TW550065B - Method of crosslinking and sterilizing a biomolecule use by using 3-hydroxypropinoaldehyde, and a biocompatible implant, substitute or wound dressing containing the crosslinked biomolecule - Google Patents

Abstract

A use of 3-hydroxypropinoaldehyde in the manufacture of a biocompatibie implant, substitute or wound dressing is disclosed, which involves crosslinking an amine-containing biomolecule including chitosan, hemoglobin and a connective-tissue protein such as collagen or gelatin derived from a collagenous source with 3-hydroxypropinoaldehyde.

Description

550065 V. Description of the invention (1) " FIELD OF THE INVENTION The present invention relates to the preparation of a biocompatible implant, a substitute or a wound dressing, and particularly to a method for preparing the biocompatible implant using 3-hydroxypropionaldehyde Of a cosmetic, substitute or wound dressing. Background of the invention

Axel ssori and co-authors reported a broad-spectrum antimicrobial agent [Axelsson, L., Chung, T.C., Dobrogosz, W.J.

Lindgren, LE ·, " Discovery of a new antimicrobial substance produced by Lactobacillus reuteri, " FEMS microbiol · rev ·, 46, 65, 1 987], which is produced by Lactobacilllas reuteri reuterin). Lactobacillus reuteri lives in the gastrointestinal tract of humans and animals. It metabolizes glycerol under oxygen pressure to produce a large amount of lactate [Axelsson, L ·, Chung, TC ·, Dobrogosz, WJ ·, Lindgren, LE ·, "Production of a broad spectrum antimicrobial substance by Lactobacillus reuteri, 丨 丨 Microb · ecol ·, 2, 131-136, 1989; Talarico, TL ·,

Dobrogosz, W.J., " Production and isolation of reuterin, a growth inhibitior produced by Lactobacillus reuteri, 1 Antimicrob · agents chemother ·, 32, 1 854-1 858, 1 988]. Lutetin is 3-hydroxypropino-aldehyde, which is a neutral water-soluble substance that can inhibit bacteria, yeast, mold, and protozoa. Lactic acid bacteria

\\ Chenlin \ 19991103 \ P11100 \ cbclll66.ptd Page 4 550065 V. Description of the invention (2)

Lactobacillas reuteri and Lutetin are only used for food preservation and sterilization. Various fixatives including formaldehyde, glutaraldehyde, dialdehyde starch and epoxides have been used to fix biological tissues. Clinically, the most commonly used fixative is glutaraldehyde [Nimi, M · E ·, Cheung, D ·, Strates, B ·, Kodama,

M ·, Sheikh, K ·, " Bioprosthesis derived from cross-linked and chemically modified collagenous tissues, " in Collagen Vol. Μ. E. N i mn i (ed.), CRC

Press, Boca Raton, Florida, 1 988, pp. 1-38]. Glutaraldehyde-fixed biological tissue has been widely used in the manufacture of prosthetics for repair of heart valves, heart repair, vascular transplantation, and ligament replacement. However, glutaric significantly alters the stiffness of the tissue and the tendency to accelerate tissue calcification is a recognized disadvantage of this fixative. 4 In order to overcome the shortcomings of glutaraldehyde-fixed biorepair molecules, the inventor and collaborators of this case published a PCT patent application with publication number W098 / 1 971 8 or a Chinese patent application number that entered the national phase of China 9 71 9 Case 9 4 5 4 · 4 discloses a biocompatible, crosslinked material suitable as an implant, wound dressing and blood substitute. The material is prepared by cross-linking biological substances such as collagen, chitosan, or hemoglobin with a natural cross-linking agent, genipin. The toxicity of the crosslinking agent is much lower than that of the conventionally used rhenium, and the crosslinked product has good thermostability and biocompatibility. The patent content is hereby incorporated by reference. Wan

550065 V. Description of the invention (3) ------- ^ In the field of biotechnology, we are still looking for a biomolecule cross-linking agent that is more advanced in terms of biocompatibility, cell performance and mechanical properties, especially Biomolecule cross-linking agent that can have sterilization effect at the same time. Summary of the Invention ^ The invention provides a formula & for cross-linking an amine-containing biomolecule, comprising contacting an amine biomolecule with 3-hydroxypropanal for a period of time. Che: ‘谠’ biomolecules are connective tissue proteins. More preferably, the connective tissue protein is collagen or a collagen-derived substance, the biomolecule is deacetylated, and the biomolecule is hemoglobin, and the biocompatible substitute is a blood substitute. . Preferably, the amine-containing molecules and 3-hydroxypropanal are contacted in an aqueous medium and at a temperature between 4-50 ° C, preferably between 25-45t, and a temperature between 5 hours and 60 hours, more preferably About 48 hours. Preferably, the aqueous medium has a 3-hydroxypropionaldehyde concentration between 0.001M and 丨 M, more preferably between 0.03M and 0.2M. Preferably, the aqueous medium has a range of 3 to 12? [1 value, more preferably the pH value is between 4 and 10.5. The present invention also provides a biocompatible implant, substitute, or wound dressing ' which comprises a crosslinked biomolecule formed by crosslinking by the method of the present invention described above. Detailed description of the invention

550065

As described in the article referred to in the background section of the invention, Rutenin has anti-pillar, mold and protozoan activity. In addition, it was also found that Rutening, 3-Jing Bingjun can react with free amino groups in biological tissues, and Ru 4 inch Temple can be used as a cross-linking agent (fixing agent) and clinically used as biological tissues, Biocides of natural products or synthetic polymers. Rutenin has the following chemical structure: Η0-CH2-CH2-CH = 0, which can be prepared by Lactobacillus reuteri under controlled conditions. Lutengine used in the following examples was identified by high performance liquid chromatography (HPLC).

In the present invention, the antibacterial activity of Lutein is studied, among which glutaraldehyde is known. The microorganisms tested in the study were Es c h e r i c h i a c ο 1 i (Αχχ C 25922), Pseudomonas aeruginosa (ATCC 27853), Staphylococcus aureus (ATCC25923), and Bacillus subt 111 is (atcc 6 633). The results showed that all tested microorganisms, including Gram-positive bacteria (Staphy lococcus aureus and Bacillus subtillis) and Gram-negative bacteria (Escherichia coli * Pseudomonas aeruginosa) were sensitive to Lutein. Generally, 2 5-3 5 ppm of Lutetine can prevent the growth of the tested microorganisms, while 40-50 Ppm of Lutetine cause the death of the tested microorganisms. However, the amount of glutaric acid was significantly greater than that of lutenin (approximately 2 to 3 times higher), which indicates that the antibacterial activity of lutenin is significantly better than glutaraldehyde. The present invention also investigated the cytotoxicity of Lutetine, in which glutaraldehyde was used again as a control. In vivo, the cytotoxicity of the test reagent (glutaraldehyde vs. lutenin) was assessed using the cell lumens of stable 3T3 fibroblasts from mice, and the test results (light microscopy and MTT analysis) were used to determine

550065 5. Description of the invention (5) Proportion of the amount of viable cells in the medium treated with the test reagent. In the test, 3T3 fibroblasts were seeded in lml Dulbecco's modified eagle medium (DMEM, Gibco) containing 10% sheep fetal serum (FCS, Hyclone Laboratories, Logan, UT ·, USA). 430-2800EG, Grand

Island, NY, USA) in 24 well container trays, each well contained 3 X 104 cells. The cell culture environment was maintained in a constant temperature and humidity environment with 37% 10% CO2 air, and the cells at the growth log stage were then exposed to a new plutonium medium with various concentrations of glutaraldehyde or mitenine as drugs. After 24 hours of incubation, the growth medium in the well-shaped container was removed, and the cells were photographed with an optical microscope. Subsequently, the cells were washed twice with phosphate buffer solution (PBS), and then the number of surviving cells was 3- (4,5-dimethylthizone) -2,5-diphenol-tetrazolium bromide (MTT, Sigma Chemical Co., St. Louis, MO, USA) indirect determination by dye reduction method. The MTT test is based on the reduction of MTT, which is a yellow soluble dye ' which forms a dark blue formazan by mitochondrial succinate dehydrogenase. Only surviving, active mitochondrial cells significantly reduced the amount of MTT and turned into methicol. In the test, 20 ml of MTT solution (0.5 g / l in the medium, filtered and sterilized) was added to the cultured well-shaped container, and the temperature was maintained at 37 ° C in a 0% CO2 atmosphere for 3 hours. The MTT reaction medium After removal, the blue 曱 σ took up 100 ml of dihydrazine (DMS0) to dissolve. Optical density readings were then measured using a multi-well scanning spectrophotometer (MRX Microplate Reader,

Dynatech Laboratories Inc., Chantilly, VA, USA) was measured at a wavelength of 5 to 70 nm.

\\ Chenlin \ 19991103 \ P11100 \ cbclll66.ptd Page 8 550065 V. Description of Invention (6) No. 5: Middle-earth. 3T3 fiber-forming micrographs without any cross-linking reagents for testing indicate that the cells cultured in the control medium are fused together, and the cells can be used to evaluate glutaric acid: dazzling six people, ☆ JL, M 6 characteristics Ning cytotoxicity control. The micrographs of dimensional cells that were cultured in the medium in which urinary acetaldehyde was administered as a drug 3 showed that: a) cells with extremely low concentrations (0 = ^ 0) were fused together, glutaric acid & Nonger = 5Ppm, all cultured cells died. In contrast, Rutening's> Chen degree rose to 15ppm, and the cells fused together. Dingtu la and lb clarified the optical density readings of 3T3 fibroblasts The result is that the glutamate is cultivated in the medium of various concentrations of glutarin and lactate obtained in the MTT analysis as a drug. As shown in the figure, with the increase of the inertia of the test agent, glutarin is used. The light density f readings of cells cultured as a drug medium are much lower than those of cells cultured with Lutetine. The MΊTμ concentration of glutaraldehyde is about 4 ppm, which is higher than that of Lutetine (about 20 ppm). ) Is much lower. Example 1: Fixative materials and methods for biological tissues In this example, a pig heart purchased from a slaughterhouse was used as a raw material, and the purchased heart was shipped in cold physiological saline. Once it arrived, the heart First rinse with fresh saline to remove excess from the tissue Blood, and then carefully remove the attached fat from the surface of the heart. The maximum time interval from recovery to initial tissue fixation is no more than 6 hours. In the first part of this example, the tissue fixation rate of Lutetin was studied.

550065 V. Description of the invention (7) Degree, glutaric acid is used as a reference. The removed heart is first placed in a glutaraldehyde or lutetine phosphate buffer solution (PBS, PBS 7.4) at room temperature (25 ° C)-block 6-X 6-cm Heart, the amount of solution used for each fixation is about 010, and each sample group under study is then at different fixation time periods (5 minutes, 1 h, 4 h, 12 h, 24 h after the initial tissue fixation, respectively) , 48h and 72h). The rate of tissue fixation of Lutein is determined by controlling the change of the fixation index and the temperature of the denaturation of the fixed tissue during fixation. In the second part of this example, the effect of fixation conditions (pH, temperature, and initial fixative concentration) on the degree of tissue fixation was studied. The degree of tissue fixation of Lutein was determined by measuring the cross-linking characteristics (fixation index and denaturation temperature) of the fixed tissue. To clarify the effect of pH on the degree of tissue fixation of Lutein, a 0. 068M Lutein solution at room temperature (25 ° C) was buffered with the following solution: citric acid / sodium citrate (PH4 · 0), PBS (PH 7 · 4), sodium borate (PH8 · 5) or sodium carbonate / sodium bicarbonate (PH 10 · 5). The effect of temperature on the degree of tissue fixation of Lutein is as follows: 4. (:, 25 ° C, 37 ° C, or 45 ° C, and a 0. 068M aqueous solution of Lutetine buffered at pH 7.4. To clarify the initial fixative concentration on the degree of tissue fixation of Lutetine The effect was to use a buffered 0.034M, 0.068M, 0.1M, or 0.2M aqueous lactate solution at a pH of 7.4 at 25 ° C, each fixed time being 72h. The obtained fixation index is defined as the percentage of free amino groups in the tissue that react with the tested crosslinker and then re-fixed. In the trione analysis, the test tissue is first hydrolyzed for 24 h, then weighed, and then hydrolyzed

\\ Chenlin \ 19991103 \ P11100 \ cbclll66.ptd Page 10 550065 V. Description of the invention (8) The tissue is heated for 20 minutes with an indone solution. After heating with Moetrione, the optical absorbance of the solution was recorded with a spectrophotometer (UV-150-2, Shi'madzu, Corp., Kyoto Japan), and various known concentrations of glycine were used as standards. It is known that, after heating with ninhydrin, the number of free amino groups in the test tissue is directly proportional to the optical absorbance of the solution. The denaturation temperature of each study group was measured using a Peerkin-Elmer thermal difference analyzer (DSC Type 7, Norwalk, Connecticut). This technique is widely used to study the thermal transformation of collagen tissue. Results Figures 2a and 2b compare the fixation index and denaturation temperature of the glutaraldehyde or Lutein solidarium obtained at different fixed durations. As shown in Figures 2a and 2b, at the beginning of fixation, the fixation index and denaturation temperature of glutaraldehyde-fixed tissues increased much more rapidly than those of lutenin-fixed tissues. However, after 48 hours of fixation, the fixation indices and denaturation temperatures of the two studied groups were similar. The pH of the fixed buffer plays an important role in affecting the cross-linking properties of the tissues fixed by Lutein. The fixed index and denaturation temperature of Lutenin-fixed tissues at different pHs were obtained. In general, the fixation index of tissues fixed by Lutetin increases with the increase of the pH of the fixative. At pH 7.4 or 8.5, the denaturation temperature of tissues fixed by lactate is relatively greater than when the pH value is 丨 0.5, and at pH 4.0, the fixed tissue is fixed. Index and denaturation temperature are the lowest. Fixation temperature significantly affects the cross-linking properties of Lutein-fixed tissue. The effect of temperature on the fixation index and denaturation temperature of tissues fixed by Mute is showing. Figures 4a and 4b show that tissues fixed at 37 ° C or 45 ° C are similar

550065 Description of the invention (9) Fixed index and denaturation temperature. In contrast, 4. The fixed index and the denaturation temperature of the fixed tissue at the lowest temperature were the lowest among all the groups studied. The effect of the concentration of the initial fixative on the cross-linking characteristics of the tissue fixed by Lutein is given in Figures 5a and 5b. As shown in Figures 5a and 5b, the fixation index increases with the increase of the initial fixative concentration. The denaturation temperatures of Lutein-fixed tissues were substantially the same at different initial fixative concentrations. Example 2: Biocompatibility study and subcutaneous study In order to evaluate the biocompatibility of biological tissues fixed with Lutetin, a growing mouse prototype was used for subcutaneous research, and fresh glutarium-fixed counterpart was used as Reference. Materials and methods Fresh pig hearts were used as raw materials and processed as in Example 1. The removed hearts were fixed with 0.068 M of glyoxal or genipin solution for 3 days. For each piece of 6-by-6-cm pig heart, the amount of solution used in each fixation process was about 200 mi, the Lutetin solution was buffered with sodium borate (pH 8.5), and the glutaraldehyde solution After buffering the solids with phosphate (0.01M, pH? 4), the test samples were divided into two groups. For the first group, wash several times with a sterilized phosphate-buffered solution of transformation and mash, the time is about 5h; for the two groups, the fixed heart is sterilized with a series of increasing concentrations of ethanol solution (20% -75%). 5h.

550065 5. Description of the invention (ίο) 3 days and 1, 4, 12 weeks of repair. The denaturation temperature of the repaired samples was measured with a thermal differential analyzer (DSC Type 7 'Norwalk, Connecticut). The # 5 concentration deposited on each repair sample was analyzed by atomic absorption spectrometry. Results Throughout the test, fresh samples were found to be more stable than other solid samples that were implanted for 1 week. After 4 weeks of surgery, fresh samples were completely degraded, while other fixed samples remained intact. The denaturation temperatures of the same study group implanted at different times are essentially the same. In the fixed samples, the denaturation temperature of the mitenite-fixed samples was similar to that of the corresponding glutaraldehyde-fixed samples. The denaturation temperature of the fixed sample was 85 ° C, which was significantly higher than that of the fresh sample (62 ° C). Photomicrographs of fresh h & e-stained glutaric acid and lactate-fixed tissue in all studied groups showed that the fresh tissue had the most significant inflammation. Inflammation of tissues fixed by glutaraldehyde and multine was not significantly different during this time. After 4 weeks of surgery, each study group was more severe than the corresponding 3-day post-surgery counterpart. The degree of inflammation observed in the tissues fixed by glutaraldehyde and multrin was not significantly different at 3 days after surgery. Micrographs of tissues fixed with glutaric acid and lactate for 12 weeks after surgery were also taken. It should be noted that due to the complete degradation of fresh tissue, micrographs of tissues repaired during this period cannot be taken photo. From the micrographs, it can be observed that all the fixed samples were less inflammatory than the 1 and 4 week postoperative repair samples. It is interesting to note that the inflammation of the cells surrounding the tissues fixed by Lutein is lighter than those fixed by glutaraldehyde. ’

550065 5. Description of the invention (11) The results of calcium content in tissues fixed by fresh glutaraldehyde and lactonin before and after implantation for 3 days, 1 and 4 weeks are shown in Table 1. It should be noted that fresh tissue that is 4 weeks after repair is not available due to complete disintegration. As shown in the table ', for all the groups studied, the difference in calcium content between samples before implantation and at different times of repair was not significant. Table 1 Calcium content (mg / mg dry tissue weight) of each study group before and at different implantation times * Implantation time 0 weeks (n = 4) 3 days (n = 4) 1 Week (n = 4) 4 weeks (n = 4) Fresh glutaraldehyde milk special ¥ 1 · 5 ± 0 · 3 1 · 2 Soil 〇 · 1 1 · 4 ± 0. 1 1. 3 ± 〇. 1 1 5 ± 0 · 3 1 · 5 ± 0 · 2 1-9 + 0.2 2 · 1 ± 0 · 9 1 · 6 ± 0 · 3 N / A # 1 · 8 soil 0 · 6 1. 7 ± 0. 5 J The standard deviation of several soils was completely degraded at 4 weeks after operation, and no data were available. In addition, the tensile strength of each repaired sample was measured using a British model (type 2) at a constant speed of 50 mm / min. = Lutenin-fixed and glutaric acid-fixed samples. The results show that the tensile strength of the postoperative repair is similar at different time periods. -In implantation and although the present invention 6 is disclosed above with reference to a preferred embodiment, it is limited to the present invention. Anyone who is familiar with the field, * does not leave ^ is not used to make various ^ changes and retouches, so the present invention "

550065 V. Description of invention (12) The scope is subject to the scope of the patent application. Η ·! Page 15 \\ Chenlin \ 19991103 \ P11100 \ cbclll66.ptd) Simple illustration of the diagram Figures 1a and 1b show the fibroblast dialdehyde (Figure 1a) and compound as shown in the figure. The concentration of MTT5G should be determined by the optical density readings. Figure 2a and 2b show the group fixed by lutenine. Figure 3a and 3b table index (Figure 3a) and Figures 4a and 4b table index (Figure 4a) and Figures 5a and 5b at fixed concentrations cannot refer to the optical density of 3T3 fibroblasts (0 · D ·) The read cells were cultured in various concentrations of pentamate and the like (Figure 1b) obtained in the MTT analysis as a drug medium and determined according to the concentration of the test reagent, which was reduced to half of the control. The fixation index (Figure 2a) and denaturation temperature (Figure 2b) of glutaraldehyde or weave obtained at different fixed durations cannot be shown. The dots indicate the tissues fixed by glutaraldehyde. Fixed denaturation temperature at different pHs (Figure 3b). Figure 1 shows the fixed denaturation temperature of tissues fixed by Lutexine at different temperatures (Figure 4b). a) and denaturation temperature (Figure 5b).

Claims (1)

550065 Amendment 6 Are there any changes to the substance of the amendment? Is it correct? Twenty-three-year-old temperature T: jk r has a 25-year-old} is small enough to apply for a patent 1. A method of cross-linking an amine-containing biomolecule, comprising an amine-containing molecule and 3-mercaptopropionic acid Contacting in an aqueous medium and at a temperature between 4-50 ° C for a time between 5 hours and 60 hours, wherein the aqueous medium has a 3-hydroxypropionaldehyde concentration between 0.01M and 1.0M. 2. The method of claim 1 in which the biomolecule is an association tissue protein. 3. The method of claim 1 in which the biomolecule is acetamidine. 4. The method according to item 2 of the patent application, wherein the connective tissue egg is collagen or gelatin derived from a collagenous substance. 5. The method of claim 1 in which the biomolecule is an albumin, and the biocompatible substitute is a blood substitute. 6. The method of claim 1 in which the aqueous medium has a pH between 3 and 12. 7. The method according to item 1 of the patent application range, wherein the temperature is between -45 ° C. 8. The method as claimed in item 1 of the patent scope, wherein the contact time is 48 hours. 9. The method of claim 1, wherein the 3-hydroxypropionic acid degree is between 0.03M and 0.2M. 10. The method according to item 6 of the patent application, wherein the pH value is between 4 10.5. cbclll66amend01.ptd Page 17