KR20180118976A - Poly(Pyridoxal 5-phosphate) which has endosomolytic activity and nuclear delivery activity, Its synthesis, and Use thereof - Google Patents

Abstract

The present invention relates to a composition having endosomolysis activity and a composition for delivering a non-virus drug which include poly(pyridoxal 5-phosphate) (pP5P), which is a polymer of a pyridoxal 5-phosphate (P5P), as an effective ingredient, or a manufacturing method of a pP5P compound. The pP5P exhibits not only endosomolysis activity but also a function to deliver a drug into a nucleus, antioxidant activity and solubility at neutral pH, so that the composition delivers a non-virus drug into a body to be usefully used as the composition for delivering a drug capable of maximizing bioavailability.

Description

(Pyridoxal 5-phosphate) Polymer Having Endosome Resolution and Nuclear Transfer Capability, Poly (Pyridoxal 5-phosphate) Polymer, Poly (Pyridoxal 5-phosphate) , ≪ / RTI > Its synthesis, and Use thereof}

The present invention relates to a poly (pyridoxal 5-phosphate) polymer having endocytotic resolution and nuclear transport capability, and a method for producing the same and a use thereof.

(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 cells and nuclear delivery restriction by nuclear membranes are important barriers to drug efficacy improvement. 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.

Furthermore, vitamin B6 (vitamin B6, pyridoxal 5-phosphate), which is used as a coenzyme in anti-oxidant and in vivo reactions, reduces the oxidative stress, . However, since vitamin B6 has a low solubility (5 g / L) in vivo pH (pH 7.4), its application is limited because it must be further added with NaOH in order to dissolve in water.

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, Does not mention the endosomal resolving ability or the intracellular delivery ability of the pyridoxal 5-phosphate polymer described in the above.

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 the poly (pyridoxal 5-phosphate) polymer compound contained in the composition.

In order to accomplish the above object, the present invention provides a poly (pyridoxal 5-phosphate) represented by the following formula (1); pP5P] polymer 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 poly (pyridoxal 5-phosphate); pP5P] polymer as an active ingredient.

In order to achieve the above-mentioned further object, the present invention provides a process for preparing a pyridoxal 5-phosphate (P5P), 1-ethyl-3- (3- dimethylaminopropyl) carbodiimide 3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS); And the polymerized mixture was dialyzed to obtain poly (Pyridoxal 5-phosphate); separating and purifying the < RTI ID = 0.0 > pP5P < / RTI > (Pyridoxal 5-Phosphoric Acid) Polymer Compounds.

[Chemical Formula 1]

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

The present invention relates to a poly (pyridoxal 5-phosphate) polymer exhibiting an endocome resolution and an enhancement of nuclear transport function, and its synthesis and use. The poly (pyridoxal 5-phosphate) The drug is delivered from a non-viral drug delivery system containing the poly (pyridoxal 5-phosphate) polymer to the cytoplasm of endosomes, the delivery efficiency of the drug delivery system in the cytoplasm is enhanced, The poly (pyridoxal 5-phosphate) polymer can enhance the cell survival rate in the presence of active oxygen through the antioxidant function. Since the polymer exhibits water solubility unlike the poorly soluble monomer, the drug effectively transports the drug in vivo And can be utilized as a novel drug delivery system.

1 shows the results of measurement of the molecular weight of poly (pyridoxal 5-phosphate) (pP5P) synthesized by gel permeation chromatography.
Figure 2 shows the results of evaluating the proton buffering capacity of pP5P.
Figure 3 shows the particle size and surface charge of the bPEI25kDa / pP5P-pDNA complex.
FIG. 4 shows the expression efficiency of luciferase gene of bPEI25kDa / pP5P-pDNA complex in various cells. More specifically, HEK293 cells (4a), dental pulp stem cells (DPSCs) -derived stem cells, adipose-derived stem cells; 4c), the expression of luciferase gene of bPEI25kDa / pP5P-pDNA complex.
FIG. 5 shows luciferase gene expression efficiency of bPEI25kDa / pP5P-pDNA complex in the presence of chloroquine or Bafilomycin A1 in HEK293 cells.
Figure 6 shows the inflow of cells and nuclei of gene drugs in the bPEI25kDa / pP5P-pDNA complex in HEK293 cells.
FIG. 7 shows the results of transfection of bPEI25kDa / pP5P-pDNA complexes in various cells. As a result, HEK293 cells (7a) were cultured in the presence or absence of fetal bovine serum (FBS) , DPSCs (7b), and ADSCs (7c), respectively, when the bPEI25kDa / pP5P-pDNA complex was transfected.
FIG. 8 shows the results of confirming the cell survival rate of DPSCs treated with bPEI25kDa / pP5P-pDNA complex under the condition of inducing active oxygen by hydrogen peroxide (H ₂ O ₂ ).

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

The inventors of the present invention have found that the pyridoxal 5-phosphate polymer has a hydrogen ion buffering ability to destabilize or destroy the endosomal membrane, thereby allowing the biofunctional drug capable of escaping the drug, which has been delivered into the cell, . Furthermore, it has been confirmed that the drug delivery system containing poly (pyridoxal 5-phosphate), which has exited Endosomes, has a function of effectively delivering drugs into the nucleus, and developed a drug delivery system using the drug.

In addition, the present invention has been completed by confirming that poly (pyridoxal 5-phosphate) has an antioxidative function like pyridoxal 5-phosphate by enhancing cell viability in an oxidative stress environment.

Accordingly, the present invention relates to poly (pyridoxal 5-phosphate) represented by the following formula (1): poly (pyridoxal 5-phosphate); < / RTI > pP5P] polymer as an active ingredient;

[Chemical Formula 1]

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

As used herein, the term " P5P "refers to pyridoxal 5-phosphate and the term" pP5P "refers to a synthesized poly (pyridoxal 5-phosphate) ≪ / RTI >

The poly (pyridoxal 5-phosphate) polymer is characterized by being negatively charged.

Since the poly (pyridoxal 5-phosphate) polymer has proton buffering activity, it can exhibit endosomal resolving ability by destabilizing or destroying the endosomal membrane to induce endosomal escape of the delivery drug have.

In addition, the poly (pyridoxal 5-phosphate) polymer can transfer a drug into the nucleus of a cell. In particular, it can solve the phenomenon of intracellular entry into the nucleus of a macromolecular drug such as protein and gene.

Furthermore, in one embodiment of the present invention, it has been confirmed that the composition comprising the poly (pyridoxal 5-phosphate) polymer improves the cell survival rate under reactive oxygen-induced conditions of oxidative stress using hydrogen peroxide. That is, the poly (pyridoxal 5-phosphate) polymer may exhibit an antioxidative effect.

In addition, since the poly (pyridoxal 5-phosphate) polymer can be solubilized at a neutral pH, that is, unlike the pyridoxal 5-phosphate, which is a monomer, easily soluble in water regardless of pH, Can exhibit low solubility in the in vivo pH (pH 7.4) and solve the disadvantage of using a basic solution together for solubilization.

Further, the present invention relates to a poly (pyridoxal 5-phosphate) represented by the following general formula (1): poly (pyridoxal 5-phosphate); pP5P] polymer as an active ingredient;

[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.

The poly (pyridoxal 5-phosphate) polymer has hydrogen ion proton buffering activity and can induce endosomal escape of the drug through endosomolysis, And can be usefully utilized as a composition for drug delivery.

In addition, the poly (pyridoxal 5-phosphate) polymer can transfer the drug into the nucleus of the cell, and can remarkably improve the bioavailability of the drug to be delivered into the nucleus of the cell, such as a nonviral gene.

Moreover, in one embodiment of the present invention, it has been confirmed that the drug composition comprising the poly (pyridoxal 5-phosphate) polymer improves the cell survival rate under reactive oxygen-induced conditions of oxidative stress using hydrogen peroxide , The poly (pyridoxal 5-phosphate) polymer may exhibit an antioxidative effect, and thus may be usefully used as a drug delivery composition.

Since the poly (pyridoxal 5-phosphate) polymer can be solubilized at a neutral pH, that is, unlike the monomer pyridoxal 5-phosphate, the poly (pyridoxal 5-phosphate) It has a low solubility at pH (pH 7.4) and can be used as a drug delivery composition which has a disadvantage of using a basic solution together for solubilization.

In addition, in an embodiment of the present invention, the drug delivery composition containing the poly (pyridoxal 5-phosphate) polymer significantly inhibits the decrease of the cell survival rate in the medium containing no bovine serum, In the medium, the amount of decrease in gene expression efficiency is remarkably reduced, and in particular, it can be usefully used to induce 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 drug delivery composition containing the poly (pyridoxal 5-phosphate) polymer may be used in stem cell culture to control the differentiation of stem cells.

In the present invention, the composition for drug delivery may further comprise at least one pharmaceutically acceptable carrier, excipient or diluent besides a pharmacologically effective amount of the drug, the poly (pyridoxal 5-phosphate) polymer and the polymer for drug delivery have.

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.

The present invention also relates to a process for the preparation of pyridoxal 5-phosphate (P5P), 1-ethyl-3- (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 (Pyridoxal 5-phosphate); separating and purifying the < RTI ID = 0.0 > pP5P < / RTI > (Pyridoxal 5-Phosphoric Acid) Polymer Compounds.

It is preferable that the synthesis and separation and purification of the pP5P polymer as described above proceed under the condition that the light is blocked.

Preferably, the mixed and stirred mixture may be a mixture of P5P: 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 (pyridoxal 5-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 Synthesis of polymer (pP5P) of pyridoxal 5-phosphate (P5P)

(P5P), 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 (pyridoxal 5-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 pyridoxal 5-phosphate. Was carried out at room temperature. Then, the aqueous solution remaining in the dialysis membrane was lyophilized to obtain poly (Pyridoxal 5-phosphate); pP5P] polymer.

[Reaction Scheme 1]

Example 2 Gel Permeation Chromatography analysis of synthesized pP5P

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 of the synthesized pP5P was shown in FIG. .

Referring to FIG. 1, the number average molecular weight of pP5P was 7.2 × 10 ⁵ Daltons, the weight average molecular weight was 9.4 × 10 ⁵ Daltons, and the polydispersity thereof was 1.3.

Example 3 Evaluation of Proton Buffering Capacity of pP5P

pP5P was dissolved in a 150 mM aqueous solution of sodium chloride to a concentration of 1 mg / mL, followed by addition of a small amount of 1 M sodium hydroxide aqueous solution to a pH of about 11. The pH change of the aqueous solution was measured while 0.1 M hydrogen chloride was added to the aqueous solution of pP5P.

As a result, as shown in FIG. 2, it was confirmed that the sodium chloride aqueous solution had no hydrogen ion buffering ability at pH 5 to 7, which is the pH range of the endosome, while pP5P had the hydrogen ion buffering ability. The ability was 3.2 μmol / mg pP5P.

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 pP5P

(bPEI25kDa, molecular weight 25 kDa) solution and a negative charge solution containing plasmid DNA (pDNA) and pP5P were prepared in order to encapsulate pP5P in a well-known polymer / gene complex, bPEI25kDa / pP5P-pDNA complex. The N / P ratio was prepared to be 5, in which phosphate of pP5P was not taken into consideration and was calculated only with the amine of pPEI25kDa and the phosphate of pDNA.

As shown in FIG. 3, the particle sizes of the bPEI25kDa / pP5P-pDNA complexes were 100 nm and 2 nmol when pP5P was 1 or 3 nmol per 1 microgram of pDNA, 300 nm. The zeta potentials were positively charged up to 1 nmol of pP5P and negatively charged above 2 nmol.

In addition, the ability of the prepared bPEI25kDa / pP5P-pDNA complex to improve the gene expression efficiency of the encapsulated pP5P was evaluated by comparing it to the bPEI25kDa / pDNA complex without pP5P. 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 and cultured for 24 hours, and 1 hour before transfection The transfection medium containing no serum or the culture medium containing bovine serum was replaced and the prepared bPEI25kDa / pP5P-pDNA complex solution was transfected into the cultured cells. After culturing for 4 hours, the culture medium was changed to a culture medium, and further cultured for 44 hours. After 48 hours of transfection, the gene expression efficiency of this cell was evaluated using the expression level of luciferase of pDNA. When the concentration of the encapsulated pP5P was 0, the control complex bPEI25kDa / pDNA complex was formed, Gene expression efficiency.

As a result, when the bPEI25kDa / pP5P-pDNA complex was transfected into HEK293 cells in the culture medium containing the right serum as shown in Fig. 4A, the gene expression efficiency was about 88 times when the pP5P concentration was 3 nmol / μg pDNA Respectively. In addition, as shown in FIG. 4B, when the bPEI25kDa / pP5P-pDNA complex was transfected into DPSCs cells cultured in a medium for transfection not containing the right serum, when the pP5P concentration was 2 nmol / μg pDNA, gene expression The efficiency was doubled compared to the transfected bPEI25kDa / pDNA, and when transfected into DPSCs cultured in culture medium containing the right serum, the concentration of pP5P was 3 nmol / μg pDNA , Which was approximately 18-fold greater than the transfected bPEI25kDa / pDNA control. Furthermore, as shown in Fig. 4C, when the bPEI25kDa / pP5P-pDNA complex was transfected into ADSCs cultured in a medium for transfection not containing the right serum, when the pP5P concentration was 3 nmol / μg pDNA, gene expression The efficiency was increased about 65-fold compared to the transfected bPEI25kDa / pDNA, and when transfected into ADSCs cultured in the culture medium containing the right serum, the concentration of pP5P was 3 nmol / μg pDNA , The gene expression efficiency was about 21 times higher than that of the control complex bPEI25kDa / pDNA.

< Example  5> Vathulomycin  A1 ( Bafilomycin  A1) or Chloroquine  When present pP5P  Evaluation of Gene Expression Efficiency of Polymer Drug Delivery System

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, chloroquine (20 μM), which is a reagent for destroying endosomes during transfection of bPEI25kDa / pP5P-pDNA complex, or Bafilomycin A1 (Bafilomycin A1; 50 nM), a reagent for preventing the pH reduction of endosomes, was added to the medium and the gene expression efficiency was evaluated according to the method of Example 4 above.

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 into HEK293 cells, the gene expression efficiency was increased 34 times when chloroquine was present, and when the barprotoxin A1 was present, Expression efficiency was decreased 3.3-fold. On the other hand, transfection of bPEI25kDa / pP5P-pDNA complex containing pP5P showed 3.8-fold increase in gene expression efficiency in the presence of chloroquine and 10-fold decrease in gene expression efficiency in the presence of barfilomycin A1.

These results suggest that the polymer gene complex containing pP5P enters the cell via endocytosis and that the bPEI25kDa / pP5P-pDNA complex escapes the endosomes by hydrogen ion buffering of pP5P.

< Example  6> pP5P  Measurement of cell and nuclear fluxes of gene drugs in the polymer gene drug delivery system

In order to investigate the effect of pP5P 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) A solution of positively charged polyethyleneimine (bPEI25kDa, molecular weight 25kDa) and a negative charge solution containing pDNA-YOYO-1 and pP5P were prepared, and then the two solutions were mixed to prepare a bPEI25kDa / pP5P-pDNA complex.

The prepared bPEI25kDa / pP5P-pDNA complex was then compared to the bPEI25kDa / pDNA complex without pP5P to evaluate the inflow of the gene drug into the nucleus by the encapsulated pP5P. 1x10 &lt; ⁵ &gt; HEK293 cells were added per well of a 6-well plate and cultured for 24 hours, and the prepared bPEI25kDa / pP5P-pDNA complex or bPEI25kDa / pDNA complex solution was transfected into the cells. 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 the bPEI25kDa / pP5P-pDNA complex through a flow cytometer.

At this time, when the concentration of the encapsulated pP5P 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 the complex in HEK293 cells.

As a result, as shown in Fig. 6, in the case of the gene drug of bPEI25kDa / pP5P-pDNA complex, when the concentration of pP5P was 2 nmol / μg pDNA, the intracellular inflow was smaller than that of the bPEI25kDa / When the concentration of pP5P was 1 nmol / μg pDNA or 3 nmol / μg pDNA, the result was similar to that of the control complex bPEI25kDa / pDNA complex. On the other hand, as the concentration of pP5P in the nucleus of the gene product of the bPEI25kDa / pP5P-pDNA complex increased, the intracellular inflow of the gene drug increased and when the concentration of pP5P was 3 nmol / μg pDNA, the control complex bPEI25kDa / The intracellular inflow was about 1.8 times higher than the pDNA complex gene drug. Furthermore, when the concentration of pP5P was 2 nmol / μg pDNA and the same amount of the gene drug as the control complex bPEI25kDa / pDNA was introduced into the cell, the intracellular inflow of the gene drug contained in the bPEI25kDa / pP5P-pDNA complex was about 2.7 Times higher.

< Example  7> pP5P  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 / pP5P-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 HEK293, DPSCs, and ADCSCs cells were added per well of a 24-well plate and cultured for 24 hours. The bPEI25kDa / pDNA complex, which is the control complex without the pP5P and the prepared bPEI25kDa / pP5P- The 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 exposed to the culture medium containing the right serum and when the survival rate of the cells not treated with the polymer gene drug transporter was 100%, when the polymer gene drug carrier containing the bPEI25kDa / pDNA complex and pP5P was transfected, Cell survival rates were compared.

As a result, as shown in Figs. 7A to 7C, when transfection was performed in a medium containing HEK293 (Fig. 7A), DPSCs (Fig. 7B) and ADSCs The bPEI25kDa / pP5P-pDNA complex showed cell viability close to 100%, whereas when transfection was performed in the medium without the right serum, the cell viability decreased to about 20% regardless of the type of complex Respectively. Therefore, there is no toxicity induced by pP5P, and pP5P can also be used for cell culture in the medium containing the right serum.

< Example  8> pP5P  Confirmation of inhibition of cell death by reactive oxygen species of polymer gene drug transporter

The bPEI25kDa / pP5P-pDNA complex prepared in the same manner as in Example 4 was compared with the bPEI25kDa / pDNA complex without pP5P to evaluate the antioxidative ability of the encapsulated pP5P.

Specifically, 5 x 10 ³ DPSCs cells per well of a 96-well plate were cultured for 24 hours, and the prepared bPEI25kDa / pP5P-pDNA complex and bPEI25kDa / pDNA complex solution were treated on the cultured cells. 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 7 .

As shown in FIG. 8, in the case of treatment with H ₂ O ₂ , cell viability was reduced by 30% when treated with the bPEI25kDa / pDNA control complex containing 12.5 μg / mL pDNA, and 25 μg / mL pDNA , The cell viability was reduced by 90% when treated with the bPEI25kDa / pDNA complex. Whereas bPEI25kDa / pP5P-pDNA complex containing 12.5 μg / mL and 25 μg / mL of pDNA showed 20% and 40% cell survival reduction, respectively. In addition, when active 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 / pP5P-pDNA complex Survival was reduced by 40-60%.

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 (15)

Poly (pyridoxal 5-phosphate) represented by the following formula (1); pP5P] polymer as an active ingredient;
[Chemical Formula 1]

In the above formula (1), n is an integer of 4 to 200. The poly (pyridoxal 5-phosphate) polymer according to claim 1, wherein the poly (pyridoxal 5-phosphate) polymer has proton buffering activity and destabilizes or destroys the endosomal membrane to induce an endosomal escape of the drug Wherein the composition has an ability to degrade endosomes. The composition of claim 1, wherein the poly (pyridoxal 5-phosphate) polymer is capable of delivering a drug into the nucleus of a cell. The composition according to claim 1, wherein the poly (pyridoxal 5-phosphate) polymer exhibits an antioxidative effect. The method of claim 1, wherein the poly (pyridoxal 5-phosphate) polymer is solubilized at neutral pH &Lt; / RTI &gt; wherein the composition has endosome resolution. Poly (pyridoxal 5-phosphate) represented by the following formula (1); pP5P] polymer as an active ingredient;
[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 drug delivery polymer of claim 8, 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 poly (pyridoxal 5-phosphate) polymer has proton buffering activity, thereby inducing endosomal escape of the drug through endosomolysis Wherein the drug delivery system is a non-viral drug delivery system. The non-viral drug delivery composition according to claim 6, wherein the poly (pyridoxal 5-phosphate) polymer transports the drug into the nucleus of the cell. The non-viral drug delivery composition according to claim 6, wherein the poly (pyridoxal 5-phosphate) polymer exhibits an antioxidative effect. 7. The composition for non-viral drug delivery according to claim 6, wherein the poly (pyridoxal 5-phosphate) polymer is solubilized at neutral pH. Pyridoxal 5-phosphate (P5P), 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 (Pyridoxal 5-phosphate); separating and purifying the &lt; RTI ID = 0.0 &gt; pP5P &lt; / RTI &gt; (Pyridoxal 5-phosphate) polymer compound. 15. The method of producing a poly (pyridoxal 5-phosphate) polymer according to claim 14, wherein the poly (pyridoxal 5-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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