WO2020224475A1 - 药物递送载体以及使用其的药物制剂 - Google Patents

药物递送载体以及使用其的药物制剂 Download PDF

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WO2020224475A1
WO2020224475A1 PCT/CN2020/087279 CN2020087279W WO2020224475A1 WO 2020224475 A1 WO2020224475 A1 WO 2020224475A1 CN 2020087279 W CN2020087279 W CN 2020087279W WO 2020224475 A1 WO2020224475 A1 WO 2020224475A1
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drug
sirna
liposome
membrane material
dspe
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English (en)
French (fr)
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魏刚
胡杨
江宽
王勇
越仮良树
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Fudan University
JSR Corp
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Fudan University
JSR Corp
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Priority to CN202080022099.9A priority Critical patent/CN114007653B/zh
Priority to JP2021565727A priority patent/JP7645601B2/ja
Publication of WO2020224475A1 publication Critical patent/WO2020224475A1/zh
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/02—Nasal agents, e.g. decongestants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids

Definitions

  • the present invention relates to a drug delivery vehicle, a method for preparing a drug preparation using the drug delivery vehicle, and a drug preparation prepared thereby.
  • the drug delivery vehicle of the present invention comprises a liposome modified with a penetrating peptide and a polymer, wherein the polymer is a positively charged polyamino acid.
  • the drug formulation prepared by using the drug delivery carrier of the present invention is a liposome complex, which is a liposome modified by a physical complex formed by a positively charged polyamino acid and one or more drug molecules and a penetrating peptide Prepared by mixing, the pharmaceutical preparation can effectively transfect cells, especially promote the absorption of the drug into the brain through the nasal cavity, and improve the efficacy of the drug.
  • Cell-penetrating peptide is a short positively charged peptide under physiological conditions that can covalently or non-covalently link drug molecules such as genes, polypeptides, and proteins, and deliver the drug molecules into cells or
  • the drug-carrying molecule penetrates the biological membrane barrier.
  • the ability of a single penetrating peptide to carry drug molecules is very limited, which results in a complex formed by the penetrating peptide and the drug molecule with a large particle size and poor stability.
  • carriers such as liposomes for delivering drug molecules (eg, genes, polypeptides, and proteins) in the prior art have problems such as low delivery efficiency and prone to tissue toxicity.
  • drug molecules eg, genes, polypeptides, and proteins
  • problems such as low delivery efficiency and prone to tissue toxicity.
  • how to use liposomes to deliver drug molecules more efficiently and make the treatment and/or prevention of diseases more specific and efficient is still a huge challenge.
  • researchers mainly improve the stability of liposomes by adding various auxiliary components to liposomes, developing and using new lipid materials, modifying the surface of liposomes, designing and using new preparation techniques, etc. Targeting and drug delivery efficiency.
  • a new drug delivery carrier using penetrating peptides which comprises a liposome modified with penetrating peptides and a polymer, wherein the polymer is a positively charged polyamino acid,
  • the polymer is used for mixing with one or more drugs to form a polymer-drug complex
  • the transmembrane peptide modified liposome is used for mixing with the polymer-drug complex to form a polymer-drug complex
  • the complex is the liposomal complex.
  • the penetrating peptide in the drug delivery vehicle of the present invention is penetratin or a lipophilic derivative of penetratin having the following amino acid sequence:
  • X 1 , X 2 and X 3 are independently selected from the amino acids glutamine (Q), asparagine (N), alanine (A), valine (valine , V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), tryptophan (W) , Methionine (M) and non-natural sources of amino acids ⁇ -aminobutyric acid ( ⁇ -aminobutyric acid), ⁇ -aminopentanoic acid ( ⁇ -aminopentanoic acid), ⁇ -aminohexanoic acid ( ⁇ -aminohexanoic acid) ), ⁇ -aminoheptanoic acid.
  • some penetrating peptides in the drug delivery vehicle of the present invention are based on the glutamine at position 2 (Q) and/or glutamine at position 8 (Q) and/or asparagine at position 9 (N) of Penetratin.
  • Penetratin derivatives mutated to hydrophobic amino acids.
  • the positively charged polyamino acid in the drug delivery vehicle of the present invention is, for example, a positively charged polyamino acid with a degree of polymerization of 2-50.
  • the spatial structure includes linear and circular, and the configuration of amino acid residues includes L-type and D-type; for example, arginine and/or lysine with a degree of polymerization of 6-12.
  • the positively charged polyamino acid optionally between the positively charged amino acid residues, 1-20 (for example, 1-6) amino acid residues that are not charged under physiological conditions are intervened
  • the positively charged polyamino acid is polyarginine with a degree of polymerization of 6-12, wherein the spatial structure of the polyarginine includes linear and cyclic structures. Types include L type and D type.
  • the penetrating peptide modified liposome in the drug delivery vehicle of the present invention contains the following membrane materials:
  • Cationic lipids including but not limited to: 1,2-di-O-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) and 2,3-dioleoyloxy-N-[2(spermine formamide)ethyl]-N,N-dimethyl-1- Propane ammonium trifluoroacetate (DOSPA) and
  • Non-cationic lipids including but not limited to: 1,2-bis-(9Z-octadecanoyl)-sn-glyceryl-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn- Glyceryl-3-phosphocholine (DOPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine Alkali (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 2-dioil Acyl-sn-glycerol-3-phosphate-(1'-rac-glycerol) (DOPG) and combinations thereof.
  • DOPE 1,2-bis-(9Z-octadecanoyl)-sn-glyceryl-3-phosphoethanolamine
  • Pegylated (PEGylated) phospholipids and polyethylene glycol phospholipids conjugated to CPP such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and sphingomyelin, preferably
  • the PEGylated phospholipid is, for example, polyethylene glycol-distearoylphosphatidylethanolamine (PEG-DSPE) and its derivative methoxy-polyethylene glycol-distearoylphosphatidylethanolamine (mPEG-DSPE),
  • the polyethylene glycol phospholipid conjugated with CPP is, for example, Penetratin/Penetratin derivative-PEG-DSPE.
  • the molar ratio of the membrane material (i):(ii):(iii):(iv) is about 20-40:20-40:20-40:1-20, and is conjugated with CPP
  • the polyethylene glycol phospholipid accounts for about 20%-80% of the molar ratio of the membrane material (iv).
  • the molar ratio of the membrane material (i):(ii):(iii):(iv) is about 27.0-31.6:27.0-31.6:31.6-39.6:1-10, and is shared with CPP
  • the conjugated polyethylene glycol phospholipid accounts for about 20%-80% of the molar ratio of the membrane material (iv).
  • the cationic liposome in the drug delivery vehicle of the present invention comprises the following membrane materials: (i) DOTAP; (ii) DOPE; (iii) cholesterol; (iv) mPEG 2000 -DSPE and 89W Penetratin -PEG 3400 -DSPE, and the molar ratio of membrane material (i):(ii):(iii):(iv) is about 20-40:20-40:20-40:1-20, and 89W Penetratin-PEG 3400- DSPE occupies about 20%-80% mole ratio in the membrane material (iv).
  • the molar ratio of the membrane material (i):(ii):(iii):(iv) is about 27.0-31.6:27.0-31.6:31.6-39.6:1-10, and 89W Penetratin-PEG 3400 -DSPE occupies about 20%-80% mole ratio in the membrane material (iv).
  • the cationic liposome in the drug delivery vehicle of the present invention is composed of the following membrane materials: (i) DOTAP; (ii) DOPE; (iii) cholesterol; (iv) mPEG 2000 -DSPE and 89W Penetratin- PEG 3400- DSPE, and the molar ratio of membrane material (i):(ii):(iii):(iv) is about 28.5:28.5:38:5, 89W Penetratin-PEG 3400- DSPE accounts for membrane material (iv) The molar ratio of about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%.
  • the present invention provides a pharmaceutical preparation, which is a pharmaceutical preparation prepared by using the drug delivery vehicle of the present invention, wherein the positively charged polyamino acid contained in the drug delivery vehicle and one or A variety of drug molecules form a physical complex, and the physical complex is mixed with the cationic liposome modified with the penetrating peptide on the surface to form a pharmaceutical preparation in the form of a liposome complex.
  • peptide drugs e.g., octreotide
  • antibodies e.g., etanercept, adalimumab
  • rituximab rituximab
  • pembrolizumab pembrolizumab
  • ranibizumab ranibizumab
  • nivolumab nivolumab
  • cytokines hormones, antibiotics (for example, the chemotherapy drug adriamycin (doxorubicin), daunorubicin (daunorubicin), epirubicin (epirubicin), actinomycin (actinomycin) and vancomycin (vancomycin)), nucleic acids, and combinations thereof.
  • doxorubicin doxorubicin
  • daunorubicin daunorubicin
  • epirubicin epirubicin
  • actinomycin actinomycin
  • vancomycin vancomycin
  • the drug molecules delivered by the drug formulation are plasmid DNA, RNA such as small interfering RNA (siRNA), sense RNA, antisense oligonucleotides (ASO), aptamers, ribozymes And other nucleic acids.
  • RNA such as small interfering RNA (siRNA), sense RNA, antisense oligonucleotides (ASO), aptamers, ribozymes And other nucleic acids.
  • the drug molecule delivered by the drug formulation is a combination of a nucleic acid and a drug (such as doxorubicin) that can be embedded in the genome of an organism.
  • the molar ratio of drug molecules (for example, chemotherapeutics) to cationic lipids (for example, DOTAP) in the pharmaceutical formulation is about 0.1-10, preferably about 0.1-5, and more preferably about 0.1-2.
  • the drug-loading amount of the drug formulation for drug molecules (for example, chemotherapeutics) is: the drug molecules (for example, chemotherapeutics) account for about 1%-30% (w/w) of the drug formulation, preferably About 1%-25% (w/w), more preferably about 3%-20% (w/w).
  • the charge ratio of the positively charged polyamino acid (for example, oligoarginine) to the drug molecule to be delivered in the pharmaceutical preparation is between 1:1 and 30:1, preferably 5:1 ;
  • the charge ratio of cationic liposomes to drug molecules ranges from 1:1 to 30:1, preferably 4:1.
  • the particle size of the pharmaceutical preparation of the present invention ranges from 50 nm to 300 nm, preferably 80 nm to 150 nm, and has good stability.
  • a method for preparing the pharmaceutical preparation of the present invention includes (a) preparing liposomes modified with penetrating peptides on the surface; (b) preparing positively charged polyamino acids and drug molecules to be delivered Physical complex; (c) mixing the liposomes obtained in step (a) with the transmembrane peptide on the surface and the physical complex obtained in step (b) at a certain molar ratio or charge ratio.
  • the preparation method of the pharmaceutical preparation of the present invention includes (a) mixing liposome membrane materials (i), (ii) and (iii) to prepare blank liposomes; (b) preparing positively charged liposomes (C) Mix the blank liposome obtained in step (a) with the physical complex obtained in step (b) at a certain molar ratio or charge ratio; (d) ) Mix and incubate the liposome obtained in step (c) with the membrane material (iv) to obtain the pharmaceutical preparation of the present invention.
  • the pharmaceutical preparations prepared using the drug delivery vehicle of the present invention can, for example, pass through the oral cavity, nasal cavity, eyes, respiratory tract, digestive tract, reproductive tract, local implantation, injection or infusion (via epidural, intraarterial, intraarticular, intrasaccular , Intracardiac, intracerebroventricular, intracranial, intradermal, intramuscular, intraorbital, intraocular, intraperitoneal, intraspine, intrasternal, intrathecal, intravenous, subarachnoid, subcapsular, subcutaneous, trachea, rectum, Sublingual and other routes) for the treatment and/or prevention of diseases.
  • injection or infusion via epidural, intraarterial, intraarticular, intrasaccular , Intracardiac, intracerebroventricular, intracranial, intradermal, intramuscular, intraorbital, intraocular, intraperitoneal, intraspine, intrasternal, intrathecal, intravenous, subarachnoid, subcapsular,
  • Figure 1 Shows the results of agarose gel electrophoresis of each complex of R8 (8-polyarginine) and siRNA with different charge ratios (expressed as the ratio of positive/negative charge) and/or CLS and siRNA with different charge ratios Particle size results.
  • Panels A and B in Figure 1 show the results of agarose gel electrophoresis and particle size of R8/siRNA complexes with different charge ratios of R8/siRNA, respectively;
  • Panels C and D show the different charge ratios of CLS/siRNA, respectively The results of agarose gel electrophoresis and particle size results of the CLS/siRNA complexes;
  • Panels E and F respectively show the CLS/R8/siRNA lipids with different charge ratios when the charge ratio of R8/siRNA is 1:1 Results of agarose gel electrophoresis and particle size results of the plastid complex.
  • Figure 2 shows the uptake of CLS/R8/siRNA liposome complexes prepared with different charge ratios of CLS and siRNA when the charge ratio of R8 to siRNA is 1:1.
  • Figure 3 shows the results of electrophoresis, particle size and stability of the CLS/siRNA/R8 complex.
  • Panels A and B in Figure 3 respectively show the results of agarose gel electrophoresis and particle size results of liposome complexes with different charge ratios of R8 and siRNA when the charge ratio of CLS to siRNA is 4:1;
  • Panels C and D show the precipitation of liposome complexes with different charge ratios of R8 and siRNA after high-speed centrifugation and the agarose coagulation of the supernatant after centrifugation when the charge ratio of CLS to siRNA is 4:1.
  • Figure 4 shows the quantitative evaluation results of cellular uptake of pharmaceutical preparations modified with different ratios of penetrating peptides on their surfaces.
  • the Lipo2000 group is a non-covalent complex of the commercially available gene transfection reagent, cationic liposome Lipofectamine 2000 and siRNA.
  • Figure 5 shows the uptake of the pharmaceutical preparations prepared by using the liposome membrane materials of different formulations shown in Table 2 of Example 8 by cells.
  • Figure 6 shows the particle size of liposome pharmaceutical preparations in which component (iv) accounts for 1%, 5%, 8%, and 10% of the liposome membrane material.
  • Figure 7 Shows the morphological results of using 35mm Dimple NanoCulture Dish to culture different cell spheroids.
  • 35mm Dimple NanoCulture Dish is a cell spheroid culture plate of Japan JSR Corporation.
  • Figure 8 shows the evaluation results of the penetration ability of the cell spheres of the penetrating peptide modified on the liposome surface.
  • Figure 9 Shows the transfection effect of GFP-siRNA-containing pharmaceutical preparations on 293T cell spheres.
  • Figure 10 Shows the transfection effect of GFP-siRNA-containing pharmaceutical preparations on U87 cell spheres.
  • Figure 11 Shows the qualitative and quantitative evaluation results of the transfection effect of different oligoarginine pharmaceutical preparations on LUC-U87 cells.
  • Figure 12 shows the particle size of the siRNA-encapsulated pharmaceutical formulation.
  • Figure 13 Shows the potential of a pharmaceutical formulation containing siRNA.
  • Figure 14 shows the evaluation results of the pro-apoptotic effect and cytotoxicity evaluation of the pharmaceutical preparations encapsulating siRNA on bEnd.3 cells and U87 cells.
  • Figure 15 shows the in vivo distribution of the drug preparation labeled with cell membrane fluorescent probe DiD after nasal administration.
  • Figure 16 shows the results of flow cytometry detection of FAM-siRNA delivered into cells by a carrier labeled with a fluorescent probe on the cell membrane DiD.
  • Figure 17 shows the distribution of the gene delivery vector labeled with DiD cell membrane fluorescent probe in U87 in situ brain tumor model mice after nasal administration.
  • Figure 18 shows the results of the pharmacodynamic evaluation of anti-U87 brain in situ tumors with siRNA-carrying delivery vector.
  • the Lipo2000/siRNA group is the non-covalent relationship between Lipofectamine 2000 and siRNA, a commercially available gene transfection reagent. Complex.
  • Figure 19 Shows the evaluation results of the in vivo pro-apoptosis ability of the delivery vector containing siRNA in U87 in situ brain tumor model mice.
  • the Lipo2000/siRNA group is a commercial gene transfection reagent, cationic liposome Lipofectamine 2000 and siRNA Of non-covalent complexes.
  • Figure 20 shows the results of in vivo nasal mucosal toxicity evaluation of delivery vehicles containing siRNA.
  • the Lipo2000/siRNA group is a non-covalent complex of Lipofectamine 2000 and siRNA, sodium deoxycholate, a commercially available gene transfection reagent cationic liposome
  • the sodium deoxycholate of the group is a commercial product of Dalian Meilun Biotechnology Co., Ltd.
  • the present invention provides a novel drug delivery vehicle, which comprises a liposome modified by penetrating peptide and a polymer, wherein the polymer is a positively charged polyamino acid.
  • the drug delivery carrier of the present invention can assemble with drug molecules to form a complex, can compact the drug molecules and encapsulate the drug molecules in liposomes, thereby utilizing liposome surface-modified penetrating peptides with membrane penetrating function Help the drug penetrate the biological membrane barrier in the subject.
  • the drug delivery carrier of the present invention has strong drug-carrying, drug-delivery, and tissue-penetrating capabilities, and has low tissue toxicity. It can efficiently deliver drug molecules to the target site in the subject through a variety of administration routes, and overcome the effects of stress.
  • the subject s biomembrane barrier causes the technical problem that the drug is difficult to reach the target site, which enhances the effectiveness of the drug in treating diseases and has good biological safety.
  • the invention also provides a pharmaceutical preparation prepared by using the drug delivery vehicle.
  • the term "subject" refers to a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and large animals). mouse). In particular, the subject is a human.
  • the term "about” is intended to be an adjustment of ⁇ 10% of the specified value.
  • the term “about 5%” is intended to encompass the range of 4.5% to 5.5%.
  • liposome refers to an artificially prepared vesicle composed of a lipid bilayer.
  • the type of liposome modified by the penetrating peptide in the present invention is not limited, and may be any liposome capable of forming lipid vesicles and encapsulating drugs.
  • the surface-modified liposomes with penetrating peptides of the present invention comprise the following membrane materials:
  • Liposomes contain one or more cationic lipids.
  • cationic lipid is a lipid that has a net positive charge at a selected pH (e.g., physiological pH).
  • 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 WO 2010/053572 and WO 2012/170930, both of which are incorporated herein by reference. .
  • the cationic lipid contained in the liposome there is no particular restriction on the cationic lipid contained in the liposome, as long as it is a lipid with a net positive charge at a selected pH (for example, physiological pH), it can be used, such as but not limited to: 1,2-di-O -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-dimethyl Ammonium propane; Dioctadecyl dimethyl ammonium chloride (DODAC), 1,2-dimyristoyloxypropyl-1,3-dimethylhydroxyethyl ammonium (DMRIE) and 2,3
  • Non-cationic lipid refers to any neutral, zwitterionic or anionic lipid.
  • anionic lipid refers to any of many lipid substances that carry a net negative charge at a selected pH, such as physiological pH.
  • non-cationic lipid contained in the liposome there is no particular limitation on the non-cationic lipid contained in the liposome, and any neutral, zwitterionic or anionic lipid can be used, such as but not limited to: 1,2-bis-(9Z-octadecanoyl)-sn- Glyceryl-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glyceryl-3-phosphocholine (DOPC), 1,2-distearoyl-sn-glycero-3-phosphocholine Alkali (DSPC), 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2- Dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 2-dioleoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol)
  • Cholesterol is an important component of mammalian cell membranes, accounting for more than 20% of cell membrane lipids.
  • the term "cholesterol” is used in the broadest sense herein and encompasses cholesterol derivatives. Studies have shown that when the temperature is high, cholesterol can prevent the disorder of the cell membrane bilayer; when the temperature is low, cholesterol can interfere with the order of the cell membrane bilayer, prevent the formation of liquid crystals, and maintain the fluidity of the cell membrane bilayer.
  • the cationic liposome of the present invention also contains cholesterol.
  • Liposomes contain PEGylated phospholipids and polyethylene glycol phospholipids conjugated with CPP.
  • PEGylated phospholipids do not include polyethylene glycol phospholipids to which CPP is attached.
  • PEGylated phospholipids are formed by covalently bonding phospholipids to one or more polyethylene glycol molecules (PEG).
  • PEGylated phospholipids include, but are not limited to, polyethylene glycol chains up to 10kDa in length, for example, polyethylene glycol chains of 1kDa, 2kDa, 3kDa, 4kDa, 5kDa, 6kDa, 7kDa, 8kDa, 9kDa, 10kDa, and any value in between.
  • Alcohol chain which is covalently bonded to phospholipids.
  • the phospholipids may be synthetic, semi-synthetic or natural phospholipids, such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and sphingomyelin.
  • the PEGylated phospholipid is, for example, polyethylene glycol-distearoylphosphatidylethanolamine (PEG-DSPE) and its derivative methoxy-polyethylene glycol-distearoylphosphatidylethanolamine (mPEG-DSPE), where the molecular weight of PEG is any value between 1kDa and 10kDa.
  • Polyethylene glycol phospholipids conjugated with CPP are formed by covalently bonding CPP to polyethylene glycol phospholipids.
  • the CPP used in the present invention not only includes the wild-type polypeptide penetratin (amino acid sequence: RQIKIWFQNRRMKWKK (SEQ ID NO: 2)), but also includes a series of lipophilic derivatives thereof, for example, combining Penetratin with glutamine (Q) at position 2 / Or Penetratin derivatives in which glutamine (Q) at position 8 and/or asparagine (N) at position 9 are mutated into hydrophobic amino acids.
  • the lipophilic derivatives of penetratin are those disclosed in Chinese invention patent application CN201710414334.7, and the derivatives have been shown to promote the passage of drugs through many eyes after intraconjunctival eye drops are administered.
  • the partial absorption barrier (cornea, conjunctiva, sclera, etc.) enters the eye, and even delivers the genes, peptides, and proteins and other biological macromolecule drugs it carries to the retina at the back of the eye.
  • the amino acid sequence of the lipophilic derivative of Penetratin disclosed in the Chinese invention patent application CN201710414334.7 is quoted as follows:
  • CPP is a compound that is linked to a part of the lipid bilayer of liposomes by covalent bonding with polyethylene glycol phospholipids.
  • the term "as part of the lipid bilayer of liposomes" is intended to indicate the fact that the phospholipids in CPP-PEG-phospholipids are integrated into the lipid bilayer.
  • the phospholipids may be synthetic, semi-synthetic or natural phospholipids, such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and sphingomyelin.
  • the polyethylene glycol chains in CPP-PEG-phospholipids include, but are not limited to, polyethylene glycol chains up to 10kDa in length, for example, 1kDa, 2kDa, 3kDa, 4kDa, 5kDa, 6kDa, 7kDa, 8kDa, 9kDa, 10kDa, and their Any value between the polyethylene glycol chain, the two ends of which are covalently bonded to phospholipids and CPP.
  • the polyethylene glycol phospholipid conjugated with CPP is, for example, a Penetratin/Penetratin derivative-PEG-DSPE, wherein the molecular weight of PEG is any value between 1kDa and 10kDa.
  • the present invention modifies CPP on the surface of cationic liposomes, aiming to enhance the penetration ability of drug delivery vehicles in tissues, thereby helping liposomes carrying more drug molecules to reach the target site, thereby exerting disease treatment and/ Or preventive effect.
  • the polyethylene glycol chain length in the PEGylated phospholipid and the polyethylene glycol phospholipid conjugated with CPP in the liposome membrane material (iv) is not equal, and the polyethylene glycol conjugated with CPP
  • the polyethylene glycol chain in the phospholipid is longer than the polyethylene glycol chain in the PEGylated phospholipid, for example, between 0.5 kDa and 5 kDa, preferably between 0.75 kDa and 4 kDa, and more preferably 1 kDa. -Any value between 2kDa.
  • the polyethylene glycol chain length of the PEGylated phospholipid in the liposome membrane material (iv) is about 1kDa, 2kDa, 3kDa, respectively, and the polyethylene glycol chain of the polyethylene glycol phospholipid conjugated with CPP The lengths are about 2.5kDa, 3.5kDa, and 4.5kDa respectively.
  • the membrane material of the cationic liposomes surface-modified with penetrating peptides of the present invention has a molar ratio of (i):(ii):(iii):(iv) of about 20-40:20-40 : 20-40:1-20, and the polyethylene glycol phospholipid conjugated with CPP accounts for about 20%-80% mole ratio in the membrane material (iv), corresponding to about about 20% to 80% in the total liposome membrane material 2%-8% molar ratio.
  • the molar ratio of the membrane material (i):(ii):(iii):(iv) is about 27.0-31.6:27.0-31.6:31.6-39.6:1-10, and is conjugated with CPP
  • the polyethylene glycol phospholipid accounts for about 20% to 80% mole ratio in the membrane material (iv), corresponding to about 2% to 8% mole ratio in the total liposome membrane material.
  • the cationic liposome in the drug delivery vehicle of the present invention comprises the following membrane materials: (i) DOTAP; (ii) DOPE; (iii) cholesterol; (iv) PEG-DSPE (for example, mPEG 2000 -DSPE) and Penetratin/Penetratin derivative-PEG-DSPE (for example, 89W Penetratin-PEG 3400 -DSPE), and the molar ratio of the membrane material (i):(ii):(iii):(iv) is about 20 -40:20-40:20-40:1-20, and Penetratin/Penetratin derivative-PEG-DSPE (for example, 89W Penetratin-PEG 3400 -DSPE) accounts for about 20%-80% of the membrane material (iv) The molar ratio of.
  • the molar ratio of the membrane material (i):(ii):(iii):(iv) is about 27.0-31.6:27.0-31.6:31.6-39.6:1-10, and the Penetratin/Penetratin derivative -PEG-DSPE (for example, 89W Penetratin-PEG 3400 -DSPE) accounts for about 20%-80% of the molar ratio of the membrane material (iv).
  • the Penetratin/Penetratin derivative -PEG-DSPE for example, 89W Penetratin-PEG 3400 -DSPE
  • the cationic liposome in the drug delivery vehicle of the present invention is composed of the following membrane materials: (i) DOTAP; (ii) DOPE; (iii) cholesterol; (iv) mPEG 2000 -DSPE and 89W Penetratin- PEG 3400- DSPE, and the molar ratio of membrane material (i):(ii):(iii):(iv) is about 28.5:28.5:38:5, 89W Penetratin-PEG 3400- DSPE accounts for membrane material (iv) The molar ratio of about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%.
  • the positively charged polyamino acid contained in the drug delivery vehicle of the present invention is used to compress drug molecules.
  • the positively charged polyamino acid is a polyamino acid having a net positive charge at a selected pH (e.g., physiological pH).
  • a selected pH e.g., physiological pH
  • There is no particular limitation on the degree of polymerization of amino acid residues in the polyamino acid for example, a positively charged polyamino acid with a degree of polymerization of 2-50 (ie, a positively charged polyamino acid has 2-50 amino acid residues)
  • arginine and/or lysine with a degree of polymerization of 6-12, and optionally 1-20 (e.g. 1--20) are inserted between the positively charged polyamino acids with a degree of polymerization of 2-50.
  • Uncharged amino acid residues under physiological conditions for example, a positively charged polyamino acid with a degree of polymerization of 2-50 (ie, a positively charged polyamino acid has 2-50 amino acid residues)
  • the positively charged polyamino acid can be linear or circular, and the configuration of amino acid residues includes L-type and D-type.
  • the positively charged polyamino acid is polyarginine with a degree of polymerization of 6-12, wherein the spatial structure of the polyarginine includes linear and circular, and the configuration of polyarginine Including L type and D type.
  • the positively charged polyamino acid is oligo-arginine, including linear polypeptides, such as 6 polyarginine (amino acid sequence is RRRRRR, R6 (SEQ ID NO: 85) ), 8-polyarginine (amino acid sequence is RRRRRRRR, R8 (SEQ ID NO: 86)), 10-polyarginine (amino acid sequence is RRRRRRRRRR, R10 (SEQ ID NO: 87)), 12 polyarginine ( The amino acid sequence is RRRRRRRRRRRRRRR, R12 (SEQ ID NO: 88)), and cyclized polypeptides, such as cyclized 6 polyarginine (c-R6), cyclized 8 polyarginine (c-R8), Cyclized 10 polyarginine (c-R10), cyclized 12 polyarginine (c-R12), and polypeptides of different configurations, such as L-type oligoarginine and D-type oligoarginine Acid
  • the following describes a method for preparing a pharmaceutical preparation using the drug delivery vehicle of the present invention, and describes the characteristics of the pharmaceutical preparation.
  • the drug delivery carrier of the present invention it is possible to prepare a drug formulation with CPP modified on the surface of the cationic liposome and with strong penetrating ability in the tissue.
  • the preparation method of the pharmaceutical preparation of the present invention is:
  • the liposome is extruded using a micro extruder (the order of passing through the nuclear pore membrane is 200 nm, 100 nm and 50 nm).
  • the preparation method of the pharmaceutical preparation of the present invention is:
  • the liposome is extruded using a micro extruder (the order of passing through the nuclear pore membrane is 200 nm, 100 nm and 50 nm).
  • the drug molecule to be delivered is not particularly limited.
  • the drug molecule includes a wide range of compounds delivered to the subject, including but not limited to: nucleic acid; anti-infective drugs such as antibiotics and antiviral drugs; analgesics and analgesics Drug combinations; Appetite suppressants; Deworming drugs; Antiarthritis drugs; Antiasthmatic drugs; Anticonvulsants; Antidepressants; Antidiabetics; Antidiarrheals; Antihistamines; Antiinflammatory drugs; Antimigraine preparations; Nausea drugs; antitumor drugs; anti-tremor paralysis drugs; antipruritic drugs; antipsychotics; antipyretics; antispasmodics; anticholinergics; sympathomimetic drugs; xanthine derivatives; cardiovascular agents including potassium channels Blockers, calcium channel blockers, ⁇ -blockers, ⁇ -blockers and antiarrhythmics; antihypertensives; diuretics and antidiuretics; vasodilators including
  • the drug molecules delivered by the pharmaceutical formulations of the present invention are, for example, peptide drugs (e.g., octreotide), antibodies (e.g., etanercept, adalimumab, ritol Rituximab (rituximab), pembrolizumab (pembrolizumab), ranibizumab (ranibizumab) and nivolumab (nivolumab)), cytokines, hormones, antibiotics (for example, the chemotherapy drug doxorubicin (doxorubicin), Daunorubicin (daunorubicin), epirubicin (epirubicin), actinomycin (actinomycin) and vancomycin (vancomycin)), nucleic acid, etc.
  • peptide drugs e.g., octreotide
  • antibodies e.g., etanercept, adalimumab, ritol Ritux
  • the drug molecules delivered by the drug formulation are plasmid DNA, RNA such as small interfering RNA (siRNA), sense RNA, antisense oligonucleotides (ASO), aptamers, ribozymes And other nucleic acids.
  • RNA such as small interfering RNA (siRNA), sense RNA, antisense oligonucleotides (ASO), aptamers, ribozymes And other nucleic acids.
  • Plasmid DNA is a gene expression system containing a DNA sequence encoding, for example, selected from interleukin-2, interleukin-4, interleukin-7, interleukin-12, and interleukin-15 , Interferon- ⁇ , interferon- ⁇ , interferon- ⁇ , colony stimulating factor, granulocyte-macrophage stimulating factor, anti-angiogenesis agent, tumor suppressor gene, thymidine kinase, eNOS, iNOS, p53, p16, TNF- ⁇ , Fas-ligand, mutated oncogene, tumor antigen, viral antigen or bacterial antigen.
  • Plasmid DNA may also encode shRNA molecules designed to inhibit proteins involved in the growth or maintenance of tumor cells or other hyperproliferative cells. Plasmid DNA can simultaneously encode a therapeutic protein and one or more shRNAs.
  • the nucleic acid in the pharmaceutical preparations of the present invention can also be a mixture of plasmid DNA and artificially synthesized RNA containing sense RNA, antisense oligonucleotides or ribozymes.
  • the molar ratio of drug molecules (e.g., chemotherapeutics) to cationic lipids (e.g., DOTAP) in the pharmaceutical formulation of the present invention is about 0.1-10, for example, about 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, preferably about 0.1-5, more preferably about 0.1-2.
  • the drug-loading amount of the drug formulation for drug molecules is: the drug molecules (for example, chemotherapeutics) account for about 1%-30% (w/w) of the drug 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 preparations of the present invention can be passed through the oral cavity, nasal cavity, eyes, respiratory tract, digestive tract, reproductive tract, local implantation, injection or infusion (via epidural, intraarterial, intraarticular, intrasaccular, intracardiac, intraventricular, Intracranial, intradermal, intramuscular, intraorbital, intraocular, intraperitoneal, intraspine, intrasternal, intrathecal, intravenous, subarachnoid, subcapsular, subcutaneous, tracheal, rectal, sublingual and other routes) administration.
  • Medical equipment known in the art can be used to administer the pharmaceutical formulation of the present invention.
  • a needle-free scalp hypodermic injection device such as that disclosed in U.S. Patent No. 5,399,163, may be used to administer the pharmaceutical formulation of the present invention.
  • the drug delivery vector of the present invention is used to prepare a pharmaceutical preparation for delivering nucleic acid, thereby exerting a gene therapy effect.
  • nucleic acid drugs have entered or completed clinical trials in the world, of which 65% are used for cancer treatment, and 181 clinical trials of gene drugs play a therapeutic effect by inhibiting tumor growth (Ginn SL et al., Genetherapy clinical trials worldwide to 2017: An update, Journal of Gene Medicine, 2018, 20(5): e3015). Based on this, the art hopes to be able to achieve the treatment of diseases (for example, tumor diseases) through the delivery of nucleic acid pharmaceutical preparations.
  • Carriers for delivering nucleic acids are one of the key technologies for gene therapy, which can protect the delivered nucleic acids from degradation by endogenous nucleases, thus making the in vivo application of gene therapy possible.
  • the nucleic acid vectors currently used in gene therapy are mainly viral vectors and non-viral vectors.
  • the transfection efficiency of viral vectors is very high, but at the same time, viral vectors also have clinical problems such as inducing immune response and causing cancer.
  • Non-viral vectors mainly include polyethyleneimine (PEI), polyamide-amine (PAMAM), cationic liposomes and so on.
  • cationic liposomes as drug carriers, have the advantages of targeting, long-acting, low toxicity, and protective drugs (Wang J et al., c-Myc is required for maintenance of glioma cancer cells, PLoS ONE, 2008,3(11):e3769).
  • Cationic liposomes are usually formed by mixing positively charged lipids and neutral auxiliary lipids. When cationic liposomes are close to negatively charged nucleic acids, the aggregated cationic lipids can play the role of "double-sided adhesive tape". Function, spontaneously form nucleic acid/cation liposome complex.
  • cationic liposomes can encapsulate nucleic acids well, promote the fusion of nucleic acid/cationic liposome complexes with cell membranes, and increase the uptake of nucleic acids by cells (Huang L et al., In vivo delivery of RNAi with lipid-based nanoparticles, Annual Review of Biomedical Engineering, August 15, 2011, 13:507-530).
  • the synthetic positively charged polyamino acids (such as oligoarginine) in the drug delivery vehicle of the present invention are used to compress nucleic acids, wherein the oligoarginine and the nucleic acid The two forms a physical complex through electrostatic interaction, and then use positively charged cationic liposomes to further compress the nucleic acid to form a liposome complex.
  • the prepared nucleic acid liposome complex has the ability to penetrate cells and tissues, and the nucleic acid liposome preparation thus constructed can be administered through a variety of ways, and the nucleic acid molecules can be efficiently delivered to the target body part, and has good Biosafety.
  • the preparation method of the nucleic acid liposome preparation of the present invention is:
  • the liposome is extruded using a micro extruder (the order of passing through the nuclear pore membrane is 200 nm, 100 nm and 50 nm).
  • the preparation method of the nucleic acid liposome preparation of the present invention is:
  • the liposome is extruded using a micro extruder (the order of passing through the nuclear pore membrane is 200 nm, 100 nm and 50 nm).
  • the positively charged polyamino acid (such as oligoarginine) and the nucleic acid molecule are mixed and incubated in a suitable solution according to a certain charge ratio to obtain a complex of the oligoarginine and the nucleic acid molecule.
  • the preparation method of the nucleic acid liposome preparation of the present invention is:
  • the liposome is extruded using a micro extruder (the order of passing through the nuclear pore membrane is 200 nm, 100 nm and 50 nm).
  • the positively charged polyamino acid (for example, oligoarginine) and the nucleic acid molecule are mixed and incubated in a suitable solution according to a certain charge ratio to obtain a complex of the oligoarginine and the nucleic acid molecule.
  • the nucleic acid molecule is siRNA
  • the preparation method of the siRNA liposome preparation of the present invention is: DOTAP, DOPE, cholesterol, mPEG 2000 -DSPE and Penetratin/Penetratin derivative-PEG 3400 -DSPE molar ratio It is about 28.5:28.5:38:5.
  • DOTAP, DOPE, and cholesterol in a round bottom flask, add chloroform to dissolve, and remove the chloroform by a rotary evaporator to obtain a uniform dry lipid film.
  • the lipid membrane is hydrated with 5% glucose aqueous solution through a water bath ultrasonically.
  • the liposome is extruded with a micro extruder (the order of passing through the nuclear pore membrane is 200nm, 100nm and 50nm) )
  • a micro extruder the order of passing through the nuclear pore membrane is 200nm, 100nm and 50nm.
  • Vortex for 30s incubate at 37°C for 30 minutes to obtain the complex Rn/siRNA, then mix the complex Rn/siRNA with the blank cationic liposome according to a certain charge ratio (the charge ratio of cationic liposome to siRNA) , Vortex for 30s, incubate at 37°C for 30 minutes to obtain the complex CLS/Rn/siRNA, and finally mix CLS/Rn/siRNA and mPEG 2000 -DSPE with Penetratin/Penetratin derivative -PEG 3400 -DSPE Incubate at 55°C for half an hour to complete the preparation of the siRNA liposome preparation.
  • the nucleic acid liposome preparation of the present invention is administered nasally, and the nucleic acid is efficiently delivered into the brain through the naso-brain pathway.
  • the nucleic acid liposome preparation can be taken up by brain tumor cells with the help of penetrating peptide and The positive charge on the surface of cationic liposomes allows endosomes to escape, release nucleic acid molecules into the cytoplasm, and play a role in gene therapy of brain tumors (for example, glioma).
  • the route of administration through the nose into the brain provides a feasible idea for the treatment of brain diseases.
  • the most direct method of the brain barrier is that the drug is absorbed through the nasal mucosa, and after being taken up by the olfactory nerve, it is transported to the olfactory bulb through axons and further reaches the olfactory brain.
  • the second is the nasal mucosal epithelial pathway.
  • the drug passes through the basement membrane and enters the lamina basement, further reaches the peripheral olfactory nerve, and then is transported to the central nervous system, and finally accumulates in the brain. Due to the non-traumatic nature of intranasal administration to the brain, the patient’s compliance is good (Agrawal M et al., Nose-to-brain drug delivery: An update on clinical challenges and progress toward approval of anti-Alzheimer drugs, Journal of Controlled Release, 2018, 281: 139-177).
  • brain tumors such as glioma are a malignant tumor of the central nervous system with a high incidence rate and low survival rate, which are extremely harmful to human health, but the clinical prognosis is not optimistic, mainly reflected in the recurrence of surgery, Chemotherapy resistance, etc., and due to the presence of the blood-brain barrier and blood-brain tumor barrier, the amount of drugs into the brain is very small after systemic administration. Therefore, people have been working hard to find an effective drug treatment.
  • the drug liposome preparation of the present invention can deliver the drug to the brain after nasal administration, thereby promoting the drug to exert a therapeutic effect.
  • the inventors took 8-polyarginine R8 as an example and used siRNA as a drug molecule model to construct a cationic liposome delivery vector carrying siRNA.
  • siRNA a drug molecule model to construct a cationic liposome delivery vector carrying siRNA.
  • the cell uptake capacity and cell sphere penetration capacity of the delivery vector constructed by the liposome modified by the penetrating peptide and the positively charged polyamino acid were investigated. The results show that, compared with commercially available cationic liposomes for nucleic acid delivery, the drug delivery vector of the present invention can significantly increase the amount of nucleic acid into cells, and has a higher delivery efficiency.
  • the advantages of the drug delivery carrier constructed by the present invention are high efficiency, low toxicity, and easy drug release.
  • the high efficiency is embodied in that the delivery vector of the present invention has high cell uptake ability and strong cell sphere penetrating ability; low toxicity is embodied in that the penetrating peptide used in the present invention is non-pathogenic and easy to degrade in the body, compared to Other drug delivery carriers such as PEI and PAMMA have better biological safety; easy-to-release drugs are reflected in the positive charge of polyamino acids and surface-modified penetrating peptides, and the positive charge of cationic liposomes can help the drug The endosome escapes, so that the drug can be successfully released into the cytoplasm to play a therapeutic role.
  • the pharmaceutical preparations prepared by using the drug delivery vehicle of the present invention can be administered to subjects through various routes, and in particular, intracerebral delivery of drugs can be achieved through the nasal route of administration, which is beneficial to improve patient compliance.
  • the resulting dried lipid film was ultrasonically hydrated with 1 mL of a 5% glucose aqueous solution in a 37°C water bath for about 10 minutes. After the lipid membrane is completely hydrated, the liposomes are extruded using a micro extruder (Hamilton, 81320) (the order of passing through the nuclear pore membrane is 200nm, 100nm and 50nm) to obtain cationic liposomes (also called in the text) For CLS).
  • a micro extruder Halton, 81320
  • the prepared cationic liposomes were mixed with 2.46 mg of mPEG 2000- DSPE (dissolved in water) and incubated at 55°C for half an hour to obtain cationic liposomes containing mPEG 2000- DSPE.
  • Agarose gel electrophoresis experiments are often used to determine the loading capacity of drug carriers for drugs such as nucleic acids.
  • R8/siRNA complexes, CLS/siRNA complexes and CLS/R8/siRNA complexes were prepared, and CLS alone, R8 alone, and CLS and R8 were determined by agarose gel electrophoresis experiments.
  • the combination of the ability to carry nucleic acids was determined by agarose gel electrophoresis experiments.
  • 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 4mg/mL oligomer Acid R8 solution, 33 ⁇ g of siRNA (in this example, the small interfering RNA against c-myc is used, which is also abbreviated as siRNA hereinafter, and its sense strand (5'-3') is AACGUUAGCUUCACCAACAdTdT (SEQ ID NO: 79), the antisense strand (5'-3') is UGUUGGUGAAGCUAACGUUdTdT (SEQ ID NO: 80)) was dissolved in 125 ⁇ L of DEPC-treated water to prepare a 20 ⁇ M siRNA solution.
  • siRNA in this example, the small interfering RNA against c-myc is used, which is also abbreviated as siRNA hereinafter
  • Liposome complexes were prepared using the above-mentioned R8/siRNA complexes and CLS with different charge ratios. Specifically, 8 ⁇ L of R8/siRNA complexes with different charge ratios (that is, 4 ⁇ L siRNA solution and 4 ⁇ L R8 solution prepared) were mixed with 2.67 ⁇ L of 16.67 ⁇ M cationic liposome CLS prepared in Example 1, and vortexed for 30 seconds Incubate at 37°C for 30 minutes to obtain a lipid complex CLS/R8/siRNA with different charge ratios of R8/siRNA.
  • the results of agarose gel electrophoresis showed that R8/siRNA complexes with different R8/siRNA charge ratios can be mixed with CLS to prepare liposome complexes.
  • the R8/siRNA complex and CLS with a charge ratio of R8/siRNA of 1:1 were used to prepare liposome complexes.
  • a CLS/siRNA complex was also prepared, in which the CLS prepared in Example 1 and the 20 ⁇ L siRNA solution prepared in this Example were compared according to the CLS/siRNA charge ratio of 2, 4, 6, 8, 10, and 12. Mix in equal volume, vortex for 30s, and incubate at 37°C for 30 minutes to obtain CLS/siRNA complexes with different CLS/siRNA charge ratios.
  • the calculation of the charge ratio of CLS/siRNA is carried out as follows: since one amino nitrogen of DOTAP has a positive charge and one phosphate of siRNA has a negative charge, 16.67 ⁇ M CLS contains 5 ⁇ M DOTAP, that is, 1 ⁇ M CLS contains 0.3 ⁇ M positive charge.
  • results of panel A in Figure 1 show that oligoarginine R8 alone cannot completely compress the gene; the results of panels C and D in Figure 1 show that although the cationic liposome CLS alone can compress genes to a certain extent, CLS The particle size of the /siRNA complex is larger; the results of panels E and F in Figure 1 show that the combination of cationic liposomes and oligoarginine R8 can effectively compress genes together.
  • the R8/siRNA complex, CLS/siRNA complex and CLS/R8/siRNA complex were prepared according to the method described in Example 2, and the particle size and potential were measured.
  • the particle size of the 89W Penetratin-modified siRNA-containing liposome complex (89W-CLS/R8/siRNA) is similar to that of CLS/R8/siRNA at 105nm.
  • the highest light intensity of 89W Penetratin-modified siRNA-containing liposome complex is lower than that of CLS/R8/siRNA, which is due to the wider particle size distribution range of the former.
  • each CLS/R8/siRNA complex prepared according to the method described in Example 2 was centrifuged at a speed of 12000 ⁇ g for 20 minutes, and the precipitation of each liposome preparation was observed.
  • the liposome pharmaceutical preparations are surface-modified by using penetrating peptides to obtain surface-modified pharmaceutical preparations.
  • DSPE-polyethylene glycol maleimide (DSPE-PEG 3400 -maleimide) (Laysan Bio, 146-123) and a penetratin derivative ( 89W Penetratin-Cys) modified with cysteine at the end
  • DSPE-PEG 3400 -maleimide (Laysan Bio, 146-123)
  • a penetratin derivative ( 89W Penetratin-Cys) modified with cysteine at the end
  • the penetrating peptide-PEG-DSPE was prepared by one-step reaction.
  • DSPE-PEG 3400 -maleimide 20 mg was dissolved in 1 mL of N,N-dimethylformamide, and 10 mL of phosphate buffer solution (10 mM, pH 7.2) was added to it under stirring. 18mg 89W Penetratin-Cys in the medium, continue to stir overnight at 25°C to complete the reaction. After the completion of the reaction, the obtained mixture was placed in pure water for 2 days in an ice bath for dialysis, and freeze-dried to obtain a white floc, which was 89W Penetratin-PEG 3400- DSPE.
  • the polypeptide 89W Penetratin is a derivative of penetratin, and the specific amino acid sequence is RQIKIWFWWRRMKWKK (SEQ ID NO: 29).
  • the penetrating peptide in the penetrating peptide-PEG-DSPE can also use other penetratin derivatives, for example, other penetratin derivatives shown in Table 1, for example, for penetratin at position 2 glutamine and/or at position 8.
  • Glutamine and/or asparagine at position 9 are the products of hydrophobic amino acid mutations (see also Chinese invention patent application CN201710414334.7).
  • cysteine residue can be added to the N-terminal or C-terminal of the penetrating peptide 89W Penetratin or other penetratin derivatives.
  • the molar ratio of DOTAP:DOPE:cholesterol:(mPEG 2000 -DSPE and 89W Penetratin-PEG 3400 -DSPE) is about 28.5:28.5:38:5.
  • the resulting dried lipid film was ultrasonically hydrated with 1 mL of a 5% glucose aqueous solution in a 37°C water bath for 10 minutes. After the lipid membrane is completely hydrated, the liposomes are extruded using a micro extruder (Hamilton, 81320) (the order of passing through the nuclear pore membrane is 200nm, 100nm and 50nm) to obtain cationic liposomes (also called in the text) For CLS).
  • a micro extruder Halton, 81320
  • oligoarginine R8 (Shanghai Xinhao Biotechnology Co., Ltd.) was dissolved in DEPC treated water (Dalian Meilun Biotechnology Co., Ltd., MA0018) to prepare a 4mg/mL oligoarginine R8 solution, and 33 ⁇ g FAM-labeled siRNA (in this example, siRNA against c-myc is used, and its sense strand (5'-3') is FAM-AACGUUAGCUUCACCAACAdTdT (SEQ ID NO: 79), that is, the 5'modification has FAM; antisense strand (5'-3') UGUUGGUGAAGCUAACGUUdTdT (SEQ ID NO: 80)) was dissolved in 125 ⁇ L of DEPC-treated water to prepare a 20 ⁇ M FAM-siRNA solution.
  • the oligoarginine R8 solution and the 20 ⁇ M FAM-siRNA solution were mixed in equal volumes at a charge ratio of 5, vortexed for 30s, and incubated at 37°C for 30 minutes to obtain the complex R8/FAM-siRNA.
  • 89W Penetratin-PEG 3400 -DSPE For the total moles of liposome 5% mPEG 2000 -DSPE and 89W Penetratin-PEG 3400 -DSPE, were formulated 89W Penetratin-PEG 3400 -DSPE accounted moles of mPEG 2000 -DSPE and 89W Penetratin-PEG 3400 - The number of moles of DSPE is a mixture of 0%, 20%, 40%, 60%, 80%, and 100%. That is, the moles of 89W Penetratin-PEG 3400 -DSPE account for 0%, 1%, 2%, 3%, 4%, and 5% of the total moles of liposome membrane material, respectively.
  • the number of moles of the prepared CLS/R8/FAM-siRNA complex and 89W Penetratin-PEG 3400 -DSPE accounted for 0%, 20%, 40% of mPEG 2000 -DSPE and 89W Penetratin-PEG 3400 -DSPE respectively. %, 60%, 80%, 100% mixtures were mixed and incubated at 55°C for half an hour to obtain lipids with surface modification ratios of 0%, 1%, 2%, 3%, 4%, and 5%. Plastid drug preparation, namely 89W-CLS/R8/FAM-siRNA.
  • U87 cells human glioblastoma cells, purchased from ATCC
  • DMEM complete medium with 10% FBS and 1% penicillin-streptomycin
  • U87 cells human glioblastoma cells, purchased from ATCC
  • DMEM complete medium supplied with 10% FBS and 1% penicillin-streptomycin. Resuspend the well-growing U87 cells in DMEM complete medium and inoculate 5 ⁇ 10 5 cells/well into a 6-well plate with 1 mL per well. After inoculation, replace the fresh culture medium once a day. After culturing for 2 to 3 days conduct experiment.
  • the liposome membrane material formulation was prepared as the liposome drug formulation shown in Table 2 (encapsulating FAM-labeled siRNA).
  • component (iv) The liposome drug formulation (encapsulating FAM-labeled siRNA) as shown in Table 3 was formulated.
  • the liposome pharmaceutical preparations with component (iv) occupying 1%, 5%, 8%, and 10% of the liposome membrane material were obtained.
  • the particle size determination of the liposomal pharmaceutical preparation is carried out. The results of particle size measurement are shown in Figure 6. "1%, 5%, 8%, 10%" on the abscissa of Fig. 6 respectively indicate that the component (iv) accounts for 1%, 5%, 8%, and 10% of the lipid of the liposome membrane material Body pharmaceutical preparations.
  • the particle size of the liposomal drug preparation is between 100nm-200nm; and as the component (iv) ) As the mole fraction of liposome membrane material increases, the particle size tends to increase.
  • U87 cells human glioblastoma cells, purchased from ATCC
  • DMEM complete medium with 10% FBS and 1% penicillin-streptomycin
  • the oligoarginine R8 solution and the 20 ⁇ M FAM-siRNA solution were mixed in equal volumes at a charge ratio of 5, vortexed for 30s, and incubated at 37°C for 30 minutes to obtain the complex R8/FAM-siRNA.
  • Example 6 a liposome pharmaceutical preparation with a surface modification of 3% penetrating peptide, that is, an 89W-CLS/R8/FAM-siRNA complex was obtained.
  • A549 cells are human non-small cell lung cancer cells
  • Caco-2 are human cloned colon adenocarcinoma cells
  • Rb-1 are human retinal glioma cells
  • U87 are human malignant cells.
  • 293T are human renal epithelial cells, all of which were purchased from ATCC.
  • A549 and Caco-2 are adherent cells with strong adherence
  • U87 and 293T are weak adherent cells
  • Rb-1 is a suspension cell.
  • sample 1 and sample 2 are respectively different magnifications of the same cell type
  • sample 3 and sample 4 are repeated experimental wells of sample 2.
  • A549 and Caco-2 are easy to adhere to the wall and grow into balls because of their good adhesion.
  • Rb-1 is a suspension cell
  • the cells are easy to aggregate into balls and are very easy to loosen during processing.
  • U87 and 293T have the best sphere-forming ability. They are roughly spherical in shape and uniform in size. They are suitable for further exploration of established cell sphere models.
  • 293T cell spheres were used to evaluate the transfection effect of GFP-siRNA-modified drug preparations with different ratios of penetrating peptides on cells.
  • GFP-siRNA liposome pharmaceutical preparations with different ratios of penetrating peptides on the surface were prepared, wherein the GFP-siRNA sense strand (5'-3') used was GCAUGUACCACGAGUCCAATT (SEQ ID NO: 81); The antisense strand (5'-3') is UUGGACUCGUGGUACAUGCTT (SEQ ID NO: 82).
  • the oligoarginine R8 solution and the 20 ⁇ M GFP-siRNA solution were mixed in equal volumes at a charge ratio of 5, vortexed for 30s, and incubated at 37°C for 30 minutes to obtain the complex R8/GFP- siRNA.
  • liposome pharmaceutical preparations with surface modification of 1%, 3%, and 5% of penetrating peptides, namely 89W-CLS/R8/GFP-siRNA complexes were obtained.
  • 1%, 3%, 5% in Figure 9 and Figure 10 respectively represent liposomal pharmaceutical preparations with 1%, 3%, and 5% surface modification peptides, namely 89W-CLS/R8/siRNA .
  • 35mm Dimple NanoCulture Dish is used to create GFP-293T cell spheres.
  • 293T cells are not tumor cells.
  • 35mm Dimple NanoCulture Dish is used to create GFP-U87 cell spheres.
  • U87 cells are glioma cells.
  • Liposome drugs have a high permeability to cell spheres.
  • U87 cells purchased from ATCC in good growth condition were inoculated into 48-well plates at 5 ⁇ 10 4 cells/well, and the medium was changed once a day after inoculation, and the experiment was carried out after culturing for 2 to 3 days.
  • 89W-CLS/R8/siRNA liposome preparation prepared in Example 6 containing 400 nmol/L siRNA was added, that is, oligoarginine and The charge ratio of siRNA is 5, the charge ratio of CLS to siRNA is 4, and the surface modification ratio is 3%.
  • 89W Penetratin-PEG 3400 -DSPE liposome pharmaceutical preparation after 6h incubation in the cell incubator, discard the liquid medicine.
  • the delivery vector designed in the present invention can specifically promote the apoptosis of tumor cells U87, but has no major influence on the growth of normal cells bEnd.3 cells.
  • liposomes labeled with lipophilic fluorescent dye DiD were prepared for tracing liposomes.
  • DiD cell membrane red fluorescent probe was purchased from Dalian Meilun Biotechnology Co., Ltd., MB6190.
  • the charge ratio of oligoarginine to siRNA was 5
  • the charge ratio of CLS to siRNA was 4, and the surface modification had a 3% ratio.
  • 89W Penetratin-PEG 3400 -DSPE DiD label The liposome drug formulation 89W-CLS/R8/siRNA.
  • ICR mice Twelve ICR mice (20g, male, 6 weeks old) were divided into 3 groups (89W-CLS/R8/siRNA group, CLS/R8/siRNA group and normal saline (NS) group).
  • 89W-CLS/R8/siRNA group 89W-CLS/R8/siRNA group
  • CLS/R8/siRNA group normal saline (NS) group
  • NS normal saline
  • the 89W-CLS/R8/siRNA group has greater fluorescence intensity in the brain, and the modification of the penetrating peptide can significantly enhance the tissue penetration ability of the delivery vector, thereby enhancing its entry into the brain Ability to increase the accumulation of genes in the brain.
  • Example 6 Similarly as described in Example 1, weigh the membrane material and add DiD cell membrane fluorescent probe (Dalian Meilun Biotechnology Co., Ltd., MB6190) as described in the kit instructions to prepare DiD cell membrane fluorescent probe labeled CLS, namely CLS /DiD. Similarly, according to the method described in Example 6, each pharmaceutical preparation labeled with DiD and/or FAM fluorescence was prepared.
  • DiD cell membrane fluorescent probe (Dalian Meilun Biotechnology Co., Ltd., MB6190) as described in the kit instructions to prepare DiD cell membrane fluorescent probe labeled CLS, namely CLS /DiD.
  • each pharmaceutical preparation labeled with DiD and/or FAM fluorescence was prepared.
  • U87 cells (purchased from ATCC) were cultured in DMEM complete medium (with 10% FBS and 1% penicillin-streptomycin). Resuspend the well-growing U87 cells in DMEM complete medium and inoculate 5 ⁇ 10 5 cells/well into a 6-well plate with 1 mL per well. After inoculation, replace the fresh culture medium once a day. After culturing for 2 to 3 days conduct experiment.
  • DMEM complete medium with 10% FBS and 1% penicillin-streptomycin
  • liposomes labeled with lipophilic fluorescent dye DiD are prepared for tracing the liposomes.
  • DiD cell membrane red fluorescent probe was purchased from Dalian Meilun Biotechnology Co., Ltd., MB6190.
  • the charge ratio of oligoarginine to siRNA was 5
  • the charge ratio of CLS to siRNA was 4, and the surface modification ratio was 3%.
  • 89W Penetratin-PEG 3400 -DSPE DiD-labeled lipid The plastid drug preparation 89W-CLS/R8/siRNA.
  • Each BALB/c nude mouse (Shanghai Xipuer-Bikai Experimental Animal Co., Ltd., China) was inoculated with 6 ⁇ 10 5 U87 cells (dispersed in 5 ⁇ L PBS buffer).
  • the nude mice were anesthetized with 7% chloral hydrate, the brain was fixed with a stereotaxic device, and the cells were inoculated into the striatum with a micro-syringe (Bregma 0.6mm forward, 1.8mm rightward, 3mm depth) to construct U87 in situ brain Tumor model.
  • the 89W-CLS/R8/siRNA group has higher fluorescence intensity at the tumor site, and the modification of the penetrating peptide can significantly enhance the tissue penetration ability of the delivery vector, thereby enhancing its penetration into the brain Ability to increase the accumulation of genes in the tumor site.
  • the liposome pharmaceutical preparations namely CLS/R8/siRNA and 89W-CLS/R8/siRNA complexes were prepared, and Lipo2000 was prepared according to the instructions of the commercial cationic gene delivery carrier Lipofectamine 2000 (Lipo2000, Invitrogen) /siRNA complex.
  • the results show that in the TUNEL stained sections, the intensity of green fluorescence indicates the number of tumor cells undergoing apoptosis.
  • the Lipo2000/siRNA group, CLS/R8/siRNA group and 89WP-CLS/R8/siRNA group can all cause tumor cell apoptosis.
  • the 89WP-CLS/R8/siRNA group has the highest fluorescence intensity, that is, the highest apoptotic ratio, indicating that the delivery vector designed in the present invention can exert an anti-tumor effect by inducing tumor cell apoptosis in vivo.
  • HE hematoxylin-eosin
  • the nasal mucosal epithelial cells of the positive control group sodium deoxycholate group and Lipo2000/siRNA group
  • the nasal mucosal epithelial cells of the positive control group showed obvious morphological changes, accompanied by nasal cilia shedding (red arrow), but in the saline group and siRNA group
  • the nasal mucosal epithelial cells are intact and neatly arranged, indicating that the delivery vector designed in the present invention has no obvious toxicity to the nasal mucosal epithelial cells, which can be considered as transnasal entry A safe carrier for brain delivery.

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Abstract

一种药物递送载体，其包含穿膜肽修饰的脂质体和聚合物，其中所述聚合物是带正电的多聚氨基酸。通过使用所述药物递送载体制备了脂质体复合物形式的药物制剂，粒径介于50nm至300nm，其是将带正电的多聚氨基酸和一种或多种药物分子形成的物理复合物与表面修饰有穿膜肽的脂质体混合而制备的，所述药物制剂能够有效地转染细胞，尤其是促进药物经鼻腔吸收入脑，提高药物的疗效。

Description

药物递送载体以及使用其的药物制剂 技术领域
本发明涉及药物递送载体、使用所述药物递送载体来制备药物制剂的方法、以及由此制备的药物制剂。具体地，本发明的药物递送载体包含穿膜肽修饰的脂质体和聚合物，其中所述聚合物是带正电的多聚氨基酸。使用本发明的药物递送载体制备的药物制剂为脂质体复合物，其是将带正电的多聚氨基酸和一种或多种药物分子形成的物理复合物与穿膜肽修饰的脂质体混合而制备的，所述药物制剂能够有效地转染细胞，尤其是促进药物经鼻腔吸收入脑，提高药物的疗效。
背景技术
穿膜肽(cell-penetrating peptide，CPP)是一种生理条件下带正电荷的短肽，能够共价或非共价连接基因、多肽和蛋白质等药物分子，并将药物分子递送至细胞内或携载药物分子穿透生物膜屏障。但是，单个穿膜肽携载药物分子的能力非常有限，这导致穿膜肽与药物分子所形成的复合物粒径较大且稳定性差。
此外，现有技术中递送药物分子(如，基因、多肽和蛋白质等药物分子)的载体如脂质体存在递送效率低而且易产生组织毒性等问题。在脂质体的研究领域，如何利用脂质体更高效地递送药物分子，使得疾病的治疗和/或预防更具有特异性、高效性仍然是一个巨大的挑战。研究人员主要通过对脂质体添加各种辅助性成分、研制并使用新型脂质材料、对脂质体表面进行结构修饰、设计并使用新的制备工艺等措施来提高脂质体的稳定性、靶向性和药物递送效率。
因此，本领域仍需要新的利用穿膜肽的药物递送载体，当药物分子(如基因、多肽、蛋白质等)与所述药物递送载体复合后具有提高的稳定性、且所制备的药物制剂粒径可控制和稳定性好，以及借助制剂表面修饰的穿膜肽 能够实现更有效的药物递送、且有助于药物从细胞的内涵体中逃逸出来而发挥药物的功效。
发明概述
本发明人通过锐意研究，开发了一种新的利用穿膜肽的药物递送载体，其包含穿膜肽修饰的脂质体和聚合物，其中所述聚合物是带正电的多聚氨基酸，所述聚合物用于与一种或多种药物混合形成聚合物-药物的复合物，所述穿膜肽修饰的脂质体用于与聚合物-药物的复合物混合形成包含聚合物-药物的复合物的脂质体复合物。
在一个实施方案中，本发明的药物递送载体中的穿膜肽是具有下述氨基酸序列的penetratin或penetratin的亲脂性衍生物：
R X 1 IKIWF X 2X 3 RRMKWKK(SEQ ID NO:1)
其中，X 1、X 2和X 3独立地选自天然来源的氨基酸谷氨酰胺(glutamine，Q)、天冬酰胺(asparagine，N)、丙氨酸(alanine，A)、缬氨酸(valine，V)、亮氨酸(leucine，L)、异亮氨酸(isoleucine，I)、脯氨酸(proline，P)、苯丙氨酸(phenylalanine，F)、色氨酸(tryptophan，W)、甲硫氨酸(methionine，M)和非天然来源的氨基酸α-氨基丁酸(α-aminobutyric acid)、α-氨基戊酸(α-aminopentanoic acid)、α-氨基己酸(α-aminohexanoic acid)、α-氨基庚酸(α-aminoheptanoic acid)。例如，本发明的药物递送载体中的一些穿膜肽是将Penetratin第2位谷氨酰胺(Q)和/或第8位谷氨酰胺(Q)和/或第9位天冬酰胺(N)突变为疏水性氨基酸的Penetratin衍生物。
在一个实施方案中，本发明的药物递送载体中的所述带正电的多聚氨基酸是例如聚合度为2-50的带正电的多聚氨基酸，所述带正电的多聚氨基酸的空间结构包括线性和环形，氨基酸残基的构型包括L型和D型；例如聚合度为6-12的精氨酸和/或赖氨酸。在所述带正电的多聚氨基酸中，任选地在带正电的氨基酸残基之间，间插有1-20个(例如1－6个)生理条件下不带电荷的氨基酸残基，优选地，所述带正电的多聚氨基酸是聚合度为6-12的多聚 精氨酸，其中所述多聚精氨酸的空间结构包括线性和环形，多聚精氨酸的构型包括L型和D型。
在一个实施方案中，本发明的药物递送载体中的穿膜肽修饰的脂质体包含如下膜材料：
(i)阳离子脂质，包括但不限于：1,2-二-O-十八烯基-3-三甲基铵丙烷(DOTMA)；二甲基双十八烷基铵(DDAB)；1,2-二油酰基-3-三甲基铵-丙烷(DOTAP)；1,2-二油酰基-3-二甲基铵-丙烷(DODAP)；1,2-二酰氧基-3-二甲基铵丙烷；1,2-二烷氧基-3-二甲基铵丙烷；双十八烷基二甲基氯化铵(DODAC)、1,2-二肉豆蔻酰氧基丙基-1,3-二甲基羟乙基铵(DMRIE)和2,3-二油酰氧基-N-[2(精胺甲酰胺)乙基]-N,N-二甲基-1-丙烷三氟乙酸铵(DOSPA)及其组合。优选为DOTMA、DOTAP、DODAC和DOSPA。最优选为DOTAP；
(ii)非阳离子脂质，包括但不限于：1,2-二-(9Z-十八酰基)-sn-甘油基-3-磷酸乙醇胺(DOPE)、1,2-二油酰基-sn-甘油基-3-磷酸胆碱(DOPC)、1,2-二硬脂酰基-sn-甘油-3-磷酸胆碱(DSPC)、1,2-二棕榈酰基-sn-甘油-3-磷酸胆碱(DPPC)、1,2-二棕榈酰基-sn-甘油-3-磷酸乙醇胺(DPPE)、1,2-二肉豆蔻酰基-sn-甘油-3-磷酸乙醇胺(DMPE)、2-二油酰基-sn-甘油-3-磷酸-(1’-rac-甘油)(DOPG)及其组合。优选为DOPE和/或DOPC。最优选为DOPE；
(iii)胆固醇；和
(iv)聚乙二醇化(PEG化)的磷脂和与CPP共轭的聚乙二醇磷脂，所述磷脂例如是磷脂酰胆碱、磷脂酰乙醇胺、磷脂酰肌醇和鞘磷脂，优选地，所述PEG化磷脂是例如聚乙二醇-二硬脂酰基磷脂酰乙醇胺(PEG-DSPE)和其衍生物甲氧基-聚乙二醇-二硬脂酰基磷脂酰乙醇胺(mPEG-DSPE))，所述与CPP共轭的聚乙二醇磷脂是例如Penetratin/Penetratin衍生物-PEG-DSPE。
在一些实施方案中，膜材料(i):(ii):(iii):(iv)的摩尔比为约20－40:20－40:20－40:1－20，且与CPP共轭的聚乙二醇磷脂占膜材料(iv)中的约20％－80％摩尔比。在一些具体的实施方案中，膜材料(i):(ii):(iii):(iv)的摩尔比为约27.0－31.6:27.0－31.6:31.6－39.6:1－10，且与CPP共轭的聚乙二醇 磷脂占膜材料(iv)中的约20％－80％摩尔比。
在一些具体的实施方案中，本发明的药物递送载体中的阳离子脂质体包含如下膜材料：(i)DOTAP；(ii)DOPE；(iii)胆固醇；(iv)mPEG 2000-DSPE和 89WPenetratin-PEG 3400-DSPE，且膜材料(i):(ii):(iii):(iv)的摩尔比为约20－40:20－40:20－40:1－20，且 89WPenetratin-PEG 3400-DSPE占膜材料(iv)中的约20％－80％摩尔比。在一些实施方案中，膜材料(i):(ii):(iii):(iv)的摩尔比为约27.0－31.6:27.0－31.6:31.6－39.6:1－10，且 89WPenetratin-PEG 3400-DSPE占膜材料(iv)中的约20％－80％摩尔比。在一些实施方案中，本发明的药物递送载体中的阳离子脂质体由如下膜材料组成：(i)DOTAP；(ii)DOPE；(iii)胆固醇；(iv)mPEG 2000-DSPE和 89WPenetratin-PEG 3400-DSPE，且膜材料(i):(ii):(iii):(iv)的摩尔比为约28.5:28.5:38:5， 89WPenetratin-PEG 3400-DSPE占膜材料(iv)中的约20％、25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％摩尔比。
在另一个方面，本发明提供了一种药物制剂，其是使用本发明的药物递送载体制备的药物制剂，其中将药物递送载体中包含的带正电的多聚氨基酸和欲递送的一种或多种药物分子形成物理复合物，并将所述物理复合物与所述的表面修饰有穿膜肽的阳离子脂质体混合形成脂质体复合物形式的药物制剂。
对欲递送的一种或多种药物分子没有特别的限定，包括但不限于：肽类药物(例如奥曲肽(octreotide))、抗体(例如，依那西普(etanercept)、阿达木单抗(adalimumab)、利妥昔单抗(rituximab)、派姆单抗(pembrolizumab)、兰尼单抗(ranibizumab)和纳武单抗(nivolumab))、细胞因子、激素、抗生素(例如，化疗药阿霉素(doxorubicin)、柔红霉素(daunorubicin)、表阿霉素(epirubicin)、放线菌素(actinomycin)和万古霉素(vancomycin))、核酸、以及它们的组合等。在一个实施方案中，所述药物制剂递送的药物分子是质粒DNA、RNA如小干扰RNA(siRNA)、有义RNA、反义寡核苷酸(ASO)、适配体(aptamers)、核酶等的核酸。在一个实施方案中，所述药物制剂递 送的药物分子是核酸和能够嵌入到生物体基因组中的药物(如阿霉素)的组合。
在一个实施方案中，所述药物制剂中药物分子(例如，化疗药)与阳离子脂质(例如，DOTAP)的摩尔比为约0.1-10，优选约0.1-5，进一步优选约0.1-2。在一个实施方案中，所述药物制剂对药物分子(例如，化疗药)的载药量为：药物分子(例如，化疗药)占药物制剂的约1％-30％(w/w)，优选约1％-25％(w/w)，进一步优选约3％-20％(w/w)。
在一个实施方案中，所述药物制剂中带正电的多聚氨基酸(例如，寡聚精氨酸)和欲递送的药物分子的电荷比介于1:1至30:1，优选5:1；且阳离子脂质体与药物分子的电荷比介于1:1至30:1，优选4:1。
在一个实施方案中，本发明的药物制剂的粒径介于50nm至300nm，优选80nm至150nm，且稳定性好。
在又一个方面，提供了本发明的药物制剂的制备方法，包括(a)制备表面修饰有穿膜肽的脂质体；(b)制备带正电的多聚氨基酸和欲递送的药物分子的物理复合物；(c)按一定的摩尔比或电荷比混合步骤(a)获得的表面修饰有穿膜肽的脂质体和步骤(b)获得的物理复合物。
在一个实施方案中，本发明的药物制剂的制备方法包括(a)混合脂质体的膜材料(i)、(ii)和(iii)，制备空白脂质体；(b)制备带正电的多聚氨基酸和欲递送的药物分子的物理复合物；(c)按一定的摩尔比或电荷比混合步骤(a)获得的空白脂质体和步骤(b)获得的物理复合物；(d)将步骤(c)获得的脂质体与膜材料(iv)混合并孵育，获得本发明的药物制剂。
使用本发明的药物递送载体制备的药物制剂能够例如通过口腔、鼻腔、眼、呼吸道、消化道、生殖道、局部植入、注射或输注(经由硬膜外、动脉内、关节内、囊内、心内、脑室内、颅内、皮内、肌内、眶内、眼内、腹膜内、脊柱内、胸骨内、鞘内、静脉内、蛛网膜下、被膜下、皮下、气管、直肠、舌下等途径)施用，用于治疗和/或预防疾病。
除非另外限定，否则本文中所用的全部技术与科学术语具有如本发明所 属领域的普通技术人员通常理解的相同含义。本文所提及的全部出版物、专利申请、专利和其他参考文献通过引用的方式完整地并入本文作为参考。此外，本文中所述的材料、方法和例子仅是说明性的并且不意在是限制性的。本发明的其他特征、目的和优点将从本说明书及附图并且从后附的权利要求书中显而易见。
附图简述：
图1：显示了R8(8聚精氨酸)与siRNA不同电荷比(表示为正电荷/负电荷的比例)和/或CLS与siRNA不同电荷比的各复合物的琼脂糖凝胶电泳结果和粒径结果。图1中的小图A和B分别显示了R8/siRNA不同电荷比的R8/siRNA复合物的琼脂糖凝胶电泳结果和粒径结果；小图C和D分别显示了CLS/siRNA不同电荷比的CLS/siRNA复合物的琼脂糖凝胶电泳结果和粒径结果；小图E和F分别显示了R8/siRNA电荷比为1:1时，CLS/siRNA不同电荷比的CLS/R8/siRNA脂质体复合物的琼脂糖凝胶电泳结果和粒径结果。
图2：显示了R8与siRNA电荷比为1:1的情况下，细胞对CLS与siRNA不同电荷比制备的CLS/R8/siRNA脂质体复合物的摄取。
图3：显示了CLS/siRNA/R8复合物的电泳、粒径和稳定性研究结果。图3中的小图A和B分别显示了在CLS与siRNA电荷比为4:1的情况下，R8与siRNA不同电荷比的脂质体复合物的琼脂糖凝胶电泳结果和粒径结果；小图C和D分别显示了在CLS与siRNA电荷比为4:1的情况下，R8与siRNA不同电荷比的脂质体复合物高速离心后的沉淀情况和离心后上清液的琼脂糖凝胶电泳结果。
图4：显示了表面修饰有不同比例穿膜肽的药物制剂的细胞摄取定量评价结果，其中，Lipo2000组为市售基因转染试剂阳离子脂质体Lipofectamine 2000与siRNA的非共价复合物。
图5：显示了细胞对使用实施例8表2中所示的不同配方脂质体膜材料 制备的药物制剂的摄取情况。
图6：显示了组分(iv)占脂质体膜材料的1％、5％、8％、10％摩尔份数的脂质体药物制剂的粒径。
图7：显示了使用35mm Dimple NanoCulture Dish培养不同细胞球的形态学结果，其中，35mm Dimple NanoCulture Dish为日本JSR Corporation的细胞球培养板。
图8：显示了修饰于脂质体表面的穿膜肽对细胞球穿透能力的评价结果。
图9：显示了包载GFP-siRNA的药物制剂对293T细胞球的转染效果。
图10：显示了包载GFP-siRNA的药物制剂对U87细胞球的转染效果。
图11：显示了使用不同寡聚精氨酸的药物制剂对LUC-U87细胞的转染效果的定性评价和定量评价结果。
图12：显示了包载siRNA的药物制剂的粒径。
图13：显示了包载siRNA的药物制剂的电势。
图14：显示了包载siRNA的药物制剂对bEnd.3细胞和U87细胞的促凋亡效果评价和细胞毒性评价结果。
图15：显示了细胞膜荧光探针DiD标记的药物制剂经鼻给药后的体内分布情况。
图16：显示了细胞膜荧光探针DiD标记的载体递送FAM-siRNA入胞的流式细胞术检测结果。
图17：显示了DiD细胞膜荧光探针标记的基因递送载体在U87原位脑瘤模型鼠中经鼻给药后的体内分布情况。
图18：显示了包载siRNA的递送载体的抗U87脑原位瘤的药效学评价结果，其中，Lipo2000/siRNA组为市售基因转染试剂阳离子脂质体Lipofectamine 2000与siRNA的非共价复合物。
图19：显示了包载siRNA的递送载体在U87原位脑瘤模型鼠中的体内促凋亡能力评价结果，其中，Lipo2000/siRNA组为市售基因转染试剂阳离子脂质体Lipofectamine 2000与siRNA的非共价复合物。
图20：显示了包载siRNA的递送载体的体内鼻粘膜毒性评价结果，其中Lipo2000/siRNA组为市售基因转染试剂阳离子脂质体Lipofectamine 2000与siRNA的非共价复合物，脱氧胆酸钠组的脱氧胆酸钠为大连美仑生物技术有限公司的市售产品。
发明详述：
本发明提供了一种新型的药物递送载体，其包含穿膜肽修饰的脂质体和聚合物，其中所述聚合物是带正电的多聚氨基酸。本发明的药物递送载体能够与药物分子组装形成复合物，可压缩(compact)药物分子并包载药物分子于脂质体中，由此利用脂质体表面修饰的具有穿膜功能的穿膜肽帮助药物穿透受试者体内的生物膜屏障。
本发明的药物递送载体具有很强的携载药物、递送药物以及组织穿透能力，且组织毒性低，可通过多种施用途径将药物分子高效递送至受试者体内的目标部位，克服因受试者的生物膜屏障导致的药物难以到达目标部位的技术问题，增强药物治疗疾病的有效性且具有良好的生物安全性。本发明还提供了使用所述药物递送载体制备的药物制剂。
如本文中所用，术语“受试者”是指哺乳动物。哺乳动物包括但不限于驯化动物(例如，牛、羊、猫、犬和马)、灵长类(例如，人和非人灵长类如猴)、兔和啮齿类(例如，小鼠和大鼠)。特别地，受试者是人。
本发明的药物递送载体的各种特征在下面进行了讨论。
如本文中所用，术语“约”旨在是指定值的±10％的调整。例如，术语“约5％”旨在包含4.5％至5.5％的范围。
如本文中所用，术语“包含”或“包括”意指包括所述的要素、整数或步骤，但是不排除任意其他要素、整数或步骤。
I.表面修饰有穿膜肽的脂质体的膜材料：
如本文所用的术语“脂质体”是指由脂质双层组成的人工制备的囊泡。不限制本发明中被穿膜肽修饰的脂质体的类型，可以是能够形成脂质囊泡并 包载药物的任何脂质体。
在一些实施方案中，本发明的表面修饰有穿膜肽的脂质体包含如下膜材料：
(i)阳离子脂质：
脂质体包含一种或多种阳离子脂质。如本文中所用，术语“阳离子脂质”是在选择的pH(例如生理pH)下具有净正电荷的脂质。许多阳离子脂质是商业可获得的。用于脂质体中的特别合适的阳离子脂质包括国际专利公开WO 2010/053572和WO 2012/170930中描述的那些阳离子脂质，所述两篇专利文献均以引用的方式并入本文作为参考。
对脂质体中包含的阳离子脂质没有特别的限制，只要是在选择的pH(例如生理pH)下具有净正电荷的脂质均可使用，例如但不限于：1,2-二-O-十八烯基-3-三甲基铵丙烷(DOTMA)；二甲基双十八烷基铵(DDAB)；1,2-二油酰基-3-三甲基铵-丙烷(DOTAP)；1,2-二油酰基-3-二甲基铵-丙烷(DODAP)；1,2-二酰氧基-3-二甲基铵丙烷；1,2-二烷氧基-3-二甲基铵丙烷；双十八烷基二甲基氯化铵(DODAC)、1,2-二肉豆蔻酰氧基丙基-1,3-二甲基羟乙基铵(DMRIE)和2,3-二油酰氧基-N-[2(精胺甲酰胺)乙基]-N,N-二甲基-1-丙烷三氟乙酸铵(DOSPA)及其组合。优选的阳离子脂质为DOTMA、DOTAP、DODAC和DOSPA。最优选的阳离子脂质为DOTAP。
(ii)非阳离子脂质：
脂质体包含一种或多种非阳离子脂质。如本文中所用，术语“非阳离子脂质”指任何中性、两性离子或阴离子脂质。如本文中所用，术语“阴离子脂质”指在选择的pH，例如生理pH下携带净负电荷的许多脂质物质中的任一种。
对脂质体中包含的非阳离子脂质没有特别的限制，可使用任何中性、两性离子或阴离子脂质，例如但不限于：1,2-二-(9Z-十八酰基)-sn-甘油基-3-磷酸乙醇胺(DOPE)、1,2-二油酰基-sn-甘油基-3-磷酸胆碱(DOPC)、1,2-二硬脂酰基-sn-甘油-3-磷酸胆碱(DSPC)、1,2-二棕榈酰基-sn-甘油-3-磷酸胆碱(DPPC)、 1,2-二棕榈酰基-sn-甘油-3-磷酸乙醇胺(DPPE)、1,2-二肉豆蔻酰基-sn-甘油-3-磷酸乙醇胺(DMPE)、2-二油酰基-sn-甘油-3-磷酸-(1’-rac-甘油)(DOPG)及其组合。优选的非阳离子脂质为DOPE和/或DOPC。最优选的非阳离子脂质为DOPE。
(iii)胆固醇：
胆固醇是构成哺乳动物细胞膜的一种重要组成成分，占细胞膜脂类的20％以上。术语“胆固醇”在本文中以最广意义使用并且涵盖胆固醇衍生物。研究表明，温度高时，胆固醇能阻止细胞膜双分子层的无序化；温度低时，胆固醇又可干扰细胞膜双分子层的有序化，阻止液晶的形成，保持细胞膜双分子层的流动性。
考虑到胆固醇在膜双分子层中的重要性，本发明的阳离子脂质体也包含胆固醇。
(iv)PEG化磷脂和与CPP共轭的聚乙二醇磷脂
脂质体包含PEG化磷脂和与CPP共轭的聚乙二醇磷脂。在本文中，PEG化磷脂不包含连接有CPP的聚乙二醇磷脂。
PEG化磷脂是将磷脂共价键合一个或多个聚乙二醇分子(PEG)而形成。PEG化磷脂包括但不限于长度高达10kDa的聚乙二醇链，例如，1kDa、2kDa、3kDa、4kDa、5kDa、6kDa、7kDa、8kDa、9kDa、10kDa、以及它们之间的任何值的聚乙二醇链，其与磷脂共价键合。所述磷脂可以是合成的、半合成的或天然的磷脂，例如是磷脂酰胆碱、磷脂酰乙醇胺、磷脂酰肌醇和鞘磷脂。在一些实施方案中，所述PEG化磷脂是例如聚乙二醇-二硬脂酰基磷脂酰乙醇胺(PEG-DSPE)和其衍生物甲氧基-聚乙二醇-二硬脂酰基磷脂酰乙醇胺(mPEG-DSPE)，其中PEG的分子量为1kDa－10kDa之间的任何值。
与CPP共轭的聚乙二醇磷脂是将CPP共价键合至聚乙二醇磷脂而形成。
本发明中使用的CPP不仅包括野生型多肽penetratin(氨基酸序列：RQIKIWFQNRRMKWKK(SEQ ID NO:2))，还包括其一系列亲脂性衍生物，例如，将Penetratin第2位谷氨酰胺(Q)和/或第8位谷氨酰胺(Q)和/或 第9位天冬酰胺(N)突变为疏水性氨基酸的Penetratin衍生物。在一些实施方案中，Penetratin亲脂性衍生物是中国发明专利申请CN201710414334.7中公开的那些衍生物，所述衍生物已被证明经结膜囊内滴眼给药后，可促进药物穿过诸多眼部吸收屏障(角膜、结膜、巩膜等)进入眼内，甚至将其携带的基因、多肽和蛋白质等生物大分子药物递送至眼后段的视网膜部位。中国发明专利申请CN201710414334.7中公开的Penetratin亲脂性衍生物的氨基酸序列此处引述如下：
表1：Penetratin衍生物的氨基酸序列
Figure PCTCN2020087279-appb-000001
Figure PCTCN2020087279-appb-000002
根据本发明，CPP是通过与聚乙二醇磷脂共价键合而被连接至作为脂质体的脂质双层的一部分的化合物。在本文中，术语“作为脂质体的脂质双层的一部分”旨在表明CPP-PEG-磷脂中的所述磷脂被整合到脂质双层中的事实。所述磷脂可以是合成的、半合成的或天然的磷脂，例如是磷脂酰胆碱、磷脂酰乙醇胺、磷脂酰肌醇和鞘磷脂。
CPP-PEG-磷脂中的聚乙二醇链包括但不限于长度高达10kDa的聚乙二醇链，例如，1kDa、2kDa、3kDa、4kDa、5kDa、6kDa、7kDa、8kDa、9kDa、10kDa、以及它们之间的任何值的聚乙二醇链，其两端分别与磷脂和与CPP共价键合。在一些实施方案中，所述与CPP共轭的聚乙二醇磷脂是例如Penetratin/Penetratin衍生物-PEG-DSPE，其中PEG的分子量为1kDa－10kDa之间的任何值。
本发明将CPP修饰在阳离子脂质体的表面，旨在增强药物递送载体在组织中的穿透能力，由此帮助携带更多药物分子的脂质体到达目标部位，从而发挥疾病的治疗和/或预防效果。
在一些实施方案中，脂质体膜材料(iv)中PEG化磷脂和与CPP共轭的聚乙二醇磷脂中的聚乙二醇链长不相等，且与CPP共轭的聚乙二醇磷脂中的聚乙二醇链较PEG化磷脂中的聚乙二醇链更长，例如，更长0.5kDa－5kDa之间，优选地更长0.75kDa－4kDa之间，更优选地更长1kDa－2kDa之间的任何值。在一个实施方案中，脂质体膜材料(iv)中PEG化磷脂的聚乙二醇链长分别是约1kDa、2kDa、3kDa，与CPP共轭的聚乙二醇磷脂的聚乙二醇链长分别是约2.5kDa、3.5kDa、4.5kDa。
在一些实施方案中，本发明的表面修饰有穿膜肽的阳离子脂质体的膜材料(i):(ii):(iii):(iv)的摩尔比为约20－40:20－40:20－40:1－20，且与CPP共轭的聚乙二醇磷脂占膜材料(iv)中的约20％－80％摩尔比，对应于占总的脂质体膜材料中的约2％－8％摩尔比。在一些实施方案中，膜材料(i):(ii):(iii):(iv)的摩尔比为约27.0－31.6:27.0－31.6:31.6－39.6:1－10，且与CPP共轭的聚乙二醇磷脂占膜材料(iv)中的约20％－80％摩尔比，对应于占总的脂质体膜材料中的约2％－8％摩尔比。
在一些具体的实施方案中，本发明的药物递送载体中的阳离子脂质体包含如下膜材料：(i)DOTAP；(ii)DOPE；(iii)胆固醇；(iv)PEG-DSPE(例如，mPEG 2000-DSPE)和Penetratin/Penetratin衍生物-PEG-DSPE(例如， 89WPenetratin-PEG 3400-DSPE)，且膜材料(i):(ii):(iii):(iv)的摩尔比为约20－40:20－40:20－40:1－20，且Penetratin/Penetratin衍生物-PEG-DSPE(例如， 89WPenetratin-PEG 3400-DSPE)占膜材料(iv)中的约20％－80％摩尔比。在一些实施方案中，膜材料(i):(ii):(iii):(iv)的摩尔比为约27.0－31.6:27.0－31.6:31.6－39.6:1－10，且Penetratin/Penetratin衍生物-PEG-DSPE(例如， 89WPenetratin-PEG 3400-DSPE)占膜材料(iv)中的约20％－80％摩尔比。在一些实施方案中，本发明的药物递送载体中的阳离子脂质体由如下膜材料组成：(i)DOTAP；(ii)DOPE；(iii)胆固醇；(iv)mPEG 2000-DSPE和 89WPenetratin-PEG 3400-DSPE，且膜材料(i):(ii):(iii):(iv)的摩尔比为约28.5:28.5:38:5， 89WPenetratin-PEG 3400-DSPE占膜材料(iv)中的约20％、25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％摩尔比。
II.带正电的多聚氨基酸
本发明的药物递送载体中包含的带正电的多聚氨基酸用于压缩药物分子。
所述带正电的多聚氨基酸是在选择的pH(例如生理pH)下具有净正电荷的多聚氨基酸。对多聚氨基酸中氨基酸残基的聚合度没有特别的限制，例如聚合度为2-50的带正电的多聚氨基酸(即，带正电的多聚氨基酸具有2-50 个氨基酸残基)，例如聚合度为6-12的精氨酸和/或赖氨酸，任选地在所述聚合度为2-50的带正电的多聚氨基酸中间插有1-20个(例如1－6个)生理条件下不带电荷的氨基酸残基。带正电的多聚氨基酸可以是线性或环形，且氨基酸残基的构型包括L型和D型。优选地，所述带正电的多聚氨基酸是聚合度为6-12的多聚精氨酸，其中所述多聚精氨酸的空间结构包括线性和环形，多聚精氨酸的构型包括L型和D型。
在一些实施方案中，带正电的多聚氨基酸是寡聚精氨酸(oligo-arginine)，包括线性的多肽，例如6聚精氨酸(氨基酸序列为RRRRRR，R6(SEQ ID NO:85))、8聚精氨酸(氨基酸序列为RRRRRRRR，R8(SEQ ID NO:86))、10聚精氨酸(氨基酸序列为RRRRRRRRRR，R10(SEQ ID NO:87))、12聚精氨酸(氨基酸序列为RRRRRRRRRRRR，R12(SEQ ID NO:88))，以及环化的多肽，例如环化的6聚精氨酸(c-R6)、环化的8聚精氨酸(c-R8)、环化的10聚精氨酸(c-R10)、环化的12聚精氨酸(c-R12)，以及不同构型的多肽，例如L型寡聚精氨酸和D型寡聚精氨酸如6聚D-精氨酸(氨基酸序列为rrrrrr，D-R6)、8聚D-精氨酸(氨基酸序列为rrrrrrrr，D-R8)、10聚D-精氨酸(氨基酸序列为rrrrrrrrrr，D-R10)、12聚D-精氨酸(氨基酸序列为rrrrrrrrrrrr，D-R12)。
以下描述了使用本发明的药物递送载体来制备药物制剂的方法，并描述了药物制剂的特征。
通过使用本发明的药物递送载体，能够制备出CPP修饰在阳离子脂质体的表面、在组织中的穿透能力强的药物制剂。
在一个实施方案中，本发明的药物制剂的制备方法为：
(1)称取上述本发明的脂质体的膜材料，其中膜材料(i):(ii):(iii):(iv)摩尔比为约20－40:20－40:20－40:1－20，且与CPP共轭的聚乙二醇磷脂占膜材料(iv)中的约20％－80％摩尔比，对应于占总的脂质体膜材料中的约2％－8％摩尔比。
(2)将膜材料(i):(ii):(iii):(iv)加入有机溶剂中溶解，然后蒸发除去有机 溶剂，获得均匀的干燥脂质膜。
(3)将所得的干燥脂质膜用含水溶液水化完全后，使用微型挤出器挤出脂质体(过核孔膜的顺序依次是200nm、100nm和50nm)。
(4)将带正电的多聚氨基酸与药物分子在合适的溶液中混合并孵育，获得带正电的多聚氨基酸与药物分子的物理复合物。
(5)以一定的摩尔比或电荷比混合上述(3)获得的脂质体和上述(4)获得的复合物，得到脂质体复合物形式的药物制剂。
又在一个实施方案中，本发明的药物制剂的制备方法为：
(1)称取上述本发明的脂质体的膜材料，其中膜材料(i):(ii):(iii):(iv)摩尔比为约20－40:20－40:20－40:1－20，且与CPP共轭的聚乙二醇磷脂占膜材料(iv)中的约20％－80％摩尔比，对应于占总的脂质体膜材料中的约2％－8％摩尔比。
(2)将膜材料(i):(ii):(iii)加入有机溶剂中溶解，然后蒸发除去有机溶剂，获得均匀的干燥脂质膜。
(3)将所得的干燥脂质膜用含水溶液水化完全后，使用微型挤出器挤出脂质体(过核孔膜的顺序依次是200nm、100nm和50nm)。
(4)将带正电的多聚氨基酸与药物分子在合适的溶液中混合并孵育，获得带正电的多聚氨基酸与药物分子的物理复合物。
(5)以一定的摩尔比或电荷比混合上述(3)获得的脂质体和上述(4)获得的复合物，然后与膜材料(iv)混合并孵育，获得本发明的药物制剂。
对欲递送的药物分子没有特别地限定，所述药物分子包括广泛类型的递送至受试者的化合物，包括但不限于：核酸；抗感染药如抗生素和抗病毒药；镇痛药和镇痛药组合；食欲抑制药；驱蠕虫药；抗关节炎药；平喘药；抗惊厥药；抗抑郁药；抗糖尿病药；止泻药；抗组胺药；抗炎药；抗偏头痛制剂；止恶心药；抗肿瘤药；抗震颤麻痹药；止痒药；抗精神药；退热药；解痉药；抗胆碱能药；拟交感神经药；黄嘌呤衍生物；心血管制剂包括钾通道阻滞剂、钙通道阻滞剂、β-阻滞剂、α-阻滞剂和抗心律失常药；抗高血压药；利尿 药和抗利尿药；血管舒张药包括全身神经、心神经、周围神经和脑神经、中枢神经系统的兴奋剂、血管抑制药；咳嗽和感冒制剂，包括减充血药；激素，如雌二醇和其它固醇类，包括皮质类固醇；催眠药；免疫抑制药；肌肉松弛药；副交感神经阻滞药；精神兴奋剂；镇静药和安定药。本发明的药物递送载体可以递送例如处于离子化的、非离子化的、游离碱、酸加成盐等所有形式的药物，可以递送高分子量或低分子量药物。
在一些实施方案中，本发明的药物制剂递送的药物分子是例如肽类药物(例如奥曲肽(octreotide))、抗体(例如，依那西普(etanercept)、阿达木单抗(adalimumab)、利妥昔单抗(rituximab)、派姆单抗(pembrolizumab)、兰尼单抗(ranibizumab)和纳武单抗(nivolumab))、细胞因子、激素、抗生素(例如，化疗药阿霉素(doxorubicin)、柔红霉素(daunorubicin)、表阿霉素(epirubicin)、放线菌素(actinomycin)和万古霉素(vancomycin))、核酸等。
在一些实施方案中，所述药物制剂递送的药物分子是质粒DNA、RNA如小干扰RNA(siRNA)、有义RNA、反义寡核苷酸(ASO)、适配体(aptamers)、核酶等的核酸。质粒DNA是含有这样的DNA序列的基因表达系统，所述DNA序列编码例如选自白细胞介素-2、白细胞介素-4、白细胞介素-7、白细胞介素-12、白细胞介素-15、干扰素-α、干扰素-β、干扰素-γ、集落刺激因子、粒细胞-巨噬细胞刺激因子、抗血管生成剂、肿瘤抑制基因、胸苷激酶、eNOS、iNOS、p53、p16、TNF-α、Fas-配体、突变的癌基因、肿瘤抗原、病毒抗原或细菌抗原。质粒DNA还可以编码shRNA分子，所述shRNA分子设计为旨在抑制参与肿瘤细胞或其它过度增生性细胞生长或维持的蛋白质。质粒DNA可以同时编码治疗性蛋白质和一种或多种shRNA。此外，本发明药物制剂中的核酸还可以是质粒DNA与包含有义RNA、反义寡核苷酸或核酶的人工合成RNA的混合物。
在一个实施方案中，本发明药物制剂中药物分子(例如，化疗药)与阳离子脂质(例如，DOTAP)的摩尔比为约0.1-10，例如，约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， 优选约0.1-5，进一步优选约0.1-2。在一个实施方案中，所述药物制剂对药物分子(例如，化疗药)的载药量为：药物分子(例如，化疗药)占药物制剂的约1％-30％(w/w)，例如，约1％、2.5％、5％、7.5％、10％、12.5％、15％、17.5％、20％、22.5％、25％、27.5％、30％(w/w)，优选约1％-25％(w/w)，进一步优选约3％-20％(w/w)。
本发明的药物制剂可通过口腔、鼻腔、眼、呼吸道、消化道、生殖道、局部植入、注射或输注(经由硬膜外、动脉内、关节内、囊内、心内、脑室内、颅内、皮内、肌内、眶内、眼内、腹膜内、脊柱内、胸骨内、鞘内、静脉内、蛛网膜下、被膜下、皮下、气管、直肠、舌下等途径)施用。
可使用本领域已知的医学设备来施用本发明的药物制剂。例如，在一个实施方案中，可用无针头皮下注射设备，例如美国专利号5,399,163中公开的设备来施用本发明的药物制剂。
在一个实施方案中，使用本发明的药物递送载体制备了递送核酸的药物制剂，由此发挥基因治疗的作用。
与传统的手术、放疗、化疗等治疗方法相比，基因治疗作为一种具有突破性意义的治疗方法，优点在于针对性强、副作用低，对正常细胞损伤小，治疗过程中痛苦少，因此在免疫缺陷病、代谢性疾病、癌症、心脏病等疾病领域显示出良好前景。目前全球有2597种核酸药物进入或完成临床试验，其中65％是用于癌症治疗，有181项临床试验的基因药物是通过抑制肿瘤生长发挥治疗作用(Ginn SL等人，Gene therapy clinical trials worldwide to 2017:An update，Journal of Gene Medicine,2018,20(5):e3015)。基于此，本领域希望能够通过递送核酸的药物制剂来实现对疾病(例如，肿瘤疾病)的治疗。
递送核酸的载体是基因治疗的关键技术之一，可保护所递送的核酸不被内源性核酸酶降解，从而使基因治疗的体内应用成为可能。目前基因治疗所使用的核酸载体主要是病毒载体和非病毒载体两类。病毒载体的转染效率很高，但同时病毒载体也存在诱导免疫反应、致癌等临床问题。非病毒载体主要包括聚乙烯亚胺(PEI)、聚酰胺-胺(PAMAM)、阳离子脂质体等。相 比较PEI和PAMAM，阳离子脂质体作为药物载体，具有靶向、长效、低毒、保护药物等优点(Wang J等人，c-Myc is required for maintenance of glioma cancer stem cells，PLoS ONE,2008,3(11):e3769)。阳离子脂质体通常由带正电荷的脂类和中性辅助脂类混合形成，当阳离子脂质体与带负电荷的核酸靠近时，聚集的阳离子脂质可发挥“双面粘胶带”的作用，自发形成核酸/阳离子脂质体复合物。对脂质体的表面基团进行特殊修饰后，阳离子脂质体能很好地包封核酸，促进核酸/阳离子脂质体复合物与细胞膜的融合，提高细胞对核酸的摄取(Huang L等人，In vivo delivery of RNAi with lipid-based nanoparticles，Annual Review of Biomedical Engineering,2011年8月15日,13:507-530)。
在制备递送核酸的药物制剂时，使用本发明的药物递送载体中的人工合成的带正电的多聚氨基酸(例如寡聚精氨酸)来压缩核酸，其中所述寡聚精氨酸和核酸这两者通过静电作用形成物理复合物，然后借助带有正电荷的阳离子脂质体进一步压缩核酸，形成脂质体复合物，进一步地由于穿膜肽修饰在阳离子脂质体的表面，可以提高所制得的核酸脂质体复合物对细胞和组织的穿透能力，由此构建的核酸脂质体制剂能够通过多种途径给药，并将核酸分子高效递送至目标身体部位，且具有良好的生物安全性。
在一个实施方案中，本发明的核酸脂质体制剂的制备方法为：
(1)称取上述本发明的脂质体的膜材料，其中膜材料(i):(ii):(iii):(iv)摩尔比为约约20－40:20－40:20－40:1－20，且与CPP共轭的聚乙二醇磷脂占膜材料(iv)中的约20％－80％摩尔比，对应于占总的脂质体膜材料中的约2％－8％摩尔比。
(2)将膜材料(i):(ii):(iii)加入有机溶剂中溶解，然后蒸发除去有机溶剂，获得均匀的干燥脂质膜。
(3)将所得的干燥脂质膜用含水溶液水化完全后，使用微型挤出器挤出脂质体(过核孔膜的顺序依次是200nm、100nm和50nm)。
(4)将带正电的多聚氨基酸(例如寡聚精氨酸)与核酸分子按照一定的 电荷比在合适的溶液中混合并孵育，获得带正电的多聚氨基酸(例如寡聚精氨酸)与核酸分子的复合物。
(5)按照一定的摩尔比或电荷比混合上述(3)获得的脂质体和上述(4)获得的复合物，获得脂质体复合物。
(6)将上述(5)获得的脂质体与膜材料(iv)混合并孵育，获得本发明的核酸脂质体制剂。
在一个实施方案中，本发明的核酸脂质体制剂的制备方法为：
(1)称取上述本发明的脂质体的膜材料，其中膜材料(i):(ii):(iii):(iv)摩尔比为约约20－40:20－40:20－40:1－20，且与CPP共轭的聚乙二醇磷脂占膜材料(iv)中的约20％－80％摩尔比，对应于占总的脂质体膜材料中的约2％－8％摩尔比。
(2)将膜材料(i):(ii):(iii)加入有机溶剂中溶解，然后蒸发除去有机溶剂，获得均匀的干燥脂质膜。
(3)将所得的干燥脂质膜用含水溶液水化完全后，使用微型挤出器挤出脂质体(过核孔膜的顺序依次是200nm、100nm和50nm)。
(4)将上述(3)获得的脂质体与膜材料(iv)混合并孵育，获得穿膜肽修饰的阳离子脂质体。
(5)将带正电的多聚氨基酸(例如寡聚精氨酸)与核酸分子按照一定的电荷比在合适的溶液中混合并孵育，获得寡聚精氨酸与核酸分子的复合物。
(6)按照一定的摩尔比或电荷比混合上述(4)获得的脂质体和上述(5)获得的复合物，获得脂质体复合物形式的本发明的核酸脂质体制剂。
在一个实施方案中，本发明的核酸脂质体制剂的制备方法为：
(1)称取上述本发明的脂质体的膜材料，其中膜材料(i):(ii):(iii):(iv)摩尔比为约约20－40:20－40:20－40:1－20，且与CPP共轭的聚乙二醇磷脂占膜材料(iv)中的约20％－80％摩尔比，对应于占总的脂质体膜材料中的约2％－8％摩尔比。
(2)将膜材料(i):(ii):(iii):(iv)加入有机溶剂中溶解，然后蒸发除去有机 溶剂，获得均匀的干燥脂质膜。
(3)将所得的干燥脂质膜用含水溶液水化完全后，使用微型挤出器挤出脂质体(过核孔膜的顺序依次是200nm、100nm和50nm)。
(4)将带正电的多聚氨基酸(例如寡聚精氨酸)与核酸分子按照一定的电荷比在合适的溶液中混合并孵育，获得寡聚精氨酸与核酸分子的复合物。
(5)按照一定的电荷比混合上述(3)获得的脂质体和上述(4)获得的复合物，获得脂质体复合物形式的本发明的核酸脂质体制剂。
在一个实施方案中，所述核酸分子是siRNA，本发明的siRNA脂质体制剂的制备方法为：DOTAP、DOPE、胆固醇、mPEG 2000-DSPE与Penetratin/Penetratin衍生物-PEG 3400-DSPE的摩尔比为约28.5:28.5:38:5，称取DOTAP、DOPE、胆固醇置于圆底烧瓶中，加入氯仿溶解，通过旋转蒸发仪除去氯仿得均匀的干燥脂质膜。将脂质膜用5％的葡萄糖水溶液通过水浴超声水化，待脂质膜水化完全后，再使用微型挤出器挤压脂质体(过核孔膜的顺序依次是200nm、100nm和50nm)，完成空白脂质体CLS的制备。然后将寡聚精氨酸Rn(R为精氨酸的单字母代码，n为例如2、3、4、5、6、7、8、9、10等整数)与siRNA按照一定电荷比混合，涡旋30s，置于37℃条件下孵育30分钟后得到复合物Rn/siRNA，再将复合物Rn/siRNA与空白阳离子脂质体按照一定电荷比(阳离子脂质体与siRNA的电荷比)混合，涡旋30s，置于37℃条件下孵育30分钟后得到复合物CLS/Rn/siRNA，最后将CLS/Rn/siRNA和mPEG 2000-DSPE与Penetratin/Penetratin衍生物-PEG 3400-DSPE混合后置于55℃孵育半小时，完成siRNA脂质体制剂的制备。
在一个实施方案中，将本发明的核酸脂质体制剂经鼻给药，由鼻脑通路将核酸高效递送进入脑部，核酸脂质体制剂被脑部肿瘤细胞摄取后能够借助穿膜肽和阳离子脂质体的表面正电荷实现内涵体逃逸，将核酸分子释放到细胞质中，发挥基因治疗脑部肿瘤(例如，脑胶质瘤)的作用。
本领域技术人员公知，脑部特殊的生理结构及功能上的复杂性，特别是血脑屏障的存在以及某些药物自身的理化性质，使治疗药物通常难以达到脑组织。然而，经鼻入脑的给药途径为脑部疾病的治疗提供了可行性的思路，药物经鼻腔入脑的直接通路有两条，一条是嗅神经通路，这是经鼻入脑绕过血脑屏障最直接的方法，药物经鼻黏膜吸收，经嗅神经摄取后，通过轴突运输至嗅球，进一步抵达嗅脑的过程。第二条是鼻黏膜上皮通路，药物穿过基膜进入固有层，进一步到达周围嗅神经，进而转运至中枢神经系统，最终实现在脑部的蓄积。由于经鼻入脑给药具有无创伤性，因此患者的顺应性良好(Agrawal M等人，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)。另一方面，脑部肿瘤例如脑胶质瘤是一种中枢神经系统恶性肿瘤，发病率高，生存率低，对人类健康的危害极大，但临床预后并不乐观，主要体现在手术复发、化疗耐药等，且由于存在血脑屏障、血脑肿瘤屏障，全身给药后药物入脑量非常少，因此，一直以来人们都在致力寻找一种有效的药物治疗方式。
本发明的药物脂质体制剂经鼻给药后，能够将药物递送至脑部，进而促进药物发挥治疗作用。
在本发明的实施例中，发明人以8聚精氨酸R8为例，以siRNA为药物分子模型，构建了携载siRNA的阳离子脂质体递送载体。通过一系列体外和体内实验，考察了由穿膜肽修饰的脂质体和带正电的多聚氨基酸共同构建的递送载体的细胞摄取能力以及细胞球穿透能力。结果表明，与商品化的递送核酸的阳离子脂质体相比，本发明的药物递送载体能够显著提高核酸的入胞量，具备较高的递送效率。
本发明构建的药物递送载体的优势在于高效、低毒，且易释放药物。高效体现在本发明的递送载体具备较高的细胞摄取能力和较强的细胞球穿透能力；低毒体现在本发明所用的穿膜肽无致病性，且易于在体内降解，相比于PEI和PAMMA等其他药物递送载体具备更好的生物安全性；易释放药物 体现在多聚氨基酸和表面修饰的穿膜肽均带有正电荷，阳离子脂质体带有的正电荷能够帮助药物的内涵体逃逸，从而使药物能够成功释放到细胞质中，发挥治疗作用。此外，使用本发明的药物递送载体制备的药物制剂可通过多种途径施用至受试者，尤其可通过经鼻给药途径实现药物的脑内递送，有利于提高患者的顺应性。
描述以下实施例以辅助对本发明的理解。不意在且不应当以任何方式将实施例解释成限制本发明的保护范围。
实施例
实施例1
阳离子脂质体的制备：
称取摩尔比为28.5:28.5:38:5的膜材，即3.5mg的DOTAP(上海艾韦特医药科技有限公司,132172-61-3)、3.72mg的DOPE(上海艾韦特医药科技有限公司,4004-05-1)、2.59mg的胆固醇(Sigma-Aldrich,C8667)和2.46mg的mPEG 2000-DSPE(上海艾韦特医药科技有限公司,147867-65-0)。
将所述量的DOTAP、DOPE、胆固醇置于50mL圆底烧瓶中，加入1mL氯仿，溶解作为脂质膜材料的所述DOTAP、DOPE、胆固醇，即16.67μM的CLS含有5μM的DOTAP，5μM的DOPE，6.67μM的DOPE。将所得溶液通过旋转蒸发仪(上海申生科技有限公司，R201L)除去氯仿，获得均匀的干燥脂质膜。
将所得的干燥脂质膜用1mL 5％的葡萄糖水溶液通过37℃水浴超声水化约10分钟。待脂质膜水化完全后，使用微型挤出器(Hamilton,81320)挤出脂质体(过核孔膜的顺序依次是200nm、100nm和50nm)，获得了阳离子脂质体(文中也称为CLS)。
将所制得的阳离子脂质体与2.46mg的mPEG 2000-DSPE(溶于水)混合后置于55℃孵育半小时，获得了包含mPEG 2000-DSPE的阳离子脂质体。
实施例2
阳离子脂质体和带正电的多聚氨基酸对药物的压缩(compact)作用：
琼脂糖凝胶电泳实验常用于测定药物载体对药物如核酸的包载能力。在本实施例中，制备了R8/siRNA复合物、CLS/siRNA复合物和CLS/R8/siRNA复合物，通过琼脂糖凝胶电泳实验分别测定了单独的CLS、单独的R8、以及CLS和R8的组合对核酸的包载能力。
将4mg的寡聚精氨酸R8(上海昕浩生物科技有限公司)作为带正电的多聚氨基酸溶于DEPC处理水(大连美仑生物技术有限公司，MA0018)中制备4mg/mL寡聚精氨酸R8溶液，将33μg的siRNA(在本实施例中，使用的是针对c-myc的小干扰RNA,下文中也缩写为siRNA，其有义链(5'-3')为AACGUUAGCUUCACCAACAdTdT(SEQ ID NO:79)，反义链(5'-3')为UGUUGGUGAAGCUAACGUUdTdT(SEQ ID NO:80))溶于125μL的DEPC处理水中制备20μM的siRNA溶液。
将寡聚精氨酸R8溶液与siRNA溶液按照电荷比为0、1、5、10、15、20、25、30的比例等体积混合(由于R8的一个氨基氮带一个正电荷，siRNA的一个磷酸根带一个负电荷，1μM的R8含8μM的氨基氮，即1μM的R8含8μM的正电荷，1μM的siRNA含有42μM的磷酸根，即1μM的siRNA含42μM的负电荷，R8与siRNA的电荷比＝(R8的μM值×8)/(siRNA的μM值×42)＝0.19×R8(μM)/siRNA(μM))，涡旋30s，置于37℃条件下孵育30分钟得到不同电荷比的R8/siRNA复合物。
使用上述不同电荷比的R8/siRNA复合物和CLS制备脂质体复合物。具体而言，取8μL不同电荷比的R8/siRNA复合物(即，4μL siRNA溶液和4μL R8溶液制备而得)与2.67μL的实施例1制备的16.67μM阳离子脂质体CLS混合，涡旋30s，置于37℃条件下孵育30分钟，得到R8/siRNA不同电荷比的脂质复合物CLS/R8/siRNA。在所述脂质复合物CLS/R8/siRNA中，以siRNA的电荷数为基准，阳离子脂质体CLS与siRNA的电荷比固定为4(由于DOTAP的一个氨基氮带一个正电荷，siRNA的一个磷酸根带一个负 电荷，16.67μM的CLS含5μM的DOTAP，即1μM的CLS含0.3μM的正电荷，1μM的siRNA含42μM的负电荷，CLS与siRNA的电荷比＝(CLS的μM值×0.3)/(siRNA的μM值×42)＝0.007×CLS(μM)/siRNA(μM))。琼脂糖凝胶电泳结果表明，使用不同R8/siRNA电荷比的R8/siRNA复合物均能够与CLS混合制备脂质体复合物。
接下来，使用R8/siRNA电荷比为1:1的R8/siRNA复合物和CLS制备脂质体复合物。具体而言，将实施例1制备的CLS与8μL如上制备的R8/siRNA电荷比为1:1的R8/siRNA复合物按照CLS/siRNA电荷比为2、4、6、8、10、12的比例混合，涡旋30s，置于37℃条件下孵育30分钟得到CLS与siRNA不同电荷比的CLS/R8/siRNA复合物。
为了比较的目的，还制备了CLS/siRNA复合物，其中将实施例1制备的CLS与本实施例制备的20μL siRNA溶液按照CLS/siRNA电荷比为2、4、6、8、10、12的比例等体积混合，涡旋30s，置于37℃条件下孵育30分钟得到不同CLS/siRNA电荷比的CLS/siRNA复合物。如下实施CLS/siRNA电荷比的计算：由于DOTAP的一个氨基氮带一个正电荷，siRNA的一个磷酸根带一个负电荷，16.67μM的CLS含5μM的DOTAP，即1μM的CLS含0.3μM的正电荷，1μM的siRNA含42μM的负电荷，因此，CLS与siRNA的电荷比＝(CLS的μM值×0.3)/(siRNA的μM值×42)＝0.007×CLS(μM)/siRNA(μM))。
将上述制备的R8/siRNA复合物、CLS/siRNA复合物和CLS/R8/siRNA复合物分别小心加入琼脂糖凝胶加样孔中，并设置游离siRNA对照组，进行电泳。电泳结束后，用GelRed工作液(Biotium Inc.)避光泡染30分钟，在紫外302nm条件下进行凝胶成像。结果如图1所示。
图1中小图A的结果表明，单独的寡聚精氨酸R8无法完全压缩基因；图1中小图C和D的结果表明，单独的阳离子脂质体CLS虽然能够一定程度地压缩基因，但是CLS/siRNA复合物的粒径较大；图1中小图E和F的结果表明，阳离子脂质体和寡聚精氨酸R8的组合能够有效地共同压缩基因。
实施例3
阳离子脂质体和带正电的多聚氨基酸压缩药物后含有药物的脂质体复合物的粒径和电势表征：
根据实施例2中所述方法分别制备R8/siRNA复合物、CLS/siRNA复合物和CLS/R8/siRNA复合物，并进行粒径与电势测定。
采用动态光散射技术直接测量粒径。粒径测定结果如图1、图3和图12所示。在图12中，来自动态光散射测量的结果以横坐标为粒径，纵坐标为光强度显示，由各粒子所贡献的光强度计算而得。
由图3中的小图B可见，当寡聚精氨酸R8/siRNA的电荷比为5、10、20时，物理复合物R8/siRNA的粒径约为200nm。当寡聚精氨酸R8/siRNA的电荷比为5，阳离子脂质体CLS与siRNA的电荷比为4时，使用阳离子脂质体和寡聚精氨酸的组合压缩siRNA后，含有siRNA的脂质体复合物(CLS/R8/siRNA)的粒径测定结果如图12所示，粒径减小至108nm。当寡聚精氨酸R8/siRNA的电荷比为5时， 89WPenetratin修饰的阳离子脂质体89W-CLS与siRNA的电荷比为4时，使用阳离子脂质体和寡聚精氨酸的组合压缩siRNA后， 89WPenetratin修饰的含有siRNA的脂质体复合物(89W-CLS/R8/siRNA)的粒径与CLS/R8/siRNA相近，为105nm。 89WPenetratin修饰的含有siRNA的脂质体复合物的最高光强度相对于CLS/R8/siRNA的最高光强度变低，这是因为前者的粒径分布范围变宽所致。
当寡聚精氨酸R8/siRNA的电荷比为5，阳离子脂质体CLS或 89WPenetratin修饰的阳离子脂质体89W-CLS与siRNA的电荷比为4时，分别制备了R8/siRNA复合物、CLS/siRNA/R8复合物和89W CLS/siRNA/R8复合物，采用ζ电势分析仪，在23℃使用5V/cm的电场强度、0.4cm的电极间距在水中测量ζ电势。ζ电势测定结果如图13所示。
由图13可见，物理复合物R8/siRNA、脂质体复合物CLS/R8/siRNA和 89WPenetratin修饰的脂质体复合物的ζ电势分别为22mV、43mV和38mV。
实施例4
含有药物的脂质体复合物的稳定性表征之一：
根据实施例2中所述方法制备DEPC处理水中的各CLS/R8/siRNA复合物，高速离心后观察各含有药物的脂质体复合物(即，脂质体制剂)的稳定性。
具体而言，将根据实施例2中所述方法制备的各CLS/R8/siRNA复合物在12000×g的速度下离心20分钟，观察各脂质体制剂的沉淀情况。
结果如图3中的小图C所示。由图3中的小图C可见，当寡聚精氨酸R8/siRNA的电荷比大于5，且当阳离子脂质体CLS与siRNA的电荷比为4时，CLS/R8/siRNA复合物经高速离心后没有明显的沉淀析出，这表明阳离子脂质体和寡聚精氨酸共同压缩siRNA，能够形成稳定的脂质体制剂。
实施例5
含有药物的脂质体复合物的稳定性表征之二：
将根据实施例2中所述方法制备的DEPC处理水中的各CLS/R8/siRNA复合物在12000×g的速度下离心20分钟，小心地将各上清加入琼脂糖凝胶加样孔中，并设置游离siRNA对照组，进行电泳。电泳结束后，用GelRed工作液避光泡染30分钟，在紫外302nm条件下进行凝胶成像。结果如图3中的小图D所示。
由图3中的小图D可见，当寡聚精氨酸R8/siRNA的电荷比大于5，且当阳离子脂质体CLS与siRNA的电荷比为4时，CLS/R8/siRNA复合物经高速离心后，CLS/R8/siRNA仍保留在上清溶液中，而且没有游离的siRNA电泳出来。这表明阳离子脂质体和寡聚精氨酸共同压缩siRNA，能够形成稳定的脂质体制剂。
实施例6
表面修饰的脂质体药物制剂的配制：
对脂质体药物制剂使用穿膜肽进行表面修饰，获得表面修饰的药物制剂。
i.穿膜肽-PEG-磷脂的制备：
由DSPE-聚乙二醇马来酰亚胺(DSPE-PEG 3400-马来酰亚胺)(Laysan Bio，146-123)与末端修饰有半胱氨酸的penetratin衍生物( 89WPenetratin-Cys)经一步反应制备得到穿膜肽-PEG-DSPE。
具体而言，将20mg DSPE-PEG 3400-马来酰亚胺溶于1mL N,N-二甲基甲酰胺中，在搅拌状态下向其中加入溶于10mL磷酸缓冲溶液(10mM,pH7.2)介质中的18mg  89WPenetratin-Cys，在25℃的条件下继续搅拌过夜使反应完全。反应结束后，将所得混合物在冰浴条件下置于纯水中透析2天，冷冻干燥得到白色絮状物，为 89WPenetratin-PEG 3400-DSPE。
其中，所述多肽 89WPenetratin是penetratin的一种衍生物，具体氨基酸序列为RQIKIWFWWRRMKWKK(SEQ ID NO:29)。穿膜肽-PEG-DSPE中的穿膜肽也可以使用其他penetratin衍生物，例如，使用表1中所示的其他penetratin衍生物，例如，对penetratin第2位谷氨酰胺和/或第8位谷氨酰胺和/或第9位天冬酰胺进行了疏水性氨基酸突变的产物(也可参见中国发明专利申请CN201710414334.7)。
为了便于与聚乙二醇末端基团反应，穿膜肽 89WPenetratin或其他penetratin衍生物的N-端或C-端可以额外添加一个半胱氨酸残基(cysteine,C)。
ii.表面修饰的脂质体药物制剂的配制：
在总的脂质体膜材料中，DOTAP:DOPE:胆固醇:(mPEG 2000-DSPE和 89WPenetratin-PEG 3400-DSPE)的摩尔比为约28.5:28.5:38:5。
称取3.5mg的DOTAP(上海艾韦特医药科技有限公司,132172-61-3)、3.72mg的DOPE(上海艾韦特医药科技有限公司,4004-05-1)、2.59mg的胆固醇(Sigma-Aldrich,C8667)。将所述量的DOTAP、DOPE、胆固醇置于50mL圆底烧瓶中，加入1mL氯仿，溶解作为脂质膜材料的所述DOTAP、DOPE、胆固醇，即16.67μM的CLS含有5μM的DOTAP，5μM的DOPE， 6.67μM的DOPE。将所得溶液通过旋转蒸发仪(上海申生科技有限公司，R201L)除去氯仿，获得均匀的干燥脂质膜。
将所得的干燥脂质膜用1mL 5％的葡萄糖水溶液通过37℃水浴超声水化10分钟。待脂质膜水化完全后，使用微型挤出器(Hamilton,81320)挤出脂质体(过核孔膜的顺序依次是200nm、100nm和50nm)，获得了阳离子脂质体(文中也称为CLS)。
将4mg的寡聚精氨酸R8(上海昕浩生物科技有限公司)溶于DEPC处理水(大连美仑生物技术有限公司，MA0018)中制备4mg/mL寡聚精氨酸R8溶液，将33μg的FAM标记的siRNA(在本实施例中，使用的是针对c-myc的siRNA，其有义链(5'-3')为FAM-AACGUUAGCUUCACCAACAdTdT(SEQ ID NO:79)，即，5'修饰有FAM；反义链(5'-3')为UGUUGGUGAAGCUAACGUUdTdT(SEQ ID NO:80))溶于125μL的DEPC处理水中制备20μM的FAM-siRNA溶液。
将寡聚精氨酸R8溶液与20μM的FAM-siRNA溶液按照电荷比为5的比例等体积混合，涡旋30s，置于37℃条件下孵育30分钟得到复合物R8/FAM-siRNA。取8μL R8/FAM-siRNA复合物，与上述制备的阳离子脂质体CLS按照脂质体与FAM-siRNA的电荷比为4的比例混合，涡旋30s，得CLS/R8/FAM-siRNA复合物。
对于占脂质体总摩尔数为5％的mPEG 2000-DSPE和 89WPenetratin-PEG 3400-DSPE，分别配制了 89WPenetratin-PEG 3400-DSPE的摩尔数占mPEG 2000-DSPE和 89WPenetratin-PEG 3400-DSPE的摩尔数为0％、20％、40％、60％、80％、100％的混合物。即， 89WPenetratin-PEG 3400-DSPE摩尔数分别占总的脂质体膜材料摩尔数的0％、1％、2％、3％、4％、5％。
将所制得的CLS/R8/FAM-siRNA复合物分别与 89WPenetratin-PEG 3400-DSPE的摩尔数占mPEG 2000-DSPE和 89WPenetratin-PEG 3400-DSPE的摩尔数为0％、20％、40％、60％、80％、100％的混合物混合后置于55℃孵育半小时，获得表面修饰有0％、1％、2％、3％、 4％、5％比例的穿膜肽的脂质体药物制剂，即89W-CLS/R8/FAM-siRNA。
实施例7
定量评价细胞对表面修饰有不同比例穿膜肽的药物制剂的摄取：
将U87细胞(人恶性胶质母细胞瘤细胞，购自ATCC)培养于DMEM完全培养基(添加10％FBS和1％青霉素-链霉素)中。取生长状态良好的U87细胞重悬于DMEM完全培养基中，按5×10 5个细胞/孔接种到6孔板中，每孔1mL，接种后每天更换新鲜培养液一次，培养2～3天后进行实验。
弃去培养液后，无菌PBS洗U87细胞三次，加入1mL实施例6制备的表面修饰有不同比例的穿膜肽的脂质体药物制剂(包载FAM标记的siRNA)的无血清DMEM培养基，37℃、5％CO 2条件下孵育4h。孵育结束后弃去培养液，用含0.02mg/mL肝素钠的PBS缓冲溶液洗去吸附在孔上或细胞表面的正电性物质。用胰酶将细胞消化下来，重悬于200μL无菌PBS缓冲溶液中，吹打均匀后对每个样品计数细胞，取10 4个细胞进行流式细胞术检测。使用无血清DMEM培养基孵育的细胞作为阴性对照组。所有实验均一式三份实施，结果如图4所示(平均值±SD，n＝3，*p<0.05，*表示两组之间在统计学上有显著性差异；**p<0.01，***p<0.001，**和***表示两组之间在统计学上有非常显著的差异)。图4横坐标上的“0％、1％、2％、3％、4％、5％”分别表示表面修饰有0％、1％、2％、3％、4％、5％比例的穿膜肽的脂质体药物制剂，即89W-CLS/R8/siRNA。
由图4可见， 89WPenetratin-PEG 3400-DSPE摩尔数占总的脂质体膜材料摩尔数的2％、3％、4％时，细胞对表面修饰有所述比例穿膜肽的药物制剂的摄取效果优于市售的基因转染试剂Lipo2000。
实施例8
定量评价细胞对使用不同配比的脂质体膜材料制备的药物制剂的摄取：
将U87细胞(人恶性胶质母细胞瘤细胞，购自ATCC)培养于DMEM 完全培养基(添加10％FBS和1％青霉素-链霉素)中。取生长状态良好的U87细胞重悬于DMEM完全培养基中，按5×10 5个细胞/孔接种到6孔板中，每孔1mL，接种后每天更换新鲜培养液一次，培养2～3天后进行实验。
按照实施例6所述方法制备脂质体膜材料配比如表2所示的脂质体药物制剂(包载FAM标记的siRNA)。
表2.脂质体膜材料的不同配比(以摩尔份数表示，％)
Figure PCTCN2020087279-appb-000003
将培养的U87细胞弃去培养液后，无菌PBS洗三次。每孔分别加入含有配方1、配方2、配方3制备的脂质体药物制剂的1ml无血清DMEM培养基中，37℃、5％CO 2条件下孵育4h。孵育结束后弃去培养液，用含0.02mg/mL肝素钠的PBS缓冲溶液洗去吸附在孔上或细胞表面的正电性物质。用胰酶将细胞消化下来，重悬于200μL无菌PBS缓冲溶液中，吹打均匀后对每个样品计数细胞，取10 4个细胞进行流式细胞术检测。使用无血清DMEM培养基孵育的细胞作为阴性对照组。所有实验均一式三份实施，结果如图5所示(平均值±SD，n＝3，*p<0.05，*表示两组之间在统计学上有显著性差异；**p<0.01，***p<0.001，**和***表示两组之间在统计学上有非常显著的差异)。
由图5可见，使用配方1、配方2、配方3制备的脂质体药物制剂均能够被细胞摄取，与对照组比较存在显著性差异，并且其中细胞对使用配方2制备的脂质体药物制剂的摄取效果最佳。
实施例9
使用组分(iv)不同配比的脂质体膜材料制备的药物制剂的粒径：
按照实施例6所述方法，在脂质体膜材料的组分(i)：组分(ii)：组分(iii)的摩尔比为3：3：4时，制备组分(iv)的配比如表3所示的脂质体药物制剂(包载FAM标记的siRNA)。
表3.组分(iv)的不同配比(以摩尔份数表示，％)
Figure PCTCN2020087279-appb-000004
获得了组分(iv)占脂质体膜材料的1％、5％、8％、10％摩尔份数的脂质体药物制剂。对所述脂质体药物制剂进行粒径测定。粒径测定结果如图6所示。图6横坐标上的“1％、5％、8％、10％”分别表示组分(iv)占脂质体膜材料的1％、5％、8％、10％摩尔份数的脂质体药物制剂。
由图6可见，当组分(iv)占脂质体膜材料的1％－10％摩尔份数时，脂质体药物制剂的粒径在100nm－200nm之间；且随着组分(iv)占脂质体膜材料的摩尔份数的提高，粒径呈现增大的趋势。
实施例10
定量评价细胞对使用阳离子脂质体/siRNA的不同电荷比制备的药物制剂的摄取：
将U87细胞(人恶性胶质母细胞瘤细胞，购自ATCC)培养于DMEM完全培养基(添加10％FBS和1％青霉素-链霉素)中。取生长状态良好的U87细胞重悬于DMEM完全培养基中，按5×10 5个细胞/孔接种到6孔板中，每孔1mL，接种后每天更换新鲜培养液一次，培养2～3天后进行实验。
按照实施例6所述方法，将寡聚精氨酸R8溶液与20μM的FAM-siRNA溶液按照电荷比为5的比例等体积混合，涡旋30s，置于37℃条件下孵育30分钟得到复合物R8/FAM-siRNA。取8μL R8/FAM-siRNA复合物，与制备的阳离子脂质体CLS按照脂质体与FAM-siRNA的电荷比为2、4、6、8、10、12的比例混合，涡旋30s，获得阳离子脂质体/siRNA的不同电荷比的CLS/R8/FAM-siRNA复合物。
进一步地，按照实施例6所述方法，获得表面修饰有3％穿膜肽的脂质体药物制剂，即89W-CLS/R8/FAM-siRNA复合物。
将培养的U87细胞弃去培养液后，无菌PBS洗三次。每孔分别加入含有阳离子脂质体/siRNA的电荷比为2、4、6、8、10、12的药物制剂的1ml无血清DMEM培养基，37℃、5％CO 2条件下孵育4h。孵育结束后弃去培养液，用含0.02mg/mL肝素钠的PBS缓冲溶液洗去吸附在孔上或细胞表面的正电性物质。用胰酶将细胞消化下来，重悬于200μL无菌PBS缓冲溶液中，吹打均匀后对每个样品计数细胞，取10 4个细胞进行流式细胞术检测。使用无血清DMEM培养基孵育的细胞作为阴性对照组。所有实验均一式三份实施，结果如图2所示(平均值±SD，n＝3，*p<0.05，*表示两组之间在统计学上有显著性差异；**p<0.01，***p<0.001，**和***表示两组之间在统计学上有非常显著的差异)。
由图2可见，使用阳离子脂质体/siRNA的电荷比为2、4、6、8、10、12制备的药物制剂均能够被细胞摄取，与对照组比较存在显著性差异，并且其中细胞对使用阳离子脂质体/siRNA的电荷比为4时制备的脂质体药物制剂的摄取效果最佳。
实施例11
表面修饰有不同比例穿膜肽的药物制剂对细胞的转染效果评价：
首先，使用35mm Dimple NanoCulture Dish培养细胞，观察细胞的成球能力和形态，建立细胞球模型。
具体而言，取1.5mL的DMEM完全培养基加入35mm Dimple NanoCulture Dish(JSR Corporation提供的细胞球培养板)中，然后将Dish放置在冷冻离心机内离心(Dish里只有培养基，没有细胞)，离心速度为1000×g，离心时间为5分钟。离心结束后，将Dish放在超净台上静置30分钟。
取生长状态良好的各种细胞，消化离心后，加入培养基调整细胞浓度为5×10 5个/mL，用移液枪吹打使细胞分散均匀，缓慢小心向Dish中加入1.5mL细胞悬液，然后放操作台上静置20分钟，放到37℃的孵箱内培育。隔天换液，取出Dish，小心吸出1.5mL的培养基(悬浮细胞与贴壁细胞的操作相同，因为换液时细胞已经初步形成细胞球，沉在Dish的底部)，加入新鲜的1.5mL DMEM完全培养基。在倒置显微镜下观察相应细胞球的形态，其中A549细胞是人非小细胞肺癌细胞，Caco-2是人克隆结肠腺癌细胞，Rb-1是人视网膜神经胶质瘤细胞，U87是人恶性胶质母细胞瘤细胞，293T是人肾上皮细胞，所述细胞均购自ATCC。A549和Caco-2是贴壁细胞，且贴壁性较强，而U87和293T贴壁性较弱的贴壁细胞，Rb-1则是悬浮细胞。结果如图7所示。在图7中，样品1和样品2分别是同一种细胞的不同放大倍数，且样品3和样品4是样品2的重复实验孔。
由图7可见，A549和Caco-2由于贴壁性较好，容易贴在壁上生长不易成球，Rb-1虽然是悬浮细胞，但细胞容易聚集成球，且处理过程中非常容易松散。相比较而言，U87和293T的成球能力最好，形态大致为球型，且大小均一，适合作为建立的细胞球模型进一步的探索。
由图8的结果可见，不同多肽对细胞球的渗透能力有显著差异，而且利用35mm Dimple NanoCulture Dish建立的细胞球模型能够对不同多肽的渗透能力进行区分。注：图8中的“S8”为8聚丝氨酸，作为阴性对照；“PE”为野生型Penetratin；89W为对penetratin第8位谷氨酰胺和第9位天冬酰胺进行了色氨酸突变的产物；289W为对penetratin第2位谷氨酰胺、第8位谷氨酰胺和第9位天冬酰胺进行了色氨酸突变的产物。野生型Penetratin的衍生物对细胞球的渗透能力显著提高。
接下来，使用293T细胞球来评估包载有GFP-siRNA的表面修饰有不同比例穿膜肽的药物制剂对细胞的转染效果。
与上述实施例6类似地，制备表面修饰有不同比例穿膜肽的GFP-siRNA脂质体药物制剂，其中使用的GFP-siRNA有义链(5'-3')为GCAUGUACCACGAGUCCAATT(SEQ ID NO:81)；反义链(5'-3')为UUGGACUCGUGGUACAUGCTT(SEQ ID NO:82)。具体而言，将寡聚精氨酸R8溶液与20μM的GFP-siRNA溶液按照电荷比为5的比例等体积混合，涡旋30s，置于37℃条件下孵育30分钟得到复合物R8/GFP-siRNA。取8μL R8/GFP-siRNA复合物，与制备的阳离子脂质体CLS按照脂质体与FAM-siRNA的电荷比为4的比例混合，涡旋30s，获得CLS/R8/GFP-siRNA复合物。
进一步地，按照实施例6所述方法，获得表面修饰有1％、3％、5％穿膜肽的脂质体药物制剂，即89W-CLS/R8/GFP-siRNA复合物。
向293T细胞球模型中加入包载有400nM GFP-siRNA的脂质体药物制剂的无血清DMEM溶液，37℃、5％CO 2条件下孵育4h，然后弃去药液，加入新鲜的DMEM完全培养基继续孵育24小时，用PBS缓冲溶液洗3次，在激光共聚焦显微镜下观察GFP荧光情况(文中也称为GFP-293T细胞球)。使用无血清DMEM培养基孵育的细胞作为阴性对照组。所有实验均一式三份实施，结果如图9所示(平均值±SD，n＝3，*p<0.05，*表示两组之间在统计学上有显著性差异；**p<0.01，***p<0.001，**和***表示两组之间在统计学上有非常显著的差异)。
类似地，进行了包载有400nM GFP-siRNA的脂质体药物制剂对U87细胞球的转染效果评价(文中也称为GFP-U87细胞球)。结果如图10所示。
图9和图10中的“1％、3％、5％”分别表示表面修饰有1％、3％、5％比例的穿膜肽的脂质体药物制剂，即89W-CLS/R8/siRNA。
图9中，使用35mm Dimple NanoCulture Dish建立GFP-293T细胞球，293T细胞不是肿瘤细胞， 89WPenetratin-PEG-DSPE在脂质体膜材料中所占摩 尔比例越高，脂质体药物对细胞球的渗透程度越高。图10中，使用35mm Dimple NanoCulture Dish建立GFP-U87细胞球，U87细胞是脑胶质瘤细胞， 89WPenetratin-PEG-DSPE在脂质体膜材料中所占摩尔比例达到3％－5％时，脂质体药物对细胞球具有较高的渗透能力。
实施例12
包含不同结构类型带正电的多聚氨基酸的药物制剂对LUC-U87细胞的转染效果的定性和定量评价：
选用R8、R10、R12、c-R8、c-R10和c-R12等寡聚精氨酸作为不同结构类型带正电的多聚氨基酸，以所述寡聚精氨酸分别与siRNA的电荷比为5，CLS与LUC-siRNA的电荷比为4，按照实施例6制备含有不同寡聚精氨酸的表面修饰有3％比例穿膜肽的脂质体药物制剂，其中使用的LUC-siRNA有义链(5'-3')为GCUGCACUCUGGCGACAUUTT(SEQ ID NO:83)；反义链(5'-3')为AAUGUCGCCAGAGUGCAGCTT(SEQ ID NO:84)。
取生长状态良好的U87细胞(购自ATCC)按5×10 4个细胞/孔分别接种到48孔板中，接种后每天换液一次，培养2～3天后进行实验。弃去DMEM完全培养基后，无菌PBS洗三次，分别加入包载400nM LUC-siRNA的脂质体药物制剂的无血清DMEM培养基，37℃、5％CO 2条件下孵育4h，然后弃去药液，加入DMEM完全培养基继续孵育24小时(文中也称为LUC-U87细胞)，用含0.02mg/mL肝素钠的PBS缓冲溶液洗去正电性吸附物质，加入萤火虫萤光光素酶底物，在小动物活体荧光/生物发光成像系统(IVIS Spectrum)内测定荧光强度。利用小动物活体光学成像系统的目的区域(Region of interest，ROI)定量圈选功能进行定性分析。结果见图11所示。
此实施例是为了探究不同结构类型带正电的多聚氨基酸的基因沉默效果，由图11可以发现，改变脂质体药物制剂中的寡聚精氨酸的聚合度和结构对基因的沉默作用没有显著影响。
实施例13
包载siRNA的递送载体对bEnd.3细胞和U87细胞的促凋亡能力评价：
取DMEM培养基中对数期生长状态良好的bEnd.3细胞和U87细胞(均购自ATCC)按5×10 4/孔的浓度分别铺于12孔板中，在37℃、5％CO2条件下培养至细胞单层铺满板底。
弃去培养液，无菌PBS缓冲液洗3次后，加入400μL含400nmol/L siRNA的实施例6制备的脂质体制剂(89W-CLS/R8/siRNA)，即，寡聚精氨酸与siRNA的电荷比为5，CLS与siRNA的电荷比为4，表面修饰有3％比例 89WPenetratin-PEG 3400-DSPE的脂质体药物制剂，细胞培养箱中孵育6h后，弃去药液，无菌PBS缓冲液洗3次后，加入完全培养基1mL继续培养18h，之后收集细胞，先用PBS清洗，无EDTA的胰酶消化1分钟，DMEM培养液终止消化后收集细胞至离心管中，按照细胞凋亡检测试剂盒(南京凯基生物科技发展有限公司，KGA108)的步骤对细胞进行双染，然后用流式细胞分析仪检测。使用无血清DMEM培养基孵育的细胞作为阴性对照组。所有实验均一式三份实施，结果如图14中的小图A和B所示(平均值±SD，n＝3，*p<0.05，*表示两组之间在统计学上有显著性差异；**p<0.01，***p<0.001，**和***表示两组之间在统计学上有非常显著的差异)。
结果表明，本发明设计的递送载体能够特异性促进肿瘤细胞U87的凋亡，而对正常细胞bEnd.3细胞的生长没有较大的影响。
实施例14
包载siRNA的递送载体对bEnd.3细胞和U87细胞的细胞毒性评价：
取对数期生长状态良好的bEnd.3(购自ATCC)和U87细胞，用DMEM完全培养基培养，按5×10 3个细胞/cm 2浓度分别铺于96孔平底板的中间60孔中，边缘孔用无菌PBS缓冲溶液填充，在37℃、5％CO 2条件下培养至细胞单层铺满板底，弃去培养液，无菌PBS缓冲液洗3次后，加入200μL含400nM siRNA的递送载体的培养液，细胞培养箱中孵育6h后，弃去药液， 无菌PBS缓冲液洗3次后，加入完全培养基继续培养12h。之后每孔加入20μL MTT溶液(5mg/mL)，继续培养4h后，小心弃去液体，PBS缓冲液洗三次后每孔加入150μL DMSO，在摇床上低速振荡(50转/分钟)20分钟后，于酶标仪(BioTek，PowerWave XS)OD 490nm处测定各孔吸光度值。同时设置空白调零孔(孔中仅添加DMEM完全培养基、MTT和DMSO)和阴性对照孔(孔中仅添加细胞、DMEM完全培养基、MTT和DMSO)。所有实验均一式三份实施，结果如图14中的小图C和D所示(平均值±SD，n＝3，*p<0.05，*表示两组之间在统计学上有显著性差异；**p<0.01，***p<0.001，**和***表示两组之间在统计学上有非常显著的差异)。
结果表明，相比商品化的阳离子基因递送载体Lipofectamine 2000(Lipo2000，Invitrogen)，本发明设计的递送载体对bEnd.3细胞和U87细胞生长状态影响较小，在所考察的浓度条件下未见细胞毒性。
实施例15
DiD细胞膜荧光探针标记的基因递送载体经鼻给药后的体内分布情况：
首先制备了亲脂性荧光染料DiD标记的脂质体，用于对脂质体进行示踪。DiD细胞膜红色荧光探针购自大连美仑生物技术有限公司,MB6190。如实施例1所述类似地，称取膜材，按试剂盒说明书所述加入DiD染料，一同溶解在氯仿中，制备DiD标记的CLS/R8/siRNA。另外，如实施例6所述类似地，制备寡聚精氨酸与siRNA的电荷比为5，CLS与siRNA的电荷比为4，表面修饰有3％比例 89WPenetratin-PEG 3400-DSPE的DiD标记的脂质体药物制剂89W-CLS/R8/siRNA。
将12只ICR小鼠(20g，雄性，6周龄)分为3个组(89W-CLS/R8/siRNA组、CLS/R8/siRNA组和生理盐水(N.S.)组)，每只小鼠以鼻腔给药的方式给予DiD标记的核酸递送载体30μL左右(DiD浓度为0.01μM)，2.5h后将动物安乐死，经灌流、固定后，收集动物的主要脏器，使用小动物活体成像系统检测各脏器荧光分布。结果如图15所示。
结果表明，相比CLS/R8/siRNA组，89W-CLS/R8/siRNA组在脑部的荧光强度更大，穿膜肽的修饰能够显著增强递送载体的组织穿透能力，进而增强其入脑能力，提高基因在脑部的蓄积量。
实施例16
探究DiD细胞膜荧光探针标记的载体递送FAM-siRNA的入胞机制：
如实施例1所述类似地，称取膜材，按试剂盒说明书所述加入DiD细胞膜荧光探针(大连美仑生物技术有限公司,MB6190)，制备DiD细胞膜荧光探针标记的CLS，即CLS/DiD。按照实施例6所述方法类似地，制备DiD和/或FAM荧光标记的各药物制剂。
将U87细胞(购自ATCC)培养于DMEM完全培养基(添加10％FBS和1％青-链霉素)中。取生长状态良好的U87细胞重悬于DMEM完全培养基中，按5×10 5个细胞/孔接种到6孔板中，每孔1mL，接种后每天更换新鲜培养液一次，培养2～3天后进行实验。
将培养的U87细胞弃去培养液后，无菌PBS洗三次。每孔分别加入含有1μM siRNA的不同荧光标记的药物制剂的1mL无血清DMEM培养基，37℃、5％CO 2条件下孵育4h。孵育结束后弃去培养液，用含0.02mg/mL肝素钠的PBS缓冲溶液洗去吸附在孔上或细胞表面的正电性物质。用胰酶将细胞消化下来，重悬于200μL无菌PBS缓冲溶液中，吹打均匀后对每个样品计数细胞，取10 4个细胞进行流式细胞术检测。实验中设置与空白的无血清DMEM培养基孵育的细胞作为阴性对照组；另外，设置了两组阳性对照组，分别为只含有FAM的89W-CLS/R8/FAM-siRNA，以及只含有DiD的89W-CLS/DiD/R8/siRNA；还设置了如下制剂组：R8/FAM-siRNA组、CLS/DiD组、CLS/DiD/R8/FAM-siRNA组、89W-CLS/DiD/R8/FAM-siRNA组。所有实验均一式三份进行。结果如图16所示。
由图16中的小图D可见，单独的R8压缩siRNA形成的药物制剂入胞量很少，然而CLS和R8共同压缩siRNA形成的药物制剂能够成功入胞(见 小图F)，且89W修饰的CLS和R8共同压缩siRNA形成的药物制剂能够显著提高细胞对基因载体的摄取量(见小图G)。通过图16的小图B和C以及小图G可见，细胞对89W-CLS/R8/FAM-siRNA药物制剂的摄取呈现DiD和FAM双荧光阳性结果，由此可以推测CLS和siRNA是以复合物的形式共同入胞的，并非以游离的形式被摄取。
实施例17
DiD细胞膜荧光探针标记的基因递送载体在U87原位脑瘤模型鼠中经鼻给药后的体内分布情况：
首先制备亲脂性荧光染料DiD标记的脂质体，用于对脂质体进行示踪。DiD细胞膜红色荧光探针购自大连美仑生物技术有限公司,MB6190。如实施例15所述类似地，称取膜材，按试剂盒说明书所述加入DiD染料，一同溶解在氯仿中，制备DiD标记的CLS/R8/siRNA。另外，如实施例6所述，制备寡聚精氨酸与siRNA的电荷比为5，CLS与siRNA的电荷比为4，表面修饰有3％比例 89WPenetratin-PEG 3400-DSPE的DiD标记的脂质体药物制剂89W-CLS/R8/siRNA。
取对数生长期的U87细胞，计数，用PBS缓冲液重悬细胞。每只BALB/c裸鼠(上海西普尔-必凯试验动物有限公司，中国)接种6×10 5个U87细胞(分散于5μL PBS缓冲液中)。将裸鼠用7％水合氯醛麻醉，脑立体定位仪固定，采用微量注射器将细胞接种于纹状体部位(前囟向前0.6mm，向右1.8mm，纵深3mm)，构建U87原位脑瘤模型。
将9只接种U87肿瘤细胞18天的荷U87脑原位瘤小鼠随机分为3组(89W-CLS/R8/siRNA组、CLS/R8/siRNA组和生理盐水组)，每只小鼠以鼻腔给药的方式给予DiD标记的核酸递送载体30μL(DiD浓度为0.01μM)，2.5h后将动物安乐死，经灌流、固定后，收集动物的脑部组织及肿瘤，进行冷冻切片处理，并在荧光显微镜(德国Leica)上观察DiD荧光信号的分布，结果如图17所示，标尺＝100μm。
结果表明，相比CLS/R8/siRNA组，89W-CLS/R8/siRNA组在肿瘤部位的荧光强度更高，穿膜肽的修饰能够显著增强递送载体的组织穿透能力，进而增强其入脑能力，提高基因在肿瘤部位的蓄积量。
实施例18
包载siRNA的递送载体的抗U87脑原位瘤的药效学评价：
按照实施例6所述方法，制备脂质体药物制剂，即CLS/R8/siRNA和89W-CLS/R8/siRNA复合物，按照商品化的阳离子基因递送载体Lipofectamine 2000(Lipo2000，Invitrogen)说明书制备Lipo2000/siRNA复合物。
按照实施例17所述方法构建U87原位脑瘤模型，U87脑原位瘤接种后将小鼠随机分为5组(n＝10)：生理盐水组、siRNA组、Lipo2000/siRNA组、CLS/R8/siRNA组和89W-CLS/R8/siRNA组。接种U87肿瘤细胞后第5天开始鼻腔给药(47μg/kg siRNA)，每天给药一次，共给药22次，记录小鼠体重及生存期，绘制体重变化曲线及生存曲线，结果如图18所示(*p<0.05，*表示两组之间在统计学上有显著性差异；**p<0.01，**表示两组之间在统计学上有非常显著的差异)。
结果表明，生理盐水组、siRNA组、Lipo2000/siRNA组、CLS/R8/siRNA组的中位生存期分别为23.5天、21.5天、24.5天和26天，89WP-CLS/R8/siRNA组则成功将中位生存时间延长到29天，并且生存曲线与其他各组均没有交叉，实现了最显著的抗脑胶质瘤效果，这些数据证实了89WP-CLS/R8/siRNA能够将药物递送至脑部肿瘤，发挥抗肿瘤效果，其独特递送优势可以转化为更高的治疗功效。
实施例19
包载siRNA的递送载体在U87原位脑瘤模型鼠中的体内促凋亡能力评价：
按照实施例18所述方法制备脂质体药物制剂，构建U87原位脑瘤模型，并进行鼻腔给药。最后一次给药后2天(第28天)，随机选择每组3只小鼠并对其实施安乐死，取脑部肿瘤进行免疫荧光组化分析。用4％多聚甲醛固定肿瘤，包埋在石蜡中并切成薄片。通过TUNEL染色检测凋亡细胞，通过CaseViewer软件分析肿瘤部位的TUNEL荧光强度，结果如图19所示，标尺＝20μm。
结果表明，在TUNEL染色切片中，绿色荧光的强度表示发生凋亡的肿瘤细胞的数量，Lipo2000/siRNA组，CLS/R8/siRNA组和89WP-CLS/R8/siRNA组均能引起肿瘤细胞发生凋亡，其中，89WP-CLS/R8/siRNA组的荧光强度最高，即细胞凋亡比例最高，表明本发明设计的递送载体能够在体内通过诱发肿瘤细胞的凋亡发挥抗肿瘤效果。
实施例20
包载siRNA的递送载体的体内鼻粘膜毒性评价：
将18只SD大鼠(上海灵畅生物科技有限公司，中国)随机分为6组(n＝3)，生理盐水组(阴性对照)，1％脱氧胆酸钠组(阳性对照)，siRNA组，CLS/R8/siRNA组，89WP-CLS/R8/siRNA组和Lipo2000/siRNA组。按照实施例6所述方法，制备脂质体药物制剂，即CLS/R8/siRNA和89W-CLS/R8/siRNA复合物，按照商品化的阳离子基因递送载体Lipofectamine 2000(Lipo2000，Invitrogen)说明书制备Lipo2000/siRNA复合物。连续7天向SD大鼠左侧鼻腔进行鼻腔给药50μL制剂(包含或不包含160nM siRNA)，最后一次给药后24h，处死大鼠，分离左鼻腔鼻粘膜并用生理盐水洗涤。样品用4％多聚甲醛固定，并切片以进行苏木精-伊红(HE)染色，结果如图20所示，标尺＝50μm。
结果表明，阳性对照组(脱氧胆酸钠组和Lipo2000/siRNA组)的鼻粘膜上皮细胞出现较为明显的形态变化，并伴有鼻纤毛脱落(红色箭头)，但是在生理盐水组，siRNA组，CLS/R8/siRNA组和89WP-CLS/R8/siRNA组中， 鼻粘膜上皮细胞形态完整且排列整齐，表明本发明设计的递送载体对鼻粘膜上皮细胞没有明显的毒性，可以认为是经鼻入脑递送的安全载体。
尽管已经出于说明本发明的目的显示了某些代表性实施方案和细节，但是本领域技术人员显而易见的是可以对它们进行多种变化和修改而不脱离主题发明的范围。在这个方面，本发明范围仅由以下权利要求限定。

Claims (13)

  1. 一种药物制剂，其是使用药物递送载体制备的药物制剂，其中所述药物递送载体包含穿膜肽修饰的脂质体和聚合物，其中所述聚合物是带正电的多聚氨基酸，其中所述穿膜肽是具有下述氨基酸序列的penetratin或penetratin的衍生物：
    R X 1 IKIWF X 2X 3 RRMKWKK
    其中，X 1、X 2和X 3独立地选自天然来源的氨基酸谷氨酰胺(Q)、天冬酰胺(N)、丙氨酸(A)、缬氨酸(V)、亮氨酸(L)、异亮氨酸(I)、脯氨酸(P)、苯丙氨酸(F)、色氨酸(W)、甲硫氨酸(M)和非天然来源的氨基酸α-氨基丁酸、α-氨基戊酸、α-氨基己酸、α-氨基庚酸，例如，X 1、X 2和X 3独立地选自疏水性氨基酸；
    所述药物制剂是通过将药物递送载体中包含的带正电的多聚氨基酸和药物分子混合形成聚合物-药物的物理复合物，并将所述物理复合物与所述穿膜肽修饰的脂质体混合形成的包含聚合物-药物的物理复合物的脂质体复合物，其粒径介于50nm至300nm之间，优选地80nm至150nm之间。
  2. 根据权利要求1所述的药物制剂，其中药物分子选自肽类药物(例如奥曲肽(octreotide))、抗体(例如，依那西普(etanercept)、阿达木单抗(adalimumab)、利妥昔单抗(rituximab)、派姆单抗(pembrolizumab)、兰尼单抗(ranibizumab)和纳武单抗(nivolumab))、细胞因子、激素、抗生素(例如，化疗药阿霉素(doxorubicin)、柔红霉素(daunorubicin)、表阿霉素(epirubicin)、放线菌素(actinomycin)和万古霉素(vancomycin))、核酸、以及它们的组合等，例如，所述药物分子是选自质粒DNA、RNA如小干扰RNA(siRNA)、有义RNA、反义寡核苷酸(ASO)、适配体(aptamers)、核酶等的核酸，例如，所述药物分子是针对脑部疾病例如脑部肿瘤(例如，脑胶质瘤)的药物分子，如，针对c-myc的siRNA。
  3. 根据权利要求1或2所述的药物制剂，所述药物制剂中药物分子(例 如，化疗药)与阳离子脂质(例如，DOTAP)的摩尔比为约0.1-10，例如，约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，优选约0.1-5，进一步优选约0.1-2；或者，所述药物制剂对药物分子(例如，化疗药)的载药量为：药物分子(例如，化疗药)占药物制剂的约1％-30％(w/w)，例如，约1％、2.5％、5％、7.5％、10％、12.5％、15％、17.5％、20％、22.5％、25％、27.5％、30％(w/w)，优选约1％-25％(w/w)，进一步优选约3％-20％(w/w)；或者，所述药物制剂中带正电的多聚氨基酸(例如，寡聚精氨酸)和药物分子的电荷比介于1:1至30:1，例如，1:1、5:1、10:1、15:1、20:1、25:1、30:1；且阳离子脂质体与药物分子的电荷比介于1:1至30:1，例如，2:1、3:1、4:1、5:1、6:1、8:1、10:1、12:1。
  4. 根据权利要求1－3中任一项所述的药物制剂，其中阳离子脂质体包含如下膜材料：(i)DOTAP；(ii)DOPE；(iii)胆固醇；(iv)mPEG 2000-DSPE和 89WPenetratin-PEG 3400-DSPE，且膜材料(i):(ii):(iii):(iv)的摩尔比为约20－40:20－40:20－40:1－20，且 89WPenetratin-PEG 3400-DSPE占膜材料(iv)中的约20％－80％摩尔比；例如，所述膜材料(i):(ii):(iii):(iv)的摩尔比为约27.0－31.6:27.0－31.6:31.6－39.6:1－10，且 89WPenetratin-PEG 3400-DSPE占膜材料(iv)中的约20％－80％摩尔比；例如为约28.5:28.5:38:5， 89WPenetratin-PEG 3400-DSPE占膜材料(iv)中的约20％、25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％摩尔比；带正电的多聚氨基酸(例如寡聚精氨酸)和药物分子(例如，核酸，如siRNA)的电荷比介于1:1至30:1，例如，1:1、5:1、10:1、15:1、20:1、25:1、30:1；且阳离子脂质体与药物分子的电荷比介于1:1至30:1，例如，2:1、3:1、4:1、5:1、6:1、8:1、10:1、12:1。
  5. 根据权利要求1－4中任一项所述的药物制剂，其为经鼻腔施用的药物制剂，例如，其为经鼻腔施用的核酸脂质体制剂(例如，siRNA脂质体制剂)，优选地，用于治疗脑部疾病，例如脑部肿瘤(例如，脑胶质瘤)。
  6. 一种药物递送载体，其包含穿膜肽修饰的脂质体和聚合物，其中所述 聚合物是带正电的多聚氨基酸，所述聚合物用于与药物混合形成聚合物-药物的复合物，所述穿膜肽修饰的脂质体用于与聚合物-药物的复合物混合形成包含聚合物-药物的复合物的脂质体复合物，其中所述穿膜肽是具有下述氨基酸序列的penetratin或penetratin的衍生物：
    R X 1 IKIWF X 2X 3 RRMKWKK
    其中，X 1、X 2和X 3独立地选自天然来源的氨基酸谷氨酰胺(glutamine，Q)、天冬酰胺(asparagine，N)、丙氨酸(alanine，A)、缬氨酸(valine，V)、亮氨酸(leucine，L)、异亮氨酸(isoleucine，I)、脯氨酸(proline，P)、苯丙氨酸(phenylalanine，F)、色氨酸(tryptophan，W)、甲硫氨酸(methionine，M)和非天然来源的氨基酸α-氨基丁酸(α-aminobutyric acid)、α-氨基戊酸(α-aminopentanoic acid)、α-氨基己酸(α-aminohexanoic acid)、α-氨基庚酸(α-aminoheptanoic acid)，例如，X 1、X 2和X 3独立地选自疏水性氨基酸。
  7. 根据权利要求6所述的药物递送载体，其特征在于，所述穿膜肽是penetratin的具有下述氨基酸序列的亲脂性衍生物：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)。
  8. 根据权利要求6或7所述的药物递送载体，其中所述带正电的多聚氨基酸是例如聚合度为2-50的带正电的多聚氨基酸，所述带正电的多聚氨基酸的空间结构包括线性和环形，氨基酸残基的构型包括L型和D型；例如聚合度为6-12的精氨酸和/或赖氨酸，任选地间插有1-20个(例如1－6个)生理条件下不带电荷的氨基酸残基，优选地，所述带正电的多聚氨基酸是聚合度为6-12的多聚精氨酸，其中所述多聚精氨酸的空间结构包括线性和环形，多聚精氨酸的构型包括L型和D型。
  9. 根据权利要求6－8中任一项所述的药物递送载体，其中所述穿膜肽修饰的脂质体包含如下膜材料：
    (i)阳离子脂质，例如但不限于：1,2-二-O-十八烯基-3-三甲基铵丙烷(DOTMA)；二甲基双十八烷基铵(DDAB)；1,2-二油酰基-3-三甲基铵-丙烷(DOTAP)；1,2-二油酰基-3-二甲基铵-丙烷(DODAP)；1,2-二酰氧基-3-二甲基铵丙烷；1,2-二烷氧基-3-二甲基铵丙烷；双十八烷基二甲基氯化铵(DODAC)；1,2-二肉豆蔻酰氧基丙基-1,3-二甲基羟乙基铵(DMRIE)和2,3-二油酰氧基-N-[2(精胺甲酰胺)乙基]-N,N-二甲基-1-丙烷三氟乙酸铵(DOSPA)和它们的任意组合；优选地为DOTMA、DOTAP、DODAC和DOSPA；最优选地为DOTAP；
    (ii)非阳离子脂质，例如但不限于：1,2-二-(9Z-十八酰基)-sn-甘油基-3-磷酸乙醇胺(DOPE)、1,2-二油酰基-sn-甘油基-3-磷酸胆碱(DOPC)、1,2-二硬脂酰基-sn-甘油-3-磷酸胆碱(DSPC)、1,2-二棕榈酰基-sn-甘油-3-磷酸胆碱(DPPC)、1,2-二棕榈酰基-sn-甘油-3-磷酸乙醇胺(DPPE)、1,2-二肉豆蔻酰基-sn-甘油-3-磷酸乙醇胺(DMPE)、2-二油酰基-sn-甘油-3-磷酸-(1’-rac-甘油)(DOPG)及其组合；优选地为DOPE和/或DOPC；最优选地为DOPE；
    (iii)胆固醇；
    (iv)聚乙二醇化(PEG化)的磷脂和与穿膜肽共轭的聚乙二醇磷脂，所述磷脂例如是磷脂酰胆碱、磷脂酰乙醇胺、磷脂酰肌醇和鞘磷脂，所述聚乙二醇(PEG)是长度高达10kDa的聚乙二醇链，例如，1kDa、2kDa、3kDa、4kDa、5kDa、6kDa、7kDa、8kDa、9kDa、10kDa、以及它们之间的任何值的聚乙二醇链；优选地，所述PEG化磷脂是聚乙二醇-二硬脂酰基磷脂酰乙醇胺(PEG-DSPE)和其衍生物甲氧基-聚乙二醇-二硬脂酰基磷脂酰乙醇胺(mPEG-DSPE))，所述与穿膜肽共轭的聚乙二醇磷脂是例如Penetratin/Penetratin衍生物-PEG-DSPE；且所述PEG化磷脂和与穿膜肽共轭的聚乙二醇磷脂中的聚乙二醇链长不相等，其中与穿膜肽共轭的聚乙二醇磷脂中的聚乙二醇链较PEG化磷脂中的聚乙二醇链更长，例如，更长0.5kDa－5kDa之间，优选地更长0.75kDa－4kDa之间，更优选地更长1kDa－2kDa 之间的任何值。
  10. 根据权利要求9所述的药物递送载体，其中所述膜材料(i):(ii):(iii):(iv)的摩尔比为约20－40:20－40:20－40:1－20，且与穿膜肽共轭的聚乙二醇磷脂占膜材料(iv)中的约20％－80％摩尔比；例如，所述膜材料(i):(ii):(iii):(iv)的摩尔比为约27.0－31.6:27.0－31.6:31.6－39.6:1－10，且与穿膜肽共轭的聚乙二醇磷脂占膜材料(iv)中的约20％－80％摩尔比；例如，所述膜材料(i):(ii):(iii):(iv)的摩尔比为约28.5:28.5:38:5，且与穿膜肽共轭的聚乙二醇磷脂占膜材料(iv)中的约20％、30％、40％、50％、60％、70％、80％摩尔比。
  11. 根据权利要求6－10中任一项所述的药物递送载体，其中阳离子脂质体包含如下膜材料：(i)DOTAP；(ii)DOPE；(iii)胆固醇；(iv)mPEG 2000-DSPE和穿膜肽-PEG 3400-DSPE，且膜材料(i):(ii):(iii):(iv)的摩尔比为约20－40:20－40:20－40:1－20，且穿膜肽-PEG 3400-DSPE占膜材料(iv)中的约20％－80％摩尔比；例如，所述膜材料(i):(ii):(iii):(iv)的摩尔比为约27.0－31.6:27.0－31.6:31.6－39.6:1－10，且穿膜肽-PEG 3400-DSPE占膜材料(iv)中的约20％－80％摩尔比；
    例如，膜材料(i):(ii):(iii):(iv)的摩尔比为约28.5:28.5:38:5，穿膜肽-PEG 3400-DSPE占膜材料(iv)中的约20％、25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％摩尔比。
  12. 制备权利要求1－4中任一项所述的药物制剂的方法，包括(a)制备表面修饰有穿膜肽的阳离子脂质体；(b)制备带正电的多聚氨基酸(例如寡聚精氨酸)和药物分子的物理复合物；(c)按一定的电荷比混合步骤(a)获得的表面修饰有穿膜肽的阳离子脂质体和步骤(b)获得的物理复合物。
  13. 制备权利要求1－4中任一项所述的药物制剂的方法，包括(a)混合脂质体的膜材料(i)、(ii)和(iii)，制备空白脂质体；(b)制备带正电的多聚氨基酸(例如寡聚精氨酸)和药物分子的物理复合物；(c)按一定的电荷比混合 步骤(a)获得的空白脂质体和步骤(b)获得的物理复合物；(d)将步骤(c)获得的脂质体与膜材料(iv)混合并孵育。
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