KR20170087585A

KR20170087585A - method for preparing porous PCL microsphere scaffolds as a cell delivery system

Info

Publication number: KR20170087585A
Application number: KR1020160007253A
Authority: KR
Inventors: 신원상; 김성열
Original assignee: 단국대학교 천안캠퍼스 산학협력단
Priority date: 2016-01-20
Filing date: 2016-01-20
Publication date: 2017-07-31
Also published as: KR101766995B1

Abstract

The present invention relates to a method for producing a biodegradable polymer solution which simultaneously contains a biodegradable polymer, a room temperature ionic liquid porogen and a sublimable solid porogen at the same time in an organic solvent to simultaneously contain an ionic liquid porogen at room temperature and a sublimable solid porogen Stage 1; A second step of preparing a gel by evaporating an organic solvent from the biodegradable polymer solution; And a third step of extracting the room temperature ionic liquid porogen from the gel using a protonic solvent, and to a porous microsphere thus prepared. According to the present invention, porous microspheres having controlled diameters and pore sizes can be prepared by controlling the ratio of the room temperature ionic liquid porogen and the sublimable solid porogen. The porous microspheres prepared according to the present invention can be applied to a cell delivery system for nerve regeneration therapy.

Description

TECHNICAL FIELD The present invention relates to a method for preparing porous microspheres for cell delivery,

More particularly, the present invention relates to a method for producing a porous microsphere from a biodegradable polymer solution simultaneously containing a room temperature ionic liquid porogen and a sublimable solid porogen, will be.

In the field of tissue engineering, the development of porous microspheres as an injectable cell delivery system has been intensively attempted. For solving many problems associated with conventional dosing methods, microspheres having diameters in the range of 300 to 500 mm are most preferred and are being developed as cell delivery systems. Cells can be adhered and propagated on porous microspheres, which can be implanted at the site of injury to induce new tissue formation.

The porous nature of the porous microsphere system allows a wider specific surface area and more efficient cell expansion compared to a smooth non-porous surface. Porous microspheres of biopolymers are typically prepared by emulsion solvent evaporation (phase separation) and gas forming pockets, Spinning disk atomization, self-assembly, and the like, which are well known to those skilled in the art. Among these, the phase separation method is the most popular technology due to the possibility of large-scale production, the uniformity of the sphere size, and the merits of a simple process without the use of a composite device.

Due to its biocompatibility and slow biodegradability properties, polycaprolactone (PCL) has been widely used in the manufacture of porous microspheres for supporting and delivering cells.

On the other hand, ionic liquids can be used as a new medium in organic synthesis, as catalyst supports and nanostructuring materials. In particular, the type of anion of an ionic liquid affects the degree of solubility (for example, hydrophilic / hydrophobic nature) of the ionic liquid. Thus, the use of anion-induced properties has become a subject of interest in surface chemistry and catalysts.

The inventors of the present invention have made it possible to obtain porous microspheres from a biodegradable polymer solution containing an ionic liquid porogen at room temperature and a sublimable solid porogen at the same time, and completed the present invention.

The present invention can be carried out at one time without any additional process by using phase separation of a hydrophilic ionic liquid and a hydrophobic biodegradable polymer, and by controlling the surface morphology by using a room temperature ionic liquid porogen and a sublimable solid porogen simultaneously, And to provide a method for producing a porous microsphere as a cell delivery system having cell adhesion.

The first aspect of the present invention relates to a biodegradable polymer solution which simultaneously dissolves a biodegradable polymer, a room temperature ionic liquid porogen and a sublimable solid porogen in an organic solvent to simultaneously contain an ionic liquid porogen at room temperature and a sublimable solid porogen ; A second step of preparing a gel by evaporating an organic solvent from the biodegradable polymer solution; And a third step of extracting the ionic liquid porogen at room temperature from the gel using a protonic solvent.

The term "biodegradable polymer" used in the present invention means a polymer substance capable of decomposing into inorganic substances such as water, carbon dioxide and the like in a relatively short period of time by the action of microorganisms or light, and eliminating environmental pollution problems.

The biodegradable polymer used in the present invention may be selected from the group consisting of polyglycolic acid, polylactic acid, poly D, L-lactic acid-co-glycolic acid, poly L-lactide-co-D, L-lactide, polyhydroxybutyrate, But are not limited to, any of those selected from the group consisting of hydroxybutyrate, hydroxyvalerate, polyvalero lactone, polycaprolactone, polydioxanone, copolymers thereof, and mixtures thereof. In one preferred embodiment of the present invention, polycaprolactone was used.

The term " ionic liquid " as used herein means ionic salts (molten salts) which are liquid at ambient temperature range including room temperature, which is composed of cations and anions. Unlike ionic salt compounds composed of cationic and nonmetal anions, such as salt, which usually dissolve at a high temperature of 800 ° C or higher, an ionic salt present as a liquid at a temperature of 100 ° C or lower is referred to as a "room temperature ionic liquid".

These ionic liquids are nonvolatile, non-toxic, non-flammable, have excellent thermal stability and ionic conductivity, have high polarity to dissolve inorganic and organometallic compounds, and exist as liquids over a wide temperature range. Separation, and electrochemistry.

Particularly, ionic liquids are ionic and ionic substances, so they are not volatile (no boiling point) and can be present in a wide range of liquid (-100 ° C to 300 ° C). Various kinds of physical properties can be changed by changing the kind of negative ions.

In the present invention, the size of the cavity of the biodegradable polymer microspheres of the porous structure can be controlled by changing the kind of the anion of the ionic liquid. The size of the cavity is related to the degree of hydrophilicity of the anion.

As the degree of hydrophilicity of the hydrophilic ionic liquid increases, the hydrophobic biodegradable polymer and the boundary energy become higher, so that the hydrophilic ion exists in a form having a larger spherical shape as the adhesion occurs, and therefore, The size of the cavity of the biodegradable polymer is further increased.

Further, the ionic liquid is formed by the combination of cation and anion in contrast to the conventional organic or aqueous solvent which is molecularly nonionic, and when it is used as a solvent, decomposition of cations and anions occurs easily as shown in Reaction Scheme 1 below.

[Reaction Scheme 1]

For this reason, ionic liquids are increasingly used to replace conventional evaporative organic compounds.

Examples of the cation of the ionic liquid include dialkylimidazolium, alkylpyridinium, quaternary ammonium, and quaternary phosphonium. The anions include Cl ^- , NO ₃ ^- , BF ₃ ^- , PF ₆ ^- , NO ₃ ^- _{^{_{, AlCl 4 -, Al 2 C}}} 7 -, AcO -, Tio - (trifluoromethanesulfonate), Tf 2 N - (trifluoromethanesulfinylamide), (Cf 3 SO 2) 2 N, CH 3 CH (OH) CO 2 - (L-lactate ).

In particular, an ionic liquid present as a liquid at a temperature of 100 ° C or lower is referred to as a room temperature ionic liquid (RTIL).

Room temperature ionic liquids (RTILs) have highly polar and non-polar moieties and can be miscible and / or immiscible with water and many organic solvents. The wettability, that is, the hydrophilic / hydrophobic characteristic, of the ionic liquid at room temperature greatly changes according to the kind of organic cations and inorganic anions.

In the present invention, the room temperature ionic liquid is used as a liquid template or a liquid porogen for microsphere formation.

In the present invention, the cation of the room temperature ionic liquid may be an imidazolium or ammonium cation substituted with C _1-10 alkyl, preferably butylmethylimidazolium or trioctylmethylammonium cation But is not limited thereto.

In the present invention, the anion of the room temperature ionic liquid is selected from the group consisting of tetrafluoroborate (BF ₄ ^- ), hexafluoroantimonate (SbF ₆ ^- ), hexafluorophosphate (PF ₆ ^- ), But are not limited to, bistriflimide (NTf ₂ ^- ), triflate (OTf ^- ), chloride (Cl ^- ), tetrafluoroborate or hexafluoroborate.

Room temperature ionic liquid captive in the invention gen is butyl methylimidazolium chloride (bmim Cl), butyl-methyl-imidazolium triflate (bmim OTf) or butyl-methyl imidazolium tetrafluoroborate (bmim BF ₄₎ and trioctyl Methylammonium chloride (TOMAC), but the present invention is not limited thereto.

For example, [bmim] BF ₄ is in liquid form at temperatures above -80 ° C, and trioctylmethylammonium chloride (TOMAC) is in liquid form at temperatures above -20 ° C.

In one preferred embodiment of the present invention, trioctylmethylammonium chloride (TOMAC) can be used.

As used herein, the term " sublimable solid porogen " refers to any structural material that can be used to produce a porous material.

The present invention allows the formation of open pores on the surface of a microsphere by using a room temperature ionic liquid porogen and a sublimable solid porogen simultaneously. The sublimable solid porogen is removed through heat treatment to form pores.

As the sublimable solid porogen, there can be used at least one compound selected from the group consisting of camphene, cyclohexanone, 2-ethylhexanol, toluene, octan-2-one, n-butylacetate, p -xylene, ethyl acetate, Dodecan-1-ol, cyclohexanol, dodecanol, heptane, cyclohexane, 1-chlorodecane, dibutyl phthalate, and the like.

In a preferred embodiment of the present invention, camphene (2,2-dimethyl-3-methylene-bicyclo [2.2.1] heptanes, C ₁₀ H ₁₆ ) was used. Kampen is particularly suitable for the present invention because it has a good melting property in an organic solvent such as ethanol and ether and therefore is removed together with the evaporation of the organic solvent.

The step of simultaneously mixing the biodegradable polymer, the room temperature ionic liquid porogen, and the sublimable solid porogen may use an organic solvent. Specifically, the organic solvent may be dichloromethane (DMC), methanol, ethanol, preferably dichloromethane, but is not limited thereto.

Wherein the first step is a step of simultaneously dissolving the biodegradable polymer, the room temperature ionic liquid porogen, and the sublimable solid porogen in an organic solvent, wherein the sublimable solid porogen is added to the room temperature ionic liquid porogens in the range of 0.5 to 2.5 By weight.

When the weight ratio of the sublimable solid porogen to the ionic liquid porogen at room temperature is less than 0.5, the surface of the microspheres is flat and smooth, so that the hole-transporting function is weakened. There is a problem in that the interval of the wave-like pores formed on the surface of the carrier is too wide and the diameter of the hole-like pores is too large,

In the second step, the organic solvent is evaporated from the biodegradable polymer solution containing the room temperature ionic liquid porogens and the sublimable solid porogen, thereby phase separation occurs between the hydrophobic biodegradable polymer and the porogen and the hydrophilic ionic liquid at room temperature Gel is formed.

Preferably, the second step proceeds under atmospheric conditions.

The third step is to remove the room temperature ionic liquid porogen from the gel, which may be a step of dissolving the gel using a protonic solvent and separating the porous microspheres from the gel dissolved in the protic solvent by centrifugation have.

The protic solvent used in the present invention means a solvent capable of providing hydrogen cations, and is preferably water or ethanol, but is not limited thereto.

In one embodiment of the present invention, the gel was dissolved in ethanol and centrifuged at 3000 rpm for 10 minutes to recover the porous microspheres from which the room temperature ionic liquid had been removed.

The third step may further include washing and drying the recovered porous microspheres.

The room temperature ionic liquid removed through the third step may be reusable.

The prepared porous microspheres have a pore channel formed therein, and a wave pore and a hole pore connected to the inner pore channel are formed on the surface.

The diameter of the porous microspheres may be 200 to 700 탆.

The interval of the wave-like pores may be 20 to 2000 nm, and the diameter of the hole-shaped pores may be 10 to 100 mu m.

The second aspect of the present invention is a method for producing the biodegradable polymer of the present invention comprising a biodegradable polymer in which a pore channel is formed and on which a wave type pore and a hole type pore connected to the internal pore channel are formed, The porous microspheres prepared according to the present invention are provided.

The diameter of the porous microspheres may be 200 to 700 탆, and the interval of the wavy pores may be 20 to 2000 nm.

The diameter of the hole-like pores may be 10 to 100 [mu] m.

A third aspect of the present invention provides a porous microsphere scaffold which is an injectable cell delivery system in which a coating layer of a nerve growth factor (NGF) and a gelatin mixture is formed on the surface of a porous microsphere of the present invention.

In one embodiment of the present invention, porous microspheres are coated with an NGF-containing gelatin solution (0.1 wt% gelatin, 0.0001 wt% NGF) and coated with an NGF / gelatin mixture for efficient cell attachment and proliferation on porous PCL microspheres Gt; PCNGpmb-2 < / RTI > (NGF / Gel / PCLpms-2) microspheres.

The porous microsphere scaffold may be a neuronal cell delivery system for nerve regeneration therapy.

According to the present invention, porous microspheres having controlled diameters and pore sizes can be produced.

According to the present invention, it is possible to produce porous microspheres having wavy pores spaced from 20 to 2000 nm on the surface and hole pores of 10 to 100 mu m connected to the internal pores.

The porous microspheres prepared according to the present invention can be applied to a cell delivery system for nerve regeneration therapy.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows a scanning electron microscope image showing the increase in size of the porous PCL microspheres: a) PCLms; b) PCLpms-1; c) PCLpms-2; d) PCLpms-3.
Figure 2 shows a scanning electron microscope image of a microbead with different pore morphologies: a) PCLms; b) PCLpms-1; c) PCLpms-2; d) PCLpms-3. The insertion figure shows the SEM image of the observed pores at higher magnification.
Figure 3 is a scanning electron microscope image showing microspheres, a) cross-section and b) internal structure of PCLpms-2.
Figure 4 shows a scanning electron microscope image of a microsphere with different surface nano-shapes: a) PCLmb; b) PCLpmb-1; c) PCLpmb-2; d) PCLpmb-3. Insertion is a SEM image of the pores observed at higher magnification, showing wave-like nanopores.
Figure 5 schematically depicts the formation of porous PCL microspheres in the presence of TOMAC and Kamppin combination.
Figure 6 shows a scanning electron microscope image of PCNGpms-2 (NGF / Gel / PCLpms-2) microspheres coated with an NGF / gelatin mixture: b) an enlarged image of the area within the blue box of a); c) is an enlarged image of the area within the green box of b); The red arrows represent small pores or depressions of <20 μm remaining after coating with the gelatin / NGF mixture.
Figure 7 shows a) FT-IR spectrum and b) TGA thermal analysis of PCNGpms-2 (NGF / Gel / PCLpms-2) microspheres coated with a pure PCLpms-2, pure gelatin and NGF / gelatin mixture .
8 is an SEM image for the morphological analysis of PC-12 neuron. A) PCLpms, b) Gel / PCLpms and c) cells attached to PCNGpms-2 at a low magnification of 500-700 × (top panel) or at a high magnification of 3000-4000 × (bottom panel).
9 is a scanning electron microscope image of microbeads when the weight ratio of camphene / TOMAC is 5;

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for describing the present invention more specifically, and the scope of the present invention is not limited by these examples.

Reference example 1: Materials and properties

Polycaprolactone (PCL) as a biodegradable polymer was purchased from Boehringer Ingelheim. Trioctylmethylammonium chloride (TOMAC), CH ₂ Cl ₂ (DCM), and camphene purchased from Korean Sigma Aldrich Co. were added to room temperature ionic liquid (RTIL), organic solvent and And was selected as a porogen. Nerve growth factor (NGF) purchased from Sigma-Aldrich, Inc. was used as a model protein important for the growth, maintenance and survival of specific target neurons. The materials were used without further purification.

The 3D morphology and physicochemical analysis of porous PCL microspheres were performed using scanning electron microscopy (SEM, JEOL and HITACHI S-3000H, Japan) and FT-IR spectrometer (Perkin-Elmer Spectrum BXII, US) Respectively. For scanning electron microscopy analysis, it was coated with about 10 nm of gold prior to analysis. IR spectra were recorded in the 16th scan 4000 to 500 cm ^-1 range 4 cm ^-1 resolution. The purity of the product was confirmed by thermogravimetric analysis (TGA) using Seiko Exstar 6000 TG / DTA6100 (SEICO INST., JAPAN) at a heating rate of 10 / min at a temperature ranging from 20 to 1000.

Example 1: Porosity PCL Microsphere Produce

Porous PCL microspheres were prepared as follows; 0.1 g of PCL, 2.5 g of TOMAC, and 2 g of camphene were dissolved in 50 mL of DCM to produce a clear solution. The organic solvent (DCM) was evaporated from the clear solution under at ambient conditions. A rubbery white gel remained on the bottom of the glass plate. To remove the TOMAC, the resulting rubbery cake was dissolved in ethanol and centrifuged at 3000 rpm for 10 minutes to recover the porous microparticles. The washing process was repeated three times and the white microspheres obtained were dried under atmospheric conditions. The recovered ionic liquid was later reused.

1-1. Kampen / TOMAC Due to rate changes Spear Resize

And to develop a porous spherical injectable cell delivery system using a PCL solution containing camptothecin as a porogen and trioctylmethylammonium chloride as a liquid phase dissolved in DCM. When the organic solvent was evaporated from the mixed solution at room temperature, a transition from a homogeneous liquid state to a gel state was observed.

The TOMAC surrounding the camppene-encapsulated or camppen-embedded PCL microspheres was selectively extracted from the polymer gel using 100% ethanol to obtain white soccer ball-shaped porous microspheres (see FIGS. 1B to 1D). Porous PCL microspheres (PCLpms-1 to PCLpms-3) similar in shape to the soccer ball showed a size of about 244 to 601 microns in diameter, as compared to the non-porous PCL microspheres (PCLms) (See Figs. 1B to 1D and Table 1).

Spear size could be controlled by various combinations of TOMAC and Kampen. As the ratio of Campan / TOMAC increased from 0.8 to 1.6 and 2.4, when the TOMAC concentration was constant, the sphere size gradually increased from about 244 μm in diameter to 290 μm and 601 μm. Compared with PCLms, the average size of PCLpms-1 to PCLpms-3 increased from about 70 to 176 times.

Name of sample
Campan / TOMAC (g)
Sphere size
(Diameter: 占 퐉)
Surface form
Wave type pore
(Inter-wave interval: nm) Hole type porosity
(탆) PCLms 1 / 2.5 3.41 ± 1.07 Flat and smooth Pore member PCLms-1 2.0 / 2.5
(= 0.8) 244 ± 45.51 20 ~ 30 10-20 PCLms-2 4.0 / 2.5
(= 1.6) 390 ± 71.51 200-300 20 to 40 PCLms-3 6.0 / 2.5
(= 2.4) 601 ± 97.23 200 ~ 2000 40-100

1-2. Kampen / TOMAC Pore size, pore shape and pore number according to the ratio change

As shown in Table 1, the weight ratio of camphene / TOMAC greatly influenced the pore shape and number.

When the ratio was zero (no camphene was used), no open pores were observed on the surface of the spherical structure of PCLms, as shown in Figure 2a. However, when the ratio increased to 0.8, a small number of open pores with a diameter of about 10 to 20 占 퐉 were observed on the spherical surface of PCLpms-1, as shown in Fig. 2b.

Furthermore, as PCLpms-2 and PCLpms-3 samples increased the ratios of camphene / TOMAC to 1.6 and 2.4, respectively, a further increase in pore size (about 20-40 urn and 40-100 urn; And 5 times), and the number of pores increased (about 3 times and 4 times of PCLpms-1).

To confirm the internal structure of the porous PCL microsphere (typically PCLpms-2), the sample is frozen in liquid nitrogen and broken into fine particles. The internal shape of the broken particles was also confirmed to be highly porous and contained interconnected pore channels (FIG. 3).

These results show that the regular and highly porous form, and the larger pore size, are due in part to higher concentrations of Kampen. It also shows that a low weight ratio of not more than 2.5 is required to form a highly porous outer shape and interconnected inner pore channels.

As shown in FIG. 9, when the weight ratio of the camphan / TOMAC was 5, the non-bead shape of the film was observed. On the surface, the open pores also disappeared and only the inner pores were observed.

1-3. Kampen / TOMAC Nano-level surface morphology change with ratio

The weight ratio of camphene / TOMAC also influenced the surface nano-form, such as the formation of wave-like nanopore microspheres at intervals of 20 to 2000 nm (see Table 1). The average pore size of the wave-like gap (gab) could be controlled by changing the weight ratio of camphene / TOMAC (or camphene concentration).

As shown in the SEM image of FIG. 4, the surface of PCLms prepared without Kampen exhibited only a flat and smooth topography without nanoporos whereas all of PCLpms-1 to PCLpms-1 produced in the presence of various Kampen / 3 The surface of the microsphere was covered with a wave-like porous nanopattern in addition to micro-sized hole-shaped pores.

In the case of PCLpms-1, a weak rough surface with an irregular wave pattern of about 20 to 30 nm gap was observed. However, the surfaces of PCLpms-2 and PCLpms-3 microspheres were further increased and covered with more regular wave pores with larger spacing of 200-2000 nm. Such surface nanopatterns became more and more intense as the concentration of camphene increased in the PCL / TOMAC precursor solution.

This is because some camphene molecules initially penetrated into the microsphere and the remainder could surround the outside during the solvent-drying process. As the excess Kampp concentration increases, more Kamppen can be regularly accumulated on the surface of the spherical PCL microbeads, and thus the pore structure of the wave pattern can be formed larger and more regularly.

1-4. Kampen / TOMAC When present PCLpms Formation mechanism

A process of forming a soccer ball-shaped porous PCL microsphere (PCLpms) according to the present invention is schematically shown in FIG.

PCL and TOMAC were homogeneously dissolved in a DCM solution containing camphene to prepare a viscous solution. As the solvent was evaporated at ambient temperature, the homogeneous fluid to gel state transitioned (or phase-separated from the microscale to the hydrophobic PCL / camphane phase and hydrophilic TOMAC phase).

In the first step, a portion of the introduced camphene migrates into the spherical PCL phase to form small-sized PCL microspheres (CLms aggregates of about 3-5 mu m in size) in which campene molecules are embedded, while Kampen molecules are hydrophobic , The remaining camphene can be deposited on the PCL microsphere surface with TOMAC molecules.

Due to the continuous evaporation of the solvent, in situ-formed nonporous PCLms complexes surrounded by hydrophobic camphor and TOMAC components are agglomerated adjacent to each other due to strong interaction between their hydrophobic surfaces to form larger sized soccer balls Of PCL microspheres (agglomerates of the PCLms complex).

When the solvent was completely evaporated, the camphene component was also volatilized to form wave-like micropores on the inner and outer surfaces, and as a result, porous PCL microspheres (PCLpms) in the form of soccer balls could be formed.

Example 2: PCL Microsphere Surface NGF / Gelatin coating

To prepare PCTpmb-2 (NGF / Gel / PCLpms-2) microspheres coated with an NGF / gelatin mixture, the NGF / gelatin mixture was coated on the microsphere surface according to the following procedure; 50 mg of the porous microspheres prepared according to Example 1 were placed in a flask and vacuumed. 5 mL of gelatin solution (0.1 wt%) containing 5 ㎍ NGF was injected into the flask while stirring at 80 rpm. The gelatin remaining on the surface of the PCL microspheres was washed with water and then lyophilized to obtain a PCL microsphere containing the gelatin-NGF mixture in the pores.

2-1. NGF / Gelatin mixture coated PCNGpms -2( NGF / Gel / PCLpms -2) Microsphere

For efficient implantation of the porous PCL microsphere-cell complex into the damaged site to induce efficient cell attachment and proliferation on porous PCL microspheres and new tissue formation, three samples (PCLpms-1 PCLpms-2 microspheres were selected as model cell carriers and their surfaces were coated with NGF-containing gelatin solution (0.1 wt% gelatin, 0.0001 wt% NGF) to form PCNGpms-2 NGF / Gel / PCLpms-2) microspheres. As shown in the SEM image of FIG. 6, the number and size of the surface pores were significantly reduced. After coating with the gelatin / NGF mixture, the original size hole pores of 20 to 40 mu m changed to small pores of less than 20 mu m or remained in a depressed form. In addition, regular wavy patterns on the original spherical surface (gap distance of about 200-300 nm) disappeared after coating and only flat, smooth surface topography was observed.

The PCNGpms-2 microspheres coated with the NGF / gelatin mixture prepared using FT-IR and TGA data were qualitatively and quantitatively analyzed (FIG. 7). The IR spectral data of PCNGpms-2 microspheres coated with NGF / gelatin mixture, and about 2939 C = O stretching of the CH stretching (stretch), 1720 cm ^-1 in 2878 cm ^-1 and about 1200-1080 cm ^- such as CO expansion and contraction of the ^first, it exhibited the typical characteristics of the polyester (Fig. 7a). Compared with the pure gelatin spectrum (Fig. 7a), the characteristic peaks of the gelatin component in the PCNGpms-2 sample were clearly observed despite the small amount on the microsphere surface and the overlap of the polyester bands and other bands. For example, NH stretching at 3440 cm ^-1 , OH stretching at about 3320 cm ^-1 , C = O stretching at 1630 cm ^-1 (amide I), and NH bending at 1522 cm ^-1 (amide I ) Could be regarded as originating from the gelatin phase on NGF / Gel / PCLpms-2. The possible signals from the NGF component were not observed in the spectrum of NGF / Gel / PCLpms-2 due to the small amount and superposition of gelatine bands and the above possible bands. For thermogravimetric analysis (TGA), the thermal degradation pattern of PCNGpms-2 microspheres was compared to that of pure PCL samples (PCLpms-2) and pure gelatin materials. Figure 7b shows that the weight loss of PCLpms-2 is in the range of about 220-420 (primary part) and 420-550 (secondary part), respectively. Gelatin showed a primary part in the range of 220 ~ 365 and a secondary part in the range of 365 ~ 675. The PCNGpms-2 microsphere showed a pattern similar to that of PCLpms. For example, a steep slope of the primary pyrolysis curve in the range of about 220-420 and a quadratic curve of gentle slope in the range of about 420-550 are shown, which is a small fraction indicating the presence of trace amounts of gelatin.

Experimental Example 1: In vitro cell culture

For the cell behavior of PCTpmb-2 (NGF / Gel / PCLpms-2) microspheres coated with NGF / gelatin mixture, rat adrenal pheochromocytoma-derived cell line, PC-12 Type Culture Collection, Manassas, Va.) Were used. At this time, by induction of neuronal phenotype, it reacts to NGF in an inverse order, stops cell division and expands neurite. PC-12 cells were 37, 5% CO ₂ wet incubator under conditions of 10% in (Thermo Scientific Inc., Waltham, MA ) (v / v) FBS (fetal bovine serum, Gibco), 100 units / mL penicillin and 100g / (Dulbecco ' s modified Eagle ' s medium, Gibco, Gaithersburg, Md.).

Experimental Example 2: To identify neuronal behavior Microsphere group Ready

The effect of NGF on cell viability, cell morphology and neural differentiation of PC-12 cells on PCLpms-2, Gel / PCLpms-2 and PCNGpms-2 (NGF / Gel / PCLpms-2) microspheres Respectively. Each microsphere group was immersed in a 1.5 mL eppendorf tube containing PBS and allowed to stand for three or four hours until the microspheres were submerged on the bottom of the tube. After washing three times with the culture medium, the cells were irradiated with ultraviolet light for 30 minutes before use And sterilized.

In this experiment, a 12 mm polycarbonate membrane cell culture insert (Millipore, Billerica, MA) with a pore size of 3.0 μm was used to maintain both microspheres and cells. The insert was placed in a 24 well cell culture plate and allowed to submerge in the culture medium for a few minutes, then 1 mg of each microsphere was transferred into the insert. PC-12 cells were plated on each microsphere in a cell culture insert at a cell density of 100,000 cells / well and cultured for 5 days.

All experiments were repeated at least 3 times and the data were expressed as mean ± SD (SD). Statistical analysis was performed using SPSS 12.0 software (SPSS, Chicago, IL). Statistical significance of differences between groups was assessed by two-tailed Student's t-test. Differences were considered statistically significant at p < 0.05.

Experimental Example 3: Cell morphology analysis

The microspheres were fixed with 2.5% glutaraldehyde with neurons and dehydrated through a series of concentrations of ethanol (50%, 75%, 95%, and 100%) and then treated with hexamethyldisilazane And the samples were dried overnight in the hood. The surface morphology of PC-12 cells on the microspheres was monitored by a field emission scanning electron microscope (MIRA II, Tescan, Czech Republic) driven at an accelerating voltage of 1.0 kV according to standard procedures.

3-1. PCNGpms -2 on cell adhesion and differentiation

Gelatin and NGF treated PCNGpms-2 microspheres showed higher cell adhesion to PC-12 neurons compared to untreated PCL microspheres (see FIG. 7). In the presence of NGF, PC-12 cells on the microspheres exhibited neuronal cell differentiation as well as high adherence (arrows in Fig. 7c). This represents a typical drug release effect of NGF on prominent cell differentiation. From these results, it can be seen that PCNGpms-2 microspheres prepared according to the present invention have a significant effect on nerve cell behavior such as adhesion and differentiation of nerve cells, and thus can be applied as a continuous cell delivery system for nerve regeneration therapy .

Claims

A first step of dissolving a biodegradable polymer, a room temperature ionic liquid porogen and a sublimable solid porogen in an organic solvent at the same time to produce a biodegradable polymer solution containing both an ionic liquid porogen at room temperature and a sublimable solid porogen at the same time;
A second step of preparing a gel by evaporating an organic solvent from the biodegradable polymer solution; And
And a third step of extracting the room temperature ionic liquid porogen from the gel using a protonic solvent.

The biodegradable polymer according to claim 1, wherein the biodegradable polymer is selected from the group consisting of polyglycolic acid, polylactic acid, poly D, L-lactic acid-co- glycolic acid, poly L-lactide-co-D, L-lactide, polyhydroxybutyrate, Wherein the porous microspheres are any one selected from the group consisting of polyhydroxyvalerate, polyvalero lactone, polycaprolactone, polydioxanone, copolymers thereof, and mixtures thereof.

According to claim 1, wherein the room temperature ionic liquid porogens butyl methylimidazolium chloride (bmim Cl), butyl-methyl-imidazolium triflate (bmim OTf) or butyl methyl borate (bmim BF ₄ as imidazolium tetrafluoroborate ) And trioctylmethylammonium chloride (TOMAC). &Lt; RTI ID = 0.0 > 11. < / RTI >

The method according to claim 1, wherein the sublimable solid porogen is camphene.

The method of claim 1, wherein the organic solvent is dichloromethane (DMC), methanol, or ethanol.

The method of claim 1, wherein the weight ratio of the sublimable solid porogen to the room temperature ionic liquid porogens in the first step is 0.5 to 2.5.

The method of claim 1, wherein the third step comprises separating the porous microspheres from the gel dissolved in the protic solvent by centrifugation.

The method of claim 1, wherein the protic solvent is water or ethanol.

The porous microspheres prepared according to the manufacturing method of claim 1, wherein the porous microspheres are formed of a biodegradable polymer having a pore channel formed therein, a pore formed on the surface thereof, and a hole- .

The biodegradable polymer according to claim 9, wherein the biodegradable polymer is selected from the group consisting of polyglycolic acid, polylactic acid, poly D, L-lactic acid-co- glycolic acid, poly L- lactide-co-D, L- lactide, polyhydroxybutyrate, Wherein the porous microspheres are any one selected from the group consisting of polyhydroxyvalerate, polyvalero lactone, polycaprolactone, polydioxanone, copolymers thereof, and mixtures thereof.

The porous microsphere according to claim 9, wherein the diameter of the porous microspheres is 200 to 700 탆.

The porous microspheres according to claim 9, wherein the interval between the wavy pores is 20 to 2000 nm, and the diameter of the hole pores is 10 to 100 μm.

A porous microsphere scaffold that is an injectable cell delivery system in which a coating layer of a nerve growth factor (NGF) and a gelatin mixture is formed on the surface of the porous microspheres according to claim 9.

14. The porous microsphere scaffold of claim 13, wherein the porous microsphere scaffold is a neuronal cell delivery system for nerve regeneration therapy.