JP7645601B2 - Drug delivery carrier and pharmaceutical preparation using same - Google Patents

Description

The present invention relates to a drug delivery carrier, a method for preparing a pharmaceutical formulation using the drug delivery carrier, and a pharmaceutical formulation prepared thereby. Specifically, the drug delivery carrier of the present invention includes a liposome modified with a cell-penetrating peptide and a polymer, and the polymer is a positively charged polyamino acid. The pharmaceutical formulation 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 formulation can effectively transfect cells, and in particular, can promote the absorption of the drug into the brain via the nasal cavity, thereby enhancing the therapeutic effect of the drug.

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 drug molecules into cells or carry drug molecules to penetrate biological membrane barriers. However, a single cell-penetrating peptide has a very limited capacity to carry drug molecules, and as a result, the complex formed by the cell-penetrating peptide and the drug molecule has a large particle size and poor stability.

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 susceptibility 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 major 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.

Therefore, there is still a need in this field for a new drug delivery carrier that utilizes cell-penetrating peptides, which can improve the 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 can be controlled, has good stability, and can achieve more effective drug delivery by the cell-penetrating peptides that modify the surface of the formulation, which helps the drug escape from the cell endosome to exert its effect.

The 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.

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:
RX ₁ IKIWFX ₂ X ₃ RRMKWKK (SEQ ID NO: 1)

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, and α-aminohexanoic acid. For example, some cell-penetrating peptides in the drug delivery carrier of the present invention are Penetratin derivatives 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 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 being linear or cyclic, and the configuration of the amino acid residues being 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 interposed between 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.

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:

(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]-N-N-dimethyl-1-propane ammonium trifluoroacetate (DOSPA) and combinations thereof. Preferred are DOTMA, DOTAP, DODAC, and DOSPA. Most preferably, it 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, and preferably, the PEGylated phospholipids are, for example, polyethylene glycol-distearoylphosphatidylethanolamine (PEG-DSPE) and its derivative, methoxy-polyethylene glycol-distearoylphosphatidylethanolamine (mPEG-DSPE), and the polyethylene glycol phospholipid conjugated with CPPs is, for example, Penetraten/Penetratin derivative-PEG-DSPE.

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 polyethylene glycol phospholipid conjugated to CPP is about 20%-80% molar relative to membrane material (iv). 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 polyethylene glycol phospholipid conjugated to CPP is about 20%-80% molar relative to membrane material (iv).

In some specific embodiments, the cationic lipid in the drug delivery carrier of the present invention comprises, as membrane materials, (i) DOTAP; (ii) DOPE; (iii) cholesterol; (iv) mPEG ₂₀₀₀ -DSPE and ^89W Penetrant-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, and the molar ratio of ^89W Penetrant-PEG ₃₄₀₀ -DSPE to the membrane material (iv) is about 20%-80%. 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 -DSPE is in a molar ratio of about 20%-80% to membrane material (iv).

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

In another aspect, the present invention provides a pharmaceutical formulation prepared using the drug delivery carrier of the present invention. A positively charged polyamino acid contained in the drug delivery carrier is physically complexed with one or more drug molecules to be delivered, and the physical complex is mixed with a cationic liposome whose surface is modified with a cell-penetrating peptide to form a pharmaceutical formulation in the form of a liposome complex.

The drug molecule or molecules delivered may 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.

In one embodiment, the drug molecule delivered by the pharmaceutical formulation is a nucleic acid such as a plasmid DNA, small interfering RNA (siRNA), sense RNA, antisense oligonucleotides (ASO), aptamers, or ribozymes. 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 genome of an organism (e.g., doxorubicin).

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.

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

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.

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

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

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 liposome obtained in step (a) with the physical complex obtained in step (b) at a certain molar ratio or charge ratio, and (d) mixing and incubating the liposome obtained in step (c) with membrane material (iv) to obtain the pharmaceutical formulation of the present invention.

The pharmaceutical preparation 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.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications, patent applications, patents, and other references mentioned herein are hereby incorporated by reference in their entirety. 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 from the appended claims.

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 with different R8/siRNA charge ratios, respectively. FIG. 1C and FIG. 1D show the agarose gel electrophoresis results and particle size results for CLS/siRNA complexes with different CLS/siRNA charge ratios, respectively. FIG. 1E and FIG. 1F show the agarose gel electrophoresis results and particle size results for CLS/R8/siRNA liposome complexes with different CLS/siRNA charge ratios, respectively, when the charge ratio of R8/siRNA is 1:1. 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. Figure 3 shows the results of electrophoresis, particle size and stability study of CLS/siRNA/R8 complex. Figures 3A and 3B show the results of agarose gel electrophoresis and particle size of liposome complexes with different charge ratios of R8 and siRNA when the charge ratio of CLS to 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 to siRNA is 4:1, respectively, and the agarose gel electrophoresis results of the supernatant after centrifugation. 4 shows the quantitative evaluation results of cellular uptake of pharmaceutical preparations 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, which is a commercially available gene transfection reagent. FIG. 5 shows the cellular uptake of drug formulations prepared using different formulations of liposome membrane materials shown in Table 2 of Example 8. FIG. 6 shows the particle size of liposomal pharmaceutical preparations in which component (iv) is 1%, 5%, 8% and 10% molar ratio of the liposomal membrane material. Figure 7 shows the morphological results of culturing different cell clusters using 35 mm Dimple NanoCulture Dish. 35 mm Dimple NanoCulture Dish is a cell cluster culture plate manufactured by JSR Corporation. FIG. 8 shows the results of evaluation of the cell mass permeability of the cell-penetrating peptides that modify the surface of the liposome. FIG. 9 shows the transfection effect of a pharmaceutical preparation encapsulating GFP-siRNA on a 293T cell mass. FIG. 10 shows the transfection effect of a pharmaceutical preparation encapsulating GFP-siRNA on a U87 cell mass. FIG. 11 shows the results of qualitative and quantitative evaluation of the transfection effect on LUC-U87 cells by pharmaceutical preparations using different oligoarginines. FIG. 12 shows the particle size of a pharmaceutical formulation encapsulating siRNA. FIG. 13 shows the potential of pharmaceutical formulations encapsulating siRNA. 14 shows the results of evaluation of the apoptosis promoting effect and cytotoxic activity of a pharmaceutical preparation encapsulating siRNA in bEnd.3 cells or U87 cells. FIG. 15 shows the distribution in the body of a pharmaceutical preparation labeled with the cell membrane fluorescent probe DiD after intranasal administration. 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. 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. 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. 19 shows the results of evaluating 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. 20 shows the results of in vivo nasal mucosal toxicity evaluation of delivery carriers encapsulating siRNA. The Lipo2000/siRNA group is a non-covalent complex of siRNA and the cationic liposome Lipofectamine2000, which is 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.

The present invention provides a novel 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 of the present invention can assemble with a drug molecule to form a complex, compact the drug molecule, and encapsulate the drug molecule in the liposome, thereby utilizing the cell-penetrating peptide on the surface of the liposome that has cell-penetrating properties to aid in the permeation of the drug into the biological membrane barrier in the subject's body.

The drug delivery carrier of the present invention has strong drug-carrying, drug-delivery, and tissue-penetrating capabilities, has low tissue toxicity, and can efficiently deliver drug molecules to target sites in the body of a subject via various administration routes, overcomes the technical problem that drugs have difficulty reaching target sites due to the biological membrane barrier of the subject, enhances the efficacy of drugs in treating diseases, and has good biological safety. The present invention also provides a pharmaceutical formulation prepared using the drug delivery carrier.

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

The various features of the drug delivery carrier of the present invention are discussed below.

As used herein, the term "about" refers to an adjustment of ±10% of the specified value. For example, the term "about 5%" is meant to include a range of 4.5% to 5.5%.

As used herein, the terms "comprise" or "comprising" mean the inclusion of the stated elements, integers, or steps, but not the exclusion of any other elements, integers, or steps.

<I. Membrane material of liposomes whose surface is modified with cell-penetrating peptide>
The term "liposome" as used herein refers to an artificially prepared vesicle composed of a lipid bilayer. The type of liposome modified with cell-penetrating peptide in the present invention is not limited, and can be any that can form lipid vesicles and encapsulate drugs.

In some embodiments, the liposomes of the present invention whose surfaces are modified with cell-penetrating peptides include the following membrane materials:

(i) Cationic lipid
Liposomes contain one or more cationic lipids. As used herein, the term "cationic lipid" refers to a lipid that has a net positive charge at a selected pH (e.g., physiological pH). Many cationic lipids are commercially available. 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.

There are no particular limitations on the cationic lipid contained in the liposome, so long as the liposome has a net positive charge at the selected pH (e.g., physiological pH), and examples thereof include 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 ... Cationic lipids that can be used include, but are not limited to, ammonium-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.

(ii) Non-cationic lipids
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 carry a net negative charge at a selected pH, such as physiological pH.

There are no particular limitations 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.

(iii) Cholesterol
Cholesterol is an important component of mammalian cell membranes, and accounts for more than 20% of cell membrane lipids.The term "cholesterol" is used in the broadest sense herein, covering cholesterol derivatives.Research has shown that cholesterol can prevent the disorder of cell membrane bilayers at high temperatures, and can prevent the ordering of cell membrane bilayers at low temperatures, inhibit the formation of liquid crystals, and maintain the fluidity of cell membrane bilayers.

Given the importance of cholesterol in membrane bilayers, the cationic liposomes of the present invention also contain cholesterol.

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

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 phospholipid is, for example, polyethylene glycol-distearoylphosphatidylethanolamine (PEG-DSPE) and its derivative methoxy-polyethylene glycol-distearoylphosphatidylethanolamine (mPEG-DSPE), and the molecular weight of PEG is any value between 1 kDa and 10 kDa.

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

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:

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.

The polyethylene glycol chain in the CPP-PEG-phospholipid includes, but is not limited to, polyethylene glycol chains up to 10 kDa in length, such as 1 kDa, 2 kDa, 3 kDa, 4 kDa, 5 kDa, 6 kDa, 7 kDa, 8 kDa, 9 kDa, 10 kDa, and any value 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, Penetrate/Penetratin derivative-PEG-DSPE, where the molecular weight of the PEG is any value between 1 kDa and 10 kDa.

The present invention intends that modifying the surface of cationic liposomes with CPPs will increase the tissue penetration ability of the drug delivery carrier, thereby helping liposomes carrying more drug molecules to reach the target site and thereby exert the effect of treating and/or preventing a disease.

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 are not 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, e.g., longer at 0.5 kDa to 5 kDa, preferably longer at 0.75 kDa to 4 kDa, and more preferably longer at any value between 1 kDa and 2 kDa.

In one embodiment, the polyethylene glycol chain lengths of the PEGylated phospholipids in the liposome membrane material (iv) are 1 kDa, 2 kDa, and 3 kDa, respectively, and the polyethylene glycol chain lengths of the polyethylene glycol phospholipids conjugated to CPP are about 2.5 kDa, 3.5 kDa, and 4.5 kDa, respectively.

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 with CPP is in a molar ratio of about 20%-80% relative to the membrane material (iv), corresponding to a molar ratio of about 2%-8% relative to the total liposome membrane material.

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 polyethylene glycol phospholipid conjugated to 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.

In some specific embodiments, the cationic liposome in the drug delivery carrier of the present invention comprises, as membrane materials, (i) DOTAP, (ii) DOPE, (iii) cholesterol, (iv) PEG-DSPE (e.g., mPEG ₂₀₀₀ -DSPE) and Penetratin/Penetratin derivative-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, and the molar ratio of Penetratin/Penetratin derivative-PEG-DSPE (e.g., ^89W Penetratin-PEG ₃₄₀₀ -DSPE) to the membrane material (iv) is about 20%-80%. In some 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 Penetraten/Penetratin derivative-PEG-DSPE (e.g., ^89W Penetraten-PEG ₃₄₀₀ -DSPE) is in a molar ratio of about 20%-80% relative to membrane material (iv). In some embodiments, the cationic liposome in the drug delivery carrier of the present invention is composed of membrane materials: (i) DOTAP, (ii) DOPE, (iii) cholesterol, (iv) mPEG ₂₀₀₀ -DSPE and ^89W Penetraten-PEG ₃₄₀₀ -DSPE, the molar ratio of the membrane materials (i):(ii):(iii):(iv) being about 28.5:28.5:38:5, and the molar ratio of ^{the 89W} Penetraten-PEG ₃₄₀₀ -DSPE to the membrane material (iv) being about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%.

II. Positively Charged Polyamino Acids
The positively charged polyamino acids contained in the drug delivery carriers of the present invention are used to compress the drug molecules.

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 particular restriction on the degree of polymerization of the amino acid residues in the polyamino acid, and for example, a positively charged polyamino acid having 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 having a degree of polymerization of 6 to 12, and the positively charged polyamino acid having a degree of polymerization of 2 to 50 may have 1 to 20 (e.g., 1 to 6) uncharged amino acid residues under physiological conditions intervening therein. The positively charged polyamino acid may be linear or cyclic, and the configuration of the amino acid residues is L-type or D-type. Preferably, the positively charged polyamino acid is a polyarginine having 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 L-type or D-type.

In some embodiments, the positively charged polyamino acid is an oligo-arginine, which includes 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 ... These include cyclized polypeptides such as cyclized 10 polyarginine (c-R8), cyclized 10 polyarginine (c-R10), and cyclized 12 polyarginine (c-R12), as well as polypeptides with different structures such as 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 rrrrrrrrrrrr, D-R12).

Below, we explain the method for preparing a pharmaceutical formulation using the drug delivery carrier of the present invention, and explain the characteristics of the pharmaceutical formulation.

By using the drug delivery carrier of the present invention, it is possible to prepare a pharmaceutical preparation in which the surface of cationic liposomes is modified with CPP and has a strong ability to penetrate tissues.

In one embodiment, the method for preparing the pharmaceutical formulation of the present invention is as follows.
(1) Weigh out the above liposome membrane material of the present invention, wherein 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.

(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.

(3) After the resulting dry lipid membrane 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, respectively).

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

(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.

In another embodiment, the method for preparing the pharmaceutical formulation of the present invention is as follows.
(1) Weigh out the above liposome membrane material of the present invention, wherein 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.

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

(3) After the resulting dry lipid membrane 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, respectively).

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

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

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, Cardiovascular drugs including β-blockers, α-blockers and antiarrhythmics; antihypertensives; diuretics and antidiuretics; vasodilators including systemic, cardiac, peripheral and cranial nerves, central nervous system agonists, vasodilators including vasopressors; cough and cold preparations including decongestants; hormones such as estradiol and other steroids including corticosteroids; hypnotics; immunosuppressants; muscle relaxants; parasympathetic blockers; psychostimulants; sedatives and tranquilizers. The drug delivery carrier of the present invention can deliver drugs in all forms, such as ionized, non-ionized, free base, acid addition salt, etc., and can deliver drugs of high or low molecular weight.

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, etc.

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, or ribozymes.

The plasmid DNA is a gene expression system that includes a DNA sequence that codes for, for example, an antigen selected from the group consisting of interleukin-2, interleukin-4, interleukin-7, interleukin-12, and interleukin-15, interferon-α, interferon-β, interferon-γ, colony-stimulating factors, granulocyte-macrophage stimulating factors, antiangiogenic agents, tumor suppressor genes, thymidine kinase, eNOS, iNOS, p53, p16, TNF-α, Fas-ligand, mutated tumor genes, tumor antigens, viral antigens, or bacterial antigens.

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 formulation of the present invention may also be a mixture of plasmid DNA and artificially synthesized RNA, including sense RNA, antisense oligonucleotides, or ribozymes.

In one embodiment, the molar ratio of drug molecule (e.g., chemotherapeutic agent) to cationic lipid (e.g., DOTAP) in the pharmaceutical formulation of the present invention is about 0.1 to 10, e.g., 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, 10, preferably about 0.1 to 5, more preferably about 0.1 to 2.

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) make up 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).

The pharmaceutical formulations of the present invention can be administered orally, nasally, ocularly, respiratory tract, digestive tract, genital tract, locally by 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 and other routes).

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. Pat. No. 5,399,163.

In one embodiment, the drug delivery carrier of the present invention can be used to prepare a pharmaceutical formulation that delivers a nucleic acid, thereby achieving the effects of gene therapy.

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, 2,597 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.

Nucleic acid delivery carriers are one of the important technologies in gene therapy, and 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. When cationic liposomes are close to negatively charged nucleic acids, the assembling cationic lipids can act as a "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).

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. Furthermore, 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 parts, and has excellent biosafety.

In one embodiment, a method for preparing a nucleic acid liposome formulation of the present invention is as follows.
(1) Weigh out the above liposome membrane material of the present invention, wherein 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.

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

(3) After the resulting dry lipid membrane 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, respectively).

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

(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.

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

In one embodiment, a method for preparing a nucleic acid liposome formulation of the present invention is as follows.
(1) Weigh out the above liposome membrane material of the present invention, wherein 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.

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

(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, respectively).

(4) The liposomes obtained in (3) above are mixed with the membrane material (iv) and incubated to obtain cationic liposomes modified with a cell-penetrating peptide.

(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 oligoarginine and the nucleic acid molecule.

(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.

In one embodiment, a method for preparing a nucleic acid liposome formulation of the present invention is as follows.
(1) Weigh out the above liposome membrane material of the present invention, wherein 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.

(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.

(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, respectively).

(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 oligoarginine and the nucleic acid molecule.

(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.

In one embodiment, the nucleic acid molecule is siRNA, and in the preparation method of the siRNA liposome formulation of the present invention, the molar ratio of DOTAP, DOPE, cholesterol, mPEG ₂₀₀₀ -DSPE and Penetratin/Penetratin derivative-PEG ₃₄₀₀ -DSPE is about 28.5:28.5:38:5, DOTAP, DOPE and cholesterol are weighed and dissolved in a round-bottom flask with 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 liposome is extruded by a mini extruder (the order of the core pore membrane is 200 nm, 100 nm and 50 nm in order), and the preparation of blank liposome CLS is completed. Then, oligoarginine Rn (R is a single letter code for arginine, and n is an integer such as 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) and siRNA were mixed at a certain charge ratio, vortexed for 30 seconds, and incubated at 37°C for 30 minutes to obtain a complex Rn/siRNA. Furthermore, complex Rn/siRNA and blank liposome were mixed at a certain charge ratio (charge ratio of cationic liposome and siRNA), vortexed for 30 seconds, and incubated at 37°C for 30 minutes to obtain a complex CLS/Rn/siRNA. Finally, CLS/Rn/siRNA and mPEG ₂₀₀₀ -DSPE were mixed with Penetrate/Penetratin derivative-PEG ₃₄₀₀ -DSPE, and then incubated at 55°C for 30 minutes to complete the preparation of the siRNA liposome formulation.

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 allow 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).

As is well 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 epithelial 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 patient compliance is good (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).

On the other hand, brain tumors such as gliomas are malignant tumors of the central nervous system with a high incidence rate and low survival rate. They cause great damage to human health and have a poor clinical prognosis, mainly manifested in recurrence after surgery and drug resistance to chemotherapy. In addition, due to the existence of the blood-brain barrier and blood-brain tumor barrier, the amount of drugs that reach the brain after systemic administration is very low. Therefore, people have been striving to find effective drug treatment methods for a long time.

The drug liposome formulation of the present invention can deliver drugs to the brain after nasal administration and can further promote the exertion of the therapeutic effect of the drug.

In the examples of the present invention, the inventors constructed a cationic liposome delivery carrier carrying siRNA using 8-polyarginine R8 as an example and siRNA as a drug molecule model. Through a series of in vitro and in vivo experiments, the cellular uptake ability and cell mass permeation ability of a delivery carrier constructed jointly with a cationic liposome modified with a cell-penetrating peptide and a positively charged polyamino acid were examined. As a result, it was 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 to commercially available cationic liposomes that deliver nucleic acids.

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 realized by the fact 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 realized by the fact 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 realized by the fact that 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 helpful for 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 for improving patient compliance.

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

[Example 1]
(Preparation of cationic liposomes)
The membrane materials were weighed out 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).

The above amounts of DOTAP, DOPE, and cholesterol were placed in a 50 mL round-bottom flask, and 1 mL of chloroform was added to dissolve the DOTAP, DOPE, and cholesterol as membrane materials, i.e., 16.67 μM CLS contains 5 μM DOTAP, 5 μM DOPE, and 6.67 μM DOPE. The resulting solution was evaporated by a rotary evaporator (Shanghai Shensheng Technology Co., Ltd., R201L) to remove chloroform and obtain a uniform dry lipid membrane.

The resulting dry lipid film was ultrasonically hydrated with 1 mL of 5% glucose aqueous solution in a 37°C water bath for about 10 minutes. After the lipid film was completely hydrated, liposomes were extruded using a mini extruder (Hamilton, 81320) (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).

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 obtain cationic liposomes containing mPEG ₂₀₀₀ -DSPE.

[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.

4 mg of oligoarginine R8 (Shanghai Xinhao 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 example, a small interfering RNA against c-myc was used, hereinafter also abbreviated as siRNA, whose sense strand (5'-3') is AACGUUAGCUUCACCAACAdTdT (sequence number 79) and whose 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.

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 (since one amino nitrogen of R8 has one positive charge and one phosphate group of siRNA has one negative charge, 1 μM R8 contains 8 μM amino nitrogen, i.e., 1 μM R8 contains 8 μM positive charge, and 1 μM siRNA contains 42 μM phosphate group, i.e., 1 μM siRNA contains 42 μM 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.

Liposome complexes were prepared using the R8/siRNA complexes and CLS with different charge ratios. 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) were mixed with 2.67 μL of 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 R8/siRNA charge ratios. In the lipid complex CLS/R8/siRNA, the charge ratio of cationic liposome CLS to siRNA is fixed at 4 based on the charge number of siRNA (one amino nitrogen of DOTAP has one positive charge, and 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, and 1 μM siRNA contains 42 μM negative charge, so the charge ratio of CLS to 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.

Next, liposome complexes were prepared using CLS and an R8/siRNA complex with an R8/siRNA charge ratio of 1:1. 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 to siRNA charge ratios.

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 = (CLS μM value x 0.3) / (siRNA μM value x 42) = 0.007 x CLS (μM) / siRNA (μM).

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.

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.

[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 measurements were performed.

Particle size was measured directly using dynamic light scattering techniques. The particle size measurements are shown in Figures 1, 3, and 12. In Figure 12, the results from the dynamic light scattering measurements are plotted as particle size on the horizontal axis and light intensity on the vertical axis, calculated from the light intensity contributed by each particle.

As can be seen from Figure 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. When the charge ratio of oligoarginine R8/siRNA is 5 and the charge ratio of cationic liposome CLS to siRNA is 4, the particle size measurement result of the liposome complex (CLS/R8/siRNA) containing siRNA after compressing siRNA with the combination of cationic liposome and oligoarginine shows that the particle size is reduced to 108 nm, as shown in Figure 12. When the charge ratio of oligoarginine R8/siRNA is 5 and the charge ratio of cationic liposome 89W-CLS modified with ^89W Penetratin to siRNA is 4, the particle size of the liposome complex (89W-CLS/R8/siRNA) modified with ^89W Penetratin and containing siRNA is 105 nm, which is close to that of CLS/R8/siRNA. The maximum light intensity of the liposome complex modified with ^89W Penetratin and containing siRNA is lower than that of CLS/R8/siRNA, because the particle size distribution range of the former is wider.

When the charge ratio of oligoarginine R8/siRNA is 5, and the charge ratio of cationic liposome CLS or cationic liposome 89W-CLS modified with ^89W Penetratin to siRNA is 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° C., 5 V/cm electric field strength, and 0.4 cm electrode spacing using a zeta potential analyzer. The zeta potential measurement results are shown in FIG.

As can be seen from FIG. 13, the ζ potentials of the physical complex R8/siRNA, the liposome complex CLS/R8/siRNA, and the liposome complex modified with ^89W Penetratin were 22 mV, 43 mV, and 38 mV, respectively.

[Example 4]
(Characteristics of stability 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.

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

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, indicating that cationic liposome and oligoarginine can jointly compress siRNA to form a stable liposome formulation.

[Example 5]
[Characteristics of stability of drug-containing liposome complex 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 an 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 in the dark for 30 minutes and imaged under UV 302 nm conditions. The results are shown in FIG. 3D.

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. This indicates that cationic liposomes and oligoarginine can both compress siRNA to form a stable liposomal formulation.

[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.

i. Preparation of cell-penetrating peptide-PEG-phospholipid
DSPE-polyethylene glycol maleimide (DSPE-PEG ₃₄₀₀ -maleimide) (Laysan Bio, 146-123) was reacted with a penetratin derivative modified with a terminal cysteine ( ^89W Penetratin-Cys) in one step to obtain the cell-penetrating peptide-PEG-DSPE.

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

Here, 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, for example, products in which the glutamine at position 2 and/or the glutamine at position 8 and/or the asparagine at position 9 of penetratin are mutated to hydrophobic amino acids (see also Chinese patent application CN2017104534.7).

To facilitate reaction with a polyethylene glycol terminal group, a cysteine residue (C) may be added to the N-terminus or C-terminus of the cell-penetrating peptide ^89W Penetratin or other penetratin derivatives.

II. Formulation of surface-modified liposomal pharmaceutical preparations
In the total liposomal membrane material, the molar ratio of DOTAP:DOPE:cholesterol:(mPEG ₂₀₀₀ -DSPE and ^89W Penetraten-PEG ₃₄₀₀ -DSPE) is about 28.5:28.5:38:5.

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) were weighed. 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 materials, 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 rotary evaporated (Shanghai Shensheng Technology Co., Ltd., R210L) to remove chloroform, and a uniform dry lipid membrane was obtained.

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 liposomes (the order of the nucleus pore membranes is 200 nm, 100 nm and 50 nm, respectively) to obtain cationic liposomes (also referred to as CLS in the specification).

4 mg of oligoarginine R8 (Shanghai Xinhao 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 example, 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.

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.

For mPEG ₂₀₀₀ -DSPE and ^89W Penetrant-PEG ₃₄₀₀ -DSPE at a 5% molar ratio relative to the total liposome, mixtures were prepared at 0%, ₂₀ %, 40%, 60%, 80% and 100% molar ratios of ^89W Penetrant-PEG _{3400 -DSPE to mPEG 2000} -DSPE and ^89W Penetrant-PEG ₃₄₀₀ -DSPE, respectively, i.e., ⁰ %, 1%, 2%, 3%, 4% and 5% molar ratios of 89W Penetrant- _{PEG 3400} -DSPE relative to the total liposome membrane material, respectively.

The obtained CLS/R8/FAM-siRNA complex was mixed with a _mixture in which ^89W Penetrate-PEG ₃₄₀₀ -DSPE was present in a molar ratio of 0%, 20%, ⁴⁰ %, 60%, 80%, or 100% relative to mPEG 2000 -DSPE and 89W Penetrate-PEG ₃₄₀₀ -DSPE, respectively, and then incubated at 55° C. for 30 minutes to obtain liposome pharmaceutical preparations whose surfaces were modified with cell-penetrating peptides in proportions of 0%, 1%, 3%, 4%, or 5%, i.e., 89W-CLS/R8/FAM-siRNA.

[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, resuspended in DMEM complete medium, and inoculated into a 6-well plate at 5 x ¹⁰⁵ cells/well, 1 mL per well. After inoculation, the culture medium was changed once a day with fresh medium, and the cells were cultured for 2-3 days before the experiment.

After discarding the culture medium, the U87 cells were washed three times with sterile PBS, and 1 mL of the liposome pharmaceutical preparation (containing siRNA labeled with FAM) prepared in Example 6, which had its surface modified with different proportions of cell-penetrating peptides, was added to the serum-free DMEM medium, and incubated for 4 h 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, and counted for each sample after uniform spraying, and ¹⁰⁴ cells were taken for flow cytometry detection. The 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 4 (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 very significant difference between the two groups). In Figure 4, "0%, 1%, 2%, 3%, 4%, 5%" on the horizontal axis represent the liposome pharmaceutical preparations whose surfaces are modified with cell-penetrating peptides at ratios of 0%, 1%, 2%, 3%, 4%, and 5%, respectively, i.e., 89W-CLS/R8/siRNA.

As can be seen from Figure 4, when the molar ratio of ^89W Penetrant-PEG ₃₄₀₀ -DSPE to the total liposome membrane material was 2%, 3%, or 4%, the cellular uptake effect of the pharmaceutical formulation whose surface was modified with a cell-penetrating peptide at the above ratio was superior to that of the commercially available gene transfection reagent Lipo2000.

[Example 8]
Quantitative evaluation of cellular uptake of drug formulations prepared using different ratios of liposome 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 inoculated into a 6-well plate at 5 x ¹⁰⁵ cells/well, 1 mL per well. After inoculation, the culture medium was changed once a day with fresh medium, and the cells were cultured for 2-3 days before the experiment.

Following the method described in Example 6, a liposome pharmaceutical preparation (containing FAM-labeled siRNA) was prepared with the mixing ratio of liposome membrane materials shown in Table 2.

After discarding the culture medium for the cultured U87 cells, the cells were washed three times with sterile PBS. 1 mL of serum-free DMEM medium containing liposome pharmaceutical formulations Formula 1, Formula 2, and Formula 3 prepared by the respective formulations was added to each well and incubated at 37°C and 5% CO2 for ₄ h. 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, and 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 5 (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 very significant statistical difference between the two groups).

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.

[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 preparation (containing FAM-labeled siRNA) was prepared in which the molar ratio of the liposome membrane material components (i):(ii):(iii) was 3:3:4 and the compounding ratio of component (iv) was as shown in Table 3.

A liposome pharmaceutical preparation was obtained in which the molar ratio of component (iv) to the liposome membrane material was 1%, 5%, 8%, and 10%. The particle size of the liposome pharmaceutical preparation was measured. The particle size measurement results are shown in Figure 6. In Figure 6, "1%, 5%, 8%, and 10%" on the horizontal axis indicates liposome pharmaceutical preparations in which the molar ratio of component (iv) to the liposome membrane material was 1%, 5%, 8%, and 10%, respectively.

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 formulation 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.

[Example 10]
Quantitative assessment of cellular uptake for 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 inoculated into a 6-well plate at 5 x ¹⁰⁵ cells/well, 1 mL per well. After inoculation, the culture medium was changed once a day with fresh medium, and the cells were cultured for 2-3 days before the experiment.

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

Furthermore, following the method described in Example 6, a pharmaceutical preparation modified with 3% cell-penetrating peptide on the surface, i.e., 89W-CLS/R8/FAM-siRNA complex, was obtained.

After discarding the culture medium for 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 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 solution, sprayed evenly, and counted for each sample, and 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).

As can be seen from Figure 2, the pharmaceutical preparations prepared with the charge ratio 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 liposomal pharmaceutical preparation prepared when the charge ratio of cationic liposome/siRNA was 4 was optimal.

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

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

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

Various types of cells in good growth state were taken, digested, centrifuged, and then medium was added to adjust the cell concentration to 5 x ¹⁰⁵ cells/mL, the cells were blown with a pipette gun to uniformly disperse the cells, and 1.5 mL of the cell suspension was slowly added to the dish, and the cells were left to stand on the operation table for 20 minutes and 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 of floating cells and wall-attached cells is the same, and the cells have already formed cell clusters initially 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.

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, whereas 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 repeat experimental wells of Sample 2.

As can be seen from Figure 7, A549 and Caco-2 have good adhesion and tend to grow by adhering to the wall, without forming cell clusters, while Rb-1 are floating cells, but tend to gather into spheres and are very easy to dissolve during processing. In comparison, U87 and 293T have the best cell cluster formation ability, are roughly spherical in morphology, and are uniform in size, making them suitable for constructing cell cluster models for further exploration.

As can be seen from the results in Figure 8, the cell mass permeation ability of the polypeptides is significantly different, and the cell mass model constructed using the 35 mm Dimple NanoCulture Dish can distinguish the permeation 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 in which glutamic acid at position 8 and asparagine at position 9 are mutated to tryptophan. 289W is the product of penetratin in which glutamine at position 2, glutamine at position 8, and asparagine at position 9 are mutated to tryptophan. Derivatives of wild-type Penetratin have significantly improved cell mass permeation ability.

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

Similar to Example 6 above, GFP-siRNA liposome pharmaceutical preparations with different surface-modified cell-penetrating peptide ratios were prepared, in which the sense strand (5'-3') of the GFP-siRNA used was GCAUGUACCACGAGUCCAATT (sequence number 81) and the antisense strand (5'-3') was UUGGACUCGUGGUACAUGCTT (sequence number 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 the 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.

Furthermore, following the method described in Example 6, liposomal pharmaceutical preparations modified with 1%, 3%, and 5% of cell-penetrating peptide on the surface, i.e., 89W-CLS/R8/GFP-siRNA complexes, were obtained.

A serum-free DMEM solution containing 400 nM of a liposomal pharmaceutical preparation encapsulating GFP-siRNA was added to the 293T cell mass model, and the model was incubated at 37°C and 5% CO2 for ₄ h. The drug solution was then discarded, and fresh DMEM complete medium was added and incubated for another 24 h. The model was 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 in 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).

Similarly, the transfection effect of a liposomal pharmaceutical preparation containing 400 nM of GFP-siRNA on U87 cell masses was evaluated (also referred to as GFP-U87 cell masses in the text). The results are shown in Figure 10.

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

In FIG. 9, a GFP-239T cell mass was constructed using a 35 mm Dimple NanoCulture Dish. 293T cells are not tumor cells, and the higher the molar ratio of ^89W Penetrate-PEG-DSPE to the liposome membrane material, the higher the degree of penetration of the liposome drug into the cell mass. In FIG. 10, a GFP-U87 cell mass was constructed using a 35 mm Dimple NanoCulture Dish. U87 cells are brain glioma cells, and when the molar ratio of ^89W Penetrate-PEG-DSPE reaches 3% to 5% to the liposome membrane material, the liposome drug has a high ability to penetrate the cell mass.

[Example 12]
(Qualitative and quantitative evaluation of transfection effect on 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, and c-R12 were used as positively charged polyamino acids of different structural types, the charge ratio between the oligoarginines and siRNA was 5, and the charge ratio between CLS and LUC-siRNA was 4, and liposome pharmaceutical preparations containing different oligoarginines and surface-modified with cell-penetrating peptide at a ratio of 3% were prepared according to Example 6. The LUC-siRNA used has a sense strand (5'-3') of GCUGCACUCUGGCGACAUUTT (SEQ ID NO: 83) and an antisense strand (5'-3') of AAUGUCGCCAGAGUGCAGCTT (SEQ ID NO: 84).

U87 cells (purchased from ATCC) in good growth condition were inoculated into 48-well plates at 5 x ¹⁰⁴ cells/well, and the culture medium was changed once a day after inoculation, and the experiment was performed after 2-3 days of culture. After discarding the complete DMEM medium, the cells were washed three times with sterile PBS, and serum-free DMEM medium containing 400 nM LUC-siRNA-encapsulated liposome pharmaceutical preparations was added, and the cells were incubated for 4 hours at 37°C and 5% _CO2 conditions. Then, the drug solution was discarded, and the complete DMEM 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). Qualitative analysis was performed using the quantitative circle selection function of the region of interest (ROI) of the small animal bio-optical imaging system, and the results are shown in FIG.

This example was conducted 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.

[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 seeded into 12-well plates at a density of 5 x ^{104 cells} /well and cultured at ₃₇ °C and 5% CO2 until a monolayer of cells covered the bottom of the wells.

The culture medium was discarded, and the cells were washed three times with a sterile PBS buffer solution. Then, 400 μL of 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, and ^89W Penetratin-PEG ₃₄₀₀ -DSPE at a ratio of 3% surface modification) was added, and the cells were incubated in an incubator for 6 hours, after which the drug solution was discarded. Then, the cells were washed three times with a sterile PBS buffer solution, and 1 mL of complete medium was added and cultured for 18 hours.

Then, the cells were collected, washed with PBS, treated with EDTA-free trypsin for 1 minute, and finished with DMEM culture medium. Then, the cells were collected into a centrifuge tube, and the cells were double-stained with a cell apoptosis test kit (Nanjing Kaiji Bioscience and Technology Development Co., Ltd., KGA108) step 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 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).

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

[Example 14]
(Evaluation of cytotoxic activity of siRNA-encapsulated delivery carriers in bEnd.3 cells and U87 cells)
bEnd.3 cells (purchased from ATCC) and U87 cells with good logarithmic growth were taken and cultured in DMEM complete medium, and placed in the middle 60 wells of a 96-well flat-bottom plate at a concentration of 5 x ¹⁰³ cells/ ^cm2 , respectively, 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. Then, the culture medium was discarded, washed three times with sterile PBS buffer solution, and 200 μL of culture medium containing 400 nM siRNA delivery carrier was added, and incubated in an incubator for 6 h, after which the drug solution was discarded and washed three times with sterile PBS buffer solution. Then, complete medium was added and cultured for 12 h. Then, 20 μL of MTT solution (5 mg/mL) was added per well, and after further incubation for 4 h, the liquid was gently discarded, washed three times with PBS buffer solution, and then 150 μL of DMSO was added per well, shaken at low speed (50 rpm) on a shaker for 20 minutes, and the absorbance value of each well at OD490 nm was measured using a microplate reader (BioTek, PowerWave XS). Blank wells (wells were added with DMEM complete medium, MTT and DMSO only) and negative control wells (wells were added with cells, DMEM complete medium, MTT and DMSO only) were placed at the same time. All experiments were performed in triplicate. The results are shown in Figures 14C and 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).

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

[Example 15]
(Distribution of gene delivery carriers labeled with DiD cell membrane fluorescent probes in vivo after intranasal administration)
First, liposomes labeled with lipophilic fluorescent dye DiD were prepared and used to track liposomes. 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, DiD dye was added as described in the kit description, and the mixture was dissolved in chloroform to prepare CLS/R8/siRNA labeled with DiD. As in Example 6, the charge ratio of oligoarginine to siRNA was 5, the charge ratio of CLS to siRNA was 4, and the surface was modified with ^89W Penetratin-PEG ₃₄₀₀ -DSPE at a ratio of 3% to prepare a liposome pharmaceutical preparation 89W-CLS/R8/siRNA labeled with DiD.

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 (N.S.) group). Each mouse was administered approximately 30 μL of DiD-labeled nucleic acid delivery carrier via nasal administration (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 in each organ was detected using a small animal bioimaging system. The results are shown in Figure 15.

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 modification with a cell-penetrating peptide significantly enhances the tissue penetration ability of the delivery carrier, further enhancing its ability to enter the brain and increasing the amount of gene accumulation in the brain.

[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.

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, resuspended in DMEM complete medium, and inoculated into a 6-well plate at 5 x ¹⁰⁵ cells/well, 1 mL per well. After inoculation, the culture medium was changed once a day with fresh medium, and the cells were cultured for 2-3 days before the experiment.

After discarding the culture medium for 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 pharmaceutical preparations with different fluorescence 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 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, and counted for each sample after uniform spraying, and 10 ⁴ cells were taken for flow cytometry detection. In the experiment, cells incubated in blank serum-free DMEM medium were set as a 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 up: 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.

From Figure 16D, it can be seen that the pharmaceutical preparation formed by compressing siRNA with only R8 has a low amount of cytoplasmic entry, while the pharmaceutical preparation formed by compressing siRNA with CLS and R8 together can be successfully cytoplasmic (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 Figures 16B, 16C and 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 the cells together in the form of a complex, not taken up in a free form.

[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, a DiD-labeled liposome pharmaceutical formulation ^{89W-CLS/R8/siRNA was prepared, in which the charge ratio of oligoarginine to siRNA was 5, the charge ratio of CLS to siRNA was 4, and the surface was modified with 89W} Penetratin-PEG ₃₄₀₀ -DSPE at a ratio of 3%.

Logarithmic growth phase U87 cells were taken, the cell number was counted, and the cells were resuspended in PBS buffer solution. Each BALB/c nude mouse (Shanghai Xipur-Bikai Experimental Animal Co., Ltd., China) was inoculated with 6 x ¹⁰⁵ U87 cells (dispersed in 5 μL of PBS buffer solution). The nude mice were anesthetized with 7% chloral hydrate, fixed in a brain stereotaxic device, and the cells were inoculated into the striatum (0.6 mm in front of bregma, 1.8 mm to the right, 3 mm deep) with a microsyringe to establish a U87 in situ brain tumor model.

Nine mice with U87 in situ brain tumors were inoculated with U87 tumor cells for 18 days and randomly divided 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 administered intranasally 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.

As a result, compared with the CLS/R8/siRNA group, the 89W-CLS/R8/siRNA group had higher fluorescence intensity at the tumor site, indicating that modification with a cell-penetrating peptide significantly enhanced the tissue penetration ability of the delivery carrier, further enhancing its ability to enter the brain and increasing the amount of gene accumulation in the brain.

[Example 18]
Pharmacodynamic evaluation of siRNA-loaded delivery carriers against 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).

The U87 in situ brain tumor model was constructed according to the method described in Example 17. After inoculation with 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 with 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 very significant difference between the two groups).

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.

[Example 19]
(Evaluation of the ability of siRNA-loaded delivery carriers to promote apoptosis in U87 in situ brain tumor mouse model)
According to the method described in Example 18, liposomal pharmaceutical preparations were prepared, and U87 in situ brain tumor models were constructed and administered intranasally. Two days after the last administration (day 28), three mice from each group were randomly selected 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.

In the TUNEL-stained sections, the intensity of green fluorescence indicates the number of tumor cells that underwent apoptosis. The results showed that the Lipo2000/siRNA group, the CLS/R8/siRNA group, and the 89WP-CLS/R8/siRNA group were all able to induce apoptosis in tumor cells, and among them, the 89WP-CLS/R8/siRNA group had the highest fluorescence intensity, i.e., the highest apoptosis rate. This indicates that the delivery carrier designed in the present invention can exert an antitumor effect by inducing apoptosis of tumor cells in the body.

[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 preparations, 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 formulation (with or without 160 nM siRNA) was administered intranasally to 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 FIG. 20. The scale bar is 50 μm.

As a result, the nasal mucosa 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), whereas the saline group, siRNA group, CLS/R8/siRNA group, and 89WP-CLS/R8/siRNA group showed intact and orderly arranged nasal mucosa epithelial cell morphology. This is believed to indicate that the delivery carrier designed in this invention has no significant toxicity to nasal mucosa epithelial cells and is a safe carrier for delivery from the nose to the brain.

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

Claims (13)

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
wherein 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) and 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. The pharmaceutical formulation according to claim 1, wherein the drug molecule is selected from the group consisting of peptide drugs, antibodies, cytokines, hormones, antibiotics, nucleic acids, and combinations thereof. 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 in the pharmaceutical formulation is 1:1 to 30:1. The cationic liposome comprises, as membrane materials, (i) DOTAP; (ii) DOPE; (iii) cholesterol; (iv) mPEG ₂₀₀₀ -DSPE and ^89W Penetraten-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 molar ratio of ^89W Penetraten-PEG ₃₄₀₀ -DSPE to the membrane material (iv) is 20%-80%,
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. The pharmaceutical preparation according to any one of claims 1 to 4, which is a pharmaceutical preparation for nasal administration. 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
In the above, X ₁ , X ₂ and X ₃ are independently selected from naturally occurring amino acids, such as 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, such as α-aminobutyric acid, α-aminopentanoic acid and α-aminohexanoic acid. a drug delivery carrier selected from α-aminoheptanoic acid; The drug delivery carrier according to claim 6, characterized in that the cell-penetrating peptide is a lipophilic derivative of penetratin having any of the following amino acid sequences: RWIKIWFQNRRMKWKK (SEQ ID NO: 24), RQIKIWFWNRRMKWKK (SEQ ID NO: 25), RQIKIWFQWRRMKWKK (SEQ ID NO: 26), RWIKIWFWNRRMKWKK (SEQ ID NO: 27), RWIKIWFQWRRMKWKK (SEQ ID NO: 28), RQIKIWFWWRRMKWKK (SEQ ID NO: 29), RWIKIWFWWRRMKWKK (SEQ ID NO: 30). 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. 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 1 kDa to 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. The drug delivery carrier according to 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 molar ratio of the polyethylene glycol phospholipid conjugated with the cell-penetrating peptide is 20%-80% relative to the membrane material (iv). The drug delivery carrier according to any one of claims 6 to 10, wherein the liposome comprises, as membrane materials, (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 molar ratio of the cell penetrating peptide-PEG ₃₄₀₀ -DSPE to the membrane material (iv) is 20%-80%. A method for preparing a 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. 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 consisting 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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