Drug delivery carrier and pharmaceutical preparation using same

Abstract

The present invention provides a drug delivery carrier comprising a liposome modified with a cell-penetrating peptide and a positively charged polyamino acid as a polymer. The drug delivery carrier is used to prepare a pharmaceutical formulation in the form of a liposome complex with a particle size of 50 nm to 300 nm. The pharmaceutical formulation is prepared by mixing a physical complex consisting of a positively charged polyamino acid and one or more drug molecules with a liposome whose surface is modified with a cell-penetrating peptide. The pharmaceutical formulation can effectively transfect cells, particularly promoting drug absorption into the brain via intranasal administration, thereby improving the therapeutic efficacy of the drug.

Description

[Technical field]

[0001] The present invention relates to a drug delivery carrier, a method for preparing a pharmaceutical preparation using the drug delivery carrier, and a pharmaceutical preparation prepared thereby. Specifically, the drug delivery carrier of the present invention comprises a liposome modified with a cell-penetrating peptide and a polymer, and the polymer is a positively charged polyamino acid. The pharmaceutical preparation prepared using the drug delivery carrier of the present invention is a liposome complex prepared by mixing a physical complex formed of a positively charged polyamino acid and one or more drug molecules with a liposome modified with a cell-penetrating peptide, and the pharmaceutical preparation can effectively transfect cells, and in particular, can promote the absorption of drugs into the brain via the nasal cavity, thereby enhancing the therapeutic effect of the drug. [Background technology]

[0002] Cell-penetrating peptides (CPPs) are short peptides that are positively charged under physiological conditions, and can be covalently or non-covalently bound to drug molecules such as genes, polypeptides, and proteins, and can deliver the drug molecules into cells or carry the drug molecules to penetrate biological membrane barriers. However, a single cell-penetrating peptide has a very limited capacity to carry drug molecules, and therefore the complex formed by the cell-penetrating peptide and the drug molecule has a large particle size and poor stability.

[0003] In addition, in the prior art, carriers for delivering drug molecules (e.g., drug molecules such as genes, polypeptides, and proteins), such as liposomes, have problems such as low delivery efficiency and prone to tissue toxicity. In the field of liposome research, how to use liposomes to deliver drug molecules more efficiently so that disease treatment and / or prevention is more specific and efficient remains a big challenge. Researchers have mainly improved the stability, targeting, and drug delivery efficiency of liposomes through measures such as adding various auxiliary components to liposomes, developing and using new liposome materials, structurally modifying the surface of liposomes, and designing and using new preparation processes.

[0004] Therefore, there is still a need in the field for new drug delivery carriers that utilize cell-penetrating peptides, which have improved stability after drug molecules (e.g., genes, polypeptides, proteins, etc.) are complexed with the drug delivery carrier, and the particle size of the prepared pharmaceutical formulation is controllable and has good stability, and more effective drug delivery can be achieved by the cell-penetrating peptides that modify the surface of the formulation, which helps the drug escape from the cell endosome to exert its effects. Summary of the Invention [Problem to be solved by the invention]

[0005] The present inventors have developed a novel drug delivery carrier that utilizes a cell-penetrating peptide through intensive research. The drug delivery carrier of the present invention comprises a liposome modified with a cell-penetrating peptide and a polymer, the polymer being a positively charged polyamino acid, the polymer being intended to be mixed with one or more drug molecules to form a polymer-drug complex, and the liposome modified with the cell-penetrating peptide being intended to be mixed with the polymer-drug complex to form a liposome complex containing the polymer-drug complex. [Means for solving the problem]

[0006] In one embodiment, the cell penetrating 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 (SEQ ID NO:1)

[0007] 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), and 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 of the cell-penetrating peptides in the drug delivery carrier of the present invention are 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 have been mutated to hydrophobic amino acids.

[0008] In one embodiment, the positively charged polyamino acid in the drug delivery carrier of the present invention is, for example, a positively charged polyamino acid having a degree of polymerization of 2 to 50, the spatial structure of the positively charged polyamino acid is linear or cyclic, and the configuration of the amino acid residue is L-type or D-type. For example, the polyamino acid is arginine and / or lysine having a degree of polymerization of 6 to 12. The positively charged polyamino acid may have 1 to 20 (for example, 1 to 6) amino acid residues that are uncharged under physiological conditions intervening among the positively charged amino acid residues. Preferably, the positively charged polyamino acid is polyarginine having a degree of polymerization of 6 to 12, in which the spatial structure of the polyarginine is linear or cyclic, and the configuration of the polyarginine is L-type or D-type.

[0009] In one embodiment, the liposome modified with a cell-penetrating peptide in the drug delivery carrier of the present invention comprises 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(sperminecarboxamido)ethyl]-NN-dimethyl-1-propane ammonium trifluoroacetate (DOSPA) and combinations thereof. Preferred are DOTMA, DOTAP, DODAC, and DOSPA. Most preferred is DOTAP; (ii) Non-cationic 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. Preferred are DOPE and / or DOPC. Most preferred is DOPE; (iii) cholesterol; and (iv) Polyethylene glycol-conjugated (PEGylated) phospholipids and polyethylene glycol phospholipids conjugated with CPPs; the phospholipids are, for example, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and sphingomyelin. Preferably, the PEGylated phospholipids are, for example, polyethylene glycol-distearoylphosphatidylethanolamine (PEG-DSPE) and its derivative, methoxy-polyethylene glycol-distearoylphosphatidylethanolamine (mPEG-DSPE). The polyethylene glycol phospholipid conjugated with CPPs is, for example, Penetratin / Penetratin derivative-PEG-DSPE.

[0011] In some embodiments, the molar ratio of membrane material (i):(ii):(iii):(iv) is about 20-40:20-40:20-40:1-20, and the molar ratio of polyethylene glycol phospholipid conjugated to CPP to membrane material (iv) is about 20%-80%. In some specific embodiments, the molar ratio of membrane material (i):(ii):(iii):(iv) is about 27.0-31.6:27.0-31.6:31.6-39.6:1-10, and the molar ratio of polyethylene glycol phospholipid conjugated to CPP to membrane material (iv) is about 20%-80%.

[0012] In some specific embodiments, the cationic lipid in the drug delivery carrier of the present invention is, as a membrane material, (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 20-40:20-40:20-40:1-20; 89W Penetratin-PEG 3400 -DSPE is in a molar ratio of about 20% to 80% relative to membrane material (iv). In some embodiments, the molar ratio of membrane materials (i):(ii):(iv) is about 27.0-31.6:27.0-31.6:31.6-39.6:1-10; and 89W Penetratin-PEG 3400 The molar ratio of the DSPE to the membrane material (iv) is about 20% to 80%.

[0013] In some embodiments, the cationic lipid in the drug delivery carrier of the present invention is: (i) DOTAP; (ii) DOPE; (iii) cholesterol; (iv) mPEG. 2000 -DSPE and 89W Penetratin-PEG 3400 -DSPE, the molar ratio of membrane materials (i):(ii):(iii):(iv) being approximately 28.5:28.5:38:5; 89W Penetratin-PEG3400 -DSPE is present in a molar ratio of about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% relative to the membrane material (iv).

[0014] In another aspect, the present invention provides a pharmaceutical preparation prepared using the drug delivery carrier of the present invention, which comprises forming a physical complex between a positively charged polyamino acid contained in the drug delivery carrier and one or more drug molecules to be delivered, and mixing the physical complex with a cationic liposome modified with a cell-penetrating peptide on the surface thereof to form a liposome complex-type pharmaceutical preparation.

[0015] The drug molecule or molecules to be delivered include, but are not limited to, peptide drugs (e.g., octreotide), antibodies (e.g., etanercept, adalimumab, rituximab, pembrolizumab, ranibizumab, and nivolumab), cytokines, hormones, antibiotics (e.g., the chemotherapeutic drugs doxorubicin, daunorubicin, epirubicin, actinomycin, and vancomycin), nucleic acids, and combinations thereof.

[0016] In one embodiment, the drug molecule delivered by the pharmaceutical formulation is a nucleic acid such as plasmid DNA, small interfering RNA (siRNA), sense RNA, antisense oligonucleotides (ASO), aptamers, ribozymes, etc. In one embodiment, the drug molecule delivered by the pharmaceutical formulation is a combination of a nucleic acid and a drug capable of being integrated into the organism's genome (e.g., doxorubicin).

[0017] In one embodiment, the molar ratio of the drug molecule (e.g., a chemotherapeutic drug) to the cationic lipid (e.g., DOTAP) in the pharmaceutical formulation is about 0.1 to 10, preferably about 0.1 to 5, and more preferably about 0.1 to 2.

[0018] In one embodiment, the amount of drug molecule (e.g., chemotherapeutic drug) encapsulated in the pharmaceutical formulation is about 1% to 30% (w / w), preferably about 1% to 25% (w / w), and more preferably about 3% to 20% (w / w), of the drug molecule (e.g., chemotherapeutic drug) relative to the pharmaceutical formulation.

[0019] In one embodiment, in the pharmaceutical preparation, the charge ratio between the positively charged polyamino acid (e.g., oligoarginine) and the drug molecule to be delivered is 1:1 to 30:1, preferably 5:1. Also, the charge ratio between the cationic liposome and the drug molecule is 1:1 to 30:1, preferably 4:1.

[0020] In one embodiment, the particle size of the pharmaceutical preparation of the present invention is 50 nm to 300 nm, preferably 80 nm to 150 nm, and has good stability.

[0021] In yet another aspect, the present invention provides a method for preparing a pharmaceutical formulation, comprising the steps of: (a) preparing liposomes having a cell-penetrating peptide modified on their surface; (b) preparing a physical complex consisting of a positively charged polyamino acid and a drug molecule to be delivered; and (c) mixing the liposomes having a cell-penetrating peptide modified on their surface obtained in step (a) with the physical complex obtained in step (b) at a certain molar ratio or charge ratio.

[0022] In one embodiment, the method for preparing the pharmaceutical formulation of the present invention includes the steps of (a) mixing liposome membrane materials (i), (ii) and (iii) to prepare blank liposomes, (b) preparing a physical complex consisting of a positively charged polyamino acid and a drug molecule to be delivered, and (c) mixing the blank liposomes obtained in step (a) and the physical complex obtained in step (b) at a certain molar ratio or charge ratio, and (d) mixing and incubating the liposomes obtained in step (c) with membrane material (iv) to obtain the pharmaceutical formulation of the present invention.

[0023] Pharmaceutical formulations prepared using the drug delivery carrier of the present invention can be administered, for example, into the oral cavity, nasal cavity, eye, respiratory tract, digestive tract, genital tract, local implant, injection, or infusion (epidural, intra-arterial, intra-articular, intracapsular, intracardiac, intraventricular, intracranial, intradermal, intramuscular, intraorbital, intraocular, intraperitoneal, intraspinal, intrasternal, intrathecal, intravenous, subarachnoid, subcapsular, subcutaneous, tracheal, rectal, sublingual, etc.) and used for the treatment and / or prevention of diseases.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. All publications, patent applications, patents and other references mentioned herein are fully incorporated herein by reference. In addition, the materials, methods and examples described herein are illustrative only and are not intended to be limiting. Other features, objects and advantages of the present invention will be apparent from the specification and drawings, and the appended claims. [Brief description of the drawings]

[0025] [Figure 1]FIG. 1 shows the agarose gel electrophoresis results and particle size results for each complex in which R8 (a polymer of eight arginines) and siRNA have different charge ratios (expressed as a percentage of positive charge / negative charge) and / or CLS and siRNA have different charge ratios. FIG. 1A and FIG. 1B show the agarose gel electrophoresis results and particle size results for R8 / siRNA complexes in which R8 / siRNA have different charge ratios, respectively. FIG. 1C and FIG. 1D show the agarose gel electrophoresis results and particle size results for CLS / siRNA complexes in which CLS / siRNA have different charge ratios, respectively. FIG. 1E and FIG. 1F show the agarose gel electrophoresis results and particle size results for CLS / R8 / siRNA liposome complexes in which R8 / siRNA has a charge ratio of 1:1 and CLS / siRNA has different charge ratios, respectively. [Diagram 2] FIG. 2 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 was 1:1. [Diagram 3] Figure 3 shows the electrophoresis, particle size and stability study results of CLS / siRNA / R8 complex. Figures 3A and 3B show the agarose gel electrophoresis and particle size results of liposome complexes with different charge ratios of R8 and siRNA when the charge ratio of CLS and siRNA is 4:1, respectively. Figures 3C and 3D show the precipitation state after high-speed centrifugation of liposome complexes with different charge ratios of R8 and siRNA when the charge ratio of CLS and siRNA is 4:1, respectively, and the agarose gel electrophoresis results of the supernatant after centrifugation. [Figure 4] Figure 4 shows the quantitative evaluation results of cellular uptake of pharmaceutical formulations whose surfaces were modified with different ratios of cell-penetrating peptides. The Lipo2000 group is a non-covalent complex of siRNA and the cationic liposome Lipofectamine2000, a commercially available gene transfection reagent. [Diagram 5] FIG. 5 shows the cellular uptake of drug formulations prepared using different formulations of liposome membrane materials as shown in Table 2 of Example 8. [Figure 6] FIG. 6 shows the particle sizes of liposome pharmaceutical preparations in which component (iv) is 1%, 5%, 8% and 10% molar ratio of the liposome membrane material. [Figure 7] Figure 7 shows the morphological results of culturing different cell clusters using the 35 mm Dimple NanoCulture Dish, a cell cluster culture plate manufactured by JSR Corporation. [Figure 8] FIG. 8 shows the results of evaluation of the cell mass permeability of the cell-penetrating peptides that modify the surface of liposomes. [Figure 9] FIG. 9 shows the transfection effect of a pharmaceutical preparation encapsulating GFP-siRNA into a 293T cell mass. [Figure 10] FIG. 10 shows the transfection effect of a pharmaceutical preparation encapsulating GFP-siRNA into a U87 cell mass. [Figure 11] FIG. 11 shows the results of qualitative and quantitative evaluation of the transfection effect on LUC-U87 cells by pharmaceutical preparations using different oligoarginines. [Figure 12] FIG. 12 shows the particle size of the pharmaceutical formulation encapsulating siRNA. [Figure 13] FIG. 13 shows the potential of pharmaceutical formulations encapsulating siRNA. [Figure 14] FIG. 14 shows the results of evaluation of the apoptosis promoting effect on bEnd.3 cells or U87 cells and the cytotoxic activity of pharmaceutical preparations encapsulating siRNA. [Figure 15] FIG. 15 shows the distribution in the body of a pharmaceutical formulation labeled with the cell membrane fluorescent probe DiD after intranasal administration. [Figure 16] FIG. 16 shows the results of flow cytometry detection of intracellular delivery of FAM-siRNA by a carrier labeled with the cell membrane fluorescent probe DiD. [Figure 17]FIG. 17 shows the biodistribution in U87 in situ brain tumor model mice after intranasal administration of a gene delivery carrier labeled with the cell membrane fluorescent probe DiD. [Figure 18] Figure 18 shows the results of pharmacodynamic evaluation of siRNA-encapsulated delivery carriers against U87 in situ brain tumors. The Lipo2000 / siRNA group is a non-covalent complex of siRNA and the cationic liposome Lipofectamine2000, a commercially available gene transfection reagent. [Figure 19] Figure 19 shows the evaluation results of the in vivo apoptosis-promoting ability of siRNA-encapsulated delivery carriers in U87 in situ brain tumor model mice. The Lipo2000 / siRNA group is a non-covalent complex of siRNA and the cationic liposome Lipofectamine2000, a commercially available gene transfection reagent. [Figure 20] Figure 20 shows the results of in vivo nasal mucosal toxicity evaluation of siRNA-encapsulated delivery carriers. The Lipo2000 / siRNA group is a non-covalent complex of siRNA and the cationic liposome Lipofectamine2000, a commercially available gene transfection reagent, and the sodium deoxycholate in the sodium deoxycholate group is a commercially available product from Dalian Meilun Biotechnology Co., Ltd. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] The present invention provides a novel drug delivery carrier comprising liposomes modified with cell-penetrating peptides and positively charged polyamino acids as polymers. The drug delivery carrier of the present invention can assemble with drug molecules to form complexes, compact the drug molecules, and encapsulate the drug molecules in liposomes, thereby utilizing the cell-penetrating peptides on the surface of the liposomes with cell-penetrating properties to help the drug penetrate the biological membrane barrier in the subject's body.

[0027] The drug delivery carrier of the present invention has strong drug loading, drug delivery, and tissue penetration capabilities, low tissue toxicity, and can efficiently deliver drug molecules to the target site in the subject's body through various administration routes, overcoming the technical problem that it is difficult for the drug to reach the target site due to the subject's biomembrane barrier, enhancing the effectiveness of the drug in the treatment of diseases, and having good biological safety. The present invention also provides a pharmaceutical preparation prepared using the above drug delivery carrier.

[0028] As used herein, the term "subject" refers to a mammal. Mammals include, but are not limited to, domesticated animals (such as cows, sheep, cats, dogs, and horses), primates (such as humans and non-human primates such as monkeys), rabbits, and rodents (such as mice and rats). In particular, the subject is a human.

[0029] The various characteristics of the drug delivery carrier of the present invention will be discussed below.

[0030] 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%.

[0031] 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.

[0032] <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 that can form lipid vesicles and encapsulate drugs.

[0033] In some embodiments, the liposome modified with a cell-penetrating peptide on the surface of the present invention comprises the following membrane materials.

[0034] [(i) Cationic lipid] Liposomes contain one or more cationic lipids. As used herein, the term "cationic lipid" refers to lipids that have a net positive charge at a selected pH (e.g., physiological pH). Many cationic lipids are commercially available. Particularly suitable cationic lipids for use in liposomes include those described in International Patent Publications WO2010 / 053572 and WO2012 / 170930, both of which are incorporated herein by reference.

[0035] The cationic lipid contained in the liposome is not particularly limited as long as the liposome has a net positive charge at a selected pH (e.g., physiological pH), and examples thereof 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(sperminecarboxamido)ethyl]-N-N-dimethyl-1-propane ammonium trifluoroacetate (DOSPA) and combinations thereof. Preferred cationic lipids are DOTMA, DOTAP, DODAC and DOSPA. The most preferred cationic lipid is DOTAP.

[0036] [(ii) Non-cationic lipid] Liposomes contain one or more non-cationic lipids. As used herein, the term "non-cationic lipid" refers to any neutral ionic, zwitterionic or anionic lipid. As used herein, the term "anionic lipid" refers to any of a number of liposomal substances that have a net negative charge at a selected pH, such as physiological pH.

[0037] There is no particular limitation on the non-cationic lipid contained in the liposome, and any neutral ionic, zwitterionic, or anionic lipid can be used, including, but 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 non-cationic lipid is DOPE and / or DOPC. The most preferred non-cationic lipid is DOPE.

[0038] [(iii) Cholesterol] Cholesterol is an important component of mammalian cell membranes, accounting for more than 20% of the cell membrane lipids. The term "cholesterol" is used in the broadest sense herein, covering cholesterol derivatives. Research has shown that cholesterol can prevent the disordering of the cell membrane bilayer at high temperatures, and can prevent the ordering of the cell membrane bilayer at low temperatures, preventing the formation of liquid crystals, and maintaining the fluidity of the cell membrane bilayer.

[0039] Given the importance of cholesterol in the membrane bilayer, the cationic liposomes of the present invention also contain cholesterol.

[0040] [(iv) PEGylated phospholipids, polyethylene glycol phospholipids conjugated with CPPs] The liposomes comprise PEGylated phospholipids and polyethylene glycol phospholipids conjugated to CPPs. As used herein, PEGylated phospholipids does not include polyethylene glycol phospholipids with CPPs linked thereto.

[0041] PEGylated phospholipids are formed by covalently attaching one or more polyethylene glycol molecules (PEG) to a phospholipid. PEGylated phospholipids are covalently attached to a phospholipid with a polyethylene glycol chain of up to 10 kDa in length, including but not limited to 1 kDa, 2 kDa, 3 kDa, 4 kDa, 5 kDa, 6 kDa, 7 kDa, 8 kDa, 9 kDa, 10 kDa, and any value therebetween. The phospholipid can be a synthetic, semi-synthetic, or natural phospholipid, such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, sphingomyelin. In some embodiments, the PEGylated phospholipids are, for example, polyethylene glycol-distearoylphosphatidylethanolamine (PEG-DSPE) and its derivatives, methoxy-polyethylene glycol-distearoylphosphatidylethanolamine (mPEG-DSPE), and the molecular weight of PEG is any value between 1 kDa and 10 kDa.

[0042] CPP-conjugated polyethylene glycol phospholipids are formed by covalently linking a CPP to a polyethylene glycol phospholipid.

[0043] The CPP used in the present 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 a Penetratin derivative in which glutamine (Q) at position 2 and / or glutamine (Q) at position 8 and / or asparagine (N) at position 9 of Penetratin are mutated to hydrophobic amino acids. In some embodiments, the Penetratin lipophilic derivative is a derivative as disclosed in Chinese patent application CN201710414334.7, which, after instillation into the conjunctival sac, can penetrate many ocular barriers (cornea, conjunctiva, sclera, etc.) to promote drug entry into the eye, and thus can deliver the biopolymer drug, such as genes, polypeptides, and proteins, carried by it, to the retina at the back of the eye. The amino acid sequence of the Penetratin lipophilic derivative disclosed in Chinese patent application CN201710414334.7 is cited as follows:

[0044] [Table 1]

[0045] According to the present invention, a CPP is a compound that is bound to a part of the lipid bilayer of a liposome by a covalent bond with a polyethylene glycol phospholipid. Hereinafter, the term "part of the lipid bilayer of a liposome" refers to the fact that the phospholipid in the CPP-PEG-phospholipid is incorporated into the lipid bilayer. The phospholipid can be a synthetic, semi-synthetic or natural phospholipid, such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, sphingomyelin.

[0046] The polyethylene glycol chain in the CPP-PEG-phospholipid is a polyethylene glycol chain up to 10 kDa in length, including but not limited to 1 kDa, 2 kDa, 3 kDa, 4 kDa, 5 kDa, 6 kDa, 7 kDa, 8 kDa, 9 kDa, 10 kDa, and any value therebetween, with both ends covalently bonded to the phospholipid and CPP, respectively. In some embodiments, the polyethylene glycol phospholipid conjugated to the CPP is, for example, Penetratin / Penetratin derivative-PEG-DSPE, and the molecular weight of PEG is any value between 1 kDa and 10 kDa.

[0047] The present invention intends that modifying the surface of cationic liposomes with CPPs will increase the tissue penetration ability of drug delivery carriers, thereby helping liposomes carrying more drug molecules to reach target sites and thereby exert the effect of treating and / or preventing diseases.

[0048] In some embodiments, in the liposome membrane material (iv), the polyethylene glycol in the PEGylated phospholipid and the polyethylene glycol phospholipid conjugated to the CPP do not have the same chain length, and the polyethylene glycol chain in the polyethylene glycol phospholipid conjugated to the CPP is longer than the polyethylene glycol chain in the PEGylated phospholipid, for example, 0.5 kDa to 5 kDa longer, preferably 0.75 kDa to 4 kDa longer, and more preferably any value between 1 kDa and 2 kDa longer.

[0049] In one embodiment, the polyethylene glycol chain length of the PEGylated phospholipid in the liposome membrane material (iv) is 1 kDa, 2 kDa, or 3 kDa, respectively, and the polyethylene glycol chain length of the polyethylene glycol phospholipid conjugated to the CPP is about 2.5 kDa, 3.5 kDa, or 4.5 kDa, respectively.

[0050] In some embodiments, the molar ratio of the cationic liposome membrane material (i):(ii):(iii):(iv) of the present invention having a cell-penetrating peptide modified on its surface is about 20-40:20-40:20-40:1-20, and the polyethylene glycol phospholipid conjugated to a CPP is in a molar ratio of about 20%-80% to the membrane material (iv), corresponding to a molar ratio of about 2%-8% to the total liposome membrane material.

[0051] In some specific 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 the polyethylene glycol phospholipid conjugated to the CPP is in a molar ratio of about 20%-80% relative to membrane material (iv), corresponding to a molar ratio of about 2%-8% relative to the total liposome membrane material.

[0052] In some specific embodiments, the cationic liposome in the drug delivery carrier of the present invention comprises, as a membrane material, (i) DOTAP, (ii) DOPE, (iii) cholesterol, (iv) PEG-DSPE (e.g., mPEG 2000 -DSPE) and Penetratin / Penetratin derivatives-PEG-DSPE (e.g., 89W Penetratin-PEG 3400 -DSPE), and the molar ratio of the membrane materials (i):(ii):(iii):(iv) is about 20-40:20-40:20-40:1-20; Penetratin / Penetratin derivative-PEG-DSPE (e.g., 89W Penetratin-PEG 3400 In some embodiments, the molar ratio of the membrane materials (i):(ii):(iii):(iv) is about 27.0-31.6:27.0-31.6:31.6-39.6:1-10, and the Penetratin / Penetratin derivative-PEG-DSPE (e.g., 89W Penetratin-PEG 3400-DSPE) is in a molar ratio of about 20% to 80% with respect to the membrane material (iv). In some embodiments, the cationic liposomes in the drug delivery carrier of the present invention are (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 is in a molar ratio of about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% with respect to the membrane material (iv).

[0053] <II. Positively charged polyamino acid> The positively charged polyamino acid contained in the drug delivery carrier of the present invention is used to compress drug molecules.

[0054] The positively charged polyamino acid is a polyamino acid having a net positive charge at a selected pH (e.g., physiological pH). There is no special restriction 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., the positively charged polyamino acid has 2 to 50 amino acid residues), for example, a polyamino acid of arginine and / or lysine with a degree of polymerization of 6 to 12. Among the positively charged polyamino acids with a degree of polymerization of 2 to 50, 1 to 20 (e.g., 1 to 6) uncharged amino acid residues under physiological conditions may be interposed. The positively charged polyamino acid can be linear or cyclic, and the configuration of the amino acid residues is of the 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, and the configuration of the polyarginine is of the L-type or D-type.

[0055] In some embodiments, the positively charged polyamino acids are oligo-arginines, including linear polypeptides such as 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), cyclized 8-polyarginine (c-R6), and cyclized 8-polyarginine (c-R8). Polypeptides of different structures include L-type oligoarginines and D-type oligoarginines 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 rrrrrrrrrr, D-R12).

[0056] Hereinafter, a method for preparing a pharmaceutical formulation using the drug delivery carrier of the present invention will be described, and the characteristics of the pharmaceutical formulation will be described.

[0057] By using the drug delivery carrier of the present invention, it is possible to prepare a pharmaceutical preparation in which the surface of a cationic liposome is modified with CPP and which has a strong tissue permeability.

[0058] In one embodiment, the method for preparing the pharmaceutical formulation of the present invention is as follows. (1) Weigh out the above-mentioned liposome membrane material of the present invention, in which the molar ratio of membrane material (i):(ii):(iii):(iv) is about 20-40:20-40:20-40:1-20, and the polyethylene glycol phospholipid conjugated with CPP is in a molar ratio of about 20%-80% relative to membrane material (iv), corresponding to a molar ratio of about 2%-8% relative to the total liposome membrane material.

[0059] (2) The membrane materials (i):(ii):(iii):(iv) are added to and dissolved in an organic solvent, and the organic solvent is removed by evaporation to obtain a uniform dry lipid membrane.

[0060] (3) After the obtained dry lipid film is completely hydrated with a water-containing solution, the liposomes are extruded using a mini-extruder (the order of the core pore membranes is 200 nm, 100 nm, and 50 nm, in order).

[0061] (4) The positively charged polyamino acid and the drug molecule are mixed and incubated in an appropriate solution to obtain a physical complex of the positively charged polyamino acid and the drug molecule.

[0062] (5) The liposome obtained in (3) above and the complex obtained in (4) above are mixed at a certain molar ratio or charge ratio to obtain a pharmaceutical preparation in the form of a liposome complex.

[0063] In another embodiment, the method for preparing the pharmaceutical formulation of the present invention is as follows. (1) Weigh out the above-mentioned liposome membrane material of the present invention, in which the molar ratio of membrane materials (i):(ii):(iii):(iv) is about 20-40:20-40:20-40:1-20, and the polyethylene glycol phospholipid conjugated with CPP is in a molar ratio of about 20%-80% relative to membrane material (iv), corresponding to a molar ratio of about 2%-8% relative to the total liposome membrane material.

[0064] (2) The membrane materials (i):(ii):(iii) are added to an organic solvent to dissolve them, and the organic solvent is removed by evaporation to obtain a uniform dry lipid membrane.

[0065] (3) After the obtained dry lipid film is completely hydrated with a water-containing solution, the liposomes are extruded using a mini-extruder (the order of the core pore membranes is 200 nm, 100 nm, and 50 nm, in order).

[0066] (4) The positively charged polyamino acid and the drug molecule are mixed and incubated in an appropriate solution to obtain a physical complex of the positively charged polyamino acid and the drug molecule.

[0067] (5) The liposome obtained in (3) above and the complex obtained in (4) above are mixed in a certain molar ratio or charge ratio, and then mixed with a membrane material (iv) and incubated to obtain the pharmaceutical preparation of the present invention.

[0068] There is no particular limitation on the drug molecule to be delivered. The drug molecule includes a wide variety of types of compounds that may be delivered to the test subject, including, but not limited to, nucleic acids; anti-infectives such as antibiotics and antivirals; analgesics and analgesic combinations; appetite suppressants; anthelmintics; anti-arthritic drugs; anti-asthmatic drugs; anticonvulsants; antidepressants; antidiabetic drugs; antidiarrheal drugs; antihistamines; anti-inflammatory drugs; antimigraine drugs; nausea drugs; antineoplastic drugs; anti-tremor paralytic drugs; antipruritic drugs; antipsychotic drugs; antipyretics; anticonvulsants; anticholinergics; sympathomimetics; xanthine derivatives; potassium channel blockers, calcium channel blockers, The drug delivery carriers of the present invention can deliver drugs in all forms, including ionized, non-ionized, free base, acid addition salts, and the like, and can deliver drugs of high or low molecular weight.

[0069] In some embodiments, the drug molecule delivered by the pharmaceutical formulation of the present invention is, for example, a peptide drug (e.g., octreotide), an antibody (e.g., etanercept, adalimumab, rituximab, pembrolizumab, ranibizumab, and nivolumab), a cytokine, a hormone, an antibiotic (e.g., the chemotherapeutic drugs doxorubicin, daunorubicin, epirubicin, actinomycin, and vancomycin), a nucleic acid, and the like.

[0070] In some embodiments, the drug molecule delivered by the pharmaceutical formulation is a nucleic acid, such as plasmid DNA, RNA such as small interfering RNA (siRNA), sense RNA, antisense oligonucleotides (ASO), aptamers, ribozymes, and the like.

[0071] The plasmid DNA is a gene expression system comprising a DNA sequence encoding, for example, interleukin-2, interleukin-4, interleukin-7, interleukin-12, and interleukin-15, interferon-α, interferon-β, interferon-γ, colony stimulating factors, granulocyte-macrophage stimulating factors, anti-angiogenic agents, tumor suppressor genes, thymidine kinase, eNOS, iNOS, p53, p16, TNF-α, Fas-ligand, mutated oncogenes, tumor antigens, viral antigens, or bacterial antigens.

[0072] The plasmid DNA encodes an shRNA molecule designed to inhibit a protein involved in the growth or maintenance of tumor cells or other hyperproliferative cells. The plasmid DNA may simultaneously encode a therapeutic protein and one or more shRNAs. The nucleic acid in the pharmaceutical preparation of the present invention may also be a mixture of plasmid DNA and artificially synthesized RNA, including sense RNA, antisense oligonucleotides, or ribozymes.

[0073] In one embodiment, the molar ratio of the drug molecule (e.g., a chemotherapeutic agent) to the cationic lipid (e.g., DOTAP) in the pharmaceutical formulation of the present invention is about 0.1 to 10, for example, about 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10, preferably about 0.1 to 5, and more preferably about 0.1 to 2.

[0074] In one embodiment, the drug loading of the pharmaceutical formulation with respect to drug molecules (e.g., chemotherapeutic agents) is as follows: the drug molecules (e.g., chemotherapeutic agents) account for about 1%-30% (w / w) of the pharmaceutical formulation, for example, about 1%, 2.5%, 5%, 7.5%, 10%, 12.5%, 15%, 17.5%, 20%, 22.5%, 25%, 27.5%, 30% (w / w), preferably about 1%-25% (w / w), more preferably about 3%-20% (w / w).

[0075] The pharmaceutical formulations of the present invention can be administered orally, nasally, ophthalmically, respiratory tract, digestive tract, genital tract, locally by implant, injection or infusion (via epidural, intra-arterial, intra-articular, intracapsular, intracardiac, intraventricular, intracranial, intradermal, intramuscular, intraorbital, intraocular, intraperitoneal, intraspinal, intrasternal, intrathecal, intravenous, subarachnoid, subcapsular, subcutaneous, tracheal, rectal, sublingual and other routes).

[0076] The pharmaceutical formulations of the present invention can be administered using medical devices known in the art. For example, in one embodiment, the pharmaceutical formulations of the present invention can be administered using a needleless scalp subcutaneous injection device, such as the device disclosed in U.S. Patent No. 5,399,163.

[0077] In one embodiment, the drug delivery carrier of the present invention can be used to prepare a pharmaceutical preparation for delivering a nucleic acid, thereby achieving the effect of gene therapy.

[0078] Compared with conventional treatment methods such as surgery, radiation therapy and chemotherapy, gene therapy, as a groundbreaking treatment method, has the advantages of high compatibility, fewer side effects, less damage to normal cells, and less pain during the treatment process, so it shows good prospects in the fields of diseases such as immunodeficiency, metabolic diseases, cancer and heart disease. Currently, 2597 nucleic acid drugs have entered or completed clinical trials worldwide, of which 65% are for cancer treatment, and 181 gene drugs in clinical trials exert their therapeutic effect by inhibiting tumor growth (Ginn SL et.al., Gene therapy clinical trials worldwide to 2017: An update, Journal of Gene Medicine, 2018, 20(5):e3015). Based on this, the field hopes to realize the treatment of diseases (e.g., tumor diseases) through pharmaceutical preparations that deliver nucleic acids.

[0079] Nucleic acid delivery carriers are one of the important technologies in gene therapy, and they can protect the delivered nucleic acid from being degraded by endogenous nucleases, making it possible to apply gene therapy in the body. Currently, there are two types of nucleic acid carriers used in gene therapy: viral vectors and non-viral vectors. The transfection efficiency of viral vectors is very high, but at the same time, there are clinical problems such as induction of immune response and carcinogenicity. Non-viral vectors mainly include polyethyleneimine (PEI), polyamidoamine (PAMAM), cationic liposomes, etc. Compared with PEI and PAMAM, cationic liposomes have the advantages of targeting, long-term action, low toxicity, drug protection, etc. as a drug carrier (Wang J et al., c-Myc is required for maintenance of glioma cancer stem cells, PLoS ONE, 2008, 3(11):e3769). Cationic liposomes are usually formed by mixing positively charged lipids with neutral co-lipids, and when cationic liposomes are close to negatively charged nucleic acids, the assembling cationic lipids can play the role of "double-sided sticky tape" to spontaneously form nucleic acid / cationic liposome complexes. After specially modifying the surface groups of liposomes, cationic liposomes can fully encapsulate nucleic acids, promote the fusion of nucleic acid / cationic liposome complexes with cell membranes, and enhance cellular nucleic acid uptake (Huang L et al., In vivo delivery of RNAi with lipid-based nanoparticles, Annual Review of Biomedical Engineering, August 15, 2011, 13:507-530).

[0080] When preparing a pharmaceutical preparation for delivering nucleic acid, the artificially synthesized positively charged polyamino acid (e.g., oligoarginine) in the drug delivery carrier of the present invention is used to compress the nucleic acid. Here, the oligoarginine and the nucleic acid form a physical complex by electrostatic action, and the positively charged cationic liposome is used to further compress the nucleic acid to form a liposome complex. In addition, since the cell-penetrating peptide modifies the surface of the cationic liposome, the ability of the prepared nucleic acid liposome complex to penetrate cells and tissues can be enhanced, and the nucleic acid liposome preparation constructed thereby can be administered by various routes, can efficiently deliver nucleic acid molecules to the target body part, and has excellent biosafety.

[0081] In one embodiment, a method for preparing a nucleic acid liposome formulation of the present invention is as follows. (1) Weigh out the above-mentioned liposome membrane material of the present invention, in which the molar ratio of membrane materials (i):(ii):(iii):(iv) is about 20-40:20-40:20-40:1-20, and the polyethylene glycol phospholipid conjugated with CPP is in a molar ratio of about 20%-80% relative to membrane material (iv), corresponding to a molar ratio of about 2%-8% relative to the total liposome membrane material.

[0082] (2) The membrane materials (i):(ii):(iii) are added to an organic solvent to dissolve them, and the organic solvent is removed by evaporation to obtain a uniform dry lipid membrane.

[0083] (3) After the obtained dry lipid film is completely hydrated with a water-containing solution, the liposomes are extruded using a mini-extruder (the order of the core pore membranes is 200 nm, 100 nm, and 50 nm, in order).

[0084] (4) A positively charged polyamino acid (e.g., oligoarginine) and a nucleic acid molecule are mixed and incubated in an appropriate solution at a specific charge ratio to obtain a complex of the positively charged polyamino acid (e.g., oligoarginine) and the nucleic acid molecule.

[0085] (5) The liposome obtained in (3) above and the complex obtained in (4) above are mixed at a certain molar ratio or charge ratio to obtain a liposome complex.

[0086] (6) The liposome obtained in (5) above is mixed with a membrane material (iv) and incubated to obtain the nucleic acid liposome preparation of the present invention.

[0087] In one embodiment, a method for preparing a nucleic acid liposome formulation of the present invention is as follows. (1) Weigh out the above-mentioned liposome membrane material of the present invention, in which the molar ratio of membrane materials (i):(ii):(iii):(iv) is about 20-40:20-40:20-40:1-20, and the polyethylene glycol phospholipid conjugated with CPP is in a molar ratio of about 20%-80% relative to membrane material (iv), corresponding to a molar ratio of about 2%-8% relative to the total liposome membrane material.

[0088] (2) The membrane materials (i):(ii):(iii) are added to an organic solvent to dissolve them, and the organic solvent is removed by evaporation to obtain a uniform dry lipid membrane.

[0089] (3) After the obtained dry lipid film is completely hydrated with a water-containing solution, the liposomes are extruded using a mini-extruder (the order of the core pore membranes is 200 nm, 100 nm, and 50 nm, in order).

[0090] (4) The liposome obtained in (3) above is mixed with a membrane material (iv) and incubated to obtain a cationic liposome modified with a cell-penetrating peptide.

[0091] (5) A positively charged polyamino acid (e.g., oligoarginine) and a nucleic acid molecule are mixed in a suitable solution at a certain charge ratio and incubated to obtain a complex of the oligoarginine and the nucleic acid molecule.

[0092] (6) The liposome obtained in (4) above and the complex obtained in (5) above are mixed at a certain molar ratio or charge ratio to obtain the nucleic acid liposome preparation of the present invention in the form of a liposome complex.

[0093] In one embodiment, a method for preparing a nucleic acid liposome formulation of the present invention is as follows. (1) Weigh out the above-mentioned liposome membrane material of the present invention, in which the molar ratio of membrane materials (i):(ii):(iii):(iv) is about 20-40:20-40:20-40:1-20, and the polyethylene glycol phospholipid conjugated with CPP is in a molar ratio of about 20%-80% relative to membrane material (iv), corresponding to a molar ratio of about 2%-8% relative to the total liposome membrane material.

[0094] (2) The membrane materials (i):(ii):(iii):(iv) are added to and dissolved in an organic solvent, and the organic solvent is removed by evaporation to obtain a uniform dry lipid membrane.

[0095] (3) After the obtained dry lipid film is completely hydrated with a water-containing solution, the liposomes are extruded using a mini-extruder (the order of the core pore membranes is 200 nm, 100 nm, and 50 nm, in order).

[0096] (4) A positively charged polyamino acid (e.g., oligoarginine) and a nucleic acid molecule are mixed in a suitable solution at a certain charge ratio and incubated to obtain a complex of the oligoarginine and the nucleic acid molecule.

[0097] (5) The liposome obtained in (3) above and the complex obtained in (4) above are mixed at a certain charge ratio to obtain the nucleic acid liposome preparation of the present invention in the form of a liposome complex.

[0098] In one embodiment, the nucleic acid molecule is siRNA, and in the method for preparing the siRNA liposome formulation of the present invention, DOTAP, DOPE, cholesterol, mPEG 2000 -DSPE and Penetratin / Penetratin Derivatives-PEG 3400The molar ratio of DOTAP to DOPE is about 28.5:28.5:38:5. DOTAP, DOPE, and cholesterol are weighed and dissolved in a round-bottom flask by adding chloroform, and the chloroform is removed by a rotary evaporator to obtain a uniform dry lipid film. The lipid film is hydrated by ultrasonication through a water bath with a 5% glucose aqueous solution, and after the lipid film is completely hydrated, the liposomes are extruded by a mini extruder (the order of the nuclear pore membrane is 200 nm, 100 nm, and 50 nm, respectively), completing the preparation of blank liposome CLS. Then, oligoarginine Rn (R is the one-letter code of arginine, and n is an integer such as 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) and siRNA are mixed at a certain charge ratio, vortexed for 30 seconds, and incubated at 37 ° C for 30 minutes to obtain the complex Rn / siRNA. Furthermore, the complex Rn / siRNA and blank liposome were mixed at a certain charge ratio (charge ratio of cationic liposome to siRNA), vortexed for 30 seconds, and incubated at 37°C for 30 minutes to obtain the complex CLS / Rn / siRNA. 2000 -DSPE and Penetratin / Penetratin derivative-PEG 3400 -DSPE and then incubated at 55°C for 30 minutes to complete the preparation of the siRNA liposome formulation.

[0099] In one embodiment, the nucleic acid liposome formulation of the present invention is administered intranasally, and the nucleic acid is efficiently delivered to the brain via the nasal-brain route; after the nucleic acid liposome formulation is taken up by brain tumor cells, the cell-penetrating peptide and the positive charge on the surface of the cationic liposome cause the endosome to escape and release the nucleic acid molecule into the cytoplasm, where the gene can play a role in treating brain tumors (e.g., brain gliomas).

[0100] As known to those skilled in the art, due to the special physiological structure and functional complexity of the brain, especially the existence of the blood-brain barrier and the physical and chemical properties of certain drugs themselves, therapeutic drugs are usually difficult to reach brain tissue.However, the route of intranasal administration to the brain provides a viable idea for the treatment of brain diseases, and there are two direct routes for intranasal administration of drugs to the brain: one is the olfactory nerve route, which is the most direct way to bypass the blood-brain barrier to enter the brain through the nasal cavity, and the drug is absorbed by the nasal mucosa and taken up by the olfactory nerve, and then transported to the olfactory bulb through the axon and further to the olfactory brain; the other is the nasal mucosa epithelium route, which allows the drug to pass through the basement membrane, enter the lamina propria, and then reach the peripheral olfactory nerve, to be delivered to the central nervous system, and finally achieve accumulation 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).

[0101] Meanwhile, brain tumors such as gliomas are malignant tumors of the central nervous system with a high incidence rate and a low survival rate. They cause great harm to human health and have poor clinical prognoses, mainly manifested in recurrence after surgery and drug resistance to chemotherapy. In addition, 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 low. Therefore, people have been making efforts to find effective drug treatment methods.

[0102] The drug liposome formulation of the present invention can deliver a drug to the brain after nasal administration, and can further enhance the exertion of the therapeutic action of the drug.

[0103] In the embodiment of the present invention, the inventors have taken 8 polyarginine R8 as an example, and siRNA as a drug molecule model to construct a cationic liposome delivery carrier carrying siRNA. Through a series of in vitro and in vivo experiments, the cell uptake ability and cell mass penetration ability of the delivery carrier jointly constructed by cationic liposomes modified with cell-penetrating peptides and positively charged polyamino acids have been investigated. As a result, it has been shown that the drug delivery carrier of the present invention can significantly increase the amount of nucleic acid in cells and has a relatively high delivery efficiency, compared with commercially available cationic liposomes that deliver nucleic acids.

[0104] The drug delivery carrier constructed by the present invention has the advantages of high efficiency, low toxicity, and easy drug release. The high efficiency is embodied in that the delivery carrier of the present invention has a relatively high cell uptake ability and a relatively strong cell mass penetration ability. The low toxicity is embodied in that the cell-penetrating peptide used in the present invention is non-pathogenic, easily degraded in the body, and has better biological safety compared with other drug delivery carriers such as PEI and PAMMA. The easy drug release is because both the polyamino acid and the cell-penetrating peptide that modifies the surface have positive charges, and the positive charges of the cationic liposomes are conducive to the endosomal escape of the drug, so that the drug can be successfully released into the cytoplasm to play a therapeutic role. In addition, the pharmaceutical preparation prepared using the drug delivery carrier of the present invention can be administered to the examinee via various routes, and in particular, the delivery of the drug to the brain can be achieved via the nasal administration route, which is beneficial to improve patient compliance. EXAMPLES

[0105] The following examples are provided to aid in the understanding of the present invention. The examples are not intended to limit the scope of protection of the present invention and should not be construed as limiting the scope of protection of the present invention in any way.

[0106] [Example 1] (Preparation of cationic liposomes) The membrane materials were prepared in a molar ratio of 28.5:28.5:38:5, i.e., 3.5 mg DOTAP (Shanghai Awite Pharmaceutical Technology Co., Ltd., 132172-61-3), 3.72 mg DOPE (Shanghai Awite Pharmaceutical Technology Co., Ltd., 4004-05-1), 2.59 mg cholesterol (Sigma-Aldrich, C8667) and 2.46 mg mPEG. 2000 -DSPE (Shanghai Awite Pharmaceutical Technology Co., Ltd., 147867-65-0) was weighed out.

[0107] The above amount of DOTAP, DOPE, and cholesterol were placed in a 50mL round-bottom flask, and 1mL of chloroform was added to dissolve the DOTAP, DOPE, and cholesterol as the membrane material, that is, 16.67μM CLS contains 5μM DOTAP, 5μM DOPE, and 6.67μM DOPE. The obtained solution was removed from the chloroform by a rotary evaporator (Shanghai Shensheng Technology Co., Ltd., R201L) to obtain a uniform dry lipid membrane.

[0108] The resulting dry lipid film was sonicated for about 10 minutes in 1 mL of 5% glucose aqueous solution in a water bath at 37° C. After the lipid film was completely hydrated, liposomes were extruded using a mini extruder (Hamilton, 81320) (the order of the core pore membranes was 200 nm, 100 nm, and 50 nm, respectively) to obtain cationic liposomes (also referred to as CLS in the specification).

[0109] The prepared cationic liposomes were mixed with 2.46 mg of mPEG 2000 -DSPE (soluble in water) and incubated at 55°C for 30 minutes to form mPEG 2000 Cationic liposomes containing -DSPE were obtained.

[0110] [Example 2] (Compact effect of cationic liposomes and positively charged polyamino acids on drugs) Agarose gel electrophoresis experiments are often used to measure the encapsulation ability of drug carriers for drugs such as nucleic acids. In this example, R8 / siRNA complexes, CLS / siRNA complexes, and CLS / R8 / siRNA complexes were prepared, and the encapsulation ability of only CLS, only R8, and the combination of CLS and R8 for nucleic acids was measured by agarose gel electrophoresis experiments.

[0111] 4 mg of oligoarginine R8 (Shanghai Xinzhao Biotechnology Co., Ltd.) as a positively charged polyamino acid was dissolved in DEPC-treated water (Dalian Meilun Biotechnology Co., Ltd., MA0018) to prepare a 4 mg / mL oligoarginine R8 solution, and 33 μg of siRNA (in this embodiment, small interfering RNA against c-myc is used, hereinafter also referred to as siRNA, its sense strand (5'-3') is AACGUUAGCUUCACCAACAdTdT (sequence number 79), and its antisense strand (5'-3') is UGUUGGUGAAGCUAACGUUdTdT (sequence number 80)) was dissolved in 125 μL of DEPC-treated water to prepare a 20 μM siRNA solution.

[0112] The oligoarginine R8 solution and the siRNA solution were mixed in equal volumes in proportions with charge ratios of 0, 1, 5, 10, 15, 20, 25, and 30 (one amino nitrogen of R8 has one positive charge, and one phosphate group of siRNA has one negative charge, so 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 group, i.e., 1 μM of siRNA contains 42 μM of negative charge, and the charge ratio of R8 to siRNA = (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 to obtain R8 / siRNA complexes with different charge ratios.

[0113] The above-mentioned R8 / siRNA complexes with different charge ratios and CLS are used to prepare liposome complexes.Specifically, 8 μL of R8 / siRNA complexes with different charge ratios (i.e., prepared by 4 μL of siRNA solution and 4 μL of R8 solution) are 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 for 30 minutes to obtain lipid complexes CLS / R8 / siRNA with different charge ratios of R8 / siRNA. In the lipid complex CLS / R8 / siRNA, the charge ratio of cationic liposome CLS and siRNA is fixed at 4 based on the charge number of siRNA (since one amino nitrogen of DOTAP has one positive charge, one phosphate group of siRNA has one negative charge, 16.67 μM CLS contains 5 μM DOTAP, i.e., 1 μM CLS contains 0.3 μM positive charge, 1 μM siRNA contains 42 μM negative charge, and the charge ratio of CLS and siRNA = (μM value of CLS × 0.3) / (μM value of siRNA × 42) = 0.007 × CLS (μM) / siRNA (μM)). The results of agarose gel electrophoresis showed that R8 / siRNA complexes with different charge ratios of R8 / siRNA can all be mixed with CLS to prepare liposome complexes.

[0114] Next, liposome complexes were prepared using the R8 / siRNA complex with an R8 / siRNA charge ratio of 1:1 and CLS. Specifically, the CLS prepared in Example 1 and 8 μL of the R8 / siRNA complex prepared as described above with an R8 / siRNA charge ratio of 1:1 were mixed in ratios of CLS / siRNA charge ratios of 2, 4, 6, 8, 10, and 12, vortexed for 30 seconds, and incubated at 37° C. for 30 minutes to obtain CLS / R8 / siRNA complexes with different CLS and siRNA charge ratios.

[0115] For comparison, CLS / siRNA complexes were also prepared by mixing equal volumes of the CLS prepared in Example 1 and 20 μL of the siRNA solution prepared in this Example in proportions of CLS / siRNA charge ratios of 2, 4, 6, 8, 10, and 12, vortexing for 30 seconds, and incubating at 37° C. for 30 minutes to obtain CLS / siRNA complexes with different CLS / siRNA charge ratios. The calculation of the CLS / siRNA charge ratio is performed as follows: one amino nitrogen of DOTAP has one positive charge, and one phosphate group of siRNA has one negative charge, so 16.67 μM CLS contains 5 μM DOTAP, i.e., 1 μM CLS contains 0.3 μM positive charge, and 1 μM siRNA contains 42 μM negative charge. Therefore, the charge ratio of CLS to siRNA = (μM value of CLS × 0.3) / (μM value of siRNA × 42) = 0.007 × CLS (μM) / siRNA (μM).

[0116] The R8 / siRNA complex, CLS / siRNA complex, and CLS / R8 / siRNA complex prepared above were gently added to the agarose gel loading hole, and a free siRNA control group was set up and electrophoresis was performed. After electrophoresis, the gel was stained with GelRed working solution (Biotium Inc.) in the dark for 30 minutes and imaged under UV 302 nm conditions. The results are shown in Figure 1.

[0117] The results in Figure 1A show that oligoarginine R8 alone cannot completely compact the gene. The results in Figures 1C and 1D show that cationic liposome CLS alone can compact the gene to some extent, but the particle size of the CLS / siRNA complex is large. The results in Figures 1E and 1F show that the combination of cationic liposome and oligoarginine R8 can effectively jointly compact the gene.

[0118] [Example 3] (Evaluation of particle size and potential characteristics of drug-containing liposome complexes after drug compression with cationic liposomes and positively charged polyamino acids) According to the method described in Example 2, an R8 / siRNA complex, a CLS / siRNA complex, and a CLS / R8 / siRNA complex were each prepared, and particle size and electric potential were measured.

[0119] Dynamic light scattering techniques were used to measure particle size directly, and the particle size measurement results are shown in Figures 1, 3, and 12. In Figure 12, the results from the dynamic light scattering measurements are shown with particle size on the horizontal axis and light intensity on the vertical axis, calculated from the light intensity contributed by each particle.

[0120] As can be seen from FIG. 3B, when the charge ratio of oligoarginine R8 / siRNA is 5, 10, and 20, the particle size of the physical complex R8 / siRNA is about 200 nm. After compressing siRNA using a combination of cationic liposome and oligoarginine, when the charge ratio of oligoarginine R8 / siRNA is 5 and the charge ratio of cationic liposome CLS and siRNA is 4, the particle size measurement result of the liposome complex containing siRNA (CLS / R8 / siRNA) shows that the particle size is reduced to 108 nm, as shown in FIG. 12. When the charge ratio of oligoarginine R8 / siRNA is 5, 89W When the charge ratio of penetratin-modified cationic liposome 89W-CLS to siRNA was 4, the siRNA was compressed using a combination of cationic liposome and oligoarginine. 89W The particle size of the penetratin-modified liposome complex containing siRNA (89W-CLS / R8 / siRNA) was close to that of CLS / R8 / siRNA, being 105 nm. 89W The maximum light intensity of the penetratin-modified liposome complex containing siRNA is lower than that of CLS / R8 / siRNA, due to the broader particle size distribution range of the former.

[0121] The charge ratio of oligoarginine R8 / siRNA is 5, and the cationic liposome CLS or 89WWhen the charge ratio of the penetratin-modified cationic liposome 89W-CLS to siRNA was 4, R8 / siRNA complex, CLS / siRNA / R8 complex and 89W-CLS / siRNA / R8 complex were prepared, respectively, and the zeta potential was measured in water at 23℃, 5V / cm electric field strength and 0.4cm electrode spacing using a zeta potential analyzer. The zeta potential measurement results are shown in Figure 13.

[0122] As can be seen from FIG. 13, the physical complex R8 / siRNA, the liposome complex CLS / R8 / siRNA, 89W The zeta potentials of the penetratin-modified liposome complexes were 22 mV, 43 mV, and 38 mV, respectively.

[0123] [Example 4] (Stability characteristics of drug-containing liposome complexes 1) Each CLS / R8 / siRNA complex was prepared in DEPC-treated water according to the method described in Example 2, and the stability of each drug-containing liposome complex (ie, liposome formulation) was observed after high-speed centrifugation.

[0124] Specifically, the CLS / R8 / siRNA complex prepared by the method described in Example 2 was centrifuged at 12,000×g for 20 minutes, and the precipitation state of each liposome preparation was observed.

[0125] The results are shown in Figure 3C. As can be seen from Figure 3C, when the charge ratio of oligoarginine R8 / siRNA is greater than 5 and the charge ratio of cationic liposome CLS to siRNA is 4, the CLS / R8 / siRNA complex has no obvious precipitation after high-speed centrifugation, which indicates that cationic liposome and oligoarginine can jointly compress siRNA to form a stable liposome formulation.

[0126] [Example 5] [Characteristics of stability of drug-containing liposome complexes 2] Each CLS / R8 / siRNA complex in DEPC-treated water prepared by the method described in Example 2 was centrifuged at 12000×g for 20 minutes, and each supernatant was gently added to the agarose gel loading hole, and a free siRNA control group was set up to perform electrophoresis. After electrophoresis, the gel was stained with GelRed working solution in the dark for 30 minutes, and imaged under UV302nm conditions. The results are shown in Figure 3D.

[0127] As can be seen from Figure 3D, when the charge ratio of oligoarginine R8 / siRNA is greater than 5 and the charge ratio of cationic liposome CLS to siRNA is 4, after the CLS / R8 / siRNA complex is centrifuged at high speed, CLS / R8 / siRNA is still retained in the supernatant solution and free siRNA does not leak out, indicating that cationic liposome and oligoarginine can both compress siRNA to form a stable liposomal formulation.

[0128] [Example 6] (Formulation of Surface-Modified Liposomal Pharmaceutical Formulations) The surface of liposomal pharmaceutical preparations was modified with cell-penetrating peptides to obtain surface-modified pharmaceutical preparations.

[0129] i. Preparation of cell-penetrating peptide-PEG-phospholipid DSPE-Polyethylene glycol maleimide (DSPE-PEG 3400 -maleimide) (Laysan Bio, 146-123) and a penetratin derivative modified with a terminal cysteine ​​( 89W The cell-penetrating peptide-PEG-DSPE was reacted with PEG-DSPE in a single step.

[0130] Specifically, 20 mg of DSPE-PEG 3400 -maleimide was dissolved in 1 mL of N,N-dimethylformamide, and 18 mg of 10 mL of phosphate buffer (10 mM, pH 7.2) was dissolved in the solvent. 89WPenetratin-Cys was added under stirring and the reaction was continued overnight at 25°C to complete the reaction. After the reaction was completed, the resulting mixture was placed in pure water under ice bath conditions, dialyzed for 2 days, and freeze-dried to obtain a white aggregate. 89W Penetratin-PEG 3400 -DSPE was obtained.

[0131] wherein the polypeptide 89W Penetratin is a derivative of penetratin, and the specific amino acid sequence is RQIKIWFWWRRMKWKK (SEQ ID NO: 29). The cell-penetrating peptide in the cell-penetrating peptide-PEG-DSPE may be other penetratin derivatives, for example, other penetratin derivatives shown in Table 1, such as penetratin products obtained by mutating the glutamine at position 2 and / or the glutamine at position 8 and / or the asparagine at position 9 of hydrophobic amino acids (see also Chinese patent application CN2017104534.7).

[0132] To facilitate reaction with polyethylene glycol end groups, cell penetrating peptides 89W A cysteine ​​residue (C) may be added to the N-terminus or C-terminus of penetratin or other penetratin derivatives.

[0133] II. Formulation of surface-modified liposome pharmaceutical preparations In the total liposome membrane material, DOTAP:DOPE:cholesterol:(mPEG 2000 -DSPE and 89W Penetratin-PEG 3400 -DSPE) is about 28.5:28.5:38:5.

[0134] Weighed out 3.5 mg of DOTAP (Shanghai Awite Pharmaceutical Technology Co., Ltd., 132172-1-3), 3.72 mg of DOPE (Shanghai Awite Pharmaceutical Technology Co., Ltd., 4004-05-1), and 2.59 mg of cholesterol (Sigma-Aldrich, C8667). 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 lipid membrane material, DOTAP, DOPE, and cholesterol, i.e., 16.67 μM CLS contains 5 μM DOTAP, 5 μM DOPE, and 6.67 μM DOPE. The resulting solution was evaporated in a rotary evaporator (Shanghai Shensheng Technology Co., Ltd., R210L) to remove chloroform, and a uniform dry lipid membrane was obtained.

[0135] The resulting dry lipid film was ultrasonically hydrated with 1 mL of 5% glucose aqueous solution in a water bath at 37° C. for about 10 minutes. After the lipid film was completely hydrated, a mini-extruder (Hamilton, 81320) was used to extrude the liposomes (the order of the core pore membranes is 200 nm, 100 nm and 50 nm, respectively) to obtain cationic liposomes (also referred to as CLS in the specification).

[0136] 4 mg of oligoarginine R8 (Shanghai Xinzhao Biotechnology Co., Ltd.) was dissolved in DEPC-treated water (Dalian Meilun Biotechnology Co., Ltd., MA0018) to prepare a 4 mg / mL oligoarginine R8 solution, and 33 μg of FAM-labeled siRNA (in this embodiment, siRNA against c-myc was used, whose sense strand (5'-3') is FAM-AACGUUAGCUUCACCAACAdTdT (sequence number 79), i.e., 5' is modified with FAM, and the antisense strand (5'-3') is UGUUGGUGAAGCUAACGUUdTdT (sequence number 80)) was dissolved in 125 μL of DEPC-treated water to prepare a 20 μM FAM-siRNA solution.

[0137] The oligoarginine R8 solution and 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 complex R8 / FAM-siRNA. 8 μL of the R8 / FAM-siRNA complex was taken and mixed with the cationic liposome CLS prepared above at a charge ratio of liposome to FAM-siRNA of 4, and vortexed for 30 seconds to obtain the CLS / R8 / FAM-siRNA complex.

[0138] 5% molar ratio of mPEG to the total liposome 2000 -DSPE and 89W Penetratin-PEG 3400 -DSPE, mPEG 2000 -DSPE and 89W Penetratin-PEG 3400 -DSPE 89W Penetratin-PEG 3400 Mixtures were prepared with molar ratios of 0%, 20%, 40%, 60%, 80% and 100% -DSPE. 89W Penetratin-PEG 3400 The molar ratio of -DSPE to the total liposome membrane material was 0%, 1%, 2%, 3%, 4%, and 5%, respectively.

[0139] The resulting CLS / R8 / FAM-siRNA complex was 89W Penetratin-PEG 3400 -DSPE is mPEG 2000 -DSPE and 89W Penetratin-PEG 3400 After mixing with mixtures having molar ratios of 0%, 20%, 40%, 60%, 80%, and 100% relative to -DSPE, respectively, and incubating at 55°C for 30 minutes, liposomal pharmaceutical formulations having cell-penetrating peptides on the surface at ratios of 0%, 1%, 3%, 4%, and 5%, i.e., 89W-CLS / R8 / FAM-siRNA, were obtained.

[0140] [Example 7] (Quantitative evaluation of cellular uptake of pharmaceutical formulations whose surfaces are modified with different proportions of cell-penetrating peptides) U87 cells (human malignant glioblastoma cells, purchased from ATCC) were cultured in DMEM complete medium (DMEM medium supplemented with 10% FBS and 1% penicillin-streptomycin). U87 cells in good growth condition were taken and resuspended in DMEM complete medium, and 5 × 10 5 The cells were inoculated into a 6-well plate at 10 cells / well, 1 mL per well. After inoculation, fresh culture medium was changed once daily, and the cells were cultured for 2-3 days before the experiment.

[0141] After discarding the culture medium, the U87 cells were washed three times with sterile PBS, and 1 mL of serum-free DMEM medium containing liposome pharmaceutical preparations (containing siRNA labeled with FAM) with different ratios of cell-penetrating peptides on the surface, prepared in Example 6, was added, and the cells were incubated for 4 hours at 37°C and 5% CO2. After the incubation was completed, the culture medium was discarded, and the positively charged substances adsorbed on the wells and cell surfaces were washed away with a PBS buffer solution containing 0.02 mg / mL sodium heparin. The cells were treated with trypsin, resuspended in 200 μL of sterile PBS buffer solution, sprayed uniformly, and the cells were counted for each sample, and 10 4 Cells were taken and subjected to flow cytometry detection. Cells incubated with serum-free DMEM medium served as the negative control group. All experiments were performed in triplicate. The results are shown in Figure 4 (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 highly statistically significant difference between the two groups). In Figure 4, "0%, 1%, 2%, 3%, 4%, 5%" on the horizontal axis represent the liposomal pharmaceutical formulations, i.e., 89W-CLS / R8 / siRNA, whose surfaces were modified with cell-penetrating peptides at the ratios of 0%, 1%, 2%, 3%, 4%, and 5%, respectively.

[0142] As can be seen from Figure 4, 89W Penetratin-PEG 3400When -DSPE is at a molar ratio of 2%, 3%, or 4% relative to the total liposome membrane material, the cellular uptake effect of the pharmaceutical preparation whose surface is modified with cell-penetrating peptide at the above ratio is superior to that of the commercially available gene transfection reagent Lipo2000.

[0143] [Example 8] (Quantitative evaluation of cellular uptake of drug formulations prepared using different ratios of liposomal membrane materials) U87 cells (human malignant glioblastoma cells, purchased from ATCC) were cultured in DMEM complete medium (DMEM medium supplemented with 10% FBS and 1% penicillin-streptomycin). U87 cells in good growth condition were taken and resuspended in DMEM complete medium, and 5 × 10 5 Cells were inoculated into a 6-well plate at 1 mL per well, and the culture medium was changed once a day with fresh medium. After inoculation, the cells were cultured for 2-3 days before the experiment.

[0144] According to the method described in Example 6, liposome pharmaceutical preparations (containing FAM-labeled siRNA) having the mixing ratios of liposome membrane materials shown in Table 2 were prepared.

[0145] [Table 2]

[0146] After discarding the culture medium from the cultured U87 cells, the cells were washed three times with sterile PBS. 1 mL of serum-free DMEM medium containing the liposomal pharmaceutical formulations Formula 1, Formula 2, and Formula 3 prepared by the respective formulations was added to each well and incubated for 4 hours at 37°C and 5% CO2. After the incubation was completed, the culture medium was discarded and the positively charged substances adsorbed on the wells and cell surfaces were washed away with a PBS buffer solution containing 0.02 mg / mL sodium heparin. The cells were treated with trypsin, resuspended in 200 μL of sterile PBS buffer solution, sprayed evenly, and counted for each sample. 10 4Cells were taken for flow cytometry detection. Cells incubated in serum-free DMEM medium were used as the negative control group. All experiments were performed in triplicate. The results are shown in Figure 5 (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 highly statistically significant difference between the two groups).

[0147] As can be seen from Figure 5, the prepared liposomal pharmaceutical preparations Formula 1, Formula 2 and Formula 3 can all be taken up by cells, with significant differences compared with the control group, and the cellular uptake effect of the prepared liposomal pharmaceutical preparation Formula 2 is optimal.

[0148] [Example 9] (Particle size of pharmaceutical preparations prepared using liposome membrane materials with different blend ratios of component (iv)) According to the method described in Example 6, a liposome pharmaceutical formulation (containing FAM-labeled siRNA) was prepared in which the molar ratio of component (i):component (ii):component (iii) of the liposome membrane material was 3:3:4 and the compounding ratio of component (iv) was as shown in Table 3.

[0149] [Table 3]

[0150] Liposomal pharmaceutical preparations were obtained in which the molar ratio of component (iv) was 1%, 5%, 8%, and 10% relative to the liposome membrane material. The liposomal pharmaceutical preparations were subjected to particle size measurement. The particle size measurement results are shown in Figure 6. In Figure 6, "1%, 5%, 8%, and 10%" on the horizontal axis indicate liposomal pharmaceutical preparations in which the molar ratio of component (iv) was 1%, 5%, 8%, and 10%, respectively, relative to the liposome membrane material.

[0151] As can be seen from Figure 6, when the molar ratio of component (iv) to the liposome membrane material is 1% to 10%, the particle size of the liposome pharmaceutical preparation is 100 nm to 200 nm. In addition, as the molar ratio of component (iv) to the liposome membrane material increases, the particle size tends to increase.

[0152] [Example 10] Quantitative evaluation of cellular uptake of pharmaceutical formulations prepared with different cationic liposome / siRNA charge ratios U87 cells (human malignant glioblastoma cells, purchased from ATCC) were cultured in DMEM complete medium (DMEM medium supplemented with 10% FBS and 1% penicillin-streptomycin). U87 cells in good growth condition were taken and resuspended in DMEM complete medium, and 5 × 10 5 Cells were inoculated into a 6-well plate at 1 mL per well, and the culture medium was changed once a day with fresh medium. After inoculation, the cells were cultured for 2-3 days before the experiment.

[0153] According to the method described in Example 6, the oligoarginine R8 solution and the 20μM FAM-siRNA solution were mixed in equal volumes at a charge ratio of 5, vortexed for 30 seconds, and incubated at 37℃ for 30 minutes to obtain the complex R8 / FAM-siRNA. 8μL of the R8 / FAM-siRNA complex was taken and mixed with the prepared cationic liposome CLS at a charge ratio of liposome to FAM-siRNA of 2, 4, 6, 8, 10, and 12, and vortexed for 30 seconds to obtain the CLS / R8 / FAM-siRNA complex with different charge ratios of cationic liposome / siRNA.

[0154] Furthermore, according to the method described in Example 6, a pharmaceutical preparation modified with 3% cell-penetrating peptide on the surface, that is, 89W-CLS / R8 / FAM-siRNA complex, was obtained.

[0155] After discarding the culture medium from the cultured U87 cells, the cells were washed three times with sterile PBS. 1 mL of serum-free DMEM medium containing pharmaceutical preparations with cationic liposome / siRNA charge ratios of 2, 4, 6, 8, 10, and 12 was added to each well, and the cells were incubated for 4 h at 37°C and 5% CO2. After incubation, the culture medium was discarded, and the positively charged substances adsorbed on the wells and cell surfaces were washed away with a PBS buffer solution containing 0.02 mg / mL sodium heparin. The cells were treated with trypsin, resuspended in 200 μL of sterile PBS buffer, sprayed evenly, and counted for each sample. 10 4 Cells were taken for flow cytometry detection. Cells incubated in serum-free DMEM medium were used as the negative control group. All experiments were performed in triplicate. The results are shown in Figure 2 (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 highly statistically significant difference between the two groups).

[0156] As can be seen from Figure 2, the pharmaceutical preparations prepared with the charge ratios of cationic liposome / siRNA of 2, 4, 6, 8, 10, and 12 were all taken up by cells, with significant differences compared with the control group, and the cellular uptake effect of the liposome pharmaceutical preparation prepared with the charge ratio of cationic liposome / siRNA of 4 is optimal.

[0157] [Example 11] (Evaluation of cell transfection effect by pharmaceutical preparations whose surfaces are modified with cell-penetrating peptides at different ratios)

[0158] First, cells were cultured using a 35mm Dimple NanoCulture Dish, and the cell sphere-forming ability and morphology were observed to construct a cell cluster model.

[0159] Specifically, 1.5 mL of DMEM complete medium was taken and added to a 35 mm Dimple NanoCulture Dish (a cell mass culture plate provided by JSR Corporation), and the dish was placed in a refrigerated centrifuge and centrifuged at a centrifuge speed of 1000 × g for 5 minutes (the dish only contained medium, no cells). After centrifugation, the dish was placed on an ultraclean desk and left to stand for 30 minutes.

[0160] Various types of cells in good growth condition were taken, digested, centrifuged, and then culture medium was added to adjust the cell concentration to 5 × 10 5 The cell concentration was adjusted to 1 / mL, the cells were blown out with a pipette gun to disperse the cells evenly, and 1.5 mL of the cell suspension was slowly added to the dish, which was then left to stand on the operation table for 20 minutes and then cultured in an incubator at 37°C. The next day, the liquid was changed, the dish was removed, and 1.5 mL of the medium was gently sucked out (the operation for floating cells and wall-attached cells is the same, and the cells have already formed initial cell aggregates when the liquid was changed, so they have sunk to the bottom of the dish), and fresh 1.5 mL of DMEM complete medium was added.

[0161] The morphology of the corresponding cell clusters was observed under an inverted microscope. A549 cells are human non-small cell lung cancer cells, Caco-2 are human clonal colon adenocarcinoma cells, Rb-1 are human retinal neuroglioma cells, U87 are human malignant glioblastoma cells, and 293T are human renal epithelial cells, all of which were purchased from ATCC. A549 and Caco-2 are adherent cells with strong adhesion, while U87 and 293T are weakly adherent cells, and Rb-1 is a floating cell. The results are shown in Figure 7. In Figure 7, Sample 1 and Sample 2 are respectively different magnifications of the same cells, and Sample 3 and Sample 4 are repeated experimental wells of Sample 2.

[0162] As can be seen from Figure 7, A549 and Caco-2 have good adhesion, tend to grow by adhering to the wall, and do not form cell clumps, while Rb-1 is a floating cell, but the cells tend to gather into spheres and are very easy to dissolve during processing. In comparison, U87 and 293T have the best cell clump forming ability, roughly spherical in morphology and uniform in size, making them suitable for constructing cell clump models for further exploration.

[0163] As can be seen from the results in Figure 8, the cell aggregate penetration ability of different polypeptides is significantly different, and the cell aggregate model constructed by the 35 mm Dimple NanoCulture Dish can distinguish the penetration ability of different polypeptides. Note: "S8" in Figure 8 is a polymer of 8 serines and was used as a negative control group. "PE" is wild-type Penetratin. 89W is the product of penetratin mutated to tryptophan for glutamic acid at position 8 and asparagine at position 9. 289W is the product of penetratin mutated to tryptophan for glutamine at position 2, glutamine at position 8, and asparagine at position 9. The wild-type Penetratin derivatives showed significantly improved cell aggregate penetration ability.

[0164] Next, using 293T cell mass, GFP-siRNA was encapsulated and the transfection effect of pharmaceutical preparations whose surfaces were modified with cell-penetrating peptides at different ratios was evaluated.

[0165] Similarly to the above Example 6, GFP-siRNA liposome pharmaceutical preparations with different ratios of cell-penetrating peptides were prepared, where the GFP-siRNA used had a sense strand (5'-3') of GCAUGUACCACGAGUCCAATT (SEQ ID NO: 81) and an antisense strand (5'-3') of UUGGACUCGUGGUACAUGCTT (SEQ ID NO: 82). Specifically, the oligoarginine R8 solution and the 20 μM GFP-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 a complex R8 / GFP-siRNA. 8 μL of the R8 / GFP-siRNA complex was taken and mixed with the prepared cationic liposome CLS at a charge ratio of liposome to FAM-siRNA of 4, and vortexed for 30 seconds to obtain a CLS / R8 / GFP-siRNA complex.

[0166] Furthermore, according to the method described in Example 6, liposome pharmaceutical preparations modified with 1%, 3%, and 5% of cell-penetrating peptide on the surface, namely, 89W-CLS / R8 / GFP-siRNA complexes, were obtained.

[0167] The 293T cell mass model was added with a serum-free DMEM solution containing 400nM of a liposomal pharmaceutical formulation encapsulating GFP-siRNA, and incubated at 37℃ and 5% CO2 for 4h. The drug solution was then discarded, and fresh DMEM complete medium was added and incubated for another 24h. The cells were washed three times with PBS buffer solution, and the GFP fluorescence status (also referred to as GFP-239T cell mass in the specification) was observed under a laser confocal microscope. Cells incubated with serum-free DMEM medium were used as a negative control group. All experiments were performed in triplicate. The results are shown in Figure 9 (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 highly statistically significant difference between the two groups).

[0168] Similarly, the transfection effect of a liposomal pharmaceutical preparation encapsulating 400 nM of GFP-siRNA on U87 cell mass was evaluated (also referred to as GFP-U87 cell mass in the text). The results are shown in FIG.

[0169] In Figures 9 and 10, "1%, 3%, 5%" indicate 89W-CLS / R8 / siRNA, which is a liposomal pharmaceutical preparation whose surface is modified with a cell-penetrating peptide at a ratio of 1%, 3%, and 5%, respectively.

[0170] In Figure 9, a GFP-239T cell mass was constructed using a 35 mm Dimple NanoCulture Dish. 293T cells are not tumor cells, 89W The higher the molar ratio of Penetratin-PEG-DSPE to the liposome membrane material, the higher the degree of penetration of the liposomal drug into the cell mass. In Figure 10, a GFP-U87 cell mass was constructed using a 35 mm Dimple NanoCulture Dish. U87 cells are brain glioma cells. 89W When the molar ratio of Penetratin-PEG-DSPE reaches 3%-5% relative to the liposome membrane material, the liposomal drug has high permeability into the cell mass.

[0171] [Example 12] Qualitative and quantitative evaluation of transfection efficiency in LUC-U87 cells by pharmaceutical preparations containing positively charged polyamino acids of different structural types. Oligoarginines such as R8, R10, R12, c-R8, c-R10, c-R12 are used as positively charged polyamino acids of different structural types, the charge ratio between the oligoarginine and siRNA is 5, and the charge ratio between CLS and LUC-siRNA is 4. According to Example 6, liposome pharmaceutical preparations containing different oligoarginines and surface-modified with cell-penetrating peptide at a ratio of 3% were prepared. The sense strand (5'-3') of the LUC-siRNA used is GCUGCACUCUGGCGACAUUTT (sequence number 83), and the antisense strand (5'-3') is AAUGUGCCAGAGUGCAGCTT (sequence number 84).

[0172] U87 cells (purchased from ATCC) in good growth condition were taken and 5 × 10 4 The cells were inoculated into a 48-well plate at 100 cells / well, and the culture medium was changed once a day after inoculation. After 2-3 days of culture, the experiment was performed. After discarding the DMEM complete medium, the cells were washed three times with sterile PBS, and serum-free DMEM medium containing 400 nM LUC-siRNA-encapsulated liposomal pharmaceutical preparations was added, and the cells were incubated for 4 hours at 37°C and 5% CO2. Then, the drug solution was discarded, and DMEM complete medium was added and incubated for 24 hours (also referred to as LUC-U87 cells in the specification). The positively charged adsorbed material was washed away with a PBS buffer solution containing 0.02 mg / mL heparin sodium, and a firefly luciferase substrate was added, and the fluorescence intensity was measured using a small animal bioluminescence / bioluminescence imaging system (IVIS Spectrum). The cells were qualitatively analyzed using the quantitative circle selection function of the region of interest (ROI) of the small animal biooptical imaging system. The results are shown in Figure 11.

[0173] This example was to explore the gene knockdown effect of positively charged polyamino acids with different structural types, and Figure 11 shows that changing the polymerization degree and structure of oligoarginine in liposomal pharmaceutical preparations does not significantly affect the gene knockdown effect.

[0174] [Example 13] (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) that were growing well in the logarithmic phase in DMEM medium were taken and then diluted to 5 × 10 4 The cells were seeded at a concentration of 1000 / well onto a 12-well plate and cultured at 37°C in 5% CO2 until a monolayer of cells covered the bottom of the well.

[0175] The culture medium was discarded, and the cells were washed three times with a sterile PBS buffer solution. Then, the cells were washed with the liposome preparation (89W-CLS / R8 / siRNA) prepared in Example 6 containing 400 nmol / L siRNA (the liposome preparation has a charge ratio of oligoarginine to siRNA of 5, a charge ratio of CLS to siRNA of 4, 89W Penetratin-PEG 3400 After incubating for 6 hours in an incubator, the solution was discarded. Then, the cells were washed three times with sterile PBS buffer solution, and 1 mL of complete medium was added and cultured for 18 hours.

[0176] After that, the cells were collected, washed with PBS, treated with EDTA-free trypsin for 1 min, and finished the treatment with DMEM culture medium. Then, the cells were collected into a centrifuge tube, and the cells were double-stained with the steps of a cell apoptosis test kit (Nanjing Kaiji Biological Science and Technology Development Co., Ltd., KGA108) and detected by flow cytometry. Cells incubated with serum-free DMEM medium served as the negative control group. All experiments were performed in triplicate. The results are shown in Figure 14A and Figure 14B (mean ± SD, n = 3, *p < 0.05, * indicates that there is a statistically significant difference between the two groups. **p < 0.01, ***p < 0.001, ** and *** indicate that there is a statistically very significant difference between the two groups).

[0177] As a result, it was shown that the delivery carrier designed according to the present invention can specifically promote apoptosis of tumor cells U87, without significantly affecting the growth of normal cells bEnd.3.

[0178] [Example 14] (Evaluation of the cytotoxic activity of siRNA-encapsulated delivery carriers in bEnd.3 and U87 cells) bEnd.3 cells (purchased from ATCC) and U87 cells in good logarithmic growth were cultured in DMEM complete medium and incubated at 5 × 10 3 Cells / cm 2The cells were placed in the middle 60 wells of a 96-well flat-bottom plate at a concentration of 100 μL each, and the other wells were filled with sterile PBS buffer solution and cultured at 37 °C and 5% CO2 until a monolayer of cells covered the bottom of the wells. The culture medium was then discarded, washed three times with sterile PBS buffer solution, and 200 μL of culture medium containing 400 nM siRNA delivery carrier was added, incubated in an incubator for 6 h, after which the drug solution was discarded and washed three times with sterile PBS buffer solution. Complete medium was then added and cultured for 12 h. Then, 20 μL of MTT solution (5 mg / mL) was added per well, and cultured for 4 h. The liquid was then gently discarded, washed three times with PBS buffer solution, and 150 μL of DMSO was added per well. The wells were shaken at low speed (50 rpm) for 20 minutes on a shaker, and the absorbance value of each well at OD490nm was measured using a microplate reader (BioTek, PowerWave XS). Blank wells (wells containing only DMEM complete medium, MTT and DMSO) and negative control wells (wells containing only cells, DMEM complete medium, MTT and DMSO) were placed simultaneously. All experiments were performed in triplicate. The results are shown in Figure 14C and Figure 14D (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 highly statistically significant difference between the two groups).

[0179] As a result, it was shown that compared with the commercially available cationic gene delivery carrier Lipofectamine 2000 (Lipo2000, Invitrogen), the delivery carrier designed according to the present invention had less effect on the growth state of bEnd.3 cells and U87 cells, and no cytotoxic activity was observed under the concentration conditions examined.

[0180] [Example 15] (Distribution of gene delivery carriers labeled with DiD cell membrane fluorescent probes in vivo after intranasal administration) First, liposomes labeled with the lipophilic fluorescent dye DiD were prepared and used to track the liposomes. The DiD cell membrane red fluorescent probe was purchased from Dalian Meilun Biotechnology Co., Ltd. and was MB6190. As in Example 1, the membrane material was weighed, and DiD dye was added as described in the kit's instructions, and the two were dissolved together in chloroform to prepare CLS / R8 / siRNA labeled with DiD. In addition, as in Example 6, the charge ratio of oligoarginine to siRNA was 5, the charge ratio of CLS to siRNA was 4, 89W Penetratin-PEG 3400 A DiD-labeled liposomal pharmaceutical formulation, 89W-CLS / R8 / siRNA, whose surface was modified with 3% -DSPE was prepared.

[0181] Twelve ICR mice (20 g, male, 6 weeks old) were divided into three groups (89W-CLS / R8 / siRNA group, CLS / R8 / siRNA group, and saline (NS) group). Approximately 30 μL of DiD-labeled nucleic acid delivery carrier was administered intranasally to each mouse (DiD concentration is 0.01 μM). After 2.5 h, the animals were euthanized, perfused, and fixed, and the major organs of the animals were collected. The fluorescence distribution of each organ was detected using a small animal bioimaging system. The results are shown in Figure 15.

[0182] As a result, the fluorescence intensity in the brain of the 89W-CLS / R8 / siRNA group was greater than that of the CLS / R8 / siRNA group, indicating that the modification with cell-penetrating peptide could significantly enhance the tissue penetration ability of the delivery carrier, further enhance its ability to enter the brain, and increase the accumulation of genes in the brain.

[0183] [Example 16] (Exploring the entry mechanism by which a carrier labeled with a DiD cell membrane fluorescent probe delivers FAM-siRNA) 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, in the same manner as in Example 1. Each pharmaceutical preparation labeled with DiD and / or FAM fluorescence was prepared in the same manner as in Example 6.

[0184] U87 cells (purchased from ATCC) were cultured in DMEM complete medium (DMEM medium supplemented with 10% FBS and 1% penicillin-streptomycin). U87 cells in good growth condition were taken and resuspended in DMEM complete medium, and 5 × 10 5 The cells were inoculated into a 6-well plate at 10 cells / well, 1 mL per well. After inoculation, fresh culture medium was changed once daily, and the cells were cultured for 2-3 days before the experiment.

[0185] After discarding the culture medium from the cultured U87 cells, the cells were washed three times with sterile PBS. 1 mL of serum-free DMEM medium containing 1 μM of siRNA labeled with different fluorescent drugs was added to each well and incubated at 37°C and 5% CO2 for 4 h. After incubation, the culture medium was discarded and the positively charged substances adsorbed on the wells and cell surfaces were washed away with PBS buffer solution containing 0.02 mg / mL sodium heparin. The cells were treated with trypsin, resuspended in 200 μL of sterile PBS buffer solution, sprayed evenly, and counted for each sample. 10 4 Cells were taken for flow cytometry detection. In the experiment, cells incubated with blank serum-free DMEM medium were set as the negative control group. In addition, two positive control groups were set, namely, 89W-CLS / R8 / FAM-siRNA containing only FAM and 89W-CLS / DiD / R8 / siRNA containing only DiD. In addition, the following formulation groups were set: R8 / FAM-siRNA group, CLS / DiD group, CLS / DiD / R8 / FAM-siRNA group, and 89W-CLS / DiD / R8 / FAM-siRNA group. All experiments were performed in triplicate. The results are shown in Figure 16.

[0186] From Figure 16D, it can be seen that the pharmaceutical preparation formed by compressing siRNA with R8 alone has a low vesicle amount, while the pharmaceutical preparation formed by compressing siRNA with CLS and R8 together can be successfully vesiculated (see Figure 16F), and the pharmaceutical preparation formed by compressing siRNA with 89W-modified CLS and R8 together can significantly increase the cellular uptake of gene carriers (see Figure 16G). From Figure 16B, Figure 16C and Figure 16G, it can be seen that cells take up the 89W-CLS / R8 / FAM-siRNA pharmaceutical preparation and show positive results of DiD and FAM dual fluorescence. This means that CLS and siRNA enter cells together in the form of a complex, not taken up in free form.

[0187] [Example 17] (Distribution of gene delivery carriers labeled with DiD cell membrane fluorescent probe in U87 in situ brain tumor model mice after intranasal administration) First, liposomes labeled with lipophilic fluorescent dye DiD were prepared and used to deposit liposomes. DiD cell membrane red fluorescent probe was MB6190, purchased from Dalian Meilun Biotechnology Co., Ltd. As in Example 15, the membrane material was weighed, DiD dye was added as described in the kit's instructions, and the mixture was dissolved in chloroform to prepare DiD-labeled CLS / R8 / siRNA. Also, as described in Example 6, the charge ratio of oligoarginine to siRNA was 5, and the charge ratio of CLS to siRNA was 4, 89W Penetratin-PEG 3400 A DiD-labeled liposomal pharmaceutical formulation, 89W-CLS / R8 / siRNA, whose surface was modified with 3% -DSPE was prepared.

[0188] Logarithmic growth phase U87 cells were harvested, the cell number was counted, and the cells were resuspended in PBS buffer. 6 × 10 cells were then transferred to each BALB / c nude mouse (Shanghai Xipur-Bikai Experimental Animal Co., Ltd., China). 5U87 cells (dispersed in 5 μL of PBS buffer solution) were inoculated into nude mice, which were anesthetized with 7% chloral hydrate and fixed in a brain stereotaxic apparatus. The cells were inoculated into the striatum (0.6 mm anterior to bregma, 1.8 mm to the right, and 3 mm behind) with a microsyringe to establish a U87 in situ brain tumor model.

[0189] Nine U87 in situ brain tumor mice inoculated with U87 tumor cells for 18 days were randomly set into three groups (89W-CLS / R8 / siRNA group, CLS / R8 / siRNA group, and saline group). Then, 30 μL of DiD-labeled nucleic acid delivery carrier (DiD concentration 0.01 μM) was intranasally administered to each mouse, and the animals were euthanized 2.5 hours later. After perfusion and fixation, the animals' brain tissues and tumors were collected and frozen sectioned, and the distribution of DiD fluorescent signals was observed under a fluorescence microscope (Leica, Germany). The results are shown in Figure 17. The scale bar is 100 μm.

[0190] The results showed that compared with the CLS / R8 / siRNA group, the 89W-CLS / R8 / siRNA group had higher fluorescence intensity at the tumor site, indicating that the modification of the cell-penetrating peptide could significantly enhance the tissue penetration ability of the delivery carrier, further enhance its ability to enter the brain, and increase the accumulation of genes in the brain.

[0191] [Example 18] Pharmacodynamic evaluation of siRNA-loaded delivery carriers for anti-U87 in situ brain tumors The liposomal pharmaceutical formulations, i.e., CLS / R8 / siRNA and 89W-CLS / R8 / siRNA complexes, were prepared according to the method described in Example 6, and the Lipo2000 / siRNA complexes were prepared according to the instructions of the commercially available cationic gene delivery carrier Lipofectamine 2000 (Lipo2000, Invitrogen).

[0192] The U87 in situ brain tumor model was constructed according to the method described in Example 17. After inoculation of U87 in situ brain tumor, the mice were randomly divided into 5 groups (n=10), namely, saline group, siRNA group, Lipo2000 / siRNA group, CLS / R8 / siRNA group and 89W-CLS / R8 / siRNA group. From the 5th day after inoculation of U87 in situ brain tumor, intranasal administration (47μg / kg siRNA) was performed once a day for a total of 22 administrations, and the body weight and survival time of the mice were recorded, and the body weight change curve and survival curve were drawn. The results are shown in Figure 18 (*p<0.05, * indicates that there is a statistically significant difference between the two groups. **p<0.01, ** indicates that there is a highly statistically significant difference between the two groups).

[0193] As a result, the median survival times of the saline group, siRNA group, Lipo2000 / siRNA group, and CLS / R8 / siRNA group were 23.5 days, 21.5 days, 24.5 days, and 26 days, respectively, while the median survival time of the 89WP-CLS / R8 / siRNA group was successfully extended to 29 days, and the survival curve did not cross with other groups, achieving the most significant anti-brain glioma effect. These data demonstrated that 89WP-CLS / R8 / siRNA can deliver drugs to brain tumors to exert anti-tumor effects, and its unique delivery advantages can be translated into higher therapeutic efficacy.

[0194] [Example 19] (Evaluation of the apoptosis-promoting ability of siRNA-encapsulated delivery carriers in U87 in situ brain tumor model mice) According to the method described in Example 18, liposomal pharmaceutical formulations were prepared, and U87 in situ brain tumor models were constructed and administered intranasally. Two days after the last administration (on the 28th day), three mice were randomly selected from each group and euthanized, and the brain tumors were collected for immunofluorescence histochemical analysis. The tumors were fixed with 4% paraformaldehyde, embedded in paraffin, and sliced. Apoptotic cells were detected by TUNEL staining, and the TUNEL fluorescence intensity at the tumor site was analyzed by CaseViewer software. The results are shown in Figure 19. The scale bar is 20 μm.

[0195] In the TUNEL stained sections, the intensity of green fluorescence indicates the number of tumor cells that have undergone apoptosis. The results show that Lipo2000 / siRNA group, CLS / R8 / siRNA group and 89WP-CLS / R8 / siRNA group can all induce apoptosis in tumor cells, among which 89WP-CLS / R8 / siRNA group has the highest fluorescence intensity, i.e., the highest apoptosis rate. This shows that the delivery carrier designed in the present invention can exert antitumor effects by inducing apoptosis of tumor cells in vivo.

[0196] [Example 20] (In vivo nasal mucosal toxicity evaluation of siRNA-encapsulated delivery carriers) Eighteen SD rats (Shanghai Lingchun Biotechnology Co., Ltd., China) were randomly divided into six groups (n=3), namely, saline group (negative control), 1% sodium deoxycholate group (positive control), siRNA group, CLS / R8 / siRNA group, 89WP-CLS / R8 / siRNA group, and Lipo2000 / siRNA group. The liposome pharmaceutical formulations, namely, CLS / R8 / siRNA and 89W-CLS / R8 / siRNA complexes, were prepared according to the method described in Example 6. Lipo2000 / siRNA complexes were also prepared according to the instructions of the commercially available cationic gene delivery carrier Lipofectamine 2000 (Lipo2000, Invitrogen). 50 μL of the formulations (with or without 160 nM siRNA) were administered intranasally into the left nasal cavity of SD rats for 7 consecutive days, and 24 hours after the last administration, the rats were euthanized, and the nasal mucosa of the left nasal cavity was isolated and washed with saline. The samples were fixed with 4% paraformaldehyde, sectioned, and stained with HE. The results are shown in Figure 20. The scale bar is 50 μm.

[0197] As a result, the nasal epithelial cells in the positive control groups (sodium deoxycholate group and Lipo2000 / siRNA group) showed relatively significant morphological changes, including loss of nasal cilia (red arrows), while the nasal epithelial cells in the saline group, siRNA group, CLS / R8 / siRNA group and 89WP-CLS / R8 / siRNA group showed intact and orderly arranged morphology. This indicates that the delivery carrier designed in the present invention is not significantly toxic to nasal epithelial cells and is a safe carrier for delivery from the nose to the brain.

[0198] While certain representative embodiments and details have been shown for the purpose of illustrating the invention, it will be apparent to those skilled in the art that various changes and modifications can be made thereto without departing from the scope of the subject invention, which in this regard is limited only by the scope of the claims that follow.

Claims

1. A pharmaceutical preparation prepared using a drug delivery carrier comprising a liposome modified with a cell-penetrating peptide, which is a penetrant or a penetrant derivative having the following amino acid sequence, and a positively charged polyamino acid as a polymer: RX １ IKIWFX ２ X ３ RRMKWKK Here, X １ , X ２ and X ３ 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; The drug delivery carrier is a liposome complex consisting of a polymer-drug physical complex, which is a complex of the positively charged polyamino acid and a drug molecule, and a liposome modified with the cell-penetrating peptide, and the particle size of the liposome complex is 50 nm to 300 nm.

2. The pharmaceutical formulation of claim 1 , wherein the drug molecule is selected from the group consisting of peptide drugs, antibodies, cytokines, hormones, antibiotics, nucleic acids, and combinations thereof.

3. The liposome is a cationic liposome comprising a cationic lipid, In the pharmaceutical formulation, the molar ratio of the drug molecule to the cationic lipid is 0.1 to 10; or In the pharmaceutical preparation, the loading amount of the drug molecule is 1% to 30% (w / w) of the drug molecule relative to the pharmaceutical preparation; or 3. The pharmaceutical formulation according to claim 1, wherein the charge ratio of the positively charged polyamino acid to the drug molecule is 1:1 to 30:

1.

4. The cationic liposome contains, as a membrane material, (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 20-40:20-40:20-40:1-20, ８９Ｗ Penetratin-PEG ３４００ - DSPE is in a molar ratio of 20% to 80% with respect to the membrane material (iv), 4. The pharmaceutical preparation according to claim 3, wherein the charge ratio between the positively charged polyamino acid and the drug molecule is 1:1 to 30:1, and the charge ratio between the cationic liposome and the drug molecule is 1:1 to 30:

1.

5. The pharmaceutical formulation according to any one of claims 1 to 4, which is a pharmaceutical formulation for nasal administration.

6. A drug delivery carrier comprising a liposome modified with a cell-penetrating peptide and a positively charged polyamino acid as a polymer, the polymer forms a polymer-drug complex with the drug; the liposome modified with the cell-penetrating peptide forms a liposome complex together with the polymer-drug complex; The cell-penetrating peptide is penetrant or a penetrant derivative having the following amino acid sequence: RX １ IKIWFX ２ X ３ RRMKWKK Here, X １ , X ２ and X ３ are independently selected from naturally occurring amino acids glutamine (Q), asparagine (N), alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), tryptophan (W), and methionine (M), and non-naturally occurring amino acids α-aminobutyric acid, α-aminopentanoic acid, α-aminohexanoic acid, and α-aminoheptanoic acid. The drug delivery carrier is selected from the group consisting of

7. The drug delivery carrier described in claim 6, characterized in that the cell-penetrating peptide is a lipophilic derivative of penetratin having any of the following amino acid sequences: RWIKIWFQNRRMKWKK (sequence number 24), RQIKIWFWNRRMKWKK (sequence number 25), RQIKIWFQWRRMKWKK (sequence number 26), RWIKIWFWNRRMKWKK (sequence number 27), RWIKIWFQWRRMKWKK (sequence number 28), RQIKIWFWWRRMKWKK (sequence number 29), RWIKIWFWWRRMKWKK (sequence number 30).

8. The drug delivery carrier according to claim 6 or 7, wherein the positively charged polyamino acid has a degree of polymerization of 2 to 50.

9. The liposome modified with the cell-penetrating peptide comprises the following membrane material: (i) cationic lipids: 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(sperminecarboxamido)ethyl]-N-N-dimethyl-1-propane ammonium trifluoroacetate (DOSPA), or combinations thereof; (ii) non-cationic lipids: 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), or combinations thereof; (iii) cholesterol; (iv) polyethylene glycol-linked (PEGylated) phospholipids and polyethylene glycol phospholipids conjugated with cell-penetrating peptides: the phospholipids are phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol or sphingomyelin, and the molecular weight of the polyethylene glycol (PEG) is between 1 kDa and 10 kDa; The drug delivery carrier according to any one of claims 6 to 8, wherein the polyethylene glycol in the PEGylated phospholipid and the polyethylene glycol phospholipid conjugated to the cell-penetrating peptide do not have the same chain length, and the polyethylene glycol chain in the polyethylene glycol phospholipid conjugated to the cell-penetrating peptide is longer than the polyethylene glycol chain in the PEGylated phospholipid.

10. The drug delivery carrier of claim 9, wherein the molar ratio of the membrane materials (i):(ii):(iii):(iv) is 20-40:20-40:20-40:1-20, and the polyethylene glycol phospholipid conjugated to the cell-penetrating peptide is in a molar ratio of 20%-80% relative to the membrane material (iv).

11. The liposome contains, as a membrane material, (i) DOTAP; (ii) DOPE; (iii) cholesterol; (iv) mPEG ２０００ -DSPE and cell penetrating peptide-PEG ３４００ -DSPE, and the molar ratio of the membrane materials (i):(ii):(iii):(iv) is 20-40:20-40:20-40:1-20, and the cell penetrating peptide-PEG ３４００ The drug delivery carrier according to any one of claims 6 to 10, wherein the DSPE is in a molar ratio of 20% to 80% relative to the membrane material (iv).

12. A method for preparing the pharmaceutical formulation according to any one of claims 1 to 4, comprising the steps of: (a) preparing a cationic liposome having a cell-penetrating peptide modified on its surface; (b) preparing a physical complex consisting of a positively charged polyamino acid and a drug molecule; (c) mixing the cationic liposome having a cell-penetrating peptide modified on its surface obtained in step (a) with the physical complex obtained in step (b) at a certain charge ratio.

13. A method for preparing the pharmaceutical formulation of claim 4, comprising the steps of: (a) mixing liposome membrane materials (i), (ii) and (iii) to prepare blank liposomes; (b) preparing a physical complex of a positively charged polyamino acid and a drug molecule; (c) mixing the blank liposome obtained in step (a) and the physical complex obtained in step (b) at a certain charge ratio; (d) mixing and incubating the liposomes obtained in step (c) with a membrane material (iv).

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