Genetic material carrier for transdermal delivery and composition for preventing or treating cancer comprising same

Abstract

The present invention relates to a genetic material carrier for transdermal delivery and a composition for preventing or treating cancer, comprising same. The genetic material carrier according to the present invention can promote the transdermal delivery of a genetic material and target cancer cells and has appropriate nano-size distribution and minimum non-specific cytotoxicity, by coating, with hyaluronic acid (HA), cationic solid lipid nanoparticles (CSLNs) that carry angiogenesis-inhibiting siVEGF, and thus can be used as a novel transdermal drug carrier for targeted cancer therapy.

Description

Genetic material delivery vehicle for transdermal delivery and composition for cancer prevention or treatment containing the same

[0001] The present invention relates to a genetic material delivery vehicle for transdermal delivery and a composition for preventing or treating cancer containing the same. More specifically, it relates to a novel siRNA delivery system that forms a stable nanoscale complex capable of targeted delivery to skin cancer tissue by coating hyaluronic acid (HA) onto cationic solid lipid nanoparticles (CSLNs) carrying angiogenesis-inhibiting siVEGF.

[0002] RNA interference (RNAi) is a cellular defense mechanism that sequence-specifically degrades foreign RNA. Short interfering RNA (siRNA) is a form of RNAi that binds to target RNA, cleaves and degrades it, and consequently downregulates the expression of target proteins. This mechanism is receiving significant attention in therapeutic applications due to its ability to downregulate specific proteins. To date, the FDA has approved six siRNA drugs (Patisiran (Onpattro), Givosiran (Givlaari), Lumasiran (Oxlumo), Inclisiran (Leqvio), Nedosiran (DCR-PHXC), and Vutrisiran (Amvutta)), most of which target the liver. These developments highlight the promising potential of siRNA for treating various diseases. However, the six approved drugs are limited to liver-targeted delivery because they primarily utilize GalNAc as a delivery carrier. While GalNAc has successfully delivered siRNA to hepatocytes, challenges remain, such as difficulties in targeting other organs, limited endocytotic escape, hepatotoxicity in non-target organs, and extensive enzymatic and systemic clearance. Currently, several other drugs are in Phase II and III clinical trials, primarily targeting organs such as the liver, eyes, lungs, kidneys, brain, and skin. However, since most are initially delivered in RNA form, the need to explore alternative delivery systems, such as lipid nanoparticles (LNPs), polymers, or biological vectors, is highlighted. Such advancements could improve delivery efficiency to a wider range of organs and increase the potential to treat more diseases.

[0003] Early strategies for siRNA delivery focused primarily on LNPs, which have been widely accepted in clinical practice due to their low immunogenicity, increased circulation time, and in vivo stability. By optimizing the lipid-nucleic acid ratio, LNPs can be customized for targeted delivery, thereby enhancing therapeutic potential. The goal of LNP design is to address issues related to the delivery of the initial nucleic acid, such as instability caused by biological degradation mechanisms and difficulty in penetrating membrane barriers. Common types of LNPs include solid lipid nanoparticles, nanostructured lipid carriers, and cationic lipid-nucleic acid complexes, which are characterized by more complex internal structures and enhanced physical stability. However, these next-generation LNPs still face several challenges: 1) RNA size, LNP formulation, size, and polydispersity index significantly affect encapsulation efficiency; 2) Existing LNPs lack targeting ability and are not effectively delivered to specific cells; and 3) storage methods, the selection of cryoprotectants, and storage temperature can affect the long-term stability of the formulation.

[0004] Therefore, it is necessary to develop genetic material delivery vehicles that solve the aforementioned problems while enhancing the ability to target specific organs and cells.

[0005] The present invention aims to provide a genetic material delivery system having a high genetic material (drug) loading capacity and capable of effectively delivering genetic material through the skin, and a composition for preventing or treating cancer comprising the same.

[0006] In addition, the present invention aims to provide a genetic material delivery vehicle capable of inhibiting tumor growth by improving transdermal delivery, effective tumor targeting, and significantly reducing VEGF mRNA levels, and a composition for preventing or treating cancer containing the same.

[0007] In addition, the present invention aims to provide a composition for the prevention or treatment of skin cancer that can effectively deliver genetic material through the dermis.

[0008] The present invention is capable of various modifications and may have various embodiments, and specific embodiments are to be illustrated and described in detail. However, this is not intended to limit the invention to specific embodiments, and it should be understood that the invention includes all modifications, equivalents, and substitutions that fall within the spirit and scope of the invention.

[0009] The terms used in this application are used merely to describe specific embodiments and are not intended to limit the invention. The singular expression includes the plural expression unless the context clearly indicates otherwise. In this application, terms such as "comprising" or "having" are intended to specify the presence of the features, numbers, steps, actions, components, parts, or combinations thereof described in the specification, and should be understood as not precluding the existence or addition of one or more other features, numbers, steps, actions, components, parts, or combinations thereof.

[0010] Where in this specification, when a quantity, concentration, or other value or parameter is given as an enumeration of a range, a preferred range, a preferred upper limit, and a preferred lower limit, it should be understood that any pair of any upper range limit or preferred value and any lower range limit or preferred value are specifically disclosed, regardless of whether the range is disclosed separately.

[0011] Where a range of numerical values ​​is mentioned in this specification, unless otherwise stated, the range and the scope of the parent invention within that range are not intended to be limited to the specific value mentioned when defining the range.

[0012] Furthermore, to prevent clutter with overlapping content, redundant details have been omitted below. In other words, the content of the invention is not limited solely to the following description, and should be interpreted in accordance with the overall context of the invention.

[0013] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as generally understood by those skilled in the art to which the present invention pertains. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and should not be interpreted in an ideal or overly formal sense unless explicitly defined in this application. Specific details for the implementation of the above invention are described below.

[0014] The present invention provides a genetic material delivery system comprising: cationic solid lipid nanoparticles having a core-shell structure; and a coating layer positioned on the cationic solid lipid nanoparticles and comprising hyaluronic acid, wherein the core comprises cholesteryl ester and triglyceride, and the shell comprises cholesterol, fusion-inducible lipids, cationic lipids, and lipid-PEG (polyethylene glycol) conjugates.

[0015] The above genetic material delivery vehicle can deliver genetic material through the skin.

[0016] The above genetic material carrier may additionally comprise genetic material, wherein the genetic material is located between the shell of the cationic solid lipid nanoparticle and the coating layer and may be bonded by electrostatic interaction with the cationic lipid of the shell.

[0017] The above genetic material may be, for example, a polynucleotide. More preferably, it may include one or more selected from the group consisting of small 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), and preferably may include siRNA.

[0018] In particular, siRNA refers to duplex RNA, or single-stranded RNA that takes the form of a double strand within single-stranded RNA. The binding between the double strands is achieved through hydrogen bonds between nucleotides, and not all nucleotides within the double strand must be completely complementary. The length of the siRNA may be about 15 to 60, specifically about 15 to 50, about 15 to 40, about 15 to 30, 15 to 25, about 16 to 25, about 19 to 25, about 20 to 25, or about 20 to 23 nucleotides. The above siRNA length refers to the number of nucleotides on one side of the duplex RNA, i.e., the number of base pairs, and in the case of single-stranded RNA, refers to the length of the double strand within the single-stranded RNA. In addition, siRNA can be composed of nucleotides with various functional groups introduced for purposes such as increasing blood stability or weakening immune responses.

[0019] Accordingly, the siRNA of the present invention may be an unmodified or modified form of typical siRNA. For example, one end of the siRNA may be modified with polyethylene glycol. Since polyethylene glycol (PEG) is a hydrophilic, flexible, and non-ionic polymer, it is one of the most common materials for modifying granular systems and imparting long-term circulation to carriers so that they are not recognized by macrophages of the monomolecular macrophage system (MPS). In one embodiment of the present invention, when the molecular weight of PEG is 3,000 to 7,000 daltons, e.g. 5,000 daltons, the siRNA can be sufficiently protected against RNase digestion while maintaining the effective transfection performance of the siRNA.

[0020] The weight ratio (dielectric material / cationic solid lipid nanoparticles) of the cationic solid lipid nanoparticles and the dielectric material may be 1 to 50, preferably 3 to 30, 5 to 20, 7 to 18, 8 to 17, 9 to 16, and more preferably 10 to 15. Specifically, in an embodiment of the present invention, the weight ratio (dielectric material / cationic solid lipid nanoparticles) of the cationic solid lipid nanoparticles and the dielectric material was set to 12, which was set as the optimal composition ratio based on the results of electrophoretic migration.

[0021] The hyaluronic acid may have a molecular weight of 5 kDa to 200 kDa, preferably 5 to 100 kDa, 5 to 50 kDa, and more preferably 5 to 10 kDa. The present invention has the effect of improving cell delivery and skin permeability by including hyaluronic acid having a molecular weight within the above range.

[0022] The above cholesteryl ester may be one in which a saturated or unsaturated fatty acid having 10 to 24 carbon atoms is ester-bonded to cholesterol, and preferably may be an ester of an unsaturated fatty acid having 16 to 18 carbon atoms, such as oleic acid. The cationic solid lipid nanoparticles of the present invention may comprise a single or multiple types of cholesteryl esters.

[0023] The above triglyceride may be a refined triglyceride having a composition of various fatty acids, or a vegetable oil having a triglyceride composed of multiple fatty acids as its main component. Specifically, the above 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, cod liver oil, etc. The above oil may be used as a single type or as a mixture of several types of oil. In a specific example, the above triglyceride may be triolein.

[0024] The above cholesteryl ester and triglyceride form the core of cationic solid lipid nanoparticles through hydrophobic bonding.

[0025] The fusogenic lipids mentioned above may be any type of neutral, cationic, or anionic lipid capable of forming the nanoparticles of the present invention, and may be a mixture of a single or multiple types of phospholipids. The fusogenic lipids mentioned above may be any type of phospholipid capable of inducing fusion, such as phosphatidylcholine (PC), phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, or their lyso forms, or may be a fully saturated or partially hardened form having an aliphatic chain having 6 to 24 carbon atoms. Specifically, the fusion-inducing lipids mentioned above are not limited thereto but include dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine (EPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), and distearoylphosphatidylethanolamine (DSPE). Phosphatidylethanolamine (PE), dipalmitoylphosphatidylethanolamine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE),It may include one or more selected from the group consisting of 1-palmitoyl-2-oleoyl-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], preferably including dioleoylphosphatidylethanolamine (DOPE), but is not limited thereto.

[0026] The fusion-inducing lipids and cholesterol forming the shell act as helper lipids that enhance transfection efficiency during gene transfection and reduce the cytotoxicity of the combined cationic lipids. Additionally, cholesterol imparts structural rigidity to the lipid packing, thereby improving the stability of the nanoparticles of the present invention along with the activity of the helper. Furthermore, the fusion-inducing lipids facilitate the passage of the nanoparticles through the cell membrane and endosomal escape, thereby enabling easy intracellular delivery.

[0027] The above cationic lipids include cationic lipids that carry a net negative charge at a specific pH, such as physiological pH. Specifically, the cationic lipids are 3-beta[N-(N',N'-dimethylaminoethane)carbamoyl]cholesterol (DC-cholesterol), 3-beta-[N-(N',N',N'-trimethylaminoethane)carbamoyl]cholesterol (TC-cholesterol), 3-beta[N-(N'-monomethylaminoethane)carbamoyl]cholesterol (MC-cholesterol), 3-beta[N-(aminoethane)carbamoyl]cholesterol (AC-cholesterol), N-(N'-aminoethane)carbamoylpropanoic tocopherol (AC-tocopherol), N-(N'-methylaminoethane)carbamoylpropanoic tocopherol (MC-tocopherol), N,N-dioleyl-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-Dioleyl)propyl)-N,N,N-Trimethylammonium Chloride (DOTMA), 1,2-Dioleyl-3-Dimethylammonium-Propane (DODAP), 1,2-Dioleylcarbamyl-3-Dimethylammonium-Propane (DOCDAP), 1,2-Dilineoyl-3-Dimethylammonium-Propane (Dilineoyl-3-Dimethylammonium-propane, DLINDAP), Dioleoyloxy-N-[2-spermincarboxamido)ethyl}-N,N-dimethyl-1-propaneamulanttrifluoroacetate (DOSPA), dioctadecylamidoglycylspermin (DOGS), 1,2-dimyristriloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DMRIE), 3-dimethylamino-2-(cholest-5-en-3-beta-oxybutane-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5-en-3-beta-oxy)-3'-oxapentoxy]-3-damethyl-1-(cis,cis-9',12'-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,It may include one or more selected from the group consisting of N'-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 1,2-diacyl-3-trimethylammonium-propane (TAP), 1,2-diacyl-3-dimethylammonium-propane (DAP), 1,2-di-O-octadeceyl-3-trimethylammonium propane, 1,2-dioleoyl-3-trimethylammonium propane, Trasfectam and 98N12-5(1), preferably may include 3-beta[N-(N',N'-dimethylaminoethane)carbamoyl]cholesterol (DC-cholesterol), but is not limited thereto.

[0028] Specifically, DC-cholesterol is less toxic than other cationic lipids, and since DC-cholesterol gene carriers have been approved for use in the clinical treatment of various diseases such as melanoma, cystic fibrosis, cervical cancer, breast cancer, and ovarian cancer, it may be desirable to use DC-cholesterol.

[0029] The above lipid-PEG (polyethyleneglycol) conjugate refers to a form in which lipids and PEG are conjugated.

[0030] The lipids in the above conjugate may be selected from the group consisting of all types of cholesterol, fusion-inducing lipids, and cationic lipids described above, for example, lipids containing amine groups. In a specific example, the lipid is cholesterol, dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine (EPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylethanolamine (DSPE), Phosphatidylethanolamine (PE), dipalmitoylphosphatidylethanolamine (DPPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine] (DOPS), 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine], 3-beta-[N-(N',N'-Dimethylaminoethane)carbamoyl]cholesterol (DC-cholesterol), 3-beta-[N-(N',N',N'-Trimethylaminoethane)carbamoyl]cholesterol (TC-cholesterol), 3-beta-[N-(N'-Monomethylaminoethane)carbamoyl]cholesterol (MC-cholesterol), 3-beta[N-(Aminonethan)carbamoyl]cholesterol (AC-cholesterol), N-(N'-Aminonethan)carbamoylpropanoic tocopherol (AC-tocopherol), N-(N'-Methylaminoethane)carbamoylpropanoic tocopherol (MC-tocopherol), N,N-Dioleyl-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-dioleyl)propyl)-N,N,N-trimethylammonium chloride (DOTMA), 1,2-dioleyl-3-dimethylammonium-propane (DODAP), 1,2-dioleylcarbamyl-3-dimethylammonium-propane (DOCDAP), 1,2-dilineoyl-3-dimethylammonium-propane (DLINDAP), dioleoyloxy-N-[2-spermincarboxamido)ethyl}-N,N-dimethyl-1-propaneamium trifluoroacetate (DOSPA), Dioctadecylamidoglycylspermin (DOGS), 1,2-Dimiristriloxypropyl-3-Dimethyl-hydroxyethylammonium bromide (DMRIE), 3-Dimethylamino-2-(Cholester-5-N-3-Beta-Oxybutane-4-Oxy)-1-(Cis,Cis-9,12-Octadecadienoxy)propane (CLinDMA), 2-[5'-(Cholester-5-N-3-Beta-Oxy)-3'-Oxapentoxy]-3-Damethyl-1-(Cis,Cis-9',12'-Octadecadienoxy)propane (CpLinDMA), N,N-Dimethyl-3,4-Dioleyloxybenzylamine (DMOBA), 1,2-N,N'-Dioleylcarbamyl-3-Dimethylaminopropane (DOcarbDAP), 1,2-diacyl-3-trimethylammonium-propane (TAP), 1,It may include one or more selected from the group consisting of 2-diacyl-3-dimethylammonium-propane (DAP), 1,2-di-O-octadeceyl-3-trimethylammonium propane, 1,2-dioleoyl-3-trimethylammonium propane, Trasfectam and 98N12-5(1). Specifically, the lipid may comprise one or more selected from the group consisting of distearoylphosphatidylethanolamine (DSPE) and dipalmitoylphosphatidylethanolamine (DPPE), and more preferably may comprise distearoylphosphatidylethanolamine (DSPE), but is not limited thereto.

[0031] The above PEG may have a weight-average molecular weight of 100 to 100,000 Da, specifically 500 to 70,000 Da, more specifically 1,000 to 50,000 Da, but is not limited thereto. In one embodiment, the PEG may be a functionalized PEG in which a functional group is attached to the side not bound to the lipid, and the functional group that can be used may be one or more selected from the group consisting of succinyl, carboxylic acid, maleimide, amine, biotin, cyanur, and folate.

[0032] The mixing ratio between lipids and PEG in the above lipid-PEG conjugate may be approximately 1:0.5 to 3 (moles of lipids:moles of PEG) in molar ratio.

[0033] In a specific example, the lipid-PEG conjugate may include one or more selected from the group consisting of distearoylphosphatidylethanolamine (DSPE)-PEG and DPPE-PEG. Such a lipid-PEG conjugate contributes to the stability of nanoparticles in serum and plays a role in protecting nucleic acids from degrading enzymes during in vivo delivery, thereby enhancing the safety of nucleic acids in the body.

[0034] In a specific example, the cationic solid lipid nanoparticle may comprise a core comprising cholesteryl oleate and triolein; and a shell comprising cholesterol, dioleoylphosphatidylethanolamine (DOPE), 3-beta-[N-(N',N'-dimethylaminoethane)carbamoyl]-cholesterol (DC-cholesterol), and DSPE-PEG.

[0035] In a specific example, the cationic solid lipid nanoparticles may comprise, based on 100 weight% of the total cationic solid lipid nanoparticles, 30 to 60 weight% of the cholesteryl ester, 0.1 to 10 weight% of the triglyceride, 5 to 20 weight% of the cholesterol, 5 to 30 weight% of the fusion-inducing lipid, 10 to 50 weight% of the cationic lipid, and 0.01 to 1 weight% of the lipid-PEG conjugate.

[0036] Preferably, the cationic solid lipid nanoparticles may comprise, based on 100 weight% of the total cationic solid lipid nanoparticles, 40 to 50 weight% of the cholesteryl ester, 1 to 5 weight% of the triglyceride, 8 to 12 weight% of the cholesterol, 12 to 16 weight% of the fusion-inducing lipid, 25 to 30 weight% of the cationic lipid, and 0.05 to 0.5 weight% of the lipid-PEG conjugate.

[0037] If the content of the lipid-PEG conjugate in the above-mentioned cationic solid lipid nanoparticles is greater than the above range, it is difficult to form a complex with nucleic acid; if it is less than the above range, it is disadvantageous in terms of the serum stability of the particles and the in vivo safety of the nucleic acid. Therefore, considering the ease of complex formation between the nanoparticles and nucleic acid, the in vivo stability of the nanoparticles, and the in vivo safety of the nucleic acid, it is preferable to set the content of the lipid-PEG conjugate within the above range.

[0038] In addition, the content ratio between the core and the shell within the nanoparticles of the present invention may be 30:70 to 70:30 by weight, specifically 40:60 to 60:40, more specifically 45:55 to 55:45.

[0039] In one embodiment of the present invention, the molar ratio between fusion-inducing lipids:cholesterol:cationic lipids among the components constituting the shell is 9.4:13:26, and the molar ratio of cationic lipids / helper lipids (fusion-inducing lipids and cholesterol) may be 1.16, which is a value providing an equimolar ratio.

[0040] The above genetic material delivery vehicle may have an average particle size of 30 to 500 nm, preferably 30 to 250 nm, 50 to 200 nm, 80 to 180 nm, and more preferably 100 to 150 nm. The above genetic material delivery vehicle has an average particle size within the above range, making it easy to introduce into cells and providing excellent efficiency in delivering genetic material to target organs and / or cells.

[0041] The above genetic material delivery vehicle may exhibit a zeta potential of -30 to -5 mV. By exhibiting a zeta potential within this range, the genetic material delivery vehicle demonstrates dispersion stability, and the hyaluronic acid coating can enhance cell delivery and skin penetration efficiency.

[0042] Compared to previous lipid nanoparticles (LNPs), solid lipid nanoparticles (SLNs) possess high physical stability, maintaining stability in both solution and tissue, making them highly suitable as drug delivery systems. While SLNs generally encapsulate hydrophobic drugs, cationic solid lipid nanoparticles (CSLNs) can also deliver water-soluble nucleotides through electrical interactions. Specifically, CSLNs are low-density lipoprotein (LDL)-like cationic solid lipid nanoparticles that lack apolipoproteins; they enhance the drug loading efficiency of RNA and, compared to bulk RNA, are highly stable in vivo and offer protection against degradation by RNase. However, when using CSLNs as drug delivery systems in in vivo experiments, targeting specific sites is often difficult, and they tend to accumulate significantly in the liver, limiting their application to liver diseases. On the other hand, the genetic material carrier (HA / CSLN) of the present invention prevents siRNA degradation by coating HA onto the surface of exposed siRNA through electrical interaction.

[0043] Specifically, the researchers of the present invention developed a hyaluronic acid (HA) / CSLN carrying siRNA to downregulate vascular endothelial growth factor (VEGF), which demonstrates excellent cell delivery capabilities and skin permeability. HA is a biomaterial with properties advantageous for treating skin-related diseases; possessing excellent biocompatibility and moisturizing properties, it promotes drug absorption and is an ideal formulation for topical administration. HA selectively targets skin tumor cells by interacting with HA receptors, such as Cluster of differentiation antigen 44 (CD44). Furthermore, the introduction of HA reduces the cytotoxicity of cationic CSLN. Here, both in vitro and in vivo studies demonstrate that siVEGF delivery effectively downregulates VEGF expression, thereby inhibiting angiogenesis and tumor growth. Therefore, based on enhanced skin absorption, excellent cell targeting, and outstanding anticancer activity, HA / CSLN / siVEGF can serve as a promising tool to advance cancer treatment and improve patient outcomes.

[0044] Figure 1a shows the results of confirming the transdermal delivery effect of hyaluronic acid labeled with Rhodamine B (RhoB) into the dermis. According to Figure 1a, the potential of this delivery pathway was proven by demonstrating that HA labeled with Rhodamine B (RhoB) can penetrate the dermis via transdermal delivery. Furthermore, because the CD44 receptor is highly expressed in cancer cells, HA can act as a major ligand to promote active tumor targeting. Targeted therapy of cancer cells through transdermal drug delivery is an effective strategy for cancer treatment.

[0045] FIG. 1b is a schematic diagram showing that the entry of a genetic material delivery vehicle according to the present invention into a cell occurs through endocytosis. Referring to FIG. 1b, the genetic material delivery vehicle of the present invention can selectively target specific cancer cells by binding to the CD44 receptor of the cell and then delivering it into the cell.

[0046] The present invention provides a pharmaceutical composition comprising: cationic solid lipid nanoparticles having a core-shell structure; a coating layer comprising hyaluronic acid located on the positively charged solid lipid nanoparticles; and a dielectric material located between the shell of the positively charged solid lipid nanoparticles and the coating layer; wherein the core comprises cholesteryl ester and triglyceride, the shell comprises cholesterol, fusion-inducible lipid, cationic lipid, and lipid-PEG (polyethylene glycol) conjugate, and the dielectric material is bonded to the cationic lipid of the shell by electrostatic interaction.

[0047] The present invention provides a pharmaceutical composition for the prevention or treatment of cancer, comprising: cationic solid lipid nanoparticles having a core-shell structure; a coating layer comprising hyaluronic acid located on the positively charged solid lipid nanoparticles; and a dielectric material located between the shell of the positively charged solid lipid nanoparticles and the coating layer; wherein the core comprises cholesteryl ester and triglyceride, the shell comprises cholesterol, fusion-inducible lipid, cationic lipid, and lipid-PEG (polyethylene glycol) conjugate, and the dielectric material is bonded to the cationic lipid of the shell by electrostatic interaction.

[0048] The present invention provides a pharmaceutical composition for a cancer vaccine comprising: cationic solid lipid nanoparticles having a core-shell structure; a coating layer comprising hyaluronic acid located on the positively charged solid lipid nanoparticles; and a genetic material located between the shell of the positively charged solid lipid nanoparticles and the coating layer; wherein the core comprises cholesteryl ester and triglyceride, the shell comprises cholesterol, fusion-inducible lipid, cationic lipid, and lipid-PEG (polyethylene glycol) conjugate, and the genetic material is bonded to the cationic lipid of the shell by electrostatic interaction.

[0049] The cationic solid lipid nanoparticles included in the above-mentioned pharmaceutical composition for cancer prevention or treatment and / or pharmaceutical composition for cancer vaccine are identical to the cationic solid lipid nanoparticles included in the aforementioned genetic material delivery vehicle.

[0050] The coating layer containing hyaluronic acid included in the above pharmaceutical composition for cancer prevention or treatment and / or pharmaceutical composition for cancer vaccine is identical to the coating layer containing hyaluronic acid included in the aforementioned genetic material delivery vehicle.

[0051] The genetic material included in the above pharmaceutical composition for cancer prevention or treatment and / or pharmaceutical composition for cancer vaccine may be a polynucleotide. More preferably, it may include one or more selected from the group consisting of small 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), and preferably siRNA, more preferably siRNA suitable for cancer prevention or treatment.

[0052] The genetic material used herein may be any genetic material known to exhibit therapeutic efficacy against cancer, more preferably any polynucleotide-based genetic material, and may be included within the scope of the genetic material of the present invention.

[0053] The above composition can deliver genetic material through the dermis.

[0054] Specifically, the above composition can be used on mammals, preferably humans, and can deliver genetic material into cells through the dermis.

[0055] The term "prevention" as used in this invention refers to any act of inhibiting cancer formation or delaying its onset through the administration of a composition.

[0056] In the present invention, "treatment" refers to any act in which the symptoms of the said disease are improved or beneficially altered by the administration of the composition.

[0057] The above cancer may be any one selected from the group consisting of skin cancer, skin or intraocular melanoma, colorectal cancer, breast cancer, brain cancer, neurocancer, lung cancer, small cell lung cancer, stomach cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, head or neck cancer, uterine cancer, ovarian cancer, rectal cancer, pro-anal cancer, colon cancer, breast cancer, fallopian tube carcinoma, endometrial carcinoma, cervical cancer, vaginal cancer, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureteral cancer, renal cell carcinoma, renopelvic carcinoma, CNS tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma and pituitary adenoma, and preferably may be skin cancer.

[0058] In one embodiment of the present invention, a gene delivery vehicle (HA / CSLN / siVEGF) containing siVEGF was transdermally delivered to a C57BL / 6J immunodeficient mouse model with a B16F10 tumor (15 μg to 30 μg applied to the skin where the tumor is located once every 2 days), and it was confirmed that the antitumor effect (inhibition of cancer cell proliferation and metastasis) was significantly superior (see FIGS. 5b to 5e).

[0059] The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier, said pharmaceutically acceptable carrier including but not limited to those commonly used in the art, such as lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. Additionally, the pharmaceutical composition of the present invention may include, but is not limited to, fillers, extenders, binders, wetting agents, disintegrants, diluents or excipients such as surfactants, and other pharmaceutically acceptable additives.

[0060] The above pharmaceutical composition may be used in various transdermal administration formulations, preferably in the form of a patch or a topical preparation. A patch may be used to deliver a controlled amount of the compound of the present invention in a continuous or discontinuous manner. The structure and use of a patch for delivering a pharmaceutical preparation are widely known in the industry and may be appropriately modified by those skilled in the art according to the delivery requirements of the pharmaceutical preparation, such as continuous, pulsed, or other methods. A topical preparation refers to a composition applied by means such as direct application to the skin, and may be formulated into ointments, gels, creams, lotions, liquids, sprays, etc., although not specifically limited thereto, and its composition and use may be appropriately modified by those skilled in the art according to the delivery requirements of the pharmaceutical preparation, such as a patch.

[0061] The effective dosage of the pharmaceutical composition of the present invention varies depending on the patient's body weight, age, gender, health condition, diet, time of administration, method of administration, excretion rate, and severity of the disease, and can be easily determined by a person skilled in the art.

[0062] The present invention also provides a method for preventing or treating cancer by administering the above composition and / or genetic material delivery vehicle to a subject who requires it.

[0063] More specifically, core-shell structured cationic solid lipid nanoparticles;

[0064] A coating layer containing hyaluronic acid located on the above positively charged solid lipid nanoparticles; and

[0065] A dielectric material located between the shell of the positively charged solid lipid nanoparticle and the coating layer; comprising

[0066] The above core comprises cholesteryl ester and triglyceride, and

[0067] The above shell comprises cholesterol, fusion-inducing lipids, cationic lipids, and lipid-PEG (polyethylene glycol) conjugates, and

[0068] A method for preventing or treating cancer is provided by administering a composition, wherein the genetic material is bonded to the cationic lipid of the shell through electrostatic interaction, to a subject in need thereof.

[0069] More specifically, cationic solid lipid nanoparticles with a core-shell structure; and

[0070] A coating layer comprising hyaluronic acid located on the above-mentioned cationic solid lipid nanoparticles; comprising

[0071] The above core comprises cholesteryl ester and triglyceride, and

[0072] The above shell provides a method for the prevention or treatment of cancer by administering a genetic material delivery vehicle, comprising cholesterol, a fusion-inducing lipid, a cationic lipid, and a lipid-PEG (polyethylene glycol) conjugate, to a subject in need thereof.

[0073] The present invention also provides the above composition and / or genetic material delivery system for use in the prevention or treatment of cancer.

[0074] The present invention also provides the use of the composition and / or genetic material delivery system according to the present invention in the manufacture of a drug for the prevention or treatment of cancer.

[0075] The present invention also provides a method for delivering genetic material by administering the composition and / or genetic material delivery vehicle to a subject in need thereof.

[0076] The present invention also provides the above composition and / or genetic material delivery system for use in the delivery of genetic material within a living organism.

[0077] The present invention also provides the use of the composition and / or genetic material delivery body according to the present invention in the manufacture of a drug for the delivery of genetic material.

[0078] Here, the above composition, the genetic material carrier, and the cancer are summarized as referring to the description in the preceding explanation.

[0079] The genetic material delivery system according to the present invention has a high genetic material (drug) loading capacity and has the effect of effectively delivering genetic material through the skin.

[0080] In addition, the genetic material delivery vehicle of the present invention and the composition for cancer prevention or treatment containing the same have the effect of inhibiting tumor growth by providing improved transdermal delivery, effective tumor targeting, and significantly reducing VEGF mRNA levels.

[0081] Specifically, the genetic material delivery vehicle according to the present invention is coated with hyaluronic acid (HA) on cationic solid lipid nanoparticles (CSLNs) that carry angiogenesis-inhibiting siVEGF, thereby promoting transdermal delivery of genetic material and targeting cancer cells. It has an appropriate nano-size distribution and minimal non-specific cytotoxicity, and can be used as a novel drug delivery vehicle for transdermal targeted cancer therapy.

[0082] In addition, the present invention can provide a composition for the prevention or treatment of skin cancer that can effectively deliver genetic material through the dermis.

[0083] In addition, the present invention is applicable to various fields, such as genetic material-based vaccines and genetic material-based therapeutic agents targeting various diseases.

[0084] Figure 1a shows the results of confirming the transdermal delivery effect of hyaluronic acid labeled with Rhodamine B (RhoB) into the dermal layer.

[0085] FIG. 1b is a schematic diagram showing that the entry of a genetic material carrier into a cell according to the present invention occurs through endocytosis.

[0086] FIG. 1c is a schematic diagram relating to a method for manufacturing a dielectric material carrier according to the present invention.

[0087] Figure 2a shows the morphology of cationic solid lipid nanoparticles (CSLN) and genetic material carriers (HA / CSLN / siRNA) as seen by TEM (scale bar = 50 μm).

[0088] Figure 2b shows the results of agarose gel electrophoresis at various weight ratios of CSLN / siRNA complexes (CSLN:siRNA = 1:3, 6, 9, 12, 15).

[0089] Figure 2c shows the size distribution of CSLN, CSLN / siRNA complex, and HA / CSLN / siRNA, respectively.

[0090] Figure 2d shows the zeta-potential (ζ-potential) measurement results for CSLN, CSLN / siRNA complex, and HA / CSLN / siRNA, respectively.

[0091] Figure 2e shows the viability of B16F10 cells according to the concentrations of siRNA, CSLN / siRNA complex and HA / CSLN / siRNA.

[0092] Figure 3a shows the results of fluorescence confocal microscopy analysis of siRNA labeled Cy3, CSLN / siRNA complex, HA / CSLN / siRNA (Example 1), and HA pretreatment (Example 2) (scale bar = 25 μm).

[0093] Figure 3b shows the flow cytometry results of siRNA labeled Cy3, CSLN / siRNA complex, HA / CSLN / siRNA (Example 1), and HA pretreatment (Example 2).

[0094] Figure 3c shows mRNA levels analyzed by RT-PCR in B16F10 cells after treatment with the CSLN / siVEGF complex (n = 3, mean ± SD, two-tailed t-test, < 0.05; **P < 0.01; ***P < 0.001).

[0095] Figure 3d shows mRNA levels analyzed by RT-PCR in B16F10 cells after cell treatment with HA / CSLN / siVEGF (n = 3, mean ± SD, two-tailed t-test, < 0.05; **P < 0.01; ***P < 0.001).

[0096] Figure 4a shows the results of fluorescence confocal microscopy analysis of the transdermal delivery of PBS, free Cy3-siRNA, CSLN / Cy3-siRNA complex, and HA / CSLN / Cy3-siRNA, respectively, into the skin of a tumor model mouse (scale bar = 100 μm).

[0097] Figure 4b shows the results of fluorescence confocal microscopy analysis of the transdermal delivery of PBS, free Cy3-siRNA, CSLN / Cy3-siRNA complex, and HA / CSLN / Cy3-siRNA, respectively, in porcine skin (scale bar = 200 μm).

[0098] Figure 4c shows the quantification of Cy3-siRNA fluorescence in the skin of a tumor mouse model, respectively, Cy3-siRNA, CSLN / Cy3-siRNA complex, and HA / CSLN / Cy3-siRNA.

[0099] Figure 4d shows the IVIS imaging results of Cy3-siRNA, CSLN / Cy3-siRNA complex, and HA / CSLN / Cy3-siRNA, respectively, in a tumor mouse model.

[0100] Figure 4e shows the quantification of Cy3-siRNA fluorescence in tumor tissue, including Cy3-siRNA, CSLN / Cy3-siRNA complex, and HA / CSLN / Cy3-siRNA.

[0101] Figure 5a is a schematic showing the in vivo experimental design using mice with B16F10 tumors.

[0102] Figure 5b shows images of tumors isolated 48 hours after final treatment in each group of PBS, siVEGF, HA / CSLN / siLuc, CSLN / siVEGF complex and HA / CSLN / siVEGF.

[0103] Figure 5c shows the results of confirming tumor growth inhibition in mice after treatment with PBS, siVEGF, HA / CSLN / siLuc, CSLN / siVEGF complex, and HA / CSLN / siVEGF, respectively.

[0104] Figure 5d shows the rate of change in body weight of mice treated with PBS, siVEGF, HA / CSLN / siLuc, CSLN / siVEGF complex, and HA / CSLN / siVEGF, respectively.

[0105] Figure 5e shows VEGF mRNA expression in tumors analyzed 48 hours after final treatment with PBS, siVEGF, HA / CSLN / siLuc, CSLN / siVEGF complex, and HA / CSLN / siVEGF, respectively (n = 3 biologically independent animals, mean ± SD, two-tailed t-test analysis, *P < 0.05; **P < 0.01; ***P < 0.001).

[0106] Figure 6 shows H&E stained images (top) and TUNEL stained images (bottom) of tumor tissue after treatment with PBS, siRNA, CSLN / siRNA complex, and HA / CSLN / siRNA (scale bar = 100 μm).

[0107] Examples are provided to aid in understanding the present invention. The following examples are provided merely to facilitate a better understanding of the invention, and the scope of the invention is not limited by these examples.

[0108] Example 1: Preparation of Genetic Material Delivery Vehicle (HA / CSNL / siRNA)

[0109] FIG. 1c relates to a method for manufacturing a genetic material delivery vehicle according to the present invention, in which a CSLN / siRNA complex is formed through electrical interaction between a cationic CSLN and a negatively charged siRNA, and subsequently, HA is coated on the surface to form an HA / CSLN / siRNA complex.

[0110] Specifically, the genetic material carrier according to the present invention comprises a core composed of cholesteryl oleate and triglycerides, and a CSLN having a shell structure modified with cholesterol, cationic lipid DC-cholesterol (DC-Chol), lipid-PEG conjugate DSPE-PEG 2k, and fusion-inducible lipid DOPE. Here, the cationic lipid is intended to provide cationicity, and the genetic material carrier according to the present invention has an external HA coating applied to enhance stability in an aqueous solution.

[0111] The genetic material carrier of Example 1 was prepared based on Fig. 1c.

[0112] Cationic solid lipid nanoparticles (CSLNs) were synthesized using a modified emulsification and solvent evaporation method. Cholesteryl oleate (22.5 mg, 45.0% w / w), glyceryl trioleate (triolein) (1.5 mg, 3.0% w / w), cholesterol hydrochloride (4.95 mg, 9.9% w / w), dioleoylphosphatidylethanolamine (DOPE) (7.0 mg, 14% w / w), 3-beta-[N-(N',N'-dimethylaminoethane)carbamoyl]cholesterol (3b-[N-(N',N'-dimethylaminoethane)carbamoyl]cholesterol (DC-Chol)) (14.0 mg, 28% w / w), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene) A lipid suspension with a total lipid concentration of 25 mg / mL was prepared by mixing [1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000], DSPE-PEG 2k] (0.05 mg, 0.1% w / w). This suspension was prepared in a chloroform / methanol mixed solvent at a volume ratio of 2:1. Subsequently, 10 mL of distilled water was added and mixed and stirred for 2 minutes, followed by dispersion treatment using a sonicator for 5 minutes. The suspension was transferred to a round-bottom flask, and the solvent was rapidly evaporated using a rotary evaporator at 60 °C. The resulting CSLN was then purified by sufficient dialyzing in distilled water with Snakeskin 10 kDa.

[0113] To prepare genetic material delivery vehicles (HA / CSNL / siRNA), CSLN was mixed with siRNA in distilled water at various weight ratios (CSLN:siRNA = 1:3, 6, 9, 12, 15) and incubated at room temperature for 15 minutes. The formed complexes were characterized by agarose gel electrophoresis at 100V for 10 minutes in 1X Tris-Borate-EDTA Buffer and TBE buffer. After staining with SYBR™ Gold Nucleic Acid Gel Stain, gel images were captured under UV illumination. Subsequently, hyaluronic acid (HA) (molecular weight 5 kDa) was mixed with the CSLN / siRNA complexes and incubated at room temperature for 15 minutes to prepare the genetic material delivery vehicles.

[0114] Example 2: Preparation of a genetic material delivery vehicle (HA pretreatment)

[0115] A genetic material delivery vehicle was prepared in the same manner as in Example 1, except that pre-treated hyaluronic acid was used. Here, the pre-treatment of hyaluronic acid was carried out by incubating hyaluronic acid (HA) (molecular weight 5 kDa, 1 mg / mL) at 37°C for 2 hours.

[0116] Experimental Example

[0117] Experimental Example 1: Characterization of Genetic Material Carriers (HA / CSNL / siRNA)

[0118] The particle size and ζ-potential of the genetic material carrier (HA / CSLN / siRNA) of Example 1 were analyzed using a Zetasizer system (MAN383-01, Malvern Instruments Co. UK). The size and morphology of the HA / CSLN / siRNA were analyzed using a High Resolution Transmission Electron Microscope (HR-TEM) at an operating voltage of 200 kV.

[0119] Figure 2a shows the morphology of cationic solid lipid nanoparticles (CSLN) and genetic material carriers (HA / CSLN / siRNA) as viewed by TEM (scale bar = 50 μm). According to Figure 2a, the formation of spherical nanostructures was confirmed for both CSLN and HA / CSLN / siRNA, and the average nanoparticle diameter of CSLN was measured to be approximately 60 nm. After HA surface coating, the nanoparticle diameter increased to approximately 120 nm, but it was confirmed that dispersibility in water was still maintained.

[0120] The formation of the CSLN / siRNA complex was evaluated via agarose gel electrophoresis. Figure 2b shows the results of agarose gel electrophoresis at various weight ratios of the CSLN / siRNA complex (CSLN:siRNA = 1:3, 6, 9, 12, 15). According to Figure 2b, it was confirmed that the electrophoretic migration speeds of CSLN nanoparticles and siRNA gradually decreased as the mass ratio of siRNA in the CSLN / siRNA complex increased. When the mass ratio exceeded 12, the migration of siRNA almost completely stopped. This result indicates that the cationic CSLN can effectively bind to negatively charged siRNA molecules through electrical interactions. Therefore, the optimal composition ratio of the CSLN / siRNA complex was determined to be a mass ratio of 12, which was applied to subsequent experiments.

[0121] Figure 2c shows the size distribution of CSLN, the CSLN / siRNA complex, and HA / CSLN / siRNA, respectively, and Figure 2d shows the zeta-potential (ζ-potential) measurement results of CSLN, the CSLN / siRNA complex, and HA / CSLN / siRNA, respectively. According to the dynamic light scattering (DLS) analysis results (Figure 2c), the sizes of CSLN, CSLN / siRNA, and HA / CSLN / siRNA complex were measured to be 65.7 ± 4.3 nm, 79.9 ± 1.3 nm, and 125.6 ± 4.2 nm, respectively. In other words, it was confirmed that the size of the cationic solid lipid nanoparticles (CSLN) increased as the surface was coated with siRNA and HA in sequence. In addition, according to Figure 2d, the ζ potential of CSLN decreased significantly from 51.1 ± 4.1 mV to 32.7 ± 0.5 mV with the addition of siRNA, and further decreased to -25.0 ± 1.4 mV with the addition of HA. This indicates the formation of an electrical complex.

[0122] Experimental Example 2: Cytotoxicity of Genetic Material Carrier (HA / CSNL / siRNA)

[0123] Cationic nanoparticles can effectively promote cell delivery. However, these nanoparticles can damage the structure of cell membranes, causing cell necrosis, or induce uncontrolled cell death by causing non-specific damage to cells. Accordingly, the cytotoxicity of siRNA, CSLN / siRNA complexes, and HA / CSLN / siRNA at concentrations ranging from 0.0 to 100 μg / mL was investigated, and the specific methods are as follows.

[0124] Mouse melanoma cell lines (B16F10) were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin at 37°C in a 5% CO2 incubator. Cells were cultured in 1 x 10 wells of a 96-well culture dish. 4 Cells were cultured at a cell / well density. After 24 hours of incubation, cells were treated with DMEM growth medium at 37°C for 2 hours to administer various concentrations of the complex (0, 20, 40, 60, 100 μg / mL). Cell viability under each condition was evaluated via CCK-8 assay, and optical density was measured from 450 nm to 655 nm.

[0125] Figure 2e shows the viability of B16F10 cells according to the concentrations of siRNA, CSLN / siRNA complex, and HA / CSLN / siRNA. According to Figure 2e, HA / CSLN / siRNA did not exhibit cytotoxicity at concentrations up to 100 μg / mL, while CSLN / siRNA significantly reduced cell viability to about 8.5% under the same conditions. These results indicate that HA can reduce the cytotoxicity of the carrier, supporting the fact that the genetic material carrier of the present invention can be used as a biocompatible and safe carrier of siRNA.

[0126] Experimental Example 3: Intracellular delivery of genetic material carriers (HA / CSNL / siRNA)

[0127] Intracellular delivery of HA / CSLN conjugated with Cy3-siRNA (HA / CSLN / Cy3-siRNA) was investigated using confocal microscopy and flow cytometry. For fluorescence microscopy analysis, B16F10 cells were placed in 24-well plates at 2.5 x 10 4Cells were cultured at a cell / well density and incubated for 24 hours. Subsequently, the culture medium was replaced with Opti-MEM containing HA / CSLN / Cy3-siRNA (100 μg / mL), and incubated at 37°C for 2 hours. Cells were fixed in 4.0% formalin solution for 15 minutes, treated with 0.25% Triton solution for 10 minutes, and washed three times with PBS. Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI). Images were captured using an LSM 900 Airyscan 2 (Carl Zeiss, Germany). To investigate the cytoplasmic internalization mechanism of HA / CSLN / Cy3-siRNA, cells were placed in 6-well plates at a density of 0.5 x 10 6 After culturing at a cell / well density and incubating for 24 hours, an inhibitor was added to Opti-MEM containing HA and incubated for 1 hour. Then, HA / CSLN / Cy3-siRNA solution (100 μg / mL) was added to Opti-MEM and incubated for 2 hours. Cells were washed twice with PBS, treated with trypsin for 4 minutes, collected, and washed twice with PBS. Cells were fixed with 4.0% paraformaldehyde aqueous solution and analyzed using FACS CytoFlex SRT (Beckman Coulter, USA).

[0128] Figure 3a shows the results of fluorescence confocal microscopy analysis of siRNA labeled with Cy3, CSLN / siRNA complex, HA / CSLN / siRNA (Example 1), and HA pretreatment (Example 2) (scale bar = 25 μm), and Figure 3b shows the results of flow cytometry analysis of siRNA labeled with Cy3, CSLN / siRNA complex, HA / CSLN / siRNA (Example 1), and HA pretreatment (Example 2).

[0129] According to Fig. 3a, a red fluorescence image of the genetic material delivery vehicle (HA / CSLN / Cy3-siRNA) of the present invention was observed in the cytoplasm of the cell, indicating that the genetic material delivery vehicle of the present invention can effectively penetrate the cell. Compared to siRNA alone, the CSLN / siRNA complex and HA / CSLN / siRNA showed higher uptake efficiency. To evaluate the targeting ability of HA / CSLN / siRNA against tumor cells, the intracellular delivery mechanism was investigated when pretreated hyaluronic acid was used (HA pretreatment, Example 2). Since tumor cells overexpress HA receptors, receptor-mediated tumor target endocytosis was reduced when pretreated hyaluronic acid was used. Intracellular uptake efficiency was significantly reduced when pretreated hyaluronic acid was used, which highlights the important role of hyaluronic acid (HA) in intracellular delivery.

[0130] Flow cytometry results in Figure 3b confirmed that fluorescence intensity decreased when pretreated hyaluronic acid was used (HA pretreatment), which reflects a decrease in HA receptor-mediated tumor target endocytosis. Conversely, the CSLN / Cy3-siRNA complex was effectively endocytized into cells by facilitating initial electrical interactions with the cell membrane due to its nanoscale size and cationic ζ potential. Meanwhile, it was confirmed that HA / CSLN / Cy3-siRNA interacts with HA receptors, such as the CD44 receptor on skin tumor cells, and is delivered to cells more effectively than the CSLN / Cy3-siRNA complex.

[0131] Experimental Example 4: In vitro gene silencing of a genetic material carrier (HA / CSNL / siRNA)

[0132] B16F10 cells were placed in a 24-well plate at a density of 0.1 x 10 6Cells were cultured at a cell / well concentration and incubated for 24 hours. The sense / antisense sequences of Cy3-siRNA, siLuc, and siVEGF are shown in Table 1 below.

[0133] siRNA sequence notation Sence-Cy3-siRNA5'-CCUACGCCAAUUUCGU-3' (Sequence No. 1) 5' end - Cy3Antisence- Cy3-siRNA5'-ACGAAAUUGGUGGCGUAGG-3' (Sequence No. 2) Sence-siLuc5'-UUGUUUUGGAGCGAAAdTdT-3' (Sequence No. 3) Antisence- siLuc5'-UUUCCUUCCAAAACAAdTdT-3' (Sequence No. 4) Sence-siVEGF5'-AUGUGAAUGCAGACCAAAGAATT-3' (Sequence No. 5) Antisence-siVEGF5'-UUCUUUGGUCUGCAUUCACAAUTT-3' (Sequence No. 6)

[0134] After treating with CSLN / siVEGF complexes and HA / CSLN / siVEGF at various weight ratios for 2 hours, the cells were washed with PBS and replaced with growth medium to induce gene silencing for 48 hours. Following the silencing studies of CSLN / siVEGF and HA / CSLN / siVEGF, mRNA transcription level analysis was performed. Total mRNA was extracted from cells using TRI Reagent® (Molecular Research Center, Inc.), and mRNA concentration was measured using NanoDrop. TMMeasurements were taken using a One / OneC Microvolume UV-Vis Spectrophotometer (Thermo Scientific). During the reverse transcription process, complementary DNA (cDNA) was synthesized using the ReverTra Ace™ qPCR RT Master kit (Toyobo, Japan), and then real-time PCR was performed using SYBR green (Bio-Rad). The same amount of cDNA was applied to the qPCR, and the gene expression level was evaluated by comparing it with a control system. The relative expression of the target gene was calculated using the 2-△△Ct equation, where △△Ct was calculated as (Gene A - GAPDH)t - (Gene A - GAPDH)t0. GAPDH is glyceraldehyde-3-phosphate dehydrogenase.

[0135] Angiogenesis is an important characteristic of tumor cells, which secrete VEGF to promote the proliferation of vascular endothelial cells and expand the vascular system. Overexpression of VEGF promotes angiogenesis and affects vascular permeability and cell migration, making VEGF targeting a promising therapeutic strategy to block the VEGF gene using siRNA.

[0136] Based on the results of intracellular uptake tests, the effects of CSLN / siVEGF complexes and HA / CSLN / siVEGF on gene silencing efficiency were evaluated. Cells were treated with various concentrations of CSLN / siVEGF and HA / CSLN / siVEGF nanoparticles, and VEGF mRNA levels were quantified via RT-PCR. siRNA released after endocytosis binds to the RNA-induced silencing complex (RISC) and degrades target mRNA.

[0137] Figure 3c shows mRNA levels via RT-PCR analyzed in B16F10 cells after cell treatment with the CSLN / siVEGF complex (n = 3, mean ± SD, two-tailed t-test, < 0.05; **P < 0.01; ***P < 0.001), and Figure 3d shows mRNA levels via RT-PCR analyzed in B16F10 cells after cell treatment with HA / CSLN / siVEGF (n = 3, mean ± SD, two-tailed t-test, < 0.05; **P < 0.01; ***P < 0.001).

[0138] As shown in Figures 3c and 3d, as the concentrations of CSLN and HA / CSLN increased, the VEGF mRNA expression level decreased significantly, and a statistically significant difference was observed. These results indicate that HA / CSLN / siRNA nanoparticles can be effectively encapsulated in cancer cells, escape from endosomes, and release siRNA into the cytoplasm to achieve efficient gene silencing.

[0139] Experimental Example 5: In vivo and ex vivo transdermal delivery of genetic material carriers (HA / CSNL / siRNA) in tumors and porcine skin tissues

[0140] B16F10 cells (1 x 10 6 A subcutaneous melanoma mouse model was established by injecting the substance into the left side. After 7 days, PBS, free Cy3-siRNA, the CSLN / Cy3-siRNA complex, and HA / CSLN / Cy3-siRNA were each applied to the tumor skin surface for 30 minutes, followed by thorough washing with distilled water. Sections were cut to a thickness of 5 µm, and transdermal delivery of the HA / CSLN / Cy3-siRNA complex was observed using an LSM 900 Airyscan 2 at a wavelength of 561 nm. In the same manner, transdermal delivery of the four complexes was observed by applying them to the surface of porcine skin tissue.

[0141] Figure 4a shows the results of fluorescence confocal microscopy analysis of the transdermal delivery of PBS, free Cy3-siRNA, CSLN / Cy3-siRNA complex, and HA / CSLN / Cy3-siRNA, respectively, into the skin of a tumor model mouse (scale bar = 100 μm), and Figure 4b shows the results of fluorescence confocal microscopy analysis of the transdermal delivery of PBS, free Cy3-siRNA, CSLN / Cy3-siRNA complex, and HA / CSLN / Cy3-siRNA, respectively, into the skin of a pig (scale bar = 200 μm). It has been reported that hairless mice can mimic human skin better than hairy skin, and pig skin is similar to human skin in thickness and hair density.

[0142] According to Figures 4a and 4b, HA-mediated transdermal delivery was enhanced, with a strong and maximum red fluorescence signal detected only in the HA / CSLN complex group. This implies that the degree of transdermal delivery was most pronounced in this group. In contrast, only very limited fluorescence signals were observed in the other three groups. The stratum corneum, the outermost layer of the skin, serves as a major barrier for transdermal drug delivery. HA possesses amphiphilic properties, allowing it to effectively penetrate the stratum corneum. Furthermore, HA receptors are highly expressed in skin cells, particularly in keratinocytes of the epidermis and fibroblasts of the dermis, thereby enhancing diffusion efficiency.

[0143] Figure 4c shows the quantification of Cy3-siRNA fluorescence in the skin of a tumor mouse model. Specifically, the degree of transdermal delivery of nanoparticles in tumor-implanted mice was quantitatively analyzed using ImageJ software. Compared to the control group, the fluorescence intensity of the HA / CSLN complex increased by more than 73.2-fold, while there were no significant changes in the other three groups.

[0144] Figure 4d shows the IVIS imaging results of a tumor mouse model. In Figure 4d, it was confirmed that the genetic material delivery vehicle (HA / CSLN / siRNA) of the present invention remained in the skin and was delivered to the tissue even after strong washing following transdermal delivery.

[0145] Figure 4e shows the quantification of Cy3-siRNA fluorescence in tumor tissue. As shown in Figure 4e, the residual fluorescence intensity after treatment with the genetic material delivery vehicle (HA / CSLN / siRNA) of the present invention was 4.0 times and 2.7 times higher than that of free siRNA and the CSLN / siRNA complex, respectively. These results confirm that HA / CSLN / siRNA can be effectively delivered transdermally to tumor tissue in the skin.

[0146] Experimental Example 6: In vivo tumor treatment using a genetic material delivery vehicle (HA / CSNL / siRNA)

[0147] The therapeutic effect of transdermal delivery of the HA / CSLN / siVEGF complex was observed using a C57BL / 6J immunodeficient mouse model with left B16F10 tumors. Figure 5a is a schematic diagram showing the in vivo experimental design using mice with B16F10 tumors. As shown in Figure 5a, to establish the tumor model, B16F10 cancer cells were subcutaneously injected into the left flank of each mouse to form a major tumor. After the major tumor had grown for 7 days, treatment was initiated by transdermal delivery of PBS or various nanoparticle complex samples (amounts of 15 μg–30 μg applied to the skin where the tumor is located every 2 days). Treatment was terminated on day 8, and tumor growth inhibition efficiency was evaluated by measuring tumor volume every 2 days.

[0148] After establishing a subcutaneous melanoma mouse model, the tumor size was approximately 73 mm 3When [the threshold] was reached, PBS was used as a control, and free siVEGF, HA / CSLN / siLuc non-specific siRNA, CSLN / siVEGF, and HA / CSLN / siVEGF were applied to the skin surface of the tumor, respectively. Tumor size was determined by measuring the longest (a) and shortest (b) diameters using calipers, and the volume was calculated using the following formula: Tumor Volume = A × B 2 / 2. Each group was transdermally administered 100 μL of the complex solution four times at two-day intervals at a dose of 15 μg siRNA per animal. Two days after the final siRNA complex treatment, three mice from each group were sacrificed, and RNA was extracted from the removed tumors using TRI-reagent. VEGF mRNA levels were evaluated by RT-PCR and normalized to GAPDH mRNA levels.

[0149] Figure 5b shows images of tumors isolated 48 hours after final treatment in each group of PBS, siVEGF, HA / CSLN / siLuc, CSLN / siVEGF complex, and HA / CSLN / siVEGF, and Figure 5c shows the results confirming tumor growth inhibition in mice after treatment with PBS, siVEGF, HA / CSLN / siLuc, CSLN / siVEGF complex, and HA / CSLN / siVEGF, respectively.

[0150] As shown in Figure 5c, compared to the PBS group, the tumor volume was 846.4 ± 100.1%, and neither the free siVEGF nor the CSLN / siVEGF complex showed a significant tumor growth inhibitory effect; the relative tumor volumes were confirmed to be 716.5 ± 150.9% and 865.1 ± 77.6%, respectively. On the other hand, co-delivery of siVEGF via HA / CSLN / siVEGF significantly inhibited tumor growth, and the relative tumor volume at the end of treatment on day 8 was confirmed to be 475.2 ± 91.9%. In particular, HA / CSLN / siLuc containing non-specific siLuc did not show a tumor growth inhibitory effect, indicating that the anti-tumor effect is specific to siVEGF.

[0151] Figure 5d shows the rate of change in body weight of mice treated with PBS, siVEGF, HA / CSLN / siLuc, CSLN / siVEGF complex, and HA / CSLN / siVEGF, respectively. According to Figure 5d, there was no significant change in the body weight of the treated mice, which suggests that the treatment has high biocompatibility.

[0152] Figure 5e shows VEGF mRNA expression in tumors analyzed 48 hours after final treatment with PBS, siVEGF, HA / CSLN / siLuc, CSLN / siVEGF complex, and HA / CSLN / siVEGF, respectively (n = 3 biologically independent animals, mean ± SD, two-tailed t-test analysis, *P < 0.05; **P < 0.01; ***P < 0.001).

[0153] To further confirm the gene silencing effect of HA / CSLN / siVEGF nanoparticles, tumor tissues were collected 48 hours after the last sample treatment and VEGF mRNA expression levels were analyzed. As shown in Figure 5e, tumors treated with PBS exhibited VEGF mRNA expression levels similar to those treated with free siVEGF, HA / CSLN / siLuc, and CSLN / siVEGF complexes, but the VEGF mRNA expression levels in tumors treated with HA / CSLN / siVEGF decreased 2.1-fold. These results strongly demonstrate that HA improves transdermal delivery efficiency, significantly enhances siRNA delivery to target tissues containing HA receptors, and contributes to VEGF gene silencing and the resulting tumor therapeutic effect.

[0154] Experimental Example 7: Histological Analysis of Tumor Cell Apoptosis

[0155] After 8 days of in vivo treatment, mice were sacrificed for histological analysis using H&E and TUNEL staining. Tumors were fixed in 4% formalin, and paraffin sections were prepared for visualization and examination under an optical microscope. Apoptosis was detected using a TUNEL assay kit, and the analysis was performed according to the manufacturer's instructions. Fluorescence images were captured using Leica Microsystems CMS GmbH and TCapture software.

[0156] Figure 6 shows H&E stained images (top) and TUNEL stained images (bottom) of tumor tissue after treatment with PBS, siRNA, CSLN / siRNA complex, and HA / CSLN / siRNA (scale bar = 100 μm).

[0157] Histological analysis was performed using hematoxylin and eosin (H&E) staining to evaluate microvascular density (MVD) in tumor tissues treated with HA / CSLN / siVEGF. VEGF-induced angiogenesis is particularly important for tumor growth and metastasis. Therefore, the inhibition of tumor growth by HA / CSLN / siVEGF may be closely related to the inhibition of neovascularization.

[0158] As shown in the H&E stained image of Fig. 6 (top), tumor tissue treated with HA / CSLN / siVEGF was noticeably disintegrated and morphologically damaged, with a significant decrease in microvascular density. This indicates that the siVEGF complex can effectively inhibit microvascular formation.

[0159] Furthermore, DNA fragmentation during apoptosis is generally associated with changes in the ultrastructure of cell morphology, and apoptotic cells resulting from DNA degradation can be identified through TUNEL analysis. As shown in the TUNEL staining image in Figure 6 (below), the HA / CSLN / Cy3-siRNA group showed a significantly increased red fluorescence signal compared to other groups, which reflects apoptosis in cancer cells. All these results confirm that HA / CSLN / Cy3-siRNA can be effectively delivered transdermally to tissues and target tumor cell death.

[0160]

[0161] In conclusion, the genetic material delivery systems of the present invention (HA / CSLN complex, HA / CSLN / siRNA) demonstrated that they can effectively encapsulate nucleic acid drugs through electrical interactions and deliver siVEGF to treat skin tumors. These delivery systems exhibit an appropriate nanoscale distribution and minimal non-specific cytotoxicity. Here, HA plays a dual role: facilitating transdermal delivery and targeting cancer cells. The HA mechanism promotes efficient uptake by cancer cells and aids in in vitro gene silencing. Confocal imaging confirmed the effective transdermal delivery of the genetic material delivery systems of the present invention. Furthermore, after transdermal administration, HA / CSLN / siVEGF targeted skin tumor tissue, significantly lowering VEGF mRNA levels and leading to tumor growth inhibition and improvement of pathological symptoms in skin cancer model mice. Overall, HA / CSLN demonstrates great potential as a novel drug carrier for transdermal targeted cancer therapy.

[0162]

[0163] The specification omits detailed descriptions of matters that can be sufficiently recognized and inferred by those skilled in the art of the present invention, and various modifications are possible within the scope of not altering the technical concept or essential configurations of the present invention, in addition to the specific examples described in this specification. Accordingly, the present invention may be implemented in a manner different from that specifically described and exemplified in this specification, and this is a matter that can be understood by those skilled in the art.

Claims

1. Cationic solid lipid nanoparticles with a core-shell structure; and A coating layer comprising hyaluronic acid located on the above-mentioned cationic solid lipid nanoparticles; comprising The above core comprises cholesteryl ester and triglyceride, and The above shell is a genetic material carrier comprising cholesterol, fusion-inducible lipids, cationic lipids, and lipid-PEG (polyethylene glycol) conjugates.

2. In Paragraph 1, The above-mentioned genetic material carrier is a genetic material carrier that delivers genetic material through the skin.

3. In Paragraph 1, The above genetic material delivery vehicle further comprises genetic material, and A dielectric material carrier in which the dielectric material is located between the shell of the cationic solid lipid nanoparticle and the coating layer, and is bonded by electrostatic interaction with the cationic lipid of the shell.

4. In Paragraph 3, A genetic material carrier comprising one or more selected from the group consisting of small 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).

5. In Paragraph 3, A genetic material carrier in which the weight ratio (genetic material / cationic solid lipid nanoparticles) of the above-mentioned cationic solid lipid nanoparticles and the above-mentioned genetic material is 1 to 50.

6. In Paragraph 1, The above hyaluronic acid is a genetic material carrier having a molecular weight of 5 to 200 kDa.

7. In Paragraph 1, The above-mentioned cholesteryl ester is a genetic material carrier in which a saturated or unsaturated fatty acid having 10 to 24 carbon atoms is ester-bonded to cholesterol.

8. In Paragraph 1, A genetic material carrier in which the above triglyceride is triolein.

9. In Paragraph 1, The above fusion-inducing lipids are dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine (EPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylethanolamine (DSPE), and phosphatidylethanolamine A genetic material carrier comprising one or more selected from the group consisting of phosphatidylethanolamine (PE), dipalmitoylphosphatidylethanolamine, 1,2-dioleyl-sn-glycero-3-phosphoethanolamine, 1-palmitoyl-2-oleyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleyl-sn-glycero-3-[phospho-L-serine] (DOPS), and 1,2-dioleyl-sn-glycero-3-[phospho-L-serine].

10. In Paragraph 1, The above cationic lipids are 3-beta[N-(N',N'-dimethylaminoethane)carbamoyl]cholesterol (DC-cholesterol), 3-beta-[N-(N',N',N'-trimethylaminoethane)carbamoyl]cholesterol (TC-cholesterol), 3-beta[N-(N'-monomethylaminoethane)carbamoyl]cholesterol (MC-cholesterol), 3-beta[N-(aminoethane)carbamoyl]cholesterol (AC-cholesterol), N-(N'-aminoethane)carbamoylpropanoic tocopherol (AC-tocopherol), N-(N'-methylaminoethane)carbamoylpropanoic tocopherol (MC-tocopherol), N,N-dioleyl-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-Dioleyl)propyl)-N,N,N-Trimethylammonium Chloride (DOTMA), 1,2-Dioleyl-3-Dimethylammonium-Propane (DODAP), 1,2-Dioleylcarbamyl-3-Dimethylammonium-Propane (DOCDAP), 1,2-Dilineoyl-3-Dimethylammonium-Propane (Dilineoyl-3-Dimethylammonium-propane, DLINDAP), Dioleoyloxy-N-[2-spermincarboxamido)ethyl}-N,N-dimethyl-1-propaneamulanttrifluoroacetate (DOSPA), dioctadecylamidoglycyl spermine (DOGS), 1,2-dimyristriloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DMRIE), 3-dimethylamino-2-(cholest-5-en-3-beta-oxybutane-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5-en-3-beta-oxy)-3'-oxapentoxy]-3-damethyl-1-(cis,cis-9',12'-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N'-Dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 1,2-diacyl-3-trimethylammonium-propane (TAP), 1,A genetic material carrier comprising one or more selected from the group consisting of 2-diacyl-3-dimethylammonium-propane (DAP), 1,2-di-O-octadeceyl-3-trimethylammonium propane, and 1,2-dioleoyl-3-trimethylammonium propane.

11. In Paragraph 1, A genetic material delivery system comprising, based on 100 weight% of total cationic solid lipid nanoparticles, 30 to 60 weight% of the cholesteryl ester, 0.1 to 10 weight% of the triglyceride, 5 to 20 weight% of the cholesterol, 5 to 30 weight% of the fusion-inducible lipid, 10 to 50 weight% of the cationic lipid, and 0.01 to 1 weight% of the lipid-PEG conjugate.

12. In Paragraph 1, A dielectric material carrier having a content ratio between the core and shell of 30:70 to 70:30 by weight.

13. In Paragraph 1, A genetic material carrier having an average particle size of 30 to 500 nm.

14. In Paragraph 1, A genetic material carrier that exhibits a zeta potential of -30 to -5 mv.

15. Core-shell structured cationic solid lipid nanoparticles; A coating layer containing hyaluronic acid located on the above positively charged solid lipid nanoparticles; and A dielectric material located between the shell of the positively charged solid lipid nanoparticle and the coating layer; comprising The above core comprises cholesteryl ester and triglyceride, and The above shell comprises cholesterol, fusion-inducing lipids, cationic lipids, and lipid-PEG (polyethylene glycol) conjugates, and A pharmaceutical composition for the prevention or treatment of cancer, wherein the above-mentioned genetic material is bonded to the cationic lipid of the shell by electrostatic interaction.

16. In Paragraph 15, The above composition is a pharmaceutical composition that delivers the genetic material through the skin.

17. In Paragraph 15, A pharmaceutical composition wherein the above cancer is any one selected from the group consisting of skin cancer, cutaneous or intraocular melanoma, colorectal cancer, breast cancer, brain cancer, neurocancer, lung cancer, small cell lung cancer, stomach cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, head or neck cancer, uterine cancer, ovarian cancer, rectal cancer, pro-anal cancer, colon cancer, breast cancer, fallopian tube carcinoma, endometrial carcinoma, cervical cancer, vaginal cancer, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureteral cancer, renal cell carcinoma, renopelvic carcinoma, CNS tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma and pituitary adenoma.

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