JP2022531010A - Drug delivery carrier and pharmaceutical product using it - Google Patents

Abstract

The present invention provides drug delivery carriers comprising liposomes modified with cell penetrating peptides and positively charged polyamino acids as polymers. The drug delivery carrier is used to prepare pharmaceutical formulations in the form of liposome complexes with a particle size of 50 nm to 300 nm. The pharmaceutical formulation is prepared by mixing a physical complex consisting of a positively charged polyamino acid, one or more drug molecules, and a liposome whose surface is modified with a cell-penetrating peptide. . Said pharmaceutical preparation can effectively transfect cells, especially facilitate the nasal absorption of drugs into the brain, and improve the therapeutic effects of drugs.

Description

The present invention relates to a drug delivery carrier, a method for preparing a pharmaceutical preparation using the drug delivery carrier, and a pharmaceutical preparation prepared thereby. Specifically, the drug delivery carrier of the invention comprises a liposome and a polymer modified with a cell permeable peptide, wherein the polymer is a positively charged polyamino acid. Pharmaceutical formulations prepared using the drug delivery carriers of the invention include a physical complex formed of positively charged polyamino acids and one or more drug molecules, and liposomes modified with a cell-permeable peptide. It is a liposome complex prepared by mixing, and the pharmaceutical preparation can effectively transfect cells, and in particular, promotes the drug to be absorbed into the brain via the nasal cavity, and has a therapeutic effect on the drug. Can be enhanced.

Cell-penetrating peptides (CPPs) are short peptides that are positively charged under physiological conditions and that covalently or non-covalently bind drug molecules such as genes, polypeptides and proteins, and drug molecules. Can be delivered intracellularly or carried a drug molecule to permeate the biomembrane barrier. However, a single cell-permeable peptide has a very finite ability to carry the drug molecule, which allows the complex formed by the cell-permeable peptide and the drug molecule to have a large particle size and stability. Will get worse.

Further, in the prior art, a carrier for delivering a drug molecule (for example, a drug molecule such as a gene, a polypeptide or a protein), for example, a liposome has a problem that the delivery efficiency is low and tissue toxicity is likely to occur. In the field of liposome research, how to utilize liposomes to deliver drug molecules more efficiently is still a major challenge, so that the treatment and / or prevention of diseases is more specific and efficient. Is. Researchers mainly take measures such as adding various auxiliary components to liposomes, developing and using new liposome materials, structurally modifying the surface of liposomes, and designing and using new preparation processes. Through this, it improves the stability, targeting and drug delivery efficiency of liposomes.

Therefore, there is still a need for new drug delivery carriers that utilize cell-permeable peptides in the art to ensure stability after drug molecules (eg, genes, polypeptides, proteins, etc.) have been combined with said drug delivery carriers. Improved and controlled particle size of the prepared pharmaceutical formulation, good stability, and more effective drug delivery can be achieved by the cell-permeable peptide that modifies the surface of the formulation, the drug. Helps to escape from the cell endosome and exert the effect of the drug.

Through diligent research, the inventor has developed a novel drug delivery carrier that utilizes cell-permeable peptides. The drug delivery carrier of the present invention comprises a liposome modified with a cell permeable peptide and a polymer, wherein the polymer is a positively charged polyamino acid, and the polymer is mixed with one or more drug molecules. The polymer-drug complex is formed, and the cell-permeable peptide-modified liposome is mixed with the polymer-drug complex to contain the polymer-drug complex. Is for forming.

In one embodiment, the cell-permeable peptide in the drug delivery carrier of the invention is a penetratin or a lipophilic derivative of penetratin having the following amino acid sequence:
RX ₁ IKIWFX ₂ X ₃ RRMKWKK (SEQ ID NO: 1)

Here, X ₁ , X ₂ , and X ₃ are independently naturally occurring amino acids such as glutamine (Q), asparagine (N), alanine (A), and valine (valine, V). Leucine, L, isoleucine, I, proline, P, phenylalanine, F, tryptophan, W, methionine, an amino acid of non-natural origin. It is selected from -aminobutyric acid, α-aminopentanoic acid, α-aminohexanoic acid, and α-aminoheptanoic acid. For example, some cell-permeable peptides in the drug delivery carriers of the invention include glutamine (Q) at position 2 and / or glutamine (Q) at position 8 and / or asparagine (N) at position 9 of Penetratin. It is a Peptlatin derivative mutated to a hydrophobic amino acid.

In one embodiment, the positively charged polyamino acid in the drug delivery carrier of the present invention is, for example, a positively charged polyamino acid having a degree of polymerization of 2 to 50, and the spatial structure of the positively charged polyamino acid. Is linear or circular, and the amino acid residue configuration is L-type or D-type. For example, polyamino acids are arginine and / or lysine with a degree of polymerization of 6-12. In the positively charged polyamino acid, uncharged amino acid residues are interposed in the positively charged amino acid residues under 1 to 20 (for example, 1 to 6) physiological conditions. May be good. Preferably, the positively charged polyamino acid is a polyarginine having a degree of polymerization of 6-12, where the spatial structure of the polyarginine is linear or cyclic and the polyarginine configuration is L-type or D. It is a type.

In one embodiment, the liposomes modified with the cell-permeable peptide in the drug delivery carrier of the invention include the following membrane materials:

(I) Cationic lipids include 1,2-di-0-octadecenyl-3-trimethylammonium-propane (DOTMA), dimethyldioctadecylammonium (DDAB), and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). ), 1,2-Dioreoil-3-dimethylammonium-propane (DODAP), 1,2-diacyloxy-3-dimethylammonium-propane, 1,2-dialkoxy-3-dimethylammonium-propane, dioctadecyldimethylammonium Chloride (DODAC), 1,2-dimyristyloxypropyl-1,3-dimethylhydroxyethylammonium (DMRIE), 2,3-dioreyloxy-N- [2 (sperminecarboxamide) ethyl] -N-N-dimethyl -1-Propanium Ammonium Trifluoroacetate (DOSPA) and combinations thereof are included, but not limited to these. DOTMA, DOTAP, DODAC and DOSPA are preferable. Most preferably DOTAP;
(Ii) Non-cationic lipids include 1,2-di- (9Z-octadecanoyl) -sn-glyceryl-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glyceryl-3-phosphocholine (ii). DOPC), 1,2-distearoyl-sn-glyceryl-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glyceryl-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glyceryl- 3-Phosphoethanolamine (DPPE), 1,2-dimyristyl-sn-glyceryl-3-phosphoethanolamine (DMPE), 2-diore oil-sn-glyceryl-3-phosphate- (1'-rac-glycerol) (DOPG) and combinations thereof are included, but not limited to these. It is preferably DOPE and / or DOPC. Most preferably DOPE;
(Iii) Cholesterol; and (iv) Polyethylene glycolylated (PEGylated) phospholipids and CPP-conjugated polyethylene glycol phospholipids; said phospholipids are, for example, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, sphingomyelin, preferably. The PEGylated phospholipid is, for example, polyethylene glycol-distearoyl phosphatidylethanolamine (PEG-DSPE) and a derivative thereof, methoxy-polyethylene glycol-distearoyl phosphatidylethanolamine (mPEG-DSPE), which is conjugated with CPP. The polyethylene glycol phospholipid obtained is, for example, Penetratin / Penetratin derivative-PEG-DSPE.

In some embodiments, the molar ratio of membrane material (i) :( ii) :( iii): (iv) is about 20-40: 20-40: 20-40: 1-20, coupled with CPP. The polyethylene glycol phospholipids obtained have a molar ratio of about 20% to 80% with respect to the membrane material (iv). In some specific embodiments, the molar ratio of membrane material (i) :( ii) :( iii): (iv) is from about 27.0 to 31.6: 27.0 to 31.6: 31. It is 6.6 to 39.6: 1 to 10, and the polyethylene glycol phospholipid conjugated with CPP has a molar ratio of about 20% to 80% with respect to the membrane material (iv).

In some specific embodiments, the cationic lipids in the drug delivery carriers of the invention are, as membrane materials, (i) DOTAP; (ii) DOPE; (iii) cholesterol; (iv) mPEG ₂₀₀₀ -DSPE and ^89W. It contains 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. ^89W Penetratin-PEG ₃₄₀₀ -DSPE has a molar ratio of about 20% to 80% with respect to the membrane material (iv). In some embodiments, the molar ratio of membrane material (i) :( ii): (iv) is about 27.0 to 31.6: 27.0 to 31.6: 31.6 to 39.6: 1. -10 and ^89W Penetratin-PEG ₃₄₀₀ -DSPE has a molar ratio of about 20% to 80% with respect to the membrane material (iv).

In some embodiments, the cationic lipids in the drug delivery carriers of the invention are referred to as (i) DOTAP; (ii) DOPE; (iii) cholesterol; (iv) mPEG ₂₀₀₀ -DSPE and ^{89W Membrane} -PEG ₃₄₀₀ -DSPE. It is composed of a membrane material, and the molar ratio of the membrane material (i) :( ii) :( iii): (iv) is about 28.5: 28.5: 38: 5, and ^89W Penetratin-PEG ₃₄₀₀ -DSPE. Is about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% mol with respect to the membrane material (iv). The ratio.

In another aspect, the invention provides a pharmaceutical formulation prepared using the drug delivery carrier of the invention. A physically complex was formed with the positively charged polyamino acid contained in the drug delivery carrier and one or more drug molecules to be delivered, and the physical complex and the surface were modified with a cell-permeable peptide. Cationic liposomes are mixed to form a liposome complex-style pharmaceutical formulation.

Not specifically limited to one or more drug molecules delivered, peptide drugs (eg, octreotide), antibodies (eg, etanercept, adalimumab), rituximab, pembrolizumab (eg, etaliximab). pembrolizumab, ranibizumab and nivolumab), cytokines, hormones, antibiotics (eg, chemotherapeutic agents doxorubicin, daunorubicin, daunorubicin, epirubicin) )), Nuclei and combinations thereof, etc., but are not limited to these.

In one embodiment, the drug molecule delivered by the pharmaceutical formulation is nucleic acid such as plasmid DNA, small interfering RNA (siRNA), sense RNA, antisense oligonucleotide (ASO), aptamers, ribozymes and the like. In one embodiment, the drug molecule delivered by the pharmaceutical product is a combination of a nucleic acid and a drug capable of integrating into the biological genome (eg, doxorubicin).

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

In one embodiment, the amount of the drug molecule (for example, a chemotherapeutic agent) encapsulated in the pharmaceutical product is about 1% to 30% (w / w /) of the drug molecule (for example, a chemotherapeutic agent) with respect to the pharmaceutical product. w), preferably about 1% to 25% (w / w), and more preferably about 3% to 20% (w / w).

In one embodiment, in the pharmaceutical product, the charge ratio of a positively charged polyamino acid (eg, oligoarginine) to the drug molecule to be delivered is 1: 1 to 30: 1, preferably 5. It is 1. The charge ratio of the cationic liposome to the drug molecule is 1: 1 to 30: 1, preferably 4: 1.

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

In yet another embodiment, the method for preparing a pharmaceutical formulation of the present invention is to (a) prepare a liposome having a cell-permeable peptide modified on the surface, and (b) deliver it with a positively charged polyamino acid. A step of preparing a physical complex consisting of a drug molecule to be used, and (c) a liposome having a cell-permeable peptide modified on the surface obtained in step (a), and a physical obtained in step (b). Provided is a method for preparing a pharmaceutical preparation, which comprises a step of mixing the complex at a constant molar ratio or charge ratio.

In one embodiment, the method for preparing a pharmaceutical preparation of the present invention comprises (a) mixing the liposome membrane materials (i), (ii) and (iii) to prepare blank liposomes, and (b) positive. The step of preparing a physical complex consisting of the polyamino acid charged in the cell and the drug molecule to be delivered, and (c) the blank liposome obtained in step (a), and the physical obtained in step (b). The step of mixing the complex at a constant molar ratio or charge ratio, and (d) the liposome obtained in step (c) and the membrane material (iv) are mixed and incubated to obtain the pharmaceutical preparation of the present invention. Including steps.

Pharmaceutical formulations prepared using the drug delivery carriers of the invention can be described, for example, in the oral cavity, nasal cavity, eyes, airways, digestive tract, reproductive tract, topical implants, injections, or infusions (epidermal, intraarterial, joint). Intra, sac, intracardiac, intraventricular, intracranial, intracutaneous, intramuscular, intraocular, intraocular, intraperitoneal, intravertebral, intrathoracic, intrathecal, intravenous, submucosal, subcapsular, subskin, It can be administered via the tracheal, rectal, sublingual, etc. routes) and used for the treatment and / or prevention of diseases.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. All publications, patent applications, patents and other references mentioned herein are fully incorporated herein by reference. Also, the materials, methods, and examples described herein are merely exemplary and are not intended to be limiting. Other features, purposes, and advantages of the invention are evident in the specification and drawings, as well as the appended claims.

FIG. 1 shows each complex in which R8 (a polymer of 8 arginines) and siRNA have different charge ratios (represented by positive / negative charge ratios) and / or CLS and siRNA have different charge ratios. The agarose gel electrophoresis result and the particle size result are shown. 1A and 1B show agarose gel electrophoresis results and particle size results of R8 / siRNA complexes having different charge ratios of R8 / siRNA, respectively. 1C and 1D show agarose gel electrophoresis results and particle size results of CLS / siRNA complexes having different charge ratios for CLS / siRNA, respectively. 1E and 1F show agarose gel electrophoresis results and particle size results of CLS / R8 / siRNA liposome complexes having different charge ratios of CLS / siRNA when the charge ratio of R8 / siRNA is 1: 1, respectively. Is shown. FIG. 2 shows the uptake of cells into a CLS / R8 / siRNA liposome complex prepared with different charge ratios for CLS and siRNA when the charge ratio of R8 to siRNA is 1: 1. FIG. 3 shows the results of studies on electrophoresis, particle size and stability of the CLS / siRNA / R8 complex. 3A and 3B show agarose gel electrophoresis results and particle size results of a liposome complex having different charge ratios of R8 and siRNA when the charge ratios of CLS and siRNA are 4: 1, respectively. 3C and 3D show the precipitation situation after high-speed centrifugation of the liposome complex having different charge ratios of R8 and siRNA when the charge ratios of CLS and siRNA are 4: 1 and the agarose gel of the supernatant after centrifugation. It shows the result of electrophoresis. FIG. 4 shows the quantitative evaluation results of cell ingestion for a pharmaceutical preparation whose surface is modified with different proportions of cell-permeable peptides. The Lipo2000 group is a non-covalent complex of cationic liposome Lipofectamine 2000 as a commercially available gene transfection reagent and siRNA. FIG. 5 shows the intake status of cells for a drug preparation prepared using the liposome membrane materials of different formulations shown in Table 2 of Example 8. FIG. 6 shows the particle size of the liposome pharmaceutical preparation in which the component (iv) is 1%, 5%, 8%, and 10% molar ratio of the liposome membrane material. FIG. 7 shows the morphological results of culturing different cell masses using a 35 mm Dish Nano Culture Dish. The 35 mm Dish NanoCulture Dish is a cell mass culture plate manufactured by JSR Corporation. FIG. 8 shows the evaluation results of the cell mass permeability of the cell-permeable peptide that modifies the surface of the liposome. FIG. 9 shows the transfection effect on the 293T cell mass by the pharmaceutical preparation containing GFP-siRNA. FIG. 10 shows the transfection effect on the U87 cell mass by the pharmaceutical preparation containing GFP-siRNA. FIG. 11 shows the results of qualitative and quantitative evaluation of the transfection effect on LUC-U87 cells by pharmaceutical preparations using different oligoarginines. FIG. 12 shows the particle size of a pharmaceutical preparation containing siRNA. FIG. 13 shows the potential of a pharmaceutical preparation containing siRNA. FIG. 14 shows the bEnd. It shows the evaluation result of the apoptosis promoting effect and the cytotoxic activity of 3 cells or U87 cells. FIG. 15 shows the distribution of the pharmaceutical product labeled with the cell membrane fluorescent probe DiD in the body after nasal administration. FIG. 16 shows the results of flow cytometric detection of carriers labeled with the cell membrane fluorescent probe DiD delivering FAM-siRNA intracellularly. FIG. 17 shows the distribution of the gene delivery carrier labeled with the cell membrane fluorescent probe DiD in the U87 in-situ brain tumor model mouse after nasal administration. FIG. 18 shows the results of pharmacodynamic evaluation of anti-U87 in-situ brain tumors of delivery carriers encapsulating siRNA. The Lipo2000 / siRNA group is a non-covalent complex of the cationic liposome Lipofectamine 2000 as a commercially available gene transfection reagent and siRNA. FIG. 19 shows the evaluation results of the in-vivo apoptosis promoting ability of a delivery carrier encapsulated with siRNA in a U87 in-situ brain tumor model mouse. The Lipo2000 / siRNA group is a non-covalent complex of the cationic liposome Lipofectamine 2000 as a commercially available gene transfection reagent and siRNA. FIG. 20 shows the results of evaluation of nasal mucosal toxicity in the body of a delivery carrier encapsulating siRNA. The Lipo2000 / siRNA group is a non-shared complex of the cationic liposome Lipofectamine 2000 as a commercially available gene transfection reagent and siRNA, and the sodium deoxycholate of the sodium deoxycholate group is produced by Dalian Mio Biotechnology Co., Ltd. It is a commercial product.

The present invention provides novel drug delivery carriers comprising liposomes modified with cell permeable peptides and positively charged polyamino acids as polymers. The drug delivery carrier of the invention can be assembled with a drug molecule to form a complex, compact the drug molecule and encapsulate the drug molecule in a liposome, whereby cells on the surface of the liposome. It utilizes a permeable cell-permeable peptide to aid the penetration of the drug into the biomembrane barrier within the subject.

The drug delivery carrier of the present invention has strong drug carrying, drug delivery, and tissue permeation ability, has low tissue toxicity, and efficiently delivers a drug molecule to a target site in a subject body via various administration routes. It can overcome the technical problem that it is difficult for the drug to reach the target site due to the subject's biological membrane barrier, enhance the efficacy of the drug in the treatment of the disease, and improve the biological safety. Have. The present invention also provides a pharmaceutical preparation prepared using the drug delivery carrier.

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

Various features of the drug delivery carrier of the present invention will be discussed below.

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

As used herein, the term "contains" or "contains" means to include the described elements, integers, or steps, but excludes any other element, integer, or step. is not.

<I, Liposome membrane material with cell-permeable peptide modified on the surface>
As used herein, the term "liposome" refers to an artificially prepared vesicle composed of a lipid bilayer. In the present invention, the type of liposome modified with the cell-permeable peptide is not limited and may be any one capable of forming lipid vesicles and encapsulating the drug.

In some embodiments, liposomes modified with a cell permeable peptide on the surface of the invention include the following membrane materials:

[(I) Cationic lipid]
Liposomes contain one or more cationic lipids. As used herein, the term "cationic lipid" is a lipid having a net positive charge at a selected pH (eg, physiological pH). Many cationic lipids are commercially available. Cationic lipids particularly suitable for use in liposomes include the cationic lipids described in International Patent Publications WO2010 / 053572 and WO2012 / 170930, both of which are described in the two patent documents by reference. Incorporated into the specification.

The cationic lipid contained in the liposome is not particularly limited as long as it is a liposome having a net positive charge at a selected pH (for example, physiological pH), and is 1,2-di-0-octadecenyl-3-trimethylammonium. -Propane (DOTMA), dimethyldioctadecylammonium (DDAB), 1,2-diore oil-3-trimethylammonium-propane (DOTAP), 1,2-diore oil-3-dimethylammonium-propane (DODAP), 1,2- Diacyloxy-3-dimethylammonium-propane, 1,2-dialkoxy-3-dimethylammonium-propane, dioctadecyldimethylammonium chloride (DODAC), 1,2-dimyristyloxypropyl-1,3-dimethylhydroxyethylammonium (DMRIE), 2,3-dioreyloxy-N- [2 (sperminecarboxamide) ethyl] -N-N-dimethyl-1-propaneammonium trifluoroacetate (DOSPA) and combinations thereof can be used. , Not limited to these. Preferred cationic lipids are DOTMA, DOTAP, DODAC and DOSPA. The most preferred cationic lipid is DOTAP.

[(Ii) Non-cationic lipid]
Liposomes contain one or more non-cationic lipids. As used herein, the term "non-cationic lipid" refers to any neutral, zwitterion or anionic lipid. As used herein, the term "anionic lipid" refers to any of the many liposomal substances having a net negative charge at a selected pH, such as physiological pH.

The non-cationic lipid contained in the liposome is not particularly limited, and any neutral ion, amphoteric ion, or anionic lipid can be used, and 1,2-di- (9Z-octadecanoyl) -sn-glyceryl- 3-Phosphorylethanolamine (DOPE), 1,2-dioleoyl-sn-glyceryl-3-phosphocholine (DOPC), 1,2-distearoyl-sn-glyceryl-3-phosphocholine (DSPC), 1,2-dipalmitoylphole -Sn-glyceryl-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glyceryl-3-phosphoethanolamine (DPPE), 1,2-dipalmitoyl-sn-glyceryl-3-phosphoethanolamine (DMPE) ), 2-Dioreoil-sn-glyceryl-3-phosphate- (1'-rac-glycerol) (DOPG) and combinations thereof can be used, but not limited to these. Preferred non-cationic lipids are DOPE and / or DOPC. The most preferred non-cationic lipid is DOPE.

[(Iii) Cholesterol]
Cholesterol is an important constituent component of mammalian cell membranes and accounts for more than 20% of cell membrane lipids. The term "cholesterol" is used in the broadest sense herein and covers cholesterol derivatives. Studies have shown that cholesterol can prevent cell membrane bilayer disordering at high temperatures, interfere with cell membrane bilayer ordering at low temperatures, block the formation of liquid crystals, and flow the cell membrane bilayer. Can maintain sex.

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

[(Iv) PEGylated phospholipid, polyethylene glycol phospholipid coupled with CPP]
Liposomes include PEGylated phospholipids and CPP-conjugated polyethylene glycol phospholipids. As used herein, PEGylated phospholipids do not contain CPP-linked polyethylene glycol phospholipids.

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

Polyethylene glycol phospholipids conjugated to CPP are formed by covalently bonding CPP to polyethylene glycol phospholipids.

The CPP used in the present invention is not only the wild-type polypeptide penetratin (amino acid sequence: RQIKIWFQNRRMKWKK (SEQ ID NO: 2)), but also glutamine (Q) at the 2-position and / or glutamine at the 8-position (Q) of, for example, Penetratin. ) And / or a series of lipophilic derivatives such as the Peptlatin derivative in which asparagine (N) at position 9 is mutated to a hydrophobic amino acid. In some embodiments, the Penetratin lipophilic derivative is a derivative as disclosed in Chinese patent application CN201710414334.7, which is a barrier for many eyes (cornea, conjunctiva) after intraconjunctival instillation. , Sclera, etc.) to promote the entry of drugs into the eye, and by extension, the biopolymer drugs such as genes, polypeptides, and proteins that carry them can be applied to the part of the retina behind the eye. Can be delivered to. The amino acid sequence of the Penetratin lipophilic derivative disclosed in Chinese patent application CN201710414334.7 is cited as follows.

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

Polyethylene glycol chains in CPP-PEG-phospholipids are polyethylene glycol chains up to 10 kDa in length, such as 1 kDa, 2 kDa, 3 kDa, 4 kDa, 5 kDa, 6 kDa, 7 kDa, 8 kDa, 9 kDa, 10 kDa and any between them. It contains, but is not limited to, polyethylene glycol chains of value, and both ends co-bond with phospholipids and CPPs, respectively. In some embodiments, the CPP-conjugated polyethylene glycol phospholipid is, for example, a Penetratin / Penetratin derivative-PEG-DSPE, with a molecular weight of PEG of any value between 1 kDa and 10 kDa.

In the present invention, modifying the surface of a cationic liposome with CPP increases the tissue permeation capacity of the drug delivery carrier so that the liposome carrying more drug molecules reaches the target site, thereby. Intended to help treat and / or prevent the disease.

In some embodiments, in the liposome membrane material (iv), the polyethylene glycol in the PEGylated phospholipid and the polyethylene glycol phospholipid coupled to the CPP is not the same chain length and the polyethylene in the polyethylene glycol phospholipid coupled to the CPP. The glycol chain is longer than the polyethylene glycol chain in the PEGylated phospholipid, eg, 0.5 kDa to 5 kDa, preferably 0.75 kDa to 4 kDa, and more preferably any of 1 kDa to 2 kDa. Longer in value.

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

In some embodiments, the molar ratio of cationic liposome membrane material (i) :( ii) :( iii) :( iv) modified with a cell-permeable peptide on the surface of the present invention is about 20-40 :. The polyethylene glycol phospholipids of 20-40: 20-40: 1-20, coupled with CPP, have a molar ratio of about 20% -80% to the membrane material (iv), relative to the total liposome membrane material. Corresponds to a molar ratio of about 2% -8%.

In some specific embodiments, the molar ratio of membrane material (i) :( ii) :( iii): (iv) is from about 27.0 to 31.6: 27.0 to 31.6: 31. The polyethylene glycol phospholipids of 6.6 to 39.6: 1 to 10 and coupled to CPP have a molar ratio of about 20% to 80% with respect to the membrane material (iv) and about about 20% to 80% of the total liposome membrane material. Corresponds to a molar ratio of 2% to 8%.

In some specific embodiments, the cationic liposomes in the drug delivery carriers of the invention are membrane materials such as (i) DOTAP, (ii) DOPE, (iii) cholesterol, (iv) PEG-DSPE (eg, iv) PEG-DSPE. It contains mPEG ₂₀₀₀ -DSPE) and Penetratin / Penetratin derivative-PEG-DSPE (eg ^89W Penetratin-PEG ₃₄₀₀ -DSPE) and has a molar ratio of membrane material (i) :( ii) :( iii) :( iv). , Approximately 20-40: 20-40: 20-40: 1-20, and the Penetratin / Penetratin derivative-PEG-DSPE (eg, ^89W Penetratin-PEG ₃₄₀₀ -DSPE) is approximately relative to the membrane material (iv). The molar ratio is 20% to 80%. In some embodiments, the molar ratio of membrane material (i) :( ii) :( iii): (iv) is from about 27.0 to 31.6: 27.0 to 31.6: 31.6 to. 39.6: 1-10, and the Penetratin / Penetratin derivative-PEG-DSPE (eg, ^89W Penetratin-PEG ₃₄₀₀ -DSPE) in a molar ratio of about 20% to 80% with respect to the membrane material (iv). be. In some embodiments, the cationic liposomes in the drug delivery carriers of the invention are referred to as (i) DOTAP, (ii) DOPE, (iii) cholesterol, (iv) mPEG ₂₀₀₀ -DSPE and ^89W Penetratin-PEG ₃₄₀₀ -DSPE. It is composed of a membrane material, and the molar ratio of the membrane material (i) :( ii) :( iii): (iv) is about 28.5: 28.5: 38: 5, and ^89W Penetratin-PEG ₃₄₀₀ -DSPE. Is about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% with respect to the membrane material (iv). It is a molar ratio.

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

The positively charged polyamino acid is a polyamino acid having a net positive charge at a selected pH (eg, physiological pH). There is no particular limitation on the degree of polymerization of amino acid residues in polyamino acids, for example, positively charged polyamino acids having a degree of polymerization of 2 to 50 (that is, positively charged polyamino acids have 2 to 50 amino acid residues. Among the positively charged polyamino acids having a degree of polymerization of, for example, arginine and / or lysine having a degree of polymerization of 6 to 12, and having a degree of polymerization of 2 to 50, 1 to 20 (eg, having). It may be mediated by uncharged amino acid residues under the physiological conditions of 1 to 6). Positively charged polyamino acids can be linear or cyclic, and the amino acid residue configuration is L-type or D-type. Preferably, the positively charged polyamino acid is a polyarginine having a degree of polymerization of 6-12, the spatial structure of the polyarginine comprises linear and cyclic, and the polyarginine configuration is L-type or D-type. be.

In some embodiments, the positively charged polyamino acid is oligo-arginine, 6-polyarginine (amino acid sequence is RRRRR, R6 (SEQ ID NO: 85)), 8 polyarginine (amino acid sequence is Linear polypeptides such as RRRRRRR, R8 (SEQ ID NO: 86), 10 polyarginine (amino acid sequence is RRRRRRRRR, R10 (SEQ ID NO: 87)), 12 polyarginine (amino acid sequence is RRRRRRRRRRRRR, R12 (SEQ ID NO: 88)). And rings such as cyclized 6-polyarginine (c-R6), cyclized 8 polyarginine (c-R8), cyclized 10 polyarginine (c-R10), cyclized 12 polyarginine (c-R12). Chemical polypeptide, L-type oligoarginine, 6-poly D-arginine (amino acid sequence is rrrrrrrr, D-R6), 8-poly D-arginine (amino acid sequence is rrrrrrrrrr, D-R8), 10-poly D-arginine (amino acid sequence is rrrrrrrrrr, D-R8) The amino acid sequence includes polypeptides of different composition such as D-type oligoarginine such as rrrrrrrrrrrr), D-R10), 12 poly-D-arginine (amino acid sequence is rrrrrrrrrrrrrrr, D-R12).

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

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

In one embodiment, the method for preparing the pharmaceutical product of the present invention is as follows.
(1) The above-mentioned liposome membrane material of the present invention is weighed, and here, the membrane material (i) :( ii) :( iii): (iv) molar ratio is about 20-40: 20-40: 20-. 40: 1-20, CPP-conjugated polyethylene glycol phospholipids have a molar ratio of about 20% -80% to membrane material (iv) and about 2% -8% to total liposome membrane material. Corresponds to the molar ratio of.

(2) Membrane material (i): (ii): (iii): (iv) is added to an organic solvent to dissolve it and evaporated to remove the organic solvent to obtain a uniform dry lipid membrane.

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

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

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

In another embodiment, the method for preparing the pharmaceutical product of the present invention is as follows.
(1) The above-mentioned liposome membrane material of the present invention is weighed, and the molar ratio of the membrane material (i) :( ii) :( iii): (iv) is about 20 to 40:20 to 40:20. The CPP-conjugated polyethylene glycol phospholipids of ~ 40: 1 to 20 have a molar ratio of about 20% to 80% with respect to the membrane material (iv) and about 2% to 8 with respect to the total liposome membrane material. Corresponds to the molar ratio of%.

(2) Membrane material (i): (ii): (iii) is added to an organic solvent to dissolve it and evaporated to remove the organic solvent to obtain a uniform dry lipid membrane.

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

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

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

The drug molecule to be delivered is not particularly limited. The drug molecule includes, but is not limited to, a wide range of types of compounds delivered to the examinee, including, but not limited to: Nucleic acid; Anti-infective drugs such as antibiotics and anti-viral drugs; Combination of painkillers and painkillers; Appetite suppressants; Insect repellents; Anti-arteritis drugs; Anti-asthma drugs; Anti-convulsants; Anti-depressants; Anti-diabetic drugs; Diarrhea Stop; anti-histamine; anti-inflammatory; anti-mitral pain; nausea; anti-tumor; anti-tremor palsy; anti-pruritus; anti-psychiatric; anti-fever; anti-convulsant; anti-cholinergic; sympathetic Neurostimulants; xanthin derivatives; potassium channel blockers, calcium channel blockers, β-blockers, α-blockers and cardiovascular drugs including anti-arrhythmic drugs; antihypertensive drugs; diuretics and antidiuretics; systemic nerves, Cardiovascular, peripheral and cerebral nerves, central nervous system agonists, vasodilators including vasopressors; cough and cold preparations including decongestants; hormones such as estradiol and other steroids including corticosteroids; hypnotics; Immunosuppressants; muscle relaxants; parasympathetic blockers; psychostimulants; sedatives and stabilizers. The drug delivery carrier of the present invention can deliver all forms of drugs such as ionized, non-ionized, free bases, acid addition salts and the like, and can deliver high molecular weight or low molecular weight drugs.

In some embodiments, the drug molecule delivered by the pharmaceutical formulation of the invention is, for example, a peptide drug (eg, octreotide), an antibody (eg, etanercept, adalimumab), rituximab. , Pembrolizumab, ranibizumab and nivolumab), cytokines, hormones, antibiotics (eg, chemotherapeutic agents doxorubicin, daunorubicin, daunorubicin, daunorubicin) Vancomycin), nucleic acids and the like.

In some embodiments, the drug molecules delivered by the pharmaceutical formulation include plasmid DNA, RNA such as small interfering RNA (siRNA), sense RNA, antisense oligonucleotides (ASOs), aptamers, ribozymes and the like. Nucleic acid.

The plasmid DNA is, for example, interleukin-2, interleukin-4, interleukin-7, interleukin-12, and interleukin-15, interferon-α, interferon-β, interferon-γ, colony stimulating factor, granulocytes. -Select from the group consisting of macrophage stimulators, antiangiogenic agents, tumor suppressor genes, thymidine kinase, eNOS, iNOS, p53, p16, TNF-α, Fas-ligands, mutant tumor genes, tumor antigens, viral antigens or bacterial antigens. It is a gene expression system containing a DNA sequence encoding what is to be.

The plasmid DNA encodes a shRNA molecule designed to inhibit proteins involved in the growth or maintenance of tumor cells or other hyperproliferative cells. The plasmid DNA may encode the therapeutic protein and one or more shRNAs at the same time. Further, the nucleic acid in the pharmaceutical preparation of the present invention may be a mixture of plasmid DNA and artificially synthesized RNA including sense RNA, antisense oligonucleotide or ribozyme.

In one embodiment, the molar ratio of the drug molecule (eg, chemotherapeutic agent) to the cationic lipid (eg, DOTAP) in the pharmaceutical formulation of the invention is about 0.1-10, eg, 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, It is 8.5, 9, 9.5, 10, preferably about 0.1 to 5, and more preferably about 0.1 to 2.

In one embodiment, the drug loading of the pharmaceutical product with respect to the drug molecule (eg, chemotherapeutic agent) is as follows. Drug molecules (eg, chemotherapeutic agents) make up about 1% to 30% (w / w) of the pharmaceutical product, eg, 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% to 25% (w / w). , More preferably about 3% to 20% (w / w).

The pharmaceutical preparation of the present invention is an oral cavity, a nasal cavity, an eye, an airway, a digestive tract, a reproductive tract, a local implant, an injection or an infusion (extradural, intraarterial, intraarticular, intracapsular, intracardiac, intraventricular, intracranial). , Intracutaneous, intramuscular, intraocular, intraocular, intraperitoneal, intravertebral, intrathoracic, intrathecal, intravenous, subspider, subcapsular, subcutaneous, trachea, rectal, sublingual and other routes Can be administered.

The pharmaceutical preparation of the present invention can be administered using a medical device known in the art. For example, in one embodiment, the pharmaceutical preparation of the present invention can be administered using a needleless scalp subcutaneous injection device, for example, the device disclosed in US Pat. No. 5,399,163.

In one embodiment, the effect of gene therapy can be exerted by preparing a pharmaceutical preparation for delivering a nucleic acid using the drug delivery carrier of the present invention.

Compared to conventional treatment methods such as surgery, radiation therapy, and chemotherapy, gene therapy is a revolutionary treatment method that is highly responsive, has few side effects, causes less damage to normal cells, and is used in the course of treatment. Due to its advantage of less pain, it offers good prospects in disease areas such as immunodeficiency, metabolic disorders, cancer and heart disease. Currently, 2597 kinds of nucleic acid drugs have entered or completed clinical trials worldwide, 65% of which are for the treatment of cancer, and the gene drug of the clinical trial of Section 181 inhibits tumor growth. (Ginn SL et.al., Gene therapy clinical trials worldwide to 2017: An update, Journal of Gene Medicine, 2018, 20 (5): e3015). Based on this, the art hopes to realize the treatment of diseases (eg, tumor diseases) through pharmaceutical formulations that deliver nucleic acids.

Nucleic acid-delivering carriers are one of the key techniques of gene therapy, and the delivered nucleic acid is not degraded by endogenous nucleases and can be protected, making it possible to apply gene therapy in the body. .. Currently, there are two main types of nucleic acid carriers used in gene therapy: viral vectors and non-viral vectors. The transfection efficiency of viral vectors is very high, but at the same time, there are clinical problems such as induction of immune response and carcinogenicity. Non-viral vectors mainly include polyethyleneimine (PEI), polyamideamine (PAMAM), cationic liposomes and the like. Compared to PEI and PAMAM, cationic liposomes have advantages as drug carriers such as targeting, long-term action, low toxicity, and drug protection (Wang J et al., C-Myc is required for maintenance of glioma cancer). stem cells, PLoS ONE, 2008, 3 (11): e3769). Cationic liposomes are usually formed by mixing positively charged lipids with neutral co-lipids, and when the cationic liposomes are close to negatively charged nucleic acids, the cationic lipids collected are of "double-sided adhesive tape". It can play a role and spontaneously form nucleic acid / cationic liposome complexes. After specially modifying the surface groups of the liposomes, the cationic liposomes can fully encapsulate the nucleic acid, facilitate the fusion of the nucleic acid / cationic liposome complex with the cell membrane, and enhance the nucleic acid uptake of the cell (Huang L). et al., In vivo delivery of RNAi with lipid-based nanoparticles, Annual Review of Biomedical Engineering, August 15, 2011, 13: 507-530).

When preparing a pharmaceutical product for delivering nucleic acid, the artificially synthesized positively charged polyamino acid (eg, oligoarginine) in the drug delivery carrier of the present invention is used to compress the nucleic acid. Here, both of the oligoarginine and the nucleic acid form a physical complex by electrostatic action, and the nucleic acid is further compressed by utilizing a positively charged cationic liposome to form a liposome complex. do. Furthermore, since the cell-permeable peptide modifies the surface of the cationic liposome, the ability of the prepared nucleic acid liposome complex to permeate cells and tissues can be enhanced, and the nucleic acid liposome preparation constructed thereby can be used. It can be administered by various routes, can efficiently deliver nucleic acid molecules to the target body part, and has excellent biosafety.

In one embodiment, the method for preparing the nucleic acid liposome preparation of the present invention is as follows.
(1) The above-mentioned liposome membrane material of the present invention is weighed, and the molar ratio of the membrane material (i) :( ii) :( iii): (iv) is about 20 to 40:20 to 40:20. The CPP-conjugated polyethylene glycol phospholipids of ~ 40: 1 to 20 have a molar ratio of about 20% to 80% with respect to the membrane material (iv) and about 2% to 8 with respect to the total liposome membrane material. Corresponds to the molar ratio of%.

(2) Membrane material (i): (ii): (iii) is added to an organic solvent to dissolve it and evaporated to remove the organic solvent to obtain a uniform dry lipid membrane.

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

(4) Positively charged polyamino acids (eg, oligoarginine) and nucleic acid molecules are mixed and incubated in an appropriate solution at a specific charge ratio to positively charged polyamino acids (eg, oligoarginine) and nucleic acid molecules. To obtain a complex with.

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

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

In one embodiment, the method for preparing the nucleic acid liposome preparation of the present invention is as follows.
(1) The above-mentioned liposome membrane material of the present invention is weighed, and the molar ratio of the membrane material (i) :( ii) :( iii): (iv) is about 20 to 40:20 to 40:20. The CPP-conjugated polyethylene glycol phospholipids of ~ 40: 1 to 20 have a molar ratio of about 20% to 80% with respect to the membrane material (iv) and about 2% to 8 with respect to the total liposome membrane material. Corresponds to the molar ratio of%.

(2) Membrane material (i): (ii): (iii) is added to an organic solvent to dissolve it and evaporated to remove the organic solvent to obtain a uniform dry lipid membrane.

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

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

(5) A positively charged polyamino acid (for example, oligoarginine) and a nucleic acid molecule are mixed in an appropriate solution at a constant charge ratio and incubated to obtain a complex of oligoarginine and the nucleic acid molecule.

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

In one embodiment, the method for preparing the nucleic acid liposome preparation of the present invention is as follows.
(1) The above-mentioned liposome membrane material of the present invention is weighed, and the molar ratio of the membrane material (i) :( ii) :( iii): (iv) is about 20 to 40:20 to 40:20. The CPP-conjugated polyethylene glycol phospholipids of ~ 40: 1 to 20 have a molar ratio of about 20% to 80% with respect to the membrane material (iv) and about 2% to 8 with respect to the total liposome membrane material. Corresponds to the molar ratio of%.

(2) Membrane material (i): (ii): (iii): (iv) is added to an organic solvent to dissolve it and evaporated to remove the organic solvent to obtain a uniform dry lipid membrane.

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

(4) A positively charged polyamino acid (for example, oligoarginine) and a nucleic acid molecule are mixed in an appropriate solution at a constant charge ratio and incubated to obtain a complex of oligoarginine and the nucleic acid molecule.

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

In one embodiment, the nucleic acid molecule is siRNA, and in the method for preparing siRNA liposome preparations of the present invention, the molar ratio of DOTAP, DOPE, cholesterol, mPEG ₂₀₀₀ -DSPE to Penetratin / Penetratin derivative-PEG ₃₄₀₀ -DSPE is. , Approximately 28.5: 28.5: 38: 5, weighed DOTAP, DOPE, cholesterol, added and dissolved chloroform in a round-bottomed flask, removed chloroform with a rotary evaporator to remove uniform dry lipids. Obtain a membrane. The lipid membrane is ultrasonically hydrated with a 5% glucose aqueous solution through a water bath, and after the lipid membrane is completely hydrated, the liposomes are extruded with a mini-extruder (the order of the nuclear pore membranes is 200 nm, 100 nm and 50 nm, respectively). ), The preparation of blank liposome CLS was completed. Then, oligoarginine Rn (R is a one-letter code of arginine, n is an integer such as 2, 3, 4, 5, 6, 7, 8, 9, 10) and siRNA are mixed at a constant charge ratio. Then, vortex for 30 seconds and incubate for 30 minutes at 37 ° C. to obtain complex Rn / siRNA. Further, the complex Rn / siRNA and the blank liposome are mixed at a constant charge ratio (charge ratio of cationic liposome to siRNA), vortexed for 30 seconds, incubated at 37 ° C. for 30 minutes, and then the complex CLS / Rn /. SiRNA was obtained. Finally, CLS / Rn / siRNA and mPEG ₂₀₀₀ -DSPE and Penetratin / Penetratin derivative-PEG ₃₄₀₀ -DSPE were mixed and then incubated at 55 ° C. for 30 minutes to complete the preparation of siRNA liposome preparation.

In one embodiment, the nucleic acid liposome formulations of the invention are administered nasally, the nucleic acids are efficiently delivered to the brain via the nasal-brain pathway, and the nucleic acid liposome formulations are cell permeable after being ingested by brain tumor cells. Positive charges on the surface of the peptides and cationic liposomes allow the endosomes to escape and release nucleic acid molecules into the cytoplasm, allowing the gene to play a role in treating brain tumors (eg, cerebral glioma).

As are well known to engineers in the art, therapeutic drugs are usually given due to the special physiological and functional complexity of the brain, especially the presence of the blood-brain barrier and the physical and chemical properties of certain drugs themselves. Hard to reach brain tissue. However, the nasal administration route to the brain provides viable ideas for the treatment of brain diseases, and there are two direct routes for nasal administration of the drug to the brain, one is , The olfactory nerve pathway, the most direct way to bypass the blood-brain barrier to enter the brain through the nasal cavity, through the axon after the drug is absorbed by the nasal mucosa and ingested by the olfactory nerve. It is the process of being carried to the olfactory bulb and further reaching the olfactory brain. The other is the nasal mucosal epithelial pathway, where the drug passes through the basement membrane into the lamina propria, reaches the peripheral olfactory nerve, is delivered to the central nervous system, and eventually accumulates in the brain. do. Patient compliance is good because nasal administration to the brain is intact (Agrawal M et al., Nose-to-brain drug delivery: An update on clinical challenges and progress towards approval of anti). -Alzheimer drugs, Journal of Controlled Release, 2018, 281: 139-177).

On the other hand, brain tumors such as glioma are malignant tumors of the central nervous system with a high incidence and low survival rate, and the damage to human health is extremely large, and the clinical prognosis is not optimistic. Due to recurrence, drug resistance of chemotherapy, etc., and the presence of blood-brain barrier and blood-brain tumor barrier, the amount of drug that arrives in the brain after systemic administration is very small, so people have traditionally been effective drugs. I'm trying to find a cure.

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

In the examples of the present invention, the present inventors constructed a cationic liposome delivery carrier carrying siRNA using 8-polyarginine R8 as an example and using siRNA as a drug molecular model. Through a series of in vitro and in vitro experiments, we investigated the cell uptake and cell mass permeability of delivery carriers jointly constructed with cationic liposomes modified with cell-permeable peptides and positively charged polyamino acids. As a result, the drug delivery carrier of the present invention can significantly increase the amount of nucleic acid into cells and has a relatively high delivery efficiency as compared with a cationic liposome that delivers nucleic acid on the market. Is shown.

The drug delivery carrier constructed by the present invention has the advantages of high efficiency, low toxicity, and easy release of the drug. High efficiency is embodied by the delivery carriers of the present invention having a relatively high cell uptake capacity and a relatively strong cell mass permeability. Low toxicity means that the cell-permeable peptide used in the present invention is non-pathogenic, easily degraded in the body, and has better biological safety compared to other drug delivery carriers such as PEI and PAMMA. It is embodied by having. The ease of drug release means that both polyamino acids and surface-modifying cell-permeable peptides have a positive charge, and the positive charge of cationic liposomes helps the drug to escape endosomes, thus making the drug cytoplasmic. Can be released and play a therapeutic role. Furthermore, pharmaceutical formulations prepared using the drug delivery carriers of the present invention can be administered to the examinee via various routes, and in particular, intracerebral delivery of the drug is via the nasal route of administration. It can be achieved and is beneficial in improving patient compliance.

The following examples will be described to aid in understanding the invention. The examples are not intended to limit the scope of protection of the invention and should not be construed as limiting the scope of protection of the invention in any way.

[Example 1]
(Preparation of cationic liposome)
Membrane material in a molar ratio of 28.5: 28.5: 38: 5, ie 3.5 mg DOTAP (Shanghai Sigma-Aldrich Pharmaceuticals Technology Co., Ltd., 132172-61-3), 3.72 mg DOPE ( Shanghai Aywaille Special Pharmaceutical Technology Co., Ltd., 4004-05-1), 2.59 mg cholesterol (Sigma-Aldrich, C8667) and 2.46 mg mPEG ₂₀₀₀ -DSPE (Shanghai Aywaille Special Pharmaceutical Technology Co., Ltd., 147867-65) -0) was weighed.

Put the above amounts of DOTAP, DOPE and cholesterol in a 50 mL round bottom flask and add 1 mL of chloroform to dissolve the DOTAP, DOPE and cholesterol as membrane material, ie 16.67 μM CLS is 5 μM DOTAP, It contains 5 μM DOPE and 6.67 μM DOPE. Chloroform was removed from the obtained solution by a rotary evaporator (Shanghai Shinsei Technology Co., Ltd., R201L) to obtain a uniform dry lipid membrane.

The obtained dry lipid membrane was ultrasonically hydrated with 1 mL of a 5% aqueous glucose solution in a water bath at 37 ° C. for about 10 minutes. After the lipid membrane is completely hydrated, the liposomes are extruded using a mini extruder (Hamilton, 81320) (the order of the nuclear pore membranes is 200 nm, 100 nm and 50 nm, respectively), cationic liposomes (as used herein). CLS) was obtained.

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

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

4 mg of oligoarginine R8 (Shanghai ▲ 忻 ▼ Hiroshi Biotechnology Co., Ltd.) as a positively charged polyamino acid is dissolved in DEPC-treated water (Dalian Mio Biotechnology Co., Ltd., MA0018), and a 4 mg / mL oligoarginine R8 solution is dissolved. 33 μg of siRNA (in this example, a small interfering RNA against c-myc is used, hereinafter also abbreviated as siRNA, and its sense strand (5'-3') is AACGUUAGCUCCACACAcadTdT (SEQ ID NO: 79). The antisense strand (5'-3') was UGUGUGGUGAAGCUAAACGUudTdT (SEQ ID NO: 80) and was dissolved in 125 μL of DEPC-treated water to prepare a 20 μM siRNA solution.

The oligoarginine R8 solution and the siRNA solution are mixed in equal volumes in proportion to a charge ratio of 0, 1, 5, 10, 15, 20, 25, 30 (one aminonitrogen in R8 has one positive charge, and one amino nitrogen in R8 has one positive charge. Since one phosphate group of siRNA has one negative charge, 1 μM R8 contains 8 μM aminonitrogen, i.e. 1 μM R8 contains 8 μM positive charge and 1 μM siRNA contains 42 μM phosphate. The group contains a group, that is, 1 μM siRNA contains 42 μM negative charge, and the charge ratio of R8 to siRNA = (μM value of R8 × 8) / (μM value of siRNA × 42) = 0.19 × R8 (μM). / SiRNA (μM)), vortexed for 30 seconds and incubated for 30 minutes at 37 ° C. to give R8 / siRNA complexes with different charge ratios.

Liposomal complexes were prepared using the R8 / siRNA complex and CLS with different charge ratios described above. Specifically, an 8 μL R8 / siRNA complex with different charge ratios (ie, prepared with 4 μL siRNA solution and 4 μL R8 solution) was prepared at 2.67 μL in Example 1 at 16.67 μM. It was mixed with cationic liposome CLS, vortexed for 30 seconds, and incubated at 37 ° C. for 30 minutes to obtain lipid complex CLS / R8 / siRNA having different charge ratios of R8 / siRNA. In the lipid complex CLS / R8 / siRNA, the charge ratio of the cationic liposome CLS to the siRNA is fixed at 4 based on the charge number of the siRNA (because one amino nitrogen of DOTAP has one positive charge). , One phosphate group of siRNA has one negative charge, 16.67 μM CLS contains 5 μM DOTAP, ie 1 μM CLS contains 0.3 μM positive charge, 1 μM siRNA contains 42 μM. It contains a negative charge, and the charge ratio of CLS to siRNA = (CLS μM value × 0.3) / (siRNA μM value × 42) = 0.007 × CLS (μM) / siRNA (μM)). The results of agarose gel electrophoresis showed that any R8 / siRNA complex with a different charge ratio of R8 / siRNA can be mixed with CLS to prepare a liposome complex.

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

For the purpose of comparison, a CLS / siRNA complex was also prepared, and the CLS prepared in Example 1 and the 20 μL siRNA solution prepared in this Example were mixed with a CLS / siRNA charge ratio of 2, 4, 6, 8, 10, and so on. Mix in equal volumes at a ratio of 12, vortex for 30 seconds and incubate for 30 minutes at 37 ° C. to give CLS / siRNA complexes with different CLS / siRNA charge ratios. The calculation of the charge ratio of CLS / siRNA is performed as follows: Since one aminonitrogen in DOTAP has one positive charge and one phosphate group in siRNA has one negative charge. 67 μM CLS contains 5 μM DOTAP, ie 1 μM CLS contains 0.3 μM positive charge and 1 μM siRNA contains 42 μM negative charge. Therefore, the charge ratio of CLS to siRNA = (μM value of CLS × 0.3) / (μM value of siRNA × 42) = 0.007 × CLS (μM) / siRNA (μM)).

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

The results in FIG. 1A show that the gene cannot be completely compressed with oligoarginine R8 alone. The results in FIGS. 1C and 1D show that the single cationic liposome CLS can compress the gene to some extent, but the particle size of the CLS / siRNA complex is large. The results in FIGS. 1E and 1F show that the combination of cationic liposomes and oligoarginine R8 can effectively co-compress genes.

[Example 3]
(Evaluation of particle size and potential characteristics of the liposome complex containing the drug after the cationic liposome and the positively charged polyamino acid compress the drug)
According to the method described in Example 2, R8 / siRNA complex, CLS / siRNA complex and CLS / R8 / siRNA complex were prepared, respectively, and the particle size and potential were measured.

The particle size was measured directly using dynamic light scattering technology. The particle size measurement results are shown in FIGS. 1, 3, and 12. In FIG. 12, the results from the dynamic light scattering measurement are calculated from the light intensity contributed by each particle, with the horizontal axis representing the particle size and the vertical axis representing the light intensity.

As can be seen from FIG. 3B, when the charge ratio of oligoarginine R8 / siRNA is 5, 10 and 20, the particle size of the physical complex R8 / siRNA is about 200 nm. When the charge ratio of oligoarginine R8 / siRNA is 5 and the charge ratio of cationic liposome CLS to siRNA is 4, siRNA is compressed using a combination of cationic liposome and oligoarginine, and then the liposome containing siRNA. As a result of measuring the particle size of the complex (CLS / R8 / siRNA), the particle size was reduced to 108 nm as shown in FIG. When the charge ratio of ^{oligoarginine} R8 / siRNA is 5 and the charge ratio of 89W-CLS modified with 89W Penestratin to siRNA is 4, the siRNA is prepared using a combination of cationic liposome and oligoarginine. After compression, the liposome complex (89W-CLS / R8 / siRNA) modified with ^89W Penetratin and containing siRNA had a particle size close to that of CLS / R8 / siRNA and was 105 nm. The maximum light intensity of the liposome complex modified with ^89W Penetratin and containing siRNA is lower than the maximum light intensity of CLS / R8 / siRNA, because the former particle size distribution range is widened.

When the charge ratio of ^{oligoarginine} R8 / siRNA is 5 and the charge ratio of the cationic liposome CLS or the cationic liposome 89W-CLS modified with 89W Penestratin to siRNA is 4, the R8 / siRNA complex, CLS / siRNA. A / R8 complex and an 89W-CLS / siRNA / R8 complex were prepared, respectively, and a ζ potential analyzer was used to obtain a ζ potential in water at an electric field strength of 5 V / cm at 23 ° C and an electrode spacing of 0.4 cm. It was measured. The ζ potential measurement result is shown in FIG.

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

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

Specifically, the CLS / R8 / siRNA complex prepared by the method described in Example 2 was centrifuged at a rate of 12000 × g for 20 minutes, and the precipitation status of each liposome preparation was observed.

The results are shown in FIG. 3C. As can be seen from FIG. 3C, when the charge ratio of oligoarginine R8 / siRNA is greater than 5 and the charge ratio of cationic liposome CLS to siRNA is 4, the CLS / R8 / siRNA complex undergoes fast centrifugation. There was no apparent precipitation over the course, indicating that cationic liposomes and oligoarginine can jointly compress siRNA to form stable liposome formulations.

[Example 5]
[Characteristics of stability of liposome complex containing drug 2]
Each CLS / R8 / siRNA complex in DEPC-treated water prepared by the method described in Example 2 was centrifuged at a rate of 12000 × g for 20 minutes, and each supernatant was gently added to an agarose gel loading hole to control free siRNA. A group was set and electrophoresis was performed. After completion of the electrophoresis, the gel was stained with GelRed working solution in a dark place for 30 minutes, and the gel was imaged under UV 302 nm conditions. The results are shown in FIG. 3D.

As can be seen from FIG. 3D, when the charge ratio of oligoarginine R8 / siRNA is greater than 5 and the charge ratio of cationic liposome CLS to siRNA is 4, after high speed centrifugation of the CLS / R8 / siRNA complex, CLS / R8 / siRNA is still retained in the supernatant solution and free siRNA does not leak. This indicates that both cationic liposomes and oligoarginine can form stable liposome control agents by compressing siRNA.

[Example 6]
(Formulation of liposomal pharmaceutical product with modified surface)
The surface of the liposome pharmaceutical product was modified with a cell-permeable peptide to obtain a surface-modified pharmaceutical product.

<< I. Preparation of Cell Permeable Peptide-PEG-Phospholipid >>
DSPE-polyethylene glycol maleimide (DSPE-PEG ₃₄₀₀ -maleimide) (Laysan Bio, 146-123) and a cysteine-modified peptideratin derivative ( ^89W Penetratin-Cys) at the end are reacted in one step to allow cell permeation. Sex peptide-PEG-DSPE was obtained.

Specifically, 20 mg of DSPE-PEG ₃₄₀₀ -maleimide was dissolved in 1 mL of N, N-dimethylformamide, and 18 mg of ^{89 W} Penetratin-Cys dissolved in a 10 mL phosphate buffer (10 mM, pH 7.2) solvent was stirred. Add in condition and continued stirring overnight at 25 ° C. to complete the reaction. After completion of the reaction, the resulting mixture was placed in pure water under ice bath conditions, dialyzed for 2 days and lyophilized to give ^89W Penetratin-PEG ₃₄₀₀ -DSPE as a white agglomerate.

Here, the polypeptide ^89W Penetratin is a derivative of penetratin, and the specific amino acid sequence is RQIKIWFWWWRRMKWKK (SEQ ID NO: 29). Cell Permeable Peptides-For cell permeabilizing peptides in PEG-DSPE, other penetratin derivatives may be used, eg, other penetratin derivatives shown in Table 1, such as glutamine at position 2 of penetratin and /. Alternatively, a product obtained by mutating a hydrophobic amino acid to glutamine at the 8-position and / or asparagine at the 9-position may be used (see also Chinese patent application CN2017104534.7).

Cysteine residues (C) may be added to the N-terminus or C-terminus of the cell-permeable peptide ^89W Penetratin or other penetratin derivatives to facilitate reaction with polyethylene glycol end groups.

<< ii. Formulation of liposomal pharmaceutical product with modified surface >>
In the whole liposome membrane material, the molar ratio of DOTAP: DOPE: cholesterol: (mPEG ₂₀₀₀ -DSPE and ^89W Penetratin-PEG ₃₄₀₀ -DSPE) is about 28.5: 28.5: 38: 5.

3.5 mg DOTAP (Shanghai Aywaille Special Pharmaceutical Technology Co., Ltd., 132172-1--3), 3.72 mg DOPE (Shanghai Aywaille Special Pharmaceutical Technology Co., Ltd., 4004-05-1), 2.59 mg cholesterol (Sigma-Aldrich, C8667) was weighed. Put the above amounts of DOTAP, DOPE and cholesterol in a 50 mL round bottom flask and add 1 mL of chloroform to dissolve the lipid membrane material DOTAP, DOPE and cholesterol, ie 16.67 μM CLS is 5 μM DOTAP. It contains 5 μM DOPE and 6.67 μM DOPE. Chloroform was removed from the obtained solution with a rotary evaporator (Shanghai Shinsei Technology Co., Ltd., R210L) to obtain a uniform dry lipid membrane.

The obtained dry lipid membrane was hydrated with 1 mL of a 5% aqueous glucose solution in a water bath at 37 ° C. for about 10 minutes by ultrasonic waves. After the lipid membrane is completely hydrated, the liposomes are extruded using a mini extruder (Hamilton, 81320) (the order of the nuclear pore membranes is 200 nm, 100 nm and 50 nm, respectively), cationic liposomes (as described herein). Is also called CLS).

4 mg of oligoarginine R8 (Shanghai ▲ 忻 ▼ Hiroshi Biotechnology Co., Ltd.) is dissolved in DEPC-treated water (Dalian Mio Biotechnology Co., Ltd., MA0018) to prepare a 4 mg / mL oligoarginine R8 solution, and 33 μg of FAM. SiRNA labeled with (in this example, siRNA for c-myc is used and its sense strand (5'-3') is FAM-AACGUAGCUUCACCAACAdTdT (SEQ ID NO: 79), ie 5'is FAM. The modified antisense strand (5'-3') was UGUGUGGUGAAGCUAAACGUudTdT (SEQ ID NO: 80) and was dissolved in 125 μL of DEPC-treated water to prepare a 20 μM FAM-siRNA solution.

The oligoarginine R8 solution and the 20 μM FAM-siRNA solution are mixed in equal volumes at a charge ratio of 5, vortexed for 30 seconds, incubated for 30 minutes at 37 ° C., and the complex R8 / FAM-siRNA. Got An 8 μL R8 / FAM-siRNA complex was taken, mixed with the cationic liposome CLS prepared above at a charge ratio of 4 between the liposome and FAM-siRNA, vortexed for 30 seconds, and then CLS / R8 / FAM-siRNA. A complex was obtained.

For mPEG ₂₀₀₀ -DSPE and 89W Penetratin-PEG ₃₄₀₀ -DSPE in 5% molar ratio to total liposomes, 0% of mPEG ₂₀₀₀ -DSPE and ^89W Penetratin-PEG ₃₄₀₀ -DSPE to ^89W Penetratin-PEG ₃₄₀₀ - ^DSPE , respectively. , 20%, 40%, 60%, 80%, 100% molar ratio mixtures were prepared. That is, ^89W Penetratin-PEG ₃₄₀₀ -DSPE has a molar ratio of 0%, 1%, 2%, 3%, 4%, and 5% to the total liposome membrane material, respectively.

The obtained CLS / R8 / FAM-siRNA complex was obtained from 89W ^{Penetran-PEG 3400-DSPE with respect to mPEG 2000-DSPE and 89W} _{Penetratin-PEG 3400 -} ^DSPE _, respectively, at 0%, 20%, 40%, 60% and 80. After mixing with the mixture having a molar ratio of%, 100%, and after incubating at 55 ° C. for 30 minutes, the surface was modified with 0%, 1%, 3%, 4%, and 5% of the cell-permeable peptide. Liposomal pharmaceutical formulations, ie 89W-CLS / R8 / FAM-siRNA, were obtained.

[Example 7]
(Quantitative evaluation of cell ingestion for pharmaceutical products whose surface is modified with different proportions of cell-permeable peptides)
U87 cells (human malignant glioblastoma cells, purchased from ATCC) were cultured in DMEM complete medium (DMEM medium supplemented with 10% FBS and 1% penicillin-streptomycin). Take well-grown U87 cells, resuspend in DMEM complete medium, inoculate 5 × 10 ⁵ cells / well, 1 mL per well into a 6-well plate, and inoculate 1 fresh culture medium daily after inoculation. The experiment was carried out after changing the times and culturing for 2 to 3 days.

After discarding the culture medium, U87 cells were washed 3 times with sterile PBS, and 1 mL of the liposome pharmaceutical preparation (FAM-labeled siRNA labeled with FAM) having a surface modified with a different proportion of the cell-permeable peptide prepared in Example 6 was used. Serum-free DMEM medium (enclosed) was added, and the cells were incubated at 37 ° C. and 5% CO ₂ for 4 hours. After incubation, the culture was discarded and a PBS buffer solution containing 0.02 mg / mL sodium heparin was used to wash away the positive electrical substances adsorbed on the wells and cell surface. The cells were treated with trypsin, resuspended in 200 μL of sterile PBS buffer, sprayed uniformly, then the cells were counted for ^each sample, and 104 cells were taken for flow cytometry detection. Cells incubated in serum-free DMEM medium were used as the negative control group. All experiments were performed in triplicate. The results are shown in FIG. 4 (mean ± SD, n = 3, * p <0.05, * indicate that there is a statistically significant difference between the two groups ** p <0. .01, *** p <0.001, ** and *** indicate that there is a statistically very significant difference between the two groups). In FIG. 4, “0%, 1%, 2%, 3%, 4%, 5%” on the horizontal axis means that the cell-permeable peptide is 0%, 1%, 2%, 3%, 4%, respectively. Represents a surface-modified liposome pharmaceutical formulation at a rate of 5%, ie 89W-CLS / R8 / siRNA.

As can be seen from FIG. 4, when ^89W Penetratin-PEG ₃₄₀₀ -DSPE had a molar ratio of 2%, 3%, 4% to the total liposome membrane material, the cell-permeable peptide modified the surface in the above proportions. The cell ingestion effect on the pharmaceutical preparation is superior to that of the commercially available gene transfection reagent Lipo2000.

[Example 8]
(Quantitative evaluation of cell intake for drug formulations prepared using liposome membrane materials with different compounding ratios)
U87 cells (human malignant glioblastoma cells, purchased from ATCC) were cultured in DMEM complete medium (DMEM medium supplemented with 10% FBS and 1% penicillin-streptomycin). Take well-grown U87 cells, resuspend in DMEM complete medium, inoculate 5 × 10 ⁵ cells / well, 1 mL per well into 6-well plates, and inoculate with fresh culture medium daily. The experiment was carried out after changing once and culturing for 2 to 3 days.

According to the method described in Example 6, a liposome pharmaceutical preparation (encapsulated with siRNA labeled with FAM) having a compounding ratio of the liposome membrane material as shown in Table 2 was prepared.

After discarding the culture medium for the cultured U87 cells, the cells were washed 3 times with sterile PBS. To each well was added 1 mL of serum-free DMEM medium containing the liposomal pharmaceutical preparations Formula 1, Formula 2 and Formula 3 prepared according to the respective formulations, and incubated at 37 ° C. and 5% CO ₂ conditions for 4 hours. After incubation, the culture was discarded and the positive electrical substances adsorbed on the wells and cell surface were washed away with a PBS buffer solution containing 0.02 mg / mL heparin sodium. The cells were treated with trypsin, resuspended in 200 μL sterile PBS buffer, sprayed evenly, the cells were counted for ^each sample, and 104 cells were taken for flow cytometry detection. Cells incubated in serum-free DMEM medium were used as a negative control group. All experiments were performed in triplicate. The results are shown in FIG. 5 (mean ± SD, n = 3, * p <0.05, * indicate that there is a statistically significant difference between the two groups ** p <0. .01, *** p <0.001, ** and *** indicate that there is a statistically very significant difference between the two groups).

As can be seen from FIG. 5, the prepared liposome pharmaceutical product Formula 1, Formula 2, and Formula 3 can all be ingested by cells, and there is a significant difference compared to the control group, and the cells for the prepared liposome pharmaceutical product Formula 2 The ingestion effect of is optimal.

[Example 9]
(Diameter size of pharmaceutical preparation prepared using liposome membrane material having different compounding ratios of component (iv))
According to the method described in Example 6, when the molar ratio of the component (i): component (ii): component (iii) of the liposome membrane material is 3: 3: 4, the compounding ratio of the component (iv) is shown in Table 3. A liposomal pharmaceutical preparation (encapsulated with FAM-labeled siRNA) as shown in the above was prepared.

A liposome pharmaceutical preparation having a molar ratio of 1%, 5%, 8%, and 10% of the component (iv) to the liposome membrane material was obtained. The particle size of the liposome pharmaceutical product was measured. The particle size measurement results are shown in FIG. In FIG. 6, “1%, 5%, 8%, 10%” on the horizontal axis means that the component (iv) is 1%, 5%, 8%, and 10%, respectively, with respect to the liposome membrane material. A liposome pharmaceutical product is shown.

As can be seen from FIG. 6, when the component (iv) has a molar ratio of 1% to 10% with respect to the liposome membrane material, the particle size of the liposome pharmaceutical preparation is 100 nm to 200 nm. In addition, the particle size tended to increase as the molar ratio of the component (iv) to the liposome membrane material increased.

[Example 10]
(Quantitative evaluation of cell intake for pharmaceutical formulations prepared with cationic liposome / siRNA at different charge ratios)
U87 cells (human malignant glioblastoma cells, purchased from ATCC) were cultured in DMEM complete medium (DMEM medium supplemented with 10% FBS and 1% penicillin-streptomycin). Take well-grown U87 cells, resuspend in DMEM complete medium, inoculate 5 × 10 ⁵ cells / well, 1 mL per well into 6-well plates, and inoculate with fresh culture medium daily. The experiment was carried out after changing once and culturing for 2 to 3 days.

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

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

After discarding the culture medium for the cultured U87 cells, the cells were washed 3 times with sterile PBS. Add 1 mL serum-free DMEM medium containing a pharmaceutical preparation having a charge ratio of cationic liposome / siRNA of 2, 4, 6, 8, 10, and 12 to each well, and incubate for 4 hours at 37 ° C. and 5% CO ₂ conditions. did. After incubation, the culture was discarded and the positive electrical substances adsorbed on the wells and cell surface were washed away with a PBS buffer solution containing 0.02 mg / mL heparin sodium. The cells were treated with trypsin, resuspended in 200 μL sterile PBS buffer, sprayed evenly, the cells were counted for ^each sample, and 104 cells were taken for flow cytometry detection. Cells incubated in serum-free DMEM medium were used as a negative control group. All experiments were performed in triplicate. The results are shown in FIG. 2 (mean ± SD, n = 3, * p <0.05, * indicate that there is a statistically significant difference between the two groups ** p <0. .01, *** p <0.001, ** and *** indicate that there is a statistically very significant difference between the two groups).

As can be seen from FIG. 2, all the pharmaceutical preparations prepared with the charge ratios of cationic liposome / siRNA of 2, 4, 6, 8, 10 and 12 were ingested by cells, and there was a significant difference compared with the control group. In addition, the effect of cell ingestion on liposome pharmaceutical formulations prepared when the charge ratio of cationic liposome / siRNA is 4 is optimal.

[Example 11]
(Evaluation of cell transfection effect by pharmaceutical preparations whose surface is modified with different proportions of cell-permeable peptides)

First, cells were cultured using a 35 mm Dish NanoCulture Dish, the sphere-forming ability and morphology of the cells were observed, and a cell mass model was constructed.

Specifically, 1.5 mL of DMEM complete medium is taken and added to a 35 mm Dish NanoCulture Dish (cell mass culture plate provided by JSR Corporation), and the Dish is placed in a freezer centrifuge and centrifuged. Centrifuged at 1000 xg for 5 minutes (Dish has only medium, no cells). After centrifugation, the dish was placed on an ultra-clean desk and allowed to stand for 30 minutes.

After taking various cells in good growth, digesting and centrifuging, add medium to adjust the cell concentration to 5 × 10 ⁵ cells / mL, blow with a pipette gun to evenly disperse the cells, and slowly Dish. 1.5 mL of the cell suspension was added to the cells, and the cells were allowed to stand on the operating table for 20 minutes and cultivated in an incubator at 37 ° C. The next day, change the liquid, remove the dish, and gently suck out 1.5 mL of medium (the operation of floating cells and wall-attached cells is the same, and when the liquid is changed, the cells already form a cell mass initially. So it is submerged in the bottom of the dish), fresh 1.5 mL DMEM complete medium was added.

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

As can be seen from FIG. 7, A549 and Caco-2 have good adhesion, adhere to the wall and easily grow and do not form a cell mass, and Rb-1 is a floating cell, but the cell easily gathers in a sphere. Very easy to unravel during processing. By comparison, U87 and 293T have the best cell mass formation capacity, are generally spherical in morphology, are uniform in size, and are suitable for construction as a cell mass model for further exploration.

As can be seen from the results of FIG. 8, the cell mass penetrating ability of the polypeptide is significantly different, and the cell mass model constructed by the 35 mm Dish NanoCulture Dish can distinguish the permeation ability of different polypeptides. Remarks: “S8” in FIG. 8 is a polymer of 8 serines and was used as a negative control group. "PE" is a wild-type polyethylene. 89W is a product mutated to tryptophan for glutamic acid at position 8 and asparagine at position 9 of penetratin. 289W is a product mutated to tryptophan for penetratin 2-position glutamine, 8-position glutamine, and 9-position asparagine. Derivatives of wild-type Penetratin have significantly improved ability to permeate cell clusters.

Next, using 293T cell mass, GFP-siRNA was encapsulated, and the transfection effect of the pharmaceutical preparation on which the surface of the cell-permeable peptide was modified at different ratios was evaluated.

Similar to Example 6 above, a GFP-siRNA liposome pharmaceutical formulation was prepared in which the cell-permeable peptide was surface-modified at different proportions, where the GFP-siRNA used was the sense strand (5'-3'. ) Was GCAUGUACCACGAGUCCAATT (SEQ ID NO: 81), and the antisense strand (5'-3') was UUGGACUCGUGGUACAUGCTT (SEQ ID NO: 82). Specifically, the oligoarginine R8 solution and the 20 μM GFP-siRNA solution are mixed in equal volumes at a charge ratio of 5, vortexed for 30 seconds, incubated at 37 ° C. for 30 minutes, and the complex R8. / GFP-siRNA was obtained. Take 8 μL of R8 / GFP-siRNA complex, mix the prepared cationic liposome CLS with a charge ratio of 4 for the liposome and FAM-siRNA, vortex for 30 seconds, and then vortex the CLS / R8 / GFP-siRNA complex. I got a body.

Further, according to the method described in Example 6, a liposome pharmaceutical preparation modified with 1%, 3%, and 5% cell-permeable peptide on the surface, that is, 89W-CLS / R8 / GFP-siRNA complex was obtained. ..

A serum-free DMEM solution containing a liposome pharmaceutical formulation encapsulating 400 nM GFP-siRNA was added to the 293T cell mass model, incubated at 37 ° C. under 5% CO ₂ conditions for 4 hours, and the drug solution was discarded to complete the fresh DMEM. The medium was added and the cells were continuously incubated for 24 hours, washed 3 times with PBS buffer solution, and the GFP fluorescence status (also referred to as GFP-239T cell mass in the specification) was observed with a laser confocal microscope. Cells incubated in serum-free DMEM medium were used as a negative control group. All experiments were performed in triplicate. The results are shown in FIG. 9 (mean ± SD, n = 3, * p <0.05, * indicate that there is a statistically significant difference between the two groups ** p <0. .01, *** p <0.001, ** and *** indicate that there is a statistically very significant difference between the two groups).

Similarly, the transfection effect of U87 cell mass by a liposome pharmaceutical preparation encapsulated with 400 nM GFP-siRNA was evaluated (also referred to as GFP-U87 cell mass in the text). The results are shown in FIG.

In FIGS. 9 and 10, "1%, 3%, 5%" is 89W-CLS / R8, which is a liposomal pharmaceutical preparation whose surface is modified with a cell-permeable peptide at a ratio of 1%, 3%, and 5%, respectively. / SiRNA is shown.

In FIG. 9, a GFP-239T cell mass was constructed using a 35 mm Dish Nano Culture Dish. The 293T cells are not tumor cells, and the higher the molar ratio of ^89W Penetratin-PEG-DSPE to the liposome membrane material, the higher the degree of permeation of the liposome drug into the cell mass. In FIG. 10, a GFP-U87 cell mass was constructed using a 35 mm Dish Nano Culture Dish. U87 cells are cerebral glioma cells, and when the molar ratio of ^89W Penetratin-PEG-DSPE reaches 3% to 5% of the liposome membrane material, the liposome drug has high permeability to the cell mass.

[Example 12]
(Qualitative and quantitative evaluation of transfection effect on LUC-U87 cells by pharmaceutical preparations containing positively charged polyamino acids of different structural types)
Oligoarginines such as R8, R10, R12, c-R8, c-R10, c-R12 are used as positively charged polyamino acids of different structural types, and the charge ratio of the oligoarginine to siRNA is 5, CLS. A liposome pharmaceutical preparation was prepared in which the charge ratio of LUC-siRNA was set to 4, and the surface of the liposome was modified according to Example 6, containing different oligoarginines and having a cell-permeable peptide at a ratio of 3%. The LUC-siRNA used is GCUGCACUCUGGCGACAUTT (SEQ ID NO: 83) for the sense strand (5'-3') and AAUGUCGCCAGAGUGCAGCTT (SEQ ID NO: 84) for the antisense strand (5'-3').

Take well-grown U87 cells (purchased from ATCC) and inoculate each 48-well plate with 5 x 10 ⁴ cells / well, and after inoculation, change the culture medium once daily and culture for 2-3 days. After that, the experiment was conducted. After discarding DMEM complete medium, wash 3 times with sterile PBS, add serum-free DMEM medium containing a liposome pharmaceutical preparation containing 400 nM LUC-siRNA, and incubate for 4 hours at 37 ° C. and 5% CO ₂ conditions. Then, discard the drug solution, add DMEM complete medium, continue to incubate for 24 hours (also referred to as LUC-U87 cells in the specification), and wash away the positively charged adsorbent with a PBS buffer solution containing 0.02 mg / mL heparin sodium. Then, a firefly luciferase substrate was added, and the fluorescence intensity was measured with a small animal biofluorescence / bioluminescence imaging system (IVIS Spectrum). Qualitative analysis was performed using the quantitative circle selection function of the target area (Region of interest, ROI) of the small animal biooptical imaging system. The results are shown in FIG.

This example is to explore the gene knockdown effect of positively charged polyamino acids of different structural types, and from FIG. 11, changing the degree of polymerization and structure of oligoarginine in liposome pharmaceutical formulations is the gene knockdown. It was found that it had no significant effect on the down action.

[Example 13]
(Evaluation of apoptosis-promoting ability of bEnd.3 cells and U87 cells by delivery carrier encapsulating siRNA)
BEnd. With good log-periodic growth in DMEM medium. 3 cells and U87 cells (both purchased from ATCC) were seeded in 12-well plates at a concentration of 5 × 10 ⁴ / well, respectively, and at 37 ° C. and 5% CO ₂ conditions, a single layer of cells was formed. The cells were cultured until the bottom of the well was covered.

The culture was discarded and washed 3 times with sterile PBS buffer. Subsequently, the liposome preparation (89W-CLS / R8 / siRNA) prepared in Example 6 containing 400 nmol / L siRNA (the liposome preparation has a charge ratio of oligoarginine to siRNA of 5, and the charges of CLS and siRNA are 5). Add 400 μL (with a ratio of 4 and ^89W Penetratin-PEG ₃₄₀₀ -DSPE modifying the surface at a rate of 3%), place in an incubator and incubate for 6 hours before discarding the drug solution. Subsequently, the cells were washed 3 times with sterile PBS buffer solution, 1 mL of complete medium was added, and the cells were continuously cultured for 18 hours.

The cells were then collected, washed with PBS, treated with trypsin without EDTA for 1 minute, and finished with DMEM culture medium. Subsequently, the cells were collected in a centrifuge tube, and the cells were double-stained at the step of the cell apoptosis test kit (Nanjing Kaisei Bioscience and Technology Development Co., Ltd., KGA108) and detected using flow cytometry. Cells incubated in serum-free DMEM medium were used as the negative control group. All experiments were performed in triplicate. The results are shown in FIGS. 14A and 14B (mean ± SD, n = 3, * p <0.05, * indicate that there is a statistically significant difference between the two groups **. p <0.01, *** p <0.001, ** and *** indicate that there is a statistically very significant difference between the two groups).

As a result, the delivery carrier designed by the present invention can specifically promote the apoptosis of the tumor cell U87, and the normal cell bEnd. It was shown that the growth of 3 cells was not significantly affected.

[Example 14]
(Evaluation of cytotoxic activity of bEnd.3 cells and U87 cells by delivery carrier encapsulating siRNA)
BEnd. With good log-periodic growth. 3 cells (purchased from ATCC) and U87 cells were taken and cultured in DMEM complete medium and placed in the middle 60 wells of a 96-well flat bottom plate at a concentration of 5 × 10 ³ cells / cm ² respectively. The cells were filled with sterile PBS buffer and cultured at 37 ° C. and 5% CO ₂ conditions until a single layer of cells covered the well bottom. Subsequently, the culture solution was discarded, washed 3 times with a sterile PBS buffer solution, 200 μL of the culture solution containing a delivery carrier of 400 nM siRNA was added, placed in an incubator and incubated for 6 hours, and then the drug solution was discarded and the sterile PBS buffer solution was discarded. Washed 3 times. Subsequently, complete medium was added and the cells were subsequently cultured for 12 hours. Then, add 20 μL of MTT solution (5 mg / mL) to each well, continue culturing for 4 hours, gently discard the liquid, wash with PBS buffer solution 3 times, add 150 μL of DMSO to each well, and place on a shaker. After vibrating at a low speed (50 rpm) for 20 minutes, the absorbance value of each well of OD490 nm was measured with a microplate reader (BioTek, PowerWave XS). Blank wells (wells supplemented with DMEM complete medium, MTT and DMSO only) and negative control wells (wells supplemented with cells, DMEM complete medium, MTT and DMSO only) were installed simultaneously. All experiments were performed in triplicate. The results are shown in FIGS. 14C and 14D (mean ± SD, n = 3, * p <0.05, * indicate that there is a statistically significant difference between the two groups **. p <0.01, *** p <0.001, ** and *** indicate that there is a statistically very significant difference between the two groups).

As a result, the delivery carrier designed by the present invention has a bEnd. It was shown that the effect on the growth state of 3 cells and U87 cells was small, and no cytotoxic activity was observed under the concentration conditions examined.

[Example 15]
(Distribution of gene delivery carriers labeled with DiD cell membrane fluorescent probe in the body after nasal administration)
First, liposomes labeled with the lipophilic fluorescent dye DiD were prepared and used to track the liposomes. The DiD cell membrane red fluorescent probe was purchased from Dalian Mio Biotechnology Co., Ltd. and was MB6190. In the same manner as in Example 1, the membrane material was weighed, a DiD dye was added as described in the kit description, and both were dissolved in chloroform to prepare DiD-labeled CLS / R8 / siRNA. .. Further, in the same manner as in Example 6, the charge ratio of oligoarginine to siRNA was 5, the charge ratio of CLS to siRNA was 4, and the surface was modified by ^89W Penetratin-PEG ₃₄₀₀ -DSPE at a ratio of 3%, DiD. Liposomal pharmaceutical formulation 89W-CLS / R8 / siRNA labeled with.

Twelve ICR mice (20 g, male, 6 weeks old) were set in three groups (89W-CLS / R8 / siRNA group, CLS / R8 / siRNA group, saline (NS) group). Approximately 30 μL of DiD-labeled nucleic interfering carrier was administered to each mouse by nasal administration (DiD concentration is 0.01 μM), the animal was euthanized after 2.5 hours, and the main organs of the animal were perfused and fixed. It was collected and the fluorescence distribution of each organ was detected using a small animal bioimaging system. The results are shown in FIG.

As a result, the fluorescence intensity in the brain of the 89W-CLS / R8 / siRNA group was higher than that of the CLS / R8 / siRNA group, and the modification of the cell-permeable peptide significantly enhanced the tissue permeability of the delivery carrier. Furthermore, it was shown that the ability to enter the brain can be enhanced and the amount of genes accumulated in the brain can be increased.

[Example 16]
(Exploration of the engraftment mechanism by which carriers labeled with DiD cell membrane fluorescent probes deliver FAM-siRNA)
In the same manner as in Example 1, the membrane material is weighed, a DiD cell membrane fluorescent probe (Dalian Mio Biotechnology Co., Ltd., MB6190) is added as described in the kit description and labeled with a DiD cell membrane fluorescent probe. CLS, that is, CLS / DiD, was prepared. The pharmaceutical formulations labeled with DiD and / or FAM fluorescence were prepared in the same manner as in Example 6.

U87 cells (purchased from ATCC) were cultured in DMEM complete medium (DMEM medium supplemented with 10% FBS and 1% penicillin-streptomycin). Take well-grown U87 cells, resuspend in DMEM complete medium, inoculate 5 × 10 ⁵ cells / well, 1 mL per well into a 6-well plate, and inoculate 1 fresh culture medium daily after inoculation. The experiment was carried out after changing the times and culturing for 2 to 3 days.

After discarding the culture medium for the cultured U87 cells, the cells were washed 3 times with sterile PBS. To each well, 1 mL of serum-free DMEM medium containing pharmaceutical formulations labeled with different fluorescence of 1 μM siRNA was added and incubated at 37 ° C. for 4 hours under 5% CO ₂ conditions. After incubation, the culture was discarded and the positive electrical substances adsorbed on the wells and cell surface were washed away with a PBS buffer solution containing 0.02 mg / mL heparin sodium. The cells were treated with trypsin, resuspended in 200 μL of sterile PBS buffer, sprayed uniformly, then the cells were counted for ^each sample, and 104 cells were taken for flow cytometry detection. In the experiment, cells incubated in blank serum-free DMEM medium were set as the negative control group. In addition, two positive control groups were set up: 89W-CLS / R8 / FAM-siRNA containing only FAM and 89W-CLS / DiD / R8 / siRNA containing only DiD. Further, the following pharmaceutical group groups: R8 / FAM-siRNA group, CLS / DiD group, CLS / DiD / R8 / FAM-siRNA group, 89W-CLS / DiD / R8 / FAM-siRNA group were set. All experiments were performed in triplicate. The results are shown in FIG.

From FIG. 16D, the pharmaceutical product formed by compressing siRNA only with R8 has a small amount of engraftment, but the pharmaceutical product formed by co-compressing siRNA with CLS and R8 can successfully engraft ( (See FIG. 16F), it was found that a pharmaceutical product formed by co-compacting siRNA with 89W-modified CLS and R8 can significantly increase the cell intake for gene carriers (FIG. 16G). reference). From FIGS. 16B, 16C and 16G, it was found that the cells ingested the 89W-CLS / R8 / FAM-siRNA pharmaceutical preparation and showed a positive result of double fluorescence of DiD and FAM. This allows CLS and siRNA to enter the cell together in the form of a complex, not in the free form.

[Example 17]
(Distribution of gene delivery carriers labeled with DiD cell membrane fluorescent probe in U87 in-situ brain tumor model mice after nasal administration)
First, liposomes labeled with the lipophilic fluorescent dye DiD were prepared and used to deposit the liposomes. The DiD cell membrane red fluorescent probe was MB6190 purchased from Dalian Mio Biotechnology Co., Ltd. In the same manner as in Example 15, the membrane material was weighed, a DiD dye was added as described in the kit description and co-dissolved in chloroform to prepare DiD-labeled CLS / R8 / siRNA. .. Further, as described in Example 6, the charge ratio of oligoarginine to siRNA is 5, the charge ratio of CLS to siRNA is 4, and ^89W Penetratin-PEG ₃₄₀₀ -DSPE modifies the surface at a ratio of 3%. The DiD-labeled liposome pharmaceutical formulation 89W-CLS / R8 / siRNA was prepared.

U87 cells in log-growth phase were taken, cell numbers were counted and the cells were resuspended in PBS buffer. Each BALB / c nude mouse (Shanghai Xihui-Shanghai Test Animal Co., Ltd., China) was inoculated with 6 × 10 ⁵ U87 cells (dispersed in 5 μL PBS buffer). Nude mice were anesthetized with 7% hydrous chloral, fixed with a brain stereostatizer, and cells were inoculated into the striatal site (0.6 mm in front of bregma, 1.8 mm on the right, 3 mm in the back) with a microsyringe, and the U87 original position. A brain tumor model was constructed.

Nine U87 in-situ brain tumor mice inoculated with U87 tumor cells for 18 days were randomly set with three groups (89W-CLS / R8 / siRNA group, CLS / R8 / siRNA group and saline group). Subsequently, 30 μL (DiD concentration 0.01 μM) of a nucleic acid delivery carrier labeled with DiD was nasally administered to each mouse, and the animal was euthanized 2.5 hours later, and after perfusion and fixation, the brain tissue and tumor of the animal were administered. Was collected, subjected to frozen section treatment, and the distribution of the DiD fluorescence signal was observed with a fluorescence microscope (Leica, Germany). The results are shown in FIG. The scale bar is 100 μm.

As a result, the 89W-CLS / R8 / siRNA group has higher fluorescence intensity at the tumor site compared to the CLS / R8 / siRNA group, and by modifying the cell-permeable peptide, the tissue permeability of the delivery carrier is enhanced. It has been shown that it can significantly enhance, further enhance the ability to enter the brain, and increase the accumulation of genes in the brain.

[Example 18]
(Pharmacodynamic evaluation of anti-U87 in-situ brain tumor by delivery carrier encapsulating siRNA)
According to the method described in Example 6, a liposome pharmaceutical preparation, that is, a CLS / R8 / siRNA and 89W-CLS / R8 / siRNA complex is prepared, and a commercially available cationic gene delivery carrier, Lipofectamine 2000 (Lipo2000,). Lipo2000 / siRNA complex was prepared according to the description of Invitrogen).

The U87 in-situ brain tumor model was constructed according to the method described in Example 17. After inoculation with U87 in-situ brain tumor, mice were randomly given 5 groups (n = 10), namely saline group, siRNA group, Lipo2000 / siRNA group, CLS / R8 / siRNA group and 89W-CLS / R8. The / siRNA group was set. Nasal administration (47 μg / kg siRNA) was performed once a day from the 5th day after inoculation with U87 in-situ brain tumor, and a total of 22 times were administered. I drew a curve. The results are shown in FIG. 18 (* p <0.05, * indicate statistically significant differences between the two groups; ** p <0.01, ** indicate the two groups. Shows that there is a very significant statistical difference between).

As a result, the median survival times of the saline group, siRNA group, Lipo2000 / siRNA group, and CLS / R8 / siRNA group were 23.5 days, 21.5 days, 24.5 days, and 26 days, respectively. The median survival time of the 89WP-CLS / R8 / siRNA group was successfully extended to 29 days, and the survival curve did not intersect with other groups, achieving the most prominent anticerebral glioma effect. These data demonstrated that 89WP-CLS / R8 / siRNA can deliver drugs to brain tumors to exert antitumor effects, and their unique delivery benefits can be translated into higher therapeutic effects.

[Example 19]
(Evaluation of the ability of delivery carriers encapsulated with siRNA to promote apoptosis in U87 in-situ brain tumor model mice)
A liposome pharmaceutical preparation was prepared according to the method described in Example 18, a U87 in-situ brain tumor model was constructed, and nasal administration was performed. Two days after the last dose (day 28), three mice from each group were randomly selected and euthanized, and brain tumors were harvested for immunofluorescent histochemical analysis. Tumors were fixed with 4% paraformaldehyde, embedded in paraffin and sliced. Apoptotic cells were detected by TUNEL staining and the TUNEL fluorescence intensity at the tumor site was analyzed with CaseViewer software. The results are shown in FIG. The scale bar is 20 μm.

In the TUNEL stained section, the intensity of green fluorescence indicates the number of apoptotic tumor cells. As a result, the Lipo2000 / siRNA group, the CLS / R8 / siRNA group, and the 89WP-CLS / R8 / siRNA group can all induce apoptosis in tumor cells, of which the 89WP-CLS / R8 / siRNA group It was shown to have the highest fluorescence intensity, i.e., the highest rate of apoptosis. This indicates that the delivery carrier designed in the present invention can exert an antitumor effect by inducing apoptosis of tumor cells in the body.

[Example 20]
(Evaluation of in-vivo nasal mucosal toxicity of delivery carrier encapsulating siRNA)
Eighteen SD rats (Shanghai Reiko Biotechnology Co., Ltd., China) were randomly given 6 groups (n = 3), namely saline group (negative control), 1% sodium deoxycholate group (positive control). ), SiRNA group, CLS / R8 / siRNA group, 89WP-CLS / R8 / siRNA group and Lipo2000 / siRNA group. Liposomal pharmaceutical formulations, ie CLS / R8 / siRNA and 89W-CLS / R8 / siRNA complexes, were prepared according to the method described in Example 6. In addition, a Lipo2000 / siRNA complex was prepared according to the description of the commercially available cationic gene delivery carrier Lipofectamine 2000 (Lipo2000, Invitrogen). A 50 μL formulation (with or without 160 nM siRNA) was nasally administered to the left nasal cavity of SD rats for 7 consecutive days, the rats were euthanized 24 hours after the last administration, and the nasal mucosa of the left nasal cavity was separated. Then, it was washed with physiological saline. The sample was fixed with 4% paraformaldehyde, sectioned and HE-stained. The results are shown in FIG. The scale bar is 50 μm.

As a result, the nasal mucosal epithelial cells of the positive control group (sodium deoxycholate group and Lipo2000 / siRNA group) showed relatively remarkable morphological changes, accompanied by nasal cilia shedding (red arrow), but in the saline group. , SiRNA group, CLS / R8 / siRNA group and 89WP-CLS / R8 / siRNA group showed that the morphology of nasal mucosal epithelial cells was complete and orderly. This is believed to indicate that the delivery carrier designed in the present invention is not significantly toxic to nasal mucosal epithelial cells and is a safe carrier for nasal to brain delivery.

Although some representative embodiments and details have been shown for purposes of exemplifying the present invention, those skilled in the art will be able to make various changes and modifications to them without departing from the scope of the subject invention. Obvious to. In this respect, the scope of the invention is limited only by the following claims.

Claims (13)

A pharmaceutical preparation prepared using a drug delivery carrier containing a liposome modified with a cell-permeable peptide having the following amino acid sequence or a derivative of penetratin and a positively charged polyamino acid as a polymer. ,
RX ₁ IKIWFX ₂ X ₃ RRMKWKK
Here, X ₁ , X ₂ and X ₃ are independently naturally occurring amino acids, glutamine (Q), asparagine (N), alanine (A), valine (V), leucine (L) and isoleucine. (I), proline (P), phenylalanine (F), tryptophan (W), methionine (M) and non-naturally occurring amino acids, α-aminobutyric acid, α-aminopentanoic acid, α-aminohexanoic acid, α -Selected from aminoheptanoic acid, for example X ₁ , X ₂ and X ₃ are independently selected from hydrophobic amino acids.
The pharmaceutical preparation is a mixture of a positively charged polyamino acid contained in a drug delivery carrier and a drug molecule to form a polymer-drug physical complex, which is modified with the physical complex and the cell-permeable peptide. A pharmaceutical preparation comprising the resulting liposomes to form a liposome complex containing a physical polymer-drug complex having a particle size of 50 nm to 300 nm, preferably 80 nm to 150 nm. The drug molecule may be a peptide drug (eg, octreotide), an antibody (eg, etanercept, adalimumab), rituximab, pembrolizumab (pembrolizumab), rabbrismab , Cyclogs, hormones, antibiotics (eg, chemotherapeutic agents doxorubicin, daunorubicin, epirubicin, actinomycin and vancomycin consisting of, and combinations thereof), nucleic acids, and the like. Selected from
For example, the drug molecule is selected from the group consisting of plasmid DNA, RNA such as small interfering RNA (siRNA), sense RNA, antisense oligonucleotide (ASO), aptamers, ribozyme and other nucleic acids.
For example, the pharmaceutical preparation according to claim 1, wherein the drug molecule is a drug molecule for a brain disease such as a brain tumor (for example, cerebral glioma), and is, for example, siRNA for c-myc. In the pharmaceutical formulation, the molar ratio of drug molecule (eg, chemotherapeutic agent) to cationic lipid (eg, DOTAP) is about 0.1-10, eg, 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, or
In the pharmaceutical product, the encapsulation amount of the drug molecule (for example, a chemotherapeutic agent) is about 1% to 30% (w / w) of the drug molecule (for example, a chemotherapeutic agent) with respect to the pharmaceutical product, for example. Approximately 1%, 2.5%, 5%, 7.5%, 10%, 12.5%, 15%, 17.5%, 20%, 22.5%, 25%, 27.5%, 30 % (W / w), preferably about 1% to 25% (w / w), more preferably about 3% to 20% (w / w), or
In the pharmaceutical preparation, the charge ratio between the positively charged polyamino acid (for example, oligoarginine) and the drug molecule is 1: 1 to 30: 1, for example, 1: 1, 5: 1, 10: 1. , 15: 1, 20: 1, 25: 1, 30: 1, and the charge ratio of the cationic liposome to the drug molecule is 1: 1 to 30: 1, for example 2: 1, 3: 1. The pharmaceutical preparation according to claim 1 or 2, characterized in that it is 4: 1, 5: 1, 6: 1, 8: 1, 10: 1, 12: 1. The cationic liposome contains (i) DOTAP; (ii) DOPE; (iii) cholesterol; (iv) mPEG ₂₀₀₀ -DSPE and ^89W Penetratin-PEG ₃₄₀₀ -DSPE as the membrane material, and the membrane material (i) :. The molar ratio of (ii) :( iii): (iv) is about 20-40: 20-40: 20-40: 1-20, and ^89W Penetratin-PEG ₃₄₀₀ -DSPE is used for the membrane material (iv). On the other hand, the molar ratio is about 20% to 80%.
For example, the molar ratio of the membrane material (i) :( ii) :( iii): (iv) is about 27.0 to 31.6: 27.0 to 31.6: 31.6 to 39.6: 1. -10, and ^89W Penetratin-PEG ₃₄₀₀ -DSPE has a molar ratio of about 20% to 80% with respect to the membrane material (iv).
For example, the molar ratio of membrane material (i) :( ii) :( iii) :( iv) is about 28.5: 28.5: 38: 5, and ^89W Penetratin-PEG ₃₄₀₀ -DSPE is the membrane material. (Iv) has a molar ratio of about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%. ,
The charge ratio of a positively charged polyamino acid (eg, oligoarginine) to a drug molecule (eg, nucleic acid such as siRNA) is 1: 1 to 30: 1, for example 1: 1, 5: 1. It is 10: 1, 15: 1, 20: 1, 25: 1, 30: 1, and the charge ratio of the cationic liposome to the drug molecule is 1: 1 to 30: 1, for example 2: 1. 3, 1, 4: 1, 5: 1, 6: 1, 8: 1, 10: 1, 12: 1, the pharmaceutical according to any one of claims 1 to 3. pharmaceutical formulation. It is a pharmaceutical product for nasal administration.
For example, a nucleic acid liposome preparation for nasal administration (for example, a siRNA liposome preparation), preferably used for the treatment of a brain disease such as a brain tumor (for example, cerebral glioma), claims 1 to 4. The pharmaceutical preparation according to any one of the above. A drug delivery carrier comprising liposomes modified with a cell-permeable peptide and positively charged polyamino acids as polymers.
The polymer mixes with the drug to form a polymer-drug complex.
The liposomes modified with the cell-permeable peptide are mixed with the polymer-drug complex to form a liposome complex containing the polymer-drug complex.
The cell-permeable peptide is a penetratin or a derivative of penetratin having the following amino acid sequence.
RX ₁ IKIWFX ₂ X ₃ RRMKWKK
Here, X ₁ , X ₂ and X ₃ are independently naturally occurring amino acids, glutamine (Q), asparagine (N), alanine (alanine, A), valine (valine, V). ), Leucine (L), isoleucine (I), proline (proline, P), phenylalanine (F), tryptophan (W), methionine (M) and non-naturally occurring amino acids. Select from α-aminobutyric acid, α-aminopentanoic acid, α-aminohexanoic acid, and α-aminoheptanoic acid. And, for example, X ₁ , X ₂ and X ₃ are independently selected from hydrophobic amino acids, drug delivery carriers. The cell-permeable peptide is a lipophilic derivative having the following amino acid sequence of penetratin: RWIKIWFQNRRMKWKK (SEQ ID NO: 24), RQIKIWFWNRRMKWKK (SEQ ID NO: 25), RQIKIWFQWRRMKWKK (SEQ ID NO: 26), RWIKR. The drug delivery carrier according to claim 6, wherein (SEQ ID NO: 28), RQIKIWFWWWRRMKWKK (SEQ ID NO: 29), RWIKIWFWWWRRMKWKK (SEQ ID NO: 30). The positively charged polyamino acid is, for example, a positively charged polyamino acid having a degree of polymerization of 2 to 50, and the spatial structure of the positively charged polyamino acid is linear or cyclic, and the composition of amino acid residues. Is L-type or D-type,
The positively charged polyamino acid having a degree of polymerization of 2 to 50 is, for example, a polyamino acid of arginine and / or lysine having a degree of polymerization of 6 to 12, and 1 to 20 (for example, 1 to 6) physiology thereof. It may be mediated by uncharged amino acid residues under certain conditions, preferably the positively charged polyamino acid is polyarginine with a degree of polymerization of 6-12, and the spatial structure of the polyarginine is linear or cyclic. The drug delivery carrier according to claim 6 or 7, wherein the polyarginine configuration is L-type or D-type. Liposomes modified with the cell-permeable peptide contain the following membrane materials:
(I) Cationic lipids: 1,2-di-0-octadecenyl-3-trimethylammonium-propane (DOTMA), dimethyldioctadecylammonium (DDAB), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) , 1,2-Dioreoil-3-dimethylammonium-propane (DODAP), 1,2-diacyloxy-3-dimethylammonium-propane, 1,2-dialkoxy-3-dimethylammonium-propane, dioctadecyldimethylammonium chloride (DODAC), 1,2-dimiristyloxypropyl-1,3-dimethylhydroxyethylammonium (DMRIE), 2,3-dioreyloxy-N- [2 (sperminecarboxamide) ethyl] -N-N-dimethyl- 1-Propylammonium trifluoroacetate (DOSPA) and combinations thereof, but not limited to these, preferably DOTMA, DOTAP, DODAC and DOSTA, most preferably DOTAP;
(Ii) Non-cationic lipids: 1,2-di- (9Z-octadecanoyl) -sn-glyceryl-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glyceryl-3-phosphocholine (DOPC) ), 1,2-Distearoyl-sn-glyceryl-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glyceryl-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glyceryl-3 -Phosphoethanolamine (DPPE), 1,2-dimyristyl-sn-glyceryl-3-phosphoethanolamine (DMPE), 2-diore oil-sn-glyceryl-3-phosphate- (1'-rac-glycerol) (1'-rac-glycerol) ( DOPG) and combinations thereof, but not limited to these, preferably DOPE and / or DOPC, and most preferably DOPE;
(Iii) Cholesterol;
(Iv) Polyethylene glycolylated (PEGylated) phospholipids and polyethylene glycol phospholipids conjugated to cell permeable peptides, said phospholipids are phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, sphingomyelin, said polyethyleneglycol (PEG). Is a polyethylene glycol chain up to 10 kDa in length, eg, 1 kDa, 2 kDa, 3 kDa, 4 kDa, 5 kDa, 6 kDa, 7 kDa, 8 kDa, 9 kDa, 10 kDa and any value between them, preferably polyethylene glycol chain. The PEGylated phospholipid is polyethylene glycol-distearoyl phosphatidylethanolamine (PEG-DSPE) and a derivative thereof, methoxy-polyethylene glycol-distearoyl phosphatidylethanolamine (mPEG-DSPE), and the cell-permeable peptide. The conjugated polyethylene glycol phospholipid is, for example, Penetratin / Penetratin derivative-PEG-DSPE.
The polyethylene glycol in the polyethylene glycol phospholipid coupled with the PEGylated phospholipid and the cell permeable peptide does not have the same chain length, and the polyethylene glycol chain in the polyethylene glycol phospholipid coupled with the cell permeable peptide is PEGylated phosphorus. It is characterized by being longer than the chain of polyethylene glycol in lipids, eg, longer at 0.5 kDa to 5 kDa, preferably longer at 0.75 kDa to 4 kDa, and even more preferably longer at any value between 1 kDa and 2 kDa. The drug delivery carrier according to any one of claims 6 to 8. The molar ratio of the membrane material (i) :( ii) :( iii): (iv) is about 20 to 40:20 to 40:20 to 40: 1 to 20, and polyethylene conjugated with a cell-permeable peptide. Glycol phospholipids have a molar ratio of about 20% to 80% with respect to the membrane material (iv).
For example, the molar ratio of the film material (i) :( ii) :( iii) :( iv) is about 27.0 to 31.6: 27.0 to 31.6: 31.6 to 39.6 :. Polyethylene glycol phospholipids of 1-10, coupled with cell-permeable peptides, have a molar ratio of about 20% to 80% with respect to the membrane material (iv).
For example, the molar ratio of the membrane material (i) :( ii) :( iii) :( iv) is about 28.5: 28.5: 38: 5, and polyethylene glycol phosphorus conjugated with a cell-permeable peptide. The drug according to claim 9, wherein the lipid is in a molar ratio of about 20%, 30%, 40%, 50%, 60%, 70%, 80% with respect to the membrane material (iv). Delivery carrier. The cationic liposome contains (i) DOTAP; (ii) DOPE; (iii) cholesterol; (iv) mPEG ₂₀₀₀ -DSPE and cell permeable peptide-PEG ₃₄₀₀ -DSPE as the membrane material, and the membrane material (i). ): (Ii): (iii): (iv) has a molar ratio of about 20-40: 20-40: 20-40: 1-20, and the cell-permeable peptide-PEG ₃₄₀₀ -DSPE is a membrane material. It is a molar ratio of about 20% to 80% with respect to (iv).
For example, the molar ratio of the film material (i) :( ii) :( iii) :( iv) is about 27.0 to 31.6: 27.0 to 31.6: 31.6 to 39.6 :. 1 to 10 and the cell permeable peptide-PEG ₃₄₀₀ -DSPE has a molar ratio of about 20% to 80% with respect to the membrane material (iv).
For example, the molar ratio of the membrane material (i) :( ii) :( iii) :( iv) is about 28.5: 28.5: 38: 5, and the cell-permeable peptide-PEG ₃₄₀₀ -DSPE is. , Approximately 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% moles with respect to the membrane material (iv). The drug delivery carrier according to any one of claims 6 to 10, characterized in that it is a ratio. The method for preparing the pharmaceutical product according to any one of claims 1 to 4.
(A) A step of preparing a cationic liposome having a cell-permeable peptide modified on the surface, and
(B) A step of preparing a physical complex consisting of a positively charged polyamino acid (eg, oligoarginine) and a drug molecule.
(C) A step of mixing the cationic liposome obtained by modifying the surface obtained in step (a) with a cell-permeable peptide and the physical complex obtained in step (b) at a constant charge ratio. A method characterized by inclusion. The method for preparing the pharmaceutical product according to any one of claims 1 to 4.
(A) A step of mixing the liposome membrane materials (i), (ii) and (iii) to prepare a blank liposome.
(B) A step of preparing a physical complex consisting of a positively charged polyamino acid (eg, oligoarginine) and a drug molecule.
(C) The step of mixing the blank liposome obtained in step (a) and the physical complex obtained in step (b) at a constant charge ratio, and
(D) A method comprising: mixing and incubating the liposomes obtained in step (c) with the membrane material (iv).

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