KR20200082719A - Composition for mixed polymeric micelles and use thereof - Google Patents

Abstract

The present invention relates to: nano-sized mixed polymer micelles consisting of a first polymer and a second polymer; and uses thereof. More specifically, the mixed polymer micelles have very excellent encapsulation content and in vivo stability of poorly soluble drugs compared to micelles made of the previously reported BAB-type triple block copolymer, and in particular, the mixed polymer micelles encapsulated with anticancer agents are confirmed to exhibit excellent tumor selectivity and superior anticancer effects. Therefore, the mixed polymer micelles can be provided as an excellent drug delivery agent and an anticancer agent.

Description

Composition for mixed polymeric micelles and use thereof

The present invention is a technique for a nano-sized mixed polymer micelle composition.

Nano-sized micelles with a core-shell structure based on amphiphilic block copolymers have been widely developed in various drug delivery systems. The micelle's hydrophobic core serves as a container for encapsulating water-insoluble drugs, and the micelle's hydrophilic shell serves to protect and stabilize the micelles.

Amphiphilic block copolymers for the production of polymer micelles developed to date are divided into those that do not contain functional groups and those that contain a plurality of functional groups. Polymers that do not contain functional groups cannot interact sufficiently with hydrophobic drugs, and also have limitations in the development and application of drug delivery systems. In the case of containing multiple functional groups, they cannot be properly encapsulated by dissolving in water. There are limits.

As a method for synthesizing a BAB-type triblock copolymer using an AB-type initial polymer, a method for preparing a BA-AB-type block copolymer using a current crosslinking agent is generally used, and a representative crosslinking agent used is hexamethylene diisocyanate. (hexamethylene diisocyanate) and adipoyl chloride.

In the BAB type block copolymer prepared by using hexamethylene diisocyanate as a crosslinking agent, a hydrophobic A block is synthesized by urethane bonding (Jeong et al., nature, 388, 860-862, 1997). In addition, when a reaction is performed using adipoyl chloride as a crosslinking agent, hydrochloric acid is generated as a by-product during the process, thereby causing the hydrophobic A block to be easily decomposed (Feng Li et al., Langmuir, 23). , 2778-2783, 2007).

In particular, it has been reported that the cross-linking agent used to prepare the BA-AB type copolymer exhibits toxicity in the human body, and there is a difficulty that decomposition of the AB type initial polymer occurs during the reaction. In addition, the BA-AB-type copolymer has a limitation in that it is difficult to have a functional group at both ends of the hydrophilic B group and is used as a drug delivery system for the purpose of simple drug delivery only.

Republic of Korea Patent Publication No. 2004-0021760 (2004.03.11 published)

The present invention is to provide a nano-sized polymer micelle capable of encapsulating a poorly soluble physiologically active substance in a high content and delivering a physiologically active substance encapsulated at a target site in a stable state in vivo, and a drug delivery system or pharmaceutical composition using the same.

The present invention is a poly(ethylene glycol) [PEG] and poly (lactic acid) [PLA] number average molecular weight ratio [PEG:PLA] is a triple block copolymer of 0.5 or less and the first polymer and poly (ethylene glycol) [PEG] Provided is a nano-sized mixed polymer micelle composition composed of a second polymer which is a triple block copolymer having a number average molecular weight ratio [PEG:PLA] of poly(lactic acid) [PLA] of 1.0 or more.

The present invention is a poly(ethylene glycol) [PEG] and poly (lactic acid) [PLA] number average molecular weight ratio [PEG:PLA] is a triple block copolymer of 0.5 or less and the first polymer and poly (ethylene glycol) [PEG] Provided is a nano-sized mixed polymer micelle-type drug delivery system composed of a second polymer that is a triple-block copolymer having a number average molecular weight ratio [PEG:PLA] of poly(lactic acid) [PLA] of 1.0 or more.

In addition, the present invention is poly(ethylene glycol) [PEG] and poly (lactic acid) [PLA] number average molecular weight ratio [PEG:PLA] is a triple block copolymer of 0.5 or less and the first polymer and poly (ethylene glycol) [PEG ] And a poly(lactic acid) [PLA] number average molecular weight ratio [PEG:PLA] is a triple block copolymer of 1.0 or more. Provide pharmaceutical composition.

According to the present invention, the nano-sized mixed polymer micelle composed of the first polymer and the second polymer has a very good encapsulation content and stability in vivo of the poorly soluble drug than the micelle made of the previously reported BAB-type triblock copolymer. In particular, as it is confirmed that the mixed polymer micelle encapsulated with an anti-cancer agent exhibits excellent tumor selectivity and excellent anti-cancer effect, the mixed polymer micelle can be provided as an excellent drug delivery agent and an anti-cancer treatment agent.

1 is a result of confirming a mixing method for an excellent drug delivery system, FIG. 1a is a result of confirming the effect of a hydrophobic part on micelle properties, and FIG. 1b is a system formed by mixing of other triblock copolymers and anticancer thereof in vivo It is a schematic diagram showing the experimental method to confirm the effect.
Figure 2 is a result of confirming the colloidal stability of the micelle system, Figure 1a is a result of checking the size change of each system every day for a week, Figure 2b and Figure 2c is a result of checking the shape of each system, Figure 2b is a micelle production It is a result of confirming the form immediately after, and FIG. 2C is a result of confirming the form after storing at room temperature for 7 days. * H.-T. Song, et.al., Development of a new tri-block copolymer with a functional end and its feasibility for treatment of metastatic breast cancer, Colloids Surf., B, 144 (2016) 73-80.
Figure 3 is the result of confirming the anti-cancer effect of kB cells in vitro , Figure 3a is the result of comparing the anti-cancer effect of DOX/B20 with free DOX, IC ₅₀ values of DOX/B20 and free DOX are 0.0357 and 0.1125 μg, respectively /mL, Figure 3b is a result of comparing the anti-cancer effect of PTX/B20 with free PTX, the results confirming that the IC ₅₀ values of PTX/B20 and free PTX are 0.1506 and 0.8587 μg/mL, respectively.
Figure 4 is a result of confirming the drug absorption in kB cells, Figure 4a is a result of comparing the absorption level of DOX / B20 with free DOX by performing flow cytometry, Figure 4b is the absorption level of DOX / B20 with a confocal microscope Is a result of comparison with glass DOX, and FIG. 4C is a result of comparing the PTX/B20 absorption level with glass PTX. Statistical analysis was performed with two-tailed Student's test.
Figure 5 is a result of confirming the in vivo effect, Figure 5a is a result of confirming the bio-distribution of nano-sized micelles in mice with tumor formation, the upper figure is an optical image, the middle figure is a fluorescent image, and the lower figure is a merged image , The right figure shows the tumor, and FIG. 5B shows the result of confirming the accumulation level of nano-sized micelles in tumors and other tissues 24 hours after injection, the upper figure is an optical image, the middle figure is a fluorescence image, and the lower figure is 5C and 5D are one-way ANOVA performed as a result of confirming tumor volume and weight of animals treated with DOX/B20, free DOX, and PBS, respectively, and the results of Dunnette's test were performed for post-analysis. (*p<0.05, **p<0.001), FIG. 5e shows the results of extracting the tumor before and 27 days after treatment of the tumor-forming mice, and the arrow indicates the tumor and (1) treated with DOX/B20 Group, (2) is free DOX treated group, (3) is PBS treated control group.

The present invention is a poly(ethylene glycol) [PEG] and poly (lactic acid) [PLA] number average molecular weight ratio [PEG:PLA] is a triple block copolymer of 0.5 or less and the first polymer and poly (ethylene glycol) [PEG] It is possible to provide a nano-sized mixed polymer micelle composition composed of a second polymer that is a triple block copolymer having a number average molecular weight ratio [PEG:PLA] of poly(lactic acid) [PLA] of 1.0 or more.

The mixed polymer micelle composition may be a mixture of 60 to 90% by weight of the first polymer and 10 to 40% by weight of the second polymer.

When the first polymer is added below the content range, there may be a problem that the amount of encapsulation and the rate of encapsulation of the drug are reduced, and when it is added above the content range, problems of phase separation, precipitation, and particle increase may appear.

In addition, when the second polymer is added below the above content range, problems of particle size increase and instability may appear, and when it is added above the above content range, there may be a problem of increasing the encapsulation amount and encapsulation rate of the drug. have.

The average diameter of the mixed polymer micelle is not particularly limited, but the average diameter range of 50 to 200 nm is preferred.

According to an embodiment of the present invention, by replacing PEG-PLA-PEG 2K-10K-2K (10 wt% of B20 composition) with FITC-PEG-PLA-PEG, FITC was marked on the B20 micelle surface and tumors were generated. As a result of confirming the biodistribution of the nano-sized B20 system by injection into the tail vein of the mouse, almost all micelles were dispersed into the liver as shown in FIG. 5A, and a small portion of the micelles were transported to the tumor. However, the fluorescence intensity of the liver decreased with time, and after 24 hours, it was very low, while a large amount of micelle accumulation was confirmed, showing a very high fluorescence intensity in the tumor 24 hours after injection.

In addition, almost no fluorescence signal was observed in other major organs except for the liver in which a small amount of micelles remained 24 hours after injection as shown in FIG. 5B.

From the above results, it was confirmed that the micelle system of the present invention shows excellent tumor selectivity.

Therefore, the present invention is a poly(ethylene glycol) [PEG] and poly (lactic acid) [PLA] number average molecular weight ratio [PEG:PLA] is a triple block copolymer of 0.5 or less and the first polymer and poly (ethylene glycol) [PEG ] And a poly(lactic acid) [PLA] number-average molecular weight ratio [PEG:PLA] of at least 1.0 is a triple-block copolymer, a nano-sized mixed polymer micelle-type drug delivery system made of a second polymer.

The mixed polymer micelle-type drug delivery system may be a mixture of 60 to 90% by weight of the first polymer and 10 to 40% by weight of the second polymer.

In addition, the present invention is poly(ethylene glycol) [PEG] and poly (lactic acid) [PLA] number average molecular weight ratio [PEG:PLA] is a triple block copolymer of 0.5 or less and the first polymer and poly (ethylene glycol) [PEG ] And a poly(lactic acid) [PLA] number average molecular weight ratio [PEG:PLA] is a triple block copolymer of 1.0 or more. Pharmaceutical compositions can be provided.

The pharmaceutical composition for treating cancer may include 70 to 99.9% by weight of a mixed polymer micelle and 0.1 to 30% by weight of an anticancer agent that can be enclosed in the mixed polymer micelle, and the anticancer agent is doxorubicin, paclitaxel, etoposide, vinca alkaloid, It can be selected from the group consisting of vinblastine and colchicine.

According to another embodiment of the present invention, in order to confirm the in vivo anticancer drug delivery efficiency of the drug delivery system (DOX/B20) in which doxorubicin (DOX) is encapsulated in the mixed polymer micelle (B20), kB tumors are formed with DOX/B20. As a result of injection into the tail vein of a nude mouse, as shown in FIGS. 5C and 5E, the administration group of DOX/B20 showed very effective tumor growth inhibition results compared to the control group DOX and saline administration groups (n=6, mean±SEM).

In more detail, the control group showed maximum tumor growth with a tumor volume of 1000 mm ³ or more, and in the DOX-administered group, tumor progression was confirmed until the study was completed, whereas in the DOX/B20-administered group, tumor suppression was observed.

Free DOX was distributed non-selectively throughout the mouse body, but DOX encapsulated in nano-sized B20 micelles selectively accumulated in the tumor tissue due to the EPR effect, so that the DOX could be maintained at a high concentration for a long time at the tumor site.

From the above results, as the mixed polymer micelle has been confirmed as an excellent drug delivery system, the mixed polymer micelle encapsulating the anticancer agent can be used as an excellent anticancer agent.

The cancer may be selected from the group consisting of liver cancer, breast cancer, colon cancer, stomach cancer, lung cancer, pancreatic cancer, kidney cancer, nasopharyngeal epidermal cancer and oral cancer.

In one embodiment of the present invention, the pharmaceutical composition for treating cancer is in the group consisting of injections, granules, powders, tablets, pills, capsules, suppositories, gels, suspensions, emulsions, drops or liquids according to conventional methods. Any one of the selected formulations can be used.

In another embodiment of the present invention, the pharmaceutical composition for the treatment of cancer is a suitable carrier, excipient, disintegrant, sweetener, coating agent, swelling agent, lubricant, lubricant, flavoring agent, antioxidant, commonly used in the manufacture of pharmaceutical compositions, Buffers, bacteriostatic agents, diluents, dispersants, surfactants, binders and lubricants may further include one or more additives selected from the group consisting of.

Specifically, carriers, excipients and diluents are lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline Cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil can be used, and solid preparations for oral administration include tablets, pills, powders, granules and capsules. Agents, and the like, and these solid preparations may be prepared by mixing at least one excipient in the composition, for example, starch, calcium carbonate, sucrose or lactose, gelatin, and the like. In addition, lubricants such as magnesium stearate and talc may be used in addition to simple excipients. Liquid preparations for oral use include suspensions, liquid solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used diluents, various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, may be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, and the like. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin butter, and glycerogelatin may be used.

According to one embodiment of the present invention, the pharmaceutical composition is intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, transdermal, intranasal, inhalation, topical, rectal, oral, intraocular or intradermal. Routes can be administered to a subject in a conventional manner.

The preferred dosage of the pharmaceutical composition for the treatment of cancer may vary depending on the condition and weight of the subject, the type and extent of the disease, the type of drug, the route and duration of administration, and may be appropriately selected by those skilled in the art. According to one embodiment of the present invention is not limited thereto, the daily dosage may be 0.01 to 200 mg/kg, specifically 0.1 to 200 mg/kg, and more specifically 0.1 to 100 mg/kg. The administration may be administered once a day or divided into several times, and the scope of the present invention is not limited thereby.

In the present invention, the'subject' may be a mammal, including a human, but is not limited to these examples.

Hereinafter, examples will be described in detail to help understanding of the present invention. However, the following examples are merely illustrative of the contents of the present invention, and the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

<Reference Example> Substance

Poly(ethylene glycol) (PEG, MW 2kDa abbreviated as 2K), methoxy poly(ethylene glycol) (mPEG, MW 2K), L-lactide((3S)-cis-3,6-dimethyl-1,4- dioxane-2,5-dione), N,N-dicyclohexylcarbodiimide (DCC), stannous octoate; Tin(II)-2-ethylhexanoate, Sn(Oct) 2), 4-dimethylaminopyridine (DMAP), dimethylsulfoxide (DMSO), succinic anhydride, pyridine, and triethylamine (TEA) Sigma-Aldrich ( St. Louis, MO, USA). Tetrahydrofuran (THF), toluene, acetone and dichloromethane (DCM) were purchased from Honeywell Burdick & Jackson® (Muskegon, MI, USA).

Doxorubicin ((DOX).HCl) was added to Boryung Co. (Seoul, South Korea), and paclitaxel (Diethyl ether) and hexane were purchased from Samchun chemical (Seoul, South Korea).

FITC-5-isothiocyanate (Fluorescein-5-isothiocyanate) was purchased from Thermo Fisher Scientific Korea Ltd (Korea). kB cells were obtained from Korean Cell Line Bank (Seoul, South Korea), RPMI-1640 medium, DPBS, penicillin-streptomycin solution, trypsin-EDTA solution and fetal bovine serum (FBS) were purchased from Welgene (South Korea). .

Cell counting Kit-8 (CCK-8) was purchased from Dojindo (USA) and PierceTM BCA Protein Assay Kit was purchased from ThemoFisher Scientific (USA) and used.

< Experimental Example 1> Triblock copolymer ( Triblock copolymer) synthesis

Previously reported method [H.-T. Song, et.al., Development of a new tri-block copolymer with a functional end and its feasibility for treatment of metastatic breast cancer, Colloids Surf., B, 144 (2016) 73-80.; N.H. Hoang, et. al., Characterization of a triblock copolymer, poly (ethylene glycol)-polylactide-poly (ethylene glycol), with different structures for anticancer drug delivery applications, Polym. Bull., (2016) 1-15.] to synthesize PEG-PLA-PEG triblock copolymers with or without functional groups, and PEG- with molecular weights of 2K-10K-2K and 5K-10K-5K. PLA-PEG triblock copolymer was synthesized.

First, PEG-PLA diblock copolymer was synthesized by ring-opening polymerization method of L-lactide at 120° C. for 24 hours using Sn(Oct) 2 and toluene solvent as catalysts in the presence of the initiator PEG.

The PEG-PLA diblock copolymer synthesized by the above method was obtained as a precipitate by pouring the reaction mixture into an excess of cold diethyl ether, and the precipitate was filtered and dried in a vacuum for 2 days.

In the presence of DMAP, TEA and pyridine, PEG and succinic anhydride were reacted to PEG carboxylate (PEG-COOH). PEG, succinic anhydride, and DMAP were dissolved in 20 mL of DCM by stirring for 30 minutes, and then TEA and pyridine were added.

The reaction was carried out overnight and precipitated in excess diethyl ether to give the final product, filtered and dried in vacuo for 2 days.

To prepare a PEG-PLA-PEG triblock copolymer, a Steglich ester reaction was performed using synthesized PEG-PLA and PEG-COOH.

PEG-PLA and PEG-COOH were dissolved in dichloromethane (DCM) by stirring for 30 minutes, and dicyclohexylcarbodiimide (DCC) as a coupling agent and dimethylaminopyridine (DMAP) as a catalyst were added to the DC solution. After the reaction was performed overnight, the final product was obtained by precipitating in excess diethyl ether, then filtered and dried in vacuo for 2 days.

FITC to PEG-PLA-PEG triblock copolymers 2K-10K-2K and 5K-10K-5K through the interaction of free -OH groups with FITC-5-isothiocyanate in the PEG-PLA-PEG triblock copolymer Combined.

Briefly, PEG-PLA-PEG and FITC-5-isothiocyanate with functional groups were added to a round bottom flask at a 1:3 molecular ratio followed by 30 mL of DMSO.

After reacting at room temperature for 24 hours, the remaining FITC-5-isothiocyanate was removed by dialysis with 1 L water for 24 hours and lyophilized to obtain FITC-PEG-PLA-PEG powder. The micelles used to obtain in vivo images contained 10 wt% FITC-PEG-PLA-PEG.

< Experimental Example 2> Self-assembled polymer micelle preparation

Polymerizable micelles were prepared by dialysis. After dissolving the triple block copolymer or a mixture of other triple block copolymers (10 mg) in 3 mL DMSO, the DMSO solution in which the triple block copolymer is dissolved is transferred to a dialysis membrane (MWCO 3.5KDa; Spectrum, USA) and buffered at pH 7.4 Dialysis was performed with 500 mL of saline (PBS) for 24 hours, and PBS pH 7.4 was changed every 3 hours.

< Experimental Example 3> Determination of critical micelle concentration

To confirm the critical micelle concentration (CMC), fluorescence was measured with a Scinco FS-2 fluorescence spectrometer (Seoul, Korea) using pyrene as a fluorescent probe.

A sample solution was prepared by adding the pyrene solution dissolved in acetone to a vial.

After evaporating acetone, a solution of triblock copolymer micelles of different concentrations was added to the vial so that the final pyrene concentration was 6×10 ⁻⁷ M.

The sample was stirred overnight at room temperature and the excitation spectrum of pyrene in the sample was recorded at λ _em =374 nm. CMC was confirmed by plotting the ratio of I336 (fluorescence intensity at 336 nm) to I334 (fluorescence intensity at 334 nm) in the excitation spectrum versus the logarithm of the copolymer concentration, and CMC was the low copolymer concentration in the above plot. Was defined as the intersection of.

< Experimental Example 4> Check particle size

The size (effective hydrodynamic size) of micelles was confirmed by photon correlation method using Zetasizer Nano-ZS (Malvern Instruments, UK) equipped with Multi Angle Sizing Option (BI-MAS).

Measurements were performed with a thermostatic cell at a 90° scattering angle and effective hydrodynamic size values were calculated using software provided by the manufacturer.

< Experimental Example 5> Morphological analysis

The morphology of the polymer micelle was confirmed using field emission scanning electron microscopy (FE-SEM) (Hitachis-4800, Japan).

A few drops of the diluted dispersion were placed on a slide glass, dried in vacuo, and subjected to FE-SEM by platinum coating on the sample.

< Experimental Example 6> Drug Loaded Michel preparation and characterization

DOX.HCl dissolved in DMSO was obtained at a concentration of 2.5 mg/mL.

HCl was separated from DOX by adding in a 2:1 molecular ratio to DOX.HCl.

The triple block copolymer was dissolved in DMSO at a concentration of 10 mg/mL. 2 mL of the DOX solution was mixed with 1 mL of the triple block copolymer solution and dialyzed against 500 mL of pH 7.4 PBS for 24 hours.

The sample was centrifuged at 5000 rpm for 5 minutes to precipitate unloaded DOX, and then the supernatant containing micelles loaded with DOX was collected.

The concentration of DOX in micelles was confirmed at λ = 481 nm using a UV-VIS spectrometer (GENESYS 10 UV, Thermo Sci., USA).

Paclitaxel (PTX) was dissolved in DMSO to obtain a concentration of 0.5 mg/mL, and the triblock copolymer was dissolved in DMSO to obtain a concentration of 10 mg/mL.

2 mL of the PTX solution and 1 mL of the triple block copolymer solution were mixed and dialyzed with 500 mL of pH 7.4 PBS for 24 hours.

The sample was centrifuged at 5000 rpm for 5 minutes to precipitate unloaded PTX, and then the supernatant containing micelles loaded with PTX was collected.

The concentration of PTX in micelles was confirmed by a UV detector with 230 nm wavelength and liquid chromatography (Agilent, USA).

The drug loading dose was calculated as follows.

Drug loading capacity (wt%) = (drug loaded micelle/copolymer and drug) × 100%

< Experimental Example 7> Intracellular Cellular uptake

One) Flow cytometry through DOX Absorption check

Absorption in DOX cells and micelles loaded with DOX were confirmed by flow cytometry.

KB cells were dispensed at 2×10 ⁵ cells/well into 6-well plates with 3 mL medium dispensed per well, and cultured at 37° C. and 5% CO ₂ for 24 hours.

After removing the medium, DOX solution or DOX loaded micelle solution was added at a concentration of 5 μg/mL and incubated at 37° C. and 5% CO ₂ for 3 hours.

The wells were washed three times with cold PBS, cells were collected with a scraper, contained in a 10 mL falcon tube, and DOX absorption was confirmed from a DOX solution or a micelle solution loaded with DOX using a BD FACS Calibur flow cytometer (BD Biosciences, USA). .

Data were obtained using Cell Quest Pro software.

2) Confocal Through a microscope DOX Absorption check

DOX uptake in kB cells in vitro was confirmed by confocal microscopy.

First, kB cells were dispensed and grown at 150,000 cells per well in a 6-well plate containing cover glass in each well.

After 24 hours, the medium containing the solution loaded with DOX or DOX loaded at a concentration of 1 μg/mL was replaced, and one well containing the medium was used as a control.

After incubation for 3 hours, the medium was removed, and the cells on the cover glass were washed three times with cold DPBS, and the cells were fixed for 15 minutes with a paraformaldehyde solution (4%) dissolved in water.

Thereafter, the cells were washed three times with cold DPBS, and the cell nuclei were stained using a protocol provided by the manufacturer using DAPI (Thermo Fisher Scientific, Korea).

Finally, it was placed on a cover glass using a Permount® mounting medium (Fisher Scientific, USA), and DOX absorption was confirmed using a confocal microscope (ZEISS, USA).

3) Paclitaxel ( PTX ) absorption

KB cells were dispensed at 2×10 ⁵ cells/well into 6-well plates with 3 mL medium dispensed per well, and cultured at 37° C. and 5% CO ₂ for 24 hours.

The medium was removed and the PTX solution or PTX loaded Michel solution was added at a concentration of 1 μg/mL, and the plate was incubated at 37° C. and 5% CO ₂ for 6 hours.

The wells were washed three times with cold PBS, and the cells were collected by scraping and placed in Eppendorf tubes.

The tube was centrifuged at 5000 rpm for 5 minutes and the supernatant was removed.

Cells were lysed with 1 mL of PBS containing 0.1% Triton X-100. After lysate was centrifuged at 15,000 rpm for 15 minutes, the supernatant was collected and the protein concentration was checked using a BCA kit.

The samples were lyophilized and the PTX concentration was confirmed by liquid chromatography (Agilent, USA) with a UV detector at a wavelength of 230 nm.

The absorbed PTX/protein ratio was used to compare the absorption of PTX solution and PTX loaded micelle solution PTX.

< Experimental Example 8> In vitro anti-cancer effect confirmation

Cytotoxicity of drug (DOX and PTX) and drug-loaded micelles was confirmed by CCK-8 viability analysis on kB cells.

5000 cells were dispensed per well of a 96-well plate containing 100 μL of RPMI-1640 medium containing 5% FBS and 1% penicillin-streptomycin and incubated at 37° C., 5% CO ₂ for 24 hours.

Thereafter, the medium was removed, and 100 μL of the drug solution or the drug-loaded micelle solution was added at different concentrations and incubated at 37° C., 5% CO ₂ for 48 hours.

The fraction of living cells was confirmed through CCK-8 cell viability analysis, and IC ₅₀ of drug alone or drug loaded micelles was calculated with GraphPad Prism 5 software.

< Experimental Example 9> In vivo (In vivo ) Research

All animal experiments were conducted under the guidelines of the protocol approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Pharmacy and the University of Pharmacy.

To construct a tumor xenograft model, 1 million kB cells were suspended in 0.1 mL of DPBS and injected into the right flank of BALB/c nude mice (Orient, Korea), and the tumor volume was calculated by the following formula.

Volume = L×W ² /2 (L is length, W is tumor depth)

Studies to confirm micelle biodistribution and anticancer effects were conducted when the tumor volume reached about 100 mm ³ .

1) Nano size Michelle's In vivo distribution ( biodistribution ) Confirm

A 1 mg/mL PEG-PLA-PEG micelle solution containing 10 wt% FITC-PEG-PLA-PEG was injected into the tail vein of a tumor-forming mouse.

After injection, the in vivo distribution of micelles was confirmed using a fluorescent in vivo imaging system (Neo Science, Korea) and a green channel of FITC at different time points.

Twenty-four hours after injection, mice were euthanized, tumors and other major organs were separated to confirm the accumulation of micelles in different sites, and quantitative analysis was performed using a program provided by the manufacturer.

2) Confirmation of anti-cancer effect in vivo

The tumor-forming nude mice were randomly separated into 3 groups, and 0.2 mL of a solution in which DOX.HCl or DOX loaded micelles were dissolved in water was injected once into the tail vein of the mice at a dose of 2 g/kg.

The mouse control group was injected with PBS (0.2 mL) intravenously into the tail vein.

Tumor size and body weight of mice were measured at 3 day intervals for 27 days.

At the end of the study, mice were euthanized and tumors were collected to confirm their size.

< Example 1> Check for mixed polymer system and optimization

In a previously reported study, micelles formed of PEG-PLA-PEG 2K-10K-2K were found to have high drug loading capacity, but very limited colloidal stability was a serious obstacle to drug delivery system development [N.H. Hoang, et. al., Characterization of a triblock copolymer, poly (ethylene glycol)-polylactide-poly (ethylene glycol), with different structures for anticancer drug delivery applications, Polym. Bull., (2016) 1-15.].

Low colloidal stability can lead to aggregation of micelles, and large particles are prone to removal by RES and cannot penetrate small capillaries and other sites, which significantly affects the overall biodistribution of the carrier.

Since the stability of the block copolymer micelle is strongly dependent on the length and density of the hydrophobic layer on the surface of the micelle, the present inventors improved micelle stability because long PEG and a small amount of PEG-PLA-PEG mixing as shown in FIG. Expected to do, after synthesizing PEG-PLA-PEG 5K-10K-5K triblock copolymer, PEG-PLA-PEG 2K-10K-2K(T2) and PEG-PLA-PEG 5K-10K-5K(T5) As shown in Table 1, a mixed polymer system composed of various ratios was prepared.

The mixed polymer system was prepared by dialysis using a semipermeable membrane (fraction molecular weight 3.5 kDa) in a phosphorylation buffer or saline at pH 7.4 after dissolving in DMSO according to the proportion of each polymer, and then confirming size and stability.

As a result, as shown in Table 1 and 2a, the size of mono5 micelles composed of 100% of PEG-PLA-PEG 5K-10K-5K(T5) was the smallest and showed very high colloidal stability, while mixing increased as the T5 ratio increased. The size of the system has been reduced.

B10 mixed with 10 wt% of T5 is a low stability micelle, stored at room temperature for a week, reached a size of about 1000 nm, and exhibited very low colloidal stability.

On the other hand, micelles with a T5 ratio of 20 wt% showed small size and high colloidal stability. Importantly, as the T5 ratio increased, the size of micelles did not decrease, and it was confirmed that 20 wt% of T5 was sufficient to prevent agglomeration by generating appropriate repulsion between micelles.

On the other hand, because the high hydrophilic-hydrophobic ratio induces a decrease in CMC, it lacks thermodynamic stability, so additional experiments were performed with the B20 system.

As a result of comparing the CMC of the B20 system with the mono2 and mono5 systems, as shown in Table 2, it was confirmed that the B20 system has a very low CMC value.

From the above results, it was confirmed that the B20 system has a high thermodynamic stability, because the B20 system has a relatively small T5 ratio, which does not significantly affect micelle assembly.

System
Weight ratio Size(nm) ^a
Stability
5K-10K-5K 2K-10K-2K Mono2* 0 One 417±24.9 Unstable ^b B10 0.1 0.9 297±11.2 Unstable ^b B20 0.2 0.8 150±4.4 Stable ^c B30 0.3 0.7 144±5.1 Stable ^c B40 0.4 0.6 149±1.6 Stable ^c Mono5 One 0 125±3.5 Stable ^c

^a Determined by Dynamic Light Scattering (DLS) at 25°C. Means ± SD, n=3.; ^b Aggregation occurred within 1 day.; ^c Stable for at least 1 week.; * H.-T. Song, et.al., Development of a new tri-block copolymer with a functional end and its feasibility for treatment of metastatic breast cancer, Colloids Surf., B, 144 (2016) 73-80.

System

CMC (μg/mL) ^a

DOX loading properties ^a DOX PTX LC ^b ( % ) Size(nm) LC ^b ( % ) Size(nm) Mono2* 5.17±0.771 17.54±0.61 550±23.3 7.19±0.33 570±35.2 B20 6.42±0.893 13.57±0.64 162±9.7 6.99±1.55 175±5.1 Mono5 17.97±1.127 4.31±0.54 150±3.2 2.21±0.97 155±6.8

^a Mean ± SD, n=3; ^b Loading capacity

< Example 2> Michelle's Morphological analysis

To confirm the relationship between the stability and the shape of micelles, the mono2, B20, and mono5 systems were stored at room temperature for a week immediately after preparation, and then the shape was confirmed using FE-SEM.

As a result, it was confirmed that the shape of the mono2 system was changed from a spherical shape to a rod shape during the storage period, as shown in FIGS. 2B and 2C, while the B20 and mono5 micelles remained spherical. Interestingly, aggregation of mono2 micelles was confirmed immediately after preparation.

From the above results, it was possible to confirm the tendency of aggregation of micelles formed of a triblock copolymer having a high hydrophobicity-hydrophilicity ratio.

The mono2 system consists of a T2 triple block copolymer capable of inducing aggregation and potential rod-to-rod transitions, indicating large differences in hydrophobic and hydrophilic moieties. However, when the T5 block copolymer having a long hydrophobic portion was added, the micelle system showed a spherical shape due to the steric stability effect of the long PEG chain.

< Example 3> Drug Loaded Micelle system

The drug loading capacity of the micelle carrier is a very important factor because it can determine the success of high dose formulation preparation, which is essential in a variety of clinical trials.

In order to confirm the effect of the polymer component on the drug loading ability of micelles, DOX and PTX, which are typically low water-soluble drugs, were loaded into mono2, B20 and mono5 systems.

As a result, as shown in Table 2, it was confirmed that the size was increased in all three systems loaded with DOX and PTX, respectively. This may be because the size of the micelle center as the above result affects the loaded drug.

The Mono2 and mono5 systems showed the highest drug loading capacity and the lowest drug loading capacity, respectively.

Although the loading capacity of DO20 and PTX of B20 was lower than that of the mono2 system, the difference was not significant, and interestingly, it was confirmed that the drug loading capacity of the B20 system was about 3 times higher than that of the mono5 system.

The drug loading capacity in the micelle is determined by several factors, such as the nature of the solute, the nature of the blocks forming the core, and the length of the core block. Because all of the systems evaluated had a core forming polymer such as PLA, the reason the mono2 and B20 systems have a higher drug loading capacity compared to the mono5 system is due to the proportion of hydrophobic moieties forming micelle cores used as drug storage space. .

In addition, the difference in the drug loading capacity thermodynamically may be the result of different free energies depending on the dilute and modified states of PEG 2K and PEG 5K.

From the above results, the combination of block copolymers can be suggested as a good strategy for increasing the loading capacity of low water soluble drugs into micelles.

Interestingly, the loading capacity of PTX was lower than the loading capacity of DOX, but compared to DOX, it was confirmed that PTX always increases the micelle size. These results are due to the low compatibility between PTX and PLA compared to between DOX and PLA.

When the compatibility between the drug and the polymer core (PLA) is low, there is a tendency to push against each other, and as they exist away from each other, a large space is formed between the drug and the polymer core, which causes low loading and a large core. .

From the results, it was confirmed that the B20 system has both drug loading capacity and stability, which are excellent characteristics of the mono2 and mono5 systems.

Thus, the B20 system can be used with good DDS as it is found to exhibit high thermodynamic, high colloidal stability, and high drug loading capacity.

< Example 4> in vitro( In vitro ) Check effect

In vitro studies were performed using a B20 system loaded with DOX and PTX (DOX/B20 and PTX/B20).

The anti-cancer effects of DOX/B20 and PTX/B20 on kB cell lines in vitro were compared with the experimental group using free DOX and PTX.

As a result, as shown in FIG. 3, the drug/B20 exhibited much better cytotoxicity than the experimental group using each free drug.

The results are similar to those of several studies in which the cytotoxicity of nanocarriers loaded with anticancer agents has been improved compared to free drugs. From these results, the nano-sized drug delivery system absorbs anti-cancer drugs that exhibit low water solubility. It has been found to improve and increase cytotoxicity.

Loading nanoparticles with anticancer agents can overcome the effects of Pgp and increase the accumulation of anticancer agents in cancer cells, so utilizing nanoparticles as an anticancer agent delivery system may be more effective in multidrug resistant cancer.

It was confirmed that the drug loaded B20 and the free drug were absorbed into kB cells in order to confirm a mechanism in which the drug loaded B20 exhibited an anticancer effect superior to that of the free drug in vitro.

As a result of flow cytometry analysis, DOX absorbed into kB cells as shown in FIG. 4A showed that DOX absorbed from the B20 system was 3-4 times higher than free DOX.

In addition, referring to Figure 4b confirming the absorption of DOX/B20 and free DOX through a confocal microscope, it was confirmed that it appeared similar to the flow cytometry results. In addition, PTX also showed a very high PTX absorbed from the B20 system compared to free PTX, as shown in FIG. 4C.

The above results are due to the way in which the free drug and the drug loaded B20 system are transported to cancer cells, and the absorption of drug loaded micelles through endocytosis is more than the free drug absorption mechanism using a simple diffusion method. Because it is fast.

< Example 5> in vivo( In vivo ) Check effect

It is well known that nano-sized particles accumulate high in tumor tissue due to EPR effects.

Accordingly, in order to confirm biodistribution of the nano-sized B20 system, B20 was replaced with FITC-PEG-PLA-PEG by replacing PEG-PLA-PEG 2K-10K-2K (10 wt% of B20 composition). FITC was marked on the micelle surface and the tumor-producing mice were injected intravenously.

As a result, almost all micelles were dispersed into the liver, as shown in FIG. 5A, and a small portion of the micelles was carried to the tumor. However, the fluorescence intensity of the liver decreased with time, and became very low after 24 hours.

On the other hand, micelles accumulated in tumors 24 hours after injection, and showed very high fluorescence intensity.

Interestingly, almost no fluorescence signal was seen in other major organs except for the liver where a small amount of micelles remained 24 hours after injection as shown in FIG. 5B.

From the above results, it was confirmed that the micelle system of the present invention is excellently selectively accumulated in tumors.

On the other hand, DOX/B20 was selected to confirm the in vivo anticancer drug delivery efficiency of the mixed system prepared in the present invention using a nude mouse with a kB tumor.

As a result, as shown in Figures 5c and 5e, the administration group of DOX/B20 showed very effective tumor growth inhibition results compared to the control group DOX and saline administration groups (n = 6, mean ± SEM). As expected, the control group showed maximum tumor growth with a tumor volume of 1000 mm ³ or more, and the tumor progression was confirmed in the DOX-administered group until the study was completed, while the tumor suppression was observed in the DOX/B20-administered group.

The superior anticancer effect of DOX/B20 compared to free DOX may be due to differences in biodistribution.

Free DOX was distributed non-selectively throughout the mouse body, but DOX encapsulated in nano-sized B20 micelles selectively accumulated in the tumor tissue due to the EPR effect, so that the DOX could be maintained at a high concentration for a long time at the tumor site.

On the other hand, as shown in Fig. 5d, there was no significant change in the body weight of the mice in all three groups during the study period, and the results are suggested to be due to a single dose of the drug.

From the above results, it was confirmed that the mixing system of the present invention was an excellent DOX drug delivery system, and thus, the mixing system was manufactured very effectively for delivery of an anticancer agent.

Since the specific parts of the present invention have been described in detail above, for those skilled in the art, it is obvious that this specific technique is only a preferred embodiment, and the scope of the present invention is not limited thereby. something to do. Therefore, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (9)

Poly(ethylene glycol) [PEG] and poly(lactic acid) [PLA] number average molecular weight ratio [PEG:PLA] is a triple block copolymer of 0.5 or less, first polymer and poly(ethylene glycol) [PEG] and poly (rock A nano-sized mixed polymer micelle composition composed of a second polymer that is a triple block copolymer having a number average molecular weight ratio [PEG:PLA] of [PLA] of 1.0 or more. The method according to claim 1, wherein the mixed polymer micelle composition is a mixed polymer micelle composition, characterized in that mixed with 60 to 90% by weight of the first polymer and 10 to 40% by weight of the second polymer. The method according to claim 1, The mixed polymer micelle is a mixed polymer micelle composition characterized in that the average diameter of 50 to 200 nm. Poly(ethylene glycol) [PEG] and poly(lactic acid) [PLA] number average molecular weight ratio [PEG:PLA] is a triple block copolymer of 0.5 or less, first polymer and poly(ethylene glycol) [PEG] and poly (rock A nano-sized mixed polymer micelle-type drug delivery system composed of a second polymer that is a triple block copolymer having a number average molecular weight ratio [PEG:PLA] of [PLA] of 1.0 or more. The method according to claim 4, The mixed polymer micelle-type drug delivery system is a mixed polymer micelle-type drug delivery system, characterized in that mixed with a first polymer of 60 to 90% by weight and a second polymer of 10 to 40% by weight. Poly(ethylene glycol) [PEG] and poly(lactic acid) [PLA] number average molecular weight ratio [PEG:PLA] is a triple block copolymer of 0.5 or less, first polymer and poly(ethylene glycol) [PEG] and poly (rock A pharmaceutical composition for the treatment of cancer, wherein an anticancer agent is encapsulated in a hydrophobic portion of a nano-sized mixed polymer micelle composed of a second polymer that is a triple block copolymer having a number average molecular weight ratio [PEG:PLA] of [PLA] of 1.0 or more. The method according to claim 6, wherein the pharmaceutical composition is a pharmaceutical composition for treating cancer, characterized in that it contains 70 to 99.9% by weight of the mixed polymer micelle and 0.1 to 30% by weight of an anticancer agent that can be enclosed in the mixed polymer micelle. The method according to claim 6, wherein the anticancer agent is a pharmaceutical composition for cancer treatment, characterized in that selected from the group consisting of doxorubicin, paclitaxel, etoposide, vinca alkaloids, vinblastine and colchicine. The method according to claim 6, wherein the cancer is cancer, pharmaceutical composition for the treatment of cancer, characterized in that selected from the group consisting of cancer, colon cancer, stomach cancer, lung cancer, pancreatic cancer, kidney cancer, nasopharyngeal epidermal cancer and oral cancer.

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