Polyplexes of nucleic acids with targeted conjugates containing polyethyleneimine and polyethylene glycol

Abstract

The present invention provides a targeting polyplex composed of (i) a nucleic acid, particularly a nucleic acid encoding a pharmaceutically active peptide or protein, and (ii) a targeting conjugate comprising an LPEI and a PEG fragment connected by distinct linkages formed by a defined chemoselective reaction. Thus, the LPEI fragment is linked to a single PEG fragment in a linear, end-to-end manner. The linear conjugate is further conjugated to a targeting fragment to enable selective interaction with specific cell types. The polyplex selectively delivers the nucleic acid to target cells, resulting in high expression and efficient protein translation and secretion of the encoded pharmaceutically active protein.

Description

[Background technology]

[0001] Cancer remains a leading cause of death worldwide. For most solid tumors after surgical resection, chemotherapy is an important treatment option for managing remaining cancer cells. The main reason for chemotherapy failure is inefficient targeting and uptake of chemotherapeutic agents by tumors (J.K. Vasir and V. Labhasetwar, Technology in Cancer Research & Treatment, 2005, 4(4):363-374). Insufficient tumor accessibility necessitates higher doses, and due to the nature of chemotherapeutic agents, this leads to nonspecific uptake and toxicity in healthy cells. Targeted drug delivery strategies, in which therapeutic agents are reversibly bound to targeting ligands and selectively delivered to cells for treatment, are currently being applied to many chemotherapeutic agents in clinical use. This strategy shows promise for maximizing the safety and efficacy of a given chemotherapeutic agent, as their selective delivery to target cells avoids nonspecific uptake and associated toxicity in healthy cells (M Srinivasarao and PS Low, Chem Rev, 2017, 117:12133-12164), which may result in a higher maximum tolerated dose.

[0002] Nanoparticle delivery systems have attracted much interest in nucleic acid therapeutics, including DNA and mRNA, particularly for their application in cancer immunotherapy (AJ Mukalel et al., 2019, Cancer Lett. 458:102-112; U Laechelt and E Wagner, 2015 Chem Rev 115(19):11043-78; RS Riley et al., 2019, Nat Rev Drug Discov 18(3):175-196; X Tan et al., 2020, J Control Release 323:240-252; and references cited therein). However, these nucleic acid therapeutics must also overcome numerous delivery obstacles to success, including rapid in vivo degradation, insufficient uptake in target cells, required nuclear entry, and potential in vivo toxicity in healthy cells and tissues. Nanoparticle delivery systems, including targeted nanoparticle delivery systems, have been designed to address and attempt to overcome some of these barriers as a means of safely and effectively delivering nucleic acid therapeutics (DE Large et al., 2018, Adv Therap, 1800091; A Patel et al., 2020, BioDrugs 34:273-293; Hj Vaughan et al., 2020, Adv Mater, 32(13):e1901081).

[0003] Cationic polymers are known to form polyplexes with negatively charged nucleic acids in solution. For example, linear polyethyleneimine (LPEI) is protonated at physiological pH and therefore has a net positive charge. When LPEI is incubated with a nucleic acid, which has a net negative charge at physiological pH, the LPEI and nucleic acid can form polyplexes held together by electrostatic interactions. These polyplexes can be internalized by cells in vivo, where they can deliver nucleic acid sequences into cells. Therefore, polyplexes containing cationic polymers and nucleic acids can be used as vectors for therapy. Despite their promise, technical challenges have arisen regarding the formation of homogeneous, well-characterized cationic polymers. Polyplexes containing only LPEI are prone to aggregation and interactions with serum proteins, potentially limiting their potential as nucleic acid delivery agents. To overcome these challenges, the polymer LPEI can be conjugated to polyethylene glycol (PEG). PEG fragments can help shield LPEI from the surrounding matrix, improving the biocompatibility and blood circulation of the resulting polyplexes. Examples of such polyethyleneimine-polyethylene glycol conjugates that further comprise targeting moieties have been described, particularly as non-viral vectors for delivering double-stranded RNA, such as polyinosinic acid:polycytidylic acid (WO2015 / 173824; WO2010 / 073247; US Patent Application Publication No. 2004 / 248842 A1; Vetter VC, Wagner EJ Control Release, 2022 346:110-135; the references cited therein).However, the coupling of PEG to LPEI in the referenced conjugates and vectors occurs through the formation of a covalent bond between the electrophilic PEG fragment(s) and the secondary amine embedded in the LPEI backbone fragment, thus resulting in branched and heterogeneous conjugates and vectors with random and undefined encapsulation of PEG fragments, characterized based on the average PEG encapsulation density.In such conjugates, a wide variety of PEG fragments are orthogonally attached to the LPEI fragment without site specificity. Such random synthesis and inaccurate characterization of LPEI-PEG conjugates can make it difficult to establish a clear structure-activity relationship (SAR) between the structure of the conjugate and the activity of the resulting polyplex.

[0004] Despite recent efforts and achievements, there remains great interest in developing novel targeted delivery platforms that can protect nucleic acids, including mRNA, and mediate their delivery to desired tissues and cells to harness the powerful therapeutic potential of these molecules (AJ Mukalel et al., 2019, Cancer Lett. 458:102-112). Therefore, there is a need for homogeneous nanoparticles, particularly homogeneous LPEI-PEG conjugates with well-defined chemical structures, that can selectively deliver nucleic acids, including mRNA or pDNA, to desired tissues and cells. Summary of the Invention

[0005] The present invention provides targeting polyplexes composed of (i) a nucleic acid encoding a peptide or protein of interest, particularly a pharmaceutically active peptide or protein such as a cytokine, interferon, or toxin, and (ii) a targeting conjugate comprising an LPEI and a PEG fragment connected by distinct linkages formed by a defined chemoselective reaction, rather than through random and uncontrolled attachment of electrophilic PEG fragments to multiple nucleophiles on the LPEI backbone fragment. Thus, the present invention provides more uniform targeting conjugates with defined chemical structures. The distinct linkages not only ensure a consistent and predictable ratio of LPEI to PEG fragments, but also ensure a defined, linear conjugate instead of a random, branched conjugate. Thus, the LPEI fragments are attached to a single PEG fragment in a linear, end-to-end manner.

[0006] The conjugate further comprises a targeting fragment linked to the PEG fragment, which allows for targeting of a specific cell type and promoting uptake of the composition of the present invention and the pharmaceutically active nucleic acid in the specific cell type. Thus, a preferred embodiment comprises a targeting fragment, such as hEGF, DUPA, or folate, specifically linked to the LPEI-PEG diconjugate to target a corresponding receptor, such as hEGFR, PSMA, or folate receptor, on a specific cell type where the receptor is highly expressed or overexpressed, typically a cancer cell type.

[0007] Even more advantageously and surprisingly, the inventors have found that the resulting preferred conjugates and polyplexes according to the present invention, which have significantly reduced heterogeneity and therefore a significantly reduced number of potentially biologically active conjugates and polyplexes due to the defined chemoselective attachment of the LPEI fragment to the PEG fragment, not only form polyplexes of appropriate size, but also maintain or even increase their overall biological activity, such as highly selective, targeted delivery of a pharmaceutically active nucleic acid and subsequent efficient translation and secretion of the encoded pharmaceutically active protein. Thus, the compositions and polyplexes of the present invention not only selectively deliver a pharmaceutically active nucleic acid encoding a pharmaceutically active peptide or protein to target cells, particularly cancer cells, but also result in high expression and efficient protein translation and secretion of the encoded pharmaceutically active protein.

[0008] Thus, in one aspect, the invention provides a composition comprising a polyplex, the polyplex comprising a conjugate and a nucleic acid, the nucleic acid preferably non-covalently bound to the conjugate, the conjugate comprising: a linear polyethyleneimine fragment comprising an alpha end and an omega end, wherein the alpha end of the polyethyleneimine fragment is an initiating residue; a polyethylene glycol fragment comprising a first end and a second end; The omega end of the polyethyleneimine fragment is linked to the first terminal end of the polyethylene glycol fragment by a divalent covalent linking group -ZX 1 -ZX 1 - is not a single bond, -Z- is not an amide, The second terminal end of the polyethylene glycol fragment is a divalent covalent linking moiety X 2 is connected to the targeting fragment by The nucleic acid is a pharmaceutically active nucleic acid, wherein the pharmaceutically active nucleic acid encodes a pharmaceutically active peptide or protein. In a preferred embodiment of this aspect, the composition consists of the polyplex.

[0009] In another aspect, the present invention provides a composition comprising a polyplex, the polyplex comprising a conjugate of formula I* or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof, and a nucleic acid, the nucleic acid preferably comprising the conjugate: R 1 -(NR 2 -CH2-CH2) n -ZX 1 -(O-CH2-CH2) m -X 2 -L(Formula I*) (In the ceremony n is an integer of 1 to 1500, preferably an integer of 2 to 1500, m is any integer from 1 to 200, preferably any integer from 2 to 200, more preferably any integer from 1 to 100, and even more preferably any integer from 2 to 100; R 1 is the starting residue, preferably R 1 is -H or -CH3, R 2 are independently -H or an organic residue, and the -(NR 2 -CH2-CH2) n -The R in question 2 at least 80%, preferably 90%, of the X1 and X 2 are independently divalent covalent linking moieties; Z is a divalent covalent linking moiety, Z is not a single bond, Z is not -NHC(O)-, L is a targeting fragment, preferably the targeting fragment is capable of binding to a cell, more preferably the targeting fragment is capable of binding to a cell surface receptor), non-covalently attached to The nucleic acid is a pharmaceutically active nucleic acid, the pharmaceutically active nucleic acid being a nucleic acid encoding a pharmaceutically active peptide or protein, and preferably the composition consists of the polyplex.

[0010] In another aspect, the present invention provides a polyplex comprising a conjugate of formula I* or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof, and a nucleic acid, wherein the nucleic acid is preferably a nucleic acid that is a conjugate of formula I* or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof, R 1 -(NR 2 -CH2-CH2) n -ZX 1 -(O-CH2-CH2) m -X 2 -L(Formula I*) (In the ceremony n is an integer of 1 to 1500, preferably an integer of 2 to 1500, m is any integer from 1 to 200, preferably any integer from 2 to 200, more preferably any integer from 1 to 100, and even more preferably any integer from 2 to 100; R 1 is the starting residue, preferably R 1 is -H or -CH3, R 2 are independently -H or an organic residue, and the -(NR 2 -CH2-CH2) n -The R in question 2 at least 80%, preferably 90%, of the X1 and X 2 are independently divalent covalent linking moieties; Z is a divalent covalent linking moiety, Z is not a single bond, Z is not -NHC(O)-, L is a targeting fragment, preferably the targeting fragment is capable of binding to a cell, more preferably the targeting fragment is capable of binding to a cell surface receptor), non-covalently attached to The nucleic acid is a pharmaceutically active nucleic acid, which is a nucleic acid that encodes a pharmaceutically active peptide or protein.

[0011] In another aspect, the present invention provides a composition comprising a polyplex, the polyplex comprising a conjugate of Formula I or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof, and a nucleic acid, the nucleic acid preferably comprising the conjugate: [ka] (In the formula, [ka] is a single or double bond, n is an integer of 1 to 1500, preferably an integer of 2 to 1500, m is any integer from 1 to 200, preferably any integer from 2 to 200, more preferably any integer from 1 to 100, and even more preferably any integer from 2 to 100; R 1 is the starting residue, preferably R 1 is -H or -CH3, R 2 are independently -H or an organic residue, and the -(NR 2 -CH2-CH2) n -The R in question 2 at least 80%, preferably at least 90%, of are H; Ring A is one or more R A1 R is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl optionally substituted at any position by A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo, or halogen, or two R A1 are combined with the atoms to which they are attached to form one or more fused C6-C 10 can form an aryl, C-C heteroaryl, or C-C cycloalkyl ring, and each fused aryl, heteroaryl, or cycloalkyl can be joined by one or more R A2 and optionally substituted with R A2 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen -SO3H or -OSO3H; X 1 is a divalent covalent linking moiety, X 2 is a divalent covalent linking moiety, L is a targeting fragment, preferably the targeting fragment is capable of binding to a cell, more preferably the targeting fragment is capable of binding to a cell surface receptor), non-covalently bound to The nucleic acid is a pharmaceutically active nucleic acid, which is a nucleic acid that encodes a pharmaceutically active peptide or protein.

[0012] Although the N-N=N fragment of the bicyclic ring in Formula I is typically depicted herein using one single bond and one double bond for simplicity, those skilled in the art will recognize that Formula I and related conjugate structures depicted herein can alternatively be depicted as shown below: Such depictions and descriptions of Formula I are used interchangeably herein. [ka] (In formula, fragment [ka] is the fragment R 1 (NR 2 CH2CH2) n Two different regioisomeric bonds, i.e., [ka] and [ka] where the wavy line represents the chemical bond to ring A). Thus, Formula I as depicted herein includes two regioisomeric embodiments, namely, fragment R 1 (NR 2 CH2CH2) n is attached to the top nitrogen atom of the above structure or the bottom nitrogen atom of the above structure, but not to the middle nitrogen atom. Those of skill in the art will recognize that the same applies to other formulas herein, including Formula IA, Formula IB, Formula IC, Formula ID, Formula IE, Formula IH, Formula IJ, Formula IK, etc.

[0013] In another aspect, the present invention provides a polyplex comprising a conjugate of Formula I, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof, and a nucleic acid, wherein the nucleic acid is preferably a conjugate of the formula I: [ka]

[0014] Formula I (In the formula, [ka] is a single or double bond, n is an integer of 1 to 1500, preferably an integer of 2 to 1500, m is an integer of 2 to 200, preferably an integer of 1 to 200, and more preferably an integer of 2 to 100; R 1 is the starting residue, preferably R 1 is -H or -CH3, R 2 are independently -H or an organic residue, and the -(NR 2 -CH2-CH2) n -The R in question 2 at least 80%, preferably at least 90%, of are H; Ring A is one or more R A1 R is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl optionally substituted at any position by A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo, or halogen, or two R A1 are combined with the atoms to which they are attached to form one or more fused C6-C 10 can form an aryl, C-C heteroaryl, or C-C cycloalkyl ring, and each fused aryl, heteroaryl, or cycloalkyl can be joined by one or more R A2 and optionally substituted with R A2 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen -SO3H or -OSO3H; X 1 is a divalent covalent linking moiety, X 2 is a divalent covalent linking moiety, L is a targeting fragment, preferably the targeting fragment is capable of binding to a cell, more preferably the targeting fragment is capable of binding to a cell surface receptor), non-covalently bound to The nucleic acid is a pharmaceutically active nucleic acid, which is a nucleic acid that encodes a pharmaceutically active peptide or protein.

[0015] In another aspect, the present invention provides a composition comprising a polyplex, the polyplex comprising a conjugate of Formula I, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof, and a nucleic acid, the nucleic acid preferably comprising the conjugate: [ka]

[0016] Formula I (In the formula, [ka] is a single or double bond, n is an integer of 1 to 1500, preferably an integer of 2 to 1500, m is any integer from 1 to 200, preferably any integer from 2 to 200, more preferably any integer from 1 to 100, and even more preferably any integer from 2 to 100; R 1 is the starting residue, preferably R 1 is -H or -CH3, R 2 are independently -H or an organic residue, and the -(NR 2 -CH2-CH2) n -The R in the part 2 at least 80%, preferably 90%, of the Ring A may have one or more R A1 is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl optionally substituted with R A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo or halogen, or two R A1 are combined with the atoms to which they are attached to form one or more fused C6-C 10can form an aryl, C5-C6 heteroaryl, or C3-C6 cycloalkyl ring, and each fused aryl, heteroaryl, or cycloalkyl can be one or more R A 2 is optionally replaced by R A2 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen -SO3H or -OSO3H; X 1 is -(Y 1 ) p where p is an integer from 1 to 20, and Y 1 Each occurrence of represents a chemical bond, -CR 11 R 12 -, -C(O)-, -O-, -S-, -NR 13 -, an amino acid residue, a divalent phenyl moiety, a divalent heterocyclic moiety, and a divalent heteroaryl moiety, each divalent phenyl or heteroaryl being independently selected from one or more R 13 and each divalent heterocycle is optionally substituted with one or more R 14 and optionally substituted with R 11 , R 12 and R 13 is independently, at each occurrence, H or C1-C6 alkyl; R 14 is a linking moiety that is independently, at each occurrence, H, C1-C6 alkyl, or oxo; X 2 is the formula -(Y 2 ) q - (wherein q is an integer from 1 to 50, and Y 2 Each occurrence of represents a chemical bond, -CR 21 R 22 -, NR 23 independently selected from -, -O-, -S-, -C(O)-, an amino acid residue, a divalent phenyl moiety, a divalent heterocyclic moiety, and a divalent heteroaryl moiety, wherein each divalent phenyl and divalent heteroaryl is selected from one or more R 23 and each divalent heterocyclic moiety is optionally substituted with one or more R 24 and optionally substituted with R 21 , R 22 and R 23are each independently, at each occurrence, -H, -COH, or C1-C6 alkyl, and each C1-C6 alkyl may be selected from one or more of -OH, oxo, C6-C 10 optionally substituted aryl, or 5- to 8-membered heteroaryl; R 24 is a linking moiety that is independently, at each occurrence, —H, —COH, C1-C6 alkyl, or oxo; L is preferably a targeting fragment capable of binding to a cell, preferably the targeting fragment is non-covalently linked to a cell surface receptor; The nucleic acid is a pharmaceutically active nucleic acid, the pharmaceutically active nucleic acid is a nucleic acid encoding a pharmaceutically active peptide or protein, and more preferably the composition consists of the polyplex.

[0017] In another aspect, the invention provides a polyplex comprising a conjugate of Formula I, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof, and a nucleic acid, wherein the nucleic acid is preferably a conjugate of Formula I: [ka]

[0018] Formula I (In the formula, [ka] is a single or double bond, n is an integer of 1 to 1500, preferably an integer of 2 to 1500, m is any integer from 1 to 200, preferably any integer from 2 to 200, more preferably any integer from 1 to 100, and even more preferably any integer from 2 to 100; R 1 is the starting residue, preferably R 1 is -H or -CH3, R2 are independently -H or an organic residue, and the -(NR 2 -CH2-CH2) n -The R in the part 2 at least 80%, preferably 90%, of the Ring A may have one or more R A1 is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl optionally substituted with R A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo or halogen, or two R A1 are combined with the atoms to which they are attached to form one or more fused C6-C 10 can form an aryl, C-C heteroaryl, or C-C cycloalkyl ring, and each fused aryl, heteroaryl, or cycloalkyl can be joined by one or more R A2 is optionally replaced by R A2 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen -SO3H or -OSO3H; X 1 is -(Y 1 ) p where p is an integer from 1 to 20, and Y 1 Each occurrence of represents a chemical bond, -CR 11 R 12 -, -C(O)-, -O-, -S-, -NR 13 -, an amino acid residue, a divalent phenyl moiety, a divalent heterocyclic moiety, and a divalent heteroaryl moiety, each divalent phenyl or heteroaryl being independently selected from one or more R 13 and each divalent heterocycle is optionally substituted with one or more R 14 and optionally substituted with R 11 , R 12 and R 13 is independently, at each occurrence, H or C1-C6 alkyl; R 14 is a linking moiety that is independently, at each occurrence, H, C1-C6 alkyl, or oxo; X2 is the formula -(Y 2 ) q - (wherein q is an integer from 1 to 50, and Y 2 Each occurrence of represents a chemical bond, -CR 21 R 22 -, NR 23 independently selected from -, -O-, -S-, -C(O)-, an amino acid residue, a divalent phenyl moiety, a divalent heterocyclic moiety, and a divalent heteroaryl moiety, wherein each divalent phenyl and divalent heteroaryl is selected from one or more R 23 and each divalent heterocyclic moiety is optionally substituted with one or more R 24 and optionally substituted with R 21 , R 22 and R 23 are each independently, at each occurrence, -H, -COH, or C1-C6 alkyl, and each C1-C6 alkyl may be selected from one or more of -OH, oxo, C6-C 10 optionally substituted aryl, or 5- to 8-membered heteroaryl; R 24 is a linking moiety that is independently, at each occurrence, —H, —COH, C1-C6 alkyl, or oxo; L is preferably a targeting fragment capable of binding to a cell, preferably the targeting fragment is non-covalently linked to a cell surface receptor; The nucleic acid is a pharmaceutically active nucleic acid, which is a nucleic acid that encodes a pharmaceutically active peptide or protein.

[0019] In a preferred embodiment, the nucleic acid is RNA. In another preferred embodiment, the nucleic acid is single-stranded RNA (ssRNA). In a further preferred embodiment, the ssRNA is messenger RNA (mRNA).

[0020] In another preferred embodiment, the nucleic acid is DNA. In a further preferred embodiment, the DNA is plasmid DNA.

[0021] In one aspect, the invention provides a pharmaceutical composition comprising a composition, the composition comprising a polyplex, the polyplex comprising a triconjugate, preferably the conjugate of Formula I* or Formula I, and a nucleic acid, the nucleic acid preferably non-covalently attached to the conjugate as described herein, the nucleic acid being a pharmaceutically active nucleic acid, the pharmaceutically active nucleic acid being a nucleic acid encoding a pharmaceutically active peptide or protein, and pharmaceutically acceptable salts thereof.

[0022] In one aspect, the present invention provides a composition or polyplex as described herein, or a pharmaceutical composition comprising said composition or polyplex as described herein, for use in the treatment of a disease or disorder, preferably cancer.

[0023] In one aspect, the invention provides the use of a composition or polyplex as described herein, or a pharmaceutical composition comprising such a composition or polyplex as described herein, for use in the manufacture of a medicament for the treatment of a disease or disorder, such as cancer.

[0024] In another aspect, the present invention provides a method of treating a disease or disorder, such as cancer, in a subject in need thereof, comprising administering to the subject an effective amount of a composition or polyplex described herein, or a pharmaceutical composition comprising such a composition or polyplex described herein.

[0025] The compositions and polyplexes of the invention comprising the linear, non-random LPEI-PEG diconjugates described herein, and thus triconjugates having targeting fragments linked to the linear, non-random LPEI-PEG diconjugates, not only ensure a consistent and predictable ratio of LPEI to PEG fragments, but also typically and preferably ensure a structurally defined, linear conjugate of LPEI fragments to PEG fragments. Thus, they offer greater batch-to-batch consistency, ease of manufacture, and more predictable SAR compared to the branched LPEI-PEG diconjugates currently prepared using the random, uncontrolled synthetic strategies described above.

[0026] Even more advantageously and surprisingly, when the linear, non-random conjugates of the present invention described herein are combined with pharmaceutically active nucleic acids, such as mRNA or plasmid DNA (pDNA) encoding pharmaceutically active peptides or proteins (e.g., cytokines, interferons, or toxins), to form polyplexes and administer them to cells, the polyplexes surprisingly not only maintain but may even increase their biological activity compared to the respective polyplexes made using random, branched conjugates. Thus, despite the significant reduction in variability and number in the structure of the conjugates and, therefore, in the structure and number of possible biological activities, including targeting and presentation of their targeting fragments to the surface of target cells and subsequent uptake, and translation and secretion of the encoded pharmaceutically active protein, there is no loss of efficacy of the linear and polyplexes of the present invention described herein. Conversely, the compositions and polyplexes of the present invention may even increase their overall biological activity.

[0027] Further features and advantages of the present technology will be apparent to those skilled in the art upon reading the following detailed description of the invention, and further aspects and embodiments of the invention will become apparent as the description continues. [Brief explanation of the drawings]

[0028] [Figure 1] Figure 1 shows DLS backscattering plots taken in triplicate of LPEI-l-[N3:DBCO]-PEG36-DUPA:DT-A) polyplexes, measured at 0.1 mg / mL in 20 mM HEPES, pH 7.2, 5% glucose, 1.0 mL volume, and an N / P ratio of 4 to measure size distribution and zeta potential. The z-average diameter was 103.4 nm, and the polydispersity index (PDI) was 0.197. The zeta potential was 44.5 mV. [Figure 2A] Figure 2A is a plot of luminescence (AU) in Renca parental and Renca EGFR M1 H cells treated with LPEI-l-[N3:DBCO]PEG36-hEGF:[Fluc mRNA] compared to the control delivery vehicle Messenger MAX. After 24 h of treatment, luminescence was measured at N / P ratios of 4, 6, and 12, and with final concentrations of LPEI-l-[N3:DBCO]PEG36-hEGF:[Fluc mRNA] and Lipofectamine Messenger MAX from 0.125 to 1.0 μg / mL. [Figure 2B] Figure 2B is a plot of luminescence (AU) in Renca parental cells and Renca EGFR M1 H cells treated with LPEI-l-[N3:DBCO]PEG36-hEGF:[Fluc mRNA] compared to the control delivery vehicle, jetPEI. After 24 h of treatment, luminescence was measured at N / P ratios of 4, 6, and 12, and at final concentrations of LPEI-l-[N3:DBCO]PEG36-hEGF:[Fluc mRNA] and jetPEI of 0.125–1.0 μg / mL. [Figure 2C]Figure 2C is a plot of the luminescence ratio (AU) between Renca parental cells and Renca EGFR M1 H cells treated with LPEI-l-[N3:DBCO]PEG36-hEGF:[Fluc mRNA] using Messenger MAX as a comparative delivery vehicle. After 24 h of treatment, luminescence was measured at N / P ratios of 4, 6, and 12, as well as with LPEI-l-[N3:DBCO]PEG36-hEGF:[Fluc mRNA] and Lipofectamine Messenger MAX at final concentrations of 0.125–1.0 μg / mL. The ratio was calculated by dividing the luminescence signal from Renca EGFR M1 H cells by the luminescence signal from Renca parental cells. [Figure 2D] Figure 2D is a plot of the luminescence ratio (AU) between Renca parental cells and Renca EGFR M1 H cells treated with LPEI-l-[N3:DBCO]PEG36-hEGF:[Fluc mRNA] using jetPEI as a comparative delivery vehicle. After 24 h of treatment, luminescence was measured at N / P ratios of 4, 6, and 12 and final concentrations of LPEI-l-[N3:DBCO]PEG36-hEGF:[Fluc mRNA] and jetPEI of 0.125–1.0 μg / mL. The ratio was calculated by dividing the average luminescence signal from Renca EGFR M1 H cells by the average luminescence signal from Renca parental cells. [Figure 2E] Figure 2E is a plot of percent survival in Renca parental and Renca EGFR M1 H cells treated with LPEI-l-[N3:DBCO]PEG36-hEGF:[Fluc mRNA] compared to the control delivery vehicle Messenger MAX. After 24 hours of treatment, percent survival was measured at N / P ratios of 4, 6, and 12, and at final concentrations of 0.125–1.0 μg / mL of LPEI-l-[N3:DBCO]PEG36-hEGF:[Fluc mRNA] and Messenger MAX. [Figure 3A]Figure 3A shows the relative luminescence (AU) in Renca parental cells and Renca EGFR M1 H cells treated with LPEI-l-[N3:DBCO]PEG36-hEGF:[Fluc mRNA] 6 hours after treatment at an N / P ratio of 4. [Figure 3B] Figure 3B shows the relative luminescence (AU) in Renca parental cells and Renca EGFR M1 H cells treated with LPEI-l-[N3:DBCO]PEG36-hEGF:[Fluc mRNA] 6 hours after treatment at an N / P ratio of 6. [Figure 3C] Figure 3C shows the relative luminescence (AU) in Renca parental cells and Renca EGFR M1 H cells treated with LPEI-l-[N3:DBCO]PEG36-hEGF:[Fluc mRNA] 22 hours after treatment at an N / P ratio of 4. [Figure 3D] Figure 3D shows the relative luminescence (AU) in Renca parental cells and Renca EGFR M1 H cells treated with LPEI-l-[N3:DBCO]PEG36-hEGF:[Fluc mRNA] 22 hours after treatment at an N / P ratio of 6. [Figure 3E] Figure 3E shows the luminescence (AU) from different densities (500–20,000 cells / well) of B16F10-hEGFR cells transfected with LPEI-l-[N3:DBCO]PEG36-hEGF:[Fluc mRNA] at an N / P ratio of 6 for 24 h. [Figure 4] Figure 4 shows luminescence normalized to viability in human prostate cell lines with differential cell surface expression of PSMA: PSMA-high-expressing LNCaP cells and PSMA-low-expressing DU145 cells, after transfection with PSMA-targeting polyplexes containing mRNA encoding luciferase. The X-axis shows the concentration of mRNA in the polyplexes (0.25, 0.5, and 1.0 μg / mL). The Y-axis shows luminescence normalized to viability in arbitrary units (AU). Selective transfection of PSMA-overexpressing cells with Luc mRNA and selective expression of luciferase were demonstrated. [Figure 5]Figure 5 shows luminescence from cancer cells with differential cell surface expression of the human folate receptor (FR) (MCF7: low folate receptor expression; SKOV3: high folate receptor expression) after treatment with FR-targeting polyplexes containing mRNA encoding Renilla luciferase (R-Luc). The X-axis indicates the concentration of mRNA in the polyplexes (0.125, 0.25, 0.5, and 1.0 μg / mL). The Y-axis indicates luminescence in arbitrary units (RLU). Standard deviations from quadruplicate samples are shown. Selective expression of Renilla Luc in folate receptor-overexpressing cells is demonstrated. [Figure 6] Figure 6 shows the levels of secreted human IL-2 normalized to survival from two cell lines with differential human EGFR (hEGFR) expression: hEGFR-high-expressing RencaEGFR M1 H cells and human EGFR-negative Renca (parent) cells after transfection with EGFR-targeting polyplexes containing hIL-2 mRNA. The X-axis shows the concentration of mRNA in the polyplexes (0.125, 0.25, 0.5, and 1.0 μg / mL). The Y-axis shows the levels of secreted human IL-2 normalized to survival, in arbitrary units (AU). Selective expression and secretion of human IL-2 from EGFR-high-expressing cells is demonstrated. [Figure 7] Figure 7 shows the levels of secreted human IL-2 from two cell lines with differential PSMA expression: PSMA-high expressing LNCaP cells and PSMA-low expressing DU145 cells after transfection with PSMA-targeting polyplexes containing IL-2 mRNA, demonstrating selective expression of human IL-2 from PSMA-overexpressing cells. [Figure 8] Figure 8 shows the levels of secreted human IFNβ from two cell lines with differential PSMA expression after transfection with PSMA-targeting polyplexes containing hIFNβ mRNA: high-PSMA-expressing LNCaP cells and low-PSMA-expressing DU145 cells. Selective expression of human IFNβ from high-PSMA-expressing cells is demonstrated. [Figure 9]Figure 9 shows the levels of secreted human IFNγ (hIFNγ) from RencaEGFR M1 H (high expression of human EGFR) and Renca (parental, negative, no expression of human EGFR) cell lines after transfection with EGFR-targeting polyplexes containing hIFNγ mRNA. Selective transfection of EGFR-overexpressing cells with hIFNγ mRNA and selective expression and secretion of hIFNγ protein are demonstrated. [Figure 10] Figure 10 shows the levels of human EPO secreted by cancer cells with differential expression of the human folate receptor (FR) (SKOV3: high FR expression; MCF7: low FR expression) after treatment with FR-targeting polyplexes containing mRNA encoding human EPO. The X-axis shows the concentration of mRNA in the polyplexes (0.125, 0.25, 0.5, and 1.0 μg / mL). The Y-axis shows the concentration of hEPO released into the medium (mIU / mL). Standard deviations from quadruplicate samples are shown. Selective expression of hEPO in folate receptor-overexpressing cells is demonstrated. [Figure 11] Figure 11 shows the inhibition of protein biosynthesis by DT-A protein in two cell lines with differential PSMA expression: high-PSMA-expressing LNCaP cells and low-PSMA-expressing DU145 cells after transfection with the PSMA-targeting polyplex LPEI-l-[N3:DBCO]PEG36-DUPA containing mRNA DT-A. Western blot analysis using anti-puromycin antibody as a probe was used to detect the inhibition of protein biosynthesis. GAPDH was used as a loading control. Selective inhibition of protein biosynthesis in PSMA-overexpressing cells is demonstrated. [Figure 12A] Figure 12A shows cell surface expression of human EGFR in various cell lines: RencaEGFR M1 H, WI-38, and MCF-7 cells. The data shown in Figures 12A and 12B are from two separate experiments using different flow cytometers. [Figure 12B]Figure 12B shows cell surface expression of human EGFR in various cell lines: WI-38, U87MG, and MCF-7 cells. The data shown in Figures 12A and 12B are from two separate experiments using different flow cytometers. [Figure 12C] Figure 12C shows luminescence levels normalized to cell viability from high EGFR-expressing RencaEGFR M1 H cells and low EGFR-expressing MCF7 cells after transfection with EGFR-targeting polyplexes containing LPEI-l-[N3:DBCO]PEG36-hEGF and a luciferase-encoding plasmid formulated at an N / P ratio of 6. Selective expression and activity of luciferase in EGFR-overexpressing cells is demonstrated. [Figure 12D] Figure 12D shows the levels of luminescence normalized to cell viability in additional cell lines: rapidly proliferating cancerous U87MG cells, which express moderate levels of EGFR; slowly proliferating noncancerous WI38 cells, which also express moderate levels of EGFR; and slowly proliferating noncancerous HUVEC cells, which express minimal or no EGFR. These cells were transfected with an EGFR-targeting polyplex containing LPEI-l-[N3:DBCO]PEG36-hEGF and a luciferase-encoding plasmid (N / P ratio 6) in the same experiment as the cells shown in Figure 12C. Selective expression of luciferase in rapidly proliferating cancerous cells expressing moderate levels of EGFR is demonstrated. [Figure 13A] Figure 13A shows the luminescence levels in two cell lines with differential human EGFR expression, i.e., high EGFR-expressing RencaEGFR M1 H cells and human EGFR-negative Renca (parental) cells, after transfection with a linear EGFR-targeting polyplex of the invention comprising LPEI-l-[N3:DBCO]PEG36-hEGF produced at an N / P ratio of 3 and a plasmid encoding luciferase (pGreenFire1-CMV). Selective expression of luciferase in EGFR-overexpressing cells is demonstrated. [Figure 13B]Figure 13B shows the luminescence levels in two cell lines with differential human EGFR expression, i.e., high EGFR-expressing RencaEGFR M1 H cells and human EGFR-negative Renca (parental) cells, after transfection with a linear EGFR-targeting polyplex of the invention comprising LPEI-l-[N3:DBCO]PEG36-hEGF produced at an N / P ratio of 4 and a plasmid encoding luciferase (pGreenFire1-CMV). Selective expression of luciferase in EGFR-overexpressing cells is demonstrated. [Figure 13C] Figure 13C shows selective luminescence from B16F10-hEGFR cells. B16F10-hEGFR and B16F10 parental cells were treated with LPEI-l-[N3:DBCO]PEG36-hEGF:[pSZL] at N / P ratios of 3 and 6. Luminescence and viability were measured after 6 days. Data are presented as relative luminescence normalized to viability. [Figure 14] Figure 14 shows luminescence from human prostate cell lines with differential cell surface expression of PSMA: high-PSMA-expressing LNCaP cells and low-PSMA-expressing DU145 cells. Cells were treated with PSMA-targeting polyplexes containing LPEI-l-[N3:DBCO]PEG36-DUPA and plasmid DNA encoding luciferase. The X-axis indicates the concentration of pGreenFire-CMV in the polyplexes (0.25, 0.5, and 1.0 μg / mL). The Y-axis indicates luminescence in arbitrary units (AU). The mean and standard deviation from triplicate samples are shown. Selective expression of luciferase after transfection of PSMA-overexpressing cells with plasmid DNA encoding luciferase (pGreenFire-CMV) is demonstrated. [Figure 15A]Figure 15A shows the levels of secreted human IL-2 (hIL-2) from two cell lines with differential human EGFR expression: high EGFR-expressing RencaEGFR M1 H cells and human EGFR-negative parental Renca cells, after transfection with an EGFR-targeting polyplex containing LPEI-l-[N3:DBCO]PEG36-hEGF and a plasmid encoding hIL-2. Selective expression of hIL-2 from EGFR-overexpressing cells is demonstrated. [Figure 15B] Figure 15B shows the levels of secreted human IL2 after transfection of a small number of high EFGR-expressing RencaEGFR M1 H cells (600 cells) with an EGFR-targeting polyplex containing LPEI-l-[N3:DBCO]PEG36-hEGF and a plasmid encoding hIL2 at the indicated concentrations of plasmid (0.125 and 0.25 μg / ml). Polyplexes were formulated at an N / P ratio of 6, and IL2 secretion was detected after 2, 3, and 4 days. [Figure 16] Figure 16 shows the levels of secreted human IL-2 normalized to cell viability in cell lines with differential PSMA expression: high-expressing LNCaP and C4-2 cells, and low-expressing DU145 cells, after transfection with PSMA-targeting polyplexes containing LPEI-l-[N3:DBCO]PEG36-DUPA and a plasmid encoding an IL-2 protein. The X-axis shows the concentration of hIL-2 plasmid DNA (0.25, 0.5, and 1.0 μg / mL) in the polyplex. The Y-axis shows the concentration of secreted IL-2 normalized to cell viability in arbitrary units (AU). Selective expression / secretion of human IL-2 after transfection of PSMA-overexpressing cells with plasmid DNA encoding hIL-2 is demonstrated. [Figure 17A]Figure 17A shows the levels of human IFNβ secreted from RencaEGFR M1 H cancer cells, which have high human EGFR expression, after transfection with EGFR-targeting polyplexes containing pCMV-hIFNβ at an N / P ratio of 3. The X-axis shows the concentration of pCMV-hIFNβ plasmid DNA (0.25, 0.5, 1.0, or 2.0 μg / mL) in the polyplexes. The Y-axis shows the concentration of secreted IFNβ protein (pg / mL), shown as the mean with standard deviation from triplicate samples. Secretion of human IFNβ from EGFR-overexpressing cancer cells was demonstrated for the delivery vectors tested, with a significant advantage of the linear triconjugate vector LPEI-l-[N3:DBCO]-PEG36-hEGF compared to random delivery vectors. [Figure 17B] Figure 17B shows the levels of human IFNβ secreted from RencaEGFR M1 H cancer cells, which have high human EGFR expression, after transfection with EGFR-targeting polyplexes containing pCMV-hIFNβ at an N / P ratio of 4. The X-axis shows the concentration of pCMV-hIFNβ plasmid DNA (0.25, 0.5, 1.0, 2.0 μg / mL) in the polyplexes. The Y-axis shows the concentration of secreted IFNβ protein (pg / mL), shown as the mean with standard deviation from triplicate samples. Secretion of human IFNβ from EGFR-overexpressing cancer cells was demonstrated for the delivery vectors tested, with a significant advantage of the linear triconjugate vector LPEI-l-[N3:DBCO]-PEG36-hEGF over random delivery vectors. DETAILED DESCRIPTION OF THE INVENTION

[0029] Detailed Description of the Invention Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The embodiments, preferred embodiments, and highly preferred embodiments described and disclosed herein should apply to all aspects and other embodiments, preferred embodiments, and highly preferred embodiments, whether or not specifically mentioned again.

[0030] As described herein and outlined below, the present invention provides polyplexes of (i) a nucleic acid encoding a peptide or protein of interest, preferably a pharmaceutically active peptide or protein such as a cytokine, interferon, or toxin, and (ii) a targeting linear conjugate of LPEI and PEG. The conjugate preferably comprises an LPEI fragment, a PEG fragment, and a targeting fragment. In preferred embodiments, the LPEI fragment and the PEG fragment are coupled separately in an end-to-end manner. In some preferred embodiments, the LPEI fragment and the PEG fragment are coupled via covalent attachment of an azide to an alkene or alkyne to form a 1,2,3-triazole or 4,5-dihydro-1H-[1,2,3]triazole.

[0031] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0032] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0033] The term "and / or" is used in this disclosure to mean either "and" or "or," unless otherwise indicated.

[0034] As used herein, the term "about" shall mean ±10%. For example, about 50% shall mean 45% to 55%. Preferably, as used herein, the term "about" shall mean ±5%. For example, about 50% shall mean 47.5% to 52.5%.

[0035] As used herein, the phrase "number X to number Y" includes number X and number Y. For example, the phrase "0.01 μmol to 50 μmol" refers to 0.01 μmol and 50 μmol, as well as values ​​therebetween. The same applies to the phrase "about number X to about number Y."

[0036] The term "optionally substituted" is understood to mean that a given chemical moiety (e.g., an alkyl group) can, but need not, be attached to other substituents (e.g., heteroatoms). For example, an optionally substituted alkyl group can be a fully saturated alkyl chain (i.e., pure hydrocarbon). Alternatively, the same optionally substituted alkyl group can have substituents other than hydrogen. For example, it can be attached at any point along the chain to a halogen atom, an alkoxy group, or any other substituent described herein. Thus, the term "optionally substituted" means that a given chemical moiety has the potential to contain other functional groups, but does not necessarily have any additional functional groups.

[0037] "Optionally replaced" is understood to refer to a situation in which a carbon atom of a methylene group (i.e., -CH-) can be replaced by a heteroatom (e.g., -NH-, -O-), but need not be. For example, a C alkylene (i.e., propylene) group in which one of the methylene groups is "optionally replaced" can have the structure -CH-O-CH- or -O-CH-CH-. Those skilled in the art will understand that a methylene group cannot be replaced if such a replacement would result in an unstable chemical moiety. For example, those skilled in the art will understand that four methylene groups cannot be replaced by oxygen atoms at the same time. Thus, in some preferred embodiments, when one methylene group of an alkylene fragment is replaced with a heteroatom, one or both of the adjacent carbon atoms are not replaced with a heteroatom.

[0038] The term "aryl" refers to cyclic aromatic hydrocarbon groups having one to two aromatic rings, including monocyclic or bicyclic groups such as phenyl, biphenyl, or naphthyl. 10 An aryl group contains 6 to 10 carbon atoms. When containing two aromatic rings (such as bicyclic rings), the aromatic rings of the aryl group may be joined at a single point (e.g., biphenyl) or fused (e.g., naphthyl). The aryl group may be optionally substituted at any point of attachment with one or more substituents, for example, 1 to 5 substituents. The substituents may themselves be optionally substituted. Furthermore, when containing two fused rings, the aryl group defined herein may have an unsaturated or partially saturated ring fused to a fully saturated ring. Exemplary ring systems of these aryl groups include indanyl, indenyl, tetrahydronaphthalenyl, and tetrahydrobenzoannulenyl. In some preferred embodiments, the aryl group is a phenyl group.

[0039] Unless otherwise specifically defined, "heteroaryl" means a monovalent monocyclic or polycyclic aromatic ring of 5 to 24 ring atoms containing one or more ring heteroatoms selected from N, S, P, or O, with the remaining ring atoms being C. A 5-10 membered heteroaryl group contains 5 to 10 atoms. Heteroaryl, as defined herein, also refers to a bicyclic heteroaromatic group in which the heteroatoms are selected from N, S, P, or O. The aromatic groups are optionally substituted independently with one or more substituents described herein. Examples include, but are not limited to, furyl, thienyl, pyrrolyl, pyridyl, pyrazolyl, pyrimidinyl, imidazolyl, isoxazolyl, oxazolyl, oxadiazolyl, pyrazinyl, indolyl, thiophen-2-yl, quinolyl, benzopyranyl, isothiazolyl, thiazolyl, thiadiazole, indazole, benzimidazolyl, thieno[3,2-b]thiophene, triazolyl, triazolyl, thiazolyl, thiadiazole, indazole, benzimidazolyl, thieno[3,2-b]thiophene, triazolyl, thiazolyl, thiazolyl, thiadiazole, thiazolyl ... indyl, imidazo[1,2-b]pyrazolyl, furo[2,3-c]pyridinyl, imidazo[1,2-a]pyridinyl, indazolyl, pyrrolo[2,3-c]pyridinyl, pyrrolo[3,2-c]pyridinyl, pyrazolo[3,4-c]pyridinyl, thieno[3,2-c]pyridinyl, thieno[2,3-c]pyridinyl, thieno[2,3-b]pyridinyl, benzothiazolyl, indolyl, indolinyl, indolinonyl, dihydrobenzyl benzothiophenyl, dihydrobenzofuranyl, benzofuran, chromanyl, thiochromanyl, tetrahydroquinolinyl, dihydrobenzothiazine, dihydrobenzoxanyl, quinolinyl, isoquinolinyl, 1,6-naphthyridinyl, benzo[de]isoquinolinyl, pyrido[4,3-b][1,6]naphthyridinyl, thieno[2,3-b]pyrazinyl, quinazolinyl, tetrazolo[1,5-a]pyridinyl, [1,2,4]thiazolinyl riazolo[4,3-a]pyridinyl, isoindolyl, pyrrolo[2,3-b]pyridinyl, pyrrolo[3,4-b]pyridinyl, pyrrolo[3,2-b]pyridinyl, imidazo[5,4-b]pyridinyl, pyrrolo[1,2-a]pyrimidinyl, tetrahydropyrrolo[1,2-a]pyrimidinyl, 3,4-dihydro-2H-1λ2-pyrrolo[2,1-b]pyrimidine, dibenzo[b,d]thiophene, pyridin-2-one, furo[3,2-c]pyridinyl, furo[2,3-c]pyridinyl, 1H-pyrido[3,4-b][1,4]thiazinyl, benzoxazolyl, benzisoxazolyl, furo[2,3-b]pyridinyl, benzothiophenyl, 1,5-naphthyridinyl, furo[3,2-b]pyridine, [1,2,4]triazolo[1,5-a]pyridinyl, benzo[1,2,3]triazolyl, imidazo[1,2-a]pyrimidinyl, [1,2,4]triazolo[4,3-b]pyridazinyl, benzo[ [c][1,2,5]thiadiazolyl, benzo[c][1,2,5]oxadiazole, 1,3-dihydro-2H-benzo[d]imidazol-2-one, 3,4-dihydro-2H-pyrazolo[1,5-b][1,2]oxazinyl, 4,5,6,7-tetrahydropyrazolo[1,5-a]pyridinyl, thiazolo[5,4-d]thiazolyl, imidazo[2,1-b][1,3,4]thiadiazolyl, thieno[2,3-b]pyrrolyl, 3H-indolyl, and derivatives thereof. Furthermore, when containing two fused rings, heteroaryl groups as defined herein can have an unsaturated or partially saturated ring fused to a fully saturated ring. Exemplary ring systems of these heteroaryl groups include indolinyl, indolinonyl, dihydrobenzothiophenyl, dihydrobenzofuran, chromanyl, thiochromanyl, tetrahydroquinolinyl, dihydrobenzothiazine, 3,4-dihydro-1H-isoquinolinyl, 2,3-dihydrobenzofuran, indolinyl, indolyl, and dihydrobenzoxanyl.

[0040] The term "alkyl" refers to a straight-chain or branched-chain saturated hydrocarbon. A C1-C6 alkyl group contains 1 to 6 carbon atoms. Examples of -C1-C6 alkyl groups include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, and neopentyl.

[0041] The term "alkylene" refers to a straight-chain or branched, saturated divalent hydrocarbon fragment. A C0-C6 alkyl group contains from 0 to 6 carbon atoms. Examples of C0-C6 alkylene groups include, but are not limited to, methylene, ethylene, propylene, butylene, pentylene, isopropylene, isobutylene, sec-butylene, tert-butylene, isopentylene, and neopentylene.

[0042] As used herein, the term "C1-C6 alkoxy" refers to a substituted hydroxyl of the formula (-OR'), where R' is an optionally substituted C1-C6 alkyl, as defined herein, and the oxygen moiety is directly attached to the parent molecule; thus, the term "C1-C6 alkoxy" as used herein refers to straight- or branched-chain C1-C6 alkoxy, which can be, for example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, straight- or branched-chain pentoxy, straight- or branched-chain hexyloxy. Preferred alkoxy is C1-C4 alkoxy, and C1-C3 alkoxy.

[0043] The term "cycloalkyl" means a monocyclic or polycyclic saturated carbocyclic ring containing 3 to 18 carbon atoms. A C3-C8 cycloalkyl contains 3 to 8 carbon atoms. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptanyl, cyclooctanyl, norboranyl, norborenyl, bicyclo[2.2.2]octanyl, or bicyclo[2.2.2]octenyl. A C3-C8 cycloalkyl is a cycloalkyl group containing 3 to 8 carbon atoms.

[0044] The term "cycloalkenyl" refers to a monocyclic non-aromatic unsaturated carbocyclic ring containing 5 to 18 carbon atoms. Examples of cycloalkenyl groups include, but are not limited to, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl, and norborenyl. A C5-C8 cycloalkenyl is a cycloalkenyl group containing 5 to 8 carbon atoms.

[0045] The terms "heterocyclyl" or "heterocycloalkyl" or "heterocycle" refer to monocyclic or polycyclic 3- to 24-membered rings containing heteroatoms taken from carbon and oxygen, nitrogen, or sulfur, and lacking delocalized π-electrons (aromaticity) shared between ring carbons or heteroatoms. 3- to 10-membered heterocycloalkyl groups contain 3 to 10 atoms. Heterocyclyl rings include, but are not limited to, oxetanyl, azetazinyl, tetrahydrofuranyl, pyrrolidinyl, oxazolinyl, oxazolidinyl, thiazolinyl, thiazolidinyl, pyranyl, thiopyranyl, tetrahydropyranyl, dioxalinyl, piperidinyl, morpholinyl, thiomorpholinyl, thiomorpholinyl S-oxide, thiomorpholinyl S-dioxide, piperazinyl, azepinyl, oxepinyl, diazepinyl, tropanyl, and homotropanyl.

[0046] The term "heterocycloalkenyl" refers to a monocyclic or polycyclic 3- to 24-membered ring containing carbon and heteroatoms derived from oxygen, nitrogen, or sulfur, where there are no delocalized π-electrons (aromaticity) shared between ring carbons or heteroatoms, but at least one unsaturated element is present within the ring. 3- to 10-membered heterocycloalkenyl groups contain 3 to 10 atoms.

[0047] As used herein, the term "halo" or "halogen" means fluoro (F), chloro (Cl), bromo (Br) or iodo (I).

[0048] The term "carbonyl" refers to a functional group composed of a carbon atom double-bonded to an oxygen atom, which may be abbreviated herein as "oxo," C(O), or C=O.

[0049] As used herein, the term "polyplex" refers to a complex of a polymer and a nucleic acid, typically and preferably formed through electrostatic interactions. In particular, as used herein, the term "polyplex" refers to a complex of a conjugate described herein for the invention and a nucleic acid, such as single-stranded RNA, preferably mRNA, or DNA, preferably plasmid DNA. The term "polyplex" also typically and preferably refers to a vector, particularly a polymeric non-viral triconjugate vector described herein for the invention, useful for carrying and delivering nucleic acids to desired target cells.

[0050] The term "overexpression" refers to increased expression of a gene or protein in a cell or on the cell surface compared to basal or normal expression. In a preferred embodiment, the targeting fragment is capable of binding to a cell that overexpresses a cell surface receptor. In one embodiment, cells that overexpress a cell surface receptor refer to an increased level of the cell surface receptor expressed in the cells of a particular tissue compared to the level of the cell surface receptor measured in normal healthy cells of the same tissue type under similar conditions. In one embodiment, cells that overexpress a cell surface receptor refer to an increased level of the cell surface receptor in a cell compared to the level in the same cell or a closely related non-malignant cell under normal physiological conditions.

[0051] As used herein, the term "polyanion" refers to a polymer, preferably a biopolymer, having two or more negatively charged sites. Typically and preferably, as used herein, the term "polyanion" refers to a polymer, preferably a biopolymer, composed of repeating units that contain residues that can carry a negative charge. In a further embodiment, the polyanion is a polymer, preferably a biopolymer, composed of repeating units that contain negatively charged residues. In another preferred embodiment, the polyanion is a nucleic acid, more preferably DNA, RNA, polyglutamic acid, or hyaluronic acid.

[0052] As used herein, the term "nucleic acid" includes deoxyribonucleic acid (DNA) and / or ribonucleic acid (RNA) or a combination thereof. In preferred embodiments, the term "nucleic acid" refers to deoxyribonucleic acid (DNA) and / or ribonucleic acid (RNA), and as used herein, refers to genomes, viruses, and recombinantly prepared and chemically synthesized molecules. Nucleic acids can be single-stranded or double-stranded and in the form of linear or covalently closed circular molecules, can include chemical derivatization of nucleic acids on the nucleotide base, sugar, or phosphate, and can include non-natural nucleotides and nucleotide analogs.

[0053] As used herein, the term "dispersity" (abbreviated as D) refers to the distribution of molar masses in a given polymer sample, such as the polymer fragments used herein for the conjugates and polyplexes of the invention. This is defined herein as D = (M w / M n ) where D is the dispersity and M w is the weight average molecular weight of the polymer sample or polymer fragment, and M n is the number average molecular weight of the polymer sample or polymer fragment.

[0054] As used herein, the term "weight average molecular weight" refers to the sum of the products of the weight fraction of a given molecule in a mixture and the molecular mass of each molecule in the mixture, and is typically and preferably represented by the symbol Mw.

[0055] As used herein, the term "number average molecular weight" refers to the total weight of a mixture divided by the number of molecules in the mixture, and is typically and preferably represented by the symbol Mn.

[0056] As used herein, the term "polydispersity index" (abbreviated as PDI) refers to the polydispersity index in dynamic light scattering measurements of polyplex nanoparticles, such as the polyplexes of the present invention. This index is a number calculated from a simple two-parameter fit to correlation data (cumulant analysis). The polydispersity index is dimensionless and is scaled so that values ​​below 0.05 are rarely observed except in highly monodisperse standards. Values ​​above 0.7 indicate a sample has a very broad size distribution and are likely unsuitable for dynamic light scattering (DLS) techniques. Various size distribution algorithms work with data that fall between these two extremes. The zeta-average diameter (z-average diameter) and polydispersity index of the polyplexes of the present invention are determined by dynamic light scattering (DLS) based on the assumption that the polyplexes are isotropic and spherical. The calculation of these parameters is defined and determined in accordance with the ISO standard document ISO 22412:2017.

[0057] The term "amino acid residue" refers to a divalent residue derived from an organic compound containing the functional groups amine (-NH) and carboxylic acid (-COOH), typically and preferably with a side chain specific to each amino acid. In a preferred embodiment of the present invention, the amino acid residue is a divalent residue derived from an organic compound containing the functional groups amine (-NH) and carboxylic acid (-COOH), the divalency being achieved by the amine and carboxylic acid functional groups, and thus the -NH- and -CO- moieties. In an alternative preferred embodiment of the present invention, the amino acid residue is a divalent residue derived from an organic compound containing the functional groups amine (-NH) and carboxylic acid (-COOH), the divalency being achieved by the amine or carboxylic acid functional group and any further functional groups present in the amino acid residue. As a preferred example and embodiment, an amino acid residue according to the present invention derived from cysteine ​​comprises the divalent structure -S-(CH)-CH(COOH)-NH-, the divalency being achieved by the amino functionality and the contained thiol functionality. As used herein, the term "amino acid residue" typically and preferably includes amino acid residues derived from natural or unnatural amino acids. Furthermore, as used herein, the term "amino acid residue" typically and preferably also includes amino acid residues derived from chemically synthesized non-natural amino acids, including α- (α-), β- (β-), γ- (γ-), or δ- (δ-) amino acids, as well as mixtures thereof in any ratio. Furthermore, as used herein, the term "amino acid residue" typically and preferably also includes amino acid residues derived from α-amino acids, any isomeric forms thereof, particularly their D- and L-stereoisomers (alternatively addressed by the (R) and (S) nomenclature), and mixtures thereof in any ratio, preferably a 1:1 racemic ratio. The terms "D-stereoisomer," "L-stereoisomer," "D-amino acid," or "L-amino acid" refer to the chiral alpha carbon of an amino acid. Thus, in a preferred embodiment, the amino acid residue is a divalent group of the structure -NH-CHR-C(O)-, where R is an amino acid side chain.Two or more consecutive amino acid residues preferably form a peptide (i.e., amide) bond at both the amine and carboxylic acid moieties of each amino acid residue. Di-, tri-, or polypeptide amino acid residues are typically (AA). a When described herein as a nucleotide, the sequence provided is presented from left to right in the NC direction. Thus, for example, Trp-Trp-Gly (AA) a shall refer to amino acid residues, with Trp corresponding to the N-terminus of the tripeptide with an -NH- valence and Gly corresponding to the C-terminus of the tripeptide with an -CO- valence.

[0058] As used herein, the terms "peptide," "polypeptide," and "protein" refer to a substance comprising about two or more consecutive amino acid residues linked together via peptide bonds. The terms "peptide," "polypeptide," and "protein" are used interchangeably herein to refer to polymers of amino acid residues of any length. In one embodiment, the term "protein" refers to large peptides, particularly peptides having at least about 151 amino acids, although in one embodiment, the term "peptide" refers to a substance containing about 2 or more, about 3 or more, about 8 or more, or about 20 or more, and up to about 50, up to about 100, or up to about 150.

[0059] As used herein, the term "disease-associated antigen" in its broadest sense refers to any antigen associated with a disease. A disease-associated antigen is a molecule containing an epitope that stimulates the host's immune system to generate a cellular antigen-specific immune response and / or a humoral antibody response against the disease. Thus, a disease-associated antigen or its epitope can be used for therapeutic purposes. A disease-associated antigen can be associated with infection by a microorganism, typically a microbial antigen, or can be associated with cancer, typically a tumor.

[0060] As used herein, the term "viral antigen" refers to any viral component that has antigenicity, i.e., is capable of eliciting an immune response in an individual. A viral antigen can be a viral ribonucleoprotein or envelope protein.

[0061] As used herein, the term "bacterial antigen" refers to any bacterial component that has antigenicity, i.e., is capable of eliciting an immune response in an individual. Bacterial antigens can be derived from the bacterial cell wall or cytoplasmic membrane.

[0062] As used herein, the term "epitope" refers to a portion or fragment of a molecule, such as an antigen, that is recognized by the immune system. For example, an epitope can be recognized by T cells, B cells, or antibodies. An epitope of an antigen preferably comprises a continuous or discontinuous portion of the protein and is preferably 5 to 100, preferably 5 to 50, more preferably 8 to 30, and most preferably 10 to 25 amino acids in length. For example, an epitope can be preferably 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. In one embodiment, an epitope is about 10 to about 25 amino acids in length. The term "epitope" includes T cell epitopes. As used herein, the term "T cell epitope" refers to a portion or fragment of a protein that is recognized by T cells when presented in the context of an MHC molecule. The term "major histocompatibility complex" and the abbreviation "MHC" refer to a complex of genes that includes MHC class I and MHC class II molecules and is present in all vertebrates. MHC proteins or molecules are important for signaling between lymphocytes and antigen-presenting or disease cells in the immune response; they bind to peptide epitopes and present them for recognition by T cell receptors on T cells. Proteins encoded by MHC are expressed on the surface of cells and present both self-antigens (peptide fragments from the cell itself) and non-self-antigens (e.g., fragments of invading microorganisms) to T cells.

[0063] The term "antibody" refers to any immunoglobulin, whether natural or wholly or partially synthetically produced, as well as derivatives and characteristic portions thereof. Antibodies can be monoclonal or polyclonal. Antibodies can be members of any immunoglobulin class, including any of the human classes: IgG, IgM, IgA, IgD, and IgE. As used herein, an antibody fragment (i.e., a characteristic portion of an antibody) refers to any derivative of an antibody that is less than full-length. Generally, an antibody fragment retains at least a significant portion of the full-length antibody's specific binding ability. Examples of antibody fragments include, but are not limited to, single- and double-chain fragments, Fab, Fab', F(ab')2, scFv, Fv, dsFv diabody, and Fd fragments. Antibody fragments can be produced by any means. For example, antibody fragments can be enzymatically or chemically produced by fragmentation of an intact antibody and / or recombinantly produced from a gene encoding a partial antibody sequence. Alternatively or additionally, antibody fragments can be wholly or partially synthetically produced. Antibody fragments may optionally include single-chain antibody fragments. Alternatively or additionally, antibody fragments may include multiple chains linked together, for example, by disulfide bonds. Antibody fragments may optionally include multimolecular complexes. Functional antibody fragments typically contain at least about 50 amino acids, more typically at least about 200 amino acids. In some embodiments, antibodies may include chimeric (e.g., "humanized") and single-chain (recombinant) antibodies. In some embodiments, antibodies may have reduced effector function and / or bispecific molecules. In some embodiments, antibodies may include fragments produced by Fab expression libraries. Single-chain Fvs (scFvs) are recombinant antibody fragments consisting only of a variable light chain (VL) and a variable heavy chain (VH) covalently connected to each other by a polypeptide linker. Either the VL or VH may contain an NH2-terminal domain. The polypeptide linker may be of variable length and composition, so long as the two variable domains are bridged without significant steric hindrance.Typically, the linker contains a stretch of primarily glycine and serine residues, with some glutamic acid or lysine residues interspersed for solubility. Diabodies are dimeric scFvs. Diabodies typically have shorter peptide linkers than most scFvs and often preferentially associate as dimers. Fv fragments are antibody fragments consisting of one VH and one VL domain held together by noncovalent interactions. As used herein, the term "dsFv" refers to an Fv with an engineered intermolecular disulfide bond to stabilize the VH-VL pair. F(ab')2 fragments are antibody fragments essentially equivalent to those obtained from immunoglobulins by digestion with the enzyme pepsin at pH 4.0-4.5. Fragments can be produced recombinantly. Fab' fragments are antibody fragments essentially equivalent to those obtained by reduction of one or more disulfide bridges linking the two heavy chain fragments in the F(ab')2 fragment. Fab' fragments can be produced recombinantly. 1. Fab fragments are antibody fragments essentially equivalent to those obtained by digesting immunoglobulins with enzymes (e.g., papain). Fab fragments can be recombinantly produced. The heavy chain segment of a Fab fragment is the Fd subfragment.

[0064] As used herein, the term "alpha end of a linear polyethyleneimine fragment" (α-end of an LPEI fragment) refers to the terminal end of an LPEI fragment at which initiation of polymerization occurs using an electrophilic initiator, as further described below for the term "initiating residue."

[0065] As used herein, the term "omega end of a linear polyethyleneimine fragment" (ω end of an LPEI fragment) refers to the terminal end of an LPEI fragment at which polymerization termination occurs using a nucleophile, such as an azide, thiol, and other nucleophiles described herein.

[0066] The term "organic residue" refers to any suitable organic group that can be bonded to a nitrogen atom embedded within an LPEI fragment. In a preferred embodiment, the organic residue is connected to the nitrogen atom via a carbonyl group to form an amide bond. Without wishing to be bound by theory, the organic residue is incorporated into the nitrogen atom of a poly(2-oxazoline) during ring-opening polymerization (see, e.g., Glassner et al., (2018), Poly(2-oxazoline)s: A comprehensive overview of polymer structures and their physical properties. Polym. Int, 67:32-45. https: / / doi.org / 10.1002 / pi.5457). Typically and preferably, the organic residue is cleaved from the poly(2-oxazoline) (i.e., the amide is typically cleaved) to yield the -(NH-CH-CH)- moiety embedded within the LPEI and LPEI fragment, and thus the conjugate of the present invention. However, if the cleavage reaction is not complete, a fraction of the organic residue will not be cleaved. Thus, in a preferred embodiment of the present invention, the R 1 -(NR 2 -CH2-CH2) n -R in part 2 at least 80%, preferably 90%, of the R of the conjugates of the invention comprising those of formula I* or I are H; 1 -(NR 2 -CH2-CH2) n -R in part 2 is at least 91%, more preferably 92%, more preferably 93%, more preferably 94%, more preferably 95%, more preferably 96%, more preferably 97%, more preferably 98%, and most preferably 99% of the

[0067] The term "initial residue" refers to the LPEI fragment and R 1 -(NR 2 -CH2-CH2) n- refers to a residue present in the - moiety, which is derived from any initiator, typically and preferably any electrophilic initiator, capable of initiating the polymerization of poly(2-oxazoline) from 2-oxazoline. As stated by Glassner et al., (2018), Poly(2-oxazoline)s: A comprehensive overview of polymer structures and their physical properties. Polym. Int, 67:32-45. https: / / doi.org / 10.1002 / pi.5457, "Different initiator systems can be used, including toluenesulfonic acid (TsOH), or alkylsulfonates such as methyl p-toluenesulfonate (MeOT), which are most frequently found in the literature; p-nitrobenzenesulfonate (nosylate) and trifluoromethanesulfonate (triflate); alkyl, benzyl, and acetyl halides; oxazolinium salts; and Lewis acids." Thus, in a preferred embodiment, R 1 is -H or -CH3, but those skilled in the art will recognize that R 1 is any other suitable residue, e.g., C where n is greater than 1. n Alkyl groups, typically C 1~6 It will be understood that these groups may also include, but are not limited to, alkyl groups, benzyl groups, or acetyl groups.

[0068] The present invention provides targeting polyplexes composed of (i) a nucleic acid, particularly a nucleic acid encoding a pharmaceutically active peptide or protein, such as a cytokine, interferon, or toxin, and (ii) a targeting conjugate comprising an LPEI and a PEG fragment connected by distinct linkages formed by a defined chemoselective reaction, rather than through random and uncontrolled attachment of electrophilic PEG fragments to multiple nucleophiles on the LPEI backbone fragment. The distinct linkages not only ensure a consistent and predictable ratio of LPEI to PEG fragments, but also ensure a defined linear conjugate instead of a random branched conjugate. Thus, the LPEI fragment is attached to a single PEG fragment in a linear, end-to-end manner. The chemoselective attachment of the LPEI fragment to the PEG fragment can be achieved using any suitable chemical precursor capable of forming a chemoselective bond. In a preferred embodiment, the chemoselective attachment of the LPEI fragment to the PEG fragment occurs via a [3+2] cycloaddition between an azide and an alkyne or alkene. Alternatively, the chemoselective attachment is via a thiol-ene reaction between a thiol and an alkene. When the chemoselective bond is between an azide and an alkyne or alkene, the resulting linkage is a 1,2,3-triazole (if an alkyne is coupled) or a 4,5-dihydro-1H-[1,2,3]triazole (if an alkene is coupled). When the chemoselective bond is between a thiol and an alkene, the resulting linkage is a thioether.

[0069] The conjugate further comprises a targeting fragment linked to the PEG fragment, which allows for targeting of a specific cell type and promoting uptake of the composition of the present invention and the pharmaceutically active nucleic acid in the specific cell type. Thus, a preferred embodiment comprises a targeting fragment, such as hEGF, DUPA, or folate, specifically linked to the LPEI-PEG diconjugate to target a corresponding receptor, such as hEGFR, PSMA, or folate receptor, on a specific cell type where the receptor is highly expressed or overexpressed, typically a cancer cell type.

[0070] Even more advantageously and surprisingly, the inventors have found that the resulting preferred conjugates and polyplexes according to the present invention, which have significantly reduced heterogeneity and therefore a significantly reduced number of potentially biologically active conjugates and polyplexes due to the defined chemoselective attachment of the LPEI fragment to the PEG fragment, not only form polyplexes of appropriate size, but also maintain or even increase their overall biological activity, such as highly selective, targeted delivery of a pharmaceutically active nucleic acid and subsequent efficient translation and secretion of the encoded pharmaceutically active protein. Thus, the compositions and polyplexes of the present invention not only selectively deliver a pharmaceutically active nucleic acid encoding a pharmaceutically active peptide or protein to target cells, particularly cancer cells, but also result in high expression and efficient protein translation and secretion of the encoded pharmaceutically active protein.

[0071] Thus, in one aspect, the invention provides a composition comprising a polyplex, the polyplex comprising a conjugate and a nucleic acid, the nucleic acid preferably non-covalently bound to the conjugate, the conjugate comprising a linear polyethyleneimine fragment comprising an alpha end and an omega end, the alpha end of the polyethyleneimine fragment being an initiating residue; a polyethylene glycol fragment comprising a first end and a second end, the omega end of the polyethyleneimine fragment being linked to a first terminal end of the polyethylene glycol fragment by a divalent covalent linking group -ZX 1 -ZX 1 - is not a single bond, -Z- is not an amide, and the second terminal end of the polyethylene glycol fragment is a divalent covalent linking moiety X 2wherein the nucleic acid is a nucleic acid encoding a peptide or protein of interest, preferably the nucleic acid is a pharmaceutically active nucleic acid, and the pharmaceutically active nucleic acid is a nucleic acid encoding a pharmaceutically active peptide or protein. In a preferred embodiment of this aspect, the composition consists of the polyplex. In a preferred embodiment, the linear polyethyleneimine fragment is a nucleic acid having the formula R 1 -(NR 2 -CH2-CH2) n -, and n is an integer of 1 to 1500. In a more preferred embodiment, the R 1 -(NR 2 -CH2-CH2) n The - moiety is a dispersed polymer moiety having about 115 to about 1150 repeating units n and a dispersity of about 5 or less, preferably about 280 to about 700 repeating units n and a dispersity of about 3 or less, more preferably about 350 to about 630 repeating units n and a dispersity of about 2 or less, and preferably R 1 is -H or -CH3.

[0072] In another aspect, the present invention provides a composition comprising a polyplex, the polyplex comprising a conjugate of formula I* or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof, and a nucleic acid, the nucleic acid preferably comprising the conjugate: R 1 -(NR 2 -CH2-CH2) n -ZX 1 -(O-CH2-CH2) m -X 2 -L(Formula I*) (In the ceremony n is an integer of 1 to 1500, preferably an integer of 2 to 1500; m is an integer of 1 to 200, preferably an integer of 2 to 200, more preferably an integer of 1 to 100, more preferably an integer of 2 to 100; R 1 is the starting residue, preferably R 1 is -H or -CH3, and R2 are independently —H or an organic residue, and the (NR 2 -CH2-CH2) n -The R in question 2 At least 80%, preferably 90%, of X are H; 1 and X 2 are independently a divalent covalent linking moiety; Z is a divalent covalent linking moiety, Z is not a single bond, Z is not -NHC(O)-; L is a targeting fragment, preferably the targeting fragment is capable of binding to a cell, more preferably the targeting fragment is capable of binding to a cell surface receptor; and the nucleic acid is a nucleic acid encoding a peptide or protein of interest; Preferably, the nucleic acid is a pharmaceutically active nucleic acid, and the pharmaceutically active nucleic acid is a nucleic acid encoding a pharmaceutically active peptide or protein. Preferably, the composition consists of the polyplex.

[0073] In another aspect, the present invention provides a polyplex comprising a conjugate of formula I* or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof, and a nucleic acid, wherein the nucleic acid is preferably a nucleic acid that is a conjugate of formula I* or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof, R 1 -(NR 2 -CH2-CH2) n -ZX 1 -(O-CH2-CH2) m -X 2 -L(Formula I*) (In the ceremony n is an integer of 1 to 1500, preferably an integer of 2 to 1500; m is an integer of 1 to 200, preferably an integer of 2 to 200, more preferably an integer of 1 to 100, more preferably an integer of 2 to 100; R 1 is the starting residue, preferably R 1 is -H or -CH3, and R 2 are independently -H or an organic residue, and the -(NR2 -CH2-CH2) n -The R in question 2 At least 80%, preferably 90%, of X are H; 1 and X 2 are independently a divalent covalent linking moiety; Z is a divalent covalent linking moiety; Z is not a single bond; Z is not -NHC(O)-; L is a targeting fragment, preferably the targeting fragment is capable of binding to a cell, more preferably the targeting fragment is capable of binding to a cell surface receptor); and the nucleic acid is a nucleic acid encoding a peptide or protein of interest, preferably the nucleic acid is a pharmaceutically active nucleic acid, and the pharmaceutically active nucleic acid is a nucleic acid encoding a pharmaceutically active peptide or protein.

[0074] In another aspect, the present invention provides a composition comprising a polyplex, the polyplex comprising a conjugate of Formula I, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof, and a nucleic acid, the nucleic acid preferably comprising the conjugate: [ka]

[0075] Formula I (In the formula, [ka] is a single or double bond, n is an integer of 1 to 1500, preferably an integer of 2 to 1500, m is an integer of 1 to 200, preferably an integer of 2 to 200, more preferably an integer of 1 to 100, and even more preferably an integer of 2 to 100; R 1 is the starting residue, preferably R 1 is -H or -CH3, R 2are independently -H or an organic residue, and the -(NR 2 -CH2-CH2) n -The R in question 2 at least 80%, preferably at least 90%, of are H; Ring A is one or more R A1 R is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl optionally substituted at any position by A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo, or halogen, or two R A1 are combined with the atoms to which they are attached to form one or more fused C6-C 10 can form an aryl, C-C heteroaryl, or C-C cycloalkyl ring, and each fused aryl, heteroaryl, or cycloalkyl can be joined by one or more R A2 and optionally substituted with R A2 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen -SO3H or -OSO3H; X 1 is a divalent covalent linking moiety, X 2 is a divalent covalent linking moiety, L is a targeting fragment, preferably the targeting fragment is capable of binding to a cell, more preferably the targeting fragment is capable of binding to a cell surface receptor), non-covalently bound to The nucleic acid is a nucleic acid that encodes a peptide or protein of interest, preferably the nucleic acid is a pharmaceutically active nucleic acid, the pharmaceutically active nucleic acid being a nucleic acid that encodes a pharmaceutically active peptide or protein.

[0076] As described herein, the depiction of Formula I above includes the fragment R 1 (NR 2 CH2CH2) n Two different regioisomeric bonds, i.e., [ka] and [ka] represents wherein the wavy line represents a chemical bond to ring A. Thus, Formula I as depicted herein includes two regioisomeric embodiments, namely, fragment R 1 (NR 2 CH2CH2) n is attached to the upper nitrogen atom of the above structure or to the lower nitrogen atom of the above structure, but not to the middle nitrogen atom. Formula I, depicted above, encompasses embodiments in which [ka] is used interchangeably herein with the equivalent depiction of Formula I, which includes:

[0077] In another aspect, the present invention provides a polyplex comprising a conjugate of Formula I, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof, and a nucleic acid, wherein the nucleic acid is preferably a polyplex comprising the conjugate: [ka]

[0078] Formula I (In the formula, [ka] is a single or double bond, n is an integer of 1 to 1500, preferably an integer of 2 to 1500, m is an integer of 2 to 200, preferably an integer of 1 to 200, and more preferably an integer of 2 to 100; R 1 is the starting residue, preferably R 1is -H or -CH3, R 2 are independently -H or an organic residue, and the -(NR 2 -CH2-CH2) n -The R in question 2 at least 80%, preferably at least 90%, of are H; Ring A is one or more R A1 R is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl optionally substituted at any position by A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo, or halogen, or two R A1 are combined with the atoms to which they are attached to form one or more fused C6-C 10 can form an aryl, C-C heteroaryl, or C-C cycloalkyl ring, and each fused aryl, heteroaryl, or cycloalkyl can be joined by one or more R A2 and optionally substituted with R A2 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen -SO3H or -OSO3H; X 1 is a divalent covalent linking moiety, X 2 is a divalent covalent linking moiety, L is a targeting fragment, preferably the targeting fragment is capable of binding to a cell, more preferably the targeting fragment is capable of binding to a cell surface receptor), non-covalently bound to The nucleic acid is a nucleic acid that encodes a peptide or protein of interest, preferably the nucleic acid is a pharmaceutically active nucleic acid, the pharmaceutically active nucleic acid being a nucleic acid that encodes a pharmaceutically active peptide or protein.

[0079] In another aspect, the present invention provides a composition comprising a polyplex, the polyplex comprising a conjugate of Formula I, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof, and a nucleic acid, the nucleic acid preferably comprising the conjugate: [ka]

[0080] Formula I (In the formula, [ka] is a single or double bond, n is an integer of 1 to 1500, preferably an integer of 2 to 1500, m is any integer from 1 to 200, preferably any integer from 2 to 200, more preferably any integer from 1 to 100, and even more preferably any integer from 2 to 100; R 1 is the starting residue, preferably R 1 is -H or -CH3, R 2 are independently -H or an organic residue, and the -(NR 2 -CH2-CH2) n -The R in the part 2 at least 80%, preferably 90%, of the Ring A may have one or more R A1 is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl optionally substituted with R A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo or halogen, or two R A1 are combined with the atoms to which they are attached to form one or more fused C6-C 10can form an aryl, C-C heteroaryl, or C-C cycloalkyl ring, and each fused aryl, heteroaryl, or cycloalkyl can be joined by one or more R A2 is optionally replaced by R A2 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen -SO3H or -OSO3H; X 1 is -(Y 1 ) p where p is an integer from 1 to 20, and Y 1 Each occurrence of represents a chemical bond, -CR 11 R 12 -, -C(O)-, -O-, -S-, -NR 13 -, an amino acid residue, a divalent phenyl moiety, a divalent heterocyclic moiety, and a divalent heteroaryl moiety, each divalent phenyl or heteroaryl being independently selected from one or more R 13 and each divalent heterocycle is optionally substituted with one or more R 14 and optionally substituted with R 11 , R 12 and R 13 is independently, at each occurrence, H or C1-C6 alkyl; R 14 is a linking moiety that is independently, at each occurrence, H, C1-C6 alkyl, or oxo; X 2 is the formula -(Y 2 ) q - (wherein q is an integer from 1 to 50, and Y 2 Each occurrence of represents a chemical bond, -CR 21 R 22 -, NR 23 independently selected from -, -O-, -S-, -C(O)-, an amino acid residue, a divalent phenyl moiety, a divalent heterocyclic moiety, and a divalent heteroaryl moiety, wherein each divalent phenyl and divalent heteroaryl is selected from one or more R 23 and each divalent heterocyclic moiety is optionally substituted with one or more R 24 and optionally substituted with R 21 , R 22 and R 23are each independently, at each occurrence, -H, -COH, or C1-C6 alkyl, and each C1-C6 alkyl may be selected from one or more of -OH, oxo, C6-C 10 optionally substituted aryl, or 5- to 8-membered heteroaryl; R 24 is a linking moiety that is independently, at each occurrence, —H, —COH, C1-C6 alkyl, or oxo; L is preferably a targeting fragment capable of binding to a cell, preferably the targeting fragment is non-covalently linked to a cell surface receptor; The nucleic acid is a nucleic acid encoding a peptide or protein of interest, preferably the nucleic acid is a pharmaceutically active nucleic acid, the pharmaceutically active nucleic acid is a nucleic acid encoding a pharmaceutically active peptide or protein. Preferably, the composition consists of the polyplex.

[0081] In another aspect, the present invention provides a polyplex comprising a conjugate of Formula I, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof, and a nucleic acid, wherein the nucleic acid is preferably a polyplex comprising the conjugate: [ka]

[0082] Formula I (In the formula, [ka] is a single or double bond, n is an integer of 1 to 1500, preferably an integer of 2 to 1500, m is any integer from 1 to 200, preferably any integer from 2 to 200, more preferably any integer from 1 to 100, and even more preferably any integer from 2 to 100; R 1 is the starting residue, preferably R 1is -H or -CH3, R 2 are independently -H or an organic residue, and the -(NR 2 -CH2-CH2) n -The R in the part 2 at least 80%, preferably 90%, of the Ring A may have one or more R A1 is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl optionally substituted with R A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo or halogen, or two R A1 are combined with the atoms to which they are attached to form one or more fused C6-C 10 can form an aryl, C-C heteroaryl, or C-C cycloalkyl ring, and each fused aryl, heteroaryl, or cycloalkyl can be joined by one or more R A2 is optionally replaced by R A2 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen -SO3H or -OSO3H; X 1 is -(Y 1 ) p where p is an integer from 1 to 20, and Y 1 Each occurrence of represents a chemical bond, -CR 11 R 12 -, -C(O)-, -O-, -S-, -NR 13 -, an amino acid residue, a divalent phenyl moiety, a divalent heterocyclic moiety, and a divalent heteroaryl moiety, each divalent phenyl or heteroaryl being independently selected from one or more R 13 and each divalent heterocycle is optionally substituted with one or more R 14 and optionally substituted with R 11 , R 12 and R 13 is independently, at each occurrence, H or C1-C6 alkyl; R 14is a linking moiety that is independently, at each occurrence, H, C1-C6 alkyl, or oxo; X 2 is the formula -(Y 2 ) q - (wherein q is an integer from 1 to 50, and Y 2 Each occurrence of represents a chemical bond, -CR 21 R 22 -, NR 23 independently selected from -, -O-, -S-, -C(O)-, an amino acid residue, a divalent phenyl moiety, a divalent heterocyclic moiety, and a divalent heteroaryl moiety, wherein each divalent phenyl and divalent heteroaryl is selected from one or more R 23 and each divalent heterocyclic moiety is optionally substituted with one or more R 24 and optionally substituted with R 21 , R 22 and R 23 are each independently, at each occurrence, -H, -COH, or C1-C6 alkyl, and each C1-C6 alkyl may be selected from one or more of -OH, oxo, C6-C 10 optionally substituted aryl, or 5- to 8-membered heteroaryl; R 24 is a linking moiety that is independently, at each occurrence, —H, —COH, C1-C6 alkyl, or oxo; L is preferably a targeting fragment capable of binding to a cell, preferably the targeting fragment is non-covalently linked to a cell surface receptor; The nucleic acid is a nucleic acid that encodes a peptide or protein of interest, preferably the nucleic acid is a pharmaceutically active nucleic acid, the pharmaceutically active nucleic acid being a nucleic acid that encodes a pharmaceutically active peptide or protein.

[0083] In another aspect, the present invention provides a composition comprising a polyplex, the polyplex comprising a conjugate of Formula I, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof, and a nucleic acid, the nucleic acid preferably comprising the conjugate: [ka]

[0084] Formula I (In the formula, [ka] is a single or double bond, n is an integer of 1 to 1500, preferably an integer of 2 to 1500, m is an arbitrary discrete number of repeating -(O-CH-CH)- units from 25 to 100, preferably from 25 to 60, and preferably the discrete number m is the discrete number of consecutive repeating -(O-CH-CH)- units, and the discrete number of consecutive repeating -(O-CH-CH)- units is an arbitrary discrete number from 25 to 100, preferably from 25 to 60; R 1 is the starting residue, preferably R 1 is -H or -CH3, R 2 are independently -H or an organic residue, and the -(NR 2 -CH2-CH2) n -The R in question 2 at least 80%, preferably at least 90%, of are H; Ring A may have one or more R A1 is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl optionally substituted with R A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo, or halogen, or two R A1 are combined with the atoms to which they are attached to form one or more fused C6-C 10 can form an aryl, C-C heteroaryl, or C-C cycloalkyl ring, and each fused aryl, heteroaryl, or cycloalkyl can be joined by one or more R A2 and optionally substituted with R A2are independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen -SO3H or -OSO3H; X 1 is a divalent covalent linking moiety, X 2 is a divalent covalent linking moiety, L is a targeting fragment, preferably the targeting fragment is capable of binding to a cell, more preferably the targeting fragment is capable of binding to a cell surface receptor), non-covalently attached to The nucleic acid is a pharmaceutically active nucleic acid, which is a nucleic acid that encodes a pharmaceutically active peptide or protein.

[0085] In another aspect, the present invention provides a polyplex comprising a conjugate of Formula I, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof, and a nucleic acid, wherein the nucleic acid is preferably a polyplex comprising the conjugate: [ka]

[0086] Formula I (In the formula, [ka] is a single or double bond, n is an integer of 1 to 1500, preferably an integer of 2 to 1500, m is an arbitrary discrete number of repeating -(O-CH-CH)- units from 25 to 100, preferably from 25 to 60, and preferably the discrete number m is the discrete number of consecutive repeating -(O-CH-CH)- units, and the discrete number of consecutive repeating -(O-CH-CH)- units is an arbitrary discrete number from 25 to 100, preferably from 25 to 60; R 1 is the starting residue, preferably R 1 is -H or -CH3, R 2 are independently -H or an organic residue, and the -(NR 2 -CH2-CH2) n -The R in question 2 at least 80%, preferably at least 90%, of are H; Ring A may have one or more R A1 is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl optionally substituted with R A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo, or halogen, or two R A1 are combined with the atoms to which they are attached to form one or more fused C6-C 10 can form an aryl, C-C heteroaryl, or C-C cycloalkyl ring, and each fused aryl, heteroaryl, or cycloalkyl can be joined by one or more R A2 and optionally substituted with R A2 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen -SO3H or -OSO3H; X 1 is a divalent covalent linking moiety, X 2 is a divalent covalent linking moiety, L is a targeting fragment, preferably the targeting fragment is capable of binding to a cell, more preferably the targeting fragment is capable of binding to a cell surface receptor), non-covalently attached to The nucleic acid is a pharmaceutically active nucleic acid, which is a nucleic acid that encodes a pharmaceutically active peptide or protein.

[0087] In another aspect, the present invention provides a composition comprising a polyplex, the polyplex comprising a conjugate of Formula I, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof, and a nucleic acid, the nucleic acid preferably comprising the conjugate: [ka]

[0088] Formula I (In the formula, [ka] is a single or double bond, n is an integer of 1 to 1500, preferably an integer of 2 to 1500, m is a discrete number of 36 repeating -(O-CH-CH)- units, preferably the discrete number m is a discrete number of consecutive repeating -(O-CH-CH)- units, and the discrete number of consecutive repeating -(O-CH-CH) units is 36; R 1 is the starting residue, preferably R 1 is -H or -CH3, R 2 are independently -H or an organic residue, and the -(NR 2 -CH2-CH2) n -The R in question 2 at least 80%, preferably at least 90%, of are H; Ring A may have one or more R A1 is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl optionally substituted with R A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo, or halogen, or two R A1 are combined with the atoms to which they are attached to form one or more fused C6-C 10 can form an aryl, C-C heteroaryl, or C-C cycloalkyl ring, and each fused aryl, heteroaryl, or cycloalkyl can be joined by one or more R A2 and optionally substituted with R A2are independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen -SO3H or -OSO3H; X 1 is a divalent covalent linking moiety, X 2 is a divalent covalent linking moiety, L is a targeting fragment, preferably the targeting fragment is capable of binding to a cell, more preferably the targeting fragment is capable of binding to a cell surface receptor), non-covalently attached to The nucleic acid is a pharmaceutically active nucleic acid, which is a nucleic acid that encodes a pharmaceutically active peptide or protein.

[0089] In another aspect, the present invention provides a polyplex comprising a conjugate of Formula I, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof, and a nucleic acid, wherein the nucleic acid is preferably a polyplex comprising the conjugate: [ka]

[0090] Formula I (In the formula, [ka] is a single or double bond, n is an integer of 1 to 1500, preferably an integer of 2 to 1500, m is a discrete number of 36 repeating -(O-CH-CH)- units, preferably the discrete number m is a discrete number of consecutive repeating -(O-CH-CH)- units, the discrete number of consecutive repeating units -(O-CH-CH) being 36; R 1 is the starting residue, preferably R 1 is -H or -CH3, R 2 are independently -H or an organic residue, and the -(NR2 -CH2-CH2) n -The R in question 2 at least 80%, preferably at least 90%, of are H; Ring A may have one or more R A1 is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl optionally substituted with R A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo, or halogen, or two R A1 are combined with the atoms to which they are attached to form one or more fused C6-C 10 can form an aryl, C-C heteroaryl, or C-C cycloalkyl ring, and each fused aryl, heteroaryl, or cycloalkyl can be joined by one or more R A2 and optionally substituted with R A2 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen -SO3H or -OSO3H; X 1 is a divalent covalent linking moiety, X 2 is a divalent covalent linking moiety, L is a targeting fragment, preferably the targeting fragment is capable of binding to a cell, more preferably the targeting fragment is capable of binding to a cell surface receptor), non-covalently attached to The nucleic acid is a pharmaceutically active nucleic acid, which is a nucleic acid that encodes a pharmaceutically active peptide or protein.

[0091] In another aspect, the invention provides a composition comprising polyplexes, each of the polyplexes comprising a conjugate and a nucleic acid, the nucleic acid preferably non-covalently bound to the conjugate, the conjugate comprising a linear polyethyleneimine fragment comprising an alpha end and an omega end, the alpha end of the polyethyleneimine fragment being an initiating residue; a polyethylene glycol fragment comprising a first end and a second end, the omega end of the polyethyleneimine fragment being linked to a first terminal end of the polyethylene glycol fragment by a divalent covalent linking group -ZX 1 -ZX 1 - is not a single bond, -Z- is not an amide, and the second terminal end of the polyethylene glycol fragment is a divalent covalent linking moiety X 2 wherein the nucleic acid is a pharmaceutically active nucleic acid, the pharmaceutically active nucleic acid being a nucleic acid encoding a pharmaceutically active peptide or protein.

[0092] In another aspect, the invention provides a composition comprising a polyplex, the polyplex comprising a conjugate and a nucleic acid, the nucleic acid preferably non-covalently bound to the conjugate, the conjugate comprising a linear polyethyleneimine fragment comprising an alpha end and an omega end, the alpha end of the polyethyleneimine fragment being an initiating residue; a polyethylene glycol fragment comprising a first end and a second end, the omega end of the polyethyleneimine fragment being linked to a first terminal end of the polyethylene glycol fragment by a divalent covalent linking group -ZX 1 -ZX 1 - is not a single bond, -Z- is not an amide, and the second terminal end of the polyethylene glycol fragment is a divalent covalent linking moiety X 2 wherein the nucleic acid is RNA, the RNA is ssRNA, and preferably the ssRNA is mRNA. In a preferred embodiment of this aspect, the composition consists of the polyplex.

[0093] In another aspect, the invention provides a composition comprising a polyplex, the polyplex comprising a conjugate and a nucleic acid, the nucleic acid preferably non-covalently bound to the conjugate, the conjugate comprising a linear polyethyleneimine fragment comprising an alpha end and an omega end, the alpha end of the polyethyleneimine fragment being an initiating residue; a polyethylene glycol fragment comprising a first end and a second end, the omega end of the polyethyleneimine fragment being linked to a first terminal end of the polyethylene glycol fragment by a divalent covalent linking group -ZX 1 -ZX 1 - is not a single bond, -Z- is not an amide, and the second terminal end of the polyethylene glycol fragment is a divalent covalent linking moiety X 2 wherein the nucleic acid is RNA, the RNA is ssRNA, preferably the ssRNA is mRNA, and more preferably the mRNA encodes a peptide or protein of interest. In a preferred embodiment of this aspect, the composition consists of the polyplex.

[0094] In another aspect, the invention provides a composition comprising polyplexes, each of the polyplexes comprising a conjugate and a nucleic acid, the nucleic acid preferably non-covalently bound to the conjugate, the conjugate comprising a linear polyethyleneimine fragment comprising an alpha end and an omega end, the alpha end of the polyethyleneimine fragment being an initiating residue; a polyethylene glycol fragment comprising a first end and a second end, the omega end of the polyethyleneimine fragment being linked to a first terminal end of the polyethylene glycol fragment by a divalent covalent linking group -ZX 1 -ZX 1 - is not a single bond, -Z- is not an amide, and the second terminal end of the polyethylene glycol fragment is a divalent covalent linking moiety X 2wherein the nucleic acid is RNA, the RNA is ssRNA, and preferably the ssRNA is mRNA.

[0095] In another aspect, the invention provides a composition comprising polyplexes, each of the polyplexes comprising a conjugate and a nucleic acid, the nucleic acid preferably non-covalently bound to the conjugate, the conjugate comprising a linear polyethyleneimine fragment comprising an alpha end and an omega end, the alpha end of the polyethyleneimine fragment being an initiating residue; a polyethylene glycol fragment comprising a first end and a second end, the omega end of the polyethyleneimine fragment being linked to a first terminal end of the polyethylene glycol fragment by a divalent covalent linking group -ZX 1 -ZX 1 - is not a single bond, -Z- is not an amide, and the second terminal end of the polyethylene glycol fragment is a divalent covalent linking moiety X 2 wherein the nucleic acid is DNA, the DNA is pDNA, and preferably the pDNA encodes a peptide or protein of interest.

[0096] In another aspect, the invention provides a composition comprising polyplexes, each of the polyplexes comprising a conjugate of Formula I, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof, and a nucleic acid, wherein the nucleic acid is preferably a conjugate of the formula I: [ka]

[0097] Formula I (In the formula, [ka] is a single or double bond, n is an integer of 1 to 1500, preferably an integer of 2 to 1500, m is an integer of 1 to 200, preferably an integer of 2 to 200, more preferably an integer of 1 to 100, and even more preferably an integer of 2 to 100; R 1 is the starting residue, preferably R 1 is -H or -CH3, R 2 are independently -H or an organic residue, and the -(NR 2 -CH2-CH2) n -The R in question 2 at least 80%, preferably at least 90%, of are H; Ring A is one or more R A1 R is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl optionally substituted at any position by A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo, or halogen, or two R A1 are combined with the atoms to which they are attached to form one or more fused C6-C 10 can form an aryl, C-C heteroaryl, or C-C cycloalkyl ring, and each fused aryl, heteroaryl, or cycloalkyl can be joined by one or more R A2 and optionally substituted with R A2 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen -SO3H or -OSO3H; X 1 is a divalent covalent linking moiety, X 2 is a divalent covalent linking moiety, L is a targeting fragment, preferably the targeting fragment is capable of binding to a cell, more preferably the targeting fragment is capable of binding to a cell surface receptor), non-covalently bound to The nucleic acid is a pharmaceutically active nucleic acid, which is a nucleic acid that encodes a pharmaceutically active peptide or protein.

[0098] In another aspect, the present invention provides a composition comprising a polyplex, the polyplex comprising a conjugate of Formula I, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof, and a nucleic acid, the nucleic acid preferably comprising the conjugate: [ka]

[0099] Formula I (In the formula, [ka] is a single or double bond, n is an integer of 1 to 1500, preferably an integer of 2 to 1500, m is any integer from 1 to 200, preferably any integer from 2 to 200, more preferably any integer from 1 to 100, and even more preferably any integer from 2 to 100; R 1 is the starting residue, preferably R 1 is -H or -CH3, R 2 are independently -H or an organic residue, and the -(NR 2 -CH2-CH2) n -The R in question 2 at least 80%, preferably at least 90%, of are H; Ring A is one or more R A1 R is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl optionally substituted at any position by A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo, or halogen, or two R A1are combined with the atoms to which they are attached to form one or more fused C6-C 10 can form an aryl, C-C heteroaryl, or C-C cycloalkyl ring, and each fused aryl, heteroaryl, or cycloalkyl can be joined by one or more R A2 and optionally substituted with R A2 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen -SO3H or -OSO3H; X 1 is a divalent covalent linking moiety, X 2 is a divalent covalent linking moiety, L is a targeting fragment, preferably the targeting fragment is capable of binding to a cell, more preferably the targeting fragment is capable of binding to a cell surface receptor), non-covalently attached to The nucleic acid is RNA, the RNA is ssRNA, preferably the ssRNA is mRNA, more preferably the mRNA encodes a peptide or protein of interest.

[0100] In another aspect, the present invention provides a composition comprising a polyplex, the polyplex comprising a conjugate of Formula I, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof, and a nucleic acid, the nucleic acid preferably comprising the conjugate: [ka]

[0101] Formula I (In the formula, [ka] is a single or double bond, n is an integer of 1 to 1500, preferably an integer of 2 to 1500, m is any integer from 1 to 200, preferably any integer from 2 to 200, more preferably any integer from 1 to 100, and even more preferably any integer from 2 to 100; R 1 is the starting residue, preferably R 1 is -H or -CH3, R 2 are independently -H or an organic residue, and the -(NR 2 -CH2-CH2) n -The R in question 2 at least 80%, preferably at least 90%, of are H; Ring A is one or more R A1 R is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl optionally substituted at any position by A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo, or halogen, or two R A1 are combined with the atoms to which they are attached to form one or more fused C6-C 10 can form an aryl, C-C heteroaryl, or C-C cycloalkyl ring, and each fused aryl, heteroaryl, or cycloalkyl can be joined by one or more R A2 and optionally substituted with R A2 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen -SO3H or -OSO3H; X 1 is a divalent covalent linking moiety, X 2 is a divalent covalent linking moiety, L is a targeting fragment, preferably the targeting fragment is capable of binding to a cell, more preferably the targeting fragment is capable of binding to a cell surface receptor), non-covalently attached to The nucleic acid is DNA, the DNA is pDNA, and preferably the pDNA encodes a peptide or protein of interest.

[0102] In another aspect, the invention provides a composition comprising polyplexes, each polyplex comprising a conjugate of Formula I, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof, and a nucleic acid, wherein the nucleic acid is preferably a conjugate of Formula I: [ka]

[0103] Formula I (In the formula, [ka] is a single or double bond, n is an integer of 1 to 1500, preferably an integer of 2 to 1500, m is any integer from 1 to 200, preferably any integer from 2 to 200, more preferably any integer from 1 to 100, and even more preferably any integer from 2 to 100; R 1 is the starting residue, preferably R 1 is -H or -CH3, R 2 are independently -H or an organic residue, and the -(NR 2 -CH2-CH2) n -The R in question 2 at least 80%, preferably at least 90%, of are H; Ring A is one or more R A1 R is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl optionally substituted at any position by A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo, or halogen, or two R A1 are combined with the atoms to which they are attached to form one or more fused C6-C 10can form an aryl, C-C heteroaryl, or C-C cycloalkyl ring, and each fused aryl, heteroaryl, or cycloalkyl can be joined by one or more R A2 and optionally substituted with R A2 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen -SO3H or -OSO3H; X 1 is a divalent covalent linking moiety, X 2 is a divalent covalent linking moiety, L is a targeting fragment, preferably the targeting fragment is capable of binding to a cell, more preferably the targeting fragment is capable of binding to a cell surface receptor), non-covalently attached to The nucleic acid is RNA, the RNA is ssRNA, preferably the ssRNA is mRNA, more preferably the mRNA encodes a peptide or protein of interest.

[0104] In another aspect, the invention provides a composition comprising polyplexes, each polyplex comprising a conjugate of Formula I or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof, and a nucleic acid, wherein the nucleic acid is preferably a conjugate of Formula I: [ka] (In the formula, [ka] is a single or double bond, n is an integer of 1 to 1500, preferably an integer of 2 to 1500, m is any integer from 1 to 200, preferably any integer from 2 to 200, more preferably any integer from 1 to 100, and even more preferably any integer from 2 to 100; R 1is the starting residue, preferably R 1 is -H or -CH3, R 2 are independently -H or an organic residue, and the -(NR 2 -CH2-CH2) n -The R in question 2 at least 80%, preferably at least 90%, of are H; Ring A is one or more R A1 R is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl optionally substituted at any position by A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo, or halogen, or two R A1 are combined with the atoms to which they are attached to form one or more fused C6-C 10 can form an aryl, C-C heteroaryl, or C-C cycloalkyl ring, and each fused aryl, heteroaryl, or cycloalkyl can be joined by one or more R A2 and optionally substituted with R A2 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen -SO3H or -OSO3H; X 1 is a divalent covalent linking moiety, X 2 is a divalent covalent linking moiety, L is a targeting fragment, preferably the targeting fragment is capable of binding to a cell, more preferably the targeting fragment is capable of binding to a cell surface receptor), non-covalently bound to The nucleic acid is DNA, the DNA is pDNA, and preferably the pDNA encodes a peptide or protein of interest.

[0105] In another aspect, the present invention provides a composition comprising a polyplex, the polyplex comprising a conjugate and a nucleic acid, the nucleic acid preferably non-covalently bound to the conjugate, the conjugate comprising a linear polyethylenimine (LPEI) fragment covalently linked to one or more polyethylene glycol (PEG) fragments, each PEG fragment covalently linked to a targeting fragment L, preferably the targeting fragment is capable of binding to a cell, the nucleic acid is single-stranded RNA (ssRNA), preferably the ssRNA is mRNA, more preferably the mRNA is a pharmaceutically active nucleic acid, the pharmaceutically active nucleic acid is a nucleic acid encoding a pharmaceutically active peptide or protein, and even more preferably the pharmaceutically active peptide or protein is selected from a cytokine, interferon, interleukin, growth factor, hormone, enzyme, toxin, tumor antigen, viral antigen, bacterial antigen, autoantigen, and allergen.

[0106] In another aspect, the present invention provides a polyplex comprising a conjugate and a nucleic acid, wherein the nucleic acid is preferably non-covalently bound to the conjugate, wherein the conjugate comprises a linear polyethylenimine (LPEI) fragment covalently linked to one or more polyethylene glycol (PEG) fragments, each PEG fragment being covalently linked to a targeting fragment L, preferably wherein the targeting fragment is capable of binding to a cell, wherein the nucleic acid is single-stranded RNA (ssRNA), preferably wherein the ssRNA is mRNA, more preferably wherein the mRNA is a pharmaceutically active nucleic acid, wherein the pharmaceutically active nucleic acid encodes a pharmaceutically active peptide or protein, and even more preferably wherein the pharmaceutically active peptide or protein is selected from a cytokine, interferon, interleukin, growth factor, hormone, enzyme, toxin, tumor antigen, viral antigen, bacterial antigen, autoantigen, and allergen.

[0107] In another aspect, the present invention provides a composition comprising polyplexes, each of the polyplexes comprising a conjugate and a nucleic acid, the nucleic acid preferably non-covalently bound to the conjugate, the conjugate comprising a linear polyethylenimine (LPEI) fragment covalently linked to one or more polyethylene glycol (PEG) fragments, each PEG fragment covalently linked to a targeting fragment L, preferably the targeting fragment is capable of binding to a cell, the nucleic acid is single-stranded RNA (ssRNA), preferably the ssRNA is mRNA, more preferably the mRNA is a pharmaceutically active nucleic acid, the pharmaceutically active nucleic acid is a nucleic acid encoding a pharmaceutically active peptide or protein, and even more preferably the pharmaceutically active peptide or protein is selected from a cytokine, interferon, interleukin, growth factor, hormone, enzyme, toxin, tumor antigen, viral antigen, bacterial antigen, autoantigen, and allergen.

[0108] In another aspect, the present invention provides a composition comprising a polyplex, the polyplex comprising a conjugate and a nucleic acid, the nucleic acid preferably non-covalently bound to the conjugate, the conjugate comprising a linear polyethylenimine (LPEI) fragment covalently linked to one or more polyethylene glycol (PEG) fragments, each PEG fragment covalently linked to a targeting fragment L, preferably the targeting fragment is capable of binding to a cell, the nucleic acid is DNA, preferably plasmid DNA (pDNA), preferably the DNA, preferably the pDNA, encodes a pharmaceutically active peptide or protein, the pharmaceutically active peptide or protein preferably being selected from a cytokine, interferon, interleukin, growth factor, hormone, enzyme, toxin, tumor antigen, viral antigen, bacterial antigen, autoantigen, and allergen.

[0109] In another aspect, the present invention provides a polyplex, the polyplex comprising a conjugate and a nucleic acid, the nucleic acid preferably non-covalently bound to the conjugate, the conjugate comprising a linear polyethylenimine (LPEI) fragment covalently linked to one or more polyethylene glycol (PEG) fragments, each PEG fragment covalently linked to a targeting fragment L, preferably the targeting fragment being capable of binding to a cell, the nucleic acid being DNA, preferably plasmid DNA (pDNA), preferably the DNA, preferably the pDNA, encoding a pharmaceutically active peptide or protein, the pharmaceutically active peptide or protein preferably being selected from a cytokine, interferon, interleukin, growth factor, hormone, enzyme, toxin, tumor antigen, viral antigen, bacterial antigen, autoantigen, and allergen.

[0110] In another aspect, the present invention provides a composition comprising polyplexes, each of the polyplexes comprising a conjugate and a nucleic acid, wherein the nucleic acid is preferably non-covalently bound to the conjugate, wherein the conjugate comprises a linear polyethylenimine (LPEI) fragment covalently linked to one or more polyethylene glycol (PEG) fragments, wherein each PEG fragment is covalently linked to a targeting fragment L, preferably wherein the targeting fragment is capable of binding to a cell, wherein the nucleic acid is DNA, preferably plasmid DNA (pDNA), and preferably the DNA, preferably the pDNA, encodes a pharmaceutically active peptide or protein, wherein the pharmaceutically active peptide or protein is preferably selected from a cytokine, interferon, interleukin, growth factor, hormone, enzyme, toxin, tumor antigen, viral antigen, bacterial antigen, autoantigen, and allergen.

[0111] In a preferred embodiment of any aspect of the invention, the nucleic acid is RNA. In another preferred embodiment of any aspect of the invention, the nucleic acid is single-stranded RNA (ssRNA). In a further preferred embodiment of any aspect of the invention, the ssRNA encodes a peptide or protein of interest. In a further preferred embodiment of any aspect of the invention, the ssRNA encodes a peptide or protein of interest, wherein the peptide or protein of interest is selected from a reporter protein and a pharmaceutically active peptide or protein. In a further preferred embodiment of any aspect of the invention, the ssRNA encodes a peptide or protein of interest, wherein the peptide or protein of interest is a reporter protein. In a further preferred embodiment of any aspect of the invention, the ssRNA encodes a peptide or protein of interest, wherein the peptide or protein of interest is a pharmaceutically active peptide or protein. In a further preferred embodiment of any aspect of the invention, the ssRNA is a pharmaceutically active nucleic acid. In a further preferred embodiment of any aspect of the invention, the ssRNA is a pharmaceutically active nucleic acid, wherein the pharmaceutically active nucleic acid encodes a pharmaceutically active peptide or protein.

[0112] In a further preferred embodiment of any aspect of the invention, the ssRNA is messenger RNA (mRNA). In a further preferred embodiment of any aspect of the invention, the mRNA encodes a peptide or protein of interest. In a further preferred embodiment of any aspect of the invention, the mRNA encodes a peptide or protein of interest, wherein the peptide or protein of interest is selected from a reporter protein and a pharmaceutically active peptide or protein. In a further preferred embodiment of any aspect of the invention, the mRNA encodes a peptide or protein of interest, wherein the peptide or protein of interest is a reporter protein. In a further preferred embodiment of any aspect of the invention, the mRNA encodes a peptide or protein of interest, wherein the peptide or protein of interest is a pharmaceutically active peptide or protein. In a further preferred embodiment of any aspect of the invention, the mRNA is a pharmaceutically active nucleic acid. In a further preferred embodiment of any aspect of the invention, the mRNA is a pharmaceutically active nucleic acid, wherein the pharmaceutically active nucleic acid encodes a pharmaceutically active peptide or protein.

[0113] In another preferred embodiment of any aspect of the invention, the nucleic acid is DNA. In a further preferred embodiment of any aspect of the invention, the DNA is plasmid DNA. In a further preferred embodiment of any aspect of the invention, the pDNA encodes a peptide or protein of interest. In a further preferred embodiment of any aspect of the invention, the pDNA encodes a peptide or protein of interest, wherein the peptide or protein of interest is selected from a reporter protein and a pharmaceutically active peptide or protein. In a further preferred embodiment of any aspect of the invention, the pDNA encodes a peptide or protein of interest, wherein the peptide or protein of interest is a reporter protein. In a further preferred embodiment of any aspect of the invention, the pDNA encodes a peptide or protein of interest, wherein the peptide or protein of interest is a pharmaceutically active peptide or protein. In a further preferred embodiment of any aspect of the invention, the pDNA is a pharmaceutically active nucleic acid. In a further preferred embodiment of any aspect of the invention, the pDNA is a pharmaceutically active nucleic acid, wherein the pharmaceutically active nucleic acid encodes a pharmaceutically active peptide or protein.

[0114] In a further preferred embodiment of any aspect of the invention, the nucleic acid is a pharmaceutically active nucleic acid, which is pharmaceutically active per se. In a further preferred embodiment of any aspect of the invention, the nucleic acid is a pharmaceutically active nucleic acid, which is a nucleic acid that encodes a pharmaceutically active peptide or protein.

[0115] In preferred embodiments, the nucleic acid encodes a peptide or protein of interest, and the peptide or protein of interest is a reporter protein. In these embodiments, the nucleic acid comprises a reporter gene. Certain genes can be chosen as reporters because the characteristics they confer on the cells or organisms that express them can be easily identified and measured, or because they are selectable markers. Reporter genes are often used as indicators of whether a particular gene has been incorporated or expressed by a population of cells or organisms. Preferably, the expression product of the reporter gene is visually detectable. Common visually detectable reporter proteins typically have fluorescent or luminescent proteins. Examples of specific reporter genes include the gene encoding the jellyfish green fluorescent protein (GFP), which causes cells expressing it to glow green under blue light; the enzyme luciferase, which catalyzes a reaction with luciferin to produce light; and red fluorescent protein (RFP). Mutants of any of these specific reporter genes are possible as long as they have visually detectable properties. For example, eGFP is a point mutant of GFP.

[0116] In some embodiments, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the LPEI in the composition are connected to the PEG fragments by a single covalent linking moiety, preferably the covalent linking moiety creates a linear end-to-end linkage between the LPEI fragments and the PEG fragments. In some embodiments, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the LPEI fragments in the composition are contained in the conjugate, preferably as determined by UV spectroscopy or mass spectrometry. In some embodiments, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the LPEI in the composition are contained in the conjugate, preferably as determined by UV spectroscopy or mass spectrometry. In some embodiments, the composition consists essentially of the conjugate. In some embodiments, the composition consists of the conjugate.

[0117] In some embodiments, at least 60% of the LPEI fragments in the composition are connected to a single PEG fragment by a single covalent linking moiety Z, preferably, the covalent linking moiety Z creates a linear end-to-end linkage between the LPEI fragment and the PEG fragment. In some embodiments, at least 60% of the LPEI fragments in the composition are connected to a PEG fragment by a single triazole linker, preferably as determined by UV spectroscopy or mass spectrometry. In some embodiments, at least 70% of the LPEI fragments in the composition are connected to a PEG fragment by a single covalent linking moiety Z, preferably, the covalent linking moiety Z creates a linear end-to-end linkage between the LPEI fragment and the PEG fragment. In some embodiments, at least 70% of the LPEI fragments in the composition are included in the conjugate, preferably, as determined by UV spectroscopy or mass spectrometry. In some embodiments, at least 80% of the LPEI fragments in the composition are connected to a PEG fragment by a single covalent linking moiety Z, preferably, the covalent linking moiety Z creates a linear end-to-end linkage between the LPEI fragment and the PEG fragment. In some embodiments, at least 80% of the LPEI fragments in the composition are comprised in the conjugate, preferably as determined by UV spectroscopy or mass spectroscopy. In some embodiments, at least 90% of the LPEI fragments in the composition are connected to the PEG fragment by a single covalent linking moiety Z, preferably, the covalent linking moiety Z creates a linear end-to-end linkage between the LPEI fragment and the PEG fragment. In some embodiments, at least 90% of the LPEI fragments in the composition are comprised in the conjugate, preferably, as determined by UV spectroscopy or mass spectroscopy. In some embodiments, at least 95% of the LPEI fragments in the composition are connected to the PEG fragment by a single covalent linking moiety Z, preferably, the covalent linking moiety Z creates a linear end-to-end linkage between the LPEI fragment and the PEG fragment. In some embodiments, at least 95% of the LPEI fragments in the composition are comprised in the conjugate, preferably, as determined by UV spectroscopy or mass spectroscopy.In some embodiments, at least 99% of the LPEI fragments in the composition are connected to the PEG fragment by a single covalent linking moiety Z, preferably the covalent linking moiety Z creates a linear end-to-end linkage between the LPEI fragment and the PEG fragment. In some embodiments, at least 99% of the LPEI fragments in the composition are comprised in the conjugate, preferably as determined by UV spectroscopy or mass spectrometry. In some embodiments, the composition consists essentially of the conjugate. In some embodiments, the composition consists of the conjugate. In some embodiments, the LPEI fragment does not include substitutions beyond its first and second terminal ends.

[0118] In some embodiments, the covalent linking moiety Z comprises a triazole.

[0119] In some embodiments, Formula I* has the structure: R 1 -(NH-CH2-CH2) n -NHC(O)-(CH2-CH2-O) m -X 2 In some embodiments, formula I* does not include the structure R 1 -(NR 2 -CH2-CH2) n -NHC(O)-X 1 -(O-CH2-CH2) m -X 2 In some embodiments, the composition does not include the structure R 1 -(NH-CH2-CH2) n -NHC(O)-X 1 -(O-CH2-CH2) m -X 2 In some embodiments, the composition does not include a conjugate of structure R 1 -(NR 2 -CH2-CH2) n -NHC(O)-(CH2-CH2-O) m -X 2 -L conjugates are not included.

[0120] In some embodiments, R 1 is -H.

[0121] In some embodiments, R in the composition 2 At least 80% of the R in the composition are —H. 2 At least 85%, preferably 90%, preferably 95%, more preferably 99% of the R 2 are independently -H or an organic residue, and the -(NR 2 -CH2-CH2) n -The R in the part 2 At least 85%, preferably 90% of R 2 are independently -H or an organic residue, and the -(NR 2 -CH2-CH2) n -The R in the part 2 At least 90% of R 2 are independently -H or an organic residue, and the -(NR 2 -CH2-CH2) n -The R in the part 2 At least 90% of R 2 are independently —H or an organic residue, and the —(NR 2 -CH2-CH2) n -The R in the part 2 At least 91%, preferably at least 92%, more preferably at least 93% of R 2 are independently —H or an organic residue, and the —(NR 2 -CH2-CH2) n -The R in the part 2 At least 94%, preferably at least 95%, more preferably at least 96% of R 2 are independently —H or an organic residue, and the —(NR 2 -CH2-CH2) n -The R in the part 2At least 95%, preferably at least 97%, more preferably at least 98%, and more preferably 99% of the total is H.

[0122] In some embodiments, ring A is an 8-membered cycloalkenyl, a 5-membered heterocycloalkyl, or a 7-8-membered heterocycloalkenyl, and each cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl may have one or more R A1 is optionally replaced by

[0123] In some embodiments, ring A is cyclooctene, maleimide, or a 7-8 membered heterocycloalkenyl, wherein the heterocycloalkyl or heterocycloalkenyl does not contain heteroatoms other than N, O, and S, and each cyclooctene, heterocycloalkyl, or heterocycloalkenyl may contain one or more R A1 is optionally replaced by

[0124] In some embodiments, ring A is cyclooctene, maleimide, or a 7-8 membered heterocycloalkenyl, wherein the heterocycloalkyl or heterocycloalkenyl contains one or more heteroatoms, preferably one or two heteroatoms selected from N, O, and S, and each cyclooctene, heterocycloalkyl, or heterocycloalkenyl contains one or more R A1 is optionally replaced by

[0125] In some embodiments, ring A is a cyclooctene, maleimide, or an 8-membered heterocycloalkene, wherein the heterocycloalkene contains exactly one heteroatom selected from N, O, and S, and each cyclooctene or heterocycloalkene contains one or more R A1 is optionally replaced by

[0126] In some embodiments, R A1 is -H, oxo or fluorine, or two R A1combine to form one or more fused phenyl rings, preferably one or two fused phenyl rings, each phenyl ring optionally substituted with one or more -OSO3H or -SO3H.

[0127] In some embodiments, ring A is a cyclooctene, maleimide, or an 8-membered heterocycloalkene, wherein the heterocycloalkene contains exactly one heteroatom selected from N, O, and S, and each cyclooctene or heterocycloalkene contains one or more R A1 and optionally substituted with R A1 is oxo or fluorine, or two R A1 combine to form one or more fused phenyl rings, preferably one or two fused phenyl rings.

[0128] In some embodiments, ring A is a cyclooctene, maleimide, or an 8-membered heterocycloalkene, wherein the heterocycloalkene contains exactly one heteroatom selected from N, and each cyclooctene or heterocycloalkene contains one or two R A1 is optionally replaced by

[0129] In some embodiments, R A1 is -H, oxo or fluorine, or two R A1 combine to form one or more fused phenyl rings, preferably one or two fused phenyl rings, each phenyl ring containing one or more R A2 is optionally replaced by

[0130] In some embodiments, ring A is a cyclooctene, maleimide, or an 8-membered heterocycloalkene, wherein the heterocycloalkene contains exactly one heteroatom selected from N, and each cyclooctene or heterocycloalkene contains one or two R A1 and optionally substituted with R A1 is H, oxo, or fluorine, or two R A1combine to form one or more fused phenyl rings, preferably one or two fused phenyl rings, each of which may be substituted with one or more -OSO3H or -SO3H.

[0131] In some preferred embodiments, ring A is a cyclooctene, maleimide, or an 8-membered heterocycloalkene, wherein the heterocycloalkene contains exactly one heteroatom selected from N, and each cyclooctene or heterocycloalkene contains one or two R A1 and optionally substituted with R A1 is H or two R A1 combine to form one or more fused phenyl rings, preferably one or two fused phenyl rings, each of which may be substituted with one or more -OSO3H or -SO3H.

[0132] Preparation of linear conjugates The conjugates of the present invention can be prepared by several methods well known to those skilled in the art of polymer synthesis.For example, the compounds of the present invention can be synthesized using the methods described below, along with synthetic methods known in the field of polymer chemistry, or variations thereof recognized by those skilled in the art.Methods include, but are not limited to, the methods described below.The conjugates of the present invention can be synthesized according to the steps outlined in general schemes 1, 2, 3, 4, 5, 6, 7 and 8, or can be prepared using an alternating sequence of assembling intermediates without departing from the present invention.The conjugates of the present invention can also be synthesized using slight variations of the steps outlined below.For example, while scheme 3 shows the use of tetrafluorophenyl ester as an electrophilic functional group for coupling with hEGF, those skilled in the art will recognize other suitable electrophilic functional groups that can be used for the same purpose.

[0133] In some preferred embodiments, the LPEI fragment and the PEG fragment are coupled via a [3+2] cycloaddition between an azide and an alkene or alkyne to form a 1,2,3 triazole or a 4,5-dihydro-1H-[1,2,3]triazole. In some preferred embodiments, the LPEI fragment contains an azide functional group and the PEG fragment contains an alkene or alkyne functional group.

[0134] LPEI fragment The conjugates of the present invention can comprise an LPEI fragment and a PEG fragment. Linear polyethyleneimine (LPEI) has the chemical formula -[NH-CH-CH]-. Thus, linear polyethyleneimine (LPEI) has a chemical formula of n repeating units of -[NH-CH-CH]-. LPEI can be synthesized according to many methods known in the art, including, in particular, polymerization of 2-oxazoline followed by hydrolysis of the pendant amide bond (see, e.g., Brissault et al., Bioconjugate Chem., 2003, 14, 581-587). As noted above, polymerization of poly(2-oxazoline) from 2-oxazoline (i.e., a suitable precursor for LPEI) can be initiated with any suitable initiator. In some embodiments, the initiator leaves an initiator residue at the alpha-terminus of the poly(2-oxazoline). In preferred embodiments, the initiator residue (i.e., Formula I* or R of Formula I) is used. 1 ) is a hydrogen atom or a C1-C6 alkyl, preferably a hydrogen or a C1-C4 alkyl, more preferably a hydrogen or a methyl group. Most preferably a hydrogen atom. In a preferred embodiment, the initial residue R of formula I 1 is a hydrogen atom or a C1-C6 alkyl, preferably a hydrogen or a C1-C4 alkyl, more preferably a hydrogen or a methyl group, and most preferably a hydrogen atom. In a preferred embodiment, the initial residue (i.e., R 1 ) is —H or —CH3, most preferably —H. In a preferred embodiment, the initial residue R of formula I* 1 In a preferred embodiment, the initial residue R of formula I is1 In a preferred embodiment, the initial residue R of formula I* is -H. 1 In a preferred embodiment, the initial residue R of formula I is 1 is —CH3. However, one skilled in the art will understand that the initiating residue can be the residue remaining from any suitable initiator capable of initiating the polymerization of poly(2-oxazoline) from 2-oxazoline.

[0135] In some embodiments, LPEI fragments can be coupled to PEG fragments via a [3+2] cycloaddition between an azide and an alkene or alkyne to form a 1,2,3 triazole or a 4,5-dihydro-1H-[1,2,3]triazole, and the LPEI fragment contains an azide (-N3) functional group at the omega-terminus of the chain. In some preferred embodiments, the LPEI fragment is not further substituted except for a single substitution at the alpha-terminus. For example, in some preferred embodiments, the LPEI fragment contains the repeating formula -[NH-CH2-CH2]- and is substituted at the omega-terminus with an azide group that can be coupled to an alkyne or alkene substituent on the PEG fragment. In some preferred embodiments, the alpha-terminus of the LPEI fragment can be substituted with a hydrogen atom or a C1-C6 alkyl, preferably a hydrogen or a C1-C4 alkyl, more preferably a hydrogen or methyl group. A hydrogen atom is most preferred.

[0136] For example, in some preferred embodiments, the LPEI fragment may be substituted at the alpha terminus with a hydrogen atom or a C1-C6 alkyl, preferably a hydrogen atom or a C1-C4 alkyl, more preferably a hydrogen atom or a methyl group, and at the omega terminus with an azide group, and in some preferred embodiments, there are no additional substitutions on the LPEI fragment. For example, the conjugates of the present invention may have the following formula: [ka] (In the formula, R 1can be prepared from an LPEI fragment of any suitable starting residue, which may be preferably hydrogen or C1-C6 alkyl, preferably hydrogen or C1-C4 alkyl, more preferably hydrogen or methyl, most preferably hydrogen.

[0137] In some embodiments, the LPEI fragment can be terminated with a thiol group, and thus in some embodiments, the omega-terminus of the LPEI fragment comprises, and preferably is, a thiol group, which can be coupled to a reactive alkene group on a PEG fragment via a thiol-ene reaction. Thus, in some embodiments, the conjugates of the present invention have the following formula: [ka] (In the formula, R 1 can be prepared from an LPEI fragment of any suitable starting residue, which may be hydrogen or methyl, preferably hydrogen.

[0138] In some embodiments, the LPEI fragment can be terminated with an alkene group, and thus in some embodiments, the omega end of the LPEI fragment comprises, and preferably is, an alkene group that can be coupled to a reactive thiol group on a PEG fragment via a thiol-ene reaction. Thus, in some embodiments, the conjugates of the invention have the following formula: [ka] (In the formula, R 1 can be prepared from an LPEI fragment of any suitable starting residue, which may be hydrogen or methyl, preferably hydrogen.

[0139] LPEI fragments can include a range of lengths (i.e., repeat units, as represented above by the variable "n"). For example, LPEI fragments can include 1 to 1000 repeat units (i.e., -NH-CH2-CH2-). In some embodiments, LPEI fragments can be present as dispersed polymer moieties and do not include discrete numbers of -NH-CH2-CH2- repeat units. For example, LPEI fragments can be present as dispersed polymer moieties having a molecular weight of about 5 to 50 KDa, preferably a dispersity of about 5 or less, preferably about 4 or less, preferably about 3 or less, preferably about 2 or less, and preferably about 1.5 or less. In some embodiments, LPEI fragments can be present as dispersed polymer moieties having a molecular weight of about 10 to 40 KDa and a dispersity of about 4 or less, preferably about 3 or less, preferably about 2 or less, and preferably about 1.5 or less. In some embodiments, LPEI fragments can be present as dispersed polymer moieties having a molecular weight of about 12 to 30 KDa and a dispersity of about 3 or less, preferably about 2 or less, and preferably about 1.5 or less. In some embodiments, LPEI fragments can be present as dispersed polymer moieties having a molecular weight of about 15-27 KDa and a dispersity of about 2 or less, preferably about 1.5 or less. In some embodiments, LPEI fragments can be present as dispersed polymer moieties having a molecular weight of about 17-25 KDa and a dispersity of about 1.2 or less.

[0140] For example, the LPEI fragments can be present as dispersed polymer portions containing about 115-1150 repeat units, preferably with a dispersity of about 5 or less, preferably about 4 or less, preferably about 3 or less, preferably about 2 or less, and preferably about 1.5 or less. In some embodiments, the LPEI fragments can be present as dispersed polymer portions containing about 230-930 repeat units, with a dispersity of about 4 or less, preferably about 3 or less, preferably about 2 or less, and preferably about 1.5 or less. In some embodiments, the LPEI fragments can be present as dispersed polymer portions containing about 280-700 repeat units, with a dispersity of about 3 or less, preferably about 2 or less, and preferably about 1.5 or less. In some embodiments, the LPEI fragments can be present as dispersed polymer portions containing about 350-630 repeat units, with a dispersity of about 2 or less, preferably about 1.5 or less. In some embodiments, the LPEI fragments can be present as dispersed polymer portions containing about 400-580 repeat units, with a dispersity of about 1.2 or less.

[0141] In some embodiments, the R 1 -(NR 2 -CH2-CH2) n The R - moiety is a dispersed polymer moiety having 115 to 1150 repeat units n and a dispersity of about 5 or less, and preferably 1 -(NR 2 -CH2-CH2) n The R moiety is a dispersed polymer moiety having 280 to 700 repeat units n and a dispersity of about 3 or less, and more preferably 1 -(NR 2 -CH2-CH2) n - moiety is a dispersed polymer moiety having repeat units n between 350 and 630 and a dispersity of about 2 or less, and even more preferably the R 1 -(NR 2 -CH2-CH2) n The - moiety is a disperse polymer moiety having 400 to 580 repeat units n and a dispersity of about 1.2 or less.

[0142] In a preferred embodiment, the polyethyleneimine fragment is a disperse polymer moiety having about 115 to about 1150 repeating units and a dispersity of about 5 or less, preferably about 230 to about 930 repeating units with a dispersity of about 4 or less, more preferably about 280 to about 700 repeating units with a dispersity of about 3 or less, even more preferably about 350 to about 630 repeating units with a dispersity of about 2 or less, and even more preferably about 400 to about 580 repeating units with a dispersity of about 1.2 or less.

[0143] In a preferred embodiment, the polyethyleneimine fragment is a dispersed polymer moiety having about 115 to about 1150 repeating units and a dispersity of about 5 or less, preferably about 4 or less, preferably about 3 or less, preferably about 2 or less, and preferably about 1.5 or less. In a preferred embodiment, the polyethyleneimine fragment is a dispersed polymer moiety having about 230 to about 930 repeating units and a dispersity of about 4 or less, preferably about 3 or less, preferably about 2 or less, and preferably about 1.5 or less. In a preferred embodiment, the polyethyleneimine fragment is a dispersed polymer moiety having about 280 to about 700 repeating units and a dispersity of about 3 or less, preferably about 2 or less, and preferably about 1.5 or less. In a preferred embodiment, the polyethyleneimine fragment is a dispersed polymer moiety having about 350 to about 630 repeating units and a dispersity of about 2 or less, preferably about 1.5 or less. In a preferred embodiment, the polyethyleneimine fragment is a dispersed polymer moiety having about 400 to about 580 repeating units and a dispersity of about 1.2 or less.

[0144] As discussed above, those skilled in the art will understand that, in some embodiments, LPEI fragments can include organic residues (i.e., pendant amide groups) connected by nitrogen atoms embedded within the LPEI chain. Those skilled in the art will understand that such organic residues (i.e., amide groups) can be formed during the ring-opening polymerization of 2-oxazolines to form poly(2-oxazolines). Without wishing to be bound by theory, LPEIs can be formed from poly(2-oxazolines) by cleavage of amide groups (e.g., using an acid such as HCl). However, in some cases, not all amide bonds may be cleaved under these conditions. Thus, in some embodiments, about 5% or less of the nitrogen atoms in LPEI fragments may be connected to organic residues to form amides. In some embodiments, about 4% or less, about 3% or less, about 2% or less, about 1% or less, about 0.5% or less, about 0.4% or less, about 0.3% or less, about 0.2% or less, or about 0.1% or less of the nitrogen atoms in LPEI fragments may be connected to organic residues to form amides. Those skilled in the art will understand that the molecular weight of an LPEI fragment includes the percentage of the LPEI fragment that is attached to an organic residue as an amide. Furthermore, those skilled in the art will understand that, although the chemical structures depicted herein show repeating -NH-CH-CH- fragments, trace amounts of residual organic residues, such as pendant amide groups (e.g., as defined above), may still be present in the resulting triconjugates or polyplexes of the present disclosure. As sometimes used herein, the term "triconguate" refers to the conjugates of the present invention. The prefix "tri-" refers to the three components included in the conjugates of the present invention: the LPEI fragment, the PEG fragment, and the targeting fragment.

[0145] PEG fragment Polyethylene glycol (PEG) has the chemical formula -[O-CH-CH]-. Thus, polyethylene glycol (PEG) has a chemical formula of m repeating units of -[O-CH-CH]-. In some preferred embodiments, PEG fragments can be coupled to LPEI fragments via a [3+2] cycloaddition between an azide and an alkene or alkyne to form a 1,2,3 triazole or a 4,5-dihydro-1H-[1,2,3]triazole, and each reactive precursor molecule comprising a PEG fragment further comprises an alkene or alkyne functional group. For example, in some preferred embodiments, a reactive precursor molecule comprising a PEG fragment comprises the repeating formula -[O-CH-CH]-, and an alkene or alkyne group (e.g., a linking moiety "X" as discussed herein) that can be coupled to the azide group of a corresponding reactive precursor molecule comprising an LPEI fragment. 1 ") is substituted at the first end (i.e., terminus).

[0146] In some preferred embodiments, the alkene or alkyne group is an activated alkene or alkyne group that can spontaneously react with an azide (e.g., without the addition of a catalyst, such as a copper catalyst). For example, an activated alkyne group can be incorporated into a 7- or 8-membered ring, resulting in a strained species that spontaneously reacts with the azide group of an LPEI fragment. The activated alkene can include a maleimide moiety, where the alkene is activated by conjugation to an adjacent carbonyl group. In some preferred embodiments, the second end (i.e., terminus) of the PEG fragment is linked to a targeting fragment (e.g., hEGF, HER2, folate, or DUPA) (e.g., a linking moiety "X" discussed herein). 2 " can be replaced with "

[0147] PEG fragments can include a range of lengths (i.e., repeating units represented by the variable "m"). In other embodiments, PEG fragments can include a discrete number of repeating -O-CH-CH- units and are not defined in terms of average chain length. In preferred embodiments, the -(O-CH-CH)m - is a dispersed polymer moiety. In a preferred embodiment, the -(O-CH-CH) m The - unit comprises, preferably consists of, a discontinuous number of repeating units m. In a preferred embodiment, the -(O-CH-CH) m -unit comprises, preferably consists of, a discrete number of consecutive repeat units m.

[0148] In some preferred embodiments, the PEG fragment is a dispersed polymer moiety comprising about 1 to about 200 repeating units, preferably about 1 to about 200 repeating units. In some preferred embodiments, the PEG fragment can comprise 1 to 100 repeating units (i.e., -O-CH2-CH2-). Preferably, the PEG fragment of the present invention comprises about 1 to about 100 repeating units, about 1 to about 90 repeating units, about 1 to about 80 repeating units, about 1 to about 70 repeating units, about 1 to about 60 repeating units, about 1 to about 50 repeating units, about 1 to about 50 repeating units, about 1 to about 40 repeating units, about 1 to about 30 repeating units, or about 1 to about 20 repeating units. In some other preferred embodiments, the PEG fragment comprises a discrete number of repeating units, m, preferably 12 repeating units or 24 repeating units. In some embodiments, the polyethylene glycol fragment is a disperse polymer moiety having about 2 to about 80 repeating units and a dispersity of about 2.0 or less, preferably about 1.8 or less, and even more preferably about 1.5 or less, and about 2 to about 70 repeating units, preferably about 1.8 or less, and even more preferably about 2 to about 50 repeating units, with a dispersity of about 1.5 or less. In some embodiments, the -(O-CH2-CH2) m The - moiety is a disperse polymer moiety having from about 2 to about 80 repeating units and a dispersity of about 2.0 or less, preferably from about 2 to about 70 repeating units with a dispersity of about 1.8 or less, and more preferably from about 2 to about 50 repeating units with a dispersity of about 1.5 or less.

[0149] In a preferred embodiment, the polyethylene glycol fragment PEG fragment comprises, preferably consists of, a discrete number of repeating units m, preferably 12 or 24 repeating units. In a preferred embodiment, the m (-(O-CH-CH) m - moiety) comprises, preferably consists of, a discrete number of repeat units m, preferably 12 or 24 repeat units.

[0150] In a preferred embodiment, the PEG fragment comprises, or preferably consists of, 2 to 100 discrete number of repeat units m, preferably 4 to 60 discrete number of repeat units m. In a preferred embodiment, the PEG fragment comprises, or preferably consists of, 4 to 60 discrete number of repeat units m, preferably 10 to 60 discrete number of repeat units m. In a preferred embodiment, the PEG fragment comprises, preferably consists of, a non-consecutive number of repeat units m of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60. In preferred embodiments, the PEG fragment comprises, or preferably consists of, 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, or 60 discrete repeat units m. In preferred embodiments, the PEG fragment comprises, or preferably consists of, 4 discrete repeat units m. In preferred embodiments, the PEG fragment comprises, or preferably consists of 12 discrete repeat units m. In preferred embodiments, the PEG fragment comprises, or preferably consists of 24 discrete repeat units m. In preferred embodiments, the PEG fragment comprises, or preferably consists of 36 discrete repeat units m.

[0151] In a preferred embodiment, the PEG fragment comprises, or preferably consists of, 2 to 100 discrete consecutive repeat units m, preferably 4 to 60 discrete consecutive repeat units m. In a preferred embodiment, the PEG fragment comprises, or preferably consists of, 4 to 60 discrete consecutive repeat units m, preferably 10 to 60 discrete consecutive repeat units m. In preferred embodiments, the PEG fragment comprises, or preferably consists of, a discrete number of consecutive repeat units m of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60. In preferred embodiments, the PEG fragment comprises, or preferably consists of, 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, or 60 discrete numbers of consecutive repeat units m. In preferred embodiments, the PEG fragment comprises, or preferably consists of, 4 discrete numbers of consecutive repeat units m. In preferred embodiments, the PEG fragment comprises, or preferably consists of, 12 discrete numbers of consecutive repeat units m. In preferred embodiments, the PEG fragment comprises, or preferably consists of, 24 discrete numbers of consecutive repeat units m. In preferred embodiments, the PEG fragment comprises, or preferably consists of, 36 discrete numbers of consecutive repeat units m.

[0152] In a preferred embodiment, the —(O—CH—CH) of formula I* or formula I m The - moiety comprises, preferably consists of, a discrete number of repeating units m of 2 to 100, preferably a discrete number of repeating units m of 4 to 60. In a preferred embodiment, the -(O-CH2-CH2) m The - moiety comprises, preferably consists of, 4 to 60 discrete repeat units m, preferably 10 to 60 discrete repeat units m. In a preferred embodiment, the -(O-CH2-CH2) mThe - moiety comprises, preferably consists of, a non-consecutive number of repeating units m of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60. In a preferred embodiment, the -(O-CH2-CH2) m The - moiety comprises, preferably consists of, a discrete number of repeating units m of 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, or 60. In a preferred embodiment, the -(O-CH-CH) m The - unit comprises, preferably consists of, a discrete number of repeating units m of 4. In a preferred embodiment, the -(O-CH-CH) m The - unit comprises, preferably consists of, 12 discrete repeating units m. In a preferred embodiment, the -(O-CH-CH) m The - unit comprises, preferably consists of, a discrete number of repeating units m of 24. In a preferred embodiment, the -(O-CH-CH) m -unit comprises, preferably consists of, 36 discrete numbers of repeating units m.

[0153] In a preferred embodiment, the —(O—CH—CH) of formula I* or formula I m The - moiety comprises, or preferably consists of, 2 to 100 discrete consecutive repeat units m, preferably 4 to 60 discrete consecutive repeat units m. In a preferred embodiment, the -(O-CH2-CH2) m The - moiety comprises, preferably consists of, 4 to 60 discrete consecutive repeat units m, preferably 10 to 60 discrete consecutive repeat units m. In a preferred embodiment, the -(O-CH2-CH2) mThe - moiety comprises, or preferably consists of, a discrete number of consecutive repeating units m of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60. In a preferred embodiment, the -(O-CH2-CH2) m The - moiety comprises, preferably consists of, a discrete number of consecutive repeating units m of 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, or 60. In a preferred embodiment, the -(O-CH-CH) m The - moiety comprises, preferably consists of, a discrete number of consecutive repeating units m of 4. In a preferred embodiment, the -(O-CH-CH) m The - moiety comprises, preferably consists of, 12 discrete consecutive repeating units m. In a preferred embodiment, the -(O-CH-CH) m The - moiety comprises, preferably consists of, a discrete number of consecutive repeating units m of 24. In a preferred embodiment, the -(O-CH-CH) m - the moiety comprises, preferably consists of, 36 discrete numbers of consecutive repeating units m.

[0154] In a preferred embodiment, the PEG fragments contained in the conjugates and compositions of the present invention comprise, and preferably consist of, a discrete number m of repeating -(O-CH-CH)- units, and are not defined in terms of average chain length. Accordingly, the PEG fragments contained in the conjugates and compositions of the present invention comprise, and preferably consist of, a discrete number m of repeating -(O-CH-CH)- units, and are not defined in terms of average chain length, but have specifically defined, distinct molecular weights associated with the discrete number m of repeating -(O-CH-CH)- units. In a preferred embodiment, the PEG fragments comprise, and preferably consist of, a discrete number m of repeating -(O-CH-CH)- units, and typically and preferably, the discrete number (m) is between 25 and 100, more preferably between 25 and 60. In a preferred embodiment, the PEG fragment comprises, preferably consists of, a discrete number m of consecutive repeating -(O-CH-CH) units, typically and preferably, the discrete number (m) is between 25 and 100, more preferably between 25 and 60.

[0155] The phrases "a polyethylene glycol fragment comprising a discrete number (m) of repeating -(O-CH2-CH2)- units" or "a PEG fragment comprising a discrete number (m) of repeating -(O-CH2-CH2)- units" refer to a fragment that comprises, and preferably consists of, a discrete number - typically herein a discrete number m - of repeating -(O-CH2-CH2)- units, wherein the discrete number (m) is discrete, i.e., a specific single, defined integer (m) between 25 and 100, preferably between 25 and 60. Thus, the phrases "polyethylene glycol fragments comprising a discrete number (m) of repeating -(O-CH-CH)- units" or "PEG fragments comprising a discrete number (m) of repeating -(O-CH-CH)- units" refer to fragments that comprise, and preferably consist of, a discrete number (m) of repeating -(O-CH-CH)- units, where the discrete number (m) is discrete, i.e., a specific, single, defined integer, a number (m) between 25 and 100, preferably between 25 and 60. Thus, the defined PEG fragments comprise, and preferably consist of, a discrete number (m) of repeating -(O-CH-CH)- units, each of which has a specifically defined, distinct molecular weight, although not defined in terms of average chain length. Reference herein to discrete numbers between 25 and 100 refers to any integer between 25 and 100, i.e., any integer between 25 and 100, including the integers and discrete numbers stated as boundaries, such as 25 and 100. As a further example, a PEG fragment containing a discrete number (m) of repeating -(O-CH-CH)- units, where the discrete number m is 36, refers to a PEG fragment containing a chain of -(O-CH-CH)- units containing exactly 36 -(O-CH-CH)- units. Such a chain of exactly 36 -(O-CH-CH)- units is a PEG fragment. 36Such PEG fragments are contrasted with "polymeric PEG fragments," "polydisperse PEG fragments," or "disperse PEG fragments," which typically refer to a heterogeneous mixture of sizes and molecular weights resulting from a polymer reaction in a Poisson distribution (J. Herzberger et al.; Chem Rev, 2016, 116:2170-2243). The PEG fragments of the present invention, which contain a discrete number (m) of repeating -(O-CH-CH)- units, are not synthesized by a polymerization process. The PEG fragments of the present invention are single-molecule fragments that contain a discrete number (m) of repeating -(O-CH-CH)- units and have a discrete, i.e., defined and specified, chain length. Thus, the PEG fragments of the present invention, which contain a discrete number (m) of repeating -(O-CH-CH)- units, are single-molecule fragments that have a discrete, i.e., defined and specified, chain length. The PEG fragments of the present invention are not a mixture of molecular entities (such as those resulting from a random polymerization reaction). The discrete nature of the distinct PEG fragments of the present invention distinguishes them from polydisperse techniques.

[0156] The PEG fragments of the present invention can comprise, and preferably consist of, homogeneous discrete PEG fragments or heterogeneous discrete PEG fragments, typically and preferably homogeneous discrete PEG fragments. As used herein, the term "homogeneous discrete PEG fragments" refers to discrete PEG structures whose entire chemical backbone is composed solely of a specific, discontinuous number of contiguous ethylene oxide units. In other words, no other functionality is present within the homogeneous discrete PEG fragments. However, the term "homogeneous discrete PEG fragments" refers to discrete PEG structures whose basic ethylene oxide backbone, comprising a discrete number of ethylene oxide units, can, and typically does, have functional groups for conjugation with PEI fragments and targeting fragments. As used herein, the term "heterogeneous discrete PEG fragments" refers to discrete PEG structures in which the basic ethylene oxide backbone, comprising a discrete number of ethylene oxide units, is divided by the inclusion of, or substituted with, other functional groups or units within the structure, such as amide or ester bonds or other functional units. In a preferred embodiment of the present invention, the PEG fragments are homogeneous discrete PEG fragments.

[0157] In some preferred embodiments, PEG fragments can be coupled to LPEI fragments via a [3+2] cycloaddition between an azide and an alkene or alkyne to form a 1,2,3 triazole or a 4,5-dihydro-1H-[1,2,3]triazole, and each reactive precursor molecule comprising a PEG fragment further comprises an alkene or alkyne functional group. For example, in some preferred embodiments, a reactive precursor molecule comprising a PEG fragment comprises a repeating formula -[O-CH-CH]-, an alkene or alkyne group (e.g., a linking moiety "X" as discussed herein) that can be coupled to an azide group of a corresponding reactive precursor molecule comprising an LPEI fragment. 1 ") at a first end (i.e., terminal end). In some preferred embodiments, the alkene or alkyne group is an activated alkene or alkyne group that can react spontaneously with an azide (e.g., without the addition of a catalyst, such as a copper catalyst). For example, an activated alkyne group can be incorporated into a 7- or 8-membered ring, resulting in a strained species that reacts spontaneously with the azide group of an LPEI fragment.

[0158] The PEG fragments contained in the conjugates and compositions of the invention comprise, and preferably consist of, a discrete number m of repeating -O-CH-CH- units, and are not defined in terms of average chain length as is the case for polymeric PEG fragments. In a preferred embodiment, the -(O-CH-CH) m The - unit comprises, preferably consists of, a discontinuous number of repeating units m. In a preferred embodiment, the -(O-CH-CH) m -unit comprises, preferably consists of, a discrete number of consecutive repeat units m.

[0159] In a preferred embodiment, the PEG fragment comprises, or preferably consists of, 25 to 100 discrete repeat units m, preferably 25 to 60 discrete repeat units m. In a preferred embodiment, the PEG fragment comprises, or preferably consists of, 25 to 60 discrete repeat units m, preferably 30 to 50 discrete repeat units m. In a preferred embodiment, the PEG fragment comprises, or preferably consists of, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 discrete repeat units m. A discrete number of repeat units m may be selected from the group consisting of -(O-CH2-CH2). m The synthesis of such PEG fragments, and thus distinct PEGs, comprising or consisting of - units is described in WO 2004 / 073620 and WO 2013 / 033476. In preferred embodiments, the PEG fragment comprises, or preferably consists of, 28, 32, 36, 40, 44, 48, 52, 56, or 60 discrete repeat units m. In preferred embodiments, the PEG fragment comprises, or preferably consists of, 28 discrete repeat units m. In preferred embodiments, the PEG fragment comprises, or preferably consists of, 32 discrete repeat units m. In preferred embodiments, the PEG fragment comprises, or preferably consists of, 36 discrete repeat units m. In preferred embodiments, the PEG fragment comprises, or preferably consists of, 40 discrete repeat units m. In preferred embodiments, the PEG fragment comprises, or preferably consists of, 44 discrete repeat units m. In preferred embodiments, the PEG fragment comprises, or preferably consists of, 48 discrete repeat units m.

[0160] In a preferred embodiment, the PEG fragment comprises, or preferably consists of, 25 to 100 discrete consecutive repeat units m, preferably 25 to 60 discrete consecutive repeat units m. In a preferred embodiment, the PEG fragment comprises, or preferably consists of, 25 to 60 discrete consecutive repeat units m, preferably 30 to 50 discrete consecutive repeat units m. In a preferred embodiment, the PEG fragment comprises, or preferably consists of, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 discrete consecutive repeat units m. In preferred embodiments, the PEG fragment comprises, or preferably consists of, 28, 32, 36, 40, 44, 48, 52, 56, or 60 discrete numbers of consecutive repeat units m. In preferred embodiments, the PEG fragment comprises, or preferably consists of, 28 discrete numbers of consecutive repeat units m. In preferred embodiments, the PEG fragment comprises, or preferably consists of, 32 discrete numbers of consecutive repeat units m. In preferred embodiments, the PEG fragment comprises, or preferably consists of, 36 discrete numbers of consecutive repeat units m. In preferred embodiments, the PEG fragment comprises, or preferably consists of, 40 discrete numbers of consecutive repeat units m. In preferred embodiments, the PEG fragment comprises, or preferably consists of, 44 discrete numbers of consecutive repeat units m. In preferred embodiments, the PEG fragment comprises, or preferably consists of, 48 discrete numbers of consecutive repeat units m.

[0161] In a preferred embodiment, the —(O—CH—CH) of formula I* or formula I m The - moiety consists of 25 to 100 discrete repeating units m, preferably 25 to 60 discrete repeating units m. In a preferred embodiment, the -(O-CH2-CH2) mThe - moiety consists of 25 to 60 discrete repeating units m, preferably 30 to 50 discrete repeating units m. In a preferred embodiment, the -(O-CH2-CH2) m The - moiety is comprised of a non-consecutive number of repeating units m of 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60. In a preferred embodiment, the -(O-CH-CH) m The - moiety consists of a non-consecutive number of repeating units m of 28, 32, 36, 40, 44, 48, 52, 56, or 60. In a preferred embodiment, the -(O-CH-CH) m The - moiety consists of 28 discrete repeating units m. In a preferred embodiment, the -(O-CH-CH) m The - moiety consists of 32 discrete repeating units m. In a preferred embodiment, the -(O-CH-CH) m The - moiety consists of 36 discrete repeating units m. In a preferred embodiment, the -(O-CH-CH) m The - moiety consists of 40 discrete repeating units m. In a preferred embodiment, the -(O-CH-CH) m The - moiety consists of a discrete number of repeating units m of 44. In a preferred embodiment, the -(O-CH-CH) m The - moiety consists of 48 discrete repeating units m.

[0162] In a preferred embodiment, the —(O—CH—CH) of formula I* or formula I m The - moiety consists of 25 to 100 discrete consecutive repeating units m, preferably 25 to 60 discrete consecutive repeating units m. In a preferred embodiment, the -(O-CH2-CH2) m The - moiety consists of 25 to 60 discrete consecutive repeating units m, preferably 30 to 50 discrete consecutive repeating units m. In a preferred embodiment, the -(O-CH2-CH2) mThe - moiety consists of a discrete number of consecutive repeating units m of 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60. In a preferred embodiment, the -(O-CH-CH) m The - moiety consists of a discrete number of consecutive repeating units m of 28, 32, 36, 40, 44, 48, 52, 56, or 60. In preferred embodiments, the -(O-CH-CH) m The - moiety consists of 28 consecutive repeating units m, which are not consecutive. In a preferred embodiment, the -(O-CH-CH) m The - moiety consists of 32 consecutive repeating units m. In a preferred embodiment, the -(O-CH-CH) m The - moiety consists of 36 consecutive repeating units m. In a preferred embodiment, the -(O-CH-CH) m The - moiety consists of 40 consecutive repeating units m. In a preferred embodiment, the -(O-CH-CH) m The - moiety consists of 44 consecutive repeating units m in a discrete number. In a preferred embodiment, the -(O-CH-CH) m The - moiety consists of 48 consecutive repeating units m, each of which is a discrete number.

[0163] In another aspect, the present invention provides a composition comprising a polyplex, the polyplex comprising a conjugate of Formula I, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof, and a nucleic acid, the nucleic acid preferably comprising the conjugate: [ka]

[0164] Formula I (In the formula, [ka] is a single or double bond, n is an integer of 1 to 1500, preferably an integer of 2 to 1500, m is a discrete number of repeating units m from 2 to 100, preferably a discrete number of repeating units m from 4 to 60, and preferably the discrete number m is the discrete number of consecutive repeating -(O-CH-CH) units, and the discrete number of consecutive repeating -(O-CH-CH)- units is an arbitrary discrete number from 2 to 100, preferably 4 to 60; R 1 is the starting residue, preferably R 1 is -H or -CH3, R 2 are independently -H or an organic residue, and the -(NR 2 -CH2-CH2) n -The R in question 2 at least 80%, preferably at least 90%, of are H; Ring A is one or more R A1 R is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl optionally substituted at any position by A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo, or halogen, or two R A1 are combined with the atoms to which they are attached to form one or more fused C6-C 10 can form an aryl, C-C heteroaryl, or C-C cycloalkyl ring, and each fused aryl, heteroaryl, or cycloalkyl can be joined by one or more R A2 and optionally substituted with R A2 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen -SO3H or -OSO3H; X 1 is a divalent covalent linking moiety, X 2 is a divalent covalent linking moiety, L is a targeting fragment, preferably the targeting fragment is capable of binding to a cell, more preferably the targeting fragment is capable of binding to a cell surface receptor), non-covalently attached to The nucleic acid is a pharmaceutically active nucleic acid, and the pharmaceutically active nucleic acid is a nucleic acid that encodes a pharmaceutically active peptide or protein. 1 is —H. In a preferred embodiment, the R 1 is -CH3.

[0165] In another aspect, the present invention provides a polyplex comprising a conjugate of Formula I, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof, and a nucleic acid, wherein the nucleic acid is preferably a conjugate of the formula I: [ka]

[0166] Formula I (In the formula, [ka] is a single or double bond, n is an integer of 1 to 1500, preferably an integer of 2 to 1500, m is a discrete number of repeating units m from 2 to 100, preferably a discrete number of repeating units m from 4 to 60, and preferably the discrete number m is the discrete number of consecutive repeating -(O-CH-CH) units, and the discrete number of consecutive repeating -(O-CH-CH)- units is an arbitrary discrete number from 2 to 100, preferably 4 to 60; R 1 is the starting residue, preferably R 1 is -H or -CH3, R 2 are independently -H or an organic residue, and the -(NR 2 -CH2-CH2)n -The R in question 2 at least 80%, preferably at least 90%, of are H; Ring A is one or more R A1 R is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl optionally substituted at any position by A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo, or halogen, or two R A1 are combined with the atoms to which they are attached to form one or more fused C6-C 10 can form an aryl, C-C heteroaryl, or C-C cycloalkyl ring, and each fused aryl, heteroaryl, or cycloalkyl can be joined by one or more R A2 and optionally substituted with R A2 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen -SO3H or -OSO3H; X 1 is a divalent covalent linking moiety, X 2 is a divalent covalent linking moiety, L is a targeting fragment, preferably the targeting fragment is capable of binding to a cell, more preferably the targeting fragment is capable of binding to a cell surface receptor), non-covalently attached to The nucleic acid is a pharmaceutically active nucleic acid, and the pharmaceutically active nucleic acid is a nucleic acid that encodes a pharmaceutically active peptide or protein. 1 is —H. In a preferred embodiment, the R 1 is -CH3.

[0167] In some preferred embodiments, the conjugates of the invention comprise an LPEI fragment present as a dispersed polymer moiety, wherein n is from about 280 to about 700 with a dispersity of about 3 or less, preferably from about 350 to about 630 with a dispersity of about 2 or less, and more preferably from about 400 to 580 with a dispersity of about 1.2 or less, and the conjugates of the invention further comprise (i) from about 2 to about 80, a dispersity of about 2 or less, preferably from about 2 to about 70, and a dispersity of about 1.8 or less, more preferably from about 2 to about 50 repeat units with a dispersity of about 1.5, or (ii) a discrete number of repeat units, m, wherein the discrete number of repeat units, m, is preferably 12 or 24 repeat units.

[0168] In some embodiments, the conjugates of the present invention comprise LPEI fragments present as dispersed polymer moieties of about 17 and 25 KDa, with a dispersity of about 1.2 or less, and PEG fragments comprising, preferably consisting of, 12 repeating units. In some preferred embodiments, the conjugates of the present invention comprise LPEI fragments present as dispersed polymer moieties of about 17-25 KDa molecular weight and a dispersity of about 1.2 or less, and PEG fragments preferably consisting of 24 repeating units.

[0169] Targeting Fragments The conjugates of the present invention comprise a targeting fragment that enables the conjugates of the present invention and the polyplexes of the present invention to be directed to a specific target cell type, cell cluster, organ, or tissue. Typically and preferably, the targeting fragment is capable of binding to the target cell, preferably its cell receptor or cell surface receptor.

[0170] As used herein, the term "cell surface receptor" refers to a protein, glycoprotein, or lipoprotein that is present on the surface of a cell and that is typically and preferably a characteristic marker for cell recognition. Typically and preferably, said cell surface receptor can bind to ligands including hormones, neurotransmitters, cytokines, growth factors, cell adhesion molecules, or nutrients in the form of peptides, small molecules, sugars and oligosaccharides, lipids, amino acids, and other binding moieties such as antibodies, aptamers, affibodies, antibody fragments, and the like.

[0171] The conjugates and polyplexes of the present invention containing targeting fragments are intended to mimic such ligand-receptor interactions. Thus, in a preferred embodiment, the targeting fragments can bind to cell surface receptors. In a preferred embodiment, the cell surface receptors are selected from growth factor receptors, extracellular matrix proteins, peripheral membrane proteins, transmembrane proteins, preferably type II transmembrane proteins, cytokine receptors, hormone receptors, glycosylphosphatidylinositol (GPI)-anchored membrane proteins, carbohydrate-binding integral membrane proteins, asialoglycoprotein receptors (ASGPr), lectins, ion channels, G protein-coupled receptors, and enzyme-linked receptors such as tyrosine kinase-coupled receptors.

[0172] In a preferred embodiment, the targeting fragment is capable of binding to a cell surface receptor. In a preferred embodiment, the cell surface receptor is selected from growth factor receptors, extracellular matrix proteins, peripheral membrane proteins, transmembrane proteins, preferably type II transmembrane proteins, cytokine receptors, hormone receptors, glycosylphosphatidylinositol (GPI)-anchored membrane proteins, carbohydrate-binding integral membrane proteins, lectins, ion channels, G protein-coupled receptors, enzyme-linked receptors such as tyrosine kinase-coupled receptors, etc. In a preferred embodiment, the cell surface receptor is a growth factor receptor. In a preferred embodiment, the cell surface receptor is an extracellular matrix protein. In a preferred embodiment, the cell surface receptor is a cytokine receptor. In a preferred embodiment, the cell surface receptor is a hormone receptor. In a preferred embodiment, the cell surface receptor is a glycosylphosphatidylinositol (GPI)-anchored membrane protein. In a preferred embodiment, the cell surface receptor is a carbohydrate-binding integral membrane protein. In a preferred embodiment, the cell surface receptor is a lectin. In a preferred embodiment, the cell surface receptor is an ion channel. In a preferred embodiment, the cell surface receptor is an enzyme-linked receptor, preferably the enzyme-linked receptor is a tyrosine kinase-coupled receptor. In a preferred embodiment, the cell surface receptor is a peripheral membrane protein. In a preferred embodiment, the cell surface receptor is a transmembrane protein. In a preferred embodiment, the cell surface receptor is a type II transmembrane protein.

[0173] In a preferred embodiment, the cell surface receptor is selected from epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), prostate-specific membrane antigen (PSMA), insulin-like growth factor 1 receptor (IGF1R), vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), and fibroblast growth factor receptor (FGFR). In a preferred embodiment, the cell surface receptor is epidermal growth factor receptor (EGFR). In a preferred embodiment, the cell surface receptor is human epidermal growth factor receptor 2 (HER2). In a preferred embodiment, the cell surface receptor is prostate-specific membrane antigen (PSMA). In a preferred embodiment, the cell surface receptor is insulin-like growth factor 1 receptor (IGF1R). In a preferred embodiment, the cell surface receptor is vascular endothelial growth factor receptor (VEGFR). In a preferred embodiment, the cell surface receptor is platelet-derived growth factor receptor (PDGFR). In a preferred embodiment, the cell surface receptor is fibroblast growth factor receptor (FGFR).

[0174] The targeting fragment according to the present invention is intended to position and deliver, particularly selectively, the polyplexes and payloads, e.g., nucleic acids, of the present invention to a desired target, particularly a desired target cell. Furthermore, the conjugates of the present invention comprising such targeting fragments not only enable the selective delivery of the conjugates and polyplexes to a target, e.g., a target cell, but also enable internalization and promote selective cellular uptake of the polyanion payload and nucleic acid payload, respectively, by the target, particularly the target cell. Thus, the targeting fragment according to the present invention represents a portion of the conjugates and polyplexes of the present invention capable of specific binding to a selected target, preferably a selected target cell, more preferably a cellular receptor.

[0175] In preferred embodiments, the targeting fragment is capable of binding to a target cell. In preferred embodiments, the targeting fragment is capable of binding to a selected target cell type. In preferred embodiments, the targeting fragment is capable of binding to a target cell receptor. In preferred embodiments, the targeting fragment is capable of binding to a target cell surface receptor.

[0176] In preferred embodiments, the targeting fragment functions to bind to a target cell. In preferred embodiments, the targeting fragment functions to bind to a selected target cell type. In preferred embodiments, the targeting fragment functions to bind to a target cell receptor, and in preferred embodiments, the targeting fragment functions to bind to a target cell surface receptor.

[0177] In preferred embodiments, the targeting fragment is capable of specifically binding to a target cell. In preferred embodiments, the targeting fragment is capable of specifically binding to a selected target cell type. In preferred embodiments, the targeting fragment is capable of specifically binding to a target cell receptor. In preferred embodiments, the targeting fragment is capable of specifically binding to a target cell surface receptor.

[0178] In one embodiment, specifically binding to the target cell, target cell receptor, or target cell surface receptor means that the targeting fragment and the conjugate of the present invention and / or the polyplex of the present invention bind to the target cell, target cell receptor, or target cell surface receptor, respectively, at least two-fold, preferably at least three-fold, more preferably at least four-fold, and even more preferably at least five-fold stronger (typically, preferably measured by dissociation constant (KD)) than they bind to other non-target cells, cell receptors, or cell surface receptors. Preferably, the targeting fragment binds to the target cell, target cell receptor, or target cell surface receptor, respectively, at least two-fold, preferably at least three-fold, more preferably at least four-fold, and even more preferably at least five-fold stronger (typically, preferably measured by dissociation constant (KD)). ... -5 Less than M, preferably 10 -6 Less than M, more preferably 10 -7M, and even more preferably less than 10 -8 Binds to selected cell surface receptors with a KD less than M.

[0179] In one embodiment, specifically binding to the target cell, target cell receptor, or target cell surface receptor means that the targeting fragment and the conjugate and / or polyplex of the present invention bind to the target cell, target cell receptor, or target cell surface receptor, respectively, at least 2-fold, preferably at least 3-fold, more preferably at least 5-fold, even more preferably at least 10-fold, and even more preferably at least 100-fold stronger than the corresponding conjugate and / or polyplex that is identical to the conjugate and / or polyplex of the present invention but contains a nonspecific fragment such as a hydroxyl group or an -OMe moiety, preferably an -OMe moiety, instead of the targeting fragment. Binding to the target cell, target cell receptor, or target cell surface receptor is typically measured by the dissociation constant (KD). Preferably, the targeting fragment binds to the target cell, target cell receptor, or target cell surface receptor at a dissociation constant greater than or equal to 10. -5 Less than M, preferably 10 -6 Less than M, more preferably 10 -7 M, and even more preferably less than 10 -8 It binds to a selected target cell surface receptor with a KD less than M. In preferred embodiments, the binding or the specific binding, and thus the binding level of the conjugates of the invention and the polyplexes of the invention, respectively, can be determined by binding or displacement assays, or by FRET or other measures demonstrating the interaction between the targeting fragment and the cell receptor, preferably a cell surface receptor.

[0180] As used herein with respect to the binding of a targeting fragment to a cell, cell receptor, or cell surface receptor, the term "binding" preferably refers to interactions via non-covalent bonds, such as electrostatic interactions, van der Waals interactions, hydrogen bonds, hydrophobic interactions, ionic bonds, charge interactions, affinity interactions, and / or dipole-dipole interactions.

[0181] In another embodiment, said specific binding to the target cell, target cell receptor or target cell surface receptor results in a biological effect caused by said specific binding of the targeting fragment and the conjugate and / or polyplex of the invention, respectively, and / or caused by the delivered conjugate and / or polyplex of the invention and the polyanion payload and nucleic acid payload, respectively, which biological effect is at least 2-fold, preferably at least 3-fold, more preferably at least 5-fold, even more preferably at least 10-fold, even more preferably at least 25-fold, at least 50-fold, or at least 100-fold greater than said biological effect of the non-target cell, non-target cell receptor or non-target cell surface receptor.

[0182] In another embodiment, said specific binding to a target cell, target cell receptor, or target cell surface receptor results in a biological effect caused by said specific binding of a targeting fragment and a conjugate and / or a polyplex of the invention, respectively, and / or caused by the delivered conjugate and / or polyplex of the invention and a polyanion payload and a nucleic acid payload, respectively, which biological effect is at least 2-fold, preferably at least 3-fold, more preferably at least 5-fold, even more preferably at least 10-fold, and even more preferably at least 25-fold, at least 50-fold, or at least 100-fold greater than the biological effect caused by a corresponding conjugate and / or polyplex identical to the conjugate and / or polyplex of the invention but comprising a non-specific fragment such as a hydroxyl group or an -OMe moiety, preferably an -OMe moiety, instead of the targeting fragment.

[0183] Binding and specific binding can also be determined by measuring activation of protein signaling, thus measuring protein phosphorylation or protein expression, mRNA expression in cells or tissues (using Western blot analysis, real-time PCR, RNAseq, IHC, etc.). The level of delivery of the polyplexes of the present invention to a particular tissue can be measured by comparing the amount of protein produced in cells with overexpression with cells with normal and low expression, by measuring protein secretion by Western blot analysis, luminescence / fluorescence assays, flow cytometry assays, or ELISA, ECLIA, etc. The level of delivery can also be measured by measuring cytotoxicity using cell viability assays or cell death assays, including MTT, methylene blue assay, CellTiter-Glo assay, and propidium iodide assay. by comparing the amount of protein produced in the tissue with the weight of the tissue, by comparing the amount of therapeutic and / or prophylactic agent in the tissue with the weight of the tissue, by comparing the amount of protein produced in the tissue with the total amount of protein in the tissue, by comparing the amount of therapeutic and / or prophylactic agent in the tissue with the total amount of therapeutic and / or prophylactic agent in the tissue. It will be understood that delivery of the polyplexes of the invention to target cells or target tissues need not be determined in the subject being treated, but can be determined in surrogates such as animal or cell models.

[0184] Thus, in preferred embodiments, the biological effect is selected from (i) activation of protein signaling, (ii) protein expression, (iii) mRNA expression in cells or tissues, (iv) expression or secretion of the delivered nucleic acid, e.g., a downstream protein from the delivered nucleic acid, in cells / tissues in which the target cell surface receptor is overexpressed compared to normal cells / tissues or cells / tissues with low expression, and (v) cytotoxicity.

[0185] In one embodiment, the target cells include, but are not limited to, hepatocytes, epithelial cells, hematopoietic cells, epithelial cells, endothelial cells, lung cells, bone cells, stem cells, mesenchymal cells, neural cells, cardiac cells, adipocytes, and vascular smooth muscle cells. Thus, in one embodiment, the target cells are cells in the liver. In one embodiment, the target cells are epithelial cells. In one embodiment, the target cells are hepatocytes. In one embodiment, the target cells are hematopoietic cells. In one embodiment, the target cells are muscle cells. In one embodiment, the target cells are endothelial cells. In one embodiment, the target cells are tumor cells or cells in the tumor microenvironment. In one embodiment, the target cells are blood cells. In one embodiment, the target cells are cells in lymph nodes. In one embodiment, the target cells are cells in the lungs. In one embodiment, the target cells are cells in the skin. In one embodiment, the target cells are spleen cells. In one embodiment, the target cells are antigen-presenting cells, such as professional antigen-presenting cells in the spleen. In one embodiment, the target cells are dendritic cells in the spleen. In one embodiment, the target cell is a T cell. In one embodiment, the target cell is a B cell. In one embodiment, the target cell is an NK cell. In one embodiment, the target cell is a monocyte.

[0186] In some embodiments, the targeting fragment interacts selectively or preferentially with a particular cell type. The targeting fragment not only serves to selectively target the conjugates and polyplexes of the present invention to a particular cell, but also typically promotes selective uptake of the conjugates and corresponding polyplexes of the present invention within a particular cell type. In some embodiments, the targeting fragment interacts selectively or preferentially with a particular cell surface receptor. When the targeting fragment of a conjugate and / or polyplex interacts selectively or preferentially with a cell surface receptor, the conjugate and / or polyplex can be selectively or preferentially taken up by cells containing that cell surface receptor.

[0187] In preferred embodiments, the targeting fragment is a peptide, a protein, a small molecule ligand, a saccharide, an oligosaccharide, a lipid, an amino acid, wherein the peptide, the protein, the small molecule ligand, the saccharide, the oligosaccharide, the lipid, or the amino acid is selected from a hormone, a neurotransmitter, a cytokine, a growth factor, a cell adhesion molecule, or a nutrient, and the targeting fragment is an antibody, an antibody fragment, an aptamer, or an affibody.

[0188] As used herein, particularly with respect to the targeting fragments of the present invention, the term "small molecule ligand" refers to a chemical moiety having a molecular weight of at least 75 g / mol, preferably at least 100 g / mol, and more preferably at least 200 g / mol, and preferably having a molecular weight of less than about 2000 g / mol. In some embodiments, the small molecule has a molecular weight of less than about 1500 g / mol, more preferably less than about 1000 g / mol. In a more preferred embodiment, the small molecule has a molecular weight of less than about 800 g / mol, and even more preferably less than about 500 g / mol. As used herein, particularly with respect to the targeting fragments of the present invention, the term "small molecule ligand" more preferably refers to such a ligand that binds, preferably specifically binds, to a target cell, a target cell receptor, or preferably a target cell surface receptor. In a preferred embodiment, the small molecule ligand has a molecular weight of at least 75 g / mol, preferably at least 100 g / mol, and more preferably at least 200 g / mol, and preferably has a molecular weight of less than about 2000 g / mol, and preferably less than about 1500 g / mol. In preferred embodiments, the small molecule ligand has a molecular weight of at least 75 g / mol, preferably at least 100 g / mol, and more preferably at least 200 g / mol, and preferably has a molecular weight of less than about 2000 g / mol, preferably less than about 1500 g / mol, and the small molecule ligand is capable of binding, preferably specifically binding, to a target cell surface receptor.

[0189] In some embodiments, the targeting fragment is a natural, natural, or modified ligand or its paralog, or a non-natural ligand such as an antibody, a single-chain variable fragment (scFv), or an antibody mimic such as an affibody. In preferred embodiments, the targeting fragment is a natural, natural, or modified cell surface antigen ligand or its paralog, or a non-natural cell surface antigen ligand, such as an antibody, a single-chain variable fragment (scFv), or an antibody mimic, such as an affibody. In preferred embodiments, the targeting fragment is a natural, natural, or modified cell surface receptor ligand or its paralog, or a non-natural cell surface receptor ligand, such as an antibody, a single-chain variable fragment (scFv), or an antibody mimic, such as an affibody. In preferred embodiments, the targeting fragment is a small molecule ligand, a peptide, a protein, an aptamer, a natural, natural, or modified ligand, and / or a paralog thereof. In a preferred embodiment, the targeting fragment is a small molecule ligand, peptide, protein, aptamer, natural, native or modified cell surface antigen ligand and / or a paralog thereof, wherein the small molecule ligand has a molecular weight of at least 75 g / mol, preferably at least 100 g / mol, more preferably at least 200 g / mol, and preferably has a molecular weight of less than about 2000 g / mol, preferably less than about 1500 g / mol. In a preferred embodiment, the targeting fragment is a small molecule ligand, peptide, protein, aptamer, natural, native or modified cell surface receptor ligand and / or a paralog thereof, wherein the small molecule ligand has a molecular weight of at least 75 g / mol, preferably at least 100 g / mol, more preferably at least 200 g / mol, and preferably has a molecular weight of less than about 2000 g / mol, preferably less than about 1500 g / mol. In preferred embodiments, the targeting fragment is a small molecule ligand, a peptide, a protein, an aptamer, a natural, natural or modified ligand and / or a paralog thereof, an antibody, a single chain variable fragment (scFv), or an antibody mimetic such as an affibody.

[0190] In a preferred embodiment, the targeting fragment is a small molecule ligand, a peptide, a protein, an aptamer, a natural, natural, or modified cell surface receptor ligand, and / or a paralog thereof. In a preferred embodiment, the targeting fragment is a small molecule ligand, a peptide, a protein, an aptamer, a natural, natural, or modified ligand, and / or a paralog thereof, and the small molecule ligand, the peptide, the protein, the aptamer, the natural, natural, or modified ligand, and / or the paralog thereof can bind to, preferably selectively bind to, a cell surface receptor. In a preferred embodiment, the targeting fragment is a small molecule ligand. In a preferred embodiment, the targeting fragment is a small molecule ligand, and the small molecule ligand can bind to, preferably selectively bind to, a cell surface receptor. In a preferred embodiment, the targeting fragment is a peptide. In a preferred embodiment, the targeting fragment is a peptide, and the peptide can bind to, preferably selectively bind to, a cell surface receptor. In a preferred embodiment, the targeting fragment is a protein. In a preferred embodiment, the targeting fragment is a protein, which can bind, preferably selectively, to a cell surface receptor. In a preferred embodiment, the targeting fragment is an aptamer. In a preferred embodiment, the targeting fragment is an aptamer, which can bind, preferably selectively, to a cell surface receptor. In a preferred embodiment, the targeting fragment is a natural, natural, or modified ligand and / or a paralog thereof, preferably a natural, natural, or modified cell surface receptor ligand and / or a paralog thereof. In a preferred embodiment, the targeting fragment is a natural, natural, or modified ligand and / or a paralog thereof, which can bind, preferably selectively, to a cell surface receptor. In a preferred embodiment, the targeting fragment is an antibody, a single-chain variable fragment (scFv), or an antibody mimetic, such as an affibody.In a preferred embodiment, the targeting fragment is an antibody, a single-chain variable fragment (scFv), or an antibody mimetic, such as an affibody, which is capable of binding, preferably selectively binding, to a cell surface receptor.

[0191] In a preferred embodiment, the targeting fragment is a small molecule ligand, a peptide, a protein, an aptamer, an antibody, an antibody fragment, preferably a single chain variable fragment (scFv), an antibody mimetic, preferably an affibody, a nanobody, a diabody, a designed ankyrin repeat protein (DARPin), a growth factor or a functional fragment thereof, preferably an antibody mimetic selected from hEGF), a hormone or a functional fragment thereof, preferably insulin, a cytokine or a functional fragment thereof, an integrin, an interleukin or a functional fragment thereof, an enzyme, a nucleic acid, a fatty acid, a carbohydrate, a monosaccharide, an oligosaccharide or a polysaccharide, a peptidoglycan, a glycopeptide, asialoorosomucoid, mannose-6-phosphate, mannose, Sialyl-Lewis x , N-acetyllactosamine, galactose, a lysosomotropic agent, and / or a nuclear localization agent, preferably a T antigen, a tumor-low pH inserting peptide (PHLIP), a p32 targeting peptide, preferably a LyP-1 tumor-homing peptide, insulin-like growth factor 1, vascular endothelial growth factor, platelet-derived growth factor, and / or fibroblast growth factor.

[0192] In some embodiments, the targeting fragment is a non-natural ligand such as an antibody or antibody fragment (e.g., a single-chain variable fragment (scFv), an antibody mimetic, e.g., an affibody, nanobody, diabody, designed ankyrin repeat protein (DARPin), or other antibody variant). In some embodiments, the targeting fragment is a growth factor or a fragment thereof, preferably a functional fragment (e.g., hEGF); a hormone or a fragment thereof, preferably a functional fragment (e.g., insulin), asialoorosomucoid, mannose-6-phosphate, mannose, Sialyl-Lewis x, N-acetyllactosamine, galactose, lysosomal inhibitors and / or nuclear localization agents (e.g., T antigens), tumor low pH inserting peptides (PHLIPs), p32 targeting peptides, such as LyP-1 tumor homing peptide, insulin-like growth factor 1, vascular endothelial growth factor, platelet-derived growth factor, and / or fibroblast growth factor. Further non-limiting examples of targeting fragments include enzymes, nucleic acids, fatty acids, carbohydrates, mono-, oligo-, or polysaccharides, peptidoglycans, and glycopeptides.

[0193] In a preferred embodiment, the targeting fragment is a small molecule ligand, a peptide, a protein, an aptamer, an antibody, an antibody fragment, preferably Fab, Fab', F(ab')2 or scFv, an antibody mimetic, preferably an affibody, nanobody, diabody, designed ankyrin repeat protein (DARPin), a growth factor or functional fragment thereof, preferably an antibody mimetic selected from hEGF, a hormone or functional fragment thereof, preferably insulin, a cytokine or functional fragment thereof, an interleukin or functional fragment thereof, an enzyme, a nucleic acid, a fatty acid, a carbohydrate, a monosaccharide, oligosaccharide or polysaccharide, peptidoglycan, a glycopeptide, asialoorosomucoid, mannose-6-phosphate, mannose, Sialyl-Lewis x , N-acetyllactosamine, galactose, a lysosomotropic agent, and / or a nuclear localization agent, preferably a T antigen, a tumor-low pH inserting peptide (PHLIP), a p32 targeting peptide, preferably a LyP-1 tumor-homing peptide, insulin-like growth factor 1, vascular endothelial growth factor, platelet-derived growth factor, and / or fibroblast growth factor.

[0194] In some embodiments, the targeting fragment L is selected from the group consisting of hEGF; an anti-HER2 peptide, preferably an anti-HER2 antibody or affibody; DUPA; a folate receptor targeting fragment, folic acid; a somatostatin receptor targeting fragment, preferably somatostatin and / or octreotide; an integrin targeting fragment, preferably an arginine-glycine-aspartic acid (RGD)-containing fragment; a low pH insertion peptide; an asialoglycoprotein receptor targeting fragment, preferably asialoorosomucoid; an insulin receptor targeting fragment, preferably insulin; a mannose-6-phosphate receptor targeting fragment, preferably mannose-6-phosphate; a mannose receptor targeting fragment, preferably mannose; or Sialyl Lewis. x selected from an antigen targeting fragment, preferably E-selectin; a sigma-2 receptor agonist, preferably N,N-dimethyltryptamine (DMT), a sphingolipid-derived amine and / or a steroid, more preferably progesterone; a p32 targeting ligand, preferably an anti-p32 antibody or a p32-binding LyP-1 tumor-homing peptide; a Trop-2 targeting fragment, preferably an anti-Trop-2 antibody and / or antibody fragment; insulin-like growth factor 1; vascular endothelial growth factor; platelet-derived growth factor; and fibroblast growth factor.

[0195] In some embodiments, the targeting fragment L is a targeting fragment derived from hEGF; an anti-HER2 peptide, preferably an anti-HER2 antibody or affibody; DUPA; folate; a somatostatin receptor targeting fragment, preferably somatostatin and / or octreotide; an integrin targeting fragment, preferably an arginine-glycine-aspartic acid (RGD)-containing fragment; a low pH insertion peptide; an asialoglycoprotein receptor targeting fragment, preferably asialoorosomucoid; an insulin receptor targeting fragment, preferably insulin; a mannose-6-phosphate receptor targeting fragment, preferably mannose-6-phosphate; a mannose receptor targeting fragment, preferably mannose; or Sialyl Lewis. xselected from an antigen targeting fragment, preferably E-selectin; a sigma-2 receptor agonist, preferably N,N-dimethyltryptamine (DMT), a sphingolipid-derived amine and / or a steroid, more preferably progesterone; a p32 targeting ligand, preferably an anti-p32 antibody or a p32-binding LyP-1 tumor-homing peptide; a Trop-2 targeting fragment, preferably an anti-Trop-2 antibody and / or antibody fragment; insulin-like growth factor 1; vascular endothelial growth factor; platelet-derived growth factor; and fibroblast growth factor.

[0196] In preferred embodiments, the targeting fragment is an EGFR targeting fragment; a PSMA targeting fragment, an anti-HER2 peptide, preferably an anti-HER2 antibody or affibody; folate; a somatostatin receptor targeting fragment, preferably somatostatin and / or octreotide; an integrin targeting fragment, preferably an arginine-glycine-aspartic acid (RGD)-containing fragment; a low pH insertion peptide; an asialoglycoprotein receptor targeting fragment, preferably asialoorosomucoid; an insulin receptor targeting fragment, preferably insulin; a mannose-6-phosphate receptor targeting fragment, preferably mannose-6-phosphate; a mannose receptor targeting fragment, preferably mannose; or Sialyl Lewis. x selected from an antigen targeting fragment, preferably E-selectin; a sigma-2 receptor agonist, preferably N,N-dimethyltryptamine (DMT), a sphingolipid-derived amine and / or a steroid, more preferably progesterone; a p32 targeting ligand, preferably an anti-p32 antibody or a p32-binding LyP-1 tumor-homing peptide; a Trop-2 targeting fragment, preferably an anti-Trop-2 antibody and / or antibody fragment; insulin-like growth factor 1; vascular endothelial growth factor; platelet-derived growth factor; and fibroblast growth factor.

[0197] In a preferred embodiment, the targeting fragment is an epidermal growth factor, such as human epidermal growth factor (hEGF), and typically and preferably, the coupling to the remainder of the conjugate is via an amino group on the hEGF, which can be selectively taken up by cells that have increased expression (e.g., overexpression) of the human epidermal growth factor receptor (EGFR).

[0198] In a preferred embodiment, the targeting fragment is capable of binding to the epidermal growth factor receptor (EGFR), also referred to herein as an EGFR targeting fragment.

[0199] EGFR is a transmembrane glycoprotein that is a member of the protein kinase superfamily and a receptor for members of the epidermal growth factor family.EGFR is a cell surface protein that binds to epidermal growth factor, thereby inducing receptor dimerization and tyrosine autophosphorylation, leading to cell proliferation.In a preferred embodiment, the EGFR targeting fragment can bind to an epitope on the extracellular domain of EGFR.

[0200] In a preferred embodiment, the targeting fragment is capable of binding to cells expressing EGFR. In a preferred embodiment, the targeting fragment is capable of binding to cells overexpressing EGFR. In one embodiment, the cells overexpressing EGFR refer to an elevated level of EGFR expressed in the cells in a particular tissue compared to the level of EGFR measured in normal healthy cells of the same type of tissue under similar conditions. In one embodiment, the cells overexpressing EGFR refer to an increase in the level of EGFR in the cells compared to the level in the same cells or closely related non-malignant cells under normal physiological conditions. In one embodiment, the cells overexpressing EGFR refer to at least 10-fold, more preferably at least 20-fold, expression of EGFR compared to the expression of EGFR in normal cells or normal tissues.

[0201] In a preferred embodiment, the targeting fragment can bind to the cell that expresses or overexpresses EGFR.For example, EGFR is overexpressed in glioma and carcinoma, and epithelial origin cancer, including head and neck, thyroid, breast, ovary, colon, gastro-colorectal, gastro-small intestine, cervix, bladder, lung, nasopharyngeal and esophageal tissue, for example, squamous cell (for example, Gan et al., J Cell Mol Med.2009 Sep;13(9b):3993-4001; Aratani et al., Anticancer Research June 2017,37(6)3129-3135), especially in glioma, non-small cell lung carcinoma, breast cancer, glioblastoma, squamous cell carcinoma, for example head and neck squamous cell carcinoma, small intestine, colorectal carcinoma, adenocarcinoma, ovarian carcinoma, bladder carcinoma or prostate carcinoma, and their metastases.

[0202] EGFR expression and overexpression are preferably detected using monoclonal antibodies targeting EGFR, for example, by immunohistochemical methods (e.g., as described in Kriegs et al., Nature, 2019, 9:13564; Prenzel et al., Endocr Relat Cancer 8, 11-31, 2001). A cutoff of 5% or more EGFR-positive cells can be used to define EGFR expression in different types of tissues or cells. Therefore, cells or tissues with less than 5% positive cells can be considered negative.

[0203] In a preferred embodiment, the targeting fragment is capable of specifically binding to EGFR. Typically, specific binding is at least about 1×10 -3 M ~ approx. 1×10 -12 Binding affinity or dissociation constant K of targeting fragments in the range of M D In a preferred embodiment, the targeting fragment is capable of specifically binding to EGFR, and typically and preferably, the affinity or specific binding is such that the dissociation constant (K D ) and the affinity or specific binding is measured by -3 Less than M, preferably 10 -4 M, more preferably less than 10-5 M, more preferably less than 10 -6 Less than M, more preferably 10 -7 M, and even more preferably less than 10 -8 M, and even more preferably less than 10 -9 K less than M D In a preferred embodiment, the targeting fragment is capable of specifically binding to EGFR, and typically and preferably, the affinity or specific binding is such that the dissociation constant (K D ) and the specific binding is measured by 10 -3 Under M, 10 -4 Under M, 10 -5 Under M, 10 -6 Under M, 10 -7 Under M, 10 -8 Less than M and 10 -9 K less than M D To detect binding or complexes or measure affinity, molecules can be analyzed using competitive binding assays, such as, typically and preferably, a Biacore 3000 instrument (Biacore Inc., Piscataway, NJ; see, for example, Wei-Ting Kuo et al., PLoS One. 2015, 10(2): e0116610 or U.S. Patent Application Publication No. 2017224620 A1). Preferably, binding results in the formation of a complex between the EGFR targeting fragment and EGFR, and the binding or complex can be detected.

[0204] In a preferred embodiment, the targeting fragment is an EGFR antibody, an EGFR affibody, an EGFR aptamer, an EGFR targeting peptide, or an EGFR targeting tyrosine kinase inhibitor. In a preferred embodiment, the EGFR targeting fragment is an EGFR antibody, an EGFR affibody, an EGFR aptamer, an EGFR targeting peptide, or an EGFR targeting tyrosine kinase inhibitor.

[0205] In a preferred embodiment, the targeting fragment is an EGFR targeting peptide. EGFR targeting peptide typically and preferably refers to a peptide ligand of EGFR. Such peptide ligands are known to those skilled in the art and are described, for example, in U.S. Patent Application Publication No. 2017224620 A1 and Gent et al., 2018, Pharmaceuticals 2018, 10, 2 (the disclosures of which are incorporated herein by reference in their entirety). EGFR targeting peptides have low immunogenicity and exhibit good penetration into solid tumor tissues.

[0206] In a preferred embodiment, the EGFR targeting peptide has a molecular weight of about 1000 g / mol to about 2000 g / mol, preferably about 1100 g / mol to about 1900 g / mol, more preferably about 1200 g / mol to about 1800 g / mol, and even more preferably about 1300 g / mol to about 1700 g / mol.

[0207] In a preferred embodiment, the EGFR targeting peptide comprises, or preferably consists of, the sequence YHWYGYTPQNVI(GE11) (SEQ ID NO: 9). In a preferred embodiment, the targeting fragment comprises, or preferably consists of, the sequence YHWYGYTPQNVI(GE11) (SEQ ID NO: 9). GE-11 has excellent affinity for EGFR and also exhibits binding specificity for EGFR (kd = 22 nM) (Ruoslahti et al., Adv. Mater. 2012, 24, 3747-3756; Li et al., J. Res. Commun. 2005, 19, 1978-1985). GE11 displaces from EGFR after the addition of the physiological ligand EGF, demonstrating both its selective binding to EGFR and its receptor affinity. GE11 has been reported to have high potential for accelerating nanoparticle endocytosis due to an alternative EGFR-dependent actin-driven pathway. (Mickeler et al., Nano Lett. 2012, 12, 3417-3423; Song et al., FASEB J. 2009, 23, 1396-1404) It has been shown that EGFR levels on the surface of cancer cells remain constant after treatment with GE11 polyplexes, indicating an EGFR recycling process with long-term receptivity of cells to circulating GE11 polyplexes.

[0208] In a preferred embodiment, the EGFR targeting fragment comprises, or preferably consists of, GE11 (SEQ ID NO: 9), particularly for use in treating solid tumors characterized by EGFR-overexpressing cells. Conjugates and polyplexes of the invention comprising, or preferably consisting of, GE11 as the targeting fragment are believed to be stable polyplexes, ensuring that the polyanion and nucleic acid payload are not released before the polyplex reaches its target cell.

[0209] In a preferred embodiment, the targeting fragment is an EGFR antibody. EGFR antibody refers to an antibody that binds to EGFR. In a preferred embodiment, the EGFR antibody is human. In a preferred embodiment, the EGFR antibody is a humanized EGFR antibody. In a preferred embodiment, the EGFR antibody is a monoclonal human. In a preferred embodiment, the EGFR antibody is a humanized EGFR antibody. In a preferred embodiment, the EGFR antibody is a monoclonal fully human EGFR antibody. In another preferred embodiment, the EGFR antibody is an scFv or Fab fragment.

[0210] EGFR antibodies are known to those skilled in the art and are described, for example, in WO 2008 / 105773 and WO 2017 / 185662 (the disclosures of which are incorporated herein by reference in their entireties), and include antibodies such as bevacizumab, panitumumab, cetuximab, tomzotuximab, futuximab, zatuximab, modetuximab, imgatuzumab, zalutumumab, matuzumab, necitumumab, nimotuzumab, CEVIAvax EGF, clone EGFR, L8A4, E6.2, TH190DS, Pep2, Pep3, LR-DM1, P1X, YC088, ratML66, FM32 9, TGM10-1, F4, 2F8, 15H8, TAB-301MZ-S(P), mAb528, 2224, E7.6.3, C225, CBL155, MR1 , MR1, L211C, N5-4, TH83DS, L2-12B, 15H8, 12Do3, 7A7, 42C11(MOB-1078z), PABL-080, HPAB-2204LY-S(P), VHH205, ABT-806, Tab-271MZ, Hu225, LA22, Fab fragment DL11, Fab fragment DX 1-6, VHH104, OA-cb6, 07D06, Fab fragment HPAB-0419-FY-F(E), Fab fragment TAB-285MZ-F(E), Fab fragment One piece TAB-293MZ-F(E), Fab fragment HPAB-0136-YJ-F(E), FGF-R2, EG-19-11, Fab fragment pSEX81-63, DX 1-4, scFv fragment DX 1-6, EG-26-11, EG-26-11, DX1-4, TAB-326MZ, scFv fragment 528, scFv fragment LA1, scFv fragment 07D06, single domain antibody VHH139, scFv fragment EG-19-11, single domain antibody VHH134, single domain antibody 9G8, ABT-414, AMG-595, and IMGN-289. Those skilled in the art will appreciate that any antibody that recognizes and / or specifically binds to EGFR may be used in accordance with the present invention.

[0211] In a preferred embodiment, the targeting fragment is an EGFR inhibitor. EGFR inhibitors refer to targeting fragments that block cell surface localization and signal transduction of EGFR, such as oligosaccharyl transferase inhibitors like nerve growth inhibitor-1, or EGFR kinase inhibitors like afatinib, erlotinib, osimertinib, and gefitinib. EGFR inhibitors are known to those skilled in the art and are described, for example, in International Publication No. 2018078076 and U.S. Patent Application Publication No. 2017224620A1 (the disclosures of which are incorporated herein by reference in their entirety).

[0212] In a preferred embodiment, the targeting fragment is an EGFR aptamer.Preferred EGFR targeting aptamers include, but are not limited to, those disclosed in Na Li et al. (PLoS One.2011;6(6):e20299), Deng-LiangWang et al. (Biochemical and Biophysical Res Com,453(4),2014,pp 681-685), Min Woo Kim et al. (Theranostics 2019;9(3):837-852), Akihiro Eguchi et al. (JACS Au 2021,1,5,578-585) or Yingpan Song et al. (RSC Adv.,2020,10,28355-28364), the disclosures of which are incorporated herein by reference in their entirety.

[0213] The term EGFR aptamer also includes EGFR aptamer derivatives and / or functional fragments of EGFR aptamers. In some embodiments, in an EGFR aptamer derivative, less than 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 nucleic acid is substituted relative to the corresponding EGFR aptamer. In some embodiments, the sequence of the EGFR aptamer derivative is at least 80%, preferably 85%, more preferably 90%, even more preferably 95%, and most preferably 99% identical to the corresponding EGFR aptamer.

[0214] In a preferred embodiment, the targeting fragment is an EGFR affibody. Preferred EGFR affibodies include, but are not limited to, ZEGFR:1907, ZEGFR:2377, or ZEGFR:03115 (available from Affibody Medical AB) or dimeric forms of these affibodies. In a preferred embodiment, the EGFR affibody has the sequence of SEQ ID NO:8.

[0215] In a preferred embodiment, the targeting fragment is the EGFR ligand epidermal growth factor (EGF). Thus, in a preferred targeting fragment, the targeting fragment is epidermal growth factor (EGF). In a preferred embodiment, the targeting fragment is human EGF (hEGF), mouse EGF (mEGF), rat EGF, or guinea pig EGF. In a highly preferred embodiment, the targeting fragment is human EGF (hEGF). In a highly preferred embodiment, the targeting fragment comprises, and preferably consists of, the sequence of SEQ ID NO: 7.

[0216] In some embodiments, EGF is modified, for example, by deleting or replacing one or more amino acids, or by truncating EGF.Modified and / or truncated EGF molecules are disclosed, for example, in International Publication No. 2019023295A1.EGF has many residues that are conserved across rat, mouse, guinea pig, and human species (Savage et al., J.Biol.Chem., 247:7612-7621, 1973; Carpenter and Cohen, Ann.Rev.Biochem., 48:193-316, 1979; Simpson et al., Eur J Biochem, 153:629-37, 1985).In particular, the six cysteine ​​residues at positions 6, 14, 20, 31, 33, and 42 are conserved because they form three disulfide bridges to provide a conserved tertiary protein structure. Also conserved across all four species are residues at positions 7, 9, 11, 12, 13, 15, 18, 21, 24, 29, 32, 34, 36, 37, 39, 41, 46, and 47. Many of these residues can be predicted to facilitate or provide important binding interactions with the corresponding EGFR. Both full-length human EGF (53 residues) and a truncated form resulting from trypsin cleavage (48 residues) have been described to retain strong binding affinity and EGFR activation (Calnan et al., 47(5):622-7, 2000; Gregory, Regul Pept, 22:217-26, 1988). Mutagenesis studies of various residues have been reported to correlate the effect of specific residue replacements on EGF binding to EGFR or EGFR activation (Campion et al., Biochemistry, 29, 9988-9993, 1990; Engler et al., J. Biol. Chem., 267:2274-2281, 1992; Tadaki and Niyogi, J. Biol. Chem., 268:10114-10119, 1993). The X-ray crystal structure of EGF bound to EGFR has been solved, showing important binding interactions and identifying residues not directly involved in binding (Ogiso et al., Cell, Vol. 110, 775-787, 2002).

[0217] In another aspect, the present invention provides a composition comprising a polyplex, the polyplex comprising a conjugate of Formula I, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof, and a nucleic acid, the nucleic acid preferably comprising the conjugate: [ka]

[0218] Formula I (In the formula, [ka] is a single or double bond, n is an integer of 1 to 1500, preferably an integer of 2 to 1500, m is the number of discrete repeating -(O-CH-CH)- units, and the discrete number m of the discrete repeating -(O-CH-CH)- units is any discrete number from 25 to 100, preferably from 25 to 60; R 1 is the starting residue, preferably R 1 is -H or -CH3, R 2 are independently -H or an organic residue, and the -(NR 2 -CH2-CH2) n -The R in question 2 at least 80%, preferably at least 90%, of are H; Ring A may have one or more R A1 is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl optionally substituted with R A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo, or halogen, or two R A1 are combined with the atoms to which they are attached to form one or more fused C6-C 10can form an aryl, C-C heteroaryl, or C-C cycloalkyl ring, and each fused aryl, heteroaryl, or cycloalkyl can be joined by one or more R A2 and optionally substituted with R A2 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen -SO3H or -OSO3H; X 1 is a divalent covalent linking moiety, X 2 is a divalent covalent linking moiety, L is a targeting fragment, wherein the targeting fragment is capable of binding to epidermal growth factor receptor (EGFR), preferably the targeting fragment is capable of binding to a cell expressing EGFR, and more preferably the targeting fragment is capable of binding to a cell surface receptor, wherein the cell surface receptor is EGFR; The nucleic acid is a pharmaceutically active nucleic acid, and the pharmaceutically active nucleic acid is a nucleic acid that encodes a pharmaceutically active peptide or protein. 1 is —H. In a preferred embodiment, the R 1 is -CH3.

[0219] In another aspect, the present invention provides a polyplex, the polyplex comprising a conjugate of Formula I, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof: [ka]

[0220] Formula I (In the formula, [ka] is a single or double bond, n is an integer of 1 to 1500, preferably an integer of 2 to 1500, m is the number of discontinuous repeating -(O-CH-CH)- units, and the discontinuous number m of repeating -(O-CH-CH)- units is any discontinuous number from 25 to 100, preferably from 25 to 60, and more preferably, the discontinuous number m of repeating -(O-CH-CH)- units is 36; R 1 is the starting residue, preferably R 1 is -H or -CH3, R 2 are independently -H or an organic residue, and the -(NR 2 -CH2-CH2) n -The R in question 2 at least 80%, preferably at least 90%, of are H; Ring A may have one or more R A1 is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl optionally substituted with R A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo, or halogen, or two R A1 are combined with the atoms to which they are attached to form one or more fused C6-C 10 can form an aryl, C-C heteroaryl, or C-C cycloalkyl ring, and each fused aryl, heteroaryl, or cycloalkyl can be joined by one or more R A2 and optionally substituted with R A2 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen -SO3H or -OSO3H; X 1 is a divalent covalent linking moiety, X 2 is a divalent covalent linking moiety, L is a targeting fragment, the targeting fragment is capable of binding to epidermal growth factor receptor (EGFR), preferably the targeting fragment is capable of binding to a cell expressing EGFR, more preferably the targeting fragment is capable of binding to a cell surface receptor, the cell surface receptor being EGFR; The nucleic acid is a pharmaceutically active nucleic acid, the pharmaceutically active nucleic acid being a nucleic acid encoding a pharmaceutically active peptide or protein. 1 is —H. In a preferred embodiment, the R 1 is -CH3.

[0221] In another aspect, the present invention provides a composition comprising a polyplex, the polyplex comprising a conjugate of Formula I, preferably a plurality of conjugates, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof, and a nucleic acid, the nucleic acid preferably comprising the conjugate: [ka]

[0222] Formula I (In the formula, [ka] is a single or double bond, n is an integer of 1 to 1500, preferably an integer of 2 to 1500, m is the number of discrete repeating -(O-CH-CH)- units, and said discrete number m of repeating -(O-CH-CH)- units is 36; R 1 is the starting residue, preferably R 1 is -H or -CH3, R 2 are independently -H or an organic residue, and the -(NR 2 -CH2-CH2) n -The R in question 2 at least 80%, preferably at least 90%, of are H; Ring A may have one or more R A1 is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl optionally substituted with R A1are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo, or halogen, or two R A1 are combined with the atoms to which they are attached to form one or more fused C6-C 10 can form an aryl, C-C heteroaryl, or C-C cycloalkyl ring, and each fused aryl, heteroaryl, or cycloalkyl can be joined by one or more R A2 and optionally substituted with R A2 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen -SO3H or -OSO3H; X 1 is a divalent covalent linking moiety, X 2 is a divalent covalent linking moiety, L is a targeting fragment, wherein the targeting fragment is capable of binding to epidermal growth factor receptor (EGFR), preferably the targeting fragment is capable of binding to a cell expressing EGFR, and more preferably the targeting fragment is capable of binding to a cell surface receptor, wherein the cell surface receptor is EGFR; The nucleic acid is a pharmaceutically active nucleic acid, and the pharmaceutically active nucleic acid is a nucleic acid that encodes a pharmaceutically active peptide or protein. 1 is —H. In a preferred embodiment, the R 1 is -CH3.

[0223] In another aspect, the present invention provides a polyplex comprising a conjugate of Formula I, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof, and a nucleic acid, wherein the nucleic acid is preferably a conjugate of the formula I: [ka]

[0224] Formula I (In the formula, [ka] is a single or double bond, n is an integer of 1 to 1500, preferably an integer of 2 to 1500, m is the number of discrete repeating -(O-CH-CH)- units, and said discrete number m of repeating -(O-CH-CH)- units is 36; R 1 is the starting residue, preferably R 1 is -H or -CH3, R 2 are independently -H or an organic residue, and the -(NR 2 -CH2-CH2) n -The R in question 2 at least 80%, preferably at least 90%, of are H; Ring A may have one or more R A1 is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl optionally substituted with R A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo, or halogen, or two R A1 are combined with the atoms to which they are attached to form one or more fused C6-C 10 can form an aryl, C-C heteroaryl, or C-C cycloalkyl ring, and each fused aryl, heteroaryl, or cycloalkyl can be joined by one or more R A2 and optionally substituted with R A2 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen -SO3H or -OSO3H; X 1 is a divalent covalent linking moiety, X 2 is a divalent covalent linking moiety, L is a targeting fragment, wherein the targeting fragment is capable of binding to epidermal growth factor receptor (EGFR), preferably the targeting fragment is capable of binding to a cell expressing EGFR, and more preferably the targeting fragment is capable of binding to a cell surface receptor, wherein the cell surface receptor is EGFR; The nucleic acid is a pharmaceutically active nucleic acid, and the pharmaceutically active nucleic acid is a nucleic acid that encodes a pharmaceutically active peptide or protein. 1 is —H. In a preferred embodiment, the R 1 is -CH3.

[0225] In another aspect, the present invention provides a composition comprising a polyplex, the polyplex comprising a conjugate of Formula I, preferably a plurality of conjugates, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof, and a nucleic acid, the nucleic acid preferably comprising the conjugate: [ka]

[0226] Formula I (In the formula, [ka] is a single or double bond, n is an integer of 1 to 1500, preferably an integer of 2 to 1500, m is the number of consecutive repeating -(O-CH-CH)- units m that is 25 to 100, preferably 25 to 60, consecutive repeating -(O-CH-CH)- units m that is not consecutive; R 1 is the starting residue, preferably R 1 is -H or -CH3, R 2 are independently -H or an organic residue, and the -(NR 2 -CH2-CH2)n -The R in question 2 at least 80%, preferably at least 90%, of are H; Ring A may have one or more R A1 is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl optionally substituted with R A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo, or halogen, or two R A1 are combined with the atoms to which they are attached to form one or more fused C6-C 10 can form an aryl, C-C heteroaryl, or C-C cycloalkyl ring, and each fused aryl, heteroaryl, or cycloalkyl can be joined by one or more R A2 and optionally substituted with R A2 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen -SO3H or -OSO3H; X 1 is a divalent covalent linking moiety, X 2 is a divalent covalent linking moiety, L is a targeting fragment, wherein the targeting fragment is capable of binding to epidermal growth factor receptor (EGFR), preferably the targeting fragment is capable of binding to a cell expressing EGFR, and more preferably the targeting fragment is capable of binding to a cell surface receptor, wherein the cell surface receptor is EGFR; The nucleic acid is a pharmaceutically active nucleic acid, and the pharmaceutically active nucleic acid is a nucleic acid that encodes a pharmaceutically active peptide or protein. 1 is —H. In a preferred embodiment, the R 1 is -CH3.

[0227] In another aspect, the present invention provides a polyplex comprising a conjugate of Formula I, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof, and a nucleic acid, wherein the nucleic acid is preferably a conjugate of the formula I: [ka]

[0228] Formula I (In the formula, [ka] is a single or double bond, n is an integer of 1 to 1500, preferably an integer of 2 to 1500, m is the number of consecutive repeating -(O-CH-CH)- units m that is 25 to 100, preferably 25 to 60, consecutive repeating -(O-CH-CH)- units m that is not consecutive; R 1 is the starting residue, preferably R 1 is -H or -CH3, R 2 are independently -H or an organic residue, and the -(NR 2 -CH2-CH2) n -The R in question 2 at least 80%, preferably at least 90%, of are H; Ring A may have one or more R A1 is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl optionally substituted with R A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo, or halogen, or two R A1 are combined with the atoms to which they are attached to form one or more fused C6-C 10can form an aryl, C-C heteroaryl, or C-C cycloalkyl ring, and each fused aryl, heteroaryl, or cycloalkyl can be joined by one or more R A2 and optionally substituted with R A2 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen -SO3H or -OSO3H; X 1 is a divalent covalent linking moiety, X 2 is a divalent covalent linking moiety, L is a targeting fragment, wherein the targeting fragment is capable of binding to epidermal growth factor receptor (EGFR), preferably the targeting fragment is capable of binding to a cell expressing EGFR, and more preferably the targeting fragment is capable of binding to a cell surface receptor, wherein the cell surface receptor is EGFR; The nucleic acid is a pharmaceutically active nucleic acid, and the pharmaceutically active nucleic acid is a nucleic acid that encodes a pharmaceutically active peptide or protein. 1 is —H. In a preferred embodiment, the R 1 is -CH3.

[0229] In another aspect, the present invention provides a composition comprising a polyplex, the polyplex comprising a conjugate of Formula I, preferably a plurality of conjugates, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof, and a nucleic acid, the nucleic acid preferably comprising the conjugate: [ka]

[0230] Formula I (In the formula, [ka] is a single or double bond, n is an integer of 1 to 1500, preferably an integer of 2 to 1500, m is the number of 36 consecutive repeating -(O-CH-CH)- units, m, that are not consecutive; R 1 is the starting residue, preferably R 1 is -H or -CH3, R 2 are independently -H or an organic residue, and the -(NR 2 -CH2-CH2) n -The R in question 2 at least 80%, preferably at least 90%, of are H; Ring A may have one or more R A1 is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl optionally substituted with R A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo, or halogen, or two R A1 are combined with the atoms to which they are attached to form one or more fused C6-C 10 can form an aryl, C-C heteroaryl, or C-C cycloalkyl ring, and each fused aryl, heteroaryl, or cycloalkyl can be joined by one or more R A2 and optionally substituted with R A2 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen -SO3H or -OSO3H; X 1 is a divalent covalent linking moiety, X 2 is a divalent covalent linking moiety, L is a targeting fragment, wherein the targeting fragment is capable of binding to epidermal growth factor receptor (EGFR), preferably the targeting fragment is capable of binding to a cell expressing EGFR, and more preferably the targeting fragment is capable of binding to a cell surface receptor, wherein the cell surface receptor is EGFR; The nucleic acid is a pharmaceutically active nucleic acid, and the pharmaceutically active nucleic acid is a nucleic acid that encodes a pharmaceutically active peptide or protein. 1 is —H. In a preferred embodiment, the R 1 is -CH3.

[0231] In another aspect, the present invention provides a polyplex comprising a conjugate of Formula I, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof, and a nucleic acid, wherein the nucleic acid is preferably a conjugate of the formula I: [ka]

[0232] Formula I (In the formula, [ka] is a single or double bond, n is an integer of 1 to 1500, preferably an integer of 2 to 1500, m is the number of 36 consecutive repeating -(O-CH-CH)- units, m, that are not consecutive; R 1 is the starting residue, preferably R 1 is -H or -CH3, R 2 are independently -H or an organic residue, and the -(NR 2 -CH2-CH2) n -The R in question 2 at least 80%, preferably at least 90%, of are H; Ring A may have one or more R A1 is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl optionally substituted with R A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo, or halogen, or two RA1 are combined with the atoms to which they are attached to form one or more fused C6-C 10 can form an aryl, C-C heteroaryl, or C-C cycloalkyl ring, and each fused aryl, heteroaryl, or cycloalkyl can be joined by one or more R A2 and optionally substituted with R A2 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen -SO3H or -OSO3H; X 1 is a divalent covalent linking moiety, X 2 is a divalent covalent linking moiety, L is a targeting fragment, wherein the targeting fragment is capable of binding to epidermal growth factor receptor (EGFR), preferably the targeting fragment is capable of binding to a cell expressing EGFR, and more preferably the targeting fragment is capable of binding to a cell surface receptor, wherein the cell surface receptor is EGFR; The nucleic acid is a pharmaceutically active nucleic acid, and the pharmaceutically active nucleic acid is a nucleic acid that encodes a pharmaceutically active peptide or protein. 1 is —H. In a preferred embodiment, the R 1 is -CH3.

[0233] In another aspect, the present invention provides a composition comprising a polyplex, the polyplex comprising a conjugate of formula I*, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof, and a nucleic acid, the nucleic acid preferably comprising the conjugate: R 1 -(NR 2 -CH2-CH2) n -ZX 1 -(O-CH2-CH2) m -X 2 -L(formula I*); (In the ceremony n is an integer of 1 to 1500, preferably an integer of 2 to 1500, m is any integer from 1 to 200, preferably, m is any integer from 1 to 100; R 1 is the starting residue, preferably R 1 is -H or -CH3, R 2 are independently -H or an organic residue, and the -(NR 2 -CH2-CH2) n -The R in question 2 at least 80%, preferably 90%, of the X 1 and X 2 are independently divalent covalent linking moieties; Z is a divalent covalent linking moiety, Z is not a single bond, Z is not -NHC(O)-, L is a targeting fragment, said targeting fragment being non-covalently linked to the sequence YHWYGYTPQNVI(GE11) (SEQ ID NO: 9), or preferably consisting of the sequence; the nucleic acid is a pharmaceutically active nucleic acid, the pharmaceutically active nucleic acid being a nucleic acid encoding a pharmaceutically active peptide or protein; Preferably, the composition consists of the conjugate.

[0234] In another aspect, the present invention provides a polyplex comprising a conjugate of formula I*, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof, and a nucleic acid, wherein the nucleic acid is preferably a conjugate of the formula I*, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof, R 1 -(NR 2 -CH2-CH2) n -ZX 1 -(O-CH2-CH2) m -X 2 -L(formula I*); (In the ceremony n is an integer of 1 to 1500, preferably an integer of 2 to 1500, m is any integer from 1 to 200, preferably, m is any integer from 1 to 100; R 1 is the starting residue, preferably R 1 is -H or -CH3, R 2 are independently -H or an organic residue, and the -(NR 2 -CH2-CH2) n -The R in question 2 at least 80%, preferably 90%, of the X 1 and X 2 are independently divalent covalent linking moieties; Z is a divalent covalent linking moiety, Z is not a single bond, Z is not -NHC(O)-, L is a targeting fragment, said targeting fragment being non-covalently linked to the sequence YHWYGYTPQNVI(GE11) (SEQ ID NO: 9), or preferably consisting of the sequence; The nucleic acid is a pharmaceutically active nucleic acid, which is a nucleic acid that encodes a pharmaceutically active peptide or protein.

[0235] In another aspect, the present invention provides a composition comprising a polyplex, the polyplex comprising a conjugate of Formula I, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof, and a nucleic acid, the nucleic acid preferably comprising the conjugate: [ka]

[0236] Formula I (In the formula, [ka] is a single or double bond, n is an integer of 1 to 1500, preferably an integer of 2 to 1500, m is an integer from 1 to 200; R 1 is the starting residue, preferably R 1 is -H or -CH3, R 2 are independently -H or an organic residue, and the -(NR 2 -CH2-CH2) n -The R in question 2 at least 80%, preferably at least 90%, of are H; Ring A is one or more R A1 R is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl optionally substituted at any position by A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo, or halogen, or two R A1 are combined with the atoms to which they are attached to form one or more fused C6-C 10 can form an aryl, C-C heteroaryl, or C-C cycloalkyl ring, and each fused aryl, heteroaryl, or cycloalkyl can be joined by one or more R A2 and optionally substituted with R A2 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen -SO3H or -OSO3H; X 1 is a divalent covalent linking moiety, X 2 is a divalent covalent linking moiety, L is a targeting fragment, said targeting fragment being non-covalently linked to the sequence YHWYGYTPQNVI(GE11) (SEQ ID NO: 9), or preferably consisting of the sequence; The nucleic acid is a pharmaceutically active nucleic acid, which is a nucleic acid that encodes a pharmaceutically active peptide or protein.

[0237] In another aspect, the present invention provides a polyplex comprising a conjugate of Formula I, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof, and a nucleic acid, wherein the nucleic acid is preferably a conjugate of the formula I: [ka]

[0238] Formula I (In the formula, [ka] is a single or double bond, n is an integer of 1 to 1500, preferably an integer of 2 to 1500, m is an integer from 1 to 200; R 1 is the starting residue, preferably R 1 is -H or -CH3, R 2 are independently -H or an organic residue, and the -(NR 2 -CH2-CH2) n -The R in question 2 at least 80%, preferably at least 90%, of are H; Ring A is one or more R A1 R is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl optionally substituted at any position by A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo, or halogen, or two R A1 are combined with the atoms to which they are attached to form one or more fused C6-C 10 can form an aryl, C-C heteroaryl, or C-C cycloalkyl ring, and each fused aryl, heteroaryl, or cycloalkyl can be joined by one or more R A2 and optionally substituted with R A2are independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen -SO3H or -OSO3H; X 1 is a divalent covalent linking moiety, X 2 is a divalent covalent linking moiety, L is a targeting fragment, said targeting fragment being non-covalently linked to the sequence YHWYGYTPQNVI(GE11) (SEQ ID NO: 9), or preferably consisting of the sequence; The nucleic acid is a pharmaceutically active nucleic acid, which is a nucleic acid that encodes a pharmaceutically active peptide or protein.

[0239] In another aspect, the present invention provides a composition comprising a polyplex, the polyplex comprising a conjugate of Formula I, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof, and a nucleic acid, the nucleic acid preferably comprising the conjugate: [ka]

[0240] Formula I (In the formula, [ka] is a single or double bond, n is an integer of 1 to 1500, preferably an integer of 2 to 1500, m is a discrete number of repeating units m of 2 to 100, preferably a discrete number of repeating units m of 4 to 60, R 1 is the starting residue, preferably R 1 is -H or -CH3, R 2 are independently -H or an organic residue, and the -(NR 2 -CH2-CH2) n -The R in question 2at least 80%, preferably at least 90%, of are H; Ring A is one or more R A1 R is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl optionally substituted at any position by A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo, or halogen, or two R A1 are combined with the atoms to which they are attached to form one or more fused C6-C 10 can form an aryl, C-C heteroaryl, or C-C cycloalkyl ring, and each fused aryl, heteroaryl, or cycloalkyl can be joined by one or more R A2 and optionally substituted with R A2 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen -SO3H or -OSO3H; X 1 is a divalent covalent linking moiety, X 2 is a divalent covalent linking moiety, L is a targeting fragment, wherein the targeting fragment is an EGFR targeting fragment, preferably the EGFR targeting fragment is capable of specifically binding to cells that express, preferably overexpress, EGFR; the targeting fragment is non-covalently linked to epidermal growth factor (EGF), preferably the targeting fragment is human EGF (hEGF), and even more preferably the targeting fragment comprises, and preferably consists of, the sequence of SEQ ID NO: 7; The nucleic acid is a pharmaceutically active nucleic acid, which is a nucleic acid that encodes a pharmaceutically active peptide or protein.

[0241] In another aspect, the present invention provides a polyplex comprising a conjugate of Formula I, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof, and a nucleic acid, wherein the nucleic acid is preferably a conjugate of the formula I: [ka]

[0242] Formula I (In the formula, [ka] is a single or double bond, n is an integer of 1 to 1500, preferably an integer of 2 to 1500, m is a discrete number of consecutive repeating units m of 2 to 100, preferably a discrete number of consecutive repeating units m of 4 to 60, R 1 is the starting residue, preferably R 1 is -H or -CH3, R 2 are independently -H or an organic residue, and the -(NR 2 -CH2-CH2) n -The R in question 2 at least 80%, preferably at least 90%, of are H; Ring A is one or more R A1 R is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl optionally substituted at any position by A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo, or halogen, or two R A1 are combined with the atoms to which they are attached to form one or more fused C6-C 10 can form an aryl, C-C heteroaryl, or C-C cycloalkyl ring, and each fused aryl, heteroaryl, or cycloalkyl can be joined by one or more R A2and optionally substituted with R A2 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen -SO3H or -OSO3H; X 1 is a divalent covalent linking moiety, X 2 is a divalent covalent linking moiety, L is a targeting fragment, wherein the targeting fragment is an EGFR targeting fragment, preferably the EGFR targeting fragment is capable of specifically binding to cells that express, preferably overexpress, EGFR; the targeting fragment is non-covalently linked to epidermal growth factor (EGF), preferably the targeting fragment is human EGF (hEGF), and even more preferably the targeting fragment comprises, and preferably consists of, the sequence of SEQ ID NO: 7; The nucleic acid is a pharmaceutically active nucleic acid, which is a nucleic acid that encodes a pharmaceutically active peptide or protein.

[0243] In another aspect, the present invention provides a polyplex comprising a conjugate of Formula I, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof, and a nucleic acid, wherein the nucleic acid is preferably a conjugate of the formula I: [ka]

[0244] Formula I (In the formula, [ka] is a single or double bond, n is an integer of 1 to 1500, preferably an integer of 2 to 1500, m is a discrete number of repeating units m of 2 to 100, preferably a discrete number of repeating units m of 4 to 60, R1 is the starting residue, preferably R 1 is -H or -CH3, R 2 are independently -H or an organic residue, and the -(NR 2 -CH2-CH2) n -The R in question 2 at least 80%, preferably at least 90%, of are H; Ring A is one or more R A1 R is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl optionally substituted at any position by A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo, or halogen, or two R A1 are combined with the atoms to which they are attached to form one or more fused C6-C 10 can form an aryl, C-C heteroaryl, or C-C cycloalkyl ring, and each fused aryl, heteroaryl, or cycloalkyl can be joined by one or more R A2 and optionally substituted with R A2 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen -SO3H or -OSO3H; X 1 is a divalent covalent linking moiety, X 2 is a divalent covalent linking moiety, L is a targeting fragment, preferably the targeting fragment is capable of binding to a cell, more preferably the targeting fragment is capable of binding to a cell surface receptor, and is non-covalently bound to The nucleic acid is a pharmaceutically active nucleic acid, which is a nucleic acid that encodes a pharmaceutically active peptide or protein.

[0245] In another aspect, the present invention provides a composition comprising a polyplex, the polyplex comprising a conjugate of Formula I, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof, and a nucleic acid, the nucleic acid preferably comprising the conjugate: [ka]

[0246] Formula I (In the formula, [ka] is a single or double bond, n is an integer of 1 to 1500, preferably an integer of 2 to 1500, m is a discrete number of consecutive repeating units m of 2 to 100, preferably a discrete number of consecutive repeating units m of 4 to 60, R 1 is the starting residue, preferably R 1 is -H or -CH3, R 2 are independently -H or an organic residue, and the -(NR 2 -CH2-CH2) n -The R in question 2 at least 80%, preferably at least 90%, of are H; Ring A is one or more R A1 R is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl optionally substituted at any position by A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo, or halogen, or two R A1 are combined with the atoms to which they are attached to form one or more fused C6-C 10 can form an aryl, C-C heteroaryl, or C-C cycloalkyl ring, and each fused aryl, heteroaryl, or cycloalkyl can be joined by one or more R A2and optionally substituted with R A2 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen -SO3H or -OSO3H; X 1 is a divalent covalent linking moiety, X 2 is a divalent covalent linking moiety, L is a targeting fragment, preferably the targeting fragment is capable of binding to a cell, more preferably the targeting fragment is capable of binding to a cell surface receptor), non-covalently bound to The nucleic acid is a pharmaceutically active nucleic acid, which is a nucleic acid that encodes a pharmaceutically active peptide or protein.

[0247] In a preferred embodiment, the targeting fragment is capable of binding to prostate-specific membrane antigen (PSMA), also referred to herein as a PSMA targeting fragment.

[0248] PSMA is a multifunctional transmembrane protein that functions as glutamic acid carboxypeptidase, and also demonstrates rapid ligand-induced internalization and recycling (Liu H, et al., 1998, Cancer Res 58:4055-4060). PSMA is primarily expressed in four tissues of the body, including prostate epithelium, renal proximal tubules, jejunal brush border of the small intestine, and ganglia of the nervous system (Mhawech-Fauceglia et al., Histopathology 2007,50:472-483). In a preferred embodiment, the targeting fragment can bind to an epitope on the extracellular domain of PSMA.

[0249] In a preferred embodiment, the targeting fragment, preferably the PSMA targeting fragment, can bind to the cells that express PSMA. In a preferred embodiment, the targeting fragment, preferably the PSMA targeting fragment, can bind to the cells that overexpress PSMA. For example, PSMA is overexpressed in neoplastic tissue and malignant prostate, especially in prostate adenocarcinoma compared with normal tissue, and the level of PSMA expression is further upregulated as the disease progresses to the metastatic stage (Silver et al., 1997, Clin. Cancer Res., 3:81). PSMA is also expressed and overexpressed in other tumor types (Mhawech-Fauceglia et al., Histopathology 2007,50:472-483; Israeli RS et al., Cancer Res 1994,54:1807-1811; Chang SS et al., Cancer Res 1999,59:3192-198).

[0250] In one embodiment, overexpressed PSMA refers to an elevated level of PSMA expressed in the cells of a particular tissue compared to the level of PSMA measured in normal, healthy cells of the same tissue type under similar conditions. In one embodiment, overexpressed PSMA refers to an increase in the level of PSMA in a cell compared to the level in the same cell or a closely related, non-malignant cell under normal physiological conditions. In one embodiment, the PSMA-overexpressing cells express PSMA at least 10-fold higher than normal cells or normal tissue. In one embodiment, the PSMA-overexpressing cells express PSMA with a cutoff of 5% or more PSMA-positive cells, as described, for example, in Mhawech-Fauceglia et al., 2007, which can be used to define PSMA expression in different types of tissues or cells. Therefore, cells or tissues with less than 5% positive cells were considered negative, as described by Hupe et al., 2018 (Hupe MC et al, Frontiers in Oncology 2018, 8(623):1-7), or PSMA expression was classified according to its intensity and scored as 0 (no expression), 1 (low expression), 2 (moderate expression), and 3 (high expression).

[0251] In a preferred embodiment, the targeting fragment can bind to cells that express or overexpress PSMA. Cells that express PSMA typically include tumor cells such as prostate, bladder, pancreatic, lung, kidney, and colon tumor cells, melanoma, and sarcoma. In a preferred embodiment, the targeting fragment can bind to cells that express or overexpress PSMA, and the cells are tumor cells, preferably selected from prostate, bladder, pancreatic, lung, kidney, and colon tumor cells, melanoma, and sarcoma. In a preferred embodiment, the targeting fragment can bind to cells that express or overexpress PSMA, and the cells are tumor cells, and the tumor cells are prostate tumor cells.

[0252] In preferred embodiments, the targeting fragment is capable of specifically binding to PSMA, and typically and preferably, the affinity or specific binding is such that the dissociation constant (K D ) and the affinity or specific binding is measured by -3 Less than M, preferably 10 -4 M, more preferably less than 10 -5 M, more preferably less than 10 -6 Less than M, more preferably 10 -7 M, and even more preferably less than 10 -8 M, and even more preferably less than 10 -9 M, and even more preferably less than 10 -10 K less than M D In preferred embodiments, the targeting fragment is capable of specifically binding to PSMA, and typically and preferably, the affinity or specific binding is such that the dissociation constant (K D ) and the affinity or specific binding is measured by -3 Under M, 10 -4 Under M, 10 -5 Under M, 10 -6 Under M, 10 -7 Under M, 10 -8 Less than M and 10 -9 K less than M D Preferably, the binding results in the formation of a complex between the targeting fragment and PSMA, and the binding or complex is detected typically and preferably using a Biacore 3000 instrument (Biacore Inc., Piscataway, NJ) or a cell-based binding assay or flow-induced dispersion analysis (FIDA), typically and preferably as described in Kularatne et al., Mol Pharm. 2009;6(3):790-800.

[0253] In preferred embodiments, the targeting fragment is a PSMA antibody, a PSMA aptamer, or a small molecule PSMA targeting fragment. In preferred embodiments, the PSMA targeting fragment is a PSMA antibody, a PSMA aptamer, or a small molecule PSMA targeting fragment. As used herein, the term "small molecule PSMA targeting fragment" refers to a chemical moiety that has a molecular weight of less than about 2000 g / mol and that is typically and preferably capable of binding to PSMA. In some embodiments, the small molecule PSMA targeting fragment has a molecular weight of less than about 1800 g / mol. In some embodiments, the small molecule PSMA targeting fragment has a molecular weight of less than about 1500 g / mol, more preferably less than about 1000 g / mol. In even more preferred embodiments, the small molecule has a molecular weight of less than about 800 g / mol, even more preferably less than about 500 g / mol.

[0254] In some embodiments, the PSMA-targeting fragment is a PSMA antibody, an antibody capable of binding to PSMA. In some embodiments, the fragment is a monoclonal antibody, a polyclonal antibody, and / or an antibody fragment, preferably a functional fragment thereof, a chimeric antibody, a recombinant antibody, and / or a bispecific or multispecific antibody. Such PSMA antibodies include scFv antibodies A5, G0, G1, G2, and G4 and mAbs 3 / E7, 3 / F11, 3 / A12, K7, K12, and D20 (Elsasser-Beile et al., 2006, Prostate, 66:1359); mAbs E99, J591, J533, and J415 (Liu et al., 1997, Cancer Res., 57:3629; Liu et al., 1998, Cancer Res., 58:4055; Fracasso et al., 2002, Prostate, 53:9; McDevitt et al., 2000, Cancer Res., 60:6095; McDevitt et al., 2001, Science, 294:1537; Smith-Jones et al., 2000, Cancer Res. Res., 60:5237; Vallabhajosula et al., 2004, Prostate, 58:145; Bander et al., 2003, J. Urol., 170:1717; Patri et al., 2004, Bioconj. Chem., 15:1174; and U.S. Patent No. 7,163,680; mAb 7E11-C5.3 (Horoszewicz et al., 1987, Anticancer Res., 7:927); antibody 7E11 (Horoszewicz et al., 1987, Anticancer Res., 7:927; and U.S. Patent No. 5,162,504); and Chang et al., 1999, Cancer Res., 59:3192; Murphy et al., 1998, J. Urol., 160:2396; Grauer et al., 1998, Cancer Res., 58:4787; and Wang et al., 2001, Int. J. Cancer, 92:871.Those skilled in the art will understand that any antibody that recognizes and / or specifically binds to PSMA can be used in accordance with the present invention. All of the foregoing documents and disclosures are incorporated herein by reference in their entirety.

[0255] In some embodiments, the targeting fragment that can bind to PSMA is an aptamer. PSMA targeting aptamers include, but are not limited to, A10 aptamer or A9 aptamer (Lupold et al., 2002, Cancer Res., 62:4029; and Chu et al., 2006, Nuc. Acid Res., 34:e73), their derivatives, and / or their functional fragments. In some embodiments, in the aptamer derivative, 30, 25, 20, 15, 10, 5, 4, 3, 2, or less than 1 nucleic acid is substituted for the aptamer. In some embodiments, the sequence of the aptamer derivative is at least 80%, preferably 85%, more preferably 90%, even more preferably 95%, and most preferably 99% identical.

[0256] In a preferred embodiment, the targeting fragment is a small molecule PSMA targeting fragment. In a preferred embodiment, the PSMA targeting fragment is a small molecule PSMA targeting fragment, preferably a small molecule PSMA-targeted peptidase inhibitor. In preferred embodiments, the small molecule PSMA peptidase inhibitors include 2-PMPA, GPI5232, VA-033, phenylalkylphosphonamidates (Jackson et al., 2001, Curr. Med. Chem., 8:949; Bennett et al., 1998, J. Am. Chem. Soc., 120:12139; Jackson et al., 2001, J Med. Chem., 44:4170; Tsukamoto et al., 2002, Bioorg. Med. Chem. Lett., 12:2189; Tang et al., 2003, Biochem. Biophys. Res. Commun., 307:8; Oliver et al., 2003, Bioorg. Med. Chem., 11:4455; and Maung et al., 2003, Bioorg. Med. Chem., 11:4455). al., 2004, Bioorg. Med. Chem., 12:4969), and / or analogs and derivatives thereof. All of the foregoing documents (scientific and other publications, patents, and patent applications) are incorporated herein by reference in their entirety. In some embodiments, the small molecule PSMA targeting fragment is a protein, peptide, amino acid, or derivative thereof. In preferred embodiments, the small molecule PSMA targeting fragment comprises thiol and indole thiol derivatives, such as 2-MPPA and 3-(2-mercaptoethyl)-1H-indole-2-carboxylic acid derivatives (Majer et al., 2003, J. Med. Chem., 4611989; and U.S. Patent Application Publication No. 2005 / 0080128). In some embodiments, the small molecule PSMA targeting fragment comprises a hydroxamate derivative (Stoermer et al., 2003, Bioorg. Med. Chem. Lett., 1312097).In a preferred embodiment, the small molecule PSMA peptidase inhibitor is one disclosed in U.S. Patent Nos. 7,026,500; 7,022,870; 6,998,500; 6,995,284; 6,838,484; 6,569,896; 6,492,554; and U.S. Patent Application Publication No. 2006 / 0287547; No. 06 / 0276540; No. 2006 / 0258628; No. 2006 / 0241180; No. 2006 / 0183931; No. 2006 / 0035966; No. 2 No. 006 / 0009529; No. 2006 / 0004042; No. 2005 / 0033074; No. 2004 / 0260108; No. 2004 / 0260092; No. 2004 / 0167103; 2004 / 0147550; 2004 / 0147489; 2004 / 0087810; 2004 / 0067979 ;No.2004 / 0052727;No.2004 / 0029913;No.2004 / 0014975;No.2003 / 0232792;No.2003 / 0232013 androgen receptor targeting agents (ARTA) such as those described in U.S. Patent Application Publication Nos. 2003 / 0225040; 2003 / 0162761; 2004 / 0087810; 2003 / 0022868; 2002 / 0173495; 2002 / 0099096; and 2002 / 0099036. In some embodiments, the small molecule PSMA targeting fragment comprises a polyamine such as putrescine, spermine, and spermidine (U.S. Patent Application Publication Nos. 2005 / 0233948 and 2003 / 0035804). All of the foregoing documents and disclosures are incorporated herein by reference in their entirety.

[0257] In preferred embodiments, the small molecule PSMA peptidase inhibitors include PBDA-based inhibitors and urea-based inhibitors, such as ZJ 43, ZJ, ZJ 17, and ZJ 38 (Nan et al., 2000, J. Med. Chem., 43:772; and Kozikowski et al., 2004, J. Med. Chem., 47, 7, 1729-1738), and / or analogs and derivatives thereof. Other agents that bind to PSMA can also be used as PSMA-targeting fragments, including, for example, those found in Clin. Cancer Res., 2008 14:3036-43, or PSMA-targeting fragments prepared by sequentially adding components to a preformed urea, such as the lysine-urea-glutamate compound described in Banerjee et al. (J. Med. Chem., vol. 51, pp. 4504-4517, 2008). In a preferred embodiment, the one or more targeting fragments capable of binding to prostate-specific membrane antigen (PSMA) are small molecule PSMA targeting fragments, more preferably small urea-based inhibitors.

[0258] In a preferred embodiment, the small molecule PSMA targeting fragment is a urea-based inhibitor (also referred to herein as a urea-based peptidase inhibitor), more preferably a urea-based inhibitor as described in Kularatne et al., Mol Pharmaceutics 2009, 6, 780; Kularatne et al., Mol. Pharmaceutics 2009, 6, 790; Kopka et al., J Nucl Med 2017, 58:17S-26S; Kozikowski et al., J Med Chem. 2001, 44:298-301; Kozikowski et al., J Med Chem. 2004, 47: 1729-1738, International Publication No. 2017 / 044936, International Publication No. 2011 / 084518, International Publication No. 2011 / 084521, International Publication No. 2011 / 084513, International Publication No. 2012 / 166923, International Publication No. 2008 / 105773, International Publication No. 2008 / 121949, WO 2012 / 135592, WO 2010 / 005740, WO 2015 / 168379, WO 03 / 045436, WO 03 / 045436, WO 2016 / 183447, U.S. Patent Application Publication No. 2015 / 258102, WO 2011 / 084513, WO and small urea inhibitors such as those disclosed in U.S. Patent Application Publication No. 2017 / 089942, U.S. Patent Application Publication No. 2010 / 278927, WO 2012 / 016188, WO 2008 / 124634, WO 2009 / 131435, U.S. Patent Application Publication No. 2007 / 225213, WO 2017 / 086467, WO 2009 / 026177, WO 2012005572, WO 2014 / 072357, and WO 2011 / 108930. All of the foregoing documents and disclosures are incorporated herein by reference in their entirety.

[0259] In a preferred embodiment, the targeting fragment is a dipeptide urea PSMA peptidase inhibitor, preferably a small molecule dipeptide urea PSMA peptidase inhibitor. In a preferred embodiment, the PSMA targeting fragment is a dipeptide urea PSMA peptidase inhibitor, preferably a small molecule dipeptide urea PSMA peptidase inhibitor.

[0260] The term "urea-based PSMA peptidase inhibitors" refers to PSMA peptidase inhibitors containing a urea group. The term "dipeptide urea-based PSMA peptidase inhibitors" refers to PSMA peptidase inhibitors containing a urea group and two peptides or amino acids independently attached to the -NH2 group of the urea group, while the term "small molecule dipeptide urea-based PSMA peptidase inhibitors" further indicates that the dipeptide urea-based PSMA peptidase inhibitors have a molecular weight of less than about 2000 g / mol and are typically and preferably capable of binding to PSMA. In some embodiments, the small molecule dipeptide urea-based PSMA peptidase inhibitors have a molecular weight of less than about 1800 g / mol, less than about 1500 g / mol, and preferably less than about 1000 g / mol. In even more preferred embodiments, the small molecule dipeptide urea-based PSMA peptidase inhibitors have a molecular weight of less than about 800 g / mol, and even more preferably less than about 500 g / mol. The PSMA peptidase inhibitor can reduce the activity of the PSMA transmembrane zinc(II) metalloenzyme, which catalyzes the cleavage of terminal glutamate. More preferably, the small molecule urea-based PSMA peptidase inhibitor has a molecular weight of less than about 500 g / mol. Even more preferably, the small molecule urea-based PSMA peptidase inhibitor is a glutamate-urea-based PSMA peptidase inhibitor, preferably as described in Kopka et al., J Nuc Med, 58(9), suppl. 2, 2017; Wirtz et al., EJNMMI Research (2018) 8:84, and the references cited therein (all of which are incorporated herein by reference in their entirety).

[0261] In a preferred embodiment, the targeting fragment, preferably the urea-based PSMA peptidase inhibitor, is of Formula 1, preferably Formula 1* [ka] and enantiomers, stereoisomers, rotamers, tautomers, diastereomers or racemates thereof, wherein R is preferably substituted or unsubstituted alkyl, substituted or unsubstituted aryl, and any combination thereof; more preferably R is C1-6-alkyl, preferably C2-C4-alkyl, substituted one or more times, preferably once, with OH, SH, NH2 or COOH, and one of said NH2, OH or SH or COOH groups is respectively X 2 The alkyl group, which serves as the point of covalent attachment to the linking moiety and the PEG fragment, is optionally interrupted by N(H), S or O. In another preferred embodiment, R is C1-6-alkyl, preferably C2-C4-alkyl, substituted once with OH, SH, NH2 or COOH, and said NH2, OH or SH or COOH group is, respectively, X 2 It serves as a linking moiety and a point of covalent attachment to the PEG fragment. In a highly preferred embodiment, R is a C2-alkyl substituted once with COOH, each COOH group being a X 2 It serves as a linking moiety and a point of covalent attachment to the PEG fragment.

[0262] In a preferred embodiment, the targeting fragment has the formula 1: [ka] a glutamic acid-urea moiety, wherein R is C1-6-alkyl, preferably C2-C4-alkyl, substituted one or more times, preferably once, with OH, SH, NH2 or COOH, and one of the NH2, OH or SH or COOH groups is selected from the group X 2The alkyl group, which serves as the point of covalent attachment to the linking moiety and the PEG fragment, is optionally interrupted by N(H), S or O. In another preferred embodiment, R is C1-6-alkyl, preferably C2-C4-alkyl, substituted once with OH, SH, NH2 or COOH, and said NH2, OH or SH or COOH group is, respectively, X 2 It serves as a linking moiety and a point of covalent attachment to the PEG fragment. In a highly preferred embodiment, R is a C2-alkyl substituted once with COOH, each COOH group being a X 2 It serves as a linking moiety and a point of covalent attachment to the PEG fragment.

[0263] In another preferred embodiment, the targeting fragment has the formula 1* [ka] is the glutamic acid-urea moiety of In the formula, R is C1-6-alkyl, preferably C2-C4-alkyl, substituted one or more times, preferably once, with OH, SH, NH2 or COOH, and one of the NH2, OH or SH or COOH groups is respectively X 2 The alkyl group, which serves as the point of covalent attachment to the linking moiety and the PEG fragment, is optionally interrupted by N(H), S or O. In another preferred embodiment, R is C1-6-alkyl, preferably C2-C4-alkyl, substituted once with OH, SH, NH2 or COOH, and said NH2, OH or SH or COOH group is, respectively, X 2 It serves as a linking moiety and a point of covalent attachment to the PEG fragment. In a highly preferred embodiment, R is a C2-alkyl substituted once with COOH, each COOH group being a X 2 It serves as a linking moiety and a point of covalent attachment to the PEG fragment.

[0264] In a further preferred embodiment, the targeting fragment comprises, or preferably consists of, a DUPA residue (HOOC-(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO-). In a further highly preferred embodiment, the targeting fragment consists of a DUPA residue (HOOC(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO-) in which both chiral C atoms have the (S)-configuration, as shown in formula 1*.

[0265] In a further preferred embodiment, the PSMA targeting fragment is a folate ligand. In a further preferred embodiment, the PSMA targeting fragment is a small molecule PSMA targeting fragment, and the small molecule PSMA targeting fragment is a folate ligand.

[0266] In a preferred embodiment, the folic acid ligand binds to a cell surface receptor, and the cell surface receptor is PSMA. As recently reported, targeting of PSMA-expressing cells has been achieved by amides of folic acid (Flores O et al., Theranostics 2017,7(9):2477-2494).

[0267] As used herein, the term "folate ligand" is understood to mean folic acid or methotrexate or a derivative or analog thereof. Preferably, the folic acid or methotrexate derivative or analog has a glutamate functional group R-NH-[CH(COOH)-CH-CH-C(O)NH] η -CH(COOH)-CH-CH-COOH, where η is an integer from 0 to 100, and R is a group represented by the formula 2 [ka] is a group of the formula R 201 is -OH or -NH2, R 202 is -H or -CH3, The wavy line indicates the point of attachment to the glutamate functional group. In a preferred embodiment, η is an integer from 0 to 10, preferably η is an integer from 0 to 5, and more preferably η is 0.

[0268] Those skilled in the art will recognize that R 201 is —OH, in a preferred embodiment, the OH tautomerizes to a carbonyl group (═O), and the R 201 It will be understood that the adjacent nitrogen atom of is protonated.

[0269] Those skilled in the art will recognize that the glutamate functional group R—NH—[CH(COOH)—CH—CH—C(O)NH] η It will be further understood that -CH(COOH)-CH-CH-COOH includes at least one alpha carboxylate group and a gamma carboxylate group. Specifically, one or more -COOH groups bonded to the same carbon as one or more -NH- groups are understood herein as an alpha carboxylate group. When η = 0, a -COOH group bonded to the same carbon as an R-NH group is understood herein as an alpha carboxylate group. A -COOH group bonded to a -(CH)- group is understood herein as a gamma carboxylate group. Furthermore, those skilled in the art will understand that the carboxylate groups discussed herein, such as alpha and gamma carboxylate groups, can be protonated or deprotonated depending on the pH of the surrounding solution. Thus, those skilled in the art will understand that while carboxylate groups are depicted as neutral species (-COOH) for simplicity and clarity, they can exist (e.g., can exist primarily) as deprotonated species, i.e., negatively charged species (-COO-) at physiological pH.

[0270] In some embodiments, the alpha carboxylate group of the glutamate functional group is X 2 In a preferred embodiment, the alpha carboxylate group of the glutamate functional group is linked to X 2 When serving as the point of attachment for a linking moiety, the alpha carboxylate group is X2 In some embodiments, the alpha carboxylate group of the glutamate functional group is condensed with the amine group of the linking moiety to form an amide. 2 When serving as the point of attachment for a linking moiety, the alpha carboxylate group is X 2 It condenses with the hydroxy group of the linking moiety to form an ester.

[0271] In a preferred embodiment, the gamma carboxylate group of the glutamate functional group is X 2 In a preferred embodiment, the gamma carboxylate group of the glutamate functional group serves as a covalent attachment point to a linking moiety. 2 When serving as the point of attachment for a linking moiety, the gamma carboxylate group is X 2 In some embodiments, the gamma carboxylate group of the glutamate functional group is condensed with the amine group of the linking moiety to form an amide. 2 When serving as the point of attachment for a linking moiety, the gamma carboxylate group is X 2 It condenses with the hydroxy group of the linking moiety to form an ester.

[0272] In a preferred embodiment, the folate ligand is folic acid: [ka] wherein either the alpha or gamma carboxylate group of the folic acid is X 2 serves as a covalent attachment point for the linking moiety).

[0273] In some embodiments, the alpha carboxylate group of the folic acid is X 2 In a preferred embodiment, the alpha carboxylate group of the folic acid is X 2 When serving as the point of attachment for a linking moiety, the alpha carboxylate group is X 2In some embodiments, the alpha carboxylate group of the folic acid is condensed with the amine group of the linking moiety to form an amide. 2 When serving as the point of attachment for a linking moiety, the alpha carboxylate group is X 2 It condenses with the hydroxy group of the linking moiety to form an ester.

[0274] In a preferred embodiment, the gamma carboxylate group of the folic acid is X 2 In a preferred embodiment, the gamma carboxylate group of the folic acid is X 2 When serving as the point of attachment for a linking moiety, the gamma carboxylate group is X 2 In some embodiments, the gamma carboxylate group of the folic acid is condensed with the amine group of the linking moiety to form an amide. 2 When serving as the point of attachment for a linking moiety, the gamma carboxylate group is X 2 It condenses with the hydroxy group of the linking moiety to form an ester.

[0275] In a preferred embodiment, the folate ligand is methotrexate: [ka] wherein either the alpha or gamma carboxylate group of the methotrexate is X 2 serves as a covalent attachment point for the linking moiety).

[0276] In some embodiments, the alpha carboxylate group of the methotrexate is X 2 In a preferred embodiment, the alpha carboxylate group of the methotrexate serves as a covalent attachment point to a linking moiety. 2 When serving as the point of attachment for a linking moiety, the alpha carboxylate group is X 2In some embodiments, the alpha carboxylate group of the methotrexate is condensed with the amine group of the linking moiety to form an amide. 2 When serving as the point of attachment for a linking moiety, the alpha carboxylate group is X 2 It condenses with the hydroxy group of the linking moiety to form an ester.

[0277] In a preferred embodiment, the gamma carboxylate group of the methotrexate is X 2 In a preferred embodiment, the gamma carboxylate group of the methotrexate serves as a covalent attachment point to a linking moiety. 2 When serving as the point of attachment for a linking moiety, the gamma carboxylate group is X 2 In some embodiments, the gamma carboxylate group of the methotrexate is condensed with the amine group of the linking moiety to form an amide. 2 When serving as the point of attachment for a linking moiety, the gamma carboxylate group is X 2 In a further aspect, the present invention provides a composition comprising a polyplex, the polyplex comprising a conjugate of Formula I* or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof, and a nucleic acid, the nucleic acid preferably comprising the conjugate: R 1 -(NR 2 -CH2-CH2) n -ZX 1 -(O-CH2-CH2) m -X 2 -L (Formula I*); (wherein n is any integer from 1 to 1500, preferably any integer from 2 to 1500, m is any integer from 1 to 200, preferably any integer from 1 to 100, and R 1 is the starting residue, preferably R 1 is -H or -CH3, and R 2 are independently —H or an organic residue, and the —(NR 2 -CH2-CH2) n -The R in the part 2at least 80%, preferably 90%, of X are H; 1 and X 2 are independently a divalent covalent linking moiety; Z is a divalent covalent linking moiety where Z is not -NHC(O)-, preferably Z is a divalent covalent linking moiety where Z is not a single bond and Z is not -NHC(O)-; L is a targeting fragment capable of binding to cells overexpressing prostate-specific membrane antigen (PSMA), preferably said L is non-covalently attached to a DUPA residue (HOOC(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO-); said nucleic acid is a pharmaceutically active nucleic acid, said pharmaceutically active nucleic acid encoding a pharmaceutically active peptide or protein; and preferably said composition consists of said conjugate.

[0278] In a further aspect, the present invention provides a composition comprising a polyplex, the polyplex comprising a conjugate of formula I* or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof, and a nucleic acid, the nucleic acid preferably comprising the conjugate: R 1 -(NR 2 -CH2-CH2) n -ZX 1 -(O-CH2-CH2) m -X 2 -L (Formula I*); (wherein n is any integer from 1 to 1500, preferably any integer from 2 to 1500, m is any integer from 1 to 200, preferably any integer from 1 to 100, and R 1 is the starting residue, preferably R 1 is -H or -CH3, and R 2 are independently —H or an organic residue, and the —(NR 2 -CH2-CH2) n -The R in the part 2 at least 80%, preferably 90%, of X are H; 1 and X 2are independently a divalent covalent linking moiety; Z is a divalent covalent linking moiety where Z is not -NHC(O)-; preferably, Z is a divalent covalent linking moiety where Z is not a single bond and Z is not -NHC(O)-; L is a targeting fragment capable of binding to prostate-specific membrane antigen (PSMA); preferably, said L is non-covalently attached to a DUPA residue (HOOC(CH)-CH(COOH)-NH-CO-NH-CH(COOH)-(CH)-CO-); said nucleic acid is a pharmaceutically active nucleic acid; said pharmaceutically active nucleic acid is a nucleic acid encoding a pharmaceutically active peptide or protein; and preferably, said composition consists of said conjugate.

[0279] In a further aspect, the present invention provides a composition comprising a polyplex, the polyplex comprising a conjugate of formula I* or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof, and a nucleic acid, the nucleic acid preferably comprising the conjugate: R 1 -(NR 2 -CH2-CH2) n -ZX 1 -(O-CH2-CH2) m -X 2 -L (Formula I*); (wherein n is any integer from 1 to 1500, preferably any integer from 2 to 1500, m is any integer from 1 to 200, preferably any integer from 1 to 100, and R 1 is the starting residue, preferably R 1 is -H or -CH3, and R 2 are independently —H or an organic residue, and the —(NR 2 -CH2-CH2) n -The R in the part 2 at least 80%, preferably 90%, of X are H; 1 and X 2are independently a divalent covalent linking moiety; Z is a divalent covalent linking moiety where Z is not -NHC(O)-; preferably, Z is a divalent covalent linking moiety where Z is not a single bond and Z is not -NHC(O)-; L is a targeting fragment; preferably, the targeting fragment L is non-covalently attached to a DUPA residue (HOOC(CH)-CH(COOH)-NH-CO-NH-CH(COOH)-(CH)-CO-); the nucleic acid is a pharmaceutically active nucleic acid; the pharmaceutically active nucleic acid is a nucleic acid encoding a pharmaceutically active peptide or protein; and preferably, the composition consists of the conjugate.

[0280] In another aspect, the present invention provides a polyplex comprising a conjugate of formula I* or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof, and a nucleic acid, wherein the nucleic acid is preferably a nucleic acid that is a conjugate of formula I* or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof, R 1 -(NR 2 -CH2-CH2) n -ZX 1 -(O-CH2-CH2) m -X 2 -L (Formula I*); (wherein n is any integer from 1 to 1500, preferably any integer from 2 to 1500, m is any integer from 1 to 200, preferably any integer from 1 to 100, and R 1 is the starting residue, preferably R 1 is -H or -CH3, and R 2 are independently —H or an organic residue, and the —(NR 2 -CH2-CH2) n -The R in the part 2 at least 80%, preferably 90%, of X are H; 1 and X 2are independently a divalent covalent linking moiety; Z is a divalent covalent linking moiety where Z is not -NHC(O)-, preferably Z is a divalent covalent linking moiety where Z is not a single bond and Z is not -NHC(O)-; L is a targeting fragment capable of binding to cells overexpressing prostate-specific membrane antigen (PSMA), preferably said L is non-covalently attached to a DUPA residue (HOOC(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO-); and the nucleic acid is a pharmaceutically active nucleic acid, which is a nucleic acid encoding a pharmaceutically active peptide or protein.

[0281] In another aspect, the present invention provides a polyplex comprising a conjugate of formula I* or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof, and a nucleic acid, wherein the nucleic acid is preferably a nucleic acid that is a conjugate of formula I* or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof, R 1 -(NR 2 -CH2-CH2) n -ZX 1 -(O-CH2-CH2) m -X 2 -L(formula I*); (wherein n is an integer of 1 to 1500, preferably an integer of 2 to 1500; m is an integer of 1 to 200, preferably an integer of 1 to 100; R 1 is the starting residue, preferably R 1 is -H or -CH3, and R 2 are independently —H or an organic residue, and the —(NR 2 -CH2-CH2) n -The R in the part 2 at least 80%, preferably 90%, of X are H; 1 and X 2are independently a divalent covalent linking moiety; Z is a divalent covalent linking moiety where Z is not -NHC(O)-, preferably Z is a divalent covalent linking moiety where Z is not a single bond and Z is not -NHC(O)-; L is a targeting fragment capable of binding to prostate-specific membrane antigen (PSMA), preferably L is non-covalently attached to a DUPA residue (HOOC(CH)-CH(COOH)-NH-CO-NH-CH(COOH)-(CH)-CO-); and the nucleic acid is a pharmaceutically active nucleic acid, which is a nucleic acid encoding a pharmaceutically active peptide or protein.

[0282] In another aspect, the present invention provides a polyplex comprising a conjugate of formula I* or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof, and a nucleic acid, wherein the nucleic acid is preferably a nucleic acid that is a conjugate of formula I* or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof, R 1 -(NR 2 -CH2-CH2) n -ZX 1 -(O-CH2-CH2) m -X 2 -L(formula I*); (wherein n is an integer of 1 to 1500, preferably an integer of 2 to 1500; m is an integer of 1 to 200, preferably an integer of 1 to 100; R 1 is the starting residue, preferably R 1 is -H or -CH3, and R 2 are independently —H or an organic residue, and the —(NR 2 -CH2-CH2) n -The R in the part 2 at least 80%, preferably 90%, of X are H; 1 and X 2are independently a divalent covalent linking moiety; Z is a divalent covalent linking moiety where Z is not -NHC(O)-, preferably Z is a divalent covalent linking moiety where Z is not a single bond and Z is not -NHC(O)-; L is a targeting fragment, preferably the targeting fragment L is non-covalently attached to a DUPA residue (HOOC(CH)-CH(COOH)-NH-CO-NH-CH(COOH)-(CH)-CO-); the nucleic acid is a pharmaceutically active nucleic acid, the pharmaceutically active nucleic acid encoding a pharmaceutically active peptide or protein.

[0283] In another aspect, the present invention provides a polyplex comprising a conjugate of Formula I, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof, and a nucleic acid, wherein the nucleic acid is preferably a conjugate of the formula I: [ka]

[0284] Formula I (In the formula, [ka] is a single or double bond, n is an integer from 1 to 1500, m is an integer of 1 to 200, preferably an integer of 2 to 1500; R 1 is the starting residue, preferably R 1 is -H or -CH3, and R 2 are independently —H or an organic residue, and the —(NR 2 -CH2-CH2) n -The R in the part 2 at least 80%, preferably at least 90% of are H; Ring A may have one or more R A1is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl optionally substituted with R A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo, or halogen, or two R A1 are combined with the atoms to which they are attached to form one or more fused C6-C 10 can form an aryl, C-C heteroaryl, or C-C cycloalkyl ring, and each fused aryl, heteroaryl, or cycloalkyl can be joined by one or more R A2 is optionally replaced by R A2 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen -SO3H or -OSO3H; X 1 is the formula -(Y 1 ) p where p is an integer from 1 to 20, and Y 1 Each occurrence of represents a chemical bond, -CR 11 R 12 -, -C(O)-, -O-, -S-, -NR 13 -, an amino acid residue, a divalent phenyl moiety, a divalent carbocyclic moiety, a divalent heterocyclic moiety, and a divalent heteroaryl moiety, each divalent phenyl or heteroaryl being independently selected from one or more R 13 and each divalent heterocycle is optionally substituted with one or more R 14 and optionally substituted with R 11 , R 12 and R 13 is independently, at each occurrence, H, —SOH, —NH, —COH, or C-C alkyl, each alkyl optionally substituted with —COH or —NH; R 14 is independently, at each occurrence, H, C1-C6 alkyl, or oxo, C6-C 10 aryl, or 5-8 membered heteroaryl), X 2 is the formula -(Y 2 ) q - (wherein q is an integer from 1 to 50, and Y2 Each occurrence of represents a chemical bond, -CR 21 R 22 -, NR 23 independently selected from -, -O-, -S-, -C(O)-, an amino acid residue, a divalent phenyl moiety, a divalent carbocyclic moiety, a divalent heterocyclic moiety, and a divalent heteroaryl moiety, wherein each divalent phenyl and divalent heteroaryl is selected from one or more R 23 and each divalent heterocyclic moiety is optionally substituted with one or more R 24 is optionally replaced by R 21 , R 22 and R 23 are each independently, at each occurrence, -H, -SO3H, -NH2, -CO2H, or C1-C6 alkyl, and each C1-C6 alkyl may be selected from one or more of -OH, oxo, -CO2H, -NH2, C6-C6 10 optionally substituted aryl or 5- to 8-membered heteroaryl; R 24 is a linking moiety that is independently, at each occurrence, —H, —COH, C1-C6 alkyl, or oxo; L is a targeting fragment, preferably the targeting fragment L is non-covalently attached to a DUPA residue (HOOC(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO-), and the nucleic acid is a pharmaceutically active nucleic acid, which is a nucleic acid encoding a pharmaceutically active peptide or protein.

[0285] In another aspect, the present invention provides a composition comprising a polyplex, the polyplex comprising a conjugate of Formula I, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof, and a nucleic acid, the nucleic acid preferably comprising the conjugate: [ka]

[0286] Formula I (In the formula, [ka] is a single or double bond, n is an integer of 1 to 1500, preferably an integer of 2 to 1500, m is a discrete number of repeating units m of 2 to 100, preferably a discrete number of repeating units m of 4 to 60, R 1 is the starting residue, preferably R 1 is -H or -CH3, R 2 are independently -H or an organic residue, and the -(NR 2 -CH2-CH2) n -The R in question 2 at least 80%, preferably at least 90%, of are H; Ring A is one or more R A1 and R is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl optionally substituted at any position by A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo, or halogen, or two R A1 are combined with the atoms to which they are attached to form one or more fused C6-C 10 can form an aryl, C-C heteroaryl, or C-C cycloalkyl ring, and each fused aryl, heteroaryl, or cycloalkyl can be joined by one or more R A2 and optionally substituted with R A2 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen -SO3H or -OSO3H; X 1 is a divalent covalent linking moiety, X 2 is a divalent covalent linking moiety, L is a targeting fragment, the targeting fragment is a PSMA targeting fragment, preferably the PSMA targeting fragment is non-covalently linked to a PSMA-expressing, preferably overexpressing, cell, and the nucleic acid is a pharmaceutically active nucleic acid, the pharmaceutically active nucleic acid encoding a pharmaceutically active peptide or protein. 1 is —H. In a preferred embodiment, the R 1 is -CH3. In a further preferred embodiment, the targeting fragment comprises, or preferably consists of, a DUPA residue (HOOC-(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO-). In a further highly preferred embodiment, the targeting fragment consists of a DUPA residue (HOOC(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO-) in which both chiral C atoms have the (S)-configuration, as shown in formula 1*.

[0287] In another aspect, the present invention provides a polyplex comprising a conjugate of Formula I, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof, and a nucleic acid, wherein the nucleic acid is preferably a conjugate of the formula I: [ka]

[0288] Formula I (In the formula, [ka] is a single or double bond, n is an integer of 1 to 1500, preferably an integer of 2 to 1500, m is a discrete number of repeating units m of 2 to 100, preferably a discrete number of repeating units m of 4 to 60, R 1is the starting residue, preferably R 1 is -H or -CH3, R 2 are independently -H or an organic residue, and the -(NR 2 -CH2-CH2) n -The R in question 2 at least 80%, preferably at least 90%, of are H; Ring A is one or more R A1 and R is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl optionally substituted at any position by A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo, or halogen, or two R A1 are combined with the atoms to which they are attached to form one or more fused C6-C 10 can form an aryl, C-C heteroaryl, or C-C cycloalkyl ring, and each fused aryl, heteroaryl, or cycloalkyl can be joined by one or more R A2 and optionally substituted with R A2 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen -SO3H or -OSO3H; X 1 is a divalent covalent linking moiety, X 2 is a divalent covalent linking moiety, L is a targeting fragment, the targeting fragment is a PSMA targeting fragment, preferably the PSMA targeting fragment is non-covalently linked to a PSMA-expressing, preferably overexpressing, cell, and the nucleic acid is a pharmaceutically active nucleic acid, the pharmaceutically active nucleic acid encoding a pharmaceutically active peptide or protein. 1 is —H. In a preferred embodiment, the R 1is -CH3. In a further preferred embodiment, the targeting fragment comprises, or preferably consists of, a DUPA residue (HOOC-(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO-). In a further highly preferred embodiment, the targeting fragment consists of a DUPA residue (HOOC(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO-) in which both chiral C atoms have the (S)-configuration, as shown in formula 1*.

[0289] In another aspect, the present invention provides a composition comprising a polyplex, the polyplex comprising a conjugate of Formula I, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof, and a nucleic acid, the nucleic acid preferably comprising the conjugate: [ka]

[0290] Formula I (In the formula, [ka] is a single or double bond, n is an integer of 1 to 1500, preferably an integer of 2 to 1500, m is a discrete number of repeating units m of 36; R 1 is the starting residue, preferably R 1 is -H or -CH3, R 2 are independently -H or an organic residue, and the -(NR 2 -CH2-CH2) n -The R in question 2 at least 80%, preferably at least 90%, of are H; Ring A is one or more R A1and R is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl optionally substituted at any position by A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo, or halogen, or two R A1 are combined with the atoms to which they are attached to form one or more fused C6-C 10 can form an aryl, C-C heteroaryl, or C-C cycloalkyl ring, and each fused aryl, heteroaryl, or cycloalkyl can be joined by one or more R A2 and optionally substituted with R A2 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen -SO3H or -OSO3H; X 1 is a divalent covalent linking moiety, X 2 is a divalent covalent linking moiety, L is a targeting fragment, the targeting fragment is a PSMA targeting fragment, preferably the PSMA targeting fragment is non-covalently linked to a PSMA-expressing, preferably overexpressing, cell, and the nucleic acid is a pharmaceutically active nucleic acid, the pharmaceutically active nucleic acid encoding a pharmaceutically active peptide or protein. 1 is —H. In a preferred embodiment, the R 1 is -CH3. In a further preferred embodiment, the targeting fragment comprises, or preferably consists of, a DUPA residue (HOOC-(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO-). In a further highly preferred embodiment, the targeting fragment consists of a DUPA residue (HOOC(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO-) in which both chiral C atoms have the (S)-configuration, as shown in formula 1*.

[0291] In another aspect, the present invention provides a polyplex comprising a conjugate of Formula I, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof, and a nucleic acid, wherein the nucleic acid is preferably a conjugate of the formula I: [ka]

[0292] Formula I (In the formula, [ka] is a single or double bond, n is an integer of 1 to 1500, preferably an integer of 2 to 1500, m is a discrete number of repeating units m of 36; R 1 is the starting residue, preferably R 1 is -H or -CH3, R 2 are independently -H or an organic residue, and the -(NR 2 -CH2-CH2) n -The R in question 2 at least 80%, preferably at least 90%, of are H; Ring A is one or more R A1 and R is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl optionally substituted at any position by A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo, or halogen, or two R A1 are combined with the atoms to which they are attached to form one or more fused C6-C 10 can form an aryl, C-C heteroaryl, or C-C cycloalkyl ring, and each fused aryl, heteroaryl, or cycloalkyl can be joined by one or more R A2 and optionally substituted with R A2are independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen -SO3H or -OSO3H; X 1 is a divalent covalent linking moiety, X 2 is a divalent covalent linking moiety, L is a targeting fragment, the targeting fragment is a PSMA targeting fragment, preferably the PSMA targeting fragment is non-covalently linked to a PSMA-expressing, preferably overexpressing, cell, and the nucleic acid is a pharmaceutically active nucleic acid, the pharmaceutically active nucleic acid encoding a pharmaceutically active peptide or protein. 1 is —H. In a preferred embodiment, the R 1 is -CH3. In a further preferred embodiment, the targeting fragment comprises, or preferably consists of, a DUPA residue (HOOC-(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO-). In a further highly preferred embodiment, the targeting fragment consists of a DUPA residue (HOOC(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO-) in which both chiral C atoms have the (S)-configuration, as shown in formula 1*.

[0293] In another aspect, the present invention provides a composition comprising a polyplex, the polyplex comprising a conjugate of Formula I, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof, and a nucleic acid, the nucleic acid preferably comprising the conjugate: [ka]

[0294] Formula I (In the formula, [ka] is a single or double bond, n is an integer of 1 to 1500, preferably an integer of 2 to 1500, m is a discrete number of consecutive repeating units m of 2 to 100, preferably a discrete number of consecutive repeating units m of 4 to 60, R 1 is the starting residue, preferably R 1 is -H or -CH3, R 2 are independently -H or an organic residue, and the -(NR 2 -CH2-CH2) n -The R in question 2 at least 80%, preferably at least 90%, of are H; Ring A is one or more R A1 and R is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl optionally substituted at any position by A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo, or halogen, or two R A1 combine with the atoms to which they are attached to form one or more fused C6-C 10 can form an aryl, C-C heteroaryl, or C-C cycloalkyl ring, and each fused aryl, heteroaryl, or cycloalkyl can be joined by one or more R A2 and optionally substituted with R A2 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen -SO3H or -OSO3H; X 1 is a divalent covalent linking moiety, X 2 is a divalent covalent linking moiety, L is a targeting fragment, the targeting fragment is a PSMA targeting fragment, preferably the PSMA targeting fragment is non-covalently linked to a PSMA-expressing, preferably overexpressing, cell, and the nucleic acid is a pharmaceutically active nucleic acid, the pharmaceutically active nucleic acid encoding a pharmaceutically active peptide or protein. 1 is —H. In a preferred embodiment, the R 1 is -CH3. In a further preferred embodiment, the targeting fragment comprises, or preferably consists of, a DUPA residue (HOOC-(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO-). In a further highly preferred embodiment, the targeting fragment consists of a DUPA residue (HOOC(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO-) in which both chiral C atoms have the (S)-configuration, as shown in formula 1*.

[0295] In another aspect, the present invention provides a polyplex comprising a conjugate of Formula I, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof, and a nucleic acid, wherein the nucleic acid is preferably a conjugate of the formula I: [ka]

[0296] Formula I (In the formula, [ka] is a single or double bond, n is an integer of 1 to 1500, preferably an integer of 2 to 1500, m is a discrete number of consecutive repeating units m of 2 to 100, preferably a discrete number of consecutive repeating units m of 4 to 60, R 1is the starting residue, preferably R 1 is -H or -CH3, R 2 are independently -H or an organic residue, and the -(NR 2 -CH2-CH2) n -The R in question 2 at least 80%, preferably at least 90%, of are H; Ring A is one or more R A1 and R is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl optionally substituted at any position by A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo, or halogen, or two R A1 combine with the atoms to which they are attached to form one or more fused C6-C 10 can form an aryl, C-C heteroaryl, or C-C cycloalkyl ring, and each fused aryl, heteroaryl, or cycloalkyl can be joined by one or more R A2 and optionally substituted with R A2 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen -SO3H or -OSO3H; X 1 is a divalent covalent linking moiety, X 2 is a divalent covalent linking moiety, L is a targeting fragment, the targeting fragment is a PSMA targeting fragment, preferably the PSMA targeting fragment is non-covalently linked to a PSMA-expressing, preferably overexpressing, cell, and the nucleic acid is a pharmaceutically active nucleic acid, the pharmaceutically active nucleic acid encoding a pharmaceutically active peptide or protein. 1 is —H. In a preferred embodiment, the R 1is -CH3. In a further preferred embodiment, the targeting fragment comprises, or preferably consists of, a DUPA residue (HOOC-(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO-). In a further highly preferred embodiment, the targeting fragment consists of a DUPA residue (HOOC(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO-) in which both chiral C atoms have the (S)-configuration, as shown in formula 1*.

[0297] In another aspect, the present invention provides a composition comprising a polyplex, the polyplex comprising a conjugate of Formula I, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof, and a nucleic acid, the nucleic acid preferably comprising th...

Claims

1. A composition comprising a polyplex, the polyplex comprising a conjugate and a nucleic acid; the conjugate is a linear polyethyleneimine fragment comprising an alpha end and an omega end, wherein the alpha end of the polyethyleneimine fragment is an initiating residue; a polyethylene glycol fragment comprising a first terminal end and a second terminal end; The omega end of the polyethyleneimine fragment is connected to the first terminal end of the polyethylene glycol fragment by a divalent covalent linking group -Z-X １ - is connected, -Z-X １ - is not a single bond, -Z- is not an amide, the second terminal end of the polyethylene glycol fragment is linked to a divalent covalent linking moiety X ２ is connected to the targeting fragment by A composition wherein said nucleic acid is a pharmaceutically active nucleic acid, said pharmaceutically active nucleic acid being a nucleic acid encoding a pharmaceutically active peptide or protein.

2. The conjugate has the formula I* R １ -(NR ２ -CH ２ -CH ２ ) ｎ -Z-X １ -(O-CH ２ -CH ２ ) ｍ -X ２ -L (Formula I*) (In the formula, n is any integer from 1 to 1500, m is any integer from 1 to 200; R １ is the initial residue, preferably R １ is -H or -CH ３ and R ２ are independently —H or an organic residue, and the —(NR ２ -CH ２ -CH ２ ) ｎ The R in - ２ at least 80%, preferably 90%, of the X １ and X ２ are independently divalent covalent linking moieties; Z is a divalent covalent linking moiety, Z is not a single bond, Z is not —NHC(O)—, 2. The composition of claim 1, wherein L is a targeting fragment, preferably wherein the targeting fragment is capable of binding to a cell, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof.

3. The conjugate has Formula I: 【Chemistry 1】 (In the formula, 【Chemistry 2】 is a single or double bond, n is any integer from 1 to 1500, m is any integer from 1 to 200; R １ is the initial residue, preferably R １ is -H or -CH ３ and R ２ are independently —H or an organic residue, and the —(NR ２ -CH ２ -CH ２ ) ｎ The R in - ２ at least 80%, preferably at least 90%, of are H; Ring A is one or more R Ａ１ is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl optionally substituted with R Ａ１ is C １ ~C ６ Alkyl, C １ ~C ６ or two R independently selected from alkoxy, oxo, or halogen; Ａ１ combine with the atoms to which they are attached to form one or more fused C ６ ~C １０ Aryl, C ５ ~C ６ Heteroaryl or C ３ ~C ６ Each fused aryl, heteroaryl, or cycloalkyl may be joined by one or more R Ａ２ and optionally substituted with R Ａ２ is C １ ~C ６ Alkyl, C １ ~C ６ Alkoxy, halogen -SO ３ H or -OSO ３ is independently selected from H, X １ is a divalent covalent linking moiety, X ２ is a divalent covalent linking moiety, 3. The composition of claim 1 or claim 2, wherein L is a targeting fragment, preferably wherein said targeting fragment is capable of binding to a cell, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or enantiomer thereof.

4. The —(O—CH ２ -CH ２ ) ｍ The - moiety is composed of 4 to 60 discrete repeating units m, preferably repeating -(O-CH ２ -CH ２ 4. The composition according to claim 2, wherein the discrete number m of 1-octyl-2-methyl-2-propanol units is 36.

5. wherein the conjugate of formula I is 【Transformation 3】 【Chemistry 4】 【Transformation 5】 【Transformation 6】 【Transformation 7】 【Transformation 8】 【Chemistry 9】 【Chemistry 10】 and 【Chemistry 11】 The composition according to any one of claims 3 to 4, selected from:

6. wherein the conjugate of formula I is 【Chemistry 12】 and 【Chemistry 13】 The composition according to any one of claims 3 to 5, wherein the composition is selected from

7. X １ but, 【Chemistry 14】 wherein r is independently in each occurrence 0 to 6, preferably 0, 1, 2 or 5; s is independently in each occurrence 0 to 6, preferably 0, 2, 3 or 4; t is independently, in each occurrence, 0 to 6, preferably 0, 1, 2, or 4; R １１ and R １２ is independently, in each occurrence, —H and —C １ ~C ２ alkyl, R １３ is —H, and preferably the wavy line closest to said integer “r” is the bond to ring A, and the wavy line closest to said integer “s” or “t” is —[OCH ２ -CH ２ ] ｍ The composition of any one of claims 3 to 6, comprising a group selected from:

8. X １ but, 【Chemistry 15】 and 【Chemistry 16】 Preferably, the wavy line on the left is a bond to ring A and the wavy line on the right is —[OCH ２ -CH ２ ] ｍ The composition of any one of claims 3 to 7, wherein the bond is to -.

9. X ２ but, 【Chemistry 17】 [Chemistry 18] and 【Chemistry 19】 (In the formula, Y ２ Each occurrence of ２１ R ２２ -, NR ２３ -, -O-, -S-, -C(O)-, an amino acid residue, a divalent phenyl moiety, a divalent carbocyclic moiety, a divalent heterocyclic moiety, and a divalent heteroaryl moiety, each divalent phenyl and divalent heteroaryl being independently selected from one or more R ２３ and each divalent heterocyclic moiety is optionally substituted with one or more R ２４ is optionally replaced by R ２１ , R ２２ and R ２３ are each independently, at each occurrence, —H, —SO ３ H, —NH ２ , -CO ２ H or C １ ~C ６ alkyl, and each C １ ~C ６ Alkyl is one or more of -OH, oxo, -CO ２ H, —NH ２ , C ６ ~C １０ optionally substituted with aryl or 5-8 membered heteroaryl; R ２４ is independently, at each occurrence, —H, —CO ２ H, C １ ~C ６ alkyl or oxo, and preferably the wavy line on the left is —[OCH ２ -CH ２ ] ｍ -, and the wavy line on the right is the bond to L) The composition according to any one of claims 3 to 8, wherein the composition is selected from

10. X ２ but, 【Chemistry 20】 【Chemistry 21】 【Chemistry 22】 【Chemistry 23】 and 【Chemistry 24】 Preferably, the wavy line on the left side is selected from -[OCH ２ -CH ２ ] ｍ The composition of any one of claims 3 to 9, wherein the wavy line on the right is a bond to - and the wavy line on the right is a bond to L.

11. 10. The composition of claim 9, wherein the targeting fragment L is capable of binding to a cell surface receptor, the cell surface receptor being selected from growth factor receptors, cytokine receptors, hormone receptors, extracellular matrix proteins, transmembrane proteins, glycosylphosphatidylinositol (GPI)-anchored membrane proteins, carbohydrate-binding integral membrane proteins, lectins, ion channels, G protein-coupled receptors, and enzyme-linked receptors such as tyrosine kinase-coupled receptors, preferably the cell surface receptor being selected from epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), prostate-specific membrane antigen (PSMA), insulin-like growth factor 1 receptor (IGF1R), vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), asialoglycoprotein receptor (ASGPr), and fibroblast growth factor receptor (FGFR).

12. the targeting fragment L is selected from the group consisting of an EGFR targeting fragment, preferably human EGF (hEGF); a PSMA targeting fragment, preferably a DUPA residue; an anti-HER2 peptide, preferably an anti-HER2 antibody or an affibody; folic acid; methotrexate; a somatostatin receptor targeting fragment, preferably somatostatin and / or octreotide; an integrin targeting fragment, preferably an arginine-glycine-aspartic acid (RGD)-containing fragment; a low pH insertion peptide; an ASGPr targeting fragment, preferably asialoorosomucoid; an insulin receptor targeting fragment, preferably insulin; a mannose-6-phosphate receptor targeting fragment, preferably mannose-6-phosphate; a mannose receptor targeting fragment, preferably mannose; ｘ 10. The composition of any one of the preceding claims, selected from: an antigen targeting fragment, preferably E-selectin; a sigma-2 receptor agonist, preferably N,N-dimethyltryptamine (DMT), a sphingolipid-derived amine and / or a steroid, more preferably progesterone; a p32 targeting ligand, preferably an anti-p32 antibody or a p32-binding LyP-1 tumor-homing peptide; a Trop-2 targeting fragment, preferably an anti-Trop-2 antibody and / or antibody fragment; insulin-like growth factor 1; vascular endothelial growth factor; platelet-derived growth factor; and fibroblast growth factor.

13. 10. The composition of any one of the preceding claims, wherein the conjugate is selected from Compound 1a, Compound 1b, Compound 4a, Compound 4b, Compound 7a, Compound 7b, Compound 10a, Compound 10b, Compound 14, Compound 17a, Compound 17b, Compound 18, Compound 19, Compound 22a, Compound 22b, Compound 28a, Compound 28b, Compound 31a, Compound 31b, Compound 38a, Compound 38b, Compound 43, Compound 47a, Compound 47b, Compound 51a, Compound 51b, Compound 56a, Compound 56b, Compound 62a, Compound 62b, Compound 70a, Compound 70b, Compound 72a, Compound 72b, Compound 75a, Compound 75b, Compound 78a, Compound 78b, Compound 81, Compound 82a, Compound 82b and / or Compound 83.

14. 10. The composition of any one of the preceding claims, wherein the nucleic acid is RNA, and the RNA is messenger RNA (mRNA).

15. 10. The composition of any one of the preceding claims, wherein the nucleic acid is DNA, and the DNA is plasmid DNA.

16. 10. The composition according to any one of the preceding claims, wherein the pharmaceutically active peptide or protein is selected from a cytokine, a growth factor, a hormone, an enzyme, a tumor antigen, a viral antigen, a bacterial antigen, an autoantigen or an allergen, preferably wherein the pharmaceutically active peptide or protein is a cytokine selected from an interleukin, an interferon and a chemokine.

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