KR100741867B1 - Preparation method of double-layered sphere using oil-in-water and solvent evaporation - Google Patents

Abstract

The present invention relates to a method for preparing double-layered microspheres using oil-in-water and solvent evaporation. More specifically, the present invention relates to an oil-in-water process using a dispersion phase of a biodegradable polymer dissolved in an organic solvent having a low boiling point and a dispersion medium of a water-soluble polymer. Next, this is a series of processes in which the low boiling point organic solvent is evaporated and precipitated by prolonged physical stirring to form microspheres having a dual structure of a hydrophilic water-soluble polymer layer inside and a hydrophobic biodegradable polymer layer outside. In addition, the present invention relates to a method for preparing double layered microspheres by using oil-in-water and solvent evaporation, which is easier to maintain than the prior art and maintains a double layer formed by preventing the collapse of the outside and the inside, and the process is simplified to improve economic efficiency.

Biodegradable polymer, water soluble polymer, oil in water, solvent evaporation, bilayer microspheres

Description

Preparation method of double-layered sphere using oil-in-water and solvent evaporation

Figure 1 shows an electron scanning micrograph of the bilayer microspheres prepared in Example 1 according to the present invention.

Figure 2 shows a fluorescence micrograph of the bilayer microspheres prepared in Example 1 according to the present invention.

Figure 3 shows a fluorescence micrograph of the bilayer microspheres prepared in Example 2 according to the present invention.

Figure 4 shows a transmission electron micrograph of the microspheres prepared in Comparative Example 1.

Figure 5 shows an electron scanning micrograph of the bilayer microspheres prepared in Example 1 according to the present invention.

The present invention relates to a method for preparing double-layered microspheres using oil-in-water and solvent evaporation. More specifically, the present invention relates to an oil-in-water process using a dispersion phase of a biodegradable polymer dissolved in an organic solvent having a low boiling point and a dispersion medium of a water-soluble polymer. Next, this is a series of processes in which the low boiling point organic solvent is evaporated and precipitated by prolonged physical stirring to form microspheres having a double structure of a hydrophilic water-soluble polymer layer inside and a hydrophobic biodegradable polymer layer outside. In addition, the present invention relates to a method for preparing double layered microspheres by using oil-in-water and solvent evaporation, which is easier to maintain than the prior art and maintains a double layer formed by preventing the collapse of the outside and the inside, and the process is simplified to improve economic efficiency.

The drug delivery system aims to optimize the drug in the treatment of disease by effectively delivering the drug to the disease site and maximizing the effect. To this end, current research is actively underway to develop more stable and effective drugs.

Dual drug release control has evolved with the advent of enteric coating, a new area of drug delivery systems.

To date, as a method for controlling the release of drugs, solid dispersions which disperse poorly soluble drugs in a polymer to a nano size to increase surface area, increase solubility and bioavailability of drugs [SC Shin, IJ Oh, and IB Koh, J. Kor, Pharm. Sci. , 20 , 193 (1990)], a clathrate, which is a solid complex surrounded by other molecules in a basket-like and lattice or layered lattice [A. Mallick, B. Haldar, and N. Chattopadhyay, J. Photochem. Photobiol. B , 78 215 (2005)], and water soluble salts [YB Jun, SI Kim, and YI Kim, J. Kor. Pharm. Sci ., 20 , 99 (1990)] are being actively researched. Such methods have the advantage of increasing solubility of poorly soluble drugs to improve solubilization rate, local directivity and release control.

Another method for controlling the release of drugs is to form bilayer microspheres.

The formation of bilayer microspheres is a method of dissolving two different polymers in a solvent, mixing them, and then using a phase separation phenomenon. The multi-emulsion double emulsion solvent evaporation method [KD Hong, YS Ahn, JT Ko, SH Yuk. HS Shin, JM Rhee, G. Khang, and HB Lee, Korea Polymer J. , (submitted)], and oil-in-oil (O / O) [JC Cho, D. Yun, G. Khang, JM Rhee, YS Kim, JS Lee and HB Lee, Biomaterials Research ., 4 , 136 (2000)] and water-in-oil (W / O) [R. Liu, G. Ma, Fan-Tao Meng, Zhi-Guo Su, J. Control. Rel., 103 , 31 (2005)].

In addition, as a method of forming the inside first, and then coating the outside using different polymers, a protein coating method using a coaxial ultrasonic atomizer [ultrasonic dismembrator model 500, Fisher Scientific, USA] have.

The bilayered microspheres formed as above have less initial release than single-layer microspheres, and have excellent advantages such as controlled release control of the drug and imparting local directivity. It is required, in particular, has the disadvantage that the size is difficult to control when preparing the nanoparticles.

Accordingly, the present inventors have tried to solve the phenomenon of collapse in a short time due to the complexity of the conventional manufacturing process of the double-layered microspheres, the size control of the prepared microsphere particles, the increase of the interfacial area between the liquid phase. As a result, after performing oil-in-water using the dispersed phase of the biodegradable polymer dissolved in the organic solvent having a low boiling point and the dispersion medium of the water-soluble polymer, it is physically stirred for a long time and the organic solvent having the low boiling point Is a series of processes for depositing particles by evaporation, which not only enables the formation of microspheres having a dual structure of a hydrophilic water-soluble polymer layer inside and a hydrophobic biodegradable polymer layer on the outside, but also decays over time. The present invention is completed by knowing that the economic efficiency is improved by a significantly simplified process compared with the related art.

Accordingly, an object of the present invention is to provide a method for producing a stable bilayer microspheres that do not collapse over time by a simple process such as oil-in-water and solvent evaporation.

The present invention comprises a step of preparing a dispersed phase by dissolving 0.005 to 0.3 parts by weight of the biodegradable polymer in 1 part by weight of an organic solvent having a boiling point of 35 to 45 ℃;

Preparing a dispersion medium by dissolving 0.01 to 0.06 parts by weight of surfactant and 0.01 to 0.05 parts by weight of water-soluble polymer in 1 part by weight of water; And

A double worm formed by dropping the dispersion phase prepared in step 1 drop by drop to the dispersion medium prepared in step 2, and then performing physical stirring for 7 to 15 hours at a speed of 300 ~ 1500 rpm to produce microspheres The manufacturing method of microspheres has the characteristics.

In addition, the present invention is a step of preparing a dispersed phase by dissolving 0.005 to 0.3 parts by weight of the biodegradable polymer in 1 part by weight of the organic solvent;

Preparing a dispersion medium by dissolving 0.01 to 0.06 parts by weight of surfactant and 0.01 to 0.05 parts by weight of water-soluble polymer in 1 part by weight of water; And

Method for producing a double-charged microspheres comprising a three-step step of producing microspheres by mixing the one-phase dispersion phase and the two-phase dispersion medium to perform ultra-high frequency grinding, then performing physical stirring for 7 to 15 hours at a speed of 300 to 1500 rpm. There is another feature.

Hereinafter, the present invention will be described in detail.

The present invention relates to a method for producing microspheres having a double-layer structure using oil-in-water and solvent evaporation processes, and has been described in the prior art, multi-oil (water-in-oil-in-water, W / O / W) and solvent evaporation [KD Hong, YS Ahn, JT Ko, SH Yuk. HS Shin, JM Rhee, G. Khang, and HB Lee, Korea Polymer J. , (submitted).

That is, multiple oils commonly used in the preparation of the conventional double-layer fine particles using oil-in-water using a dispersed phase of a biodegradable polymer dissolved in an organic solvent having a low boiling point and a dispersion medium of a water-soluble polymer. -in-oil-in-water (SIN), which is a simplified process, which shows the difference in tension between the aqueous solution in which the dispersion medium is dissolved and the organic solvent in which the dispersion phase is dissolved, and the phase separation effect between the biodegradable polymer and the polysaccharide used in the dispersion medium and the dispersion phase, respectively. After performing the above oil-in-water drop, the organic solvent having a low boiling point is evaporated by a long time mixing process using a physical stirrer to make the hydrophilic water-soluble polymer layer inside and the hydrophobic biodegradable polymer layer outside. Microspheres having a double structure of are formed.

This solvent evaporation method has a characteristic that the procedure is simplified as compared with the conventional one, which has a simple and easy production effect. In addition, when the double-layer microspheres are manufactured by the conventional multi-drop method, the free energy of the entire system is increased due to the increase of the interfacial area between the liquid phases, which causes the O / W emulsion to break down quickly. Yoo, and SY Lim, Korea Polymer J. , 21 , 490 (1997)], while using the above-mentioned underwater reclamation method, the interfacial area between liquid phases does not change, resulting in invariant fractures of the total energy of the system. It is therefore more effective in the production of double layer.

Looking at the method for producing a double-worm microspheres according to the present invention in more detail.

First, a biodegradable polymer is dissolved in an organic solvent having a low boiling point to prepare a dispersed phase.

At this time, the boiling point of the organic solvent has a range of 35 to 45 ℃, specifically looking at, it is preferable to use methylene chloride, hexafluoroisopropanol, acetone, tetrahydrofuran, ethyl acetate and chloroform. If the boiling point is less than 35 ℃, the remaining of the organic solvent requires a stirring time of 15 hours or more, and if the boiling point exceeds 45 ℃, the volatility is reduced under the conditions of normal pressure and room temperature, the formation of the double-layer fine particles is maintained, so the above range is maintained It is desirable to.

The biodegradable polymer that forms the outside of the bilayer microspheres is commonly used in the art, but is not particularly limited, but is decomposed into a material that does not harm in the human body and has biocompatibility and blood compatibility. Lactide, polyglycolide, poly (lactide-glycolide) copolymer, polycaprolactone, polyisoester, polyiminocarbonate, polyanhydride (poly (SA-HDA anhydride)), polyethyl glutamate and It is preferable to select and use among polydioxanone. Such biodegradable polymers are used in an amount of 0.005 to 0.3 parts by weight based on 1 part by weight of an organic solvent, and when the amount is less than 0.005 parts by weight, a monolayer microsphere is not formed that does not contain polysaccharides. It is preferable to maintain the above range because the viscosity of the compound increases to form bilayer microspheres of 1 mm or more and causes a problem in which an association phenomenon occurs.

Next, the dispersion medium is prepared by dissolving the surfactant and the water-soluble polymer in water. At this time, when dissolving the surfactant and the water-soluble polymer at the same time, it is preferable to form an emulsion, so that the two components are not dissolved, so as to dissolve each. In addition, the surfactant is preferably carried out by heating to 45 ~ 60 ℃, if out of the temperature range bubbles of the surfactant is generated, it shows a phenomenon that precipitates on the bottom.

It is preferable to use pure water without an impurity added to the water as usual, and in the present invention, it is preferable to use tertiary distilled water.

The surfactant serves to form and maintain the microspheres from the dispersed phase and the dispersion medium, and to maintain and stabilize the formed microspheres, and is not particularly limited to those commonly used in the art. Specifically, for example, polyvinyl, which is a high molecular surfactant, Alcohols, tweens 20, 40, 60, and 80, natural polymer gelatin, higher fatty acid alkali salts and the like can be selected and used. The surfactant is used in an amount of 0.01 to 0.06 parts by weight based on 1 part by weight of water. If the amount is less than 0.01 parts by weight, the amount is too small to serve as a surfactant, and when the amount is more than 0.06 parts by weight, it is washed. When the ultracentrifugation is carried out in the process, the surfactant is precipitated and the association with the bilayer microspheres occurs, so it is preferable to maintain the above range.

The water-soluble polymer that forms the inside of the bilayer microspheres is commonly used in the art, but is not particularly limited. It is recommended to use among sulfuric chondroitin and hyaluronic acid. The water-soluble polymer is used in 0.01 to 0.05 parts by weight based on 1 part by weight of water, the amount is less than 0.01 parts by weight of the biodegradable polymer does not contain polysaccharides to form a monolayer microspheres, the amount is too small, If the amount exceeds 0.05 parts by weight, the biodegradable polymer may not be dispersed evenly due to the high viscosity of the aqueous solution, so it is preferable to maintain the above range.

After performing the oil-in-water process using the dispersed phase and the dispersion medium formed as described above to form the double-layer fine particles, there is a feature of the technical configuration to precipitate the formed double-layer fine particles by performing a solvent evaporation process using a physical stirrer, The oil-in-water process can be carried out in the following two ways.

First, a method of forming a double layer of fine particles by dropping a dispersed phase in which a biodegradable polymer is dissolved drop by drop into the dispersion medium in which the water-soluble polymer is dissolved may be used. In this case, as a method of dropwise adding the dispersed phase dropwise, a syringe, pipette, and dropper generally used in the art may be used.

Secondly, after the dispersion medium and the dispersed phase are mixed, ultra-high frequency grinding may be performed to form the double layer fine particles. At this time, the ultra-high frequency grinding used is preferably performed for 30 seconds to 1 minute using a grinder having an output range of 30 to 50%, in the present invention is performed using a Sonic dismembrator (Model 500, Fisher sceintific). When the pulverization time is performed for less than 30 seconds for a short time, the dispersing phase is not evenly dispersed, thereby causing an association phenomenon, which is a aggregation phenomenon between biodegradable polymers.

  The bilayer fine particles formed by the above method evaporate the solvent having a low boiling point using a long time physical stirrer to precipitate the fine particles. The physical stirrer is generally used in the art and is not particularly limited. Unlike the stirring using a magnetic bar, the physical stirrer forms a propeller for stirring directly, and thus has a stronger stirring effect. The angle is formed, and particles can be formed more finely. The physical stirrer used in the present invention uses a mechanical stirrer (RZR 2102 control, Heidolph Instruments GmbH & Co. KG).

Such agitation is preferably performed at a speed of 300 rpm or more, more preferably at least 7 hours in a speed range of 500 to 1500 rpm, more preferably 9 to 12 agitation times, and when the stirring speed is less than 300 rpm, the biodegradable polymer is included. Due to the concentration of the dispersed phase, which is an organic solvent, it is not evenly distributed in the dispersion medium containing polysaccharides, so that the interaction between the formed bilayer fine particles does not form a spherical shape. It becomes smaller and cannot contain polysaccharides, resulting in a problem of monolayer microspheres. In addition, when the stirring time is less than 7, the action of stabilization of the double-layered microspheres by the surfactant is incomplete, the phenomenon that the form of the microspheres collapses when washing the distilled water, and when exceeding 12 hours, the double-layered microspheres due to precipitation of the surfactant Association phenomenon occurs.

After the bilayer microspheres prepared above are subjected to a physical stirring process, the microspheres are settled using a centrifuge and then discarded the supernatant and washed with fresh distilled water. At this time, washing is carried out 5 times or more using distilled water, centrifugation is performed for 4 to 6 minutes at 1500 ~ 2000 rpm. The washed bilayer microspheres are dried using a lyophilizer. The lyophilization is preferably carried out for 5 to 7 days at -49 to -56 ℃, 80 to 9 mtorr conditions.

As described above, it is possible to form microspheres having a size of 400 nm to 500 μm having a dual structure of a hydrophilic water-soluble polymer layer inside and a hydrophobic biodegradable polymer layer outside by using oil-in-water and solvent evaporation according to the present invention. Rather, it is economically improved with a significantly simplified process compared with the conventional art. In addition, the bilayer structure can reduce side effects caused by excessive initial release of water-soluble drugs and the economic and time loss caused by them.

Hereinafter, the present invention will be described in more detail based on the following examples, but the present invention is not limited thereto.

Example 1

To 1 part by weight of tertiary distilled water, 0.01 part by weight of dextran and 0.03 part by weight of polyvinyl alcohol were mixed with respect to 1 part by weight of distilled water to prepare an aqueous dispersion medium. Also, 1 part by weight of dichloromethane was mixed with 0.04 part by weight of poly (lactide-glycolide) copolymer based on 1 part by weight of dichloromethane to prepare a dispersed phase. While dropping the dispersed phase drop by drop in the dispersion medium at the same time using a physical stirrer was continuously stirred for more than 7 hours at 300 rpm to prepare a bilayer microspheres.

The prepared microspheres were washed 5 times or more using tertiary distilled water. At this time, the bilayer microspheres were precipitated using a centrifuge each time, and the supernatant was filtered off and replaced with new tertiary distilled water. The filtered bilayer microspheres were lyophilized for one week at -56 ° C. and 80 mtorr to extract bilayer microspheres to produce purified bilayer microspheres.

Example 2

To 1 part by weight of tertiary distilled water, 0.01 part by weight of dextran and 0.03 part by weight of polyvinyl alcohol were mixed with respect to 1 part by weight of distilled water to prepare an aqueous dispersion medium. Also, 1 part by weight of dichloromethane was mixed with 0.04 part by weight of poly (lactide-glycolide) copolymer based on 1 part by weight of dichloromethane to prepare a dispersed phase. After mixing the powdered phase medium and the dispersed phase, the ultra-high frequency grinding at 30% output for 30 seconds and then continuously stirred for 7 hours or more to prepare a bilayer microspheres.

The prepared microspheres were washed 5 times or more using tertiary distilled water. At this time, the bilayer microspheres are precipitated using a centrifuge each time, and the supernatant is filtered off and replaced with new tertiary distilled water. The filtered bilayer microspheres were lyophilized for one week at -56 ° C. and 80 mtorr to extract bilayer microspheres to produce purified bilayer microspheres.

Comparative Example 1

As a conventional multi-drop and solvent evaporation method, an aqueous solution in which 100 parts by weight of dextran is mixed with respect to 1 part by weight of phosphate buffer solution and 1 part by weight of phosphate buffer solution is prepared, and then 0.0003 parts by weight of the polyvinyl alcohol is mixed. An aqueous mixture was prepared. In addition, 15.6 parts by weight of poly (lactide-glycolide) copolymer was dissolved in 1 part by weight of dichloromethane to 1 part by weight of dichloromethane to prepare an organic solution mixture. Thereafter, the aqueous solution mixture and the organic solution mixture were mixed and then subjected to ultra-high frequency grinding for 5 seconds, followed by stirring while adding 1666.67 parts by weight of the phosphate buffer solution to 1 part by weight of the phosphate buffer solution. At this time, the reaction temperature was 20 ℃, humidity was 65%.

The solution prepared above was stirred for 3.5 hours. When stirring, the opening of the vessel was necessarily opened to evaporate dichloromethane. After the agitation was stopped while dichloromethane did not occur in the phosphate buffer solution, the mixture was filtered, distilled water washing and lyophilization were performed to extract bilayer microspheres.

Experimental Example 1 Observation of Morphology

In order to observe the cross-section and the external morphology of the bilayer microspheres, platinum coating was performed using the bilayer microspheres prepared in Example 1 and observed at × 500 using an electron scanning microscope. The electron scanning micrograph is shown in FIG.

Experimental Example 2 Observation of Confocal Slight Microscope

In order to identify the internal and external materials of the bilayer microspheres, the water-soluble polymers in which the fluorescent materials were bound to Examples 1 and 2 and Comparative Example 1 were observed under a confocal laser microscope at a × 200 magnification.

At this time, Figure 2 is a fluorescence micrograph of a bilayer microspheres prepared without ultra-high frequency pulverization, Figure 3 shows a fluorescence micrograph of a bilayer microspheres prepared by performing a high frequency pulverization, Figure 4 is a bilayer microspheres prepared by a multi-emulsion method The transmission electron micrograph is shown and Figure 5 is an electron transmission microscope of the bilayer microspheres prepared in Example 1 of the present invention was confirmed that the double layer microspheres of the present invention is a spherical surface having a smooth surface and distinctly separated from the outer wall, It was confirmed that the diameter of the double worm microspheres forms a range of 400 nm to 500 µm.

As described above, the oil-in-water and solvent evaporation methods according to the present invention not only enable the formation of microspheres having a dual structure of an internal hydrophilic water-soluble polymer layer and an external hydrophobic biodegradable polymer layer, but also decay over time. It forms a spherical shape that is not stable and can significantly reduce the economic and time loss by significantly simplifying the process compared to the conventional method.

Claims (7)

1 step of dissolving 0.005 to 0.3 parts by weight of a biodegradable polymer in 1 part by weight of an organic solvent having a boiling point of 35 to 45 ° C .; Preparing a dispersion medium by dissolving 0.01 to 0.06 parts by weight of surfactant and 0.01 to 0.05 parts by weight of water-soluble polymer in 1 part by weight of water; And After dropping the dispersion phase prepared in step 1 dropwise to the dispersion medium prepared in step 2, and performing physical stirring at 300 to 1500 rpm for 7 to 15 hours, the hydrophilic water-soluble polymer inner layer and the hydrophobic biodegradable polymer Three steps to manufacture microspheres with a double structure of the outer layer Method for producing a double worm microspheres comprising a. Dissolving 0.005 to 0.3 parts by weight of the biodegradable polymer in 1 part by weight of the organic solvent to prepare a dispersed phase; Preparing a dispersion medium by dissolving 0.01 to 0.06 parts by weight of surfactant and 0.01 to 0.05 parts by weight of water-soluble polymer in 1 part by weight of water; And Ultra high frequency pulverization was performed by mixing the first and second dispersion phases, followed by physical stirring at 300 to 1500 rpm for 7 to 15 hours to double the hydrophilic water-soluble polymer inner layer and the hydrophobic biodegradable polymer outer layer. Three steps to produce microspheres with a structure Method for producing a double worm microspheres comprising a. The method according to claim 1 or 2, wherein the biodegradable polymer is polylactide, polyglycolide, poly (lactide-glycolide) copolymer, polycaprolactone, polyisoester, polyiminocarbonate, polyanhydride , Polyethyl glutamate and polydioxanone. The method according to claim 1 or 2, wherein the organic solvent is selected from methylene chloride, hexafluoroisopropanol, acetone, tetrahydrofuran, ethyl acetate, and chloroform. According to claim 1 or 2, wherein the water-soluble polymer is selected from alginic acid, dextran, starch, glycogen, cellulose, amylopectin, chitin, galactan, laminanan, heparin, sulfur chondroitin and hyaluronic acid Method of producing microspheres. The method according to claim 1 or 2, wherein the surfactant is selected from polyvinyl alcohol, tin 20, 40, 60, and 80, which are polymer surfactants, gelatin, which is a natural polymer, and alkali salts of higher fatty acids. Method for producing double insect microspheres. The method of claim 1 or 2, wherein the diameter of the double-chambered microspheres is in the range of 400 nm to 500 µm.

Images