Multi-layer nanoparticles for nucleic acid delivery

Abstract

The present invention relates to multi-layer nanoparticles for nucleic acid delivery, a method for preparing same, a nucleic acid delivery composition comprising same, an antibacterial composition, and a pharmaceutical composition for preventing or treating infectious diseases. The multi-layer nanoparticles for nucleic acid delivery comprise: gold nanoparticles; a polyethyleneimine modified on the surface of the gold nanoparticles; and chitosan applied on the gold nanoparticles modified with the polyethyleneimine. According to the present invention, the introduction of nucleic acids (e.g., ASOs) into target bacterial cells is maximized, and highly efficiently loaded nucleic acids can be effectively delivered into bacteria to maximize the effect of inhibiting target nucleic acids, and thus the present invention can be useful as an antibacterial agent and a gene therapeutic agent.

Description

Multilayer nanoparticles for nucleic acid delivery

[0001] The present invention relates to multilayer nanoparticles for nucleic acid delivery and their use, and more particularly, to multilayer nanoparticles for nucleic acid delivery comprising gold nanoparticles; polyethyleneimine modified on the surface of the gold nanoparticles; and chitosan coated on the gold nanoparticles modified with polyethyleneimine, a method for preparing the same, a nucleic acid delivery composition comprising the same, an antibacterial composition, and a pharmaceutical composition for preventing or treating infectious diseases.

[0002]

[0003] Gene therapy is a groundbreaking technology that can be applied in a variety of ways, including as a treatment or vaccine for rare genetic diseases, various cancers, central nervous system diseases, and infectious diseases, by specifically designing and expressing, suppressing, or regulating disease-related target genes. Gene therapy agents come in various forms, including viruses, plasmid DNA, messenger RNA (mRNA), which express specific genes, or antisense oligonucleotides (ASOs) and small interfering RNA (siRNA) that suppress or regulate them (Bulaklak, K., Gersbach, CA, Nat Commun, 2020, 11, 5820).

[0004] Gene therapy can be categorized into in vivo and ex vivo treatment methods depending on the delivery method. It is reported that the in vivo delivery market will reach 10.4512 billion dollars and the ex vivo delivery market will reach 2.5545 billion dollars. Excluding vaccines, representative gene therapy products approved by the Ministry of Food and Drug Safety to date include viral vectors such as Spark Therapeutics' Luxturna and Avexis / Novartis' Zolgensma, and non-viral treatments such as Alnylam's Oxlumo, Givlaari, and Onpattro, Novartis' Leqvio, Biogen's Spinraza, and Sarepta Therapeutics' Vyondys and Exondys.

[0005] For virus-based gene therapies, there are Ebola virus vaccines such as Merck's Ervebo® and Johnson & Johnson's Zabdeno® / Mvabea®, and COVID-19 vaccines such as Johnson & Johnson's JNJ-78436735.

[0006] Among gene therapies, viral-based delivery methods offer high in vivo delivery efficiency, but they are plagued by issues such as immune side effects and carcinogenicity due to mutations. Conversely, non-viral delivery methods are relatively safe, but their rapid in vivo degradation and low tissue and cell permeability necessitate the study of delivery systems utilizing synthetic carriers.

[0007] A variety of carriers have been developed, including polymers, lipids, cell-penetrating peptides, and dendrimers, primarily used for non-viral gene delivery, such as plasmid DNA and siRNA. Among them, the lipid-based nanoparticle (LNP) of Onpattro, a carrier approved by the Ministry of Food and Drug Safety as an siRNA therapeutic, is the carrier.

[0008] Drug and genome delivery using nanoparticles not only enhances therapeutic efficacy by increasing solubility, improving stability, enhancing permeability and bioavailability, extending half-life, targeting tissues, and minimizing side effects compared to conventional formulations, but also enables the release of therapeutic agents in response to changes in hydrogen ion concentration or enzymes in the surrounding environment.

[0009] Among the therapeutics being developed using LNP as a delivery vehicle are Translate Bio's MRT5005 for the treatment of cystic fibrosis, BioNTech's BNT111 (Lipo-MERIT) for the treatment of melanoma, and Moderna's mRNA-2416 for the treatment of solid tumors and lymphoma. Vaccine therapeutics include Moderna's VAL-506440 for the flu vaccine, Moderna's mRNA-1653 for the parainfluenza vaccine, and Moderna's mRNA-1325 for the Zika virus vaccine. Also, there are BioNTech / Pfizer's Comirnaty (BNT162b2) and Moderna's Spikevax (mRNA-1273), which were recently approved as COVID-19 vaccines.

[0010] For bacteria, there are 51 antibiotic delivery nanoparticles approved by the US Food and Drug Administration, and research is underway to treat bacterial wound infections in the skin, subcutaneous areas, or lungs using antibiotics and delivery using gold, silver, iron oxide nanoparticles, polylactate-co-glyclate (PLGA), and liposomes.

[0011] ASOs are used as a gene therapy method in bacteria due to their high specificity, low risk of affecting human gene expression, and ease of design and synthesis, allowing them to target all possible genes. Various non-viral gene vectors, including gold nanoparticles, cell-penetrating peptides (CPPs), lipid nanoparticles, and DNA nanostructures, have been utilized. While significant efficacy of ASOs conjugated to CPPs has been demonstrated in animal infection models, CPPs have the potential to activate inflammatory responses and cause pruritus.

[0012] For fungi, antibiotics such as azoles, echinocandins, polyenes, allylamines, and antimetabolites are used, and while their antibacterial action is effective, their therapeutic efficacy is limited due to high toxicity and resistance. For the treatment of tinea versicolor, the University of Minia has conducted phase 2 clinical trials using nanoemulsion gels, and for the treatment of candidiasis, Matinas BioPharma has conducted phase 2 clinical trials using cochleate lipid-crystal nanoparticles. For the treatment of onychomycosis, Celtic Pharma has conducted phase 3 clinical trials using transfersomes for drug delivery.

[0013]

[0014] Meanwhile, there is a research result that reported an antibacterial effect by modifying the surface of gold nanoparticles with aminophenol to produce a nanocarrier that mimics an antibiotic and treating it with various strains (Le Wang, et al., Nano Lett. 2022 May 11;22(9):3576-3582), but it has high cytotoxicity when introduced into mammalian cells and has side effects that can affect mammalian cell activity. In addition, there is a prior literature (Marcel Janis Beha, et al., Materials Science & Engineering C 126 (2021) 112167) that discloses a multilayer nanocarrier for ASO delivery, but it has a side effect of showing cytotoxicity to mammalian cells due to the severe toxicity of PEI itself.

[0015]

[0016] Under these technical backgrounds, the present inventors have made efforts to develop a gene delivery system that maximizes the antibacterial effect by effectively delivering genes (especially, ASO) to target bacteria without cytotoxicity to mammalian cells, and enables multiple targets and has structural stability. As a result, they have developed multilayer nanoparticles containing gold nanoparticles and polymers (branched PEI, chitosan) with high biocompatibility, and confirmed that the nanoparticles exhibit increased bacterial biofilm inhibition effect, increased nucleic acid loading efficiency, long-term storage through a stable structure, increased intracellular delivery effect, increased antibacterial effect, restored antibiotic sensitivity of strains, and showed low toxicity effect on infected individuals, thereby completing the present invention.

[0017]

[0018] The above information described in this background section is solely intended to enhance understanding of the background of the present invention and may not include information that constitutes prior art already known to a person of ordinary skill in the art to which the present invention pertains.

[0019]

[0020] Summary of the invention

[0021] An object of the present invention is to provide multi-layer nanoparticles for nucleic acid delivery that exhibit increased delivery efficiency and reduced mammalian cell toxicity.

[0022] Another object of the present invention is to provide a method for producing multilayer nanoparticles for nucleic acid delivery.

[0023] Another object of the present invention is to provide a composition for nucleic acid delivery comprising the multilayer nanoparticles.

[0024] Another object of the present invention is to provide an antibacterial composition comprising the multilayer nanoparticles.

[0025] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating infectious diseases and a method for preventing or treating infectious diseases, comprising the multilayer nanoparticles.

[0026] Another object of the present invention is to provide a use of the multilayer nanoparticles for preventing or treating infectious diseases and a use of the multilayer nanoparticles for preparing a medicament for preventing or treating infectious diseases.

[0027]

[0028] To achieve the above object, the present invention provides multi-layer nanoparticles for nucleic acid delivery, comprising: gold nanoparticles; polyethyleneimine (PEI) modified on the surface of the gold nanoparticles; and chitosan coated on the gold nanoparticles modified with polyethyleneimine.

[0029] The present invention also provides a method for producing multilayer nanoparticles for nucleic acid delivery, comprising the steps of (a) modifying the surface of gold nanoparticles with polyethyleneimine; (b) loading nucleic acids; and (c) coating chitosan to produce multilayer nanoparticles.

[0030] The present invention also provides a composition for nucleic acid delivery comprising the multilayer nanoparticles.

[0031] The present invention also provides an antibacterial composition comprising the multilayer nanoparticles.

[0032] The present invention also provides a pharmaceutical composition for preventing or treating infectious diseases comprising the multilayer nanoparticles.

[0033] The present invention also provides a method for preventing or treating an infectious disease, comprising a step of administering the multilayer nanoparticles.

[0034] The present invention also provides the use of the multilayer nanoparticles for preventing or treating infectious diseases and the use of the multilayer nanoparticles for preparing a medicament for preventing or treating infectious diseases.

[0035]

[0036] Figure 1 is a schematic diagram showing the manufacturing process of a gene (ASO) nanocarrier (MLGNP).

[0037] Figure 2 is a diagram showing the results of size analysis of gene delivery vehicles (MLGNPs) according to ASO loading concentration and modified polymer.

[0038] Figure 3 is a drawing showing the results of comparing the inhibitory effects of the existing gene delivery system and the novel gene delivery system (MLGNP) according to the present invention on MSSA (Methicillin-sensitive Staphylococcus aureus) biofilm formation over time using a microtiter plate assay and a Congo-red assay.

[0039] Figure 4 is a graph showing the results of a comparative analysis of the particle size and biofilm formation inhibition effect of gene delivery vehicles whose surfaces are modified with various positively charged polymers.

[0040] Figure 5 is a diagram showing the results of particle size and zeta potential measurements according to the process of polymer multi-coating and gene (ASO) loading on gold nanoparticles.

[0041] Figure 6 is a graph showing the results of stability analysis according to the solution of the gene delivery vehicle (MLGNP).

[0042] Figure 7 is a diagram showing the results of introduction of a gene vector (MLGNP) into various fungal species.

[0043] Figure 8 is a drawing showing the results of a comparative evaluation with a gene delivery vehicle (MLGNP) by treating cultured C. albicans with a 5'FAM fluorescently labeled gene (ASO) loaded onto various types of nanocarriers and observing them using a confocal microscope.

[0044] Figure 9 is a graph showing the results of the inhibition effect of target genes (ftsZ, mecA, and icaA) of MRSA strains by a gene delivery vector (MLGNP).

[0045] Figure 10 is a graph showing the results of recovery of antibiotic susceptibility of MRSA strains by gene delivery vector (MLGNP).

[0046] Figure 11 is a graph showing the results of measuring biomass within a biofilm by a gene delivery vehicle (MLGNP).

[0047] Figure 12 is a drawing showing the results of confocal microscopic observation of cells and extracellular polymer substances (EPS) in a biofilm by a gene delivery vehicle (MLGNP).

[0048] Figure 13 is a diagram showing the results of cytotoxicity evaluation for mammalian cells (NIH-3T3) and the results of evaluation of the degree of infiltration into mammalian cells according to treatment with a gene delivery vehicle (MLGNP).

[0049]

[0050] Detailed description of the invention and preferred embodiments

[0051] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Generally, the nomenclature used herein is well known and commonly used in the art.

[0052]

[0053] Unlike many nanocarriers previously developed for gene delivery, the present invention uses gold nanoparticles and polymers (PEI, chitosan) with high biocompatibility to produce multilayer nanoparticles, and in particular, minimizes the influx of mammalian cells and the resulting toxicity, thereby enabling use as a gene delivery agent and gene therapy agent that selectively acts on target bacteria.

[0054]

[0055] Accordingly, the present invention relates, in one aspect, to multi-layer nanoparticles for nucleic acid delivery, comprising: gold nanoparticles; polyethyleneimine (PEI) modified on the surface of the gold nanoparticles; and chitosan coated on the gold nanoparticles modified with polyethyleneimine.

[0056] In the present invention, the multilayer nanoparticle may be characterized in that gold nanoparticles and polyethyleneimine are combined in a core-shell form, a nucleic acid to be delivered is combined in the core-shell form, and chitosan is coated in a shell form on the core-shell form to which the nucleic acid is combined to form a multilayer form.

[0057] In the present invention, the polyethyleneimine may be characterized as being branched or linear polyethyleneimine, and preferably branched polyethyleneimine, but is not limited thereto.

[0058] In the present invention, the gold nanoparticles may be characterized as having a size of 1 to 100 nm, preferably 1 to 80 nm, more preferably 2 to 50 nm, even more preferably 3 to 30 nm, and most preferably 4 to 10 nm, but are not limited thereto.

[0059] In the present invention, the gold nanoparticles may be characterized by having a surface modified with a functional group. The functional groups may include, but are not limited to, N-hydroxysuccinimide (NHS), amine (-NH2), carboxyl (-COOH), thiol (-SH), hydroxyl (-OH), maleimide, ethylene dichloride (EDC), azide, alkyne, and the like. In one embodiment of the present invention, the gold nanoparticles may be characterized by being modified with an NHS ester, but are not limited thereto.

[0060] In the present invention, the molecular weight of the polyethylene imine may be 0.5 to 100 kDa, preferably 0.5 to 50 kDa, more preferably 1 to 30 kDa, and most preferably 2 to 25 kDa, but is not limited thereto.

[0061] In the present invention, the molecular weight of the chitosan may be 1 to 1000 kDa, preferably 2 to 700 kDa, more preferably 3 to 500 kDa, and most preferably 5 to 310 kDa, but is not limited thereto.

[0062] In one embodiment of the present invention, the molecular weight of the polyethyleneimine may be 25 kDa, and the molecular weight of the chitosan may be 5 kDa, but is not limited thereto.

[0063] In the present invention, the concentration ratio of the polyethyleneimine and chitosan may be 1:0.1 to 1:20, more preferably 1:1 to 1:10, more preferably 1:1 to 1:5, and most preferably 1:2, but is not limited thereto.

[0064] In one embodiment of the present invention, the concentration of polyethyleneimine may be 1 μg / ml and the concentration of chitosan may be 2 μg / ml based on 100 pmole / ml of the gold nanoparticles, but is not limited thereto.

[0065] In this specification, the term nucleic acid is used interchangeably with the term gene.

[0066] In the present invention, the nucleic acid may be delivered by being loaded onto a multilayer nanoparticle to reduce the expression of a target gene and protein or induce the expression of a desired protein both in vitro and in vivo.

[0067] In the present invention, the nucleic acid may be characterized by being at least one selected from the group consisting of siRNA, rRNA, RNA, DNA, cDNA, plasmid, aptamer, mRNA, tRNA, lncRNA, piRNA, circRNA, saRNA, antisense oligonucleotide (ASO), shRNA, miRNA, ribozyme, PNA, and DNAzyme, but is not limited thereto.

[0068] Antisense oligonucleotide (ASO) technology modulates the transfer of information from genes to proteins by altering the intermediate metabolism of mRNA via single-stranded RNA or DNA. Specifically, by selecting a sufficiently complementary and specific base sequence that hybridizes, the desired suppression of target protein expression is achieved. Because ASOs bind sequence-specifically to the target gene, they do not affect the expression of other genes. Therefore, ASO technology is not only a useful tool for analyzing the in vivo role of specific proteins, but also has potential for use as a gene therapy for specific diseases (KJ Scanlon, et al., FASEBJ. 1995 Oct;9(13):1288-96).

[0069] Antisense DNA binds to the target mRNA to form an RNA / DNA double helix, and this RNA / DNA double helix structure is attacked and degraded by RNase H (RNase H; a type of ribonuclease that specifically degrades mRNA in which RNA / DNA hybrid double helix is ​​formed) existing in the body. Antisense RNA forms an RNA / RNA double helix and is attacked by RNase L to cause degradation of the target mRNA. RNase L is a ribonuclease that preferentially degrades single-stranded RNA around the double-stranded RNA strand (ST Crooke, Annu Rev Pharmacol Toxicol. 1992:32:329-76).

[0070] In this specification, "antisense oligonucleotide (ASO)" is interpreted to include an oligonucleotide capable of hybridizing with a target nucleic acid sequence by hydrogen bonding. Antisense oligonucleotides include, but are not limited to, oligonucleotides, oligonucleotide analogs, oligonucleotide mimetics, siRNA, single-stranded siRNA (ss siRNA), short hairpin RNA (shRNA), microRNA mimics, ribozymes, external guide sequence oligonucleotides, and other oligonucleotides that hybridize with a target nucleic acid sequence to regulate its expression, and the antisense oligonucleotides are interpreted as a concept including single-stranded and double-stranded oligonucleotides.

[0071]

[0072] In one embodiment of the present invention, the surface of a 5 nm sized gold nanoparticle was modified with branched polyethyleneimine (PEI), and after loading a bacterial target nucleic acid (Anti-icaA, Anti-mecA, and Anti-ftsZ ASO), the surface was finally modified with chitosan to manufacture a multi-layer nanoparticle, and it was confirmed that the nucleic acid was stably and efficiently loaded onto the nanoparticle.

[0073] Accordingly, from another perspective, the present invention relates to a method for producing multilayer nanoparticles for nucleic acid delivery, comprising the steps of (a) reacting gold nanoparticles with polyethyleneimine (PEI) to modify the surface of gold nanoparticles with polyethyleneimine; (b) loading nucleic acids; and (c) coating chitosan to produce multilayer nanoparticles.

[0074] In the present invention, the gold nanoparticles, polyethyleneimine, chitosan, and genes are as described above, and redundant description is omitted.

[0075] In one embodiment of the present invention, step (a) may be characterized by reacting 1 μg / ml of polyethyleneimine having a molecular weight of 25 kDa with 100 pmole / ml of gold nanoparticles, and step (c) may be characterized by coating 2 μg / ml of chitosan having a molecular weight of 5 kDa with 100 pmole / ml of gold nanoparticles, but is not limited thereto.

[0076]

[0077] In one embodiment of the present invention, the manufactured multilayer nanoparticles were confirmed to maximize nucleic acid entry into target bacterial cells as nucleic acid delivery vehicles, effectively delivering the nucleic acid into the bacterial cells and maximizing the target nucleic acid inhibition effect. Furthermore, it was confirmed that entry into mammalian cells was minimized, thereby minimizing toxicity. Consequently, it was confirmed that a maximized antibacterial effect due to the nucleic acid delivered into the bacterial cells could be achieved, while minimizing entry into mammalian cells and exhibiting very low toxicity.

[0078]

[0079] Therefore, the present invention, from another aspect, relates to a composition for nucleic acid delivery comprising the multilayer nanoparticles.

[0080] In this specification, the nucleic acid delivery composition is used interchangeably with the nucleic acid delivery agent.

[0081]

[0082] In another aspect, the present invention relates to an antibacterial composition comprising the multilayer nanoparticles.

[0083] In the present invention, the term "antibacterial" means the ability to resist bacteria, and refers to any mechanism that is performed to inhibit the growth or proliferation of microorganisms such as bacteria or fungi or to defend against the action of microorganisms.

[0084] In the present invention, the multilayer nanoparticles may be characterized by having excellent antibacterial activity specifically against Gram-positive bacteria, Gram-negative bacteria, and antibiotic-resistant strains, and more specifically, antibacterial activity against fungi including Candida species (Candida albicans, Candida glabrata, Candida tropicalis, Candida krusei), Staphylococcus aureus, Staphylococcus epidermidis, Bacillus subtilis, Escherichia coli, and Pseudomonas aeruginosa, but are not limited thereto.

[0085] In the present invention, the target strain may vary depending on the type of nucleic acid that can be loaded onto the multilayer nanoparticle, so it is expected that an antibacterial effect will be exhibited against all types of bacteria, and thus the effect is not limited to the pathogenic bacteria or fungi listed above.

[0086] The antibacterial composition of the present invention can be used as a single agent, and can be manufactured and used as a composite agent by additionally including a composition known to have a recognized antibacterial effect.

[0087] According to one embodiment of the present invention, an antibacterial agent or antibacterial food composition comprising the antibacterial composition is provided.

[0088]

[0089] In another aspect, the present invention relates to a pharmaceutical composition for preventing or treating infectious diseases comprising the multilayer nanoparticles.

[0090] In another aspect, the present invention relates to a method for preventing or treating an infectious disease, comprising a step of administering the multilayer nanoparticle to a subject.

[0091] In another aspect, the present invention relates to the use of the multilayer nanoparticles for preventing or treating infectious diseases.

[0092] In another aspect, the present invention relates to the use of the multilayer nanoparticles for the manufacture of a medicament for the prevention or treatment of infectious diseases.

[0093] In the present invention, the multilayer nanoparticles have excellent antibacterial activity specifically against gram-positive bacteria, gram-negative bacteria, and antibiotic-resistant strains, and more specifically, have an antibacterial effect against pathogenic bacteria or fungi including Candida species (Candida albicans, Candida glabrata, Candida tropicalis, Candida krusei), Staphylococcus aureus, Staphylococcus epidermidis, Bacillus subtilis, Escherichia coli, and Pseudomonas aeruginosa, and the pharmaceutical composition may be characterized by having a preventive or therapeutic effect against a disease caused by the pathogenic bacteria or fungi.

[0094] In the present invention, the target strain may vary depending on the type of nucleic acid that can be loaded onto the multilayer nanoparticle, so it is expected that an antibacterial effect will be exhibited against all types of fungi. Therefore, the diseases caused by the pathogenic fungi are not limited to the diseases caused by the pathogenic bacteria or fungi listed above.

[0095] In the present invention, an infectious disease is a disease caused by infection with a pathogen, and includes, but is not limited to, bacterial diseases, viral diseases, fungal diseases, and protozoan diseases. Preferably, the infectious disease is characterized as being a fungal disease, but is not limited thereto.

[0096] In this specification, the term “prevention” means any act of inhibiting or delaying the progression of a disease caused by a pathogenic bacteria by administering the composition of the present invention, and “treatment” means inhibiting the development of a disease caused by a pathogenic bacteria, alleviating or eliminating symptoms.

[0097] The pharmaceutical composition according to the present invention may contain a pharmaceutically effective amount of the multilayer nanoparticles alone, or may contain one or more pharmaceutically acceptable carriers, excipients, or diluents. The pharmaceutically effective amount herein refers to an amount sufficient to prevent, improve, or treat the target disease.

[0098] The above “pharmaceutically acceptable” means physiologically acceptable and does not typically cause allergic reactions such as gastrointestinal upset or dizziness or similar reactions when administered to humans. Examples of the carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil. In addition, the pharmaceutical composition may further include fillers, anticoagulants, lubricants, wetting agents, fragrances, emulsifiers and preservatives.

[0099] The term "carrier" is defined as a compound that facilitates the introduction of a nucleic acid structure into cells or tissues. For example, dimethyl sulfoxide (DMSO) is a commonly used carrier that facilitates the introduction of many organic compounds into the cells or tissues of living organisms.

[0100] The term "diluent" is defined as a compound that stabilizes the biologically active form of the target compound and is diluted in water to dissolve the compound. Salts dissolved in buffer solutions are used as diluents in the field. A commonly used buffer solution is phosphate-buffered saline, as it mimics the salt content of human body fluids. Because buffer salts can control the pH of a solution at low concentrations, buffer diluents rarely alter the biological activity of a compound.

[0101] A pharmaceutical composition comprising multilayer nanoparticles according to the present invention may be administered to a patient as such or as a pharmaceutical composition mixed with other active ingredients or with suitable carriers or excipients, such as in combination therapy.

[0102] The pharmaceutical composition may further comprise, in addition to the above ingredients, lubricants, humectants, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0103] The pharmaceutical composition according to the present invention may be formulated as an injectable formulation, such as an aqueous solution, suspension, or emulsion, or as a lyophilized formulation, but is not limited thereto. Furthermore, the composition may be formulated as desired for each disease or ingredient using an appropriate method in the art or a method disclosed in Remington's Pharmaceutical Sciences.

[0104] The term "administration" of the present invention means introducing the pharmaceutical composition of the present invention to a patient by any appropriate method, and the pharmaceutical composition of the present invention can be administered orally or parenterally, and can be administered by, for example, infusion, intravenous injection, intramuscular injection, subcutaneous injection, intraperitoneal injection, intrarectal administration, topical administration, intranasal injection, etc., but is not limited thereto.

[0105] The appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as the formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity. A physician of ordinary skill can easily determine and prescribe an amount effective to achieve the intended purpose of desired treatment or prevention. More specifically, a therapeutically effective amount refers to an amount effective to prolong the survival of a subject to be treated, or to prevent, alleviate, or relieve the symptoms of a disease. Determination of a therapeutically effective amount is within the capabilities of a person of ordinary skill in the art, especially in light of the detailed disclosure provided herein.

[0106] In the present invention, “subject” means a mammal suffering from or at risk of a condition or disease that can be alleviated, suppressed or treated by administering a multilayer nanoparticle according to the present invention, and preferably means a human.

[0107] The pharmaceutical composition according to the present invention can be used in combination with conventional therapeutic agents. This means that the multilayer nanoparticles according to the present invention and the pharmaceutical compositions containing them can be administered simultaneously with, or sequentially or in reverse order with, conventional therapeutic agents such as antifungal agents. These agents can be administered in combination in an appropriate effective amount within the scope of those skilled in the art.

[0108]

[0109] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0110]

[0111] Example 1: Fabrication and property analysis of a gene delivery system loaded with a gene (ASO)

[0112] Example 1-1: Production of gene (ASO) delivery system (MLGNP)

[0113] Multilayer-coated gold nanoparticles (MLGNPs) were manufactured using 5 nm-sized gold nanoparticles as core materials and modified with multiple polymer layers to deliver an antigen-specific sequence (ASO).

[0114] First, the size is 5 nm and the surface is N-hydroxysuccinimide (NHS) ester (5 nm, ~1 NHS group / nm 2 Gold nanoparticles (100 pmole in 1 ml) functionalized with polyethylene glycol (PEG) spacer (5,000 Da) were reacted with branched polyethyleneimine (PEI) polymer having a molecular weight of 25,000 Da at a concentration of 1 μg / ml at room temperature for 1 hour, modified in a 1 mM NaCl aqueous solution, and purified several times with a 10 mM NaCl aqueous solution through centrifugation (4000 g, 10 min) using a 100 kDa amicon filter.

[0115] Next, ASOs (Anti-ftsZ, Anti-mecA, and Anti-icaA ASOs) targeting fungal target resistance genes and essential growth genes were synthesized on branched PEI-modified nanoparticles, prepared at concentrations of 0.1 μM, 1 μM, 10 μM, 50 μM, and 100 μM, loaded in a 10 mM NaCl aqueous solution at room temperature for 30 minutes, and then purified several times with a 10 mM NaCl aqueous solution through centrifugation (4000 g, 10 min) using a 100 kDa amicon filter. The sequence of the ASOs used in the present invention is as follows:

[0116]

[0117] Finally, MLGNPs were manufactured by reacting chitosan (5 kDa) in a 10 mM NaCl aqueous solution containing 0.01 M acetic acid at concentrations of 0.2 μg / ml, 1 μg / ml, 2 μg / ml, and 10 μg / ml for 1 hour at room temperature, and then purified several times with a 10 mM NaCl aqueous solution through centrifugation (4000 g, 10 min) using a 100 kDa amicon filter.

[0118] By controlling the concentration and amount of the polymer (branched PEI, Chitosan) used for modification and controlling the reaction time and temperature, etc., a gene delivery vehicle (MLGNP) with optimal properties and stability was developed (Fig. 1).

[0119]

[0120] Example 1-2: Optimization of gene (ASO) delivery system (MLGNP)

[0121] The optimal conditions selected are a formulation in which a branched polyethyleneimine (PEI) polymer with a molecular weight of 25,000 Da is modified at a concentration of 1 μg / ml based on gold nanoparticles (100 pmole in 1 ml), a gene (ASO) is loaded at a desired concentration, and finally chitosan with a molecular weight of 5 kDa is modified at a concentration of 2 μg / ml (Fig. 2).

[0122] In particular, in order to confirm the type and concentration conditions of the optimal modified polymer, the gene carrier was treated to the cultured S. aureus strain for 24 hours, and the biofilm formation ability was confirmed over time using a microtiter plate assay and a Congo-red assay. As a result of comparing the existing gene carrier (MLGNP (PEI)) whose surface was modified with PEI and the novel gene carrier (MLGNP (Chitosan)) according to the present invention whose surface was modified with Chitosan, the gene carrier according to the present invention was found to have the greatest range of inhibitory effects and also the greatest long-term inhibitory effects for up to 48 hours (Fig. 3A). In addition, when qualitatively analyzed using the Congo-red assay, it was confirmed that the novel gene carrier showed the best biofilm inhibition effect for a long period of time (Fig. 3B).

[0123] In addition, as a result of analyzing the formulation with the best efficiency by comparing and analyzing the particle size and function according to the modified polymer, it was confirmed that the formulation with the best biofilm inhibition effect was shown when the surface was modified with Chitosan with a molecular weight of 5 kDa among various positively charged polymers (Fig. 4).

[0124]

[0125] Example 1-3: Confirmation of successful production of gene (ASO) delivery system (MLGNP)

[0126] As a result of analyzing the particle size according to the multilayer coating process of the gene delivery vehicle (MLGNP) developed according to Example 1-2 using dynamic light scattering (DLS), when 50 μM of multi-target ASO was loaded, the diameters of the nanoparticles were measured to be 53.7 nm, 106.0 nm, and 143 nm, confirming that the diameters of the nanoparticles sequentially increased (Fig. 5A).

[0127] Next, the surface charge (zeta potential) according to the multilayer coating process of MLGNP was measured, and when 50 μM of multi-target ASO was loaded, the surface charge was sequentially confirmed to be +4.12 mV, -9.37 mV, and +37 mV, verifying that each coating process was successfully performed (Fig. 5B).

[0128] In addition, as a result of analyzing the loading efficiency according to ASO loading, it was confirmed that MLGNP showed a high loading efficiency of 65.6%, which was 4.3 times higher than the loading efficiency of the control group, gold nanoparticles, which was approximately 15.2% (Fig. 5C).

[0129] In particular, the particle morphology was analyzed using a scanning electron microscope (SEM), and it was confirmed that the particle size was uniformly distributed at approximately 140 nm, similar to the DLS results, confirming the successful production of multi-target ASO nanocarriers (MLGNPs) (Fig. 5D).

[0130]

[0131] Example 1-4: Confirmation of the stability of gene (ASO) delivery system (MLGNP)

[0132] To confirm the particle stability of MLGNP nanoparticles loaded with a gene (ASO), the particles were stored at 4°C for 7 days in 0.01 M acetic acid solution and PBS solution, which are the particle synthesis conditions.

[0133] When stored in a 0.01 M acetic acid solution for 7 days, the particle size increased slightly on the first day after synthesis, but the size of the nanoparticles for the next 7 days was confirmed to be maintained within the error range of approximately 110 nm when 10 μM of ASO was loaded and approximately 142 nm when 100 μM was loaded, indicating that they were stable (Fig. 6A).

[0134] When the gene delivery carrier (MLGNP) particles manufactured in a PBS solution were stored for 7 days, the particle size was maintained to some extent from immediately after manufacture to the first day, but from the second day, the particle size was confirmed to be maintained at a reduced size of 58.2 nm when 10 μM ASO was loaded and 80.9 nm when 100 μM ASO was loaded (Fig. 6B).

[0135] Therefore, it was concluded that the manufactured gene delivery vehicle (MLGNP) could be stored for a long period of time when stored in a 0.01 M acetic acid solution.

[0136]

[0137] Example 2: Analysis of the intracellular uptake efficiency of a fungal-targeted gene delivery system (MLGNP)

[0138] In order to analyze the influx efficiency of the gene delivery vehicle (MLGNP) developed in Example 1, representative strains were selected and the degree of influx was compared and analyzed.

[0139] The target genes of ASOs to be loaded onto the gene delivery vehicle (MLGNP) were ftsZ, which plays a direct role in bacterial proliferation and growth, mecA, an antibiotic resistance biomarker, and icaA, a key gene in forming bacterial biofilm EPS (extracellular polymeric substance) (Anti-ftsZ ASO (SEQ ID NO: 1), Anti-mecA ASO (SEQ ID NO: 2), and Anti-icaA ASO (SEQ ID NO: 3)).

[0140]

[0141] The MLGNPs, which were surface-modified with Chitosan having a molecular weight of 5 kDa and loaded with ASO having a FAM fluorescent label at the 5' end, were treated for 4 hours to cultured strains. The efficiency of cell entry into Candida species (Candida albicans, Candida glabrata, Candida tropicalis, Candida krusei) and various fungal species (Methicillin-sensitive Staphylococcus aureus (MSSA), Methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus epidermidis, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa) was analyzed using a confocal microscope. As a result, a significantly higher fluorescent signal was observed compared to the control group, confirming that the MLGNPs entered the bacteria with high efficiency (Fig. 7).

[0142]

[0143] Next, in order to compare and evaluate the efficacy of the developed gene delivery vehicle (MLGNP), various types of nanocarriers commonly used as gene delivery vehicles were treated to cultured strains to compare the cell uptake efficiency.

[0144] For comparison, nanocarriers were developed, including nanocarriers with silver nanoparticles as cores (MLSNP), lipid-based nanocarriers (Liposome, MC3 Lipid nanoparticle), and complexes composed of only polymers without a gold core (CS+PEI NP), and were compared and evaluated with the gene carrier (MLGNP).

[0145] When each nanocarrier loaded with ASO fluorescently labeled with 5'FAM was treated for 2 hours in cultured Candida strain (C. albicans), it was confirmed that MLGNPs were introduced into the cells with the highest efficiency compared to other nanocarriers (Fig. 8).

[0146] In the case of MLSNPs with silver nanoparticle cores, there was almost no intracellular uptake, and in the case of lipid-based nanocarriers such as Liposome and MC3 lipid nanoparticles, there were cases where intracellular uptake was partially observed, but no significant intracellular uptake was observed compared to MLGNPs. In addition, when a complex consisting only of polymers (CS+PEI NPs) was treated without a gold nanoparticle core, there was almost no intracellular uptake, similar to the control group treated with a gene (ASO) alone. Therefore, it can be confirmed that the combination of the gold nanoparticle core of MLGNPs and the polymer used plays an important role in the intracellular uptake.

[0147]

[0148] Example 3: Analysis of intrabacterial delivery effect and antibacterial effect of a fungal-targeted gene delivery vehicle (MLGNP)

[0149] Example 3-1: Analysis of target gene suppression effect

[0150] To observe the target gene suppression effect by the gene delivery vehicle (MLGNP), the MRSA (Methicillin-resistant Staphylococcus aureus) strain was cultured, and the gene delivery vehicle (MLGNP) prepared according to Example 1 was treated to the strain, and the suppression efficiency of the target gene was analyzed by RT-qPCR.

[0151] The target genes of ASO loaded on the gene delivery vehicle (MLGNP) were ftsZ, which plays a direct role in bacterial proliferation and growth, mecA, an antibiotic resistance biomarker, and icaA, a key gene in forming bacterial biofilm EPS (extracellular polymeric substance) (Anti-ftsZ ASO (SEQ ID NO: 1), Anti-mecA ASO (SEQ ID NO: 2), and Anti-icaA ASO (SEQ ID NO: 3)).

[0152] When the gene vector (MLGNP) carrying an ASO targeting ftsZ, which plays a direct role in bacterial proliferation and growth, was treated to the MRSA strain, the ftsZ expression level of the strain was inhibited with a decrease of approximately 31% (Fig. 9A). When the gene vector (MLGNP) carrying an ASO targeting mecA, an antibiotic resistance biomarker, was treated to the MRSA strain, the mecA expression level was confirmed to be inhibited with a decrease of approximately 60% (Fig. 9B). In addition, when the gene vector (MLGNP) carrying an ASO targeting icaA, a key gene in forming bacterial biofilm EPS, was treated to the strain, the icaA expression level of the MRSA strain was confirmed to be inhibited with a decrease of approximately 51% (Fig. 9C).

[0153] As a result, it was confirmed that the gene delivery vehicle (MLGNP) was effectively delivered into the bacteria and acted.

[0154]

[0155] Example 3-2: Evaluation of antibiotic susceptibility recovery

[0156] To evaluate the restoration of antibiotic susceptibility by the gene delivery vector (MLGNP), an ASO targeting mecA, a representative gene associated with methicillin resistance in MRSA, was loaded onto the gene delivery vector (MLGNP).

[0157] The degree of antibiotic susceptibility recovery was confirmed by treating cultured MRSA strains with the manufactured gene vector (MLGNP) using the WST-8 assay. The WST-8 assay is a colorimetric assay in which the WST-8 reagent reacts with dehydrogenase in living cells to form orange formazan, which exhibits absorbance at 450 nm. Therefore, it is widely used as an analytical method for quantifying living cells in a sample.

[0158] When the cultured MRSA strain was cultured in a medium containing oxacillin, a methacillin-based antibiotic, the degree of recovery of antibiotic susceptibility was evaluated using the WST-8 assay (Fig. 10).

[0159] In the case of the medium containing 0 μg / ml of oxacillin, no antibacterial effect was observed in any experimental group, and in the case of the medium containing 512 μg / ml, which is the minimal inhibition concentration (MIC) of MRSA, an antibacterial effect was observed in all experimental groups. When the strain treated with the gene vector (MLGNP) was cultured in the medium containing 32 μg / ml of oxacillin, which is 1 / 16 of the MIC, the antibiotic susceptibility of the strain treated with the gene vector (MLGNP) recovered by approximately 43.7% compared to the control group. In addition, in the case of the medium containing 64 μg / ml of oxacillin, which is 1 / 8 of the MIC, the antibiotic susceptibility of the strain treated with the gene vector recovered by 28.6% compared to the control group.

[0160] As a result, it was confirmed that the gene vector (MLGNP) effectively acts on methicillin-resistant strains, restoring the strains' antibiotic susceptibility.

[0161]

[0162] Example 3-3: Analysis of biofilm inhibition effect

[0163] To confirm the biofilm inhibition effect of the gene delivery vector (MLGNP), analytical methods such as microtiter plate assay and confocal microscopy were used. In the microtiter plate assay, the total biomass of the biofilm in the sample can be quantified by measuring the absorbance at 570 nm after staining the biofilm sample with crystal violet. The target gene of the ASO loaded on the gene delivery vector (MLGNP) was icaA, which plays a key role in the formation of bacterial biofilm EPS (extracellular polymeric substance).

[0164] When cultured MRSA strains were treated with gene vectors (MLGNPs) and the total biomass within the sample was checked after crystal violet staining, the gene vectors (MLGNPs) loaded with icaA showed a maximum reduction of 80.5%. In particular, a very significant reduction in biomass was confirmed compared to the control group, and the inhibitory effect was confirmed to last up to 72 hours (Fig. 11A).

[0165] In addition, the biofilm inhibition effect was confirmed by treating the cultured strain with various concentrations of the gene vector (MLGNP). When the vector was treated at 10 nM, 25 nM, and 50 nM, the maximum biomass reduction effect was observed to be approximately 45.4%, 75.4%, and 86.2%, respectively, confirming that the higher the concentration, the greater the biomass reduction effect (Fig. 11B).

[0166]

[0167] Next, the degree of reduction in cells and extracellular polymeric substances (EPS) within the sample was qualitatively confirmed using a confocal microscope.

[0168] The cultured MRSA strain was treated with a gene vector (MLGNP) loaded with anti-icaA ASO, and the sample was stained with DAPI (4′,6-diamidino-2-phenylindole), which binds to cellular DNA in the sample, and Sypro Ruby fluorescence, which binds to polysaccharides in extracellular polymeric substances (EPS), to visually observe the degree of reduction in cells and extracellular polymeric substances (EPS) in the sample.

[0169] In the strains treated with each control group, the cells and extracellular polymer substances (EPS) each showed strong fluorescence signals, whereas in the experimental group treated with the gene vector (MLGNP) loaded with anti-icaA ASO, the fluorescence signals of the cells and extracellular polymer substances (EPS) were significantly reduced (Fig. 12).

[0170]

[0171] Therefore, it was confirmed quantitatively and qualitatively that the developed gene delivery vehicle (MLGNP) effectively penetrated into bacteria and inhibited MRSA biofilm.

[0172]

[0173] Example 4: Evaluation of mammalian cell toxicity of gene delivery vehicles (MLGNPs)

[0174] To predict the toxicity that may occur when the newly developed gene delivery system (MLGNP) is administered in vivo, MLGNP was treated in NIH-3T3 mammalian cell line, and cell viability was measured using cell counting kit-8 (CCK-8) assay, and the degree of influx into mammalian cells was confirmed.

[0175] First, the cell viability was measured by treating a mammalian cell line (NIH-3T3) cultured with a carrier (MLGNP) loaded with a gene (ASO), and the cell viability was as low as 95.2% for up to 72 hours, confirming that MLGNP has very low toxicity to mammalian cells (Fig. 13A).

[0176] Next, since off-target effects that suppress gene expression in mammalian cells as well as bacteria may occur when the antibiotic resistance gene targeted by the ASO has homology in the genome of mammalian cells, the extent of mammalian cell entry of the developed gene delivery vehicle (MLGNP) was confirmed using confocal microscopy.

[0177] A gene (ASO) with a FAM fluorescent label at the 5' end was loaded onto MLGNPs, and the degree of cell uptake was analyzed using a confocal microscope after treating a mammalian cell line (NIH-3T3). As a result, it was confirmed that no significant cell uptake occurred even after 6 hours (Fig. 13B).

[0178] Accordingly, the developed gene delivery vehicle (MLGNP) was verified to have very low toxicity toward mammalian cells and very little off-target effect as it did not significantly enter mammalian cells.

[0179]

[0180] The present invention relates to a multilayer nanoparticle manufactured by modifying a polymer material (PEI and chitosan) with a gold nanoparticle as a core and controlling the content and ratio thereof, thereby maximizing the introduction of a gene (e.g., ASO) into a target bacterial cell and effectively delivering the loaded gene into the bacterial cell with high efficiency, thereby maximizing the target gene suppression effect, and thus can be usefully used as an antibacterial agent and gene therapy agent.

[0181]

[0182] While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0183]

[0184] Electronic file attached.

Claims

1. Multi-layer nanoparticles for nucleic acid delivery comprising gold nanoparticles; polyethyleneimine (PEI) modified on the surface of the gold nanoparticles; and chitosan coated on the gold nanoparticles modified with polyethyleneimine.

2. A multilayer nanoparticle characterized in that in the first paragraph, the gold nanoparticle and polyethyleneimine are combined in a core-shell form, a nucleic acid to be delivered is combined in the core-shell form, and chitosan is coated in a shell form on the core-shell form to which the nucleic acid is combined to form a multilayer form.

3. A multilayer nanoparticle according to claim 1, characterized in that the polyethyleneimine is branched or linear polyethyleneimine.

4. A multilayer nanoparticle according to claim 1, wherein the gold nanoparticle has a size of 1 to 100 nm.

5. In the first paragraph, the gold nanoparticle is a multilayer nanoparticle characterized in that the surface is modified with one or more functional groups selected from the group consisting of N-hydroxysuccinimide (NHS), amine (-NH2), carboxyl (-COOH), thiol (-SH), hydroxyl (-OH), maleimide, ethylene dichloride (EDC), azide, and alkyne.

6. A multilayer nanoparticle according to claim 1, characterized in that the molecular weight of the polyethyleneimine is 0.5 to 100 kDa, and the molecular weight of the chitosan is 1 to 1000 kDa.

7. A multilayer nanoparticle according to claim 6, characterized in that the concentration ratio of polyethyleneimine and chitosan is 1:0.1 to 1:

20.

8. A multilayer nanoparticle according to claim 1, characterized in that the nucleic acid is at least one selected from the group consisting of siRNA, rRNA, RNA, DNA, cDNA, plasmid, aptamer, mRNA, tRNA, lncRNA, piRNA, circRNA, saRNA, antisense oligonucleotide (ASO), shRNA, miRNA, ribozyme, PNA, and DNAzyme.

9. A method for manufacturing multilayer nanoparticles for nucleic acid delivery comprising the following steps: (a) A manufacturing step of reacting gold nanoparticles with polyethyleneimine (PEI) to modify the surface of the gold nanoparticles with polyethyleneimine; (b) a step of loading nucleic acid; and (c) A step of manufacturing multilayer nanoparticles by coating chitosan.

10. A manufacturing method according to claim 9, characterized in that the polyethyleneimine is a branched or linear polyethyleneimine.

11. A manufacturing method according to claim 9, wherein the gold nanoparticles have a size of 1 to 100 nm.

12. A manufacturing method according to claim 9, wherein the gold nanoparticles are characterized in that the surface is modified with one or more functional groups selected from the group consisting of N-hydroxysuccinimide (NHS), amine (-NH2), carboxyl (-COOH), thiol (-SH), hydroxyl (-OH), maleimide, ethylene dichloride (EDC), azide, and alkyne.

13. A manufacturing method according to claim 9, characterized in that the molecular weight of the polyethyleneimine is 0.5 to 100 kDa, and the molecular weight of the chitosan is 1 to 1000 kDa.

14. A manufacturing method according to claim 13, characterized in that the concentration ratio of polyethyleneimine and chitosan is 1:0.1 to 1:

20.

15. A manufacturing method according to claim 9, characterized in that the nucleic acid is at least one selected from the group consisting of siRNA, rRNA, RNA, DNA, cDNA, plasmid, aptamer, mRNA, tRNA, lncRNA, piRNA, circRNA, saRNA, antisense oligonucleotide (ASO), shRNA, miRNA, ribozyme, PNA, and DNAzyme.

16. A composition for nucleic acid delivery comprising a multilayer nanoparticle according to any one of claims 1 to 8.

17. An antibacterial composition comprising a multilayer nanoparticle according to any one of claims 1 to 8.

18. A pharmaceutical composition for preventing or treating an infectious disease, comprising a multilayer nanoparticle according to any one of claims 1 to 8.

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