Tumor-targeted peptide nanoparticle delivery system for nucleic acid therapy

Abstract

A new nucleic acid delivery system is provided, which comprises a linear peptide rich in histidine-lysine and containing cysteine ​​with a targeting function, and a four-branched histidine-lysine-rich polypeptide. The delivery system comprises a nucleic acid, such as siRNA. These components form a stable nanoparticle complex through non-covalent interactions between the phosphate of the siRNA and the histidine / lysine of the polypeptide, which has reduced toxicity and selectively delivers genetic material to cells. The targeting function enhances the efficiency of nucleic acid delivery and transfection. A carrier molecule is also provided, which can deliver therapeutic molecules to specific cells. The carrier molecule is modified with a targeting ligand, which can bind to a specific receptor present on the targeted cell. The therapeutic molecule is siRNA, miRNA or other oligonucleotides. The targeting moiety is a small molecule, peptide or protein that shows affinity for the receptor present on the targeted cell.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application Serial No. 62 / 910,760, filed on October 4, 2019, and U.S. Provisional Patent Application Serial No. 62 / 915,450, filed on October 15, 2019, the entire contents of which are incorporated herein by reference. Technical Field

[0003] Provided are delivery systems and methods of use for nucleic acids, including methods for targeted or local delivery of nucleic acid molecules. Background Art

[0004] Targeted delivery of therapeutic agents has attracted considerable attention and holds promise for improving tumor therapy by increasing efficacy and reducing side effects. Nanoparticles (NPs) are believed to accumulate in tumors through enhanced permeability and retention (EPR) effects (Maeda, Bioconjugate Chemistry, 21:797–802 (2010)). Therefore, tumor delivery can be enhanced by coating the particles with tumor-localizing ligands. The mechanisms by which ligands increase the antitumor efficacy of their cargo, such as siRNA, remain under investigation. Enhanced NP binding to tumor surface markers can increase NP accumulation in tumors compared to nontargeted tissues. Other researchers have claimed that the accumulation of targeted and nontargeted NPs within tumor cells is comparable. It has been suggested that the enhanced efficacy of targeted NPs is due to enhanced receptor-mediated endocytosis and improved localization of siRNA therapeutics into cells. Bartlett et al., (2007): Proc. Nat'l Acad. Sci. USA, 104: 15549-15554 (2007). Most likely, both mechanisms play a crucial role in the therapeutic and therapeutic efficacy of ligand-targeted drugs.

[0005] Since siRNA is degraded by serum nucleases and quickly cleared, nanoparticles (NP) are subject to endosomal entrapment and innate immune stimulation, and targeted delivery of siRNA in vivo is challenging. Until recently, very limited methods have been developed in preclinical and clinical trials for targeted delivery of siRNA. One approach is Alnylam. It has developed a GalNAc-siRNA conjugate, in which a synthetic triantennary N-acetylgalactosamine ligand (GaLNAc) is coupled to a chemically modified siRNA. This enables efficient, ASGPR-mediated delivery to hepatocytes. Maja et al.; Nature Communications, 9:723 (2018). GaLNAc targets the hepatocyte-specific asialoglycoprotein receptor (ASGPR) in the liver. An example is Sanofi Genzyme's Fitusiran (ALN-AT3, Phase II clinical trial, Alnylam) for the treatment of hemophilia and rare bleeding disorders (RBD). It is administered subcutaneously, and RNAi therapeutics (therapeutic) are intended to target antithrombin (AT). In another case, when multiple components are co-assembled with siRNA, the targeting ligand has been incorporated into a liposome formulation. This type of system faces many challenges in terms of stability, biocompatibility, toxicity, production and large-scale long-term storage of liposomes. Leng et al., J.Drug Delivery, ID6971297, (2017). Recently, nanoparticles formed by polypeptide / polymer and siRNA have been effectively delivered in vivo to siRNA, 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 their targets to achieve therapeutic efficacy. A leading drug is being studied in a clinical phase IIa trial. See: Zhou et al., Oncotarget, 8:80651-80665 (2017); WO2011 / 140285. BRIEF DESCRIPTION OF THE DRAWINGS

[0006] Figure 1 Tumor-targeting HKC / HKP or HKP(+H) polypeptide nanoparticle delivery system. The figure illustrates the formation of tumor-targeting polypeptide nanoparticles and HKC polypeptide-siRNA nanocomplexes between (A) a branched polypeptide H3K4B(HKP) or H3K(+H)4b or (HKP(+H)) with a specific histidine / lysine sequence, (B) a linear polypeptide functionalized by terminal cysteine ​​linkage to a tumor-targeting ligand (e.g., RGD, folic acid, or SmAb), and a selected siRNA.

[0007] Figure 2 HKC-PEG-functionalized polypeptide targeting ligand (HKC = HKC1, HKC2 or HK2C, see Figure 3 HKC contains a terminal cysteine, which is coupled to a maleimide-functionalized PEG-linked targeting motif (such as folate, RGD, mAb, etc.) via a thiol / maleimide addition reaction under mild conditions.

[0008] Figure 3 The structures of a branched peptide of H3K4b (abbreviated as HKP), an H3K4C (abbreviated as HKC1 or HKC) with a single cysteine ​​at its terminal position, and HKC2 with two cysteines in its sequence. The two-branched cysteine-containing peptide HK2C has the sequence [(KHHH)4]2KXC. Figure 3 (b) discloses SEQ ID No. 15, and Figure 3 (c) discloses SEQ ID No.16.

[0009] Figure 3B HPLC chromatography of HKC was performed on a reverse phase Alltima™ column C-18 (4.6×250 mm) at RT=15.196, eluting with a gradient of >91% water (0.065% TFA) and acetonitrile (0.05% TFA).

[0010] Figure 3C The mass spectrum of HKC1 compound (ESI-MS, positively charged) is 1335.6[M] 2+ A doubly charged molecular ion peak was observed at .

[0011] Figure 4 HKC2-Peg is shown 1000 - Preparation of folic acid, HKC1 and maleimide-PEG-folic acid react under alkaline conditions through thiol / maleimide addition reaction to obtain the coupling product. The solvent is removed and then purified by dialysis to obtain HKC1-PEG 1000 -Folic acid.

[0012] Figure 5 .pass 1 H NMR characterization of HKC2-PEG1k-Folic acid. (Top) HKC2 in D2O, (Middle) HKC2-PEG1k-Folic acid in DMSO-d6, and (Bottom) Folic acid-PEG1k-Mal. HKC2 was covalently coupled to Folic acid-PEG1k-Mal. After reaction with cysteine, the signal characteristic of the maleimide double bond at 7.0 ppm disappeared.

[0013] Figure 6 The UV / Vis (water, 25° C.) spectra of aqueous solutions of HKC2-PEG1k-Folic acid (top red curve) and Folic acid-PEG1k-Mal (bottom grey curve) are shown. Characteristic absorbances of the peptide at 220 nm and folic acid at 275 nm were observed in the product spectra.

[0014] Figure 7 . Shows the MALDI-MS (positive charge) spectrum of HKC2-PEG1k-folic acid at about 4302M + The molecular ion peak at indicated that HKC2 was successfully converted from the coupling reaction.

[0015] Figure 8 The preparation of HKC2-PEG2k-RGD is a two-step process. In the first step, c(RGDfk) is coupled to a bifunctional PEG molecule (containing N-hydroxysuccinimide (NHS) and maleimide (Mal) functional groups) to form an amide bond via coupling between the amine and NHS groups. In the second step, the thiol in HKC (SEQ ID No. 17) reacts with the maleimide of RGD-PEG2000-Mal to yield the RGD-linked PEG linker peptide HKC2-PEG2000-RGD.

[0016] Figure 9 .Show by 1 H NMR spectroscopy (DMSO-d6, 25°C) characterized the RGD-PEG2k-Mal intermediate. RGD signals were observed at 8.5-7.2 ppm, maleimide signals at 7.00 ppm, and a broad peak of PEG ethylene at approximately 3.5 ppm.

[0017] Figure 10 .Show by 1 Comparison of H NMR spectroscopy stack plots characterizing HKC2-PEG-RGD and derivatives of the RGD targeting ligand of HKC2.

[0018] Figure 11 The figure shows the preparation of HKC1-PEGn-GalNAc (n=6, 12, 24) by coupling HKC1 and a trivalent GalNAc-PEG molecule (containing a maleimide (Mal) functional group) to form an SC bond via coupling between the thiol on the cysteine ​​and Mal. KHHHKHHHKHHHKHHHKSSSC is disclosed in the figure as SEQ ID No. 11.

[0019] Figure 12HKC:HKP: Formulation of TGFβ1 in nanoparticle formation and its size distribution. HKC = HKC2 = K(HHHK)4CSSC (SEQ ID No. 1), HKP = H3K4b.

[0020] Figure 13 HKC:HKP: The formulation of TGFβ1 in nanoparticle formation and its polydispersity index: HKC = HKC2 = K(HHHK)4CSSC (SEQ ID No. 1), HKP = H3K4b.

[0021] Figure 14 Effects of cell-killing siRNAs formulated with HKP alone or in combination with varying 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 defined ratios and incubated at room temperature for 30 minutes. The transfection complex was diluted with OPTI-MEM and added to the cells in 100 μL of medium supplemented with fresh medium. After 6 hours, the transfection medium was replaced with 10% FBS / DMEM or EMEM. 72 hours after transfection, the number of viable cells was assessed using the CellTiter-Glo Luminescent Cell Viability Assay (Promega). Values ​​derived from untreated cells (blank) were set to 100%. All values ​​are expressed as the mean ± SD of four replicates. NS = non-silencing siRNA, CD = cell-killing siRNA.

[0022] Figure 15.The effect of treatment with cell-killing siRNA alone formulated with HKP, or in combination with different amounts of HKP and HKC2, on the viability of human hepatocellular carcinoma HepG2 cells. An aqueous solution of a mixture of HKC2 (160 ng / μL), HKP (320 ng / μL) and siRNA (80 ng / μL) was incubated at room temperature for 30 minutes. The transfection complex was diluted with OPTI-MEM and then added to the cells in 100 μL of medium supplemented with fresh medium. After 6 hours, the transfection medium was replaced with 10% FBS / DMEM or EMEM. 72 hours after transfection, the number of viable cells was assessed using the CellTiter-Glo luminescent cell viability assay (Promega). The value derived from untreated cells (blank) was set to 100%. All values ​​are expressed as the mean ± SD of four replicates, NS-non-silencing siRNA, CD-cell-killing siRNA.

[0023] Figure 16 Nanoparticles were formulated and formed by self-assembly between HKC2-PEG1k-folate, H3K4b (HKP), and siRNA. TGFβ1 was used at 80 ng / μL in aqueous solution and mixed with equal volumes of HKC and HKP aqueous solutions.

[0024] Figure 17 Polydispersity index of nanoparticles formed by self-assembly between HKC1-PEG-folate, H3K4b (HKP) and siRNA TGFβ1 (80 ng / μL in aqueous solution) was mixed with equal volumes of HKC and HKP aqueous solutions. Summary of the Invention

[0025] A novel method for delivering tumor-targeting nucleic acids in vitro and in vivo is provided. As used herein, histidine (H)-lysine (K)-rich polypeptide (HKP) is used to describe a positively charged peptide with four branched repeating (H3K)4 units, which includes a nucleic acid binding domain and provides a non-cell-specific transduction function (e.g., the ability to non-selectively pass through the cell membrane). Chou et al., Biomaterials, 35, 846-855 (2014). A linear peptide (abbreviated as HKC) with four repeating units of H3K and a targeting ligand located at the terminal site is used. The peptide includes a nucleic acid binding domain and a cell-specific targeting function, so it can help materials pass through the cell membrane and specifically deliver nucleic acids to specific cell types.

[0026] In some embodiments, compositions and methods for delivering nucleic acids to target cells are provided. In some embodiments, the composition comprises a branched polypeptide (HKP) and a linear peptide (HKC). In another embodiment, the composition comprises one or more nucleic acids. In some embodiments, the composition comprises a pharmaceutically acceptable carrier.

[0027] In some embodiments, a tetra-branched histidine-lysine-rich polypeptide is used in a formulation, wherein the linear peptide has certain structural and functional properties to serve as an efficient carrier for: a) targeting nucleic acids to one or more specific cell types, and b) delivering the targeted nucleic acid to a specific intracellular location. In some embodiments, the linear peptide contains a cell-specific targeting ligand (e.g., a small molecule or a homing domain based on a cyclic peptide), which is coupled to a positively charged linear HKC peptide that can bind to the nucleic acid and provide cell guidance and transport properties, thereby helping to deliver the nucleic acid to the cytoplasm of the targeted cell.

[0028] In other aspects, a method for coupling a targeting ligand to a delivery vector via a direct covalent attachment scheme is provided. In some embodiments, a targeting ligand (e.g., folic acid, RGD, or peptide) is effectively coupled by chemical reaction to form a covalent bond with a linear histidine-lysine-rich cysteine ​​(HKC) peptide. This method provides a general platform for introducing various targeting ligands into a delivery system to protect target nucleic acids. The chemical coupling between the positively charged peptide HKC and the ligand can be a disulfide bond, a sulfur-carbon bond of thiol / maleimide, or any other covalent bond or biodegradable bond (e.g., hydrazine and amide), but is not necessarily limited to this type.

[0029] In other aspects, novel methods for targeting tumors using nanoparticle formulations of polypeptides (HKPs), linear peptides containing targeting ligands, and siRNA are provided. In some embodiments, nucleic acids are delivered as complexes comprising targeted linear polypeptides (containing a motif that binds to a cellular target) and branched polypeptides. The two peptides are mixed at a defined ratio and formulated with the target nucleic acid into nanoparticles. In some embodiments, the ratio of negative charge (e.g., from the nucleic acid) to positive charge (e.g., in the peptide and polypeptide) of the peptide / nucleic acid complex can affect the strength of the non-cell-specific transduction properties of the complex.

[0030] In other aspects, compositions and methods for delivering one or more nucleic acids to cellular targets are provided. In some embodiments, one or more nucleic acids are simultaneously delivered in nanoparticles within a peptide / nucleic acid complex. In some embodiments, chemotherapeutic drugs can be co-formulated within the nanoparticle complex. This provides advantages and benefits for combined therapies for treating tumors.

[0031] In some embodiments, the present invention provides a delivery platform, which is a system that can introduce any type of targeting motif to target any cell of interest. In some embodiments, a stepwise method for coupling targeting ligands to peptides by a joint (e.g., PEG or polymer) has been developed and provided in the present application. Various targeting ligands provide the characteristic that can be specifically transduced to any cell of interest. In some embodiments, the binding domain of the positively charged repeat unit with HK in the peptide is combined with negatively charged nucleic acid by hydrogen bond (between histidine and phosphoric acid) and ion-ion interaction (between protonated lysine and phosphonic acid). Nucleic acid is protected and delivered to the region of the targeted cell of interest.

[0032] In terms of targeting ligands, the peptide can be 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, targeting ligands can be incorporated into a bivalent or trivalent combination of homologous or heterologous peptide ligands in a system to achieve better therapeutic efficacy.

[0033] Therefore, aspects of the present invention provide a kind of modular delivery platform, it can be applicable to any nucleic acid delivery to any target cell.In some embodiments, provide and comprise multivalent peptide component and siRNA, mRNA or DNA composition, it forms nanoparticle.The formation of complex effectively protects siRNA, mRNA or DNA, and delivers it to cell.In some embodiments, nucleic acid and peptide carrier reversibly combine, and this allows carrier to penetrate into specific tumor cell, and releases nucleic acid from endosome to reach the target gene of nucleic acid.

[0034] The production of siRNA delivery vectors as described herein can be performed by combining a branched polypeptide (HKP), a linear peptide (HKC), and siRNA, and can be carried out by a method comprising the following steps: (a) preparing a positively charged linear peptide, such as a peptide HKC, having a functional group for attaching a targeting group or other functional moiety; (b) covalently attaching a targeting ligand to the linear peptide HKC, and recovering the product; (c) stably combining the branched polypeptide (HKP), the linear peptide HKC carrying the targeting ligand in step (b), and the siRNA to produce homogenous nanoparticles. In the above method, these steps can also be performed simultaneously to allow for better interaction and nanoparticle formation. The plyometric nanoparticles obtained by this method effectively form a composition with various siRNAs in aqueous solution to form poly nanoparticles, which can selectively accumulate in specific diseases through targeting. Preferably, based on the production method, the size of the poly nanoparticles described herein can range from 10 nm to 3000 nm. Based on preclinical studies, the preferred size determined by dynamic light scattering is 40 nm to 300 nm.

[0035] Furthermore, the HKC polypeptide-nucleic acid delivery system described herein can be used as an active ingredient in a pharmaceutical composition. Thus, a pharmaceutical composition comprising a therapeutically effective dose of an HKC peptide and a nucleic acid in a mixed form is provided. In addition to the HKC polypeptide-nucleic acid delivery system described herein, it can also include one or more pharmaceutically compatible polymers or carriers, as well as methods for administering the same.

[0036] The resulting product can be formulated into forms such as powder, liquid, solid, capsule, injectable, etc., which can be mixed with one or more effective components (such as saline solution, buffer solution or other compatible components) to maintain the stability and effectiveness of the nucleic acid-peptide polymer nanoparticles.

[0037] The pharmaceutical compositions described herein can be administered by standard methods, including oral or parenteral administration. DETAILED DESCRIPTION

[0038] Example:

[0039] Example 1. Synthesis of peptides HKC1 and HKC2.

[0040] The designed HKC1 peptide sequence (sequence: KHHHKHHHKHHHKHHHKSSSC (SEQ ID No. 11)) was synthesized by a solid-state synthesizer, as Figure 3 The product was purified by HPLC using water (0.065% TFA) and acetonitrile (0.05% TFA). The HPLC chromatogram is shown in Figure 3A. The structure of H3K4C (abbreviated as HKC1) has a cysteine ​​at the terminal position. The structure was further verified by mass spectrometry, such as Figure 3B shown.

[0041] The second designed peptide sequence of HK2C (sequence: (KHHHKHHHKHHHKHHH)2KCSSC) was synthesized by a solid-state synthesizer in a similar manner, as Figure 3B shown.

[0042] The third designed peptide sequence of HKC2 (sequence: KHHHKHHHKHHHKHHHKCSSC (SEQ ID No. 12)) was synthesized by a solid-state synthesizer, such as described in US Pat. Nos. 7,070,807, 7,163,695, and 7,772,201.

[0043] Example 2. Cross-linking of HKC2 peptides via sulfonated maleimide coupling reaction

[0044] Figure 2 The general scheme for coupling is shown. To prepare functionalized polypeptides of H3K4C-PEG-targeting ligands, functionalized PEG with a targeting motif is used. Such PEG with a targeting motif (such as folic acid, RGD and / or monoclonal antibodies) is commercially available or can be pre-prepared using methods known in the art. Figure 2 As shown, HKC with a terminal cysteine ​​was coupled to a maleimide-functionalized PEG-linked targeting motif (e.g., folate, RGD, mAb, etc.) via a thiol / maleimide addition reaction under mild conditions.

[0045] Example 3. Cross-linking of HKC2 peptide with folic acid.

[0046] The targeting ligand was installed on the HKC peptide by forming a covalent bond between the thiol and maleimide in a coupling reaction. Figure 4A scheme for preparing HKC2-PEG1000-Folic Acid is shown. Folic Acid-PEG1000-Mal (6.0 mg, 3.7 mmol) was dissolved in anhydrous DMF (2.0 mL), followed by the addition of trimethylamine in anhydrous DMF (52 μL, 0.726 g / mL). A mixture of HKC (10.0 mg, 3.7 mmol) in degassed water (100 μL) and DMF (300 μL) was added to the mixture under nitrogen at 25°C with sonication and stirring. The resulting mixture was stirred in the dark at 25°C under nitrogen for 15 hours. HPLC analysis indicated that the starting material, Folic Acid-PEG1000-Mal, was completely consumed, indicating the reaction was complete. The reaction mixture was poured into a cold diethyl ether solution, producing a yellow precipitate. The mixture was centrifuged at 4000 rpm for 10 minutes, and the clear supernatant was discarded. The yellow precipitate was washed with acetone (5.0 mL), and the supernatant was discarded and centrifuged again to collect the product. The product was further purified by preparative RP-HLPC or dialysis in water to obtain the pure product.The product solution was lyophilized to afford the product as a yellow powder (12 mg, 75% yield).

[0047] Example 4. By 1 H NMR characterization of HKC2-PEG-folic acid.

[0048] The structure of HKC2-PEG-folic acid was determined in DMSO-d6 by 1 H NMR characterization, the results are as follows Figure 5 As shown. Approximately 5 mg of sample was dissolved in D2O or DMSO-d6, and the NMR spectra were recorded at 400 MHz. The three spectra were superimposed to clearly see the differences. HKC2 in D2O is at the top, HKC2-PEG-folic acid in DMSO-d6 is in the middle, and folic acid-Peg1000-Mal is present at the bottom. HKC is covalently coupled to folic acid-Peg1000-Mal, and the characteristic signal of the maleimide double bond at 7.0 ppm disappears after reaction with cysteine. The CH2 protons of the PEG group are present in the 3.5 ppm region, and the peptide protons are located at 6.0-9.0 ppm in HKC2-PEG-folic acid.

[0049] Example 5. Characterization of HKC2-PEG-Folic Acid by UV / Vis Spectroscopy.

[0050] The structure of HKC2-PEG-folic acid was further characterized by UV / Vis spectroscopy. Figure 6 As shown. The UV / Vis spectra of HKC2-PEG-Folic Acid (top red curve) and Folic Acid-PEG-Mal (bottom gray curve) were measured in water at room temperature. Characteristic absorbances of the peptide at 220 nm and folic acid at 275 nm were observed in the product spectra.

[0051] Example 6. Characterization of HKC2-PEG-Folic Acid by Mass Spectrometry.

[0052] The MALDI-MS (positive charge) spectra of HKC2-PEG-folate were recorded using a Bruker Autoflex Speed ​​spectrometer. + The presence of a molecular ion peak nearby indicates successful conversion of HKC1 from the coupling reaction. Figure 7 .

[0053] Example 7. Preparation of HKC2 containing RGD ligand as HKC2-PEG2k-RGD

[0054] Step 1: Coupling of c(RGDfk) with a bifunctional PEG molecule (carrying N-hydroxysuccinimide (NHS) and maleimide (Mal) functional groups) to form an amide bond via coupling between amine and NHS ester. 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 stirred at room temperature under N2 for 30 minutes, after which Mal-PEG2k-NHS (10 mg, 8.28 μmol) was added in a portion and stirred at 25°C for 12 hours. The reaction mixture was poured into cold diethyl ether (20 mL). The mixture was centrifuged at 4000 rpm at 5°C for 10 minutes, and the clear supernatant at the top was discarded. The white precipitate was resuspended in acetone, cold diethyl ether (10 mL) was added, and then sonicated for 5 minutes. The white precipitate was collected by centrifugation again at 4000 rpm at 5°C for 10 minutes. RGD-PEG2k-Mal (12 mg, 80% yield) was obtained by drying under vacuum. 1 The H NMR spectrum (400 MHz, DMSO-d6) showed a peak corresponding to maleimide at 7.0 ppm, but no peak corresponding to NHS ester (2.8 ppm). Figure 9 .

[0055] This material was used directly in the second step, in which the thiol in HKC reacted with the maleimide of RGD-PEG2k-Mal to provide the RGD-linked PEG linker peptide 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 HKC2 solution was added to RGD-PEG2k-Mal (5.0 mg, 1.69 μmol) dissolved in anhydrous DMF (1 mL) with stirring. Triethylamine (100 μL, 10 μg / μL in anhydrous DMF) was then added, and the mixture was stirred at 25°C under N2 for 15 hours. The reaction mixture was poured into cold diethyl ether (20 mL). The mixture was centrifuged at 4000 rpm at 5°C for 10 minutes, and the clear supernatant was discarded. The crude product was dialyzed against water for 2 days, with the water replaced. After vacuum drying, the product HKC2-PEG2k-RGD (7.1 mg, 75% yield) was obtained. 1 H NMR (see Figure 10 The products were characterized by spectroscopic methods including HPLC and mass spectrometry.

[0056] Example 8. Preparation of HKC containing trivalent GalNAc ligand as HKC-PEGn-GalNAc.

[0057] HKC1 ((KHHH)4KSSC (SEQ ID No. 13)), 18.0 mg, 6.75 μmol) was dissolved in pH 7.2 phosphate buffer in a glass vial. GalNAc3-PEG6-Mal (29.3 mg, 1.56 μmol) in anhydrous DMF (300 μL) was added to the HKC1 solution via syringe over 5 minutes. The resulting mixture was stirred under a nitrogen atmosphere for 16 hours. After HPLC monitoring indicated complete consumption of the starting material GalNAc, the crude product was purified using a Pierce Dextran desalting column to yield pure GalNAc3-PEG6-HKC1 (19 mg, 80% yield) as a white solid. The product was characterized by mass spectrometry (MALDI-TOF-MS positive charge) m / z 4595.824 [M+H], calculated MW = 4595.9. HPLC analysis indicated a 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 see Figure 11 ).

[0058] Example 9. Formulation of HKC:HKP:TGFβ1 in nanoparticle formation and its size distribution. HKC = HKC2 = K(HHHK)4CSSC (SEQ ID No. 1). HKP = H3K4b. ( Figure 13 ).

[0059] Nanoparticle formation of HKC2, HKP and siRNA (TGFβ1) at various ratios was evaluated. Compared with the control HKP / siRNA (N:P mass ratio = 4:1), the addition of HKC2 to the HKP / siRNA formulation maintained similar nanoparticle size but significantly reduced the polydispersity index (PDI). HKC2 / HKP / siRNA was formulated at mass ratios of 0:4:1, 1:4:1, 1:3:1, 2:3:1, 2:2:1, 3:1:1. Aqueous solutions of HKC2 (160 ng / μL), HKP (320 ng / μL) and siRNA (80 ng / μL) were mixed at a defined ratio and incubated at room temperature for 30 minutes. The resulting samples were then measured by dynamic light scattering using a Nanoplus 90. The dynamic radius and polydispersity index were recorded as Figure 11 and Figure 12 As shown. Figure 12 It can be seen that the size slightly decreases 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 increases to 2:2:1 and 3:1:1, the nanoparticle size also increases to 140 nm and 180 nm. From another perspective, the PDI decreases from 0.22 to 0.11-0.17, which is the benefit of HKC filling coverage (see Figure 13 ).

[0060] Example 10. Effects of cell apoptosis siRNA alone formulated with HKP or in combination with different amounts of HKP and HKC2 on the viability of human glioblastoma T98G cells.

[0061] Aqueous solutions of HKC2 (160 ng / μL), HKP (320 ng / μL), and siRNA (80 ng / μL) were mixed at defined ratios (HKC2 / HKP / siRNA were prepared 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 minutes. The transfection complex was diluted in OPTI-MEM and then added to the cells in 100 μL of medium supplemented with fresh medium. After 6 hours, the transfection medium was replaced with 10% FBS / DMEM or EMEM. 72 hours after transfection, the number of viable cells was assessed using the CellTiter-Glo luminescent cell viability assay (Promega). Values ​​derived from untreated cells (blank) were set to 100%. All values ​​are expressed as the mean ± SD of four replicates. NS = non-silencing siRNA, CD = cell-killing siRNA. Lipofectamine and HKP / siRNA (4:1) were used as positive controls. The addition of HKC2 in the 2:3:1 and 2:2:1 formulations showed comparable or even higher cell death in terms of cell viability compared to the controls 0:3:1 and 0:2:1 ( Figure 14 ).

[0062] Example 11. Effects of cell apoptosis siRNA alone and in combination with HKP, or in combination with different amounts of HKP and HKC2, on the viability of human hepatocellular carcinoma HepG2 cells.

[0063] An aqueous solution of a mixture of HKC2 (160 ng / μL), HKP (320 ng / μL) and siRNA (80 ng / μL) was mixed at a defined ratio (HKC2 / HKP / siRNA was prepared at mass ratios of 0:4:1, 0:3:1, 1:3:1, 2:3:1, 0:2:1, 2:2:1) and incubated at room temperature for 30 minutes. The transfection complex was diluted with OPTI-MEM and then added to the cells in 100 μL of medium supplemented with fresh medium. After 6 hours, the transfection medium was replaced with 10% FBS / DMEM or EMEM. 72 hours after transfection, the number of living cells was assessed using the CellTiter-Glo luminescent cell viability assay (Promega). The value derived from untreated cells (blank) was set to 100%. All values ​​are expressed as the mean ± SD of four replicates, NS-non-silencing siRNA, CD-cell-killing siRNA. Lipofectamine and HKP / siRNA (4:1) were used as positive controls ( Figure 15Addition of HKC2 in the 2:3:1 and 2:2:1 formulations showed comparable or even higher percentages of cell death in terms of cell viability compared to the controls 0:3:1 and 0:2:1, although overall cell viability was higher than that of the human glioblastoma T98G cell line.

[0064] All publications identified herein, including issued patents and published patent applications, and all database entries identified by URL addresses or accession numbers, are incorporated by reference in their entirety.

[0065] Although the present invention has been described in conjunction with certain embodiments thereof and many details have been set forth for illustrative purposes, it will be apparent to those skilled in the art that the present invention may have other embodiments and that some of the details described herein may be changed without departing from the basic principles of the invention.

Claims

1. A peptide, characterized in that The peptide comprises a nucleic acid binding domain and a cell-specific targeting ligand, the sequence of the peptide is KHHHKHHHKHHHKHHHKCSSC, and the cell-specific targeting ligand is connected to the C-terminal cysteine ​​of the peptide sequence.

2. The peptide according to claim 1, characterized in that The cell-specific targeting ligand is covalently linked to the C-terminal cysteine ​​of the peptide sequence via a sulfur-carbon bond.

3. The peptide according to claim 2, characterized in that The peptide and the cell-specific targeting ligand are connected via a spacer molecule, which comprises polyethylene glycol.

4. The peptide according to claim 1, characterized in that The targeting ligand includes folic acid, RGD or galactose.

5. The peptide according to claim 4, characterized in that The number of the targeting ligands is 1 to 4.

6. A composition, characterized in that The method comprises the peptide of any one of claims 1 to 5 and a branched polypeptide having repeating units rich in histidine (H) and lysine (K), wherein the branched polypeptide comprises four branched K(HHHK)4 (SEQ ID No. 14) or KHHHKHHHHKHHHKHHHK-repeating units (SEQ ID No. 18).

7. The composition according to claim 6, characterized in that The branched polypeptide is selected from HKP and HKP(+H), and the structural formula of HKP is: , where R = KHHHKHHHKHHHKHHHK; The structural formula of the HKP(+H) is: , where R = KHHHKHHHHKHHHKHHHK.

8. A composition, characterized in that It comprises the peptide according to any one of claims 1 to 5 and a nucleic acid.

9. The composition according to claim 8, characterized in that The nucleic acid includes siRNA.

10. A method for preparing a composition containing nucleic acid, characterized in that: It includes the steps of: a) mixing the peptide of any one of claims 1 to 5 with a nucleic acid to form a complex, b) adding a branched polypeptide having repeating units rich in histidine (H) and lysine (K) to the mixture in a defined ratio to form nanoparticles, and c) recovering the nanoparticles; The branched polypeptide comprises four branched K(HHHK)4 (SEQ ID No. 14) or KHHHKHHHHKHHHKHHHK-repeating units (SEQ ID No. 18).

11. The method according to claim 10, wherein The nanoparticles have a size of 50 to 300 nm.

12. The method according to claim 10, characterized in that The nucleic acid includes siRNA.

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