KR101647178B1 - Stabilized Plasmid-Lipid Particles Using polyethylen glycol-lipid composed of an Enzymatically Cleavable Linker or oligo-lysines - Google Patents

Abstract

The present invention relates to a non-viral gene delivery system that utilizes "stabilized plasmid-lipid particles" (SPLPs), wherein a particular oligopeptide linker that can be cleaved by an enzyme in a particular PEG- To a gene transfer conjugate that significantly increases gene transfer efficiency and reduces cytotoxicity, and uses thereof.

Description

Stabilized Plasmid-Lipid Particles Using Polyethylene Glycol-Lipid Comprising An Enzyme Cleavable Linker or Oligo-Lysines Using a Polyethylene Glycol-Lipid Containing an Enzyme Cleavable Linker or Oligyl Lysine [

The present invention relates to a non-viral gene delivery system that utilizes "stabilized plasmid-lipid particles" (SPLPs), wherein a particular oligopeptide linker that can be cleaved by an enzyme in a particular PEG- To a gene transfer conjugate that significantly increases gene transfer efficiency and reduces cytotoxicity, and uses thereof.

Nucleic acid transporters for delivering nucleic acid materials into cells can be roughly divided into viral vectors and non-viral vectors.

As the non-viral vector, various formulations such as liposomes, cationic polymers, micelles, emulsions, nanoparticles and the like can be used. Cationic lipids in these formulations are the key material in nucleic acid delivery design because they provide the ability to electrostatically bind to negative nucleic acid material [lipofection]. Cationic lipids form complex particles by stable ionic bond with anionic nucleic acid material, and the complex thus formed is transported into cells by cell membrane fusion or endocytosis.

The cationic lipids conventionally developed include amines such as primary amines, secondary amines, tertiary amines, or quaternary ammonium salts in the neutral fatty acid chain A method of bonding the compounds to impart cationic properties was used.

These cationic lipids include 1,2-bis (2,2-bis (diphenylphosphino) ethyl) propionate, including N- [1- Oleyloxy) 3-3- (trimethylammonium) propane (DOTAP), 1,2-bis (dimyristoyloxy) 3-3- (trimethylammonium) propane (DMTAP), 1,2-dimyristyloxy Propyl-3-dimethylhydroxyethylammonium bromide (DMRIE).

It has been reported that the lipids in the stomach are relatively high in cytotoxicity. To overcome this cytotoxicity, lipids have been synthesized using amino acid linkers instead of non-amino acid linkers. Quay and colleagues describe cationic and neutral anionic lipids synthesized using several amino acids in US2008 / 0317839 A1. In addition, Korean Patent No. 10-0807060 reports that anionic amino acids are bonded to amine groups of fatty acid amine derivatives to synthesize cationic lipids, thereby enhancing the transport of nucleic acid drugs into cells. In addition, Korean Patent Registration No. 10-0909786 discloses a cationic lipid in which fatty acid amines are bonded to amino acid sites where three to six lysines are bonded to improve oligonucleic acid delivery efficiency.

Recently, it has been reported that cationic lipids prepared by bonding carboxyl groups of fatty acid amines with amino acids have cytotoxic properties unlike expected. Especially, most of the produced lipid lipids are transported to cells by oligonucleic acid It has been reported that the delivery efficiency of the target substance is very low and thus has no practical value. This is because it is difficult to obtain the intracellular delivery efficiency by merely composing the lipid carrier with only the combination of the amino acid and the fatty acid amine, and the delivery efficiency is determined according to the specific structure. Therefore, (Akin Akinc et al., A combinatorial library of lipid-like materials for delivery of RNAi therapeutics, Nature Biotechnology, 2008, vol. 26, No. 5, pp 561-569).

In the technical field of the present invention, as described above, the cationic lipid liposome should be prepared considering the transfer efficiency of the substance to be delivered and various metabolism in the cells. For this purpose, various types of cationic lipid liposomes And to develop a modified liposome that can improve the physical, chemical and physiological properties of cationic lipid liposomes.

That is, the conventional method and structure of the cationic lipid have structural limitations in the modification of the compound for improving the physical, chemical and physiological characteristics, and thus various modifications can be made to improve the intracellular toxicity and the intracellular delivery efficiency. It is necessary to develop a cationic lipid and a new structure of the cationic lipid obtained thereby.

The present inventors have found that by binding a specific oligopeptide linker that can be enzymatically cleaved in the PEG-lipid structure of the stabilized plasmid-lipid particle (SPLP), it is possible to increase the transfection efficiency by promoting the endosome escape Confirming the function of the enhanced SPLP to reduce cytotoxicity.

1. M. E. Davis, Non-viral gene delivery systems. Current Opinion in Biotechnology 13, 128 (Apr, 2002). 2. D. Fischer, T. Bieber, Y. Li, H.-P. Elsasser, T. Kissel, A Novel Non-Viral Vector for DNA Delivery Based on Low Molecular Weight, Branched Polyethylenimine: Effect of Molecular Weight on Transfection Efficiency and Cytotoxicity. Pharm Res 16, 1273 (1999/08/01, 1999). F. Liu, L. Huang, Development of non-viral vectors for systemic gene delivery. J Control Release 78, 259 (1/17/2002).

The main object of the present invention is to provide stabilized plasmid-lipid particles (SPLP) with improved transfection efficiency and reduced cytotoxicity and a method for producing the same.

It is another object of the present invention to provide a method for transfecting a gene into cells with high efficiency using the stabilized plasmid-lipid particles (SPLP).

In order to solve the above-mentioned object,

Cationic lipids; Fusogenic non-cationic lipids; And a PEG-lipid conjugate as a bilayer stabilizing component (BSC). The stabilized plasmid-lipid particle (SPLP)

In particular, the bilayer stabilizing component (BSC) is characterized by being a PEG-lipid conjugate conjugated with a glycine-leucine-phenylalanine-glycine (GLFG) -lysine (K) linker or an oligolysin K7 linker.

In this case, the glycine-leucine-phenylalanine-glycine (GLFG) linker is cleaved by Cathepsin-B enzyme, whereby endosome escape after intracellular internalization, . In addition, the oligo lysine K7 linker functions to increase the gene-containing efficiency when the nanoparticles are formed.

The cationic lipid may be selected from the group consisting of N, N-dioleyl-N, N-dimethylammonium chloride (DODAC), N, N-distearyl-N, N-dimethylammonium bromide (DDAB) N, N-trimethylammonium chloride (DOTAP), N- (1- (2,3-dioleyloxy) propyl) -N, N, N-trimethylammonium chloride DOTMA) and N, N-dimethyl-2,3-dioleyloxy) propylamine (DODMA) and mixtures thereof, preferably N- (1- Yl) propyl) -N, N, N-trimethylammonium chloride (DOTAP).

The fusogenic non-cationic lipid is selected from the group consisting of lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, cephalin, cardiolipin, (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioloylphosphatidylglycerol (DOPG), palmitoyl oleoylphosphatidylglycerol (POPG), dicyclohexylphosphatidylglycerol (DOPE), palmitoyl oleoyl phosphatidylcholine (POPC), palmitoyl oleoyl-phosphatidylethanolamine (POPE), and diolauryl-phosphatidylethanolamine 4- (N-maleimidomethyl) -cyclohexane-1-carboxylate (DOPE-mal), preferably dioleoyl- Phosphatidylethanolamine (DOPE).

In addition, it is preferred that the PEG has an average molecular weight of 750 to 7,000 daltons, and the terminal hydroxyl group of the PEG is preferably substituted with methoxy.

In particular, the PEG-lipid conjugate is selected from the group consisting of PEG-dilauroyl glycerol (C ₁₂ ), PEG-dimyristoyl glycerol (C ₁₄ ), PEG-dipalmitoyl glycerol (C ₁₆ ), and PEG- PEG-diacylglycerol (DAG) selected from the group consisting of monoglycerol ( _C18 ) and combinations thereof; PEG-phospholipids; PEG-dialkyloxypropyl (DAA); PEG-ceramide; And PEG-distearoyl glycerol (C ₁₈ ). In the present invention, therefore, the PEG-lipid conjugate is preferably a PEG-K7- (C ₁₆ ) ₂ or Characterized by having a PEG-GLFG-K- structure of (C _{16) 2.}

Particularly, the SPLP of the present invention is characterized in that the loading efficiency is excellent when the DOPE: DOTPA: PEG-conjugate is contained at a ratio of 80:10:10, and the diameter is 200 to 230 nm.

In one embodiment of the present invention, 1,2-dioleoyloxy-3-trimethylammonium propane (DOTAP), 1,2-dioleyl-sn-glycero-3-phosphoethanolamine (DOPE), and mPEG ₅₀₀₀ -K7- (C _{16) 2} stabilized nucleic acid transfer plasmid, characterized in that consisting of - providing a lipid particle (SPLP _1), or represented by the following formula (2) in another exemplary embodiment, (DOTAP), 1,2-dioleyl-sn-glycero-3-phosphoethanolamine (DOPE), and mPEG ₅₀₀₀ -GLFG-K- (C provides lipid particles (SPLP) - the plasmid for stabilizing nucleic acid delivery according to claim ₁₆ consisting of a) _2,

≪ Formula 1 >

(2)

In another aspect, the present invention provides a complex for intracellular nucleic acid delivery formed by binding the stabilized plasmid-lipid particle (SPLP) described above and a nucleic acid, and a method for delivering the nucleic acid into a cell using the same. In this case, it is particularly preferable that the nucleic acid is pDNA (plasmid DNA). The detailed explanation is as described above.

Thus, the present invention encompasses both stabilized plasmid-lipid particles (SPLPs) and their uses as intracellular nucleic acid delivery vehicles with high transfection efficiency, low cytotoxicity.

According to the present invention, stabilized plasmid-lipid particles having enhanced intracellular or in vivo delivery efficiency of a multi-anionic target compound such as a drug, an anti-cancer agent, or nucleic acid and having no intracellular toxicity and increased stability, , SPLP), so that it can be used as an effective nonviral gene delivery system.

1 is a schematic diagram of SPLP (stabilized plasmid-lipid particle) which is an embodiment of the present invention.
Figure 2 is a schematic diagram for the synthesis of PEG ₅₀₀₀ -GFLG-KC ₁₆ .
Figure 3 is a 1 H nuclear magnetic resonance spectrum of PEG ₅₀₀₀ -GFLG-KC ₁₆ .
FIG. 4 is a MALDI-TOF (matrix-assisted laser desorption ionization time-of-flight) mass spectrum of PEG ₅₀₀₀ -GFLG-KC ₁₆ .
Figure 5 shows the UV absorbance at 260 nm as a result of DEAE-Sepharose CL-6B ion-exchange chromatography.
Figure 6 shows the uniformly distributed diameter size measurement results of the SPLP of the present invention.
Figure 7 shows the results of DLS (dynamic light scattering) experiments on the enzyme-reactive SPLP size.
Figure 8 shows cytotoxicity results of SPLP in HEK293 cells [PEI 25 kDa (●); SPLP containing PEG _5000- GLFG-K (C ₁₆ ) ₂ encapsulated with pDNA ({circle over ( ₂ )}); Only PEG ₅₀₀₀ -GLFG-K (C ₁₆ ) ₂ lipid only ()).
Figure 9 shows the results of transfection efficiency measurement using luciferase assay in HEK293 cells.
10 is a schematic diagram of a general detergent dialysis method used in the manufacture of SPLP.

The terms used in the present invention are defined as follows.

"Nucleic acid" is meant to include any DNA or RNA, PNA, chromosome, mitochondria, virus and / or bacterial nucleic acid present in a tissue sample, for example. Includes one or both strands of a double-stranded nucleic acid molecule and includes any fragment or portion of the intact nucleic acid molecule.

"Gene" means any nucleic acid sequence or portion thereof that has a functional role at the time of protein coding or transcription, or in the control of other gene expression. The gene may consist of only a portion of the nucleic acid encoding or expressing any nucleic acid or protein that encodes the functional protein. The nucleic acid sequence may comprise an exon, an intron, an initiation or termination region, a promoter sequence, another regulatory sequence, or a gene abnormality within a particular sequence adjacent to the gene.

What intracellular delivery is meant is transfection of cells with natural or modified polynucleotides or plasmids that confer therapeutic activity (gene transfer), or introduce drugs or immunogenic peptides into cells.

"Transfection or transfection" refers to a method of expressing a genetic trait in a cell by directly introducing nucleic acid (DNA, PNA, etc.) into a cultured animal cell. The introduced nucleic acid is generally introduced by introducing the desired gene into a medium such as a plasmid. When the introduced gene was stabilized in the cells, it was often interrupted by the chromosome. Cells into which the nucleic acid has been introduced are called transgenic plants. Since transfection efficiency is very low, several methods have been developed to increase efficiency. Among them, calcium phosphate coprecipitation method, DEAE-text method. Electroporation, and redistribution (a method of fusing a membrane called a liposome with a cell that produces a DNA complex).

"Lipid" refers to a group of organic compounds that are characterized by being soluble in water but insoluble in many organic solvents, including but not limited to esters of fatty acids.

"Cationic lipid" is an amphipathic molecule having a lipophilic region generally comprising one or more hydrocarbon groups and a hydrophilic region comprising at least one positively charged polar head group. Cationic lipids are useful because macromolecules such as cationic lipids and nucleic acids form a positively charged complex as a total charge, and macromolecules such as nucleic acids are likely to enter the cytoplasm through the plasma membrane of the cell. This process, which can be done in vitro and in vivo, is known as transfection.

"Encapsulation" is used for the arrangement of a substance, particularly a hydrophilic substance, in the aqueous core of the liposome or between two adjacent shells of the liposome. The amount of entrapped material in liposomes is determined by centrifugation as described in "Liposomes, A Practical Approach, edited by P. Torchilin and VolkmarWeissig, Oxford University Press (2002) Dialysis as described in " RRC New, IRL Press (1990) ", or methods such as those described in the examples of the present invention.

"Lipid encapsulation" may refer to a lipid formulation that provides full encapsulated, partially encapsulated, or both compounds. In a preferred embodiment, the nucleic acid is fully encapsulated during lipid formulation.

"Linker bond" means a bond between a hydrocarbon chain and the remainder of the molecule. This bond determines the chemical stability and biodegradability of the cationic lipids.

"Gene therapy" refers to treating a genetic disease by correcting the mutated gene. In other words, it is a method of treating a disease by transplanting a normal gene from the outside into a patient's cell and changing the phenotype of the cell. Currently, gene therapy for sperm, oocyte, and embryo in humans has raised ethical issues around the world. However, after the "Guidelines for Clinical Research in Gene Therapy" was designated in the 1993s, the age of gene therapy began. In gene therapy, research on gene vectors and systems that transduce genes into the body is essential.

"Systemic delivery" refers to delivery which results in a broad-spectrum biodistribution of compounds within an organism. Some administration techniques may induce systemic delivery to certain compounds and others may not. Systemic delivery means that a useful, preferably therapeutic, amount of a compound is exposed to the majority of the body. Generally, in order to obtain a global bio-distribution, it is desirable that the compound not be rapidly degraded or removed prior to reaching the diseased site far from the site of administration (e.g., by first passage organ (liver, lung, etc.) ) Is required. The systemic delivery of nucleic acid-lipid particles may be by any means known in the art, including intravenous, subcutaneous, intraperitoneal, and in a preferred embodiment, systemic delivery of nucleic acid- Delivery.

"Local delivery" refers to direct delivery of a compound to a target site within an organism. For example, a compound can be delivered locally by direct injection into a diseased site, such as a tumor or other target site, such as an inflamed site or target organ such as liver, heart, pancreas, kidney,

"About" means that the reference quantity, level, value, number, frequency, percentage, dimension, size, quantity, weight or length is 30, 25, 20, 25, 10, 9, 8, 7, , Level, value, number, frequency, percent, dimension, size, quantity, weight, or length that varies from one to three, two, or one percent.

Throughout this specification, the words " comprising "and" comprising ", unless the context requires otherwise, include the stated step or element, or group of steps or elements, but not to any other step or element, And that they are not excluded.

Hereinafter, the present invention will be described in detail.

The present invention relates to a non-viral gene delivery system that utilizes "stabilized plasmid-lipid particles" (SPLP), which increases the efficiency of gene delivery by binding specific linkers that can be enzymatically cleaved And reduces the cytotoxicity, and a use thereof.

SPLP - PEG  Use

The present invention relates to a system for encapsulating nucleic acid and delivering the gene using "stabilized plasmid-lipid particles" (SPLP).

Stabilized plasmid-lipid particles (SPLP) are small (about 70 nm diameter) particles (Wheeler, et al., Gene Therapy 6: 271 (1999)) consisting of a single plasmid encapsulated in bilayer lipid vesicles, Cationic lipids; Fusogenic non-cationic lipids; And a bilayer stabilizing component (BSC), such as conjugated lipids that inhibit the aggregation of SPLP.

A variety of suitable cationic lipids can be used alone or in combination with one or more other cationic lipid species or neutral lipid species in the SPLPs described herein. (DODAC), N, N-distearyl-N, N-dimethylammonium bromide (DDAB), N- (1- (2,3- N, N-trimethylammonium chloride (DOTMA), N- (1- (2,3-diolyloxy) propyl) And N, N-dimethyl-2,3-dioleyloxy) propylamine (DODMA), and mixtures thereof. Most preferred in the present invention is N- (1- (2,3-dioloyloxy) propyl) -N, N, N-trimethylammonium chloride (DOTAP).

The cationic lipids comprise from about 2% to about 60%, preferably from about 5% to about 45%, of the total lipid present in the particles. Depending on the intended use of the nucleic acid-lipid particles, the proportions of the components may vary. For example, in the case of systemic delivery, the cationic lipid may comprise from about 5% to about 15% of the total lipid present in the particle, and in the case of local or local delivery, the cationic lipid may comprise the total Can account for from about 40% to about 50% of lipids

In addition, the non-cationic lipid component may be any of a variety of neutral non-degradable, zwitterionic, or anionic lipids capable of producing a stable complex as a fusogenic lipid.

"Fusogenic" refers to the ability of a liposome, SPLP or other drug delivery system to fuse with a membrane of a cell. Membranes may be membranes surrounding plasma membranes or organelles, such as endosomes, nuclei, and the like.

Examples of non-cationic lipids useful in the present invention include phospholipid-related substances such as lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioloylphosphatidylglycerol (DOPG), palmitoyl oleate (DOPC), phosphatidylserine Phosphatidylethanolamine (POPE), palmitoyl oleoylphosphatidylcholine (POPC), palmitoyl oleoyl-phosphatidylethanolamine (POPE) and diol leuyl-phosphatidylglycerol (POPP), dipalmitoyl phosphatidylglycerol Phosphatidylethanolamine 4- (N-maleimidomethyl) -cyclohexane-1-carboxylate (DOPE-mal). Most preferably, diolauryl-phosphatidylethanolamine (DOPE) is used.

The non-cationic lipids may comprise from about 5% to about 90%, preferably from about 20% to about 85%, of the total lipid present in the particles.

Stabilized plasmid-lipid particles (SPLP) also include a bilayer stabilizing component (BSC). This provides a hydrophilic coating that stabilizes the structure of the particle by interfering with rapid systemic clearance and protects the positive charge of the surface.

Suitable bi-layer stabilizing components (BSC) include, but are not limited to, polyamide oligomers, peptides, proteins, detergents, lipid-derivatives, PEG-lipids such as PEG (PEG-DAA) coupled to dialkyloxypropyl, diacylglycerol PEG (PEG-PE) coupled to PEG (PEG-DAG), phosphatidylethanolamine (PE) or PEG conjugated to ceramides, or mixtures thereof. Preferably in the present invention, the bi-layer stabilizing component is a PEG-lipid, and most preferably a PEG-lipid conjugate.

"PEG" refers to polyethylene glycol, which refers to a linear water soluble polymer of ethylene PEG repeat units having two terminal hydroxyl groups. PEGs are classified by their molecular weight, and in the present invention, PEG is polyethylene glycol having an average molecular weight of about 550 to about 10,000 daltons, preferably about 750 to about 7,000 daltons. In one embodiment of the present invention, PEG having a molecular weight of 5000 was used. The PEG may be optionally substituted with alkyl, alkoxy, acyl or aryl. In a preferred embodiment, the terminal hydroxyl group is substituted with a methoxy or methyl group. PEG can be conjugated directly to lipids or linked to lipids via linker residues. Preferably, the PEG is substituted with methyl at the terminal hydroxyl position.

The PEG-lipid conjugate can be one or more of PEG-dialkyloxypropyl (DAA), PEG-diacylglycerol (DAG), PEG-phospholipids, PEG-ceramides and combinations thereof. The PEG-DAG conjugate is a mixture of PEG-diureoyl glycerol (C ₁₂ ), PEG-dimyristoyl glycerol (C ₁₄ ), PEG-dipalmitoyl glycerol (C ₁₆ ), and PEG-distearoyl glycerol C ₁₈ ), and combinations thereof. Preferably PEG-dipalmitoyl glycerol (C ₁₆ ).

By controlling the composition and concentration of the bilayer stabilizing component, the rate at which the bilayer stabilizing component is exchanged from the liposome, and thus the rate at which the liposome is fused, can be controlled. By changing the molecular weight of PEG (polyethylene glycol) or by changing the chain length and the degree of saturation of the acyl chain group on the phosphatidyl ethanolamine or the ceramide. It can also be used to change and / or control the rate at which the liposomes are fused, for example, using other parameters, including pH, temperature, ionic strength, and the like.

Linker

In particular, the present invention is characterized by binding a specific oligopeptide linker to a PEG-lipid or PEG-lipid conjugate of the components of the stabilized plasmid-lipid particle (SPLP).

Oligonucleotide used in the present invention the peptide linker "glycine-leucine-phenylalanine-glycine-lysine (GLFG-K)" peptide or oligonucleotide consists of a lysine (K7): PEG-GLFG- K- (C 16) 2. or PEG -K7- (C _{16) 2.}

In particular, the oligopeptide linker GLFG is cleaved by the Cathepsin-B enzyme, thereby promoting endosome escape after intracellular internalization.

Generally, a gene is introduced into a cell through the formation of an endosome (endosome), and in order to express and treat an effective gene, a gene in the endosome escapes the endosome and moves to the nucleus or performs its function in the cytoplasm Should be able to do. At this time, fusogenic groups or ligands which cause receptor mediated endocytosis can be used to help endosomal escape in order to obtain a high expression rate.

In the present invention, the above-mentioned specific peptide is used for endosomal escape to release the encapsulated nucleic acid by increasing the osmotic pressure inside the endosome or destabilizing the membrane of the endosome. Particularly, in the present invention, this object is achieved by using the peptide linker "glycine-leucine-phenylalanine-glycine (GLFG)" which is cleaved by a specific enzyme cathepsin B. Therefore, the present invention provides, as another embodiment, a PEG-GLFG-K conjugate to which the oligopeptide linker GLFG is conjugated.

As another oligopeptide linker of the present invention, oligo lysine may also be used. This oligo lysine enhances gene-containing efficiency in nanoparticle formation. Therefore, the present invention provides a PEG-K7 conjugate in which the oligopeptide linker K7 (Lys7) is coupled as another embodiment.

It is contemplated that the PEG-GLFG conjugates or PEG-K7 conjugates of the present invention may be applied to drug delivery systems such as liposomes, micelles, virosomes, etc., as well as other drug delivery systems, .

Thus, in one aspect, the present invention provides stabilized plasmid-lipid particles (SPLP) characterized by modifying the structure of PEF-lipid conjugation with an enzymatically cleavable peptide linker.

SPLPs of the present invention include cationic lipids; Fusogenic non-cationic lipids; And bilayer stabilizing component (BSC) as GLFG-K K7 linker or linker-bonded PEG- lipid conjugate [PEG-GLFG-K- (C 16) 2 or And a _{PEG-K7- (C 16) 2} ].

In one most preferred embodiment, the cationic lipid is N- (1- (2,3-diolooyloxy) propyl) -N, N, N-trimethylammonium chloride (DOTAP), a fusible non-cationic lipid diol Leo yl-phosphatidylethanolamine (DOPE) and a suitable bilayer stabilizing component as GLFG-K K7 linker or linker is bonded PEG- lipid conjugate [PEG-GLFG-K- (C 16) 2 or And a _{PEG-K7- (C 16) 2} ].

In one embodiment of the invention the 1,2-diol Leo yloxy-3-trimethylammonium propane (DOTAP), 1,2- dioleyl to -sn- glycero-3-phospho-ethanolamine (DOPE) and PEG ₅₀₀₀ - K7- (C _{16) 2} or _{PEG 5000 -GLFG-K- (C 16} ) 2 was used. The respective "DOTAP-DOPE-PEG-K7- (C 16) 2" thereof in the specification, [Chemical Formula _{1: SPLP 1] "DOTAP-} DOPE-PEG-GLFG-K- (C 16) 2" [ Chemical Formula _2: SPLP 2 ].

≪ Formula 1 >

(2)

Manufacturing method

On the other hand, the stabilized plasmid-lipid particles (SPLP) of the present invention can be prepared using any method known in the art including a detergent dialysis method. A general schematic diagram of the detergent dialysis method is shown in Fig.

Without wishing to be bound by any particular formation mechanism, the plasmid or nucleic acid is contacted with a detergent solution of cationic lipid to form a coated nucleic acid complex. These coated nucleic acids can aggregate and precipitate. However, the presence of a detergent reduces this aggregation and causes the coated nucleic acid to react with an excess of lipid (typically, non-cationic lipid) such that the plasmid or other nucleic acid forms encapsulated particles in the lipid bilayer.

The present invention also encompasses lipid-nucleic acid particles produced through hydrophobic nucleic acid-lipid intermediate complexes prepared in the process. The complex is preferably charge neutralized. Operating these complexes in a detergent substrate or organic solvent based system may cause particle formation where the nucleic acid is protected.

The necessary processes may be appropriately modified and carried out by those skilled in the art.

The particles formed in the present invention are preferably neutral or negative at physiological pH. In vivo applications, neutral particles are advantageous, and negatively charged particles are more preferred when applied in vitro. This provides the additional advantage that the coagulation is reduced compared to a positive-charged liposomal formulation in which the nucleic acid can be encapsulated in a cationic lipid.

Usage

The present invention also relates to the use of a vector for delivering a nucleic acid (gene) into a cell of a stabilized plasmid-lipid particle (SPLP) of the present invention produced through such a process from another viewpoint. Specific embodiments include stabilized plasmid-lipid particles (SPLPs) comprising the gene of interest and their use.

The positively charged SPLP of the present invention forms a complex (polyplex) with various nucleic acids including negatively charged DNA to make it totally neutral or cationic to facilitate penetration into cells. Therefore, the invention in another aspect is DOTAP-DOPE-PEG-K7- ( C 16) 2 or DOTAP-DOPE-PEG-GLFG- K- (C 16) relates to a composite for transfer, cellular nucleic acid is combined with the target nucleic acid to the _second SPLP.

Suitable nucleic acids include, but are not limited to, plasmids, antisense oligonucleotides, ribozymes, as well as other polynucleotides and oligonucleotides. In a preferred embodiment, the nucleic acid encodes a product of interest, for example a therapeutic product. Suitable classes of gene products include, but are not limited to, cytotoxic / suicide genes, immunomodulators, cell receptor ligands, tumor suppressors, and anti-angiogenic genes. The particular gene selected depends on the intended purpose or treatment.

As described above, in some embodiments of the present invention, the SPLP is a nucleic acid encoding a therapeutic product, such as a tumor suppressor gene, an immunomodulator gene, a cell receptor ligand gene, an anti-angiogenic gene, and a cytotoxic / Lt; / RTI >

The SPLPs of the present invention may be administered alone or in admixture with physiologically acceptable carriers (e. G., Physiological saline or phosphate buffer) selected according to the route of administration and standard pharmaceutical practice. In general, normal saline is used as a pharmaceutically acceptable carrier. Other suitable carriers include, for example, water, buffered water, 0.4% saline, 0.3% glycine and the like, including stability enhancing glycoproteins such as albumin, lipoprotein, globulin and the like.

In general, the pharmaceutical carrier is added after particle formation. Thus, after the particles are formed, the particles may be diluted with a pharmaceutically acceptable carrier, for example, normal saline. The concentration of the particles in the pharmaceutical formulation may vary widely, i.e., less than about 0.05% by weight, usually from about 2% to about 5% by weight and from 10% to 30% by weight and is selected primarily by fluid volume, do.

SPLPs can be incorporated in a wide variety of topical dosage forms including, but not limited to, gels, oils, emulsions, and the like. For example, suspensions containing nucleic acid-lipid particles can be formulated and administered as topical creams, pastes, ointments, gels, lotions, and the like.

In addition, the stabilized plasmid-lipid particles (SPLPs) of the present invention are useful for introducing nucleic acid into cells after formation.

Thus, the present invention also provides a method for introducing a nucleic acid (e.g., a plasmid) into a cell. The method may be performed in vitro or in vivo by first forming the particles as described above, and then contacting the particles with the cells for a time sufficient to cause the particles to undergo transfection

The SPLPs of the present invention can be adsorbed to almost any cell type that is mixed or contacted. After the particles are adsorbed, they can be intracellularly transferred by a part of the cell, exchanged for lipids with the cell membrane, or fused with cells. Transformation or incorporation of the nucleic acid portion of the particle can be accomplished through any of these pathways. In particular, when fusion occurs, the particle film is incorporated into the cell membrane, and the content of the particles is combined with the intracellular fluid.

Thus, the present invention provides _two high transfection efficiency and lower cytotoxicity for cells having a nucleic acid delivery system as in DOTAP-DOPE-PEG-K7- ( C 16) 2 or DOTAP-DOPE-PEG-GLFG- K- (C 16) Structure SPLPs and methods of using the same, and can be used as an effective non-viral gene delivery system.

<Examples>

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Experimental Preparation and Experimental Methods

1. Materials

DOPE (1,2-dioleoyl-sn-glycero-3-phoshphoethanolamine) and DOTAP (1,2-dioleoyl-3-trimethylammonium-propane) were purchased from Avanti polar lipids (Alabaster, AL, USA) propyl-ω-methoxy, polyoxyehylene, MW5000) were purchased from Sunbright (Ebisu, shinbuya-ku, Tokyo). Fmoc-L-leucine, Fmoc-Gly-OH, N-α-Fmoc-L-leucine and Fmoc- -Hydroxybenzotriazole (HOBt) was obtained from Anaspec (Fremont, CA, USA). L-lysine (Fmoc-Lys (Fmoc) -OH), 2- (lH-benzotriazole-l-yl) -1,1,3,3-tetra methyluronium (HBTU) Novabiochem. N-dimethylformamide (DMF), N-diisopropylethylamine (DIPEA), Thiazolyl blue tetrazolium bromide, L-glutathione reduced, dimethyl sulfoxide-d6, DEAE- Aldrich (Seoul, Korea) was added to the reaction mixture. The reaction was carried out in the same manner as in Example 1, except that the reaction was carried out in the same manner as in Example 1, . Luciferase kits were purchased from Promega (Madison, Wis.) And FBS (Fetal bovine serum), DMEM (Dulbeccos modified Eagles medium), 100 x antibiotic-antimycotic agent and DPBS (Dulbecco's Phosphate Buffered Saline) were purchased from Gibco , USA). Acetic acid was purchased from Merck (Darmstadt, Germany). A 500 mM solution of ethylenediaminetetraacetic acid (EDTA) and sodium chloride (NaCl) were purchased from Calbiochem (San Diego, California), palmitic acid was purchased from JUNSEI (Nihonbashi-honcho, Chuo SPECTRA / POR 7 (10,000 MWCO) and SPECTRA / POR 7 (3,500 MWCO) were purchased from Spectrum Labs (Rancho Dominguez, CA) and Triton X-100 was purchased from Shinyo Pure Chemicals (Osaka, PicoGreen Assay Kit and Lipofectamine 2000 were obtained from Invitrogen (Carlsbad, Calif.) Micro BCA Protein Assay Kit was purchased from Thermo Scientific (Hudson, NH, USA) , And luciferase expression plasmid DNA (pCN-Luci) was prepared by a known method [MJ Lee et al., Gene Ther 9, 859 (Jul, 2002)

2. PEG  Lipid synthesis

Peptide monomers were synthesized in polyethylene glycol (PEGcol) - lipid using Fmoc chemistry method.

After dissolving methoxy-PEG (molecular weight 5000) in dimethylformamide, N-hydroxybenzotriazole and 2- (1H-benzotriazole-1-yl) -1 , 1,3,3-tetra methyluronium and 8 moles of base were reacted at room temperature for 16 hours using N, N-diisopropylethylamine. The synthesized Fmoc-glycine-PEG was precipitated with cold diethyl ether, and the precipitate was separated through a centrifuge. After repeating the above procedure twice, the precipitate in white powder state was dissolved in 30% piperidine solution (7: 3 v / v) and reacted at room temperature for 1 hour and 30 minutes to remove Fmoc. The mixture was then resuspended in cold diethyl ether.

Fmoc-leucine-OH, Fmoc-phenylalanine-OH, Fmoc-G glycine-OH, DiFmoc-lysine-OH, and palmitic acid were sequentially carried out in the order of the remaining peptide monomers.

The final compound was placed in a 3.5 kDa membrane and dialyzed against 4 L of distilled water for 2 days to remove impurities. After the dialysis process, the compound was obtained as a solid using a freeze dryer. (Voyager-TOF Mass Spectrometer, Applied Biosystems Inc., USA) and matrix 2,5-dihydroxybenzoic acid to determine the structure of the final composite. And the structure was confirmed by H1 NMR spectrum (Fourier 300, Bruker, USA) using DMSO (solvent dimethyl sulfoxide) -d6. The measurement results are plotted in Fig. 3 and Fig.

3. Stabilized plasmid lipid particles ( SPLP ) formation

PDNA was sutured into the SPLP using the detergent dialysis method. First, DOPE and DOTAP are dissolved in chloroform, and then the solvent is blown using N 2 gas. And stored overnight at low pressure. Solubilize the synthesized PEG lipid, 50 μg pDNA, and 0.2 Moctyl-β-D-glucopyranoside together with the solvent. The final lipid concentration was 10 mg / mL. The prepared lipid mixture is stored for 1 hour and dialyzed at room temperature overnight using a molecular weight of 10,000 to obtain SPLP ₁ of formula ( ₁₎ and SPLP ₂ of formula (2). After the dialysis process, purification of SPLP ₁ and SPLP ₂ was carried out using ion-exchange DEAE-Sepharose chromatography.

4. Stabilized plasmid lipid particles ( SPLP )

Unsealed DNA was isolated using DEAE-Sepharose ion-exchange chromatography. The sample obtained after the dialysis step was injected into the column and allowed to stand for 30 minutes. The running buffer was 5 mM HEPES (pH 7.4) and the flow rate was 1 mL / min. And treated with 1 M NaCl to obtain unsealed pDNA.

After the chromatographic procedure, each fraction was measured at 260 nm absorbance using an ultraviolet / visible light spectrophotometer (Optizen POP, Mecasys, Korea). Purified SPLP ₁ and SPLP ₂ were disrupted by treatment with detergent Triton X-100, and the concentration of pDNA stuck inside was measured using GreenPico Green reagent.

As a result, we calculated the loading efficiency (%) of the pDNA sealed in SPLP using the equation of loading efficiency (%) = (F ° -F) / F ° × 100. F and F ° were obtained by using 0.4% detergent Triton X-100 treated and untreated fluorescence intensity, respectively.

5. Enzyme digestion experiment ( Enzyme - cleavage release experiment )

Experiments were conducted to observe the changes by human liver cathepsin B using SPLP ₂ produced. The enzyme was prepared at a concentration of 0.5 × 10 -6 M in stock solution (50 mM sodium acetate with 1 mM EDTA [ethylenediaminetetraacetic acid], pH 5.0). The prepared SPLPs and enzymes were treated with an activation solution (0.1 M acetate buffer with 0.01 M EDTA and 0.05 M reduced glutathione, pH 5.0), followed by 0 (control), 2, 4 The reaction took place over a period of time. After the reaction proceeded, the samples were analyzed by dynamic light scattering (DLS) (ELS-Z2, Photal, Otsuka Electronics, Otsuka, Japan).

6. Cell culture

Human embryonic kidney 293 cells were cultured in DMEM supplemented with 10% FBS and antibiotic / antimycotic (0.1 mg / mL) in T25 flasks. Cell culture was maintained in an incubator at 37 ° C, 5% CO ₂ , 95% humidity, and cultured at the second culture time with 0.25% trypsin / EDTA solution.

7. Cytotoxicity analysis

Toxicity of SPLP ₂ was conducted using a 3- (4,5-dimethylthiazol-2- yl) -2,5-diphenyl-tetrazolium bromide (MTT). Briefly, cells were seeded at 1.5 × 10 ³ cells / well in a 96-well microplate and cultured for one day to maintain a density of about 70-80%. The control group (treated with polyethylenimine 25kDa [PEI25KD] and _{PEG-GLFG-K- (C 16} ) with various concentrations to the cells using a _second liposome. MTT solution (phosphate-buffered saline melting, 2 mg / Ml After a day) to each well The MTT material can measure the degree of enzyme activity and thus form an insoluble formazan. Remove the supernatant, dissolve in 150 μL DMSO and measure with a VERASmax microplate reader do.

8. Transfection  analysis( Transfection 분석 )

5.0 × 10 ⁴ cells / well are dispensed for transfection and cultured for 24 hours. We compared SPLP ₁ and SPLP ₂ with Lipofectamine 2000 and GLFG-removed SPLP (formula 3). Lipofectamine 2000, a control, was mixed with pDNA (PCN-Luc) in a weight ratio of 3: 1 and left in the cell culture for 30 minutes. SPLP was used to measure the concentration of DNA clipped by fluorescence quantification method using Triton X-100 and Pico Green assay. Lipofectamine 2000 and SPLP were treated at a concentration of 0.25-0.5 μg / well. After the particle treatment, the cells were cultured for luciferase activity for 24 hours. The culture solution was removed, washed twice with PBS, and then treated with a reporter lysis buffer for 30 minutes. For protein purification, centrifugation was carried out at 12000 rpm, 4 ° C for 20 minutes. Luciferase activity was measured with a Lumat 9507 instrument (Berthold Technology, Bad Wildbad, Germany) and protein quantification was performed with a Micro BCA protein assay kit (Pierce, Rockford, IL, USA).

Example  One: SPLP ₂  [ PEG ₅₀₀₀ - GLFG -K- ( C ₁₆ ) ₂ ] And SPLP _One [ PEG ₅₀₀₀ - K7 - ( C ₁₆ ) ₂ ] Synthesis of

PEG lipids were synthesized according to known methods.

mPEG contains modified ester groups in ω-end (methoxy-PEG-CONH2) and was synthesized sequentially by Fmoc-based chemistry. The N-terminal amine group was activated with a peptide to obtain a PEG-peptide conjugate (mPEG-GLFG-K, 1 eq). Finally, the compound was synthesized as shown in Figure 2 with palmitic acid (2 eq).

Characterization of SPLP ₂ [PEG ₅₀₀₀ -GLFG-K- (C ₁₆ ) ₂ ] with 1 H NMR (300 Mz; solvent, DMSO-d 6) and MALDI-TOF mass spectrometer (matrix, 2,5-dihydroxybenzoic acid) : Δ 0.86 (-CH 3 of palmitic acid), 3.23 (-OCH 3 of mPEG), 7.22 (-C 6 H 5 phenyl of phenylalanine). Functional groups were identified in the &lt; 1 &gt; H NMR spectrum of palmitic acid, mPEG chains and phenylalanine

The mPEG spectra showed high intensity peaks for the repeating functional groups while the palmitic acid and phenylalanine spectra showed relatively low intensity peaks. These data show that the synthesized compound is composed of three blocks (mPEG, oligopeptide, palmitic acid) (Fig. 3).

The average mass of the synthesized PEG lipid observed with a MALDI-TOF mass spectrometer was 5994.69 Da (theoretical molecular weight, 6098.88 Da). The mass spectrum of the synthesized PEG lipid shows increased molecular weight compared to the case of the mPEG spectrum (observed molecular weight, 5191.38 Da) due to the synthesis of the oligopeptide linker and palmitic acid (Figure 4).

Example  2: Characteristics of stabilized plasmid lipid particles

To isolate the non-entrapped DNA, liposomes were purified using DEAE-Sepharose chromatography and the absorbance at 260 nm was determined by analyzing each fraction. The peak of the encapsulated DNA was eluted. Non-entrapped DNA was eluted from the column using 1 M NaCl solution (Figure 5). The loading efficiency was calculated as the perspective fluorescence value of the PicoGreen reagent.

In addition, purified SPLP ₁ and SPLP ₂ were digested with Triton X-100 and stained with PicoGreen reagent. The fluorescence values were measured with a Quantech Filter fluorometer (Thermo Scientific Korea) using filters NB490 and SC515.

As a result, under DOPE 80, DOTAP 10, and PEG lipid 10 conditions, the prepared SPLP ₂ exhibited a high loading efficiency (74.1%).

In addition, SPLP ₁ (135 ± 8 nm) and SPLP ₂ (135 ± 8 nm) were measured through size dispersion measurement using dynamic lighting scattering (215 8 nm) particles were stable (Fig. 6).

Example  3: Enzyme-cleavage release experiment Enzyme - cleavage release experiment )

The properties of GFLG linker in SPLP ₂ [PEG ₅₀₀₀ -GLFG-K- (C ₁₆ ) ₂ ] were examined by enzymatic cleavage experiments.

In the presence of cathepsin B, degradation and aggregation predicted the pattern of GFLG SPLP and increased the final particle size. DLS (dynamic light scattering) was used to confirm that there was a change in particle diameter after the enzyme treatment (Fig. 7)

Size dispersion showed increased aggregation depending on incubation time. The control sample was maintained at a diameter of about 215.1 8 nm. However, at 2 and 4 hours, the diameter increased (20-40 μm). These data show that the cultured samples were truncated and aggregated, suggesting an enzyme-sensitive function. The present inventors predicted that SPLP would readily release pDNA through cellular cathepsin B treatment.

Example  4: Cytotoxicity analysis and luciferase expression

In order to examine SPLP ₁ and SPLP ₂ cytotoxicity, MTT analysis was performed using HEK293 cells. Cells were cultured with liposomes or polymers at 4-point concentration in DMEM in the presence of 10% serum for 24 hours. The results are compared to the SPLP in the bleaching PEI25KD and _{PEG-GLFG-K- (C 16} ) 2 self-assembled the various liposome concentration was shown in Fig.

As shown in FIG. 8, PEG-GLFG-K- (C ₁₆ ) ₂ liposomes and SPLPs showed relatively low cytotoxicity although PEI25KD showed high toxicity with increasing polymer concentration. These results imply the function of PEG coating nanoparticle surfaces. Biodegradable PEG-modified liposomes have the advantage of reducing charge density and reducing cell membrane damage on liposomal surfaces. Cytotoxicity assay results suggest that SPLP may be a good candidate for safe and stable gene transfer therapy.

In addition, we attempted to enhance transfection efficiency in an enzymatically oligopeptide-modified SPLP. The luciferase expression efficiency was analyzed in HEK293 cells with various amounts of pDNA. Transfection efficiency was measured by relative light intensity divided by the amount of protein

The SPLP ₁ and SPLP ₂ particles were prepared and purified by DEAE-Sepharose chromatography, followed by quantitative analysis of the encapsulated pDNA using picogreen.

In particular, the transfection efficiency of GFLG-modified SPLP (Formula 2) was compared with the two standard transfection reagent, lipofectamine 2000 and GFLG oligopeptide-removed SPLP (Formula 3) , And the transfection efficiency of lipofectamine 2000 and oligo lysine-modified SPLP (Formula 1) is shown in the graph of FIG. 9 below.

(3)

As shown in Figure 9, the level of gene expression induced by SPLP ₁ and SPLP ₂ in HEK293 cells increased with increasing pDNA.

In particular, to show enzyme sensitive the SPLP SPLP GFLG- ₂ is removed (PEG ₅₀₀₀ -K- _[16 C] ₂₎ and enhance the transfection capability compared-rescue, which may be described by the enzyme cleavage properties . In the endosomal state, the GFLG-modified SPLP is degraded by the cathepsin B enzyme and contributes to the release of pDNA in the cytoplasm. As a result, it can be seen that the ability of the enzyme to enhance gene expression levels.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Claims (20)

Cationic lipids; Fusogenic non-cationic lipids; And a PEG-lipid conjugate to which a glycine-leucine-phenylalanine-glycine (GLFG) -lysine (K) linker or an oligo lysine K7 linker is coupled as a bilayer stabilizing component (BSC) SPLP). delete The method according to claim 1,
Wherein the glycine-leucine-phenylalanine-glycine (GLFG) is cleaved by Cathepsin-B enzyme. The method according to claim 1,
The cationic lipid may be selected from the group consisting of N, N-dioleyl-N, N-dimethylammonium chloride (DODAC), N, N-distearyl-N, N-dimethylammonium bromide (DDAB) N, N-trimethylammonium chloride (DOTAP), N- (1- (2,3-dioleyloxy) propyl) -N, N, N-trimethylammonium chloride DOTMA) and N, N-dimethyl-2,3-diolyloxy) propylamine (DODMA) and mixtures thereof. 5. The method of claim 4,
Characterized in that the cationic lipid is N- (1- (2,3-dioloyoyloxy) propyl) -N, N, N-trimethylammonium chloride (DOTAP) stabilized plasmid- SPLP). The method according to claim 1,
The fusogenic non-cationic lipid is selected from the group consisting of lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, cephalin, cardiolipin, (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioloylphosphatidylglycerol (DOPG), palmitoyl oleoylphosphatidylglycerol (POPG), dicyclohexylphosphatidylglycerol (DOPE), palmitoyl oleoyl phosphatidylcholine (POPC), palmitoyl oleoyl-phosphatidylethanolamine (POPE), and diolauryl-phosphatidylethanolamine 4- (N-maleimidomethyl) -cyclohexane-1-carboxylate (DOPE-mal). The purified plasmid-lipid particles (SPLP). The method according to claim 6,
Stabilized plasmid-lipid particle (SPLP) for nucleic acid delivery, characterized in that the fusogenic non-cationic lipid is Dioleoyl-phosphatidylethanolamine (DOPE). The method according to claim 1,
Wherein the PEG has an average molecular weight of 750 to 7,000 Daltons. 9. The method of claim 8,
Wherein the PEG is substituted with methoxy at the terminal hydroxyl group. &Lt; RTI ID = 0.0 &gt; 11. &lt; / RTI &gt; The method according to claim 1,
The PEG-lipid conjugate is selected from the group consisting of PEG-dilaurylglycerol (C ₁₂ ), PEG-dimyristoyl glycerol (C ₁₄ ), PEG-dipalmitoyl glycerol (C ₁₆ ), and PEG-distearoyl glycerol ( _C18 ), and combinations thereof; PEG-diacylglycerol (DAG); PEG-phospholipids; PEG-dialkyloxypropyl (DAA); PEG-ceramide; And combinations thereof. &Lt; RTI ID = 0.0 &gt; 11. &lt; / RTI &gt; The stabilized plasmid-lipid particle (SPLP) for nucleic acid delivery. 11. The method of claim 10,
Wherein the PEG-lipid conjugate is PEG-distearoyl glycerol ( _C18 ). The stabilized plasmid-lipid particle (SPLP) for nucleic acid delivery. 12. The method of claim 11,
PEG- lipid conjugate is a PEG-K7- (C _{16) 2} or _{PEG-GLFG-K- (C 16} ) , characterized in that with the structure of Figure _2, the stabilized plasmid for nucleic acid delivery-lipid particles (SPLP). The method according to claim 1,
(DOTAP), 1,2-dioleyl-sn-glycero-3-phosphoethanolamine (DOPE), and mPEG ₅₀₀₀ -K7- (C ₁₆₎ of stabilized nucleic acid delivery according to claim ₂ consisting of a plasmid-lipid particle (SPLP _1):
[Chemical Formula 1]

. The method according to claim 1,
(DOTAP), 1,2-dioleyl-sn-glycero-3-phosphoethanolamine (DOPE), and mPEG ₅₀₀₀ Stabilized plasmid-lipid particle (SPLP ₂ ) for nucleic acid delivery characterized in that it is composed of -GLFG-K- (C ₁₆ ) ₂ .
(2)

. The method according to claim 13 or 14,
Wherein the DOPE: DOTAP: PEG-conjugate is contained in a ratio of 80:10:10. The method according to claim 1,
Wherein the stabilized plasmid-lipid particle (SPLP) has a diameter ranging from 200 to 230 nm. A complex for intracellular nucleic acid delivery formed by binding the stabilized plasmid-lipid particle (SPLP) of claim 1 with a nucleic acid. 18. The method of claim 17,
Wherein the nucleic acid is pDNA (plasmid DNA). delete delete

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