KR20180024459A - Sponge-like porous microspheres and method for preparing the same - Google Patents

Abstract

The present invention relates to a method for manufacturing fine polymer particles, and specifically, to a method for manufacturing fine polymer particles having a porous sponge-like structure while using methyl propionate, ethyl acetate or isopropyl formate as a non-halogenated solvent by using chemical inducers such as methylamine, dimethylamine or ethylamine, and to a composition for delivering medicines, which comprises the manufactured sponge-like structure fine particles.

Description

[0001] Sponge-like porous microspheres and methods for preparing same [0002]

More particularly, the present invention relates to a method for producing fine particles having a porous structure like a sponge by using a chemical inducing factor and a method for producing a physiologically active substance containing microparticles having the same structure as the sponge ≪ / RTI >

Commercially available PLGA microparticles have a long-acting depot functionality that slowly releases a variety of drugs from one week to six months. Examples of such PLGA particulate products include Bydureon®, Risperdal Consta®, Vivitrol®, Sandostatin LAR®, and Lupron Depot®. . All of these particulate products have a non-porous matrix. In general, in the case of nonporous PLGA microparticles, the degradation rate of PLGA is a crucial factor in determining drug release rate. For this reason, the drug release rate of the fine particles having a non-porous matrix is disadvantageously difficult to adjust slowly and precisely. In order to overcome such disadvantages, studies on the production of fine particles having a new shape instead of the conventional nonporous polymer fine particles have been actively conducted. Typical examples of the fine particles having such a new shape include a porous structure, an island-sea, a core-shell, an acorn, a porous layer, a porous layer- dense core, golf ball, and Janus (Y. Cai, Y. Chen, X. Hong, Z. Liu and W. Yuan, Int . J. Nanomedicine , 8 , 1111-11120; C. Takai, T. Hotta, S. Shiozaki, Y. Boonsongrit and H. Abe, Chem . Commun ., 2009, 37 , 5533-5535; D. Cheng, X. Cao, H. Gao Kim, KH Hwangbo, JH Lee and KY Cho, RSC Adv . , 2014, 4 , 46536-46540, MR.J., W. Li and Y. Wang, RSC Adv. , 2014, 4 , 9031-9038 Kim, S. Lee, JK Park and KY Cho, Chem. Commun ., 2010, 46 , 7433-7435 and C. Huang and X. Shen, Chem . Commun ., 2014, 50 , 2646-2649, etc.). Among these peculiar morphologies, porous PLGA microparticles are particularly recognized as biodegradable scaffolds, cell delivery, sorbents, nanoparticle-particulate composites, pulp drug delivery and long-term sustained depot applications in tissue repair and regeneration (W. Xu, L. Wang, Y. Ling, K. Wei and S. Zhong, RSC Adv ., 2014, 4 , 13495-13501). Porous polymer microparticles are also used as templates for making functional metal-polymer hybrid composites (HY Son, DJ Lee, JB Lee, CH Park, M. Seo, J. Jang, SJ Kim, MS Yoon and YS Nam, RSC Adv ., 2014, 4 , 55604-55609).

Important quality characteristics of the porous polymer microparticles include pore size distribution, pore position and internal connection, and bulk density. Many researchers have proposed a strategy for manufacturing porous PLGA microparticles, the most representative of which is the double emulsion using porogens, specifically W1 / O / W2 (water-in-oil-in-water) This is due to the large osmotic pressure difference between W1 and W2 of the W1 / O / W2 emulsion, the porogen present in W1 diffuses into W2 and the water of W2 penetrates into W1, It is a method using the production of polymer fine particles. A number of such processes for producing fine particles using such a double emulsion have been reported (Int J Pharm 2007; 333: 103, J Controlled Release 133 (2009) 37, Angew Chem Int Ed 51 (2012) 10800, ACS Macro Lett 1 (2012) 697 and Ch Chem Lett 24 (2013) 710).

As porogens mainly added to W1, gaseous substances such as sodium bicarbonate, calcium carbonate or ammonium bicarbonate, osmolytes such as salts or polymers to maximize osmosis between W1 and W2, A protein or a surfactant for stabilizing a very small droplet of W1 which is shaped. Porogens added to the oil phase also act to increase the porosity of fine particles as they are leached during or after the manufacturing process of the fine particles. These microparticle manufacturing processes generally use methylene chloride, a halogenated solvent, as a dispersing solvent for dissolving PLGA and require a high-shear or high-energy mixer to make a W1 / O primary emulsion. Solvent removal techniques are also based on physical approaches utilizing solvent evaporation and / or extraction principles.

However, methylene chloride as an organic solvent used as a dispersion solvent is an organic solvent which is strongly volatile and does not mix well with water and has a boiling point much lower than that of water, but is highly toxic to ICH Class 2 solvents It is a substance that is a carcinogen and has a serious problem of causing environmental toxicity. Therefore, it is recommended not to use it for the manufacture of medicines because of the problem of solvent toxicity. Currently, the drug tolerance limit for methylene chloride is 600 ppm (0.06%). Therefore, when methylene chloride is used as a solvent, it is necessary to stir the emulsion overnight to remove it from the emulsion (U.S. Patent No. 6,884,435), requiring a large-scale reactor having a large volume as a large amount of water is used, In order to shorten the manufacturing time, a troublesome process of raising the temperature of the reactor or introducing a reduced pressure condition is required (see U.S. Patent Nos. 3,691,090, 3,891,570, 6,270,700, and 6,572,894). Furthermore, there is a problem that the drug encapsulated in the fine particles is denatured as well as the complicated process steps . Further, in order to ensure porosity, a high concentration of porogen must be added to W1. The osmotic pressure generated at this time causes a problem that the drug dissolved in W1 escapes to W2, which is a trauma, And the drug encapsulation rate is lowered. Furthermore, a high-shear mixer (such as a homogenizer) or a high-energy mixer (e.g., a sonicator or a microfluidizer), which is used to produce the W1 / The scale-up of the process using a mixing apparatus such as a centrifuge has a disadvantage that it involves various difficulties, making mass production required in the pharmaceutical industry difficult. Moreover, even when porogens are used, the porosity of PLGA microparticles often shows low porosity which does not meet expectation. Sometimes the surface of the particulate has a large number of large porous pores, but the interior of the particulate still has a non-porous matrix form

On the other hand, solvents that exhibit low toxicity to humans are classified as ICH Class 3 solvents in the pharmaceutical industry, allowing them to be present in pharmaceuticals by less than 0.5% by global regulatory agencies. Ethyl acetate is an organic solvent that is currently preferred for the development of microparticle manufacturing processes because it belongs to ICH Class 3 solvent. However, when the general production method using ethyl acetate is followed, the amount of residual microparticles is often more than 2% (U.S. Patent No. 6,884,372). Therefore, various attempts have been made to achieve the residual organic solvent content (0.5%) of the particulate that meets regulatory agency acceptance criteria. For example, the residual organic solvent present in the fine particles may be reduced to a non-aqueous cleaning system using ethanol or the like (U.S. Patent No. 8,187,672). Methylpropionate, which has the same molecular weight as ethyl acetate and has a similar structure, is a safe organic solvent currently approved by the world regulatory authorities such as the US FDA and the European Medicines Agency for use as additives in human food.

In this connection, the present inventors have recently found that the use of an O / W emulsion technique based on an ammonia decomposition reaction enables a micro-particle which exhibits an appropriate drug release rate without a problem of drug encapsulation rate and drug denaturation, A very porous PLGA fine particle has been produced (Korean Patent Application No. 10-2015-0129183). This is characterized in that an ammonia solution is added to the emulsion after preparing an oil-in-water (O / W) emulsion, wherein the ammonia diffuses into the emulsion droplet to decompose the dispersed solvent to form a hydrophilic decomposition by- And has the advantage of being able to produce microparticles having porosity while being leached out as an aqueous phase. However, in this method, it is necessary to use isopropyl formate, methyl formate, ethyl formate, propyl formate, and the like as a dispersion solvent. In the pharmaceutical industry, ethyl acetate or methyl propionate Is used as a dispersion solvent, it can not be applied. In addition, when the polymer PLGA is used as a particulate forming material, it has a disadvantage that its porosity is remarkably reduced or nonporous fine particles are formed.

Under these circumstances, the inventors of the present invention have found that, among the non-halogenated solvents, methyl propionate and ethyl acetate, widely used in the pharmaceutical industry, can be used as a dispersion solvent, and even if PLGA having a high molecular weight is used, In order to develop a process for producing sponge-like and porous PLGA microparticles according to an underwater type emulsion-based method, various studies have been repeated. As a result, when methylamine, ethylamine, or dimethylamine is used as a chemical trigger capable of decomposing PLGA and a dispersion solvent into a water-soluble solvent, the preferred dispersion solvent and high molecular weight PLGA are used, It was first confirmed that PLGA fine particles having a sponge-like porosity can be produced by the method of the present invention.

Accordingly, one object of the present invention is to provide a method for producing porous polymer microparticles having a structure such as a sponge, and polymer microparticles having the same structure as the sponge produced thereby.

Furthermore, another object of the present invention is to provide a composition for delivering a physiologically active substance comprising the porous polymer microparticles as an active ingredient.

In order to achieve the above object, the present invention relates to a method for producing porous polymer microparticles having a sponge-like structure.

Specifically, the manufacturing method according to the present invention is characterized in that it has the following structure to have a sponge-like structure:

(a) dissolving the hydrophobic polymer in at least one organic solvent selected from the group consisting of methyl propionate, ethyl acetate, propyl acetate, isopropyl acetate, propyl formate and isopropyl formate as a dispersion solvent to prepare a dispersed phase;

(b) an emulsion preparation step of preparing an oil-in-water (O / W) emulsion by adding an aqueous solvent to the dispersed phase prepared in step (a); And

(c) adding at least one chemical inducer selected from the group consisting of methylamine, ethylamine and dimethylamine to the oil-in-water type emulsion prepared in the step (b) to react with the polymer in the dispersion phase and the dispersion solvent to form a water- ≪ / RTI >

Hereinafter, the present invention will be described in more detail

In the step (a) of the present invention, a hydrophobic polymer can be used. Any hydrophobic polymer that can be used as a material for forming porous polymer fine particles can be used without limitation. Specifically, poly (diene) s such as poly (butadiene) and derivatives thereof; Poly (acrylates) such as poly (acrylic acid), poly (methacrylates) such as poly (methylene methacrylate), poly (vinyl esters) such as poly (vinyl acetate) , Poly (ester), poly (ertheraimide), poly (anhydride), poly (lactone-co-glycolide) Cellulose esters such as ethyl cellulose, and cellulose esters such as cellulose acetate may be used alone or as a mixture, preferably poly (lactic acid), poly (urethane), poly (Co-glycolic acid) (PLGA) and ethyl cellulose can be used. The polymer may be selected from polymers having appropriate molecular weights depending on the physical properties of the fine particles. In the conventional Korean Patent Application No. 10-2015-0129183, the by-product of the ammonia reaction, that is, the water-soluble components, is effectively dispersed in the water phase and the polymer having a relatively low molecular weight is used in order to prevent the production of the nonporous polymer fine particles. , The decomposition reaction of the dispersion solvent can be effectively performed by the chemical inducing factor even if a polymer having a relatively high molecular weight is used and at the same time the by-product of the decomposition reaction, that is, the water-soluble components can be effectively diffused into the water phase, It is possible to manufacture porous polymer microparticles having a structure such as a sponge.

Such a polymer compound is contacted or mixed with a dispersion solvent to prepare a dispersed phase. The dispersion solvent used herein is a non-halogen organic solvent containing no halogen in the molecule, and examples thereof include methyl propionate, ethyl acetate, propyl acetate, isopropyl acetate, propyl formate, isopropyl formate and mixtures of these organic solvents Is selected from the group consisting of methyl propionate, ethyl acetate and isopropyl formate. In Korean Patent No. 910892 and US Patent No. 8,202,525, the organic solvents used for the ammonia reaction are all halogenated organic solvents, which is a solvent that is practically difficult to use in the pharmaceutical industry due to toxicity of the solvent itself In this case, only non-porous polymer fine particles are produced. In Korean Patent Application No. 10-2015-0129183, methyl propionate, which is widely used in the pharmaceutical industry although it uses a non-halogenated organic solvent, , And ethyl acetate were used, ammonia reaction did not proceed effectively. Therefore, only ethyl formate, methyl formate, ethyl formate and propyl formate had to be used. However, in the present invention, methyl propionate, ethyl acetate, propyl acetate , Isopropyl acetate, It is possible to produce microparticles having a relatively low toxicity as well as a porosity of an ultra-high strength and can be used as a non-halogenated organic solvent widely used in the pharmaceutical industry, It can be applied directly to industry.

Furthermore, the dispersed phase prepared by using the polymer compound and the dispersion solvent is mixed with an aqueous solvent in step (b) to produce a single emulsion of the water type (O / W). As the aqueous solvent used herein, an aqueous solution containing a hydrophilic emulsifier, for example, an emulsifier such as polyvinyl alcohol or Tween series, or a co-solvent thereof may be used. The concentration of the emulsifier contained in the dispersion solvent may be 0.1 to 4% (w / w), preferably 0.2 to 2.0% (w / v).

In the step (a), the ratio of the polymer compound to the dispersion solvent for producing the dispersed phase is preferably from 3 (w / v) to 30 (w / v)%, (w / v) to 20 (w / v)%. In this regard, when the concentration of the polymer exceeds 30 (w / v)%, the viscosity of the dispersed phase increases and the decomposition reaction of the dispersion solvent by the chemical inducing factor is suppressed in the subsequent step (c) Diffusion of decomposition reaction byproducts is suppressed to reduce the degree of porosity formation, and when the content is less than 3% (w / v), the mechanical strength of the fine particles is weak, resulting in poor practicality. In one specific example, 0.25 g of PLGA was dissolved in 4 ml of methyl propionate to prepare a dispersion.

Further, when the dispersed phase prepared in step (a) is mixed with an aqueous solvent in step (b), the mixing volume ratio thereof is 1: 1 to 1: 100, preferably 1: 4 to 1:10. In this regard, when the amount of the aqueous solvent is too large, solvent extraction occurs, and the porosity of the fine particles may be significantly reduced due to the suppression of the porous forming chemical reaction. Also, in step (c), a chemical inducer is added to the oil-in-water type single emulsion prepared in step (b) to perform the decomposition reaction of the dispersion solvent. In the present invention, such a chemical inducing factor is characterized by using at least one selected from the group consisting of methylamine, ethylamine and dimethylamine. Due to the use of such a chemical inducing factor, a high molecular weight polymer compound is used, Methyl propionate or ethyl acetate which can not be used in the conventional method of producing fine particles using the ammonia reaction can be used. At this time, the amount of the chemical inducing factor to be used is determined based on the volume of the dispersion solvent used in the step (a), and the molar ratio of the dispersion solvent to the chemical inducing factor is preferably 1: 0.25 to 1: 5 . In this connection, when a small amount of a chemical inducing agent is used to deviate from the above range, voids are not formed, or when an excessive amount of the chemical inducing agent outside the above range is used, a lot of voids are formed, The reaction between the drug and the chemical inducer may occur, and the mechanical strength of the fine particles may become weak.

According to the method for producing fine particles according to the present invention, unlike conventional processes for preparing porous polymer microparticles, a polymer compound is preferably used in a non-halogen organic solvent such as methyl propionate, ethyl acetate or isopropyl formate, Is added to the O / W emulsion, and the resultant dispersion solvent constituting the emulsion droplets is decomposed into a water-soluble decomposition product (For example, when methylamine is added, methyl propionate is decomposed into methanol and N-methylpropionamide). Since these water-soluble decomposition products act as an anti-solvent for the polymer compound, the polymer compound is not dissolved and thus the polymer compound-rich domain and the polymer compound-free domain are divided into the emulsion droplets, The water-soluble decomposition product acts as an anti-solvent for the polymer compound, and then spontaneously diffuses into the water phase, resulting in the formation of fine particles of a highly porous polymer compound having a sponge-like structure. As a result, the solvent extraction using a large amount of quenching liquid or the step of evaporating the solvent is not required at all, and an O / W type emulsion can be easily produced as a low energy emulsifier This eliminates the need for special machinery, high-energy or high-shear mixing equipment. In particular, in the case of biomacromolecules, due to shearing by a high-energy mixing device used for forming processes or emulsions exposed to an organic solvent in a dispersed phase or exposed to an organic solvent / water interface There is a risk of denaturation, aggregation or degradation of the biomacromolecule itself. However, the use of the present porous polymer microparticles has an advantage that bioactive molecules such as biomolecules can be safely encapsulated under 100% aqueous conditions. Further, in the case of producing the fine particles by the production method according to the present invention, unlike the conventional production method using ammonia reaction without using the porogens essential in the conventional multi-processing fine particle manufacturing technique, It has an advantage that a remarkable porous structure can be secured while using methyl propionate or ethyl acetate which is preferred.

Further, in another aspect, the present invention relates to a sponge-like fine particle prepared according to the above-described method.

Specifically, the microparticles according to the present invention have voids or voids in the form of a sponge that exhibits extreme porosity, in which many voids are distributed evenly inside and outside the microparticles, unlike conventional porous PLGA microparticles, And has a unique characteristic of being connected to form a network.

In the present invention, the polymer fine particles may have an average diameter of 20 to 1,000 μm, preferably 50 to 350 μm, but are not limited thereto and may be freely deformed according to a desired application. At this time, the average diameter of the fine particles can be determined by the viscosity of the emulsion, the concentration of the polymer compound in the dispersed phase, the molecular weight of the polymer compound, the volume ratio of the dispersed phase and the dispersed solvent,

As described above, the microparticles according to the present invention have the advantage of exhibiting excellent drug encapsulation rate because they can release drugs continuously for a long time when they are used as a drug delivery device due to the porous nature of sponge.

In another aspect, the present invention relates to a composition for delivering a physiologically active substance comprising porous polymer microparticles and a physiologically active substance having the same structure as the sponge.

Specifically, the physiologically active substance refers to any substance that promotes or suppresses the function of a living body when living organisms live, including drugs capable of exerting a desired effect in vivo. The physiologically active material may be biomacromolecules and may be, for example, but is not limited to, peptides, polypeptides, polynucleotides, proteins, nanoparticles, DNA, RNA, monoclonal antibodies or vaccines . In addition, when hydrophobic drugs are encapsulated in PLGA fine particles, nonporous fine particles are mainly used. However, the porous polymer fine particles according to the present invention provide a basis for encapsulating hydrophobic drugs into the fine particles.

Preferred examples of such physiologically active substances include growth factors, growth hormones, peptides or protein drugs, anti-inflammatory analgesics, anticancer agents, antiviral agents, sex hormones, antibiotics, antibacterial agents or low molecular compounds.

In another preferred embodiment, the composition for delivering a physiologically active substance according to the present invention may contain carriers, excipients, additives and the like commonly used in pharmaceutical compositions, in addition to the porous polymer microparticles and the physiologically active substance.

Further, in another aspect, the present invention relates to a method for producing the composition for delivering a physiologically active substance. Specifically, the method for producing the composition for delivering a physiologically active substance includes the following steps:

Preparing porous polymer microparticles having the same structure as the sponge; And

Encapsulating the microparticles with a physiologically active substance.

In a preferred embodiment, the step of enclosing the physiologically active substance may include a step of contacting the microparticles with the physiologically active substance and a step of raising the temperature. In a preferred embodiment, the temperature raising step can be performed by raising the temperature to around the glass transition temperature of the fine particles.

When the porous polymer microparticles are prepared according to the preparation method of the present invention, organic solvents preferred in the pharmaceutical industry such as non-halogenated methyl propionate and the like using chemical inducing factors such as methylamine, dimethylamine, Solubilization of hydrophobic polymers for the production of fine particles Polymeric microparticles having a structure such as sponge with extreme porosity can be produced by simple o / w emulsion-template technology platform without the aid of porogens, Compared with the conventional techniques for preparing porous polymer fine particles, non-halogenated methyl propionate, ethyl acetate, propyl acetate, propyl formate, and isopropyl formate can be used as a dispersion solvent. , The extreme porosity can be achieved It has the advantage of being capable of establishing the manufacturing process over the liver and meeting the criterion of residual particulate organic solvent required by the regulatory authorities without any special additional equipment or treatment. As a result, long-acting drug depots, sorbents, and biodegradable scaffolds.

Figure 1 is a GC chromatogram showing the concentration of water soluble methyl propionate over time in a methyl propionate-water two-layer system. No chemical inducers were added to the water. The arrow indicates the peak of methyl propionate. The peak appearing at 6 minutes represents the internal standard isopropanol.
2 is a GC chromatogram showing the concentration of water-soluble methyl propionate and methanol over time in a methyl propionate-water two-layer system. Ammonia was added to the water. Arrows 1 and 2 indicate methyl propionate and methanol peak, respectively. Only a small amount of methanol is formed and the concentration of water soluble methanol tends to increase with time.
Figure 3 shows the concentration of methyl propionate in water (arrow 1), methanol (arrow 2) and N -methyl propionamide (arrow 3) over time in the methyl propionate- GC chromatogram. Methylamine was added to the water. A very small amount of methyl propionate is observed, and the concentration of methanol and N - methyl propionamide, which are degradation products of this solvent, in water increases with time.
Figure 4 shows the time course of the concentration of (a) methyl propionate and (b) methanol in water. (□, □) indicates that when no addition was made as the water phase of the methyl propionate-water two-layer system, (Δ, ▲) indicates the case where ammonia was added, to be. The results indicate that methylamine is an excellent chemoattractant for removing methyl propionate dissolved in aqueous phase.
Figure 5 shows the change in water concentration of N -methyl propionamide observed in a methyl propionate-water two-layer system. After methylamine was added to the aqueous phase, the concentration of N -methylpropionamide in water was analyzed as a function of time.
Fig. 6 is a view of the means-III containing water-in-oil emulsion as a function of time. (a) shows the case where nothing is added to the emulsion, (b) when ammonia is added, and (c) methylamine is added. The color of the methylamine-treated water-in-oil emulsion was clear, demonstrating that methylamine can completely remove methyl propionate.
7 is a photograph showing a dynamic change in color of Sudan III-containing water-in-oil type emulsion treated with methylamine. The molar ratio of methyl propionate to methylamine was set at 1: 0.5.
8 is a view showing the state of various oil-in-water type emulsions stirred for 1 hour. (a) is an untreated group, (b) is ammonia, and (c) to (f) are groups in which methylamine is treated with an emulsion. Methyl propionate-methylamine molar ratio was set to 1: 0.75, (d) 1: 1, (e) 1: 1.25, and (f) 1: 1.5.
9 is a SEM micrograph showing a typical sponge-like internal structure of PLGA microparticles prepared by a solvent removal method using methylamine.
10 shows a mechanism of forming fine particles having a structure such as non-porous polymer fine particles and sponge. In a typical evaporation / extraction process, as in (a), the slow diffusion of dispersed solvent molecules outside the emulsion droplets causes their shrinkage into the interior, resulting in a non-process matrix. (b) Methylamine diffuses into the emulsion in order to react with methyl propionate. As a result, it acts as an anti-solvent for PLGA to decompose methyl propionate with water-soluble methanol and N -methyl propionamide. This chemical reaction leads to the conversion of the emulsion droplets into fine particles and at the same time induces the porosity of the fine particles.
Fig. 11 is a SEM micrograph of (a) exterior and (b) and (c) internal shapes of porous PLGA microparticles having different skeletons such as sponges. The molar ratio of methyl propionate to methylamine used for the preparation of the fine particles is from 1: 0.5 to 1: 1.25. The particles were processed in two ways to identify their internal form. (a) The surface of the fine particles was peeled off using a tape, and (b) the epoxy resin was impregnated before the crossing.
12 is a graph showing the change in the activity of PLGA microparticles prepared using different methyl propionate-methylamine molar ratios (a) 1: 0.5, (b) 1: 0.75, (c) 1: The result of thermogravimetric analysis is shown.
Fig. 13 is a photograph of a microparticle according to the present invention having a sponge-like structure prepared by using ethyl cellulose (4cp) as a polymeric compound and ethylamine as a chemical inducing factor, and observing its inner and outer shapes with an electron microscope.
Fig. 14 is a photograph of a microparticle according to the present invention having a sponge-like structure prepared by using ethyl cellulose (10 cP) as a polymeric compound and ethylamine as a chemical inducing factor, and observing the inside and outside shapes thereof with an electron microscope.
15 shows the gas chromatogram of the aqueous samples taken at various conditions of the ethyl acetate-water two-layer system. (a) is a control test, that is, when no chemical inducer is added to the water phase. (b) when ammonia is used as a chemical inducer. (c) when methylamine is used. 1 and 2 indicate ethyl acetate and ethanol peaks, respectively.
Fig. 16 is a photograph of Sulu-III containing water-in-oil type emulsion as a function of time. (a) shows the case where nothing is added to the emulsion, (b) when ammonia is added, and (c) methylamine is added. The methylamine-treated oil-in-water type emulsion is clear, indicating that methylamine can completely remove ethyl acetate.
Fig. 17 is a photograph of Sulu-III containing water-in-oil type emulsion as a function of time. (a) shows the case where nothing is added to the emulsion, (b) ammonia is added, and (c) methylamine, (d) dimethylamine and (e) ethylamine are added. Thus, it is confirmed that amines such as methylamine, dimethylamine and ethylamine are chemically induced factors that can be effectively degraded even when isopropyl formate is used as a solvent.

Hereinafter, the present invention will be described in more detail by way of examples. However, these examples are for illustrating the present invention, and the present invention is not limited by these examples.

Example  1: Materials used

Lactide: glycolide ratio of 75:25 (inherent viscosity = 0.71 dL / g in CHCl ₃ ) Poly (lactide-co-glycolide) was purchased from Evonik Corporation (Theodore, AL, USA). This polymer is abbreviated PLGA unless otherwise stated. Methyl propionate and ethyl acetate were supplied from Alfa Aesar (Ward Hill, MA, USA) and JT Baker (Phillipsburg, PA, USA). A 28% ammonia solution was purchased from Merck (Darmstadt, Germany), 40% methylamine solution and Sudan III from Sigma-Aldrich (St. Louis, Mo., USA).

N -propionamide was purchased from Tokyo Chemical Industry Co., Ltd. (PVA; 88% hydrolyzed, Mw = 25,000 g / mol) were purchased from Polysciences Inc. (Tokyo, Japan), and methanol was purchased from Burdick & Jackson (Muskegon, MI, USA). (Warrington, PA, USA).

Example  2: Methyl propionate - the concentration of aqueous solvent in the two-layer system of water monitoring

30 ml of 0.5% aqueous PVA solution was placed in a 50 ml beaker and 3.2 ml of 40% methylamine solution was added thereto. 4 ml of methyl propionate was carefully added and stirred slowly at 110 rpm to avoid mixing of the two phases. 0.2 ml of aqueous solution was taken from the water layer at predetermined time intervals and 200 mg of MgSO ₄ and 10 ml of butanol were vortexed. Then 0.2 ml of internal standard solution was added to 0.4 ml of filtrate obtained by using a 0.45 mm syringe filter to prepare a GC injection sample solution. The internal standard solution used above was prepared by dissolving 100 ml of isopropanol in 1.9 ml of butanol and diluting it 10 times with butanol. The above experiment was conducted by adding 2.5 ml of 28% ammonia solution instead of methylamine. In addition, a control experiment in which no methylamine or ammonia was added was also conducted.

Aliquots of each sample were injected into a model GC-2010 gas chromatograph (Shimadzu Corporation, Kyoto, Japan) equipped with a flame ionization detector. Zebron ZB-624 column (30 m x 0.32 mm x 1.80 mm; Phenomenex, Torrance, CA, USA) was used as a stationary phase. The flow rate of the nitrogen gas into the column was set at 1.1 mL / min. The injector and detector temperatures were set at 200 ° C and 220 ° C, respectively. The oven temperature was maintained at 80 ° C for 6.3 minutes and increased to 180 ° C at a rate of 200 ° C / min. Standard solutions of known concentrations of methanol, methyl propionate and N - methyl propionamide were prepared using butanol and the isopropanol internal standard solution was added. Their standard calibration curves were constructed to determine unknown sample concentrations.

Thus, when 4 ml of PLGA-free methyl propionate was placed on 30 ml of a 0.5% aqueous PVA solution to form two kinds of phases, and the water-soluble methyl propionate concentration was observed at each time point using GC, The concentration of water soluble methyl propionate gradually increased with time as shown in the GC chromatogram shown in FIG. This is caused by the gradual diffusion of methyl propionate from the organic solvent layer into the water phase. Similar results were observed when 1: 1 equivalent of ammonia was added to the aqueous phase in comparison to methyl propionate under the same conditions (FIG. 2). However, the concentration of water - soluble methyl propionate was remarkably decreased when 10 minutes elapsed after adding 1: 1 equivalent of methylamine to methyl propionate in the aqueous phase. Even after more time, the water-soluble methyl propionate concentration was still observed to be very low. Instead, the concentration of N - methylpropionamide and methanol in water produced by the reaction of methylamine and methyl propionate with each other increased with time (FIG. 3). This suggests that the methylamine added to the aqueous phase migrates to the organic solvent layer and reacts with methyl propionate, and the resulting reaction decomposition products, N - methylpropionamide and methanol, are diffused into the aqueous phase.

Based on the results obtained by the above GC experiment, the concentrations of methyl propionate and methanol in the water phase observed under various conditions were determined, and as a result, nothing was added on the methyl propionate-water two-layer As the time elapsed, the water soluble methyl propionate concentration gradually increased and saturation reached. When ammonia was added to the water phase, the concentration of water soluble methyl propionate tended to decrease slightly. Very different from these results, when methylamine was added, the concentration of water-soluble methylpropionate was very small. The results of measuring the concentration of methanol in the aqueous phase under the above three conditions are shown in FIG. 4B. Ammonia added to the aqueous phase of the methyl propionate-water bilayer did not effectively remove the methyl propionate present in the organic solvent layer, but methylamine decomposes very efficiently methyl propionate to produce methanol have. When the water-soluble N -methylpropionamide concentration was measured over time under the same test conditions, the same results as those shown in Fig. 4b were observed (Fig. 5). All of these results indicate that ammonia does not effectively remove methyl propionate, whereas methylamine is a highly effective chemoattractant to remove methyl propionate.

Example  3: Solvent with different chemical inducers ( Methyl propionate ) Comparison of removal processes

GC experiments according to Example 2 confirmed that methylamine decomposes methyl propionate into water-soluble solvents. The following experiment was designed to investigate whether solvent removal can be achieved in the microparticle manufacturing process using this reaction.

Specifically, the water-soluble red dye, Sudan III, was dissolved in methyl propionate to a concentration of 0.25 mg / ml. 4 ml of this dye solution was added to 30 ml of 0.5% aqueous PVA solution and stirred at 300 rpm. Since the Sudan III was dissolved in the oil phase, the oil type emulsion showed red color. After 3 minutes, ammonia or methylamine was added to the oil. The chemical inducer (ammonia or ethylamine) was added in a 1: 0.5 or 1: 1 ratio to the organic solvent. As a control, no emulsion was added. The state of each emulsion was monitored as a function of time.

Sudan III, a red hydrophobic dye, was dissolved in methyl propionate and stirred in water to form an o / w emulsion, which turned red. These results were due to the fact that the hydrophobic dye, Sudan III, was dissolved in the oil phase. As long as the organic solvent constituting the oil phase was not removed, the emulsion was considered to show red color continuously. Indeed, a red o / w emulsion was consistently observed in the control test conditions, which allowed the emulsion to continue to agitate without any chemical inducers (FIG. 6). A similar result was observed when the molar ratio of methyl propionate to ammonia was 1: 1. However, when methylamine was added, the color of the emulsion was surprisingly changed to clear color after 5 minutes. As methyl propionate is decomposed and removed by methylamine, Sudan III is precipitated and adsorbed on the beaker walls, and some of them are dispersed in water as very small particles, resulting in the disappearance of red color and a clear color. If this is true, the rate of change in the emulsion color will be influenced by the methylamine capacity added to the o / w emulsion. When 0.5 equivalents of methylamine was added to the methyl propionate actually used, the color change rate of the emulsion was observed to be lower than expected when one equivalent was used (Fig. 7).

Example  4: On optical microscope  Various emulsions by Droplet / Observation of early particulate matter

It is expected that methylamine can be used as a chemical inducer to remove methyl propionate through GC and Sudan III dye experiments and that PLGA microspheres can be effectively produced using this technique. Thus, the o / w emulsion prepared by emulsifying the dispersed phase of PLGA in methyl propionate into water phase was treated under various conditions and the status of emulsion was monitored by optical microscope.

Specifically, PLGA (0.25 g) was dissolved in 4 ml of methyl propionate and added to 0.5% PVA aqueous solution with stirring at 450 rpm. Ammonia or methylamine corresponding to 1: 1 equivalent to the organic solvent used after 3 minutes of stirring was added to the oil type emulsion. In another control test, stirring was continued without any chemical inducer. After 1 hour of stirring, the physical state of the emulsion was observed using a model S16C optical microscope (MiCro Scopes, Inc., St. Louis, Mo., USA) and the results are shown in FIG.

In the case of FIG. 8A, o / w emulsion was prepared as described in the experimental conditions, and a portion of the emulsion was sampled after 1 hour without adding any chemical inducer. When the emulsion droplets stopped stirring, they were immediately fused to polymeric films / aggregates. A similar phenomenon was observed after 1 hour of adding 1: 1 equivalent of ammonia to methyl propionate after forming an o / w emulsion (Fig. 8B). These results were due to the fact that methyl propionate was not effectively removed from the droplets. In complete contrast with these results, emulsion droplets showed an opaque / dark image when methyl ophthalate of greater than 0.75 equivalent to the methyl propionate used for emulsion preparation was added to the o / w emulsion (Fig. 8 (c) f)). These results show that when ammonia is used, emulsion droplets are not cured well as fine particles (FIG. 8). When methylamine is used, methylpropionate is easily decomposed and methylpropionate in an oil-in-water emulsion droplet is effectively decomposed And the emulsion droplets were hardened with PLGA fine particles.

Example  5: Sponge and  same PLGA  Manufacture of fine particles

0.25 g of the dispersed phase in which PLGA was dissolved in 4 ml of methyl propionate was emulsified in 30 ml of 0.5% aqueous PVA solution. At this time, the water phase was stirred at 450 rpm using a digital magnetic plate for emulsification. After 3 minutes, an amount of methylamine equivalent to 0.5 to 1.5 equivalents of methyl propionate equivalent to the water-in-oil emulsion was added. After stirring for 1 hour, the formed microsphere suspension was put into a dialysis bag and dialyzed with 1 L of water for 8 hours. The fine particles obtained through filtration were dried at room temperature overnight.

Example  6: Observation of particle shape

The external and internal shapes of the Spongy microspheres were observed using SEM. Two methods were used to view the interior of the particles. In order to observe the internal shape biased toward the surface of the fine particles, the fine particles were dispersed in a double-sided adhesive tape adhered to a metal rod, adsorbed thereon, pressed with a new adhesive tape, and then peeled off. At this time, the surface portions of the spongy microspheres were peeled off and the inside was exposed. In order to view the deep inside of Spongy microspheres, the particle sample was first dispersed in epoxy resin and cured, and then cut with a nipper. All processed microparticle samples were sputter-coated with platinum and observed with SEM (model JSM-5200; Jeol Inc., MA, USA).

9 is an SEM photograph showing the inside of typical microparticles prepared according to the microparticle manufacturing process developed in this study. As can be seen from these photographs, it is proved that sponge PLGA microspheres having an extremely strong polarity can be effectively produced by using the manufacturing method according to the present invention. What is very surprising is that the porosity of the microparticles is very good, despite the use of a simple o / w emulsion based manufacturing process without the w / o / w emulsion microencapsulation process, which is supported by porogen. FIG. 10 shows a mechanism of manufacturing a porous PLGA microspheres having a sponge-like structure based on a new solvent removal technique according to the present invention. A methyl propionate solution in which PLGA is dissolved is emulsified in water to prepare an o / w emulsion. After that, the methylamine added to the water phase diffuses into the dispersed phase of the emulsion and reacts with methyl propionate to form methanol and N - methyl propionamide. They act as anti-solvents for PLGA as a hydrophilic solvent, and the inside of the emulsion is divided into numerous small PLGA-rich domains and PLGA-free anti-solvent domains. As the methyl propionate is gradually depleted, a skeletal structure such as a sponge of a particulate is formed due to PLGA precipitation. As the anti-solvents are diffused into the water phase, a lot of voids are left in the fine particles. In contrast, most solvent removal techniques that induce diffusion and evaporation of organic solvents in emulsion droplets form microparticles with non-porous matrices. The reason for this can be inferred from the fact that when the solvent diffusion from the emulsion droplet to the traumatic phase occurs slowly, the emulsion droplet is continuously contracted until the PLGA precipitation occurs.

The effect of methylamine added to the o / w emulsion on the shape of the sponge microparticles was determined. SEM micrographs of PLGA microparticles prepared using 0.5, 0.75, 1, and 1.25 equivalents of methylamine relative to the methyl propionate used were observed (Figure 11). When the surface of the fine particles was peeled off using a double adhesive tape, the properties of the fine particles observed were all extremely porous in sponge form. However, when the nipper was used to observe the deep interior of the microparticles, it was found that the amount of methylamine used was influenced by the amount of microparticles in the microparticles. That is, in the case of 1: 0.5 equivalent ratio, the inside of the deep layer of the fine particles maintained the matrix shape, and the sponge-like porosity was shown in the outwardly biased region. Even at a 1: 0.75 equivalent ratio, some of the deep layers showed a matrix form, but the domain was much smaller than in the case of 1: 0.5. At a 1: 1 equivalence ratio, no significant amount of matrix domains were observed within the microparticles and the pores were homogeneously distributed in all the microparticles.

Example  7: Thermogravimetry ( TGA )

Thermal mass spectrometry (TGA) was performed to obtain information on the residual organic solvent after the preparation of the fine particles. Specifically, a thermogravimetric analyzer (model Q50; TA Instruments, DE, USA) was used to investigate the thermal behavior of the particulate samples. Microparticle samples (5.078 to 9.483 mg) were platinum pan. After equilibrium at 30 ° C for 5 minutes, the temperature was increased to 550 ° C at a rate of 20 ° C / min. The change of sample mass with temperature was automatically measured by TA Universal Analysis 2000 software program.

Considering that the boiling point of methyl propionate is 79 ° C, the mass change observed from 30 ° C to 150 ° C was measured using TGA and the results are shown in FIG. As can be seen in FIG. 10, the observed mass reduction for PLGA fine particles prepared using 1: 0.5 and 1: 0.75 equivalent reaction conditions was 1.32 to 1.38%. At an equivalence ratio of 1: 1 or more, the amount of residual organic solvent was observed to be less than 0.40%. From these results it can be seen that the solvent removal technique used in the process according to the invention is effective and at the same time shows a very low residual solvent level of the microparticles.

It is necessary to consider why the porous polymer microparticles having a structure similar to the sponge according to the present invention contain a relatively low residual organic solvent in spite of not using the special residual organic solvent removal process. When PLGA microparticles were prepared by using ammonium chloroacetate or ethyl fluoroacetate as an organic solvent and ammonia-based microparticle production process, the microparticles showed a non-porous shape and residual organic solvent was 2.67% (References, and HY Im, H Sah, IJP). In this case, since the melting point and aqueous solubility of chloroacetamide, which is a decomposition product produced by the ammonia reaction, are 120 ° C and 9%, respectively, so that they are not effectively leached into the water phase and the nonporous matrices of the fine particles inhibit their leaching will be. On the other hand, the melting point and the solubility in water of N-methylpropionamide produced by the reaction of methylamine and methyl propionate are -3 ° C and 19%, respectively, and thus tend to leach / diffuse farther into the water phase . In addition, the microparticles according to the present invention are non-porous, and have a structure such as a sponge having a very dense matrix and a very high porosity, thus making it possible to easily remove residual organic solvent. Therefore, much smaller amount of residual solvent will be present in the microparticles prepared through this study than the microparticles formed through the ammonolysis-based microencapsulation previously reported.

Example  8: Ethyl cellulose  And Ethylamine  Used Sponge  Production of the same fine particles (4 cP)

A dispersed phase of 0.25 g of ethyl cellulose (viscosity of 4 cP; 48% ethoxyl when dissolved in toluene / ethanol 80:20 mixture at 5% concentration) in 4 ml of methyl propionate was dissolved in 30 ml of 0.5% aqueous PVA solution And emulsified. At this time, the water phase was stirred at 450 rpm using a digital magnetic plate for emulsification. After 3 minutes, ethylamine was added to the o / w emulsion. At this time, the molar ratio of methyl propionate to ethyl amine was 1: 1. After stirring for 1 hour, the microsphere suspension was placed in a dialysis bag and dialyzed for 8 hours using 1 liter of water. The fine particles obtained through filtration were dried overnight at room temperature. The external and internal shapes of the particles after drying were observed using an electron microscope. As can be seen from FIG. 13, it is proved that when ethylamine is used as a chemical inducer for methylpropionate decomposition, fine particles having sponge-like porosity can be produced.

Example  9: Ethyl cellulose  And Ethylamine  Used Sponge  Production of the same fine particles (10 cP)

To a solution of 0.25 g of ethyl cellulose (viscosity of 10 cP; 48% ethoxyl when dissolved in toluene / ethanol 80:20 mixture at 5% concentration) in 4 ml of methyl propionate was added 30 ml of 0.5% aqueous PVA solution And emulsified. At this time, the water phase was stirred at 450 rpm using a digital magnetic plate for emulsification. After 3 minutes, ethylamine was added to the o / w emulsion. At this time, the molar ratio of methyl propionate to ethyl amine was 1: 1. After stirring for 1 hour, the microsphere suspension was placed in a dialysis bag and dialyzed for 8 hours using 1 liter of water. The fine particles obtained through filtration were dried overnight at room temperature. The external and internal shapes of the particles after drying were observed using an electron microscope and the results are shown in Fig. As can be seen from FIG. 14, when ethylamine was used as a chemical inducer for the decomposition of methyl propionate, fine particles having a sponge-like porous structure were prepared. Ethylamines were obtained in the same manner as in Example 8 except that ethyl Even if cellulose is used, the porous polymer microparticles can be prepared by using the chemical induction factors of amines by the production method according to the present invention.

Example  10: Ethyl acetate - water The concentration of aqueous solvent in the two-layer system monitoring

30 ml of 0.5% aqueous PVA solution was added to a 50 ml beaker and 3.2 ml of the 40% methylamine solution was added. 4 ml of ethyl acetate was carefully added and the mixture was slowly stirred at 110 rpm so that the two phases did not mix. After stirring for 50 minutes, 0.2 ml of aqueous solution was taken from the aqueous layer, and 200 mg of MgSO ₄ and 10 ml of butanol were vortexed. Then 0.2 ml of internal standard solution was added to 0.4 ml of filtrate obtained by using a 0.45 mm syringe filter to prepare a GC injection sample solution. The internal standard solution used above was prepared by dissolving 100 ml of isopropanol in 1.9 ml of butanol and diluting it 10 times with butanol. The above experiment was conducted by adding 2.5 ml of 28% ammonia solution instead of methylamine. A control experiment was also carried out in which no methylamine or ammonia was added at all. The samples were analyzed by gas chromatography.

Figure 15 shows that a significant amount of ethyl acetate is dissolved in water when no chemical inducer is added to the water phase. Even when ammonia was added, a high concentration of ethyl acetate was observed in the water phase and a very small amount of ethanol peak was observed. These results show that ethyl acetate is not effectively removed by ammonia. However, when methylamine was used, ethyl acetate was hardly observed in the aqueous phase, but a high concentration of ethanol was observed. In other words, the efficient reaction between methylamine and ethylacetate shows that the ethanol degradation products are well formed, and thus ethyl acetate can be easily removed.

Example  11: Comparison of Solvent (Ethyl Acetate) Removal Process with Different Chemical Inducers

Sudan III, a hydrophobic dye, was dissolved in ethyl acetate to a concentration of 0.25 mg / ml. 4 ml was taken and placed in 30 ml of 0.5% aqueous PVA solution stirred at 300 rpm. The o / w emulsion formed at this time showed red color because Sudan III was dissolved in the oil phase. After 3 minutes, 1 molar equivalent of ammonia or methylamine was added to ethyl acetate, and the state of the emulsion over time was observed. As a control test, no chemical inducer was used.

In Figure 16, a red o / w emulsion was continuously observed in the control test conditions in which no chemical inducer was added and the emulsion was only agitated continuously. Similar results were observed when ammonia was added, and a red emulsion was observed. However, when methylamine was added, emulsion became clear color after 5 minutes. This is because ethyl acetate is decomposed and removed by methylamine, Sudan III is precipitated and adsorbed on the beaker walls, and a part of the Sudan III is dispersed in the water phase as a small particle, so that the red color disappears and the color becomes clear. That is, methylamine has proven to be very useful as a chemical inducer to remove ethyl acetate.

Example  12: Solvents with different chemical inducers (isopropyl Formate ) Comparison of removal processes

Sudan III, a hydrophobic dye, was dissolved in isopropyl formate to a concentration of 0.25 mg / ml. 4 ml was taken and placed in 30 ml of 0.5% aqueous PVA solution stirred at 300 rpm. The o / w emulsion formed at this time showed red color because Sudan III was dissolved in the oil phase. After 3 minutes, 1 molar equivalent of ammonia, methylamine, dimethylamine or ethylamine was added to the isopropyl formate, and the state of the emulsion over time was observed. No chemical inducer was used as a control test.

As can be seen in FIG. 17, dimethylamine and ethylamine, as well as methylamine, also play a role as good chemical inducers. That is, it has an ability to remove organic solvents in the emulsion droplets, and it is suggested that microparticle hardening and porous polymer microparticles can be produced by using the same.

As described above, according to the production method of the present invention, it is possible to provide a method for producing a porous polymer fine particle which is completely different from conventional porous polymer microparticle production methods and which is difficult to use in the ammonia reaction, , Ethyl acetate, and the like as a dispersing solvent, sponge-like PLGA microparticles could be produced first as a very simple underwater type emulsion technique. Nevertheless, unlike conventional porous PLGA microparticles, PLGA microparticles having a sponge-like structure exhibiting extreme porosity distributed evenly inside and outside many void microparticles can be produced.

Claims (13)

(a) dissolving a hydrophobic polymer in at least one organic solvent selected from the group consisting of methyl propionate, ethyl acetate, propyl acetate, isopropyl acetate, propyl formate and isopropyl formate as a dispersion solvent to prepare a dispersed phase ;
(b) an emulsion preparation step of preparing an oil-in-water (O / W) emulsion by adding an aqueous solvent to the dispersed phase prepared in step (a); And
(c) adding at least one chemical inducer selected from the group consisting of methylamine, ethylamine and dimethylamine to the oil-in-water type emulsion prepared in the step (b) to react with the polymer in the dispersion phase and the dispersion solvent to form a water- , ≪ / RTI >
Wherein the sponge-like porous polymer microparticles are dispersed in water. The method according to claim 1,
Wherein the concentration of the polymer compound in the dispersed phase prepared in the step (a) is 3 to 30 (w / v)%. The method according to claim 1,
Wherein the volume ratio of the dispersed phase: dispersed solvent in the step (b) is 1: 1 to 1: 100. The method according to claim 1,
Wherein the aqueous solvent used in step (b) is an aqueous solution containing polyvinyl alcohol. The method according to claim 1,
Wherein the molar ratio of the dispersion solvent to the chemical inducing factor is 1: 0.25 to 1: 5. The method according to claim 1,
The hydrophobic polymer may be selected from the group consisting of a poly (diene), a poly (alkene), a poly (acrylic), a poly (methacryl), a poly (vinyl ester), a poly (styrene) (Amides), cellulose ethers, and cellulose esters from the group consisting of poly (anhydrides), poly (orthoesters), poly (erthera mides), poly Wherein at least one selected from the group consisting of the porous polymer fine particles and the porous polymer fine particles is selected. The process for producing porous polymer microparticles according to claim 1, wherein the hydrophobic polymer is at least one selected from the group consisting of PLGA and ethyl cellulose.  8. Sponge-like porous polymeric microparticles prepared by the method of any one of claims 1 to 7, characterized in that they have voids and the openings have a sponge shape of an open structure interconnected in the microparticles. The sponge-like porous polymer microparticle according to claim 8, wherein the fine particles have a diameter of 20 to 1000 μm. A method for producing a physiologically active substance, which comprises the steps of: sponge-like porous polymer microparticles and a physiologically active substance produced by the method of any one of claims 1 to 7, wherein the physiologically active substance is encapsulated in the polymer microparticles Composition. 11. The method of claim 10,
Wherein the physiologically active substance is a biomacromolecule or a hydrophobic drug. 12. The method of claim 11,
Wherein the biomacromolecule is a peptide, a polypeptide, a polynucleotide, a protein, a DNA, an RNA, a nanoparticle, a monoclonal antibody or a nucleic acid. 8. A method for manufacturing a porous polymer fine particle, comprising: preparing sponge-like porous polymer microparticles by the method according to any one of claims 1 to 7; And injecting the physiologically active substance into the microparticles.

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