KR101929828B1 - Reductive degradable poly(ε-caprolactone) polymer with hydrophilic carboxyl group at both ends and drug delivery composition using the same - Google Patents

Abstract

The present invention relates to reductive degradable multiple disulfide poly(ε-caprolactone) (MSPCL) or multiple diselenide poly(ε-caprolactone) (MSePCL) polymer, in which both terminals are carboxyl groups, and to a drug delivery composition using the polymer. The synthesized MSPCL polymer and MSePCL polymer form nanoparticles through self-assembly in a water phase, and can also serve as a carrier capable of delivering various drugs into cells.

Description

(Epsilon-caprolactone) polymer having a hydrophilic carboxyl group at both ends and a drug delivery composition using the polymer, and a composition for drug delivery using the polymer }

The present invention relates to a reductive degradable poly (epsilon-caprolactone) polymer having hydrophilic carboxyl groups at both ends and a composition for drug delivery using the polymer.

Polymers are used in many fields because they can form various kinds of structures such as straight line, branching and star shape by different composition and shape. The amphipathic polymer capable of self-assembly in an aqueous solution has a high molecular weight of 5 kDa or more since a hydrophobic polymer and a hydrophilic polymer are linked in various forms, and a macromolecular polymeric macromolecule and a micelle- And thus it is possible to efficiently encapsulate various chemicals and biologics. In addition, commonly used amphipathic polymers of 5 kDa or more mainly have hydrophilic moieties of about 27% to about 50% by weight, and in particular lipids commonly used in liposomal formulations have a weight ratio of about 20% to about 26% % Level of hydrophilic moiety. However, high molecular weight substances can cause the possibility of intracellular accumulation and undesired interaction with the intracellular major components due to the slow degradation rate in vivo, and thus, such as phospholipids and lipids, which are low molecular weight amphipathic substances constituting the cell membrane A method of simulating the characteristics of components in vivo, or a method using a substance having biocompatibility and irritation reactivity.

The hydrolysis of polyester in polyester-based nanostructures is difficult to induce rapid release of drugs in the drug organs. In order to overcome these problems, a chemical bond or a component that specifically reacts to intracellular stimuli such as reducing conditions, light, pH, enzymes and the like may be added to promote rapid drug release in the intracellular environment. However, the weakly acidic pH condition has a disadvantage in that it is difficult to clearly distinguish the endosomes in the endosomes and the pH environment inside the lysosome as well as the pathological environment such as the extracellular solid tumor periphery and the infection site. In addition, drug release from the drug delivery system can be promoted by various enzymes present in the rysosome, but if the released drug does not escape the lysosome, the therapeutic effect due to the degradation of the drug by the degradation enzyme may be reduced.

Recently, many researchers have reported that the extracellular concentration of glutathione, a biological thiol (~ SH, thiol), is 0.02 ~ 2 μM, the concentration in the cytoplasm and nucleus within the cell is 1-11 mM, the disulfide bond (~ Se-Se ~) and chiol (~ SH). In contrast to pH and enzymes, the difference in glutathione concentration between the extracellular environment and the intracellular environment causes the drug carrier to be stable in the extracellular environment, allowing the encapsulated drug to be delivered to the cell without loss, (~ SH) by disruption of disulfide bonds and diselenide bonds present in the drug delivery system may lead to rapid drug release.

The present invention relates to a disulfide-bonded or multi-disulfide bond having a carboxyl group at both ends by linking a low molecular weight carboxyl group as a hydrophilic moiety and a reducing degradable poly (epsilon-caprolactone) having a poly-disulfide bond or a multiple diselenide bond as a hydrophobic moiety Carboxylated multiple disulfide poly (ε-caprolactone) with a selenide bond; MSPCL and carboxylated multiple diselenide poly (ε-caprolactone); The MSPCL polymer and the MSePCL polymer synthesized MSPCL nanoparticles and MSePCL nanoparticles by self-assembly under a water environment, even though the hydrophilic block had about 5 ~ 15% .

The multiple disulfide poly (ε-caprolactone), which inhibits intracellular accumulation and nonspecific interactions using low-molecular-weight biodegradable polymers and has both terminal carboxyl groups. MSPCL and multiple diselenide poly (ε-caprolactone); MSePCL] polymer, and the carboxylate at both ends of the MSPCL polymer or MSePCL polymer at the acidic pH of endosomes and lysosomes, particularly in the periplasmic environment of the cancer cells, Carboxylic acid, which can easily enter into cancer cells or escape into the cytoplasm from endosomes. Also, low molecular weight poly (epsilon-caprolactone) [poly (ε-caprolactone); (~SH ~) and des-selenide bond (~Se-Se ~), which are bonded between the MSPCL polymer and the MSEPCL polymer, are converted to the polyol (~ SH) by the high concentration of glutathione present in the cells, Is decomposed into low molecular weight PCL. The low molecular weight biodegradable polymer produced in this process can be biocompatible because it reduces intracellular accumulation and nonspecific interaction, and MSPCL nanoparticles or MSePCL nanoparticles loaded with drug can rapidly Release from the cytoplasm to improve the effectiveness of the drug.

Korean Registered Patent No. 1208192 (Notice of April 4, 2012)

An object of the present invention is to provide a reductive decomposable poly (epsilon-caprolactone) polymer having hydrophilic carboxyl groups at both ends.

Another object of the present invention is to provide a drug delivery composition comprising the polymer as nanoparticles and nanoparticles self-assembled in an aqueous phase as an active ingredient.

In order to achieve the above object, the present invention provides a method for producing a poly (epsilon-caprolactone) comprising the step of reacting a poly (epimeric-caprolactone) with a dithiodipropionate or diselenodipropionate (Epsilon-caprolactone) polymer having a hydrophilic carboxyl group at the terminal thereof.

[Chemical Formula 1]

Wherein A is sulfur (S) or selenium (Se), x is an integer of 3 to 20, and y is an integer of 2 to 50.

The present invention also provides nanoparticles in which the polymer is self-assembled in an aqueous phase.

The present invention also provides an anticancer composition comprising the nanoparticles as an active ingredient.

The present invention also provides a drug delivery composition comprising the nanoparticles as an active ingredient.

The present invention relates to a reductive degradable poly (epsilon-caprolactone) polymer having hydrophilic carboxyl groups at both ends and a composition for drug delivery using the polymer. The synthesized polymer can form nanoparticles through self-assembly in an aqueous phase , And can also serve as a carrier capable of inducing the death of cancer cells by encapsulating various drugs and delivering them into cells.

1 is a schematic diagram of a multiple disulfide poly (ε-caprolactone) with multiple disulfide bonds having carboxyl groups at both ends; MSPCL "]< / RTI >polymer;
Figure 2 is a schematic diagram of a multiple diselenide poly (ε-caprolactone) with multiple diselenide bonds with both terminal carboxyl groups; MSePCL "]< / RTI >polymer;
3 is a ¹ H-NMR spectrum of PCL _0.5 material and MSPCL _0.5 polymer;
4 is a ¹ H-NMR spectrum of PCL _1.25 material and MSPCL _1.25 polymer;
5 is a ¹ H-NMR spectrum of PCL _2.0 material and MSPCL _2.0 polymer;
6 is a ¹ H-NMR spectrum of PCL _0.5 material and MSePCL _0.5 polymer;
7 is a ¹ H-NMR spectrum of PCL _1.25 material and MSePCL _1.25 polymer;
8 is a ¹ H-NMR spectrum of PCL _2.0 material and MSePCL _2.0 polymer;
9 is a graph showing the results of GPC analysis of MSPCL _0.5 , MSPCL _1.25 and MSPCL _2.0 polymer;
10 is a graph showing the results of GPC analysis of MSePCL _0.5 , MSePCL _1.25 and MSePCL _2.0 polymers;
Figure 11 shows the critical micelle concentration of MSPCL _0.5 , MSPCL _1.25 and MSPCL _2.0 block copolymer;
Figure 12 shows the critical micelle concentration of MSePCL _0.5 , MSePCL _1.25 and MSePCL _2.0 block copolymer;
13 is a graph showing changes in particle size with time of MSPCL _0.5 nanoparticles under reducing conditions such as chiol (~ SH);
14 is a graph showing changes in particle size with time of MSPCL _1.25 nanoparticles under reducing conditions such as chiol (~ SH);
15 is a graph showing changes in particle size with time of MSPCL _2.0 nanoparticles under reducing conditions such as chiol (~ SH);
FIG. 16 is a graph showing changes in particle size with time of MSePCL _0.5 nanoparticles under reducing conditions such as chiol (~ SH);
17 is a graph showing changes in particle size with time of MSePCL _1.25 nanoparticles under reducing conditions such as chiol (~ SH);
18 shows changes in particle size with time of MSePCL _2.0 nanoparticles under reducing conditions such as chiol (~ SH);
19 shows the results of evaluating the toxicity of MSPCL _0.5 , MSPCL _1.25 and MSPCL _2.0 nanoparticles on HepG2 cells;
20 shows the results of evaluating the anticancer properties of MSePCL _0.5 , MSePCL _1.25 and MSePCL _2.0 nanoparticles on HepG2 cells;
Figure 21 shows drug release over time for DOX @ MSPCL _0.5 , DOX @ MSPCL _1.25 and DOX @ MSPCL _2.0 nanoparticles;
Figure 22 depicts drug release over time for DOX @ MSPCL _0.5 , DOX @ MSPCL _1.25 and DOX @ MSPCL _2.0 nanoparticles under reducing conditions such as chiol (~ SH);
Figure 23 shows drug release over time for DOX @ MSPCL _0.5 and DOX @ MSePCL _0.5 nanoparticles depending on the presence or absence of cholol (~ SH);
Figure 24 shows drug release over time for DOX @ MSePCL _0.5 , DOX @ MSePCL _1.25 and DOX @ MSePCL _2.0 nanoparticles under reducing conditions such as chiol (~ SH);
FIG. 25 shows the results of evaluating the anticancer properties of DOX @ MSPCL _0.5 , DOX @ MSPCL _1.25 and DOX @ MSPCL _2.0 nanoparticles on HepG2 cells;
26 is a graph showing the results of evaluating anticancer properties of DOX @ MSePCL _0.5 , DOX @ MSePCL _1.25 and DOX @ MSePCL _2.0 nanoparticles on HepG2 cells;
FIG. 27 is a graph showing drug fluorescence intensities of DOX @ MSPCL _0.5 , DOX @ MSPCL _1.25 and DOX @ MSPCL _2.0 nanoparticles introduced into HepG2 cells;
FIG. 28 shows drug fluorescence intensities of DOX @ MSPCL _0.5 , DOX @ MSPCL _1.25 and DOX @ MSPCL _2.0 nanoparticles introduced into the nucleus of HepG2 cells;
FIG. 29 shows the drug fluorescence intensities of DOX @ MSPCL _0.5 , DOX @ MSPCL _1.25 and DOX @ MSPCL _2.0 nanoparticles introduced into the mitochondria of HepG2 cells;
FIG. 30 is a graph showing drug fluorescence intensities of DOX @ MSePCL _0.5 , DOX @ MSePCL _1.25 and DOX @ MSePCL _2.0 nanoparticles introduced into HepG2 cells;
31 is a graph showing drug fluorescence intensities of DOX @ MSePCL _0.5 , DOX @ MSePCL _1.25 and DOX @ MSePCL _2.0 nanoparticles introduced into the nucleus of HepG2 cells;
FIG. 32 shows drug fluorescence intensities of DOX @ MSePCL _0.5 , DOX @ MSePCL _1.25 and DOX @ MSePCL _2.0 nanoparticles introduced into mitochondria of HepG2 cells;
33 is a confocal microscope image of the drug fluorescence intensity distribution of DOX @ MSPCL _0.5 , DOX @ MSPCL _1.25 and DOX @ MSPCL _2.0 nanoparticles introduced into HepG2 cells;
Figure 34 is a line profile for the distribution of drugs in the nucleus, mitochondria or cytoplasm of DOX @ MSPCL _0.5 , DOX @ MSPCL _1.25 and DOX @ MSPCL _2.0 nanoparticles for the white arrow in FIG. 33;
Figure 35 is a confocal microscope image of the drug fluorescence intensity distribution of drug-enriched MSPCL _0.5 and MSePCL _0.5 nanoparticles introduced into HepG2 cells; And
FIG. 36 is a diagram showing the line profile of the distribution of DOX @ MSPCL _0.5 and DOX @ MSePCL _0.5 nanoparticles in the nucleus, mitochondria, or cytoplasm of drug in the white arrow in FIG.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, a reductive degradable poly (Epsilon-caprolactone) polymer having hydrophilic carboxyl groups at both ends and a composition for drug delivery using the polymer will be described in more detail.

The inventors of the present invention synthesized a reductive decomposable poly (Epsilon-caprolactone) polymer having hydrophilic carboxyl groups at both ends. The synthesized polymer can form nanoparticles through self-assembly in a water phase, The present invention has been completed based on this finding.

The present invention relates to a process for producing a poly (dicumyl-caprolactone) having a hydrophilic carboxyl group at both terminals represented by the formula (1) in which dithiodipropionate or diselenodipropionate is covalently bonded to both ends of a poly Degradable poly (epsilon-caprolactone) polymer.

[Chemical Formula 1]

Wherein A is sulfur (S) or selenium (Se), x is an integer of 3 to 20, and y is an integer of 2 to 50.

In one embodiment of the present invention, the polymer represented by Formula 1 may be a reductive decomposable poly (epsilon-caprolactone) (MSPCL) polymer having multiple disulfide bonds having hydrophilic carboxyl groups at both terminals , And the polymer represented by the following formula (2) may be one in which dithiodipropionate is covalently bonded to both ends of poly (epsilon-caprolactone).

(2)

M is an integer of 3 to 20, and n is an integer of 2 to 50. [

In one embodiment of the present invention, the polymer represented by the formula (1) is a reductive decomposable poly (epsilon-caprolactone) (MSePCL) having multiple diselenide bonds having hydrophilic carboxyl groups at both terminals, And the polymer represented by the following general formula (3) may be one in which diselenodipropionate is covalently bonded to both ends of poly (epsilon-caprolactone).

(3)

In the above formula (3), o is an integer of 3 to 20, and p is an integer of 2 to 50.

The present invention also provides nanoparticles in which the polymer is self-assembled in an aqueous phase.

The nanoparticles may have an average particle diameter of 20 to 200 nm, but are not limited thereto.

In one embodiment of the present invention, the MSPCL polymer represented by Formula 2 and the MSePCL polymer represented by Formula 3 may be self-assembled in an aqueous phase to prepare MSPCL nanoparticles and MSePCL nanoparticles. MSPCL nanoparticles and MSePCL nanoparticles Is prepared by the following method, but is not limited thereto.

According to one embodiment of the present invention, the MSPCL polymer represented by Formula 2 or the MSePCL polymer represented by Formula 3 is dissolved in dimethyl sulfoxide (DMSO), stirred for 30 minutes, purified water is added Stirring was continued for another 30 minutes. The dispersion containing MSPCL nanoparticles or MSePCL nanoparticles is placed in a dialysis membrane to remove DMSO from the aqueous phase by dialysis. The dispersion remaining in the dialysis membrane was filtered with a syringe filter having a pore size of 0.45 mu m, and MSPCL nanoparticles or MSePCL nanoparticles were finally obtained. In the present invention, the PCLs having molecular weights of 530 Da, 1250 Da, and 2000 Da were named PCL _0.5 , PCL _1.25 , and PCL _2.0 , respectively. MSPCL and MSePCL polymers thereof were named MSPCL _0.5 , MSPCL _1.25 , MSPCL _2.0 , and MSePCL _0.5 , MSePCL _1.25 , and MSePCL _2.0 . The nanoparticles prepared by the above method were named MSPCL _0.5 nanoparticles (NPs), MSPCL _1.25 nanoparticles, and MSPCL _2.0 nanoparticles, MSePCL _0.5 nanoparticles, MSePCL _1.25 Nanoparticles, and MSePCL _2.0 nanoparticles.

The present invention also provides an anticancer composition comprising the nanoparticles as an active ingredient.

The cancer may be any one selected from the group consisting of thyroid cancer, stomach cancer, colon cancer, lung cancer, liver cancer, breast cancer, prostate cancer, gallbladder cancer, biliary cancer, pancreatic cancer, oral cancer, esophageal cancer, bladder cancer, colon cancer and cervical cancer. But is not limited to.

The present invention also provides a drug delivery composition comprising the nanoparticles as an active ingredient.

The drug may be an anti-cancer agent, but is not limited thereto.

The anticancer agent may be selected from the group consisting of doxorubicin, paclitaxel, epirubicin, gemcitabine, silorimus, ethoposide, vinblastine, vinca alkaloid, docetaxel, cisplatin, glivec, adriamycin, cyclophosphamide, But are not limited to, uracil, camptothecin, tamoxifen, anastrozole, flocuridine, lufurolide, flotamide, zoledronate, vincristine, streptozotocin, carboplatin, Which consists of irinotecan, vinorelbine, hydoroxyurea, valvicin, methotrexate, mechlorethamine, chlorambucil, bisulfan, doxifluridine, prednisone, testosterone, mitosartron, docetaxel, vinorelbine, and prednisolone And the like, but is not limited thereto.

According to one embodiment of the present invention, MSPCL nanoparticles and MSePCL nanoparticles self-assembled with the MSPCL polymer represented by Chemical Formula 2 or the MSePCL polymer represented by Chemical Formula 3 can carry a water-soluble chemical substance and a water-insoluble chemical substance, It is applicable as a carrier. In addition, the reduction reaction characteristics of MSPCL nanoparticles or MSePCL nanoparticles can be applied as a drug delivery system using the surrounding environment.

The anticancer composition or composition for drug delivery according to the present invention may contain a pharmaceutically effective amount of a drug alone or may include one or more pharmaceutically acceptable carrier, excipient or diluent. The pharmaceutically effective amount as used herein refers to an amount sufficient for a drug to be administered to an animal or a human to exhibit a desired physiological or pharmacological activity. However, the pharmaceutically effective amount may be appropriately changed depending on the age, body weight, health condition, sex, administration route and treatment period of the subject to be administered.

The term "pharmaceutically acceptable" as used herein means physiologically acceptable and does not generally cause an allergic reaction such as gastrointestinal disorder, dizziness, or the like when administered to a human. 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. Further, it may further include a filler, an anticoagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent and an antiseptic agent.

The anticancer composition or drug delivery composition 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 vary depending on the route of administration, the age, sex, The severity of the disease, and the like. In addition, the polymer composition for drug delivery of the present invention may be administered in combination with a known compound capable of enhancing the desired effect of the drug.

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 human, and is a chemical or biological substance (1) prevent undesired biological effects such as infection prevention and have a preventive effect on the organism, (2) alleviate the condition caused by the disease, for example, the pain or infection resulting from the disease And (3) mitigate, reduce or completely eliminate disease from organic matter.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in more detail with reference to the following Examples and Experimental Examples, which are a reductive degradable poly (Epsilon-caprolactone) polymer having hydrophilic carboxyl groups at both ends and a composition for drug delivery using the polymer. However, the present invention is not limited by these Examples and Experimental Examples.

Example 1 Synthesis and Analysis of MSPCL Polymer and MSePCL Polymer

As shown in Figure 1, the MSPCL polymer was prepared by mixing 1 mmol of polycaprolactone diol (PCL diol) and 1.17 mmol of 3,3'-dithiodipropionic acid (DTPA) in 10 ml of tetrahydrofuran The solution was prepared by dissolving in tetrahydrofuran (hereinafter referred to as 'A solution').

3.6 mmol of dicyclohexylcarbodiimide (DCC) and 3.6 mmol of 4-dimethylaminopyridine (DMAP) were dissolved in 5 ml of THF to prepare a solution (hereinafter referred to as " B solution ').

B solution was added to the solution A, 0.1 ml of triethylamine (hereinafter, referred to as 'TEA') was added, and the mixture was stirred at room temperature for 2 days under nitrogen charging.

After completion of the reaction, dicyclohexylurea (DCU), which is a by-product produced by using a filter paper, was removed and THF as a solvent was removed using a rotary evaporator. The dried product was redissolved in dichloromethane (DCM), precipitated in n-hexane, and the precipitate was vacuum dried to obtain a high molecular weight MSPCL polymer (see Chemical Formula 2 below).

(2)

As shown in Figure 2, the MSePCL polymer was prepared by mixing 1 mmol of polycaprolactone diol (PCL diol) and 1.17 mmol of 3,3'-diselenodipropionic acid (DSePA) in 10 ml of THF (Hereinafter referred to as " C solution ").

3.6 mmol of DCC and 3.6 mmol of DMAP were dissolved in 5 ml of THF to prepare a solution (hereinafter referred to as 'D solution').

D solution was added to the C solution, 0.1 ml of TEA was added, and the mixture was reacted at room temperature for 2 days under a nitrogen purge.

After completion of the reaction, DCU, which is a by-product produced by using a filter paper, is removed, and THF as a solvent is removed by using a rotary evaporator. The dried product was again dissolved in DCM, precipitated in n-hexane, and the precipitate was vacuum dried to obtain a high molecular weight MSePCL polymer (see Chemical Formula 3 below).

(3)

As shown in FIGS. 3, 4 and 5, the chemical structure of MSPCL polymers was confirmed by ¹ H-NMR, and the number of low molecular weight PCL blocks existing in MSPCL _0.5 polymer, MSPCL _1.25 polymer and MSPCL _2.0 polymer was 8 , 5, and 3, and the number of disulfide bonds was 9, 6, and 4, respectively.

6, 7 and 8, the chemical structure of the MSePCL polymer was confirmed by using ¹ H-NMR. The number of low molecular weight PCL blocks existing in the MSePCL _0.5 polymer, MSePCL _1.25 polymer and MSePCL _2.0 polymer was 3 , 3, and 3, and the number of diselenide bonds was 4, 4, and 4, respectively.

As shown in FIG. 9, the number average molecular weights of MSPCL _0.5 polymer, MSPCL _1.25 polymer and MSPCL _2.0 polymer were 6270 Da, 6520 Da, and 6880 Da, respectively, through gel permeation chromatography (hereinafter, referred to as "GPC" The dispersions were 1.66, 1.71, and 1.69, respectively.

As shown in FIG. 10, the number average molecular weights of MSePCL _0.5 polymer, MSePCL _1.25 polymer and MSePCL _2.0 polymer were 2590 Da, 4820 Da, and 7130 Da, respectively, and the polydispersities were 1.52, 1.72, and 1.55, respectively.

The MSPCL polymer and MSePCL polymer can be self-assembled according to the concentration under an aqueous solution, and the concentration dependent self-assembling property is confirmed by using pyrene. Fluorescence analysis of pyrene is an analytical method that utilizes the principle that a pyrene molecule showing hydrophobicity penetrates selectively into a hydrophobic environment under an aqueous solution to exhibit very strong fluorescence characteristics.

It does not show fluorescence below the critical micelle concentration, but it has a very strong fluorescence property and a characteristic peak of pyrene (373 nm, 393 nm) above the critical micelle concentration.

As shown in FIGS. 11 and 12, the fluorescence intensity ratio (I ₃₇₃ / I ₃₉₃ ) of the characteristic peak shows a constant value at a low concentration, and the fluorescence intensity ratio decreases sharply as the concentration increases.

The critical micelle concentration of the MSPCL _0.5 polymer, the MSPCL _1.25 polymer, and the MSPCL _2.0 polymer were 0.50 ㎍ / ㎖, 0.48 ㎍ / ㎖, and 0.44 ㎍ / ㎖, respectively, Mu] g / ml. The critical micelle concentrations of MSePCL _0.5 polymer, MSePCL _1.25 polymer and MSePCL _2.0 polymer were 1.41 ㎍ / ㎖, 1.20 ㎍ / ㎖ and 0.71 ㎍ / ㎖ respectively.

Example 2 Preparation, Physicochemical Properties and Cytotoxicity of MSPCL Nanoparticles and MSePCL Nanoparticles

To prepare MSPCL nanoparticles and MSePCL nanoparticles, MSPCL polymer and MSePCL polymer were dissolved in DMSO, and purified water was added while stirring the polymer solution. MSPCL polymers and MSePCL polymers were self assembled with water to form MSPCL nanoparticles and MSePCL nanoparticles. The dispersion containing MSPCL nanoparticles and MSePCL nanoparticles was placed in a dialysis membrane to remove DMSO from the aqueous phase by dialysis. The dispersion remaining in the dialysis membrane was filtered with a syringe filter having a pore size of 0.45 mu m, and MSPCL nanoparticles and MSePCL nanoparticles were finally obtained.

The mean particle sizes of MSPCL _0.5 nanoparticles, MSPCL _1.25 nanoparticles and MSPCL _2.0 nanoparticles were 95 nm, 72 nm, and 62 nm, respectively, and the average zeta potentials were -20 mV, -15 mV, and -12 mV.

The average particle sizes of MSePCL _0.5 nanoparticles, MSePCL _1.25 nanoparticles and MSePCL _2.0 nanoparticles were 65 nm, 51 nm, and 43 nm, respectively. The average zeta potentials were -18 mV, -13 mV, and -11 mV .

To confirm the particle size changes of MSPCL nanoparticles and MSePCL nanoparticles under reducing conditions, a solution of dithiothreitol (pH 7.4) was added to a phosphate buffer solution (pH 7.4) containing MSPCL nanoparticles and MSePCL nanoparticles (DL-dithiothreitol; hereinafter referred to as 'DTT') was added to make 10 mM.

As shown in FIG. 13, the size of the MSPCL _0.5 nanoparticles before exposure to DTT was 95 nm, but it increased to about 150 nm when it was exposed to DTT for 30 minutes and to 220 nm when it passed 120 minutes. As shown in FIG. 14, the size of the MSPCL _1.25 nanoparticles was 72 nm, but it was increased to about 300 nm when the sample was exposed to DTT for 30 minutes, and about 450 nm when the sample was 120 minutes after exposure to DTT. As shown in FIG. 15, the size of the MSPCL _2.0 nanoparticles was 62 nm, but it was increased to about 450 nm at 30 minutes after exposure to DTT and to about 600 nm at 120 minutes after exposure to DTT.

As shown in FIG. 16, the size of the MSePCL _0.5 nanoparticles before the exposure to DTT was 65 nm, but it increased to about 100 nm when elapsed after 5 minutes from DTT and about 300 nm after 60 minutes. As shown in FIG. 17, the size of the MSePCL _1.25 nanoparticles was 51 nm, but it was agglomerated to about 300 nm when elapsed 5 minutes after DTT exposure and about 550 nm after 20 minutes elapsed. As shown in FIG. 18, the size of the MSePCL _2.0 nanoparticles was 43 nm, but it was agglomerated to about 700 nm when passed 5 minutes after DTT exposure and about 1050 nm after 10 minutes.

The particle size changes of MSPCL nanoparticles and MSePCL nanoparticles under reducing conditions appeared to be due to the decomposition of MSPCL and MSePCL polymers by the polyol (~ SH), resulting in the generation of hydrophobic low molecular weight PCLs.

The anticancer effect of MSPCL nanoparticles and MSePCL nanoparticles was determined by incubating for 24 hours in 5,000 hepatocarcinoma cells (hereinafter, referred to as "HepG2 cells") on a 96-well plate and then exposing them to MSPCL nanoparticles and MSePCL nanoparticles for 48 hours At the nanoparticle concentration, cytotoxicity of cancer cells was evaluated using MTT method.

As shown in Fig. 19, the MSPCL nanoparticles were not observed at a concentration of 50% viability in HepG2 cells at an IC50 of 500 ㎍ / ml or less. As shown in Fig. 20, the IC50 values of MSePCL _0.5 nanoparticles, MSePCL _1.25 nanoparticles and MSePCL _2.0 nanoparticles were 68.7 쨉 g / ml, 167.3 쨉 g / ml, and 417 쨉 g / ml, respectively.

<Example 3> Preparation and physicochemical properties of DOX @ MSPCL nanoparticles and DOX @ MSePCL nanoparticles encapsulating doxorubicin anticancer agent

To dissolve doxorubicin (DOX) in MSPCL nanoparticles and MSePCL nanoparticles, doxorubicin hydrochloride (DOX · HCl) was dissolved in DMSO and TEA was added to DOX · HCl 3 times, and stirred for 16 hours or more to prepare hydrogen chloride-free DOX.

0.5 mg of DOX solution was mixed with 2 mg of MSPCL polymer and 0.1 ml of DMSO solution in which MSePCL polymer was dissolved, 4 ml of purified water was added while stirring the drug-polymer solution, and further stirred for 1 hour. DMSO and DOX not contained in the nanoparticles were removed by dialysis in the aqueous phase, and the remaining unoxidized DOX was removed by filtration using a syringe filter having a pore size of 0.45 mu m.

MSPCL _0.5 nanoparticles, DOX @ MSPCL _1.25 nanoparticles, and DOX @ MSPCL _2.0 nanoparticles according to their PCL molecular weights. The nanoparticles synthesized as described above were named DOX @ MSPCL and DOX @ MSePCL, And DOX @ MSePCL _0.5 nanoparticles, DOX @ MSePCL _1.25 nanoparticles and DOX @ MSePCL _2.0 nanoparticles.

The average particle sizes of the prepared DOX @ MSPCL _0.5 nanoparticles, DOX @ MSPCL _1.25 nanoparticles and DOX @ MSPCL _2.0 nanoparticles were 120 nm, 179 nm, and 193 nm, respectively, and the average zeta potentials were 1.7 mV, 3.3 mV, And 4.9 mV.

The average particle sizes of the prepared DOX @ MSePCL _0.5 nanoparticles, DOX @ MSePCL _1.25 nanoparticles and DOX @ MSePCL _2.0 nanoparticles were 242 nm, 284 nm, and 293 nm, respectively, and the average zeta potentials were 4.8 mV and 6.1 mV, and 7.5 mV, respectively.

When DOX was enclosed within MSPCL nanoparticles and MSePCL nanoparticles at a target weight of drug of 20 wt%, the actual drug content of DOX @ MSPCL _0.5 nanoparticles, DOX @ MSPCL _1.25 nanoparticles and DOX @ MSPCL _2.0 nanoparticles The average weight contents were 3.1 wt%, 4.1 wt%, and 4.5 wt%, respectively, and the average filling efficiency of the drug was 15.7%, 20.3%, and 22.4%, respectively.

In addition, the average weight content of actual drugs of DOX @ MSePCL _0.5 nanoparticles, DOX @ MSePCL _1.25 nanoparticles and DOX @ MSePCL _2.0 nanoparticles were 4.6 wt%, 5.9 wt%, and 6.2 wt%, respectively, Were 22.9%, 27.9%, and 30.8%, respectively.

The in vitro release experiment of DOX was carried out by shaking (100 rpm) at 37 ° C in a constant temperature shaker. Under the conditions similar to the intracellular reducing environment of the blood environment and cancer cells as shown in FIGS. 21, 22, 23, SH) DTT on the drug release of nanoparticles.

At pH 7.4 without DTT, the DOX @ MSPCL nanoparticles released 5-7% DOX for 24 hours (see FIG. 21) and the DOX @ MSPCL nanoparticles at pH 7.4 with 10 mM DTT 34 to 38% of the drug was released (see Fig. 22).

Regardless of the presence or absence of DTT, the drug release rate was independent of PCL molecular weight. When DTT is not, DOX @ MSPCL _0.5 nanoparticles and DOX @ MSePCL _0.5 were similar for 24 hours as the drug release rate of the nano-particles 23 (about 7% release), when DTT is present in 10 mM, DOX @ MSePCL _0.5 The release rate of nanoparticles was faster than the release rate of DOX @ MSPCL _0.5 nanoparticles (54% versus 34% released over 24 hours). That is, nanoparticles with diselenide bonds rather than disulfide bonds are more likely to release drugs according to DTT. In Figure 24, the drug released faster in the order of DOX @ MSePCL _2.0 nanoparticles, DOX @ MSePCL _1.25 nanoparticles, and DOX @ MSePCL _0.5 nanoparticles.

<Example 4> Anticancer effect of DOX @ MSPCL nanoparticles and DOX @ MSePCL nanoparticles encapsulating doxorubicin anticancer agent

To evaluate the anticancer effect of DOX @ MSPCL nanoparticles and DOX @ MSePCL nanoparticles prepared from Example 3, 5,000 HepG2 cells were cultured on a 96-well plate for 24 hours, and then cancer cells were treated with DOX @ MSPCL nanoparticles and DOX @ MSePCL nanoparticles were treated for 48 hours to evaluate the effect of the drug by the MTT method.

As shown in FIG. 25, the IC50 of DOX to DOX @ MSPCL _0.5 nano particles, DOX @ MSPCL _1.25 nano particles and DOX @ MSPCL _2.0 nano particles were 2.14 .mu.g / ml and 3.71 Mu] g / ml, and 6.88 [mu] g / ml. These results indicate that the anticancer effects of DOX @ MSPCL _0.5 nanoparticles, DOX @ MSPCL _1.25 nanoparticles, and DOX @ MSPCL _2.0 nanoparticles are about 2.1, 3.6 and 6.7 times lower than those of DOX, respectively.

As shown in FIG. 26, the IC50 values of DOX @ MSePCL _0.5 nanoparticles, DOX @ MSePCL _1.25 nanoparticles and DOX @ MSePCL _2.0 nanoparticles were 3.35, 2.78, and 1.32 .mu.g / Respectively, and 3.3 times, 2.7 times, and 1.3 times lower, respectively.

Example 5 Drug Infusion and Drug Distribution of DOX @ MSPCL Nanoparticles and DOX @ MSePCL Nanoparticles

Evaluation of intracellular drug uptake (CU), nuclear uptake (NU) and mitochondrial uptake (MU) of DOX @ MSPCL nanoparticles and DOX @ MSePCL nanoparticles prepared from Example 3 HepG2 cells ^{(5 × 10 5 cells in 2} ㎖) to and then incubated for 6-well plates to spread 24 hours, the DOX and DOX-loaded nanoparticles ([DOX] = 5 ㎍ / ㎖) for 4 hours to And analyzed the drug infusion rate by their cell and organelle.

To assess the intracellular flux (CU) of DOX and DOX loaded nanoparticles, cells were washed twice with DPBS and separated with trypsin-EDTA. Nuclear inflow (MU) and mitochondrial inflow (MU) were separated using a FACSCanto ^™ flow cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA) and primary argon laser using the Nuclei PURE Prep Kit and the Mitochondrial Fractionation Kit And the fluorescence of the drug introduced into the cell was measured and analyzed.

As shown in FIG. 27, the intracellular drug inflow (CU) of DOX @ MSPCL _0.5 nanoparticles, DOX @ MSPCL _1.25 nanoparticles, and DOX @ MSPCL _2.0 nanoparticles, when the intracellular drug inflow (CU) Were 0.4 ± 0.04, 0.55 ± 0.01, and 0.61 ± 0.03, respectively. As shown in FIG. 28 and FIG. 29, when DOX nuclear drug inflow (NU) and mitochondrial drug inflow (MU) were set at 1, DOX @ MSPCL _0.5 nanoparticles, DOX @ MSPCL _1.25 nanoparticles, and The nuclear inflow (NU) of DOX @ MSPCL _2.0 nanoparticles was 0.23 ± 0.03, 0.36 ± 0.03, and 0.50 ± 0.04, respectively, and the influx of drug in the mitochondria was 0.56 ± 0.04, 0.67 ± 0.02, and 0.70 ± 0.02. When the DOX and DOX @ MSPCL nanoparticles DOX have the same intracellular inflow (CU), the anticancer effect of DOX @ MSPCL _0.5 nanoparticles is about 1.2 times better than DOX and DOX @ MSPCL _1.25 nanoparticles and DOX @ MSPCL The anticancer effect of _2.0 nanoparticles is about 2.0 times and 4.1 times lower, respectively. The DOX @ MSPCL _0.5 nanoparticles, DOX @ MSPCL _1.25 nanoparticles, and DOX @ MSPCL _2.0 nanoparticles were about 1.4, 1.5, and 1.5, respectively, when the DOX was 1, compared to the intracellular drug inflow (CU) About 1.2, and about 1.1.

For DOX @ MSePCL nanoparticles, the intracellular drug inflow (CU) of DOX @ MSePCL _0.5 nanoparticles, DOX @ MSePCL _1.25 nanoparticles, and DOX @ MSePCL _2.0 nanoparticles were 0.52 (MU) in the mitochondria were 0.65 ± 0.01, 0.74 ± 0.02, and 0.75 ± 0.02, respectively, and the intracellular drug infusions (NU) were 0.26 ± 0.00, 0.37 ± 0.02, and 0.53 ± 0.03, 0.0 &gt; 0.01, &lt; / RTI &gt; and 0.78 + 0.01. When DOX and DOX @ MSePCL nanoparticles DOX had the same intracellular inflow (CU), the anticancer effects of DOX and DOX @ MSePCL _2.0 nanoparticles were similar, and DOX @ MSePCL _0.5 nanoparticles and DOX @ MSePCL _2.0 nanoparticles The anticancer effect is about 1.7 times and 1.8 times lower, respectively. The DOX @ MSePCL _0.5 nanoparticles, DOX @ MSePCL _1.25 nanoparticles, and DOX @ MSePCL _2.0 nanoparticles were about 1.3 .mu.l, respectively, when the DOX was 1, the mitochondrial drug inflow (MU) versus the intracellular drug inflow (CU) , About 1.1, and about 1.0.

HepG2 cells were plated on a confocal dish for 24 h and then treated with DOX, DOX @ MSPCL nanoparticles and DOX @ MSePCL nanoparticles in order to confirm the distribution of cellular organelles in the delivered DOX according to the drug delivery system. And cultured for another 4 hours. Before completion of the experiment, Hoechst 33342 (5 μg / ml) was treated for 10 minutes to stain nuclei and mitochondria were stained by treatment with MitoTracker Deep Red FM (200 nM) for 15 minutes. The cells were washed twice with DPBS and the intracellular distribution and fluorescence of doxorubicin were measured by excitation laser (405 nm for diode, 543 nm for HeNe, 633 nm for HeNe) and bandpass emission filters (LSM710; Carl Zeiss, Oberkochen, Germany) .

33 is a fluorescence image of DOX @ MSPCL nanoparticles introduced into nucleus, mitochondria, of HepG2 cells. 34 is data showing the fluorescence intensities of the DOX @ MSPCL nanoparticles introduced into the nucleus and mitochondria of HepG2 cells by the white arrows in FIG. Figures 33 and 34 show that DOX delivered by DOX @ MSPCL nanoparticles as well as DOX are distributed predominantly in mitochondria rather than nuclei.

Figure 35 is a fluorescence image for comparison of DOX @ MSPCL and DOX @ MSePCL nanoparticles infused into nucleus, mitochondria, of HepG2 cells. 36 is data showing the fluorescence intensities of DOX @ MSPCL and DOX @ MSePCL nanoparticles introduced into the nucleus and mitochondria of HepG2 cells in the white arrow in FIG. Figures 35 and 36 show that DOX delivered by DOX @ MSePCL nanoparticles as well as DOX is distributed predominantly in mitochondria rather than nuclei.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. It is to be understood that various modifications and changes may be made without departing from the scope of the appended claims.

Claims (8)

A method for producing a reducing degradable poly (meth) acrylate having hydrophilic carboxyl groups at both terminals represented by formula (1), wherein dithiodipropionate or diselenodipropionate is covalently bonded to both terminals of poly (epsilon-caprolactone) Epsilon-caprolactone) Polymers:
[Chemical Formula 1]

Wherein A is sulfur (S) or selenium (Se), x is an integer of 3 to 20, and y is an integer of 2 to 50. A nanoparticle according to claim 1, wherein the polymer is self-assembled in an aqueous phase. The method of claim 2,
The nano-
Wherein the nanoparticles have an average particle diameter of 20 to 200 nm. An anticancer composition comprising nanoparticles according to claim 2 as an active ingredient. The method of claim 4,
The arm
Wherein the gastric cancer is any one selected from the group consisting of thyroid cancer, stomach cancer, colon cancer, lung cancer, liver cancer, breast cancer, prostate cancer, gallbladder cancer, biliary cancer, pancreatic cancer, oral cancer, esophageal cancer, bladder cancer, colon cancer and cervical cancer. Composition. A composition for drug delivery comprising nanoparticles according to claim 2 as an active ingredient. The method of claim 6,
The medicament,
A composition for drug delivery, characterized by being an anticancer agent. The method of claim 7,
The anti-
But are not limited to, doxorubicin, paclitaxel, epirubicin, gemcitabine, silolimus, etoposide, vinblastine, vinca alkaloid, docetaxel, cisplatin, glivec, adriamycin, cyclophosphamide, But are not limited to, corticosteroids, streptomycin, streptomycin, streptozotocin, carbaplastine, ifosfamide, topotecan, velotecan, irinotecan, Selected from the group consisting of: angiotensin converting enzyme inhibitors selected from the group consisting of: angiotensin converting enzyme inhibitors selected from the group consisting of angiotensin converting enzyme, angiotensin converting enzyme inhibitor, angiotensin converting enzyme inhibitor, angiotensin converting enzyme inhibitor, angiotensin converting enzyme inhibitor, Or a pharmaceutically acceptable salt thereof.

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