KR101920284B1 - Heparin nanosponge for controlled release of growth factors and metho for manufacturing thereof - Google Patents

Abstract

The present invention relates to a heparin nano-sponge for effectively delivering growth factors. The heparin nano-sponge comprises: a thiolated heparin; and a pluronic polymer bonded with the thiolated heparin. The present invention provides a nano-sponge by combining heparin and a pluronic polymer, so the growth factors can be mounted and efficiently delivered in vivo.

Description

TECHNICAL FIELD [0001] The present invention relates to a heparin nano sponge for sustained delivery of a growth factor and a method for producing the heparin nano sponge,

The present invention relates to a heparin nano-sponge for effectively delivering growth factors, and more particularly, to a heparin-substituted heparin and a photo-crosslinkable polymer through a simple photopolymerization reaction. The heparin nano- The present invention relates to a nano-carrier capable of minimizing phosphorus and hemorrhage and maximizing the effective activity of a growth factor.

Growth factors are signaling molecules that regulate cell proliferation, cell differentiation, and cell survival, and are a class of water-soluble proteins. These biomolecules are essential for promoting tissue growth and tissue repair. The biomolecule has a very short half-life, but may have an influence even at low concentrations. Therefore, growth factors have great potential as biotherapeutics in a variety of clinical applications. It has great potential in the clinical restoration of various tissue defects due to trauma, degenerative disease, oncological resection, etc. Growth factors are essential to the success of clinical trials of tissue engineering to repair or replace damaged tissue in the human body. To mimic the natural tissue regeneration process using an engineered scaffold, complex growth factors must be delivered at optimal rates and controlled release rates.

Advances in protein purification and recombinant DNA technology enable the production of a variety of natural and recombinant growth factors for clinical applications. However, there are very few growth factors approved and commercialized for use in treating humans. The disappointing rate for clinical trial success is mainly due to instability in solution, self-aggregation, poor bioavailability, short in vivo hal-life, and in vivo delivery Adventures for the main reason. Generally, growth factors are delivered to the site of the disease through bolus injection or systemic administration. It is difficult to maintain the bioactive state of growth factors for a long time under a physiological dose. Due to this, very high doses of administration are required to achieve therapeutic effects in vivo. However, administration of these very high doses often results in undesirable side effects such as hypotension, retinopathy or progression of malignant tumors due to abnormal activity at the distant site.

Therefore, there is a need to optimize delivery of growth factors in order to improve the success rate of clinical trials of growth factors and to reduce unwanted side effects. This should maintain the local concentration of the growth factor delivered to the target tissue for a long time, maintain the bioactivity, and minimize the action in the normal tissue. Over the past decade, there have been many attempts to develop emission control systems for growth factor delivery. Micro / nanoparticles, hydrogels, and porous 3D scaffolds are examples of various vehicles for the delivery of growth factors. The release behavior is controlled by the adjustment of physico-chemical properties such as porosity, degree of cross-linking and degradation rate of the carrier. In addition, the release system is designed to provide distinct release behavior, control release in different time and space, stimulation of immediate release. However, previous efforts to capture growth factors within a sustained release vehicle have only partially succeeded, since they resulted in low bioactivity of the growth factors.

Heparin is a naturally occurring persulfated anionic polysaccharide that is clinically used as an anticoagulant. Heparin has specific interactions with a variety of proteins with heparin-binding domains containing most growth factors. Due to these properties, several systems including heparin have been developed for the sustained release of growth factors. Biodegradable poly (D, L-lactic-co-glycolic acid) (PLGA) microspheres and nanoparticles functionalized with heparin are known as basic fibroblast growth factor (bFGF) And vascular endothelial growth factor (VEGF). In addition, heparin binds to various implantable three-dimensional scaffolds such as collagen, alginate and chitosan for controlled delivery of growth factors. However, since the anticoagulant properties of heparin can cause hemorrhage, it can be a concern or limited in many in vivo applications.

Eduardo Anitua, Mikel Sa'nchez, Gorka Orive, Lsabel Andia. &Quot; Delivering growth factors for therapeutics ", Trends in Pharmacological Sciences, 2008, 29, 37-41. Prakriti Tayalia, David J. Mooney. 'Controlled growth factor delivery for tissue engineering', Advanced maerials, 2009, 21, 3269-3285.

It is an object of the present invention to provide a photo-crosslinked thermosensitive heparin nano sponge as a chemically stabilized carrier for long term sustained release of a variety of growth factors.

In order to achieve the above object, the present invention provides a pharmaceutical composition comprising thiolated heparin; And a heparin nano sponge comprising a pluronic polymer combined with the thiolate heparin.

The thiolate heparin may be 3 to 24 wt% based on 100 wt% of the total weight.

The size of the heparin nano sponge may be 600 nm to 50 nm at 5 to 37 ° C, more preferably 600 nm or less at 5 ° C, and 50 nm or more at 37 ° C.

The surface charge of the heparin nano sponge may be -4.0 mV to -40.0 mV.

The growth factors include basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), bone morphogenic protein-2 (BMP-2) And hepatocyte growth factor (HGF).

(A) synthesizing thiolated heparin (Hep-SH) by introducing a thiol group (-SH) into heparin; (b) mixing the thiolate heparin and the pluronic polymer to prepare a mixture; And (c) adding a photoinitiator to the mixture and treating the mixture with ultraviolet light. The present invention further provides a method for producing a heparin nano sponge.

The pluronic polymer may include a photo-crosslinkable functional group.

The photocrosslinkable functional groups include, but are not limited to, acrylate, diacrylate, oligo-acrylate, methacrylate, dimethacrylate, oligomethacrylate oligo (meth) acylate, coumarin, thymine or cinnamate.

According to the present invention, heparin and pluronic polymer are combined with each other to provide a nano-sponge, so that a growth factor can be loaded and efficiently delivered in vivo.

In addition, bleeding which is a side effect of heparin can be minimized, the release of the growth factor can be maintained for a long time, and the bioactivity of the original growth factor can be maintained, thereby maximizing the effective activity of the growth factor.

In addition, by providing a nanocomposer which is easy to change in volume with a change in temperature, it is possible to provide a nanocomposer having an excellent ability to mount a growth factor.

1 is a schematic view of a method of manufacturing a heparin nanospond according to an embodiment of the present invention.
FIG. 2 shows the particle size (a), surface charge (FIG. 2b) and TEM image (FIG. 2c) of a heparin nanospond according to an embodiment of the present invention.
FIG. 3 shows binding affinity (FIG. 3 a) and cytotoxicity (FIG. 3 b) measurement results of AT-III of heparin nano sponge according to an embodiment of the present invention.
FIG. 4 illustrates the in vivo growth factor release profile of a heparin nano sponge according to an embodiment of the present invention.
FIG. 5 illustrates the growth factor release profile of a heparin nano sponge according to an embodiment of the present invention.
FIG. 6 is a graph showing a bioactivity measurement result of heparin nano sponge according to an embodiment of the present invention.

Hereinafter, the present invention will be described in detail.

In the present invention, a nano-carrier containing heparin having photo-crosslinked thermosensitive properties, which is a chemically stable carrier that constantly releases various growth factors for a long period of time, was synthesized. The specification is hereinafter referred to as " heparin nano sponge ". The heparin nano sponge is prepared by thiol (-SH) -substituted heparin and diacrylated pluronic, and exhibits lower anticoagulant activity than natural heparin. The heparin nano sponge has a volume change characteristic in response to a temperature change useful for high loading of the growth factor. The heparin nano-sponge contains basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), bone morphogenic protein-2 (BMP-2 ) And hepatocyte growth factor (HGF), and can be used for growth factors other than the growth factors described above.

The amount of heparin in each heparin nano sponge was different and the release of growth factors was observed. In addition, the bioactivity of growth factors was analyzed.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

sample.

Heparin (soduim salt, extracted from intestinal mucosa of pigs, molecular weight (Mw) = 12,000 Da) (Cellsus Inc., cincinnati, OH, USA), Pluronic F 127, PF 127) (PEO ₁₀₀ PPO ₆₅ PEO ₁₀₀ (PEO: poly (propylene oxide), Mw = 12,600 Da) (BASF Corp., Seoul, Korea) (HOBT), cysteamine, dithiotreitol (DTT), sodium chloride, potassium phosphate monobasic, sodium phosphate dibasic, sodium azide, Azurea chloride, heptocyte growth factor (HGF (2-hydroxy-2-propyl) ketone (Irgacure 2959) (Ciba Specialty Chemicals Inc., Basel, Switzerland) (Sigma-Aldrich Corp., St. Louis, ), Recombinant human vascular endothelial growth factor (VEGF), recombinant human bone morphogenetic protein 2 (BMP-2), recombinant human fibroblast (BMP-2), Ellman's reagent (Pierce, Rockford, USA), COATEST heparin kit (Chromogenix Inc., Milano, Italy) growth factor basic (bFGF), development ELISA kits (PeproTech, Rocky Hill, NJ, USA).

Culture medium ultraMEM (containing reduced proteins) (LONZA Walkersville, Inc., Basel, MA), Dulbecco's Eagle's medium, Fetal bovine serum (FBS), penicillin- streptomycin, trypsin ethylenediaminetetraacetic acid (EDTA) , Switzerland), Balb / c3T3 fibroblast cell (Korea Cell Line Bank, Seoul, Korea).

Example 1. Synthesis of thiol-functionalized heparin

Thiolated heparin (Hep-SH) is synthesized by the reaction of a carboxylic group of heparin with an amine group of cysteamine. Specifically, heparin is dissolved in deionized water to a concentration of 10 mg / mL. Thereafter, EDC, HOBT and excess cysteamine were sequentially added to prepare a reaction solution having a molar ratio of (heparin: HOBT: EDC: cysteamine = 1: 0.75: 0.75: 2) For 24 hours. Thereafter, the reaction solution was dialyzed against a dialysis membrane (MWCO of 3500 Da, Spectrum lab., CA, USA), and then disulfide groups (disulfide groups) were added to obtain free thiol groups. Lt; RTI ID = 0.0 > (10x molar ratio) < / RTI > Heparin having a free thiol group is purified by dialysis and then lyophilized under reduced pressure. The degree of thiol group substitution in heparin is measured by Ellman's assay at 412 nm.

Example 2. Synthesis of heparin nano sponge

The heparin nano sponge is prepared by photo-polymerization of diacrylated Pluronic F127 (DA-PF 127) and thiolate heparin (Hep-SH). Specifically, the mixture is prepared by diluting DA-PF 127 solution (10 wt%, 154 μL) with 300 μL of deionized water and adding thiolate heparin (~12%). For the manufacture of nanoclubers with various heparin contents, the amount of thiolate heparin is set at 0.5 to 7 mg. Then, 1546 L of deionized water was added to the mixture to prepare a 0.77 wt% mixed solution of DA-PF127. Then, a photoinitiator (Irgacure 2959, 0.05 wt%) was slowly added to 2 mL of the mixed solution, and then UV lamp (unfiltered) (VL -4.LC, 8 W, Vilber Lourmat, France) was added to the mixed solution UV treatment at a strength of 1.3 mW / m < ² > for 15 minutes. Finally, the purified heparin nano sponge is obtained by spin filtration using deionized water (MWCO of 300 kDa) and Nanosep ^® centrifugal devices (MWCO of 300 kDa, Pall Life Sciences, Ann Arbor, MI, USA). The prepared heparin nano sponge is lyophilized under reduced pressure and stored at -80 ° C.

Experimental Example 1. Characteristics of heparin nano sponge

The amount of heparin introduced into the heparin nano sponge is analyzed by spectrophotometry using a cationic dye Azure A capable of binding to an anionic heparin molecule. The lyophilized heparin nano sponge prepared in Example 2 was resuspended in deionized water and then 20 μL was injected into a 96-well plate. Then, the mixed solution is mixed with 180 μL (150 μM) Azure A solution, and the mixed solution is incubated at room temperature for 20 minutes. The absorbance of the mixed solution was measured using a multiwell spectrophotometer (Spectra Max M2, Molecular Devices Inc., USA), and the absorbance was measured at 595 nm. The concentration of heparin introduced into the heparin nano sponge is calculated using the calibration curve of heparin.

Electrophoretic light scattering spectrophotometer (ELS-Z2, manufactured by Otsuka Electronics Co., Japan) equipped with a laser diode light source (638 nm) and a photomultiplier tube detector (165 ° scattering angle) ), The particle diameter (hydrodynamic diameter) and the surface charge (zeta potential) of the heparin nano sponge are measured at 5, 25 and 37 ° C under deionized water, respectively. The morphologies of the heparin nano sponge are measured using a transmission electron microscope (JEM-2100, JEOL, Japan) driven at an accelerating voltage of 200 kV. All measurements were performed 3 times.

Experimental Example 2: Anticoagulant bioactivity of heparin nano-carrier

The anticoagulant bioactivity of heparin nano-carriers was measured using Coatest Heparin kit, which measures the binding affinity of antithrombine Ⅲ (AT Ⅲ) and heparin, and intact heparin and thiolated heparin heparin).

To prepare the precursor solution, 25 μL of samples present in deionized water (intact heparin, thiolate heparin, heparin nano sponge) were mixed with 25 μL of AT III and 200 μL of 0.1 M Tris buffer solution , Followed by incubation at 37 DEG C for 4 minutes. Then, 25 μL of Factor Xa (FXa) is added to 50 μL of each precursor to prepare a mixture and incubated at 37 ° C. for 30 seconds. The mixture is mixed with 50 μL of chromogenic peptide S-2222 (chomogenic peptide S-2222) and incubated at 37 ° C. for 3 minutes. Finally, the reaction is terminated by the addition of 75 μL of acetic acid (20% v / v) and the absorbance of the solution is measured at 405 nm using a multiwell spectrophotometer.

Experimental Example 3: In vitro ( in vitro ) Serum stability and cytotoxic measurement

To determine the serum stability of the heparin nano sponge, the heparin nano sponge was suspended in a DMEM medium containing 10% FBS and 0.05% NaN ₃ , and then incubated in a shaking rocker at 37 ° C and 100 rpm. The size and polydispersity of the heparin nano sponge is then measured for two weeks.

Cytotoxicity of heparin nano sponge and free heparin is analyzed using Balb / c3T3 fibroblast cells. Balb / c3T3 fibroblast cells (passage 2 upon use) were transplanted into a 96-well tissue culture plate (a density 1 × 10 ⁴ cells per well) and cultured in DMEM medium (10% fetal bovine serum ) and 1% penicillin-streptomycin) at 37 ° C for 8 hours under 5% CO ₂ . Each of the intact heparin, thiolate heparin, and heparin nano sponge is then added to the cultured cells in an amount of 10 to 500 μg / mL (based on hepatatin amount). After 24 hours of incubation, the supernatant is removed and the cells are washed with PBS (pH 7.4). Subsequently, the medium is replaced with fresh medium containing 10-time-diluted CCK-8 (cell counting kit-8, Dojindo Laboratories, Kumamoto, Japan) and cultured at 37 ° C for 1 hour. The absorbance of the colored medium is measured at 450 nm using a multiwell spectrophotometer. All measurements were performed in triplicate.

EXPERIMENTAL EXAMPLE 4: In vivo growth factor release profile from heparin nano sponge

Growth factors such as HGF, VEGF, BMP-2 and bFGF are used to analyze whether heparin nanospores can adequately transfer macromolecularly bound heparin.

For the release experiment, add lyophilized powder (200 μg) of heparin nano-sponge to each protein solution (200 ng / 100 μL) and incubate at 4 ° C for over 12 hours. The protein-leaded nanosponge suspension was then placed in dialysis bags (cellulose ester, MWCO of 300 kD) and the dialysis bag was shaken at 37 < 0 > C and 100 rpm Of 0.01% BSA and 0.05% NaN ₃ . To maintain the infinite sink condition, at every measurement time, all release batches were replaced with fresh media. The amount of protein released from the heparin nano sponge is measured using a development ELISA kit. The encapsulation efficiency of the protein in the heparin nano sponge is calculated by measuring the dialysis at 100 rpm for 5 minutes at 37 ° C.

EXPERIMENTAL EXAMPLE 5. Bioactivity analysis of growth factors in heparin or heparin nano sponge

To analyze the bioactivity of bFGF, bFGF-dependent Balb / c3T3 fibroblast cells are used. bFGF-dependent Balb / c3T3 fibroblast cells (passage 2 upon use) were implanted in a 96-well tissue culture plate (a density 1 x 10 ⁴ cells per well) and cultured in DMEM medium (10% fetal bovine serum (FBS) and 1% antibiotics) for 8 hours. After incubation, the cells were washed with PBS and incubated with bFGF (5 or 10 ng / mL) itself (bFGF alone), bFGF (1 μg / mL based on heparin amount) mixed with heparin or heparin And placed in each ultraMEM (1% antibiotics) containing bFGF (1 ug / mL based on heparin amount) mixed with a sponge. After 40 hours of incubation, the supernatant is removed and the cells are washed with PBS. Cell proliferation is measured using the CCK-8 assay at 450 nm in a multiwell spectrophotometer.

Having described specific portions of the present invention in detail, those skilled in the art will appreciate that these specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Results and discussion

1. Preparation and characteristics of heparin nano sponge (Experimental Example 1)

Heparin nano sponge (Hep-NS), which is a stable carrier capable of efficiently transferring growth factors, was synthesized. Referring to FIG. 1, the heparin nano sponge was synthesized by UV-initiated thiol-ene reaction of thiolate heparin (Hep-SH) and diacrylate pluronic F127. This single-step photopolymerization method produces a mono-dispersed nano-carrier with relatively high binding reaction yield. The content of heparin in the heparin nano sponge can be appropriately adjusted to 3 to 24 wt% by adjusting the concentration of Hep-SH (mass ratio of thiolate heparin to pluronic polymer) input during the reaction. When the content of heparin exceeds 24 wt%, aggregation as well as unstable heparin nano sponge are synthesized.

Hep (3) -NS, Hep (6) -NS, Hep (15) -NS, Hep (24) -NS) with a heparin content of 3 to 24 wt% (Heparin nano sponge) at 51 ° C ± 57 nm at 5 ° C, 141 ± 12 nm at 25 ° C and 58 ± 6 nm at 37 ° C, And thermo-sensitive properties. The content of heparin in the heparin nano sponge does not affect the temperature dependence of size and volume change.

However, referring to FIG. 2 (b), the surface charge of the heparin nano sponge gradually increases from -4.1 ± 0.6 mV to -29.1 ± 1.5 mV as the content of heparin increases. This means that the high negatively charged heparin is located on the surface of the nano sponge.

Referring to FIG. 2 (3), the TEM image of the heparin nano sponge shows similar size and morphology, and the presence of heparin does not affect the morphology and size.

2. Anticoagulant bioactivity of heparin nano sponge (Experimental Example 2)

Anticoagulant bioactivity of heparin itself (intact heparin, thiolate heparin) or heparin nano sponge was measured by FXa inhinition assay. Referring to FIG. 3 (a), the anticoagulant bioactivity of thiolate heparin (Hep (-SH)) was somewhat reduced compared to intact heparin (Hep). This means that the binding affinity with AT-III is reduced. However, still significant bioactivity of the anticoagulant is maintained. This is consistent with the conventional result that the sulfate group of heparin affects the bioactivity of the adherent solids.

Heparin nano sponge (Hep (3) -NS, Hep (6) -NS, Hep (15) -NS, Hep (24) -NS) shows that the bioactivity of the adherent solids was significantly reduced below 35%. This means that the side effects of heparin (hyperhidrosis) associated with anticoagulants are effectively inhibited.

3. In vitro ( in vitro ) Serum Stability and Cytotoxic Measurement (Experimental Example 3)

The in vitro serum stability of heparin-bound nano-carriers (NC) and heparin nano-sponge (Hep-NS) was measured at 100 rpm and 37 ° C in serum containing cell culture medium. As shown in Table 1 below, the size and dispersion of all heparin nano sponges remained constant for two weeks. This means that the heparin nano sponge can be stabilized without forming major aggregation in the in vivo environment.

weeks 0 One 2 NC 58 ± 6 nm
(0.25 + - 0.01) 61 ± 1 nm
(0.26 0.02) 60 ± 2 nm
(0.27 ± 0.02) Hep (3) -NS 63 ± 8 nm
(0.27 ± 0.02) 65 ± 6 nm
(0.28 + - 0.01) 67 ± 6 nm
(0.28 0.02) Hep (6) -NS 63 ± 9 nm
(0.28 0.02) 62 ± 3 nm
(0.28 0.02) 65 ± 3 nm
(0.29 0.02) Hep (15) -NS 67 ± 4 nm
(0.30 + - 0.01) 64 ± 3 nm
(0.29 0.02) 69 ± 4 nm
(0.29 + 0.03) Hep (24) -NS 74 ± 6 nm
(0.31 + - 0.01) 71 ± 5 nm
(0.30 + - 0.01) 73 ± 4 nm
(0.29 0.02)

In addition, the acute cytotoxic effect of heparin nano-sponge with intact heparin (intact heparin, thiolate heparin) or various heparin concentrations in Balb / c3T3 fibroblast cells was measured by the CCK-8 assay.

3 (b), nano sponge (Hep (24) -NS) conjugated with intact heparin (Hep), thiolate heparin (Hep (-SH) mL did not affect the metabolic activity of the cells up to the baseline on heparin amount. Heparin itself (intact heparin, thiolate heparin) can induce apoptosis at a high concentration (500 ㎍ / mL), which is somewhat cytotoxic, but heparin nano sponge has cell viability of over 90% , Indicating that it does not cause cytotoxicity even at high concentrations. Heparin nano-sponges with different amounts of heparin were also found to be less cytotoxic than heparin itself.

These results suggest that heparin nanosponses can minimize the potential problems associated with the safety of heparin in vivo applications.

4. Growth factor release profile in vivo from heparin nano sponge (Experimental Example 4)

In order to investigate the release efficiency of growth factors according to heparin content in heparin nano sponge, hepatocyte growth factor (HGF) was analyzed as a heparin-binding growth factor. The acidic growth factor (HGF) was more than 90% efficiently loaded on the heparin nano sponge and did not cause a change in the size and surface charge of the heparin nano sponge.

Referring to Figure 4, the pattern of release of growth factors from heparin nanospons in aqueous buffer (PBS) depends on the concentration of heparin. (Hep (3) -NS, Hep (6) -NS, Hep (15) -NS) and heparin nano-sponge at different concentrations of heparin Compared to one month (1 month) or more.

5, VEGF, HGF, BMP-2, and HGF were analyzed to analyze the release pattern of bare nano-carrier (NC) and heparin (24 wt% Four kinds of growth factors of bFGF were used. Heparin nano sponge (Hep-NS) shows a constant release profile compared to bare nano-carrier (NC). This means that the growth factor is superior to the heparin nano-sponge in interaction with heparin. Among the four growth factors, VEGF and HGF, which have relatively higher acceptance than BMP-2 and bFGF, show very rapid release from heparin nano sponge. This is because the low water soluble protein has low water solubility for pluronic based nanocarriers with rich PEG.

5. Analysis of bioactivity of growth factors in heparin nano sponge (Experimental Example 5)

To investigate the enhanced bioactivity of the growth factors by heparin polymer, Balb / c3T3 fibroblast cells were incubated with bFGF alone or in combination with bFGF or heparin (24 wt%) mixed with heparin, as in Example 5, Lt; RTI ID = 0.0 > bFGF < / RTI > And then analyzed by CCK-8 assay.

Referring to FIG. 6, the bioactivity of bFGF mixed with heparin (Hep (24) -NS) combined with heparin (24 wt%) was maintained at 5 and 10 ng / mL, respectively. On the other hand, bFGF alone showed very low bioactivity compared to the results containing heparin polymer. This means that heparin can maintain and enhance the bioactivity of bFGF. To increase cellular metabolic activity compared to natural heparin, heparin nanosponge exhibits almost similar biological effects. This means that the heparin nano sponge maintains the biological function of heparin.

Sintering.

The heparin nano sponge containing heparin according to the present invention exhibits the following effective characteristics as a whole. 1) It shows excellent volume change behavior with temperature change to enable easy loading of active ingredient and excellent loading ability. 2) Minimal anticoagulant bioactivity and heparin bleeding compared with heparin. 3) induce low apoptosis compared with heparin polymer, 4) maintain the release profile of the growth factor, and maintain the bioactivity of the growth factor.

Claims (8)

The present invention relates to a heparin nano sponge for sustained release of growth factors,
The heparin nano sponge may be thiolated heparin; And a pluronic polymer bonded with the thiolate heparin,
Wherein the thiolate heparin is present in an amount of 24 wt% based on the total weight of 100 wt%.
delete The method according to claim 1,
Wherein the heparin nano sponge has a size of from 600 nm to 50 nm at 5 to 37 占 폚.
The method according to claim 1,
Wherein the heparin nano sponge has a surface charge of -4.0 mV to -40 mV.
The method according to claim 1,
The growth factors include basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), bone morphogenic protein-2 (BMP-2) And hepatocyte growth factor (HGF). ≪ RTI ID = 0.0 > 18. < / RTI >
(a) synthesizing thiolated heparin (Hep-SH) by introducing a thiol group (-SH) into heparin;
(b) mixing the thiolate heparin and the pluronic polymer to prepare a mixture; And
(c) adding a photoinitiator to the mixture and treating the mixture with ultraviolet light. 6. The method according to claim 1,
The method according to claim 6,
Wherein the pluronic polymer comprises a photo-crosslinkable functional group. ≪ RTI ID = 0.0 > 18. < / RTI >
8. The method of claim 7,
The photocrosslinkable functional groups include, but are not limited to, acrylate, diacrylate, oligo-acrylate, methacrylate, dimethacrylate, oligomethacrylate wherein the heparin is selected from the group consisting of oligo (meth) acylate, coumarin, thymine, and cinnamate.

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