Drug delivery carriers and pharmaceutical formulations that use them to co-deliver multiple therapeutic agents.

Abstract

To provide a novel drug delivery carrier.SOLUTION: There is provided a drug delivery carrier comprising: a cationic liposome modified with a cell permeable peptide; and a cationic material which is selected from a polyamino acid which charges positive, a PEI, a penetratin or a derivative of penetratin and a PAMAM. There are also provided: use of the drug delivery carrier for delivering one kind or many kinds of therapeutic agents; a pharmaceutical preparation which is prepared using the drug delivery carrier and delivers in common, many kinds of the therapeutic agents; and a method for preparing the pharmaceutical preparation for delivering in common the many kinds of therapeutic agents using the drug delivery carrier. The pharmaceutical preparation has a grain diameter of 50 nm to 300 nm, has stability, and can simultaneously deliver many kinds of therapeutic agents to a same lesion site for exhibiting a synergistic therapeutic effect of many kinds of therapeutic agents and significantly enhancing the therapeutic effect.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This invention relates to a drug delivery carrier, a method for preparing a pharmaceutical formulation using the drug delivery carrier, and a pharmaceutical formulation prepared thereby. Specifically, the drug delivery carrier of the present invention comprises a cationic liposome modified with a cell-permeable peptide containing a cell-permeable peptide conjugated with polyethylene glycol phospholipid, and a cationic material. Using the drug delivery carrier of the present invention, a pharmaceutical formulation can be prepared that co-delivers multiple therapeutic agents. The pharmaceutical formulation has a particle size of 50 nm to 300 nm, good stability, and can simultaneously deliver multiple therapeutic agents to the same lesion site, thereby exhibiting a synergistic therapeutic effect and significantly improving the therapeutic effect. [Background technology]

[0002] Cell-penetrating peptides (CPPs) are short peptides that are positively charged under physiological conditions. They can conjugate or unconjugate to drug molecules such as genes, polypeptides, and proteins, delivering the drug molecules into cells or allowing them to carry drug molecules and pass through biological membrane barriers. However, a single cell-penetrating peptide has a very limited ability to carry drug molecules, resulting in complexes formed by cell-penetrating peptides and drug molecules that are large in size and have poor stability.

[0003] Traditionally, carriers for delivering drug molecules (e.g., genes, polypeptides, and proteins), such as liposomes, have problems such as poor permeability to biological membrane barriers, low delivery efficiency, and failure to achieve desired therapeutic effects. In the field of liposome research, a major challenge remains how to improve the permeability of liposomes to biological membrane barriers and encapsulate multiple different therapeutic drugs simultaneously within liposomes, in order to deliver multiple types of drug molecules more efficiently to the same lesion site, thereby enhancing the therapeutic effect of the disease through the synergistic action of multiple drugs. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] Chinese Patent Application Publication No. 108976288 Specification [Overview of the project] [Problems that the invention aims to solve]

[0005] Therefore, in this field, there is a need for novel drug delivery carriers that utilize cell-permeable peptides to enhance therapeutic effects by co-delivering multiple types of therapeutic agents and allowing them to exert synergistic therapeutic effects after simultaneous delivery to the same lesion site. [Means for solving the problem]

[0006] The inventors have developed a novel drug delivery carrier utilizing cell-permeable peptides through diligent research. The drug delivery carrier of the present invention comprises a cationic liposome modified with a cell-permeable peptide, which includes a cell-permeable peptide conjugated to polyethylene glycol phospholipid, and a cationic material. The cationic material is selected from positively charged polyamino acids, polyethyleneimine (PEI), penetratin or a derivative of penetratin, and polyamide-amine (PAMAM).

[0007] In one embodiment, the cell-permeable peptide in the drug delivery carrier of the present invention is penetratin or a lipophilic derivative of penetratin having the following amino acid sequence. RX1IKIWFX2X3RRMKWKK (Sequence ID 1)

[0008] Here, X1, X2, and X3 are independently selected from the naturally occurring amino acids glutamine (Q), asparagine (N), alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), tryptophan (W), methionine (M), and the non-naturally occurring amino acids α-aminobutyric acid, α-aminopentanoic acid, α-aminohexanoic acid, and α-aminoheptanoic acid. For example, some cell-permeable peptides in the drug delivery carrier of the present invention are penetratin derivatives in which the glutamine (Q) at position 2 and / or glutamine (Q) at position 8 and / or asparagine (N) at position 9 are mutated with hydrophobic amino acids.

[0009] In one embodiment, the liposomes modified with a cell-permeable peptide in the drug delivery carrier of the present invention include the following membrane material.

[0010] (i) Cationic lipids include, but are not limited to, 1,2-di-0-octadecenyl-3-trimethylammonium-propane (DOTMA), dimethyldioctadecylammonium (DDAB), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), 1,2-diacyloxy-3-dimethylammonium-propane, 1,2-dialkoxy-3-dimethylammonium-propane, dioctadecyldimethylammonium chloride (DODAC), 1,2-dimyristoyloxypropyl-1,3-dimethylhydroxyethylammonium (DMRIE), 2,3-dioleyloxy-N-[2(sperminecarboxamide)ethyl]-NN-dimethyl-1-propaneammonium trifluoroacetate (DOSPA) and combinations thereof. Preferably, DOTMA, DOTAP, DODAC, and DOSPA. Most preferably, DOTAP; (ii) Noncationic lipids include, but are not limited to, 1,2-di-(9Z-octadecanoyl)-sn-glyceryl-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glyceryl-3-phosphocholine (DOPC), 1,2-distearoyl-sn-glyceryl-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glyceryl-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glyceryl-3-phosphoethanolamine (DPPE), 1,2-dimyristoyl-sn-glyceryl-3-phosphoethanolamine (DMPE), 2-dioleoyl-sn-glyceryl-3-phosphate-(1'-rac-glycerol) (DOPG) and combinations thereof. Preferably, DOPE and / or DOPC. Most preferably, DOPE; (iii) cholesterol; and (iv) polyethylene glycolated (PEGylated) phospholipids and polyethylene glycol phospholipids conjugated with CPP; the phospholipids are, for example, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and sphingomyelin, and preferably the PEGylated phospholipids are, for example, polyethylene glycol-distearoylphosphatidylethanolamine (PEG-DSPE) and its derivatives methoxy-polyethylene glycol-distearoylphosphatidylethanolamine (mPEG-DSPE), and the polyethylene glycol phospholipids conjugated with CPP are, for example, penetratin / penetratin derivative-PEG-DSPE.

[0011] In the present invention, cationic liposomes not modified with cell-permeable peptides may be abbreviated as cationic liposomes (abbreviation: CLS), and include the above membrane materials (i), (ii), and (iii).

[0012] In some embodiments, the molar ratio of membrane materials (i):(ii):(iii):(iv) is approximately 20-40:20-40:20-40:1-20, and the molar ratio of polyethylene glycol phospholipid conjugated with CPP to membrane material (iv) is approximately 20%-80%. In some specific embodiments, the molar ratio of membrane materials (i):(ii):(iii):(iv) is approximately 27.0-31.6:27.0-31.6:31.6-39.6:1-10, and the molar ratio of polyethylene glycol phospholipid conjugated with CPP to membrane material (iv) is approximately 20%-80%.

[0013] In some specific embodiments, the cationic liposomes modified with cell-permeable peptides in the drug delivery carrier of the present invention are (i) DOTAP; (ii) DOPE; (iii) Cholesterol; (iv) mPEG as the membrane material. 2000 -DSPE and 89W Penetratin-PEG 3400-Containing DSPE, and the molar ratio of membrane materials (i):(ii):(iii):(iv) is about 20-40:20-40:20-40:1-20, and with respect to membrane material (iv) 89W Penetratin-PEG 3400 -The molar ratio of DSPE is about 20%-80%.

[0014] In some embodiments, the molar ratio of membrane materials (i):(ii):(iii):(iv) is about 27.0-31.6:27.0-31.6:31.6-39.6:1-10, and with respect to membrane material (iv) 89W Penetratin-PEG 3400 -The molar ratio of DSPE is about 20%-80%.

[0015] In some embodiments, the cationic liposome in the drug delivery carrier of the present invention uses, as membrane materials, (i) DOTAP; (ii) DOPE; (iii) cholesterol; (iv) mPEG 2000 -DSPE and 89W Penetratin-PEG 3400 -DSPE, and the molar ratio of membrane materials (i):(ii):(iii):(iv) is about 28.5:28.5:38:5, and with respect to membrane material (iv) 89W Penetratin-PEG 3400 -The molar ratio of DSPE is about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%.

[0016] In another aspect, the present invention provides a pharmaceutical preparation for co-delivering multiple types of therapeutic agents, the multiple types of therapeutic agents include at least one non-nucleic acid therapeutic agent and at least one nucleic acid therapeutic agent, and it is prepared using the drug delivery carrier of the present invention.

[0017] In one embodiment, a cationic material as a component contained in the drug delivery carrier and at least one nucleic acid therapeutic agent are mixed to form a physical complex composed of the cationic material - at least one nucleic acid therapeutic agent.

[0018] Liposomes containing at least one non-nucleic acid therapeutic agent were prepared using a cationic liposome membrane material and at least one non-nucleic acid therapeutic agent.

[0019] A lipid complex was prepared by mixing a physical complex consisting of the cationic material and at least one nucleic acid therapeutic agent with liposomes containing at least one non-nucleic acid therapeutic agent.

[0020] The aforementioned lipid complex was modified with a cell-permeable peptide to prepare the pharmaceutical formulation. For example, the lipid complex was mixed with polyethylene glycol (PEGylated) phospholipid and polyethylene glycol phospholipid conjugated with a cell-permeable peptide to prepare the pharmaceutical formulation.

[0021] In one embodiment, a cationic material as a component contained in a drug delivery carrier was mixed with at least one nucleic acid therapeutic agent to form a physical composite consisting of the cationic material and at least one nucleic acid therapeutic agent.

[0022] Using all the membrane materials of cationic liposomes modified with cell-permeable peptides and at least one non-nucleic acid therapeutic agent, liposomes encapsulating at least one non-nucleic acid therapeutic agent were prepared.

[0023] The pharmaceutical formulation was prepared by mixing a physical complex consisting of the cationic material and at least one nucleic acid therapeutic agent with liposomes containing at least one non-nucleic acid therapeutic agent.

[0024] The at least one non-nucleic acid therapeutic agent to be delivered includes, but is not limited to, chemotherapeutic agents such as platinum-based drugs (e.g., cisplatin, carboplatin, oxaliplatin), taxane-based drugs (e.g., paclitaxel, docetaxel (DTX)), etoposide, irinotecan, pemetrexand, gemcitabine, melphalan, carmatine (BCNU), doxorubicin (DOX), bortezomib, methotrexate, imatinib, bleomycin, and vinca alkaloids (e.g., vinblastine).

[0025] The at least one nucleic acid therapeutic agent to be delivered is not particularly limited and includes nucleic acids such as plasmid DNA, RNA such as small interfering RNA (siRNA), miRNA, sense RNA, antisense oligonucleotide (ASO), aptamers, and ribozymes. For example, the nucleic acid therapeutic agent is RNA for brain diseases such as brain tumors (e.g., gliomas), and for example, siRNA for c-myc.

[0026] In one embodiment, the molar ratio of the non-nucleic acid therapeutic agent (e.g., chemotherapeutic agent) to the cationic lipid in the cationic liposome (e.g., DOTAP) in the pharmaceutical formulation is about 1:1500 to 2000:1, for example, about 1:1200, 1:1000, 1:500, 1:200, 1:100, 1:5, 5:1, 100:1, 200:1, 500:1, 1000:1, 1200:1, 1400:1, 1600:1, and 1800:1, preferably about 1:1000 to 2000:1, and more preferably about 1:500 to 500:1.

[0027] In one embodiment, the charge ratio of the cationic material (e.g., oligoarginine, PEI, penetratin or a derivative of penetratin, PAMAM) to the nucleic acid therapeutic agent in the pharmaceutical formulation is 1:1 to 30:1, for example, 1:1, 5:1, 10:1, 15:1, 20:1, 25:1, or 30:1.

[0028] In one embodiment, the charge ratio of cationic liposomes to nucleic acid therapeutic agents in the pharmaceutical formulation is 1:1 to 30:1, for example, 2:1, 3:1, 4:1, 5:1, 6:1, 8:1, 10:1, and 12:1.

[0029] In one embodiment, the amount of the non-nucleic acid therapeutic agent (e.g., chemotherapeutic agent) encapsulated in the pharmaceutical formulation is approximately 0.01% to 40% (w / w), preferably approximately 0.02% to 25% (w / w), of the pharmaceutical formulation. In one specific embodiment, the amount of the pharmaceutical formulation encapsulated in docetaxel (DTX) is 0.04% w / w.

[0030] In one embodiment, the charge ratio between the cationic material in the pharmaceutical formulation (e.g., oligoarginine, PEI, penetratin or a derivative of penetratin, PAMAM) and the nucleic acid therapeutic agent to be delivered is 1:1 to 30:1, preferably 5:1, and the charge ratio between the cationic liposome and the nucleic acid therapeutic agent is 1:1 to 30:1, preferably 4:1.

[0031] In one embodiment, the pharmaceutical formulation of the present invention has a particle size of 50 nm to 300 nm, preferably 80 nm to 150 nm, and is highly stable, can provide better protection to various therapeutic agents it contains, and is also advantageous for delivering siRNA and drugs to deep tissues at the lesion site.

[0032] In yet another aspect, the present invention provides a method for preparing a pharmaceutical formulation, comprising: (a) mixing a cationic material as a component contained in the drug delivery carrier of the present invention with at least one nucleic acid therapeutic agent to form a physical complex consisting of the cationic material and at least one nucleic acid therapeutic agent; (b) preparing liposomes encapsulating at least one non-nucleic acid therapeutic agent using a cationic liposome membrane material and at least one non-nucleic acid therapeutic agent; (c) preparing a lipid complex by mixing the physical complex consisting of the cationic material and at least one nucleic acid therapeutic agent with the liposomes encapsulating at least one non-nucleic acid therapeutic agent; and (d) modifying the lipid complex with polyethylene glycol (PEGylated) phospholipid and polyethylene glycol phospholipid conjugated with a cell-permeable peptide.

[0033] In another aspect, the present invention provides a method for preparing a pharmaceutical formulation, comprising: (a) mixing a cationic material as a component contained in the drug delivery carrier of the present invention with at least one nucleic acid therapeutic agent to form a physical complex consisting of the cationic material and at least one nucleic acid therapeutic agent; (b) preparing liposomes encapsulating at least one non-nucleic acid therapeutic agent using all membrane materials of a cationic liposome modified with a cell-permeable peptide and at least one non-nucleic acid therapeutic agent; and (c) mixing the physical complex consisting of the cationic material and at least one nucleic acid therapeutic agent with the liposomes encapsulating the at least one non-nucleic acid therapeutic agent to prepare the pharmaceutical formulation.

[0034] Pharmaceutical preparations prepared using the drug delivery carrier of the present invention can be administered, for example, via the oral cavity, nasal cavity, eye, airway, digestive tract, reproductive tract, local implantation, injection, or infusion (through routes such as epidural, intra-arterial, intra-articular, intra-capsular, intracardiac, intraventricular, intracranial, intradermal, intramuscular, intraorbital, intraocular, intraperitoneal, intravertebral, intrasternal, intrathecal, intravenous, subarachnoid, subcapsular, subcutaneous, trachea, rectum, sublingual, etc.) and used for the treatment and / or prevention of diseases.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as generally understood by those skilled in the art. All publications, patent applications, patents, and other references referenced herein are incorporated herein in their entirety by reference. Furthermore, the materials, methods, and examples described herein are illustrative and not intended to limit the invention. Other features, purposes, and advantages of the invention are evident from this specification and the drawings, as well as from the appended claims. [Brief explanation of the drawing]

[0036] [Figure 1]Figure 1 shows the results of agarose gel electrophoresis of cationic material / siRNA complexes. The left side shows agarose gel electrophoresis images of R8 (a polymer of 8 arginine molecules) and siRNA with different charge ratios (the numbers in the figure indicate the ratio of positive charge to negative charge), and the right side shows agarose gel electrophoresis images of penetratin and siRNA with different charge ratios (the numbers in the figure indicate the ratio of positive charge to negative charge). [Figure 2] Figure 2 shows the results of agarose gel electrophoresis of cationic material / siRNA complexes. The left side shows agarose gel electrophoresis images of CLS and siRNA with different charge ratios (the numbers in the figure indicate the ratio of positive charge to negative charge), and the right side shows agarose gel electrophoresis images of PEI and siRNA with different charge ratios (the numbers in the figure indicate the ratio of positive charge to negative charge). [Figure 3] Figure 3 shows agarose gel electrophoresis images of PAMAM and siRNA at different charge ratios (the numbers in the figure indicate the ratio of positive charge to negative charge). [Figure 4] Figure 4 shows the particle size results for R8 and siRNA with different charge ratios (the numbers on the horizontal axis in the figure indicate the ratio of positive charge to negative charge). [Figure 5] Figure 5 shows the potential results for different charge ratios of R8 and siRNA (the values ​​on the horizontal axis in the figure indicate the ratio of positive charge to negative charge). [Figure 6] Figure 6 shows the particle size results for penetratin and siRNA with different charge ratios (the numbers on the horizontal axis in the figure indicate the ratio of positive charge to negative charge). [Figure 7] Figure 7 shows the potential results for different charge ratios of penetratin and siRNA (the numbers on the horizontal axis in the figure indicate the ratio of positive charge to negative charge). [Figure 8] Figure 8 shows the particle size results for PEI and siRNA with different charge ratios (the numbers on the horizontal axis in the figure indicate the ratio of positive charge to negative charge). [Figure 9] Figure 9 shows the potential results for different charge ratios of PEI and siRNA (the values ​​on the horizontal axis in the figure indicate the ratio of positive charge to negative charge). [Figure 10]Figure 10 shows the particle size results for CLS and siRNA with different charge ratios (the numbers on the horizontal axis in the figure indicate the ratio of positive charge to negative charge). [Figure 11] Figure 11 shows the potential results for CLS and siRNA with different charge ratios (the numbers on the horizontal axis in the figure indicate the ratio of positive charge to negative charge). [Figure 12] Figure 12 shows the particle size results for PAMAM and siRNA with different charge ratios (the numbers on the horizontal axis in the figure indicate the ratio of positive charge to negative charge). [Figure 13] Figure 13 shows the potential results for PAMAM and siRNA with different charge ratios (the numbers on the horizontal axis in the figure indicate the ratio of positive charge to negative charge). [Figure 14] Figure 14 shows the particle sizes of CLS / cationic material / siRNA complexes. Group 1: CLS / siRNA complex, CLS / PEI / siRNA liposome complex, PEI / siRNA complex; Group 2: CLS / siRNA complex, CLS / Penetratin / siRNA liposome complex, Penetratetin / siRNA complex; Group 3: CLS / siRNA complex, CLS / PAMAM / siRNA liposome complex, PAMAM / siRNA complex. [Figure 15] Figure 15 shows the change in particle size of the CLS / cationic material / siRNA composite during the standing process. [Figure 16] Figure 16 shows the quantitative evaluation results of cell uptake of pharmaceutical formulations modified on their surface with cell-permeable peptides in different proportions. The Lipo2000 group is a non-covalently bound complex of the commercially available cationic liposome Lipofectamine 2000, a gene transfection reagent, and siRNA. [Figure 17] Figure 17 shows the cell uptake of pharmaceutical formulations prepared using membrane materials with different formulation ratios as components of liposomes modified with cell-permeable peptides as shown in Table 2 of Example 7. [Figure 18]Figure 18 shows the particle sizes of liposome pharmaceutical formulations modified with cell-permeable peptides, where the molar ratio of component (iv) to the membrane material is 1%, 5%, 8%, and 10%. [Figure 19] Figure 19 shows the cellular uptake of CLS / R8 / siRNA liposome complexes prepared with different charge ratios of CLS and siRNA, when the charge ratio of R8 to siRNA is 1:1. [Figure 20] Figure 20 shows the results of qualitative and quantitative evaluations of the transfection effects of pharmaceutical formulations using different oligoarginines on LUC-U87 cells. [Figure 21] Figure 21 shows the results of the evaluation of the apoptosis-promoting effect and cytotoxicity of siRNA-encapsulated pharmaceutical formulations in bEnd.3 and U87 cells. [Figure 22] Figure 22 shows the measured IC50 values ​​for the half-maximum inhibitory concentration of each chemotherapy drug or gene drug. [Figure 23] Figure 23 shows the results of in vitro antitumor pharmacodynamic evaluations when gene drugs are used in combination with different chemotherapeutic agents. [Figure 24] Figure 24 shows the results of the combination index and dose reduction index for combining gene drugs with different chemotherapeutic agents. [Figure 25] Figure 25 shows the results of in vitro antitumor drug efficacy screening when gene drugs are used in combination with different chemotherapeutic agents. [Figure 26] Figure 26 shows the in vitro antitumor pharmacodynamic results of the pharmaceutical formulation of the present invention, in combination with the chemotherapeutic drug docetaxel (DTX). [Figure 27] Figure 27 shows the results of ingesting FAM-siRNA by U87 cells or Calu-3 cells after carriers labeled with the cell membrane fluorescent probe DiD have been encapsulated. [Figure 28]Figure 28 shows the evaluation effect of the gene drug in combination with the chemotherapy drug docetaxel (DTX) of the present invention on the ability to promote apoptosis in U87 cells. [Figure 29] Figure 29 shows the results of the pharmacodynamic evaluation of the pharmaceutical formulation of the present invention, in which the gene drug is combined with the chemotherapy drug docetaxel (DTX), against situ brain tumors with U87. [Figure 30] Figure 30 shows the animal body weight curve in a pharmacodynamic study of the present invention, in which the gene drug is combined with the chemotherapy drug docetaxel (DTX), for the treatment of situ brain tumors with U87. [Modes for carrying out the invention]

[0037] The present invention provides a novel drug delivery carrier comprising cationic liposomes modified with cell-permeable peptides and a cationic material selected from positively charged polyamino acids, PEI, penetratin or a derivative of penetratin, and PAMAM.

[0038] In the process of preparing a pharmaceutical formulation containing at least one non-nucleic acid therapeutic agent and at least one nucleic acid therapeutic agent using the aforementioned drug delivery carrier, the cationic material contained in the drug delivery carrier compresses the nucleic acid therapeutic agent once, and the cationic liposomes contained in the drug delivery carrier perform secondary compression on the physical complex consisting of the cationic material and at least one nucleic acid therapeutic agent. As a result, the particle size of the physical complex consisting of the cationic material and at least one nucleic acid therapeutic agent is significantly reduced, the stability is significantly improved, and the cell-permeable peptide that modifies the liposome surface and has cell permeability helps the pharmaceutical formulation to penetrate the biological membrane barrier in the subject's body.

[0039] The drug delivery carrier of the present invention can carry multiple types of drugs, deliver multiple types of drugs, has strong tissue penetration ability, low tissue toxicity, and can efficiently deliver multiple types of drug molecules to the target site in the subject's body through various administration routes, overcoming the technical problem that it is difficult for multiple types of drugs to reach the target site due to the biological membrane barrier of the subject, enhancing the effectiveness of the combined use of multiple types of drugs for treating diseases, and having good biological safety. Therefore, the present invention further provides a pharmaceutical preparation for co-delivering multiple types of therapeutic agents prepared using the above drug delivery carrier.

[0040] The various characteristics of the drug delivery carrier of the present invention will be examined below.

[0041] As used herein, the term "about" means an adjustment of ±10% of the specified value. For example, the term "about 5%" means including the range of 4.5% to 5.5%.

[0042] As used herein, the term "comprising" or "including" means including the recited element, integer, or step, but not excluding any other element, integer, or step.

[0043] As used herein, the term "cationic material" refers to a cationic material selected from positively charged polyamino acids, PEI, penetratin or derivatives of penetratin, and PAMAM.

[0044] <I. Membrane material of liposome modified with cell-penetrating peptide on the surface> As used herein, the term "liposome" refers to an artificially prepared vesicle composed of a lipid bilayer. In the present invention, the type of liposome modified with a cell-penetrating peptide is not limited and can be any one that can form a lipid vesicle and encapsulate a drug.

[0045] As used herein, the term “lipid” refers to hydrophobic or amphoteric small molecules, including, but not limited to, fatty acids, phospholipids, glycerides, glycerophospholipids, sphingolipids, glycolipids, or polyketides.

[0046] In some embodiments, cationic liposomes modified with cell-permeable peptides on the surface of the present invention include the following membrane materials:

[0047] [(i) Cationic lipids] Liposomes contain one or more cationic lipids. As used herein, the term “cationic lipid” refers to a lipid that has a net positive charge at a selected pH (e.g., physiological pH). Many cationic lipids are commercially available. Cationic lipids particularly suitable for use in liposomes include those described in International Patent Publications WO2010 / 053572 and WO2012 / 170930, both of which are incorporated herein by reference.

[0048] Regarding the cationic lipids contained in liposomes, there are no particular restrictions as long as the liposomes have a net positive charge at the selected pH (e.g., physiological pH). 1,2-di-0-octadecenyl-3-trimethylammonium-propane (DOTMA), dimethyldioctadecylammonium (DDAB), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), 1,2-diacyloxy-3-dimethylammonium-propane, 1,2-dialkoxy-3-dimethylammonium-propane, dioctadecyldimethylammonium chloride (DODAC), 1,2-dimyristoyloxypropyl-1,3-dimethylhydroxyethylammonium (DMRIE), 2,3-dioleyloxy-N-[2(sperminecarboxamide)ethyl]-NN-dimethyl-1-propaneammonium trifluoroacetate (DOSPA), and combinations thereof can be used, but are not limited to these. Preferred cationic lipids are DOTMA, DOTAP, DODAC, and DOSPA. The most preferred cationic lipid is DOTAP.

[0049] [(ii) Noncationic lipids] Liposomes contain one or more noncationic lipids. As used herein, the term “noncationic lipid” refers to any neutral, zwitterionic, or anionic lipid. As used herein, the term “anionic lipid” refers to any of many lipid substances that have a net negative charge at a selected pH, such as physiological pH.

[0050] There are no special restrictions on the noncationic lipids contained in liposomes; any neutral, amphoteric, or anionic lipids can be used, including, but are not limited to, 1,2-di-(9Z-octadecanoyl)-sn-glyceryl-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glyceryl-3-phosphocholine (DOPC), 1,2-distearoyl-sn-glyceryl-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glyceryl-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glyceryl-3-phosphoethanolamine (DPPE), 1,2-dimyristoyl-sn-glyceryl-3-phosphoethanolamine (DMPE), 2-dioleoyl-sn-glyceryl-3-phosphate-(1'-rac-glycerol) (DOPG), and combinations thereof. The preferred noncationic lipids are DOPE and / or DOPC. The most preferred noncationic lipid is DOPE.

[0051] [(iii) Cholesterol] Cholesterol is a crucial component of mammalian cell membranes, accounting for more than 20% of cell membrane lipids. The term "cholesterol" is used in its broadest sense herein and covers cholesterol derivatives. Studies have shown that cholesterol can prevent disordering of the cell membrane bilayer at high temperatures, hinder ordering of the cell membrane bilayer at low temperatures, inhibit liquid crystal formation, and maintain the fluidity of the cell membrane bilayer.

[0052] Considering the importance of cholesterol in the liposome membrane bilayer, the cationic liposomes of the present invention also contain cholesterol.

[0053] [(iv) PEGylated phospholipids, polyethylene glycol phospholipids conjugated with CPP] The liposomes contain PEGylated phospholipids and polyethylene glycol phospholipids conjugated with CPP. In this specification, PEGylated phospholipids do not include polyethylene glycol phospholipids linked to CPP.

[0054] PEGylated phospholipids are formed by conjugating a phospholipid to one or more polyethylene glycol molecules (PEG). PEGylated phospholipids include, but are not limited to, polyethylene glycol chains of lengths up to 10 kDa, such as 1 kDa, 2 kDa, 3 kDa, 4 kDa, 5 kDa, 6 kDa, 7 kDa, 8 kDa, 9 kDa, 10 kDa, and any value in between, that are conjugated to a phospholipid. The phospholipid can be synthetic, semi-synthetic, or naturally occurring phospholipids, such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and sphingomyelin. In some embodiments, the PEGylated phospholipid is, for example, polyethylene glycol-distearoylphosphatidylethanolamine (PEG-DSPE) and its derivative methoxy-polyethylene glycol-distearoylphosphatidylethanolamine (mPEG-DSPE), where the molecular weight of PEG is any value between 1 kDa and 10 kDa.

[0055] Polyethylene glycol phospholipids conjugated with CPP are formed by the conjugation of CPP to polyethylene glycol phospholipids.

[0056] The CPP used in this invention includes not only the wild-type polypeptide penetratin (amino acid sequence: RQIKIWFQNRRMKWKK (SEQ ID NO: 2)), but also a series of lipophilic derivatives, such as penetratin derivatives in which the glutamine (Q) at position 2 and / or the glutamine (Q) at position 8 and / or the asparagine (N) at position 9 of penetratin are mutated to hydrophobic amino acids. In some embodiments, the penetratin lipophilic derivative is a derivative such as that disclosed in Chinese Patent Application CN201710414334.7, which, after intraconjunctival sac administration, can penetrate many ocular barriers (cornea, conjunctiva, sclera, etc.) and promote drug entry into the eye, thereby delivering the biomolecular drugs it carries, such as genes, polypeptides, and proteins, to the retinal region at the back of the eye. The amino acid sequence of the penetratin lipophilic derivative disclosed in Chinese Patent Application CN201710414334.7 is cited below.

[0057] [Table 1]

[0058] According to the present invention, CPP is a compound that is conjugated to a portion of the lipid bilayer of a liposome by a polyethylene glycol phospholipid. In this specification, the term "part of the lipid bilayer of a liposome" refers to the fact that the phospholipid in CPP-PEG-phospholipid is incorporated into the lipid bilayer. The phospholipid may be a synthetic, semi-synthetic, or native phospholipid, such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, or sphingomyelin.

[0059] The polyethylene glycol chain in CPP-PEG-phospholipids includes, but is not limited to, polyethylene glycol chains with lengths up to 10 kDa, such as 1 kDa, 2 kDa, 3 kDa, 4 kDa, 5 kDa, 6 kDa, 7 kDa, 8 kDa, 9 kDa, 10 kDa, and any value in between, where both ends are conjugated to the phospholipid and CPP, respectively. In some embodiments, the polyethylene glycol phospholipid conjugated to CPP is, for example, Penetratin / Penetratin derivative-PEG-DSPE, where the molecular weight of PEG is any value between 1 kDa and 10 kDa.

[0060] In this invention, modifying the surface of cationic liposomes with CPP is intended to enhance the tissue penetration ability of drug delivery carriers, thereby helping liposomes carrying various drug molecules reach target sites and exert therapeutic and / or preventive effects on diseases.

[0061] In some embodiments, in the cell-permeable peptide-modified liposome membrane material (iv), the polyethylene glycol in the PEGylated phospholipid and the polyethylene glycol phospholipid conjugated with CPP is not the same in length, and the polyethylene glycol chain in the polyethylene glycol phospholipid conjugated with CPP is longer than the polyethylene glycol chain in the PEGylated phospholipid, for example, by 0.5 kDa to 5 kDa, preferably by 0.75 kDa to 4 kDa, and more preferably by any value between 1 kDa and 2 kDa.

[0062] In one embodiment, the polyethylene glycol chain lengths of the PEGylated phospholipids in the cell-permeable peptide-modified liposome membrane material (iv) are 1 kDa, 2 kDa, and 3 kDa, respectively, while the polyethylene glycol chain lengths of the CPP-conjugated polyethylene glycol phospholipids are approximately 2.5 kDa, 3.5 kDa, and 4.5 kDa, respectively.

[0063] In some embodiments, the molar ratio of the cationic liposome membrane materials (i):(ii):(iii):(iv) modified with cell-permeable peptides on the surface of the present invention is approximately 20-40:20-40:20-40:1-20, the molar ratio of polyethylene glycol phospholipid conjugated with CPP to membrane material (iv) is approximately 20-80%, and the molar ratio of liposomes modified with cell-permeable peptides to the total membrane material is approximately 2-8%.

[0064] In some embodiments, the molar ratio of membrane materials (i):(ii):(iii):(iv) is approximately 27.0-31.6:27.0-31.6:31.6-39.6:1-10, with polyethylene glycol phospholipids conjugated with CPP accounting for approximately 20%-80% of membrane material (iv), and the cell-permeable peptide-modified liposomes accounting for approximately 2%-8% of the total membrane material.

[0065] In some specific embodiments, the cationic liposomes in the drug delivery carrier of the present invention are, as membrane materials, (i) DOTAP, (ii) DOPE, (iii) cholesterol, and (iv) PEG-DSPE (e.g., mPEG). 2000 -DSPE) and Penetrate / Penetratin derivative-PEG-DSPE (for example, 89W Penetratin-PEG 3400 It contains -DSPE), and the molar ratio of the membrane material (i):(ii):(iii):(iv) is approximately 20-40:20-40:20-40:1-20, and Penetratin / Penetratin derivative-PEG-DSPE (for example, 89W Penetratin-PEG 3400 -DSPE) has a molar ratio of approximately 20% to 80% of the film material (iv).

[0066] In some embodiments, the molar ratio of film materials (i):(ii):(iii):(iv) is approximately 27.0~31.6:27.0~31.6:31.6~39.6:1~10, and Penetratin / Penetratin derivative-PEG-DSPE (e.g.,89W Penetratin-PEG 3400 -DSPE) has a molar ratio to the membrane material (iv) of about 20% to 80%.

[0067] In some embodiments, the cationic liposomes in the drug delivery carrier of the present invention use, as the membrane material, (i) DOTAP, (ii) DOPE, (iii) cholesterol, (iv) mPEG 2000 -DSPE and 89W Penetratin-PEG 3400 -DSPE, and the molar ratio of the membrane materials (i):(ii):(iii):(iv) is about 28.5:28.5:38:5, 89W Penetratin-PEG 3400 -DSPE has a molar ratio to the membrane material (iv) of about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%.

[0068] <II. Cationic Material> The cationic material as a component included in the drug delivery carrier of the present invention is used to compress nucleic acid therapeutics. The cationic material is selected from positively charged polyamino acids, polyethyleneimine (PEI), penetratin or derivatives of penetratin, and polyamidoamine (PAMAM).

[0069] In some embodiments, the cationic material is a positively charged polyamino acid, which has a net positive charge at a selected pH (e.g., physiological pH). There are no special restrictions on the degree of polymerization of amino acid residues in the polyamino acid; for example, a positively charged polyamino acid with a degree of polymerization of 2 to 50 (i.e., a positively charged polyamino acid has 2 to 50 amino acid residues), such as an arginine and / or lysine polyamino acid with a degree of polymerization of 6 to 12, where 1 to 20 (e.g., 1 to 6) uncharged amino acid residues under physiological conditions are interposed within the positively charged polyamino acid with a degree of polymerization of 2 to 50. The positively charged polyamino acid may be linear or cyclic, and the configuration of amino acid residues is L-type or D-type. Preferably, the positively charged polyamino acid is a polyarginine with a degree of polymerization of 6 to 12, the spatial structure of the polyarginine includes linear and cyclic structures, and the configuration of the polyarginine is L-type or D-type.

[0070] In some embodiments, the positively charged polyamino acids are oligoarginines, such as linear polypeptides like 6-polyarginine (amino acid sequence RRRRRR,R6 (SEQ ID NO: 85)), 8-polyarginine (amino acid sequence RRRRRRRR,R8 (SEQ ID NO: 86)), 10-polyarginine (amino acid sequence RRRRRRRRRR,R10 (SEQ ID NO: 87)), and 12-polyarginine (amino acid sequence RRRRRRRRRRRR,R12 (SEQ ID NO: 88)), as well as cyclized 6-polyarginine (c-R6) and cyclized 8-polyarginine. It includes cyclized polypeptides such as ginine (c-R8), cyclized 10-polyarginine (c-R10), and cyclized 12-polyarginine (c-R12), as well as polypeptides with different compositions such as L-type oligoarginine and D-type oligoarginine such as 6-poly-D-arginine (amino acid sequence rrrrrr, D-R6), 8-poly-D-arginine (amino acid sequence rrrrrrrr, D-R8), 10-poly-D-arginine (amino acid sequence rrrrrrrrrr, D-R10), and 12-poly-D-arginine (amino acid sequence rrrrrrrrrrrr, D-R12).

[0071] The following describes a method for preparing a pharmaceutical formulation that co-delivers multiple therapeutic agents using the drug delivery carrier of the present invention, and then describes the characteristics of the pharmaceutical formulation.

[0072] By using the drug delivery carrier of the present invention, it is possible to prepare pharmaceutical formulations that have a strong tissue penetration ability and co-deliver multiple therapeutic agents by modifying the surface of cationic liposomes with CPP.

[0073] In one embodiment, the method for preparing the pharmaceutical formulation of the present invention is as follows. (1) The liposome membrane material of the present invention described above and at least one non-nucleic acid therapeutic agent are weighed, where the molar ratio of membrane material (i):(ii):(iii):(iv) is approximately 20-40:20-40:20-40:1-20, the molar ratio of polyethylene glycol phospholipid conjugated with CPP to membrane material (iv) is approximately 20-80%, and the molar ratio of liposomes modified with cell-permeable peptides is approximately 2-8% of the total membrane material.

[0074] (2) The membrane materials (i):(ii):(iii):(iv) and at least one non-nucleic acid therapeutic agent are dissolved in an organic solvent, evaporated to remove the organic solvent, and a uniform dry lipid membrane is obtained.

[0075] (3) After completely hydrating the obtained dried lipid membrane with a water-containing solution, the liposomes are extruded using a mini extruder (the order of the pore membranes is 200 nm, 100 nm, and 50 nm, respectively).

[0076] (4) A cationic material (e.g., positively charged polyamino acids, PEI, penetratin or a derivative of penetratin, PAMAM) and at least one nucleic acid therapeutic agent are mixed and incubated in a suitable solution to obtain a physical complex consisting of the cationic material and at least one nucleic acid therapeutic agent.

[0077] (5) The liposomes obtained in (3) above and the physical complex consisting of the cationic material and at least one nucleic acid therapeutic agent obtained in (4) above are mixed in a certain molar ratio or charge ratio to obtain a pharmaceutical formulation in the form of a liposome complex containing at least one non-nucleic acid therapeutic agent and at least one nucleic acid therapeutic agent.

[0078] In one embodiment, the method for preparing the pharmaceutical formulation of the present invention is as follows. (1) The liposome membrane material of the present invention described above is weighed, where the molar ratio of membrane materials (i):(ii):(iii):(iv) is approximately 20-40:20-40:20-40:1-20, the molar ratio of polyethylene glycol phospholipid conjugated with CPP to membrane material (iv) is approximately 20-80%, and the molar ratio of liposomes modified with cell-permeable peptides is approximately 2-8% of the total membrane material.

[0079] (2) The membrane materials (i):(ii):(iii) and at least one non-nucleic acid therapeutic agent are dissolved in an organic solvent, evaporated to remove the organic solvent, and a uniform dry lipid membrane is obtained.

[0080] (3) After completely hydrating the obtained dried lipid membrane with a water-containing solution, the liposomes are extruded using a mini extruder (the order of the pore membranes is 200 nm, 100 nm, and 50 nm, respectively).

[0081] (4) A cationic material (e.g., positively charged polyamino acids, PEI, penetratin or a derivative of penetratin, PAMAM) and at least one nucleic acid therapeutic agent are mixed and incubated in a suitable solution to obtain a physical complex consisting of the cationic material and at least one nucleic acid therapeutic agent.

[0082] (5) The liposomes obtained in (3) above and the cationic material-at least one nucleic acid therapeutic agent obtained in (4) above are mixed in a certain molar ratio or charge ratio, and then mixed with membrane material (iv) and incubated to obtain the pharmaceutical formulation of the present invention.

[0083] The non-nucleic acid therapeutic agent to be delivered is not particularly limited. The non-nucleic acid therapeutic agent includes, but is not limited to, a wide range of compounds delivered to the examinee, including: anti-infective agents such as antibiotics and antivirals; analgesics and combinations of analgesics; appetite suppressants; anthelmintics; antiarthritis agents; anti-asthma agents; anticonvulsants; antidepressants; antidiabetic agents; antidiarrheals; antihistamines; anti-inflammatory agents; anti-migraine agents; nausea agents; antitumor agents; anti-tremor paralytic agents; antipruritics; antipsychotics; antipyretics; anticonvulsants; anticholinergic agents; sympathomimetic agents; xanthin Electrolyte derivatives; cardiovascular drugs including potassium channel blockers, calcium channel blockers, β-blockers, α-blockers and antiarrhythmic agents; antihypertensive drugs; diuretics and antidiuretics; vasodilators including agonists and vasosuppressants for systemic nerves, cardiac nerves, perineurotic nerves and cranial nerves, and the central nervous system; cough and cold preparations including decongestants; hormones such as estradiol and other steroids including corticosteroids; hypnotics; immunosuppressants; muscle relaxants; parasympathetic nerve blockers; psychostimulants; sedatives and tranquilizers. The drug delivery carrier of the present invention can deliver all forms of drugs, such as ionized, non-ionized, free bases, and acid addition salts, and can deliver high molecular weight or low molecular weight drugs.

[0084] The at least one nucleic acid therapeutic agent to be delivered is not particularly limited and includes nucleic acids such as plasmid DNA, RNA such as small interfering RNA (siRNA), miRNA, sense RNA, antisense oligonucleotide (ASO), aptamers, and ribozymes. For example, the nucleic acid therapeutic agent is RNA for brain diseases such as brain tumors (e.g., glioma), and for example, siRNA for c-myc.

[0085] The pharmaceutical formulations of the present invention can be administered via oral cavity, nasal cavity, eye, airway, digestive tract, reproductive tract, local implantation, injection, or infusion (via epidural, intra-arterial, intra-articular, intra-capsular, intracardiac, intraventricular, intracranial, intradermal, intramuscular, intraorbital, intraocular, intraperitoneal, intravertebral, intrasternal, intrathecal, intravenous, subarachnoid, subcapsular, subcutaneous, trachea, rectum, sublingual, and other routes).

[0086] The pharmaceutical formulation of the present invention can be administered using medical devices known in the art. For example, in one embodiment, the pharmaceutical formulation of the present invention can be administered using a needle-free scalp subcutaneous injection device, such as the device disclosed in U.S. Patent No. 5,399,163.

[0087] In one embodiment, by preparing a pharmaceutical formulation for co-delivering a chemotherapy drug and a nucleic acid drug siRNA using the drug delivery carrier of the present invention, a synergistic effect in treating cancer can be achieved.

[0088] Chemotherapy remains one of the most commonly used cancer treatments. However, while chemotherapy drugs actively target dividing cells (a characteristic of cancer cells), they also affect healthy dividing cells such as blood cells, as well as cells in the gut, mouth, and hair, leading to severe side effects. Scientists have been working to improve the administration and combination of chemotherapy drugs to minimize these side effects.

[0089] Furthermore, nucleic acid therapies are attracting considerable attention for their use in treating diseases (e.g., cancer). While nucleic acid therapies have potential efficacy in cancer treatment, they can be easily degraded by enzymes ubiquitous in the environment, and nucleic acids themselves cannot penetrate cells, and existing delivery systems only have low delivery efficiency.

[0090] The use of the drug delivery carrier of the present invention minimizes the side effects of chemotherapy drugs, allows nucleic acid therapeutics to efficiently enter cells, and prevents nucleic acid therapeutics from being easily attacked by enzymes ubiquitous in the environment.

[0091] In one embodiment, the pharmaceutical formulation for co-delivering the chemotherapeutic agent and nucleic acid drug siRNA of the present invention is administered intranasally, efficiently delivering the chemotherapeutic agent and nucleic acid drug siRNA to the brain via the nasal-brain pathway. After the chemotherapeutic agent and nucleic acid drug siRNA are taken up by brain tumor cells, they escape endosomes with the help of cell-permeable peptides and the positive surface charge of cationic liposomes, releasing the chemotherapeutic agent and nucleic acid drug siRNA into the cytoplasm to play a role in the chemical death of brain tumors (e.g., gliomas) and gene therapy.

[0092] As engineers in this field are well aware, the unique physiological structure and functional complexity of the brain, particularly the presence of the blood-brain barrier and the physical and chemical properties of certain drugs themselves, make it difficult for therapeutic drugs to reach brain tissue. However, nasal administration routes offer a viable idea for treating brain diseases, and there are two direct routes for nasal administration of drugs to the brain: one is the olfactory pathway, the most direct way to bypass the blood-brain barrier to enter the brain through the nasal cavity, where the drug is absorbed by the nasal mucosa, taken up by the olfactory nerve, transported via axons to the olfactory bulb, and then to the olfactory brain; the other is the nasal mucosal epithelial pathway, where the drug passes through the basement membrane into the lamina propria, then reaches the peripheral olfactory nerve, is delivered to the central nervous system, and ultimately accumulates in the brain. Nasal administration to the brain is non-invasive and results in good patient compliance (Agrawal M et al., Nose-to-brain drug delivery: An update on clinical challenges and progress towards approval of anti-Alzheimer drugs, Journal of Controlled Release, 2018, 281:139-177).

[0093] On the other hand, brain tumors such as gliomas are malignant tumors of the central nervous system with a high incidence and low survival rate, causing significant harm to human health, and the clinical prognosis is not optimistic. These prognoses mainly manifest as recurrence after surgery and drug resistance to chemotherapy. Furthermore, due to the existence of the blood-brain barrier and the blood-brain tumor barrier, the amount of drugs that reach the brain after systemic administration is very small. For this reason, people have traditionally been striving to find effective drug treatment methods.

[0094] The pharmaceutical formulation of the present invention for co-delivering a chemotherapeutic agent and a nucleic acid drug siRNA can deliver the chemotherapeutic agent and the nucleic acid drug siRNA to the brain simultaneously after intranasal administration, thereby synergistically exerting a therapeutic effect against brain tumors. Furthermore, because the pharmaceutical formulation of the present invention allows for intracerebral administration via intranasal administration, it is beneficial in improving patient compliance. [Examples]

[0095] The following embodiments are provided to aid in understanding the present invention. These embodiments should not be construed as limiting the scope of protection of the present invention.

[0096] [Example 1] (Preparation of cationic liposomes) The membrane materials were in a molar ratio of 28.5:28.5:38:5, namely 3.5 mg of DOTAP (Shanghai AWEITE Pharmaceutical Technology Co., Ltd., 132172-61-3), 3.72 mg of DOPE (Shanghai AWEITE Pharmaceutical Technology Co., Ltd., 4004-05-1), 2.59 mg of cholesterol (Sigma-Aldrich, C8667), and 2.46 mg of mPEG. 2000 -DSPE (Shanghai AWEITE Pharmaceutical Technology Co., Ltd., 147867-65-0) was weighed.

[0097] The above amounts of DOTAP, DOPE, and cholesterol were placed in a 50 mL round-bottom flask, and 1 mL of chloroform was added to dissolve the DOTAP, DOPE, and cholesterol as the lipid membrane material. Specifically, 16.67 μmol of CLS contained 5 μmol of DOTAP, 5 μmol of DOPE, and 6.67 μmol of cholesterol. The resulting solution was evaporated using a rotary evaporator (Shanghai Shensheng Technology Co., Ltd., R201L) to remove the chloroform and obtain a homogeneous dried lipid membrane.

[0098] The obtained dried lipid membrane was hydrated in a 37°C water bath with 1 mL of 5% glucose aqueous solution using sonication for approximately 10 minutes. After the lipid membrane was completely hydrated, liposomes were extruded using a mini extruder (Hamilton, 81320) (the order of the pore membranes was 200 nm, 100 nm, and 50 nm, respectively) to obtain cationic liposomes (hereinafter also referred to as CLS) that were not modified with cell-permeable peptides.

[0099] The prepared cationic liposomes were placed in 2.46 mg of mPEG. 2000 - Mix with DSPE (water soluble), incubate at 55°C for 30 minutes, and then mPEG 2000 -Cationic liposomes containing DSPE were obtained.

[0100] [Example 2] (Compression effect of gene drugs by cationic materials) Agarose gel electrophoresis is commonly used to measure the encapsulation ability of cationic materials contained in drug delivery carriers for drugs such as nucleic acids. In this example, R8 / siRNA complexes, penetratin / siRNA complexes, CLS / siRNA complexes, PEI / siRNA complexes, and PAMAM / siRNA complexes were prepared, and the encapsulation ability of R8, penetratin, CLS, polyethyleneimine (PEI, molecular weight: 25000), and third-generation polyamidoamine (PAMAM) for nucleic acids was measured by agarose gel electrophoresis.

[0101] 33 μg of siRNA (in this example, a small interfering RNA against c-myc was used, hereafter abbreviated as siRNA, with its sense strand (5'-3') being AACGUUAGCUUCACCAACAdTdT (SEQ ID NO: 79) and its antisense strand (5'-3') being UGUUGGUGAAGCUAACGUUdTdT (SEQ ID NO: 80)) was dissolved in 125 μL of DEPC-treated water to prepare a 20 μM siRNA solution.

[0102] Oligoarginine R8 (Shanghai Xinhao Biotechnology Co., Ltd.), Penetratin (Shanghai Xinhao Biotechnology Co., Ltd.), CLS, PEI (Sigma-Aldrich (Shanghai)), and PAMAM (Weihai Chenyuan Molecular Materials Co., Ltd.) prepared in Example 1 were each dissolved in pure water, mixed with siRNA at a specific charge ratio, vortexed for 30 seconds, and incubated at 37°C for 30 minutes to obtain R8 / siRNA complexes, Penetratin / siRNA complexes, CLS / siRNA complexes, PEI / siRNA complexes, and PAMAM / siRNA complexes.

[0103] The prepared R8 / siRNA complexes, penetratin / siRNA complexes, CLS / siRNA complexes, PEI / siRNA complexes, and PAMAM / siRNA complexes were gently added to the loading holes of the agarose gel, and a free siRNA control group was established. Electrophoresis was then performed. After electrophoresis, the gels were stained with GelRed working solution (Biotium Inc.) in the dark for 30 minutes, and the gels were imaged under UV 302nm conditions. The results are shown in Figures 1 to 3.

[0104] Particle size and zeta potential measurements were performed on the fabricated R8 / siRNA complex, penetratin / siRNA complex, CLS / siRNA complex, PEI / siRNA complex, and PAMAM / siRNA complex. Particle size was measured directly using dynamic light scattering technology. Zeta potential was measured in water at 23°C with an electric field strength of 5 V / cm and an electrode spacing of 0.4 cm using a zeta potential analyzer. The results are shown in Figures 4 to 13.

[0105] Since one amino nitrogen in R8 has one positive charge and one phosphate group in siRNA has one negative charge, 1 μM of R8 contains 8 μM of amino nitrogen, i.e., 1 μM of R8 contains 8 μM of positive charge, and 1 μM of siRNA contains 42 μM of phosphate groups, i.e., 1 μM of siRNA contains 42 μM of negative charge. The charge ratio of R8 to siRNA is (μM value of R8 × 8) / (μM value of siRNA × 42) = 0.19 × R8 (μM) / siRNA (μM).

[0106] Since one amino nitrogen in penetratin has one positive charge and one phosphate group in siRNA has one negative charge, 1 μM of penetratin contains 7 μM of amino nitrogen, i.e., 1 μM of penetratin contains 7 μM of positive charge, and 1 μM of siRNA contains 42 μM of phosphate groups, i.e., 1 μM of siRNA contains 42 μM of negative charge. The charge ratio of penetratin to siRNA is (value of penetratin in μM × 7) / (value of siRNA in μM × 42) = 0.17 × penetratin (μM) / siRNA (μM).

[0107] Since one amino nitrogen in DOTAP has one positive charge and one phosphate group in siRNA has one negative charge, 16.67 μM of CLS contains 5 μM of DOTAP, meaning that 1 μM of CLS contains 0.3 μM of positive charge and 1 μM of siRNA contains 42 μM of negative charge. The charge ratio of CLS to siRNA is (μM value of CLS × 0.3) / (μM value of siRNA × 42) = 0.007 × CLS(μM) / siRNA(μM).

[0108] Since one amino nitrogen in PEI has a positive charge, primary and secondary amines containing nitrogen atoms are attached to every two carbon atoms in its repeating monomer structure. In other words, in PEI, there is one N atom per 43 molecular weights. In siRNA, one phosphate group has a negative charge, the average molecular weight of siRNA is 13300, and 1M siRNA contains 42M phosphate groups. That is, in siRNA, there is one phosphate group per 317 molecular weights. The charge ratio of PEI to siRNA is (mass of PEI mg) / (mass of siRNA mg) × (317 / 43) = 7.37 × PEI(mg) / siRNA(mg).

[0109] Since one amino nitrogen in PAMAM has a positive charge and one phosphate group in siRNA has a negative charge, according to the formula in the literature, the charge ratio of PAMAM to siRNA = 1.53 × PAMAM(mg) / siRNA(mg).

[0110] [Example 3] (Particle size and potential characteristics after double compression using cationic liposomes) Using the Penetratin / siRNA complex, PEI / siRNA complex, and PAMAM / siRNA complex prepared in Example 2, CLS and liposome complexes were prepared, respectively. Specifically, 8 μL each of the Penetratin / siRNA complex, PEI / siRNA complex, and PAMAM / siRNA complex were mixed with 2.67 μL each of the 16.67 μM cationic liposome CLS prepared in Example 1, vortexed for 30 seconds, and incubated at 37°C for 30 minutes to obtain the liposome complexes CLS / Penetratin / siRNA, CLS / PEI / siRNA, and CLS / PAMAM / siRNA.

[0111] In the liposome complexes CLS / Penetratin / siRNA, CLS / PEI / siRNA, and CLS / PAMAM / siRNA, the charge ratio of cationic liposome CLS to siRNA was fixed at 4 based on the number of charges of siRNA. (Since one amino nitrogen in DOTAP has one positive charge and one phosphate group in siRNA has one negative charge, 16.67 μM of CLS contains 5 μM of DOTAP, meaning that 1 μM of CLS contains 0.3 μM of positive charge, and 1 μM of siRNA contains 42 μM of negative charge. The charge ratio of CLS to siRNA is (μM value of CLS × 0.3) / (μM value of siRNA × 42) = 0.007 × CLS(μM) / siRNA(μM).)

[0112] The particle size of the prepared liposome complexes CLS / Penetratin / siRNA, CLS / PEI / siRNA, and CLS / PAMAM / siRNA was measured, and the change in particle size during the standing period of the liposome complexes was observed. The results are shown in Figures 14 and 15.

[0113] The results indicate that, during the preparation of the CLS / cationic material / siRNA liposome complex, the particle size is significantly reduced after double compression of CLS compared to the CLS / siRNA complex alone or the cationic material / siRNA complex alone. Therefore, the CLS / cationic material / siRNA liposome complex is more stable than the CLS / siRNA complex alone or the cationic material / siRNA complex alone, can provide better protection for siRNA and drugs, and is advantageous for delivering siRNA and drugs to deep tissues at lesion sites.

[0114] [Example 4] (Preparation of pharmaceutical formulations in which cationic liposomes and cationic materials encapsulate gene drugs) A 4 mg / mL oligoarginine R8 solution was prepared by dissolving 4 mg of positively charged polyamino acid, i.e., oligoarginine R8 (Shanghai Xinhao Biotechnology Co., Ltd.), as a cationic material in DEPC-treated water (Dalian Meilun Biotechnology Co., Ltd., MA0018).

[0115] 33 μg of siRNA (in this example, a small interfering RNA against c-myc was used, hereafter abbreviated as siRNA, with its sense strand (5'-3') being AACGUUAGCUUCACCAACAdTdT (SEQ ID NO: 79) and its antisense strand (5'-3') being UGUUGGUGAAGCUAACGUUdTdT (SEQ ID NO: 80)) was dissolved in 125 μL of DEPC-treated water to prepare a 20 μM siRNA solution.

[0116] Oligoarginine R8 solution and siRNA solution were mixed in equivolume to obtain an R8 / siRNA complex (since one amino nitrogen in R8 has one positive charge and one phosphate group in siRNA has one negative charge, 1 μM of R8 contains 8 μM of amino nitrogen, i.e., 1 μM of R8 contains 8 μM of positive charge, and 1 μM of siRNA contains 42 μM of phosphate groups, i.e., 1 μM of siRNA contains 42 μM of negative charge, so the charge ratio of R8 to siRNA is (μM value of R8 × 8) / (μM value of siRNA × 42) = 0.19 × R8 (μM) / siRNA (μM)), vortexed for 30 seconds, and incubated at 37°C for 30 minutes.

[0117] The prepared R8 / siRNA complex was mixed with CLS to prepare the CLS / R8 / siRNA lipid complex. Specifically, 8 μL of the R8 / siRNA complex (i.e., prepared with 4 μL of siRNA solution and 4 μL of R8 solution) was taken and mixed with 2.67 μL of the 16.67 μM cationic liposome CLS prepared in Example 1, vortexed for 30 seconds, and incubated at 37°C to obtain the lipid complex CLS / R8 / siRNA. In the aforementioned lipid complex CLS / R8 / siRNA, the charge ratio of cationic liposome CLS to siRNA was fixed at 4 based on the charge number of siRNA (since one amino nitrogen in DOTAP has one positive charge and one phosphate group in siRNA has one negative charge, 16.67 μM of CLS contains 5 μM of DOTAP, i.e., 1 μM of CLS contains 0.3 μM of positive charge, and 1 μM of siRNA contains 42 μM of negative charge, so the charge ratio of CLS to siRNA is (μM value of CLS × 0.3) / (μM value of siRNA × 42) = 0.007 × CLS(μM) / siRNA(μM)). Similarly, FAM-siRNA solution and CLS / R8 / FAM-siRNA complex were prepared in which FAM was modified at the 5' of the sense strand of the siRNA.

[0118] [Example 5] (Preparation of liposome pharmaceutical formulations with modified surfaces) Surface modification was performed on liposome pharmaceutical formulations using cell-permeable peptides to obtain pharmaceutical formulations with modified surfaces.

[0119] i. Preparation of cell-permeable peptide-PEG-phospholipids DSPE-Polyethylene Glycol Maleimide (DSPE-PEG 3400 -maleimide) (Laysan Bio, 146-123) and penetratin derivatives modified with cysteine ​​at the terminal end ( 89W Penetratin-Cys) reacted in a single step to obtain the cell-permeable peptide-PEG-DSPE.

[0120] Specifically, 20 mg of DSPE-PEG 3400- Dissolve maleimide in 1 mL of N,N-dimethylformamide, and then dissolve 18 mg in 10 mL of phosphate buffer (10 mM, pH 7.2). 89W Penetratin-Cys was added under stirring conditions, and the reaction was continued overnight at 25°C to complete. After the reaction was complete, the resulting mixture was dialyzed in pure water under ice bath conditions for 2 days, lyophilized, and obtained as a white aggregate. 89W Penetratin-PEG 3400 -DSPE obtained.

[0121] Here, the polypeptide 89W Penetratin is a derivative of penetratin, with the specific amino acid sequence RQIKIWFWWRRMKWKK (in this specification, 89W represents the product of a mutation in which tryptophan is replaced with glutamine at position 8 and asparagine at position 9 of penetratin). The cell-permeable peptide in cell-permeable peptide-PEG-DSPE may be other penetratin derivatives, for example, see Chinese Patent Application CN201710414334.7.

[0122] To facilitate reaction with polyethylene glycol terminal groups, a cell-permeable peptide is used. 89W A cysteine ​​residue (cysteine,C) may be added to the N-terminus or C-terminus of penetratin or another penetratin derivative.

[0123] 《ii. Preparation of liposome pharmaceutical formulations with modified surfaces》 mPEG, where the molar ratio of liposomes modified with cell-permeable peptides is 5% of the total liposomes. 2000 -DSPE and 89W Penetratin-PEG 3400 -Regarding DSPE, each is mPEG 2000 -DSPE and 89W Penetratin-PEG 3400 -The molar ratio to DSPE is 0%, 20%, 40%, 60%, 80%, and 100%. 89W Penetratin-PEG 3400-A mixture of DSPE was prepared. That is, 89W Penetratin-PEG 3400 -DSPE represented 0%, 1%, 2%, 3%, 4%, and 5% of the total liposomes modified with cell-permeable peptides, respectively.

[0124] The CLS / R8 / FAM-siRNA complexes obtained in Example 4 were each treated with mPEG. 2000 -DSPE and 89W Penetratin-PEG 3400 -The molar ratio to DSPE is 0%, 20%, 40%, 60%, 80%, and 100%. 89W Penetratin-PEG 3400 After mixing with the DSPE mixture and incubating at 55°C for 30 minutes, liposomal pharmaceutical formulations, namely 89W-CLS / R8 / FAM-siRNA, were obtained, in which the surface was modified with cell-permeable peptides in proportions of 0%, 1%, 3%, 4%, and 5%.

[0125] [Example 6] (Quantitative evaluation of cell uptake of pharmaceutical formulations modified on their surface with cell-permeable peptides in different proportions) U87 cells (human glioma cells, purchased from ATCC) were cultured in DMEM complete medium (DMEM medium supplemented with 10% FBS and 1% penicillin-streptomycin). Well-developed U87 cells were selected, resuspended in DMEM complete medium, and cultured in 5 × 10⁶ units. 5 Cells were seeded in a 6-well plate with 1 mL of culture medium per well. After seeding, the culture medium was changed once daily, and the cells were cultured for 2-3 days before the experiment was performed.

[0126] After discarding the culture medium, the U87 cells were washed three times with sterile PBS, and serum-free DMEM medium was added to 1 mL of the liposomal pharmaceutical preparation prepared in Example 5 (containing FAM-labeled siRNA), which had different proportions of cell-permeable peptides modified on its surface. The cells were incubated at 37°C, 5%, and CO2 for 4 hours.

[0127] Next, the culture medium was discarded, and positively charged substances adsorbed to the wells and cell surfaces were washed away with PBS buffer solution containing 0.02 mg / mL heparin sodium. The cells were treated with trypsin, resuspended in 200 μL of sterile PBS buffer solution, and after uniform spraying, the cells were counted for each sample. 4 Individual cells were isolated and detected by flow cytometry. Cells incubated in serum-free DMEM medium served as the negative control group. All experiments were performed in triple replication, and the results are shown in Figure 16 (mean ± SD, n=3, *p<0.05, * indicates a statistically significant difference between the two groups; **p<0.01, ***p<0.001, ** and *** indicate a statistically very significant difference between the two groups).

[0128] In Figure 16, the "0%, 1%, 2%, 3%, 4%, 5%" on the horizontal axis represent liposomal pharmaceutical formulations, i.e., 89W-CLS / R8 / siRNA, in which cell-permeable peptides are modified on the surface at rates of 0%, 1%, 2%, 3%, 4%, and 5%, respectively.

[0129] As can be seen from Figure 16, the entire membrane material of liposomes modified with cell-permeable peptides, 89W Penetratin-PEG 3400 -When the molar ratio of DSPE was 2%, 3%, and 4%, the cell uptake effect of pharmaceutical formulations modified with cell-permeable peptides in the aforementioned proportions was superior to that of the commercially available gene transfection reagent Lipo 2000.

[0130] [Example 7] (Quantitative evaluation of cell uptake of pharmaceutical formulations prepared using liposome membrane materials modified with cell-permeable peptides in different formulation ratios) U87 cells (human glioma cells, purchased from ATCC) were cultured in DMEM complete medium (DMEM medium supplemented with 10% FBS and 1% penicillin-streptomycin). Well-developed U87 cells were selected, resuspended in DMEM complete medium, and cultured in 5 × 10⁶ units. 5Cells were seeded in a 6-well plate with 1 mL of culture medium per well. After seeding, the culture medium was changed once daily, and the cells were cultured for 2-3 days before the experiment was performed.

[0131] According to the method described in Example 5, pharmaceutical formulations (containing FAM-labeled siRNA) were prepared with the following proportions of cell-permeable peptide-modified liposome membrane materials as shown in Table 2.

[0132] [Table 2]

[0133] After discarding the culture medium of U87 cells, the cells were washed three times with sterile PBS. Each well was then filled with 1 mL of serum-free DMEM medium containing liposomal pharmaceutical formulations prepared according to Formula 1, Formula 2, and Formula 3, respectively, and incubated at 37°C, 5%, and CO2 for 4 hours.

[0134] Next, the culture medium was discarded, and positively charged substances adsorbed to the wells and cell surfaces were washed away with PBS buffer solution containing 0.02 mg / mL heparin sodium. The cells were treated with trypsin, resuspended in 200 μL sterile PBS buffer solution, and uniformly sprayed, with the cells counted for each sample. 4 Individual cells were isolated and detected by flow cytometry. Cells incubated in serum-free DMEM medium were used as a negative control group. All experiments were performed in triple replication, and the results are shown in Figure 17 (mean ± SD, n=3, *p<0.05, * indicates a statistically significant difference between the two groups; **p<0.01, ***p<0.001, ** and *** indicate a statistically very significant difference between the two groups).

[0135] As can be seen from Figure 17, the liposomal pharmaceutical formulations prepared by Formula 1, Formula 2, and Formula 3 were all able to be taken up by cells, showing a significant difference compared to the control group. Furthermore, the liposomal pharmaceutical formulation prepared by Formula 2 showed the optimal effect on cell uptake.

[0136] [Example 8] (Particle size of pharmaceutical formulations prepared using liposome membrane materials modified with cell-permeable peptides in different proportions of component (iv)) Following the method described in Example 5, liposome pharmaceutical formulations (containing FAM-labeled siRNA) were prepared with a cell-permeable peptide-modified liposome membrane material having a molar ratio of component (i):component (ii):component (iii) of 3:3:4s and a component (iv) ratio as shown in Table 3.

[0137] [Table 3]

[0138] Liposome pharmaceutical formulations were obtained with cell-permeable peptides, in which the molar ratio of component (iv) to the membrane material was 1%, 5%, 8%, and 10%. The particle size of these liposome pharmaceutical formulations was measured.

[0139] The particle size measurement results are shown in Figure 18. In Figure 18, "1%, 5%, 8%, 10%" on the horizontal axis represent liposome pharmaceutical formulations in which the molar ratio of component (iv) to the membrane material of the liposome modified with a cell-permeable peptide is 1%, 5%, 8%, and 10%, respectively.

[0140] As can be seen from Figure 18, when the molar ratio of component (iv) to the membrane material of liposomes modified with cell-permeable peptides is 1% to 10%, the particle size of the pharmaceutical formulation is 100 nm to 200 nm. Furthermore, the particle size tended to increase as the molar ratio of component (iv) to the membrane material of liposomes modified with cell-permeable peptides increased.

[0141] [Example 9] (Quantitative evaluation of cellular uptake of pharmaceutical formulations prepared with cationic liposomes / siRNA at different charge ratios) U87 cells (human glioma cells, purchased from ATCC) were cultured in DMEM complete medium (DMEM medium supplemented with 10% FBS and 1% penicillin-streptomycin). Well-developed U87 cells were selected, resuspended in DMEM complete medium, and cultured in 5 × 10⁶ units. 5 Cells were seeded in a 6-well plate with 1 mL of culture medium per well. After seeding, the culture medium was changed once daily, and the cells were cultured for 2-3 days before the experiment was performed.

[0142] According to the method described in Example 5, an oligoarginine R8 solution and a 20 μM FAM-siRNA solution were mixed in equal volumes at a charge ratio of 5, vortexed for 30 seconds, and incubated at 37°C for 30 minutes to obtain the R8 / FAM-siRNA complex.

[0143] 8 μL of R8 / FAM-siRNA complex was taken and mixed with prepared cationic liposome CLS in charge ratios of liposome to FAM-siRNA of 2, 4, 6, 8, 10, and 12, and vortexed for 30 seconds to obtain CLS / R8 / FAM-siRNA complexes with different charge ratios of cationic liposome / siRNA.

[0144] Furthermore, a pharmaceutical formulation, namely the 89W-CLS / R8 / FAM-siRNA complex, was obtained by modifying the surface with 3% of a cell-permeable peptide according to the method described in Example 5.

[0145] After discarding the culture medium of U87 cells, the cells were washed three times with sterile PBS. Each well was then filled with 1 mL of serum-free DMEM medium containing pharmaceutical formulations with cationic liposome / siRNA charge ratios of 2, 4, 6, 8, 10, and 12, and incubated at 37°C, 5% CO2 for 4 hours.

[0146] Next, the culture medium was discarded, and positively charged substances adsorbed to the wells and cell surfaces were washed away with PBS buffer solution containing 0.02 mg / mL heparin sodium. The cells were treated with trypsin, resuspended in 200 μL sterile PBS buffer solution, and uniformly sprayed, with the cells counted for each sample. 4Individual cells were isolated and detected by flow cytometry. Cells incubated in serum-free DMEM medium were used as a negative control group. All experiments were performed in triple replication, and the results are shown in Figure 19 (mean ± SD, n=3, *p<0.05, * indicates a statistically significant difference between the two groups; **p<0.01, ***p<0.001, ** and *** indicate a statistically very significant difference between the two groups).

[0147] As shown in Figure 19, all pharmaceutical formulations prepared with cationic liposome / siRNA charge ratios of 2, 4, 6, 8, 10, and 12 were taken up by cells, showing significant differences compared to the control group. Of these, the liposomal pharmaceutical formulation prepared with a cationic liposome / siRNA charge ratio of 4 showed the optimal effect on cell uptake.

[0148] [Example 10] (Qualitative and quantitative evaluation of transfection effects on LUC-U87 cells by pharmaceutical formulations containing cationic materials of different structural types) Oligoarginines such as R8, R10, R12, c-R8, c-R10, and c-R12 were used as positively charged polyamino acids of different structural types. The charge ratio between the oligoarginine and siRNA was set to 5, and the charge ratio between CLS and LUC-siRNA was set to 4. Following Example 5, liposomal pharmaceutical formulations containing different oligoarginines and modified with a cell-permeable peptide at a rate of 3% on the surface were prepared. The LUC-siRNA used had a sense strand (5'-3') of GCUGCACUCUCGGCGACAUUTT (SEQ ID NO: 83) and an antisense strand (5'-3') of AAUGUCGCCAGAGUGCAGCTT (SEQ ID NO: 84).

[0149] Take U87 cells in good growth condition (purchased from ATCC) and divide them into 5 × 10 4Cells were seeded in 48-well plates at a rate of one cell / well. After seeding, the culture medium was changed once daily, and the cells were incubated for 2-3 days before the experiment. After discarding the complete DMEM medium, the cells were washed three times with sterile PBS, and serum-free DMEM medium containing liposomal pharmaceutical preparations encapsulating 400 nM LUC-siRNA was added to each medium. The cells were incubated at 37°C, 5%, and CO2 for 4 hours. Subsequently, the drug solution was discarded, and complete DMEM medium was added for a further 24 hours of incubation (referred to herein as LUC-U87 cells). Positively charged adsorbents were washed away with PBS buffer solution containing 0.02 mg / mL heparin sodium, and a firefly luciferase substrate was added. Fluorescence intensity was measured using a small animal biofluorescence / bioluminescence imaging system (IVIS Spectrum).

[0150] The region of interest (ROI) was qualitatively analyzed using the quantitative circle selection function of the small animal biooptical imaging system. The results are shown in Figures 20A and 20B.

[0151] This example explores the effects of different structural types of positively charged polyamino acids on gene knockdown. Figures 20A and 20B show that altering the degree of polymerization and structure of oligoarginine in liposomal pharmaceutical formulations does not significantly affect the gene knockdown effect.

[0152] [Example 11] (Evaluation of the ability of siRNA-encapsulated delivery carriers to promote apoptosis in bEnd.3 and U87 cells) bEnd.3 cells and U87 cells (both purchased from ATCC) exhibiting good logarithmic growth in DMEM medium were selected and measured in 5 × 10⁶ units. 4 Seeds were seeded at a concentration of / well into 12-well plates and cultured at 37°C, 5% CO2 until a monolayer of cells covered the bottom of the wells.

[0153] After discarding the culture medium and washing three times with sterile PBS buffer solution, the pharmaceutical formulation prepared in Example 5 (89W-CLS / R8 / siRNA) containing 400 nmol / L siRNA (the pharmaceutical formulation has an oligoarginine-to-siRNA charge ratio of 5, and a CLS-to-siRNA charge ratio of 4, and on the surface) 89W Penetratin-PEG 3400 -400 μL of DSPE modified at a rate of 3% was added and incubated in an incubator for 6 hours. Subsequently, the drug solution was discarded, the cells were washed three times with sterile PBS buffer solution, and then 1 mL of complete medium was added and cultured for another 18 hours. Subsequently, the cells were collected, washed with PBS, treated with EDTA-free trypsin for 1 minute, and the treatment was terminated with DMEM culture medium.

[0154] Next, cells were collected in centrifuge tubes, double-stained using a cell apoptosis testing kit (Nanjing Kaiji Biotechnology Development Co., Ltd., KGA108), and detected by flow cytometry. Cells incubated in serum-free DMEM medium served as the negative control group. All experiments were performed in triple replication, and the results are shown in Figures 21A-D (mean ± SD, n=3, *p<0.05, * indicates a statistically significant difference between the two groups; **p<0.01, ***p<0.001, ** and *** indicate a statistically very significant difference between the two groups).

[0155] As a result, it was revealed that the delivery carrier designed by the present invention can specifically promote apoptosis in tumor cells U87 without significantly affecting the growth of normal cells bEnd.3.

[0156] [Example 12] (half maximum inhibitory concentration IC of each chemotherapy drug and gene drug) 50 (Measurement of values) Half the maximum inhibitory concentration IC of chemotherapy drugs 50 The value was measured. That is, the density was 3 × 10 4A U87 single-cell suspension was prepared at a cell / mL concentration and seeded in 100 μL per well in a 96-well plate. Edge wells were filled with sterile PBS buffer, and the plate was transferred to a CO2 incubator (37°C, 5% CO2, saturated humidity) and incubated for 24 hours.

[0157] Next, the culture medium was discarded and replaced with 200 μL of culture medium containing, or without, the following chemotherapeutic agents: carmustine (BCNU, Dalian Meilun Biotechnology Co., Ltd., MB130), docetaxel (DTX, Dalian Meilun Biotechnology Co., Ltd., MB1081), gemcitabine (Gemcitabine, Dalian Meilun Biotechnology Co., Ltd., MB5386), imatinib (Imatinib, Dalian Meilun Biotechnology Co., Ltd., MB2031), cisplatin (Cisplatin, Dalian Meilun Biotechnology Co., Ltd., MB1055), and doxorubicin (DOX, Dalian Meilun Biotechnology Co., Ltd., MB1087), and the cells were cultured.

[0158] After 48 hours, 10 μL of cck-8 solution (Shanghai Biyuntian Biotechnology Co., Ltd., C0042) was added to each well, and incubation was continued at 37°C for 2 hours. The OD value of each well was measured at a wavelength of 490 nm using a microplate reader (BioTek, PowerWave XS).

[0159] Half the maximum inhibitory concentration IC of gene drugs 50 The value was measured. That is, the density was 1 × 10⁻⁶. 5 A U87 single-cell suspension was prepared at a concentration of cells / mL, seeded in 500 μL per well in a 24-well plate, and incubated in a CO2 incubator (37°C, 5% CO2, saturated humidity) for 24 hours.

[0160] Next, the culture medium was discarded, and 400 μL of the liposome formulation (89WP-CLS / R8 / siRNA) prepared in Example 5, containing siRNA, was added. In this liposome formulation, the charge ratio of oligoarginine to siRNA was 5, and the charge ratio of CLS to siRNA was 4, and on the surface 89W Penetratin-PEG 3400 -It was a liposomal pharmaceutical formulation modified with DSPE at a rate of 3%.

[0161] After 6 hours, the drug solution was discarded, the cells were treated again, and seeded at a concentration of 3000 cells / well in a 96-well plate. Fresh DMEM complete medium (DMEM medium supplemented with 10% FBS and 1% penicillin-streptomycin) was added, and cultivation was continued. After 48 hours, 10 μL of cck-8 solution was added to each well, and the cells were incubated at 37°C for 2 hours. The OD value of each well was then measured at a wavelength of 490 nm using a microplate reader (BioTek, PowerWave XS).

[0162] Cell viability (%) = (OD s -OD blank ) / (OD control -OD blank ) × 100% [In the formula, O.D. s This refers to the absorbance of the drug group, OD. control The absorbance of the blank control group (each well contained only cells, DMEM complete medium, and cck-8), OD blank This represents the absorbance of the blank well (only DMEM complete medium and cck-8 were added to the well).

[0163] All experiments were performed in triples, and the results were analyzed using GraphpadPrism software. The results are shown in Figure 22.

[0164] The results showed that the half-maximal inhibitory concentration IC of the chemotherapy drugs BCNU, DTX, Gemcitabine, Imatinib, Cisplatin, and DOX was the IC25. 50 The values ​​were 92.04 μM, 15.52 nM, 0.11 μM, 41.59 μM, 5.62 μM, and 0.17 μM, respectively. (Half maximum inhibitory concentration IC of gene drug siRNA) 50 The value was 90.99 nM.

[0165] [Example 13] (In vitro antitumor pharmacodynamic evaluation by combining gene drugs with different chemotherapy drugs) Density 1 × 10 5A U87 single-cell suspension was prepared at a cell / mL concentration and seeded in 500 μL per well in a 24-well plate. The plate was then placed in a CO2 incubator (37°C, 5% CO2, saturated humidity). After 24 hours of incubation, the medium was discarded, and 400 μL of the liposomal formulation (89WP-CLS / R8 / siRNA) prepared in Example 5, containing siRNA, was added.

[0166] The liposome formulation has a charge ratio of 5 between oligoarginine and siRNA, and a charge ratio of 4 between CLS and siRNA, and on the surface 89W Penetratin-PEG 3400 -It was a liposomal pharmaceutical formulation modified with DSPE at a rate of 3%.

[0167] After 6 hours, the drug solution was discarded, the cells were treated again, seeded at a concentration of 3000 cells / well in 96-well plates, and cultured for another 12 hours in fresh DMEM complete medium (DMEM medium supplemented with 10% FBS and 1% penicillin-streptomycin). Subsequently, the medium was replaced with complete medium containing different chemotherapeutic agents (BCNU, DTX, Gemcitabine, Imatinib, Cisplatin, DOX) and cultured further (the molar concentration ratio of gene drugs to chemotherapeutic agents was half of the maximum inhibitory concentration IC50). 50 (This is the ratio of the values). After 48 hours, 10 μL of cck-8 solution was added to each well and incubated at 37°C for 2 hours, after which the OD value of each well was measured at a wavelength of 490 nm using a microplate reader.

[0168] Cell viability (%) = (OD s -OD blank ) / (OD control -OD blank ) × 100% [In the formula, O.D. s This refers to the absorbance of the drug group, OD. control The absorbance of the blank control group (each well contained only cells, DMEM complete medium, and cck-8), OD blank This represents the absorbance of the blank well (only DMEM complete medium and cck-8 were added to the well).

[0169] All experiments were performed in triplicate. The results are shown in Figure 23. The results were further analyzed using CompuSyn software for the purpose of obtaining combination indices and dose reduction indices for the combined use of different chemotherapeutic drugs with gene drugs.

[0170] The calculation results are shown in Figures 24A and 24B. As shown in Figures 24A and 24B, different chemotherapeutic drugs, BCNU, DTX, Gemcitabine, Imatinib, Cisplatin, and DOX, respectively played a role in synergistically reducing cell viability with the gene drug siRNA. In particular, the combination indices of the chemotherapeutic drugs DTX, Gemcitabine, Cisplatin, and DOX with the gene drug were less than 1, and the dose reduction indices were greater than 1, indicating a favorable combined therapeutic effect with a synergistic effect.

[0171] Also, the combination indices of the chemotherapeutic drugs BCNU and Imatinib with the gene drug were greater than 1, and the dose reduction indices were less than 1, playing a role in exerting a synergistic effect, but the dose reduction used in the combined therapy was not as much as that of the chemotherapeutic drugs DTX, Gemcitabine, Cisplatin, and DOX. Therefore, the chemotherapeutic drugs DTX, Gemcitabine, Cisplatin, and DOX were used in further experiments to screen for the optimal chemotherapeutic drugs for combination with gene drugs at a specific total drug dose.

[0172] [Example 14] (In vitro Antitumor Pharmacodynamic Screening by Combining Different Chemotherapeutic Drugs with Gene Drugs) A U87 single-cell suspension with a density of 1×10 5 cells / mL was prepared, seeded into a 24-well plate at 500 μL per well, transferred to a CO2 incubator (37 °C, 5% CO2, saturated humidity), cultured for 24 hours, the original medium was discarded, and 400 μL of the liposome formulation (89WP-CLS / R8 / siRNA) prepared in Example 5 containing siRNA was added.

[0173] The liposome formulation had a charge ratio of 5 between oligoarginine and siRNA, a charge ratio of 4 between CLS and siRNA, and was a liposome pharmaceutical formulation modified with 89W Penetratin-PEG 3400 -DSPE at a ratio of 3%.

[0174] After 6 hours, the chemical solution was discarded, the cells were treated again, seeded in a 96-well plate at a concentration of 3000 cells / well, and fresh DMEM complete medium (DMEM medium supplemented with 10% FBS and 1% penicillin-streptomycin) was added and cultured for an additional 12 hours. Subsequently, the medium was replaced with DMEM complete medium containing different chemotherapeutic drugs (DTX, Gemcitabine, Cisplatin, DOX) and the culture was continued (the concentration ratio of the gene drug to the chemotherapeutic drug was the ratio of their half-maximal inhibitory concentration IC 50 values, and the total concentration of the gene drug and the chemotherapeutic drug was 200 nM).

[0175] After 48 hours, 10 μL of cck-8 solution was added to each well, incubated at 37 °C for 2 hours, and then the OD value of each well was measured at a wavelength of 490 nm using a microplate reader.

[0176] Cell viability (%) = (OD s - OD blank ) / (OD[[ID=​​​​​​​​​​​​​​All experiments were performed in triplicate, and the results were analyzed using GraphpadPrism software. The results are shown in Figure 25 (mean ± SD, n=3, *p<0.05, * indicates a statistically significant difference between the two groups; ***p<0.001, *** indicates a statistically very significant difference between the two groups; ns indicates no statistically significant difference between the two groups).

[0178] The results showed that the concentration ratio of gene drugs to chemotherapy drugs was half of the maximum inhibitory concentration IC. 50 The ratio of values ​​indicates that when the combined concentration of the gene drug and the chemotherapy drug is 200 nM, the combination of the gene drug and the chemotherapy drug DTX has the greatest inhibitory effect on cell viability, demonstrating the optimal in vitro antitumor effect. Based on these results, the combination of the gene drug and the chemotherapy drug DTX at specific total drug doses was used in subsequent experimental studies.

[0179] [Example 15] (Preparation of pharmaceutical formulations in which cationic liposomes and positively charged polyamino acids encapsulate gene drugs and chemotherapeutic agents) As described in Example 1, the membrane material, namely 3.5 mg of DOTAP, 3.72 mg of DOPE, and 2.59 mg of cholesterol, was weighed, 4 μg of the DTX drug substance (Dalian Meilun Biotechnology Co., Ltd., MA1081) was added, and the mixture was dissolved in chloroform.

[0180] The obtained solution was dechloroformed using a rotary evaporator (Shanghai Shensheng Technology Co., Ltd., R201L), and the resulting dried lipid film was hydrated in a 37°C water bath with 1 mL of 5% glucose aqueous solution using sonication for approximately 10 minutes. After the lipid membrane was completely hydrated, liposomes were extruded using a mini extruder (Hamilton, 81320) (the order of the pore membranes was 200 nm, 100 nm, and 50 nm, respectively) to obtain CLS encapsulated with DTX (CLS / DTX).

[0181] Similar to Example 4, the liposome complex CLS / DTX / R8 / siRNA was prepared using the prepared R8 / siRNA complex and CLS / DTX.

[0182] Similar to Example 5, the prepared CLS / DTX / R8 / siRNA complex and mPEG 2000 -DSPE and 89W Penetratin-PEG 3400 - The molar ratio of DSPE to the total is 60%. 89W Penetratin-PEG 3400 -A mixture of DSPE was incubated at 55°C for 30 minutes to obtain a liposomal pharmaceutical formulation, namely 89WP-CLS / DTX / R8 / siRNA, on which a cell-permeable peptide was modified on the surface in a certain percentage. The molar concentration ratio of the gene drug siRNA and the chemotherapeutic drug DTX in the liposomal pharmaceutical formulation was such that the IC of both was 50 The ratio of the values ​​was approximately 6, and the molar concentration of the gene drug siRNA was approximately 6.20 μM, while the molar concentration of the chemotherapy drug DTX was 1.03 μM.

[0183] [Example 16] (In vitro antitumor pharmacodynamic evaluation of the combined use of gene therapy drugs and docetaxel (DTX)) Density 5 × 10 4 A U87 single-cell suspension was prepared at a concentration of cells / mL and seeded into a 24-well plate at 500 μL per well. The plate was then transferred to a CO2 incubator (37°C, 5% CO2, saturated humidity) and incubated for 24 hours. Subsequently, the medium was discarded, and either 400 μL of the liposome formulation 89WP-CLS / DTX / R8 / siRNA or CLS / DTX / R8 / siRNA prepared in Example 15, or 400 μL of 89WP-CLS / R8 / siRNA+DTX was added.

[0184] The liposome formulation has a charge ratio of 5 between oligoarginine and siRNA, and a charge ratio of 4 between CLS and siRNA, and on the surface 89W Penetratin-PEG 3400-DSPE was modified at a rate of 3% in a liposomal pharmaceutical formulation. The aforementioned 89WP-CLS / R8 / siRNA+DTX was obtained by physically mixing 89WP-CLS / R8 / siRNA and DTX. The molar concentration ratios of the gene drug siRNA and the chemotherapy drug DTX were both the IC25 of the two. 50 It was a ratio of values.

[0185] After 6 hours, the drug solution was discarded, the cells were treated again, and seeded at a concentration of 3000 cells / well in a 96-well plate. Fresh DMEM complete medium (DMEM medium supplemented with 10% FBS and 1% penicillin-streptomycin) was added, and cultivation was continued. After 48 hours, 10 μL of cck-8 solution was added to each well, incubated at 37°C for 2 hours, and then the OD value of each well was measured at a 490 nm wavelength using a microplate reader.

[0186] Cell viability (%)=(ODs-ODblank) / (ODcontrol-ODblank)×100% [In the formula, O.D. s This refers to the absorbance of the drug group, OD. control The absorbance of the blank control group (each well contained only cells, DMEM complete medium, and cck-8), OD blank This represents the absorbance of the blank well (only DMEM complete medium and cck-8 were added to the well).

[0187] All experiments were performed in three copies, and the results were analyzed using GraphpadPrism software. The results are shown in Figure 26 (mean ± SD, n=3, ns indicates no statistically significant difference between the two groups).

[0188] As shown in Figure 26, the liposome formulation 89WP-CLS / DTX / R8 / siRNA showed half the maximum inhibitory concentration IC of DTX. 50 The value was 8.9 nM, and for the liposomal formulation CLS / DTX / R8 / siRNA, the maximum inhibitory concentration IC50 was half that of DTX. 50The value was 24.7 nM, and when the liposome formulation 89WP-CLS / R8 / siRNA was used in combination with DTX, the maximum inhibitory concentration IC50 was half that of DTX. 50 The value was 10.3 nM. As can be seen from the above, when DTX is only functional for 6 hours, the combined use of siRNA and DTX in the liposomal formulation of the present invention significantly reduced the half-maximal inhibitory concentration of DTX, resulting in a more effective tumor inhibitory effect.

[0189] [Example 17] (Exploring the situation in which carriers labeled with DiD cell membrane fluorescent probes are taken up by cells after encapsulating FAM-siRNA) Similar to Example 1, the membrane material was weighed and the DiD cell membrane fluorescent probe (Dalian Meilun Biotechnology Co., Ltd., MB6190) was added as described in the kit instructions to prepare CLS labeled with the DiD cell membrane fluorescent probe, i.e., CLS / DiD.

[0190] 33 μg of FAM-labeled siRNA (in this example, siRNA against c-myc was used, with its sense strand (5'-3') being FAM-AACGUUAGCUUCACCAACAdTdT, i.e., 5' being modified with FAM and the antisense strand (5'-3') being UGUUGGUGAAGCUAACGUUdTdT) was dissolved in 125 μL of DEPC-treated water to prepare a 20 μM FAM-siRNA solution, and each pharmaceutical formulation labeled with DiD and / or FAM fluorescence was prepared.

[0191] U87 cells (human glioma cells, purchased from ATCC) or Calu-3 cells (human lung adenocarcinoma cells, purchased from ATCC) were cultured in DMEM complete medium (DMEM medium supplemented with 10% FBS and 1% penicillin-streptomycin). Well-developed U87 or Calu-3 cells were selected, resuspended in DMEM complete medium, and 5 × 10⁶ cells were cultured. 5 Cells were seeded in a 6-well plate with 1 mL of culture medium per well. After seeding, the culture medium was changed once daily, and the cells were cultured for 2-3 days before the experiment was performed.

[0192] After discarding the culture medium of U87 cells or Calu-3 cells, the wells were washed three times with sterile PBS. 1 mL of serum-free DMEM medium containing 1 μM of a different fluorescently labeled pharmaceutical formulation of siRNA was added to each well, and the wells were incubated at 37°C and 5% CO2 for 4 hours.

[0193] Next, the culture medium was discarded, and positively charged substances adsorbed to the wells and cell surfaces were washed away with PBS buffer solution containing 0.02 mg / mL heparin sodium. The cells were treated with trypsin, resuspended in 200 μL of sterile PBS buffer solution, and after uniform spraying, the cells were counted for each sample. 4 Individual cells were taken and detected using flow cytometry.

[0194] In the experiment, cells incubated in blank serum-free DMEM medium were set as the negative control group. Furthermore, two positive control groups were established: 89WP-CLS / R8 / FAM-siRNA containing only FAM, and 89WP-CLS / DiD containing only DiD. In addition, the following formulation groups were established: 89WP-CLS / DiD / R8 / siRNA group, CLS / DiD / R8 / FAM-siRNA group, and 89WP-CLS / DiD / R8 / FAM-siRNA group.

[0195] All experiments were performed in triple replication. The dose of all formulations was 1 mL, of which the siRNA concentration was approximately 1 μM and the DTX concentration was approximately 0.1667 μM (the molar concentration ratio of DTX to siRNA was IC). 50 The ratio of the values ​​is 6), the charge ratio of oligoarginine to siRNA is 5, and the charge ratio of CLS to siRNA is 4, and on the surface at a rate of 3% 89W Penetratin-PEG 3400 -DSPE was modified.

[0196] The results are shown in Figure 27 (mean ± SD, n=3, ***p<0.001, *** indicates a statistically significant difference between the two groups).

[0197] The results in Figure 27 show that ingestion of the 89WP-CLS / DiD / R8 / FAM-siRNA drug formulation in U87 cells and Calu-3 cells resulted in stronger bifluorescence positivity for both DiD and FAM. This indicates that the fluorescent probe DiD embedded in the liposome membrane and the siRNA encapsulated in the liposome are not ingested in a free form, but rather co-ingested as a lipid complex. Given the similar physical and chemical properties of the chemotherapeutic drug DTX and the fluorescent probe DiD, it is hypothesized that when the 89WP-CLS / DTX / R8 / siRNA drug formulation, which co-encapsulates the gene drug and the chemotherapeutic drug, is ingested by cells, the gene drug siRNA and the chemotherapeutic drug DTX are also co-ingested and ingested as a lipid complex.

[0198] [Example 18] (Evaluation of the ability of U87 cells to promote apoptosis by combining gene therapy drugs and DTX chemotherapy drugs) U87 cells (purchased from ATCC) with good logarithmic growth status in DMEM medium were selected and 5 × 10⁶ cells were taken. 4 Seeds were seeded at a concentration of / well into 12-well plates and cultured at 37°C and 5% CO2 until a monolayer of cells covered the bottom of each well.

[0199] After discarding the culture medium and washing it three times with sterile PBS buffer solution, 400 μL of the liposome formulations 89WP-CLS / DTX / R8 / siRNA and CLS / DTX / R8 / siRNA prepared in Example 15, 89WP-CLS / R8 / siRNA prepared in Example 5, and a physical mixture of 89WP-CLS / R8 / siRNA and DTX, each containing 150 nmol / L siRNA, were added. In other words, the dose of each formulation was 400 μL. The siRNA concentration was diluted to 150 nmol / L.

[0200] The liposome formulation has a charge ratio of 5 between oligoarginine and siRNA, and a charge ratio of 4 between CLS and siRNA, with a concentration of 3% on the surface. 89W Penetratin-PEG 3400 -DSPE is modified, and the molar concentration ratio of DTX to siRNA is IC50 It was the ratio of the values, which was 6.

[0201] After incubation in an incubator for 6 hours, the reagent solution was discarded, the cells were washed three times with sterile PBS buffer solution, and then 1 mL of complete culture medium was added and cultured for another 18 hours. Subsequently, the cells were collected, washed with PBS, treated with EDTA-free trypsin for 1 minute, and then treated with DMEM culture medium. Next, the cells were collected in a centrifuge tube, double-stained using a cell apoptosis test kit (Nanjing Kaiji Biotechnology Development Co., Ltd., KGA108), and detected by flow cytometry.

[0202] Cells incubated in serum-free DMEM medium served as the negative control group. All experiments were performed in triple replication, and the results are shown in Figure 28 (mean ± SD, n=3, **p<0.01, ***p<0.001, ** and *** indicate statistically significant differences between the two groups).

[0203] The results showed that the pharmaceutical formulation, which co-delivers the gene drug and chemotherapy agent designed in this invention, significantly improved the apoptosis rate of tumor cells U87 and demonstrated a more efficient tumor suppression effect.

[0204] [Example 19] (Pharmacodynamic evaluation of anti-U87 situ brain tumors by combining gene therapy and DTX chemotherapy) Logarithmic growth phase U87 cells were taken, counted, and resuspended in PBS buffer. 6 × 10⁶ cells were placed in each BALB / c nude mouse (Shanghai Xipuel-Bikai Test Animals Co., Ltd., China). 5 We inoculated the mice with U87 cells (dispersed in 5 μL of PBS buffer solution). Nude mice were anesthetized with 7% chloral water, fixed with a stereotactic brain device, and the cells were inoculated into the striatal region (0.6 mm anterior to bregma, 1.8 mm to the right, and 3 mm posterior) using a microsyringe to construct a U87 situ brain tumor model.

[0205] According to the methods described in the foregoing embodiments, liposomal pharmaceutical preparations, namely, CLS / DTX / R8 / siRNA, 89WP-CLS / R8 / siRNA, 89WP-CLS / DTX / R8 / siRNA, and physical mixtures of 89WP-CLS / R8 / siRNA + DTX, were prepared respectively.

[0206] After inoculating U87 orthotopic brain tumors, the mice were randomly divided into six groups (n = 10), namely, the normal saline group, the DTX group, the CLS / DTX / R8 / siRNA group, the 89WP-CLS / R8 / siRNA group, the 89WP-CLS / DTX / R8 / siRNA group, and the 89WP-CLS / R8 / siRNA + DTX group.

[0207] Transnasal administration was performed from the 5th day after inoculating U87 orthotopic brain tumors. The dosages were 0.66 mg / kg siRNA (CLS / DTX / R8 / siRNA group, 89WP-CLS / R8 / siRNA group, 89WP-CLS / DTX / R8 / siRNA group, 89WP-CLS / R8 / siRNA + DTX group), 6.66 μg / kg DTX (DTX group, CLS / DTX / R8 / siRNA group, 89WP-CLS / DTX / R8 / siRNA group, 89WP-CLS / R8 / siRNA + DTX group).

[0208] In all administered preparations, the charge ratio of oligoarginine to siRNA was 5, the charge ratio of CLS to siRNA was 4, and Penetratin-PEG 89W -DSPE was modified at a ratio of 3% on the surface, and the molar concentration ratio of DTX to siRNA was the ratio of the IC 3400 value, that is, 6. 50 The administration was performed once a day for a total of 22 times. The body weight and survival period of the mice were recorded, and the body weight change curve and survival curve were drawn. The results are shown in FIGS. 29 and 30 (**p < 0.01, ***p < 0.001, ** and *** indicate that there are very significant statistical differences between the two groups).

[0209] The administration was carried out once a day for a total of 22 times. The body weight and survival period of the mice were recorded, and the body weight change curve and survival curve were plotted. The results are shown in FIGS. 29 and 30 (**p < 0.01, ***p < 0.001, ** and *** indicate that there are statistically very significant differences between the two groups).

[0210] As a result, the median survival periods of the normal saline group, DTX group, CLS / DTX / R8 / siRNA group, 89WP-CLS / R8 / siRNA group, and 89WP-CLS / R8 / siRNA+DTX group were 20 days, 21 days, 27 days, 27 days, and 28.5 days, respectively.

[0211] The median survival period of the 89WP-CLS / DTX / R8 / siRNA group was successfully extended to 33.5 days, the survival curve did not cross that of other groups, and the most significant anti-glioblastoma effect was achieved. These data demonstrated that 89WP-CLS / DTX / R8 / siRNA can deliver drugs to brain tumors to exert an antitumor effect, and its unique delivery advantages can be converted into a higher therapeutic effect.

[0212] Although several representative embodiments and details have been shown for the purpose of illustrating the present invention, it will be apparent to those skilled in the art that various changes and modifications can be made to them without departing from the scope of the subject invention. In this regard, the scope of the present invention is limited only by the following claims.

Claims

1. A pharmaceutical formulation that delivers multiple types of therapeutic agents together, It includes various therapeutic agents and drug delivery carriers. The drug delivery carrier comprises a cationic liposome modified with a cell-permeable peptide and a cationic material. The cell-permeable peptide is penetratin or RX having the amino acid sequence represented by Sequence ID No.

2. １ IKIWFX ２ X ３ RRMKWKK (Sequence No. 1, where X １ , X ２ , X ３ Each of these is an amino acid independently selected from the group consisting of glutamine (Q), asparagine (N), alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), tryptophan (W), methionine (M), α-aminobutyric acid, α-aminopentanoic acid, α-aminohexanoic acid, and α-aminoheptanoic acid. The amino acid sequence represented by (excluding the amino acid sequence represented by Sequence ID No. 2) is It is a derivative of penetratin, The cationic material is a compound selected from the group consisting of positively charged polyamino acids, polyethyleneimine (PEI), penetratin, derivatives of penetratin, and polyamidoamines (PAMAM). The aforementioned variety of therapeutic agents is a pharmaceutical formulation comprising at least one non-nucleic acid therapeutic agent and at least one nucleic acid therapeutic agent.

2. The nucleic acid therapeutic agent is a therapeutic agent selected from the group consisting of plasmid DNA, small interfering RNA (siRNA), miRNA, sense RNA, antisense oligonucleotide (ASO), aptamers, and ribozymes. The pharmaceutical formulation according to claim 1, wherein the non-nucleic acid therapeutic agent is a chemotherapeutic agent.

3. The aforementioned cationic liposome contains cationic lipids, The molar ratio of the non-nucleic acid therapeutic agent to the cationic lipid is 1:1500 to 2000:

1. The charge ratio between the cationic material and the nucleic acid therapeutic agent is 1:1 to 30:

1. The pharmaceutical formulation according to claim 1 or 2, wherein the charge ratio of the cationic liposome to the nucleic acid therapeutic agent is 1:1 to 30:

1.

4. The pharmaceutical formulation according to any one of claims 1 to 3, wherein the cationic liposome comprises a cationic lipid, a non-cationic lipid, cholesterol, polyethylene glycol (PEG)-conjugated phospholipid, and polyethylene glycol phospholipid conjugated with a cell-permeable peptide.

5. The pharmaceutical formulation according to claim 4, wherein the cationic lipid is 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), the non-cationic lipid is 1,2-di-(9Z-octadecanoyl)-sn-glyceryl-3-phosphoethanolamine (DOPE), the PEGylated phospholipid is methoxy-polyethylene glycol-distearoylphosphatidylethanolamine (mPEG-DSPE), and the polyethylene glycol phospholipid conjugated with the cell-permeable peptide is penetratin-PEG-DSPE or a derivative of penetratin-PEG-DSPE.

6. The cationic lipid is DOTAP, the non-cationic lipid is DOPE, the PEGylated phospholipid is mPEG-DSPE, and the polyethylene glycol phospholipid conjugated with the cell-permeable peptide is the penetratin derivative-PEG-DSPE. The molar ratio of (the DOTAP):(the DOPE):(the cholesterol):(the sum of the mPEG-DSPE and the penetratein derivative-PEG-DSPE) is approximately 28.5:28.5:38:

5. The molar ratio of the penetratin derivative -PEG-DSPE to the sum of the mPEG-DSPE and the penetratin derivative -PEG-DSPE is 60%. The molar ratio of the non-nucleic acid therapeutic agent to DOTAP is approximately 1:1000. The charge ratio of the cationic material to the nucleic acid therapeutic agent is approximately 5:

1. The charge ratio of the cationic liposome to the nucleic acid therapeutic agent is approximately 4:

1. The molar concentration ratio of the non-nucleic acid therapeutic agent and the nucleic acid therapeutic agent is the IC of both. ５０ The ratio of the values A pharmaceutical preparation according to claim 4 or 5.

7. A pharmaceutical preparation according to any one of claims 1 to 6, for intranasal administration.

8. It comprises cationic liposomes modified with cell-permeable peptides and cationic materials, The cell-permeable peptide is penetratin or RX having the amino acid sequence represented by SEQ ID NO: 2 １ IKIWFX ２ X ３ RRMKWK K (SEQ ID NO: 1, where X １ , X ２ , X ３ is independently an amino acid selected from the group consisting of glutamine (Q), asparagine (N), alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), tryptophan (W), methionine (M), α-aminobutyric acid, α-aminopentanoic acid, α-aminohexanoic acid, and α-aminoheptanoic acid.)) (however, excluding the amino acid sequence represented by SEQ ID NO: 2). It is a derivative of penetratin, The cationic material is a compound selected from the group consisting of positively charged polyamino acids, polyethyleneimine (PEI), penetratin, derivatives of penetratin, and polyamidoamines (PAMAM), and is a drug delivery carrier.

9. The cell-permeable peptide is a peptide consisting of an amino acid sequence represented by RWIKIWFQNRRMKWKK (SEQ ID NO: 24), RQIKIWFWNRRMKWKK (SEQ ID NO: 25), RQIKIWFQWRRMKWKK (SEQ ID NO: 26), RWIKIWFWNRRMKWKK (SEQ ID NO: 27), RWIKIWFQWRRMKWKK (SEQ ID NO: 28), RQIKIWFWWRRMKWKK (SEQ ID NO: 29), or RWIKIWFWWRRMKWKK (SEQ ID NO: 30). The drug delivery carrier according to claim 8, wherein the cationic liposome comprises a cationic lipid, a non-cationic lipid, cholesterol, PEGylated phospholipid, and polyethylene glycol phospholipid conjugated with the cell-permeable peptide.

10. A drug delivery carrier according to claim 8 or 9 for co-delivering multiple types of therapeutic agents.

11. A method for producing a pharmaceutical preparation according to any one of claims 4 to 6, (a) A step of mixing the cationic material and the nucleic acid therapeutic agent to form a physical composite consisting of the cationic material and at least one nucleic acid therapeutic agent, (b) A step of preparing liposomes containing at least one non-nucleic acid therapeutic agent using the cationic lipid, the non-cationic lipid, the cholesterol, and the non-nucleic acid therapeutic agent as materials, (c) A step of preparing a lipid complex by mixing a physical complex consisting of the cationic material and at least one nucleic acid therapeutic agent with liposomes containing at least one non-nucleic acid therapeutic agent, (d) The step of modifying the lipid complex with the PEGylated phospholipid and the polyethylene glycol phospholipid conjugated with the cell-permeable peptide, or (a') A step of mixing the cationic material and the nucleic acid therapeutic agent to form a physical composite consisting of the cationic material and at least one nucleic acid therapeutic agent, (b') A step of preparing liposomes containing at least one non-nucleic acid therapeutic agent using the cationic lipid, the non-cationic lipid, the cholesterol, the PEG-modified phospholipid, the polyethylene glycol phospholipid conjugated with the cell-permeable peptide, and the non-nucleic acid therapeutic agent as materials, (c') A manufacturing method comprising the step of mixing a physical complex consisting of the cationic material and at least one nucleic acid therapeutic agent with liposomes containing at least one non-nucleic acid therapeutic agent to obtain the pharmaceutical formulation.

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