KR19990024622A - Biodegradable nanoparticles and preparation method thereof - Google Patents

Abstract

The present invention relates to a biodegradable nanoparticles (nanoparticle) and a method for producing the same, and more particularly, a) preparing a diblock copolymer of a heterocyclic ester compound of the lactones and polyethylene glycol in the absence of a catalyst; b) dissolving the diblock copolymer in an organic solvent; c) adding a solution of a hydrophobic substance or drug in an organic solvent to the diblock copolymer of step b); And d) dialysis of the additive in water to form particles having hydrophobic substances or drugs introduced into the diblock copolymer. The inclusion efficiency of the hydrophobic drug in the prepared nanoparticles was obtained up to about 40wt%, and the release test resulted in continuous release for more than two weeks, and the release rate was increased as the amount of drug introduced and the size of the nanoparticles were large.

Description

Biodegradable nanoparticles and preparation method thereof

The present invention relates to a biodegradable nanoparticles (nanoparticle) and a method for producing the same, and more particularly to a biodegradable lactone heterocyclic ester compound and polyethylene glycol (PEG) diblock copolymer, The present invention relates to a biodegradable nanoparticle having a small particle size of 0.2 μm (200 nm) or less and a method for preparing the same, by introducing a hydrophobic substance or drug into the same.

Copolymerization of a PEG with a heterocyclic ester compound of lactones such as ε-caprolactone yields a biodegradable aliphatic polyester. This aliphatic polyester is usually biodegraded by hydrolysis. ε-caprolactone is hydrophobic and PEG is hydrophilic, and copolymers made of these two monomers are unique structures with hydrophilic-hydrophobic linkages that can form associations while self-organizing in water. That is, when dialysis in water together with a hydrophobic substance or drug, spherical nanoparticles are formed by rearranging the hydrophobic portion and the drug inward and the hydrophilic portion outward.

In the past, studies on microparticles (microparticles) that are several orders of magnitude larger than nanoparticles have been conducted, but there is an advantage in that they can continuously release drugs containing particles of smaller size than those of large particles, and more continuously. Particles larger than 0.2 μm (200 nm) at the time of administration have problems such as accumulation in organs and phagocytosis by epithelial reticulum cells (Biomaterial, 13, 1093 (1992), and J. Pharm. Sci., 68). , 1443 (1979).

In the conventional particle system, various biodegradable polymers such as polyglycolide, polylactide, or copolymers of lactide and glycolide, and natural polymers have been used, but are prepared by emulsifying and crosslinking or polymerizing Particles are prepared by emulsifying the containing organic phase in an aqueous solution containing a surfactant (Pharm. Rel. 10, 750 (1993)) and the like. Accumulation, phagocytosis by epithelial reticulum cells, and rapid removal in blood. In addition, bio-derived materials such as lipoproteins and liposomes have high biocompatibility and can produce nano-sized carriers, but they do not maintain their original properties for a long period of time stably in storage and thus change in size over time. There was a problem (Advanced Drug Delivery Reviews, 20, 113 (1996)).

On the other hand, aliphatic polyester has been widely known as a medical material due to its biocompatibility, non-toxicity, biodegradability, and the like, and as a catalyst for initiating ring-opening polymerization during synthesis, tin oxide (stannous octoate) and tin chloride (stannous) Polymerization was carried out using a catalyst such as cloride, tetraphenyltin, zinc oxide or metal alkoxide.However, in the case of preparing a polymer micelle required for human body as in the present invention, the catalyst remains after synthesis. Toxicity in the human body can be a problem.

Accordingly, it is an object of the present invention to provide a method for producing biodegradable nanoparticles which are harmless to human body and have excellent biotransporting ability.

Another object of the present invention to provide a biodegradable nanoparticles prepared by the above method.

The production method of the present invention for achieving the above object comprises the steps of preparing a diblock copolymer of a lactone heterocyclic ester compound and polyethylene glycol (PEG) under a catalyst; Adding a solution of a hydrophobic substance or drug in an organic solvent to the diblock copolymer; And dialysis of the additive in water to form particles into which the hydrophobic material or drug is introduced into the diblock copolymer.

Looking at the present invention in more detail as follows.

The present invention does not use a catalyst which remains in the synthesis of a dicyclic copolymer of a lactone heterocyclic ester compound such as ε-caprolactone, D, L-lactide or glycolide and a polyethylene glycol, which may be harmful to the human body. In the presence of a PEG homopolymer in which one end is substituted with methoxy under a non-catalytic condition, a ring-opening reaction of the ε-caprolactone monomer is caused by a hydroxyl group at one end to prepare a copolymer, and a drug or a hydrophobic substance is prepared by using the same. Introduced, but a nanoparticle having a small particle size of less than 0.2㎛ (200nm) was prepared.

The molar ratio of monomer (M) to initiator (I) used in the present invention ([M] / [I], [M] = ε-caprolactone, [I] = PEG homopolymer) was varied from 35 to 150 Copolymers were prepared in bulk. At this time, as [M] / [I] increases, that is, as the length of the hydrophobic block ε-caprolactone increases, the amount of drug introduced can be increased, and the rate of drug release can be decreased, but the particle size is increased. In particular, in excess of 150, the hydrophobicity becomes so large that no particles are formed. The diblock copolymer thus prepared was dialyzed in water to prepare uniform nanoparticles having a size of 200 nm or less, into which a hydrophobic drug was introduced.

For example, 2-10 mg of the block copolymer is dissolved in 10-20 ml of an organic solvent such as dimethylformamide, dimethylacetamide, tetrahydrofuran, or dichloromethane, and then indomethacin, ketoprofen, doxorubicin, and cyclosporin. 2-50 mg of hydrophobic substances or drugs such as cyclosporine, lidocaine, lidocaine, metipranolol, betaxolol, or tetracycline are dissolved separately in 10-20 ml of the organic solvent and stirred Mix. If the solution is dialyzed for 6 to 72 hours in ultrapure water using, for example, a cellulose dialysis membrane, the hydrophobic part forms a core and the hydrophilic part forms a tissue at a certain concentration (CMC) or more to form a hydrophobic drug. Introduced fine polymer micelles of the core-shell structure are formed. The micelle solution was sonicated, centrifuged and lyophilized to obtain dried nanoparticles.

In the present invention, the polymer micelle formed by dialysis of a block copolymer composed of hydrophilicity and hydrophobicity in water has a low structural dissociation rate from the micelles of the polymer chain constituting the micelles compared to the low molecular micelles. In addition, the outer shell is hydrophilic and plays an important role in maintaining the excellent stability and solubility of the polymer micelles, and PEG, which has extremely high terminal motility and biocompatibility, is used to recognize the epidermal reticulum cells in vivo. Can be avoided.

According to the present invention, the inclusion efficiency of the hydrophobic drug was obtained up to about 40wt%, and was released continuously for more than two weeks as a result of the release experiment, the more the drug introduced amount and the larger the size of the nanoparticles was released slowly.

Since the particle size of the present invention is 200 nm or less, it is possible to prevent the accumulation in the epithelial reticulum tissues including the Cooper cells of the liver, so that the drug can reach tissue cells through the capillary walls of the liver, cancer lesions, and inflammatory sites. It may be possible to increase the distribution in vivo as a carrier. In addition, anticancer drugs or AIDS therapeutic drugs, which are potent drugs in general, have been severely restricted in use because they are toxic to other cells even when the appropriate concentrations of the drug's effective concentrations are exceeded. New drugs using these biodegradable polymer nanoparticles The development of the vehicle is expected to solve the above problems.

Hereinafter, the method and effect of the present invention will be described in more detail with reference to Examples and Comparative Examples, but the scope of the present invention is not limited to the following Examples.

Preparation Examples 1 to 4

While changing the supply molar ratio of ε-caprolactone and methoxy polyethylene glycol (MePEG) as shown in Table 1 below, the mixture was stirred and mixed in a flask, cooled to 0 ° C., made into a vacuum state using a vacuum pump, and then stirred at 160 ° C. under vacuum. The reaction was allowed to proceed for 12 hours in an oven. The product was cooled to room temperature, dissolved in 5 ml of dimethylformamide, and added to propanol to remove unreacted monomer, MePEG homopolymer, and the like, followed by filtration. The product is then extracted with excess diethyl ether and filtered to collect the product. The product obtained was dried in a vacuum oven at 40 ° C. for 3 days. The molecular weight of the synthesized copolymer was confirmed by gel permeation chromatography (GPC) and nuclear magnetic resonance (NMR) spectroscopy.

Preparation Example 5

Instead of ε-caprolactone used in Preparation Examples 1 to 4, D, L-lactide was reacted in the same manner as in Preparation Example to prepare a copolymer of D, L-lactide and methoxy polyethylene glycol (MePEG). The molecular weight of the synthesized copolymer was confirmed by gel permeation chromatography (GPC) and nuclear magnetic resonance (NMR) spectroscopy.

Preparation Example 6

Instead of ε-caprolactone used in Preparation Examples 1 to 4, a glycolide was reacted in the same manner as in Preparation Example to prepare a copolymer of glycolide and methoxy polyethylene glycol (MePEG). The molecular weight of the synthesized copolymer was confirmed by gel permeation chromatography (GPC) and nuclear magnetic resonance (NMR) spectroscopy.

Examples 1-7

Ε-caprolactone and methoxy polyethylene glycol (MePEG) block copolymers prepared in Preparation Examples 1 to 4, D, L-lactide and MePEG block copolymers of Preparation Example 5, glycolide and MePEG of Preparation Example 6 After dissolving 10 mg of block copolymer in 20 ml of dimethylformamide, a solution of 10 to 30 mg of indomethacin is added to 20 ml of dimethylformamide, followed by stirring. At this time, the drug is produced by changing the weight ratio of the copolymer and its effect is shown in Table 2 below. The micelles are formed and the solution is dialyzed for 48 hours in one liter of ultrapure water using a cellulose dialysis membrane to remove indomethacin and organic solvents not introduced into the micelles. The polymer micelle solution is sonicated and then centrifuged to remove particles not incorporated with the drug. The micelle solution obtained through this process was lyophilized at -50 ° C. for 2 days to obtain a dried nanoparticle product.

Example 8

10 mg of the ε-caprolactone and methoxy polyethylene glycol (MePEG) block copolymer prepared in Preparation Example 3 were dissolved in 20 ml of dimethylformamide, and a solution of 30 mg of doxorubicin dissolved in 20 ml of dimethylformamide was added and stirred. The micelles are formed and the solution is dialyzed for 48 hours in 1 liter of ultrapure water using a cellulose dialysis membrane to remove doxorubicin and organic solvents not introduced into the micelles. The polymer micelle solution is sonicated and then centrifuged to remove particles not incorporated with the drug. The micelle solution obtained through this process was lyophilized at -50 ° C. for 2 days to obtain a dried nanoparticle product. The particle diameter of the produced particles was measured by dynamic light scattering.

Comparative Example 1

After stirring and mixing ε-caprolactone and methoxy polyethylene glycol (MePEG) in the feed molar ratio as in Preparation Example 3 above, the catalyst tin oxide was added and stirred at a molar ratio of [M] / [tin oxide] = 5000. Mix The mixture was cooled, vacuumed using a vacuum pump, and stirred for 12 hours in a vacuum oven at 160 ° C. while stirring. The product is cooled to room temperature, dissolved in dimethylformamide, and then added to propanol to extract unreacted monomer, MePEG homopolymer, and the like. The product is then extracted with excess diethyl ether and filtered to collect the product. The product obtained was dried in a vacuum oven at 40 ° C. for 3 days. The molecular weight of the synthesized copolymer was confirmed by gel permeation chromatography (GPC) and nuclear magnetic spectroscopy (NMR).

Comparative Example 2

10 mg of a block copolymer prepared by preparing a reaction molar ratio of ε-caprolactone and methoxy polyethylene glycol (MePEG) in 160 was dissolved in 20 ml of dimethylformamide, and 30 mg of indomethacin in 20 ml of dimethylformamide. Add the dissolved solution and mix by stirring. The micelles are formed and the solution is dialyzed for 48 hours in one liter of ultrapure water using a cellulose dialysis membrane to remove indomethacin and organic solvents not introduced into the micelles. The polymer micelle solution is sonicated and then centrifuged to remove particles not incorporated with the drug. The micelle solution obtained through this process was lyophilized at -50 ° C. for 2 days to obtain a dried nanoparticle product. The particle diameter of the produced particles was measured by dynamic light scattering.

Comparative Example 3

10 mg of ε-caprolactone and methoxy polyethylene glycol (MePEG) block copolymer prepared in Preparation Example 3 were dissolved in 20 ml of dimethylformamide, and a solution of 30 mg of cyanocobalamine (hydrophilic) in 20 ml of ultrapure water was added thereto. Stir and mix. The micelles are formed and the solution is dialyzed for 48 hours in 1 liter of ultrapure water using a cellulose dialysis membrane to remove cyanocobalamin and organic solvents not introduced into the micelles. The polymer micelle solution is sonicated and then centrifuged to remove particles that have not been incorporated with the drug. The micelle solution obtained through this process was lyophilized at -50 ° C. for 2 days to obtain a dried nanoparticle product.

Experimental Example 1

The release behavior of indomethacin itself, which was not introduced into nanoparticles, was examined. Accurately weigh indomethacin and disperse in phosphate buffer (PBS, 0.1M, pH 7.4). The indomethacin solution is placed in a dialysis bag and placed in a phosphate buffer solution, which is a release medium, and stirred at 37 ° C. 0.5 to 5 ml of the medium was taken at regular intervals and the absorbance of the material was measured using an ultraviolet spectrometer to examine the emission behavior.

The amount of material introduced into the nanoparticles prepared in Examples 1 to 8 and Comparative Example 3 was investigated using an ultraviolet spectrometer. That is, after sonication and centrifugation in order to remove particles accumulated with drugs not introduced into the nanoparticles, they are lyophilized at -50 ° C. After a certain amount of the dried product is destroyed by addition of ethanol / tetrahydrofuran (1: 1 v / v) solution, the absorbance of the introduced material is measured by using an ultraviolet spectrometer to measure the amount of introduction into the nanoparticles. The introduction efficiency of the drug was calculated by the following equation.

The release behavior of the hydrophobic material introduced in the nanoparticles prepared in Examples 1 to 8 and the amount of introduction of the material was calculated by Equation 1 was examined. Accurately weigh the dried nanoparticles into which the hydrophobic material is introduced and disperse in phosphate buffer solution (PBS, 0.1M, pH 7.4). The nanoparticle solution is placed in a dialysis bag and placed in a phosphate buffer solution, which is a release medium, and stirred at 37 ° C. 3 ml of the release medium was taken at regular intervals and the absorbance of the material was measured using an ultraviolet spectrometer to examine the emission behavior.

The molecular weight and particle size of the copolymers prepared in Examples and Comparative Examples are shown in Table 1 below. In Table 1, the molecular weight of the copolymer was measured by gel permeation chromatography, nuclear magnetic resonance spectroscopy, and particle size of the particles by dynamic light scattering method, respectively.

As can be seen in Table 1, by measuring the molecular weight using GPC and NMR, ε-caprolactone and methoxy polyethylene glycol (MePEG) block copolymer having a molecular weight similar to the theoretical value was synthesized, the catalyst Compared with Comparative Example 1 using stannous octoate, it was possible to synthesize a copolymer having a molecular weight similar to that of using a catalyst without using a catalyst.

In addition, copolymers of D, L-lactide / methoxy polyethylene glycol (MePEG) block copolymers and glycolide / methoxy polyethylene glycol (MePEG) block copolymers synthesized in Preparation Examples 5 and 6 also had molecular weights similar to theoretical values. It was confirmed that is synthesized. As shown in Table 1, as the molecular weight of the copolymer increased, the size of the particles produced increased, and in Comparative Example 2, where the chain length of ε-caprolactone was relatively longer than that of PEG, nanoparticles were formed by dialysis. This was not generated.

Molecular Weight of Polymer and Size of Particle Yes Supply molar ratio [M] / [I] Number average molecular weight Dispersion Particle size (nm) Theoretical value ^a measured value ^b Mw / Mn Avg. S.D. Preparation Example 1 35 8995 8037 7971 1.256 54 0.082 Preparation Example 2 50 10707 10116 10317 1.250 77 0.010 Preparation Example 3 70 12990 11734 13367 1.102 114 0.089 Preparation Example 4 100 16414 14839 14401 1.102 130 0.105 Preparation Example 5 35 9324 8588 8318 1.127 101 0.107 50 11207 10783 10200 1.152 124 0.113 70 15089 12103 13035 1.179 139 0.156 100 19413 15257 15340 1.112 165 0.201 Preparation Example 6 35 9070 8250 8265 1.213 89 0.118 50 10800 10051 10190 1.117 103 0.231 70 13100 12135 11295 1.215 118 0.189 100 16600 14056 13720 1.256 147 0.120 Comparative Example 1 70 12990 12305 13890 1.131 129 0.114 Comparative Example 2 150 22121 18085 18205 1.178 N.D.

a is the molecular weight measured by gel permeation chromatography (GPC)

b is the molecular weight measured by ¹ H nuclear magnetic resonance (NMR) spectroscopy

In addition, the results of measuring the amount of the drug introduced into the nanoparticles through the procedure of Examples 1 to 4 are shown in Table 2 below. As can be seen in Examples 1 to 4 of Table 2, the introduction efficiency of the drug increased as the molecular weight and the length of the hydrophobic block of the copolymer used increased, and as shown in Examples 5 to 7 and 4, As the molecular weight of was constant and the feed weight ratio of the drug to the copolymer was increased, the introduction efficiency of the drug increased. In addition, although not shown in Table 2, the size of the nanoparticles also increased as the introduction efficiency of the drug increased.

In addition, looking at the characteristics of the drug to be introduced, both the hydrophobic materials indomethacin and doxorubicin were able to produce a nanoparticle having an introduction efficiency of more than 40wt%. However, in Comparative Example 3, the hydrophilic material cyanocobalamine was confirmed to be hardly introduced into the nanoparticles.

The amount of drug introduced into the nanoparticles Yes Supply weight ratio Introduction efficiency (%) ^a Type and amount of copolymer: drug amount Example 1 Preparation Example 1, 1.00: 3.00 25.83 Example 2 Preparation Example 2, 1.00: 3.00 34.08 Example 3 Preparation Example 3, 1.00: 3.00 41.98 Example 4 Preparation Example 4, 1.00: 3.00 42.03 Example 5 Preparation Example 3, 1.00: 1.00 16.33 Example 6 Preparation Example 3, 1.00: 1.50 20.99 Example 7 Preparation Example 3, 1.00: 2.00 31.96 Example 8 Preparation Example 3, 1.00: 3.00 40.57 Comparative Example 3 Preparation Example 3, 1.00: 3.00 N.D.

a is a value calculated by Equation 1

On the other hand, although not shown in Table 2, the copolymer prepared according to Preparation Examples 5 and 6 also showed similar results to the introduction efficiency described in Table 2.

And the results of measuring the amount of release of the hydrophobic material introduced from the nanoparticles calculated by the introduction amount of the material by the formula 1 shown in Table 3 below.

Release Behavior over Time of Hydrophobic Materials Introduced from Nanoparticles Yes The cumulative discharge amount of the drug over time (in days) ^a 0 0.13 0.25 0.5 One 2 4 6 8 10 12 14 Example 1 0 6.88 8.38 8.96 9.72 14.02 21.51 26.74 28.65 29.65 33.76 34.89 Example 2 0 5.64 7.77 9.01 12.26 14.36 18.16 24.42 24.83 25.59 27.05 27.97 Example 3 0 6.40 8.97 9.48 10.35 11.32 16.54 19.46 21.40 23.41 26.42 26.86 Example 4 0 8.47 10.21 11.19 11.96 12.67 15.93 18.50 18.82 19.53 20.73 22.04 Example 5 0 0.86 2.12 3.56 3.63 4.01 10.38 22.98 37.162 38.04 39.62 39.94 Example 6 0 4.50 7.75 12.41 16.97 22.55 25.31 33.80 34.09 35.50 38.56 39.62 Example 7 0 7.96 9.01 10.06 11.46 13.76 18.18 19.01 23.38 26.98 28.03 28.06 Example 8 0 6.54 7.43 8.97 9.62 12.79 15.83 22.59 23.47 24.96 25.61 25.92 Comparative Example 4 0 10.81 42.24 71.29 93.05 97.00 97.00 97.00 97.00 97.00 97.00 97.00

a is the cumulative amount of drug released 〓 (accumulated amount of released drug / amount of drug introduced into nanoparticles) × 100

In Comparative Example 4 in which the drug was not introduced into the nanoparticles in Table 3, almost 100% of the drug was released within 24 hours, while the introduction efficiency of the drug was 25.85 to 42.03 as can be seen in Examples 1 to 4. As the wt% (in Table 2) was increased, the sustained release behavior was shown more slowly. In terms of the molecular weight of the copolymer used, in the case of Examples 3 and 4, the drug introduction efficiency is similar to 41.98 and 42.03 wt%, but the copolymer having a molecular weight of 166414 in Example 3 prepared from a copolymer having a molecular weight of 12990 It can be seen that the release behavior faster than Example 4 using. Furthermore, in Examples 2 and 7, although the introduction efficiency of Example 2 (34.08%) was greater than that of Example 7 (31.96%), Example 2 using a copolymer having a molecular weight of 10707 gave a copolymer having a molecular weight of 12990. It was released faster than Example 7 used. In addition, the release of drug from the prepared nanoparticles showed a sustained release behavior of more than two weeks.

Claims (7)

a) preparing a diblock copolymer of a heterocyclic ester compound of lactones and polyethylene glycol under a catalyst; b) dissolving the diblock copolymer in an organic solvent; c) adding a solution of a hydrophobic substance or drug in an organic solvent to the diblock copolymer of step b); And d) dialysis of the additive in water to form particles having hydrophobic substances or drugs introduced into the diblock copolymer. The method for producing biodegradable nanoparticles according to claim 1, wherein the heterocyclic ester compound of lactones is ε-caprolactone, D, L-lactide or glycolide. The method for producing biodegradable nanoparticles according to claim 1, wherein the reaction molar ratio of the heterocyclic heterocyclic ester compound / polyethylene glycol of the lactones is 35 to 150. The method of claim 1, wherein the organic solvent is dimethylformamide, dimethylacetamide, tetrahydrofuran or dichloromethane. The method according to claim 1, wherein the hydrophobic drug or substance is indomethacin, ketopropene, doxorubicin, cyclosporine, lidocaine, metipranolol, betasolol or betaxolol, or tetracycline ( method of producing biodegradable nanoparticles, characterized in that tetracycline). The method of claim 1, wherein the particle size is 0.2㎛ (200nm) or less. Biodegradable nanoparticles prepared by the method of any one of claims 1 to 6.