KR101818377B1 - Block copolymer containing diselenide-crosslinked for reactive oxygen species-related diagnosis/treatment and manufacturing method therof - Google Patents

Abstract

The present invention relates to a block copolymer containing a diselenide-crosslinking bond for diagnosing and treating reactive oxygen species-related diseases and a production method of the block copolymer. More specifically, the purpose of the present invention is to apply the produced block copolymer as a drug carrier selectively enabling drug delivery by producing a block copolymer which is capable of forming a diselenide-crosslinking bond having excellent biostability. The block copolymer of the present invention is a block copolymer comprising (A) a second block, (B) a first block which is coupled to one end of the second block, and (C) a third block which is coupled to the other end of the second block, wherein the first block is a polymer block which is biocompatible and hydrophilic, the second block as a polypeptide block which is biocompatible and hydrophilic includes a side chain of which a terminal is a selenol (SeH) or chlorine (Cl), and the third block as a polypeptide block which is biocompatible and hydrophobic includes a side chain including a hydrophobic functional group. According to various embodiments of the present invention, the block copolymer which can form a diselenide-crosslinking bond having excellent biostability, and a production method of the block copolymer are provided. The block copolymer can be applied as the drug carrier since the block copolymer is effective in selectively releasing a diagnosis/treatment agent according to the collapse of the crosslinking bond by converting a hydrophobic diselenide-crosslinking bond which has excellent biostability and responds to active oxygen existing at a high concentration in a cancer tissue into a hydrophilic selenic acid.

Description

TECHNICAL FIELD The present invention relates to a block copolymer containing diselenide crosslinking for diagnosis / treatment of reactive oxygen species-related diseases, and a process for producing the block copolymer containing diselenide-crosslinked reactive oxygen species-

The present invention relates to a block copolymer containing a diselenide crosslinking for diagnosing / treating an active oxygen species-related disease and a method for producing the block copolymer, and more particularly, to a block copolymer capable of achieving diselenide crosslinking with excellent biostability And to apply the copolymer as a drug delivery agent capable of selectively delivering a drug.

In recent years, nano-medicine for diagnosis and treatment has been actively studied in order to solubilize poorly soluble drugs and improve drug delivery efficiency to lesion tissues.

In particular, micelles of biocompatible and biodegradable polymers have been widely used as delivery modules for the delivery of customized hydrophobic anti-cancer drugs in nanotechnology for cancer treatment. Due to the amphipathic nature of such polymeric micelles, polymeric micelles can include drugs that are poorly water soluble in their hydrophobic core. In addition, since the size of the polymerizable micelle is nanometer, it can passively accumulate in the tumor epilepsy by passing through the permeable neovasculature, and can directly interact with cancer cells.

However, in the case of conventional drug delivery nano-drugs, conventional polymeric micelles when administered intravenously become unstable immediately after being exposed to very low diluted conditions in the blood, and form a self-assembly by physical interaction, When exposed to the environment, the structure is very unstable due to dilution or excessive amphiphiles, and thus the drug and contrast agent are released early due to the collapse.

To solve this problem, a crosslinking strategy has been applied to the core, shell, or core-shell interface of the micelle. At this time, while maintaining the micelle structure intact and efficiently, the cross-linked portion of the micelle serves as a physical barrier to prevent premature drug release during the systemic cycle.

However, such cross-linking has limitations in retarding drug release in lesion tissue and may also limit the desired drug release at the tumor site. For example, cross-linked micelles often release drugs in a continuous manner, failing to provide cytotoxic concentrations of drug to the site of tumor tissue. In order to improve overall therapeutic efficacy by induction and promotion of tumor-specific drug release, cross-linked micelles with stimulus-sensitive properties have been studied. These micelles can be tailored to specific physiological stimuli that distinguish tumor parts from normal tissues. Changes in the levels of various endogenous stimuli such as pH, glutathione, protease, and reactive oxygen species (ROS) may act as a trigger for drug release.

Such ROS is one of the most important physiological stimuli associated with a wide range of diseases including cancer, rheumatoid arthritis, diabetes, atherosclerosis, and Alzheimer's disease. Specifically, high ROS levels are common in almost all types of cancer and play an important role in cancer initiation and proliferation. Regarding the cell signaling pathway, ROS can penetrate cell membranes through various channels. Cellular ROS is mainly produced by mitochondria due to incomplete reduction of oxygen molecules. Hydrogen peroxide (H ₂ O ₂ ) is the most abundant molecule in the cell and is stable ROS. The H ₂ O ₂ level in normal cells is well maintained at about 20 nM, while in cancer cells it increases to 50-100 μM, which is due to excessive H ₂ O ₂ production that exceeds the capacity of existing antioxidant systems It is by accumulation. Because of this high concentration difference, H ₂ O ₂ is a promising molecule that can distinguish normal and cancer cells more effectively than other physiological stimuli.

Accordingly, existing active oxygen species-sensitive nano-drugs have been studied mainly on self-assemblies incorporating thioketal, boronic ester, and peroxylate ester. However, due to the above-described in-vivo stability problems, It is essential to develop a substance which is excellent in biostability and capable of releasing a drug at a desired position.

Korean Patent Publication No. 10-2015-0104401

Accordingly, an object of the present invention to solve the conventional problems is to provide a block copolymer capable of achieving cross-linking of diselenide having excellent biostability and a method for producing the block copolymer.

Also, it is an object of the present invention to provide a drug delivery system capable of responding to a high active oxygen concentration of a lesion tissue using the block copolymer, thereby selectively delivering a drug.

According to an exemplary aspect of the present invention, there is provided a method of manufacturing a semiconductor device, comprising (A) a second block, (B) a first block coupled to one end of the second block, and (C) As a block copolymer,

Wherein the first block is a biocompatible and hydrophilic polymer block,

Wherein the second block is a biocompatible, hydrophilic polypeptide block comprising a side chain whose terminal is selenol (SeH) or chlorine (Cl)

Wherein the third block is a biocompatible and hydrophobic polypeptide block comprising a side chain comprising a hydrophobic functional group.

According to another exemplary aspect of the present invention, as a combination of two or more block copolymers,

And the selenol group of the second block binds to the selenol group of the second block of the adjacent block copolymer to form a diselenide bond.

According to another exemplary aspect of the present invention, there is provided a micelle structure including a core, a first shell surrounding the core, and a second shell surrounding the first shell,

Wherein the micelle structure comprises the block copolymer combination,

Wherein the core comprises the third block,

The first shell is composed of the second block,

And the second shell is composed of the first block.

According to another exemplary aspect of the present invention, there is provided a drug delivery system comprising a micelle structure and a drug, wherein the drug is trapped in the core.

According to another exemplary aspect of the present invention, there is provided a pharmaceutical composition for treating diseases comprising the drug delivery system, wherein the disease is a disease that causes active oxygen species.

According to another exemplary aspect of the present invention, there is provided a process for preparing a polymer comprising the steps of (i) reacting a first monomer and a second monomer, and

(Ii) adding selenium to the product produced through the reaction and reacting,

Wherein the first monomer and the second monomer are different from each other and represented by the following formula (2).

(2)

(Wherein A is a halogen or a phenyl group, R ₁ , R ₂ and R ₃ are the same or different and independently selected from O, S and N, n is an integer of 1-7, m is an integer of 1-7.)

According to various embodiments of the present invention, there is provided a block copolymer capable of achieving bis-selenide crosslinking excellent in biostability and a method for producing the block copolymer.

The block copolymer is excellent in biostability and is sensitive to active oxygen present in cancer tissues at a high concentration to convert the hydrophobic diselenide bond into hydrophilic selenic acid to collapse the crosslinking, It can be applied as a drug delivery system since it is effective in selectively releasing the agent.

FIG. 1 is a graph showing stability due to cross-linking of a block copolymer and a micelle prepared through an embodiment of the present invention and a reaction process applied to a drug delivery system.
Fig. 2 shows the results of ¹ H NMR spectroscopic analysis of the monomers used in the synthesis of the block copolymers of Production Examples 1 and 2.
FIG. 3 (a) shows the results of ¹ H NMR spectral analysis of CP 1 and CP 2 'in Production Examples 1 and 2, and b shows FT-IR spectra of CP 2' and CP 2 in Production Examples 1 and 2, cm ^-1 and 765.9 cm ^-1 indicates a peak value, c, d is a graph showing the respective embodiments 1 and 2 of the TEM images and size distribution.
4A and 4B are graphs showing the CMC data values of CP 1 and CP 2 'of Production Examples 1 and 2, respectively, and c and d are graphs showing pyrene excitation spectrum values.
5 (a) is a graph showing the stability of micelles in Examples 1 and 2 in a solution state, (b) is a graph showing the stability of micelles of Examples 1 and 2 in 2.5% SDS, 2 is a graph showing the size distributions of Examples 1 and 2.
6 is a graph showing DOX-NCMs and DOX-DCMs DOX emission profiles in PBS at 37 ° C.
7 (a) and 7 (b) are graphs showing the results of MTT analysis of the cytotoxicity of the micelles not containing the drug and the DOX-encapsulated micelles treated for 24 hours, respectively.
FIGS. 8A and 8B are graphs showing the results of analysis of cell death of PC3 cells induced by DOX-NCMs and DOX-DCMs, respectively, by flow cytometry for 24 hours.
FIG. 9 (a) is an image observed with a confocal microscope of PC3 incubated with DOX-DCMs and DOX-NCMs at a temperature of 37 ° C., and b and c are DOX-DCMs and DOX- The results are shown by the flow cytometry.
10 shows the DOX content in tumor lysates, b denotes the DOX content in the main organs, c denotes the change in tumor volume in the treated group, d denotes the weight of the extracted tumor after treatment, An H & E stained image of the extracted tumor is shown.

Hereinafter, various aspects and various embodiments of the present invention will be described in more detail.

According to an aspect of the present invention, there is provided a method of manufacturing a semiconductor device, comprising (A) a second block, (B) a first block coupled to one end of the second block, and (C) Wherein the first block is a biocompatible and hydrophilic polymer block and the second block is a biocompatible and hydrophilic polypeptide block having a side chain of selenol (SeH) or chlorine (Cl) Wherein the third block is a biocompatible and hydrophobic polypeptide block comprising a side chain comprising a hydrophobic functional group.

Wherein the first block is formed by repeating repeating units of CR ₁₁ R ₁₂ -R ₁₃ -O or CO-CR ₁₆ -NH with a degree of polymerization of 22 to 227, wherein R ₁₁ and R ₁₂ are the same or different from each other independently represent an alkyl group of hydrogen, or a C1 to C7, wherein R ₁₃ is (CR ₁₄ R _15), and n _1, wherein R ₁₄ and R ₁₅ are the same or are different and each represents a hydrogen, or a C1 to C7, each independently from each other , R ₁₆ is R ₁₇ -COOH, R ₁₇ is a C 1 to C 5 alkyl group, and n ₁ is preferably 22 to 227.

The second block is formed by repeating repeating units of 4 to 8 of CO-CX-NH, and X is preferably selenol at the terminal.

Specifically, the X is R ₂₁ -COO-R ₂₂ -SeH, and wherein R ₂₁ is (CR ₂₃ R ₂₄₎ n _21, and the R ₂₃ and R ₂₄ are independently hydrogen, or C1 same as or different from each other and each to an alkyl group of C7, and wherein n ₂₁ is 2 to 4, wherein R ₂₂ is (CR ₂₅ R ₂₆₎ n _22, and the R ₂₅ and R ₂₆ are the same or different and are each independently hydrogen together, or C7 to C7 Lt; ₂₂ > is an alkyl group of 2 to 5 carbon atoms.

It is preferable that the third block is formed by repeating repeating units of CO-CY-NH with a degree of polymerization of 10 to 25, and Y is a side chain containing a hydrophobic functional group.

Specifically, Y is R ₃₁ -COO-R ₃₂ -Z, or R ₃₇ -A, A is an alkyl group of C 1 to C 5 or SR ₃₈ , Z is selected from phenyl, benzyl, and benzoyl, R ₃₁ is (CR ₃₃ R _34), and n _31, said R ₃₃ and R ₃₄ are the same or are different and each represents a hydrogen, C1 to C7, each independently from each other, wherein n ₃₁ is 1 to 5, wherein R ₃₂ is (CR ₃₅ R ₃₆ ) n ₃₂ , wherein R ₃₅ , R _36, R ₃₇ and R ₃₈ are the same or different from each other and are independently hydrogen or a C 1 to C 7 alkyl group, and n ₃₂ is 1 to 5 desirable.

It is more preferable that the block copolymer has one structure of the following formula. It was confirmed that the block copolymer having the structure of the following formula (1) was formed most stably when a micelle structure was formed.

[Formula 1a]

[Chemical Formula 1b]

[Chemical Formula 1c]

≪ RTI ID = 0.0 &

[Formula 1e]

(Provided that m = 22-227, n = 4-8, p = 10-25).

According to another aspect of the present invention, there is provided a block copolymer comprising two or more block copolymers, wherein the seleno group of the second block is bonded to the seleno group of the second block of the adjacent block copolymer to form a diselenide bond. Copolymer combination.

The diselenide bond is characterized in that it is converted to hydrophilic selenic acid in response to hydrogen peroxide which is an active oxygen species, which plays a role in enabling selective drug release at a specific tumor site.

Particularly, the diselenide bond (crosslinking) is formed through the reaction of the selenium group of the polymer with oxygen in the air without addition of a specific crosslinking agent, and the block copolymer forms a self-assembly in an aqueous solution.

A detailed description of the block copolymer is the same as that described above, and therefore, it will be omitted.

According to another aspect of the present invention there is provided a micelle structure comprising a core, a first shell surrounding the core, and a second shell surrounding the first shell, wherein the micelle structure comprises the block copolymer combination, Wherein the first shell is constituted by the third block, the first shell is constituted by the second block, and the second shell is constituted by the first block.

According to another aspect of the present invention, there is provided a drug delivery system comprising a micelle structure and a drug, wherein the drug is trapped in the core.

The drug is a hydrophobic drug, and is a drug exhibiting activity at a site where active oxygen species are present.

Specifically, the drug is preferably at least one selected from doxorubicin, paclitaxel, docetaxel, camptothecin, and methotrexate.

In the present invention, the site in which active oxygen species are present refers to all parts of the body such as lung, liver, large intestine, kidney, stomach, brain, uterus, breast and prostate where cancer cells may be present. Quot; refers to all parts where there may be.

According to another aspect of the present invention, there is provided a process for preparing a polymer comprising the steps of (i) reacting a first monomer and a second monomer, and (ii) adding selenium to a product produced through the reaction, Wherein the monomer and the second monomer are different from each other and represented by the following formula (2).

(2)

(Wherein A is a halogen or a phenyl group, R ₁ , R ₂ and R ₃ are the same or different and independently selected from O, S and N, n is an integer of 1-7, m is an integer of 1-7.)

The step (i) is preferably a step of reacting the first monomer with the second monomer to synthesize a triblock copolymer, and the reaction is preferably carried out by further adding an initiator.

The first monomer and the second monomer are different from each other and preferably represented by the following Formula 2, more preferably the first monomer is represented by the following Formula 3, and the second monomer is represented by the following Formula 4 .

(3)

[Chemical Formula 4]

The initiator is preferably? -Methoxy-? -Amino polyethylene glycol.

At this time, it is preferable that the reaction is carried out at a temperature of 20 to 70 ° C for 20 to 60 hours. If the temperature is out of the above range, it is not preferable because of a decrease in polymerization degree due to the stability problem of the NCA type monomer. If it is less than the time range, the polymerization is difficult to be sufficiently carried out, and the polymerization efficiency and the polymerization degree are reduced.

More specifically, the initiator and the first monomer are first reacted at a temperature of 30 to 60 ° C. for 10 to 30 hours, the reaction is completed, a second monomer is added to the resulting product, and the reaction is carried out at a temperature of 30 to 60 ° C. For 10 to 30 hours.

The first monomer and the second monomer are preferably reacted at a molar ratio (M) of 1: 1.5 to 3.5. If the molar ratio is out of the range, the stability of the micelles or the sensitivity of the active oxygen species is unfavorable. When the reaction is carried out at a molar ratio of less than 1: 1.5, the stability increases but the sensitivity to active oxygen decreases. When the reaction is carried out at a molar ratio of 1: 3.5 or more, the content of the selenoyl group is relatively decreased, There is a problem that the active oxygen sensitivity decreases.

More preferably, the initiator, the first monomer and the second monomer were reacted at a molar ratio of 1: 8: 20, and when reacted at the above-mentioned molar ratio, they were most stable as micelles, and the most effective drug delivery system was confirmed.

In the step (ii), selenium is added to the product generated through the reaction, and the chlorine formed at the end of the product is replaced with selenium.

At this time, the reaction is preferably performed at a temperature of 40 to 80 ° C for 1 to 10 hours. If the reaction temperature is higher than the reaction temperature, selenium is added in an aqueous solution, which is not preferable due to the nature of the solvent. The hydrolysis of the -COO- group in the second block is a concern, which is not preferable.

The selenium is used for forming the above-described diselenide bond. The selenium powder may be added in powder form. It is more preferable that the selenium powder is dispersed in a solvent and added in a solution state, in order to improve reactivity.

Hereinafter, the present invention will be described in more detail with reference to Examples and the like, but the scope and content of the present invention can not be construed to be limited or limited by the following Examples. It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit and scope of the present invention as set forth in the following claims. It is natural that it belongs to the claims.

In addition, the experimental results presented below only show representative experimental results of the embodiments and the comparative examples, and the respective effects of various embodiments of the present invention which are not explicitly described below will be specifically described in the corresponding part.

(Reagents and apparatus)

_{selenium powder, Sodium borohydride (NaBH 4} ), triphosgene, L-glutamic acid γ-benzyl ester, L-glutamic acid, doxorubicin o hydrochloride (DOX · HCl), triethylamine (TEA), 2 ', 7'-dichlorofluorescin diacetate (DCFH -DDA), DL-dithiothreitol (DTT), 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide, anhydrous tetrahydrofuran (THF), propedium iodide and anhydrous dimethylformamide Amino-polyethylene glycol (mPEG-NH ₂ , Mn = 3.4 KDa) was purchased from ID biochem (ulsan, Korea). 3-chloropropanol, and chlorotrimethylsilane were purchased from Tokyo Chemical Industries, Inc. (Japan).

All reagents were used as purchased without further purification and distilled water (DW) prepared from AquaMax-Ultra water purification system (Younglin Co., Anyang, Korea) was used.

Human prostate cancer (PC3) cells were purchased from the American Type Culture Collection (ATCC, Rockville, Md., USA) and cultured in RPMI-1640 medium, trypsin-EDTA, penicillin-streptomycin and fetal bovine serum Cell-culture-related reagents were obtained from Welgene Inc. (Daegu, Korea).

Annexin V (Alexa Fluor 488) conjugate and annexin binding buffer were purchased from Life Tecchnologies (CA, USA).

All test results were analyzed using one way ANOVA and statistical significance was considered when p value <0.05 (indicated by asterisk (*) in the drawing) Respectively.

(Preparation Example 1: DC-NPs tri-block copolymer synthesis-CP 2)

Preparation of PEG-b-poly (γ-3-chloropropanyl-L-glutamate) -b-poly (γ-benzyl-L-glutamate)

0.142 mmol of mPEG-NH ₂ (500 mg) was dissolved in 12 ml of anhydrous DMF under a nitrogen atmosphere, followed by the addition of 1.142 mmol of γ-3-chloropropanyl-L-glutamate N-carboxy anhydride (CG- NCA) The mixture was reacted at 45 DEG C for 24 hours with stirring. To this was added 751 mg of 2.85 mmol of L-glutamate N-carboxy anhydride (G-NCA) and further stirred at 45 ° C for 24 hours. After the reaction was terminated, the product was precipitated with 300 ml of diethyl ether, filtered and dried under vacuum to synthesize CP 2 'of the following scheme.

Then, a suspension of 5 mg of selenium powder and 3 ml of water was prepared and placed in a cooling bath under nitrogen atmosphere and stirred. Then, 2.5 mg of NaBH ₄ was added thereto, and the solution waited until the solution became transparent. Then, 5 mg of selenium powder was stirred . The solution was heated to 110 &lt; 0 &gt; C and then heated for about 20 minutes until it turned reddish brown, and the solution was used as a sodium diselenide stock solution.

The CP 2 '(500 mg) was dissolved in 5 ml of dry acetonitrile, and 1 ml of the sodium diselenide stock solution was added. The mixture was stirred at 60 ° C for 4 hours under a nitrogen atmosphere. The product was then precipitated with cold diethyl ether and washed with methanol. The precipitate was filtered and dried in vacuo to produce a DC-NPs tri-block copolymer having the CP 2 (b) structure of the following scheme.

(Production Example 2: NC-NPs block copolymer synthesis-CP 1)

Preparation of PEG-b-poly (γ-benzyl-L-glutamate)

500 mg of 0.142 mmol of mPEG-NH ₂ was dissolved in 12 ml of anhydrous DMF under nitrogen atmosphere, and 751 mg of γ-benzyl-L-glutamate N-carboxy anhydride (G-NCA) Lt; / RTI &gt; for 24 hours. After the reaction was completed, the product was precipitated with 300 ml of diethyl ether, filtered and dried under vacuum to prepare NC-NPs block copolymer having the CP 1 (a) structure of the following reaction formula.

[Reaction Scheme]

(Example 1: preparation of drug carrier using DC-NPs)

Preparation Example 1 (50 mg) was dissolved in 2 ml of acetonitrile, to which 50 ml of distilled water was added dropwise, followed by vigorous stirring. The suspension was dialyzed against distilled water using a dialysis membrane (MW cut off 3.5 KDa) for 24 hours.

(Example 2: preparation of drug carrier using NC-NPs)

The same procedure as in Example 1 was carried out except that Production Example 2 was used instead of Production Example 1.

(Test Example 1: Structural Analysis)

Each monomer and polymer was subjected to chemical structural analysis using ¹ H NMR and FT-IR, and the molecular weight was calculated. The results are shown in FIGS. 2 to 5.

As shown in the above reaction formula, the block copolymers of Preparation Examples 1 and 2 were synthesized through ring opening polymerization and then substituted with selenium. ^{By 1} H NMR spectrum, the monomers clearly showed their characteristic peaks (NCAs) and the structures of CP 1 and CP 2 'were observed, as shown in Figures 2 to 3, It is a result of showing that it is synthesized successfully. The calculated degree of polymerization and the molecular weight of the polymer are summarized in Table 1 below.

The FT-IR spectrum confirmed that the chlorine was completely replaced with selenium, indicating the formation of CP 2 from CP 2 '(FIG. 3b). Each of the NCMs and DCMs was formed by suspending amphipathic copolymers CP 1 and CP 2 in the aqueous solution. When exposed to ambient air, the seleno groups in CP 2 naturally self-crosslink to form diselenide bonds without any crosslinking agent. The DLS and TEM results show that both NCMs and DCMs are spherical and have a size distribution of unimpeded shapes with average diameters of 65.48 ± 2.1 nm and 85.10 ± 1.9 nm, respectively (FIGS. 3c and d). In general, nanoparticles that are spherical and have a size of less than 100 nm have been shown to have minimal absorption by the RES system.

That is, the CMC values of CP1 and CP2 'were 8.24 / / ml and 6.34 / / ml, respectively (Fig. 4). Preferably, both types of micelles were stable enough to maintain their size for at least 6 days under physiological conditions (Fig. 5A).

division Feed ratio ^a DP ^b Mn ^c CP 1 1: 8: 20 1: 5: 14 7497.23 CP 2 1:20 1:17 7244.08

( ^a molar feed ratio = PEG-NH ₂ : CG-NCA: G-NCA

^b Degree of polymerization calculated using ¹ H NMR.

^c Number average molecular weight determined using ¹ H NMR.

In addition, particle size and morphology were analyzed by dynamic light scattering (DLS) and transmission electron microscopy (TEM), and the excitation spectrum of pyrene was measured by a spectrofluorometer to calculate the critical micelle concentration (CMC) The results are shown in Table 2 below.

division Size (nm) The polydispersity index (PDI) NC-NPs 65.5 ± 2.1 0.15 DC-NPs 85.1 ± 1.9 0.18

Referring to Table 2 above, it was confirmed that NC-NPs and DC-NPs were spherical nanoparticles by observing the morphology of the particles by the TEM.

(Test Example 2: Evaluation of in-vivo stability)

For the in vivo stability evaluation of CCNSs, the mixture was mixed with a solution containing 2.5% of sodium dodecyl sulfate or 50% of dimethylsulfoxide and stored at 37 ° C., and the change of light scattering intensity was observed at regular intervals through DLS. The results are shown in Fig.

Referring to FIG. 5, in both cases of the control group, Comparative Example 1 (NC-NPs), a sharp decrease in the scattering intensity was observed, whereas in DC-NPs of Example 1, It can be confirmed that the structural stability is improved by introducing crosslinking bonds.

In addition, in Comparative Example 1, the relative dispersion strength rapidly decreased to 43% after 5 hours due to dissociation of the micelle structure induced by SDS. On the other hand, it can be seen that the DCMs of Example 1 maintain the intensity higher than 92% of the initial dispersion intensity for 5 hours, thereby keeping the micellar structure thermodynamically well fixed. In addition, the micelle structure was evaluated for resistance to organic solvents such as 50% DMSO. Only DCMs exhibited a size distribution with a significant intensity count rate (251 Kcps), while NCMs had a strength ratio of 1.4 Kcps And it was confirmed that the structure as a micelle was not maintained. These results clearly show that the crosslinked shell contributed to the excellent structural stability of the DCMs. Thus, DCMs can provide the best therapeutic effect with minimal adverse drug reaction (ADR) by preventing the early release of unintended drugs, especially when the drug loaded on the transporter is cytotoxic And can function as a solid drug carrier.

(Test Example 3: In vitro cytotoxicity and cell uptake studies)

Experiments were conducted to confirm the cytotoxicity of Example 1 (DC-NPs) through prostate cancer cell line PC3, and the results are shown in FIGS. 6 to 9 and Table 3 below.

However, the test method, first the PC3 cells 10% (v / v) FBS and 1% (v / v) penicillin (penicillin) - streptomycin (streptomycin) CO ₂ at 37 ℃ using RPMI 1640 medium, containing the Lt; / RTI &gt; The cytotoxic effects of the NCMs of Comparative Example 1 and the DCMs of Example 1, which were internally assessed, were evaluated by MTT analysis. 1 x 10 &lt; ⁴ &gt; PC3 cells were inoculated into each well of a 96-well culture dish and cultured for 24 hours. The cells were then washed twice with DPBS and incubated with micelles free of internal micelles and DOX at various concentrations for 24 hours Lt; / RTI &gt; After the used medium was removed and washed twice with DPBS, the cells were cultured for 3 hours in a medium (10%, v / v) containing 10% MTT solution (5 mg / ml in DPBS) , The medium was removed, MTT crystals were dissolved in 100 [mu] l of DMSO and absorbance was measured at 570 nm using a microplate reader (BioTek, Korea). For the analysis of cellular senescence, cells were cultured cell SCC7 inoculated at a density of 5 × 10 ⁵ cells / well in 6-well culture plate and the cells until the formation of a single layer. The cells were then washed twice with DPBS and replaced with medium containing 100 μg / ml of test sample. After incubation, the cells were harvested and incubated with FITC-labeled annexin V conjugate and propidium iodide in annexin binding buffer for 30 minutes. Finally, cells were analyzed using flow cytometry (guava easyCyte, USA).

Referring to FIG. 7, the in vitro cytotoxicity of micelles and DOX-loaded micelles were analyzed by MTT assay and cell death assay by flow cytometry for 24 hours. MTT analysis indicated that the internal micelle was non-toxic up to a concentration of 200 μg / ml, the main cause of which is the biocompatible polymer main chain of the internal micelle (FIG. 7a). Although selenium metal has high toxicity, the organic selenite derivatives exhibit negligible toxicity at low concentrations. It should be noted that the organic forms of selenium such as selenomethionine and selenocysteine are essential micronutrients in the human body. As shown in FIG. 7B, both DOX-loaded micelles and DOX showed dose-dependent cytotoxicity. Specifically, higher concentrations of DOX-DCMs in excess of 25 μg / ml were toxic to cancer cells slightly higher than DOX-NCMs, probably due to improved DOX release behavior of DOX-DCMs. In addition, the cell aging analysis result of the micelles loaded with DOX was in agreement with the MTT analysis result (FIG. 8). Interestingly, DOX-DCMs induced higher levels of apoptosis than DOX-NCMs. This difference may be due to the inherent physical properties of the organic selenium, and the organic selenium causes cytotoxicity when used in combination with an anticancer agent. Because of these unique properties, the selenium derivatives have been studied as modulators for chemotherapy, which are in clinical trials. Cancer cell death is highly advantageous compared to cell necrosis because tissue necrosis can result in host inflammation and further complications of treatment. In vitro cell fluorescence images imaged that DOX-loaded micelles released DOX from intracellular levels (Fig. 9A). The DOX values accumulated in PC3 cells were slightly higher in DOX-NCMs than in DOX-DCMs when imaged at 6 hours after incubation with the micelles. However, after 12 hours, slightly more drug accumulation was observed in DOX-DCMs treated cells than in DOX-NCMs. In contrast, drugs derived from DOX-NCMs are released at a steady rate. A similar pattern was also confirmed in the analysis by flow cytometry showing qualitative intracellular DOX content (Fig. 9b and c). The difference in fluorescence intensity between 6 hours and 12 hours after DOX-DCMs was greater than DOX-NCMs. During the initial treatment time, the cross-linked shell of DOX-DCMs appeared to limit drug release. However, long-term incubation resulted in a sufficient number of diselenide bond cleavages in response to ROS and then triggered rapid release of the drug from DOX-DCMs.

Intracellular drug release was also monitored using a confocal microscope (LSM 510 META NLO, Germany). PC3 cells (5000 cells / well) were inoculated into 8-well microarrays (Ibidi, Germany) and cultured for various time intervals with media containing DOX-NCMs and DOX-DCMs (non-lethal dose of DOX) Respectively. After incubation with the above samples, the cells were washed with PBS and incubated for 1 hour with 100 μl of serum-free medium containing 1 μM DCFH-DA. The cells were then washed twice with PBS and fixed with 4% paraformaldehyde solution. The fixed cells were stained with DAPI and then observed with a microscope.

The DOX-NCMs and DOX-DCMs showed appropriate loading efficiencies of 79% and 72%, respectively, and specific values are shown in Table 3 below. Referring to this, the size of the drug loaded micelles was slightly increased, which may have increased the volume of the hydrophobic compartment with loaded drug molecules. The drug release behavior was confirmed in PBS (pH 7.4) with or without H ₂ O ₂ (100 μM) as a representative ROS for 3 days at 37 ° C. (FIG. 6). DOX-NCMs did not show any rapid change in DOX release behavior due to the reaction to H ₂ O ₂ . After 3 days, the doses of DOX released from DOX-NCMs were found to be 53.28% and 45.83%, respectively, in the presence and absence of H ₂ O ₂ . In contrast, the release profile of DOX from DOX-DCMs varied greatly depending on the presence in H ₂ O ₂ . In the presence of H ₂ O ₂ , only 17.3% of the DOX was released for 3 days and the release rate tended to be relatively retarded. This shows that the diselenide cross-linking of DOX-DCMs effectively suppressed DOX release as a diffusion barrier. However, in the presence of H ₂ O ₂ (100 μM), the dose of DOX released from DOX-DCMs increased significantly up to 65% for 3 days at an increased DOX release rate. Also, compared to DOX-NCMs, DOX-DCMs showed a 12% increase in cumulative drug release over 3 days in the presence of H ₂ O ₂ . Upon exposure to ROS-rich conditions such as tumor microenvironment, the hydrophilic conversion of the diselenide bond present in the shell of the DOX-DCMs to the aforementioned selenic acid occurs resulting in a change in the molecular structure of the shell, Thereby triggering and facilitating release. This unique behavior has been shown to affect the excellent suitability of DCMs as cancer target drug transport and improved drug release has been shown to lower the rate of excretion of p-glycoprotein, making it very useful for treating cells with resistance to anti-cancer drugs .

division Size
(nm) ^a PDI ^b Drug loading (%) Loading content ^c Loading efficiency ^c NCMs 65.48 ± 2.1 0.151 - - - DCMs 85.10 ± 1.9 0.179 - - - DOX-NCMs 71.56 + - 3.4 0.181 10 7.9 79% DOX-DCMs 92.85 + 4.1 0.212 10 7.3 73%

(Test Example 4: Biological Distribution and Anti-Tumor Efficacy of DOX in the Body)

To evaluate the distribution of DOX in the body, a PC3 tumor animal model was prepared by inoculating 1 x 10 &lt; ⁶ &gt; cells present in serum-free medium (80 [mu] l) to the left side of 5-week-old male rats without hair. When the tumor volume became about 100 mm ^3, were divided animals into three experimental groups as follows: (i) DOX, (ii ) DOX-NCMs, and (iii) DOX-DCMs (n = 4, each group 4 Of experimental mice). The mice were then dosed intravenously with DOX (5 mg / kg), DOX-NCMs (DOX 5 mg / kg), and DOX-DCMs (DOX 5 mg / kg). After 12 hours of intravenous injection, the tumor and major organs were harvested and weighed to freeze the tissue in liquid nitrogen. The frozen tissue was then homogenized with 1 ml of dissolution buffer (50 mM Tris-HCl, pH 7.5, 0.2 M NaCl, 5 mM CaCl ₂ , 1% Triton X-100). To the prepared homogenate was added 200 μl of 33% (w / v) silver nitrate solution and stirred for 10 minutes. Silver nitrate facilitates the extraction of DOX bound to DNA and improves protein precipitation. Thereafter, 1 ml of acidified isopropanol (0.5 N HCl) was added to the homogenate and stored at 4 占 폚 overnight. The tissue extracts were centrifuged at 15,000 rpm for 15 minutes, and then the absorbance of the confluent solution was measured at 485 nm using an ultraviolet / visible ray spectrometer (Optizen 3220UV, Mecasys Co., Ltd., Korea) The concentrations were quantified.

PC3 tumor animal models were prepared in the same manner as described above and the tumor animal models were divided into four groups as follows: (i) saline, (ii) DOX (5 mg / kg) -NCMs (DOX 5 mg / kg), and (iv) DOX-DCMs (DOX 5 mg / kg). When the tumor volume reached 100 mm ³ , the rats were intravenously injected (n = 4, 4 mice per group) every 3 days. Tumor volume was calculated as a × b ^2/2 (a is the largest diameter, b is the smallest diameter). Tumor volume was observed every 2 days. Values are expressed as mean + standard deviation for an experimental group containing at least 4 animals.

To confirm tumor-specific DOX release, the body biomass distribution in the PC3 tumor animal model was assessed 24 hours after intravenous administration of DOX loaded micelles and DOX. The results shown in Fig. 10a show that the tumor DOX content (18.03 [mu] g / g tissue) in an animal model treated with DOX-DCMs was significantly higher in DOX-NCMs (10.66 [mu] g / g tissue) and DOX (4.82 [ Lt; RTI ID = 0.0 &gt; DOX &lt; / RTI &gt; On the other hand, DOX content in major organs (especially liver) was much lower in DOX-DCMs injected animal models compared to DOX-NCMs and DOX (FIG. 10b). This may be due to the improved structural stability of DOX-DCMs, which can facilitate tumor accumulation of DOX-DCMs with intact micelle structure.

Finally, anti-tumor efficacy of the DOX-loaded micelles and the DOX was confirmed by recording tumor volume and weight for 29 days after injecting micelles and DOX loaded with DOX into experimental mice with PC3 tumors. Saline injected experimental rats were used as a control and tumor volume and weight were most significantly decreased in the experimental group injected with DOX-DCMs (Fig. 10c and d), as expected, as compared to the other experimental groups. At the end of the anti-tumor efficacy assays, the tumor volumes of DOX, DOX-NCMs and DOX-DCMs injected groups were 1053.40, 680.86, 490.52, and 210.19 cm ³ , respectively. These observations clearly show that the cross-linking allows DOX-DCMs to better retain DOX in the systemic circulation, to deliver drugs more efficiently to tumor sites, and ultimately to treat cancer efficiently. Compared with other experimental groups through immunohistochemical staining, DOX-DCMs caused more apoptosis in tumors (Fig. 10e), confirming that damage to other major organs during treatment was negligible. Both DOX and DOX-NCMs showed cytotoxicity similar to DOX-DCMs in tumor tissues. On the other hand, significant toxicity was observed in the experimental group injected with DOX.

Therefore, according to various embodiments of the present invention, there is provided a block copolymer capable of achieving bis-selenide crosslinking excellent in biostability and a method for producing the block copolymer.

The block copolymer is excellent in biostability and is sensitive to active oxygen present in cancer tissues at a high concentration to convert the hydrophobic diselenide bond into hydrophilic selenic acid to collapse the crosslinking, It can be applied as a drug delivery system since it is effective in selectively releasing the agent.

Claims (19)

(A) a second block, (B) a first block coupled to one end of the second block, and (C) a third block coupled with the other end of the second block,
Wherein the first block is a biocompatible and hydrophilic polymer block,
Wherein the second block is a biocompatible, hydrophilic polypeptide block comprising a side chain whose terminal is selenol (SeH) or chlorine (Cl)
Wherein the third block is a biocompatible and hydrophobic polypeptide block comprising a side chain comprising a hydrophobic functional group.
The method according to claim 1,
The first block is formed by repeating repeating units of CR ₁₁ R ₁₂ -R ₁₃ -O or CO-CR ₁₆ -NH with a degree of polymerization of 22 to 227,
And R &lt; ₁₁ &gt; and R &lt; ₁₂ &gt; are the same or different from each other and are independently hydrogen or a C1 to C7 alkyl group,
Wherein R ₁₃ is (CR ₁₄ R ₁₅ ) n ₁ , R ₁₄ and R ₁₅ are the same or different and each independently hydrogen or a C 1 to C 7 alkyl group,
Wherein R ₁₆ is R ₁₇ -COOH, R ₁₇ is a C 1 to C 5 alkyl group,
Wherein n &lt; ₁ &gt; is from 22 to 227.
The method according to claim 1,
Wherein the second block is formed by repeating repeating units of CO-CX-NH with a degree of polymerization of 4 to 8,
And X is a selenol terminus.
The method of claim 3,
Wherein X is R ₂₁ -COO-R ₂₂ -SeH,
Wherein R ₂₁ is (CR ₂₃ R ₂₄ ) n ₂₁ , R ₂₃ and R ₂₄ are the same or different from each other and are each independently hydrogen or a C 1 to C 7 alkyl group, n ₂₁ is 2 to 4,
Wherein R ₂₂ is (CR ₂₅ R ₂₆ ) n ₂₂ , R ₂₅ and R ₂₆ are the same or different and each independently hydrogen or a C 7 to C 7 alkyl group, and n ₂₂ is 2 to 5 &Lt; / RTI &gt;
The method according to claim 1,
The third block is formed by repeating the repeating units of CO-CY-NH with a degree of polymerization of 10 to 25,
And Y is a side chain containing a hydrophobic functional group.
6. The method of claim 5,
Y is R ₃₁ -COO-R ₃₂ -Z, or R ₃₇ -A,
Wherein A is an alkyl group or an SR ₃₈ of C1 to C5,
Wherein Z is selected from phenyl, benzyl, and benzoyl,
Wherein R ₃₁ is (CR ₃₃ R ₃₄ ) n ₃₁ , R ₃₃ and R ₃₄ are the same or different from each other and are independently hydrogen or a C 1 to C 7 alkyl group, n ₃₁ is 1 to 5,
Wherein R ₃₂ is (CR ₃₅ R ₃₆ ) n ₃₂ , R ₃₅ , R _36, R ₃₇ and R ₃₈ are the same or different and each independently hydrogen or a C 1 to C 7 alkyl group,
And n &lt; ₃₂ &gt; is 1 to 5.
The method according to claim 1,
Wherein the block copolymer has one of the following structures:
[Formula 1a]

[Chemical Formula 1b]

[Chemical Formula 1c]

&Lt; RTI ID = 0.0 &

[Formula 1e]

M = 22-227, n = 4-8, and p = 10-25.
As a combination of two or more block copolymers,
Wherein said block copolymer is a block copolymer according to any one of claims 1 to 7,
Wherein the seleno group of the second block is bonded to the seleno group of the second block of the adjacent block copolymer to form a diselenide bond.
A micelle structure comprising a core, a first shell surrounding the core, and a second shell surrounding the first shell,
Wherein the micelle structure comprises the block copolymer combination according to claim 8,
Wherein the core comprises the third block,
The first shell is composed of the second block,
And the second shell is composed of the first block.
A drug delivery system comprising a micelle structure and a drug,
Wherein the micelle structure is the micelle structure according to claim 9,
Wherein the drug is trapped in the core.
11. The method of claim 10,
The drug is a hydrophobic drug,
Wherein the drug is a drug exhibiting activity at a site where active oxygen species are present.
12. The method of claim 11,
Wherein the drug is an anticancer agent.
13. The method of claim 12,
Wherein the drug is at least one selected from doxorubicin, paclitaxel, docetaxel, camptothecin, and methotrexate.
11. A pharmaceutical composition for treating diseases comprising a drug delivery system according to claim 10,
Wherein the disease is a disease that causes active oxygen species.
(I) reacting a first monomer and a second monomer, and
(Ii) adding selenium to the product produced through the reaction and reacting,
Wherein the first monomer and the second monomer are different from each other and are represented by the following Formula 2:
(2)

Wherein A is a halogen or a phenyl group, R ₁ , R ₂ and R ₃ are the same or different from each other and independently selected from O, S and N, n is an integer of 1-7, It is an integer of -7.
16. The method of claim 15,
Wherein the first monomer is represented by the following general formula (3)
Wherein the second monomer is represented by the following formula (4): &lt; EMI ID =
(3)

[Chemical Formula 4]

16. The method of claim 15,
The step (i) is characterized in that an initiator is further added to react,
Wherein the initiator is? -Methoxy-? -Amino polyethylene glycol.
16. The method of claim 15,
Wherein the step (i) is carried out at a temperature of 20 to 70 ° C for 20 to 60 hours.
16. The method of claim 15,
Wherein the step (ii) is carried out at a temperature of 40 to 80 ° C for 1 to 10 hours.

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