Tumor-targeted polypeptide nanoparticle delivery systems for nucleic acid therapeutics

Abstract

A novel nucleic acid delivery system is provided, comprising a linear histidine-lysine-rich cysteine-containing peptide with a targeting function and a tetraantennary histidine-lysine-rich polypeptide. The delivery system contains a nucleic acid such as siRNA. The components form a stable nanoparticle complex through non-covalent interactions between the phosphate of the siRNA and the histidine / lysine of the polypeptide, resulting in reduced toxicity and selective delivery of genetic material to cells. The targeting function improves the efficiency of nucleic acid delivery and transfection. Also provided are carrier molecules capable of delivering therapeutic molecules to specific cells. The carrier molecules are modified with targeting ligands that can bind to specific receptors on the targeted cells. The therapeutic molecules are siRNAs, miRNAs, or other oligonucleotides. The targeting moieties are small molecules, peptides, or proteins that exhibit affinity for receptors present on the targeted cells.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 62 / 910,760, filed October 4, 2019, and U.S. Provisional Patent Application No. 62 / 915,450, filed October 15, 2019, the entire contents of which are incorporated herein by reference. Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated by reference in its entirety. Said ASCII copy, created on December 11, 2020, is named 4690_0026i_SL.txt and is 5,732 bytes in size.

[0002] Nucleic acid delivery systems and methods of use are provided, including methods for targeted or localized delivery of nucleic acid molecules. [Background technology]

[0003] Targeted delivery of therapeutics has attracted great interest and benefits to improve tumor treatment while increasing efficacy and reducing side effects. Accumulation of nanoparticles (NPs) in tumors is believed to be due to the enhanced vascular permeability retention (EPR) effect (Maeda, Bioconjugate Chemistry, 21:797-802 (2010)). Therefore, tumor delivery may be improved by coating particles with tumor-localizing ligands. The mechanism by which ligands increase the antitumor efficacy of cargo (e.g., siRNA) is still under debate. Enhanced binding to tumor surface markers may increase the accumulation of NPs in tumors compared to non-targeted tissues. Other researchers have argued that the accumulation of targeted and non-targeted NPs in tumor cells is comparable. It has been suggested that the increased efficacy of targeted NPs is caused by enhanced receptor-mediated endocytosis and increased intracellular localization of siRNA therapeutics. Bartlett et al., (2007): Proc. Nat'l Acad. Sci. USA, 104:15549-15554 (2007). Both mechanisms most likely play essential roles in ligand-targeted therapy and efficacy.

[0004] Targeted delivery of siRNA in vivo is challenging due to degradation and rapid clearance by serum nucleases, endosomal trapping, and innate immune stimulation by nanoparticles (NPs). In recent years, very limited methods have been developed in preclinical and clinical trials for targeted delivery of siRNA. One approach is by Alnylam, which developed GalNAc-siRNA, in which a synthetic triantennary N-acetylgalactosamine (GaLNAc)-based ligand is conjugated to a chemically modified siRNA. This allows efficient delivery to hepatocytes mediated by ASGPR. Maja et al.; Nature Communications, 9:723 (2018). GaLNAc targets the liver-specific asialoglycoprotein receptor (ASGPR) in the liver. Another example is Fitusiran (ALN-AT3, Phase II clinical trial, Alnylam) by Sanofi Genzyme for the treatment of hemophilia and rare bleeding disorders (RBD). It is administered subcutaneously, and the RNAi therapeutic is aimed at targeting antithrombin (AT). In another case, a targeting ligand was incorporated into a liposomal formulation when multiple components were co-assembled with siRNA. This kind of system holds many challenges in terms of liposome stability, biocompatibility, toxicity, large-scale manufacturing and long-term storage. Leng et al., J. Drug Delivery, ID6971297, (2017). Most recently, nanoparticles formed by polypeptides / polymers and siRNA have efficiently delivered siRNA in vivo, and some of these products have entered early clinical trials. For example, histidine (H)-lysine (K)-rich polypeptides have safely and effectively delivered dual siRNA to its target and achieved its therapeutic efficacy. One of the lead drugs is being tested in a phase IIa clinical trial. See: Zhou et al., Oncotarget, 8:80651-80665 (2017); WO 2011 / 140285. Summary of the Invention [Brief description of the drawings]

[0005] [Figure 1] 1 shows a tumor-targeting HKC / HKP or HKP(+H) polypeptide nanoparticle delivery system. The graph shows the formation of tumor-targeting polypeptide nanoparticles and HKC polypeptide-siRNA nanoplex formation between (A) branched polypeptides H3K4B(HKP) or H3K(+H)4b or (HKP(+H)) with specific histidine / lysine sequences, (B) linear polypeptides functionalized by terminal cysteines with tumor-targeting ligands such as RGD, folate, or SmAb, and selected siRNA. [Diagram 2] Schematic diagram of the preparation of HKC-PEG-targeting ligand functionalized polypeptides (HKC=HKC1, HKC2 or HK2C, see FIG. 3). HKC has a terminal cysteine ​​conjugated to a maleimide-functionalized PEG-linked targeting motif, such as (folate, RGD, mAb, etc.) via a thiol / maleimide addition reaction under mild conditions. [Diagram 3] 3 shows the structure of H3K4b (abbreviation HKP) branched peptide, the structure of H3K4C (abbreviation HKC1 or HKC) with one cysteine ​​at the terminal position, and the structure of HKC2 with two cysteines in the sequence. Bi-branched cysteine-containing peptide HK2C with the sequence [(KHHH)4]2KXC. FIG. 3(a) discloses SEQ ID NO: 15, and FIG. 3(c) discloses SEQ ID NO: 16. [Figure 3B] FIG. 1 shows HPLC chromatogram of HKC, reversed phase Alltima™ column C-18 (4.6×250 mm), elution=15.196 at room temperature, >91% with a gradient of water (0.065% TFA) and acetonitrile (0.05% TFA). [Figure 3C] FIG. 1 shows the mass spectrometry (ESI-MS, positive) of HKC1 compound, in which a doubly charged molecular ion peak was observed at 1335.6 [M] 2+ . [Figure 4]1 shows the preparation route of HKC2-Peg1000-folate, HKC1 reacted with maleimide-PEG-folate via thiol / maleimide addition reaction under basic condition to obtain the conjugation product. After removing the solvent and then purifying by dialysis, HKC1-Peg1000-folate was obtained. [Diagram 5] Figure 1 shows the characterization of HKC2-PEG1k-folate by 1H NMR: (top) HKC2 in D2O, (middle) HKC2-PEG1k-folate in DMSO-d6, and (bottom) folate-PEG1k-Mal. HKC was covalently conjugated to folate-PEG1k-Mal, and the characteristic signal of the maleimide double bond at 7.0 ppm disappeared after reaction with cysteine. [Figure 6] Figure 1 shows UV / Vis (water, 25°C) spectroscopic analysis of HKC2-PEG1k-folate (top, red curve) and folate-PEG1k-Mal (bottom, grey curve) in water. Characteristic absorbances at 220 nm for the peptide and 275 nm for folate were observed in the product spectra. [Figure 7] FIG. 1 shows MALDI-MS (positive) spectroscopy analysis of HKC2-PEG1k-folate. The molecular ion peak around 4302 M+ indicates successful conversion of HKC2 from the coupling reaction. [Figure 8] 2 shows the two-step preparation of HKC2-PEG2k-RGD. The first step is the coupling between c(RGDfk) and a bifunctional PEG molecule bearing N-hydroxysuccinimide (NHS) and maleimide (Mal) functional groups, where an amide bond is formed via coupling between the amine and NHS. In the second step, the thiol of HKC (SEQ ID NO: 17) reacts with the maleimide of RGD-PEG2000-Mal to obtain the RGD-linked PEG linker polypeptide HKC2-PEG2000-RGD. [Figure 9]FIG. 1 shows the characterization of the RGD-PEG2k-Mal intermediate by 1H NMR (DMSO-d6, 25° C.) spectroscopy. RGD signals at 8.5-7.2 ppm and a maleimide signal at 7.00 ppm, as well as a broad peak of PEG ethylene at about 3.5 ppm, can be observed. [Figure 10] FIG. 13 shows stack plot comparison of HKC2-PEG-RGD characterization by 1H NMR spectroscopy of HKC2 derivatization with RGD targeting ligand. [Figure 11] FIG. 1 shows the preparation of HKC1-PEG-GalNAc (n=6, 12, 24) from the conjugation of HKC1 with a trivalent GalNAc-PEG molecule bearing a maleimide (Mal) functional group, forming a SC bond via coupling between the thiol on the cysteine ​​and Mal. The figure discloses KHHHKHHHKHHHKHHHKSSSC as SEQ ID NO:11. [Figure 12] Figure 1 shows the formulation of HKC:HKP:TGFβ1 in nanoparticle form and its size distribution, HKC=HKC2=K(HHHK)4CSSC (SEQ ID NO: 1), HKP=H3K4b. [Figure 13] Figure 1 shows the formulation of HKC:HKP:TGFβ1 in nanoparticle form and its polydispersity index, HKC=HKC2=K(HHHK)4CSSC (SEQ ID NO: 1), HKP=H3K4b. [Figure 14]Figure 1 shows the effect of treatment with Cell Death siRNA formulated with HKP alone or in combination with various amounts of HKP and HKC2 on the viability of human glioblastoma T98G cells. Aqueous solutions of HKC2 (160 ng / μL), HKP (320 ng / μL) and siRNA (80 ng / μL) were mixed at the specified ratios and incubated at room temperature for 30 min. The transfection complex was diluted in OPTI-MEM and added to the cells in 100 μL of medium supplemented with fresh medium. The transfection medium was replaced with 10% FBS / DMEM or EMEM after 6 h. 72 h after transfection, the number of viable cells was assessed using a CellTiter-Glo Luminescent cell viability assay (Promega). The value obtained from untreated cells (blank) was set to 100%. All values ​​represent the mean ± SD of four replicates: NS-non-silencing siRNA, CD-Cell Death siRNA. [Figure 15] Figure 1 shows the effect of treatment with Cell Death siRNA formulated with HKP alone or in combination with various amounts of HKP and HKC2 on the viability of human hepatocellular carcinoma HepG2 cells. Aqueous solutions of a mixture of HKC2 (160 ng / μL), HKP (320 ng / μL) and siRNA (80 ng / μL) were incubated at room temperature for 30 min. The transfection complexes were diluted in OPTI-MEM and added to the cells in 100 μL of medium supplemented with fresh medium. The transfection medium was replaced with 10% FBS / DMEM or EMEM after 6 h. 72 h after transfection, the number of viable cells was assessed using a CellTiter-Glo Luminescent cell viability assay (Promega). The value obtained from untreated cells (blank) was set to 100%. All values ​​represent the mean ± SD of four replicates: NS-non-silencing siRNA, CD-Cell Death siRNA. [Figure 16] Figure 1 shows the formulation and nanoparticle formation via self-assembly between HKC2-PEG1k-folate, H3K4b (HKP) and siRNA. TGFβ1 was used at 80 ng / μL in water and mixed with equal volumes of HKC and HKP in water. [Figure 17] 1 shows the polydispersity index of nanoparticle formation via self-assembly between HKC1-PEG-folate, H3K4b (HKP) and siRNA. Equal volumes of HKC and HKP in water were mixed with TGFβ1 (80 ng / μL in water). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0006] A novel approach is provided for tumor targeting of nucleic acids for in vitro and in vivo delivery. As used herein, histidine (H)-lysine (K) rich polypeptide (HKP) is used to describe a positively charged peptide that contains a nucleic acid binding domain and four branched repeats (H3K) that provide non-cell specific transduction functions (e.g., the ability to cross cell membranes non-selectively). Chou et al., Biomaterials, 35, 846-855 (2014). A linear peptide (abbreviated HKC) with four repeats of H3K and a terminal targeting ligand was used. This peptide contains both a nucleic acid binding domain and a cell specific targeting function, and thus may help the agent cross the cell membrane and specifically deliver nucleic acid to a particular cell type.

[0007] In some embodiments, compositions and methods are provided for delivering nucleic acids to a target cell of interest. In some embodiments, the composition comprises a branched polypeptide (HKP) and a linear peptide (HKC). In yet another embodiment, the composition comprises one or more nucleic acids. In some embodiments, the composition comprises a pharma- ceutically acceptable carrier.

[0008] In some embodiments, the 4-antennary histidine-lysine rich polypeptide is used in a formulation with a linear peptide having specific structural and functional properties that are effective carriers for a) targeting nucleic acid to one or more specific cell types, and b) delivering the targeted nucleic acid to a specific intracellular interaction site. In some embodiments, the linear peptide comprises a cell-specific targeting ligand (e.g., a small molecule or cyclic peptide-based homing domain) that is conjugated to a positively charged linear HKC peptide that provides cell orientation and transport properties that help bind to the nucleic acid and deliver the nucleic acid to the cytosol of the targeted cell.

[0009] In another aspect, a method is provided for conjugating targeting ligands for delivery to carriers by direct covalent linkage strategy. In some embodiments, targeting ligands (e.g., folic acid, RGD or peptides) have been effectively conjugated with linear histidine-lysine rich cysteine-containing (HKC) peptides through chemical reaction by forming covalent bonds. This method provides a versatile platform for introducing various targeting ligands into delivery systems for protecting target nucleic acids. The chemical conjugation between the positively charged peptide HKC and the ligands can be disulfide bonds, sulfur-carbon bonds from thiol / maleimide, or any other covalent bonds or biodegradable bonds such as hydrazine and amide, but is not necessarily limited to this type.

[0010] In yet another aspect, a novel method is provided for nanoparticle formulation of polypeptides (HKPs), which are linear peptides with targeting ligands, and siRNA for tumor targeting. In some embodiments, the nucleic acid is delivered in a complex that includes a targeting linear polypeptide and a branched polypeptide that includes a motif that binds to a cellular target. The two peptides are formulated in a nanoparticle form in a mixture with the targeting nucleic acid at a defined ratio. In some embodiments, the ratio of negative charges (e.g., from the nucleic acid) to positive charges (e.g., peptide and polypeptide) of the peptide / nucleic acid complex can affect the strength of the non-cell-specific transduction properties of the complex.

[0011] In another aspect, compositions and methods are provided for delivering one or more nucleic acids to cell targets.In some embodiments, in peptide / nucleic acid complexes, one or more nucleic acids are delivered simultaneously in nanoparticles.In some embodiments, chemotherapy drugs can be co-formulated in nanoparticle complexes.This provides both advantages and benefits to the combination for the treatment of tumors.

[0012] Thus, these and other aspects provide a delivery platform, a system that can introduce any kind of targeting motif to target any cell of interest. In some embodiments, a stepwise method of conjugating targeting ligands to peptides via linkers (e.g., PEG or polymers) has been developed and presented in this application. Various targeting ligands provide specific transduction properties for any cell type of interest. In some embodiments, the binding domain in a peptide with a positive HK repeat unit binds to negatively charged nucleic acid through hydrogen bonds between histidine and phosphate and ion-ion interactions between protonated lysine and phosphonate. The nucleic acid is protected and delivered to the targeted region of the cell of interest.

[0013] In terms of targeting ligands, peptides include cyclic (c)RGD, APRPG, (SEQ ID NO:2) , NGR, F3 peptide, CGKRK (SEQ ID NO:3), LyP-1, iRGD, iNGR, T7 peptide (HAIYPRH (SEQ ID NO:4) ), MMP2-cleavable octapeptide (GPLGIAGQ (SEQ ID NO:5) ), CP15(VHLGYAT SEQ ID NO:6 ), FSH (FSH-β, 33-53 amino acids, YTRDLVKDPARPKIQKTCTF (SEQ ID NO:7) ), LHRH(QHTSYkcLRP (SEQ ID NO:8) ), gastrin releasing peptide (GRP) (CGGNHWAVGHLM (SEQ ID NO:9) ), RVG(YTWMPENPRPGTPCDIFTNSRGKRASNG (SEQ ID NO:10) In some embodiments, the targeting ligands can be incorporated into combinations of bivalent or trivalent homo- or hetero-peptide ligands within one system for added efficacy.

[0014] Thus, aspects of the present invention provide a delivery platform that is modular and can be applied to deliver any nucleic acid to any cell of interest. In some embodiments, compositions are provided that include a multivalent peptide component and siRNA, mRNA or DNA to form nanoparticles. The complex formation effectively protects and delivers siRNA, mRNA or DNA to cells. In some embodiments, the nucleic acid is reversibly bound to the peptide carrier, which allows it to penetrate tumor-specific cells and release the nucleic acid from endosomes to reach the target gene.

[0015] The production of the siRNA delivery carrier described herein may occur by combining a branched polypeptide (HKP), a linear peptide (HKC) and an siRNA, and may be carried out by a method including the steps of: (a) preparing a positively charged linear peptide, such as a peptide HKC having a functional group for linking a targeting group or other functional moiety; (b) attaching a targeting ligand to the linear peptide HKC via covalent binding and recovery of the product; and (c) stably combining the branched polypeptide (HKP), the linear peptide HKC having the targeting ligand of step (b) and the siRNA to produce a uniform nanoparticle. In the above method, the steps may be performed simultaneously, thereby allowing for favorable interactions and nanoparticle formation. The polymeric nanoparticles from this method can effectively form complexes with various siRNAs in aqueous solution to form polynanoparticles, which can be selectively accumulated in a specific disease via a targeting effect. Preferably, the size of the polynanoparticles described herein may range from 10 nm to 3000 nm based on the production method described. According to preclinical studies, the preferred size would be 40-300 nm as determined by dynamic light scattering.

[0016] Furthermore, the HKC polypeptide-nucleic acid delivery system described herein can be used as an active ingredient of pharmaceutical composition.Therefore, a pharmaceutical composition is provided that comprises a therapeutically effective dose of HKC peptide and nucleic acid in a mixture form.This can include one or more types of pharmaceutically compatible polymers or carriers in addition to the HKC polypeptide-nucleic acid delivery system described herein together with the administration method.

[0017] The resulting product may be formulated, for example, in the form of a powder, liquid, solid phase, capsule, or injectable form, which may be mixed with one or more active ingredients, such as saline solution, buffer solution, or other compatible ingredients, to maintain the stability and effectiveness of the nucleic acid-peptide polynanoparticles.

[0018] The pharmaceutical compositions described herein may be administered in standard manner, for example by oral or parenteral administration. EXAMPLES

[0019] Example 1. Synthesis of peptides HKC1 and HKC2 The designed peptide sequence of HKC1 (sequence: KHHHKHHHKHHHKHHHKSSSC SEQ ID NO:11 ) was synthesized by solid phase synthesis apparatus as described in Figure 3. The product was purified by HPLC using water (0.065% TFA) and acetonitrile (0.05% TFA) and the HPLC chromatogram is shown in Figure 3A. The structure of H3K4C (abbreviation HKC1) has one cysteine ​​at the terminal position. The structure was further confirmed by mass spectrometry as shown in Figure 3B.

[0020] The second designed peptide sequence of HK2C (sequence: (KHHHKHHHKHHHKHHH) 2 KCSSC) was synthesized in a similar manner as shown in Figure 3B by a solid-phase synthesizer.

[0021] The third designed peptide sequence of HKC2 (sequence: KHHHKHHHKHHHKHHHKCSSC (SEQ ID NO:12) ) was synthesized on a solid phase as described, for example, in U.S. Pat. Nos. 7,070,807, 7,163,695, and 7,772,201.

[0022] Example 2. Cross-linking of HKC2 peptide via sulfide maleimide coupling reaction Figure 2 shows a general scheme of coupling. For the preparation of H3K4C-PEG-targeting ligand functionalized polypeptides, PEG functionalized with a targeting motif is used. Such PEGs with targeting motifs, such as folate, RGD and / or monoclonal antibodies, can be commercially available or pre-prepared using methods well known in the art. HKC with a terminal cysteine ​​was conjugated to maleimide-functionalized PEG-linked targeting motifs, such as (folate, RGD, mAb, etc.) via thiol / maleimide addition reaction under mild conditions as shown in Figure 2.

[0023] Example 3. Cross-linking of HKC2 peptide with folic acid The targeting ligand was attached onto the HKC peptide via the formation of a covalent bond between a thiol and a maleimide in a coupling reaction. Figure 4 shows the scheme for preparing HKC2-PEG1000-folate. Folate-PEG1000-Mal (6.0 mg, 3.7 mmol) was dissolved in anhydrous DMF (2.0 mL), followed by the addition of trimethylamine (52 uL, 0.726 g / mL) in anhydrous DMF. HKC (10.0 mg, 3.7 mmol) in a mixture of degassed water (100 μL) and DMF (300 μL) was added to the mixture at 25° C. under nitrogen by sonication and stirring. The resulting mixture was stirred at 25° C. under nitrogen in the dark for 15 h. HPLC analysis showed that the starting material, Folate-PEG1000-Mal, was completely consumed and the reaction was complete. The reaction mixture was poured into a cold diethyl ether solution to obtain a yellow precipitate. The mixture was centrifuged at 4000 rpm for 10 min and the top clear supernatant was discarded. The yellow precipitate was washed with acetone (5.0 mL) and centrifuged again to recover the product after discarding the supernatant. The product was further purified by preparative RP-HLPC or dialysis in water to obtain the pure product. The product solution was lyophilized to obtain the product as a yellow powder (12 mg, 75% yield).

[0024] Example 4. 1 Characterization of HKC2-PEG-folate by H NMR The structure of HKC2-PEG-folate is shown in DMSO-d 6 In 1 The sample was characterized by H NMR and the results are shown in FIG. 2 O or DMSO-d 6 and the nmr spectrum was recorded at 400 MHz. The three spectra were overlaid and the differences were clearly seen. 2 HKC2 in O is on the top, DMSO-d 6HKC2-PEG-folate is shown in the center, and folate-Peg1000-Mal is shown at the bottom. HKC was covalently bound to folate-Peg1000-Mal, and the characteristic signal of the maleimide double bond at 7.0 ppm disappeared after reaction with cysteine. The CH of the PEG group 2 The protons are in the 3.5 ppm region and the peptide protons are located at 6.0-9.0 ppm for HKC2-PEG-folate.

[0025] Example 5. Characterization of HKC2-PEG-Folate by UV / Vis Spectroscopy The structure of HKC2-PEG-folate was further characterized by UV / Vis spectroscopy, and the results are shown in Figure 6. UV / Vis spectroscopy of HKC2-PEG-folate (top, red curve) and folate-PEG-Mal (bottom, grey curve) was measured in water at room temperature. Characteristic absorbance at 220 nm for the peptide and 275 nm for folate was observed in the product spectrum.

[0026] Example 6. Characterization of HKC2-PEG-Folate by Mass Spectrometry MALDI-MS (positive) spectroscopic analysis of HKC2-PEG-folate was recorded using a Bruker Autoflex Speed ​​spectrometer. + The presence of a nearby molecular ion peak indicates successful conversion of HKC1 from the coupling reaction, see FIG.

[0027] Example 7. Preparation of HKC2 containing RGD ligand as HKC2-PEG2k-RGD First step: c(RGDfk) was coupled to a bifunctional PEG molecule bearing N-hydroxysuccinimide (NHS) and maleimide (Mal) functional groups to form an amide bond via coupling between the amine and the NHS ester. See Figure 8. c(RGDfk) (5.0 mg, 8.28 μmol) was dissolved in anhydrous DMF (1 mL) and triethylamine (10 μL) was added. The resulting mixture was incubated at room temperature for 2 h at 4°C for 1 h at 20°C. 2After stirring for 30 min under reduced pressure, Mal-PEG2k-NHS (10 mg, 8.28 μmol) was added in one portion and stirred for 12 h at 25° C. The reaction mixture was poured into cold diethyl ether (20 mL). The mixture was centrifuged at 4000 rpm for 10 min at 5° C. and the top clear supernatant was discarded. The white precipitate was resuspended in acetone, cold diethyl ether (10 mL) was added and sonicated for 5 min. Centrifugation again at 4000 rpm for 10 min at 5° C. allowed recovery of the white precipitate. Drying under reduced pressure gave RGD-PEG2k-Mal (12 mg, 80% yield). 1 H NMR spectrum (400 MHz, DMSO-d 6 ) showed the presence of a peak assigned to the maleimide at 7.0 ppm, but the absence of a peak for the NHS ester (2.8 ppm). See FIG.

[0028] This material was used directly in the second step, where the thiol in HKC reacted with the maleimide of RGD-PEG2k-Mal to give the RGD-linked PEG linker polypeptide HKC2-PEG2k-RGD. HKC2 (5.4 mg, 2.0 μmol) was dissolved in a mixture of DMF (0.6 mL) and degassed water (100 μL). The solution of HKC2 was added to RGD-PEG2k-Mal (5.0 mg, 1.69 μmol) dissolved in anhydrous DMF (1 mL) with stirring. Triethylamine (100 uL, 10 μg / μL in anhydrous DMF) was then added and the mixture was immersed in N 2 The mixture was stirred at 25° C. for 15 h under reduced pressure. The reaction mixture was poured into cold diethyl ether (20 mL). The mixture was centrifuged at 4000 rpm at 5° C. for 10 min, and the top clear supernatant was discarded. The crude product was dialyzed against water for 2 days with water exchange. After drying under reduced pressure, the product HKC2-PEG2k-RGD was obtained (7.1 mg, 75% yield). 1 It was characterized by spectroscopic techniques including 1 H NMR (see FIG. 10) and mass spectrometry.

[0029] Example 8. Preparation of HKC containing trivalent GalNAc ligand as HKC-PEG-GalNAc HKC1((KHHH)4KSSC (SEQ ID NO:13) ), 18.0 mg, 6.75 μmol) was dissolved in phosphate buffer, pH=7.2 in a glass vial. GalNAc3-PEG6-Mal (29.3 mg, 1.56 μmol) in anhydrous DMF (300 μL) was added to the HKC1 solution by spring needle over 5 min. The resulting mixture was stirred under nitrogen atmosphere for 16 h. After HPLC monitoring showed that the starting material GalNAc was completely consumed, the crude product was purified using a Pierce Dextran Desalting Column to obtain the pure product GalNAc3-PEG6-HKC1 as a white solid, 19 mg, 80% yield. The product was characterized by mass spectrometry (MALDI-TOF-MS positive) m / z 4595.824 [M+H], calculated MW=4595.9. HPLC analysis showed purity >90%. GalNAc-PEG12-HKC1 and GalNAc-PEG24-HKC1 were prepared in a similar manner by replacing GalNAc3-PEG6-Mal with the corresponding GalNAc-PEG12-Mal and GalNAc-PEG24-Mal (reaction scheme shown in Figure 11).

[0030] Example 9. Formulation of HKC:HKP:TGFβ1 in the formation of nanoparticles and their size distribution. HKC=HKC2=K(HHHK) 4 CSSC (SEQ ID NO:1) . HKP=H3K4b. (Figure 13). Nanoparticle formation of HKC2, HKP, and siRNA (TGFβ1) was evaluated at various ratios. The addition of HKC2 to the HKP / siRNA formulation maintained a comparable nanoparticle size, but significantly narrowed the polydispersity index (PDI) compared to the control HKP / siRNA (N:P mass ratio = 4:1). HKC2 / HKP / siRNA were formulated at mass ratios of 0:4:1, 1:4:1, 1:3:1, 2:3:1, 2:2:1, and 3:1:1. Aqueous solutions of HKC2 (160 ng / μL), HKP (320 ng / μL), and siRNA (80 ng / μL) were mixed in the specified ratios and incubated at room temperature for 30 min. The resulting samples were then measured by dynamic light scattering using a Nanoplus90. The dynamic radius and polydispersity index were recorded and are shown in Figures 11 and 12. From Figure 12, the size slightly decreased from 120 nm (HKP:siRNA=4:1) to 100-113 nm (HKC2 / HKP / siRNA=1:4:1, 1:3:1, 2:3:1). When the ratio increased to 2:2:1, 3:1:1, the nanoparticle size also increased to 140 nm and 180 nm. In another perspective, the PDI decreased from 0.22 to 0.11-0.17, which is an advantage of HKC filling the coating surface (see Figure 13).

[0031] Example 10. Effect of treatment with Cell Death siRNA formulated with HKP alone or in combination with various amounts of HKP and HKC2 on the viability of human glioma T98G cells Aqueous solutions of HKC2 (160 ng / μL), HKP (320 ng / μL) and siRNA (80 ng / μL) were mixed at a defined ratio (HKC2 / HKP / siRNA were formulated at mass ratios of 0:4:1, 0:3:1, 1:3:1, 2:3:1, 0:2:1, and 2:2:1) and incubated at room temperature for 30 min. Transfection complexes were diluted in OPTI-MEM and added to cells in 100 μL of medium supplemented with fresh medium. Transfection medium was replaced with 10% FBS / DMEM or EMEM after 6 h. 72 h after transfection, the number of viable cells was assessed by CellTiter-Glo Luminescent cell viability assay (Promega). Values ​​derived from untreated cells (blank) were set as 100%. All values ​​represent the mean ± SD of four replicates: NS-non-silencing siRNA, CD-CellDeath siRNA. Lipofectamine and HKP / siRNA (4:1) were used as positive controls. Addition of HKC2 in 2:3:1 and 2:2:1 formulations showed comparable or even better cell death in terms of cell viability compared to the controls 0:3:1 and 0:2:1 (Figure 14).

[0032] Example 11. Effect of treatment with Cell Death siRNA formulated with HKP alone or in combination with various amounts of HKP and HKC2 on the viability of human hepatocellular carcinoma HepG2 cells Aqueous solutions of a mixture of HKC2 (160 ng / μL), HKP (320 ng / μL) and siRNA (80 ng / μL) were mixed at a defined ratio (HKC2 / HKP / siRNA were formulated at mass ratios of 0:4:1, 0:3:1, 1:3:1, 2:3:1, 0:2:1, and 2:2:1) and incubated at room temperature for 30 min. Transfection complexes were diluted in OPTI-MEM and added to cells in 100 μL of medium supplemented with fresh medium. Transfection medium was replaced with 10% FBS / DMEM or EMEM after 6 h. The number of viable cells was assessed 72 h after transfection using a CellTiter-Glo Luminescent cell viability assay (Promega). Values ​​derived from untreated cells (blank) were set as 100%. All values ​​represent the mean ± SD of four replicates: NS-non-silencing siRNA, CD-CellDeath siRNA. Lipofectamine and HKP / siRNA (4:1) were used as positive controls (Figure 15). The addition of HKC2 to the 2:3:1 and 2:2:1 formulations showed similar or higher cell death percentages in terms of cell viability compared to the controls 0:3:1 and 0:2:1, but overall cell viability was higher compared to the human glioblastoma T98G cell line study.

[0033] All publications identified herein, including issued patents and published patent applications, and all database entries identified by URL address or deposit number, are hereby incorporated by reference in their entirety.

[0034] While the present invention has been described in relation to specific embodiments thereof and numerous details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the present invention is susceptible to additional embodiments and that some of the details described herein may be varied without departing from the underlying principles of the invention.

Claims

1. A linear or branched peptide comprising a nucleic acid binding domain, a polymeric spacer molecule and a cell-specific targeting ligand, wherein the nucleic acid binding domain comprises KHHHKHHHKHHHKHHHKSSSC (SEQ ID NO:11), (KHHH)4KSSC (SEQ ID NO:13), (KHHHKHHHKHHHKHHH)2KCSSC, KHHHKHHHKHHHKHHHKCSSC (SEQ ID NO:12), or K(HHHK)4CSSC (SEQ ID NO:1).

2. The peptide described in claim 1, wherein a polymer spacer molecule comprises polyethylene glycol (PEG) and is positioned between the nucleic acid binding domain and the cell-specific targeting ligand.

3. 3. The peptide of claim 1 or 2, wherein the cell-specific targeting ligand is selected from the group consisting of small molecules, peptides, proteins, antibodies and aptamers.

4. The peptide of claim 3, wherein the number of targeting ligands is 1 to 4.

5. The targeting ligands include cyclic (c)RGD, APRPG (SEQ ID NO:2), NGR, F3 peptide, CGKRK (SEQ ID NO:3), LyP-1, iRGD, iNGR, T7 peptide (HAIYPRH (SEQ ID NO:4)), MMP2-cleavable octapeptide (GPLGIAGQ (SEQ ID NO:5)), CP15 (VHLGYAT (SEQ ID NO:6)), FSH (FSH-β, 33-5 3 amino acids), YTRDLVKDPARPKIQKTCTF (SEQ ID NO: 7), LHRH (QHTSYkcLRP (SEQ ID NO: 8)), gastrin releasing peptide (GRP) (CGGNHWAVGHLM (SEQ ID NO: 9)) and RVG (YTWMPENPRPGGTPCDIFTNSRGKRASNG (SEQ ID NO: 10)).

6. The peptide described in claim 4, wherein the targeting ligand is folic acid, RGD or GalNAc.

7. A composition comprising the peptide of claim 1 and a branched polypeptide having a histidine (H) and lysine (K) rich repeating unit, wherein the branched polypeptide comprises four branches of K(HHHK)4 (SEQ ID NO: 14) or KHHHKHHHHKHHHKHHHK repeating units (SEQ ID NO: 18).

8. A composition comprising the peptide of claim 1 and a nucleic acid.

9. The composition of claim 8, wherein the nucleic acid is selected from the group consisting of siRNA, miRNA, antisense oligonucleotide, plasmid, mRNA, RNAzyme, DNAzyme and aptamer sequences.

10. The composition of claim 8 or 9, further comprising a second nucleic acid.

11. The composition of claim 10, wherein the first nucleic acid sequence is an siRNA and the second nucleic acid is an siRNA, miRNA, antisense oligo, plasmid, mRNA, RNAzyme, DNAzyme or aptamer sequence.

12. A kit for delivering a nucleic acid to a mammalian cell, comprising the peptide of claim 1 and a nucleic acid.

13. The kit of claim 12 , wherein the nucleic acid is delivered to a cell in vitro.

14. The kit of claim 12 , wherein the nucleic acid is delivered to a cell in vivo.

15. The kit of claim 12, wherein the mammalian cells are human cells.

16. 12. A medicament for treating a mammal comprising a therapeutically effective amount of a composition according to any one of claims 8 to 11.

17. The method of claim 16, wherein the mammal is a human.

18. 9. A method for producing the composition of claim 8, comprising the steps of: a) mixing the peptide of claim 1 with a nucleic acid to form a complex; b) adding a branched polypeptide having histidine (H) and lysine (K) rich repeating units, the branched polypeptide including 4 branches of K(HHHK)4 (SEQ ID NO: 14) or KHHHKHHHHKHHHKHHHK repeating units (SEQ ID NO: 18) to the mixture in a specified ratio to form nanoparticles; and c) recovering the nanoparticles.

19. The method of claim 18, wherein the nanoparticle size is between 50 and 300 nm.

20. 20. The method of claim 18 or 19, wherein the nucleic acid is an siRNA, miRNA, antisense oligo, plasmid, mRNA, RNAzyme, DNAzyme or aptamer sequence.

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