KR101664415B1 - A Collagen Wound Dressing Containing Drugs and Methods for Preparing thereof - Google Patents

Abstract

The present invention relates to a method for producing a collagen wound dressing containing a drug and a collagen wound dressing prepared thereby. The wound dressing prepared by the method of the present invention has a high biocompatibility and can carry a drug, which can prevent infection from foreign substances, which is a disadvantage of conventional wound dressings, and can release the loaded drug stepwise And thus it is possible to reduce the adverse reaction in the tissue caused by the drug. In addition, the method of the present invention is advantageous in that a wound dressing can be produced easily and at a low cost compared to a conventional collagen wound dressing preparation method.

Description

Technical Field [0001] The present invention relates to a collagen wound dressing containing a drug and a method for producing the collagen wound dressing,

The present invention relates to a collagen wound dressing material capable of releasing a drug and a method for producing the same.

Collagen is a structural protein component that constitutes soft tissues, bones and hard tissues such as dermis, tendon / ligament and blood vessels, and in mammals it accounts for about one-third of the total protein .

Collagen is known to have more than 20 forms, and type I collagen, which makes up the skin, tendons / tendons and bones, accounts for about 90% of the collagen.

Collagen is a 300,000 daltons protein with a structure consisting of three proteins with a molecular weight of 100,000 daltons. Glycine (Gly), which has the smallest molecular weight among amino acids, is repeatedly linked.

Currently, collagen is used as a medical material in fields such as hemostatic agents, wound dressings, artificial blood vessels and wrinkle reducing materials.

The wound dressing of the above medical materials is applicable to localized wounds or wounds of less severe depth of cut. It can be wound for 3-4 weeks to protect the wounds during the period until the food is ready for use or to complete the self-cultured skin However, there is a problem that the risk of secondary infections due to foreign substances such as microorganisms and bacteria increases during 3-4 weeks when the wound dressing is transferred.

Gentamicin is an aminoglycoside antibiotic that is used to treat bacterial infections caused by Gram-negative bacteria and is irreversibly bound to bacterial ribosomes, which inhibits protein synthesis (Robert and Melanie, 2010) Are limited to low doses (Cabanes et al., 1998). (Megoulas and Koupparis, 2004), efforts are underway to reduce the systemic toxicity of gentamicin (Lovering and Sunderland, 2010; Hayes et al., 2013). In addition, gentamicin is a new form of gentamicin with low systemic toxicity and less local irritation due to severe local irritation after injection (Rubinstein, 1983; Abdelbary and El-Gendy, 2008).

Collagen sponges containing antibiotics have been proven effective in treating soft tissue and bone infections and have the advantage of minimizing the risk of systemic toxicity (Stemberger et al., 1997). Despite the fact that the main function of gentamicin-containing collagen implants is the hemostatic effect, many studies have reported a significant reduction in infection at the surgical site (Rutten and Nijhuis, 1997; Friberg et al., 2005) (Singisetti and Ashcroft, 2009; Lovering and Sunderland, 2012; Simpson et al., 2012), which has been used to minimize systemic toxicity with a gentamicin-containing collagen sponge.

Conventionally, a collagen dispersion is dispensed to a predetermined thickness and then lyophilized to produce a porous collagen sponge. Then, a porous collagen sponge is crosslinked using a crosslinking agent to prepare a collagen wound dressing. Such a conventional method requires washing of the collagen sponge prepared by freeze-drying after crosslinking to remove the crosslinking agent, and there is a problem that freeze-drying must be performed again after cleaning.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The inventors of the present invention have found that a method of protecting wounds due to surgical operation, infection, wound, hemorrhage and inflammation and preventing wounds from external substances, which is a disadvantage of the conventional wound dressings described above, and further making wound dressings easy and at low cost We have tried to develop our example. As a result, when a collagen wound dressing material is produced by crosslinking collagen in a dispersion state and then lyophilized to form a porous collagen sponge, a porous collagen sponge prepared by a conventional freeze-drying method is prepared and crosslinked It is possible to reduce the manufacturing time by carrying out lyophilization once in comparison with the method, and it is possible to carry a drug capable of preventing the infection, and the collagen wound dressing bearing the drug can continuously release the drug over a certain period of time Thereby confirming the completion of the present invention.

It is therefore an object of the present invention to provide a method for producing a collagen wound dressing comprising a drug.

Another object of the present invention is to provide a collagen wound dressing comprising a drug.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention provides a method of making a collagen wound dressing comprising a drug comprising the steps of:

(a) immersing collagen in an acidic solvent to prepare a collagen solution;

(b) pulverizing the collagen solution to prepare a collagen dispersion, and crosslinking the collagen in the collagen dispersion;

(c) adding a drug to the result of step (b); And

(d) lyophilizing the result of step (c) to produce a porous collagen sponge comprising the drug.

According to another aspect of the present invention, there is provided a collagen wound dressing comprising a drug prepared by the method of the present invention.

The inventors of the present invention have found that a method of protecting wounds due to surgical operation, infection, wound, hemorrhage and inflammation and preventing wounds from external substances, which is a disadvantage of the conventional wound dressings described above, and further making wound dressings easy and at low cost We have tried to develop our example. As a result, when a collagen wound dressing material is produced by crosslinking collagen in a dispersion state and then lyophilized to form a porous collagen sponge, a porous collagen sponge prepared by a conventional freeze-drying method is prepared and crosslinked It is possible to reduce the manufacturing time by carrying out lyophilization once in comparison with the method, and it is possible to carry a drug capable of preventing the infection, and the collagen wound dressing bearing the drug can continuously release the drug over a certain period of time Respectively.

Accordingly, the present invention is directed to a method of making a collagen wound dressing comprising a drug, particularly a collagen wound dressing comprising an infection-preventing drug.

As used herein, the term "wound dressing" refers to a dressing or matrix that can adhere to a wound due to surgical procedures, infection, wound, bleeding and inflammation.

The method of the present invention will be described in detail in accordance with the respective steps as follows:

Step (a): The collagen is immersed in an acidic solvent

According to the present invention, collagen is first immersed in an acidic solvent.

According to one embodiment of the present invention, the pH of the acidic solvent of the present invention is 1.0-3.0. According to another embodiment of the present invention, the pH of the acidic solvent of the present invention is 1.5-3.0, 2.0-3.0, 2.3-3.0 or 2.3-2.8.

According to one embodiment of the present invention, the acidic solvent of the present invention is hydrochloric acid (HCl) or acetic acid.

The collagen of the present invention includes any collagen in the art used as a raw material for wound dressings, including, but not limited to, collagen extracted from skin or ligaments such as pigs, horses, and cows. According to one embodiment of the present invention, the collagen of the present invention is collagen extracted from cattle. According to another embodiment of the present invention, the collagen is a collagen extracted from a bovine ligament. According to an embodiment of the present invention, Is a type I collagen extracted from bovine ligaments.

According to one embodiment of the present invention, the collagen solution of the present invention has a collagen content of 0.3-1.0% (w / v). According to another embodiment of the present invention, the collagen solution of the present invention has a collagen content of 0.3-0.7, 0.4-0.7, 0.4-0.6 or 0.5-0.6% (w / v).

According to one embodiment of the present invention, the collagen solution of the present invention further comprises hyaluronic acid. Hyaluronic acid is a biomaterial commonly used in tissue engineering and drug delivery system (DDS), and has the function of increasing cell migration when bound to collagen fibers.

Step (b): Preparation and crosslinking of the collagen dispersion

After step (a) is carried out, the collagen solution of step (a) is pulverized to prepare a collagen dispersion.

The pulverization of the present invention includes any method of pulverizing collagen in the art and can be, for example, pulverized chemically or mechanically. According to one embodiment of the present invention, the grinding of the present invention is performed by mechanical grinding, and according to another embodiment of the present invention, a homogenizer and ultrasonic waves are used. According to some embodiments of the present invention, the grinding by the homogenizer is performed at a speed of 10000-30000, 15000-30000, 15000-25000, 18000-25000 or 18000-22000 rpm. According to some embodiments of the present invention, ultrasonic pulverization is performed at 25-50, 30-50, 30-45, 30-40, or 35-40 kHz. According to some embodiments of the present invention, the milling is carried out for 30-120 minutes, 30-90, 45-90, 45-75, 50-75, 50-70, 55-70 or 55-65 minutes.

In the present invention, the collagen is crosslinked after preparation of the collagen dispersion and before freeze-drying.

The crosslinking of the present invention includes any collagen crosslinking method in the art including, but not limited to, physical crosslinking DHT (dehydrothermal treatment) and chemical crosslinking (aldehyde or EDC treatment).

According to one embodiment of the present invention, the crosslinking of the present invention comprises: (b-1) centrifuging the collagen dispersion to remove the supernatant; (b-2) removing the supernatant by adding buffer solution to the result of step (b-1) and centrifuging; And (b-3) adding a cross-linking agent to the resultant of step (b-2), and performing a cross-linking reaction, followed by centrifugation to remove the supernatant.

According to one embodiment of the present invention, the crosslinking of the present invention is carried out after the step (b-3), the result of step (b-3) is immersed in a NaH ₂ PO ₄ solution to stop the crosslinking reaction, (B-4).

According to one embodiment of the present invention, the crosslinking of the present invention further comprises a step (b-5) of washing after step (b-4).

According to one embodiment of the present invention, the crosslinking agent is EDC [1-ethyl-3- (3-dimethyl aminopropyl) carbodiimide].

According to one embodiment of the present invention, the buffer solution is a MES buffer solution.

According to an embodiment of the present invention, the crosslinking agent is added in the step (b-3), and the crosslinking agent is added for 60-180 minutes, 60-150 minutes, 90-150 minutes, 105-150 minutes, 105-135 minutes, 115-135 minutes Or crosslinking reaction for 115-125 minutes.

The cross-linking is determined by the ratio of collagen to cross-linker, depending on the form and strength of EDC. According to one embodiment of the present invention, the EDC of the present invention has a particle size of 0.1-10, 0.1-9, 0.1-8, 0.5-7, 0.5-6, 0.5-5, 0.5-4, 0.5-3, 0.5-2 or 0.5-7 parts by weight is used.

Step (c): Drug addition

After step (b) is carried out, the drug is treated in step (b). The result of step (c) can carry the treated drug, which can prevent infection against foreign substances.

The medicament to be treated with the result of step (b) of the present invention includes any medicament that can be included in wound dressings in the art, including, but not limited to, antibiotics and antibodies.

According to one embodiment of the present invention, the drug of the present invention is an antibiotic. The antibiotics of the present invention include any antibiotic in the art and include, for example, tetracycline, doxycycline, aureomycin, penicillin V, ampicillin, amoxicillin, bacampicillin, carbenicillin, clock factin, , Oxacillin, cephalexin, cephradine, cephadoxil, sephaclo, sefrocim, axetil, cephofoxin, loracabef, cefixime, gentamycin sulfate, tobramycin, amikacin, It has been shown that antibiotics such as polymyxin B, mepenide, silver sulfadiazine, sulfasalazine, cell membrane formation inhibitors teicoplanin, bacitracin, novobiocin, clindamycin, nofloxacin, enox Gospel, pheoproxacin, cyproproxacin, and oproxacin.

According to some embodiments of the present invention, the drug of the present invention is gentamicin sulfate. The term "gentamycin sulfate" herein is used interchangeably with "gentamycin" in the present specification. As demonstrated in the following examples, when gentamicin alone is administered into a tissue, an adverse reaction such as edema may occur histopathologically. However, the collagen wound dressing containing gentamicin prepared by the method of the present invention It is understood that a certain amount of drug is continuously released from the wound dressing material in a stepwise manner over a certain period of time, thereby significantly reducing the adverse reaction in the tissue.

According to a specific embodiment of the present invention, the gentamicin sulfate of the present invention is added in an amount of 0.2-0.7, 0.2-0.5, 0.3-0.5 or 0.3-0.4% (w / v).

Step (d): Freeze-drying to produce porous collagen sponge

After step (c), the resultant of step (c) is lyophilized to prepare a drug-containing porous collagen sponge.

According to one embodiment of the present invention, the result of step (c) is divided into 0.1-5, 0.1-3, 0.1-1, 0.3-1 or 0.3-0.7 cm thick, and lyophilized.

According to one embodiment of the present invention, the freeze-drying of the present invention is carried out for 48-96 hours after gradual descent at -40 to -20 占 폚 for 1-2 hours.

According to an embodiment of the present invention, the porous collagen sponge containing the drug has a pore size of 50-200 mu m.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a method for producing a collagen wound dressing comprising a drug.

(b) The present invention provides a collagen wound dressing comprising a drug prepared by the method of the present invention.

(c) The wound dressing prepared by the method of the present invention has a high biocompatibility and can carry a drug, so that infection can be prevented from external substances, which is a disadvantage of the conventional wound dressing, It is possible to release the drug stepwise so that the adverse reaction in the tissue caused by the drug can be reduced.

(d) The method of the present invention is advantageous in that it is possible to perform the lyophilization once more than the conventional method of manufacturing a collagen wound dressing material, so that the wound dressing can be manufactured at low cost because it is simple and efficient.

FIG. 1 shows the shape of a collagen wound dressing containing a drug prepared by the method of the present invention.
Fig. 2 shows images observed histologically to confirm the biocompatibility of a rat animal model after applying the control group, gentamicin (Comparative Example 1) or Gentaci (experimental example) to the muscle tissue of the animal. A: Control group, B: Intramuscular injection of gentamicin (Comparative Example 1), C: Gentacue (Experimental Example).
FIG. 3 is a graph showing the release of drug in the local tissues after implantation of the wound dressing materials of Comparative Example 1 and Experimental Example into animal tissues in a rat animal model. The abscissa represents the number of days and the ordinate represents the concentration of gentamicin released in the local tissues.
FIG. 4 shows photographs and wound healing effects of the wound healing process of the animals to which Gentaci was implanted, respectively.
Fig. 5 shows the results of the evaluation of the efficacy of gentacay on the inhibition of the increase of IL-1? Levels after 3, 9 and 15 days of wound induction.
Fig. 6 shows the results of the evaluation of the efficacy of Gentaci on inhibition of IL-6 concentration increase after wound induction 3, 9 and 15 days.
FIG. 7 shows the results of the evaluation of the efficacy of Gentaci on the inhibition of TNF-a concentration increase after 3, 9, and 15 days of wound induction.
Fig. 8 shows the results of the evaluation of the efficacy of Gentaci on the inhibition of the increase of eNOS after 3, 9 and 15 days of wound induction.
FIG. 9 shows the results of evaluation of the efficacy of Gentacay on the inhibition of VEGF uptake at 3, 9 and 15 days after wound induction.
Fig. 10 shows the results of evaluation of the efficacy of Gentaci on inhibition of ERK expression increase at 3, 9 and 15 days after injury induction.
Figure 11 shows a micrograph (x400, bar = 100 [mu] m) by H & E staining 15 days after wound induction in each group. A: Normal control group, B: Sham group, C: Genta-Q recipient group.
12 shows an SEM image of a Genta cue.
Fig. 13 shows the results of the measurement of the solubility of Gentaci.
Fig. 14 shows the result of measurement of collagen enzyme degradation rate of Gentaci.
Fig. 15 shows the tensile force measurement results of the Genta cue.

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 embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Throughout this specification, "%" used to denote the concentration of a particular substance is intended to include solids / solids (wt / wt), solid / liquid (wt / The liquid / liquid is (vol / vol)%.

Example 1: Preparation of drug-containing collagen sponge

Preparation of collagen solution

Bovine Type I collagen Tendon, Southern Lights Biomaterials, Newzealand) was soaked in 2 mM HCl (pH 2.65) at 0.56% concentration for 10 hours, respectively.

Mechanical grinding of collagen solution

The prepared collagen solution was pulverized at a speed of 15,000 rpm for 30 minutes (homogenize, Ultra turrax T-25, IKA) and sonicated at 37.1 kHz using a sonicator (WUC-N60H, DAIHAN-SCI) To obtain a collagen dispersion.

EDC crosslinking of collagen

The prepared collagen dispersion was centrifuged to remove the supernatant. The precipitate was added with the same amount of 0.05 M MES buffer (pH 5.0) as that of the collagen dispersion, followed by reaction at room temperature for 2 hours, followed by centrifugation with 10000 g (RCF (Relative Centrifugal Force) ) To discard the supernatant, and a precipitate was obtained.

The same amount of EDC solution as that of the collagen dispersion was added to the precipitate, and the reaction was allowed to proceed for 2 hours so that the cross-linking reaction of collagen occurred. The resultant was again centrifuged at 10,000 g to discard the supernatant, and a precipitate was obtained. The precipitate was immersed in a NaH ₂ PO ₄ solution for 2 hours to terminate the crosslinking reaction, followed by centrifugation at 10000 g to discard the supernatant, and a precipitate was obtained. The same procedure was repeated three times for 30 minutes using distilled water.

Drug (antibiotic) treatment

0.4% (w / v) of gentamicin sulfate was dissolved in the resultant product of the collagen after EDC crosslinking, and the pH was adjusted to 4.5 with 0.5 M NaOH.

Manufacture of collagen sponge

The resultant dissolved in the gentamicin was dispensed into a mold (sus tray, CY-1180, CHUNYANGSA) at a height of 0.5 cm or more, started at room temperature, lowered to -30 캜 for 80 minutes, And dried to prepare a collagen sponge.

Example 2: Biocompatibility study of collagen sponge containing gentamicin

Transplantation and tissue harvesting

Biocompatibility was confirmed by using a collagen sponge Gentaky and gentamicin powder (Fujian Fukang Pharmaceutical Co., Ltd.) prepared in Example 1.

The rats were anesthetized with Zoletil (25 mg / kg, Zoletile 50 ^™ ; VirbacLab., France), and the left femur was removed. The skin was incised to a depth of 3 cm. Respectively. Gentamicin 0.5 x 1 cm ² (2.4 mg total) was transplanted onto each wound (Experimental Example 1) and intramuscularly injected with gentamicin solution dissolved in water for injection (Comparative Example 1). The skin was treated with 3-0 nylon , Inc.). Sixteen muscle samples from the transplantation site were taken from the control group, Comparative Example 1 and Experimental Example 1 for histological examination after 14 days.

Histological observation

The muscle samples taken from just below the implant site were trimmed transversely (Lee et al., 2006). After trimming, it was fixed to a 10% formalin solution (Sigma-Aldrich Co. LLC). After paraffin embedding, the sections were cut to a thickness of 3 μm and stained with H & E to perform histological evaluation of each sample. In order to observe more detailed histologic changes, the evaluation system of tissue damage and edema was applied and graded into four stages [3+: Severe, 2+: Moderate, 1+: Slight, 0: not detected-normal appearances (Max = 3)]. In addition, the number of inflammatory cells (polymorphonuclear leukocytes, lymphocytes, and macrophages) was counted with reference to previous studies (Lee et al., 2006; Hwang et al., 2010).

Statistical analysis

All histomorphological results were expressed as mean ± standard deviation of 6 muscle samples. Changes in uniformity were observed with the Levene test (Levene, 1981). One-way ANOVA test was used to compare significant changes in the two groups when no significant change was observed in the Leven test. In the case of significant changes in the Leven test, Kruskal-Wallis H test, a non-parametric test, was performed. Mann-Whitney U (MW) tests were performed when significant changes were observed in the Kruskal-Wallis H test, and significant changes in both groups were observed. Statistical analysis was performed using SPSS. Statistical significance was considered significant when P <0.05. In addition, changes in the control and Comparative Example 1 were calculated to determine the local stimulation of gentamicin injection. The changes in Comparative Example 1, Comparative Example 2 and Experimental Example 1 were calculated to understand the local stimulation of each preparation.

result

The results of histomorphometric analysis of Comparative Example 1 and Experimental Example 1 are shown in Table 1 below, and typical tissue states were as shown in FIG.

^a : p <0.01 compared to control by LSD test

^b : p <0.01 compared with Comparative Example 1 by LSD test; ^c : p <0.05 compared with Comparative Example 1 by LSD test

^d : P <0.01 compared with the control group by MW test

^e : In comparison with Comparative Example 1 by MW test, p <0.01

In Comparative Example 1, infiltration of inflammatory cells including polymorphonuclear leukocytes, lymphocytes and macrophages was moderate to severe, and muscle fibers showed mild to moderate swelling (Fig. 2). The scores for tissue injury, edema and inflammation were significantly increased in Comparative Example 1 compared to the control, and polymorphonuclear leukocytes, lymphocytes and macrophages were also significantly increased (Table 1). However, histological changes due to local stimulation of Comparative Example 1 were significantly reduced in Experimental Example 1 (Fig. 2). Histologic score and number of inflammatory cells were significantly decreased in Experimental Example 1 compared to Comparative Example 1 (Table 1).

The numbers of tissue injury, edema, inflammation score, polymorphonuclear leukocyte, lymphocyte, and macrophage were increased to 650.00, 800.00, 650.00, 228.57, 3325.00 and 1725.00% in Comparative Example 1, respectively. Compared with Comparative Example 1, -46.67, -66.67, and -73.33 in Experimental Example 1. -78.26, -93.43 and -91.78%, respectively.

Example 3: Study on drug release

Treatment and sampling

The rats were treated with Zoletile (25 mg / kg, Zoletile 50 ^™ ; VirbacLab., France). The left femur was removed under anesthesia, the femur was incised 3 cm, and the quadriceps muscle was incised 3 cm. Thereafter, Gentaci was transplanted into each size of 1 X 2 cm ² (total of 4 mg) (Experimental Example 2, 3 each), and the skin was sutured with 3-0 nylon. Blood samples were taken from the abdominal vein for 7 days from the first day after transplantation and the muscle at the transplantation site was sampled. The whole procedure of transplantation and sampling was performed by a veterinarian as a blind.

Muscle tissue sampling and calibration

The frozen muscle (0.4 g) sampled in Experimental Example 2 was placed in a 50 ml tube and homogenized in a phosphate buffer containing 0.4 mM EDTA and 2% trichloroacetic acid. Shaking for 10 minutes followed by centrifugation at 4500 rpm for 10 minutes. The supernatant was transferred to a 50 ml tube and 15 ml of TCA phosphate buffer was added to the tissue-remaining tube and the supernatant was added. In addition, 0.2 ml of 30% sodium hydroxide solution was added and the pH was adjusted to 7.5 with 1 N hydrochloric acid or 1 N sodium hydroxide solution. In the vacuum manifold, the HLB cartridge and the WCX cartridge were activated with methanol and distilled water. Extracts were added to the cartridge, passed through at a rate of 1-3 mL / min, and washed with distilled water. The water in the cartridge was removed under vacuum. The test solution was eluted by elution at a rate of 1-3 mL / min under 1.6% TCA and filtration through a 0.45 μm PTFE filter. The stock solution was prepared by dissolving gentamycin in 90% methanol and diluting the standard solution to a concentration of 0.5-100 g / ml in 90% methanol.

Plasma sample preparation and calibration

Plasma samples from each group were dissolved and vortexed, and 500 μL was transferred to an Enpenden tube. Plasma standards were prepared by adding 100 μL of diluted gentamycin to the plasma at a concentration of 0.02 to 20 μg / mL. Blank was prepared in 0.5 ml of plasma. The tube was vortexed to add 100 μL of the precipitation solution. The tube was vortexed to completely destroy the precipitate. Samples were centrifuged at 14,000 rpm (20,000 RCF) for 5 minutes to separate the layers. 300 μL of the supernatant was separated and analyzed in 5 μL increments.

LC-MS / MS conditions

Gentamicin concentrations in rat plasma and muscle LC-MS / MS method. Chromatographic analysis was performed on an Agilent 1100 Series HPLC (Agilent Technologies, Palo Alto, USA), including on-line degasser, binary pump, autosampler and column compartment Respectively. Potential detachment of analytes from interferents was carried out on a VDSpher PUR100 C18-E column (2.0 X 75 mm, 3.5 μm) (VDS Optilab Chromatographietechnik GmbH, Germany) at 40 ° C in a column oven. The mobile phase used for chromatographic separation consisted of 50% acetonitrile / 50% distilled water (20 mM HFBA) in 5% acetonitrile / 95% distilled water (20 mM heptafluorobutyric acid) (AB Sciex Pte. Ltd) at 400 ° C under the following conditions: (1) the sample was passed through a turbo-ion spray (AB Sciex Pte. A high pressure of 4.0 kV was applied to the ion spray. Nitrogen was used in nebulizer gas, curtain gas, and collision gas at the settings of 7.5, 10, and 4. Detection of gentamicin was performed using MRM detection method, Retention time: 7.5 min, Retention time: 7.4 min, Retention time: 7.3 min, Retention time: 7.3 min, Gentamicin Calibration curves are measured in the range of r2> 0.999 (C) the lowest concentration was 20 ng / ml. Reagents used in the assay were purchased from Sigma-Aldrich (USA).

Results and Discussion

Gentamicin was continuously measured in Experimental Example 2 for 7 days. The gentamicin content in Experimental Example 2 gradually decreased (Table 2 and Fig. 3).

This result means that gentamicin is effectively absorbed into the local region by the presence of the collagen sponge of the present invention.

The concentration of gentamicin in plasma during the same experimental period was not measured beyond the lowest measured concentration value (20 ng / ml) of this method (Table 3). The above results indicate that gentamicin is not absorbed into the whole body in Experimental Example 2.

As a result, it was found that the gentamicin-containing collagen sponge produced by the method of the present invention was locally absorbed locally to the graft site without systemic exposure.

Example 4. Evaluation of pre-clinical efficacy of collagen sponge containing gentamicin

Materials and Experiments

Experimental animal

All experimental animals were male Sprague-Dawley (SD) rats weighing 190-210 g at 7 weeks of age and purchased from Korea-Japan experimental animals [source: Coatec Co., Ltd.]) and 48 rats (12 rats per group) Respectively. The animals were housed at 22 ° C for 12 hours and 12 hours (12: 12 h light-dark cycle). The humidity maintained the optimum condition (35-55%) during the experiment. This experiment was conducted after passing the deliberation of the Animal Experiment Ethics Committee of Chonbuk National University.

Induce wound

The animals were ex- cised in the dorsal region using a 10 mm circular punch under suction inhalation. After wound induction, each one was placed in a cage. In order to inhibit pain and fever, 6.0 mg / kg dipyrone was dissolved in 250 ml of water for 3 days.

Collagen sponge (Gentaci) implantation

The animals were randomly selected and divided into four groups.

① Normal control group (N / C, n = 12): normal group without incision

② Shame group (n = 12): group with 10 mm incision on the dorsal skin

③ Genta Qi transplant group (n = 12): Ten mm incision was made on the dorsal skin,

Evaluation of wound healing

In order to evaluate the wound healing process, all the animals were photographed with a digital camera (Sony Cybershot) at 0, 3, 6, 9, 12 and 15 days after injury induction at 2:00 pm. The wound area was quantitatively evaluated using a digital software image. The evaluation of wound area was calculated as follows.

Equation

Wound area (%) = [size of wound area (3, 6, 9, 12, 15 days) / size of wound area (0 day)] X 100

Effect of Gentacay on the Regulation of IL-1β, IL-6, IL-10, TNF-α and eNOs

(IL-1β, IL-6, IL-10 and TNF-α) and eNOs associated with epithelial cell regeneration (endothelial NO synthase) was measured using an ELISA method.

Evaluation of wound healing efficacy of Gentaci by histological evaluation

After the end of the experiment (15 days after injury induction), the injured area was excised from the experimental animals, fixed with 10% formaldehyde fixative, washed with PBS, dehydrated with ethanol at a sequential concentration, embedded in paraffin, , 5 ㎛ in thickness, and then paraffin was removed with xylene and hydrophilized with 100%, 95%, 90%, 80%, 70% alcohol. As a staining method, dehydration process was performed by H & E staining. After that, it was sealed with Canadian balsam and observed with an optical microscope (Olympus, Japan). Tissue determinations were obtained by observing and averaging three independent observers without information on the control and experimental groups.

Data analysis and statistical processing

All experimental results are expressed as mean ± standard error. Statistical significance was compared with the normal control group (N / C) by Student's t-test and it was judged that there was a significant difference when P <0.05.

result

Measurement of area of wound area (Planimetry)

The area was measured using a microscope program to evaluate the degree of wound healing through the photographs. The wound site was restored in a time-dependent manner after 3, 6, 9, 12 and 15 days after wound induction, which was significantly reduced in the Gentaky graft group compared to the Sham group.

FIG. 4 shows the results of evaluating the wound healing efficacy of Gentaci, which showed significant wound healing effect when transplanted with Gentaci (** P <0.01). In the Sham group, the recovery effect was 96.76 ± 2.8, 92.43 ± 4.73, 85.73 ± 3.64, 71.63 ± 5.65 and 57.83 ± 8.97, respectively, after 3, 6, 9, 12 and 15 days after wound induction (100% In the Gentaci graft group, the recovery effect was 97.43 ± 2.15, 82.41 ± 3.95, 64.83 ± 4.73, 43.75 ± 8.43 and 26.94 ± 5.95 (** P <0.01). These results show that Gentacillin grafting has a very significant effect on wound healing.

Evaluation of the efficacy of Gentacay on the expression of IL-1β, IL-6, IL-10, TNF-α and eNOs

After 3, 9, and 15 days after wound induction, the blood of each group of experimental animals was collected and the inflammation related indicators were measured as follows.

① Inhibitory effect of IL-1β

IL-1 is a cytokine mainly produced by monocytes and macrophages. IL-1, IL-1 and IL-1RA (IL-1 receptor antagonist) are found in the IL-1 family. IL-1β, a pro-inflammatory cytokine, is known to be the most important inflammatory regulator and is involved in inflammation. Therefore, we have identified the mechanism of action of Gentaci grafting to inhibit IL-1β. The results are shown in FIG. 5 below.

 FIG. 5 shows the inhibitory effect of Gentaci on inflammation-induced cytokines. When Gentaci was transplanted, IL-1β was significantly inhibited (FIG. 5). At 3 days after induction of wound injury, the AU (arbitrary units) value for IL-1β was 4.37 ± 0.11 in the Sham group, while the Gentacyl group showed a reduction of 3.25 ± 0.13 (** P <0.01).

At 9 days after injury induction, the AU (arbitrary units) value in the Sham group was 3.15 ± 0.074, while the Gentaci graft group had a 2.32 ± 0.096 decrease (p <0.01).

In the Sham group after 15 days of injury induction, the difference was 2.24 ± 0.087, while in the Gentaci graft group, the difference was 1.87 ± 0.079 (** P <0.01).

These results show that Gentacillin-induced transplantation reduces IL-1β, an inflammatory index.

② Inhibitory effect of IL-6

IL-6 is a signaling molecule that mediates the inflammatory response. It is an activating factor secreted by T lymphocytes and macrophages, stimulating B lymphocytes and promoting various inflammatory responses. Therefore, we investigated the mechanism of action of Gentaci grafting on the inhibition of IL-6 secretion. The result was as shown in Fig.

FIG. 6 shows the inhibitory effect of IL-6 by Gentaci graft, and the inhibition of IL-6 was significantly inhibited when Gentaci was transplanted into the sham group (** P <0.01) . At 3 days after injury induction, the AU (arbitrary units) value for IL-6 was 6.15 ± 0.098 in the Sham group, but decreased to 4.52 ± 0.14 in the Gentacillin group (** P <0.01).

At 9 days after wound induction, the arbitrary units (AU) value of the Sham group was 4.27 ± 0.085, while that of the Gentaci graft group was 2.91 ± 0.093 (** P <0.01).

In the Sham group after 15 days of injury induction, the difference was 3.98 ± 0.094, whereas in the Gentaci graft group, the difference was 1.96 ± 0.076 (** P <0.01).

These results show that Gentacillin-induced transplantation reduces IL-6, an inflammatory marker.

③ TNF-α inhibitory effect

TNF-α is a 'tumor necrosis factor' that signals tumor cells to commit suicide, inhibits the intracellular replication of viruses, stimulates macrophages, promotes inflammatory responses, It is a signal molecule involved in the innate immune response and is an indicator of the inflammatory response. Therefore, the efficacy was evaluated as to whether the transplantation of Gentacule inhibits the secretion of TNF- ?. The result was as shown in Fig.

FIG. 7 shows the inhibitory effect of TNF-α upon transplantation of Gentaci cue. When Gentaci was transplanted, TNF-α was significantly inhibited compared to Sham group (** P <0.01) . At 3 days after injury induction, the AU (arbitrary units) value for TNF-α was 5.14 ± 0.079 in the Sham group, but decreased to 3.56 ± 0.099 in the Gentacillin group (** P <0.01).

At 9 days after wound induction, the arbitrary units (AU) value in the Sham group was 4.73 ± 0.088, while in the Gentacillin group, it was 2.43 ± 0.091 (** P <0.01).

In the Sham group after 15 days of injury induction, the difference was 3.23 ± 0.073, whereas in the Gentaci graft group, the difference was 1.95 ± 0.094 (** P <0.01).

These results demonstrate that Gentacillin Gene reduces TNF-α, an inflammatory index.

④ eNOS increase efficacy

eNOS (Endothelial NOS), known as NOS3, is involved in vascular function by producing NO in blood vessels. Increases in NO (nitric oxide) synthase (eNOS) have been shown to relax the blood vessels and aid wound healing. Therefore, we clarified the mechanism of action of Gentaci grafting to increase secretion of eNOS. The result was as shown in Fig.

FIG. 8 shows the results of evaluating the increase of eNOS by transplantation of Gentaci. In the case of transplanting Gentaci, eNOS was significantly increased by at least 9 days compared to Sham group (** P <0.01). At 3 days after injury induction, the AU (arbitrary units) value for eNOS was 1.98 ± 0.089 in the Sham group, but increased to 2.87 ± 0.11 in the Gentacillin group (** P <0.01).

At 9 days after injury induction, the AU (arbitrary units) value in the Sham group increased from 3.13 ± 0.12 to 4.94 ± 0.15 (** P <0.01).

Specifically, it was 2.84 ± 0.22 in the Sham group after 15 days of injury induction, while it was 1.98 ± 0.076 in the Gentaci graft group (** P <0.01).

These results show that Gentaci grafting can increase wound healing by increasing eNOS, a vasodilator.

⑤ VEGF increase efficacy

VEGF is the most potent cytokine involved in angiogenesis and is produced during wound healing. Thus, in this procedure, we have identified whether Gentaci grafting increases secretion of VEGF. The results are shown in Fig.

FIG. 9 shows the results of evaluating the increase of VEGF by transplantation of Gentaci. In the case of transplanting Gentaci, VEGF was significantly increased (** P <0.01) as compared with Sham group until at least 9 days. At 3 days after injury induction, the AU (arbitrary units) value for VEGF was 4.01 ± 0.31 in the Sham group, whereas it increased to 5.89 ± 0.27 in the Gentaky graft group (** P <0.01).

At 9 days after injury induction, the arbitrary units (AU) value in the Sham group increased from 4.76 ± 0.29 to 6.37 ± 0.32 in the Gentacyl group (** P <0.01).

As in the case of eNOS, the sham group was 3.98 ± 0.34 after 15 days of injury induction, while the Gentaci graft group showed a significant decrease of 2.32 ± 0.17 (** P <0.01).

These results show that Gentacillin grafting significantly increases VEGF, a vasodilator, and may affect wound healing.

⑥ ERK increase efficacy

Activation of ERK (extracellular signal - regulated kinase) proteins at the wound site plays an important role in collagen synthesis and has been shown to be an important key to wound healing. Therefore, in this course, we have clarified whether transplantation of Gentacake increases the secretion of ERK. The results were as shown in Fig.

FIG. 10 shows the results of evaluating the increase of ERK caused by transplantation of Gentaci. In the case of transplanting Gentaci, ERK was significantly increased (** P <0.01) as compared with Sham group until at least 9 days. At 3 days after injury induction, the AU (arbitrary units) value for ERK was 2.32 ± 0.12 in the Sham group, but increased to 3.75 ± 0.19 (** P <0.01) in the Gentaky graft group.

At 9 days after wound induction, the arbitrary units (AU) value in the Sham group increased from 2.85 ± 0.15 to 5.27 ± 0.13 in the Gentacillin group (** P <0.01).

As in the case of VEGF, the sham group was 2.32 ± 0.09 after 15 days of wound induction, while the gentacity group showed a significant decrease of 1.64 ± 0.07 (** P <0.01).

These results show that Gentacillin grafting significantly increases ERK associated with collagen biosynthesis and can affect wound healing.

Histological evaluation

To evaluate the wound healing efficacy of Gentaci, histological evaluation was performed on the affected area 15 days after injury induction. After 15 days of Gentaci grafting, H & E staining was performed in each group. The degree of differentiation of epithelial cells and fibroblasts, inflammatory cell infiltration, degree of edema of the epidermis layer, degree of neovascularization and epithelialization epithelization) and the like. Histological evaluation findings were as shown in Fig.

As shown in FIG. 11, in the normal control group, the arrangement of the skin was normal, and inflammatory cell infiltration, epithelial cell dislocation, subcutaneous edema, fibroblasts, and redness were not observed (A). In the Sham group, epithelial detachment (yellow arrow), irregular arrangement of tissue, inflammatory cell infiltration (red arrow) and edema were observed (B). On the other hand, the Gentaci graft group showed histologic findings similar to the normal controls (C, D). These results show that the transplantation of Gentacu cage on the injured area can significantly improve wound healing in experimental animals.

Example 5 Analysis of Physical Properties of Collagen Sponge Containing Drug

SEM image

FIG. 12 shows an SEM image of the Gentaci, a collagen sponge prepared in Example 1. FIG. The control group is an SEM image of a collagen sponge prepared without crosslinking.

Solubility

According to the following formula, the solubility of the collagen sponge, GENTAQUE manufactured in Example 1, was compared by comparing the weight of collagen eluted for 4 hours at 37 DEG C relative to the weight of collagen sponge before elution. A collagen sponge prepared without crosslinking was used as a control, and the results are shown in Table 4 and FIG.

Equation

Solubility (%) = (sample eluted at 37 ° C in water for 4 hours, filtered and dried) Sample weight before elution X 100

division EDC bridging (EDC sol R 0.5) Non-crossing (control) Solubility (%) 3.64 3.97 10.81 10.83

Collagen enzyme degradation rate

Collagen enzyme (100 units / mL dissolved in Tris buffer) was added to 8.4 mg (2.8 mg / cm ² x 3 EA) of the sample. After reacting for 1 hour or 3 hours in a constant temperature water bath at 37 ° C, , And the collagen enzyme degradation rate of the collagen sponge, Gentaci, prepared in Example 1, was compared with the weight of the collagen before the enzyme reaction. As a control, a collagen sponge prepared without crosslinking was used, and the results are shown in Table 5 and FIG.

Equation

Enzyme decomposition rate (%) = (initial sample weight - weight after enzyme reaction) / initial weight X 100

division EDC bridging (EDC sol R 0.5) Non-crossing (control) 1hr 24.3% 35.8% 3hr 31.9% 42.7%

Tensile force

The tensile load of the collagen sponge, Gentaci, manufactured in Example 1 was measured by the Korean Polymer Test Research Institute. The tensile load was measured using a universal testing machine. The test was performed at a test speed of 5 mm / min, a span distance of 20 mm, a load cell of 20 N, a test environment of 25 ± 2 ° C, 45 ± 5 RH, And a length of 50 mm. A collagen sponge prepared without crosslinking was used as a control, and the results are shown in FIG.

The above solubility, collagenase degradation rate and tensile strength test results show excellent durability of the collagen sponge produced according to the present invention.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Claims (10)

A method for preparing a collagen wound dressing comprising a drug comprising the steps of:
(a) immersing collagen in an acidic solvent to prepare a collagen solution;
(b) pulverizing the collagen solution to prepare a collagen dispersion, and crosslinking the collagen in the collagen dispersion,
(b-1) centrifuging the collagen dispersion to remove the supernatant;
(b-2) removing the supernatant by adding buffer solution to the result of step (b-1) and centrifuging; And
(b-3) adding a cross-linking agent to the resultant of step (b-2) to effect crosslinking reaction and centrifuging to remove the supernatant;
(c) adding a drug to the result of step (b); And
(d) lyophilizing the result of step (c) to produce a porous collagen sponge comprising the drug.
The method of claim 1, wherein the pH of the acidic solvent is 1.0-3.0.
The method of claim 1, wherein the acidic solvent is hydrochloric acid or acetic acid.
The method according to claim 1, wherein the collagen solution has a collagen content of 0.3-0.7% (w / v).
delete The method according to claim 1, wherein the crosslinking agent is EDC [1-ethyl-3- (3-dimethyl aminopropyl) carbodiimide].
The method of claim 1, wherein the drug is gentamicin sulfate.
The method of claim 1, wherein step (d) comprises dispensing the result of step (c) to a thickness of 0.1-5 cm and freeze-drying to produce a porous collagen sponge comprising the drug.
The method according to claim 1, wherein the lyophilization of step (d) is performed at -40 to -20 캜 for 48-96 hours.
A collagen wound dressing comprising the drug according to any one of claims 1 to 4 and 6 to 9.

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