KR20180108190A - Ascorbic acid 2-phosphate, Its polymers having endosomolytic activity and nuclear delivery activity, and Use thereof - Google Patents

Abstract

The present invention relates to a composition having endosomolytic activity, a composition for non-viral drug delivery comprising an ascorbic acid 2-phosphate (AAP) or a polymer thereof (poly(ascorbic acid 2-phosphate)) (pAAP) as an active ingredient, or a method for manufacturing a poly(ascorbic acid 2-phosphate) polymer compound which is a polymer of ascorbic acid 2-phosphate. Since the AAP and pAAP exhibit not only the ability to decompose endosomes but also the function of delivering the drug into the nucleus and the antioxidant activity, the composition comprising the same can deliver the non-viral drug in vivo to maximize the bioavailability, and thus can be usefully used as a composition for a drug delivery.

Description

Ascorbic acid 2-phosphate, its polymers and their uses {Endosomolytic activity and nuclear delivery activity of Ascorbic acid 2-phosphate, and its use}

The present invention relates to ascorbic acid 2-phosphoric acid, a polymer thereof and a use thereof, which have endo-somatic resolution and nuclear transport capability.

(Eg, high molecular weight peptides, proteins and genetic material as well as low molecular weight chemical drugs and contrast agents) to specific organs, tissues, cells, cytoplasm, mitochondria, perinuclear regions, There is a growing interest in developing an effective system to do so. Unlike conventional formulations, position-specific nanotherapeutics are designed to maximize bioavailability of the therapeutic delivered to the target site, and can be used to treat the disease with low side effects and improve the therapeutic effect. This drug delivery technology is a high value added technology and it is increasingly used as a method to improve the compliance of patients and the convenience of taking drugs.

Endosomal sequestration of chemical and biological agents delivered into the cell and nuclear delivery restriction by the nuclear membrane are important obstacles to the improvement of drug efficacy. Endosome sequestration can be overcome through endosome-membrane destabilization or destruction induced by small molecules or polymers with amines, sulfonamides, carboxylic acids, or phosphate groups. These small molecules / polymers have improved results in nonviral gene delivery and cytoplasmic delivery of chemical drugs. Also, at endosomal pH, proton buffering and conformational transition characteristics are known to induce lipid membrane destabilization or endosomolysis. Nuclear transfer ability is also known as a low molecular ligand substance such as a nuclear localization signal (NLS), a peptide containing a large amount of lysine or arginine, dexamethasone to expand the nucleus, / RTI > and / or < RTI ID = 0.0 > a < / RTI > These methods lead to improved efficacy in non-viral gene delivery and nuclear delivery of chemical drugs.

In addition, vitamin C (vitamin C, ascorbic acid), an anti-oxidant, is used as an adjunct to reduce oxidative stress, which is a side effect of anticancer drugs and various disease environments in which active oxygen occurs. However, vitamin C can be easily degraded in vivo, so its application is limited. Therefore, an improved effect can be obtained by using ascorbic acid 2-phosphate, which is a phosphate derivative thereof, and ascorbyl palmitate, which is a lipid-conjugate thereof, to prevent degradation in vivo.

Korean Patent No. 10-1547635, No. 10-1547636 and No. 10-1577274 use nucleotides, reducing or non-reducing nucleotide polymers to improve the efficiency of drug delivery by improving endocytic resolution, Quot; does not mention the ability of the ascorbic acid 2-phosphoric acid and its polymer to hydrolyze endosomes or to transfer them into the nucleus.

1. Korean Registered Patent No. 10-1547635 (registered on August 20, 2015). 2. Korean Registered Patent No. 10-1547636 (registered on August 20, 2015). 3. Korean Patent No. 10-1577274 (Registered on Dec. 2015).

It is an object of the present invention to provide a novel composition having endosomal resolution ability through endosomal membrane destabilization or destructive action.

Another object of the present invention is to provide a non-viral drug delivery composition capable of enhancing endo-somatic resolution and transmembrane delivery, thereby improving the drug delivery rate.

It is still another object of the present invention to provide a process for preparing a polymeric compound of ascorbic acid 2-phosphoric acid contained in the composition.

In order to accomplish the above object, the present invention provides an aspherical acid-2-phosphate (AAP) or a polymer thereof, such as poly (ascorbic acid 2-phosphate); pAAP] as an active ingredient.

To achieve these and other advantages and in accordance with the purpose of the present invention, as embodied and broadly described herein, the present invention provides a method for producing ascorbic acid 2-phosphate (AAP) or poly (ascorbic acid 2-phosphate) pAAP] as an active ingredient.

In order to accomplish the above object, the present invention relates to a method for producing an optically active 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide compound of the present invention, which comprises Ascorbic acid 2-phosphate (AAP) (3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS); And the polymerized mixture was dialyzed to obtain poly (ascorbic acid 2-phosphate); (ascorbic acid 2-phosphoric acid) polymer compound comprising separating and purifying a pAAP polymer.

At this time, the poly (ascorbic acid 2-phosphate) polymer may be represented by the following formula (1);

[Chemical Formula 1]

In the above formula (1), n is an integer of 4 to 200.

The present invention relates to ascorbic acid 2-phosphoric acid, a polymer thereof, and a use thereof, which exhibit an endocome resolution and an enhancement of intracellular transport capability. The ascorbic acid 2-phosphate and its polymer have hydrogen ion buffering function and nuclear transport capability , The drug delivered from the non-viral drug delivery vehicle containing ascorbic acid 2-phosphate or its polymer is excreted from the endosome into the cytoplasm, the delivery efficiency of the drug delivery system in the cytoplasm is enhanced, and ascorbic acid 2- And its polymer can enhance the cell survival rate in the state where active oxygen is induced through the antioxidant function, so that it can be utilized as a novel drug delivery system that effectively delivers the drug in vivo.

1 shows the result of measurement of the molecular weight of synthesized poly (ascorbic acid 2-phosphoric acid) [pAAP] by gel permeation chromatography.
2 is a result of evaluating proton buffering capacity of ascorbic acid 2-phosphate (AAP) and its polymer (pAAP).
Figure 3 shows the particle size and surface charge of bPEI25kDa / AAP-pDNA complex and bPEI25kDa / pAAP-pDNA complex.
4 shows the expression efficiency of luciferase gene of bPEI25kDa / AAP-pDNA complex and bPEI25kDa / pAAP-pDNA complex in various cells. More specifically, HEK293 cells (4 (a)), DPSCs (dental pulp AAP-pDNA complexes and bPEI25kDa / pAAP-pDNA complexes in the adipose tissue-derived stem cells (ADSCs) 4 (b) Expression efficiency.
FIG. 5 shows the expression efficiency of luciferase gene of bPEI25kDa / AAP-pDNA complex and bPEI25kDa / pAAP-pDNA complex in the presence of chloroquine or Bafilomycin A1 in HEK293 cells.
Figure 6 shows the cell and nuclear intakes of gene drugs in the bPEI25kDa / AAP-pDNA complex and the bPEI25kDa / pAAP-pDNA complex in HEK293 cells.
Fig. 7 shows the results of observation of intracellular distribution of gene drug delivered by bPEI25kDa / pDNA complex and bPEI25kDa / AAP-pDNA complex in HEK293 cells by confocal microscopy.
FIG. 8 shows the results of evaluating the intracellular and intracellular inflow of gene drugs in the bPEI25kDa / AAP-pDNA complex in the presence of Geldanamycin in HEK293 cells.
FIG. 9 shows the results of confirming the cell survival rate according to the presence or absence of fetal bovine serum (FBS) when transfection of bPEI25kDa / AAP-pDNA complex or bPEI25kDa / pAAP-pDNA complex in HEK293 cells.
FIG. 10 shows the results of confirming the cell survival rate changes of DPSCs treated with bPEI25kDa / AAP-pDNA complex and bPEI25kDa / pAAP-pDNA complex under reactive oxygen-induced conditions with hydrogen peroxide (H ₂ O ₂ ).
Fig. 11 shows the results of evaluation of the degree of reactive oxygen species production of bPEI25kDa / AAP-pDNA complex and bPEI25kDa / pAAP-pDNA complex in HEK293 cells.

Hereinafter, the present invention will be described in more detail.

The inventors of the present invention have found that the ascorbic acid 2-phosphate and its polymer have a hydrogen ion buffering ability, destabilizing or destroying the endosomal membrane, and allowing the drug to escape from the endosome to other organelles in the cell biofunctional. Furthermore, it has been confirmed that the drug delivery system containing ascorbic acid 2-phosphate or its polymer escaping endosomes has a function of effectively transferring the drug into the nucleus by binding to the glucocorticoid receptor in the cytoplasm. .

In addition, the present inventors completed the present invention by ascertaining that the ascorbic acid 2-phosphate polymer has antioxidative properties like ascorbic acid 2-phosphate through the enhancement of cell viability in an oxidative stress environment.

Accordingly, the present invention relates to ascorbic acid 2-phosphate (AAP) or a polymer thereof, such as poly (ascorbic acid 2-phosphate); pAAP] as an active ingredient.

At this time, the pAAP may be represented by the following formula (1);

[Chemical Formula 1]

In the above formula (1), n is an integer of 4 to 200.

The pAAP is characterized by being negatively charged.

As used herein, the term "AAP" refers to ascorbic acid 2-phosphate, pAAP refers to synthesized poly (ascorbic acid-2-phosphate) ≪ / RTI >

The AAP or pAAP has proton buffering activity and can exhibit endosomal resolution by destabilizing or destroying the endosomal membrane to induce endosomal escape of the delivery drug.

In addition, the AAP or pAAP can transfer the drug into the nucleus of the cell. In particular, it is possible to solve the phenomenon of intracellular influx by the nuclear membrane and nucleus of the macromolecular drug such as protein and gene.

Furthermore, in one embodiment of the present invention, it has been confirmed that the composition comprising the AAP or pAAP improves the cell survival rate under reactive oxygen-induced conditions of oxidative stress using hydrogen peroxide.

That is, since pAAP can exhibit an antioxidative effect, it possesses the antioxidant activity of AAP, which is a monomer, and has an advantage of lowering the level of reactive oxygen during bioavailability.

In addition, the present invention relates to ascorbic acid 2-phosphate (AAP) or a polymer thereof, such as poly (ascorbic acid 2-phosphate); pAAP] as an active ingredient.

At this time, the pAAP may be represented by the following formula (1);

[Chemical Formula 1]

In the above formula (1), n is an integer of 4 to 200.

The drug used in the present invention is a substance capable of inducing a desired biological or pharmacological effect by promoting or inhibiting a physiological function in the body of an animal or a human and is a chemical or biological substance or compound , And it has a preventive effect on organic matter by preventing unwanted biological effects such as infection prevention, alleviating the condition caused by disease, for example, relieving the pain or infection caused as a result of disease, Mitigate, or completely eliminate it.

More preferably, the non-viral drug may be any one or more selected from the group consisting of a hydrophilic chemical drug, a hydrophobic chemical drug, a diagnostic drug, a peptide, and a gene. More specifically, the non-viral drug may be a non- It is not limited thereto as long as it can act as a drug or a physiologically active substance.

In addition, the composition may further comprise a polymer for drug delivery to form a drug delivery complex.

The drug delivery polymer may be selected from the group consisting of polyethylenimine (bPEI), polylysine (PLL), poly (arginine), polyhistidine, poly (amido amine) , Poly (beta amino ester), chitosan, protamine, histone, poly (tertiary amine methacrylate), poly (2- (dimethylamino) ethyl methacrylate), poly (N- [N- (2-aminoethyl) -2-aminoethyl] asphaltamide (poly A nanoparticle-derived nanostructure, a nanoparticle-derived nanostructure, a carbon-derived nanostructure, a calcium phosphate nanostructure, a porous silica nanostructure, and a complex thereof. , And more preferably polyethyleneimine (bPEI). , Without being limited thereto.

Since the AAP or pAAP has a proton buffering activity, endosomal escape of a drug through endosomolysis can be induced, and thus it is useful as a drug delivery composition .

In addition, the AAP or pAAP can deliver a drug into the nucleus of a cell, and more specifically, the AAP or pAAP can transfer a drug into the nucleus by binding to a glucocorticoid receptor in the cytoplasm, The bioavailability of the drug, such as a viral gene, that is to be delivered into the nucleus of the cell can be significantly improved.

Further, in one embodiment of the present invention, it was confirmed that the drug composition containing the AAP or pAAP improves the cell survival rate under an oxidative stress-induced oxidative stress condition using hydrogen peroxide. The pAAP has antioxidative effects Therefore, it can be used as a composition for drug delivery because the degree of induction of active oxygen is low.

In addition, in one embodiment of the present invention, the drug delivery composition containing the AAP or pAAP significantly inhibits the decrease of cell viability in the medium containing no bovine serum, and in the medium containing bovine serum, The present invention can be effectively utilized for inducing the expression of a desired gene in stem cells by simultaneously maintaining the survival rate and gene expression efficiency of stem cells. That is, the composition for drug delivery comprising AAP or pAAP may be used in stem cell culture to control stem cell differentiation.

In the present invention, the composition for drug delivery may further comprise at least one pharmaceutically acceptable carrier, excipient or diluent in addition to a pharmaceutically effective amount of the drug, the AAP or pAAP, and the polymer for drug delivery.

The "pharmaceutically effective amount" in the present invention means an amount sufficient for the drug to exhibit the desired physiological or pharmacological activity to be administered to an animal or a human, and includes an age, weight, health condition, And the duration of treatment.

In addition, the term "pharmaceutically acceptable " in the present invention means physiologically acceptable and when administered to humans, does not normally cause an allergic reaction such as gastrointestinal disorder, dizziness, or the like.

Examples of the carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

In addition, the drug delivery composition may further include a filler, an anti-coagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifier, and an antiseptic agent.

The composition for drug delivery according to the present invention may be administered through various routes including oral, transdermal, subcutaneous, intravenous, or muscular, and the dose of the drug may be varied depending on the administration route, age, sex, And can be appropriately selected according to various factors. In addition, the composition for drug delivery of the present invention may be administered in combination with a known compound capable of raising the desired effect of the drug.

In addition, the present invention relates to a process for the preparation of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (1-ethyl-3-dimethylaminopropyl) carbodiimide; EDC) and N-hydroxysuccinimide (NHS) are mixed and stirred to effect polymerization reaction; And the polymerized mixture was dialyzed to obtain poly (ascorbic acid 2-phosphate); (ascorbic acid 2-phosphoric acid) polymer compound comprising separating and purifying a pAAP polymer.

It is preferable that the synthesis and separation and purification of the pAAP polymer proceed under the condition that the light is blocked.

Preferably, the mixed and stirred mixture may be a mixture of AAP: EDC: NHS in a molar ratio of 1: 2: 1, and the polymerization reaction may be carried out at 5 ° C to 50 ° C for 0.5 to 50 hours .

At this time, the poly (ascorbic acid 2-phosphate) polymer may be represented by the following formula (1);

[Chemical Formula 1]

In the above formula (1), n is an integer of 4 to 200.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. It is to be understood that both the foregoing description and the following detailed description are exemplary and explanatory and are intended to provide further explanation of the invention as claimed. no.

< Example  1> Polymers of ascorbic acid 2-phosphate (AAP) pAAP ) synthesis

The monomer ascorbic acid 2-phosphate (AAP) is reacted with 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (N-hydroxysuccinimide (NHS)) and triethylamine (TEA) in an aqueous solution, thereby polymerizing the monomers. After completion of the reaction, the synthesized polymer and byproducts were separated and purified by dialysis and lyophilized to obtain poly (ascorbic acid 2-phosphate). The above procedure was performed with the light blocked.

More specifically, the molar ratio of monomers and reactants in the above reaction was monomer: EDC: NHS = 1: 2: 1, and 200 mg of monomer was dissolved in 5 mL of water. At this time, the amount of TEA used was 100 μL, and the stirring reaction proceeded at room temperature for 48 hours. The synthesized polymer aqueous solution was put into a dialysis membrane having a molecular weight cut-off (MWCO) of 1000 daltons and the water was changed at least 10 times for 48 hours to remove by-products and low molecular weight ascorbic acid 2-phosphate. Was carried out at room temperature. Then, the aqueous solution remaining in the dialysis membrane was freeze-dried to obtain poly (ascorbic acid-2-phosphate); pAAP].

[Reaction Scheme 1]

< Example  2> pAAP  Gel Permeation Chromatography Analysis

The aqueous solution of polymer (0.5 mg / mL) was passed through a column equipped with a water-soluble solvent column at a flow rate of 1 mg / mL, and the molecular weight of the polymer was calculated in a GPC analysis mode. The GPC results of the synthesized pAAP are shown in FIG. .

Referring to FIG. 1, the number average molecular weight of pAAP was 3.43 × 10 ⁴ daltons, the weight average molecular weight was 5.63 × 10 ⁴ daltons, and the polydispersity thereof was 1.63.

< Example  3> With AAP pAAP  Evaluation of proton buffering capacity

AAP or pAAP was dissolved in a 150 mM aqueous solution of sodium chloride to a concentration of 1 mg / mL, followed by adding a small amount of 1 M aqueous sodium hydroxide solution to a pH of about 11. The pH of the aqueous solution was measured while 0.1 M hydrogen chloride was added to the AAP or pAAP aqueous solution.

As a result, as shown in FIG. 2, it was confirmed that the aqueous sodium chloride solution had no hydrogen ion buffering ability at pH 5 to 7, which is the pH range of the endosome, but that AAP and pAAP had hydrogen ion buffering ability. The ion buffer capacities were 2.3 μmol / mg AAP and 2.74 μmol / mg pAAP, respectively.

Therefore, it can be seen that due to the hydrogen ion buffering ability in the pH environment of the endosomes, the endosomal membrane can be destabilized or destroyed by the impact due to the osmotic pressure difference like the proton sponge effect.

<Example 4> Evaluation of gene expression efficiency of a polymer gene drug carrier containing AAP and pAAP

In order to encapsulate AAP and pAAP in a well-known polymer / gene complex, a solution of positively charged polyethyleneimine (bPEI25kDa, molecular weight 25 kDa), a plasmid DNA (pDNA) and a negative charge solution containing AAP or pDNA and pAAP After that, the two solutions were mixed to make a bPEI25kDa / AAP-pDNA complex and a bPEI25kDa / pAAP-pDNA complex. The N / P ratio was prepared to be 5, at which time the phosphate of AAP and pAAP was not taken into account and was calculated only with the phosphate of amine and pDNA of bPEI25 kDa.

As shown in FIG. 3, the particle size of the bPEI25kDa / AAP-pDNA complex was 200 nm, 4 nmol to 8 nmol when the AAP concentration was 2 nmol per microgram of pDNA , The surface charge was positive at 4 nmol of AAP concentration and negative at 8 nmol. The particle size of the bPEI25kDa / pAAP-pDNA complex was 200 nm to 1 nmol of pAAP and 300-400 nm to 4 to 16 nmol per 1 microgram of pDNA. The surface charge was positive to 8 nmol In the above, it has a negative charge.

In addition, the ability of the prepared bPEI25kDa / AAP-pDNA complex and bPEI25kDa / pAAP-pDNA complexes to improve the gene expression efficiency of the encapsulated AAP and pAAP was evaluated by comparing the bPEI25kDa / pAAP-pDNA complex with the bPEI25kDa / pDNA complex without AAP and pAAP. Specifically, 1 × 10 ⁵ HEK293 cells per well of a 6-well plate, 1 × 10 ⁵ DPSCs or ADSCs cells per well of a 12-well plate were added, cultured for 24 hours, and the prepared bPEI25kDa / AAP-pDNA complex Or bPEI25kDa / pAAP-pDNA complex solution was transfected into the cultured cells. After incubation for 4 hours, the medium was shaved and further incubated for 44 hours. After 48 hours of transfection, the gene expression efficiency of this cell was evaluated using the expression level of luciferase in the pDNA. When the concentration of the encapsulated AAP and pAAP was 0, the control complex bPEI25kDa / pDNA complex was formed, And compared with the gene expression efficiency of this complex.

As shown in FIG. 4 (a), transfection of bPEI25kDa / AAP-pDNA complex in HEK293 cells resulted in about 120-fold increase in gene expression efficiency when the AAP concentration was 4 nmol / μg pDNA and bPEI25kDa / When the pAAP concentration was 8 nmol / μg pDNA, the gene expression efficiency was increased about 100 times when transfected with the pDNA complex. In addition, as shown in Fig. 4 (b), transfection of the bPEI25kDa / AAP-pDNA complex in DPSCs increased gene expression efficiency by about 29 times when the AAP concentration was 4 nmol / μg pDNA and bPEI25kDa / When the pAAP concentration was 2 nmol / μg pDNA, the gene expression efficiency was increased about 85 times when the pDNA complex was transfected. Furthermore, when transfected with bPEI25kDa / AAP-pDNA complexes in ADSCs cells as shown in Fig. 4 (c), gene expression efficiency was increased about 200 times when AAP concentration was 4 nmol / μg pDNA and bPEI25kDa / pAP- When the pAAP concentration was 2 nmol / μg pDNA, the gene expression efficiency was increased about 5200 times when the pDNA complex was transfected.

< Example  5> Vathulomycin  A1 ( Bafilomycin  A1) or Chloroquine  Evaluation of gene expression efficiency of polymer drug delivery system containing AAP or pAAP in the presence

In general, when a polymer gene drug transporter enters the cell through endocytosis, the endosome excretion of the drug transporter plays an important role in determining gene expression efficiency. In order to evaluate whether the polymer gene drug delivery system of the present invention is introduced into cells using endocytosis, a reagent for destroying cells and endosomes upon transfection of bPEI25kDa / AAP-pDNA and bPEI25kDa / pAAP- Bafilomycin A1 (Bafilomycin A1; 50 nM), a reagent for preventing the pH decrease of chloroquine (20 μM) or endosomes, was added to the medium, and the gene expression efficiency was evaluated according to the method of Example 4 .

Theoretically, when a polymer gene drug transporter is introduced into a cell via endocytosis, compared with a polymer gene carrier in a control experiment in which neither chloroquine nor vathomycin A1 is added, when chloroquine is added, a polymer drug carrier Since the gene expression efficiency increases rapidly as it escapes from endosomes, the efficiency of gene expression is lowered or is not affected because it is difficult to escape the endomocyte of the polymer drug delivery system by adding barfilomycin A1.

In this regard, as shown in FIG. 5, when the bPEI25kDa / pDNA complex was transfected, the gene expression efficiency was increased 50-fold when chloroquine was present, and when the barprotoxin A1 was present, gene expression efficiency 11 times. On the other hand, transfection of the bPEI25kDa / AAP-pDNA complex containing the AAP and pAAP and the bPEI25kDa / pAAP-pDNA complex showed gene expression efficiencies of 2-fold and 8-fold increased in the presence of chloroquine, respectively, When present, showed 100 and 25 fold reduction in gene expression efficiency, respectively.

This result suggests that the polymer gene complex containing AAP or pAAP enters the cell through endocytosis and that the bPEI25kDa / AAP-pDNA complex and the bPEI25kDa / pAAP-pDNA complex are bound to endosomes by hydrogen ion buffering of AAP and pAAP I could see that I had escaped.

< Example  6> with AAP pAAP  Measurement of cell and nuclear fluxes of gene drugs in the polymer gene drug delivery system

To investigate the effect of AAP and pAAP on the cell and nucleus inflow of gene drug carriers, a fluorescent substance such as YOYO-1 was inserted into plasmid DNA (pDNA) at a ratio of 10: 1 (pDNA: YOYO-1) (BPEI25kDa, pAAP-pAAP-pAAP) was prepared by mixing a solution of polyethylenimine (bPEI25kDa, molecular weight 25kDa) and a negative charge solution containing pDNA-YOYO-1 and AAP or pAAP, pDNA complexes.

We then evaluated the inflow of the intracellular gene drug by the AAP and pAAP with the prepared bPEI25kDa / AAP-pDNA complex and bPEI25kDa / pAAP-pDNA complexes compared with the bPEI25kDa / pDNA complex without AAP and pAAP. 1 x 10 &lt; ⁵ &gt; HEK293 cells were added per well of a 6-well plate, followed by culturing for 24 hours and transfection of the prepared bPEI25kDa / AAP-pDNA complex, bPEI25kDa / pAAP-pDNA complex or bPEI25kDa / pDNA complex solution Respectively. After incubation for 4 hours, the cells were detached from the well plate using trypsin-EDTA (trypsin-EDTA) to separate the nuclei. Finally, the inflow of the cell and nucleus gene drug was evaluated by confirming the fluorescence value of cells and nuclei transfected with bPEI25kDa / AAP-pDNA complex and bPEI25kDa / pAAP-pDNA complex through a flow cytometer.

At this time, when the concentration of the encapsulated AAP and pAAP was 0, the control complex, bPEI25kDa / pDNA complex, was formed and set to 1 in order to relatively compare the intracellular inflow of the gene drug in this complex in HEK293 cells.

As a result, as shown in Fig. 6, the gene drug of bPEI25kDa / AAP-pDNA complex was reduced at a concentration twice as much as that of the control drug bPEI25kDa / pDNA complex at all concentrations. On the other hand, the intracellular flux of the gene product of the bPEI25kDa / AAP-pDNA complex was about twice that of the gene product of the control complex bPEI25kDa / pDNA complex. Assuming that the same amount of gene drug was introduced into the cell, the intracellular inflow of the gene drug of the AAP-containing complex increased about four times. In the case of the bPEI25kDa / pAAP-pDNA complex gene drug, the intracellular inflow was 2.5 times lower than that of the control complex bPEI25kDa / pDNA complex when pAAP concentration was 2-4 nmol pAAP / μg pDNA. On the other hand, when the concentration of pAAP in the bPEI25kDa / pAAP-pDNA complex was 2-4 nmol pAAP / μg pDNA, the intracellular flux of the gene drug was 1.6 times higher than that of the bPEI25kDa / pDNA complex. Assuming that the same amount of gene drug has been introduced into the cell, the intracellular inflow of the gene drug in the pAAP-containing complex increased about four times.

Example 7 Confirmation of Intracellular Distribution of Gene Drugs in Polymer Gene Drug Delivery System Containing AAP

In order to examine the effect of AAP on the intracellular distribution of the gene drug delivery vehicle, a polymer gene drug complex was formed in the same manner as in Example 4 above. The prepared bPEI25kDa / AAP-pDNA complex was compared with the bPEI25kDa / pDNA complex without AAP, and the intracellular distribution of the gene drug by AAP encapsulated gene drug was evaluated. First, 1x10 ⁵ HEK293 cells were placed in a confocal microscope measuring dish and cultured for 24 hours, and the prepared bPEI25kDa / AAP-pDNA complex or bPEI25kDa / pDNA complex solution was transfected into the cultured cells. After 4 hours, the distribution of the intracellular gene drug was evaluated using a confocal microscope.

As a result, as shown in FIG. 7, the gene drug of the control complex bPEI25kDa / pDNA complex was mainly distributed in the cytoplasm, but the gene drug of bPEI25kDa / AAP-pDNA complex was distributed mainly in the nucleus rather than the cytoplasm.

< Example  8> Geldanamycin ( Geldanamycin ), The presence of AAP in polymeric gene drug delivery vehicles

Some substances bind to receptors present in the cytoplasm and migrate to the nucleus, regulating various cellular responses in the nucleus. Among them, the glucocorticoid receptor, one of the steroid hormone receptors, is known to bind to a ligand such as dexamethasone to form a receptor-ligand complex and the complex to migrate to the nucleus. In the present invention, in order to evaluate whether the polymer gene drug carrier containing AAP binds to the glucocorticoid receptor and is introduced into the nucleus, in the presence of geladinamycin (2.5 μM) which is a substance inhibiting the transfer of the glucocorticoid receptor to the nucleus, The changes in the intracellular and intracellular inflow of the bPEI25kDa / AAP-pDNA complex were observed according to the method described in Example 6.

As a result, as shown in Fig. 8, in the case of the control bPEI25kDa / pDNA complex without AAP, there was no change in the cell and nuclear intakes of the gene drug in the presence of gelanamycin. On the other hand, in the case of the bPEI25kDa / AAP-pDNA complex gene drug containing AAP, the intracellular inflow was not changed but the intracellular inflow was reduced by about 24% in the presence of geladenamycin.

< Example  9> AAP or pAAP  Included polymeric gene drug delivery Transfection  Determination of cell survival rate by method

In general, when a cationic polymer is used as a polymer gene drug delivery vehicle, it can be coagulated with an anionic serum protein present in the right serum (FBS). Therefore, a medium containing no serum is used for transfection for 4 hours. However, it is important to increase the gene expression efficiency in the culture medium containing bovine serum because the cell survival rate decreases when the cells are exposed to the medium without bovine serum.

Thus, in the case of the bPEI25kDa / AAP-pDNA complex and the bPEI25kDa / pAAP-pDNA complex used in the present invention, the change in the gene expression efficiency in the culture medium containing the right serum was confirmed. In fact, in the case of using the culture medium containing bovine serum during the transfection process, the transfection medium containing the serum for transfection and the culture containing the bovine serum Cell viability was evaluated after exposing the cells to the medium.

Specifically, 5 × 10 ⁴ DPSCs cells per well of a 24-well plate were cultured for 24 hours, and the control complex without the bPEI25kDa / AAP-pDNA complex, bPEI25kDa / pAAP-pDNA complex and AAP or pAAP RTI ID = 0.0 &gt; bPEI25kDa / pDNA &lt; / RTI &gt; complex solution was transfected into the cells. One hour before the transfection, the medium was replaced with a culture medium containing no serum-free transfection medium or bovine serum. After 4 hours of transfection, the culture medium was replaced with a culture medium, followed by further incubation for 44 hours. At 48 hours after transfection, cell viability was assessed using MTT assay. At this time, when the cells were transfected with the bPEI25kDa / pDNA complex and the polymer gene drug carrier containing AAP or pAAP when the cells were exposed to the culture medium containing the right serum and the survival rate of the cells not treated with the polymer gene drug transporter was 100% The cell viability was compared with the presence of the right serum.

As a result, as shown in Fig. 9, when transfection was carried out in a medium containing the right serum in DPSCs cells, both bPEI25kDa / pDNA complex, bPEI25kDa / AAP-pDNA complex and bPEI25kDa / pAAP- Cell survival rate was decreased to about 20% regardless of the type of complex when transfection was performed in a medium containing no serum. Therefore, there was no toxicity induced by AAP or pAAP, and it was found that AAP or pAAP could be used for cell culture in the medium containing the right serum.

< Example  10> AAP or pAAP  Confirmation of inhibition of cell death by reactive oxygen species of polymer gene drug transporter

The bPEI25kDa / AAP-pDNA complex and the bPEI25kDa / pAAP-pDNA complex prepared in the same manner as in Example 4 were compared with the bPEI25kDa / pDNA complex without AAP or pAAP to evaluate the antioxidative capacity of the encapsulated AAP and pAAP.

Specifically, 5 x 10 ³ DPSCs cells per well of a 96-well plate were cultured for 24 hours and the prepared bPEI25kDa / AAP-pDNA complex, bPEI25kDa / pAAP-pDNA complex and bPEI25kDa / pDNA complex solution were treated Respectively. Then, after 20 hours of cultivation, hydrogen peroxide (H ₂ O ₂ ) was treated with 200 μM to further generate active oxygen, and after further incubation for 4 hours, cell viability was evaluated according to the method in Example 9.

As a result, cell viability was reduced by 30% when the bPEI25kDa / pDNA control complex containing 12.5 μg / mL pDNA was treated with H ₂ O ₂ , as shown in FIG. 10, and 25 μg / mL of pDNA , The cell viability was reduced by 90% when treated with the bPEI25kDa / pDNA complex. On the other hand, the bPEI25kDa / AAP-pDNA complex containing 12.5 μg / mL and 25 μg / mL of pDNA and the bPEI25kDa / pAAP-pDNA complex showed cell viability decreased by 10% and 20%, respectively. In addition, when activated oxygen was induced using 200 μM H ₂ O ₂ , the cell viability of cells treated with bPEI25kDa / pDNA control complex was reduced by 60-90%, but the cells of cells treated with bPEI25kDa / AAP-pDNA complex The survival rate was reduced by 40-60% and the cell survival rate of cells treated with bPEI25kDa / pAAP-pDNA complex decreased by 20-30%.

<Example 11> Inhibition of active oxygen production of a polymer gene drug carrier containing AAP

In order to confirm the amount of active oxygen induced by the polymer gene drug carrier depending on the presence or absence of AAP, 1x10 ⁵ HEK293 cells per well of a 6-well plate were cultured for 24 hours. The prepared bPEI25kDa / AAP-pDNA complex and bPEI25kDa / pDNA complex solution was then treated with cells and incubated for 4 hours. After that, the active oxygen (DCFDA) was treated and incubated for 1 hour. At this time, N-acetyl-L-cysteine, which removes active oxygen, was treated to determine whether the measured fluorescence means active oxygen.

As a result, as shown in Fig. 11, the bPEI25kDa / AAP-pDNA complex containing AAP showed 15% lower active oxygen production than the control complex bPEI25kDa / pDNA complex.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. Do. That is, the practical scope of the present invention is defined by the appended claims and their equivalents.

Claims (16)

Ascorbic acid 2-phosphate (AAP) or its polymer [poly (Ascorbic acid 2-phosphate)]; pAAP] as an active ingredient. The composition according to claim 1, wherein the pAAP is represented by the following formula (1):
[Chemical Formula 1]

In the above formula (1), n is an integer of 4 to 200. [Claim 2] The method according to claim 1, wherein the AAP or pAAP has proton-buffering activity, thereby destabilizing or destroying the endosomal membrane to induce endosomal escape of the delivery drug, Wherein the composition has endosome resolution. The composition of claim 1, wherein the AAP or pAAP delivers the drug into the nucleus of the cell. The composition according to claim 1, wherein the pAAP exhibits an antioxidative effect. Ascorbic acid 2-phosphate (AAP) or its polymer [poly (Ascorbic acid 2-phosphate)]; pAAP] as an active ingredient. 7. The non-viral drug delivery composition according to claim 6, wherein the pAAP is represented by the following formula (1).
[Chemical Formula 1]

In the above formula (1), n is an integer of 4 to 200. The non-viral drug delivery composition according to claim 6, wherein the non-viral drug is at least one selected from the group consisting of a hydrophilic chemical, a hydrophobic chemical, a diagnostic drug, a peptide, and a gene. The composition for delivering a drug according to claim 6, wherein the composition further comprises a polymer for drug delivery to form a drug delivery complex. The method of claim 9, wherein the drug delivery polymer is selected from the group consisting of polyethyleneimine (bPEI), polylysine (PLL), poly (arginine), polyhistidine, poly (amidoamine) amido amine], poly (beta amino ester), chitosan, protamine, histone, poly (tertiary amine methacrylate) ), Poly (2- dimethylamino) ethyl methacrylate), poly (N- [N- (2-aminoethyl) -2-aminoethyl] asphaltamide The present invention relates to a method for producing a nanoparticle-containing nanostructure, comprising the steps of: (1) preparing a nanoparticle-containing nanostructure (N- [N- (2-aminoethyl) -2-aminoethyl] aspartamide), lipid, phospholipid, Complex, and a non-viral drug delivery carrier Water. 7. The method of claim 6, wherein the AAP or pAAP has a proton buffering activity, thereby inducing an endosomal escape of the drug through endosomolysis. A composition for viral drug delivery. 7. The composition according to claim 6, wherein the AAP or pAAP delivers the drug into the nucleus of the cell. 13. The non-viral drug delivery composition according to claim 12, wherein the AAP or pAAP binds to glucocorticoid receptor in the cytoplasm to deliver the drug into the nucleus. The non-viral drug delivery composition according to claim 6, wherein the pAAP exhibits an antioxidative effect. Ascorbic acid 2-phosphate (AAP), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) Mixing and stirring N-hydroxysuccinimide (NHS) to effect polymerization; And the polymerized mixture was dialyzed to obtain poly (ascorbic acid 2-phosphate); (ascorbic acid 2-phosphoric acid) polymer compound comprising the step of separating and purifying the pAAP polymer. 16. The method according to claim 15, wherein the poly (ascorbic acid-2-phosphate) polymer is represented by the following formula (1):
[Chemical Formula 1]

In the above formula (1), n is an integer of 4 to 200.

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