KR102184768B1 - Composition for mixed polymeric micelles and use thereof - Google Patents

Abstract

The present invention relates to a nano-sized mixed polymer micelle consisting of a first polymer and a second polymer, and to a use thereof, and in more detail, the mixed polymer micelle is less than the micelles made of the previously reported BAB-type triple block copolymer. The encapsulation content of soluble drugs and stability in vivo were very excellent.In particular, as it was confirmed that the mixed polymer micelles encapsulated with an anticancer agent exhibit excellent tumor selectivity and excellent anticancer effects, the mixed polymer micelles are excellent drug delivery agents and anticancer drugs. Can be provided.

Description

Composition for mixed polymeric micelles and use thereof

The present invention relates to a nano-sized mixed polymer micelle composition.

Nano-sized micelles having a core-shell structure based on amphiphilic block copolymers have been widely developed as various drug delivery systems. The hydrophobic core of micelles acts as a container for encapsulating drugs that are hardly soluble in water, and the hydrophilic shell of micelles protects and stabilizes the micelles.

The amphiphilic block copolymers for preparing polymer micelles developed to date are classified into ones that do not contain a functional group and one that contains a plurality of functional groups. Polymers that do not contain functional groups cannot sufficiently interact with hydrophobic drugs, and there are limitations in the development and application of drug delivery systems, and in the case of containing multiple functional groups, they are themselves dissolved in water, which prevents adequately encapsulating drugs. There is a limit.

As a method of synthesizing a BAB-type triple block copolymer using an AB-type initial polymer, a method of preparing a BA-AB-type block copolymer using a crosslinking agent is currently 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 the reaction is performed using adipoyl chloride as a crosslinking agent, hydrochloric acid is generated as a by-product during the process, and thus the hydrophobic A block is easily decomposed (Feng Li et al., Langmuir, 23). , 2778-2783, 2007).

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

Republic of Korea Patent Publication No. 2004-0021760 (published on Mar 11, 2004)

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 the physiologically active substance encapsulated in a target site in a stable state in vivo, and a drug delivery system or pharmaceutical composition using the same.

The present invention relates to the first polymer and poly(ethylene glycol) [PEG], which are triple block copolymers having a number average molecular weight ratio [PEG:PLA] of 0.5 or less of poly(ethylene glycol) [PEG] and poly(lactic acid) [PLA] It provides a nano-sized mixed polymer micelle composition consisting of a second polymer, 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 relates to the first polymer and poly(ethylene glycol) [PEG], which are triple block copolymers having a number average molecular weight ratio [PEG:PLA] of 0.5 or less of poly(ethylene glycol) [PEG] and poly(lactic acid) [PLA] It provides a nano-sized mixed polymer micelle-type drug delivery system 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.

In addition, the present invention is the first polymer and poly(ethylene glycol) [PEG] which are triple block copolymers having a number average molecular weight ratio [PEG:PLA] of 0.5 or less between poly(ethylene glycol) [PEG] and poly(lactic acid) [PLA] ] And poly(lactic acid)[PLA] with an anticancer agent encapsulated in the hydrophobic part of a nano-sized mixed polymer micelle consisting of a second polymer, a triple block copolymer having a number average molecular weight ratio [PEG:PLA] of 1.0 or more. Provide pharmaceutical compositions.

According to the present invention, the nano-sized mixed polymer micelle consisting of the first polymer and the second polymer has superior encapsulation content and in vivo stability of poorly soluble drugs than micelles made of the previously reported BAB-type triple block copolymer. In particular, as it was confirmed that the mixed polymer micelles encapsulated with anticancer agents exhibit excellent tumor selectivity and excellent anticancer effects, the mixed polymer micelles can be provided as excellent drug delivery agents and anticancer drugs.

Figure 1 is a result of confirming the mixing method for an excellent drug delivery system, Figure 1a is the result of confirming the effect of the hydrophobic part on the micelle properties, Figure 1b is a system formed by mixing other triblock copolymers and its anticancer 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 the result of confirming the size change of each system every one week, Figures 2b and 2c is a result of confirming the shape of each system, Figure 2b is a micelle fabrication It is the result of confirming the shape immediately after, and FIG. 2C is the result of confirming the shape after storage at room temperature for 7 days. * Means 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 a result of confirming the anticancer effect of kB cells in vitro , Figure 3a is a result of comparing the anticancer effect of DOX/B20 with free DOX, the IC ₅₀ values of DOX/B20 and free DOX are 0.0357 and 0.1125 μg, respectively /mL, and Figure 3b is a result of comparing the anticancer effect of PTX/B20 with free PTX, and the IC ₅₀ values of PTX/B20 and free PTX are respectively 0.1506 and 0.8587 μg/mL.
Figure 4 is a result of confirming 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 under a confocal microscope Is a result of comparing with free DOX, and FIG. 4c is a result of comparing the PTX/B20 absorption level with free PTX. Statistical analysis was performed with a two-tailed Student's test.
5 is a result of confirming the effect in vivo, and FIG. 5a is a result of confirming the biodistribution of nano-sized micelles in mice with tumors. The upper drawing is an optical image, the middle drawing is a fluorescent image, and the lower drawing is a merged image. The right figure shows a tumor, and FIG. 5B is a result of confirming the accumulation level of nano-sized micelles in tumors and other tissues 24 hours after injection. The upper drawing is an optical image, the middle drawing is a fluorescent image, and It is a merged image, and Figures 5c and 5d are the results of one-way ANOVA as a result of confirming the tumor volume and weight of animals treated with DOX/B20, free DOX and PBS, respectively, and Dunnette's test was performed for post-mortem analysis. (*p<0.05, **p<0.001), Figure 5e is a result of extracting and confirming tumors before and 27 days after treatment of mice with tumors. Arrows denote tumors and (1) DOX/B20 treatment Group, (2) is a free DOX-treated group, and (3) is a PBS-treated control group.

The present invention relates to the first polymer and poly(ethylene glycol) [PEG], which are triple block copolymers having a number average molecular weight ratio [PEG:PLA] of 0.5 or less of poly(ethylene glycol) [PEG] and poly(lactic acid) [PLA] It is possible to provide 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 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 above content range, there may be a problem in that the amount and rate of encapsulation of the drug decrease, and when it is added above the above content range, problems of phase separation, precipitation, and particle increase may occur.

In addition, when the second polymer is added below the above content range, the particle size increases and problems of instability may occur, and when the second polymer is added above the above content range, there may be a problem in that the amount and rate of the drug are increased. have.

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

According to an embodiment of the present invention, FITC is marked on the surface of B20 micelles by replacing PEG-PLA-PEG 2K-10K-2K (10 wt% of B20 composition) with FITC-PEG-PLA-PEG, and tumors are 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 distributed to the liver, and a small portion of the micelles were transported to the tumor. However, with the passage of time, the fluorescence intensity of the liver decreased, and after 24 hours, the fluorescence intensity was very low, whereas the tumor showed a very high fluorescence intensity 24 hours after the injection, and a large amount of micelle accumulation was confirmed.

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

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

Accordingly, the present invention relates to a first polymer and a poly(ethylene glycol) [PEG] triblock copolymer having a number average molecular weight ratio [PEG:PLA] of 0.5 or less between poly(ethylene glycol) [PEG] and poly(lactic acid) [PLA]. ] And poly(lactic acid)[PLA] can provide a nano-sized mixed polymer micelle-type drug delivery system composed of a second polymer which is a triple block copolymer having a number average molecular weight ratio [PEG:PLA] of 1.0 or more.

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 the first polymer and poly(ethylene glycol) [PEG] which are triple block copolymers having a number average molecular weight ratio [PEG:PLA] of 0.5 or less between poly(ethylene glycol) [PEG] and poly(lactic acid) [PLA] ] And poly(lactic acid)[PLA] with an anticancer agent encapsulated in the hydrophobic part of a nano-sized mixed polymer micelle consisting of a second polymer, a triple block copolymer having a number average molecular weight ratio [PEG:PLA] of 1.0 or more. Pharmaceutical compositions can be provided.

The cancer treatment pharmaceutical composition may include 70 to 99.9% by weight of mixed polymer micelles and 0.1 to 30% by weight of an anticancer agent that may be encapsulated in the mixed polymer micelles, and the anticancer agents doxorubicin, paclitaxel, etoposide, vincalkaloid, It may be selected from the group consisting of vinblastine and colchicine.

According to another embodiment of the present invention, in order to confirm the delivery efficiency of an anticancer agent in vivo of a drug delivery system (DOX/B20) containing doxorubicin (DOX) in the mixed polymer micelle (B20), a kB tumor is formed. As a result of injection into the tail vein of nude mice, as shown in FIGS. 5C and 5E, the DOX/B20 administration group showed very effective tumor growth inhibition results compared to the control group DOX and saline administration (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 tumor progression was confirmed until the study was completed in the DOX-treated group, while tumor suppression was observed in the DOX/B20-treated group.

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

From the above results, as the advantages of the mixed polymer micelles as an excellent drug delivery system were confirmed, the mixed polymer micelles containing an anticancer agent may be used as an excellent anticancer treatment 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 cancer treatment is in the group consisting of injections, granules, powders, tablets, pills, capsules, suppositories, gels, suspensions, emulsions, drops or liquids according to a conventional method. Any one of the selected formulations can be used.

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

Specifically, carriers, excipients and diluents are lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, 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, capsules. And the like, and these solid preparations may be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, and the like in the composition. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral use include suspensions, liquid solutions, emulsions, syrups, etc.In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as humectants, sweeteners, fragrances, and preservatives may be included. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, suppositories, and the like. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc. may be used as the non-aqueous solvent and suspension. As a base material for the suppository, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used.

According to an 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 It can be administered to a subject in a conventional manner via the route.

The preferred dosage of the pharmaceutical composition for cancer treatment may vary depending on the condition and weight of the subject, the type and degree of the disease, the form of the drug, the route and duration of administration, and may be appropriately selected by those skilled in the art. According to an embodiment of the present invention, although 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. Administration may be administered once a day or may be divided into several doses, whereby the scope of the present invention is not limited.

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 aid understanding of the present invention. However, the following examples are for illustrative purposes only, and the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those of ordinary skill 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 (N,N-dicyclohexylcarbodiimide; DCC), stannous octoate; Tin(II)-2-ethylhexanoate, Sn(Oct) 2), 4-dimethylaminopyridine (DMAP), dimethylsulfoxide (DMSO), succinic anhydride, pyridine and triethylamine (TEA) were added to Sigma-Aldrich ( St. Louis, MO, USA). Tetrahydrofuran (THF), toluene, acetone and dichloromethane (DCM) were purchased and used from Honeywell Burdick & Jackson® (Muskegon, MI, USA).

Doxorubicin ((DOX) HCl) was added to Boryung Co. (Seoul, South Korea), and paclitaxel (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), and 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.] synthesized a PEG-PLA-PEG triple block copolymer with or without a functional group, and PEG- so that the molecular weights were 2K-10K-2K and 5K-10K-5K. The PLA-PEG triple block copolymer was synthesized.

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

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

In the presence of DMAP, TEA and pyridine, PEG was reacted with succinic anhydride to PEG carboxylation (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 an excess of diethyl ether to obtain the final product, filtered and dried in a vacuum for 2 days.

To prepare a PEG-PLA-PEG triple block copolymer, Steglich esterification was performed using the 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 carried out overnight, the final product was obtained by precipitating in an excess of diethyl ether, which was then filtered and dried under vacuum for 2 days.

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

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

After reacting at room temperature for 24 hours, dialysis with 1L water for 24 hours to remove the remaining FITC-5-isothiocyanate and freeze-drying to obtain FITC-PEG-PLA-PEG powder. The micelles used to obtain an in vivo image contained 10 wt% of FITC-PEG-PLA-PEG.

< Experimental example 2> Preparation of self-assembled polymer micelles

Polymerizable micelles were prepared by dialysis. After dissolving the triblock copolymer or a mixture of other triblock copolymers (10 mg) in 3mL DMSO, the DMSO solution in which the triblock copolymer was dissolved was transferred to a dialysis membrane (MWCO 3.5KDa; Spectrum, USA) and pH 7.4 phosphate buffered 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

In order to confirm the critical micelle concentration (CMC), pyrene was used as a fluorescent probe, and fluorescence was measured with a Scinco FS-2 fluorescence Spectrometer (Seoul, Korea).

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

After evaporation of 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 at room temperature overnight and the excitation spectrum of pyrene in the sample was recorded with 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) of the excitation spectrum to the logarithm of the copolymer concentration, and CMC was determined by plotting the low copolymer concentration in the plot. Was defined as the intersection of

< Experimental example 4> Check particle size

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

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

< Experimental example 5> Morphological analysis

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

A few drops of the diluted dispersion were put on a slide glass, dried in a vacuum state, and coated with platinum on the sample to perform FE-SEM.

< Experimental example 6> drugs Loaded Micelle preparation and characterization

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

HCl was isolated from doxorubicin (DOX) in 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 triblock copolymer solution and dialyzed against 500 mL of PBS, pH 7.4 for 24 hours.

The sample was centrifuged at 5000 rpm for 5 minutes to precipitate the unloaded DOX, and then the supernatant including the DOX-loaded micelles 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 triblock copolymer solution were mixed, and dialyzed with 500 mL of PBS, pH 7.4 for 24 hours.

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

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

The drug loading dose was calculated according to the following formula.

Drug loading dose (wt%) = (drug-loaded micelles/copolymers and drugs) × 100%

< Experimental example 7> Intracellular Cellular uptake

One) Flow cytometry through DOX Absorption check

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

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

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

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

Data were obtained using Cell Quest Pro software.

2) Confocal Through the microscope DOX Absorption check

DOX uptake in kB cells in vitro was confirmed with a confocal microscope.

First, in a 6-well plate containing a cover glass in each well, kB cells were distributed and grown at 150000 cells per well.

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

After incubation for 3 hours, the medium was removed, 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 with 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 in a 6-well plate dispensed with 3 mL medium per well, and cultured for 24 hours at 37° C. and 5% CO ₂ .

The medium was removed, a PTX solution or a PTX-loaded micelle 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.

Wells were washed three times with cold PBS, cells were collected with a scraper 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 centrifuging the lysate at 15,000 rpm for 15 minutes, the supernatant was collected and the protein concentration was confirmed using a BCA kit.

The sample was lyophilized and the PTX concentration was checked using 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 the PTX solution and the micelle solution PTX loaded with PTX.

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

The cytotoxicity of drug (DOX and PTX) and drug-loaded micelles was confirmed through CCK-8 viability analysis for kB cells.

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

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

The fraction of live cells was confirmed through CCK-8 cell viability analysis, and the IC ₅₀ of the 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 performed under the guidelines of a protocol approved by the Republic of Korea and the Institutional Animal Care and Use Committee (IACUC) of the University of Pharmacy.

In order 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 the length, W is the depth of the tumor)

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

1) Nano size Micellar In vivo distribution ( biodistribution ) Confirm

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

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

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

2) Confirmation of anticancer effect in vivo

3 groups of nude mice with tumors were randomly separated, and 0.2 mL of a solution obtained by dissolving DOX.HCl or DOX-loaded micelles in water was injected once into the tail vein of the mice at a dose of 2 g/kg.

The control mice were injected with PBS (0.2 mL) intravenously into the tail vein.

The tumor size and the weight of the mice were measured every 3 days for 27 days.

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

< Example 1> Confirmation of mixed polymer system and optimization

In a previously reported study, micelles formed with 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 the aggregation of micelles, and large particles are easy to remove 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 micelles strongly depends on the length and density of the hydrophobic layer on the micelle surface, the present inventors suggested that mixing a long PEG and a small amount of PEG-PLA-PEG improves the PEG density as shown in FIG. 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 manufactured using.

The mixed polymer system was prepared by dissolving in DMSO according to the ratio of each polymer and dialysis using a semipermeable membrane (fractional molecular weight of 3.5 kDa) in a phosphorylation buffer solution or saline solution having a pH of 7.4, and then the size and stability were confirmed.

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

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

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

On the other hand, since the high hydrophilicity-hydrophobicity 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, it was confirmed that the B20 system had a very low CMC value as shown in Table 2.

From the above results, it was confirmed that the B20 system has high thermodynamic stability, which is because the B20 system has a relatively small T5 ratio that 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℃. Means±SD, n=3.; ^b Aggregation occurred within 1 day.; ^c Stable for at least 1 week.; * Means 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) Prepared by the method 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> Micellar Morphological analysis

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

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

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

The mono2 system consists of a T2 triple block copolymer that exhibits a large length difference between the hydrophobic and hydrophilic moieties, which can induce agglomeration and potential spheroid to rod-like transitions. However, when a T5 block copolymer having a long hydrophobic moiety was added, the micelle system exhibited a spherical shape due to the steric stability effect of the long PEG chain.

< Example 3> drugs Loaded Micelle system

The drug loading capacity of the micelle carrier is a very important factor as it can determine the success of manufacturing high-dose formulations, which are essential in various clinical trials.

In order to confirm the effect of the polymer component on the drug loading ability of micelles, representatively low water-soluble drugs, DOX and PTX, 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. The size of the center of micelles as described above may be due to the influence of the loaded drug.

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

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

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

In addition, the difference in drug loading dose thermodynamically may be a result of different free energies depending on the dilution and modification 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, although the loading capacity of PTX was lower than that of DOX, it was confirmed that compared to DOX, PTX always increased the micelle size. This result is due to the low compatibility between PTX and PLA compared to DOX and PLA.

When the compatibility between the drug and the polymer core (PLA) is low, they tend to push each other and exist far apart, resulting in a large space between the drug and the polymer core, which causes low loading and large cores. .

From the above 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 as an excellent DDS as it has been found to exhibit high thermodynamics, high colloidal stability, and high drug loading capacity.

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

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

The anticancer 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, drug/B20 exhibited very superior cytotoxicity compared to the experimental group using each free drug.

The above results are similar to several studies that have reported that the cytotoxicity of the anticancer drug-loaded nanocarrier is improved compared to the free drug. It was confirmed to increase cytotoxicity by improving.

Since loading an anticancer agent into nanoparticles can overcome the effect of Pgp and increase the accumulation of anticancer agents in cancer cells, using nanoparticles as an anticancer agent delivery system may be more effective in multidrug resistant cancer.

In order to confirm the mechanism in which the drug-loaded B20 exhibits superior anticancer effect than the free drug in vitro, it was confirmed that the drug-loaded B20 and the free drug were absorbed into kB cells.

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

In addition, referring to FIG. 4B, which confirmed the absorption of DOX/B20 and free DOX through a confocal microscope, it was confirmed that the results were similar to the flow cytometric analysis results. In addition, PTX also showed very high absorption of PTX from the B20 system compared to free PTX as shown in FIG. 4C.

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

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

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

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

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

On the other hand, micelles accumulated in a large amount in the tumor 24 hours after injection, and showed very high fluorescence intensity.

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

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

Meanwhile, DOX/B20 was selected to confirm the in vivo anticancer drug delivery efficiency of the mixed system manufactured in the present invention using nude mice with kB tumors formed thereon.

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

Compared to free DOX, the superior anticancer effect of DOX/B20 may be due to differences in biodistribution.

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

On the other hand, as shown in FIG. 5D, there was no significant change in the weight of mice in all three groups during the study period, and the result is suggested to be due to a single dose of the drug.

From the above results, as the advantages of the mixed system of the present invention as an excellent DOX drug delivery system were confirmed, it was confirmed that the mixed system was produced very effectively for the delivery of anticancer drugs.

As described above, specific parts of the present invention have been described in detail, and for those of ordinary skill in the art, it is obvious that these specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereby. something to do. Therefore, it will be said that the practical scope of the present invention is defined by the appended claims and their equivalents.

Claims (9)

Poly(ethylene glycol)[PEG] 2kDa(2K) and poly(lactic acid)[PLA] 10kDa(10K) of PEG-PLA-PEG triple block copolymer, the first polymer and poly(ethylene glycol)[PEG] 5kDa( 5K) and poly (lactic acid) [PLA] 10kDa (10K) PEG-PLA-PEG triple block copolymer consisting of a second polymer nano-sized mixed polymer micelle composition consisting of a copolymer. The mixed polymer micelle composition according to claim 1, wherein the mixed polymer micelle composition is mixed with 60 to 90% by weight of the first polymer and 10 to 40% by weight of the second polymer. The mixed polymer micelle composition of claim 1, wherein the mixed polymer micelle has an average diameter of 50 to 200 nm. Poly(ethylene glycol)[PEG] 2kDa(2K) and poly(lactic acid)[PLA] 10kDa(10K) of PEG-PLA-PEG triple block copolymer, the first polymer and poly(ethylene glycol)[PEG] 5kDa( 5K) and poly(lactic acid) [PLA] 10kDa (10K) PEG-PLA-PEG triple block copolymer consisting of a second polymer of a nano-sized mixed polymer micelle type drug delivery system. The mixed polymer micelle type drug delivery system according to claim 4, wherein the mixed polymer micelle type drug delivery system is mixed with 60 to 90% by weight of the first polymer and 10 to 40% by weight of the second polymer. Poly(ethylene glycol)[PEG] 2kDa(2K) and poly(lactic acid)[PLA] 10kDa(10K) of PEG-PLA-PEG triple block copolymer, the first polymer and poly(ethylene glycol)[PEG] 5kDa( 5K) and poly (lactic acid) [PLA] 10kDa (10K) PEG-PLA-PEG triple block copolymer consisting of a second polymer consisting of a nano-sized mixed polymer micelles with an anticancer agent encapsulated in the hydrophobic portion Composition. The pharmaceutical composition for cancer treatment according to claim 6, wherein the pharmaceutical composition comprises 70 to 99.9% by weight of mixed polymer micelles and 0.1 to 30% by weight of an anticancer agent that can be encapsulated in the mixed polymer micelles. The pharmaceutical composition for cancer treatment according to claim 6, wherein the anticancer agent is selected from the group consisting of doxorubicin, paclitaxel, etoposide, vinca alkaloid, vinblastine and colchicine. The pharmaceutical composition for cancer treatment according to claim 6, wherein the cancer is selected from the group consisting of liver cancer, breast cancer, colon cancer, gastric cancer, lung cancer, pancreatic cancer, kidney cancer, nasopharyngeal epidermal cancer, and oral cancer.

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