KR100715815B1 - Method for preparing chitosan-poloxama complex - Google Patents

Abstract

The present invention relates to a method for producing a chitosan-poloxama complex, and more specifically, (a) adding triethylamine and acryloyl chloride to benzene containing poloxama and stirring; (b) filtering the stirred solution to remove triethylamine-hydrochloride, and then adding the solution to a hexane solution to form poloxama macroma; And (c) adding a photoinitiator to the solution of chitosan mixed with poloxama macroma, irradiating with UV, and lyophilizing to form a chitosan-poloxama complex. It is about.

The chitosan-poloxama complex according to the present invention has an effect of providing a wound coating agent having fast water absorption, high mechanical strength, and excellent wound healing ability by stabilized hydrogen bonding of chitosan and poloxama.

Chitosan, Poloxama Macroma, Complex

Description

Method for Preparing Chitosan-Poloxama Composite {Method for Preparing a Chitosan-Poloxamer Composite}

Figure 1 illustrates the synthesis of poloxama macroma according to the present invention.

Figure 2 illustrates the structure of Semi-interpenetrating Polymer Networks according to the present invention.

Figure 3 shows the H-NMR spectrum of the poloxama macroma of the present invention.

Figure 4 shows the FT-IR spectrum for the chitosan-poloxama complex according to the present invention (a: chitosan, b: poloxama macroma, c: chitosan-poloxama (1/1) mixed, d: chitosan- Poloxama (1/1) complex).

Figure 5 shows the X-ray diffraction spectrum of the chitosan-poloxama complex according to the present invention (a: chitosan, b: poloxama macroma, c: chitosan-poloxama (1/1) mixed, d: chitosan- Poloxama (1/1) complex, e: chitosan-poloxama (1/2) complex, f: chitosan-poloxama (1/3) complex).

6 is a graph showing the compressive strength of chitosan, chitosan-poloxama mixture, chitosan-poloxama complex (a: chitosan, b: chitosan-poloxama (1/1) mixture, c: chitosan-poloxama ( 1/1) complex, d: chitosan-poloxama (1/2) complex, e: chitosan-poloxama (1/3) complex).

7 is a graph showing the swelling degree of the chitosan-poloxama complex with time (□: chitosan, ○: chitosan-poloxama (1/1) complex, Δ: chitosan-poloxama (1/2) complex,?) Chitosan-poloxama (1/3) complex).

FIG. 8 is a 200-fold magnification of SEM showing the microstructure of the surface and cross-section of chitosan-poloxama composites prepared in different configurations after freezing at −20 ° C. (a and d: chitosan-poloxama (1/1) Complex, c and e: chitosan-poloxama (1/2) complex, e and f: chitosan-poloxama (1/3) complex).

9 is a 200-fold magnification of SEM showing the microstructure of chitosan-poloxama complex prepared according to freezing temperature (a: -20 ° C, b: -70 ° C, c: -196 ° C).

Field of invention

The present invention relates to a method for producing a chitosan-poloxama complex, and more specifically, (a) adding triethylamine and acryloyl chloride to benzene containing poloxama and stirring; (b) filtering the stirred solution to remove triethylamine-hydrochloride, and then adding the solution to a hexane solution to form poloxama macroma; And (c) adding a photoinitiator to the solution of poloxama macroma and chitosan, irradiating with UV, and lyophilizing to form a chitosan-poloxama complex. It is about.

Background of the Invention

Chitosan is a kind of aminopolysaccharide existing in nature. It is a natural polymer composed of glucosamine and acetyl glucosamine and is the most abundant polysaccharide in nature. Chitosan can also be easily obtained from crustaceans, including shrimp and crabs, which produce 7 billion tonnes per year, and deacetylates chitin contained in the cell walls of microorganisms such as shells, squid bones, mold, mushrooms, and bacteria. It is a natural substance obtained by non-toxic and biodegradable and biocompatible.It has biological properties such as cell binding and tissue culture, antimicrobial activity, hemostatic action, cholesterol lowering effect, intestinal metabolism, and immune enhancement. It is known to have physiological effects such as anticancer action, liver function improvement and hypoglycemic action, and heavy metal detoxification. Chitin is contained in the cell walls of fungi such as crustaceans, insect uppers, and fungal yeast mushrooms, and consists of tissues such as ash, proteins, lipids, and pigments, centered on potassium carbonate.

Chitin has an acetyl amino group in the molecule, so the hydrogen bond between molecules is very strong, resistant to chemicals, insoluble in water and organic solvents, it is difficult to form a useful form of fiber, film and the like. Therefore, by deacetylating chitin and converting it into water-soluble chitosan in a weakly acidic liquid, chitosan is industrially used more usefully than chitin.

Chitosan is widely used and studied in a variety of fields, including drug delivery systems, chromatography materials, surgical operating rooms, scaffolds and wound coating agents used in tissue engineering. In particular, chitosan induces collagen synthesis by fibroblasts to accelerate the elasticity of wound skin, promote homeostasis and tissue regeneration. In addition, chitosan has an antimicrobial effect against bacteria and fungi. For these reasons, chitosan has recently been in the spotlight as one of the important biomaterials for wound healing.

However, in spite of many of the advantages described above, chitosan itself has a low mechanical strength, which limits its use as a wound coating agent. In recent years, there has been an effort to overcome the low mechanical strength of chitosan by mixing or crosslinking with other synthetic and natural polymers such as polyvinyl pyrrolidone, polycaprolactone, polyethylene glycol, collagen, and the like.

Conventional patents related to chitosan include 'moisturizing chitosan-hydroxy acid complex compounds and aqueous solution compositions thereof' (Korean Patent Application No. 10-2005-0053904, 2005.08.22) and chitosan-acetylsalicylic acid (aspirin) salt compounds. Method and chitosan-acetylsalicylate compound prepared therefrom (Korean Patent Application No. 10-0467764, 2005.01.13) and the like. However, the patent provides a chitosan-hydroxy acid complex compound in which the drug release rate is uniform in the process of biodegradation and dissolution, and the amount of drug release is controlled, but still has a problem of low mechanical strength.

Accordingly, the present inventors have made diligent efforts to solve the shortcomings of the prior art. As a result, the poloxama macromama was prepared and mixed with chitosan, and then irradiated with UV in the presence of a photoinitiator to form the poloxama network and chitosan as SIPNs. The present invention was completed by preparing a chitosan-poloxama composite and confirming that the composite not only had excellent mechanical strength, but also had an excellent wound coating effect.

It is a main object of the present invention to provide a chitosan-poloxama composite having excellent mechanical strength and a method for producing the same.

Another object of the present invention to provide a wound coating agent containing chitosan-poloxama complex as an active ingredient.

In order to achieve the above object, the present invention (a) adding triethylamine and acryloyl chloride to benzene containing poloxama and stirring; (b) filtering the stirred solution to remove triethylamine-hydrochloride, and then adding the solution to a hexane solution to form poloxama macroma; And (c) adding a photoinitiator to the solution of chitosan mixed with poloxama macroma, irradiating with UV, and lyophilizing to form a chitosan-poloxama complex. do.

In the present invention, after the step (b) it may be characterized in that it further comprises the step of drying the formed poloxama macroma at 40 ℃ for 24 hours.

In the present invention, after the step (c) it may be characterized in that it further comprises the step of washing the distilled water after the chitosan-poloxama complex formed in a base solution.

In the present invention, the molecular weight of the chitosan is 5000 ~ 1000000 g / mol, deacetylation degree can be characterized in that 50 to 98%, the photoinitiator 2.2-dimethoxy-2phenyl acetophenone and N- It may be characterized by containing vinyl pyrrolidone.

The present invention also provides a chitosan-poloxama complex prepared by the above method and having a SIPNs structure and a wound coating agent containing the chitosan-poloxama complex as an active ingredient.

Hereinafter, the present invention will be described in detail.

In order to prepare the chitosan-poloxama complex according to the present invention, poloxama macroma was first prepared by the following method. The poloxama was dissolved in a round flask containing benzene, and triethylamine and acryloyl chloride were added thereto, stirred at 80 ° C. for 3 hours, and then the stirred solution was filtered to remove triethylamine-hydrochloride, and hexane Poloxama macroma was added to the solution (Fig. 1).

Next, chitosan was added to the obtained poloxama macroma, and then a photoinitiator was added to the mixed solution, and exposed to an LWUV lamp for 5 minutes to obtain a chitosan-poloxama complex.

Chitosan used in the present invention is a deacetylated chitin, a molecular weight of 5000 ~ 1000000 g / mol, a deacetylation degree of 50 to 98% was used. In general, the deacetylation degree is at least 50% to become chitosan, and when the deacetylation degree is at least 79%, it is very difficult to obtain chitosan, and when the molecular weight of chitosan is 1,000 g / mol or less, the characteristics of the polymer chitosan as oligosaccharides are reduced. do.

Lyophilization used in the method for producing the chitosan-poloxama complex is a method of freezing the material and then drying it by sublimation with gaseous vapor without passing through a liquid state in a high vacuum apparatus. The freeze-drying method consists of three stages of pre-freezing, sublimation drying, and dehumidification drying, and can obtain a much higher quality product than the general drying method, and since the moisture is removed in the frozen state, the dried product has a light film structure. It is currently used in the drying of food, pharmaceuticals and microorganisms, and it is a drying method to obtain high quality products.

Semi-interpenetrating polymer networks (SPINs), or composites made from silk fibroin and poloxama macroma, are those in which one or more polymers form a branched or linear network. It is formed by a crosslinking method by radiation or chemical method or a polymerization method of a protomeric monomer using a crosslinking agent.

Such a composite according to the present invention has the advantage of improving the mechanical strength of the network and mixing the materials that are phase-separated, the chitosan-poloxama composite has a high water absorption, high mechanical strength, due to the porosity of the composite It can be used as a wound coating agent having excellent wound healing ability.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and the scope of the present invention will not be construed as being limited by these examples.

Example 1 Synthesis of Poloxama Macroma

Poloxama 407 (25.2 g, 2 mmol) was dissolved in a round flask containing 75 ml benzene, and triethylamine (0.59 ml, 4.23 mmol) and acryloyl chloride (0.34 ml, 4.18 mmol) were added. Stir at 3 ° C. for 3 hours. Subsequently, after filtering to remove triethylamine-hydrochloride, the solution was added to hexane solution to obtain poloxama macroma, and the prepared polosama macroma was dried at 40 ° C for 24 hours.

Example 2: Preparation of Chitosan-Poloxama Complex

100 mg 2.2-dimethoxy-2phenyl acetophenone and 1 mL of N- in a solution of 1 to 3 wt% poloxama macroma and 1 wt% chitosan prepared in Example 1 in a solvent of acetic acid solution (1 wt%). 25 μl photoinitiator containing vinyl pyrrolidone was added. And after exposure to LWUV lamp (Toshiba Chemical Lamp FL 20 LB; wave range, 300-400nm; maximum intensity, 360nm) for 5 minutes, and lyophilized to prepare a chitosan-poloxama complex, the prepared composite 2wt% The solution was added to NaOH solution for 30 minutes and then washed well with distilled water.

Example  3: chitosan- according to the present invention Poloxama  Complex spectrum analysis

(1) H- NMR spectrum

The H-NMR spectrum of the chitosan-poloxama complex prepared in Example 2 was measured at 600 MHz with an AVAVCE 600 spectrometer (Bruker, Rheinstten, Germany) (FIG. 3).

As a result, as shown in Figure 3, the typical hydrogen signal according to the methyl group of the polypropylene oxide appeared at 1.19ppm of poloxama, the hydrogen in the acrylate of the poloxama macroma sock end was found in 6.02 ~ 6.50ppm. The H-NMR analysis showed that poloxama macroma was synthesized through the reaction between the terminal hydroxyl group (-OH) of poloxama and acryloyl chloride, and the degree of substitution of poloxama macroma was calculated as 87.2%. .

(2) FT - IR spectrum

Infrared spectrophotometer (FT-IR) spectrophotometer used Nicoloet Magna 550 series II spectrometer (Midac, Atlanta, GA, USA), because the stretching vibration of chitosan hydroxyl group and -NH group overlaps at 3000 ~ 3600cm ^-1 It was measured by focusing on 500 ~ 2000cm ^-1 (Fig. 4).

 As a result, as shown in FIG. 4, the intermolecular interaction occurs between the two polymers during the preparation of the complex. After formation of the complex, hydrogen bonding occurs between C = O of chitosan and hydroxyl groups of poloxama. This can be seen as the result of the overlap of C-O stretching vibrations of chitosan and poloxama compared to chitosan before poloxama was added. In addition, it was found that after the chitosan and poloxama were mixed, the formation of the chitosan-poloxama complex caused an intermolecular hydrogen bond between them.

On the other hand, amide and amine bands of chitosan were found at 1630 and 1524 cm ⁻¹ , respectively, and ester peaks of CO stretching vibration in the main chain of poloxama were found at 1152 and 1090 cm ⁻¹ , respectively. C = O stretching vibration of the SIPNs after forming, chitosan (1630㎝ ^-1) showed a little high in 1633㎝ ^-1. This can be seen taken place the hydrogen bonding has occurred between the chitosan results from the C = O and -OH Pollock Sima Yi there was. In addition, the peak around 1110 cm ^-1 indicates COC stretching vibration, which is a characteristic band of poloxama, as shown in (c) and (d) of FIG. 4, respectively, 1108 and 1106 cm ^-1 . This is higher than 1090 cm ⁻¹ shown in (a) of FIG. 4, which is a superimposed result of CO stretching vibration of chitosan and poloxama as compared to chitosan before poloxama was added. These results confirm that the formation of SIPNs by mixing chitosan and poloxama induces intermolecular hydrogen bonds between them.

(3) X-ray Diffraction analysis  Measure

X-ray patterns of chitosan, poloxama macroma, and chitosan-poloxama complexes were analyzed using CuKα radiation (GADDS; Bruker-AXS D5005, Karlstuhe, Germany) by radiation of wide-angle X-rays in the general diffraction range It was. The X-ray source was operated at 35kV and 20Hz, and the diffraction was measured by reflectance angle through 2 ° / min scan rate. X-ray diffraction analysis was carried out to analyze the crystal structure affected by the various elements of the polymer in the solid state, it was possible to observe the change in the crystal structure of chitosan and poloxama (Fig. 5).

As shown in FIG. 5, the poloxama macroma and chitosan-poloxama complex showed typical diffraction peaks at Bragg angles (2θ) of 18.9, 23.0, and 26.5 °, respectively, for crystal spacing of 4.62, 3.86, and 3.40 mm 3. Moreover, this peak was observed even after complex formation from chitosan. However, the crystallinity peak of poloxama in the chitosan-poloxama complex decreased due to the increase in the content of chitosan. This result indicates that crystallization of poloxama is hampered by chitosan penetrated into the crosslinked poloxama.

On the other hand, the crystallinity of the chitosan-poloxama (1/1) mixture was higher than that of the chitosan-poloxama complex under the same conditions, even though the mixing of the two polymers did not affect the diffraction pattern. In general, rearrangement of polymers by crosslinking increases amorphos activity, which impedes crystallization. Therefore, even though the interference by chitosan was more dominant than the crosslinking of poloxama macroma, the crystallization of chitosan-poloxama complex was prevented by these two phenomena.

Example  4: chitosan- according to the present invention Poloxama  Property analysis of the complex

(1) chitosan- according to the present invention Poloxama  Composite thermal properties

Melting point and thermal decomposition point were measured using DSC 2910 (TA Instrument Co., Leatherhead, UK) to measure the thermal properties of the chitosan-poloxama complex according to the present invention. Chitosan, poloxama macroma, a mixture of the two polymers and the thermal properties of the chitosan-poloxama complex are shown in Table 1 below.

Thermal Properties of Chitosan, Poloxama Macroma, Chitosan-Poloxama Blend, and Chitosan-Poloxama Complex Sample (w / w) Td (℃) Tm (℃) Melting enthalpy (J / g) Chitosan 255.4 Not measured Not measured Poloxama Macroma Not measured 53.9 135.0 Chitosan-Poloxama (1/1) Blend 246.6 50.5 64.7 Chitosan-Poloxama (1/1) Complex 232.3 38.3 10.2 Chitosan-Poloxama (1/2) Complex 219.2 42.3 40.2 Chitosan-Poloxama (1/3) Complex 203.0 45.5 49.1

As shown in Table 1, the melting point of poloxama in chitosan-poloxama mixture and chitosan-poloxama complex was lower than that of poloxama alone. In general, the melting point of the previously crystallized crystal component is higher than when it is taut, thus reducing the crosslinking of poloxama macroma and melting point into the penetrated chitosan.

On the other hand, the melting point of poloxama in the chitosan-poloxama complex increased with increasing poloxama content. In the chitosan-poloxama mixture or complex, the thermal decomposition point of chitosan was lower than that of chitosan alone. In addition, the thermal decomposition point of the chitosan-poloxama complex decreased with increasing poloxama.

(2) chitosan- according to the present invention Poloxama  Composite mechanical strength

When applied to medical applications, including wound coatings and artificial skin, mechanical strength is the most important factor to withstand various types of stress.

The compressive strength of chitosan, poloxama macroma, and chitosan-poloxama complex was measured using Minimat (Rheometric Scientic, Piscataway, NJ, USA) (FIG. 6). As a result, as shown in Figure 6, the chitosan-poloxama mixture and the resistance to pressure of chitosan-poloxama complex prepared under the same conditions showed a big difference. This is because a network of poloxama macromas is formed. In addition, the compressive strength also increased because the crosslinking density increased with increasing poloxama content in the chitosan-poloxama complex. These results show that by the formation of chitosan-poloxama complexes, there is a significant difference in mechanical strength according to the poloxama content in the chitosan-poloxama complex.

(3) chitosan- according to the present invention Poloxama  Composite swelling degree

The swelling degree of the wound coating agent is an important factor for the rapid absorption of the exudate. The swelling degree of the chitosan-poloxama complex was measured with different conditions to measure the swelling degree of the chitosan-poloxama complex.

In order to investigate the swelling degree of the chitosan-poloxama complex prepared in Example 2, the dried sample was placed in PBS (pH 7.4) at 37 ℃, and weighed at regular intervals, the swelling degree is Calculated (FIG. 7).

Swelling degree (%) = Swelled  Weight-Initial Weight / Initial Weight X 100

Chitosan is a cationic polysaccharide with good binding to water, and has abundant hydroxyl groups necessary for hydrogen bonding, and the poloxama network can contain sufficient water in its structure as an aqueous sulfide gel. The poloxama complex swells rapidly and reached equilibrium within 5 minutes. In addition, due to the increased crosslink density of poloxama in the chitosan-poloxama complex, the degree of swelling in equilibrium decreased with increasing poloxama content.

(4) chitosan- according to the present invention Poloxama  Surface and cross-sectional shape of the composite

In order to confirm the morphology of the surface and cross-section of the chitosan-poloxama complex, the chitosan-poloxama complex was first plated with gold and then observed with a scanning probe microscope (JSM-5410LV, JEOL, Japan). The cross section of the sample was observed after being completely frozen through liquid nitrogen (Fig. 8, Fig. 9).

FIG. 8 is a 200-fold magnification of SEM showing the microstructure of the surface and cross-section of chitosan-poloxama composites prepared by different configurations after freezing at −20 ° C., and (a) and (d) are chitosan-poloxama (1/1) complex, (b) and (e) are chitosan-poloxama (1/2) complexes, and (c) and (f) are chitosan-poloxama (1/3) complexes and surface It shows the form of.

As the poloxama content of the chitosan-poloxama complex increased, the micropore size decreased while the micropore density increased slightly. Thus, from the above results, the degree of porosity is determined by the poloxama content since ice nuclei are formed through the freezing point drop caused by energy exchange. The faster the freezing, the shorter the time the ice cores form and grow. The micropore size is determined by the size of the ice nucleus, since the evaporation of the ice from the frozen chitosan-poloxama complex leaves a micropore of the same size as the ice.

FIG. 9 is a 200-fold magnification of SEM showing the microstructure of chitosan-poloxama complex prepared according to freezing temperature, showing the cross-sectional shape according to temperature under the same conditions of chitosan-poloxama complex, (a) Is the cross-sectional shape of the chitosan-poloxama complex at -20 ° C, (b) is the cross-sectional shape of the chitosan-poloxama complex at -70 ° C, and (c) is the cross-sectional shape of the chitosan-poloxama complex at -196 ° C. .

The micropore diameter decreases with decreasing freezing point, and if the temperature between the ice cores is low, the ice cubes do not grow well because the faster freezing, the less chance of ice particles growing in the chitosan-poloxama complex. .

Having described the specific parts of the present invention in detail, it will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. will be. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

As described in detail above, the present invention has an effect of providing a chitosan-poloxama composite having excellent mechanical strength and a method of manufacturing the same. The chitosan-poloxama complex according to the present invention is fast in water absorption, has high mechanical strength, and has excellent wound healing ability by stabilized hydrogen bonding of chitosan and poloxama, and thus is useful as a wound coating agent. In addition, it can be widely used in all fields such as food, medical medicine, biotechnology, cosmetics, agriculture, chemicals, and environment.In particular, the purpose of protecting or treating skin wounds such as the development of artificial skin for tissue engineering Can be used effectively.

Claims (7)

Method for preparing chitosan-poloxama complex comprising the following steps: (a) adding triethylamine and acryloyl chloride to benzene containing poloxama and reacting with stirring; (b) filtering the reaction solution to remove triethylamine-hydrochloride, and then precipitating poloxama macroma in hexane solution; (c) drying the precipitated poloxama macroma at 40 ° C. for 24 hours; And (d) A photoinitiator containing 2.2-dimethoxy-2phenyl acetophenone and / or N-vinyl pyrrolidone is added to the solution of chitosan mixed with the poloxama macroma, irradiated with UV, and then lyophilized. Forming a poloxama complex. delete The method of claim 1, further comprising the step of (d) following the step of the chitosan-poloxama complex formed in a base solution and then washing with distilled water. The method of claim 1, wherein the chitosan has a molecular weight of 5000 to 1000000 g / mol and a degree of deaselthiation of 50 to 98%. delete delete A wound coating agent prepared by the method of any one of claims 1, 3, and 4, and containing a chitosan-poloxama complex having an SIPNs (Semi-Interpenetrating Polymer Networks) structure as an active ingredient.

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