KR101720458B1 - Self-assembled polymeric micelles composed of Glycol chitosan-dequalinium, preparation method and use thereof - Google Patents

Abstract

The present invention relates to self-assembled polymeric micelles consisting of glycol chitosan-dequalinium (GC-DQA), a preparation method thereof and uses thereof. The micelles made of GC-DQA polymers have low cytotoxicity compared to a control group DQAsome, easily load hydrophobic drugs in the micelles, can be stably maintained at room temperature and when being kept under refrigeration, and have excellent targeting effects with respect to mitochondria.

Description

Glycol chitosan-Dicuarinium self-assembling polymer micelle, a process for its preparation and its use {

The present invention relates to a glycol chitosan-dicuarinium self-assembling polymeric micelle, a method for producing the same, and a use thereof, and as such a pharmaceutical composition or a drug delivery vehicle for preventing or treating cancer.

The technologies of biopharmaceuticals in accordance with environmental and social changes are getting better as the generation passes. In recent years, development of therapeutic gene carriers and drug delivery vehicles is actively under way.

The dequalinium used in the present invention has been studied as a liposome-type DQAsome ™ and various gene delivery systems. Dicualinium is an antimicrobial, anticancer drug that is functionally used as a treatment for malaria and is known to be effective in mitochondria. Also structurally, it has a bolaform morphology in which two hydrophilic quinaldinium units are connected by a series of ten hydrophobic methylene chains. DicuAluminium is a cationic bola-amphiphile with an amide group of quadrandinium, which is also known as a bolaform surfactant, and can play an effective role as an emulsifier in emulsions.

Curcumin is a substance extracted from the roots of curcuma longa Linn and is structurally a hydrophobic polyphenol material containing a ring structure of phenol and an unsaturated carbonyl group. It is also known to have antioxidant and anti-inflammatory actions and antitumor and anti-amyloid action. Hydrophobic curcumin will not readily dissolve in aqueous solution, but will readily dissolve in the oil phase and allow drug delivery of mitochondrial targets.

Glycol chitosan is well known as a substance exhibiting hydrophilicity and non-toxicity.

The inventors of the present invention have found that micelles formed from glycol chitosan-dicaluminium (GC-DQA) polymer have significantly lower cytotoxicity than conventional DQAsome, can easily mount hydrophobic drugs in micelles, State, and the mitochondrial targeting effect is excellent, and the present invention has been completed.

U.S. Published Patent Application 2001/0001067 A1

It is an object of the present invention to provide a glycol chitosan-dicuarinium (GC-DQA) polymer.

It is another object of the present invention to provide a process for preparing the glycol chitosan-dicaluminium (GC-DQA) polymer.

It is another object of the present invention to provide a method for producing a hydrophobic drug-loaded dicuarinium emulsion.

Another object of the present invention is to provide glycol chitosan-dicaluminium (GC-DQA) micelles formed of the above-mentioned glycol chitosan-dicaluminium (GC-DQA) polymer.

Another object of the present invention is to provide a drug delivery system comprising the above-mentioned glycol chitosan-dicuarinium (GC-DQA) micelles.

It is another object of the present invention to provide a pharmaceutical composition for preventing or treating cancer, which comprises a drug delivery vehicle in which curcumin is loaded in the glycol chitosan-dicaluminium (GC-DQA) micelle.

In order to achieve the above object,

The present invention provides glycol chitosan-dicaluminium (GC-DQA) polymers in which Dequalinium and glycol chitosan are linked by a linker. Here, the linker may be methyl acrylate or the like.

The present invention also relates to a process for producing a compound represented by the formula (1)

(Step 1) of preparing glycol chitosan-methyl acrylate (GC-MA) by Michael addition of methyl acrylate (MA) to the amine group of glycol chitosan (GC) in a solvent; And

Reacting the GC-MA prepared in step 1 with decahalinium (DQA) chloride to prepare a glycol chitosan-dicuaninium (GC-DQA) polymer (step 2);

(GC-DQA) polymer, comprising the steps of:

[Reaction Scheme 1]

In the above Reaction Scheme 1,

a and b are independently an integer of 1-100000.

Further, the present invention provides a glycol chitosan-dicaluminium (GC-DQA) micelle formed from the glycol chitosan-dicaluminium (GC-DQA) polymer. Here, the micelles can be loaded with a hydrophobic drug therein, and examples of the hydrophobic drug include curcumin.

In addition, the present invention provides a drug delivery system comprising the glycol chitosan-dicaluminium (GC-DQA) micelle.

Furthermore, the present invention provides a pharmaceutical composition for preventing or treating cancer, which comprises a drug carrier having curcumin on the inside of the glycol chitosan-dicaluminium (GC-DQA) micelle. Here, the cancer may be cervical cancer or liver cancer.

The micelles formed from the glycol chitosan-dicaluminium (GC-DQA) polymer according to the present invention have significantly lower cytotoxicity than the control DQAsome, can easily mount the hydrophobic drug in the micelle, It remains stable for a long time and has excellent mitochondrial targeting effect.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a block diagram illustrating self-assembled GC-DQA micelles and their mitochondrial targeting steps.
FIG. 2 is an image showing the step of synthesizing the GC-DQA polymer prepared in Example 1. FIG.
Figure 3 is ¹ H NMR data for glycol chitosan (GC), dicaluminium (DQA) chloride, and GC-DQA polymer of Example 1.
Figure 4 is FT-IR data for glycol chitosan (GC), dicaluminium (DQA) chloride, and GC-DQA polymer of Example 1.
5 is (a) AFM image and (b) FE-SEM image of GC-DQA micelle.
FIG. 6 is a graph showing the fluorescence intensities according to the concentration of Nile Red-loaded GC-DQA polymer and (b) the fluorescence intensity (left Y-axis) indicating the critical aggregation concentration of GC-DQA polymer and dynamic light scattering ) Size (right Y axis).
FIG. 7 shows the cytotoxicity of the GC-DQA polymer, the control glycol chitosan (GC) and the control DQAsome (Comparative Example 1) by MTT evaluation and LDH evaluation (above: HDF cells, below: HeLA cells).
FIG. 8 is a confocal scanning laser microscope image showing mitochondrial targeting of Nile Red-loaded GC-DQA micelles in HeLa cells.
Figure 9 is a confocal scanning laser microscope image showing lysosome escape of Nile Red-loaded GC-DQA in HeLa cells.
10 is a photograph of a curcumin formulation loaded on curcumin and GC-DQA.
11 is a graph showing cytotoxicity of curcumin and general curcumin loaded on GC-DQA in HeLa cells.
12 is a confocal image showing the potential fluctuation of mitochondrial membrane in HeLa cells by staining with GC-DQA-loaded curcumin (GC-DQA Curcumin) and various control groups with JC-1.

Hereinafter, the present invention will be described in detail.

Glycol chitosan - Diquariuminium ( GC - DQA ) Polymer

The present invention provides glycol chitosan-dicaluminium (GC-DQA) polymers in which Dequalinium and glycol chitosan are linked by a linker.

The linker may be methyl acrylate (all of the linkers available) and the like.

The glycol chitosan-dicaluminium (GC-DQA) polymer according to the present invention is characterized by being mitochondria-targeted.

The micelles formed from the glycol chitosan-dicaluminium (GC-DQA) polymer according to the present invention have significantly lower cytotoxicity than the control DQAsome, can easily mount the hydrophobic drug in the micelle, It remains stable for a long time and has excellent mitochondrial targeting effect.

quite

As shown in the following Reaction Scheme 1,

(Step 1) of preparing glycol chitosan-methyl acrylate (GC-MA) by Michael addition of methyl acrylate (MA) to the amine group of glycol chitosan (GC) in a solvent; And

Reacting the GC-MA prepared in step 1 with decahalinium (DQA) chloride to prepare a glycol chitosan-dicuaninium (GC-DQA) polymer (step 2);

(GC-DQA) polymer, comprising the steps of:

[Reaction Scheme 1]

In the above Reaction Scheme 1,

a and b are independently an integer of 1-100000.

In the preparation process according to the present invention, step 1 is a step of preparing glycol chitosan-methyl acrylate (GC-MA) by Michael addition of methyl acrylate (MA) to an amine group of glycol chitosan (GC) in a solvent.

The solvent may be water, methanol, ethanol, acetone, dimethylformamide (DMF), dimethyl sulfoxide (DMSO), acetonitrile, THF (tetrahydrofuran) or the like.

The reaction temperature may be 20-60 캜, preferably 30-40 캜, and the reaction time is 1-5 days, preferably 2-4 days.

In the production process according to the present invention, the step 2 is a step of reacting the GC-MA prepared in the step 1 with decanoylium (DQA) chloride to prepare a glycol chitosan-dicuaninium (GC-DQA) polymer.

The reaction temperature may be 20-60 캜, preferably 30-40 캜, and the reaction time is 1-5 days, preferably 2-4 days.

Glycol chitosan - Diquariuminium ( GC - DQA ) Micelle

The present invention provides glycol chitosan-dicaluminium (GC-DQA) micelles formed from the glycol chitosan-dicaluminium (GC-DQA) polymer.

The micelle may contain a hydrophobic drug therein, and the hydrophobic drug may include curcumin or the like.

Drug delivery vehicle

The present invention provides a drug delivery system comprising the glycol chitosan-dicaluminium (GC-DQA) micelles.

Pharmaceutical composition

The present invention provides a pharmaceutical composition for preventing or treating cancer, which comprises a drug delivery vehicle in which curcumin is loaded in the glycol chitosan-dicaluminium (GC-DQA) micelle.

The cancer may be cervical cancer, liver cancer, stomach cancer, breast cancer, lung cancer, brain cancer, glioma, prostate cancer, uterine cancer, skin cancer and the like.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are illustrative of the present invention, and the present invention is not limited by the following examples.

material

(≥ 60%, titration, crystalline), methyl acrylate (99%), methanol (anahydrous 99.8%), dicaluminium chloride hydrate (≥95%), and 3- (4,5-dimethylthiazol- 2-yl) -2,5-diphenyl-tetrazolium bromide (MTT) was purchased from Sigma Aldrich (Seoul, Korea). Curcumin (95%) was purchased from Alfa Aesar (Seoul, Korea). The LDH cytotoxicity evaluation kit was purchased from Daeillab service (Seoul, Korea). DMEM (Dulbecco's modified Eagle's medium), FBS (fetal bovine serum) and 100 × antibiotic-antimycotic reagent were purchased from Gibco (Gaithersburgs, MD, USA). An innovative Pure Water System (DW) was used for the preparation and testing of all solutions.

< Example  1> Glycol chitosan - Diquariuminium  ( GC - DQA ) Preparation of polymer

Glycol chitosan was subjected to Michael addition reaction with methyl acrylate and Michael to prepare glycol chitosan-dicuaninium polymer.

Specifically, a solution prepared by dissolving glycol chitosan (10 mg) in distilled water (DW, 5 mL) was prepared. Next, a 100-fold excess of methyl acrylate was mixed with anhydrous methanol (45 mL) into a round bottom flask, and then the glycol chitosan solution prepared above was dropwise added. The reaction was carried out for 3 days under an inert atmosphere at 37 ° C. Next, methanol and excess methyl acrylate were evaporated under reduced pressure and purified by dialysis for 24 hours. The resulting glycol chitosan-methyl acrylate (5 mL in DW) was added to a 20-fold excess of dicaluminium chloride (45 mL in MeOH) and reacted under an inert atmosphere for 3 days. Next, the desired glycol chitosan-dicaluminium (GC-1) was dialyzed against cellulose membrane (MWCO = 1 kD) for 3 days (against MeOH / DQA) amphipathic polymer solution. The GC-DQA polymer solution was lyophilized to obtain a white sample.

< Example  2> Nile Red (dye) and Curcumin  loading GC - DQA Micelle  Produce

Curcumin / dye loading GD-DQa micelles were prepared using a simple dialysis method. Specifically, anhydrous methanol in which curcumin / Nile Red (500 μM) was dissolved was mixed with 5 mg of GC-DQA-prepared distilled water prepared in Example 1. A MeOH / DW (9: 1) solution was used for all formulations to a final volume of 2.5 mL. The prepared solution was ultrasonicated for 2 minutes and dialyzed for 12 hours using a dialysis membrane (MWCO = 1 kD). To remove the residual curcumin, the solution obtained above was filtered through a syringe filter (0.8 탆 pore size), and the curcumin / dye-loaded micelles obtained were lyophilized and the curcumin concentration was determined using a curcumin standard curve.

< Comparative Example  1> Diquariuminium Liposome  ( DQAsome &Lt; / RTI &gt;

DQAsome was used as a control group. 5 ml of methanol and 0.0528 g of dicaluminium were weighed into a round bottom flask to completely dissolve dicualuminium and then all of the methanol solvent was blown off using a vacuum pump. Then, 5 ml of HEPES buffer (N- (2-Hydroxyethyl) piperazine-N '- (2-ethanesulfonic acid)) (pH 7.4, 5 mM) was added and sonicated at 25 ° C or lower for 1 hour. After the ultrasonic treatment, the undissolved dicuarinium was removed by centrifugation at 10,000 rpm for 5 minutes. The prepared DQAsome was filtered with a 0.8 μm filter and stored at room temperature in a glass vial.

< Experimental Example  1> Glycol chitosan - Diquariuminium  ( GC - DQA ) Identification of polymer

The GC-DQA polymer prepared in Example 1 was confirmed by H ¹ NMR.

Specifically, glycol chitosan (GC), dicaluminium (DQA) and GC-DQA polymers were dissolved in D ₂ O, methanol-d4 and D ₂ O / methanol-d4 (1: 1 v / v) ¹ NMR (400 MHz) sample was prepared. The conjugation yield was calculated based on the respective material peaks. The size of the self-assembled nanoparticles was determined by the dynamic light scattering technique through a particle size analyzer ELS-Z (Photal, Otuka Electonics, Japan) and the zeta potential using a Zetasizer Nano ZS (Malvern Instruments, UK). GC (glycol chitosan) and DQAsome (Comparative Example 1) were used as controls. The measurement was carried out at 25 캜 in distilled water at a total concentration of 1 mg / mL. All measurements were performed at least 3 times and averaged. The morphology of the nanoparticles was confirmed using a Nanoscope MultiMode Atomic Force Microscope and FE-SEM (JSM-7500F; JEOL, Tokyo, Japan).

In conventional studies, amphiphilic glycol chitosan has been applied to various anticancer drug delivery systems. To obtain self-assembled nanoparticles, FITC, 5β-cholanic acid and cholesterol were chemically bonded to glycol chitosan as hydrophobic moieties.

FIG. 2 is an image showing the step of synthesizing the GC-DQA polymer prepared in Example 1. FIG.

Initially, methyl acrylate (MA) reacted covalently with primary amine functional groups of glycol chitosan dissolved in methanol. Next, the product reacted with dicualinium (DQA) to form an amphiphilic GC-DQA polymer.

Figure 3 is ¹ H NMR data for glycol chitosan (GC), dicaluminium (DQA) chloride, and GC-DQA polymer of Example 1.

As shown in Fig. 3, characteristic peaks at 7.5-8.5 ppm and 1.3 ppm corresponding to the CH protons of the benzene ring and the methylen proton of the dicaluminium, respectively, as compared with the glycol chitosan.

Figure 4 is FT-IR data for glycol chitosan (GC), dicaluminium (DQA) chloride, and GC-DQA polymer of Example 1.

As shown in Figure 4, the peaks of glycol chitosan are as follows: 1057 (CO stretch), 1420-1376 (CH bend), 1619 (amide I band, C = O stretch of acetyl group, 2879 3341 (OH stretch overlapped with NH stretch).

However, in GC-DQA, a new peak was identified at 1726 cm &lt; ^-1 &gt; indicating carbonyl stretching of the amine bond formed between the methyl acyl functional group of methyl acrylate and the amine functional group of dicaluminium.

< Experimental Example  2> GC - DQA Micelle  Physicochemical characterization and critical aggregation concentration (Critical Aggregate Concentration, CAC ) Calculation

Size rating

The sizes of GC-DQA micelles, glycol chitosan (GC) as a control group and DQAsome (control 1) as a control group were confirmed by dynamic light scattering (DLS), scanning electron microscope (SEM) and atomic force microscope (AFM) Are shown in Table 1 below.

Through DLS (dynamic light scattering), aggregates of 315 nm in 1 mg / ml (in DW) solution were identified. Agglomerates of about 260 nm smaller than SEM and AFM were observed. This is a common result of analyzing the dryness of the sample.

5 is (a) AFM image and (b) FE-SEM image of GC-DQA micelle.

As shown in Fig. 5, the morphology of the GC-DQA micelles in (a) AFM image and (b) FE-SEM image was confirmed to be spherical.

Zeta potential evaluation

The zeta potential was measured and is shown in Table 1 below.

Size (nm) Zeta potential (mV) Control group
(Glycol chitosan, GC) N / A 23.7 ± 2 Comparative Example 1
(DQAsome) 169.9 ± 2 62.2 ± 3 Example 1
(GC-DQA micelle) 315.1 ± 1 29.1 ± 1

As shown in Table 1, there was no significant difference between the GC and GC-DQA micelles at the zeta potential, but a high positive charge of 62 mV was shown for the DQAsome of Comparative Example 1. Thus, when glycol chitosan and dicualinium are combined, dicaluminium (DQA) appears to have no contribution to the surface charge of the GC-DQA polymer. This is because DQA is located inside the core of the micelle due to the hydrophobicity of dicalcium aluminium (DQA) and there is no contribution to the surface charge of the whole polymer.

GC - DQA Micelle  Critical aggregation concentration

The critical aggregation concentration of GC-DQA micelles was calculated using the fluorescent protein Nile Red through the fluorescent technique.

Specifically, 5 μl of Nile Red (25 μl in MeOH) was added to 1 mL of GC-DQA solution at a concentration of 0.05-0.5 mg / mL. Next, the solution was vortexed and analyzed using Cary Eclipse fluorescence spectroscopy (Santa Clara, Unites states) (excitation wavelength 550 nm, emission wavelength 560-760 nm). The fluorescence intensity at 650 nm was calculated as the critical aggregation concentration of GC-DQA micelles. In addition, the critical aggregation concentration of the GC-DQA suspension was measured by DLS (dynamic light scattering) technique. The same GC-DQA suspension concentration was measured to confirm coagulation formation at specific concentrations. All DLS measurements were averaged at least three measurements.

FIG. 6 is a graph showing the fluorescence intensities according to the concentration of Nile Red-loaded GC-DQA polymer and (b) the fluorescence intensity (left Y-axis) and dynamic light scattering (DLS) representing the critical aggregation concentration of GC- ) Size (right Y axis).

As shown in Fig. 6 (a), a sharp fluorescence intensity of Nile Red was observed at polymer concentrations between 0.15 and 0.2 mg / ml.

As shown in FIG. 6 (b), DLS (dynamic light scattering) showed that the GC-DQA polymer started to aggregate at a concentration of 0.01 mg / ml. Thus, the critical aggregation concentration of the GC-DQA polymer was calculated to be approximately 0.097 mg / mL, which is similar to the conventional cholesterol-modified glycol chitosan.

< Experimental Example  3> MTT  Evaluation and Cytotoxicity Assessment

MTT  evaluation

The cell viability of GC-DQA micelles on cancer cells and normal cells was calculated using the MTT colorimetric procedure, and the results are shown in FIG.

Specifically, human cervical cancer cells (HeLa cell) and human skin fibroblast cells (HDF cells) (source: ATCC, Rockville, MD, USA) were inoculated into 10% (v / v) FBS (Gibco, Grand Island, and cultured in DMEM medium containing 10% (w / v) penicillin / streptomycin in a humidified atmosphere of 5% CO ₂ at 37 ° C for 24 hours. To assess cytotoxicity, cells were seeded at a density of 1.5x10 ⁴ cells / well in a 96-well bottom well plate (SPL Life Sciences, Seoul, Korea) and the same conditions were maintained for 24 hours. Next, after adding the GC-DQA polymer, the control glycol chitosan (GC) and the control DQAsome (Comparative Example 1) at various concentrations and culturing for 24 hours, the medium was removed and the cells were washed twice with DPBS. The MTT solution (5 mg / ml) was diluted to a concentration of 0.5 mg / ml in the fresh medium, and then 100 μl of the DMEM medium containing the MTT reagent was added to the cells and cultured at 37 ° C for 2 hours. Next, the blue formazan crystals were dissolved in DMSO by discarding the medium. UV absorbance was measured using a microplate reader (VERSAMA XTM, Molecular Devices Corp., Sunnyvale, Calif.) And the data were interpreted as a percentage of viable cells as compared to the control.

In addition, the curcumin-loaded micelles in HeLa cells and the narrow-cut toxicity of curcumin alone as a control group were measured. DQAsome (Comparative Example 1) was excluded from the control group due to low encapsulation efficiency (EE), while performing a cytotoxicity assay of drug loaded GC-DQA micelles. In addition, DQAsome (Comparative Example 1) itself without drug loading showed high toxicity and was not suitable for drug efficacy measurement.

Dicualinium is positively charged, highly toxic, and well known as a mitochondrial target gene delivery vehicle. It is a practical challenge to use the target capacity of dicaluminium without toxicity. Thus, biocompatible polymers such as glycol chitosan (GC) appeared to inhibit the toxicity of dicaluminium.

In conventional studies, cytotoxic effects of dicaluminium chloride in cancer cells have been reported to be related to changes in mitochondrial potential and K ⁺ channel occlusion. However, the overproduction of active oxygen in mitochondria, which mediate the cellular apoptosis of cancer cells in the presence of dicu- alinium chloride, also plays an important role. In addition, the Warburg effect hypothesis, which is associated with mitochondria and apoptosis resistance, can be influenced by high toxicity in DQAsome and cancer cells. In our study, GC-DQA polymers are non-toxic in both mitochondria mediated and membrane disruptive pathways. Biocompatibility of glycol chitosan in the micelle form and dicu- alium core can inhibit the toxicity of GC-DQA polymer.

LDH (Lactate dehydrogenase ) evaluation

In order to examine the toxicity of nanoparticles (micelles) in HeLa cells and HDF cells, LDH release measurements were taken together with the control group, and the results are shown in FIG. LDH assessment measures membrane perturbation. The release of LDH means elution of the membrane and the increase in LDH release means high toxicity.

Specifically, the human cervical cancer cells (HeLa cell) and human dermal fibroblasts (HDF cells) (Source: ATCC, Rockville, MD, USA) to 10% (v / v) FBS and 1 to 1.0x10 ⁴ cells / well density and cultured in DMEM medium containing 10% (w / v) penicillin / streptomycin in a humidified atmosphere of 5% CO ₂ at 37 ° C for 24 hours. GC-DQA micelles, control glycol chitosan (GC), and control DQAsome (Comparative Example 1) were treated at various concentrations in 10 μl. After 24 hours of incubation, 10 [mu] l supernatant was added to a 100 [mu] l aliquot of the working reagent containing 0.2 mM NADH and 2.5 mM sodium pyruvate in wells of a 96 well plate. Untreated cells and 20% Tween-20 treated cells were regarded as 100% and 0%, respectively, and their LDH activity was measured. The evaluation was carried out three times for each sample to obtain an average. The viability of cells treated with nanoparticles (micelles) was expressed as a percentage of untreated cells.

FIG. 7 shows the cytotoxicity of the GC-DQA polymer, the control glycol chitosan (GC) and the control DQAsome (Comparative Example 1) by MTT evaluation and LDH evaluation (above: HDF cells, below: HeLA cells).

As shown in Fig. 7, GC-DQA micelles were non-toxic in both cancer cells (HeLa) and normal cells (HDF). However, DQAsome (Comparative Example 1) showed lower cell viability in HeLa cell line than HDF cell line.

Curcumin  loading GC - DQA  ( GC - DQA -Cur) Micelle  Cytotoxicity

The cytotoxic effect of curcumin alone and curcumin loading GC-DQA was evaluated in HeLa cells. As described above, the GC-DQA micelles not loaded with drug showed non-toxicity in both cancer cells and normal cells, while DQ Asome (Comparative Example 1) showed high toxicity (see FIG. 7). Previous studies have reported that curcumin can impair the mitochondrial potential of cancer cells that interfere with energy production mechanisms and lead to apoptosis. However, low concentrations of curcumin have no obvious effect.

10 is a photograph of a curcumin formulation loaded on curcumin and GC-DQA.

11 is a graph showing cytotoxicity of curcumin and general curcumin loaded on GC-DQA in HeLa cells.

As shown in Fig. 11, when curcumin was encapsulated in GC-DQA micelles and treated at a concentration of 5 [mu] M, 50% viability was observed. However, curcumin alone at the same concentration showed a slight effect in HeLa cells. These results clearly demonstrate that GC-DQA-Cur micelles effectively deliver hydrophobic curcumin and improve the bioavailability of curcumin. In addition, it is considered that the toxicity from dicuarinium is synergistic with curcumin, which facilitates the apoptosis mechanism of cancer cells.

< Experimental Example  4> Cell uptake and mitochondria Targeting  evaluation

Cellular uptake of Nile Red loading nanoparticles (micelles) was assessed by confocal laser scanning microscopy. To confirm the dispersion of the nanoparticles, HeLa cells were suspended in 5 x 10 &lt; ³ &gt; cells were cultured in DMEM medium containing 10% (v / v) FBS and 1% (w / v) penicillin / streptomycin in a confluent dish (μ slide 8 well, ibiTreat, South Korea) And cultured in a humidified atmosphere of% CO ₂ at 37 캜 for 24 hours. Next, 20 μl of Nile Red loading nanoparticles (micelle) was added and cultured for 4 hours. Mitochondria were stained with MitoTracker Green, and nuclei were stained with bisbenzimide (Hoechst 33342). After the medium was removed, the cells were washed with DPBS (Dulbecco's phosphate buffered saline) and the cells were observed using a Zeiss LSM 5 Live confocal laser microscope.

Previously reported studies on glycol chitosan (GC) have shown that GC-based nanoreversors have a high affinity for cell membranes and some pathways of phagocytosis (eg, clathrin-mediated endocytosis, caveolae-mediated endocytosis, and micropinocytosis) Lt; / RTI &gt; Nonetheless, intracellular trafficking studies of GC-based nanotransports have found particles in the cytoplasm shortly after 1 hour incubation and rarely captured by lysosomes. Thus, cell uptake and mitochondrial targeting of GC-DQA micelles were confirmed by confocal laser scanning microscopy.

FIG. 8 is a confocal scanning laser microscope image showing mitochondrial targeting of Nile Red-loaded GC-DQA micelles in HeLa cells.

As shown in FIG. 8, Nile Red loaded GC-DQA micelles were absorbed after 4 hours of incubation in HeLa cells and selectively accumulated in mitochondria. The fluorescence signal from MiTo-tracker green is fully integrated with the red fluorescence from Nile Red and marked with a yellow dot. In addition, cells were observed with elliptical nuclei indicating the non-toxicity of GC-DQA micelles.

< Experimental Example  5> same position ( colocalization Research and nano particle tracking

Nanomaterials after internalization by the phagocyte process tend to degrade in acidic lysosomal compartments. For effective therapeutic efficacy, nanomaterials should be able to escape easily from the endosome. To assess the intracellular fate of GC-DQA micelles after cell uptake, HeLa cells were plated at 5 x 10 &lt; ³ &gt; cells were cultured in DMEM medium containing 10% (v / v) FBS and 1% (w / v) penicillin / streptomycin in a confluent dish (μ slide 8 well, ibiTreat, South Korea) And cultured in a humidified atmosphere of% CO ₂ at 37 캜 for 24 hours. Next, 20 μl of GC-DQA nanoparticles, control glycol chitosan (GC) and DQAsome (Comparative Example 1) were added, respectively. After 4 hours of incubation, the cells were washed, stained with LysoTracker Green for 30 minutes and stained with acridine orange (AO, 5 μM) for 15 minutes. Cells were washed with DPBS (Dulbecco's phosphate buffered saline) and the cells were observed using a Zeiss LSM 5 Live confocal laser microscope.

In nanosuper design for therapeutic applications, the fate of nanoparticles is an important factor. The degradation of extracellular material in lysosomal compartments is a common cleaning mechanism of cells. Colocalization studies were performed using Nile Red loading GC-DQA micelles to confirm endosomal escape. Lysosomes and nuclei were stained with LysoTracker green and Hoechst, respectively. After 4 hours of incubation, Nile Red loaded GC-DQA micelles were observed in the cytoplasm without being captured inside the lysosomes. When the nanoparticles were in the same position in the lysosomes, the green and red fluorescence were combined and appeared as yellow dots.

Figure 9 is a confocal scanning laser microscope image showing lysosome escape of Nile Red-loaded GC-DQA in HeLa cells.

As shown in Fig. 9, fluorescence of lysosome was not combined with Nile Red fluorescence, and endosomal escape was confirmed after incubation for 4 hours. Previous reports on hydrophobically modified glycol chitosan nanoparticles have shown that about 80% of the nanoparticles are observed in the cytoplasm after only 1 hour of incubation.

< Experimental Example  6> JC -1 staining of mitochondrial translocation (△ Ψm )

The mitochondrial membrane potential was measured in HeLa cells using fluorescent probe JC-1 (5,59,6,69-tetrachloro-1,19,3,39-tetraethylbenzimidazole carbocyanine iodide). JC-1 is a cationic dye present in j-aggregates when the mitochondrial potential is intact, yielding red fluorescence (590 ± 10 nm). When the mitochondrial membrane potential is low, the probe monomers can not efficiently be incorporated into the mitochondria, resulting in green fluorescence (525 ± 10 nm) in the cytoplasm. To evaluate the mitochondrial membrane potential of curcumin loading GC-DQA micelles, HeLa cells were plated at 5 x 10 &lt; ³ &gt; cells were cultured in DMEM medium containing 10% (v / v) FBS and 1% (w / v) penicillin / streptomycin in a confluent dish (μ slide 8 well, ibiTreat, South Korea) And cultured in a humidified atmosphere of% CO ₂ at 37 캜 for 24 hours. After 4 hours of treatment, the cells were washed and incubated with 10 mM JC-1 for 10 minutes at 37 ° C. Next, the cells were washed twice with PBS and analyzed using a Zeiss LSM 5 Live confocal laser microscope.

The mitochondrial membrane potential (MMP, △ Ψm) is considered a 'master signal' between cell survival and death. In conventional reports, anti-apoptotic signals from cancer cells arising from damaged mitochondrial membrane potentials due to several simultaneously occurring mechanisms have been identified. Therefore, a variety of extrinsic factors / reagents are available that can induce MMP and subsequent cell suicide. It has been reported that lipophilic cations accumulate in the inner membrane of mitochondria causing MMP reduction.

Fluorescence probe JC-1 was used to detect MMP in GC-DQA polymer and GC-DQA-Cur treated HeLa cells.

12 is a confocal image showing the potential variation of the mitochondrial membrane in HeLa cells by staining with GC-DQA-loaded curcumin (GC-DQA Curcumin) and various control groups with JC-1.

As shown in Fig. 12, the cells treated with GC-DQA-Cur and DQAsome (Comparative Example 1) showed stronger green fluorescence intensity after incubation at 4 o'clock compared to the other control. The toxicity of GC-DQA-Cur and DQAsome can affect MMP, a major directive of apoptosis. As confirmed in the cytotoxicity assays, GC, GC-DQA micelles and curcumin (10 μM) alone did not show any significant effect on MMP. These results demonstrate that curcumin, which causes mitochondrial mediated cell suicide in cancer cells, is efficiently delivered to the mitochondria by GC-DQA micelles.

Therefore, the combination of the mitochondrial targeting site and the drug according to the present invention is highly likely to be applied in the field of cancer treatment.

< Experimental Example  7> GC - DQA Micelle  Drug capture efficiency (Encapsulation Efficiency, EE ) evaluation

As shown in the above-mentioned experimental results, the GC-DQA micelle can form an aggregate of about 315 nm in an aqueous solvent which can be stabilized at room temperature and in a refrigerated state for 6 months or more. Since the stability of the drug delivery or formulation is a prerequisite, GC-DQA-Cur micelles are prepared which can further capture curcumin. Curcumin is a polyphenolic compound that exhibits various therapeutic effects such as antioxidant, anti-inflammatory and anti-cancer. However, curcumin is not soluble in water and is an obstacle to its application as a therapeutic agent.

GC-DQA-Cur was prepared as in Example 2, and its drug loading efficiency was evaluated. The results are shown in Table 2 below.

GC-DQA polymer content (mg) of Example 1 Curcumin (μM) Capture efficiency
(Encapsulation Efficiency, EE,%) Loading efficiency Size (nm) One 100 1.23 6 0.0006 352 2 100 3.3 ± 3 0.0016 349 3 100 2.1 ± 5 0.001 280 4 100 1.7 ± 3 0.0005 299 5 100 10 ± 5 0.005 308 3 200 2.18 ± 6 0.017 N / A 5 200 5.34 ± 3 0.008 N / A 5 500 25.7 ± 4 0.18 N / A

As shown in Table 2, 25% efficiency was exhibited when the trapping efficiency was the highest. Based on the results of this experiment, the above-mentioned in vitro experiments used the formulation with the highest capture efficiency.

&Lt; Formulation Example 1 > Preparation of pharmaceutical preparation

<1-1> Preparation of powder

GC-DQA-Cur Micelle 2 g

Lactose 1 g

After mixing the above components, the mixture was packed in an airtight container to prepare a powder.

<1-2> Preparation of tablets

GC-DQA-Cur micelles 100 mg

Corn starch 100 mg

100 mg of milk

2 mg of magnesium stearate

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

&Lt; 1-3 > Preparation of capsules

GC-DQA-Cur micelles 100 mg

Corn starch 100 mg

100 mg of milk

2 mg of magnesium stearate

After mixing the above components, the capsules were filled in gelatin capsules according to the conventional preparation method of capsules.

<1-4> Preparation of Injection Solution

GC-DQA-Cur micelles 10 占 퐂 / ml

Until dilute hydrochloric acid BP pH 3.5

Sodium chloride BP injected up to 1 ml

The compound according to the invention was dissolved in a suitable volume of injected sodium chloride BP and the pH of the resulting solution was adjusted to pH 3.5 using dilute hydrochloric acid BP and the volume was adjusted using injectable sodium chloride BP and mixed thoroughly. The solution was filled in 5 ml type I ampoule made of transparent glass, sealed in the upper lattice of the air by dissolving the glass, and sterilized by autoclave at 120 캜 for 15 minutes or longer to prepare an injection solution.

Claims (11)

Dequalinium and glycol Chitosan-Dicuallinium (GC-DQA) polymers with glycol chitosan attached to a methyl acrylate linker.
delete The method according to claim 1,
Wherein the glycol chitosan-dicalaninium (GC-DQA) polymer is mitochondrial-targeted.
As shown in Scheme 1 below,
(Step 1) of preparing glycol chitosan-methyl acrylate (GC-MA) by Michael addition of methyl acrylate (MA) to the amine group of glycol chitosan (GC) in a solvent; And
Reacting the GC-MA prepared in step 1 with decahalinium (DQA) chloride to prepare a glycol chitosan-dicuaninium (GC-DQA) polymer (step 2);
(GC-DQA) &lt; / RTI &gt; polymer comprising:
[Reaction Scheme 1]

In the above Reaction Scheme 1,
a and b are independently an integer of 1-100000.
5. The method of claim 4,
Wherein the solvent is at least one selected from the group consisting of water, methanol, ethanol, acetone, dimethylformamide (DMF), dimethyl sulfoxide (DMSO), acetonitrile and THF (tetrahydrofuran).
A glycol chitosan-dicaluminium (GC-DQA) micelle formed from the glycol chitosan-dicaluminium (GC-DQA) polymer of claim 1.
The method according to claim 6,
Wherein the micelles are loaded with a hydrophobic drug therein.
8. The method of claim 7,
Wherein the hydrophobic drug is curcumin.
A drug delivery system comprising the glycol chitosan-dicuaninium (GC-DQA) micelle of claim 6.
A pharmaceutical composition for preventing or treating cancer, which comprises a drug carrier having curcumin on the inside of the glycol chitosan-dicaluminium (GC-DQA) micelle of claim 6.
11. The method of claim 10,
Wherein said cancer is cervical cancer, liver cancer, stomach cancer, breast cancer, lung cancer, brain cancer, glioma, prostate cancer, uterine cancer or skin cancer.

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