Lipoprotein-mimetic solid lipid nanoparticles and their applications for drug delivery

Abstract

According to the present invention, a drug delivery vehicle with excellent bioavailability and improved drug encapsulation efficiency can be provided by producing lipoprotein-mimetic solid lipid nanoparticles with a core-shell structure composed of albumin-bound cholesterol, fusogenic lipid, cationic lipid, triglyceride and cholesteryl ester.

Description

[Technical field]

[0001] The present invention relates to lipoprotein-mimetic solid lipid nanoparticles for drug delivery and their use as drug delivery vehicles. [Background technology]

[0002] The K-ras gene (KRAS) is one of the ras genes that are frequently mutated in some carcinomas, and mutations in codons 12 and 13 of the KRAS gene result in functional changes in the p21-ras protein, which is the product of this gene, and as a result, it promotes cell growth and division by excessively transmitting growth signals to the cell nucleus, and is involved in the carcinogenesis process. KRAS mutations are very common in human cancers, appearing in approximately 90% of pancreatic cancers, approximately 50% of colorectal cancers, and approximately 30% of non-small cell lung cancers, and mutation profiles have confirmed that the majority of these mutations are concentrated in codons 12 and 13 (Non-Patent Document 1).

[0003] For this reason, research into the development of anticancer drugs related to KRAS has been attempted for several decades using various methods, but no anticancer drugs have been clinically available, so KRAS has been considered an "undruggable" target. KRAS is activated when it binds to GTP (guanosine triphosphate), but it has a very high binding affinity with GTP and has almost no pockets on its surface that allow it to bind, making it very difficult to develop new drugs targeting it. In addition, because KRAS is an intracellular protein, it is also problematic in that it is not possible to use antibodies with large molecular weights.

[0004] In addition, nanoparticle paclitaxel (trade name Abraxane), which is a drug in which the anticancer drug paclitaxel is attached to albumin using albumin-fusion technology that takes advantage of the various binding abilities of albumin, has been shown to improve the tumor response of paclitaxel. However, Abraxane's pharmacokinetic properties and biodistribution are problematic in that it is less effective than the Taxol dosage form.

[0005] Therefore, there is a need to develop a drug delivery system that directly targets KRAS while being biocompatible and having high drug delivery efficiency. [Prior art documents] [Patent documents]

[0006] [Non-Patent Document 1] Samowitz WS,et al.,Cancer Epidemiol.Biomarkers Prev.9:1193-7,2000 Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide lipoprotein-mimetic solid lipid nanoparticles as a drug delivery vehicle having excellent bioavailability and highly efficient drug delivery effect, and uses thereof. [Means for solving the problem]

[0008] The present inventors have confirmed that solid lipid nanoparticles having a composition similar to that of natural lipoproteins can be used as drug delivery vehicles with excellent bioavailability while being capable of encapsulating drugs with high efficiency, thereby completing the present invention.

[0009] Therefore, the present invention relates to lipoprotein-mimetic solid lipid nanoparticles (SLNs) and their applications for biocompatible and highly efficient drug delivery. Effect of the Invention

[0010] According to the present invention, by producing core-shell lipoprotein-mimetic solid lipid nanoparticles composed of cholesteryl esters, triglycerides, albumin-bound cholesterol, fusogenic lipids and cationic lipids, it is possible to prevent aggregation between particles and ensure structural stability, and to provide a drug delivery vehicle with excellent bioavailability and improved drug encapsulation efficiency. [Brief description of the drawings]

[0011] [Figure 1] FIG. 1 is a schematic diagram of the preparation of lipoprotein-mimetic solid lipid nanoparticles according to the present invention and their form as drug delivery vehicles. [Diagram 2] FIG. 2 is a diagram showing Z-average values ​​of lipoprotein-mimicking solid lipid nanoparticles according to the content of their components. [Diagram 3] 3a and 3b show TEM images of lipoprotein-mimetic solid lipid nanoparticles. [Figure 4] FIG. 4 is a schematic diagram of the synthesis of monomeric siRNAs into reducible dimeric siRNAs. [Diagram 5] FIG. 5 shows the results of confirming the reducibility of reducible dimeric siRNA. [Figure 6] FIG. 6 shows the results of flow cytometry analysis confirming intracellular uptake of the siRNA / SLN complex. [Figure 7] FIG. 7 shows the results of confirming Kras mRNA levels using reverse transcriptase-polymerase chain reaction (RT-PCR) to confirm the gene silencing effect of the siRNA / SLN complex. [Figure 8]FIG. 8 shows the results of quantification of Kras mRNA levels to confirm the gene silencing effect of the siRNA / SLN complex. [Figure 9] FIG. 9 shows the results of confirming Kras mRNA levels using reverse transcriptase-polymerase chain reaction (RT-PCR) to confirm the gene silencing effect of the siRNA / SLN complex in an animal model. [Figure 10] FIG. 10 shows the results of quantification of Kras mRNA levels to confirm the gene silencing effect of the siRNA / SLN complex in an animal model. [Figure 11] FIG. 11 shows the results of confirming the blood removal rate of the siRNA / SLN complex formed by dimeric siRNA in a mouse model. [Figure 12] FIG. 12 shows the results of confirming the biodistribution of siRNA / SLN complexes formed by dimeric siRNA in a mouse model. BEST MODE FOR CARRYING OUT THEINVENTION

[0012] The configuration of the present invention will be specifically described below. In the present invention, numerical limitations indicate greater than or equal to, unless otherwise specified.

[0013] The present invention provides low density lipoprotein-mimetic solid lipid nanoparticles comprising: a core comprising cholesteryl esters and triglycerides; and a shell comprising albumin-bound cholesterol, a fusogenic lipid, and a cationic lipid.

[0014] In the present invention, the term "solid lipid nanoparticle (SLN)" refers to a particle having a size of nanometers (nm) from the morphological viewpoint, and existing in the form of a solid matrix, unlike liposomes and emulsions. The solid lipid nanoparticle of the present invention has a core-shell structure, and may be structurally configured to capture water-insoluble drugs and / or nucleic acids in the center (core) or surface (shell). The solid lipid nanoparticle according to the present invention can significantly increase the in vivo delivery efficiency of the water-insoluble drugs and / or nucleic acids by capturing the water-insoluble drugs and / or nucleic acids. The solid lipid nanoparticle can be produced by a conventional method, and the method can be appropriately modified as necessary.

[0015] Natural high density lipoproteins (HDL) are 5-17 nm in size and have the highest density of 1.063-1.21 g / dL due to the largest protein composition of all lipoproteins. High density lipoproteins are composed of approximately 50% protein, 17% cholesterol, and 22% phospholipids. The major proteins are apoA-I and apoA-II. High density lipoproteins are structurally composed of two lipid phases: a polar lipid phase (phospholipids, apolipoproteins, and free cholesterol) and a non-polar lipid phase (cholesteryl esters and triglycerides). Phospholipids have the advantage of emulsifying non-polar lipids and stabilizing the surface of nanoparticles.

[0016] The solid lipid nanoparticles according to the present invention are high density lipoprotein (HDL) mimicking solid lipid nanoparticles (SLN). Unlike natural high density lipoproteins, the high density lipoprotein mimicking solid lipid nanoparticles according to the present invention may be substantially free of apolipoproteins. Also, unlike natural high density lipoproteins containing apolipoproteins, the high density lipoprotein mimicking solid lipid nanoparticles according to the present invention may contain a high content of cationic lipids instead of apolipoproteins. By imparting cationicity to the surface of the solid lipid nanoparticles by the cationic lipids, they can be bound to drugs and used as drug delivery vehicles.

[0017] In addition, unlike natural high density lipoproteins, the high density lipoprotein-mimicking solid lipid nanoparticles of the present invention may contain free cholesterol in the form of albumin-bound cholesterol. As described above, by including albumin-bound cholesterol, the solid lipid nanoparticles can have a cancer cell targeting ability when used as a drug delivery vehicle. Such cancer cell targeting ability is due to albumin bound to cholesterol, and in order for albumin to exert its cancer cell targeting ability, the particle size must be at a level that can pass through the cell membrane, i.e., 20 to 50 nm. Therefore, the size of the high density lipoprotein-mimicking solid lipid nanoparticles according to the present invention is preferably 20 to 50 nm.

[0018] Natural low-density lipoproteins (LDL) are composed of two lipid phases, i.e., polar components (phospholipids and apolipoproteins) and non-polar neutral lipids pre-composed of cholesterol esters and triglycerides, with a size of approximately 18 to 25 nm and a zeta potential of -11.4 ± 1.9 mV. Specifically, the core is composed of 45% cholesterol esters and 3% triglycerides, and the surface is composed of 10% cholesterol, 22% phospholipids, and 20% apolipoprotein B-100.

[0019] The solid lipid nanoparticles according to the present invention are low density lipoprotein (LDL) mimicking solid lipid nanoparticles (SLN). Unlike natural low density lipoproteins, the low density lipoprotein mimicking solid lipid nanoparticles according to the present invention may be substantially free of apolipoproteins. Also, unlike natural low density lipoproteins containing apolipoproteins, the low density lipoprotein mimicking solid lipid nanoparticles according to the present invention may contain fusogenic lipids and cationic lipids instead of phospholipids and apolipoproteins. The cationic lipids impart cationicity to the surface of the solid lipid nanoparticles, allowing them to be bound to drugs and used as drug delivery vehicles.

[0020] Additionally, the low density lipoprotein-mimetic solid lipid nanoparticles of the present invention may contain free cholesterol in the form of albumin-bound cholesterol, unlike native low density lipoproteins.

[0021] As described above, the inclusion of albumin-bound cholesterol can provide cancer cell targeting capability when the low density lipoprotein mimicking solid lipid nanoparticles are used as drug delivery vehicles, and can emulsify non-polar lipids to provide surface stability, prevent inter-particle aggregation, and form structurally stable nanoparticles.

[0022] Such cancer cell targeting ability and permeability are due to albumin bound to cholesterol, and in order for albumin to exert its cancer cell targeting ability, the particle size must be at a level that can pass through the cell membrane. That is, due to the EPR (Enhanced permeability and retention) effect, the neovascularization of tumor tissue is incomplete, and the gaps widen to about 200 nm, and particles of 200 nm or less can penetrate the tumor blood vessels without entering normal tissue (Enhanced permeability), or tumor tissue has an underdeveloped lymphatic system, so that leaked substances are likely to remain. Therefore, the size of the solid lipid nanoparticles according to the present invention is 60 to 200 nm, and the EPR effect can stably transport drugs to cancer tissues, etc.

[0023] In the present invention, the high density lipoprotein-mimicking solid lipid nanoparticles and / or low density lipoprotein-mimicking solid lipid nanoparticles may be high density and / or low density lipoprotein-mimicking cationic solid lipid nanoparticles in the form of cationic solid lipid nanoparticles.The nanoparticles are reconstituted to mimic the components of natural high density and / or low density lipoproteins, and nucleic acid therapeutic agents such as proteins, peptides, aptamers, or siRNAs have very low absorption rates into the circulatory system due to their physicochemical properties. In addition, solid lipid nanoparticles are degraded by proteolytic enzymes, peptidases, or nucleases, and are removed through the reticuloendothelial system (RES), and then rapidly excreted through the kidneys. To improve this, drugs can be encapsulated in natural high-density lipoprotein-mimicking nanoparticles, which inhibit enzymatic decomposition, promote absorption into target organs and cells, inhibit decomposition by the reticuloendothelial system (RES), and extend the blood retention time. As they mimic natural lipoproteins in the body, they have the advantage of being highly stable and bioavailable in the body, and can encapsulate and deliver drugs with high efficiency.

[0024] In the following examples, high density lipoprotein mimicking solid lipid nanoparticles and low density lipoprotein mimicking solid lipid nanoparticles were prepared, and it was confirmed that the lipoprotein mimicking nanoparticles had excellent drug encapsulation rates. More specifically, the average particle size was about 100 nm for low density lipoprotein mimicking solid lipid nanoparticles and about 30 nm for high density lipoprotein mimicking solid lipid nanoparticles, and it was confirmed that the drug encapsulation rates were at similar levels.

[0025] In the present invention, lipoprotein-mimetic solid lipid nanoparticles can be referred to as solid lipid nanoparticles, and unless otherwise indicated, the disclosure should be understood to apply to both low density and / or high density lipoprotein-mimetic solid lipid nanoparticles.

[0026] In the present invention, the core of the solid lipid nanoparticles is in a solid state at room temperature and body temperature and can capture water-insoluble drugs and / or nucleic acid genes through hydrophobic interactions within the core. The core of the solid lipid nanoparticles includes cholesteryl esters and triglycerides.

[0027] The cholesteryl ester, which is one of the components constituting the core of the nanoparticles, is formed by esterifying cholesterol with a saturated or unsaturated fatty acid having 10 to 24 carbon atoms, and may be an ester of an unsaturated fatty acid having 16 to 18 carbon atoms, such as oleic acid. The solid lipid nanoparticles according to the present invention may contain a single or multiple types of cholesteryl esters.

[0028] In one embodiment, the cholesteryl ester may be one or more selected from the group consisting of cholesteryl stearate, cholesteryl palmitate, cholesterol hydroxystearate, soybean sterol, or a derivative thereof, and preferably cholesteryl oleate.

[0029] The cholesteryl ester may be 5 to 60 parts by weight based on 100 parts by weight of the whole lipoprotein-mimicking solid lipid nanoparticles.

[0030] For example, when the lipoprotein-mimicking solid lipid nanoparticles are high-density lipoprotein-mimicking solid lipid nanoparticles, the cholesteryl ester may be included in an amount of 5 to 30 parts by weight, 10 to 25 parts by weight, or 15 to 20 parts by weight based on 100 parts by weight of the high-density lipoprotein-mimicking solid lipid nanoparticles. When the cholesteryl ester constitutes the core of the high-density lipoprotein-mimicking solid lipid nanoparticles in the above range, it is possible to produce solid lipid nanoparticles that can pass through cell membranes while encapsulating a large amount of drug, and have excellent intracellular drug delivery efficiency. In addition, by including the cholesteryl ester in the above content, it is possible to produce solid lipid nanoparticles similar to natural high-density lipoproteins.

[0031] In addition, for example, when the lipoprotein-mimicking solid lipid nanoparticles are low-density lipoprotein-mimicking solid lipid nanoparticles, the cholesteryl ester may be contained in an amount of 30 to 60 parts by weight, more than 30 to 50 parts by weight, or 40 to 50 parts by weight based on 100 parts by weight of the low-density lipoprotein-mimicking solid lipid nanoparticles. When the cholesteryl ester constitutes the core of the low-density lipoprotein-mimicking solid lipid nanoparticles in the above range, it is possible to produce low-density lipoprotein-mimicking solid lipid nanoparticles that can pass through cell membranes while encapsulating a large amount of drug and have excellent intracellular drug delivery efficiency. In addition, by containing the cholesteryl ester in the above content, it is possible to produce solid lipid nanoparticles similar to natural low-density lipoproteins.

[0032] In addition, the triglyceride, which is one of the components forming the core of the nanoparticles, may be a refined triglyceride having various fatty acid compositions, or a vegetable oil mainly composed of triglycerides composed of a large number of fatty acids. For example, the triglyceride may be an animal or vegetable oil, and may be one or more selected from the group consisting of soybean oil, olive oil, cottonseed oil, sesame oil, and cod liver oil, but is not limited thereto. More specifically, the triglyceride may be one molecule of glycerol esterified with three molecules of saturated or unsaturated fatty acids having 10 to 24 carbon atoms.

[0033] In one embodiment, the triglyceride may be one or more selected from the group consisting of triheptanoin, trimyristin, tripalmitin, tristearin, and trilinolein, or a derivative thereof, and preferably may be triolein.

[0034] The triglyceride may be included in an amount of 0.01 to 5 parts by weight, 0.05 to 3.5 parts by weight, 0.1 to 2.5 parts by weight, or 0.5 to 1.5 parts by weight based on 100 parts by weight of the total lipoprotein-mimicking solid lipid nanoparticles. When the triglyceride constitutes the core of the lipoprotein-mimicking solid lipid nanoparticles in the above range, the triglyceride can hydrophobically bond with the cholesteryl ester to form nanoparticles having a size that can pass through a cell membrane.

[0035] The cholesteryl esters and triglycerides can form the core of lipoprotein-mimetic solid lipid nanoparticles through hydrophobic bonds.

[0036] In the present invention, the shell of the solid lipid nanoparticle is bound to the upper surface of the core by hydrophobic interaction, and the shell has exposed cationic lipids capable of electrostatic interaction with drugs, particularly water-insoluble drugs and / or nucleic acid genes. The nanoparticles according to the present invention can bind to drugs, particularly water-insoluble drugs and / or nucleic acid genes, by electrostatic interaction through the cationic lipids exposed to the shell to form complexes, and can stably deliver drugs, particularly water-insoluble drugs and / or nucleic acid genes, into cells. Thus, the shell of the nanoparticles includes albumin-binding cholesterol, fusogenic lipids and cationic lipids.

[0037] In the present invention, albumin-bound cholesterol can be prepared by binding albumin, a hydrophilic protein, to cholesterol, a hydrophobic moiety and a natural lipid. In particular, the present invention can tightly bind cholesterol and albumin by covalently binding albumin to cholesterol. Through this, it is possible to provide cholesterol-albumin in which albumin is tightly bound to cholesterol at a certain ratio, unlike a complex in which two components are simply mixed and non-specifically bound through interaction. In the present invention, the albumin-bound cholesterol can be used interchangeably with the term "cholesterol-albumin".

[0038] The albumin-bound cholesterol of the present invention can be prepared by various methods generally known in the art. In one embodiment of the present invention, the albumin-bound cholesterol may be formed by reacting cholesterol chloroformate with albumin, but is not limited thereto. This may be formed by reacting the chloroformate group of cholesterol chloroformate with the amine group of albumin to bind the cholesterol chloroformate.

[0039] The "albumin" is one of the proteins that constitute the basic substances of cells, and is the protein with the smallest molecular weight among the simple proteins existing in the natural state. Albumin is water-soluble and is a major protein found in blood plasma. It binds to various ligands such as water, cations such as calcium, sodium, and potassium, fatty acids, hormones, bilirubin, and drugs, and plays a role in regulating and transmitting the colloid osmotic pressure of blood. In particular, the binding of drugs to albumin contributes greatly to the expression of the drug's efficacy. The albumin can be obtained in a significant amount from egg white (ovalbumin), bovine serum, or human serum, and can also be extracted from soybeans, milk, and grains. Alternatively, recombinant albumin obtained from a transformant produced to express a polypeptide having the same amino acid sequence as naturally occurring albumin can be used. Moreover, the "cholesterol" is a type of sterol, a combination of steroid and alcohol, a lipid present in the cell membrane of all animal cells, and an organic molecule transported through the blood. Cholesterol is absorbed through food or synthesized in the body, and is present in high concentrations in organs with many cell membranes, providing cells with appropriate membrane permeability and fluidity. Since it contains multiple fused ring structures within the molecule, it can exhibit improved interaction with substances that contain ring structures.

[0040] As mentioned above, the present invention may include albumin-bound cholesterol as a component thereof, instead of the free cholesterol of natural lipoproteins.

[0041] The albumin-bound cholesterol may be 1 to 20 parts by weight based on 100 parts by weight of the whole solid lipid nanoparticles.

[0042] For example, when the lipoprotein-mimicking solid lipid nanoparticles are high-density lipoprotein-mimicking solid lipid nanoparticles, the albumin-bound cholesterol may be included in an amount of 1.5 to 8 parts by weight, 2 to 5 parts by weight, or 3.5 to 4.5 parts by weight based on 100 parts by weight of the high-density lipoprotein-mimicking solid lipid nanoparticles. Also, for example, when the lipoprotein-mimicking solid lipid nanoparticles are low-density lipoprotein-mimicking solid lipid nanoparticles, the albumin-bound cholesterol may be included in an amount of 3 to 15 parts by weight, 5 to 12 parts by weight, or 9 to 11 parts by weight based on 100 parts by weight of the low-density lipoprotein-mimicking solid lipid nanoparticles. When the albumin-bound cholesterol constitutes solid lipid nanoparticles in the above range, it is possible to form solid lipid nanoparticles with excellent drug encapsulation rate by preventing interparticle aggregation while mimicking natural lipoproteins. In the above solid lipid nanoparticles, it is possible to prevent the decomposition of nanoparticles due to phagocytosis while encapsulating a large amount of drug, thereby maximizing the drug delivery efficiency to the target site. Furthermore, by including albumin-bound cholesterol in the above content, the albumin-bound cholesterol can form a strong shell of the solid lipid nanoparticles, improving the efficiency of gene transfection for gene transfer, and has the advantage of reducing the cytotoxicity of the cationic lipid described below, thereby inhibiting phagocytosis and improving the drug delivery efficiency of the solid lipid nanoparticles.

[0043] Albumin has endosomolytic activity that can promote the release of nanoparticles in the endosomal-lysosomal compartment, and therefore can contribute to improving drug delivery efficiency. For example, the following examples show that such endosomolytic activity contributes to improving the efficiency of in vivo gene transfer (silencing) of solid lipid nanoparticles and siRNA complexes (albumin_SLN / siRNA complexes). In addition, the nanoparticles can maintain their nanostructure even during vascular circulation, and therefore have the advantage of being preferentially distributed in tumor tissues, minimizing side effects and maximizing the therapeutic efficacy of drugs.

[0044] The fusogenic lipid may be any kind of neutral, cationic, or anionic lipid capable of forming solid lipid nanoparticles, or may be a single or mixture of two or more kinds of phospholipids, but is not limited thereto. The fusogenic lipid may be any kind of phospholipid capable of inducing fusion, such as 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylethanolamine (POPC), egg phosphatidylcholine (egg phosphatidylcholine), or 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylethanolamine (POPC), egg phosphatidylcholine (egg phosphatidylcholine), or 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylethanolamine (POPC), egg phosphatidylcholine (EGG), or 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine ... phosphatidylcholine (EPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylethanolamine (distearoyl The phosphatidylethanolamine (DSPE), phosphatidylethanolamine (PE), dipalmitoylphosphatidylethanolamine (dipalmitoylphosphatidylethanolamine), 1-palmitoyl-2-oleyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine] (DOPS), and 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine] may be one or more selected from the group consisting of, but is not limited to, 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine].

[0045] In one embodiment, the fusogenic lipid may be 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE).

[0046] The fusogenic lipid may be included in an amount of 5 to 40 parts by weight, 15 to 40 parts by weight, 20 to 35 parts by weight, or 20 to 30 parts by weight based on the weight of the entire solid lipid nanoparticle. When the fusogenic lipid constitutes the solid lipid nanoparticle in the above range, it is advantageous in that it improves the efficiency of gene transfection and reduces the cytotoxicity of the cationic lipid described below. In addition, the fusogenic lipid facilitates the passage of the solid lipid nanoparticle through the cell membrane and endosomal escape, facilitating intracellular drug delivery.

[0047] The cationic lipid may include cationic lipids that carry a net negative charge at a particular pH, such as physiological pH. For example, the cationic lipid may be 3β-[N-(N',N'-dimethylaminoethane)carbamoyl]cholesterol (DC-cholesterol), 3β-[N-(N',N',N'-trimethylaminoethane)carbamoyl]cholesterol (TC-cholesterol), 3β-[N-(N'-monomethylaminoethane)carbamoyl]cholesterol (MC-cholesterol), 3β-[N-(aminoethane)carbamoyl]cholesterol (AC-cholesterol), N-(N'-amino N-(1-(2,3-dioleoyloxy)propyl-N,N,N-trimethylammonium chloride (DOTAP), N,N-dimethyl, ... -(2,3-Dioleoyloxy)propylamine (DODMA), N-(1-(2,3-dioleoyl)propyl)-N,N,N-trimethylammonium chloride (DOTMA), 1,2-Dioleoyl-3-dimethylammonium-propane (DODAP), 1,2-Dioleoylcarbamyl-3-dimethylammonium-propane (DOCDAP), 1,2-Dilineoyl-3-dimethylammonium-propane (Dilineoyl-3-Dimethylammonium-propane opane,DLINDAP), dioleoyloxy-N-[2-sperminecarboxamido)ethyl}-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), dioctadecylamidoglycylspermine (DOGS), 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DMRIE), 3-dimethylamino-2-(cholest-5-ene-3-beta-oxybutane-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5-ene-3-beta-oxy)-3'-oxapentoxy]-3-dimethyl-1-(cis,cis-9',12'-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N'-dioleoylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 1,2-diaza-3-trimethylammonium-propane (TAP), 1,2-diaza-3-dimethylammonium-propane (DAP), 1,2-di-O-octadecenyl-3-trimethylammoniumpropane, and 1,2-dioleoyl-3-trimethylammoniumpropane, but are not limited thereto.

[0048] In one embodiment, the cationic lipid may be 3β-[N-(N′,N′-dimethylaminoethane)carbamoyl]cholesterol (DC-cholesterol). DC-cholesterol is particularly preferred because it is less toxic than other cationic lipids and DC-cholesterol-based gene carriers have been approved for use in the clinical treatment of various diseases, such as melanoma, cystic fibrosis, cervical cancer, breast cancer, or ovarian cancer.

[0049] The cationic lipid may be included in an amount of 10 to 60 parts by weight based on 100 parts by weight of the total solid lipid nanoparticles.

[0050] For example, when the lipoprotein-mimicking solid lipid nanoparticles are high density lipoprotein-mimicking solid lipid nanoparticles, the cationic lipid may be contained in an amount of 10 to 60 parts by weight, 35 to 58 parts by weight, or 40 to 50 parts by weight based on 100 parts by weight of the high density lipoprotein-mimicking solid lipid nanoparticles. When the cationic lipid constitutes the shell of the solid lipid nanoparticles in the above content range, the solid lipid nanoparticles are formed in a size that can pass through the cell membrane while encapsulating a large amount of drug, and the drug can be delivered efficiently.

[0051] For example, when the lipoprotein-mimicking solid lipid nanoparticles are low density lipoprotein-mimicking solid lipid nanoparticles, the cationic lipid may be contained in an amount of 10 to 40 parts by weight, 10 to 30 parts by weight, or 12 to 28 parts by weight based on 100 parts by weight of the low density lipoprotein-mimicking solid lipid nanoparticles. When the cationic lipid constitutes the shell of the solid lipid nanoparticles in the above content range, the solid lipid nanoparticles are formed in a size that can pass through the cell membrane while encapsulating a large amount of drug, and the drug can be delivered efficiently.

[0052] In the present invention, the shell contains albumin-binding cholesterol, which exposes albumin on the shell surface, thereby maximizing intracellular drug delivery efficiency. The shell may also be substantially free of apolipoprotein. The solid lipid nanoparticles according to the present invention are different from conventional solid lipid nanoparticles in that they contain cationic lipids instead of apolipoproteins, which impart cationic properties to the surface (shell) of the solid lipid nanoparticles, allowing drugs to be bound to the solid lipid nanoparticles, and allowing the solid lipid nanoparticles to be formed in a size that can pass through cell membranes.

[0053] In the present invention, the term "substantially free" means that the amount is less than 3 parts by weight, or less than 1 part by weight, based on 100 parts by weight of the total solid lipid nanoparticles.

[0054] Moreover, the solid lipid nanoparticles may have an average particle diameter of 20 to 200 nm to facilitate intracellular drug delivery.

[0055] For example, when the lipoprotein-mimetic solid lipid nanoparticles are high density lipoprotein-mimetic solid lipid nanoparticles, the average particle diameter may be 20 to 50 nm, 20 to 45 nm, 25 to 40 nm, or 20 to 40 nm.

[0056] Furthermore, for example, when the lipoprotein-mimicking solid lipid nanoparticles are low density lipoprotein-mimicking solid lipid nanoparticles, the average particle diameter may be 60 to 200 nm, 60 to 150 nm, 25 to 40 nm, or 20 to 40 nm.

[0057] The solid lipid nanoparticles may have a uniform particle size distribution. The solid lipid nanoparticles having the above-mentioned diameter range and uniform particle size distribution can be useful for systemic drug delivery.

[0058] In the present invention, the high-density lipoprotein-mimetic solid lipid nanoparticles may contain 5 to 30 parts by weight of cholesteryl ester, 0.01 to 5 parts by weight of triglyceride, 1 to 20 parts by weight of albumin-bound cholesterol, 5 to 40 parts by weight of fusogenic lipid, and 10 to 60 parts by weight of cationic lipid, based on 100 parts by weight of the total high-density lipoprotein-mimetic solid lipid nanoparticles.

[0059] In addition, in the present invention, the low density lipoprotein-mimetic solid lipid nanoparticles may contain 30 to 60 parts by weight of cholesteryl ester, 0.01 to 5 parts by weight of triglyceride, 1 to 20 parts by weight of albumin-bound cholesterol, 5 to 40 parts by weight of fusogenic lipid, and 10 to 40 parts by weight of cationic lipid, based on 100 parts by weight of the total low density lipoprotein-mimetic solid lipid nanoparticles.

[0060] The present invention also provides a method for producing the lipoprotein solid lipid nanoparticles. The method includes mixing cholesteryl esters, triglycerides, albumin-bound cholesterol, fusogenic lipids and cationic lipids in an organic solvent to produce an aqueous dispersion of lipoprotein-mimetic solid lipid nanoparticles.

[0061] The method may further include a step of producing albumin-bound cholesterol; and / or a step of purifying and concentrating the aqueous dispersion by dialysis.

[0062] In the preparation method, all of the descriptions of the cholesteryl ester, triglyceride, albumin-bound cholesterol, fusogenic lipid, cationic lipid, and the lipoprotein-mimicking solid lipid nanoparticles can be applied as they are as described above.

[0063] More specifically, the method comprises the steps of: i) producing albumin-bound cholesterol; ii) mixing cholesteryl esters, triglycerides, albumin-bound cholesterol, fusogenic lipids and cationic lipids in an organic solvent to produce an aqueous dispersion of solid lipid nanoparticles; and iii) The method may include a step of dialyzing the aqueous dispersion against distilled water for 12 to 36 hours at regular intervals, and then concentrating the dialysate.

[0064] The step i) may include the steps of dissolving cholesterol chloroformate in an organic solvent, dropping the solution into the albumin solution to react with the solution, and dialysis of the reaction solution against distilled water for 12 to 36 hours, followed by freeze-drying. In this case, the cholesterol chloroformate and albumin may be used in a molar ratio of 1:1 to 5:1, but is not limited thereto.

[0065] In step ii), the organic solvent may be selected from, but is not limited to, dimethylformamide, tetrahydrofuran, C1-C4 lower alcohols, methylene chloride, chloroform, acetone, dimethylsulfoxide, N-methylpyrrolidone, dioxane, ethyl acetate, methyl ethyl ketone, acetonitrile, and mixtures thereof. The organic solvent is completely removed in the preparation step of the solid lipid nanoparticles, so toxicity of the organic solvent is not a consideration.

[0066] In one embodiment, the organic solvent in step ii) may be a mixture of chloroform and methanol.

[0067] In addition, in step ii), the mixture of the cholesteryl ester, triglyceride, albumin-bound cholesterol, fusogenic lipid and cationic lipid may be mixed with the organic solvent, and the mixture may further include a step of sonicating the mixture and / or drying the mixture under reduced pressure.

[0068] The lipoprotein-mimetic solid lipid nanoparticles of the present invention can be used as a carrier for various water-insoluble drugs and / or drugs such as nucleic acid genes through electrostatic interactions with the cationic lipids exposed on the surface of the shell. Thus, the present invention provides a drug delivery composition comprising the lipoprotein-mimetic solid lipid nanoparticles.

[0069] In the present invention, the drug may include a water-insoluble drug, a nucleic acid, or a water-insoluble drug and a nucleic acid at the same time.

[0070] In the present invention, the water-insoluble drug may be, but is not limited to, a poorly soluble drug, an anion-bearing peptide, a protein, a hyaluronic acid-peptide conjugate, or a hyaluronic acid-protein conjugate. The poorly soluble drug may be paclitaxel, docetaxel, amphotericin, doxorubicin, nifedipine, propofol, diazepam, estradiol, cyclosporine, ritonavir, saquinavir, biphenyl dimethyl dicarboxylate, coenzyme Q10, ursodeoxycholic acid, ilaprazole. , imatinib, mesylate, acyclovir, allopurinol, amiodarone, azathioprine, benazepril, calcitriol, candesartan, eprosartan, carbidopa / levodopa, clarithromycin, clozapine, desmopressin acetate, diclofenac, enalapril, famotidine, felodipine, fenofibrate, fentanyl, fexofenadine, fosinopril, furosemide, glyburide, hyoscyamine, imipramine, It may be selected from, but is not limited to, itraconazole, levothyroxine, atorvastatin, lovastatin, meclizine, megestrol, mercaptopurine, metolazone, mometasone, nabumetone, omeprazole, paroxetine, propafenone, quinapril, simvastatin, sirolimus, tacrolimus, tizanidine, fluvastatin, pitavastatin, pravastatin, rosuvastatin, epirubicin, or mixtures thereof.

[0071] In one embodiment, the water-insoluble drug may be a poorly soluble drug. In the composition, the water-insoluble drug and / or nucleic acid and the solid lipid nanoparticles can form a complex at a weight ratio of 1:1 to 1:10, or 1:3 to 1:8.

[0072] The complex may have a water-insoluble drug and / or nucleic acid bound to the shell of the solid lipid nanoparticle through an interaction, or may have a form in which the water-insoluble drug and / or nucleic acid is trapped in the core.

[0073] The nucleic acid may be one or more selected from the group consisting of, but is not limited to, short interfering ribonucleic acid (siRNA), ribosomal ribonucleic acid (rRNA), ribonucleic acid (RNA), deoxyribonucleic acid (DNA), complementary deoxyribonucleic acid (cDNA), aptamer, messenger ribonucleic acid (mRNA), transfer ribonucleic acid (tRNA) and antisense oligodeoxynucleotide (AS-ODN).

[0074] In one embodiment, the nucleic acid may be an siRNA. In particular, siRNA refers to double-stranded RNA (duplex RNA) or single-stranded RNA that takes a double-stranded form within a single-stranded RNA. The bond between the double strands is formed through hydrogen bonds between nucleotides, and not all nucleotides within the double strand should be completely bound in a complementary manner. The length of the siRNA may be about 15 to 60, about 15 to 50, about 15 to 40, about 15 to 30, about 15 to 25, 16 to 25, 19 to 25, 20 to 25, or 20 to 23 nucleotides. The siRNA length refers to the number of nucleotides on one side of the double-stranded RNA, that is, the number of base pairs, and in the case of single-stranded RNA, it refers to the length of the double strand within the single-stranded RNA. In addition, siRNA may be composed of nucleotides to which various functional groups have been introduced for the purpose of increasing stability in blood or weakening immune responses.

[0075] Thus, the siRNAs of the present invention may include both unmodified and modified versions of typical siRNAs.

[0076] The present invention also provides a method for delivering water-insoluble drugs and / or nucleic acids to target cells using said lipoprotein-mimetic solid lipid nanoparticles.

[0077] The method comprises: i) forming a complex of a water-insoluble drug and / or a nucleic acid with said lipoprotein-mimetic solid lipid nanoparticles; and ii) transfecting a target cell with the complex.

[0078] In step i), the complex is formed by mixing the lipoprotein-mimetic solid lipid nanoparticles in the presence of a water-insoluble drug and / or nucleic acid in, for example, phosphate buffered saline (PBS) or demineralized water, where the PBS has a pH of 7.0 to 8.0 and may contain NaCl, but is not limited thereto.

[0079] In step i), the water-insoluble drug and / or nucleic acid may form a complex with the solid lipid nanoparticles in a weight ratio of 1:1 to 1:10, or 1:3 to 1:8.

[0080] The present invention also provides a pharmaceutical composition for preventing or treating cancer caused by KRAS gene mutation, comprising the lipoprotein-mimicking solid lipid nanoparticles that entrap a water-insoluble drug or nucleic acid that targets KRAS.

[0081] In the pharmaceutical composition, the water-insoluble drug, the nucleic acid, and the high density lipoprotein-mimicking solid lipid nanoparticles may be the same as or similarly described above.

[0082] The cancer caused by the KRAS gene mutation may be, for example, non-small cell lung cancer, colon cancer, or pancreatic cancer, but is not limited thereto.

[0083] In the pharmaceutical composition, the lipoprotein-mimicking solid lipid nanoparticles entrapping the water-insoluble drug or nucleic acid targeting KRAS may be in the form of a complex between the water-insoluble drug or nucleic acid and the high density lipoprotein-mimicking solid lipid nanoparticles.

[0084] The water-insoluble drug contained in the complex may be a poorly soluble drug. Furthermore, the KRAS-targeting nucleic acid contained in the complex may be a reducible nucleic acid dimer.

[0085] The advantages and features of the present invention, as well as the methods for achieving them, will become clearer with reference to the following detailed examples. However, the present invention is not limited to the following examples, and may be embodied in various different forms. The following examples are provided to fully disclose the present invention and to fully inform those skilled in the art of the present invention of the scope of the invention. The present invention is defined only by the scope of the claims. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0086] Working Example The manufacturing process of lipoprotein-mimetic solid lipid nanoparticles and the morphology of lipoprotein-mimetic solid lipid nanoparticles as drug delivery vehicles are illustrated in Figure 1.

[0087] [Production Example 1] Production of albumin-bound cholesterol (cholesterol-albumin) To prepare albumin-bound cholesterol, human serum albumin (HSA, Sigma-Aldrich, Germany) and cholesteryl chloroformate (Sigma-Aldrich, Germany) were used.

[0088] First, 0.5 mmol of human serum albumin was dissolved in 2 mL of phosphate buffered saline (PBS, pH 8.0). 1.5 mmol of cholesterol chloroformate was dissolved in 1 mL of DMF, and this solution was added dropwise to the human serum albumin solution. At this time, cholesteryl chloroformate and human serum albumin were used in a molar ratio of 3:1. Then, after reacting overnight at room temperature with weak shaking, the reaction solution was dialyzed against 3 L of distilled water using a molecular cutoff 12000 g / mol dialysis tube for 3 hours, and the distilled water was replaced at 3-4 hour intervals for 24 hours. Then, the product solution was freeze-dried to produce albumin-bound cholesterol (hereinafter referred to as cholesterol-albumin).

[0089] [Production Example 2] Production of high density lipoprotein (HDL) mimicking solid lipid nanoparticles (SLN) HDL-mimicking SLN was prepared by a modified solvent-emulsification method.

[0090] Cholesteryl oleate and triolein were used as core structure lipids, and DOPE, cholesterol-albumin prepared in Preparation Example 1, and DC-cholesterol were used as surface (shell) structure lipids. The components in Tables 1 and 2 below were dissolved in 2 mL of a chloroform / methanol mixture (2:1, v / v). 10 mL of deionized water was added thereto, and the mixture was thoroughly vortexed. The suspension was sonicated for 5 minutes using a Branson Sonifier (registered trademark) (20 kHz, duty cycle = 40, output control = 3.5). The emulsified solution was transferred to a rotary evaporator, and the solvent was removed by evaporation at a temperature of 52 ° C or higher, which is the melting point of cholesteryl oleate. Finally, the aqueous dispersion of HDL-mimicking SLN was purified by dialysis (MWCO: 5,000) overnight and concentrated to a maximum of 5 mg particle / mL through vacuum evaporation.

[0091] [Table 1]

[0092] [Table 2]

[0093] In addition, the particle size and zeta potential value of the produced HDL-mimicking SLN (Examples 1-1 to 1-12) were compared depending on the composition.

[0094] In this case, the solid lipid nanoparticles (SLN) prepared according to the composition of Example 1-2, but using cholesterol instead of cholesterol-albumin, were used as the negative control group. The SLN was used as the HDL negative control group in the following Experimental Examples 1 to 5.

[0095] As a result, as is clear from Tables 1 and 2, the size of HDL-mimicking SLN tended to decrease as the amount of cholesteryl oleate and albumin-bound cholesterol (cholesterol_albumin) decreased. In addition, as shown in FIG. 2, the size of HDL-mimicking SLN tended to decrease as the amount of DC-Chol increased.

[0096] The results in FIG. 2 show that the Z-average value of HDL-mimicking SLN tends to increase as the content of cholesteryl oleate and cholesterol (cholesterol_albumin) increases and as the content of DC-Chol decreases. Based on this, Example 1-2 was selected as the optimal composition of HDL-mimicking SLN.

[0097] [Production Example 3] Production of low density lipoprotein (LDL) mimicking solid lipid nanoparticles (SLN) An LDL-mimicking SLN was produced in the same manner as in Production Example 2, except that the ingredients and compositions in Tables 3 and 4 below were used.

[0098] [Table 3]

[0099] [Table 4]

[0100] In addition, the particle size and zeta potential value of the produced LDL-mimicking SLN (Examples 2-1 to 2-10) were compared depending on the composition.

[0101] In this case, solid lipid nanoparticles (SLN) prepared using the composition of Examples 2-5 but using cholesterol instead of cholesterol-albumin were used as the negative control group. The SLN was used as the LDL negative control group in the following Experimental Examples 1 to 5.

[0102] As a result, as can be seen from Tables 3 and 4, it can be seen that the size of LDL-mimicking SLN tends to decrease as the amount of cholesteryl oleate and albumin-bound cholesterol (cholesterol-albumin) decreases, and it can also be seen that the size of LDL-mimicking SLN decreases as the amount of DC-Chol increases (FIG. 2).

[0103] The results in FIG. 2 show that the Z-average value of LDL-mimicking SLN tends to increase as the content of cholesteryl oleate and cholesterol (cholesterol-albumin) increases and as the content of DC-Chol decreases. Based on this, Examples 2-5 were selected as the optimal composition of LDL-mimicking SLN.

[0104] [Experimental Example 1] Comparison of drug encapsulation rates between optimal HDL-mimicking SLN and optimal LDL-mimicking SLN The drug encapsulation rates of HDL-mimicking SLN (composition of Examples 1-2) and LDL-mimicking SLN (composition of Examples 2-5) having the compositions shown in Tables 5 and 6 below were compared.

[0105] [Table 5]

[0106] [Table 6]

[0107] [Table 7]

[0108] [Table 8]

[0109] As a result, as is clear from Tables 7 and 8, it can be confirmed that both HDL-mimicking SLN and LDL-mimicking SLN have excellent drug encapsulation rates.

[0110] More specifically, the average particle size of LDL-mimicking SLN nanoparticles was measured to be 2.5 times larger than that of HDL-mimicking SLN, but the drug encapsulation rates were found to be at similar levels, confirming that HDL-mimicking SLN has a superior drug encapsulation effect.

[0111] Also, Figure 3a shows TEM photographs of the LDL-negative comparison group (left photograph) and the LDL-mimicking SLN (right photograph) of the SLN having the composition of Table 5. Also, Figure 3b shows TEM photographs of the HDL-negative comparison group (left photograph) and the HDL-mimicking SLN (right photograph) of the SLN having the composition of Table 6. As is clear from Figure 3, it can be confirmed that the HDL-mimicking SLN and the LDL-mimicking SLN according to the present invention are uniform nanoparticles with excellent dispersibility without interparticle aggregation.

[0112] [Production Example 4] Production of dimeric siRNA 1) Selection of Kras siRNA Kras siRNA sequences were selected from three libraries for research. The three sequences are not publicly available, and Kras itself is a natural gene, but it is up to the researcher to choose which sequence among the entire base sequence constituting the Kras gene to use as the target sequence of siRNA. The present inventors used the sequences in Table 1 below. All double-stranded monomer siRNAs used in this experiment were purchased by requesting synthesis from Bioneer (Daejeon, Korea).

[0113] [Table 9]

[0114] 2) siRNA dimer synthesis To synthesize reducible dimeric siRNAs linked via a cleavable coupling agent, a double-stranded monomeric siRNA (anti-Kras, scrambled) modified with a thiol group at the 3' end (sense) was used.

[0115] Briefly, 50 nmol of double-stranded monomeric siRNA in 500 μl of diethylpyrocarbonate (DEPC)-treated deionized water (DW) was activated with 50 nmol of the cleavable coupling agent dithiobismaleimideethane (DTME, 15.6 μg) in dimethyl sulfoxide (siRNA:DTME molar concentration 1:1). To minimize charge repulsion between the highly anionic double-stranded siRNA, 750 mM NaCl was added to the solution (optimal solution pH range, 6.5-7.5). After overnight incubation at room temperature with weak shaking, the resulting dimeric siRNA was purified by dialysis (MWCO: 10 kDa) against DW overnight and concentrated to 1 nmol / μl through vacuum evaporation. The process of synthesizing monomeric siRNA to reducible dimeric siRNA is diagrammed in Figure 4.

[0116] 3) Dissociation of dimeric siRNA First, the dimeric siRNA synthesized in 2) acts in the form of a monomer in cells, so if it can be further separated into monomeric siRNA, it can act as an siRNA in cells. Therefore, an experiment was carried out to prove whether it is a reducible dimeric siRNA using a reducing agent, dithiothreitol (DTT).

[0117] To confirm the cleavage of disulfide bonds for dimeric siRNA, 100 nmol of dimeric siRNA sample (100 μl) was incubated with 2 μmol of dithiothreitol (DTT, 100 μl) overnight at room temperature. The final pH of the solution was adjusted to 8.0 using 5 M NaOH. The resulting cleaved monomeric siRNA was purified by dialysis (MWCO: 10 kDa) against DW overnight and concentrated up to 1 nmol / μl via vacuum evaporation.

[0118] In addition, uncoupled dimeric siRNAs with sequences for scrambled monomeric siRNAs were obtained from Bioneer (Daejeon, Korea).

[0119] All resulting products, monomeric siRNA, dimeric siRNA linked through a cleavable disulfide bond, cleaved monomeric siRNA and unlinked dimeric siRNA, were analyzed by 10% polyacrylamide gel electrophoresis (PAGE).

[0120] As a result, as is clear from FIG. 5, it was confirmed that the dimeric siRNA was cleaved into monomeric siRNAs.

[0121] [Production Example 5] Production of a complex of dimeric siRNA and lipoprotein-mimicking SLN Complexes of monomer, dimeric siRNA linked through a cleavable disulfide bond, and non-coupling dimeric siRNA were formed using lipoprotein-mimicking SLN (Examples 1-2 and 2-5). Each siRNA (1.5 μg, 100 pmol) was complexed with solid lipid nanoparticles (SLN) at a weight ratio (siRNA / SLN) of 6 for 10 min (siRNA / SLN complex).

[0122] To investigate the stability of the complexes, heparin was added to the complexes at various heparin / siRNA weight ratios (0, 1, 2, 5, 10, 20, and 50) and further incubated for 30 min at 37°C. After incubation, the amount of degraded siRNA was analyzed by 1% agarose gel electrophoresis.

[0123] To analyze serum stability, the complexes were incubated at 37°C in 50% v / v serum for predetermined times (0, 2, 4, 8, 12, 24, and 48 h), and the resulting samples were further treated with heparin (100 μg) and DTT solution (100 mM) before agarose gel electrophoresis of the released siRNA fraction.

[0124] The hydrodynamic diameter and surface ζ potential values ​​of the siRNA / SLN complexes were measured using a Zetasizer nano-series nano-ZS with a He-Ne laser beam (Malvern Instruments Ltd., Malvern, UK) at a wavelength of 663 nm and a fixed scattering angle of 90°. Measurements were performed in triplicate for all samples at 25°C.

[0125] The morphology and size of the siRNA / SLN complexes were observed by AFM (XE-100, Park Systems, Korea). For AFM analysis, the siRNA / SLN complexes were placed on a fresh mica surface and dried in air, after which images were recorded in non-contact mode with an acquisition frequency of 312 kHz.

[0126] [Experimental Example 2] Confirmation of intracellular absorption of siRNA / SLN complex In order to confirm and compare the intracellular absorption of the siRNA / SLN complex produced in Production Example 5, FITC-labeled siRNA was used for complexation, and the intracellular absorption was analyzed with a flow cytometry analyzer (FACSCalibur, USA). The H441 cell line was used as the cell.

[0127] For FITC-labeled monomeric siRNA, the monomeric siRNA was end-labeled with FITC at the 5' end (sense) with a thiol group modification at the 3' end (sense) with a sequence for the scrambled monomeric siRNA, and was purchased by synthesis from Bioneer (Daejeon, Korea). FITC-labeled dimeric siRNA was synthesized as described in the Dimeric siRNA Synthesis section and analyzed by 10% PAGE.

[0128] 2 x 10 cells per well 4After seeding at a density of 100 cells in 6-well plates for 24 h, the dimer siRNA / SLN complexes were transfected (128 nm siRNA) at a predetermined time (30 min) at 37 °C, respectively. The culture medium was removed to interrupt cell absorption, and the transfected cells were washed smoothly three times with PBS and then dispersed in PBS. The fluorescence of the cells was measured using a FACSCalibur flow cytometry analysis system (BD Bioscience) and CellQuest software (PharMinutesgen).

[0129] The results are shown in Figure 6. In Figure 6, dimeric siRNA / albumin HDL is the case where the HDL-mimicking SLN according to Examples 1 to 2 was used, and dimeric siRNA / LDL is the case where the HDL-negative comparison group was used. Furthermore, dimeric siRNA / albumin LDL is the case where the LDL-mimicking SLN according to Examples 2 to 5 was used, and dimeric siRNA / LDL is the case where the LDL-negative comparison group was used.

[0130] As a result, as is clear from FIG. 6, excellent intracellular uptake was observed in both dimeric siRNA / albumin HDL and dimeric siRNA / albumin LDL, with dimeric siRNA / albumin HDL showing the highest intracellular uptake.

[0131] These data suggest that the albumin-fused HDL-mimicking siRNA / SLN complexes formed with nanoparticle sizes of approximately 50 nm have the best intracellular uptake and can be used as effective drug delivery vehicles.

[0132] [Experimental Example 3] Confirmation of in vitro activity of siRNA / SLN complex The gene silencing effects of siRNA / SLN complexes formed by two types of dimeric siRNA, Kras (endogenous) and scramble (control group), were confirmed and compared.

[0133] First, the lipoprotein-mimicking SLN prepared in Examples 1-2 and 2-5 was complexed with the cells, and then reverse transcriptase-polymerase chain reaction (RT-PCR) was performed at the cellular level of Kras mRNA to confirm the gene silencing effect. The H441 cell line was used as the cell.

[0134] Briefly, cells were plated at 2 x 10 per well. 4 Cells were seeded at a density of 100 cells per well in 6-well plates for 24 h and then transfected with dimeric siRNA / SLN complexes at various siRNA concentrations (n=3 per group). After 3 days of culture, cells were harvested and total RNA was isolated using Trizol reagent according to the manufacturer's protocol.

[0135] cDNA synthesis was performed using the High Capacity RNA-to-cDNA kit according to the manufacturer's protocol.

[0136] DNA amplification for the target gene (human Kras) and control gene (human β-actin) was performed with primers and Prime Taq Premix under the following thermocycling conditions: 1 cycle of 94°C for 2 min; 30 cycles of 94°C for 30 s, 57°C for 30 s, 72°C for 1 min; and 1 cycle of 72°C for 5 min. The primer sequences used were those shown in Table 10 below.

[0137] [Table 10]

[0138] The PCR products were analyzed by 1% agarose gel electrophoresis, and the intensity of each band was quantified using the ImageJ program.

[0139] The results are shown in Figures 7 and 8. In Figures 7 and 8 (and Figures 9 to 12 described below), albumin HDL represents the case where the LDL-mimicking SLN according to Examples 1 to 2 was used (i.e., the HDL-mimicking siRNA / SLN complex was used), and HDL represents the case where the HDL-negative comparison group was used. Furthermore, albumin LDL represents the case where the LDL-mimicking SLN according to Examples 2 to 5 was used (i.e., the LDL-mimicking siRNA / SLN complex was used), and LDL represents the case where the LDL-negative comparison group was used.

[0140] As a result, as is clear from Figures 7 and 8, it can be seen that the HDL-mimicking siRNA / SLN complex according to the present invention exhibits the most excellent kras silencing effect, thereby confirming the excellent drug delivery effect of the HDL-mimicking siRNA / SLN complex.

[0141] [Experimental Example 4] Confirmation of in vivo activity of siRNA / SLN complex 1) Construction of animal models To establish the xenograft mouse model, cancer cells (5 × 10 5 The tumors were injected subcutaneously with an average volume of 50–100 mm. 3 Once this was reached, treatment began.

[0142] To investigate and compare the gene silencing effects of two types of monomer or dimer siRNA, Kras (endogenous) and scramble (control group), they were complexed with the lipoprotein-mimicking SLN prepared in Examples 1-2 and Examples 2-5 and then administered intravenously via tail vein injection to female C57BL / 6 mice, and the gene silencing effect of mouse Kras mRNA in tumor tissue was evaluated by RT-PCR.

[0143] Briefly, the formulation was diluted in PBS at the siRNA concentration to be administered to each mouse. After 24 h treatment, tumor tissues were ground and tissue lysates were prepared using a homogenizer (Tissuelyser, QIAGEN, Germany). Total RNA was then isolated using TRIzol reagent according to the manufacturer's protocol, and Kras mRNA levels normalized to β-actin were measured.

[0144] For RT-PCR, DNA amplification of the target gene (mouse Kras) and control gene (mouse β-actin) was performed with primers and Primer Taq Premix under the following thermal cycling conditions: 1 cycle of 94°C for 2 min; 26 cycles of 94°C for 30 s, 57°C for 30 s, 72°C for 1 min; and 1 cycle of 72°C for 5 min. The primer sequences used were those shown in Table 11 below.

[0145] [Table 11]

[0146] The PCR products were analyzed by 1% agarose gel electrophoresis, and the intensity of each band was quantified using the ImageJ program.

[0147] As can be seen from the results of Figures 9 and 10, it can be confirmed that the HDL-mimicking siRNA / SLN complex of the present invention showed the best gene transfer effect in animal models, similar to the above results (Figures 7 and 8).

[0148] [Experimental Example 5] Confirmation of tissue distribution and pharmacokinetic profile of siRNA To confirm and compare the biodistribution and blood clearance of siRNA / SLN complexes formed by dimeric siRNA in mice, 125I-labeled siRNA was prepared using the Bolton-Hunter method and used for complexation.

[0149] First, for the synthesis of radiolabeled monomeric siRNA, the Bolton-Hunter reagent N-succinimidyl-3-(4-hydroxyphenyl)propionate (SHPP) was dissolved in DMSO at a concentration of 10 mg / ml, and 10 μl of 125I solution (1 mCi) was added to 10 μl of SHPP solution. Chloramine-T was dissolved in PBS at a concentration of 10 mg / ml, and 50 μl of the chloramine-T solution was vigorously mixed for 15 s for iodination of SHPP and immediately added to the prepared 20 μl SHPP / 125I mixture. The iodized SHPP solution was extracted with 1 mL of benzene / DMF mixture (40:1, v / v), and the organic phase was transferred to a clean tube. The organic solvent was removed by evaporation, and the dried iodized SHPP was dissolved and reacted with 10 nmol of aminated monomeric siRNA (100 μl) for 2 h at 4 °C to covalently bond the NHS group of the iodized SHPP phase to the amine group modified at the 5' end of the monomeric siRNA. The 125I-labeled monomeric siRNA was purified by overnight dialysis (MWCO: 5 kDa) (molar ratio of NHS groups, NHS groups of SHPP / amine groups of monomeric siRNA).

[0150] For aminated monomeric siRNA, monomeric siRNA was end-labeled with an amine group at the 5' end (sense) together with a thiol group modification at the 3' end (sense) with the sequence for scrambled siRNA, and was purchased from Bioneer (Daejeon, Korea). 125I-labeled dimeric siRNA was synthesized as described in the dimeric siRNA synthesis section and analyzed by 10% PAGE.

[0151] Pharmacokinetic profiles corrected for radioactive decay up to the time of injection were measured by collecting blood samples in groups of female SPC C57BL / 6 mice. After intravenous (iv) injection of siRNA / SLN complexes formed by 125I-labeled monomeric or dimeric siRNA, sampling was performed at the indicated times, and the radioactivity of the harvested samples was measured using a gamma counter (1470 Auto Gamma Counter, Perkin Elmer, USA). The time points used in the experiment were 0.25, 0.5, 1, 2, 4, 24 and 48 hours, and approximately 2 μl of blood was collected from the mouse tail vein and stored in 8 μl of EDTA solution to prevent blood clotting (n=3 for each group).

[0152] To evaluate the in vivo siRNA tissue distribution, siRNA / SLN complexes formed by 125I-labeled dimeric siRNA were injected into female SPF C57BL / 6 mice via intravenous (iv) injection. After injection, mice (n=3 mice / time point) were sacrificed at specific time points (1, 4, 24, and 48 hours) and sample organs were harvested and weighed. The in vivo siRNA tissue distribution was calculated from the radioactivity of each tissue measured using a γ-counter, compared with the radioactivity at the time of the initial injection (% of injection dose / gram).

[0153] As a result, as shown in Figure 11, the HDL-mimicking siRNA / SLN complex (albumin HDL) showed the best blood stability and the longest blood half-life among all the experimental groups, and it was confirmed that the siRNA was distributed more in the cancer tissue as time passed (Figure 12). Through this, it was confirmed that the HDL-mimicking siRNA / SLN complex according to the present invention has the best drug delivery efficiency to the cancer tissue.

[0154] [Experimental Example 6] Preparation of water-insoluble drug delivery system 1) Preparation of lipoprotein-mimicking SLNs loaded with paclitaxel (Taxol) as a water-insoluble drug (Taxol-loaded SLNs) As shown in Tables 12 and 13 below, the components were dissolved in 2 mL of chloroform:methanol (2:1) solution contained in a glass bottle. 10 mL of distilled water was added to the glass bottle and mixed by vortexing for 1 minute, and then the solution was sonicated for 3 minutes with a Branson sonicator 450 (20 kHz, duty cycle = 40, output control = 3.5). The solution was transferred to a rotary evaporator, and the solvent chloroform:methanol (2:1, v / v) solution was removed at a temperature above 60 ° C, which is the melting point of cholesteryl oleate. The solid lipid nanoparticle solution was purified in distilled water overnight using a dialysis membrane with a molecular weight cut-off (MWCO) of 5000, and the purified solid lipid nanoparticle solution encapsulating a water-insoluble drug (taxol) was stored at 4 ° C to prepare solid lipid nanoparticles encapsulating a water-insoluble drug (taxol) (Taxol containing SLN).

[0155] Here, when HDL-mimicking SLN was used, it was expressed by HDL-mimicking SLN encapsulated with Taxol, and when LDL-mimicking SLN was used, it was expressed by LDL-mimicking SLN encapsulated with Taxol.

[0156] [Table 12]

[0157] [Table 13]

[0158] 2) Confirmation of the physicochemical properties of Taxol-loaded SLN The average size and zeta potential of the Taxol-loaded HDL-mimicking SLN and Taxol-loaded LDL-mimicking SLN prepared in Experimental Example 6-1) were measured by laser light scattering using a dynamic light scattering device (DSL) (Zeta-Plus, Brookhaven Instruments, NY) equipped with a He-Ne laser with a wavelength of 632 nm and a detection angle of 90°. The size of the Taxol-loaded HDL-mimicking SLN and Taxol-loaded LDL-mimicking SLN dispersed in distilled water at 25°C at a concentration of 5 mg / ml was measured three times, and high performance liquid chromatography (HPLC) was used to evaluate the drug content and encapsulation rate contained in the Taxol-loaded HDL-mimicking SLN and Taxol-loaded LDL-mimicking SLN. The HDL-mimicking SLN and LDL-mimicking SLN solutions containing Taxol were freeze-dried to remove distilled water. The freeze-dried solid lipid nanoparticles containing the water-insoluble drug (taxol) were dispersed in 20 mL of methanol to completely dissolve the solid lipid nanoparticles, and the water-insoluble drug was extracted using a filter (Millex SR 0.45 um filter unit), and the amount of the water-insoluble drug encapsulated was analyzed using high performance liquid chromatography. The results are shown in Tables 14 and 15 below. In this case, the amount of the water-insoluble drug was quantitatively analyzed by comparing with a calibration curve according to the drug concentration, and the amount of the encapsulated water-insoluble drug (%, w / w) and the encapsulation rate (%) were analyzed together using the following Equations 1 and 2.

[0159]

number

[0160]

number

[0161] [Table 14]

[0162] [Table 15]

[0163] Table 14 shows the results for HDL-mimicking SLN loaded with Taxol, and Table 15 shows the results for LDL-mimicking SLN loaded with Taxol.

[0164] As shown in Tables 14 and 15, it was confirmed that Taxol-loaded HDL-mimicking SLN and Taxol-loaded LDL-mimicking SLN have stable physicochemical properties in the aqueous solution phase.

[0165] More specifically, in the case of HDL-mimicking SLN encapsulating Taxol, the drug was encapsulated at an efficiency of 11% (w / w) relative to the particle weight, and it was confirmed that the HDL-mimicking SLN encapsulating Taxol had a size of 115-125 nm and a surface charge of 65-85 mV.

[0166] In addition, in the case of LDL-mimicking SLN encapsulating Taxol, the drug was encapsulated at an efficiency of 14.9% (w / w) relative to the particle weight, and it was confirmed that the LDL-mimicking SLN encapsulating Taxol had a size of 100-130 nm and a surface charge of 65-85 mV. [Industrial Applicability]

[0167] According to the present invention, by producing core-shell lipoprotein-mimetic solid lipid nanoparticles composed of cholesteryl esters, triglycerides, albumin-bound cholesterol, fusogenic lipids and cationic lipids, it is possible to prevent aggregation between particles and ensure structural stability, and to provide a drug delivery vehicle with excellent bioavailability and improved drug encapsulation efficiency.

Claims

1. a core comprising triglycerides and cholesteryl esters; A lipoprotein-mimetic solid lipid nanoparticle comprising: a shell comprising albumin-bound cholesterol, a fusogenic lipid, and a cationic lipid.

2. The lipoprotein-mimetic solid lipid nanoparticle according to claim 1, wherein the cholesteryl ester is formed by esterifying cholesterol with a saturated or unsaturated fatty acid having 10 to 24 carbon atoms.

3. The lipoprotein-mimetic solid lipid nanoparticle according to claim 1, wherein the triglyceride is one molecule of glycerol to which three molecules of saturated or unsaturated fatty acid having 10 to 24 carbon atoms are ester-bonded.

4. Albumin-bound cholesterol is the reaction product of cholesteryl chloroformate and albumin. The lipoprotein-mimetic solid lipid nanoparticle of claim 1 , wherein the chloroformate group of the cholesteryl chloroformate is bound to an amine group of albumin.

5. The lipoprotein-mimetic solid lipid nanoparticles of claim 4, wherein the molar ratio of cholesteryl chloroformate to albumin in the albumin-bound cholesterol is 1:1 to 5:

1.

6. The fusogenic lipids were 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylethanolamine (POPC), egg phosphatidylcholine (egg phosphatidylcholine), and 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE). phosphatidylcholine (EPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylethanolamine (distearoylphosphatidylethanolamine) 2. The lipoprotein-mimetic solid lipid nanoparticle of claim 1, which is one or more selected from the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine] (DOPS), and 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine].

7. Cationic lipids include 3β-[N-(N',N'-dimethylaminoethane)carbamoyl]cholesterol (DC-cholesterol), 3β-[N-(N',N',N'-trimethylaminoethane)carbamoyl]cholesterol (TC-cholesterol), 3β-[N-(N'-monomethylaminoethane)carbamoyl]cholesterol (MC-cholesterol), 3β-[N-(aminoethane)carbamoyl]cholesterol (AC-cholesterol), and N-(N'-aminoethane)carbamoylpropanoic tocopherol (AC-tocopherol). , N-(N'-methylaminoethane)carbamoylpropanoic tocopherol (MC-tocopherol), N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleoyloxy)propyl-N,N,N-trimethylammonium chloride (DOTAP), N,N-dimethyl-(2,3-dioleoyloxy)propylamine (DODMA), N-(1-(2,3-dioleoyl)propyl)-N,N,N-trimethyl dioleoyl ammonium chloride (DOTMA), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), 1,2-dioleoylcarbamyl-3-dimethylammonium-propane (DOCDAP), 1,2-dilineoyl-3-dimethylammonium-propane (DLINDAP), dioleoyloxy-N-[2-sperminecarboxamido)ethyl}-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), dioctadecyl Amidoglycylspermine (DOGS), 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DMRIE), 3-dimethylamino-2-(cholest-5-ene-3-beta-oxybutane-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5-ene-3-beta-oxy)-3'-oxapentoxy]-3-dimethyl-1-(cis,cis-9',12'-octadecadienoxy)propane (CpLinDMA), N,The lipoprotein-mimetic solid lipid nanoparticles according to claim 1, which are one or more selected from the group consisting of N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N'-dioleoylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 1,2-diaza-3-trimethylammonium-propane (TAP), 1,2-diaza-3-dimethylammonium-propane (DAP), 1,2-di-O-octadecenyl-3-trimethylammoniumpropane, and 1,2-dioleoyl-3-trimethylammoniumpropane.

8. The lipoprotein-mimetic solid lipid nanoparticles of claim 1, wherein the lipoprotein-mimetic solid lipid nanoparticles have a diameter of 20 to 200 nm.

9. Lipoprotein-mimicking solid lipid nanoparticles are high density lipoprotein-mimicking solid lipid nanoparticles, The lipoprotein-mimetic solid lipid nanoparticle of claim 1, comprising 5 to 30 parts by weight of cholesteryl ester, 0.01 to 5 parts by weight of triglyceride, 1 to 20 parts by weight of albumin-bound cholesterol, 5 to 40 parts by weight of fusogenic lipid, and 10 to 60 parts by weight of cationic lipid, based on 100 parts by weight of the total high density lipoprotein-mimetic solid lipid nanoparticle.

10. Lipoprotein-mimicking solid lipid nanoparticles are low density lipoprotein-mimicking solid lipid nanoparticles, The lipoprotein-mimetic solid lipid nanoparticle of claim 1, comprising 30 to 60 parts by weight of cholesteryl ester, 0.01 to 5 parts by weight of triglyceride, 1 to 20 parts by weight of albumin-bound cholesterol, 5 to 40 parts by weight of fusogenic lipid, and 10 to 40 parts by weight of cationic lipid, based on 100 parts by weight of the total low density lipoprotein-mimetic solid lipid nanoparticle.

11. A method for producing lipoprotein-mimetic solid lipid nanoparticles, comprising the step of mixing a cholesteryl ester, a triglyceride, an albumin-bound cholesterol, a fusogenic lipid, and a cationic lipid in an organic solvent to produce an aqueous dispersion in which the lipoprotein-mimetic solid lipid nanoparticles are dispersed.

12. The method for producing lipoprotein-mimetic solid lipid nanoparticles according to claim 11, further comprising a step of purifying and concentrating the produced aqueous dispersion by dialysis.

13. The method for producing lipoprotein-mimetic solid lipid nanoparticles according to claim 11, wherein the albumin-bound cholesterol is prepared by mixing cholesteryl chloroformate dissolved in an organic solvent with an aqueous albumin solution.

14. The method for producing lipoprotein-mimetic solid lipid nanoparticles according to claim 12, wherein the molar ratio of cholesterol to albumin in the albumin-bound cholesterol is 1:1 to 5:

1.

15. The method for producing lipoprotein-mimetic solid lipid nanoparticles according to claim 11, wherein the organic solvent is selected from the group consisting of dimethylformamide, tetrahydrofuran, C1-C4 lower alcohols, methylene chloride, chloroform, acetone, dimethylsulfoxide, N-methylpyrrolidone, dioxane, ethyl acetate, methyl ethyl ketone, acetonitrile, and mixtures thereof.

16. The method for producing lipoprotein-mimetic solid lipid nanoparticles according to claim 11, wherein the diameter of the lipoprotein-mimetic solid lipid nanoparticles is 20-200 nm.

17. Lipoprotein-mimicking solid lipid nanoparticles are high density lipoprotein-mimicking solid lipid nanoparticles, The method for producing lipoprotein-mimetic solid lipid nanoparticles according to claim 11, comprising 5 to 30 parts by weight of cholesteryl ester, 0.01 to 5 parts by weight of triglyceride, 1 to 20 parts by weight of albumin-bound cholesterol, 5 to 40 parts by weight of fusogenic lipid, and 10 to 60 parts by weight of cationic lipid, based on 100 parts by weight of the total high density lipoprotein-mimetic solid lipid nanoparticles.

18. Lipoprotein-mimicking solid lipid nanoparticles are low density lipoprotein-mimicking solid lipid nanoparticles, The method for producing lipoprotein-mimetic solid lipid nanoparticles according to claim 11, comprising 30 to 60 parts by weight of cholesteryl ester, 0.01 to 5 parts by weight of triglyceride, 1 to 20 parts by weight of albumin-bound cholesterol, 5 to 40 parts by weight of fusogenic lipid, and 10 to 40 parts by weight of cationic lipid, based on 100 parts by weight of the total low density lipoprotein-mimetic solid lipid nanoparticles.

19. A drug delivery composition comprising the lipoprotein-mimetic solid lipid nanoparticles of claim 1.

20. The drug delivery composition of claim 19 , wherein the drug comprises one or more of a nucleic acid and a water-insoluble drug.

21. The nucleic acid is one or more selected from the group consisting of siRNA, rRNA, RNA, DNA, cDNA, aptamer, mRNA, tRNA and antisense-oligodeoxynucleotide (AS-ODN); 21. The drug delivery composition of claim 20, wherein the nucleic acid is bound to the cationic lipids in the shell of the solid lipid nanoparticles by electrostatic interactions.

22. The drug delivery composition of claim 20, wherein the water-insoluble drug is a poorly soluble drug, an anion-bearing peptide, a protein, a hyaluronic acid-peptide conjugate, or a hyaluronic acid-protein conjugate.

23. one or more of a nucleic acid targeting KRAS and a water-insoluble drug; A pharmaceutical composition for preventing or treating cancer caused by KRAS gene mutation, comprising the lipoprotein-mimetic solid lipid nanoparticles of claim 1.

24. The pharmaceutical composition for preventing or treating cancer caused by KRAS gene mutation according to claim 23, wherein the nucleic acid targeting KRAS is a reducible nucleic acid dimer.

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