein, a lipid, a hormone, a drug, or a prodrug.

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

55. The composition of claim 54, wherein the DNA encodes a C7 protein, a TGM1 protein, a MBTPS1 protein, a COL1A1 protein, a COL3A1 protein, a silk fibroin protein, a Cas9 protein, an ADAM17 protein, a TGF-beta 1 protein, an ABCD1 protein, a P53 protein, an RPE65 protein, an SMN1 protein, an AADC protein, an FVIII protein, a VEGF protein, and a HGF protein.

56. The composition of claim 54, wherein the RNA is an mRNA or an siRNA.

57. The composition of claim 56, wherein the RNA encodes a C7 protein, a TGM1 protein, a MBTPS1 protein, a COL1A1 protein, a COL3A1 protein, a silk fibroin protein, a Cas9 protein, an ADAM17 protein, a TGF-beta 1 protein, an ABCD1 protein, a P53 protein, an RPE65 protein, an SMN1 protein, an AADC protein, an FVIII protein, a VEGF protein, and a HGF protein.

58. The composition of claim 53, wherein the protein is a serum protein, a saponin, a ribonuclease, a beta-galactosidase, a horseradish peroxidase, a yellow fluorescent protein, a R-phycoerythrin, a green fluorescent protein, a trypsin, a lysozyme, an alpha-chymotrypsin, a superoxide dismutase, an egg white protein, a C7 protein, a TGM1 protein, a MBTPS1 protein, a COL1A1 protein, a COL3A1 protein, a silk fibroin protein, a Cas9 protein, an ADAM17 protein, a TGF-beta 1 protein, an ABCD1 protein, a P53 protein, an RPE65 protein, an SMN1 protein, an AADC protein, an FVIII protein, a VEGF protein, and a HGF protein.

59. The composition of any one of claims 52-58, wherein the mass ratio of the branched poly(beta-amino ester) to the biomolecule is in the range of 1:1 to 200:

1.

60. The composition of claim 59, wherein the mass ratio of the branched poly(beta-amino ester) to the biomolecule is in the range of 5:1 to 120:

1.

61. The composition of any one of claims 52-60, having a form of a nanoparticle.

62. The composition of claim 61, wherein the nanoparticle has a particle size in the range of 50 nm to 500 nm.

63. The composition of claim 62, wherein the nanoparticle has a particle size in the range of 150 nm to 400 nm.

64. A method of delivering a biomolecule to a subject, comprising administering to the subject a composition of any one of claims 52-63.

65. A method of treating a disease in a subject, comprising administering to the subject a composition of any one of claims 52-63.

66. The method of claim 64 or 65, wherein the composition is administered to the subject by a transdermal, subcutaneous injection, or intravenous injection.

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