Polymer complexes of targeting conjugates comprising polyethyleneimine and polyethylene glycol and nucleic acids

Abstract

The present invention provides targeted polymer complexes comprising: (i) a nucleic acid, in particular a nucleic acid encoding a peptide or protein having pharmaceutical activity; and (ii) a targeting conjugate comprising LPEI and PEG fragments linked by discrete bonds formed by a defined chemically selective reaction. Thus, the LPEI fragment is bonded to a single PEG fragment in a linear end-to-end manner. The linear conjugate is further conjugated to a targeting fragment to achieve selective interaction with a particular cell type. The polymer complexes selectively deliver nucleic acids to target cells, thereby achieving high expression and efficient protein translation and secretion of the encoded pharmaceutically active proteins.

Description

Background Art

[0001] Cancer remains the leading cause of death worldwide. For most solid tumors, chemotherapy is a key treatment option for controlling residual cancer cells after surgical resection. One of the main reasons for chemotherapy failure is the low targeting and uptake efficiency of chemotherapeutic drugs by tumors (JK Vasir and V Labhasetwar, Technology in Cancer Research & Treatment, 2005, 4 (4): 363-374). Chemotherapeutic drugs are difficult to approach tumors, resulting in the need for higher doses, and due to the characteristics of chemotherapeutic drugs, this can lead to nonspecific uptake and toxicity of healthy cells. Targeted drug delivery strategies (wherein therapeutic drugs are reversibly bound to targeting ligands and selectively delivered to cells for treatment) have been applied to many clinically used chemotherapeutic drugs. This strategy is expected to maximize the safety and effectiveness of specific chemotherapeutic drugs because they are selectively delivered to target cells, avoiding nonspecific uptake and related toxicity of healthy cells (M Srinivasarao and PS Low, Chem Rev, 2017, 117: 12133-12164), thereby achieving a higher maximum tolerated dose.

[0002] With regard to nucleic acid therapies (including DNA and mRNA), nanoparticle delivery systems have attracted much attention due to their applications, particularly 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; XTan et al., 2020, J Control Release 323: 240-252; and references cited therein). However, for these nucleic acid therapies to be successful, they must overcome many delivery barriers, including rapid degradation in vivo, poor uptake by target cells, the need for nuclear entry, and potential in vivo toxicity to healthy cells and tissues. Nanoparticle delivery systems (including targeted nanoparticle delivery systems) have been designed to address and attempt to overcome some of these obstacles 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 polymer complexes (polyplexes) with negatively charged nucleic acids in solution. For example, linear polyethyleneimine (LPEI) is protonated at physiological pH and therefore carries a net positive charge. When LPEI is incubated with nucleic acids that have a net negative charge at physiological pH, LPEI and the nucleic acids can form polymer complexes that are bound together by electrostatic interactions. These polymer complexes can be taken up by cells in vivo and deliver nucleic acid sequences into cells. Therefore, polymer complexes comprising cationic polymers and nucleic acids can be used as therapeutic carriers. Despite their promising prospects, there are still technical challenges in forming homogeneous and well-characterized cationic polymers. Polymer complexes containing only LPEI easily aggregate and interact with serum proteins, limiting their potential as nucleic acid delivery agents. To overcome these challenges, polymerized LPEI can be coupled with polyethylene glycol (PEG). The PEG segment can help protect LPEI from the effects of the surrounding matrix and improve the biocompatibility and blood circulation of the resulting polymer complex. Examples of such polyethyleneimine-polyethylene glycol conjugates that also include a targeting moiety and are used as non-viral vectors for delivery, in particular, of double-stranded RNA (e.g., polyinosinic acid:polycytidylic acid) have been described (WO2015 / 173824, WO2010 / 073247, US2004 / 248842A1, Vetter VC, Wagner EJ Control Release, 2022 346: 110-135, and references cited therein). However, in the referenced conjugates and carriers, the conjugation of PEG to LPEI is achieved by forming a covalent bond between an electrophilic PEG segment and a secondary amine embedded in the LPEI backbone segment, resulting in branched, heterogeneous conjugates and carriers that randomly and indefinitely contain PEG segments and are characterized based on average PEG inclusion density. In such conjugates, a large number of different PEG segments are bonded to the LPEI segments in an orthogonal manner and without site specificity. This random synthesis and imprecise characterization of LPEI-PEG conjugates makes it difficult to establish a clear structure-activity relationship (SAR) between the conjugate structure and the activity of the resulting polymer complexes.

[0004] Although efforts have been made in recent years and results have been achieved, the development of novel targeted delivery platforms has received great attention, which can protect nucleic acid (including mRNA) and deliver it to target tissues and cells, thereby giving play to the powerful therapeutic potential of these molecules (AJ Mukalel et al., 2019, Cancer Lett.458:102-112). Therefore, it is necessary to have a homogeneous nanoparticle that can selectively deliver nucleic acid (including mRNA or pDNA) to target tissues and cells, particularly with a homogeneous LPEI-PEG conjugate of a clear chemical structure. Summary of the Invention

[0005] The present invention provides targeted polymer complexes, which include: (i) nucleic acids encoding target peptides or proteins, particularly nucleic acids encoding peptides or proteins (such as cytokines, interferons or toxins) with pharmaceutical activity; and (ii) targeted conjugates, wherein the conjugates include LPEI segments and PEG segments connected by discrete bonds, wherein the discrete bonds are formed by a determined chemical selective reaction rather than by the random and uncontrolled bonding of multiple nucleophiles of electrophilic PEG segments and LPEI main chain segments. Therefore, the present invention provides more homogeneous targeted conjugates with a determined chemical structure. Discrete bonds not only ensure the consistent and predictable ratio of LPEI segments to PEG segments, but also ensure a determined linear conjugate rather than a randomly branched conjugate. Therefore, the LPEI segments are bonded to a single PEG segment in a linear end-to-end manner.

[0006] The conjugate also includes a targeting segment connected to the PEG segment, which is capable of targeting a specific cell type and promoting the uptake of the compositions of the present invention and pharmaceutically active nucleic acids in the specific cell type. Therefore, preferred embodiments include a targeting segment (e.g., hEGF, DUPA, or folic acid) that is specifically linked to the LPEI-PEG diconjugate to target a corresponding receptor (e.g., hEGFR, PSMA, or folate receptor) on a specific cell type (typically a cancer cell type) that exhibits high expression and overexpression on these cell types.

[0007] More advantageously and unexpectedly, the inventors have found that the resulting preferred conjugates and polymer complexes of the present invention have significantly reduced heterogeneity due to the determined chemoselective bonding between the LPEI segment and the PEG segment, and therefore have a significantly reduced number of potentially bioactive conjugates and polymer complexes, not only forming polymer complexes of suitable size, but also maintaining or even improving their overall bioactivity, such as highly selective targeted delivery of pharmaceutically active nucleic acids and subsequent efficient translation and secretion of the encoded pharmaceutically active proteins. Thus, the compositions and polymer complexes of the present invention not only selectively deliver pharmaceutically active nucleic acids encoding pharmaceutically active peptides or proteins to target cells (particularly cancer cells), but also achieve high expression, efficient protein translation, and secretion of the encoded pharmaceutically active proteins.

[0008] Thus, in one aspect, the present invention provides a composition comprising a polymer complex comprising a conjugate and a nucleic acid, wherein the nucleic acid is preferably non-covalently bound to the conjugate, the conjugate comprising:

[0009] a linear polyethyleneimine segment comprising an α terminus and an ω terminus, wherein the α terminus of the polyethyleneimine segment is an initiation residue;

[0010] a polyethylene glycol segment comprising a first terminus and a second terminus; and

[0011] The ω end of the polyethyleneimine segment is connected by a divalent covalent linker -ZX 1 - is linked to the first end of the polyethylene glycol segment, wherein -ZX 1 is not a single bond and -Z- is not an amide;

[0012] The second end of the polyethylene glycol segment is covalently linked to a moiety X via a divalent 2 ligated with the targeting fragment, and

[0013] The nucleic acid is a nucleic acid with pharmaceutical activity, and the nucleic acid with pharmaceutical activity is a nucleic acid encoding a peptide or protein with pharmaceutical activity. In a preferred embodiment of this aspect, the composition consists of the polymer complex.

[0014] In another aspect, the present invention provides a composition comprising a polymer complex, wherein the polymer complex comprises a conjugate of Formula I* or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or enantiomer thereof and a nucleic acid, preferably the nucleic acid is non-covalently bound to the conjugate:

[0015] R 1 -(NR 2 -CH2-CH2) n -ZX 1 -(O-CH2-CH2) m -X 2 -L (Formula I*);

[0016] Where:

[0017] n is any integer from 1 to 1500, preferably any integer from 2 to 1500;

[0018] m is any integer from 1 to 200, preferably any integer from 2 to 200, preferably any integer from 1 to 100, more preferably any integer from 2 to 100;

[0019] R 1 is an initiating residue, preferably, R 1 is -H or -CH3;

[0020] R 2 are independently -H or an organic residue, the -(NR 2 -CH2-CH2) n - At least 80%, preferably 90% of R2 is H;

[0021] X 1 and X 2 are independently divalent covalently linking moieties;

[0022] Z is a divalent covalent linking moiety, at least 80%, preferably 90%, of the moieties in Z are single bonds and at least 80%, preferably 90%, of the moieties in Z are -NHC(O)-;

[0023] L is a targeting fragment, preferably, the targeting fragment can bind to a cell, more preferably, the targeting fragment can bind to a cell surface receptor; and

[0024] The nucleic acid is a nucleic acid with pharmaceutical activity, and the nucleic acid with pharmaceutical activity is a nucleic acid encoding a peptide or protein with pharmaceutical activity. Preferably, the composition consists of the polymer complex.

[0025] In another aspect, the present invention provides a polymer complex comprising a conjugate of Formula I* or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or enantiomer thereof and a nucleic acid, preferably wherein the nucleic acid is non-covalently bound to the conjugate:

[0026] R 1 -(NR 2 -CH2-CH2) n -ZX 1 -(O-CH2-CH2) m -X 2 -L (Formula I*);

[0027] Where:

[0028] n is any integer from 1 to 1500, preferably any integer from 2 to 1500;

[0029] m is any integer from 1 to 200, preferably any integer from 2 to 200, preferably any integer from 1 to 100, more preferably any integer from 2 to 100;

[0030] R 1 is an initiating residue, preferably, R 1 is -H or -CH3;

[0031] R 2 are independently -H or an organic residue, the -(NR 2 -CH2-CH2) n - At least 80%, preferably 90% of R 2 is H;

[0032] X 1 and X2 are independently divalent covalently linking moieties;

[0033] Z is a divalent covalent linking moiety, wherein Z is not a single bond and Z is not -NHC(O)-;

[0034] L is a targeting fragment, preferably, the targeting fragment can bind to cells, more preferably, the targeting fragment can bind to cell surface receptors;

[0035] The nucleic acid is a nucleic acid with pharmaceutical activity, and the nucleic acid with pharmaceutical activity is a nucleic acid encoding a peptide or protein with pharmaceutical activity.

[0036] In another aspect, the present invention provides a composition comprising a polymer complex comprising a conjugate of Formula I or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or enantiomer thereof and a nucleic acid, preferably the nucleic acid is non-covalently bound to the conjugate:

[0037]

[0038] Where:

[0039] is a single bond or a double bond;

[0040] n is any integer from 1 to 1500, preferably any integer from 2 to 1500;

[0041] m is any integer from 1 to 200, preferably any integer from 2 to 200, preferably any integer from 1 to 100, more preferably any integer from 2 to 100;

[0042] R 1 is an initiating residue, preferably, R 1 is -H or -CH3;

[0043] R 2 are independently -H or an organic residue, the -(NR 2 -CH2-CH2) n - at least 80%, preferably at least 90% of R 2 is H;

[0044] Ring A is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl group, optionally substituted at any position with one or more R A1 Replacement; R A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo or halogen; or, two R A1 Together with the atoms to which they are attached, they can form more than one fused C6-C 10aryl, C5-C6 heteroaryl or C3-C6 cycloalkyl ring, wherein each fused aryl, heteroaryl or cycloalkyl is optionally substituted by one or more R A2 Replacement; R A2 Independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, -SO3H or -OSO3H;

[0045] X 1 is a divalent covalent linking moiety;

[0046] X 2 is a divalent covalent linking moiety; and

[0047] L is a targeting fragment, preferably, the targeting fragment can bind to a cell, more preferably, the targeting fragment can bind to a cell surface receptor; and

[0048] The nucleic acid is a nucleic acid with pharmaceutical activity, and the nucleic acid with pharmaceutical activity is a nucleic acid encoding a peptide or protein with pharmaceutical activity.

[0049] Although for simplicity, the bicyclic NN=N fragments in Formula I are generally drawn herein using one single bond and one double bond, those skilled in the art will appreciate that Formula I and its related coupled structures described herein can also be drawn as shown below. Such descriptions and illustrations of Formula I are used interchangeably herein:

[0050]

[0051] In the formula, the fragment Represents fragment R 1 (NR 2 CH2CH2) n The two different regioisomers of

[0052] wherein the wavy line represents the chemical bond to Ring A. Thus, Formula I as drawn herein includes two regioisomeric embodiments, namely, wherein the fragment R 1 (NR 2 CH2CH2) n In the above structure, it is bonded to the top nitrogen atom or in the above structure, it is bonded to the bottom nitrogen atom, but not to the middle nitrogen atom. Those skilled in the art will appreciate that this also applies to other formulae herein, including Formula IA, Formula IB, Formula IC, Formula ID, Formula IE, Formula IH, Formula IJ, Formula IK, etc.

[0053] In another aspect, the present invention provides a polymer complex comprising a conjugate of Formula I or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or enantiomer thereof and a nucleic acid, preferably wherein the nucleic acid is non-covalently bound to the conjugate:

[0054]

[0055] Where:

[0056] is a single bond or a double bond;

[0057] n is any integer from 1 to 1500, preferably any integer from 2 to 1500;

[0058] m is any integer from 2 to 200, preferably any integer from 1 to 200, more preferably any integer from 2 to 100;

[0059] R 1 is an initiating residue, preferably, R 1 is -H or -CH3;

[0060] R 2 are independently -H or an organic residue, the -(NR 2 -CH2-CH2) n - at least 80%, preferably at least 90% of R 2 is H;

[0061] Ring A is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl group, optionally substituted at any position with one or more R A1 Replacement; R A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo or halogen; or, two R A1 Together with the atoms to which they are attached, they can form more than one fused C6-C 10 aryl, C5-C6 heteroaryl or C3-C6 cycloalkyl ring, wherein each fused aryl, heteroaryl or cycloalkyl is optionally substituted by one or more R A2 Replacement; R A2 Independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, -SO3H or -OSO3H;

[0062] X 1 is a divalent covalent linking moiety;

[0063] X 2 is a divalent covalent linking moiety; and

[0064] L is a targeting fragment, preferably, the targeting fragment can bind to a cell, more preferably, the targeting fragment can bind to a cell surface receptor; and

[0065] The nucleic acid is a nucleic acid with pharmaceutical activity, and the nucleic acid with pharmaceutical activity is a nucleic acid encoding a peptide or protein with pharmaceutical activity.

[0066] In another aspect, the present invention provides a composition comprising a polymer complex comprising a conjugate of Formula I or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or enantiomer thereof and a nucleic acid, preferably the nucleic acid is non-covalently bound to the conjugate:

[0067]

[0068] Where:

[0069] is a single bond or a double bond;

[0070] n is any integer from 1 to 1500, preferably any integer from 2 to 1500;

[0071] m is any integer from 1 to 200, preferably any integer from 2 to 200, preferably any integer from 1 to 100, more preferably any integer from 2 to 100;

[0072] R 1 is an initiating residue, preferably, R 1 is -H or -CH3;

[0073] R 2 are independently -H or an organic residue, the -(NR 2 -CH2-CH2) n - At least 80%, preferably 90% of said R 2 is H;

[0074] Ring A is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl group, optionally substituted at any position with one or more R A1 replace;

[0075] R A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo or halogen; or, two R A1 Together with the atoms to which they are attached, they can form more than one fused C6-C 10 aryl, C5-C6 heteroaryl or C3-C6 cycloalkyl ring, wherein each fused aryl, heteroaryl or cycloalkyl is optionally substituted by one or more R A2 replace;

[0076] R A2 Independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, -SO3H or -OSO3H;

[0077] X 1 Formula -(Y 1 ) p- a connecting portion, wherein p is an integer from 1 to 20, Y 1 is independently selected at each occurrence from a chemical bond, -CR 11 R 12 -、-C(O)-、-O-、-S-、-NR 13 -, amino acid residues, divalent phenyl moieties, divalent heterocyclic moieties and divalent heteroaryl moieties, each divalent phenyl or heteroaryl group being optionally substituted by one or more R 13 Each divalent heterocyclic ring may be substituted with one or more R 14 Replacement, R 11 、R 12 and R 13 is independently H or C1-C6 alkyl at each occurrence, R 14 is independently H, C1-C6 alkyl or oxo at each occurrence;

[0078] X 2 Formula -(Y 2 ) q - a connecting portion, wherein q is an integer from 1 to 50, Y 2 is independently selected at each occurrence from a chemical bond, -CR 21 R 22 -、NR 23 -, -O-, -S-, -C(O)-, amino acid residues, divalent phenyl moieties, divalent heterocyclic moieties and divalent heteroaryl moieties, each of which is optionally substituted by one or more R 23 Each divalent heterocyclic moiety is optionally substituted with one or more R 24 Replacement, R 21 、R 22 and R 23 Each occurrence is independently -H, -CO2H or C1-C6 alkyl, each C1-C6 alkyl optionally substituted by one or more -OH, oxo, C6-C 10 aryl or 5 to 8 membered heteroaryl substituted, R 24 is independently at each occurrence -H, -CO2H, C1-C6 alkyl, or oxo; and

[0079] L is a targeting fragment, preferably, the targeting fragment is capable of binding to a cell, preferably, the targeting fragment is capable of binding to a cell surface receptor; and

[0080] The nucleic acid is a nucleic acid with pharmaceutical activity, and the nucleic acid with pharmaceutical activity is a nucleic acid encoding a peptide or protein with pharmaceutical activity. More preferably, the composition consists of the polymer complex.

[0081] In another aspect, the present invention provides a polymer complex comprising a conjugate of Formula I or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or enantiomer thereof and a nucleic acid, preferably wherein the nucleic acid is non-covalently bound to the conjugate:

[0082]

[0083] Where:

[0084] is a single bond or a double bond;

[0085] n is any integer from 1 to 1500, preferably any integer from 2 to 1500;

[0086] m is any integer from 1 to 200, preferably any integer from 2 to 200, preferably any integer from 1 to 100, more preferably any integer from 2 to 100;

[0087] R 1 is an initiating residue, preferably, R 1 is -H or -CH3;

[0088] R 2 are independently -H or an organic residue, the -(NR 2 -CH2-CH2) n - At least 80%, preferably 90% of said R 2 is H;

[0089] Ring A is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl group, optionally substituted at any position with one or more R A1 replace;

[0090] R A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo or halogen; or, two R A1 Together with the atoms to which they are attached, they can form more than one fused C6-C 10 aryl, C5-C6 heteroaryl or C3-C6 cycloalkyl ring, wherein each fused aryl, heteroaryl or cycloalkyl is optionally substituted by one or more R A2 replace;

[0091] R A2 Independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, -SO3H or -OSO3H;

[0092] X 1 Formula -(Y 1 ) p - a connecting portion, wherein p is an integer from 1 to 20, Y 1is independently selected at each occurrence from a chemical bond, -CR 11 R 12 -、-C(O)-、-O-、-S-、-NR 13 -, amino acid residues, divalent phenyl moieties, divalent heterocyclic moieties and divalent heteroaryl moieties, each divalent phenyl or heteroaryl group being optionally substituted by one or more R 13 Each divalent heterocyclic ring may be substituted with one or more R 14 Replacement, R 11 、R 12 and R 13 is independently H or C1-C6 alkyl at each occurrence, R 14 is independently H, C1-C6 alkyl or oxo at each occurrence;

[0093] X 2 Formula -(Y 2 ) q - a connecting portion, wherein q is an integer from 1 to 50, Y 2 is independently selected at each occurrence from a chemical bond, -CR 21 R 22 -、NR 23 -, -O-, -S-, -C(O)-, amino acid residues, divalent phenyl moieties, divalent heterocyclic moieties and divalent heteroaryl moieties, each of which is optionally substituted by one or more R 23 Each divalent heterocyclic moiety is optionally substituted with one or more R 24 Replacement, R 21 、R 22 and R 23 Each occurrence is independently -H, -CO2H or C1-C6 alkyl, each C1-C6 alkyl optionally substituted by one or more -OH, oxo, C6-C 10 aryl or 5 to 8 membered heteroaryl substituted, R 24 is independently at each occurrence -H, -CO2H, C1-C6 alkyl, or oxo; and

[0094] L is a targeting fragment, preferably, the targeting fragment is capable of binding to a cell, preferably, the targeting fragment is capable of binding to a cell surface receptor; and

[0095] The nucleic acid is a nucleic acid with pharmaceutical activity, and the nucleic acid with pharmaceutical activity is a nucleic acid encoding a peptide or protein with pharmaceutical activity.

[0096] In one preferred embodiment, the nucleic acid is RNA. In another preferred embodiment, the nucleic acid is single-stranded RNA (ssRNA). In another preferred embodiment, the ssRNA is messenger RNA (mRNA).

[0097] In another preferred embodiment, the nucleic acid is DNA. In another preferred embodiment, the DNA is plasmid DNA.

[0098] In one aspect, the present invention provides a pharmaceutical composition comprising a composition comprising a polymer complex comprising a ternary conjugate (preferably a conjugate of Formula I* or Formula I) or a pharmaceutically acceptable salt thereof and a nucleic acid, preferably the nucleic acid is non-covalently bound to the conjugate, as described herein, the nucleic acid is a nucleic acid having pharmaceutical activity, and the nucleic acid having pharmaceutical activity is a nucleic acid encoding a peptide or protein having pharmaceutical activity.

[0099] In one aspect, the present invention provides a composition or polymer complex as described herein or a pharmaceutical composition comprising a composition or polymer complex as described herein for use in treating a disease or disorder, preferably treating cancer.

[0100] In one aspect, the present invention provides a use of a composition or polymer complex as described herein, or a pharmaceutical composition comprising a composition or polymer complex as described herein, in the preparation of a medicament for treating a disease or disorder (eg, cancer).

[0101] In another aspect, the present invention provides a method of treating a disease or disorder (e.g., cancer) in a subject in need thereof, comprising administering to the subject an effective amount of a composition or polymer complex described herein, or a pharmaceutical composition comprising a composition or polymer complex described herein.

[0102] The linear non-random LPEI-PEG binary conjugates described herein, as well as the compositions and polymer complexes of the present invention comprising ternary conjugates having targeting segments attached thereto, not only ensure a consistent and predictable ratio of LPEI segments to PEG segments, but also typically and preferably ensure a linear conjugate with defined LPEI and PEG segments. Thus, they have greater batch-to-batch consistency, ease of manufacture, and more predictable SAR compared to the branched LPEI-PEG binary conjugates currently prepared using random, uncontrolled synthetic strategies.

[0103] More advantageously and surprisingly, when the linear non-random conjugates of the present invention described herein are combined with pharmaceutically active nucleic acids (e.g., mRNA or plasmid DNA (pDNA)) encoding pharmaceutically active peptides or proteins (e.g., cytokines, interferons, or toxins) to form polymer complexes and administered to cells, these polymer complexes not only unexpectedly maintain biological activity but may even enhance its biological activity compared to corresponding polymer complexes prepared using randomly branched conjugates. Thus, while the variability and number of conjugate structures are significantly reduced, and thus the variability and number of structures that may be responsible for biological activity (including targeting and presentation of the targeted fragment to the target cell surface, and subsequent uptake, translation, and secretion of the encoded pharmaceutically active protein), the efficacy of the linear polymer complexes of the present invention described herein is not compromised. On the contrary, the compositions and polymer complexes of the present invention can even enhance their overall biological activity.

[0104] After reading the detailed description of the invention, additional features and advantages of the present technology will be apparent to those skilled in the art. As the description continues, the following and other aspects and embodiments of the invention will become apparent. BRIEF DESCRIPTION OF THE DRAWINGS

[0105] Figure 1 The polymer complex LPEI-1-[N3:DBCO]PEG 36 -DLS backscatter profiles of triplicate DUPA:DT-A particles were measured for particle size distribution and zeta potential in 20 mM HEPES (5% glucose, pH 7.2) at 0.1 mg / mL (in 5% glucose), a volume of 1.0 mL, and an N / P ratio of 4. The z-average diameter was 103.4 nm, the polydispersity index (PDI) was 0.197, and the zeta potential was 44.5 mV.

[0106] Figure 2A Compared with the control delivery vehicle Messenger MAX, LPEI-1-[N3:DBCO]PEG 36 - Luminescence (AU) graphs in Renca parental and RencaEGFR M1 H cells treated with hEGF:[firefly luciferase mRNA]. LPEI-1-[N3:DBCO]PEG was measured at final concentrations of 0.125 to 1.0 μg / mL 24 hours after treatment. 36 - Luminescence of hEGF:[firefly luciferase mRNA] (N / P ratios of 4, 6, and 12) and lipofectamine Messenger MAX.

[0107] Figure 2BCompared with the control delivery vehicle jetPEI, LPEI-1-[N3:DBCO]PEG 36 - Luminescence (AU) graphs of Renca parental cells and RencaEGFR M1 H cells treated with hEGF:[firefly luciferase mRNA]. LPEI-1-[N3:DBCO]PEG was measured at final concentrations of 0.125 to 1.0 μg / mL 24 hours after treatment. 36 - Luminescence of hEGF:[firefly luciferase mRNA] (N / P ratios of 4, 6, and 12) and jetPEI.

[0108] Figure 2C Messenger MAX was used as a control delivery vehicle and LPEI-1-[N3:DBCO]PEG 36 - Graph of the luminescence (AU) ratio between hEGF:[firefly luciferase mRNA]-treated RencaEGFR M1 H cells and Renca parental cells. LPEI-1-[N3:DBCO]PEG was measured at final concentrations of 0.125 to 1.0 μg / mL 24 hours after treatment. 36 - Luminescence of hEGF:[firefly luciferase mRNA] (N / P ratios of 4, 6, and 12) and Lipofectamine Messenger MAX. The ratio was calculated by dividing the luminescence signal of RencaEGFR M1 H cells by the luminescence signal of Renca parental cells.

[0109] Figure 2D To use jetPEI as a control delivery vehicle, LPEI-1-[N3:DBCO]PEG 36 - Graph of the luminescence (AU) ratio between RencaEGFR M1 H cells and Renca parental cells treated with hEGF:[firefly luciferase mRNA]. 36 - Luminescence of hEGF:[firefly luciferase mRNA] (N / P ratios of 4, 6, and 12) and jetPEI. The ratio was calculated by dividing the mean luminescence signal of RencaEGFR M1 H cells by the mean luminescence signal of Renca parental cells.

[0110] Figure 2E Compared with the control delivery vehicle Messenger MAX, LPEI-1-[N3:DBCO]PEG 36- Graph of the percent survival of Renca parental and RencaEGFR M1 H cells treated with hEGF:[firefly luciferase mRNA]. LPEI-1-[N3:DBCO]PEG was measured at final concentrations of 0.125 to 1.0 μg / mL 24 hours after treatment. 36 - Percent survival of hEGF:[firefly luciferase mRNA] (N / P ratios of 4, 6, and 12) and Messenger MAX.

[0111] Figure 3A Shown with LPEI-1-[N3:DBCO]PEG 36 - Relative luminescence (AU) of Renca parental cells and RencaEGFR M1 H cells 6 hours after treatment with hEGF:[firefly luciferase mRNA] (N / P ratio of 4).

[0112] Figure 3B Shown with LPEI-1-[N3:DBCO]PEG 36 - Relative luminescence (AU) of Renca parental cells and RencaEGFR M1 H cells 6 hours after treatment with hEGF:[firefly luciferase mRNA] (N / P ratio of 6).

[0113] Figure 3C Shown with LPEI-1-[N3:DBCO]PEG 36 - Relative luminescence (AU) of Renca parental cells and RencaEGFR M1 H cells 22 hours after treatment with hEGF:[firefly luciferase mRNA] (N / P ratio of 4).

[0114] Figure 3D Shown with LPEI-1-[N3:DBCO]PEG 36 - Relative luminescence (AU) of Renca parental cells and RencaEGFR M1 H cells 22 hours after treatment with hEGF:[firefly luciferase mRNA] (N / P ratio of 6).

[0115] Figure 3E Shown with LPEI-1-[N3:DBCO]PEG 36 - Luminescence (AU) of B16F10-hEGFR cells at different densities (500-20,000 cells / well) 24 hours after transfection with hEGF:[firefly luciferase mRNA] (N / P ratio of 6).

[0116] Figure 4The luminescence of human prostate cell lines with different cell surface PSMA expression (PSMA-high-expressing LNCaP cells and PSMA-low-expressing DU145 cells) normalized by viability is shown after transfection with PSMA-targeted polymer complexes containing mRNA encoding luciferase. The X-axis represents the concentration of mRNA in the polymer complex (0.25, 0.5, and 1.0 μg / mL). The Y-axis represents the luminescence expressed in arbitrary units (AU) normalized by viability. Selective transfection of luciferase mRNA into PSMA-overexpressing cells and selective expression of luciferase are demonstrated.

[0117] Figure 5 Shown are luminescence measurements of cancer cells with varying human folate receptor (FR) surface expression (MCF7: FR low expression; SKOV3: FR high expression) after treatment with a human folate receptor (FR)-targeted polymer complex containing mRNA encoding Renilla luciferase (R-Luc). The x-axis represents the concentration of mRNA in the polymer complex (0.125, 0.25, 0.5, and 1.0 μg / mL). The y-axis represents luminescence in arbitrary units (RLU). The standard deviation of four replicates is shown. This demonstrates selective expression of Renilla luciferase in folate receptor-overexpressing cells.

[0118] Figure 6 Figure 20: After transfection with an EGFR targeting polymer complex containing hIL-2 mRNA, the level of secreted human IL-2 normalized to survival rate was measured in two cell lines (RencaEGFR M1 H cells with high hEGFR expression and Renca cells (parents) with negative human EGFR) expressing different human EGFR (hEGFR). The X-axis represents the concentration (0.125, 0.25, 0.5, and 1.0 μg / mL) of mRNA in the polymer complex. The Y-axis represents the level of secreted human IL-2 normalized to survival rate, expressed in arbitrary units (AU). The selective expression and secretion of human IL-2 in EGFR high-expressing cells were demonstrated.

[0119] Figure 7 Figure 2 shows the levels of secreted human IL-2 in two cell lines with different PSMA expression (PSMA-high-expressing LNCaP cells and PSMA-low-expressing DU145 cells) after transfection with PSMA-targeted polymer complexes containing hIL-2 mRNA. This demonstrates the selective expression of human IL-2 in PSMA-overexpressing cells.

[0120] Figure 8Figure 2 shows the levels of secreted human IFNβ in two cell lines with different PSMA expression (PSMA-high-expressing LNCaP cells and PSMA-low-expressing DU145 cells) after transfection with PSMA-targeted polymer complexes containing hIFNβ mRNA. This demonstrates the selective expression of human IFNβ in PSMA-high-expressing cells.

[0121] Figure 9 Shown are the levels of human IFNγ (hIFNγ) secreted by RencaEGFR M1H (human EGFR overexpressing) and Renca (parental, human EGFR negative) cell lines following transfection with EGFR-targeted polymer complexes containing hIFNγ mRNA. The figure demonstrates the selective transfection of EGFR-overexpressing cells with hIFNγ mRNA and the selective expression and secretion of hIFNγ protein.

[0122] Figure 10 Plotted are the levels of human EPO secreted by cancer cells with varying human folate receptor (FR) expression (SKOV3: high FR expression; MCF7: low FR expression) after treatment with a folate receptor (FR)-targeted polymer complex containing mRNA encoding human EPO. The X-axis represents the concentration of mRNA in the polymer complex (0.125, 0.25, 0.5, and 1.0 μg / mL). The Y-axis represents the concentration of hEPO released into the culture medium (mIU / mL). The standard deviation of four replicate samples is shown. This demonstrates the selective expression of hEPO in cells overexpressing the folate receptor.

[0123] Figure 11 The PSMA targeting polymer complex LPEI-1-[N3:DBCO]PEG containing DT-A mRNA was shown. 36 Following transfection with -DUPA, the DT-A protein inhibited protein biosynthesis in two cell lines with varying PSMA expression: LNCaP cells with high PSMA expression and DU145 cells with low PSMA expression. Inhibition of protein biosynthesis was assessed by Western blot analysis using an anti-puromycin antibody probe. GAPDH was used as a loading control. This demonstrated selective inhibition of protein biosynthesis in PSMA-overexpressing cells.

[0124] Figure 12A The cell surface expression of human EGFR on various cell lines (RencaEGFR M1 H, WI-38 and MCF-7 cells) is shown. Figure 12A and Figure 12B The data shown are from two independent experiments performed using different flow cytometers.

[0125] Figure 12B The cell surface expression of human EGFR on various cell lines (WI-38, U87MG and MCF-7 cells) is shown. Figure 12A and Figure 12B The data shown are from two independent experiments performed using different flow cytometers.

[0126] Figure 12C The results show that the LPEI-1-[N3:DBCO]PEG 36 Luminescence levels normalized to cell viability in RencaEGFR M1 H cells (overexpressing EGFR) and MCF7 cells (underexpressing EGFR) after transfection with an EGFR-targeted polymer complex containing hEGF and a luciferase-encoding plasmid. This demonstrates the selective expression and activity of luciferase in EGFR-overexpressing cells.

[0127] Figure 12D Luminescence levels normalized to cell viability are shown for other 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 little to no EGFR). Figure 12C In the same experiment as shown, cells were treated with LPEI-1-[N3:DBCO]PEG 36 These cells were transfected with an EGFR-targeted polymer complex containing hEGF and a plasmid encoding luciferase (N / P ratio of 6). The selective expression of luciferase in rapidly proliferating cancer cells expressing moderate levels of EGFR was demonstrated.

[0128] Figure 13A The results show that the LPEI-1-[N3:DBCO]PEG prepared with an N / P ratio of 3 36 After transfection of the linear EGFR-targeted polymer complex of the present invention with hEGF and a plasmid encoding luciferase (pGreenFire1-CMV), the luminescence levels in two cell lines with different human EGFR expression (i.e., EGFR-overexpressing RencaEGFR M1 H cells and human EGFR-negative Renca (parental) cells) demonstrated the selective expression of luciferase in EGFR-overexpressing cells.

[0129] Figure 13B Depicted is the preparation of LPEI-1-[N3:DBCO]PEG 36After transfection of the linear EGFR-targeted polymer complex of the present invention with hEGF and a plasmid encoding luciferase (pGreenFire1-CMV), the luminescence levels of two cell lines with different human EGFR expression (i.e., EGFR-overexpressing RencaEGFR M1 H cells and human EGFR-negative Renca (parental) cells) demonstrated the selective expression of luciferase in EGFR-overexpressing cells.

[0130] Figure 13C Shows the selective luminescence of B16F10-hEGFR cells. 36 B16F10-hEGFR and B16F10 parental cells were treated with hEGF:[pSZL] (N / P ratios of 3 and 6). Luminescence and viability were measured after 6 days. Data are presented as relative luminescence intensity normalized to viability.

[0131] Figure 14 The luminescence of human prostate cell lines with different PSMA cell surface expression (PSMA high-expressing LNCaP cells and PSMA low-expressing DU145 cells) is shown. 36 -DUPA and plasmid DNA encoding luciferase) were used to treat cells. The X-axis represents the concentration of pGreenFire-CMV in the polymer complex (0.25, 0.5, and 1.0 μg / mL). The Y-axis represents luminescence in arbitrary units (AU). The mean and standard deviation of three replicate samples are shown. Selective expression of luciferase was demonstrated after transfection of PSMA-overexpressing cells with plasmid DNA encoding luciferase (pGreenFire1-CMV).

[0132] Figure 15A It shows that the 36 The levels of human IL-2 (hIL-2) secreted in two cell lines with different human EGFR expression (EGFR-overexpressing RencaEGFRM1 H cells and human EGFR-negative parental Renca cells) after transfection with an EGFR-targeted polymer complex containing hEGF and a plasmid encoding hIL-2. This demonstrates the selective expression of hIL-2 in EGFR-overexpressing cells.

[0133] Figure 15B The results show that the EGFR-targeted polymer complex (containing LPEI-1-[N3: DBCO]PEG 36Levels of human IL-2 secretion were measured after transfection of a small number of EFGR-overexpressing RencaEGFRM1H cells (600 cells) with hEGF and a plasmid encoding hIL-2 at the indicated plasmid concentrations (0.125 and 0.25 μg / ml). Polymer complexes were formulated at an N / P ratio of 6, and IL-2 secretion was measured after 2, 3, and 4 days.

[0134] Figure 16 It shows that the 36 Figure 2: Levels of secreted human IL-2 normalized to cell viability in cell lines with varying PSMA expression (high-expressing LNCaP and C4-2 cells, and low-expressing DU145 cells) following transfection of PSMA-targeted polymer complexes containing hIL-2-DUPA and a plasmid encoding the IL-2 protein. The x-axis represents the concentration of hIL-2 plasmid DNA in the polymer complex (0.25, 0.5, and 1.0 μg / mL). The y-axis represents the concentration of secreted IL-2 normalized to cell viability, expressed in arbitrary units (AU). This demonstrates the selective expression / secretion of human IL-2 following transfection of PSMA-overexpressing cells with plasmid DNA encoding hIL-2.

[0135] Figure 17A The figure shows the level of human IFNβ secreted by RencaEGFR M1 H cancer cells with high expression of human EGFR after transfection with an EGFR targeting polymer complex containing pCMV-hIFNβ (N / P ratio of 3). The X-axis represents the concentration of pCMV-hIFNβ plasmid DNA in the polymer complex (0.25, 0.5, 1.0 and 2.0 μg / mL). The Y-axis represents the concentration of secreted IFNβ protein (in pg / mL), which is represented by the mean and standard deviation of three replicate samples. The results show that for the delivery vector tested, the linear ternary conjugate carrier LPEI-l-[N3:DBCO]PEG 36 -hEGF is significantly superior to random delivery vectors in secreting human IFNβ from EGFR-overexpressing cancer cells.

[0136] Figure 17B The figure shows the level of human IFNβ secreted by RencaEGFR M1 H cancer cells with high expression of human EGFR after transfection with an EGFR targeting polymer complex containing pCMV-hIFNβ (N / P ratio of 4). The X-axis represents the concentration of pCMV-hIFNβ plasmid DNA in the polymer complex (0.25, 0.5, 1.0 and 2.0 μg / mL). The Y-axis represents the concentration of secreted IFNβ protein (in pg / mL), which is represented by the mean and standard deviation of three replicate samples. The results show that for the delivery vector tested, the linear ternary conjugate carrier LPEI-l-[N3:DBCO]-PEG36 -hEGF EGFR is significantly superior to random delivery vectors in secreting human IFNβ from EGFR-overexpressing cancer cells. DETAILED DESCRIPTION

[0137] 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 very preferred embodiments described and disclosed herein should apply to all aspects and other embodiments, preferred embodiments and very preferred embodiments, whether or not specifically mentioned again.

[0138] The present invention provides a polymer complex of (i) a nucleic acid encoding a target peptide or protein, preferably a nucleic acid encoding a pharmaceutically active peptide or protein (e.g., a cytokine, interferon, or toxin) and (ii) a targeted linear conjugate of LPEI and PEG, as described herein and below. The conjugate preferably comprises an LPEI segment, a PEG segment, and a targeting segment. In a preferred embodiment, the LPEI segment and the PEG segment are coupled in a discrete end-to-end manner. In some preferred embodiments, the LPEI segment and the PEG segment are coupled by covalently linking an azide to an olefin or alkyne to form 1,2,3-triazole or 4,5-dihydro-1H-[1,2,3]triazole.

[0139] definition

[0140] Unless defined otherwise, 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.

[0141] As used in this disclosure, the articles "a" and "an" refer to one or more than one (ie, at least one) of the grammatical object of the article. For example, "an element" means one element or more than one element.

[0142] Unless stated otherwise, the term "and / or" used in this disclosure means "and" or "or".

[0143] As used herein, the term "about" shall have a meaning of + / - 10%. For example, about 50% shall mean 45% to 55%. Preferably, as used herein, the term "about" shall have a meaning of + / - 5%. For example, about 50% shall mean 47.5% to 52.5%.

[0144] As used herein, the phrase "between number X and number Y" shall include number X and number Y. For example, the phrase "between 0.01 μmol and 50 μmol" shall include 0.01 μmol and 50 μmol and values therebetween. The phrase "between approximately number X and approximately number Y" also applies.

[0145] The term "optionally substituted" is understood to mean that a given chemical moiety (e.g., an alkyl group) may, but is not required to, be bonded to other substituents (e.g., heteroatoms). For example, an optionally substituted alkyl group may be a fully saturated alkyl chain (i.e., a pure hydrocarbon group). Alternatively, the same optionally substituted alkyl group may have substituents other than hydrogen. For example, it may be bonded to a halogen atom, an alkoxy group, or any other substituent described herein at any point along the chain. Thus, the term "optionally substituted" means that a given chemical moiety may contain other functional groups, but does not necessarily have any other functional groups.

[0146] Term " optional replacement " should be construed as referring to the situation that the carbon atom of methylene radical (i.e. -CH 2 -) can but not necessarily be replaced by heteroatom (such as -NH-, -O-).For example, the C 3 alkylidene (i.e. propylene) of a methylene radical " optional replacement " can have -CH 2 -O-CH 2 -or-O-CH 2 -CH 2 -structure.It will be appreciated by those skilled in the art that when this type of replacement causes unstable chemical part, methylene radical can not be replaced.For example, it will be appreciated by those skilled in the art that four methylene radicals can not be replaced by oxygen atom simultaneously.Therefore, in some preferred embodiments, when a methylene radical of alkylidene fragment is replaced by heteroatom, adjacent one or two carbon atoms are not replaced by heteroatom.

[0147] The term "aryl" refers to a cyclic aromatic hydrocarbon group having 1 to 2 aromatic rings, including monocyclic or bicyclic groups, such as phenyl, biphenyl or naphthyl. 10 Aryl comprises 6 to 10 carbon atoms. When comprising two aromatic rings (bicyclic etc.), the aromatic ring of aryl can be connected (for example biphenyl) at single point, or can be fused (for example naphthyl). Aryl can be replaced by more than one substituent (for example 1 to 5 substituents) at any connection point alternatively. The substituent itself can be replaced alternatively. In addition, when comprising two fused rings, aryl defined herein can have an unsaturated or partially saturated ring fused with a fully saturated ring. The exemplary ring systems of these aryls include indanyl, indenyl, tetrahydronaphthyl and tetrahydrobenzocycloalkenyl. In some preferred embodiments, aryl is phenyl.

[0148] Unless specifically defined otherwise, "heteroaryl" refers to a monovalent monocyclic aromatic ring or polycyclic aromatic ring having 5 to 24 ring atoms, which contains one or more ring heteroatoms selected from N, S, P, or O, with the remaining ring atoms being C. 5- to 10-membered heteroaryl groups contain 5 to 10 atoms. As defined herein, heteroaryl also refers to bicyclic heteroaromatic groups in which the heteroatoms are selected from N, S, P, or O. Aromatic groups are optionally substituted independently with one or more substituents described herein.Examples include, but are not limited to, furyl, thienyl, pyrrolyl, pyridinyl, pyrazolyl, pyrimidinyl, imidazolyl, isoxazolyl, oxazolyl, oxadiazolyl, pyrazinyl, indolyl, thien-2-yl, quinolyl, benzopyranyl, isothiazolyl, thiazolyl, thiadiazole, indazole, benzimidazolyl, thieno[3,2-b]thiophene, triazolyl, triazinyl, 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[3,2-b]thiophene, triazolyl, triazinyl, 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, dihydroindolinyl, indolinonyl, dihydrobenzothienyl, dihydrobenzofuranyl, benzofuran, chromanyl, thiochromanyl, tetrahydroquinolinyl, dihydrobenzothiazine, dihydrobenzooxanyl, 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]triazolo[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. Additionally, when containing two fused rings, a heteroaryl group as defined herein may 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 dihydrobenzooxanyl.

[0149] The term "alkyl" refers to a straight 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.

[0150] The term "alkylene" refers to a straight or branched chain saturated divalent hydrocarbon fragment. A C0-C6 alkyl group contains 0 to 6 carbon atoms. Examples of C0-C6 alkylene groups include, but are not limited to, methylene, ethylene, propylene, butylene, pentylene, isopropylene, isobutylene, secondary butylene, tertiary butylene, isopentylene, and neopentylene.

[0151] As used herein, the term "C1-C6 alkoxy" refers to a substituted hydroxyl group of the formula (-OR'), wherein R' is an optionally substituted C1-C6 alkyl group, as defined herein, and the oxygen moiety is directly attached to the parent molecule. Thus, as used herein, the term "C1-C6 alkoxy" refers to a linear or branched C1-C6 alkoxy group, which can be, for example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, linear or branched pentoxy, linear or branched hexyloxy, preferably C1-C4 alkoxy and C1-C3 alkoxy.

[0152] The term "cycloalkyl" refers to a monocyclic or polycyclic saturated carbocyclic ring containing 3 to 18 carbon atoms. A C3-C8 cycloalkyl group contains 3 to 8 carbon atoms. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, norboranyl, norborenyl, bicyclo[2.2.2]octyl, or bicyclo[2.2.2]octenyl. A C3-C8 cycloalkyl group is a cycloalkyl group containing 3 to 8 carbon atoms.

[0153] 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 norbornenyl. C5-C8 cycloalkenyl groups are cycloalkenyl groups containing 5 to 8 carbon atoms.

[0154] The term "heterocyclyl" or "heterocycloalkyl" or "heterocycle" refers to a monocyclic or polycyclic 3 to 24-membered ring containing carbon and heteroatoms from oxygen, nitrogen or sulfur, wherein there are no shared delocalized pi electrons between the ring carbons or heteroatoms (aromaticity). 3-10 membered heterocycloalkyl groups contain 3 to 10 atoms. Heterocyclyl rings include, but are not limited to, oxetanyl, azetadinyl, tetrahydrofuranyl, pyrrolidinyl, oxazolinyl, oxazolidinyl, thiazolinyl, thiazolidinyl, pyranyl, thiopyranyl, tetrahydropyranyl, dioxolinyl, piperidinyl, morpholinyl, thiomorpholinyl, thiomorpholinyl S-oxide, thiomorpholinyl S-dioxide, piperazinyl, azepinyl, oxepinyl, diazepinyl, tropanyl, and homotropanyl.

[0155] The term "heterocycloalkenyl" refers to a monocyclic or polycyclic 3 to 24-membered ring containing carbon and heteroatoms from oxygen, nitrogen or sulfur, wherein there are no shared delocalized pi electrons between the ring carbons or heteroatoms (aromaticity), but there is at least one unsaturated element within the ring. 3-10 membered heterocycloalkenyl groups contain 3 to 10 atoms.

[0156] As used herein, the term "halo" or "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br), or iodine (I).

[0157] The term "carbonyl" refers to a functional group consisting of a carbon atom and an oxygen atom double-bonded together, which may be abbreviated herein as "oxo," C(O), or C=O.

[0158] As used herein, the term "polymer complex" refers to a complex of a polymer and a nucleic acid, typically and preferably formed by electrostatic interactions. In particular, as used herein, the term "polymer complex" refers to a complex of a conjugate for use in the present invention as described herein and a nucleic acid (e.g., single-stranded RNA, preferably mRNA, or DNA, preferably plasmid DNA). The term "polymer complex" also typically and preferably refers to a vector, particularly a polymeric non-viral ternary conjugate vector for use in the present invention as described herein, for carrying and delivering nucleic acids to desired target cells.

[0159] The term "overexpression" refers to an increase in the expression of a gene or protein within a cell or on the cell surface relative 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, the cell that overexpresses a cell surface receptor refers to an increase in the level of the cell surface receptor expressed in the cell of a specific tissue compared to the level of the cell surface receptor measured in normal healthy cells of the same type of tissue under similar conditions. In one embodiment, the cell that overexpresses a cell surface receptor refers to an increase in the level of the cell surface receptor in the cell relative to the level in the same cell or closely related non-malignant cells under normal physiological conditions.

[0160] As used herein, the term "polyanion" refers to a polymer, preferably a biopolymer, having more than one negatively charged site. Typically and preferably, as used herein, the term "polyanion" refers to a polymer, preferably a biopolymer, consisting of repeating units comprising a residue capable of carrying a negative charge. In a further embodiment, the polyanion is a polymer, preferably a biopolymer, consisting of repeating units comprising a residue capable of carrying a negative charge. In another preferred embodiment, the polyanion is a nucleic acid, more preferably DNA, RNA, polyglutamic acid or hyaluronic acid.

[0161] As used herein, the term "nucleic acid" includes deoxyribonucleic acid (DNA) and / or ribonucleic acid (RNA) or a combination thereof. In a preferred embodiment, "nucleic acid" refers to deoxyribonucleic acid (DNA) and / or ribonucleic acid (RNA), and thus refers to genomic, viral, recombinantly produced and chemically synthesized molecules. Nucleic acids can be in the form of single-stranded or double-stranded, linear or covalently closed circular molecules, can include chemical derivatization of nucleic acids on nucleotide bases, sugars or phosphates, and can include non-natural nucleotides and nucleotide analogs.

[0162] As used herein, the term "dispersity" (abbreviated as D) refers to the distribution of molar masses in a given polymer sample (e.g., polymer segments used herein in the conjugates and polymer complexes of the present invention). w / M n ), where D is the dispersion, M w is the weight average molecular weight of the polymer sample or polymer fragment, M n is the number average molecular weight of a polymer sample or polymer fragment.

[0163] 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 multiplied by the mass of each molecule in the mixture, and is typically and preferably represented by the symbol Mw.

[0164] 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.

[0165] As used herein, the term "polydispersity index" (abbreviated as PDI) refers to the polydispersity index of polymer composite nanoparticles (such as the polymer composites of the present invention) in dynamic light scattering measurements. The index is a numerical value calculated by performing a simple 2-parameter fit (cumulant analysis) on the relevant data. The polydispersity index is dimensionless and is scaled, so values less than 0.05 are rarely seen except when using highly monodisperse standards. Values greater than 0.7 indicate that the size distribution of the sample is very broad and may not be suitable for dynamic light scattering (DLS) technology. Various size distribution algorithms are applicable to data between these two extremes. The zeta average diameter (z average diameter) and polydispersity index of the polymer composites of the present invention are determined by dynamic light scattering (DLS) based on the assumption that the polymer composite is isotropic and spherical. The calculation of these parameters is defined and determined according to the ISO standard document ISO 22412:2017.

[0166] The term "amino acid residue" refers to a divalent residue derived from an organic compound containing an amine (-NH2) and a carboxylic acid (-COOH) functional group, 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 an amine (-NH2) and a carboxylic acid (-COOH) functional group, wherein the divalency is achieved through the amine and the carboxylic acid functional group, thus through 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 an amine (-NH2) and a carboxylic acid (-COOH) functional group, wherein the divalency is achieved by the amine or the carboxylic acid functional group and other functional groups present in the amino acid residue. As a preferred example and embodiment, the amino acid residue derived from cysteine according to the present invention comprises a divalent structure -S-(CH2)-CH(COOH)-NH-, wherein the divalency is achieved by an amino functionality and a thiol functionality contained. As used herein, the term "amino acid residue" generally and preferably includes amino acid residues derived from naturally occurring or non-naturally occurring amino acids. In addition, as used herein, the term "amino acid residue" generally and preferably also includes amino acid residues derived from chemically synthesized non-natural amino acids, including alpha-(α-) amino acids, beta-(β-) amino acids, gamma-(γ-) amino acids or delta-(δ-) amino acids, and mixtures thereof in any ratio. In addition, as used herein, the term "amino acid residue" generally and preferably also includes amino acid residues derived from α amino acids (including any isomeric form thereof, in particular its D-stereoisomer and L-stereoisomer (or represented by (R) and (S) nomenclature) and mixtures thereof in any ratio (preferably a racemic ratio of 1:1)). The terms "D-stereoisomer", "L-stereoisomer", "D-amino acid" or "L-amino acid" refer to the chiral α carbon of an amino acid. Therefore, in a preferred embodiment, the amino acid residue is a divalent group of the structure -NH-CHR-C(O)-, wherein R is an amino acid side chain. Two or more consecutive amino acid residues preferably form a peptide bond (i.e., an amide bond) at the amine portion and the carboxylic acid portion of the amino acid residue, respectively. When a dipeptide, tripeptide, or polypeptide is described herein as an amino acid residue, it is usually represented by (AA). a Indicates that the provided sequence is described from left to right in NC direction. Thus, for example, (AA) a Trp-Trp-Gly refers to an amino acid residue, wherein Trp corresponds to the N-terminus of the tripeptide and has a valence of -NH-, and Gly corresponds to the C-terminus of the tripeptide and has a valence of -CO-.

[0167] As used herein, the terms "peptide," "polypeptide," and "protein" refer to a substance comprising about two or more consecutive amino acid residues interconnected by 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; while in one embodiment, the term "peptide" refers to a substance comprising about 2 or more, about 3 or more, about 8 or more, about 20 or more, and up to about 50, about 100, or about 150 amino acids.

[0168] As used herein, the term "disease-associated antigen" broadly refers to any antigen associated with a disease. A disease-associated antigen is a molecule containing an epitope that can stimulate the host immune system to produce a cellular antigen-specific immune response and / or humoral antibody response against the disease. Therefore, a disease-associated antigen or its epitope can be used for therapeutic purposes. A disease-associated antigen may be associated with a microbial infection (usually a microbial antigen) or with a cancer (usually a tumor).

[0169] As used herein, the term "viral antigen" refers to any viral component that has antigenic properties (ie, is capable of eliciting an immune response in an individual). A viral antigen may be a viral ribonucleoprotein or an envelope protein.

[0170] As used herein, the term "bacterial antigen" refers to any bacterial component that has antigenic properties (ie, is capable of eliciting an immune response in an individual). Bacterial antigens may be derived from the bacterial cell wall or cytoplasmic membrane.

[0171] As used herein, the term "epitope" refers to a portion or fragment of a molecule (e.g., an antigen) that can be recognized by the immune system. For example, an epitope can be recognized by T cells, B cells, or antibodies. An antigenic epitope preferably comprises a continuous or discontinuous portion of the protein, preferably 5 to 100 amino acids in length, preferably 5 to 50 amino acids, more preferably 8 to 30 amino acids, and most preferably 10 to 25 amino acids in length. For example, the epitope is 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, the epitope is about 10 to 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 by an MHC molecule. "Major histocompatibility complex" and its abbreviation "MHC" include MHC class I and MHC class II molecules, and refer to a gene complex present in all vertebrates. MHC proteins or molecules are crucial for signaling between lymphocytes and antigen-presenting cells or disease cells during immune responses, where they bind peptide epitopes and present them to T cell receptors on T cells for recognition. MHC-encoded proteins are expressed on the cell surface and display both self-antigens (peptide fragments from the cell itself) and non-self-antigens (such as fragments of invading microorganisms) to T cells.

[0172] The term "antibody" refers to any immunoglobulin (whether natural or wholly or partially synthetically produced) and its derivatives and characteristic portions. An antibody can be a monoclonal antibody or a polyclonal antibody. An antibody can be a member of any immunoglobulin class (including any human class: IgG, IgM, IgA, IgD and IgE). As used herein, an antibody fragment (i.e., a characteristic portion of an antibody) refers to any antibody derivative that is shorter than the full length. Typically, an antibody fragment retains at least most of the specific binding ability of a full-length antibody. Examples of antibody fragments include, but are not limited to, single-chain and double-chain fragments, Fab, Fab', F(ab')2, scFv, Fv, dsFv diabodies and Fd fragments. Antibody fragments can be produced in any manner. For example, antibody fragments can be produced by enzymatic or chemical fragmentation of intact antibodies, and / or can be produced by genetic recombination encoding partial antibody sequences. Alternatively or additionally, antibody fragments can be produced by wholly or partially synthetic synthesis. Antibody fragments can optionally comprise single-chain antibody fragments. Alternatively or additionally, an antibody fragment may comprise multiple chains linked together (e.g., linked together by disulfide bonds). An antibody fragment may optionally comprise a multimolecular complex. A functional antibody fragment typically comprises at least about 50 amino acids, more typically at least about 200 amino acids. In some embodiments, antibodies may include chimeric (e.g., "humanized") antibodies and single-chain (recombinant) antibodies. In some embodiments, antibodies may have reduced effector functions and / or bispecific molecules. In some embodiments, antibodies may include fragments produced by a Fab expression library. Single-chain Fv (scFv) is a recombinant antibody fragment consisting solely of a variable light chain (VL) and a variable heavy chain (VH) covalently linked to each other by a polypeptide linker. Either VL or VH may comprise an NH2-terminal domain. The length and composition of the polypeptide linker may vary, as long as the two variable domains are bridged and there is no significant spatial interference. Typically, the linker primarily comprises a fragment of glycine and serine residues, interspersed with some glutamic acid or lysine residues for solubility purposes. Diabodies are dimerized scFvs. Diabodies typically have shorter peptide linkers than most scFvs, and they generally tend to bind in the form of dimers. Fv fragments are antibody fragments that are bound together by a VH domain and a VL domain through non-covalent interactions. As used herein, the term "dsFv" refers to an Fv in which the VH-VL pair is stabilized by an engineered intermolecular disulfide bond. The F(ab')2 fragment is an antibody fragment that is substantially equivalent to a fragment obtained by digesting an immunoglobulin with pepsin at pH 4.0-4.5. This fragment can be produced by recombination. The Fab' fragment is an antibody fragment that is substantially equivalent to a fragment obtained by reducing the disulfide bridge connecting the two heavy chain fragments in the F(ab')2 fragment. The Fab' fragment can be produced by recombination.A Fab fragment is an antibody fragment that is essentially equivalent to a fragment obtained by digesting an immunoglobulin with an enzyme such as papain. Fab fragments can be produced recombinantly. The heavy chain fragment of a Fab fragment is the Fd subfragment.

[0173] As used herein, the term "alpha end of a linear polyethyleneimine segment" (alpha end of an LPEI segment) refers to the end of an LPEI segment where polymerization is initiated using an electrophilic initiator, as further described below for the term "initiating residue."

[0174] As used herein, the term "ω end of a linear polyethyleneimine segment" (ω end of an LPEI segment) refers to the end of an LPEI segment where polymerization is terminated using nucleophiles described herein (eg, azides, thiols, and other nucleophiles).

[0175] The term "organic residue" refers to any suitable organic group capable of binding to the nitrogen atom embedded in the LPEI segment. In a preferred embodiment, the organic residue is connected to the nitrogen atom via a carbonyl group to form an amide bond. Without being bound by theory, the organic residue is bound to the nitrogen atom of the poly(2-oxazoline) during the ring-opening polymerization of 2-oxazoline (see, for example, 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., typically the amide is cleaved) to produce LPEI and LPEI fragments, thereby producing -(NH-CH2-CH2)- moieties embedded in the conjugates of the present invention. However, if the cleavage reaction is not completed, some of the organic residues will not be cleaved. Therefore, in a preferred embodiment of the present invention, R of the conjugates of the present invention (including conjugates of Formula I* and I) is 1 -(NR 2 -CH2-CH2) n - At least 80%, preferably 90% of the R 2 is H. Preferably, R 1 -(NR 2 -CH2-CH2) n - at least 91%, more preferably 92%, more preferably 93%, more preferably 94%, more preferably 95%, more preferably 96%, more preferably 97%, more preferably 98%, most preferably 99% of the R 2 For H.

[0176] The term "priming residue" refers to the LPEI segment and R 1 -(NR 2 -CH2-CH2) n -a residue present in the moiety, the residue being derived from any initiator capable of initiating polymerization of 2-oxazoline to poly(2-oxazoline), typically and preferably any electrophilic initiator. As described in "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 (MeOTs), p-nitrobenzenesulfonates (nosylates) and trifluoromethanesulfonates (triflates), alkyl, benzyl and acetyl halides, oxazolinium salts and Lewis acids. " Therefore, although in a preferred embodiment R 1 is -H or -CH3, but those skilled in the art will understand that R 1 Other suitable residues may also be included, but are not limited to, for example, C n Alkyl (where n is greater than 1), usually C 1-6 alkyl, benzyl or acetyl.

[0177] The present invention provides a targeted polymer complex comprising: (i) a nucleic acid, particularly a nucleic acid encoding a pharmaceutically active peptide or protein (e.g., a cytokine, interferon, or toxin); and (ii) a targeted conjugate comprising an LPEI segment and a PEG segment connected by a discrete bond, the discrete bond being formed by a defined chemoselective reaction rather than by random and uncontrolled bonding of multiple nucleophiles of the electrophilic PEG segment to the LPEI backbone segment. The discrete bond not only ensures a consistent and predictable ratio of the LPEI segment to the PEG segment, but also ensures a defined linear conjugate rather than a randomly branched conjugate. Thus, the LPEI segment is bonded to a single PEG segment in a linear end-to-end manner. The chemoselective bonding of the LPEI segment to the PEG segment can be carried out using any suitable chemical precursor capable of forming a chemoselective bond. In a preferred embodiment, the chemoselective bonding of the LPEI segment to the PEG segment is carried out by a [3+2] cycloaddition reaction between an azide and an alkyne or alkene. Alternatively, the chemoselective bonding is carried out by a thiol-ene reaction between a thiol and an alkene. When chemoselective bonding occurs between an azide and an alkyne or alkene, the resulting bond is a 1,2,3-triazole (when coupling an alkyne) or a 4,5-dihydro-1H-[1,2,3]triazole (when coupling an alkene). When chemoselective bonding occurs between a thiol and an alkene, the resulting bond is a thioether.

[0178] The conjugate also comprises a targeting segment connected to the PEG segment, which is capable of targeting a specific cell type and promoting the uptake of the compositions of the present invention and pharmaceutically active nucleic acids in the specific cell type. Therefore, preferred embodiments include a targeting segment (e.g., hEGF, DUPA, or folic acid) that is specifically connected to the LPEI-PEG diconjugate to target a corresponding receptor (e.g., hEGFR, PSMA, or folate receptor) on a specific cell type (typically a cancer cell type) that is highly expressed and overexpressed on these cell types.

[0179] More advantageously and unexpectedly, the inventors have found that the resulting preferred conjugates and polymer complexes of the present invention have significantly reduced heterogeneity due to the determined chemoselective bonding between the LPEI segment and the PEG segment, and therefore have a significantly reduced number of potentially bioactive conjugates and polymer complexes, not only forming polymer complexes of suitable size, but also maintaining or even improving their overall bioactivity, such as highly selective targeted delivery of pharmaceutically active nucleic acids and subsequent efficient translation and secretion of the encoded pharmaceutically active proteins. Thus, the compositions and polymer complexes of the present invention not only selectively deliver pharmaceutically active nucleic acids encoding pharmaceutically active peptides or proteins to target cells (particularly cancer cells), but also achieve high expression, efficient protein translation, and secretion of the encoded pharmaceutically active proteins.

[0180] Therefore, in one aspect, the present invention provides a composition comprising a polymer complex comprising a conjugate and a nucleic acid, preferably the nucleic acid is non-covalently bound to the conjugate, wherein the conjugate comprises: a linear polyethyleneimine segment comprising an α-terminus and an ω-terminus, wherein the α-terminus of the polyethyleneimine segment is a priming residue; a polyethylene glycol segment comprising a first terminus and a second terminus; wherein the ω-terminus of the polyethyleneimine segment is linked to a divalent covalent linking group -ZX 1 - is linked to the first end of the polyethylene glycol segment, -ZX 1 is not a single bond and -Z- is not an amide; the second end of the polyethylene glycol segment is divalently linked to the moiety X 2 The nucleic acid is connected to the targeting segment; the nucleic acid is a nucleic acid encoding a target peptide or protein, preferably, the nucleic acid is a nucleic acid with pharmaceutical activity, and the nucleic acid with pharmaceutical activity is a nucleic acid encoding a peptide or protein with pharmaceutical activity. In a preferred embodiment of this aspect, the composition consists of the polymer complex. In a preferred embodiment, the linear polyethyleneimine segment is a nucleic acid of formula R 1 -(NR 2 -CH2-CH2) n -, n is any integer from 1 to 1500. In another preferred embodiment, the R 1 -(NR 2 -CH2-CH2) n - portion is a dispersing polymer portion, wherein the repeating unit n is from about 115 to about 1150 and the dispersity is about 5 or less; preferably, the repeating unit n is from about 280 to about 700 and the dispersity is about 3 or less; more preferably, the repeating unit n is from about 350 to about 630 and the dispersity is about 2 or less; preferably, R 1 It is -H or -CH3.

[0181] In another aspect, the present invention provides a composition comprising a polymer complex comprising a conjugate of Formula I* or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or enantiomer thereof and a nucleic acid, preferably the nucleic acid is non-covalently bound to the conjugate:

[0182] R 1 -(NR 2 -CH2-CH2) n -ZX 1 -(O-CH2-CH2) m -X 2 -L (Formula I*);

[0183] In the formula, 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 2 to 200, preferably any integer from 1 to 100, more preferably any integer from 2 to 100; R 1 is an initiating residue, preferably, R 1 is -H or -CH3; R 2 are independently -H or an organic residue, the -(NR 2 -CH2-CH2) n - at least 80%, preferably 90% of the R 2 H; X 1 and X 2 are independently divalent covalently linked moieties; Z is a divalent covalently linked moiety, Z is not a single bond and is not -NHC(O)-; L is a targeting segment, preferably, the targeting segment is capable of binding to a cell, more preferably, the targeting segment is capable of binding to a cell surface receptor; the nucleic acid is a nucleic acid encoding a target peptide or protein, preferably, the nucleic acid is a nucleic acid with pharmaceutical activity, and the nucleic acid with pharmaceutical activity is a nucleic acid encoding a peptide or protein with pharmaceutical activity. Preferably, the composition consists of the polymer complex.

[0184] In another aspect, the present invention provides a polymer complex comprising a conjugate of Formula I* or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or enantiomer thereof and a nucleic acid, preferably wherein the nucleic acid is non-covalently bound to the conjugate:

[0185] R 1 -(NR 2 -CH2-CH2) n -ZX 1 -(O-CH2-CH2) m -X 2 -L (Formula I*);

[0186] 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 2 to 200, preferably any integer from 1 to 100, more preferably any integer from 2 to 100; R 1 is an initiating residue, preferably, R 1 is -H or -CH3; R 2 are independently -H or an organic residue, the -(NR 2 -CH2-CH2) n - At least 80%, preferably 90% of R 2 H; X 1 and X 2 are independently divalent covalently linked moieties; Z is a divalent covalently linked moiety, Z is not a single bond and 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; the nucleic acid is a nucleic acid encoding a target peptide or protein, preferably, the nucleic acid is a nucleic acid having pharmaceutical activity, and the nucleic acid having pharmaceutical activity is a nucleic acid encoding a peptide or protein having pharmaceutical activity.

[0187] In another aspect, the present invention provides a composition comprising a polymer complex comprising a conjugate of Formula I or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or enantiomer thereof and a nucleic acid, preferably the nucleic acid is non-covalently bound to the conjugate:

[0188]

[0189] Where:

[0190] is a single bond or a double bond;

[0191] n is any integer from 1 to 1500, preferably any integer from 2 to 1500;

[0192] m is any integer from 1 to 200, preferably any integer from 2 to 200, preferably any integer from 1 to 100, more preferably any integer from 2 to 100;

[0193] R 1 is an initiating residue, preferably, R 1 is -H or -CH3;

[0194] R 2 are independently -H or an organic residue, the -(NR 2 -CH2-CH2) n - at least 80%, preferably at least 90% of R 2 is H;

[0195] Ring A is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl group, optionally substituted at any position with one or more R A1 Replacement; R A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo or halogen; or, two R A1 Together with the atoms to which they are attached, they can form more than one fused C6-C 10 aryl, C5-C6 heteroaryl or C3-C6 cycloalkyl ring, wherein each fused aryl, heteroaryl or cycloalkyl is optionally substituted by one or more R A2 Replacement; R A2 Independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, -SO3H or -OSO3H;

[0196] X 1 is a divalent covalent linking moiety;

[0197] X 2 is a divalent covalent linking moiety; and

[0198] L is a targeting fragment, preferably, the targeting fragment can bind to a cell, more preferably, the targeting fragment can bind to a cell surface receptor; and

[0199] The nucleic acid is a nucleic acid encoding a target peptide or protein. Preferably, the nucleic acid is a nucleic acid having pharmaceutical activity. The nucleic acid having pharmaceutical activity is a nucleic acid encoding a peptide or protein having pharmaceutical activity.

[0200] As described herein, the above description of Formula I represents fragment R 1 (NR 2 CH2CH2) n The two different regioisomers of

[0201]

[0202] wherein the wavy line represents the chemical bond to Ring A. Thus, Formula I as drawn herein includes two regioisomeric embodiments, i.e., wherein the fragment R 1 (NR 2 CH2CH2) n In the above structure, it is bonded to the top nitrogen atom or in the above structure, it is bonded to the bottom nitrogen atom, but not to the middle nitrogen atom. The above-drawn Formula I can be used interchangeably herein with the following equivalently drawn Formula I containing the fragment HNN=N, namely:

[0203]

[0204] In another aspect, the present invention provides a polymer complex comprising a conjugate of Formula I or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or enantiomer thereof and a nucleic acid, preferably wherein the nucleic acid is non-covalently bound to the conjugate:

[0205]

[0206] Where:

[0207] is a single bond or a double bond;

[0208] n is any integer from 1 to 1500, preferably any integer from 2 to 1500;

[0209] m is any integer from 2 to 200, preferably any integer from 1 to 200, more preferably any integer from 2 to 100;

[0210] R 1 For initiating residues, preferably, R1 is -H or -CH3;

[0211] R 2 are independently -H or an organic residue, the -(NR 2 CH2CH2) n - at least 80%, preferably at least 90% of R 2 is H;

[0212] Ring A is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl group, optionally substituted at any position with one or more R A1 Replacement; R A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo or halogen; or, two R A1 Together with the atoms to which they are attached, they can form more than one fused C6-C 10 aryl, C5-C6 heteroaryl or C3-C6 cycloalkyl ring, wherein each fused aryl, heteroaryl or cycloalkyl is optionally substituted by one or more R A2 Replacement; R A2 Independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, -SO3H or -OSO3H;

[0213] X 1 is a divalent covalent linking moiety;

[0214] X 2 is a divalent covalent linking moiety; and

[0215] L is a targeting fragment, preferably, the targeting fragment can bind to a cell, more preferably, the targeting fragment can bind to a cell surface receptor; and

[0216] The nucleic acid is a nucleic acid encoding a target peptide or protein. Preferably, the nucleic acid is a nucleic acid having pharmaceutical activity. The nucleic acid having pharmaceutical activity is a nucleic acid encoding a peptide or protein having pharmaceutical activity.

[0217] In another aspect, the present invention provides a composition comprising a polymer complex comprising a conjugate of Formula I or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or enantiomer thereof and a nucleic acid, preferably the nucleic acid is non-covalently bound to the conjugate:

[0218]

[0219] Where:

[0220] is a single bond or a double bond;

[0221] n is any integer from 1 to 1500, preferably any integer from 2 to 1500;

[0222] m is any integer from 1 to 200, preferably any integer from 2 to 200, preferably any integer from 1 to 100, more preferably any integer from 2 to 100;

[0223] R 1 is an initiating residue, preferably, R 1 is -H or -CH3;

[0224] R 2 are independently -H or an organic residue, the -(NR 2 -CH2-CH2) n - At least 80%, preferably 90% of said R 2 is H;

[0225] Ring A is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl group, optionally substituted at any position with one or more R A1 replace;

[0226] R A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo or halogen; or, two R A1 Together with the atoms to which they are attached, they can form more than one fused C6-C 10 Aryl, C 5- C6 heteroaryl or C3-C6 cycloalkyl ring, wherein each fused aryl, heteroaryl or cycloalkyl is optionally substituted by one or more R A2 replace;

[0227] R A2Independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, -SO3H or -OSO3H;

[0228] X 1 Formula -(Y 1 ) p - a connecting portion, wherein p is an integer from 1 to 20, Y 1 is independently selected at each occurrence from a chemical bond, -CR 11 R 12 -、-C(O)-、-O-、-S-、-NR 13 -, amino acid residues, divalent phenyl moieties, divalent heterocyclic moieties and divalent heteroaryl moieties, each divalent phenyl or heteroaryl group being optionally substituted by one or more R 13 Each divalent heterocyclic ring may be substituted with one or more R 14 Replacement; R 11 、R 12 and R 13 is independently H or C1-C6 alkyl at each occurrence, R 14 is independently H, C1-C6 alkyl or oxo at each occurrence;

[0229] X 2 Formula -(Y 2 ) q - a connecting portion, wherein q is an integer from 1 to 50, Y 2 is independently selected at each occurrence from a chemical bond, -CR 21 R 22 -、NR 23 -, -O-, -S-, -C(O)-, amino acid residues, divalent phenyl moieties, divalent heterocyclic moieties and divalent heteroaryl moieties, each of which is optionally substituted by one or more R 23 Each divalent heterocyclic moiety is optionally substituted with one or more R 24 Replacement; R 21 、R 22 and R 23 Each occurrence is independently -H, -CO2H or C1-C6 alkyl, each C1-C6 alkyl optionally substituted by one or more -OH, oxo, C6-C 10 aryl or 5 to 8 membered heteroaryl substituted, R 24 is independently at each occurrence -H, -CO2H, C1-C6 alkyl, or oxo; and

[0230] L is a targeting fragment, preferably, the targeting fragment is capable of binding to a cell, preferably, the targeting fragment is capable of binding to a cell surface receptor; and

[0231] The nucleic acid is a nucleic acid encoding a target peptide or protein. Preferably, the nucleic acid is a nucleic acid having pharmaceutical activity. The nucleic acid having pharmaceutical activity is a nucleic acid encoding a peptide or protein having pharmaceutical activity.

[0232] Preferably, the composition consists of the polymer composite.

[0233] In another aspect, the present invention provides a polymer complex comprising a conjugate of Formula I or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or enantiomer thereof and a nucleic acid, preferably wherein the nucleic acid is non-covalently bound to the conjugate:

[0234]

[0235] Where:

[0236] is a single bond or a double bond;

[0237] n is any integer from 1 to 1500, preferably any integer from 2 to 1500;

[0238] m is any integer from 1 to 200, preferably any integer from 2 to 200, preferably any integer from 1 to 100, more preferably any integer from 2 to 100;

[0239] R 1 is an initiating residue, preferably, R 1 is -H or -CH3;

[0240] R 2 are independently -H or an organic residue, the -(NR 2 -CH2-CH2) n - At least 80%, preferably 90% of said R 2 is H;

[0241] Ring A is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl group, optionally substituted at any position with one or more R A1 replace;

[0242] R A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo or halogen; or, two R A1 Together with the atoms to which they are attached, they can form more than one fused C6-C 10 aryl, C5-C6 heteroaryl or C3-C6 cycloalkyl ring, wherein each fused aryl, heteroaryl or cycloalkyl is optionally substituted by one or more R A2 replace;

[0243] R A2Independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, -SO3H or -OSO3H;

[0244] X 1 Formula -(Y 1 ) p - a connecting portion, wherein p is an integer from 1 to 20, Y 1 is independently selected at each occurrence from a chemical bond, -CR 11 R 12 -、-C(O)-、-O-、-S-、-NR 13 -, amino acid residues, divalent phenyl moieties, divalent heterocyclic moieties and divalent heteroaryl moieties, each divalent phenyl or heteroaryl group being optionally substituted by one or more R 13 Each divalent heterocyclic ring may be substituted with one or more R 14 Replacement; R 11 、R 12 and R 13 R is independently H or C1-C6 alkyl at each occurrence; 14 is independently H, C1-C6 alkyl or oxo at each occurrence;

[0245] X 2 Formula -(Y 2 ) q - a connecting portion, wherein q is an integer from 1 to 50, Y 2 is independently selected at each occurrence from a chemical bond, -CR 21 R 22 -、NR 23 -, -O-, -S-, -C(O)-, amino acid residues, divalent phenyl moieties, divalent heterocyclic moieties and divalent heteroaryl moieties, each of which is optionally substituted by one or more R 23 Each divalent heterocyclic moiety is optionally substituted with one or more R 24 Replacement; R 21 、R 22 and R 23 Each occurrence is independently -H, -CO2H or C1-C6 alkyl, each C1-C6 alkyl optionally substituted by one or more -OH, oxo, C6-C 10 aryl or 5 to 8 membered heteroaryl substituted; R 24 is independently at each occurrence -H, -CO2H, C1-C6 alkyl, or oxo; and

[0246] L is a targeting fragment, preferably, the targeting fragment is capable of binding to a cell, preferably, the targeting fragment is capable of binding to a cell surface receptor; and

[0247] The nucleic acid is a nucleic acid encoding a target peptide or protein. Preferably, the nucleic acid is a nucleic acid having pharmaceutical activity. The nucleic acid having pharmaceutical activity is a nucleic acid encoding a peptide or protein having pharmaceutical activity.

[0248] In another aspect, the present invention provides a composition comprising a polymer complex comprising a conjugate of Formula I or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or enantiomer thereof and a nucleic acid, preferably the nucleic acid is non-covalently bound to the conjugate:

[0249]

[0250] Where:

[0251] is a single bond or a double bond;

[0252] n is any integer from 1 to 1500, preferably any integer from 2 to 1500;

[0253] m is any discrete number of -(O-CH2-CH2)- repeating units from 25 to 100, preferably from 25 to 60; preferably, the discrete number m is a discrete number of -(O-CH2-CH2)- continuous repeating units, and the discrete number m of -(O-CH2-CH2)- continuous repeating units is any discrete number from 25 to 100, preferably from 25 to 60;

[0254] R 1 is an initiating residue, preferably, R 1 is -H or -CH3;

[0255] R 2 are independently -H or an organic residue, the -(NR 2 -CH2-CH2) n - at least 80%, preferably at least 90% of R 2 is H;

[0256] Ring A is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl group, optionally substituted at any position with one or more R A1 Replacement; R A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo or halogen; or, two R A1 Together with the atoms to which they are attached, they can form more than one fused C6-C 10 aryl, C5-C6 heteroaryl or C3-C6 cycloalkyl ring, wherein each fused aryl, heteroaryl or cycloalkyl is optionally substituted by one or more R A2 Replacement; R A2Independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, -SO3H or -OSO3H;

[0257] X 1 is a divalent covalent linking moiety;

[0258] X 2 is a divalent covalent linking moiety; and

[0259] L is a targeting fragment, preferably, the targeting fragment can bind to a cell, more preferably, the targeting fragment can bind to a cell surface receptor; and

[0260] The nucleic acid is a nucleic acid with pharmaceutical activity, and the nucleic acid with pharmaceutical activity is a nucleic acid encoding a peptide or protein with pharmaceutical activity.

[0261] In another aspect, the present invention provides a polymer complex comprising a conjugate of Formula I or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or enantiomer thereof and a nucleic acid, preferably wherein the nucleic acid is non-covalently bound to the conjugate:

[0262]

[0263] Where:

[0264] is a single bond or a double bond;

[0265] n is any integer from 1 to 1500, preferably any integer from 2 to 1500;

[0266] m is any discrete number of -(O-CH2-CH2)- repeating units from 25 to 100, preferably from 25 to 60, preferably, the discrete number m is a discrete number of -(O-CH2-CH2)- continuous repeating units, the discrete number m of -(O-CH2-CH2)- continuous repeating units is any discrete number from 25 to 100, preferably from 25 to 60;

[0267] R 1 is an initiating residue, preferably, R 1 is -H or -CH3;

[0268] R 2 are independently -H or an organic residue, the -(NR 2 -CH2-CH2) n - at least 80%, preferably at least 90% of R 2 is H;

[0269] Ring A is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl group, optionally substituted at any position with one or more R A1Replacement; R A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo or halogen; or, two R A1 Together with the atoms to which they are attached, they can form more than one fused C6-C 10 aryl, C5-C6 heteroaryl or C3-C6 cycloalkyl ring, wherein each fused aryl, heteroaryl or cycloalkyl is optionally substituted by one or more R A2 Replacement; R A2 Independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, -SO3H or -OSO3H;

[0270] X 1 is a divalent covalent linking moiety;

[0271] X 2 is a divalent covalent linking moiety; and

[0272] L is a targeting fragment, preferably, the targeting fragment can bind to a cell, more preferably, the targeting fragment can bind to a cell surface receptor; and

[0273] The nucleic acid is a nucleic acid with pharmaceutical activity, and the nucleic acid with pharmaceutical activity is a nucleic acid encoding a peptide or protein with pharmaceutical activity.

[0274] In another aspect, the present invention provides a composition comprising a polymer complex comprising a conjugate of Formula I or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or enantiomer thereof and a nucleic acid, preferably the nucleic acid is non-covalently bound to the conjugate:

[0275]

[0276] Where:

[0277] is a single bond or a double bond;

[0278] n is any integer from 1 to 1500, preferably any integer from 2 to 1500;

[0279] m is a discrete number of -(O-CH2-CH2)- repeating units, and the discrete number m of -(O-CH2-CH2)- repeating units is 36; preferably, the discrete number m is a discrete number of -(O-CH2-CH2)- continuous repeating units, and the discrete number m of -(O-CH2-CH2)- continuous repeating units is 36;

[0280] R 1 is an initiating residue, preferably, R 1 is -H or -CH3;

[0281] R 2 are independently -H or an organic residue, the -(NR 2 -CH2-CH2) n - at least 80%, preferably at least 90% of said R 2 is H;

[0282] Ring A is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl group, optionally substituted at any position with one or more R A1 Replacement; R A1 Independently selected from C1-C6 alkyl, C 1- C6 alkoxy, oxo or halogen; or, two R A1 Together with the atoms to which they are attached, they can form more than one fused C6-C 10 aryl, C5-C6 heteroaryl or C3-C6 cycloalkyl ring, wherein each fused aryl, heteroaryl or cycloalkyl is optionally substituted by one or more R A2 Replacement; R A2 Independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, -SO3H or -OSO3H;

[0283] X 1 is a divalent covalent linking moiety;

[0284] X 2 is a divalent covalent linking moiety; and

[0285] L is a targeting fragment, preferably, the targeting fragment can bind to a cell, more preferably, the targeting fragment can bind to a cell surface receptor; and

[0286] The nucleic acid is a nucleic acid with pharmaceutical activity, and the nucleic acid with pharmaceutical activity is a nucleic acid encoding a peptide or protein with pharmaceutical activity.

[0287] In another aspect, the present invention provides a polymer complex comprising a conjugate of Formula I or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or enantiomer thereof and a nucleic acid, preferably wherein the nucleic acid is non-covalently bound to the conjugate:

[0288]

[0289] Where:

[0290] is a single bond or a double bond;

[0291] n is any integer from 1 to 1500, preferably any integer from 2 to 1500;

[0292] m is a discrete number of -(O-CH2-CH2)- repeating units, and the discrete number m of -(O-CH2-CH2)- repeating units is 36; preferably, the discrete number m is a discrete number of -(O-CH2-CH2)- continuous repeating units, and the discrete number m of -(O-CH2-CH2)- continuous repeating units is 36;

[0293] R 1 is an initiating residue, wherein, preferably, R 1 is -H or -CH3;

[0294] R 2 are independently -H or an organic residue, the -(NR 2 -CH2-CH2) n - at least 80%, preferably at least 90% of said R 2 is H;

[0295] Ring A is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl group, optionally substituted at any position with one or more R A1 Replacement; R A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo or halogen; or, two R A1 Together with the atoms to which they are attached, they can form more than one fused C6-C 10 aryl, C5-C6 heteroaryl or C3-C6 cycloalkyl ring, wherein each fused aryl, heteroaryl or cycloalkyl is optionally substituted by one or more R A2 Replacement; R A2 Independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, -SO3H or -OSO3H;

[0296] X 1 is a divalent covalent linking moiety;

[0297] X 2 is a divalent covalent linking moiety; and

[0298] L is a targeting fragment, preferably, the targeting fragment can bind to a cell, more preferably, the targeting fragment can bind to a cell surface receptor; and

[0299] The nucleic acid is a nucleic acid with pharmaceutical activity, and the nucleic acid with pharmaceutical activity is a nucleic acid encoding a peptide or protein with pharmaceutical activity.

[0300] On the other hand, the present invention provides a composition comprising a polymer complex, each of the polymer complexes comprising a conjugate and a nucleic acid, preferably the nucleic acid is non-covalently bound to the conjugate, the conjugate comprising: a linear polyethyleneimine segment comprising an α-terminus and an ω-terminus, the α-terminus of the polyethyleneimine segment being a priming residue; a polyethylene glycol segment comprising a first terminus and a second terminus; wherein the ω-terminus of the polyethyleneimine segment is linked to a divalent covalent linking group -ZX 1 - is linked to the first end of the polyethylene glycol segment, -ZX 1 is not a single bond and -Z- is not an amide; the second end of the polyethylene glycol segment is divalently linked to the moiety X 2 Connected to the targeting fragment; the nucleic acid is a nucleic acid with pharmaceutical activity, and the nucleic acid with pharmaceutical activity is a nucleic acid encoding a peptide or protein with pharmaceutical activity.

[0301] On the other hand, the present invention provides a composition comprising a polymer complex, wherein the polymer complex comprises a conjugate and a nucleic acid, preferably the nucleic acid is non-covalently bound to the conjugate, wherein the conjugate comprises: a linear polyethyleneimine segment comprising an α-terminus and an ω-terminus, wherein the α-terminus of the polyethyleneimine segment is a priming residue; a polyethylene glycol segment comprising a first terminus and a second terminus; wherein the ω-terminus of the polyethyleneimine segment is connected via a divalent covalent linking group -ZX 1 - is linked to the first end of the polyethylene glycol segment, -ZX 1 is not a single bond and -Z- is not an amide; the second end of the polyethylene glycol segment is divalently linked to the moiety X 2 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 polymer complex.

[0302] On the other hand, the present invention provides a composition comprising a polymer complex, wherein the polymer complex comprises a conjugate and a nucleic acid, preferably the nucleic acid is non-covalently bound to the conjugate, wherein the conjugate comprises: a linear polyethyleneimine segment comprising an α-terminus and an ω-terminus, wherein the α-terminus of the polyethyleneimine segment is a priming residue; a polyethylene glycol segment comprising a first terminus and a second terminus; wherein the ω-terminus of the polyethyleneimine segment is connected via a divalent covalent linking group -ZX 1 - is linked to the first end of the polyethylene glycol segment, -ZX 1 is not a single bond and -Z- is not an amide; the second end of the polyethylene glycol segment is divalently linked to the moiety X 2The nucleic acid is connected to the targeting fragment, the RNA is ssRNA, preferably the ssRNA is mRNA, more preferably the mRNA encodes a target peptide or protein. In a preferred embodiment of this aspect, the composition consists of the polymer complex.

[0303] On the other hand, the present invention provides a composition comprising a polymer complex, each of the polymer complexes comprising a conjugate and a nucleic acid, preferably the nucleic acid is non-covalently bound to the conjugate, the conjugate comprising: a linear polyethyleneimine segment comprising an α-terminus and an ω-terminus, the α-terminus of the polyethyleneimine segment being a priming residue; a polyethylene glycol segment comprising a first terminus and a second terminus; wherein the ω-terminus of the polyethyleneimine segment is linked to a divalent covalent linking group -ZX 1 - is linked to the first end of the polyethylene glycol segment, -ZX 1 is not a single bond and -Z- is not an amide; the second end of the polyethylene glycol segment is divalently linked to the moiety X 2 Connected to the targeting fragment; the nucleic acid is RNA, wherein the RNA is ssRNA, preferably the ssRNA is mRNA.

[0304] On the other hand, the present invention provides a composition comprising a polymer complex, each of the polymer complexes comprising a conjugate and a nucleic acid, preferably the nucleic acid is non-covalently bound to the conjugate, the conjugate comprising: a linear polyethyleneimine segment comprising an α-terminus and an ω-terminus, the α-terminus of the polyethyleneimine segment being a priming residue; a polyethylene glycol segment comprising a first terminus and a second terminus; wherein the ω-terminus of the polyethyleneimine segment is linked to a divalent covalent linking group -ZX 1 - is linked to the first end of the polyethylene glycol segment, -ZX 1 is not a single bond and -Z- is not an amide; the second end of the polyethylene glycol segment is divalently linked to the moiety X 2 Connected to the targeting fragment; the nucleic acid is DNA, the DNA is pDNA, and preferably the pDNA encodes the target peptide or protein.

[0305] In another aspect, the present invention provides a composition comprising a polymer complex, each of the polymer complexes comprising a conjugate of Formula I or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or enantiomer thereof and a nucleic acid, preferably the nucleic acid is non-covalently bound to the conjugate:

[0306]

[0307] Where:

[0308] is a single bond or a double bond;

[0309] n is any integer from 1 to 1500, preferably any integer from 2 to 1500;

[0310] m is any integer from 1 to 200, preferably any integer from 2 to 200, preferably any integer from 1 to 100, more preferably any integer from 2 to 100;

[0311] R 1 is an initiating residue, preferably, R 1 is -H or -CH3;

[0312] R 2 are independently -H or an organic residue, the -(NR 2 -CH2-CH2) n - at least 80%, preferably at least 90% of R 2 is H;

[0313] Ring A is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl group, optionally substituted at any position with one or more R A1 Replacement; R A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo or halogen; or, two R A1 Together with the atoms to which they are attached, they can form more than one fused C6-C 10 aryl, C5-C6 heteroaryl or C3-C6 cycloalkyl ring, wherein each fused aryl, heteroaryl or cycloalkyl is optionally substituted by one or more R A2 Replacement; R A2 Independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, -SO3H or -OSO3H;

[0314] X 1 is a divalent covalent linking moiety;

[0315] X 2 is a divalent covalent linking moiety; and

[0316] L is a targeting fragment, preferably, the targeting fragment can bind to a cell, more preferably, the targeting fragment can bind to a cell surface receptor; and

[0317] The nucleic acid is a nucleic acid with pharmaceutical activity, and the nucleic acid with pharmaceutical activity is a nucleic acid encoding a peptide or protein with pharmaceutical activity.

[0318] In another aspect, the present invention provides a composition comprising a polymer complex comprising a conjugate of Formula I or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or enantiomer thereof and a nucleic acid, preferably the nucleic acid is non-covalently bound to the conjugate:

[0319]

[0320] Where:

[0321] is a single bond or a double bond;

[0322] n is any integer from 1 to 1500, preferably any integer from 2 to 1500;

[0323] m is any integer from 1 to 200, preferably any integer from 2 to 200, preferably any integer from 1 to 100, more preferably any integer from 2 to 100;

[0324] R 1 is an initiating residue, preferably, R 1 is -H or -CH3;

[0325] R 2 are independently -H or an organic residue, the -(NR 2 -CH2-CH2) n - at least 80%, preferably at least 90% of R 2 is H;

[0326] Ring A is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl group, optionally substituted at any position with one or more R A1 Replacement; R A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo or halogen; or, two R A1 Together with the atoms to which they are attached, they can form more than one fused C6-C 10 aryl, C5-C6 heteroaryl or C3-C6 cycloalkyl ring, wherein each fused aryl, heteroaryl or cycloalkyl is optionally substituted by one or more R A2 Replacement; R A2 Independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, -SO3H or -OSO3H;

[0327] X 1 is a divalent covalent linking moiety;

[0328] X 2 is a divalent covalent linking moiety; and

[0329] L is a targeting fragment, preferably, the targeting fragment can bind to a cell, more preferably, the targeting fragment can bind to a cell surface receptor; and

[0330] The nucleic acid is RNA, the RNA is ssRNA, preferably, the ssRNA is mRNA, more preferably, the mRNA encodes a target peptide or protein.

[0331] In another aspect, the present invention provides a composition comprising a polymer complex comprising a conjugate of Formula I or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or enantiomer thereof and a nucleic acid, preferably the nucleic acid is non-covalently bound to the conjugate:

[0332]

[0333] Where:

[0334] is a single bond or a double bond;

[0335] n is any integer from 1 to 1500, preferably any integer from 2 to 1500;

[0336] m is any integer from 1 to 200, preferably any integer from 2 to 200, preferably any integer from 1 to 100, more preferably any integer from 2 to 100;

[0337] R 1 is an initiating residue, preferably, R 1 is -H or -CH3;

[0338] R 2 are independently -H or an organic residue, the -(NR 2 -CH2-CH2) n - at least 80%, preferably at least 90% of R 2 is H;

[0339] Ring A is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl group, optionally substituted at any position with one or more R A1 Replacement; R A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo or halogen; or, two R A1 Together with the atoms to which they are attached, they can form more than one fused C6-C 10 aryl, C5-C6 heteroaryl or C3-C6 cycloalkyl ring, wherein each fused aryl, heteroaryl or cycloalkyl is optionally substituted by one or more R A2 Replacement; R A2 Independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, -SO3H or -OSO3H;

[0340] X 1 is a divalent covalent linking moiety;

[0341] X 2 is a divalent covalent linking moiety; and

[0342] L is a targeting fragment, preferably, the targeting fragment can bind to a cell, more preferably, the targeting fragment can bind to a cell surface receptor; and

[0343] The nucleic acid is DNA, and the DNA is pDNA. Preferably, the pDNA encodes a target peptide or protein.

[0344] In another aspect, the present invention provides a composition comprising a polymer complex, each of the polymer complexes comprising a conjugate of Formula I or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or enantiomer thereof and a nucleic acid, preferably the nucleic acid is non-covalently bound to the conjugate:

[0345]

[0346]

[0347] Where:

[0348] is a single bond or a double bond;

[0349] n is any integer from 1 to 1500, preferably any integer from 2 to 1500;

[0350] m is any integer from 1 to 200, preferably any integer from 2 to 200, preferably any integer from 1 to 100, more preferably any integer from 2 to 100;

[0351] R 1 is an initiating residue, preferably, R 1 is -H or -CH3;

[0352] R 2 are independently -H or an organic residue, the -(NR 2 -CH2-CH2) n - at least 80%, preferably at least 90% of R 2 is H;

[0353] Ring A is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl group, optionally substituted at any position with one or more R A1 Replacement; R A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo or halogen; or, two R A1 Together with the atoms to which they are attached, they can form more than one fused C6-C 10aryl, C5-C6 heteroaryl or C3-C6 cycloalkyl ring, wherein each fused aryl, heteroaryl or cycloalkyl is optionally substituted by one or more R A2 Replacement; R A2 Independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, -SO3H or -OSO3H;

[0354] X 1 is a divalent covalent linking moiety;

[0355] X 2 is a divalent covalent linking moiety; and

[0356] L is a targeting fragment, preferably, the targeting fragment can bind to a cell, more preferably, the targeting fragment can bind to a cell surface receptor; and

[0357] The nucleic acid is RNA, the RNA is ssRNA, preferably, the ssRNA is mRNA, more preferably, the mRNA encodes a target peptide or protein.

[0358] In another aspect, the present invention provides a composition comprising a polymer complex, each of the polymer complexes comprising a conjugate of Formula I or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or enantiomer thereof and a nucleic acid, preferably the nucleic acid is non-covalently bound to the conjugate:

[0359]

[0360] Where:

[0361] is a single bond or a double bond;

[0362] n is any integer from 1 to 1500, preferably any integer from 2 to 1500;

[0363] m is any integer from 1 to 200, preferably any integer from 2 to 200, preferably any integer from 1 to 100, more preferably any integer from 2 to 100;

[0364] R 1 is an initiating residue, preferably, R 1 is -H or -CH3;

[0365] R 2 are independently -H or an organic residue, the -(NR 2 -CH2-CH2) n - at least 80%, preferably at least 90% of R 2 is H;

[0366] Ring A is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl group, optionally substituted at any position with one or more R A1 Replacement; R A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo or halogen; or, two R A1 Together with the atoms to which they are attached, they can form more than one fused C6-C 10 aryl, C5-C6 heteroaryl or C3-C6 cycloalkyl ring, wherein each fused aryl, heteroaryl or cycloalkyl is optionally substituted by one or more R A2 Replacement; R A2 Independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, -SO3H or -OSO3H;

[0367] X 1 is a divalent covalent linking moiety;

[0368] X 2 is a divalent covalent linking moiety; and

[0369] L is a targeting fragment, preferably, the targeting fragment can bind to a cell, more preferably, the targeting fragment can bind to a cell surface receptor; and

[0370] The nucleic acid is DNA, and the DNA is pDNA. Preferably, the pDNA encodes a target peptide or protein.

[0371] On the other hand, the present invention provides a composition comprising a polymer complex, wherein the polymer complex comprises a conjugate and a nucleic acid, preferably the nucleic acid is non-covalently bound to the conjugate, the conjugate comprises a linear polyethyleneimine (LPEI) segment covalently linked to one or more polyethylene glycol (PEG) segments, each PEG segment is covalently linked to a targeting segment L, preferably the targeting segment is capable of binding to cells; 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 cytokines, interferons, interleukins, growth factors, hormones, enzymes, toxins, tumor antigens, viral antigens, bacterial antigens, autoantigens and allergens.

[0372] On the other hand, the present invention provides a polymer complex, comprising a conjugate and a nucleic acid, preferably the nucleic acid is non-covalently bound to the conjugate, the conjugate comprises a linear polyethyleneimine (LPEI) segment covalently linked to one or more polyethylene glycol (PEG) segments, each PEG segment is covalently linked to a targeting segment L, preferably the targeting segment is capable of binding to cells; the nucleic acid is single-stranded RNA (ssRNA), preferably the ssRNA is mRNA, more preferably the mRNA is a nucleic acid with pharmaceutical activity, the nucleic acid with pharmaceutical activity is a nucleic acid encoding a peptide or protein with pharmaceutical activity, and even more preferably the peptide or protein with pharmaceutical activity is selected from cytokines, interferons, interleukins, growth factors, hormones, enzymes, toxins, tumor antigens, viral antigens, bacterial antigens, autoantigens and allergens.

[0373] On the other hand, the present invention provides a composition comprising a polymer complex, each of the polymer complexes comprising a conjugate and a nucleic acid, preferably the nucleic acid is non-covalently bound to the conjugate, the conjugate comprising a linear polyethyleneimine (LPEI) segment covalently linked to one or more polyethylene glycol (PEG) segments, each PEG segment being covalently linked to a targeting segment L, preferably the targeting segment being capable of binding to cells; 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 cytokines, interferons, interleukins, growth factors, hormones, enzymes, toxins, tumor antigens, viral antigens, bacterial antigens, autoantigens and allergens.

[0374] On the other hand, the present invention provides a composition comprising a polymer complex, wherein the polymer complex comprises a conjugate and a nucleic acid, preferably the nucleic acid is non-covalently bound to the conjugate, the conjugate comprises a linear polyethyleneimine (LPEI) fragment covalently linked to one or more polyethylene glycol (PEG) fragments, each PEG fragment is covalently linked to a targeting fragment L, preferably the targeting fragment is capable of binding to cells; the nucleic acid is DNA, preferably plasmid DNA (pDNA), preferably the DNA (preferably the pDNA) encodes a peptide or protein with pharmaceutical activity, and the peptide or protein with pharmaceutical activity is preferably selected from cytokines, interferons, interleukins, growth factors, hormones, enzymes, toxins, tumor antigens, viral antigens, bacterial antigens, autoantigens and allergens.

[0375] On the other hand, the present invention provides a polymer complex, which comprises a conjugate and a nucleic acid, preferably the nucleic acid is non-covalently bound to the conjugate, the conjugate comprises a linear polyethyleneimine (LPEI) fragment covalently linked to one or more polyethylene glycol (PEG) fragments, each PEG fragment is covalently linked to a targeting fragment L, preferably the targeting fragment is capable of binding to cells; the nucleic acid is DNA, preferably plasmid DNA (pDNA), preferably the DNA (preferably the pDNA) encodes a peptide or protein with pharmaceutical activity, and the peptide or protein with pharmaceutical activity is preferably selected from cytokines, interferons, interleukins, growth factors, hormones, enzymes, toxins, tumor antigens, viral antigens, bacterial antigens, autoantigens and allergens.

[0376] On the other hand, the present invention provides a composition comprising a polymer complex, each of the polymer complexes comprising a conjugate and a nucleic acid, preferably the nucleic acid is non-covalently bound to the conjugate, the conjugate comprises a linear polyethyleneimine (LPEI) fragment covalently linked to one or more polyethylene glycol (PEG) fragments, each PEG fragment is covalently linked to a targeting fragment L, preferably the targeting fragment is capable of binding to cells; the nucleic acid is DNA, preferably plasmid DNA (pDNA); preferably the DNA (preferably the pDNA) encodes a peptide or protein with pharmaceutical activity, and the peptide or protein with pharmaceutical activity is preferably selected from cytokines, interferons, interleukins, growth factors, hormones, enzymes, toxins, tumor antigens, viral antigens, bacterial antigens, autoantigens and allergens.

[0377] In a preferred embodiment of any aspect of the present invention, the nucleic acid is RNA. In another preferred embodiment of any aspect of the present invention, the nucleic acid is single-stranded RNA (ssRNA). In another preferred embodiment of any aspect of the present invention, the ssRNA encodes a target peptide or protein. In another preferred embodiment of any aspect of the present invention, the ssRNA encodes a target peptide or protein, wherein the target peptide or protein is selected from a reporter protein and a peptide or protein with pharmaceutical activity. In another preferred embodiment of any aspect of the present invention, the ssRNA encodes a target peptide or protein, wherein the target peptide or protein is a reporter protein. In another preferred embodiment of any aspect of the present invention, the ssRNA encodes a target peptide or protein, wherein the target peptide or protein is a peptide or protein with pharmaceutical activity. In another preferred embodiment of any aspect of the present invention, the ssRNA is a pharmaceutically active nucleic acid. In another preferred embodiment of any aspect of the present invention, the ssRNA is a pharmaceutically active nucleic acid, and the pharmaceutically active nucleic acid is a nucleic acid encoding a pharmaceutically active peptide or protein.

[0378] In another preferred embodiment of any aspect of the present invention, the ssRNA is a messenger RNA (mRNA). In another preferred embodiment of any aspect of the present invention, the mRNA encodes a target peptide or protein. In another preferred embodiment of any aspect of the present invention, the mRNA encodes a target peptide or protein, and the target peptide or protein is selected from a reporter protein and a peptide or protein with pharmaceutical activity. In another preferred embodiment of any aspect of the present invention, the mRNA encodes a target peptide or protein, wherein the target peptide or protein is a reporter protein. In another preferred embodiment of any aspect of the present invention, the mRNA encodes a target peptide or protein, and the target peptide or protein is a peptide or protein with pharmaceutical activity. In another preferred embodiment of any aspect of the present invention, the mRNA is a nucleic acid with pharmaceutical activity. In another preferred embodiment of any aspect of the present invention, the mRNA is a nucleic acid with pharmaceutical activity, and the nucleic acid with pharmaceutical activity is a nucleic acid encoding a peptide or protein with pharmaceutical activity.

[0379] In another preferred embodiment of any aspect of the present invention, the nucleic acid is DNA. In another preferred embodiment of any aspect of the present invention, the DNA is plasmid DNA. In another preferred embodiment of any aspect of the present invention, the plasmid DNA encodes a target peptide or protein. In another preferred embodiment of any aspect of the present invention, the plasmid DNA encodes a target peptide or protein, and the target peptide or protein is selected from a reporter protein and a peptide or protein with pharmaceutical activity. In another preferred embodiment of any aspect of the present invention, the plasmid DNA encodes a target peptide or protein, and the target peptide or protein is a reporter protein. In another preferred embodiment of any aspect of the present invention, the plasmid DNA encodes a target peptide or protein, and the target peptide or protein is a peptide or protein with pharmaceutical activity. In another preferred embodiment of any aspect of the present invention, the plasmid DNA is a pharmaceutically active nucleic acid. In another preferred embodiment of any aspect of the present invention, the pDNA is a pharmaceutically active nucleic acid, and the pharmaceutically active nucleic acid is a nucleic acid encoding a pharmaceutically active peptide or protein.

[0380] In another preferred embodiment of any aspect of the present invention, the nucleic acid is a pharmaceutically active nucleic acid, and the pharmaceutically active nucleic acid itself has pharmaceutical activity. In another preferred embodiment of any aspect of the present invention, 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.

[0381] In a preferred embodiment, the nucleic acid encodes a target peptide or protein, which is a reporter protein. In these embodiments, the nucleic acid comprises a reporter gene. Certain genes may be selected as reporter genes because they confer characteristics on cells or organisms expressing them that can be easily identified and measured, or because they are selection markers. Reporter genes are typically used to indicate whether a gene has been taken up by a cell or organism colony or expressed in a cell or organism colony. Preferably, the expression product of the reporter gene can be visually detected. Common visually detectable reporter proteins typically have fluorescent or luminescent proteins. Examples of specific reporter genes include genes encoding jellyfish green fluorescent protein (GFP), which causes cells expressing the protein to emit green light under blue light; genes encoding luciferase, which catalyzes a reaction with luciferin to produce light; and genes encoding red fluorescent protein (RFP). Variants of any of these specific reporter genes are possible, as long as these variants have visually detectable characteristics. For example, eGFP is a point mutation variant of GFP.

[0382] 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 is connected to the PEG segment via a single covalent linking moiety, preferably, the covalent linking moiety creates a linear end-to-end connection between the LPEI segment and the PEG segment. In some embodiments, at least 60%, at least 70%, or at least 80%, at least 90%, at least 95% or at least 99% of the LPEI segment contained in the composition consists of the conjugate, preferably as determined by UV spectroscopy or mass spectrometry. In some embodiments, at least 60%, at least 70%, or at least 80%, at least 90%, at least 95% or at least 99% of the LPEI contained in the composition consists of 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.

[0383] In some embodiments, at least 60% of the LPEI in the composition is linked to a single PEG segment via a single covalent linking moiety Z, preferably, the covalent linking moiety Z creates a linear end-to-end connection between the LPEI segment and the PEG segment. In some embodiments, at least 60% of the LPEI segments contained in the composition are linked to the PEG segment via a single triazole linking group, preferably as determined by UV spectroscopy or mass spectrometry. In some embodiments, at least 70% of the LPEI in the composition is linked to the PEG segment via a single covalent linking moiety Z, preferably, the covalent linking moiety Z creates a linear end-to-end connection between the LPEI segment and the PEG segment. In some embodiments, at least 70% of the LPEI segments contained in the composition are composed of the conjugate, preferably as determined by UV spectroscopy or mass spectrometry. In some embodiments, at least 80% of the LPEI in the composition is linked to the PEG segment via a single covalent linking moiety Z, preferably, the covalent linking moiety Z creates a linear end-to-end connection between the LPEI segment and the PEG segment. In some embodiments, at least 80% of the LPEI segments contained in the composition are composed of the conjugate, preferably as determined by UV spectroscopy or mass spectrometry. In some embodiments, at least 90% of the LPEI in the composition are linked to the PEG segments via a single covalent linking moiety Z, preferably, the covalent linking moiety Z creates a linear end-to-end connection between the LPEI segments and the PEG segments. In some embodiments, at least 90% of the LPEI segments contained in the composition are composed of the conjugate, preferably as determined by UV spectroscopy or mass spectrometry. In some embodiments, at least 95% of the LPEI in the composition are linked to the PEG segments via a single covalent linking moiety Z, preferably, the covalent linking moiety Z creates a linear end-to-end connection between the LPEI segments and the PEG segments. In some embodiments, at least 95% of the LPEI segments contained in the composition are composed of the conjugate, preferably as determined by UV spectroscopy or mass spectrometry. In some embodiments, at least 99% of the LPEI in the composition is linked to the PEG segment via a single covalent linker Z, preferably, the covalent linker Z creates a linear end-to-end linkage between the LPEI segment and the PEG segment. In some embodiments, at least 99% of the LPEI segment contained in the composition consists of 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 segment does not contain substitutions outside of its first and second termini.

[0384] In some embodiments, the covalently linking moiety Z comprises a triazole.

[0385] In some embodiments, Formula I* does not include the following structure: R 1-(NH-CH2-CH2) n -NHC(O)-(CH2-CH2-O) m -X 2 -L. In some embodiments, Formula I* does not contain the following structure: R 1 -(NR 2 -CH2-CH2) n -NHC(O)-X 1 -(O-CH2-CH2) m -X 2 -L. In some embodiments, the composition does not contain a structure of R 1 -(NH-CH2-CH2) n -NHC(O)-X 1 -(O-CH2-CH2) m -X 2 In some embodiments, the composition does not contain a conjugate of structure R 1 -(NR 2 -CH2-CH2) n -NHC(O)-(CH2-CH2-O) m -X 2 -L conjugate.

[0386] In some embodiments, R 1 is -H.

[0387] In some embodiments, at least 80% of the R 2 In some embodiments, at least 85%, preferably 90%, preferably 95%, and more preferably 99% of the R 2 In a preferred embodiment, R 2 are independently -H or an organic residue, the -(NR 2 -CH2-CH2) n - At least 85%, preferably 90% of said R 2 is H. In another preferred embodiment, R 2 are independently -H or an organic residue, the -(NR 2 -CH2-CH2) n - At least 90% of the R 2 is H. In another preferred embodiment, R 2 are independently -H or an organic residue, the -(NR 2 -CH2-CH2) n - At least 90% of the R 2 is H. In another preferred embodiment, R 2are independently -H or an organic residue, the -(NR 2 -CH2-CH2) n - at least 91%, preferably at least 92%, more preferably at least 93% of said R 2 is H. In another preferred embodiment, R 2 are independently -H or an organic residue, the -(NR 2 -CH2-CH2) n - at least 94%, preferably at least 95%, more preferably at least 96% of said R 2 is H. In another preferred embodiment, R 2 are independently -H or an organic residue, the -(NR 2 -CH2-CH2) n - at least 95%, preferably at least 97%, more preferably at least 98%, more preferably 99% of said R 2 For H.

[0388] In some embodiments, ring A is an 8-membered cycloalkenyl, a 5-membered heterocycloalkyl, or a 7- to 8-membered heterocycloalkenyl, and each cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group is optionally substituted at any position with one or more R A1 replace.

[0389] In some embodiments, ring A is cyclooctene, maleimide or 7-8 membered heterocycloalkenyl, the heterocycloalkyl or heterocycloalkenyl does not contain heteroatoms other than N, O and S, and each cyclooctene, heterocycloalkyl or heterocycloalkenyl is optionally substituted at any position with one or more R A1 replace.

[0390] In some embodiments, ring A is cyclooctene, maleimide or 7-8 membered heterocycloalkenyl, the heterocycloalkyl or heterocycloalkenyl group contains one or more heteroatoms, preferably one or two heteroatoms selected from N, O and S, and each cyclooctene, heterocycloalkyl or heterocycloalkenyl group is optionally substituted at any position by one or more R A1 replace.

[0391] In some embodiments, Ring A is a cyclooctene, a maleimide, or an 8-membered heterocyclen containing exactly one heteroatom selected from N, O, and S, each cyclooctene or heterocyclen optionally substituted by one or more R A1 replace.

[0392] In some embodiments, R A1 is -H, oxo or fluorine, or two R A1 The groups are combined to form one or more fused benzene rings, preferably one or two fused benzene rings, each of which is optionally substituted with one or more -OSO3H or -SO3H.

[0393] In some embodiments, Ring A is a cyclooctene, a maleimide, or an 8-membered heterocyclen containing exactly one heteroatom selected from N, O, and S, each cyclooctene or heterocyclen optionally substituted by one or more R A1 Substitute, where R A1 is oxo or fluorine, or two R A1 The groups are combined to form one or more fused benzene rings, preferably one or two fused benzene rings.

[0394] In some embodiments, Ring A is a cyclooctene, a maleimide, or an 8-membered heterocyclen containing exactly one heteroatom selected from N, each cyclooctene or heterocyclen optionally being replaced by one or two R A1 replace.

[0395] In some embodiments, R A1 is -H, oxo or fluorine, or two R A1 Combined to form one or more fused benzene rings, preferably one or two fused benzene rings, each of which is optionally substituted by one or more R A2 replace.

[0396] In some embodiments, Ring A is a cyclooctene, a maleimide, or an 8-membered heterocyclen containing exactly one heteroatom selected from N, each cyclooctene or heterocyclen optionally being replaced by one or two R A1 Substitute, where R A1 is -H, oxo or fluorine, or two R A1 The groups are combined to form one or more fused benzene rings, preferably one or two fused benzene rings, each of which is optionally substituted with one or more -OSO3H or -SO3H.

[0397] In some preferred embodiments, ring A is a cyclooctene, a maleimide or an 8-membered heterocyclen containing exactly one heteroatom selected from N, each cyclooctene or heterocyclen optionally being replaced by one or two R A1 Substitute, where R A1 -H, or two R A1 The groups are combined to form one or more fused benzene rings, preferably one or two fused benzene rings, each of which is optionally substituted with one or more -OSO3H or -SO3H.

[0398] Preparation of linear conjugates

[0399] The conjugates of the present invention can be prepared by a variety of 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 following methods and synthetic methods known in the field of polymer chemistry or variations thereof as contemplated by those skilled in the art. These methods include, but are not limited to, the following methods. 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 alternative sequence of assembly intermediates without departing from the present invention. The conjugates of the present invention can also be synthesized using slight variations of the following steps. For example, Scheme 3 shows the use of a tetrafluorophenyl ester as an electrophilic functional group for coupling to hEGF, and those skilled in the art will recognize other suitable electrophilic functional groups that can be used for the same purpose.

[0400] In some preferred embodiments, the LPEI segment and the PEG segment are coupled via a [3+2] cycloaddition reaction 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 segment comprises an azide functional group and the PEG segment comprises an alkene or alkyne functional group.

[0401] LPEI fragment

[0402] The conjugate of the present invention may comprise an LPEI segment and a PEG segment. Linear polyethyleneimine (LPEI) has the chemical formula -[NH-CH2-CH2]-. Therefore, linear polyethyleneimine (LPEI) has the chemical formula of the repeating unit n of -[NH-CH2-CH2]-. LPEI can be synthesized according to various methods known in the art, including in particular 2-oxazoline polymerization, followed by hydrolysis of the side chain amide bond (see, for example, Brissault et al., Bioconjugate Chem., 2003, 14, 581-587). As mentioned above, the polymerization from 2-oxazoline to poly (2-oxazoline) (i.e., a suitable precursor of LPEI) can be initiated using any suitable initiator. In some embodiments, the initiator will leave an initiating residue at the α end of the poly (2-oxazoline). In a preferred embodiment, the initiating residue (i.e., R in Formula I* or Formula I) is 1 ) is a hydrogen atom or a C1-C6 alkyl group, preferably a hydrogen atom or a C1-C4 alkyl group, more preferably a hydrogen atom or a methyl group, and most preferably a hydrogen atom. In a preferred embodiment, the initiating residue R in Formula I 1 is a hydrogen atom or a C1-C6 alkyl group, preferably a hydrogen atom or a C1-C4 alkyl group, more preferably a hydrogen atom or a methyl group, and most preferably a hydrogen atom. 1 ) is -H or -CH3, most preferably -H. In a preferred embodiment, the initiating residue R in formula I* 1In a preferred embodiment, the initiating residue R in Formula I 1 In a preferred embodiment, the initiating residue R in formula I* 1 In a preferred embodiment, the initiating residue R in formula I 1 However, it will be understood by those skilled in the art that the initiator residue may be any residue left by a suitable initiator capable of initiating the polymerization of 2-oxazoline to poly(2-oxazoline).

[0403] In some embodiments, the LPEI segment and the PEG segment can be coupled via a [3+2] cycloaddition reaction between an azide and an olefin or alkyne to form a 1,2,3 triazole or a 4,5-dihydro-1H-[1,2,3] triazole, wherein the LPEI segment comprises an azide (-N3) functional group at the ω terminus of the chain. In some preferred embodiments, the LPEI segment has no substitutions other than a single substitution at the α terminus. For example, in some preferred embodiments, the LPEI segment comprises a repeating formula -[NH-CH2-CH2]- and is substituted at the ω terminus with an azide group that can couple to the alkyne or olefin substituent on the PEG segment. In some preferred embodiments, the α terminus of the LPEI segment can be substituted with a hydrogen atom or a C1-C6 alkyl group, preferably hydrogen or a C1-C4 alkyl group, more preferably hydrogen or a methyl group, and most preferably a hydrogen atom.

[0404] For example, in some preferred embodiments, the LPEI segment can be substituted at the α-terminus with a hydrogen atom or a C1-C6 alkyl group, preferably with a hydrogen atom or a C1-C4 alkyl group, more preferably with a hydrogen atom or a methyl group, and can be substituted at the ω-terminus with an azido group. In some preferred embodiments, no other substitutions are present on the LPEI segment. For example, the conjugate of the present invention can be prepared from the LPEI segment of the following formula:

[0405]

[0406] Where R 1 It can be any suitable initiating residue, preferably hydrogen or C1-C6 alkyl, preferably hydrogen or C1-C4 alkyl, more preferably hydrogen or methyl, most preferably hydrogen.

[0407] In some embodiments, the LPEI segment can be terminated with a thiol group. Thus, in some embodiments, the ω end of the LPEI segment includes a thiol group (preferably a thiol group) that can be coupled to a reactive olefin group on the PEG segment via a thiol-ene reaction. Thus, in some embodiments, the conjugate of the present invention can be prepared from an LPEI segment of the following formula:

[0408]

[0409] Where R 1 It can be any suitable initiating residue, preferably hydrogen or methyl, more preferably hydrogen.

[0410] In some embodiments, the LPEI segment can be terminated with an olefin group. Thus, in some embodiments, the ω end of the LPEI segment includes an olefin group (preferably an olefin group) that can be coupled to a reactive thiol group on a PEG segment via a thiol-ene reaction. Thus, in some embodiments, the conjugate of the present invention can be prepared from an LPEI segment of the following formula:

[0411]

[0412] Where R 1 It can be any suitable initiating residue, preferably hydrogen or methyl, more preferably hydrogen.

[0413] The LPEI segments can have a range of lengths (i.e., repeat units represented by the variable "n" above). For example, the LPEI segments can comprise from 1 to 1000 repeat units (i.e., -NH-CH2-CH2-). In some embodiments, the LPEI segments can exist as a dispersing polymer portion and do not comprise a discrete number of -NH-CH2-CH2- repeat units. For example, the LPEI segments can exist as a dispersing polymer portion having a molecular weight of about 5 to 50 KDa, preferably with a dispersity of about 5 or less, preferably about 4 or less, preferably about 3 or less, preferably about 2 or less, preferably about 1.5 or less. In some embodiments, the LPEI segments can exist as a dispersing polymer portion having a molecular weight of about 10 to 40 KDa, with a dispersity of about 4 or less, preferably about 3 or less, preferably about 2 or less, preferably about 1.5 or less. In some embodiments, the LPEI fragments may be present as a dispersing polymer portion having a molecular weight of about 12 to 30 KDa and a dispersity of about 3 or less, preferably about 2 or less, and more preferably about 1.5 or less. In some embodiments, the LPEI fragments may be present as a dispersing polymer portion having a molecular weight of about 15 to 27 KDa and a dispersity of about 2 or less, preferably about 1.5 or less. In some embodiments, the LPEI fragments may be present as a dispersing polymer portion having a molecular weight of about 17 to 25 KDa and a dispersity of about 1.2 or less.

[0414] For example, the LPEI segment may be present as a dispersing polymer portion comprising about 115 to 1150 repeating units, preferably having 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 segment may be present as a dispersing polymer portion comprising about 230 to 930 repeating units, having 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 segment may be present as a dispersing polymer portion comprising about 280 to 700 repeating units, having a dispersity of about 3 or less, preferably about 2 or less, and more preferably about 1.5 or less. In some embodiments, the LPEI segment may be present as a dispersing polymer portion comprising about 350 to 630 repeating units, having a dispersity of about 2 or less, preferably about 1.5 or less. In some embodiments, the LPEI segment may be present as a dispersing polymer portion comprising about 400 to 580 repeating units and having a dispersity of about 1.2 or less.

[0415] In some embodiments, the R 1 -(NR 2 -CH2-CH2) n - portion is a dispersing polymer portion having 115 to 1150 repeating units n and a dispersity of about 5 or less, preferably, the R 1 -(NR 2 -CH2-CH2) n - portion is a dispersing polymer portion having 280 to 700 repeating units n and a dispersity of about 3 or less, more preferably, the R 1 -(NR 2 -CH2-CH2) n - portion is a dispersing polymer portion having 350 to 630 repeating units n and a dispersity of about 2 or less, and more preferably, the R 1 -(NR 2 -CH2-CH2) n The - portion is a dispersing polymer portion having 400 to 580 repeating units n and a dispersity of about 1.2 or less.

[0416] In a preferred embodiment, the polyethyleneimine segment is a dispersing polymer portion having from about 115 to about 1150 repeating units with a dispersity of about 5 or less; preferably from about 230 to about 930 repeating units with a dispersity of about 4 or less; more preferably from about 280 to about 700 repeating units with a dispersity of about 3 or less; still more preferably from about 350 to about 630 repeating units with a dispersity of about 2 or less; and still more preferably from about 400 to about 580 repeating units with a dispersity of about 1.2 or less.

[0417] In a preferred embodiment, the polyethyleneimine segment is a dispersing polymer portion 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 segment is a dispersing polymer portion 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 segment is a dispersing polymer portion 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 segment is a dispersing polymer portion 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 segment is a dispersing polymer portion having a number of repeating units ranging from about 400 to about 580 and a dispersity of about 1.2 or less.

[0418] As described above, it will be understood by those skilled in the art that, in some embodiments, the LPEI fragments may include organic residues (i.e., side chain amide groups) that are attached to nitrogen atoms embedded in the LPEI chain. It will be understood by those skilled in the art that such organic residues (i.e., amide groups) can be formed during the ring-opening polymerization of 2-oxazoline to form poly(2-oxazoline). Without being bound by theory, LPEI can be formed by the cleavage of amide groups from poly(2-oxazoline) (e.g., using an acid such as HCl). However, in some cases, not every amide bond can be cleaved under these conditions. Therefore, in some embodiments, less than about 5% of the nitrogen atoms in the LPEI fragments may be attached to form amides with organic residues. In some embodiments, less than about 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.5%, less than about 0.4%, less than about 0.3%, less than about 0.2%, or less than about 0.1% of the nitrogen atoms in the LPEI fragments may be attached to form amides with organic residues. It will be understood by those skilled in the art that the molecular weight of the LPEI fragments includes the percentage of LPEI fragments attached to the organic residues in the form of amides. In addition, it will be understood by those skilled in the art that, although the chemical structures depicted herein show repeated -NH-CH2-CH2- fragments, trace amounts of residual organic residues, such as side chain amide groups (e.g., those defined above), may still be present in the resulting ternary conjugates or polymer complexes disclosed herein. The term "ternary conjugate" as occasionally used herein refers to the conjugates of the present invention. The prefix "ternary" is because the conjugates of the present invention comprise three parts, namely, the LPEI segment, the PEG segment, and the targeting segment.

[0419] PEG fragment

[0420] Polyethylene glycol (PEG) has the chemical formula -[O-CH2-CH2]-. Thus, polyethylene glycol (PEG) has the chemical formula of a repeating unit m of -[O-CH2-CH2]-. In some preferred embodiments, the PEG segment can be coupled with the LPEI segment 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, wherein the corresponding reactive precursor molecule comprising the PEG segment further comprises an alkene or alkyne functional group. For example, in some preferred embodiments, the reactive precursor molecule comprising the PEG segment comprises a repeating formula -[O-CH2-CH2]- and is substituted at the first end (i.e., the first terminal) with an alkene or alkyne group (e.g., via a linking portion "X" as described herein). 1 ”), the alkene or alkyne group can be coupled to the azide group of the corresponding reactive precursor molecule comprising the LPEI segment.

[0421] In some preferred embodiments, the olefin or alkyne group is an activated olefin or alkyne group that can react spontaneously with azide (e.g., without adding a catalyst, such as a copper catalyst). For example, the activated alkyne group can be incorporated into a 7-membered or 8-membered ring to produce a strained species that reacts spontaneously with the azide group of the LPEI segment. The activated olefin can include a maleimide moiety, wherein the olefin is activated by binding to an adjacent carbonyl group. In some preferred embodiments, the second end (i.e., the second terminal) of the PEG segment can be substituted (e.g., by a linking portion "X" as described herein) with a targeting fragment (e.g., hEGF, HER2, folic acid, or DUPA). 2 ”).

[0422] The PEG segments can have a range of lengths (i.e., the repeating units represented by the variable "m"). In other embodiments, the PEG segment can comprise a discrete number of repeating -O-CH2-CH2- units and is not defined by an average chain length. In a preferred embodiment, the -(O-CH2-CH2) m - is a dispersing polymer part. In a preferred embodiment, the -(O-CH2-CH2) m -part comprises, preferably consists of, a discrete number m of repeating units. In a preferred embodiment, the -(O-CH2-CH2) m The -part comprises, preferably consists of, a discrete number m of consecutive repeating units.

[0423] In some preferred embodiments, the PEG fragment is a dispersing polymer portion comprising from about 1 to about 200 repeating units, preferably from about 1 to about 200 repeating units. In some preferred embodiments, the PEG fragment may comprise from 1 to 100 repeating units (i.e., -O-CH2-CH2-). Preferably, the PEG fragment of the present invention comprises from about 1 to about 100 repeating units, from about 1 to about 90 repeating units, from about 1 to about 80 repeating units, from about 1 to about 70 repeating units, from about 1 to about 60 repeating units, from about 1 to about 50 repeating units, from about 1 to about 50 repeating units, from about 1 to about 40 repeating units, from about 1 to about 30 repeating units, or from about 1 to about 20 repeating units. In some other preferred embodiments, the PEG fragment comprises a discrete number m of repeating units, preferably 12 repeating units or 24 repeating units. In some embodiments, the polyethylene glycol segment is a dispersing polymer portion having about 2 to about 80 repeating units with a dispersity of about 2.0 or less, preferably about 1.8 or less, more preferably about 1.5 or less; preferably about 2 to about 70 repeating units with a dispersity of about 1.8 or less, preferably about 1.5 or less; 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 - portion is a dispersing polymer portion having from about 2 to about 80 repeating units and a dispersity of about 2.0 or less; preferably having from about 2 to about 70 repeating units and a dispersity of about 1.8 or less; more preferably having from about 2 to about 50 repeating units and a dispersity of about 1.5 or less.

[0424] In a preferred embodiment, the polyethylene glycol segment (PEG) comprises, preferably consists of, a discrete number m of repeating units, preferably 12 or 24 repeating units. In a preferred embodiment, the m (the -(O-CH2-CH2) m The moiety m) comprises, preferably consists of, a discrete number m of repeating units, preferably 12 or 24 repeating units.

[0425] In a preferred embodiment, the PEG fragment comprises, preferably consists of, a discrete number m of repeating units ranging from 2 to 100, preferably a discrete number m of repeating units ranging from 4 to 60. In a preferred embodiment, the PEG fragment comprises, preferably consists of, a discrete number m of repeating units ranging from 4 to 60, preferably a discrete number m of repeating units ranging from 10 to 60. In a preferred embodiment, the PEG segment comprises, preferably consists of, a discrete number m of repeating units 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 PEG segment comprises, preferably consists of, a discrete number m of repeating units of 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56 or 60. In a preferred embodiment, the PEG segment comprises, preferably consists of, a discrete number m of repeating units of 4. In a preferred embodiment, the PEG segment comprises, preferably consists of, a discrete number m of repeating units of 12. In a preferred embodiment, the PEG segment comprises, preferably consists of, a discrete number m of repeating units of 24. In a preferred embodiment, the PEG segment comprises, preferably consists of, a discrete number m of repeating units of 36.

[0426] In a preferred embodiment, the PEG fragment comprises, preferably consists of, a discrete number m of consecutive repeating units ranging from 2 to 100, preferably a discrete number m of consecutive repeating units ranging from 4 to 60. In a preferred embodiment, the PEG fragment comprises, preferably consists of, a discrete number m of consecutive repeating units ranging from 4 to 60, preferably a discrete number m of consecutive repeating units ranging from 10 to 60. In a preferred embodiment, the PEG segment comprises, preferably consists of, a discrete number m of consecutive repeating units 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 PEG segment comprises, preferably consists of, a discrete number m of consecutive repeating units of 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56 or 60. In a preferred embodiment, the PEG segment comprises, preferably consists of, a discrete number m of consecutive repeating units of 4. In a preferred embodiment, the PEG segment comprises, preferably consists of, a discrete number m of consecutive repeating units of 12. In a preferred embodiment, the PEG segment comprises, preferably consists of, a discrete number m of consecutive repeating units of 24. In a preferred embodiment, the PEG segment comprises, preferably consists of, a discrete number m of consecutive repeating units of 36.

[0427] In a preferred embodiment, the -(O-CH2-CH2) of formula I* or formula I m -part comprises, preferably consists of, a discrete number of repeating units ranging from 2 to 100, preferably a discrete number of repeating units ranging from 4 to 60. In a preferred embodiment, the -(O-CH2-CH2) m -part comprises, preferably consists of, a discrete number m of repeating units ranging from 4 to 60, preferably consists of a discrete number m of repeating units ranging from 10 to 60. In a preferred embodiment, the -(O-CH2-CH2) m -part comprises, preferably consists of, a discrete number m of repeating units 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 -part comprises, preferably consists of, a discrete number m of repeating units of 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56 or 60. In a preferred embodiment, the -(O-CH2-CH2) m -part comprises, preferably consists of, a discrete number m of repeating units of 4. In a preferred embodiment, the -(O-CH2-CH2) m -part comprises, preferably consists of, a discrete number m of repeating units of 12. In a preferred embodiment, the -(O-CH2-CH2) m -part comprises, preferably consists of, a discrete number m of repeating units of 24. In a preferred embodiment, the -(O-CH2-CH2) m The -part comprises, preferably consists of, a discrete number m of 36 repeating units.

[0428] In a preferred embodiment, the -(O-CH2-CH2) of formula I* or formula I m -part comprises, preferably consists of, a discrete number m of consecutive repeating units ranging from 2 to 100, preferably a discrete number m of consecutive repeating units ranging from 4 to 60. In a preferred embodiment, the -(O-CH2-CH2) m -part comprises, preferably consists of, a discrete number m of consecutive repeating units ranging from 4 to 60, preferably a discrete number m of consecutive repeating units ranging from 10 to 60. In a preferred embodiment, the -(O-CH2-CH2) m -part comprises, preferably consists of, a discrete number m of consecutive repeating units 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 -part comprises, preferably consists of, a discrete number m of consecutive repeating units of 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56 or 60. In a preferred embodiment, the -(O-CH2-CH2) m -part comprises, preferably consists of, a discrete number m of consecutive repeating units of 4. In a preferred embodiment, the -(O-CH2-CH2) m -part comprises, preferably consists of, a discrete number m of consecutive repeating units of 12. In a preferred embodiment, the -(O-CH2-CH2)m -part comprises, preferably consists of, a discrete number m of consecutive repeating units of 24. In a preferred embodiment, the -(O-CH2-CH2) m The -part comprises, preferably consists of, a discrete number m of 36 consecutive repeating units.

[0429] In a preferred embodiment, the PEG fragment contained in the conjugates and compositions of the present invention comprises, preferably consists of, a discrete number m of repeating units -(O-CH2-CH2)- and is not defined by an average chain length. Thus, the PEG fragment contained in the conjugates and compositions of the present invention comprises, preferably consists of, a discrete number m of repeating units -(O-CH2-CH2)- and is not defined by an average chain length, but rather has a specifically defined discrete molecular weight associated with the discrete number m of -(O-CH2-CH2)- repeating units. In a preferred embodiment, the PEG fragment comprises, preferably consists of, a discrete number m of repeating units -(O-CH2-CH2)-, typically and preferably, the discrete number (m) is between 25 and 100 discrete numbers (m), more preferably between 25 and 60 discrete numbers (m). In a preferred embodiment, the PEG fragment comprises, preferably consists of, a discrete number m of consecutive repeating -(O-CH2-CH2)- units, typically and preferably the discrete number (m) is from 25 to 100 discrete numbers (m), more preferably from 25 to 60 discrete numbers (m).

[0430] The expression "a polyethylene glycol fragment comprising a discrete number (m) of -(O-CH2-CH2)- repeating units" or "a PEG fragment comprising a discrete number (m) of -(O-CH2-CH2)- repeating units" shall mean a fragment comprising, preferably consisting of, a discrete number (generally referred to herein as the discrete number m) of -(O-CH2-CH2)- repeating units, said discrete number (m) being a discrete (i.e., specific, single defined, integer) number (m) ranging from 25 to 100, preferably from 25 to 60. Thus, the expression "a polyethylene glycol segment comprising a discrete number (m) of -(O-CH2-CH2)-repeating units" or "a PEG segment comprising a discrete number (m) of -(O-CH2-CH2)-repeating units" shall mean a segment comprising, preferably consisting of, a discrete number m of -(O-CH2-CH2)-repeating units, said discrete number (m) being a discrete (i.e., specific, singly defined, integer) number (m) ranging from 25 to 100, preferably from 25 to 60, such that the defined PEG segment comprises, preferably consists of, a discrete number m of -(O-CH2-CH2)-repeating units and is not defined by an average chain length, but rather each of them has a specifically defined discrete molecular weight. Reference herein to discrete numbers ranging from 25 to 100 shall mean any integer from 25 to 100, i.e. any integer from 25 to 100 including the integers and discrete numbers mentioned as boundaries (e.g., 25 and 100 herein). By further example, a PEG fragment comprising a discrete number (m) of -(O-CH2-CH2)- repeating units, wherein the discrete number m is 36, refers to a PEG fragment comprising a chain of -(O-CH2-CH2)- units containing exactly 36 -(O-CH2-CH2)- units. Such a chain of exactly 36 -(O-CH2-CH2)- units is abbreviated as PEG 36 . Such PEG fragments are different from "polymeric PEG fragments", "polydisperse PEG fragments" or "dispersed PEG fragments", which refer to heterogeneous mixtures of sizes and molecular weights produced due to polymer reactions, typically with a Poisson distribution (J Herzberger et al., Chem Rev, 2016, 116: 2170-2243). The PEG fragments of the present invention comprising a discrete number (m) of -(O-CH2-CH2)-repeat units are not synthesized by a polymerization process. The present invention comprises a discrete number (m) of -(O-CH2-CH2)-repeat units and is a single molecular fragment with a discrete (i.e., determined and specific) chain length. Therefore, the PEG fragments of the present invention comprising a discrete number (m) of -(O-CH2-CH2)-repeat units are single molecular fragments with a discrete (i.e., determined and specific) chain length. The PEG fragments of the present invention are not a mixture of molecular entities (e.g., molecular entities produced by random polymerization reactions). The discreteness of the discrete PEG fragments of the present invention distinguishes them from polydispersity technology.

[0431] The PEG segments of the present invention may comprise, and preferably consist of, homogeneous discrete PEG segments or heterogeneous discrete PEG segments, typically and preferably homogeneous discrete PEG segments. As used herein, the term "homogeneous discrete PEG segment" refers to a discrete PEG structure whose entire chemical backbone consists only of a continuous, contiguous, and specific discrete number of ethylene oxide units. In other words, no other functional groups are present in the homogeneous discrete PEG segment. However, the ends of the corresponding reactive precursor molecules comprising the homogeneous discrete PEG segments may and typically have functional groups to bind to the PEI segment and the targeting segment. As used herein, the term "heterogeneous discrete PEG segment" refers to a discrete PEG structure in which the basic ethylene oxide backbone comprising a discrete number of ethylene oxide units is disconnected or substituted by other functional groups or units within its structure, for example, comprising amide bonds or ester bonds or other functional units. In a preferred embodiment of the present invention, the PEG segments are homogeneous discrete PEG segments.

[0432] In some preferred embodiments, the PEG segment can be coupled to the LPEI segment 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, wherein the corresponding reactive precursor molecule comprising the PEG segment further comprises an alkene or alkyne functional group. For example, in some preferred embodiments, the reactive precursor molecule comprising the PEG segment comprises a repeating formula -[O-CH2-CH2]- and is substituted at the first end (i.e., the first terminal) with an alkene or alkyne group (e.g., via a linker "X" as described herein). 1 ”), the alkene or alkyne group can be coupled to the azide group of the corresponding reactive precursor molecule comprising the LPEI segment. In some preferred embodiments, the alkene or alkyne group is an activated alkene or alkyne group that can react spontaneously with azide (e.g., without the addition of a catalyst, such as a copper catalyst). For example, the activated alkyne group can be incorporated into a 7-membered or 8-membered ring, thereby generating a strained species that reacts spontaneously with the azide group of the LPEI segment.

[0433] The PEG segments included in the conjugates and compositions of the present invention comprise, and preferably consist of, a discrete number m of repeating -O-CH2-CH2- units and are not defined by an average chain length as is the case with polymeric PEG segments. In a preferred embodiment, the -(O-CH2-CH2) m -unit comprises, preferably consists of, a discrete number m of repeating units. In a preferred embodiment, the -(O-CH2-CH2) m - unit comprises, preferably consists of, a discrete number m of consecutively repeating units.

[0434] In a preferred embodiment, the PEG fragment comprises, preferably consists of, a discrete number m of repeating units ranging from 25 to 100, preferably a discrete number m of repeating units ranging from 25 to 60. In a preferred embodiment, the PEG fragment comprises, preferably consists of, a discrete number m of repeating units ranging from 25 to 60, preferably a discrete number m of repeating units ranging from 30 to 50. In a preferred embodiment, the PEG fragment comprises, preferably consists of, a discrete number m of repeating units ranging from 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. WO2004 / 073620 and WO2013 / 033476 describe compounds comprising or consisting of a discrete number of repeating -(O-CH2-CH2) m -units and thus the synthesis of discrete PEGs. In preferred embodiments, the PEG segment comprises, preferably consists of, a discrete number m of repeating units of 28, 32, 36, 40, 44, 48, 52, 56 or 60. In preferred embodiments, the PEG segment comprises, preferably consists of, a discrete number m of repeating units of 28. In preferred embodiments, the PEG segment comprises, preferably consists of, a discrete number m of repeating units of 32. In preferred embodiments, the PEG segment comprises, preferably consists of, a discrete number m of repeating units of 36. In preferred embodiments, the PEG segment comprises, preferably consists of, a discrete number m of repeating units of 40. In preferred embodiments, the PEG segment comprises, preferably consists of, a discrete number m of repeating units of 44. In preferred embodiments, the PEG segment comprises, preferably consists of, a discrete number m of repeating units of 48.

[0435] In a preferred embodiment, the PEG fragment comprises, preferably consists of, a discrete number m of consecutive repeating units ranging from 25 to 100, preferably a discrete number m of consecutive repeating units ranging from 25 to 60. In a preferred embodiment, the PEG fragment comprises, preferably consists of, a discrete number m of consecutive repeating units ranging from 25 to 60, preferably a discrete number m of consecutive repeating units ranging from 30 to 50. In a preferred embodiment, the PEG fragment comprises, preferably consists of, a discrete number m of consecutive repeating units ranging from 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 PEG segment comprises, preferably consists of, a discrete number m of consecutive repeating units of 28, 32, 36, 40, 44, 48, 52, 56 or 60. In a preferred embodiment, the PEG segment comprises, preferably consists of, a discrete number m of consecutive repeating units of 28. In a preferred embodiment, the PEG segment comprises, preferably consists of, a discrete number m of consecutive repeating units of 32. In a preferred embodiment, the PEG segment comprises, preferably consists of, a discrete number m of consecutive repeating units of 36. In a preferred embodiment, the PEG segment comprises, preferably consists of, a discrete number m of consecutive repeating units of 40. In a preferred embodiment, the PEG segment comprises, preferably consists of, a discrete number m of consecutive repeating units of 44. In a preferred embodiment, the PEG segment comprises, preferably consists of, a discrete number m of consecutive repeating units of 48.

[0436] In a preferred embodiment, the -(O-CH2-CH2) of Formula I* or Formula I m -part consists of a discrete number m of repeating units ranging from 25 to 100, preferably a discrete number m of repeating units ranging from 25 to 60. In a preferred embodiment, the -(O-CH2-CH2) m -part consists of a discrete number m of repeating units ranging from 25 to 60, preferably a discrete number m of repeating units ranging from 30 to 50. In a preferred embodiment, the -(O-CH2-CH2) m - portion consists of a discrete number m of repeating units 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-CH2-CH2) m-part is composed of a discrete number m of repeating units of 28, 32, 36, 40, 44, 48, 52, 56 or 60. In a preferred embodiment, the -(O-CH2-CH2) m -part consists of a discrete number m of repeating units of 28. In a preferred embodiment, the -(O-CH2-CH2) m -part consists of a discrete number m of repeating units of 32. In a preferred embodiment, the -(O-CH2-CH2) m -part consists of a discrete number m of repeating units of 36. In a preferred embodiment, the -(O-CH2-CH2) m -part consists of a discrete number m of repeating units of 40. In a preferred embodiment, the -(O-CH2-CH2) m -part consists of a discrete number m of repeating units of 44. In a preferred embodiment, the -(O-CH2-CH2) m The -part consists of a discrete number m of 48 repeating units.

[0437] In a preferred embodiment, the -(O-CH2-CH2) of Formula I* or Formula I m -part consists of a discrete number m of continuous repeating units ranging from 25 to 100, preferably a discrete number m of continuous repeating units ranging from 25 to 60. In a preferred embodiment, the -(O-CH2-CH2) m -part consists of a discrete number m of continuous repeating units ranging from 25 to 60, preferably a discrete number m of continuous repeating units ranging from 30 to 50. In a preferred embodiment, the -(O-CH2-CH2) m - portion consists of a discrete number m of consecutive repeating units 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-CH2-CH2) m -part consists of a discrete number m of consecutive repeating units of 28, 32, 36, 40, 44, 48, 52, 56 or 60. In a preferred embodiment, the -(O-CH2-CH2) m -part consists of a discrete number m of consecutive repeating units of 28. In a preferred embodiment, the -(O-CH2-CH2) m -part consists of a discrete number m of consecutive repeating units of 32. In a preferred embodiment, the -(O-CH2-CH2) m-part consists of a discrete number m of consecutive repeating units of 36. In a preferred embodiment, the -(O-CH2-CH2) m -part consists of a discrete number m of consecutive repeating units of 40. In a preferred embodiment, the -(O-CH2-CH2) m -part consists of a discrete number m of consecutive repeating units of 44. In a preferred embodiment, the -(O-CH2-CH2) m The -part consists of a discrete number m of 48 consecutive repeating units.

[0438] In another aspect, the present invention provides a composition comprising a polymer complex comprising a conjugate of Formula I or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or enantiomer thereof and a nucleic acid, preferably the nucleic acid is non-covalently bound to the conjugate:

[0439]

[0440] Where:

[0441] is a single bond or a double bond;

[0442] n is any integer from 1 to 1500, preferably any integer from 2 to 1500;

[0443] m is a discrete number of repeating units ranging from 2 to 100, preferably a discrete number of repeating units ranging from 4 to 60, preferably, the discrete number m is a discrete number of consecutive repeating units of -(O-CH2-CH2)-, and the discrete number of consecutive repeating units of -(O-CH2-CH2)- is any discrete number ranging from 2 to 100, preferably from 4 to 60;

[0444] R 1 is an initiating residue, preferably, R 1 is -H or -CH3;

[0445] R 2 are independently -H or an organic residue, the -(NR 2 -CH2-CH2) n - at least 80%, preferably at least 90% of R 2 is H;

[0446] Ring A is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl group, optionally substituted at any position with one or more R A1 Replacement; R A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo or halogen; or, two R A1Together with the atoms to which they are attached, they can form more than one fused C6-C 10 aryl, C5-C6 heteroaryl or C3-C6 cycloalkyl ring, wherein each fused aryl, heteroaryl or cycloalkyl is optionally substituted by one or more R A2 Replacement; R A2 Independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, -SO3H or -OSO3H;

[0447] X 1 is a divalent covalent linking moiety;

[0448] X 2 is a divalent covalent linking moiety; and

[0449] L is a targeting fragment, preferably, the targeting fragment can bind to a cell, more preferably, the targeting fragment can bind to a cell surface receptor; and

[0450] The nucleic acid is a nucleic acid with pharmaceutical activity, and the nucleic acid with pharmaceutical activity is a nucleic acid encoding a peptide or protein with pharmaceutical activity. 1 In a preferred embodiment, the R 1 is -CH3.

[0451] In another aspect, the present invention provides a polymer complex comprising a conjugate of Formula I or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or enantiomer thereof and a nucleic acid, preferably the nucleic acid is non-covalently bound to the conjugate:

[0452]

[0453] Where:

[0454] is a single bond or a double bond;

[0455] n is any integer from 1 to 1500, preferably any integer from 2 to 1500;

[0456] m is a discrete number of repeating units ranging from 2 to 100, preferably a discrete number of repeating units ranging from 4 to 60, preferably, the discrete number m is a discrete number of consecutive repeating units of -(O-CH2-CH2)-, and the discrete number of consecutive repeating units of -(O-CH2-CH2)- is any discrete number ranging from 2 to 100, preferably from 4 to 60;

[0457] R 1 is an initiating residue, preferably, R 1 is -H or -CH3;

[0458] R2 are independently -H or an organic residue, the -(NR 2 -CH2-CH2) n - at least 80%, preferably at least 90% of R 2 is H;

[0459] Ring A is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl group, optionally substituted at any position with one or more R A1 Replacement; R A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo or halogen; or, two R A1 Together with the atoms to which they are attached, they can form more than one fused C6-C 10 aryl, C5-C6 heteroaryl or C3-C6 cycloalkyl ring, wherein each fused aryl, heteroaryl or cycloalkyl is optionally substituted by one or more R A2 Replacement; R A2 Independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, -SO3H or -OSO3H;

[0460] X 1 is a divalent covalent linking moiety;

[0461] X 2 is a divalent covalent linking moiety; and

[0462] L is a targeting fragment, preferably, the targeting fragment can bind to a cell, more preferably, the targeting fragment can bind to a cell surface receptor; and

[0463] The nucleic acid is a nucleic acid with pharmaceutical activity, and the nucleic acid with pharmaceutical activity is a nucleic acid encoding a peptide or protein with pharmaceutical activity. 1 In a preferred embodiment, the R 1 is -CH3.

[0464] In some preferred embodiments, the conjugates of the present invention comprise LPEI segments present as dispersing polymer moieties, wherein n is from about 280 to about 700 and has a dispersity of about 3 or less; preferably n is from about 350 to about 630 and has a dispersity of about 2 or less; more preferably n is from about 400 to 580 and has a dispersity of about 1.2 or less. The conjugates of the present invention further comprise PEG segments present as follows: (i) as dispersing polymer moieties, wherein m is from about 2 to about 80 and has a dispersity of about 2 or less; preferably m is from about 2 to about 70 and has a dispersity of about 1.8 or less; still more preferably m is from about 2 to about 50 repeating units and has a dispersity of about 1.5; or (ii) as a discrete number m of repeating units, preferably, the discrete number m of repeating units is 12 or 24 repeating units.

[0465] In some embodiments, the conjugates of the present invention comprise an LPEI segment present as a dispersing polymer portion having a molecular weight of about 17 to 25 kDa and a dispersity of about 1.2 or less, and a PEG segment comprising, preferably consisting of, 12 repeating units. In some preferred embodiments, the conjugates of the present invention may comprise an LPEI segment present as a dispersing polymer portion having a molecular weight of about 17 to 25 kDa and a dispersity of about 1.2 or less, and a PEG segment preferably consisting of 24 repeating units.

[0466] Targeting fragment

[0467] The conjugates of the present invention comprise a targeting segment that can direct the conjugates of the present invention and the polymer complexes of the present invention to a specific target cell type, cell collection, organ or tissue. Typically and preferably, the targeting segment is capable of binding to a target cell, preferably to a cell receptor or cell surface receptor thereof.

[0468] As used herein, the term "cell surface receptor" refers to a protein, glycoprotein, or lipoprotein present on the cell surface, which is typically and preferably a unique marker for identifying the cell. Typically and preferably, the cell surface receptor is capable of binding to a ligand, which includes 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.

[0469] The conjugates and polymer complexes comprising the targeting fragments of the present invention are intended to simulate such ligand-receptor interactions. Therefore, 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-bound integral membrane proteins, asialoglycoprotein receptors (ASGPr), lectins, ion channels, G protein-coupled receptors, and enzyme-linked receptors (e.g., tyrosine kinase-coupled receptors).

[0470] 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, and enzyme-linked receptors (e.g., tyrosine kinase-coupled receptors). 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.

[0471] 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).

[0472] The targeting fragment according to the present invention is intended to locate and deliver (particularly selectively deliver) the polymer complex of the present invention and a payload (e.g., a nucleic acid) to a desired target, particularly a desired target cell. In addition, the conjugate of the present invention comprising the targeting fragment is not only capable of selectively delivering the conjugate and the polymer complex to a target (e.g., a target cell), but is also capable of internalizing and promoting selective cellular uptake of the polyanionic payload and the nucleic acid payload, respectively, by the target (particularly a target cell). Thus, the targeting fragment according to the present invention represents a portion of the conjugate and polymer complex of the present invention that is capable of specifically binding to a selected target, preferably to a selected target cell, and more preferably to a cell receptor.

[0473] In a preferred embodiment, the targeting fragment is capable of binding to a target cell. In a preferred embodiment, the targeting fragment is capable of binding to a selected target cell type. In a preferred embodiment, the targeting fragment is capable of binding to a target cell receptor. In a preferred embodiment, the targeting fragment is capable of binding to a target cell surface receptor.

[0474] In a preferred embodiment, the targeting fragment is used to bind to a target cell. In a preferred embodiment, the targeting fragment is used to bind to a selected target cell type. In a preferred embodiment, the targeting fragment is used to bind to a target cell receptor. In a preferred embodiment, the targeting fragment is used to bind to a target cell surface receptor.

[0475] In a preferred embodiment, the targeting fragment can specifically bind to a target cell. In a preferred embodiment, the targeting fragment can specifically bind to a selected target cell type. In a preferred embodiment, the targeting fragment can specifically bind to a target cell receptor. In a preferred embodiment, the targeting fragment can specifically bind to a target cell surface receptor.

[0476] In one embodiment, the specific binding to a target cell, a target cell receptor or a target cell surface receptor refers to the binding strength of the targeting fragment, the conjugate of the present invention and / or the polymer complex of the present invention to the target cell, the target cell receptor or the target cell surface receptor, respectively, which is at least two times, preferably at least three times, more preferably at least four times, more preferably at least five times, of the binding strength to other non-target cells, cell receptors or cell surface receptors, typically and preferably with a dissociation constant (K D ) is measured. Preferably, the K of the targeting fragment binding to the selected cell surface receptor is D Less than 10 -5 M, preferably less than 10 - 6 M, more preferably less than 10 -7 M, still more preferably less than 10 -8 M.

[0477] In one embodiment, the specific binding to a target cell, a target cell receptor or a target cell surface receptor refers to a binding strength of the targeting fragment, the conjugate of the present invention and / or the polymer complex of the present invention to the target cell, the target cell receptor or the target cell surface receptor, respectively, that is at least two times, preferably at least three times, more preferably at least five times, more preferably at least ten times, more preferably at least one hundred times that of the corresponding conjugate and / or polymer complex (the corresponding conjugate and / or polymer complex is the same as the conjugate of the present invention and / or the polymer complex of the present invention, but contains a non-specific fragment, such as a hydroxyl group or an -OMe moiety, preferably an -OMe moiety, instead of the targeting fragment). Binding to a target cell, a target cell receptor or a target cell surface receptor is typically and preferably determined by a dissociation constant (K D ) is measured. Preferably, the K of the targeting fragment binding to the selected target cell surface receptor is D Less than 10 -5 M, preferably less than 10 -6 M, more preferably less than 10 -7 M, still more preferably less than 10 -8 In a preferred embodiment, the binding or the specific binding and thus the binding level of the conjugates according to the invention and the polymer complexes according to the invention, respectively, can be determined by a binding assay or a displacement assay or by FRET or other measurement methods indicating an interaction between a targeting moiety and a cell receptor, preferably a cell surface receptor.

[0478] The term "binding" as used herein with respect to the binding of a targeting fragment to a cell, a cell receptor or a cell surface receptor preferably refers to interactions through non-covalent binding, such as electrostatic interactions, van der Waals interactions, hydrogen bonds, hydrophobic interactions, ionic bonds, charge interactions, affinity interactions and / or dipole-dipole interactions.

[0479] In another embodiment, specific binding to a target cell, a target cell receptor, or a target cell surface receptor results in a biological effect caused by said specific binding of the targeting fragment and the conjugate of the invention and / or the polymer complex of the invention, respectively, and / or caused by the delivered conjugate and / or polymer complex of the invention and the polyanionic payload and the nucleic acid payload, respectively, that is at least 2-fold, preferably at least 3-fold, more preferably at least 5-fold, still more preferably at least 10-fold, yet more preferably at least 25-fold, at least 50-fold, or at least 100-fold greater than the biological effect on a non-target cell, a non-target cell receptor, or a non-target cell surface receptor.

[0480] In another embodiment, specific binding to a target cell, a target cell receptor, or a target cell surface receptor results in a biological effect caused by said specific binding of the targeting moiety and the conjugate and / or polymer complex of the invention, respectively, and / or caused by the delivered conjugate and / or polymer complex of the invention and the polyanionic payload and the nucleic acid payload, respectively, that is at least 2 times, preferably at least 3 times, more preferably at least 5 times, even more preferably at least 10 times, even more preferably at least 25 times, at least 50 times, or at least 100 times greater than the biological effect caused by the corresponding conjugate and / or polymer complex that is the same as the conjugate and / or polymer complex of the invention, but comprises a non-specific moiety (e.g., a hydroxyl or -OMe moiety, preferably an -OMe moiety) in place of the targeting moiety.

[0481] Binding and specific binding can also be determined by measuring the activation of protein signaling, and thus can be measured by measuring protein phosphorylation or protein expression, mRNA expression (using Western blot analysis, real-time PCR, RNA sequencing IHC, etc.) in cells or tissues. The level of delivery of the polymer complex of the present invention to a specific tissue can be measured by comparing the amount of protein produced in overexpressing cells with that produced in normal and low-expressing cells by Western blot analysis or luminescence / fluorescence assay, flow cytometry, or by measuring protein secretion by methods such as ELISA, ECLIA, etc.; by comparing the expression or secretion of downstream proteins (from the delivered nucleic acid, such as poly IC) in cells / tissues overexpressing the target receptor with normal cells / tissues or low-expressing cells / tissues by Western blot analysis or luminescence / fluorescence assay, flow cytometry, or by measuring protein secretion by methods such as ELISA, ECLIA, etc. Delivery levels can also be measured by cytotoxicity, which is measured using cell viability assays or cell death assays, including MTT, methylene blue assay, CellTiter-Glo assay, propidium iodide assay. By comparing the amount of protein produced in a tissue to the weight of the tissue, the amount of the therapeutic and / or prophylactic agent in a tissue to the weight of the tissue, the amount of protein produced in a tissue to the amount of total protein in the tissue, or the amount of the therapeutic and / or prophylactic agent in a tissue to the amount of the total therapeutic and / or prophylactic agent in the tissue. It should be understood that delivery of the polymer complexes of the present invention to target cells or target tissues need not be determined in a subject undergoing treatment, but can be determined in alternative models such as animal models or cell models.

[0482] Thus, in a preferred embodiment, the biological effect is selected from the group consisting of: (i) activation of protein signaling, (ii) protein expression, (iii) mRNA expression in cells or tissues, (iv) expression or secretion of downstream proteins from the delivered nucleic acid (e.g., the delivered nucleic acid in cells / tissues that overexpress the target cell surface receptor compared to normal cells / tissues or underexpressing cells / tissues), and (v) cytotoxicity.

[0483] 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 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 the 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 cells are T cells. In one embodiment, the target cells are B cells. In one embodiment, the target cells are NK cells. In one embodiment, the target cell is a monocyte.

[0484] In some embodiments, the targeting fragment selectively or preferentially interacts with a specific cell type. The targeting fragment is not only used to selectively target the conjugate of the present invention and polymer complex to specific cells, but also generally helps specific cell types to selectively absorb the conjugate of the present invention and the corresponding polymer complex. In some embodiments, the targeting fragment selectively or preferentially interacts with a specific cell surface receptor. When the targeting fragment of the conjugate and / or polymer complex selectively or preferentially interacts with a cell surface receptor, the conjugate and / or polymer complex can be selectively or preferentially absorbed into a cell with the cell surface receptor.

[0485] In a preferred embodiment, the targeting fragment is a peptide, a protein, a small molecule ligand, a sugar, an oligosaccharide, a lipid, or an amino acid, wherein the peptide, the protein, the small molecule ligand, the sugar, the oligosaccharide, the lipid, or the amino acid is selected from hormones, neurotransmitters, cytokines, growth factors, cell adhesion molecules, or nutrients, and the targeting fragment is an antibody, an antibody fragment, an aptamer, or an affibody.

[0486] As used herein, the term "small molecule ligand", particularly when referring to the targeting fragment of the present invention, refers to a chemical moiety having a molecular weight of at least 75 g / mol, preferably at least 100 g / mol, more preferably at least 200 g / mol, and preferably a molecular weight of less than about 2000 g / mol. In some embodiments, the molecular weight of the small molecule is less than about 1500 g / mol, more preferably less than about 1000 g / mol. In another preferred embodiment, the molecular weight of the small molecule is less than about 800 g / mol, more preferably less than about 500 g / mol. As used herein, the term "small molecule ligand", particularly when referring to the targeting fragment of the present invention, also preferably refers to a ligand that is capable of binding (preferably specifically binding) 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, more preferably at least 200 g / mol, and preferably a molecular weight of less than about 2000 g / mol, preferably less than about 1500 g / mol. In a preferred embodiment, 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, and is capable of binding (preferably specifically binding) to a target cell surface receptor.

[0487] In some embodiments, the targeting fragment is a natural, natural or modified ligand or its horizontal homologue (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 a preferred embodiment, the targeting fragment is a natural, natural or modified cell surface antigen ligand or its horizontal homologue, 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 a preferred embodiment, the targeting fragment is a natural, natural or modified cell surface receptor ligand or its horizontal homologue, 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 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 its horizontal homologue. 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 its horizontal homologue, 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 its horizontal homologue, 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, a peptide, a protein, an aptamer, a natural, native or modified ligand and / or its horizontal homolog, an antibody, a single chain variable fragment (scFv) or an antibody mimetic (e.g., an affibody).

[0488] In a preferred embodiment, the targeting fragment is a small molecule ligand, a peptide, a protein, an aptamer, a natural, native or modified cell surface receptor ligand and / or a transverse homologue thereof. In a preferred embodiment, the targeting fragment is a small molecule ligand, a peptide, a protein, an aptamer, a natural, native or modified ligand and / or a transverse homologue thereof, wherein the small molecule ligand, the peptide, the protein, the aptamer, the natural, native or modified ligand and / or a transverse homologue thereof is capable of binding, preferably selectively binding, 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, wherein the small molecule ligand is capable of binding, preferably selectively binding, 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, wherein the peptide is capable of binding, preferably selectively binding, 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, wherein the protein is capable of binding, preferably selectively binding, 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, wherein the aptamer can bind to, preferably selectively bind to, a cell surface receptor. In a preferred embodiment, the targeting fragment is a natural, natural or modified ligand and / or its horizontal homologue, preferably a natural, natural or modified cell surface receptor ligand and / or its horizontal homologue. In a preferred embodiment, the targeting fragment is a natural, natural or modified ligand and / or its horizontal homologue, wherein the natural, natural or modified ligand and / or its horizontal homologue can bind to, preferably selectively bind to, a cell surface receptor. In a preferred embodiment, the targeting fragment is an antibody, a single chain variable fragment (scFv) or an antibody mimic (e.g., an affibody). In a preferred embodiment, the targeting fragment is an antibody, a single chain variable fragment (scFv) or an antibody mimic (e.g., an affibody), wherein the antibody, a single chain variable fragment (scFv) or an antibody mimic (e.g., an affibody) can bind to, preferably selectively bind to, a cell surface receptor.

[0489] In a preferred embodiment, the targeting moiety 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 selected from affibodies, nanobodies, diabodies, designed ankyrin repeat proteins (DARPins)), a growth factor or a functional fragment thereof (preferably 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, an asialoorosomucoid, mannose-6-phosphate, mannose, sialyl Lewis X, N-acetyllactosamine, galactose, a lysosomal chemoattractant and / or a nuclear localizer (preferably a T antigen), a tumor low pH inserting peptide (PHLIP), a p32 targeting peptide (preferably LyP-1 tumor homing peptide), insulin-like growth factor 1, vascular endothelial growth factor, platelet-derived growth factor and / or fibroblast growth factor.

[0490] 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, such as an affibody, a nanobody, a diabody, a designed ankyrin repeat protein (DARPin), or other antibody variants). In some embodiments, the targeting fragment is a growth factor or a fragment thereof, preferably a functional fragment thereof (e.g., hEGF), a hormone or a fragment thereof, preferably a functional fragment thereof (e.g., insulin), asialo-seromucoid, mannose-6-phosphate, mannose, sialylated Lewis X, N-acetyllactosamine, galactose, a lysosomal chemoattractant and / or a nuclear localizer (e.g., T antigen), a tumor low pH insert peptide (PHLIP), a p32 targeting peptide (e.g., LyP-1 tumor homing peptide), insulin-like growth factor 1, vascular endothelial growth factor, platelet-derived growth factor, and / or fibroblast growth factor. Other non-limiting examples of targeting fragments include enzymes, nucleic acids, fatty acids, carbohydrates, monosaccharides, oligosaccharides or polysaccharides, peptidoglycans, and glycopeptides.

[0491] 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 fragment), an antibody mimetic (preferably selected from affibodies, nanobodies, diabodies, designed ankyrin repeat proteins (DARPin)), a growth factor or a functional fragment thereof (preferably hEGF), a hormone or a functional fragment thereof (preferably insulin), a cytokine or a functional fragment thereof, 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, asialo-seromucoid, mannose-6-phosphate, mannose, sialyl Lewis X, N-acetyllactosamine, galactose, a lysosomal chemoattractant and / or a nuclear localizer (preferably a T antigen), a tumor low pH inserting peptide (PHLIP), a p32 targeting peptide (preferably LyP-1 tumor homing peptide), insulin-like growth factor 1, vascular endothelial growth factor, platelet-derived growth factor and / or fibroblast growth factor.

[0492] In some embodiments, the targeting fragment L is selected from: hEGF; anti-HER2 peptide, preferably anti-HER2 antibody or affinity body; DUPA; folate receptor targeting fragment, folic acid; somatostatin receptor targeting fragment, preferably somatostatin and / or octreotide; integrin targeting fragment, preferably arginine-glycine-aspartic acid (RGD) containing fragment; low pH insertion peptide; asialoglycoprotein receptor targeting fragment, preferably asialo serum mucin; insulin receptor targeting fragment, preferably insulin; mannose-6-phosphate receptor targeting fragment, preferably mannose-6-phosphate. phosphate; a mannose receptor targeting fragment, preferably mannose; a sialyl Lewis X 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 LyP-1 tumor homing peptide that binds to p32; 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.

[0493] In some embodiments, the targeting fragment L is selected from: a targeting fragment derived from hEGF; an anti-HER2 peptide, preferably an anti-HER2 antibody or affinity body; DUPA; folic acid; a somatostatin receptor targeting fragment, preferably somatostatin and / or octreotide; an integrin targeting fragment, preferably a fragment containing arginine-glycine-aspartic acid (RGD); a low pH insertion peptide; an asialoglycoprotein receptor targeting fragment, preferably an asialo-seromucoid; an insulin receptor targeting fragment, preferably insulin; a mannose-6-phosphate receptor targeting fragment, preferably mannose-6-phosphate. acid; a mannose receptor targeting fragment, preferably mannose; a sialyl Lewis X 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 LyP-1 tumor homing peptide that binds to p32; 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.

[0494] In a preferred embodiment, the targeting fragment is selected from the group consisting of: EGFR targeting fragment; PSMA targeting fragment; anti-HER2 peptide, preferably anti-HER2 antibody or affibody; folic acid; somatostatin receptor targeting fragment, preferably somatostatin and / or octreotide; integrin targeting fragment, preferably arginine-glycine-aspartic acid (RGD) containing fragment; low pH inserting peptide; asialoglycoprotein receptor targeting fragment, preferably asialo-seromucoid; insulin receptor targeting fragment, preferably insulin; mannose-6-phosphate receptor targeting fragment, preferably mannose-6-phosphate; acid; a mannose receptor targeting fragment, preferably mannose; a sialyl Lewis X 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 LyP-1 tumor homing peptide that binds to p32; 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.

[0495] In a preferred embodiment, the targeting fragment is an epidermal growth factor, such as human epidermal growth factor (hEGF); typically and preferably, the conjugation to the rest of the conjugate is achieved through an amino group of hEGF. hEGF can be selectively taken up by cells that have increased expression (e.g., overexpression) of the human epidermal growth factor receptor (EGFR).

[0496] In a preferred embodiment, the targeting fragment is capable of binding to epidermal growth factor receptor (EGFR), which is also referred to herein as an EGFR targeting fragment.

[0497] EGFR is a transmembrane glycoprotein belonging to 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, inducing receptor dimerization and tyrosine autophosphorylation, leading to cell proliferation. In a preferred embodiment, the EGFR targeting fragment is capable of binding to an epitope on the extracellular domain of EGFR.

[0498] 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 that overexpress EGFR. In one embodiment, cells that overexpress EGFR refer to cells that have increased expression of EGFR in a particular tissue compared to the EGFR levels measured in normal, healthy cells of the same type of tissue under similar conditions. In one embodiment, cells that overexpress EGFR refer to cells in which the EGFR levels are increased relative to the EGFR levels in the same cells or closely related non-malignant cells under normal physiological conditions. In one embodiment, cells that overexpress EGFR refer to cells in which the EGFR expression is increased by at least 10 times, more preferably by at least 20 times, compared to the EGFR expression in normal cells or normal tissues.

[0499] In a preferred embodiment, the targeting fragment can bind to cells expressing or overexpressing EGFR. For example, EGFR is overexpressed in tumor tissues and cancer types, such as gliomas and epithelial carcinomas or carcinomas derived from epithelium, including head and neck cancer, thyroid cancer, breast cancer, ovarian cancer, colon cancer, gastric colorectal cancer, gastric small intestine cancer, cervical cancer, bladder cancer, lung cancer, nasopharyngeal and esophageal tissue cancer, such as squamous cell carcinoma (for example, Gan et al., J Cell Mol Med. 2009 September; 13 (9b): 3993-4001; Aratani et al., Anticancer Research 2017 June, 37 (6) 3129-3135), particularly gliomas, non-small cell lung cancer, breast cancer, glioblastoma, squamous cell carcinoma (head and neck squamous cell carcinoma), small intestine cancer, colorectal cancer, adenocarcinoma, ovarian cancer, bladder cancer or prostate cancer and their metastatic cancers.

[0500] The expression and overexpression of EGFR are preferably detected using monoclonal antibodies targeting EGFR, for example, by immunohistochemistry (e.g., "Kriegs et al., Nature, 2019, 9: 13564", "Prenzel et al., Endocr Relat Cancer 8, 11-31, 2001"). A cutoff value 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 <5% positive cells can be considered negative.

[0501] In a preferred embodiment, the targeting fragment is capable of specifically binding to EGFR. Typically, specific binding refers to the binding affinity or dissociation constant K of the targeting fragment. D In about 1×10 -3 M is about 1×10 -12 In a preferred embodiment, the targeting fragment is capable of specifically binding to EGFR, typically and preferably, the affinity or specific binding is measured by the dissociation constant (K D ) is measured, and the affinity or specific binding is referred to as K D Less than 10 -3 M, preferably less than 10 -4 M, more preferably less than 10 - 5 M, more preferably less than 10 -6 M, more preferably less than 10 -7 M, still more preferably less than 10 -8 M, still more preferably less than 10 -9 In preferred embodiments, the targeting fragment is capable of specifically binding to EGFR, typically and preferably, with affinity or specific binding measured by the dissociation constant (K D ) is measured, the specific binding refers to K D Less than 10 -3 M, less than 1 0-4 M, less than 10 -5 M, less than 10 -6 M, less than 10 -7 M, less than 10 -8 M and less than 10 -9 M. To detect binding of the complex or measure affinity, the molecule can be analyzed using a competitive binding assay, typically and preferably, for example, using a Biacore 3000 instrument (Biacore Inc., Piscataway, New Jersey; e.g., as described in "Wei-Ting Kuo et al., PLoS One. 2015, 10(2): e0116610" or US2017224620A1). Preferably, binding results in the formation of a complex between the EGFR targeting fragment and the EGFR, wherein the binding or complex can be detected.

[0502] 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 targeting fragment is an EGFR antibody, an EGFR affibody, an EGFR aptamer, an EGFR targeting peptide or an EGFR targeting tyrosine kinase inhibitor.

[0503] In a preferred embodiment, the targeting fragment is an EGFR targeting peptide. EGFR targeting peptides generally and preferably refer to peptide ligands of EGFR. Such peptide ligands are known to those skilled in the art and are described in, for example, US2017224620A1 and "Gent et al., 2018, Pharmaceutics 2018, 10, 2" (their disclosures are incorporated herein by reference in their entirety). EGFR targeting peptides have low immunogenicity potential and demonstrate good solid tumor tissue permeability.

[0504] In a preferred embodiment, the molecular weight of the EGFR targeting peptide is 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.

[0505] 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 shows binding specificity to EGFR (kd = 22 nM) (Ruoslahtiet et al., Adv. Mater. 2012, 24, 3747-3756; Li et al., J. Res. Commun. 2005, 19, 1978-1985). After addition of the physiological ligand EGF, GE11 moves away from EGFR, indicating its selective binding to EGFR and its receptor affinity. It is reported that GE11 has great potential to accelerate 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). Studies have shown that after treatment with GE11 polymer complexes, EGFR levels on the surface of cancer cells remain constant, indicating that the EGFR recycling process has a prolonged acceptance of the cells for circulating GE11 polymer complexes.

[0506] In a preferred embodiment, the EGFR targeting fragment comprises or preferably consists of GE11 (SEQ ID NO: 9), and is particularly useful for treating solid tumors characterized by cells overexpressing EGFR. The conjugates and polymer complexes of the present invention comprising or preferably consisting of GE11 as a targeting fragment are believed to be stable polymer complexes, ensuring that the polyanion and nucleic acid payload are not released before the polymer complex reaches its target cell.

[0507] In a preferred embodiment, the targeting fragment is an EGFR antibody. An EGFR antibody refers to an antibody that binds to EGFR. In a preferred embodiment, the EGFR antibody is a human antibody. In a preferred embodiment, the EGFR antibody is a humanized EGFR antibody. In a preferred embodiment, the EGFR antibody is a monoclonal human antibody. 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.

[0508] EGFR antibodies are known to those skilled in the art and are described, for example, in WO2008 / 105773 and WO2017 / 185662 (the disclosures of which are incorporated herein by reference in their entirety), including: Bevacizumab, Panitumumab, Cetuximab, Tomuzotuximab, Vortuximab, Zatuximab, Modotuximab, Imgatuzumab, Zalutumumab, Matuzumab, Necitumumab, Nimotuzumab, CEVIAvax EGF, EGFR clone, L8A4, E6.2, TH190DS, Pep2, Pep3, LR-DM1, P1X, YC088, ratML66, FM329, 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 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. It will be understood by those skilled in the art that any antibody capable of recognizing and / or specifically binding to EGFR can be used according to the present invention.

[0509] 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, such as neurotrophic inhibitor-1; or EGFR kinase inhibitors, such as afatinib, erlotinib, osimertinib, and gefitinib. EGFR inhibitors are known to those skilled in the art, for example, as described in WO2018078076 and US2017224620A1 (their disclosures are incorporated herein by reference in their entirety).

[0510] In a preferred embodiment, the targeting fragment is an EGFR aptamer. Preferred EGFR targeting aptamers include, but are not limited to, those disclosed in the following documents: Na Li et al. (PLoS One. 2011; 6(6):e20299), Deng-Liang Wang 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.

[0511] The term "EGFR aptamer" also includes EGFR aptamer derivatives and / or functional fragments of EGFR aptamers. In some embodiments, in the 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%, more preferably 95%, and most preferably 99% identical to the corresponding EGFR aptamer.

[0512] 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.

[0513] In a preferred embodiment, the targeting fragment is epidermal growth factor (EGF), a ligand of EGFR. Thus, in a preferred embodiment, 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 very preferred embodiment, the targeting fragment is human EGF (hEGF). In a very preferred embodiment, the targeting fragment comprises, and preferably consists of, the sequence of SEQ ID NO: 7.

[0514] In some embodiments, EGF is modified, for example, by deleting or replacing one or more amino acids or truncating EGF. Modified and / or truncated EGF molecules are disclosed, for example, in WO2019023295A1. EGF has many conserved residues in rats, mice, guinea pigs, and human species (Savage et al., J.Biol.Chem., 247:7612-7621, 1973; Carpenterh 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 residues because they form three disulfide bonds, thereby providing a conserved tertiary protein structure. Residues 7, 9, 11, 12, 13, 15, 18, 21, 24, 29, 32, 34, 36, 37, 39, 41, 46, and 47 are also conserved in all four species. Many of these residues may contribute to or provide key binding interactions with the corresponding EGFR. It has been reported that both the full-length human EGF (53 residues) and the truncated form (48 residues) produced by trypsin cleavage retain strong binding affinity and activation to EGFR (Calnan et al., 47(5):622-7, 2000; Gregory, Regul Pept, 22:217-26, 1988). Mutagenesis studies targeting various residues have been reported to correlate the effects of replacement of specific residues 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 resolved, which shows key binding interactions and identifies residues not directly involved in binding (Ogiso et al., Cell, Vol. 110, 775-787, 2002).

[0515] In another aspect, the present invention provides a composition comprising a polymer complex comprising a conjugate of Formula I or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or enantiomer thereof and a nucleic acid, preferably the nucleic acid is non-covalently bound to the conjugate:

[0516]

[0517] Where:

[0518] is a single bond or a double bond;

[0519] n is any integer from 1 to 1500, preferably any integer from 2 to 1500;

[0520] m is a discrete number of -(O-CH2-CH2)- repeating units, wherein the discrete number m of -(O-CH2-CH2)-repeating units is any discrete number from 25 to 100, preferably from 25 to 60;

[0521] R 1 is an initiating residue, preferably, R 1 is -H or -CH3;

[0522] R 2 are independently -H or an organic residue, the -(NR 2 -CH2-CH2) n - at least 80%, preferably at least 90% of R 2 is H;

[0523] Ring A is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl group, optionally substituted at any position with one or more R A1 Replacement; R A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo or halogen; or, two R A1 Together with the atoms to which they are attached, they can form more than one fused C6-C 10 aryl, C5-C6 heteroaryl or C3-C6 cycloalkyl ring, wherein each fused aryl, heteroaryl or cycloalkyl is optionally substituted by one or more R A2 Replacement; R A2 Independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, -SO3H or -OSO3H;

[0524] X 1 is a divalent covalent linking moiety;

[0525] X 2 is a divalent covalent linking moiety; and

[0526] L is a targeting fragment, and the targeting fragment can bind to epidermal growth factor receptor (EGFR). Preferably, the targeting fragment can bind to cells expressing EGFR. More preferably, the targeting fragment can bind to a cell surface receptor, and the cell surface receptor is EGFR; and

[0527] The nucleic acid is a nucleic acid with pharmaceutical activity, and the nucleic acid with pharmaceutical activity is a nucleic acid encoding a peptide or protein with pharmaceutical activity. 1In a preferred embodiment, the R 1 is -CH3.

[0528] In another aspect, the present invention provides a polymer complex comprising a conjugate of Formula I or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or enantiomer thereof:

[0529]

[0530] Where:

[0531] is a single bond or a double bond;

[0532] n is any integer from 1 to 1500, preferably any integer from 2 to 1500;

[0533] m is a discrete number of -(O-CH2-CH2)- repeating units, and the discrete number m of the -(O-CH2-CH2)- repeating units is any discrete number from 25 to 100, preferably from 25 to 60, more preferably, the discrete number m of the -(O-CH2-CH2)- repeating units is 36;

[0534] R 1 For initiating residues, preferably, R1 is -H or -CH3;

[0535] R 2 are independently -H or an organic residue, the -(NR 2 -CH2-CH2) n - at least 80%, preferably at least 90% of R 2 is H;

[0536] Ring A is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl group, optionally substituted at any position with one or more R A1 Replacement; R A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo or halogen; or, two R A1 Together with the atoms to which they are attached, they can form more than one fused C6-C 10 aryl, C5-C6 heteroaryl or C3-C6 cycloalkyl ring, wherein each fused aryl, heteroaryl or cycloalkyl is optionally substituted by one or more R A2 Replacement; R A2 Independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, -SO3H or -OSO3H;

[0537] X 1 is a divalent covalent linking moiety;

[0538] X2 is a divalent covalent linking moiety; and

[0539] L is a targeting fragment, and the targeting fragment can bind to epidermal growth factor receptor (EGFR). Preferably, the targeting fragment can bind to cells expressing EGFR. More preferably, the targeting fragment can bind to a cell surface receptor, and the cell surface receptor is EGFR; and

[0540] The nucleic acid is a nucleic acid with pharmaceutical activity, and the nucleic acid with pharmaceutical activity is a nucleic acid encoding a peptide or protein with pharmaceutical activity. 1 In a preferred embodiment, the R 1 is -CH3.

[0541] In another aspect, the present invention provides a composition comprising a polymer complex comprising a conjugate (preferably a plurality of conjugates) of Formula I or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or enantiomer thereof and a nucleic acid, preferably the nucleic acid is non-covalently bound to the conjugate:

[0542]

[0543] Where:

[0544] is a single bond or a double bond;

[0545] n is any integer from 1 to 1500, preferably any integer from 2 to 1500;

[0546] m is the discrete number of -(O-CH2-CH2)- repeating units, and the discrete number m of -(O-CH2-CH2)- repeating units is 36;

[0547] R 1 is an initiating residue, preferably, R 1 is -H or -CH3;

[0548] R 2 are independently -H or an organic residue, the -(NR 2 -CH2-CH2) n - at least 80%, preferably at least 90% of R 2 is H;

[0549] Ring A is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl group, optionally substituted at any position with one or more R A1 Replacement; R A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo or halogen; or, two R A1Together with the atoms to which they are attached, they can form more than one fused C6-C 10 aryl, C5-C6 heteroaryl or C3-C6 cycloalkyl ring, wherein each fused aryl, heteroaryl or cycloalkyl is optionally substituted by one or more R A2 Replacement; R A2 Independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, -SO3H or -OSO3H;

[0550] X 1 is a divalent covalent linking moiety;

[0551] X 2 is a divalent covalent linking moiety; and

[0552] L is a targeting fragment, and the targeting fragment can bind to epidermal growth factor receptor (EGFR). Preferably, the targeting fragment can bind to cells expressing EGFR. More preferably, the targeting fragment can bind to a cell surface receptor, and the cell surface receptor is EGFR; and

[0553] The nucleic acid is a nucleic acid with pharmaceutical activity, and the nucleic acid with pharmaceutical activity is a nucleic acid encoding a peptide or protein with pharmaceutical activity. 1 In a preferred embodiment, the R 1 is -CH3.

[0554] In another aspect, the present invention provides a polymer complex comprising a conjugate of Formula I or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or enantiomer thereof and a nucleic acid, preferably the nucleic acid is non-covalently bound to the conjugate:

[0555]

[0556] Where:

[0557] is a single bond or a double bond;

[0558] n is any integer from 1 to 1500, preferably any integer from 2 to 1500;

[0559] m is a discrete number of -(O-CH2-CH2)- repeating units, and the discrete number m of -(O-CH2-CH2)- repeating units is 36;

[0560] R 1 is an initiating residue, preferably, R 1 is -H or -CH3;

[0561] R 2are independently -H or an organic residue, the -(NR 2 -CH2-CH2) n - at least 80%, preferably at least 90% of R 2 is H;

[0562] Ring A is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl group, optionally substituted at any position with one or more R A1 Replacement; R A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo or halogen; or, two R A1 Together with the atoms to which they are attached, they can form more than one fused C6-C 10 aryl, C5-C6 heteroaryl or C3-C6 cycloalkyl ring, wherein each fused aryl, heteroaryl or cycloalkyl is optionally substituted by one or more R A2 Replacement; R A2 Independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, -SO3H or -OSO3H;

[0563] X 1 is a divalent covalent linking moiety;

[0564] X 2 is a divalent covalent linking moiety; and

[0565] L is a targeting fragment, and the targeting fragment can bind to epidermal growth factor receptor (EGFR). Preferably, the targeting fragment can bind to cells expressing EGFR. More preferably, the targeting fragment can bind to a cell surface receptor, and the cell surface receptor is EGFR.

[0566] The nucleic acid is a nucleic acid with pharmaceutical activity, and the nucleic acid with pharmaceutical activity is a nucleic acid encoding a peptide or protein with pharmaceutical activity. 1 In a preferred embodiment, the R 1 is -CH3.

[0567] In another aspect, the present invention provides a composition comprising a polymer complex comprising a conjugate (preferably a plurality of conjugates) of Formula I or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or enantiomer thereof and a nucleic acid, preferably the nucleic acid is non-covalently bound to the conjugate:

[0568]

[0569] Where:

[0570] is a single bond or a double bond;

[0571] n is any integer from 1 to 1500, preferably any integer from 2 to 1500;

[0572] m is a discrete number of 25 to 100 consecutive repeating units of -(O-CH2-CH2)-, preferably a discrete number of 25 to 60 consecutive repeating units of -(O-CH2-CH2)-;

[0573] R 1 is an initiating residue, preferably, R 1 is -H or -CH3;

[0574] R 2 are independently -H or an organic residue, the -(NR 2 -CH2-CH2) n - at least 80%, preferably at least 90% of R 2 is H;

[0575] Ring A is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl group, optionally substituted at any position with one or more R A1 Replacement; R A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo or halogen; or, two R A1 Together with the atoms to which they are attached, they can form more than one fused C6-C 10 aryl, C5-C6 heteroaryl or C3-C6 cycloalkyl ring, wherein each fused aryl, heteroaryl or cycloalkyl is optionally substituted by one or more R A2 Replacement; R A2 Independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, -SO3H or -OSO3H;

[0576] X 1 is a divalent covalent linking moiety;

[0577] X 2 is a divalent covalent linking moiety; and

[0578] L is a targeting fragment, and the targeting fragment can bind to epidermal growth factor receptor (EGFR). Preferably, the targeting fragment can bind to cells expressing EGFR. More preferably, the targeting fragment can bind to a cell surface receptor, and the cell surface receptor is EGFR.

[0579] The nucleic acid is a nucleic acid with pharmaceutical activity, and the nucleic acid with pharmaceutical activity is a nucleic acid encoding a peptide or protein with pharmaceutical activity. 1 In a preferred embodiment, the R 1is -CH3.

[0580] In another aspect, the present invention provides a polymer complex comprising a conjugate of Formula I or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or enantiomer thereof and a nucleic acid, preferably the nucleic acid is non-covalently bound to the conjugate:

[0581]

[0582] Where:

[0583] is a single bond or a double bond;

[0584] n is any integer from 1 to 1500, preferably any integer from 2 to 1500;

[0585] m is a discrete number of consecutive repeating -(O-CH2-CH2)- units from 25 to 100, preferably a discrete number of consecutive repeating -(O-CH2-CH2)- units from 25 to 60;

[0586] R 1 is an initiating residue, preferably, R 1 is -H or -CH3;

[0587] R 2 are independently -H or an organic residue, the -(NR 2 -CH2-CH2) n - at least 80%, preferably at least 90% of R 2 is H;

[0588] Ring A is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl group, optionally substituted at any position with one or more R A1 Replacement; R A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo or halogen; or, two R A1 Together with the atoms to which they are attached, they can form more than one fused C6-C 10 aryl, C5-C6 heteroaryl or C3-C6 cycloalkyl ring, wherein each fused aryl, heteroaryl or cycloalkyl is optionally substituted by one or more R A2 Replacement; R A2 Independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, -SO3H or -OSO3H;

[0589] X 1 is a divalent covalent linking moiety;

[0590] X 2 is a divalent covalent linking moiety; and

[0591] L is a targeting fragment, and the targeting fragment can bind to epidermal growth factor receptor (EGFR). Preferably, the targeting fragment can bind to cells expressing EGFR. More preferably, the targeting fragment can bind to a cell surface receptor, and the cell surface receptor is EGFR.

[0592] The nucleic acid is a nucleic acid with pharmaceutical activity, and the nucleic acid with pharmaceutical activity is a nucleic acid encoding a peptide or protein with pharmaceutical activity. 1 In a preferred embodiment, the R 1 is -CH3.

[0593] In another aspect, the present invention provides a composition comprising a polymer complex comprising a conjugate (preferably a plurality of conjugates) of Formula I or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or enantiomer thereof and a nucleic acid, preferably the nucleic acid is non-covalently bound to the conjugate:

[0594]

[0595] Where:

[0596] is a single bond or a double bond;

[0597] n is any integer from 1 to 1500, preferably any integer from 2 to 1500;

[0598] m is a discrete number of consecutive repeating units of -(O-CH2-CH2)-, said discrete number being 36;

[0599] R1 is an initiating residue, preferably, R 1 is -H or -CH3;

[0600] R 2 are independently -H or an organic residue, the -(NR 2 -CH2-CH2) n - at least 80%, preferably at least 90% of R 2 is H;

[0601] Ring A is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl group, optionally substituted at any position with one or more R A1 Replacement; R A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo or halogen; or, two R A1 Together with the atoms to which they are attached, they can form more than one fused C6-C 10aryl, C5-C6 heteroaryl or C3-C6 cycloalkyl ring, wherein each fused aryl, heteroaryl or cycloalkyl is optionally substituted by one or more R A2 Replacement; R A2 Independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, -SO3H or -OSO3H;

[0602] X 1 is a divalent covalent linking moiety;

[0603] X 2 is a divalent covalent linking moiety; and

[0604] L is a targeting fragment, and the targeting fragment can bind to epidermal growth factor receptor (EGFR). Preferably, the targeting fragment can bind to cells expressing EGFR. More preferably, the targeting fragment can bind to a cell surface receptor, and the cell surface receptor is EGFR; and

[0605] The nucleic acid is a nucleic acid with pharmaceutical activity, and the nucleic acid with pharmaceutical activity is a nucleic acid encoding a peptide or protein with pharmaceutical activity. 1 In a preferred embodiment, the R 1 is -CH3.

[0606] In another aspect, the present invention provides a polymer complex comprising a conjugate of Formula I or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or enantiomer thereof and a nucleic acid, preferably the nucleic acid is non-covalently bound to the conjugate:

[0607]

[0608] Where:

[0609] is a single bond or a double bond;

[0610] n is any integer from 1 to 1500, preferably any integer from 2 to 1500;

[0611] m is a discrete number of consecutive repeating units of -(O-CH2-CH2)-, said discrete number being 36;

[0612] R 1 is an initiating residue, preferably, R 1 is -H or -CH3;

[0613] R 2 are independently -H or an organic residue, the -(NR 2 -CH2-CH2) n- at least 80%, preferably at least 90% of R 2 is H;

[0614] Ring A is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl group, optionally substituted at any position with one or more R A1 Replacement; R A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo or halogen; or, two R A1 Together with the atoms to which they are attached, they can form more than one fused C6-C 10 aryl, C5-C6 heteroaryl or C3-C6 cycloalkyl ring, wherein each fused aryl, heteroaryl or cycloalkyl is optionally substituted by one or more R A2 Replacement; R A2 Independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, -SO3H or -OSO3H;

[0615] X 1 is a divalent covalent linking moiety;

[0616] X 2 is a divalent covalent linking moiety; and

[0617] 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 cells expressing EGFR, more preferably, the targeting fragment is capable of binding to a cell surface receptor, and the cell surface receptor is EGFR; and

[0618] The nucleic acid is a nucleic acid with pharmaceutical activity, and the nucleic acid with pharmaceutical activity is a nucleic acid encoding a peptide or protein with pharmaceutical activity. 1 In a preferred embodiment, the R 1 is -CH3.

[0619] In another aspect, the present invention provides a composition comprising a polymer complex comprising a conjugate of Formula I* or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or enantiomer thereof and a nucleic acid, preferably the nucleic acid is non-covalently bound to the conjugate:

[0620] R 1 -(NR 2 -CH2-CH2) n -ZX 1 -(O-CH2-CH2) m -X 2 -L (Formula I*);

[0621] Where:

[0622] n is any integer from 1 to 1500, preferably any integer from 2 to 1500;

[0623] m is any integer from 1 to 200, preferably any integer from 1 to 100;

[0624] R 1 is an initiating residue, preferably, R 1 is -H or -CH3;

[0625] R 2 are independently -H or an organic residue, the -(NR 2 -CH2-CH2) n - At least 80%, preferably 90% of R 2 is H;

[0626] X 1 and X 2 are independently divalent covalently linking moieties;

[0627] Z is a divalent covalent linking moiety, wherein Z is not a single bond and Z is not -NHC(O)-;

[0628] L is a targeting fragment, said targeting fragment comprising or preferably consisting of the sequence YHWYGYTPQNVI(GE11) (SEQ ID NO: 9), and

[0629] The nucleic acid is a nucleic acid having pharmaceutical activity, and the nucleic acid having pharmaceutical activity is a nucleic acid encoding a peptide or protein having pharmaceutical activity, and

[0630] Said composition preferably consists of said conjugate.

[0631] In another aspect, the present invention provides a polymer complex comprising a conjugate of Formula I* or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or enantiomer thereof and a nucleic acid, preferably the nucleic acid is non-covalently bound to the conjugate:

[0632] R 1 -(NR 2 -CH2-CH2) n -ZX 1 -(O-CH2-CH2) m -X 2 -L (Formula I*);

[0633] Where:

[0634] n is any integer from 1 to 1500, preferably any integer from 2 to 1500;

[0635] m is any integer from 1 to 200, preferably any integer from 1 to 100;

[0636] R 1 is an initiating residue, preferably, R 1 is -H or -CH3;

[0637] R 2 are independently -H or an organic residue, the -(NR 2 -CH2-CH2) n - At least 80%, preferably 90% of R 2 is H;

[0638] X 1 and X 2 are independently divalent covalently linking moieties;

[0639] Z is a divalent covalent linking moiety, wherein Z is not a single bond and Z is not -NHC(O)-;

[0640] L is a targeting fragment, said targeting fragment comprising or preferably consisting of the sequence YHWYGYTPQNVI(GE11) (SEQ ID NO: 9), and

[0641] The nucleic acid is a nucleic acid with pharmaceutical activity, and the nucleic acid with pharmaceutical activity is a nucleic acid encoding a peptide or protein with pharmaceutical activity.

[0642] In another aspect, the present invention provides a composition comprising a polymer complex comprising a conjugate of Formula I or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or enantiomer thereof and a nucleic acid, preferably the nucleic acid is non-covalently bound to the conjugate:

[0643]

[0644] Where:

[0645] is a single bond or a double bond;

[0646] n is any integer from 1 to 1500, preferably any integer from 2 to 1500;

[0647] m is any integer from 1 to 200;

[0648] R 1 is an initiating residue, preferably, R 1 is -H or -CH3;

[0649] R 2 are independently -H or an organic residue, the -(NR 2 -CH2-CH2) n- at least 80%, preferably at least 90% of R 2 is H;

[0650] Ring A is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl group, optionally substituted at any position with one or more R A1 Replacement; R A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo or halogen; or, two R A1 Together with the atoms to which they are attached, they can form more than one fused C6-C 10 aryl, C5-C6 heteroaryl or C3-C6 cycloalkyl ring, wherein each fused aryl, heteroaryl or cycloalkyl is optionally substituted by one or more R A2 Replacement; R A2 Independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, -SO3H or -OSO3H;

[0651] X 1 is a divalent covalent linking moiety;

[0652] X 2 is a divalent covalent linking moiety; and

[0653] L is a targeting fragment, said targeting fragment comprising or preferably consisting of the sequence YHWYGYTPQNVI(GE11) (SEQ ID NO: 9), and

[0654] The nucleic acid is a nucleic acid with pharmaceutical activity, and the nucleic acid with pharmaceutical activity is a nucleic acid encoding a peptide or protein with pharmaceutical activity.

[0655] In another aspect, the present invention provides a polymer complex comprising a conjugate of Formula I or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or enantiomer thereof and a nucleic acid, preferably the nucleic acid is non-covalently bound to the conjugate:

[0656]

[0657]

[0658] Where:

[0659] is a single bond or a double bond;

[0660] n is any integer from 1 to 1500, preferably any integer from 2 to 1500;

[0661] m is any integer from 1 to 200;

[0662] R 1is an initiating residue, preferably, R 1 is -H or -CH3;

[0663] R 2 are independently -H or an organic residue, the -(NR 2 -CH2-CH2) n - at least 80%, preferably at least 90% of R 2 is H;

[0664] Ring A is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl group, optionally substituted at any position with one or more R A1 Replacement; R A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo or halogen; or, two R A1 Together with the atoms to which they are attached, they can form more than one fused C6-C 10 aryl, C5-C6 heteroaryl or C3-C6 cycloalkyl ring, wherein each fused aryl, heteroaryl or cycloalkyl is optionally substituted by one or more R A2 Replacement; R A2 Independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, -SO3H or -OSO3H;

[0665] X 1 is a divalent covalent linking moiety;

[0666] X 2 is a divalent covalent linking moiety; and

[0667] L is a targeting fragment, said targeting fragment comprising or preferably consisting of the sequence YHWYGYTPQNVI(GE11) (SEQ ID NO: 9), and

[0668] The nucleic acid is a nucleic acid with pharmaceutical activity, and the nucleic acid with pharmaceutical activity is a nucleic acid encoding a peptide or protein with pharmaceutical activity.

[0669] In another aspect, the present invention provides a composition comprising a polymer complex comprising a conjugate of Formula I or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or enantiomer thereof and a nucleic acid, preferably the nucleic acid is non-covalently bound to the conjugate:

[0670]

[0671] Where:

[0672] is a single bond or a double bond;

[0673] n is any integer from 1 to 1500, preferably any integer from 2 to 1500;

[0674] m is a discrete number of repeating units ranging from 2 to 100, preferably a discrete number of repeating units ranging from 4 to 60;

[0675] R 1 is an initiating residue, preferably, R 1 is -H or -CH3;

[0676] R 2 are independently -H or an organic residue, -(NR 2 -CH2-CH2) n - at least 80%, preferably at least 90% of R 2 is H;

[0677] Ring A is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl group, optionally substituted at any position with one or more R A1 Replacement; R A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo or halogen; or, two R A1 Together with the atoms to which they are attached, they can form more than one fused C6-C 10 aryl, C5-C6 heteroaryl or C3-C6 cycloalkyl ring, wherein each fused aryl, heteroaryl or cycloalkyl is optionally substituted by one or more R A2 Replacement; R A2 Independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, -SO3H or -OSO3H;

[0678] X 1 is a divalent covalent linking moiety;

[0679] X 2 is a divalent covalent linking moiety; and

[0680] L is a targeting fragment, which is an EGFR targeting fragment. Preferably, the EGFR targeting fragment can specifically bind to cells expressing (preferably overexpressing) EGFR; and

[0681] The targeting fragment is epidermal growth factor (EGF), preferably, the targeting fragment is human EGF (hEGF), more preferably, the targeting fragment comprises the sequence of SEQ ID NO: 7, preferably consists of the sequence of SEQ ID NO: 7; and

[0682] The nucleic acid is a nucleic acid with pharmaceutical activity, and the nucleic acid with pharmaceutical activity is a nucleic acid encoding a peptide or protein with pharmaceutical activity.

[0683] In another aspect, the present invention provides a polymer complex comprising a conjugate of Formula I or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or enantiomer thereof and a nucleic acid, preferably the nucleic acid is non-covalently bound to the conjugate:

[0684]

[0685] Where:

[0686] is a single bond or a double bond;

[0687] n is any integer from 1 to 1500, preferably any integer from 2 to 1500;

[0688] m is a discrete number of consecutive repeating units ranging from 2 to 100, preferably a discrete number of consecutive repeating units ranging from 4 to 60;

[0689] R 1 is an initiating residue, preferably, R 1 is -H or -CH3;

[0690] R 2 are independently -H or an organic residue, -(NR 2 -CH2-CH2) n - at least 80%, preferably at least 90% of R 2 is H;

[0691] Ring A is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl group, optionally substituted at any position with one or more R A1 Replacement; R A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo or halogen; or, two R A1 Together with the atoms to which they are attached, they can form more than one fused C6-C 10 aryl, C5-C6 heteroaryl or C3-C6 cycloalkyl ring, wherein each fused aryl, heteroaryl or cycloalkyl is optionally substituted by one or more R A2 Replacement; R A2 Independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, -SO3H or -OSO3H;

[0692] X 1 is a divalent covalent linking moiety;

[0693] X 2 is a divalent covalent linking moiety; and

[0694] L is a targeting fragment, which is an EGFR targeting fragment. Preferably, the EGFR targeting fragment can specifically bind to cells expressing (preferably overexpressing) EGFR; and

[0695] The targeting fragment is epidermal growth factor (EGF), preferably, the targeting fragment is human EGF (hEGF), more preferably, the targeting fragment comprises the sequence of SEQ ID NO: 7, preferably consists of the sequence of SEQ ID NO: 7, and

[0696] The nucleic acid is a nucleic acid with pharmaceutical activity, and the nucleic acid with pharmaceutical activity is a nucleic acid encoding a peptide or protein with pharmaceutical activity.

[0697] In another aspect, the present invention provides a polymer complex comprising a conjugate of Formula I or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or enantiomer thereof and a nucleic acid, preferably the nucleic acid is non-covalently bound to the conjugate:

[0698]

[0699] Where:

[0700] is a single bond or a double bond;

[0701] n is any integer from 1 to 1500, preferably any integer from 2 to 1500;

[0702] m is a discrete number of repeating units ranging from 2 to 100, preferably a discrete number of repeating units ranging from 4 to 60;

[0703] R 1 is an initiating residue, preferably, R 1 is -H or -CH3;

[0704] R 2 are independently -H or an organic residue, -(NR 2 -CH2-CH2) n - at least 80%, preferably at least 90% of R 2 is H;

[0705] Ring A is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl group, optionally substituted at any position with one or more R A1 Replacement; R A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo or halogen; or, two R A1 Together with the atoms to which they are attached, they can form more than one fused C6-C 10aryl, C5-C6 heteroaryl or C3-C6 cycloalkyl ring, wherein each fused aryl, heteroaryl or cycloalkyl is optionally substituted by one or more R A2 Replacement; R A2 Independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, -SO3H or -OSO3H;

[0706] X 1 is a divalent covalent linking moiety;

[0707] X 2 is a divalent covalent linking moiety; and

[0708] L is a targeting fragment, preferably, the targeting fragment can bind to a cell, more preferably, the targeting fragment can bind to a cell surface receptor; and

[0709] The nucleic acid is a nucleic acid with pharmaceutical activity, and the nucleic acid with pharmaceutical activity is a nucleic acid encoding a peptide or protein with pharmaceutical activity.

[0710] In another aspect, the present invention provides a composition comprising a polymer complex comprising a conjugate of Formula I or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or enantiomer thereof and a nucleic acid, preferably the nucleic acid is non-covalently bound to the conjugate:

[0711]

[0712] Where:

[0713] is a single bond or a double bond;

[0714] n is any integer from 1 to 1500, preferably any integer from 2 to 1500;

[0715] m is a discrete number of consecutive repeating units ranging from 2 to 100, preferably a discrete number of consecutive repeating units ranging from 4 to 60;

[0716] R 1 is an initiating residue, preferably, R 1 is -H or -CH3;

[0717] R 2 are independently -H or an organic residue, -(NR 2 -CH2-CH2) n - at least 80%, preferably at least 90% of R 2 is H;

[0718] Ring A is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl group, optionally substituted at any position with one or more R A1Replacement; R A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo or halogen; or, two R A1 Together with the atoms to which they are attached, they can form more than one fused C6-C 10 aryl, C5-C6 heteroaryl or C3-C6 cycloalkyl ring, wherein each fused aryl, heteroaryl or cycloalkyl is optionally substituted by one or more R A2 Replacement; R A2 Independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, -SO3H or -OSO3H;

[0719] X 1 is a divalent covalent linking moiety;

[0720] X 2 is a divalent covalent linking moiety; and

[0721] L is a targeting fragment, preferably, the targeting fragment can bind to a cell, more preferably, the targeting fragment can bind to a cell surface receptor; and

[0722] The nucleic acid is a nucleic acid with pharmaceutical activity, and the nucleic acid with pharmaceutical activity is a nucleic acid encoding a peptide or protein with pharmaceutical activity.

[0723] In a preferred embodiment, the targeting fragment is capable of binding to prostate-specific membrane antigen (PSMA), which is also referred to herein as a PSMA targeting fragment.

[0724] PSMA is a multifunctional transmembrane protein that functions as a glutamate carboxypeptidase and exhibits rapid ligand-induced internalization and circulation (Liu H et al., 1998, Cancer Res 58:4055-4060). PSMA is primarily expressed in four tissues of the human body, including prostate epithelium, renal proximal tubules, jejunal brush border of the small intestine, and nervous system ganglia (Mhawech-Fauceglia et al., Histopathology 2007, 50:472-483). In a preferred embodiment, the targeting fragment is capable of binding to an epitope on the extracellular domain of PSMA.

[0725] In a preferred embodiment, the targeting fragment, preferably the PSMA targeting fragment, is capable of binding to cells expressing PSMA. In a preferred embodiment, the targeting fragment, preferably the PSMA targeting fragment, is capable of binding to cells overexpressing PSMA. For example, PSMA is overexpressed in tumor tissue and malignant prostate, particularly in prostate adenocarcinoma, relative to normal tissue; as the disease progresses to the metastatic stage, the expression level of PSMA is further upregulated (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).

[0726] In one embodiment, overexpressing PSMA refers to cells in a particular tissue expressing elevated levels of PSMA compared to levels of PSMA measured in normal, healthy cells of the same type of tissue under similar conditions. In one embodiment, overexpressing PSMA refers to cells expressing elevated levels of PSMA relative to levels in the same cells or closely related non-malignant cells under normal physiological conditions. In one embodiment, cells overexpressing PSMA refer to cells expressing PSMA at levels at least 10-fold higher than those in normal cells or normal tissues. In one embodiment, cells overexpressing PSMA refer to cells expressing PSMA at a cutoff value of 5% or more PSMA-positive cells (e.g., as described in "Mhawech-Fauceglia et al., (2007)"), which can be used to define PSMA expression in different types of tissues or cells. Thus, cells or tissues with <5% positive cells are considered negative, or PSMA expression is categorized based on its intensity and scored as 0 (no expression), 1 (low expression), 2 (moderate expression), and 3 (high expression), as described in "Hupe et al., 2018 (Hupe MC et al., Frontiers in Oncology 2018, 8(623):1-7)".

[0727] In a preferred embodiment, the targeting fragment is capable of binding to a cell that expresses or overexpresses PSMA. Cells that express PSMA typically include tumor cells, such as prostate, bladder, pancreas, lung, kidney, colon, melanoma, and sarcoma tumor cells. In a preferred embodiment, the targeting fragment is capable of binding to a cell that expresses or overexpresses PSMA, and the cell is a tumor cell, preferably a tumor cell selected from the group consisting of prostate, bladder, pancreas, lung, kidney, colon, melanoma, and sarcoma tumor cells. In a preferred embodiment, the targeting fragment is capable of binding to a cell that expresses or overexpresses PSMA, and the cell is a tumor cell, and the tumor cell is a prostate tumor cell.

[0728] In preferred embodiments, the targeting fragment is capable of specifically binding to PSMA, typically and preferably with affinity or specific binding as measured by the dissociation constant (K D ) is measured, the affinity or specific binding refers to K D Less than 10 -3 M, preferably less than 10 -4 M, more preferably less than 10 -5 M, more preferably less than 10 -6 M, more preferably less than 10 -7 M, still more preferably less than 10 -8 M, still more preferably less than 10 - 9 M, still more preferably less than 10 -10 In preferred embodiments, the targeting fragment is capable of specifically binding to PSMA, typically and preferably with an affinity or specific binding determined by a dissociation constant (K D ) is measured, the affinity or specific binding refers to K D Less than 10 -3 M, less than 10 -4 M, less than 10 -5 M, less than 10 -6 M, less than 10 -7 M, less than 10 -8 M and less than 10 -9 Preferably, binding results in formation of a complex between the targeting fragment and PSMA, wherein the binding or complex can be detected, typically and preferably using a Biacore 3000 instrument (Biacore Inc., Piscataway, NJ) or a cell-based binding assay or flow-induced dispersion assay (FIDA), typically and preferably as described in "Kularatne et al., Mol Pharm. 2009; 6(3): 790-800".

[0729] In a preferred embodiment, the targeting fragment is a PSMA antibody, a PSMA aptamer, or a small molecule PSMA targeting fragment. In a preferred embodiment, 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 having a molecular weight of less than about 2000 g / mol 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 another preferred embodiment, the small molecule has a molecular weight of less than about 800 g / mol, more preferably less than about 500 g / mol.

[0730] In some embodiments, the PSMA-targeting fragment is a PSMA antibody capable of binding to PSMA. In some embodiments, the antibody 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, but are not limited to, 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., 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 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 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". It will be understood by those skilled in the art that any antibody capable of recognizing and / or specifically binding to PSMA can be used according to the present invention. All of the aforementioned documents and disclosures are incorporated herein by reference in their entirety.

[0731] In some embodiments, the targeting fragment capable of binding to PSMA is an aptamer. PSMA-targeting aptamers include, but are not limited to, the A10 aptamer or the A9 aptamer (Lupold et al., 2002, Cancer Res., 62:4029; and Chu et al., 2006, Nuc. Acid Res., 34:e73), derivatives thereof, and / or functional fragments thereof. In some embodiments, fewer than 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 nucleic acid residues are substituted in the aptamer derivative relative to the aptamer. In some embodiments, the sequence of the aptamer derivative is at least 80%, preferably 85%, more preferably 90%, more preferably 95%, and most preferably 99% identical.

[0732] 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 targeting peptidase inhibitor. In a preferred embodiment, the small molecule PSMA peptidase inhibitor includes 2-PMPA, GPI5232, VA-033, phenylalkylphosphonamide (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., 2004, Bioorg. Med. Chem., 12:4969) and / or their analogs and derivatives. All of the aforementioned references (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 a 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., 461:1989; and U.S. Patent Publication No. 2005 / 0080128). In some embodiments, the small molecule PSMA targeting fragment comprises a hydroxamic acid derivative (Stoermer et al., 2003, Bioorg. Med. Chem. Lett., 131:2097).In a preferred embodiment, the small molecule PSMA peptidase inhibitor includes an androgen receptor targeting agent (ARTA), such as those described in U.S. Patents 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 Publication Nos. 2006 / 0287547, 2006 / 0276540, 2006 / 0258628, 2006 / 0241180, 2006 / 0183931, 2006 / 0035966, 2006 / 0009529, 2006 / 0004042, 2005 / 0033074, 2006 / 0033075, 2006 / 0033076, 2006 / 0033077, 2006 / 0033078, 2006 / 0033079, 2006 / 0033078, 2006 / 0033079, 2006 / 0033079, 2006 / 0033079, 2006 / 0033079, 2006 / 0033079, 2006 / 0033079 4 / 0260108, 2004 / 0260092, 2004 / 0167103, 2004 / 0147550, 2004 / 0147489, 2004 / 0087810, 2004 / 0067979, 2004 / 0052727, 2004 / 0029913, 2004 / 001497 5, 2003 / 0232792, 2003 / 0232013, 2003 / 0225040, 2003 / 0162761, 2004 / 0087810, 2003 / 0022868, 2002 / 0173495, 2002 / 0099096, 2002 / 0099036. In some embodiments, the small molecule PSMA targeting fragment comprises a polyamine, such as putrescine, spermine, and spermidine (U.S. Patent Publication Nos. 2005 / 0233948 and 2003 / 0035804). All of the aforementioned documents and disclosures are incorporated herein by reference in their entirety.

[0733] In preferred embodiments, small molecule PSMA peptidase inhibitors include PBDA and urea-based inhibitors, such as ZJ43, ZJ, ZJ17, ZJ38 (Nan et al., 2000, J. Med. Chem., 43:772; and Kozikowski et al., 2004, J. Med. Chem., 47, 7, 1729-1738) and / or their analogs and derivatives. Other PSMA-binding agents may 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 preformed urea, such as the lysine-urea-glutamate compounds described by 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 inhibitors.

[0734] In a preferred embodiment, the small molecule PSMA targeting fragment is a urea inhibitor (also referred to herein as a urea peptidase inhibitor), more preferably a small urea inhibitor, such as those disclosed in the following documents: 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; WO2017 / 044936; WO2011 / 084518; WO2011 / 084521; WO2011 / 084513; WO2012 / 166923; WO 2008 / 105773; WO2008 / 121949; WO2012 / 135592; WO2010 / 005740; WO2015 / 168379; WO03 / 045436; WO03 / 045436; WO20 16 / 183447; US2015 / 258102; WO2011 / 084513; WO2017 / 089942; US2010 / 278927; WO2012 / 016188; WO2008 / 124634; WO2009 / 131435; US2007 / 225213; WO2017 / 086467; WO2009 / 026177; WO2012005572; WO2014 / 072357 and WO2011 / 108930. All of the foregoing documents and disclosures are incorporated herein by reference in their entirety.

[0735] 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.

[0736] The term "urea-based PSMA peptidase inhibitor" refers to a PSMA peptidase inhibitor that contains a urea group. The term "dipeptide-urea-based PSMA peptidase inhibitor" refers to a PSMA peptidase inhibitor that contains a urea group and two peptides or amino acids (each peptide or amino acid independently linked to the -NH2 group of the urea group); and the term "small molecule dipeptide-urea-based PSMA peptidase inhibitor" further refers to a dipeptide-urea-based PSMA peptidase inhibitor that has a molecular weight of less than about 2000 g / mol and is typically and preferably capable of binding to PSMA. In some embodiments, the small molecule dipeptide-urea-based PSMA peptidase inhibitor has 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 another preferred embodiment, the small molecule dipeptide-urea-based PSMA peptidase inhibitor has a molecular weight of less than about 800 g / mol, more preferably less than about 500 g / mol. PSMA peptidase inhibitors are capable of reducing the activity of the PSMA transmembrane zinc(II) metalloenzyme that catalyzes the cleavage of terminal glutamic acid. More preferably, the molecular weight of the small molecule urea-based PSMA peptidase inhibitor is less than about 500 g / mol. Still 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 references cited therein", all of which are incorporated herein by reference in their entirety.

[0737] In a preferred embodiment, the targeting fragment, preferably the urea-based PSMA peptidase inhibitor, is a glutamic acid-urea moiety of Formula 1, preferably Formula 1*, and its enantiomers, stereoisomers, rotamers, tautomers, diastereomers or racemates:

[0738]

[0739] In the formula, R is preferably a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, or any combination thereof; more preferably, R is C 1-6 Alkyl, preferably C2-C4 alkyl, which is substituted more than once, preferably once, by OH, SH, NH2 or COOH, one of the NH2, OH or SH or COOH groups being present as X 2 The covalent attachment point for the linking moiety and the PEG segment, wherein the alkyl group is optionally interrupted by N(H), S or O. In another preferred embodiment, R is C 1-6 Alkyl, preferably C2-C4 alkyl, which is substituted once by OH, SH, NH2 or COOH, wherein the NH2, OH or SH or COOH group is respectively as 2In a very preferred embodiment, R is a C2-alkyl group substituted once with COOH, wherein the COOH groups are respectively used as the covalent attachment point for X 2 The covalent attachment point to which the linking moiety and the PEG segment are attached.

[0740] In a preferred embodiment, the targeting fragment is a glutamate-urea moiety of Formula 1:

[0741]

[0742] Where R is C 1-6 Alkyl, preferably C2-C4 alkyl, which is substituted more than once, preferably once, by OH, SH, NH2 or COOH, one of said NH2, OH, SH or COOH groups being respectively substituted with X 2 The covalent attachment point for the linking moiety and the PEG segment, wherein the alkyl group is optionally interrupted by N(H), S or O. In another preferred embodiment, R is C 1-6 Alkyl, preferably C2-C4 alkyl, which is substituted once by OH, SH, NH2 or COOH, wherein the NH2, OH, SH or COOH group is respectively as 2 In a very preferred embodiment, R is a C2 alkyl group substituted once with COOH, wherein the COOH group serves as a covalent attachment point for X 2 The covalent attachment point to which the linking moiety and the PEG segment are attached.

[0743] In another preferred embodiment, the targeting fragment is a glutamate-urea moiety of formula 1*:

[0744]

[0745] Where R is C 1-6 Alkyl, preferably C2-C4 alkyl, which is substituted more than once, preferably once, by OH, SH, NH2 or COOH, one of said NH2, OH, SH or COOH groups being respectively substituted with X 2 The covalent attachment point for the linking moiety and the PEG segment, wherein the alkyl group is optionally interrupted by N(H), S or O. In another preferred embodiment, R is C 1-6 Alkyl, preferably C2-C4 alkyl, which is substituted once by OH, SH, NH2 or COOH, wherein the NH2, OH, SH or COOH group is respectively as 2 In a highly preferred embodiment, R is a C2 alkyl group substituted once with COOH, wherein the COOH groups serve as covalent attachment points for X 2 The covalent attachment point to which the linking moiety and the PEG segment are attached.

[0746] In another 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 very preferred embodiment, the targeting fragment consists of a DUPA residue (HOOC(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO-), wherein both chiral C atoms have the (S) configuration, as shown in Formula 1*.

[0747] In another preferred embodiment, the PSMA targeting fragment is a folate ligand. In another preferred embodiment, the PSMA targeting fragment is a small molecule PSMA targeting fragment, and the small molecule PSMA targeting fragment is a folate ligand.

[0748] In a preferred embodiment, the folate ligand binds to a cell surface receptor, which is PSMA. According to recent reports, folate amide has been shown to target cells expressing PSMA (Flores O et al., Theranostics 2017, 7(9): 2477-2494).

[0749] 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 comprises a glutamic acid functional group R-NH-[CH(COOH)-CH2-CH2-C(O)NH] η -CH(COOH)-CH2-CH2-COOH, wherein n is an integer from 0 to 100, and R is a group of formula 2:

[0750] Where:

[0751] R 201 is -OH or -NH2;

[0752] R 202 is -H or -CH3; and

[0753] Wavy line represents the point of attachment with the glutamic acid functional group.In a preferred embodiment, n is an integer of 0 to 10, preferably n is an integer of 0 to 5, more preferably n is 0.

[0754] It should be understood by those skilled in the art that when R 201 When -OH, in a preferred embodiment, the OH will tautomerize to a carbonyl (=O), and the R 201 The adjacent nitrogen atoms will be protonated.

[0755] It will also be understood by those skilled in the art that the glutamic acid functional group R-NH-[CH(COOH)-CH2-CH2-C(O)NH]η-CH(COOH)-CH2-CH2-COOH comprises at least one α-carboxylic acid group and one γ-carboxylic acid group. Specifically, one or more -COOH groups bonded to the same carbon atom as the -NH- group are understood herein as α-carboxylic acid groups. When n=0, the -COOH group bonded to the same carbon atom as the R-NH group is understood herein as an α-carboxylic acid group. The -COOH group bonded to the -(CH2)2- group is understood herein as a γ-carboxylic acid group. In addition, it will be understood by those skilled in the art that the carboxylic acid groups discussed herein, such as α-carboxylic acid groups and γ-carboxylic acid groups, can be protonated or deprotonated depending on the pH value of the surrounding solution. Thus, one skilled in the art will appreciate that, although carboxylic acid groups are drawn as neutral species (-COOH) for simplicity and clarity, they can exist as (e.g., can exist primarily as) deprotonated species (i.e., negatively charged species (-COO-)) at physiological pH.

[0756] In some embodiments, the α-carboxylic acid group of the glutamic acid functional group is used as a 2 In a preferred embodiment, when the α-carboxylic acid group of the glutamic acid functional group is connected to X 2 When the connection point of the connecting part is 2 In some embodiments, when the α-carboxylic acid group of the glutamic acid functional group is used as the amino group of X 2 When the connection point of the connecting part is 2 The hydroxyl groups of the linking moiety are condensed to form an ester.

[0757] In a preferred embodiment, the γ-carboxylic acid group of the glutamic acid functional group is used as a 2 In a preferred embodiment, when the gamma-carboxylic acid group of the glutamic acid functional group is connected to X 2 When the connection point of the connecting part is 2 In some embodiments, when the γ-carboxylic acid group of the glutamic acid functional group is used as a ligand to bind to X 2 When the connection point of the connecting part is 2 The hydroxyl groups of the linking moiety are condensed to form an ester.

[0758] In a preferred embodiment, the folate ligand is folic acid:

[0759]

[0760] In the formula, the α-carboxyl group or γ-carboxyl group of folic acid acts as 2 The point of covalent attachment to which the linking moiety is attached.

[0761] In some embodiments, the α-carboxylic acid group of folic acid acts as a 2 In a preferred embodiment, when the α-carboxylic acid group of the folic acid is connected to X 2 When the connection point of the connecting part is 2 In some embodiments, when the α-carboxylic acid group of the folic acid is used as a 2 When the connection point of the connecting part is 2 The hydroxyl groups of the linking moiety are condensed to form an ester.

[0762] In a preferred embodiment, the γ-carboxylic acid group of folic acid acts as a 2 In a preferred embodiment, when the gamma-carboxylic acid group of folic acid is used as a covalent attachment point for X 2 When the connection point of the connecting part is 2 In some embodiments, when the γ-carboxylic acid group of the folic acid is used as a 2 When the connection point of the connecting part is 2 The hydroxyl groups of the linking moiety are condensed to form an ester.

[0763] In a preferred embodiment, the folate ligand is methotrexate:

[0764]

[0765] In the formula, the α-carboxylic acid group or γ-carboxylic acid group of methotrexate is used as the 2 The point of covalent attachment to which the linking moiety is attached.

[0766] In some embodiments, the alpha carboxylic acid group of methotrexate acts as a 2 In a preferred embodiment, when the alpha carboxylic acid group of the methotrexate is used as the covalent attachment point for the X 2 When the connection point of the connecting part is 2 In some embodiments, when the α-carboxylic acid group of the methotrexate is used as a 2 When the connection point of the connecting part is 2 The hydroxyl groups of the linking moiety are condensed to form an ester.

[0767] In a preferred embodiment, the γ-carboxylic acid group of methotrexate acts as a 2 In a preferred embodiment, when the gamma-carboxylic acid group of methotrexate is used as a covalent attachment point for X 2 When the connection point of the connecting part is 2 In some embodiments, when the γ-carboxylic acid group of methotrexate is used as a 2 When the connection point of the connecting part is 2 The hydroxyl groups of the linking moiety are condensed to form an ester.

[0768] In another aspect, the present invention provides a composition comprising a polymer complex comprising a conjugate of Formula I* or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or enantiomer thereof and a nucleic acid, preferably the nucleic acid is non-covalently bound to 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; R 1 is the initiating residue, preferably R 1 is -H or -CH3; R 2 are independently -H or an organic residue, the -(NR 2 -CH2-CH2) n - At least 80%, preferably 90% of the R 2 H; X 1 and X 2 are independently divalent covalently linked moieties; Z is a divalent covalently linked moiety, wherein Z is not -NHC(O)-, preferably a divalent covalently linked moiety, wherein 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 a DUPA residue (HOOC(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO-), the nucleic acid is a nucleic acid with pharmaceutical activity, and the nucleic acid with pharmaceutical activity is a nucleic acid encoding a peptide or protein with pharmaceutical activity, and preferably the composition consists of the conjugate.

[0769] In another aspect, the present invention provides a composition comprising a polymer complex comprising a conjugate of Formula I* or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or enantiomer thereof and a nucleic acid, preferably the nucleic acid is non-covalently bound to 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; R 1 is an initiating residue, preferably, R 1 is -H or -CH3; R 2 are independently -H or an organic residue, the -(NR 2 -CH2-CH2) n - At least 80%, preferably 90% of the R 2 H; X 1 and X 2 are independently divalent covalently linked moieties; Z is a divalent covalently linked moiety, wherein Z is not -NHC(O)-, preferably Z is a divalent covalently linked moiety, wherein 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 a DUPA residue (HOOC(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO-); said nucleic acid is a nucleic acid having pharmaceutical activity, and said nucleic acid having pharmaceutical activity is a nucleic acid encoding a peptide or protein having pharmaceutical activity; preferably said composition consists of said conjugate.

[0770] In another aspect, the present invention provides a composition comprising a polymer complex comprising a conjugate of Formula I* or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or enantiomer thereof and a nucleic acid, preferably the nucleic acid is non-covalently bound to 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; R 1 is the initiating residue, preferably R1 is -H or -CH3; R 2 are independently -H or an organic residue, the -(NR 2 -CH2-CH2) n - At least 80%, preferably 90% of the R 2 H; X 1 and X 2 are independently divalent covalent linking moieties; Z is a divalent covalent linking moiety, wherein Z is not -NHC(O)-, preferably Z is a divalent covalent linking moiety, wherein Z is not a single bond and Z is not -NHC(O)-; L is a targeting fragment, wherein the targeting fragment L is a DUPA residue (HOOC(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO-); the nucleic acid is a nucleic acid with pharmaceutical activity, and the nucleic acid with pharmaceutical activity is a nucleic acid encoding a peptide or protein with pharmaceutical activity; preferably, the combination consists of the conjugate.

[0771] In another aspect, the present invention provides a polymer complex comprising a conjugate of Formula I* or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or enantiomer thereof and a nucleic acid, preferably the nucleic acid is non-covalently bound to the conjugate:

[0772] R 1 -(NR 2 -CH2-CH2) n -ZX 1 -(O-CH2-CH2) m -X 2 -L (Formula I*);

[0773] In the formula, 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; R 1 is an initiating residue, preferably, R 1 is -H or -CH3; R 2 are independently -H or an organic residue, the -(NR 2 -CH2-CH2) n - At least 80%, preferably 90% of the R 2 H; X 1 and X 2are independently divalent covalently linked moieties; Z is a divalent covalently linked moiety, wherein Z is not -NHC(O)-, preferably Z is a divalent covalently linked moiety, wherein 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 a DUPA residue (HOOC(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO-); the nucleic acid is a nucleic acid having pharmaceutical activity, and the nucleic acid having pharmaceutical activity is a nucleic acid encoding a peptide or protein having pharmaceutical activity.

[0774] In another aspect, the present invention provides a polymer complex comprising a conjugate of Formula I* or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or enantiomer thereof and a nucleic acid, preferably the nucleic acid is non-covalently bound to the conjugate:

[0775] R 1 -(NR 2 -CH2-CH2) n -ZX 1 -(O-CH2-CH2) m -X 2 -L (Formula I*);

[0776] In the formula, 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; R 1 is an initiating residue, preferably, R 1 is -H or -CH3; R 2 are independently -H or an organic residue, the -(NR 2 -CH2-CH2) n - At least 80%, preferably 90% of the R 2 H; X 1 and X 2 are independently divalent covalently linked moieties; Z is a divalent covalently linked moiety, wherein Z is not -NHC(O)-, preferably, Z is a divalent covalently linked moiety, wherein 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 a DUPA residue (HOOC(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO-); the nucleic acid is a nucleic acid having pharmaceutical activity, and the nucleic acid having pharmaceutical activity is a nucleic acid encoding a peptide or protein having pharmaceutical activity.

[0777] In another aspect, the present invention provides a polymer complex comprising a conjugate of Formula I* or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or enantiomer thereof and a nucleic acid, preferably the nucleic acid is non-covalently bound to the conjugate:

[0778] R 1 -(NR 2 -CH2-CH2) n -ZX 1 -(O-CH2-CH2) m -X 2 -L (Formula I*);

[0779] In the formula, 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; R 1 is an initiating residue, preferably, R 1 is -H or -CH3; R 2 are independently -H or an organic residue, the -(NR 2 -CH2-CH2) n - At least 80%, preferably 90% of the R 2 H; X 1 and X 2 are independently divalent covalent linking moieties; Z is a divalent covalent linking moiety, wherein Z is not -NHC(O)-, preferably Z is a divalent covalent linking moiety, wherein Z is not a single bond and Z is not -NHC(O)-; L is a targeting fragment, wherein the targeting fragment L is a DUPA residue (HOOC(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO-); the nucleic acid is a nucleic acid having pharmaceutical activity, and the nucleic acid having pharmaceutical activity is a nucleic acid encoding a peptide or protein having pharmaceutical activity.

[0780] In another aspect, the present invention provides a polymer complex comprising a conjugate of Formula I or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or enantiomer thereof and a nucleic acid, preferably the nucleic acid is non-covalently bound to the conjugate:

[0781]

[0782] Where:

[0783] is a single bond or a double bond;

[0784] n is any integer from 1 to 1500;

[0785] m is any integer from 1 to 200, preferably any integer from 2 to 1500;

[0786] R 1 is an initiating residue, preferably, R 1 is -H or -CH3;

[0787] R 2 are independently -H or an organic residue, the -(NR 2 -CH2-CH2) n - At least 80%, preferably at least 90% of the R 2 is H;

[0788] Ring A is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl group, optionally substituted at any position with one or more R A1 replace;

[0789] R A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo or halogen; or, two R A1 Together with the atoms to which they are attached, they can form more than one fused C6-C 10 aryl, C5-C6 heteroaryl or C3-C6 cycloalkyl ring, wherein each fused aryl, heteroaryl or cycloalkyl is optionally substituted by one or more R A2 replace;

[0790] R A2 Independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, -SO3H or -OSO3H;

[0791] X 1 Formula -(Y 1 ) p - a connecting portion, wherein p is an integer from 1 to 20, Y 1 is independently selected at each occurrence from a chemical bond, -CR 11 R 12 -、-C(O)-、-O-、-S-、-NR 13 -, amino acid residues, divalent phenyl moieties, divalent carbocyclic moieties, divalent heterocyclic moieties and divalent heteroaryl moieties, each divalent phenyl or heteroaryl group being optionally substituted by one or more R 13 Each divalent heterocyclic ring may be substituted with one or more R 14 Replacement; R 11 、R 12 and R 13 R is independently H, -SO3H, -NH2, -CO2H or C1-C6 alkyl at each occurrence, each alkyl group being optionally substituted with -CO2H or -NH2; 14Each occurrence is independently H, C1-C6 alkyl, oxo, C6-C 10 aryl or 5- to 8-membered heteroaryl;

[0792] X 2 Formula -(Y 2 ) q - a connecting portion, wherein q is an integer from 1 to 50, Y 2 is independently selected at each occurrence from a chemical bond, -CR 21 R 22 -、NR 23 -, -O-, -S-, -C(O)-, amino acid residues, divalent phenyl moieties, divalent carbocyclic moieties, divalent heterocyclic moieties and divalent heteroaryl moieties, each divalent phenyl and divalent heteroaryl group being optionally substituted by one or more R 23 Each divalent heterocyclic moiety is optionally substituted with one or more R 24 Replacement; R 21 、R 22 and R 23 Each occurrence is independently -H, -SO3H, -NH2, -CO2H or C1-C6 alkyl, each C1-C6 alkyl group is optionally replaced by one or more -OH, oxo, -CO2H, -NH2, C6-C 10 aryl or 5 to 8 membered heteroaryl substituted; R 24 is independently at each occurrence -H, -CO2H, C1-C6 alkyl, or oxo; and

[0793] L is a targeting fragment, preferably, the targeting fragment L is a DUPA residue (HOOC(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO-); the nucleic acid is a nucleic acid with pharmaceutical activity, and the nucleic acid with pharmaceutical activity is a nucleic acid encoding a peptide or protein with pharmaceutical activity.

[0794] In another aspect, the present invention provides a composition comprising a polymer complex comprising a conjugate of Formula I or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or enantiomer thereof and a nucleic acid, preferably the nucleic acid is non-covalently bound to the conjugate:

[0795]

[0796] Where:

[0797] is a single bond or a double bond;

[0798] n is any integer from 1 to 1500, preferably any integer from 2 to 1500;

[0799] m is a discrete number of repeating units ranging from 2 to 100, preferably a discrete number of repeating units ranging from 4 to 60;

[0800] R 1 is an initiating residue, preferably, R 1 is -H or -CH3;

[0801] R 2 are independently -H or an organic residue, wherein -(NR 2 -CH2-CH2) n - at least 80%, preferably at least 90% of R 2 is H;

[0802] Ring A is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl group, optionally substituted at any position with one or more R A1 Replacement; R A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo or halogen; or, two R A1 Together with the atoms to which they are attached, they can form more than one fused C6-C 10 aryl, C5-C6 heteroaryl or C3-C6 cycloalkyl ring, wherein each fused aryl, heteroaryl or cycloalkyl is optionally substituted by one or more R A2 Replacement; R A2 Independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, -SO3H or -OSO3H;

[0803] X 1 is a divalent covalent linking moiety;

[0804] X 2 is a divalent covalent linking moiety; and

[0805] L is a targeting fragment, which is a PSMA targeting fragment. Preferably, the PSMA targeting fragment can specifically bind to cells expressing (preferably overexpressing) PSMA; the nucleic acid is a nucleic acid with pharmaceutical activity, which is a nucleic acid encoding a peptide or protein with pharmaceutical activity. In a preferred embodiment, the R 1 In a preferred embodiment, the R 1=-CH3. In another 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 another very preferred embodiment, the targeting fragment consists of a DUPA residue (HOOC(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO-), wherein both chiral C atoms have the (S) configuration, as shown in Formula 1*.

[0806] In another aspect, the present invention provides a polymer complex comprising a conjugate of Formula I or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or enantiomer thereof and a nucleic acid, preferably the nucleic acid is non-covalently bound to the conjugate:

[0807]

[0808] Where:

[0809] is a single bond or a double bond;

[0810] n is any integer from 1 to 1500, preferably any integer from 2 to 1500;

[0811] m is a discrete number of repeating units ranging from 2 to 100, preferably a discrete number of repeating units ranging from 4 to 60;

[0812] R 1 is an initiating residue, preferably, R 1 is -H or -CH3;

[0813] R 2 are independently -H or an organic residue, -(NR 2 -CH2-CH2) n - at least 80%, preferably at least 90% of R 2 is H;

[0814] Ring A is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl group, optionally substituted at any position with one or more R A1 Replacement; R A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo or halogen; or, two R A1 Together with the atoms to which they are attached, they can form more than one fused C6-C 10 aryl, C5-C6 heteroaryl or C3-C6 cycloalkyl ring, wherein each fused aryl, heteroaryl or cycloalkyl is optionally substituted by one or more R A2 Replacement; R A2Independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, -SO3H or -OSO3H;

[0815] X 1 is a divalent covalent linking moiety;

[0816] X 2 is a divalent covalent linking moiety; and

[0817] L is a targeting fragment, which is a PSMA targeting fragment. Preferably, the PSMA targeting fragment can specifically bind to cells expressing (preferably overexpressing) PSMA; the nucleic acid is a nucleic acid with pharmaceutical activity, which is a nucleic acid encoding a peptide or protein with pharmaceutical activity. In a preferred embodiment, the R 1 In a preferred embodiment, the R 1 =-CH3. In another 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 another very preferred embodiment, the targeting fragment consists of a DUPA residue (HOOC(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO-), wherein both chiral C atoms have the (S) configuration, as shown in Formula 1*.

[0818] In another aspect, the present invention provides a composition comprising a polymer complex comprising a conjugate of Formula I or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or enantiomer thereof and a nucleic acid, preferably the nucleic acid is non-covalently bound to the conjugate:

[0819]

[0820] Where:

[0821] is a single bond or a double bond;

[0822] n is any integer from 1 to 1500, preferably any integer from 2 to 1500;

[0823] m is the discrete number of repeating units, which is 36;

[0824] R 1 is an initiating residue, preferably, R 1 is -H or -CH3;

[0825] R 2 are independently -H or an organic residue, the -(NR2 -CH2-CH2) n - at least 80%, preferably at least 90% of R 2 is H;

[0826] Ring A is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl group, optionally substituted at any position with one or more R A1 Replacement; R A1 Independently selected from C1-C6 alkyl, C 1- C6 alkoxy, oxo or halogen; or, two R A1 Together with the atoms to which they are attached, they can form more than one fused C6-C 10 aryl, C5-C6 heteroaryl or C3-C6 cycloalkyl ring, wherein each fused aryl, heteroaryl or cycloalkyl is optionally substituted by one or more R A2 Replacement; R A2 Independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, -SO3H or -OSO3H;

[0827] X 1 is a divalent covalent linking moiety;

[0828] X 2 is a divalent covalent linking moiety; and

[0829] L is a targeting fragment, which is a PSMA targeting fragment, preferably, the PSMA targeting fragment can specifically bind to cells expressing (preferably over-expressing) PSMA; the nucleic acid is a nucleic acid with pharmaceutical activity, which is a nucleic acid encoding a peptide or protein with pharmaceutical activity. In a preferred embodiment, the R 1 In a preferred embodiment, the R 1 =-CH3. In another 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 another very preferred embodiment, the targeting fragment consists of DUPA (HOOC(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO-), wherein both chiral C atoms have the (S) configuration, as shown in Formula 1*.

[0830] In another aspect, the present invention provides a polymer complex comprising a conjugate of Formula I or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or enantiomer thereof and a nucleic acid, preferably the nucleic acid is non-covalently bound to the conjugate:

[0831]

[0832] Where:

[0833] is a single bond or a double bond;

[0834] n is any integer from 1 to 1500, preferably any integer from 2 to 1500;

[0835] m is the discrete number of repeating units, which is 36;

[0836] R 1 is an initiating residue, preferably, R 1 is -H or -CH3;

[0837] R 2 are independently -H or an organic residue, the -(NR 2 -CH2-CH2) n - at least 80%, preferably at least 90% of R 2 is H;

[0838] Ring A is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl group, optionally substituted at any position with one or more R A1 Replacement; R A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo or halogen; or, two R A1 Together with the atoms to which they are attached, they can form more than one fused C6-C 10 aryl, C5-C6 heteroaryl or C3-C6 cycloalkyl ring, wherein each fused aryl, heteroaryl or cycloalkyl is optionally substituted by one or more R A2 Replacement; R A2 Independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, -SO3H or -OSO3H;

[0839] X 1 is a divalent covalent linking moiety;

[0840] X 2 is a divalent covalent linking moiety; and

[0841] L is a targeting fragment, which is a PSMA targeting fragment. Preferably, the PSMA targeting fragment can specifically bind to cells expressing (preferably overexpressing) PSMA; the nucleic acid is a nucleic acid with pharmaceutical activity, which is a nucleic acid encoding a peptide or protein with pharmaceutical activity. In a preferred embodiment, the R 1 In a preferred embodiment, the R 1=-CH3. In another 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 another very preferred embodiment, the targeting fragment consists of a DUPA residue (HOOC(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO-), wherein both chiral C atoms have the (S) configuration, as shown in Formula 1*.

[0842] In another aspect, the present invention provides a composition comprising a polymer complex comprising a conjugate of Formula I or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or enantiomer thereof and a nucleic acid, preferably the nucleic acid is non-covalently bound to the conjugate:

[0843]

[0844] Where:

[0845] is a single bond or a double bond;

[0846] n is any integer from 1 to 1500, preferably...

Claims

1. A composition comprising a polymer complex comprising a conjugate and a nucleic acid, wherein the conjugate comprises: A linear polyethyleneimine segment comprising an α-terminus and an ω-terminus; the α-terminus of the polyethyleneimine segment is an initiation residue; a polyethylene glycol segment comprising a first terminus and a second terminus; and in, The ω end of the polyethyleneimine segment is connected by a divalent covalent linking group -ZX 1 - is linked to the first end of the polyethylene glycol fragment, wherein -ZX 1 - is not a single bond and -Z- is not an amide; The second end of the polyethylene glycol segment is covalently linked to a divalent moiety X 2 ligated with a targeting fragment; as well as The nucleic acid is a nucleic acid with pharmaceutical activity, and the nucleic acid with pharmaceutical activity is a nucleic acid encoding a peptide or protein with pharmaceutical activity.

2. The composition according to claim 1, wherein The conjugate is a conjugate of formula I* or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or enantiomer thereof: R 1 -(NR 2 -CH2-CH2) n -Z-X 1 -(O-CH2-CH2) m -X 2 -L (Formula I*); Where: n is any integer from 1 to 1500; m is any integer from 1 to 200; R 1 is an initiating residue, preferably, R 1 is -H or -CH3; R 2 are independently -H or an organic residue, the -(NR 2 -CH2-CH2) n - at least 80%, preferably 90% of said R 2 is H; X 1 and X 2 are independently divalent covalently linking moieties; Z is a divalent covalent linking moiety, wherein Z is not a single bond and Z is not -NHC(O)-; L is a targeting fragment, and preferably, the targeting fragment is capable of binding to cells.

3. The composition according to claim 1 or 2, wherein The conjugate is a conjugate of formula I or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or enantiomer thereof: Where: is a single bond or a double bond; n is any integer from 1 to 1500; m is any integer from 1 to 200; R 1 is an initiating residue, preferably, R 1 is -H or -CH3; R 2 are independently -H or an organic residue, the -(NR 2 -CH2-CH2) n - at least 80%, preferably at least 90% of said R 2 is H; Ring A is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl group, optionally substituted by one or more R A1 Replacement; R A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo or halogen; or, two R A1 Together with the atoms to which they are attached, they can form more than one fused C6-C 10 aryl, C5-C6 heteroaryl or C3-C6 cycloalkyl ring, wherein Each fused aryl, heteroaryl or cycloalkyl group is optionally substituted by one or more R A2 Replacement; R A2 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; and L is a targeting fragment, and preferably, the targeting fragment is capable of binding to cells.

4. The composition according to any one of claims 2 to 3, wherein The -(O-CH2-CH2) m The - portion consists of a discrete number m of repeating units ranging from 4 to 60, preferably, the discrete number m of repeating -(O-CH2-CH2)- units is 36.

5. The composition according to any one of claims 3 to 4, wherein The conjugate of formula I is selected from:

6. The composition according to any one of claims 3 to 5, wherein The conjugate of formula I is selected from:

7. The composition according to any one of claims 3 to 6, wherein X 1 Contains a group selected from the following: Where: r is independently 0-6 at each occurrence, preferably 0, 1, 2 or 5; s is independently 0-6 at each occurrence, preferably 0, 2, 3 or 4; t is independently 0-6 at each occurrence, preferably 0, 1, 2, 4; R 11 and R 12 is independently selected at each occurrence from -H and -C1-C2 alkyl; and R 13 Preferably, the wavy line closest to the integer "r" is the bond to ring A, and the wavy line closest to the integer "s" or "t" is the bond to -[OCH2-CH2] m - The key to connect.

8. The composition according to any one of claims 3 to 7, wherein X 1 Selected from: Preferably, the wavy line on the left is the bond to ring A, and the wavy line on the right is the bond to -[OCH 2- CH2] m - The key to connect.

9. The composition according to any one of claims 3 to 8, wherein X 2 Selected from: Where Y 2 is independently selected at each occurrence from a chemical bond, -CR 21 R 22 -、NR 23 -, -O-, -S-, -C(O)-, amino acid residues, divalent phenyl moieties, divalent carbocyclic moieties, divalent heterocyclic moieties and divalent heteroaryl moieties, each divalent phenyl and divalent heteroaryl group being optionally substituted by one or more R 23 Each divalent heterocyclic moiety is optionally substituted with one or more R 24 Replacement; R 21 、R 22 and R 23 Each occurrence is independently -H, -SO3H, -NH2, -CO2H or C1-C6 alkyl, each C1-C6 alkyl group is optionally replaced by one or more -OH, oxo, -CO2H, -NH2, C6-C 10 aryl or 5- to 8-membered heteroaryl substitution; and R 24 is independently at each occurrence -H, -CO2H, C1-C6 alkyl or oxo; Preferably, the wavy line on the left is the same as -[OCH2-CH2] m - is the bond connected to the , and the wavy line on the right is the bond connected to the L.

10. The composition according to any one of claims 3 to 9, wherein X 2 Selected from: Preferably, the wavy line on the left is -[OCH2-CH2] m - is the bond connected to the , and the wavy line on the right is the bond connected to the L.

11. A composition according to any one of the preceding claims, wherein The targeting fragment L is capable of binding to a cell surface receptor, and the cell surface receptor is selected from the group consisting of 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 is selected from the group consisting of 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. A composition according to any one of the preceding claims, wherein 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 affinity body; folic acid; methotrexate; a somatostatin receptor targeting fragment, preferably somatostatin and / or octreotide; an integrin targeting fragment, preferably a fragment containing arginine-glycine-aspartic acid (RGD); a low pH insertion peptide; an ASGPr targeting fragment, preferably desialylated serum mucoid; an insulin receptor targeting fragment, preferably insulin; a mannose-6-phosphate receptor targeting fragment, preferably The invention further comprises the following: mannose-6-phosphate; a mannose receptor targeting fragment, preferably mannose; a sialyl Lewis X 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 LyP-1 tumor homing peptide that binds to p32; 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. A composition according to 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. A composition according to any one of the preceding claims, wherein The nucleic acid is RNA, and the RNA is messenger RNA (mRNA).

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

16. A composition according to any one of the preceding claims, wherein The pharmaceutically active peptide or protein is selected from: cytokines, growth factors, hormones, enzymes, tumor antigens, viral antigens, bacterial antigens, autoantigens or allergens; preferably, the pharmaceutically active peptide or protein is a cytokine selected from interleukins, interferons and chemokines.

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