KR20110096333A - Phv-block-mpeg diblock copolymer and amphiphilic nanocontainers for hydrophobic drug delivery using thereof - Google Patents

Abstract

PURPOSE: A PHV-mPEG diblock copolymer is provided to obtain amphiphilic diblock copolymer nanoparticles without toxicity to nerve blastocyte, which is stably dispersed in an aqueous solution. CONSTITUTION: A PHV-mPEG(poly(3-hydroxyvalerate)-monomethoxy polyethylene glycol) diblock copolymer has the average molecular weight of 10,000-15,000. An amphiphilic PHV-mPEG nanocontainer for drug delivery is prepared by the PHV-mPEG(poly(3-hydroxyvalerate)-monomethoxy polyethylene glycol) diblock copolymer. The amphiphilic PHV-mPEG nanocontainer has a PHV core and an mPEG shell surrounding the core. The average diameter of the nano container is 190-220 nm.

Description

PhV-mPEGE diblock copolymer and amphiphilic nanocontainer for hydrophobic drug delivery using the same {PHV-block-mPEG diblock copolymer and amphiphilic nanocontainers for hydrophobic drug delivery using

The present invention relates to novel amphiphilic nanoparticles for drug delivery. More specifically, it relates to amphiphilic PHV-mPEG diblock copolymer nanoparticles for delivering hydrophobic drugs.

In the pharmaceutical field, nanotechnology has been steadily developed, particularly as a skeleton in drug delivery systems and tissue design. The use of nanoparticles for drug delivery increases biocompatibility and allows for delayed release of the main therapeutic drug. Several types of polymers have been used to synthesize nanoparticles, but using nanoparticles made from biodegradable polymers is not suitable for delayed release because it is rapidly removed from the blood by opsonization and phagocytosis. Therefore, recently, biodegradable polymer nanoparticles have been spotlighted as drug delivery systems.

Polyhydroxyalkanoate (PHA) is a biodegradable thermoplastic polymer produced by microorganisms in the form of inclusion bodies under unbalanced growth conditions. While a wide variety of biologicals have been produced as potential drug carriers to date, PHA is still a choice. Various types of PHA homopolymers and copolymers produced by microorganisms can be used as tissue design materials and controlled release drug vectors. Poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) is attracting attention because of its biocompatible, biodegradable and non-toxic properties. Moreover, degradation products (monomers and oligomers) of PHA polymers are also known to be safe with no toxicity to tissues.

It has been found that microbial poly (3-hydroxybutyrate) (PHB) is resistant without causing any inflammatory response in the tissue of the animal. In some cases, however, chemically unmodified microbial PHB was observed to exacerbate the inflammatory response in animal tissues. There is a need for the development of non-toxic substances for drug delivery that do not trigger the body's immune response.

High molecular weight polyesters are difficult to use as skeletons of amphiphilic block copolymers and inhibit drug diffusion efficiency, so only low molecular weight polyesters in the form of block copolymers can be used as additives or adjuvants of drug delivery. The hydrophilic portion in the amphiphilic block copolymer has a strong covalent bond with the hydrophobic polymer. In an aqueous environment, the hydrophobic core of the polymer is buried inside the exposed hydrophilic portion. Hydrophobic biodegradable aliphatic polyesters such as PHB, poly (L-lactide) and poly (D, L-lactide-co-glycolide) because poly (ethyleneglycol) (PEG) is low toxicity and does not cause an immune response It is widely used as a hydrophilic part with. The introduction of a hydrophilic mPEG (monomethoxy polyethylene glycol) group on the surface of the nanoparticles counter opsonization, and longer residence time in the blood than nanoparticles without the mPEG group. The longer residence time of mPEG coated particles in the blood is due to the thickness of the high molecular weight mPEG, which protects the nanoparticles from opsonization and phagocytosis. In addition, mPEG released upon decomposition of the polyester-mPEG block polymer is easily excreted out of the body through the kidneys.

It is believed that low molecular weight biodegradable block copolymers in the form of amphiphilic nanoparticles can be used for delayed release of various types of drugs. Putting the drug in the central hydrophobic domain of the amphiphilic nanoparticles depends on the size of the block copolymer. The use of biodegradable amphiphilic nanoparticles as drug carriers is preferred because the drug is released in two stages: first diffusion released from the mPEG moiety and then degradation release from the biodegradable polymer moiety. The size of the nanoparticles is also considered to be an important factor in determining the properties of the nanoparticles. Large particle sizes, high molecular weight polymers and high polymer concentrations are known to inhibit drug diffusion efficiency.

Most studies to date have focused solely on the use of PHA copolymers at the micron-sized particle level, using either high molecular weight unmodified PHA copolymers as one component or mixed with other polyesters. However, microparticles made from such copolymers are large and difficult to pass through the thin biological membranes of most bodies. For nanoparticles with small diameters, lower clearance rates and longer blood residence times were observed compared to those with larger diameters. Therefore, it is necessary to make a drug delivery vehicle from nanoparticles made of low molecular weight polymer.

As a result of repeated studies to solve the above problems, the inventors of the present invention are advantageous for drug delivery when using nanoparticles made of low molecular weight PHV-mPEG (poly (3-hydroxyvalerate) -monomethoxy polyethylene glycol) diblock copolymer. I found that.

Accordingly, the present invention provides a PHV-mPEG diblock copolymer and an amphiphilic nanocontainer using the same.

Preferably the PHV-mPEG diblock copolymer of the present invention has a weight average molecular weight of 10,000 to 15,000. More preferably, the PHV-mPEG diblock copolymer of the present invention has a weight average molecular weight of 12,000 to 13,500.

Also preferably, the amphiphilic PHV-mPEG nanocontainer of the present invention has an average diameter of 190 to 220 nm, more preferably has an average diameter of 200 to 215 nm.

Also preferably the amphipathic PHV-mPEG nanocontainers of the present invention have an average zeta potential of -10 to -20 mV. More preferably -12 to -16 mV.

Hereinafter, the present invention will be described in detail.

The process of synthesizing the PHV-mPEG diblock copolymer of the present invention is schematically illustrated in FIG. 1. The high molecular weight PHV having a melting point (Tm) of 110 ° C was decomposed into low molecular weight macromolecules by transesterification at 190 ° C for 20-30 minutes. Terminal hydroxyl groups of PHV sensitive to high temperature disappear and olefin groups are formed. Low molecular weight macromolecules react with the OH groups of mPEG in the presence of bis (2-ethylhexanoate) tin, which acts as a catalyst for the transesterification reaction. Thus, PHV-mPEG diblock copolymer is produced by transesterification.

Table 1 shows the polymer yield, number average molecular weight (Mn), weight average molecular weight (Mw) and polydispersity (Mw / Mn) of the diblock copolymers made in one embodiment of the present invention. GPC analysis was conducted to determine the molecular weight and molecular weight distribution of the diblock copolymer. PHV-mPEG diblock copolymers showed broader peaks at relatively early elution times in comparison to mPEG prepolymers on GPC chromatographs (FIG. 2). The peak of this unimodal peak and the relatively narrow molecular weight distribution (PI = 1.24) indicate that there is almost no unreacted glass precursor prepolymer in the pure product.

Molecular Weight and Thermal Properties of PHV-mPEG Diblock Copolymer sample M _w ^a M _n ^a PI (M _w / M _n ^a ) Yield ^b (%) T _m ^c (℃)
PHV Block T _m ^c (℃)
mPEG block PHV-mPEG 12,900 10,400 1.24 78 96.7 42.2

^a Measured by GPC. ^b Weight ratio of the formed product to mPEG and PHV put at the beginning of the reaction. ^c melting point of the diblock copolymer measured by DSC; The PHV block shows a beekeeping peak due to melt recrystallization. T _m showed the main second peak.

The chemical structure of the PHV-mPEG diblock copolymer was determined by ¹ H NMR spectroscopy. The spectrum at the top of FIG. 3 is that of PHV-mPEG, the bottom is that of pure mPEG, with peaks a to h related to PHV, and i, j and k peaks related to mPEG. The absorption at 3.71 ppm indicated by arrows at the bottom of the spectrum in FIG. 3 is thought to be due to both methylene bound to the hydroxyl terminus of the free mPEG. In the spectrum of PHV-mPEG there is no absorption signal and instead a peak at 4.16 ppm (peak i), indicating that both methylenes have formed ester bonds at the end groups of ethylene glycol, resulting in a binding site between PHV and mPEG. Similar to PHB-mPEG, PHV-mPEG also showed two small peaks at 6.95 and 5.74 ppm (peaks c and d, respectively) associated with olefin end groups by thermal hydrolysis of hydroxyl end groups. Both methyl and methylene associated with the terminal pentenoyl groups of the PHV block show resonances (peaks a and b) at 1.00 and 2.15 ppm, respectively. Resonance (j) at 3.60 ppm and resonance (k) at 3.31 ppm are related to both methylene and methyl of the mPEG block, respectively.

In one embodiment of the present invention, the thermal transition was measured by DSC analysis of the diblock copolymer. The scan range for the PHV-mPEG diblock copolymer was 50 to 200 ° C. and for mPEG was measured at 35 to 100 ° C. Since the Tm values are 42.2 ° C for the mPEG block and 96.7 ° C for the PHV block (see Table 1), two distinct melt endotherms are observed for the PHV and mPEG blocks in the PHV-mPEG diblock copolymer. . Two melt endotherms indicated that separate crystal phases for the two blocks were formed in the diblock copolymer. Beekeeping melt endotherms also appear in the PHV moiety in PHV-mPEG, which is believed to be the cause for this double melt peak for PHA polymers. Compared with the precursor prepolymers, the thermal properties of both blocks shifted to lower temperatures (Table 1). This lowering of the melting point is in part due to the coupling reaction of the hydroxyl group and the carboxyl group by the transesterification reaction, which means that the crystallinity of the sample is reduced.

In one embodiment of the present invention, the biodegradable nanoparticles were prepared by the o / w solvent evaporation method with the diblock copolymer. 4a and b show FE-SEM micrographs of PHV (control) and PHV-mPEG nanoparticles, respectively. The nanoparticles thus made are well dispersed and have a smooth surface shape. However, some of the particles collapsed and flattened while drying on the microscope studs, slightly deforming the surface. The average size of PHV nanoparticles measured by ELS is 370 ± 3 nm and the PHV-mPEG nanoparticles are 208 ± 1 nm. Table 2 describes the average size, polydispersity, and zeta potential of the nanoparticles. The average size of the nanoparticles measured by ELS was consistent with that measured by FE-SEM. The relatively large size of PHV nanoparticles compared to mPEG-grafted nanoparticles is due to the suspension being prepared without filtration for particle size analysis. As a result, some aggregation occurred in the PHV particles. On the other hand, PEGylated nanoparticles did not aggregate even after freeze drying and formed a stable suspension in aqueous solution. This is because the mPEG portion of the nanoparticles electrostatically prevents the aggregation of the nanoparticles. PEG has been widely used as a stabilizer in proteins to prevent aggregation during lyophilization. The zeta potential of PHV particles is 25 ± 2 mV and the value of mPEG-grafted particles is 14 ± 1 mV (Table 2). This clearly indicates that PEGylation reduced the surface charge of the nanoparticles.

Nanoparticle size and surface charge sample Particle size ^a (nm) ± SD Magnitude polydispersity ^a ± SD Zeta Potential ^a (mV) ± SD PHV-mPEG 208 ± 1 0.26 ± 0.01 -14 ± 1 PHV (control) 370 ± 3 0.23 ± 0.20 -25 ± 2

Particle size, polydispersity and surface charge of ^a nanoparticle were measured by ELS.

In one embodiment of the present invention, in vitro cytotoxicity of the nanoparticles of the present invention was tested. Five diluting samples (20, 50, 100, 200, 400 / mL) were used for MTT analysis by diluting the sample using Dulbecco's modified Eagle medium (DMEM). Cytotoxicity of nanoparticles was assessed for dose and duration of action. After preparing suspensions of the desired concentration of nanoparticles, toxicity experiments were carried out with neural embryonic cells highly sensitive to foreign substances. 5A shows cell viability after treatment with PEGylated nanoparticles and non-PEGylated nanoparticle controls and incubation at 37 ° C. for 24 hours. It can be seen that both the nanoparticle and non-PEGylated nanoparticle samples of the present invention show similar cell viability at low concentrations and thus have little effect on cell viability. When cells were exposed to higher concentrations (more than 200 μg / mL) of the nanoparticles for 48 hours, a slight increase in cytotoxicity was observed in non-PEGylated nanoparticle samples (FIG. 5B). The increased cytotoxicity in PHV nanoparticles is believed to be due to the inflammatory response induced by HV units.

The novel amphiphilic diblock copolymer nanoparticles of the present invention can be used as an effective drug delivery system for hydrophobic drugs because they are not only stably dispersed in an aqueous solution but have little toxicity to toxic neurons.

1 is a schematic diagram showing a process of synthesizing a PHV-mPEG diblock copolymer of the present invention.
2 is a GPC chromatograph of PHV-mPEG diblock copolymer and mPEG prepolymer.
3 is a ¹ H-NMR graph of PHV-mPEG diblock copolymer and mPEG.
4 is a graph showing the FE-SEM micrographs of PHV (control) (a) and PHV-mPEG nanoparticles (b) and the size distribution of PHV (control) (c) and PHV-mPEG nanoparticles (d).
5 is a graph showing the survival rate of embryonic hippocampal neuron cells treated with PHV and PHV-mPEG nanoparticles of the present invention. Nanoparticles of the desired concentration (25400 μg / mL) were added in DMEM medium for 24 hours (FIG. 5A) and 48 hours (FIG. 5B) and cultured at an initial cell concentration of 1 × 10 ⁶ / well.

Hereinafter, the present invention will be described in more detail by way of examples. It should be noted, however, that the following examples are provided to further illustrate the present invention and are not intended to limit the scope of the present invention.

≪ Example 1 >

Preparation of Diblock Copolymers

1.1 Preparation of Materials

PHV homopolymers were prepared by incubating Paracoccus denitrificans KCTC 2530 (Korean Culture Type Collection) with 90 mM n-valeric acid as a single carbon source.

Cells pre-cultured in nutrient medium were transferred to PHA synthetic mineral medium containing 10 mL / L of n-valeric acid and incubated for 60 hours. Cells were harvested by centrifugation with Hanil-Supra 22K (Rotor A250S-6, 10 min at 6,000 rpm). Polyester was extracted with hot chloroform in a Pyrex Soxhlet apparatus. mPEG [number average molecular weight (Mn) = 2000], bis (2-ethylhexanoate) tin catalyst, polyvinyl alcohol (PVA) (Mw, 9,00010,000 Da, 80% hydrated) and dichloromethane (DCM ) Was purchased from Sigma-Aldrich Korea Ltd. Other chemicals used analytical grades.

1.2 Preparation of Diblock Copolymers

The PHV-mPEG copolymer was synthesized by a slightly modified Marchessault's method.

First, in a 25 mL round bottom flask equipped with a magnetic stirrer, the polymer and mPEG prepared in Example 1.1 were added in a weight ratio of 1: 1. The reaction was carried out in vacuum in an oil bath preheated to 190 ° C. About 70 mg of bis-ethylhexanoate tin catalyst was introduced through a rubber septum using a syringe while flowing nitrogen. The reaction was continued while stirring for 20-30 minutes. After the reaction was completed, the flask was taken out and cooled to ice or room temperature to yield a waxy product. The obtained modified diblock copolymer was dissolved in 5 mL chloroform and then dropped into 100 mL of distilled water to form an emulsion. The resulting emulsion was vigorously stirred with a magnetic stirrer to allow the organic solvent to fly away. The colloidal suspension was dialyzed for 1 week in distilled water using a 30,00035,000 Da molecular weight removal membrane. The polymer was then lyophilized to give a light white powder (yield 70-80%).

1.3 Properties of Copolymer

Three PL gel columns (10 ⁵ , 10 ³ and 10 ² ), one Agilent G1310A isocratic pump, one Agilent 1047A RI detector, one Agilent G1316A column portion and one Agilent G1311A at 30 ° C. The molecular weight of the diblock copolymer was measured using an Agilent 1100 series gel permeation chromatography (GPC) system (Agilent, Santa Clara, Calif.) Configured with a vacuum degasser. Chloroform was used as the eluent at a flow rate of 1.0 mL per minute. Standard polystyrene with low polydispersity (Polymer Laboratories, Amherst, Mass.) Was used for system calibration.

The molecular structure of the diblock copolymer was obtained using ¹ H-NMR with a Bruker-DRX-500 MHz spectrometer. Spectra of the samples were recorded in CDCl ₃ at room temperature. The spectral signal divided by standard software is integrated.

Measure the thermal transition of the diblock copolymer while purging with nitrogen using a differential scanning calorimeter (TA DSC) Q10 V6.21 (TA Instruments, New Castle, DE) equipped with a data station It was. Scanning was in the range of 50 to 200 ° C. while heating at a rate of 10 ° C. per minute.

<Example 2>

Synthesis of Nanoparticles

2.1 Synthesis of Nanoparticles

Amphiphilic nanoparticles were synthesized from diblock copolymers by slightly modified o / w solvent evaporation.

2 mL DCM containing 50 mg of diblock copolymer was added to a 1% PVA solution (60 mL) and the mixture was mixed at 1-% using a microtip probe sonicator D-12207 (Bandelin Electronics, Germany) at 15% output. Probe sonication for 2 minutes. The resulting o / w emulsion was gently stirred in a fume hood at room temperature to allow the organic solvent to evaporate completely. The nanoparticles were purified by centrifugation at 12,000 rpm for 10 minutes and washed three times with water to remove excess emulsion. The nanoparticle suspension was freeze dried to obtain a fine powder of nanoparticles. PHV homopolymer nanoparticles were made in the same way.

2.2 Characteristics of Nanoparticles

The morphology of the nanoparticles was observed using a field emission scanning electron microscopy (FE-SEM) model XL30S (Philips, The Netherlands). The nanoparticle suspension was diluted with distilled water to make a sample and dropped onto a carbon stud. The nanoparticles were dried in air and coated with gold using a sputter coater (JFC-1100E, ion sputtering apparatus, JEOL, Japan) under vacuum for 300 seconds at a current intensity of 10 mA.

The size and zeta potential of the nanoparticles were measured at 90 ° and 20 ° with an electrophoretic light scattering spectrophotometer (ELS-8000; Otsuka Electronics, Osaka, Japan) at 25 ° C. About 100 mg of lyophilized nanoparticles were ultra-sonicated for 1 minute and redispersed in 10 mL of PBS (phosphate buffered saline) at pH 7.4. Nanoparticle suspensions diluted with PBS were used to determine particle size and zeta potential without filtration.

<Example 3>

Cytotoxicity experiments ( in vitro )

3.1 Hippocampal Neuron Cell Culture

Twenty 250 gram female Sprague-Dawley rats were randomly eaten in a temperature-controlled environment and lit up to 06: 0020: 00 in the Gyeongsang National University Neurobiology Laboratory. On the 17th day of fertilization, pregnant rats were subjected to a migraine and the fetus was removed.

Hippocampal neurons of fetal rats removed from the pregnant rats were cultured.

The harvested hippocampal tissue was treated with 0.25% trypsin-EDTA for 20 minutes and mechanically ground in ice-free, calcium-free magnesium free Hanks' balanced salt solution (pH 7.4). Cells (1 × 10 ⁶ cells / mL) were planted in a chamber slide culture plate coated with polylysine (0.02 g / L) after pelleting via centrifugation. DMEM (Dulbecco's) with 10% heat-inactivated fetal bovine serum, 1 mM pyruvate, 4.2 mM sodium bicarbonate, 20 mM HEPES, 0.3 g / L bovine serum albumin, 50 U / mL penicillin and 50 mg / L of streptomycin In the modified Eagle's medium, the cells were incubated in a humid environment consisting of 5% CO ₂ and 95% air at 37 ° C.

3.2 MTT Analysis

To determine the toxicity of the nanoparticles of the invention to in vitro cells, a 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) assay was performed. Was performed. The MTT assay is based on the reducing ability of the reductase enzyme (succinate dehydrogenase) of living cell mitochondria, which reduces the yellow water-soluble MTT dye to the purple insoluble formazan product. Formazan production, measured by absorbance at 570 nm, is directly proportional to the number of living cells in the medium.

Cells were first planted in 96-well plates at a rate of 1 × 10 ⁶ cells / well and then incubated for 24 hours at 37 ° C. in a humid 5% CO ₂ atmosphere to allow cells to adhere and grow completely. After 24 hours, the medium in the wells was changed to a medium containing the desired concentration of nanoparticle dispersion and further incubated for 24 and 48 hours. After 24 hours, cells were washed twice with PBS (pH 7.4) and 20 μL of MTT solution (5 mg / mL PBS) was added to each well and incubated for 4 hours. After the medium was aspirated off, 100 μL of dimethylsulfoxide per well was added and the plate was incubated for 1 hour on an orbital shaker at room temperature to allow formazan crystals to dissolve. Absorbance of cell viability was measured using a microplate reader (Anthos 2020, Anthos Labtech Instruments, Wals., Austria) at 570 nm.

Cell viability was calculated from the ratio of OD 570 to OD 570 of cells treated with control nanoparticles of cells treated with nanoparticles of the invention. All results are expressed as mean ± standard deviation from three experiments. Statistical error was assessed using ANOVA as a student's t-test. The error has statistical significance at the level of P <0.05.

Although embodiments of the present invention have been described above with reference to the accompanying drawings, those skilled in the art to which the present invention pertains may be embodied in other specific forms without changing the technical spirit or essential features of the present invention. I can understand that. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

Claims (9)

Poly (3-hydroxyvalerate) -monomethoxy polyethylene glycol (PHV-mPEG) diblock copolymer.
The diblock copolymer according to claim 1, having a weight average molecular weight of 10,000 to 15,000.
The diblock copolymer according to claim 2, having a weight average molecular weight of 12,000 to 13,500.
Amphiphilic PHV-mPEG nanocontainer for drug delivery made of PHV-mPEG diblock copolymer and having structure of PHV core and mPEG shell surrounding it.
The nanocontainer of claim 4, wherein the nanocontainer has an average diameter of 190 to 220 nm.
The nanocontainer of claim 5, having an average diameter of 200 to 215 nm.
6. The nanocontainer of claim 5, having an average zeta potential of -10 to -20 mV.
8. The nanocontainer of claim 7, wherein the nanocontainer has an average zeta potential of -12 to -16 mV.
The nanocontainer of claim 4, wherein the nanocontainer is made by emulsifying solvent evaporation.

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