KR20190026448A - POLYMER CONTAINING pH-RESPONSIVE HYDROPHOBIC MONOMER AND MICELLE COMPRISING SAME - Google Patents

Abstract

The present invention relates to a polymer including a pH-sensitive hydrophobic monomer having excellent stability, carrying capacity, drug release ability, and degradability, and to a micelle including the polymer. The polymer comprises a repeating unit represented by chemical formula 1 and is a homopolymer.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention [0001] The present invention relates to a polymer containing a pH-sensitive hydrophobic monomer and a micelle comprising the polymer.

The present invention relates to a polymer which can be used as a carrier in a biological / biomedical field by including a pH-sensitive hydrophobic monomer excellent in flexibility and biocompatibility, and a micelle containing the polymer.

Amphiphilic molecules can be applied to a wide range of fields, from self-assembling properties, to surfactants, templates and catalysts to drug delivery. In particular, nanostructures derived from amphiphilic polymers, such as amphiphilic block copolymers, have attracted attention in that stability and functionality can be adjusted because of the high physical and chemical coordination of each of the monomers making up the block copolymer. Polymer micelles self-assembled in aqueous solution have been studied as ideal drug delivery carriers and some of them have been developed to clinical environments such as pluronics, paclical and genexol-PM .

The self-assembly properties of polymer micelles depend on the choice of hydrophobic blocks, in addition to the stabilization of hydrophilic blocks. In addition, the properties of the hydrophobic block are important factors for determining the stability, degradability, and supporting efficiency of the generated micelle. For example, polymeric micelles are dynamically dissolved in systemic injection into the bloodstream often at high dilutions and exposed to changes in pH and salt concentration. Therefore, studies for improving the stability of micelles using crystallinity, stereoregularity, molecular weight, and substitution of hydrophobic blocks have been actively conducted. In addition, the elaborate transfer of polymeric micelles, which are drug delivery carriers, is also an important property. With the development of micelles that are sensitive to external stimuli such as pH, light, redox reaction and temperature, control over the release profile of the internal support is being studied by introducing new structures into the hydrophobic blocks. Thus, the hydrophobic block serves to control important parameters of the polymer micelle. As a result, efforts have been made to develop new hydrophobic monomers to improve the properties of micelles in drug delivery systems.

Korean Patent Publication No. 2017-0060998

Accordingly, an object of the present invention is to provide a polymer having excellent stability, supported capacity, drug releasing ability and degradability, and micelles containing the same, including monomers having excellent flexibility and biocompatibility.

According to the above object, the present invention provides a polymer comprising a repeating unit represented by the following formula (1): < EMI ID =

The present invention also provides a micelle comprising the block copolymer, wherein the polymer is a block copolymer.

Further, the present invention provides a process for producing the polymer, which comprises polymerizing a first monomer represented by the following formula (4) and a second monomer having a hydroxy group:

The polymer according to the present invention includes a monomer having excellent flexibility and biocompatibility, and is excellent in stability, supporting capacity, drug releasing ability and decomposability. Thus, the polymers of the present invention and micelles comprising them are suitable for use as carriers in the field of biology / biology.

1 is a ¹³ C NMR spectrum (100 MHz, CDCl ₃ ) of PGE (pyranyl glycidyl ether) monomer.
2 is a ¹ H- ¹ H correlation analysis (COSY) NMR spectrum of PGE measured in CDCl ₃ .
3 is a HSQC NMR spectrum of PGE measured in CDCl ₃ .
Figure 4 is a schematic diagram of an embodiment of the present invention comprising (a) PGE monomers, (b) PPGE (homopolymer of PGE) (item 3 of Table 1), (c) PPGE homopolymer after deprotection, and (d) PEG- block copolymers of PGE) is a ¹ H NMR spectrum of (5 entries in Table 1). All spectra except (c) recorded in D ₂ O were collected in CDCI ₃ .
Figure 5 shows the results of the GPC-elution using (a) homopolymer PPGE _n , (b) block copolymer PEG ₁₁₄ -b-PPGE _n , and (c) PEG ₁₁₄ -b-PEEGE _n using RI signal and PS Results.
6 is an expanded MALDI-ToF mass spectrum of a PPGE ₃₈ homopolymer (item 3 in Table 1) of 3000 Da to 4500 Da. The signal spacing corresponds to the mass of the PGE monomers in the homopolymer (158.09 g / mol).
7 is a 400 MHz ¹ H NMR spectrum of EEGE (ethoxyethyl glycidyl ether) measured in CDCl ₃ .
8 is a 400 MHz ¹ H NMR spectrum of PEG-b-PEEGE (block copolymer of PEG and EEGE) measured in CDCl ₃ .
Figure 9 shows a block copolymer PEG ₁₁₄ - b- PPGE _n ( n = 18, 37, and 72) and (c) a homopolymer PPGE _n ( n = 14,20,38 and 118) ) Differential scanning calorimetry (DSC) thermogram of the block copolymer PEG ₁₁₄ - b - PEEGE _n ( n = 9, 22, and 60). The melting point of the PEG block was 50-54 占 폚.
10 is the result of CMC measurement of the amphiphilic block copolymer micelle through measurement of I ₃ / I ₁ as a function of polymer concentration using the fluorescence excitation spectrum of pyrene (λ _em = 372 nm). Specifically, (a) P1 (n = 18), P2 (n = 37) and P3 (n = 72), and (b) E1 (n = 9 ), E2 (n = 22) and E3 (n = 60 ), n is PEG ₁₁₄ - indicates the number of each repeating unit in the b _n -PEEGE (see note in Table 1) - b -PPGE _n and PEG _114. The dashed line was added to indicate the inflection point.
FIG. 11 shows (a) P1, (b) P2, (c) P3, (d) E1, (e) E2, and (f) excitation spectrum of pyrene in an aqueous solution of E3 micelle (emission wavelength: 372 nm).
Figure 12 is a cryogenic-TEM image of (a) E2 and (b) E3 micelles.
13 is a schematic diagram of a P2 micelle having (a) P1, (b) P2, and (c) P3 micelles as typical cryogenic TEM images and (d) each block size in micelles.
14 is a particle size distribution diagram of (a) P1 micelles and (b) P2 micelles measured by the dynamic light scattering method (DLS).
15 is a particle size distribution diagram of (a) P1 micelles and (b) P2 micelles measured by dynamic light scattering method (DLS).
Figure 16 is an atomic force microscope (AFM) image and height of (a) P1, (b) P2, and (c) E2 micelles with average particle sizes of 77.2, 58.7 and 79.0 nm.
FIG. 17 shows various encapsulation efficiencies of micelles containing Nile Red as a result of measurement of encapsulation efficiency of Nile Red in all micelles.
(A) P1, (b) P2, (c) P3, and (d) E3 micelles in PBS (left) and 100% serum (right) as a result of the encapsulation stability analysis based on FRET of various micelles under biological conditions, . At this time, DiO (FRET donor) can confirm the emission peak at 510 nm and DiI (FRET receptor) at 570 nm.
Figure 19 shows FRET-based encapsulation stability analysis results of (a) P1, (b) P2, (c) P3 and (d) E3 in 10 and 50% serums. At this time, the excitation peak of DiO (FRET donor) was observed at 505 nm and the excitation peak of DiI (FRET receptor) was observed at 570 nm.
Figure 20 shows the results of an in vitro FRET study of various micelles showing that the concentration of HeLa cells treated with 10% fetal bovine serum (FBS) and (a) P1, (b) P2, (c) P3, FRET results. At this time, the excitation and emission wavelengths for DiO were 488 nm and 535 nm, respectively, and DiI was set to 543 nm and 620 nm, respectively.
Figure 21 shows the results of in vitro FRET loss studies after incubation of HeLa cancer cells treated with 10% fetal bovine serum (FBS) and (a) P1, (b) P2, (c) P3 and (d) Results. At this time, the excitation and emission wavelengths for DiO were 488 nm and 535 nm, respectively. The excitation and emission wavelengths for the languages were 543 nm and 620 nm, respectively.
Figure 22 is a schematic diagram of (a) treating the PEG-b-PPGE micelle with an acid to release the pyrene to form a PEG-b-linear PG polymer. I ₃ / I ₁ plot for time under acidic conditions for (b) P1, (c) P2, (d) P3, (e) E2, (f) E2 and (g) E3. All polymers have a concentration of 1.0 mg / mL except for the sample in (f) with a concentration of 5.0 mg / mL. The gray line in each graph represents the I ₃ / I ₁ value of the free fluorene in water.
Figure 23 shows the results of the change measurements of pyrene encapsulated by (a) P1, (b) P2, (c) P3, (d) E2, (e) E2 and (f) E3 micelles after acid treatment in the excitation spectrum. All samples contained a polymer concentration of 1.0 mg / mL, and (e) contained a polymer concentration of 5.0 mg / mL.
24 shows the in vitro survival analysis results of the block copolymer micelles measured by MTT analysis using HeLa cells.
25 shows (a) the paclitaxel (PTX) encapsulation efficiency of P1, P2, P3, E2 and E3 micelles at a polymer concentration of 5 mg / mL, (b) the in vitro cell viability of PTX- Results.

The present invention provides a polymer comprising a repeating unit represented by the following Formula 1:

[Chemical Formula 1]

.

.

Homopolymer ( homopolymer )

The polymer may be a homopolymer composed of the repeating unit represented by the formula (1). Specifically, the homopolymer may be represented by the following general formula (2).

In the general formula (2), n is an integer of 5 to 200. Specifically, in Formula 2, n may be an integer of 12 to 150, 12 to 130, or 13 to 120.

The homopolymer may have a number average molecular weight of 1,500 to 20,000 g / mol. Specifically, the homopolymer may have a number average molecular weight of 2,000 to 20,000 g / mol, 2,000 to 19,000 g / mol, or 2,200 to 19,000 g / mol.

The weight average molecular weight (Mw / Mn) of the homopolymer to the number average molecular weight may be 1 to 3. Specifically, the weight average molecular weight (Mw / Mn) of the homopolymer to the number average molecular weight may be 1 to 2, or 1 to 1.5.

The homopolymer may have a glass transition temperature of -60 to 0 占 폚. Specifically, the homopolymer may have a glass transition temperature of -50 to 0 占 폚, -45 to -2 占 폚, or -40 to -10 占 폚.

Block copolymer

The polymer may be a block copolymer comprising a repeating unit represented by the formula (1) and an alkylene glycol repeating unit. The alkylene may be ethylene.

The block copolymer may be represented by the following Formula 3:

In Formula 3, n is an integer of 5 to 200, and m is an integer of 50 to 500. Specifically, in Formula 3, n is an integer of 10 to 150, or 10 to 100, and m may be an integer of 50 to 400, or 80 to 300. [

In the above formula (3), when m is 100, n may be an integer of 5 to 200. Specifically, when m is 100 in the formula (3), n may be an integer of 10 to 200, 13 to 150, or 14 to 140.

The block copolymer may have a critical micelle concentration of 0.1 to 100 mg / l. Specifically, the block copolymer may have a critical micelle concentration of 0.1 to 70 mg / l, 0.2 to 50 mg / l, or 0.3 to 40 mg / l.

The block copolymer may have a number average molecular weight of 5,000 to 30,000 g / mol. Specifically, the block copolymer may have a number average molecular weight of 5,000 to 28,000 g / mol, 6,000 to 25,000 g / mol, or 7,000 to 20,000 g / mol.

The block copolymer may have a weight average molecular weight (Mw / Mn) to number-average molecular weight of 1 to 3. Specifically, the block copolymer may have a weight average molecular weight (Mw / Mn) based on the number average molecular weight of 1.0 to 2.8, 1.0 to 2.5, or 1.0 to 2.0.

The block copolymer may have a glass transition temperature of -60 to 0 占 폚. Specifically, the block copolymer may have a glass transition temperature of -50 to 0 占 폚, -45 to -5 占 폚, or -40 to -10 占 폚.

Micelle

The present invention also provides a micelle comprising the block copolymer as described above.

The micelle may have an average diameter of 5 to 1,000 nm. Specifically, the micelle may have an average diameter of 10 to 800 nm, or 20 to 500 nm.

The micelle may be in the form of a core-shell. Specifically, the micelle may include a hydrophobic core and a hydrophilic shell. More specifically, the micelle may include a hydrophobic core of the repeating unit represented by the formula (1) and a hydrophilic shell of an alkylene glycol repeating unit. The repeating unit represented by the formula (1) and the alkylene glycol repeating unit are as defined in the block copolymer.

As described above, micelles including a hydrophobic core and a hydrophilic shell are excellent in self-assembly ability, stability, drug retention ability, drug release efficiency, and biocompatibility, and thus can be utilized as drug delivery materials in various biological / biomedical fields.

In addition, the micelle may include a core having a diameter of 5 to 500 nm and a shell having a diameter of 5 to 500 nm. Specifically, the micelle may include a core having a diameter of 5 to 300 nm and a shell having a diameter of 8 to 300 nm.

The micelle may further comprise a drug encapsulated by the copolymer. The drug may be hydrophobic or hydrophilic. Specifically, the drug may be hydrophobic.

Further, the present invention provides a drug delivery composition comprising the micelle.

Method of producing polymer

In addition, the present invention provides a process for producing a polymer comprising a repeating unit represented by the above formula (1), comprising the step of polymerizing a first monomer represented by the following formula (4) and a second monomer having a hydroxy group:

[Chemical Formula 4]

.

.

(PGE), 2- (2-oxiranylmethoxy) tetrahydro-2 H-pyran, CAS 64244-53 (2-oxiranylmethoxy) tetrahydro- -7) is pH sensitive, hydrophobic and flexible. Therefore, polymers polymerized from the above monomers are excellent in biostability, drug-retaining ability, drug releasing ability and degradation degree.

In the polymerization, the epoxy group at the end of the first monomer represented by the formula (4) reacts with the hydroxy group at the end of the second monomer to cause a ring-opening polymerization reaction, and the newly generated hydroxy group functions as another polymerization initiation site due to the ring opening of the epoxy group A ring-opening polymerization reaction with another epoxy group at the end of the first monomer may occur.

The second monomer is not particularly limited as long as it contains a hydroxy group at the terminal. For example, the second monomer may be a compound represented by the following formula (5) or a compound represented by the following formula (6)

When the second monomer is a compound represented by the formula 5, the polymer to be produced may be a compound represented by the following formula 2:

(2)

In the general formula (2), n is an integer of 10 to 200.

When the second monomer is a compound represented by Formula 6, the polymer may be a compound represented by Formula 3:

(3)

In Formula 3,

n is an integer from 5 to 200,

and m is an integer of 50 to 500.

The polymerization can also be carried out at 20 to 40 占 폚, or at 20 to 25 占 폚 and under a strong base catalyst.

The strong base catalyst may not contain a metal. Specifically, the strong base catalyst may be at least one member selected from the group consisting of t-Bu-P ₄ , t-Bu-P ₁ , t-Bu-P ₂ and triisobutyl phosphate (TiBP). More specifically, the strong base catalyst may be a t-Bu-P _4.

The polymerization may be carried out in a solvent. The solvent is not particularly limited as long as it is used for polymerization using monomers. For example, the solvent may be at least one selected from the group consisting of toluene, tetrahydrofuran, and diglyme.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[ Example ]

The abbreviations used in the following Examples and Test Examples have the following meanings:

- PGE: pyranyl glycidyl ether.

- PPGE: homopolymer of PGE

- EEGE: Ethoxyethyl glycidyl ether.

- PG: polyglycerol

- PEG: Poly (ethylene glycol) (poly (ethylene glycol))

- PEG-b-PPGE: block copolymer of PEG and PGE

- PEG-b-PEEGE: Block copolymer of PEG and EEGE

The materials used in the following Examples and Test Examples are as follows:

- Deuterated NMR solvents (CDCl ₃ and D ₂ O) were purchased from Cambridge Isotope Laboratory, and all reagents and solvents were purchased from Sigma-Aldrich or Acros.

Reference example . Character analysis

^One H-NMR and ¹³ C-NMR measurement method

¹ H-NMR (400 MHz) and ¹³ C-NMR (100 MHz) were measured using a 400-MR DD2 spectrometer. All spectra were recorded in ppm using tetramethylsilane (TMS) as the internal standard in deuterated solvents CDCl ₃ and D ₂ O.

Number average molecular weight (Mn) and weight average molecular weight (Mw)

The number average molecular weight (M _n), weight average molecular weight (M _w), and molecular weight distribution (M _w / M _n) was measured using gel permeation chromatography (GPC, Agilent 1200 series). GPC was also measured in the eluent (tetrahydrofuran, THF) at room temperature using a refractive index (RI) detector at a flow rate of 1.0 mL / min and calibrated based on polystyrene.

MAG-assisted laser desorption and ionization time-of-flight mass spectrometry, MALDI - ToF  Mass analysis)

A-cyano-4-hydroxycinnamic acid was used as the medium and mass spectrometry assisted laser desorption / excitation time-of-flight mass spectrometry was performed using an Ultraflex III MALDI mass spectrometer.

Manufacturing example  One. PGE  Synthesis of monomers

Dichloromethane (350 mL), glycidol (10.1 g, 68.0 mmol) and 3,4-dihydro-2H-pyran (16.8 g, 200 mmol) were added to a round bottom flask and stirred at room temperature for 30 minutes Respectively. To this solution, p -toluenesulfonic acid ( p- TsOH, 0.26 g, 1.38 mmol) was slowly added and stirred vigorously overnight. 100 mL of saturated NaHCO ₃ solution was added thereto, followed by stirring at room temperature for 1 hour. The aqueous layer was then extracted with dichloromethane (DCM), and the organic layer, dichloromethane, was extracted with water. The extracted organic layer was dried over Na ₂ SO ₄ and concentrated under reduced pressure. The residue was purified by flash column chromatography with an eluent (ethyl acetate / hexane = 1: 8 (v / v)) to give 6.45 g (60%) of PGE monomers as colorless liquid.

Then, ¹ H-NMR (400 MHz) and ¹³ C-NMR (100 MHz) spectra, correlation spectroscopy (COZY) -NMR, heteronuclear single-quantum correlation (HSQC) The chemical structure of PGE monomers was analyzed using ESI-MS. ¹ H-NMR and ¹³ C-NMR were measured in the same manner as above, and the measurement results are shown in FIGS. 1 to 4.

NMR results

^{1 H NMR (400 MHz, CDCl} 3): σ ppm 4.73 - 4.59 (m, 1H, d), 3.95 (dd, J = 11.7, 3.1 Hz, 0.5H, c), 3.87 (ddd, J = 15.3, 7.8 , 3.8 Hz, 1H, e) , 3.76 - 3.66 (m, 1H, c), 3.51 (dt, J = 5.1, 4.4 Hz, 1H, e), 3.40 (dd, J = 11.7, 6.4 Hz, 0.5H, (dd, J = 5.2, 2.7 Hz, 1H, a), 2.60 (dd, J = 7.7, 3.9 Hz, 1H, b), 2.83-2.7 J = 5.0, 2.7 Hz, 0.5H , a), 1.84 (dd, J = 17.4, 8.6 Hz, 1H, f 3), 1.78 - 1.67 (m, 1H, f 1), 1.67 - 1.48 (m, 4H, f ₁ -f ₃ ).

¹³ C NMR (100 MHz, CDCl ₃ ):? Ppm 98.8, 67.9, 62.1, 50.7, 44.5, 30.50, 25.4, 19.3.

^{MS (m / z + Na +} , ESI +) C 8 H 14 O 3 calculated for, 181.1; Peak confirmation, 180.6.

Example  One. PPGE  Synthesis of homopolymers

0.25 mL of n-hexane solution in which t-Bu-P ₄ (0.8 M, 0.20 mmol, strong base) was dissolved was added to 6.94 mL of toluene solution in which benzyl alcohol (initiator: 20.7 μL, 0.20 mmol) Respectively. Subsequently, PGE (1.27 g, 8.00 mmol) was added to the solution by a syringe pump in a dropping manner to carry out the polymerization reaction over 6 hours. After stirring at room temperature for 24 hours, an excessive amount of benzoic acid was added to terminate the polymerization reaction. After precipitation in hexane, the mixture was passed through an alumina pad using tetrahydrofuran (THF). Thereafter, the polymer solution was dried to obtain PPGE (851 mg, yield: 69.8%) which was poly (pyranyl glycidyl ether).

The M _n of PPGE was calculated from the NMR data of FIG. 4 using the following equations (1) and (2). As shown in FIG. 4 b, the ¹ H NMR spectrum of PPGE showed an aromatic proton (7.30 to 7.38 ppm), a methylene proton (4.50 to 4.54 ppm), a polyether skeleton (3.24 to 4.05 ppm) and a pyranyl proton 4.62 ppm, and 1.44 to 1.91 ppm). The number average molecular weight (M _n , NMR) was also calculated by measuring the ratio of the peak integral to the methylene group of the initiator (benzyl alcohol) relative to the pyranyl proton (4.55 to 4.62 ppm) in ¹ H NMR.

[Equation 1]

Number of repeating units (PGE) = 18.9 (NMR integral value) X 2 (number of protons of methylene in benzyl alcohol) = 38

&Quot; (2) "

M _n = 158.20 g / mol (molecular weight of PGE monomer) X 38 (number of repeating units of PGE) + 108.14 g / mol (molecular weight of benzyl alcohol) = 6119.74 g / mol.

Considering the error of the calculated NMR integration, the M _n of PPGE was 6,100 g / mol.

To confirm the presence of the initiator and the incorporation of PGE into the PPGE homopolymer, MALDI-ToF mass spectrometry was performed and the results are shown in FIG.

As shown in FIG. 6, the molecular weight of 3450.7 g / mol was determined to be PPGE having Na ⁺ as the counterion and 21 repeating units (benzyl alcohol (108.06 g / mol) + PGE (158.09 g / mol) Na < ⁺ & gt ^; (22.99 g / mol)). As a result, the signal interval coincided with the molecular weight of the PGE monomer (158.09 g / mol) and the polymerization reaction was confirmed.

Manufacturing example  2. PPGE Deprotection  Linear by processing PG  Produce

A solution of HCl / MeOH (1.25 M, 0.13 mL) in which PPGE (0.50 g, M _n = 6,100 g / mol) was dissolved was stirred at room temperature for 2 hours. The reaction mixture was neutralized with potassium carbonate, filtered and precipitated in excess cold diethyl. The resulting polymer was then dried under vacuum for 24 hours to obtain linear polyglycerol (linear PG; 210 mg, yield: 82.3%). The M _n of the resulting polymer was calculated to be 3,100 g / mol using NMR data.

As shown in Fig. 4c, the removal of the pyranyl moiety (deprotection treatment) was confirmed by ¹ H NMR spectrum in which the peak corresponding to the pyranyl group disappeared at 1.44 to 1.91 ppm.

Example  2. PEG-b- Of PPGE (amphiphilic block copolymer, P series)  synthesis

A block copolymer was synthesized in the same manner as in Example 1 using poly (ethylene glycerol) methyl ether (mPEG) as a macro initiator.

Specifically, mPEG (0.50 mg, 0.10 mmol) was added to the flask in the presence of argon, and toluene (3.47 mL) was added to the flask and heated to 60 ° C. After cooling to room temperature, n-hexane (0.8 M, 0.10 mmol) in which 0.13 mL of t-Bu-P ₄ dissolved was added to the solution. Then, PGE of Production Example 1 was added to the solution by a syringe pump in a dropwise manner for 6 hours. After stirring at room temperature for 24 hours, benzoic acid was added to terminate the polymerization reaction. The mixture was then precipitated in hexane and passed through alumina pad with THF. Thereafter, the polymer solution was dried and evaporated to obtain PEG-b-PPGE (647 mg, yield: 74.4%) which was poly (ethylene glycol) -b-poly (pyranyl glycidyl ether).

The M _n of PEG-b-PPGE was calculated from NMR data (see FIG. 4) using the following equations (3) and (4).

&Quot; (3) "

Number of repeating units (PGE) = 5.89 (NMR integral value) X 3 (number of protons of methyl of mPEG) = 18

&Quot; (4) "

M _n = 158.20 (molecular weight of PGE monomer) X 18 + 5,900 (molecular weight of mPEG macromonomer) = 8747.6 g / mol.

Considering the error range of NMR integration, the M _n value of PEG-b-PPGE was 8,700 g / mol.

As shown in FIG. 4 c, the ¹ H NMR spectrum of PEG-b-PPGE, which is an amphiphilic block copolymer, showed that the methyl protons of mPEG (3.88 ppm), the polyether skeleton (3.41 to 3.97 ppm), and the pyranyl protons To 4.62 ppm and 1.44 to 1.91 ppm), respectively, (Fig. 2 (d)).

In addition, block copolymers of different molecular weights (8,700 to 18,000 g / mol) were prepared based on peak enrichments between methyl and piranyl protons of mPEG.

Manufacturing example  3. PEG-b- PEEGE (Amphiphilic Block Copolymer, E Series)  synthesis

PEG-b-PEEGE was synthesized in the same manner as in Example 2.

Specifically, mPEG (0.50 mg, 0.10 mmol) was added to the flask in the presence of argon, and toluene (3.47 mL) was added to the flask and heated to 60 ° C. After cooling to room temperature, n-hexane (0.8 M, 0.10 mmol) in which 0.13 mL of t-Bu-P ₄ dissolved was added to the solution. Then, EEGE (see NMR in FIG. 7) was added to the solution by a dropping method using a syringe pump for 6 hours to perform polymerization reaction. After stirring at room temperature for 24 hours, benzoic acid was added to terminate the polymerization reaction. The mixture was precipitated in hexane and passed through alumina pad with THF. The polymer solution was dried and evaporated to obtain 672 mg (yield: 73.8%) of poly (ethylene glycol) -b-poly (ethoxyethyl glycidyl ether) PEG-b-PEEGE.

The M _n of PEG-b-PEEGE was calculated from NMR data (see FIG. 8) using the following equations (5) and (6).

&Quot; (5) "

Number of repeating units (EEGE) = 7.26 (NMR integral value) X 3 (number of protons of methyl of mPEG) = 22

&Quot; (6) "

M _n = 146.19 g / mol (molecular weight of EEGE monomer) X 22 + 5,900 g / mol (molecular weight of mPEG macromonomer) = 9116.2 g / mol.

Considering the error range of NMR integration, the M _n value of PEG-b-PEEGE was then used as 9,100 g / mol.

a: Measured by ¹ H NMR spectroscopy. b: Measured using GPC measurement (THF, RI signal, PS standard, c: T _g measured by differential scanning calorimetry at a rate of 10 ° C / min d: Fluorescence spectroscopy Calculation of critical micelle concentration (CMC) from e: Not measured.

As shown in Table 1, the PPGE homopolymer exhibited a single mode dispersion and a narrow polydispersity index (Mw / Mn = 1.03 to 1.19).

In addition, PEG-b-PPGE, an amphiphilic block copolymer, exhibited a single mode dispersion with a narrow polydispersity index of 1.05 to 1.23. Furthermore, it was clearly observed that the GPC of PEG-b-PPGE was shifted to a higher molecular weight region than that of the mPEG macromonomer, confirming that the copolymerization reaction occurred (see FIG. 5 b).

Example  3. Micelle  Produce

5.0 mg of PEG-b-PPGE as an amphiphilic block copolymer of Example 2 was dissolved in 200 μl of DMF, and then 5 ml of water was added dropwise through a syringe pump for 1 hour to form micelles. After stirring overnight, the polymer solution was dialyzed against deionized water for 2 days to remove residual DMF. The resulting solution was then passed through a 0.45 [mu] m syringe filter and filtered.

Manufacturing example  4. Micelle  Produce

Micelles were prepared in the same manner as in Example 3, except that the polymer of Preparation Example 3 was used.

Experimental Example  1. Characterization

The properties of the polymers or micelles of Examples 1 to 3 and 4 were analyzed in the following manner.

(1-1) Differential scanning Heat ridge (differential scanning calorimetry, DSC )

The differential scanning calorie was measured in a nitrogen atmosphere using a differential scanning calorimeter (Q200 model, TA Instruments) at a heating rate of 10 DEG C / min in a temperature range of -80 to 65 DEG C, and the results are shown in FIG.

As shown in Figure 9, the PEG block T _g was not detected because of its high crystalline property, T _m was observed at about 50 to 54 ° C. In addition, the glass transition temperature (T _g ) (-17 to -26 ° C) of PEG-b-PPGE (P series) is higher than that of PEG-b-PEEGE (E series) About -59 ℃). This is believed to be due to the lower spatial conformational freedom of the sterically large cyclic moiety. Also, the glass transition temperature difference is judged to be the result of the chair-to-chair stacking between the side chains increasing the energy barrier for the twisting motion of both the polyether skeleton and the side chain.

(1-2) Critical micelle concentration ( CMC ) (Self-assembling property)

The critical micelle concentration (CMC) is an important indicator of the self-assembly and stability of micelles, measured using the excitation spectrum of pyrene, a hydrophobic drug.

Specifically, a solution of 10 占 퐇 of pyrene (concentration: 5.2 mg / L, solvent: DMF) was added to the acetone solution in which PEG-b-PPGE was dissolved and the mixture was stirred at room temperature for 30 minutes. A total of 5 mL of deionized water was then added to the solution at a rate of 0.5 mL / min using a syringe pump. The solution was allowed to equilibrate overnight. The fluorescence of each pyrene-containing polymer micelle solution was measured at 372 nm emission wavelength through a 1 cm x 1 cm quartz cell using a fluorescence analyzer (RF-6000, Shimadzu). The following parameters were selected: emission wavelength = 372 nm, excitation wavelength range = 360 to 372 nm, scan rate = 600 nm / min, and data interval = 0.5 nm. The fluorescence intensity ratio at 339.5 and 332.5 nm wavelengths was plotted against the polymer concentration and the critical micelle concentration was determined from the inflection point.

The pyrene can be encapsulated within the hydrophobic core of the micelle to dramatically increase excitation band strength (I ₃₃₉ / I ₃₃₂ , also expressed as I ₃ / I ₁ ). At this time, the excitation spectrum was recorded using a fluorescence meter (RF-6000, Shimadzu). The results of plotting the I ₃ / I ₁ ratio as a function of the concentration of each polymer are shown in FIGS. 10 and 11.

As can be seen in Figures 10 and 11, clear intersections were observed in the pyrene excitation spectrum of the PEG-b-PPGE block copolymer, indicating that the CMC of 18.1 (P1), 9.79 (P2), and 0.89 mg / L (P3) Suggesting the formation of micelles. In addition, the CMC value of PEG-b-PPGE decreased as the number of repeating units of PGE, the hydrophobic block, increased. On the other hand, E1 did not show any signs of micelle formation because the degree of hydrophobicity of the PEEGE block was decreased, while the other polymers with longer PEEGE blocks showed CMC of 96.0 (E2) and 10.3 mg / mL (E3).

In addition, the CMC value of PEG-b-PPGE was about 10 times lower than that of PEG-b-PEEGE, indicating that the micelle prepared from PEG-b-PPGE containing more hydrophobic cyclic pyranyl groups It is more stable than PEG-b-PEEGE, which is a similar chain, and maintains its integrity when diluted after systemic injection. In addition, the CMC value of PEG-b-PPGE was significantly lower than that of the broad commercial Pluronic copolymer (4.0 mg / L to 1 X 10 ⁴ mg / L).

(1-3) Cryogenic-transmission electronic microscopy, cryo - TEM )

Micelles were observed using a cryo-transmission electron microscope to analyze the morphology of the prepared micelles, and the results are shown in FIGS. 12 and 13. The cryogenic TEM is able to directly observe the micellar structure in a solution state with relatively no artificial product.

Specifically, a thin film of an aqueous solution (3 μl) of PEG-b-PPGE block copolymer or PEG-b-PEEGE block copolymer was transferred to a lace carbon supported lattice by a plunge-dipping method Respectively. The thin aqueous film was prepared at a humidity of 97 to 99% at ambient temperature in a customized environmental chamber to prevent water from evaporating from the sample solution. The excess solution was poured off with filter paper for 3 seconds and the thin aqueous film was rapidly vitrified by placing it in liquid ethane (cooled with liquid nitrogen) at its freezing point. The lattice was transferred to a Gatan 626 cryo-holder using a cryo-moving device. Using a JEM-3011 HR microscope (JEOL, Tokyo, Japan), direct imaging was performed at an accelerating voltage of 300 kV at a temperature of about -175 ° C. Micrographs were obtained using an SC 1000 CCD camera (Gatan, Inc., Pennsylvania) and the data were analyzed using a Gatan Digital Micrograph program.

As shown in Figures 12 and 13, the micelles of P1 and P2 exhibited spherical shapes with core sizes of approximately 20.4 ± 2.9 nm and 13.4 ± 1.6 nm, respectively. In addition, the core size of the PEG-b-PPGE micelles decreased as the length of the hydrophobic PPGE block increased. As a result, it was determined that the packing of the side chains in the hydrophobic core of the micelle was enhanced when the degree of hydrophobicity of PPGE was increased. In particular, the cryo-TEM image of P2 micelles exhibited a uniform spherical shape. Also, the diameter of the measured P2 micelles was 26.7 nm with a 14.0 nm hydrophobic core and a 6.4 nm hydrophilic corona.

(1-4) Micelle diameter  analysis

The size distribution of self-assembled micelles was analyzed using a dynamic light scattering method (DLS, BI-APD, Brookhaven Instrument) at angles of 90 ° and 30 °. The results are shown in FIG.

As shown in FIG. 14, the hydrodynamic diameter (D _h ) of the P2 micelles measured by the dynamic light scattering method (DLS) was 26.2 ± 4.6 nm and the diameter of the micelles measured through the ultra- .

The size and structure of the micelles were analyzed using X-ray small scattering (SAXS). The results are shown in FIG. Specifically, SAXS measurements were performed on Beamline 4C SAXS of the Pohang Acceleration Laboratory (PAL) using 16.9 keV radiation corresponding to a wavelength of lambda of 0.734 A. The distance between the detectors in the sample is set to 4.3 m to cover the q range of 0.007 Å ^-1 <q <0.12 Å ^-1 , where q is a scattering vector defined by q = 4πλ ^-1 sin (θ / 2) . The micellar solution was supported, placed in a capillary tube, sealed, and exposed to X-rays for 1 to 2 minutes. The two-dimensional images were averaged azimuthally to obtain a one-dimensional intensity plot for q. The solvent, i.e., water, was removed from solution scattering.

Based on the cryogenic TEM image and DLS data, P1 and P2 The SAXS profile obtained from the micelles was simulated by a model comprising a spherical core having a constant shell thickness of: &lt; EMI ID = 7.0 &gt; The Schulz distribution of the core radius was used to account for polydisperse core radii.

&Quot; (7) &quot;

이고,In Equation (7)

ego,

V _c , V _s , R _c , and R _s are the volume of the core and shell, respectively, and the radius of the core and shell,

p _c , p _s, and p _sol are the electron density of the core, shell, and solvent, respectively.

As shown in FIG. 15, the P1 and P2 micelles had core diameters of 16.6 nm and 11.0 nm, respectively, and total micelle diameters of 31.4 nm and 26.4 nm.

Further, the structure of the micelles was observed by atomic force microscopy (AFM), and the results are shown in FIG.

As shown in FIG. 16, the diameters of P1, P2, and E2 micelles measured by AFM were 77.2 ± 9.4 nm, 58.7 ± 9.1 nm, and 79.0 ± 14.8 nm, respectively.

On the other hand, the difference between the diameter of the micelles measured by the ultra-low temperature TEM and the diameter of the micelles of the AFM is a difference caused by the increase in diameter of the micelles attached to the substrate during AFM measurement.

(1-5) Encapsulation efficiency

The encapsulation efficiency of micelles was measured by the emission spectrum of Nile Red. At this time, the emission spectrum was recorded using a fluorescence meter (RF-6000, Shimadzu), and the measurement results are shown in Fig.

Specifically, 0.50 mL of an acetone solution (concentration 50 / / mL) containing nile red was added to the PEG-b-PPGE solution (solvent: acetone), and the mixture was stirred at room temperature for 30 minutes. A total of 5 mL of deionized water was then added to the mixture at a rate of 0.5 mL / min using a syringe pump, the mixture was allowed to equilibrate overnight, and the lid was opened to allow the acetone to evaporate. After filtration using a 0.45 [mu] m syringe filter, the solution was lyophilized and redissolved in acetone. The amount of nile red supported on the micelle was passed through a 1 cm x 1 cm quartz cell by a fluorescence meter measurement (RF-6000, Shimadzu) and the encapsulation efficiency was calculated by the following equation (8). At this time, excitation wavelength = 480 nm, emission wavelength range = 500 to 800 nm, scan speed = 2000 nm / min, and data interval = 0.5 nm were used.

&Quot; (8) &quot;

As shown in FIG. 17, the P1, P2, P3, and E3 micelles exhibited encapsulation efficiencies of 8.2, 14.9, and 16.6% for Nile Red, respectively. In particular, E3 micelles exhibited lower encapsulation efficiency than P2 micelles, indicating that the ability to support block copolymers is associated with hydrophobic PPGE blocks.

Experimental Example  2. Stability and Emissivity

The polymers or micelles of Examples 2 and 3 and Preparation Examples 2 and 4 were characterized in the following manner.

(2-1) Evaluation of encapsulation stability

Of the various factors that determine the suitability of the drug delivery carrier, the encapsulation stability under biological conditions is an important factor. To investigate the stability of micelles under harsh biological conditions, we used a method based on the Zerstor resonance energy transfer (FRET) as reported in previous studies. The donor, 3,3'-dioctadecyloxycabbocyanine perchlorate (DiO) and the acceptor 1,1'-dioctadecyl-3,3,3 ', 3'-tetramethyldicarbocyanine perchlorate (DiI) To evaluate the loading stability of the micelles prepared using the FRET pair. When the micelle is stable, the FRET pair will exhibit strong release (release of DiI receptor) at 564 nm due to effective energy transfer by nearby molecules in the micelle. On the other hand, if the micelle tends to be unstable, the release of FRET molecules will eliminate the FRET effect and only the release of the FRET donor DiO will be observed at 501 nm. The evaluation results are shown in Figs. 18 and 19.

Specifically, the dye in the micelle was carried in the same manner as in item (1-5), except that DiO and DiI were used instead of nile red. The stability of the DiI and DiO loaded micellar structures was measured in fetal bovine serum (FBS) in phosphate buffered saline (PBS) and different serum concentrations (10% FBS, 50% FBS and 100% FBS) The characteristics were analyzed in vitro. The excitation was set at 450 nm and emission was detected at 460 nm to 700 nm using a spectrophotometer.

As shown in Figure 18, P1, P2, and P3 micelles exhibited near-intact FRET release in phosphate-buffered saline (PBS), as the spectrum occupied by the release of DiI at the FRET receptor at 570 nm . As mentioned, the spectral profile is due to the intermolecular energy transfer between the adjacent DiO and DiI in the micelle core, confirming the excellent stability of micelles in PBS for 24 hours. On the other hand, E3 micelles showed significant changes in FRET pattern over time, which is a result of structural instability in PBS.

18 and 19, the fluorescence intensity of P1 and P3 micelles was remarkably changed as a result of the analysis of stability in pure serum. As a result, it was confirmed that the fluorescence intensity of the inner and the inner microparticles of albumin and globulin Indicating rapid distribution. In particular, P3 micelles resisted release of the support for 24 hours, which blocked the hydrophobic molecules inside the core from contact with serum components and showed a variety of controllability of emission kinetics under biological conditions, And 50% serum.

(2-2) Intracellular Micelle  stability

Human epithelial cancer cells (purchased from Korea Cell Line Bank, HeLa cells) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Invitrogen Life Technologies, Lt; / RTI &gt; The cells were maintained in a humidified atmosphere containing 5% CO ₂ at 37 &lt; 0 &gt; C, exchanging the medium every other day. The cells were cultured on eight wells of a Lab-Tek glass chamber slide (Thermo Fisher Scientific, Korea).

Then, the cells were treated with 0.5 μg / mL of the DiI and DiO-carrying micelles of item (2-1) and incubated for 0.5, 6, or 12 hours. Images were captured using an Olympus confocal laser scanning microscope model FV1000 using an excitation filter set at 473 nm. The results are shown in FIGS. 20 and 21. FIG.

As shown in FIGS. 20 and 21, the red signal from the cell membrane shows strong red fluorescence in DiI due to FRET generated in P1, P2, and P3 micelles, while E3 Micelles lost FRET signal in the cell membrane before internalization. This indicates that micelles (P series) having PPGE blocks exhibit excellent stability even under in vitro conditions in HeLa cells.

In addition, when cultured for 6 hours, some noticeable differences were observed between micelles of the P series. Specifically, P1 rapidly released the inner support, while P2 and P3 showed slow release even in cytoplasm. This represents a successful cytoplasmic delivery potential of an active therapeutic agent with an adjustable encapsulation stability depending on the choice of hydrophobic block and length.

(2-3) Decomposition in Acid

0.10 mL of 1 M HCl was slowly added to the pyrene-containing polymer micelle solution prepared in the same manner as in item (1-2) above, and the excitation spectrum change was recorded. The results are shown in FIGS. 22 and 23, and FIG. 22 plotted the ratio of fluorescence intensities (I ₃ / I ₁ ) at 339 and 332 nm as a function of time.

As shown in FIGS. 22 and 23, after lowering the pH, the intensity of the fluorescent excitation band shifted with time and decreased.

As shown in FIGS. 22b-d, the I ₃ / I ₁ plot for the time of the P1 and P2 micelles showed sustained release, indicating that the P series is capable of effective drug delivery over a long period of time. In particular, P3 micelles with the longest hydrophobic blocks did not release internal support even after 7 days in an ideal acidic environment (FIG. 22d).

On the other hand, as shown in Figs. 22 (e) and (f), E2 micelles showed burst release only 5 minutes after the acid treatment, even when the polymer concentration was increased to 5 mg / mL. In addition, E3 micelles with longer hydrophobic blocks exhibited rapid release of encapsulated pyrene compared to E2 micelles, but emission kinetics were much faster than P series micelles (see g in Figure 22). This is presumed to be due to the dense packing of the hydrophobic PPGE blocks in the micelle core, which makes it difficult for the acid to penetrate the hydrophobic domains and thus to reduce the acid approach to the hydrolysis of unstable acetal bonds.

(2-4) Evaluation of cytotoxicity

The cell viability of micelles was evaluated to determine its potential for use as a drug delivery carrier.

Specifically, the HeLa cells in 96-well plate in DMEM, 10% FBS, and 1% penicillin-using growth medium containing streptomycin and grown at a density of 3 X 10 ⁴ cells per well. After incubation for 24 hours in a humidified atmosphere of 95% air and 5% CO ₂ , the cells were stabilized and HeLa cells were treated with micelles carrying the respective micelles and paclitaxel. After 24 hours, 10 占 퐇 of MTT was added to each well (final concentration of 0.5 mg / ml) and incubated for 3 hours. Then, the culture medium was removed and 100 μl of dimethyl sulfoxide (DMSO) was added to each well to dissolve the residual MTT reagent. The plates were then stirred at room temperature for 15 minutes to dissolve the MTT in DMSO. Absorbance was measured at a wavelength of 540 nm based on 620 nm. The measurement results are shown in Figs. 23 and 24.

The encapsulation efficiency of the paclitaxel (non-aqueous anticancer drug) was measured by high pressure liquid chromatography (HPLC), and the measurement results are shown in FIG.

Specifically, 0.3 mL of acetonitrile containing paclitaxel (concentration 1 mg / mL) was added to a PEG-b-PPGE solution (solvent: acetonitrile) and the mixture was stirred at room temperature for 30 minutes. A total of 5 mL of deionized water was then added to the mixture at a rate of 0.5 mL / min using a syringe pump, the mixture was allowed to equilibrate overnight and acetonitrile was removed by dialysis with water. It was then filtered using a 0.45 um syringe filter. The amount of paclitaxel carried on the micelle was calculated by high pressure liquid chromatography measurement (Prominence, Shimadzu). At this time, the excitation wavelength was 228 nm, and the solvent condition was water: acetonitrile = 20: 80.

As shown in FIG. 24, cell viability after treatment with micellar solutions of various concentrations was almost 100% in P1, P2 and E2 micelles even at a high concentration of 500 μg / mL. However, the cell viability of P3 and E3 micelles (micelles with longer hydrophobic chains) decreased slightly at 250 ug / mL concentration. This can be explained by the fact that long hydrophobic chains can have a negative impact on biocompatibility.

As shown in FIG. 25, after culturing the micelles carrying paclitaxel for 24 hours, cell viability was decreased in all the micelles, and the P series micelles exhibited particularly excellent efficiency as compared with the micelles of the E series. This indicates that the micelles of the P series have higher loading efficiency than the micelles of the E series. This indicates that the micelles of the P series as the drug delivery material are more useful than the micelles of the E series.

Claims (16)

1. A polymer comprising a repeating unit represented by the following formula (1): &lt; EMI ID =
[Chemical Formula 1]

.
The method according to claim 1,
Wherein the polymer is a homopolymer comprising repeating units represented by the general formula (1).
3. The method of claim 2,
Wherein the homopolymer is represented by the following formula (2): &lt; EMI ID =
(2)

In the general formula (2), n is an integer of 5 to 200.
The method of claim 3,
Wherein the homopolymer has a number average molecular weight of 1,500 to 20,000 g / mol.
The method according to claim 1,
Wherein the polymer is a block copolymer comprising a repeating unit represented by the formula (1) and an alkylene glycol repeating unit.
6. The method of claim 5,
Wherein the block copolymer is represented by the following Formula 3:
(3)

In Formula 3,
n is an integer from 5 to 200,
and m is an integer of 50 to 500.
6. The method of claim 5,
Wherein, when m is 100 in the above formula (3), n is an integer of 5 to 200.
6. The method of claim 5,
Wherein the block copolymer has a critical micelle concentration of 0.1 to 100 mg / l.
A micelle comprising a polymer according to any one of claims 5 to 8.
10. The method of claim 9,
Wherein the micelles have an average diameter of 5 to 1,000 nm.
10. The method of claim 9,
Wherein the micelle further comprises a drug encapsulated by a polymer.
12. A drug delivery composition comprising the micelle of claim 9.
A process for producing a polymer according to claim 1, which comprises polymerizing a first monomer represented by the following formula (4) and a second monomer having a hydroxy group:
[Chemical Formula 4]

.
14. The method of claim 13,
Wherein the polymerization is carried out at 20 to 40 占 폚 and under a strong base catalyst.
14. The method of claim 13,
Wherein the second monomer is a compound represented by the following formula (5)
Wherein the polymer is a compound represented by the following formula (2): &lt; EMI ID =
[Chemical Formula 5]

(2)

In the general formula (2), n is an integer of 10 to 200.
14. The method of claim 13,
Wherein the second monomer is a compound represented by the following formula (6)
Wherein the polymer is a compound represented by the following formula (3): &lt; EMI ID =
[Chemical Formula 6]

(3)

In this formula,
n is an integer from 5 to 200,
and m is an integer of 50 to 500.

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