KR20120054279A - Ph sensitive block copolymer, preparation method thereof and drug delivery system using the same - Google Patents

Abstract

PURPOSE: A pH-reactive block copolymer is provided to improve overall yield more than existing histidine, and to easily control molecular weight, thereby capable of manufacturing a polymer with high molecular weight. CONSTITUTION: A pH-reactive block copolymer is formed by copolymerization of a polyethylene glycol-based compound, and poly(aspartic acid-graft-imidazole) in which imidazole is grafted into an aspartic acid. The block copolymer is a compound in chemical formula 1, m is an integer from 10-45, and n is an integer from 15-35, and the sum of m and n is an integer from 18-60. A drug delivery system comprises the pH sensitive block copolymer, and physiologically active material capable of sealing into the block copolymer.

Description

pH sensitive block copolymer, preparation method thereof and drug delivery agent using the same

The present invention relates to a pH-reactive block copolymer comprising an aspartic acid polymer containing an imidazole group reacting with an acidic pH, a method for preparing the same, and a drug carrier using the same.

Micelle generally refers to a thermodynamically stable and uniform spherical structure of low molecular weight materials having amphiphilic, for example, hydrophilic and hydrophobic groups, and dissolves and injects a water-insoluble drug into the compound having the micelle structure. In this case, the drug is present in the micelle, and since the micelle can release the target-oriented drug in response to a change in temperature or pH in the body, there is a high possibility of application as a carrier for drug delivery.

It is generally known that the pH of normal tissues and blood is 7.4 while the pH of cancer cells is on average between 7.0-6.5. Therefore, when it is noted that most solid cancers have a weakly acidic pH around the cell, the development of a pH-sensitive polymer material that can target the pH of cancer cells can be widely used for diagnosis and treatment of cancer. It may be used as a material for the development of medicines for the diagnosis and treatment of weakly acidic lesions and rheumatoid arthritis.

Meanwhile, N-carnoxyanhydride (NCA), which is required for synthesizing histidine polymers, which is widely known as a pH-sensitive polymer, is very vulnerable to air and reacts with moisture in the air, resulting in low yield and high molecular weight. Difficult to synthesize polymers (molecular weight of 5000 Da or more), which leads to difficulty in synthesizing polymers targeting endosomal pH for multi-drug-resistant chemotherapy, and also because they are insoluble in volatile solvents, micelle formation is possible only by dialysis. There was inconvenience of operation and difficulty of mass production.

Therefore, the inventors of the present invention can increase the yield than the histidine NCA using aspartic acid as a mother nucleus instead of histidine used in the prior art, the molecular weight can be easily controlled to synthesize a polymer having a high molecular weight (5000 Da or more), By controlling the introduction of imidazole, it is possible to change the physical properties at the desired pH, and because it has a property of dissolving in volatile solvent when forming micelles, it can form micelles by various methods such as bottom flask method other than dialysis. The present invention was completed by revealing that it is possible to control the introduction of the imidazole group and to control the water solubility / lipidity and to mount the anticancer agent and the contrast agent.

Accordingly, an object of the present invention is to introduce a group of imidazoles with aspartic acid having excellent biocompatibility and biodegradability, which is not toxic to a living body, and exhibits a pH-sensitivity similar to that of histidine, while simulating synthesis. The present invention provides a pH-sensitive block copolymer and a method for preparing the same, which can introduce various methods to form a mass and micelles.

In addition, another object of the present invention to provide a drug carrier using the pH sensitive block copolymer.

In order to solve the problems of the prior art, the present invention is a poly (aspartic acid-graft-imidazole) grafted imidazole to aspartic acid; And it provides a pH reactive block copolymer formed by copolymerizing a polyethylene glycol-based compound.

Preferably, the pH-reactive block copolymer may be a compound represented by Formula 1 below:

[Formula 1]

In Formula 1, m is an integer of 10 to 45, n is an integer of 15 to 35, the sum of m and n may be an integer of 18 to 60.

The pH-reactive block copolymer may form or disintegrate micelles according to pH change, preferably, micelles are formed at a pH of 7.4 or more, and micelles formed may be disintegrated at a pH of 7.0 or less, and more preferably. At pH 7.4 to 14.0, micelles are formed, and at pH 7.0 to 3.0 the micelles formed can collapse. Accordingly, the bioactive material may be used as a drug carrier capable of encapsulating the bioactive material into the micelles of the pH-reactive block copolymer to release the bioactive material at the target site.

The pH reactive block copolymer may comprise 5 to 95% by weight of poly (aspartic acid-graft-imidazole) grafted imidazole to aspartic acid; And it may be formed by copolymerizing polyethylene glycol compound 5 to 95% by weight.

The poly (aspartic acid-graft-imidazole) has an imidazole introduction ratio of 1 to 95%, and the molecular weight range of the poly (aspartic acid-graft-imidazole) is not particularly limited, but is preferably in the range of 4,000 to 10,000. . If the molecular weight is out of the above range may cause a problem that the micelle does not collapse between pH 6.5 ~ 7.0.

In addition, the molecular weight range of the polyethylene glycol compound is not particularly limited, but is preferably in the range of 500 to 8,000. If the molecular weight is out of the above range, the length of the lipophilic portion may be longer than that of the hydrophilic portion, thereby causing a problem of decreasing the critical micelle formation concentration (CMC) and increasing the size.

The pH-reactive block copolymer according to the present invention is excellent in biocompatibility and biodegradability, and aspartic acid in which an imidazole group having a surface composed of hydrophilic polyethylene glycol (PEG) in an aqueous solution and having a hydrophobic interior is introduced. As a result, spherical aggregates of several tens to hundreds of nanometers were formed, and the properties of the pH-reactive block copolymers were confirmed through the critical micelle formation concentration (CMC), particle distribution, zeta potential, and scanning electron microscope observation.

In addition, the present invention comprises the steps of: i) synthesizing the aspartic acid polymer by ring opening polymerization using a polymerization initiator after N-carboxyhydride (NCA) of aspartic acid (first step); ii) reacting the aspartic acid polymer obtained in the first step with activated polyethylene glycol (PEG) (second step); iii) removing unactivated benzene from the reactants obtained in the second step (third step); And iv) introducing an imidazole ring into the reactant obtained in the third step.

Preferably, the pH-reactive block copolymer may be a compound represented by Formula 1 below:

[Formula 1]

In Formula 1, m is an integer of 10 to 45, n is an integer of 15 to 35, the sum of m and n may be an integer of 18 to 60.

The pH-reactive block copolymer may be produced alone or in parallel with a method such as agitation, heating, ultrasonic scanning, solvent evaporation using an emulsification method, matrix formation, or dialysis using an organic solvent, preferably using an organic solvent. It can be mass-produced by the bottom flask method.

The diameter of the pH-reactive block copolymer micelles prepared in the present invention is not particularly limited, but is preferably in the range of 10 to 1000 nm.

In addition, the present invention is the pH reactive block copolymer; And it provides a drug carrier comprising a bioactive material that can be encapsulated in the block copolymer.

The pH-reactive block copolymer is in the form of a polymer micelle, and the bioactive substance is not particularly limited as long as it is a substance for treating, preventing or diagnosing a disease, and particularly in the group consisting of peptide or protein medicine, antimicrobial agent, anticancer agent, contrast agent and anti-inflammatory agent. It may be any one or more than one selected.

In addition, it is possible to effectively encapsulate the tracer or gene into the polymer micelles, so that the pH-reactive block copolymer may be usefully used as a tracer and gene transporter that can be used molecularly.

The pH-reactive block copolymer according to the present invention can improve yield than conventional histidine by using aspartic acid as a mother nucleus instead of histidine, and it is possible to easily synthesize a polymer having a high molecular weight (more than 5000 Da) by controlling molecular weight. In addition, it is possible to change the physical properties at the desired pH by controlling the introduction of the imidazole group, and it is soluble in a volatile solvent when forming micelles, and thus micelles can be formed by various methods such as a bottom flask method other than dialysis. Therefore, it is possible to mass production, and also to control the introduction of the imidazole group to control the water solubility / fat solubility can be used as a drug carrier by mounting a bioactive substance such as anticancer agent and contrast agent.

1 is a conceptual diagram showing a synthetic route for the preparation of P (Asp-g-Im) -PEG according to the present invention,
Figure 2 shows the structure of pH-reactive micelles based on P (Asp-g-Im) -PEG according to the present invention,
Figure 3 shows the chemical structure and ¹ H NMR analysis of (a) PBLA, (b) PBLA-PEG, (c) P (Asp) -PEG and (d) P (Asp-g-Im) -PEG. ,
4 shows pH profiles of NaCl (○), P (Asp) -PEG (■) and P (Asp-g-Im) -PEG (●),
Figure 5 shows the effect of pH on CMC of P (Asp-g-Im) -PEG according to the present invention,
6 shows particle diameters of micelles prepared from P (Asp-g-Im) -PEG according to the present invention at various pHs,
7 shows (a) FE-SEM images of (p) pH 7.4 and (b) pH 6.0 of P (Asp-g-Im) -PEG according to the present invention, and (c) at pH 7.4 and (d) pH 6.0. Shows the particle size distribution of micelles by DLS of
8 shows the zeta potential of P (Asp-g-Im) -PEG according to the invention at various pHs.

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

Example 1 P (Asp- g -Im) -PEG Manufacturing

1. Preparation of β-benzyl-L-aspartic acid N-carboxyhydride (BLA-NCA)

BLA-NCA was synthesized by Fuchs-Farthing method using triphosgen. That is, L-aspartic acid-β-benzyl ester (5 g) was suspended in anhydrous 1,4-dioxane (100 ml) containing triphosgen (2.2 g) and stirred at 60 ° C. until the solution became clear. The synthesized BLA-NCA was precipitated by addition of hexane, obtained by filtration and dried under vacuum. Activated monocarboxylated PEG is prepared according to previously known methods (Zalipsky, et al., Facile synthesis of α-hydroxy-ω -carboxymethylpolyethylene oxide , Sage, London, ROYAUME-UNI, 1990). Ready.

2.P (Asp- g -Im) -PEG Manufacturing

P (Asp- g -Im) -PEG was synthesized as shown in FIG. First, poly (β-benzyl-L-aspartic acid) (PBLA; a) was synthesized by ring-opening polymerization of BLA-NCA using hexylamine (HA) as an initiator. That is, BAL-NCA (6 mmol) was dissolved in anhydrous 1,4-dioxane in the presence of HA (0.14 mmol) and stirred at 45 ° C. for 3 days under nitrogen atmosphere. PBLA precipitated with hexanes and dried in vacuo for 2 days. Its molecular weight was confirmed by ¹ H NMR analysis in DMSO-d ₆ using TMS and 300-MHz units as internal standards. The degree of polymerization (DP) of PBLA was calculated to be 35 and a yield of 92.6% was obtained.

PBLA-PEG was synthesized by coupling PBLA with activated monocarboxylated PEG ( Macromol Biosci, 5, 1118-1124, 2005) . To remove benzyl groups from PBLA, PBLA-PEG was dissolved in a DMF / MeOH mixture (1: 1 volume ratio) and Pd / C catalyst was added to the solution. The solution was then stirred under hydrogen atmosphere at room temperature for 1 hour. The catalyst was then removed by filtration using celite, the filtrate was precipitated with cold diethyl ether and the filtrate was obtained and dried in vacuo. The presence of deprotected carboxyl groups on P (Asp) -PEG was confirmed by ¹ H NMR analysis in DMSO-d ₆ using TMS standards. p (Asp) -PEG was obtained in 60.5% yield.

Deprotected P (Asp) -PEG is then preactivated by N-hydroxysuccinimide (NHS) and N, N'-dicyclohexylcarbodiimide (DCC) dissolved in dichloromethane (DCM) , Dissolved in DMF in the presence of 1- (3-aminopropyl) -imidazole (1.5 molar excess relative to carboxyl groups on P (Asp) -PEG) and stirred at 30 ° C. for 24 hours. After completion of the reaction, the solution was transferred to a preswollen dialysis membrane tube (Spectra / Por; MWCO 2K) and dialyzed with deionized water for 3 days to remove unbound 1- (3-aminopropyl) -imidazole. Then, it was confirmed by δ 6.88-7.63 (imidazole group) of ¹ H NMR (DMSO-d ₆ with TMS) that the obtained solution was a lyophilized 1- (3-aminopropyl) -imidazole conjugate (FIG. 3).

Experimental Example 1 Acid-Base Titration Analysis

Proper experiment was carried out to confirm the reactivity according to the pH of the prepared polymer. That is, a titration graph of P (Asp) -PEG, P (Asp-g-Im) -PEG and NaCl (control) was obtained using the potentiometric method. The block copolymer or NaCl was dissolved in deionized water (30 mmol / L) and then adjusted to pH 11 with 1N NaOH. This solution was titrated with 0.1N HCl to obtain a pH profile.

As a result, as shown in FIG. 2, the polymer after the imidazole group was introduced into the buffer zone longer than the polymer immediately after the deprotection process, and thus the polymer in each state was known.

Experimental Example 2 micelle formation and analysis

1. Formation of micelles

P (Asp-g-Im) -PEG was dissolved in 10 ml of a 50:50 EtOH / DCM mixture. For micelle formation, the organic phase was removed using a rotary evaporator (EYELA n-1000, Tokyo) to form a film in a round bottom flask. The film was rehydrated with HCl (or NaOH) -Na ₂ B ₄ O ₇ buffer (1 mM, pH 8.5) to form micelles. The pH value of the micelle solution was adjusted with phosphate buffered saline (pH 8.0-6.0) and citric acid buffer (pH 5.0-4.0).

2. Measurement of Size Distribution of Micell Particles Using Dynamic Light Scattering (DLS)

The size change was measured using DLS (Dynamic Light Scattering, Zetanano) while changing the previously prepared micelle to pH 6.0-8.0. More specifically, the effective hydrodynamic diameter (D) of the particles is determined by photon correlation spectroscopy (PCS) using Zetasizer Nano-ZS (Malvern Instruments, UK) with Multi Angle Sizing Option (BI-MAS). _eff ) was measured. Sizes were measured in thermostat cells at a scattering angle of 90 °. D _eff was calculated using the software provided by the manufacturer. Polymer micelles (0.1 wt.%, Ionic strength: 0.15) were prepared at various pH values (pH 8.0-6.0) in PBS buffer from the stock micelle suspension suspended in borate buffer (pH 8.5).

As a result, it can be seen that micelles having a certain size exist at pH 7.0 or higher as shown in FIG. 2, but at pH below 7.0, imidazole groups are ionized to increase in size.

3. Zeta potential measurement

Zeta potential of P (Asp-g-Im) -PEG solution (0.1 mg / ml) at various pH values (pH 8.0-4.0, ionic strength: 0.15) was measured using Zetasizer Nano-ZS (Malvern Instruments, UK) It was. The P (Asp-g-Im) -PEG solution was stabilized at room temperature for at least 3 hours before measurement.

It can be seen that the ionization starts from pH 7.0 as shown in Figure 3 is slightly lower than the value above pH 7.0, the value is replaced by the + charge to the -charge of the remaining carboxyl functional groups with a fully + charge at pH 4.0 It can be seen that.

4. Critical micelle formation concentration (CMC) analysis

Using the Scinco FS-2 fluorescence spectrometer, the previously prepared micelles were measured for CMC up to pH 6.0-pH 8.0. The fluorometer is equipped with polarizing plates for excitation (339 nm) and emission (374 nm) rays. Pyrene was used as the fluorescent probe. Micelles samples were prepared at various pH range values of 8.0 to 6.0 using PBS buffer, pyrene mixed (pyrene concentration 6.0 × 10 ⁻⁷ M) and stirred overnight at room temperature. All measurements were performed at room temperature using air-equilibrated solutions. CMC values were determined by plotting the ratio of I ₁ (the intensity of the first peak) to I ₃ (the intensity of the third peak) of the emission analysis profile against the log ₁₀ value of the micelle concentration. CMC values were defined as the intersection of low polymer concentrations on these graphs.

As a result, as shown in Figure 4, the block copolymer of the present invention formed a stable micelle at pH 7.4, it was confirmed that the change in concentration occurs as the imidazole ring is broken at pH 7.0. This is due to an increase in the degree of ionization of the tertiary amine present in the imidazole at low pH (below pH 7.0), resulting in the polymer becoming water soluble and the copolymer being unable to form micelles. By expressing this means forming the micelle by self-assembly.

5. Shape of micelles

To observe the shape of P (Asp-g-Im) -PEG micelles, cast a P (Asp-g-Im) -PEG solution (0.1 g / l) diluted at pH 7.4 or pH 6.0 on a glass slide and Prepared by drying in vacuo. At this time, the shape was measured by using a field emission scanning electron microscope (FE-SEM, Hitachi-4800, Japan).

As a result, as shown in FIG. 5, the results of the similar size and the shape of the micelle were changed as compared with the size distribution.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Claims (12)

Poly (aspartic acid-graft-imidazole) grafted imidazole to aspartic acid; And a pH reactive block copolymer formed by copolymerizing a polyethylene glycol compound. The pH-reactive block copolymer of claim 1, wherein the block copolymer is a compound represented by the following Chemical Formula 1:
[Formula 1]

In Formula 1, m is an integer of 10 to 45, n is an integer of 15 to 35, the sum of m and n is an integer of 18 to 60. The pH-reactive block copolymer according to claim 1 or 2, wherein the block copolymer forms micelles at a pH of 7.4 or more, and micelles formed at pH of 7.0 or less are disrupted. The method according to claim 1 or 2, 5 to 95% by weight of poly (aspartic acid-graft-imidazole) grafted imidazole to aspartic acid; And a pH reactive block copolymer formed by copolymerizing 5 to 95% by weight of a polyethylene glycol compound. The pH-reactive block copolymer according to claim 1 or 2, wherein the poly (aspartic acid-graft-imidazole) has an imidazole introduction rate of 1 to 95%. The pH-reactive block copolymer according to claim 1 or 2, wherein the poly (aspartic acid-graft-imidazole) has a molecular weight of 4,000 to 10,000. The pH-reactive block copolymer according to claim 1 or 2, wherein the polyethylene glycol compound has a molecular weight of 500 to 8,000. i) synthesizing aspartic acid polymer by ring-opening polymerization using N-carboxyhydride (NCA) of aspartic acid (first step);
ii) reacting the aspartic acid polymer obtained in the first step with activated polyethylene glycol (PEG) (second step);
iii) removing unactivated benzene from the reactants obtained in the second step (third step); And
iv) introducing the imidazole ring into the reactant obtained in the third step. The method of claim 8, wherein the pH-reactive block copolymer is a compound represented by Formula 1 below.
[Formula 1]

In Formula 1, m is an integer of 10 to 45, n is an integer of 15 to 35, the sum of m and n is an integer of 18 to 60. PH reactive block copolymers according to claim 1; And a bioactive substance which may be encapsulated in the block copolymer. The drug carrier according to claim 10, wherein the pH reactive block copolymer is in the form of a polymer micelle. The drug carrier according to claim 10 or 11, wherein the physiologically active substance is any one or two or more selected from the group consisting of peptide or protein pharmaceuticals, antibacterial agents, anticancer agents, contrast agents and anti-inflammatory agents.

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