Adsorbing material for multiple pathogenic factors of sepsis as well as preparation method and application thereof
10898632 ยท 2021-01-26
Assignee
Inventors
Cpc classification
A61M1/0259
HUMAN NECESSITIES
A61M1/38
HUMAN NECESSITIES
A61M1/3609
HUMAN NECESSITIES
A61M1/3486
HUMAN NECESSITIES
A61M1/3633
HUMAN NECESSITIES
B01J20/26
PERFORMING OPERATIONS; TRANSPORTING
B01J20/30
PERFORMING OPERATIONS; TRANSPORTING
International classification
A61M1/00
HUMAN NECESSITIES
B01J20/26
PERFORMING OPERATIONS; TRANSPORTING
B01J20/30
PERFORMING OPERATIONS; TRANSPORTING
A61M1/36
HUMAN NECESSITIES
B01J20/00
PERFORMING OPERATIONS; TRANSPORTING
A61M1/34
HUMAN NECESSITIES
Abstract
An adsorbing material for multiple pathogenic factors of sepsis as well as a preparation method and an application thereof are provided. The adsorbing material is formed by coupling a carrier with good mechanical performance and blood compatibility and a ligand with the capacity to adsorb multiple pathogen-associated molecular patterns, and is capable of effectively adsorbing bacterial endotoxin, bacterial genomic DNA, peptidoglycan, lipoteichoic acid, virus RNA, and zymosan from fluids such as blood and the like, and in particular has application value in blood purification for treatment of sepsis.
Claims
1. A preparation method of a ligand of adsorbing materials for adsorbing multiple pathogenic factors of sepsis in fluids, comprising the following steps: 1) In dichloromethane, compound 1 reacts with di-tert-butyl dicarbonate to generate compound 2, reaction temperature is 2030 C., the equivalence ratio of compound 1 and di-tert-butyl dicarbonate is 1:0.52, reaction equation is ##STR00023## 2) In a saturated solution of ammonia in methanol, compound 3 is generated from compound 2 through hydrogenation under the existence of raney nickel and hydrogen, reaction temperature is 2050 C., pressure is 110 Mpa, the mass of raney nickel is 10%50% of the mass of compound 2, reaction equation is: ##STR00024## 3) In ethanol or methanol, compound 3 reacts with , -unsaturated nitrile to generate compound 4, reaction temperature is 2050 C., the equivalence ratio of compound 3 and , -unsaturated nitrile is 1:23, reaction equation is: ##STR00025## 4) In dichloromethane, compound 5 reacts with N-Hydroxysuccinimide to generate compound 6 under the existence of N,N-dicyclohexylcarbodiimide and 4-dimethylaminopyridine, reaction temperature is 2030 C., the equivalence ratio of compound 5 and N-Hydroxysuccinimide is 1:12, reaction equation is: ##STR00026## 5) In dioxane, compound 4 reacts with compound 6 to generate compound 7, reaction temperature is 3050 C., the equivalence ratio of compound 4 and compound 6 is 1:12, reaction equation is: ##STR00027## 6) In dioxane, compound 7 reacts with N-Carbobenzoxyoxysuccinimide to generate compound 8, reaction temperature is 3050 C., the equivalence ratio of compound 7 and N-Carbobenzoxyoxysuccinimide is 1:12, reaction equation is: ##STR00028## 7) In ethanol or methanol, compound 8 reacts with di-tert-butyl dicarbonate to generate compound 9 under the existence of raney nickel and hydrogen, reaction temperature is 3050 C., the equivalence ratio of compound 8 and di-tert-butyl dicarbonate is 1:0.53, pressure is 110 Mpa, the mass of the raney nickel is 10%50% of the mass of compound 8, reaction equation is: ##STR00029## 8) In methanol, compound 10 is generated from compound 9 under the existence of Palladium on carbon and hydrogen, reaction temperature is 2050 C., pressure ranges from atmospheric pressure to 10 MPa, the mass of the Palladium on carbon is 10%30% of the mass of compound 9, reaction equation is: ##STR00030## 9) In dichloromethane, compound 10 reacts with succinic anhydride to generate compound 11 under the existence of 4-dimethylaminopyridine, reaction temperature is 2030 C., the equivalence ratio of compound 10 and succinic anhydride is 1:12, reaction equation is: ##STR00031## 10) In ethyl acetate, compound 11 reacts with N-Hydroxysuccinimide to generate compound 12, reaction temperature is 2030 C., the equivalence ratio of compound 11 and N-Hydroxysuccinimide is 1:12, reaction equation is: ##STR00032## wherein n.sub.1-n.sub.4 is an integer between 1-6, and n.sub.5 is an integer between 0-3.
2. The method according to claim 1, wherein the multiple pathogenic factors of sepsis include bacterial endotoxin, bacterial genomic DNA, peptidoglycan, lipoteichoic acid, virus RNA and/or zymosan.
3. The method according to claim 1, wherein the fluids include human blood or blood plasma or drug injection or liquid biological reagent.
4. An adsorbing material for multiple pathogenic factors of sepsis, wherein the material is formed by coupling the ligand prepared through the method of claim 1 and a carrier, whose molecular structure is shown as follows ##STR00033##
5. The adsorbing material according to claim 4, wherein the carrier is amino-functionalized agarose or amino-functionalized polystyrene resin.
6. A preparation method of an adsorbing material for adsorbing multiple pathogenic factors of sepsis, comprising following steps: 1) In tetrahydrofuran or tetrahydrofuran aqueous solution or ethanol aqueous solution, compound 12 reacts with carrier M to generate compound 13, the mass ratio of compound 12 and carrier M is 0.011:100, reaction equation is: ##STR00034## 2) Blockade of residual amino of carrier: In N,N-Diisoprolethylaamine, acetic anhydride is added into compound 13, and reacts with compound 13 to obtain crude product in which the residual amino of carrier are blocked, the equivalence ratio of compound 13 and acetic anhydride is 1:12, reaction equation is: ##STR00035## 3) Preparation of end product: In methanol, 26M hydrochloric acid in methanol is added into the crude product in ice bath, the reaction generates the end product MTAM, the volume ratio of the crude product to hydrochloric acid in methanol is 1:0.51.5, reaction equation is: ##STR00036## wherein n.sub.1-n.sub.4 is an integer between 1-6, and n.sub.5 is an integer between 0-3.
7. A blood purification device for treatment of sepsis comprising the adsorbing material according to claim 4.
8. A blood purification device for treatment of sepsis comprising the adsorbing material according to claim 5.
Description
DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION
(9) Following embodiments are only preferred embodiments to specify the present invention, which do not limit the present invention in any forms.
(10) Chemical reagents used in embodiments were analytically pure purchased from Sigma-Aldrich Co. LLC. LPS, LTA and zymosan was purchased from Sigma-Aldrich Co. LLC, CpG DNA was purchased from Sangon Biotech (Shanghai) Co. LTD, PGN and virus RNA were purchased from InvivoGen Inc. Other reagents are commercially available analytical grade reagents without special description. English abbreviations in embodiments have following meaning.
(11) TABLE-US-00001 abbrevi- abbrevi- ation meaning ation meaning DCM dichloromethane SOCl.sub.2 Thionyl chloride Boc.sub.2O di-tert-butyl dicarbonate Et.sub.3N triethylamine MeOH/NH.sub.3 saturated solution of THF tetrahydrofuran ammonia in methanol RaneyNi Raney nickel H.sub.2O water H.sub.2 hydrogen Pd/C Palladium on carbon MPa Megapascal K.sub.2CO.sub.3 Potassium carbonate EtOH Ethanol BnCl benzyl chloride DMF N,N-Dimethylformamide NaOH sodium hydroxide LPS lipopolysaccharide CpG DNA bacterial genomic DNA PGN peptidoglycan LTA lipoteichoic acid ssRNA Single-stranded RNA dsRNA Double-stranded RNA
Embodiment 1: Preparation of Ligand 1
1.1 Experimental Method
(12) ##STR00015## ##STR00016##
(13) 6 g of Di(2-cyanoethyl) amine (compound 1) was dissolved in 60 ml of dichloromethane under room temperature, and dichloromethane with equivalent amounts of di-tert-butyl dicarbonate was added dropwise to the solution, after 10 hours of reaction, the solution was dried by rotary evaporation, water and ethyl acetate was added for extracting for 3 times, the reaction solution was dried by anhydrous sodium sulfate, the solution was dried by rotary evaporation to obtain compound 2. 11 g of compound 2 was dissolved in 400 ml of saturated solution of ammonia in methanol, 1 g of raney nickel was added, the reactor was filled with hydrogen under the pressure of 4 MPa, after 72 hours of reaction under room temperature, the reaction solution was filtered and dried by rotary evaporation to obtain compound 3. 5.2 g of compound 3 was dissolved into 40 ml of ethanol, and 15 M acrylonitrile dissolved in ethanol was added dropwise in ice bath, after 10 hours of reaction at 40 C., the solution was dried by rotary evaporation to obtain compound 4. 4.2 g of 3,4-Dimethoxy hydrocinnamic acid (compound 5) was dissolved in dichloromethane, and 2.3 g N-Hydroxysuccinimide, 2.8 g dicyclohexylcarbodiimide and 0.5 g 4-dimethylaminopyridine was added, after 12 hours of reaction under room temperature, the reaction solution was filtered and dried by rotary evaporation to obtain compound 6. 2 g of compound 4 was dissolved in 15 ml of dioxane, and 1.76 g of compound 6 was added, after 24 hours of reaction at 50 C., compound 7 was obtained, then 1.48 g of N-(Benzyloxycarbonyloxy) succinimide was added, after 20 hours of reaction, water and ethyl acetate was added for extracting for 3 times, the reaction solution was dried by anhydrous sodium sulfate and rotary evaporation to obtain compound 8. 1 g of compound 8 was dissolved in ethanol, 1 g of di-tert-butyl dicarbonate and 0.1 g of raney nickel was added, the reactor was filled with hydrogen under the pressure of 2 MPa, after 48 hours of reaction at 45 C., the reaction solution was filtered and dried by rotary evaporation to obtain compound 9. 0.44 g of compound 9 was dissolved in methanol, 45 mg of palladium on carbon was added, and the reactor was filled with hydrogen, after 48 hours of reaction at 30 C., the reaction solution was filtered and dried by rotary evaporation to obtain compound 10. 0.4 g of compound 10 was dissolved in dichloromethane, 12 mg of 4-dimethylaminopyridine and 80 mg of succinic anhydride were added, after 48 hours of reaction at 25 C., the solution was dried by rotary evaporation to obtain compound 11, then 5 ml of ethyl acetate was added to dissolve it, and 93 mg of N-Hydroxysuccinimide was added, after 72 hours of reaction at 25 C., the solution was dried by rotary evaporation to obtain compound 12 (ligand 1).
1.2 Experimental Result
(14) ligand 1 was obtained, mass spectrum: [M+Na].sup.+ m/z=957.5; .sup.1H NMR spectrum: 6.80-7.54 (m, 3H), 3.86 (s, 3H), 3.85 (s, 3H), 3.40 (brs, 2H), 3.34-3.28 (m, 3H), 3.23-3.21 (m, 2H), 3.15 (brs, 5H), 3.09-2.97 (m, 5H), 2.93-2.89 (m, 2H), 2.82 (brs, 4H), 2.74-2.64 (m, 3H), 2.59-2.57 (m, 2H), 1.90 (s, 2H), 1.81-1.75 (m, 5H), 1.65-1.63 (m, 3H), 1.44-1.41 (m, 27H), the chemical structure was identified as
(15) ##STR00017##
Embodiment 2: Preparation of Ligand 2
2.1 Experimental Method
(16) the preparation method of embodiment 1 was employed, reaction is carried out under the same scale and condition, except that 3,4-Dimethoxyhydrocinnamic acid (compound 5) was replaced by Hydrocinnamic acid.
2.2 Experimental Result
(17) ligand 2 was obtained, mass spectrum: [M+Na].sup.+ m/z=897.5, the chemical structure was identified as
(18) ##STR00018##
Embodiment 3: Preparation of Ligand 3
3.1 Experimental Method
(19) the preparation method of embodiment 1 was employed, reaction was carried out under the same scale and condition, except that acrylonitrile was replaced by 3-Butene nitrile.
3.2 Experimental Result
(20) ligand 3 was obtained, mass spectrum: [M+Na].sup.+ m/z=985.5, the chemical structure was identified as
(21) ##STR00019##
Embodiment 4: Preparation of Ligand 4
4.1 Experimental Method
(22) the preparation method of embodiment 1 was employed, reaction carried out under the same scale and condition, except that 3,4-Dimethoxyhydrocinnamic acid (compound 5) was replaced by 3,4-Dimethoxybenzoic acid.
4.2 Experimental Result
(23) ligand 4 was obtained, mass spectrum: [M+Na].sup.+ m/z=929.5, the chemical structure was identified as
(24) ##STR00020##
Embodiment 5: Preparation of Ligand 5
5.1 Experimental Method
(25) the preparation method of embodiment 1 was employed, reaction was carried out under the same scale and condition, except that Di(2-cyanoethyl) amine (compound 1) was replaced by Iminodiacetonitrile.
5.2 Experimental Result
(26) ligand 5 was obtained, mass spectrum: [M+Na].sup.+ m/z=929.5, the chemical structure was identified as
(27) ##STR00021##
Embodiment 6: Preparation of Adsorbing Material for Multiple Pathogenic Factors (MTAM01S) of Sepsis with Agarose as Carrier
6.1 Experimental Method
(28) ##STR00022##
(29) 5 ml of amino-functionalized agarose gel (purchased from Beijing wei shi bo hui chromatography technology co. LTD.) was dispersed in 2 ml of Tetrahydrofuran, then 2 mg of ligand 1 was dissolved into a small amount of Tetrahydrofuran and added dropwise in this solution, after 48 hours of reaction under room temperature, the reaction solution was filtered and washed with water to obtain compound 13. 0.5 ml of 10 mM N,N-Diisopropylethylamine was added in compound 13, then 0.8 ml of acetic anhydride was added, after 8 hours of reaction under room temperature, the reaction solution was filtered, and crude product of which the amino groups were blocked was obtained. The crude product was dissolved into 3 ml of methanol, 2 ml of 6 M hydrochloric acid in methanol was added dropwise into the solution in ice bath, after about 2 hours of reaction under room temperature, the reaction solution was filtered and washed with water to obtain the end product MTAM01S.
6.2 Experimental Result
(30) adsorbing material MTAM01S was obtained, and saved in 20% ethanol, structure was shown in
Embodiment 7: Preparation of Adsorbing Material for Multiple Pathogen-Associated Molecular Patterns (MTAM01P) with Polystyrene Resin as Carrier
7.1 Experimental Method
(31) the preparation method of embodiment 6 was employed, reaction was carried out under the same scale and condition, except that amino-functionalized agarose gel was replaced by (Aminomethyl)poly(styrene-co-divinylbenzene) (purchased from Sigma-Aldrich)
7.2 Experimental Result
(32) adsorbing material MTAM01P was obtained, saved in 20% ethanol, structure was shown in
Embodiment 8: The Static Adsorption of Bacterial Endotoxin (LPS) by MTAM01S and MTAM01P in Water
8.1 Experimental Method
(33) 0.5 ml of endotoxin (1 g/ml) was isovolumetrically mixed with 0.5 ml of agarose resin (S carrier), polystyrene resin (P carrier), MTAM01S or MTAM01P respectively, the mixture was shaken and the reaction was carried out for 1 hour at 37 C. The reaction solution was centrifuged to collect the supernatant for the quantitative determination of endotoxin. Detection method was referred to bacterial endotoxins test of Appendix XI E of Chinese Pharmacopoeia (Volume II) and literature Wei Guo, Zheng Jiang. Analysis and countermeasure of influence factors of quantitative detection of bacterial endotoxin. Journal of Regional Anatomy and Operative Surgery, 2003, 12:215-216.. The experimental result was expressed by measured endotoxin value and converted to adsorption rate.
Experimental Result
(34) adsorption rate of the agarose and the polystyrene resin on endotoxin in water were only 8.31% and 8.39%, indicating the carriers themselves almost had no adsorption capacity. MTAM01S and MTAM01P have good adsorption activity on endotoxin, the adsorption rates reached to 93.83% and 89.41% respectively, the results were shown in
Embodiment 9: The Static Adsorption of Bacterial Endotoxin (LPS) by MTAM01S and MTAM01P in Blood Plasma
9.1 Experimental Method
(35) 0.5 ml of endotoxin (1 g/ml) dissolved in human blood plasma was isovolumetrically mixed with 0.5 ml of agarose resin (S carrier), polystyrene resin (P carrier), MTAM01S or MTAM01P respectively, the mixture was shaken and the reaction was carried out for 1 hour at 37 C. The reaction solution was centrifuged to collect the supernatant for the quantitative determination of endotoxin. Detection method was the same as embodiment 8. The experimental result was expressed by measured endotoxin value and converted to adsorption rate.
9.2 Experimental Result
(36) adsorption rate of the agarose and the polystyrene resin on endotoxin in blood plasma were only 7.61% and 8.20%, indicating the carriers themselves almost had no adsorption capacity. MTAM01S and MTAM01P had good adsorption activity on endotoxin in blood plasma, the adsorption rates reached to 92.67% and 88.10% respectively, the result was shown in
Embodiment 10: The Dynamic Adsorption of Bacterial Endotoxin (LPS) by MTAM01S and MTAM01P in Blood Plasma
10.1 Experimental Method
(37) 10 ml of agarose resin (S carrier), polystyrene resin (P carrier), MTAM01S or MTAM01P were separately added into a chromatographic column with a diameter of 3 cm and a height of 15 cm, 10 ml of endotoxin (1 g/ml) dissolved in human blood plasma was loaded on the column, and then the percolate was repeatedly loaded for 8 times, the level of endotoxin in each percolate was detected. Detection method is the same as embodiment 8. The experimental result was expressed by measured endotoxin value and converted to adsorption rate.
10.2 Experimental Result
(38) the agarose and polystyrene resin almost has no adsorption capacity on endotoxin, but MTAM01S and MTAM01P had good adsorption effects on endotoxin, and the adsorption effect was in proportion to times of adsorption, the final adsorption rates reached to 92.83% and 85.90% respectively, the results were shown in
Embodiment 11: The Static Adsorption of Bacterial Endotoxin (LPS), Bacterial Genomic DNA (CpG DNA), Peptidoglycan (PGN), Lipoteichoic Acid (LTA), Virus ssRNA, Virus dsRNA and Zymosan by MTAM01S and MTAM01P in Blood Plasma
11.1 Experimental Method
(39) 0.5 ml of bacterial endotoxin (1 g/ml), Bacterial genomic DNA (10 g/ml), peptidoglycan (10 g/ml), lipoteichoic acid (10 g/ml), virus ssRNA (10 g/ml), virus dsRNA (10 g/ml) or zymosan (10 g/ml) dissolved in human blood plasma, were separately isovolumetrically mixed with 0.5 ml of agarose resin (S carrier), polystyrene resin (P carrier), MTAM01S or MTAM01P, the mixture was shaken and the reaction was carried out for 1 hour at 37 C. The reaction solution was centrifuged and 20 l of the supernatant was collected and added into murine macrophage RAW 264.7 cells (110.sup.6/ml) cultured in vitro, after 12 hours of incubation, the stimulation of inflammatory cells by blood plasma that includes pathogen-associated molecular patterns before and after adsorption was detected. The detailed detection method was carried out according to the operating manual of mouse ELISA kit of eBioscience, the main steps included: {circle around (1)} the supernatant of RAW 264.7 cell culture medium was added into 96-well plate coated with capture antibody, and incubated for 2 hours under room temperature, washed 5 times with PBS; {circle around (2)} primary antibody marked with biotin was added, and incubated for 1 hour under room temperature, washed 5 times with PBS; {circle around (3)} Horseradish Peroxidase marked with avidin was added, and incubated for half an hour under room temperature, washed 5 times with PBS; {circle around (4)} coloring solution was added, and incubated for 10 minutes at 37 C., then stop solution was added; {circle around (5)} Optical density value was measured by microplate reader at 450 nm wavelength. Experimental result reflected the adsorption capacity of adsorbing materials on pathogen-associated molecular patterns by inhibition ratio of TNF- release in inflammatory cells.
11.2 Experimental Result
(40) the agarose and polystyrene resin had no absorption effects on any pathogen-associated molecular patterns, manifesting as no inhibiting effect on TNF- release in RAW 264.7 cells stimulated by pre- and post-treatment of blood plasma. However the stimulation of inflammatory cells by blood plasma was significantly attenuated after the treatment of MTAM01S and MTAM01P, indicating that after the adsorption by MTAM01S and MTAM01P, the level of pathogen-associated molecular patterns in blood plasma was significantly reduced. Results were shown in
Embodiment 12: The Dynamic Adsorption of Bacterial Endotoxin (LPS), Bacterial Genomic DNA (CpG DNA), Peptidoglycan (PGN), Lipoteichoic Acid (LTA), Virus ssRNA, Virus dsRNA and Zymosan by MTAM01S and MTAM01P in Blood Plasma
12.1 Experimental Method
(41) 10 ml of agarose resin (S carrier), polystyrene resin (P carrier), MTAM01S or MTAM01P were separately added into a chromatographic column with a diameter of 3 cm and a height of 15 cm, 10 ml of endotoxin (1 ug/ml), bacterial genomic DNA (10 g/ml), peptidoglycan (10 g/ml), lipoteichoic acid (10 g/ml), virus ssRNA (10 g/ml), virus dsRNA (10 g/ml) and zymosan (10 g/ml) dissolved in human blood plasma were loaded on the column, then it's the percolate was repeatedly loaded for 5 times, 20 l of the first, third and fifth percolates were added into RAW 264.7 cells, the stimulation effect of inflammatory cells by blood plasma containing pathogen-associated molecular patterns before and after adsorption was detected according to the method described in embodiment 11.
12.2 Experimental Result
(42) the agarose resin and polystyrene resin had no absorption effect on any pathogen-associated molecular patterns, MTAM01S and MTAM01P could significantly adsorb various pathogen-associated molecular patterns, manifesting as the stimulation of inflammatory cells by blood plasma was significantly attenuated after absorption (significant decrease in release of TNF-), indicating that after adsorption of MTAM01S and MTAM01P, the level of pathogen-associated molecular patterns in blood plasma was significantly reduced, results were shown in
Embodiment 13: The Static Adsorption of Bacterial Lysate (Mixture of Multiple Pathogen-Associated Molecular Patterns) by MTAM01S and MTAM01P
13.1 Experimental Method
(43) the cultured Escherichia coli and Staphylococcus aureus was separately added with Lysis Buffer (50 mM Tris pH 8.0, 10% glycine, 0.1% triton-X100, 100 ug/ml Lysozyme, 1 mM PMSF) in a volume ratio of 2:1, broke down the cell membrane by sonication (3 times, 20 seconds for each), the bacterial cleavage product treated by sonication was diluted with human blood plasma, the concentration was 110.sup.8 CFU/ml (Escherichia coli) and 510.sup.8 CFU/m (Staphylococcus aureus) according to bacteria count. The cleavage products were isovolumetrically mixed with 0.5 ml of agarose resin (S carrier), polystyrene resin (P carrier), MTAM01S or MTAM01P, respectively, the mixture was shaken and the reaction was carried out for 1 hour at 37 C. The reaction solution was centrifuged and 20 l of supernatant was collected and added into murine macrophage RAW 264.7 cells (110.sup.6/ml) cultured in vitro, after 12 hours of incubation, the stimulation of inflammatory cells by blood plasma before and after adsorption was detected, detection method was the same as embodiment 11.
13.2 Experimental Result
(44) the agarose resin and polystyrene resin themselves had no absorption effect on various bacterial pathogen-associated molecular patterns, MTAM01S and MTAM01P had good adsorption activity on pathogen-associated molecules mixture derived from Escherichia coli (gram-negative bacteria) and Staphylococcus aureus (gram-positive bacteria), manifesting as stimulating activity of inflammatory cells by bacterial lysate significantly attenuated after adsorption of MTAM01S and MTAM01P (significant decrease in release of TNF-), results were shown in
Embodiment 14: The Dynamic Adsorption of Bacteria Lysate (Mixture of Multiple Pathogen-Associated Molecular Patterns) by MTAM01S and MTAM01P
14.1 Experimental Method
(45) 10 ml of agarose resin (S carrier), polystyrene resin (P carrier), MTAM01S or MTAM01P were separately added into a chromatographic column with a diameter of 3 cm and a height of 15 cm. The Escherichia coli and Staphylococcus aureus lysate were prepared according to the method of embodiment 10. 10 ml of bacterial lysate diluted with human blood plasma was loaded on the column, then the percolate was repeatedly loaded for 5 times, 20 l of the first, third and fifth percolates were added into RAW 264.7 cells, the stimulation effect of inflammatory cells by bacteria lysate before and after adsorption was detected according to the method described in embodiment 11.
14.2 Experimental Result
(46) the agarose resin and polystyrene resin themselves had no absorption effect on various pathogen-associated molecular patterns, MTAM01S and MTAM01P were capable of adsorbing the mixture of pathogen-associated molecular patterns derived from Escherichia coli (gram-negative bacteria) and Staphylococcus aureus (gram-positive bacteria), manifesting as stimulating activity of inflammatory cells by bacterial lysate significantly attenuated after adsorption of MTAM01S and MTAM01P (significant decrease in release of TNF-), results were shown in
Embodiment 15: Preparation of Adsorbing Material Based on Ligands 25
15.1 Experimental Method
(47) the preparation method of embodiment 6 was employed, reaction was carried out under the same scale and condition, except that ligand 1 was replaced by ligand 2, ligand 3, ligand 4 or ligand 5, and ligand 25 was coupled with amino-functionalized agarose gel or amino-functionalized polystyrene resin respectively.
15.2 Experimental Method
(48) below-mentioned adsorbing materials were obtained: an adsorbing material MTAM02S with agarose gel as carrier, ligand 2 as ligand; an adsorbing material MTAM03S with agarose gel as carrier, ligand 3 as ligand; an adsorbing material MTAM04S with agarose gel as carrier, ligand 4 as ligand; an adsorbing material MTAM05S with agarose gel as carrier, ligand 5 as ligand; an adsorbing material MTAM02P with polystyrene resin as carrier, ligand 2 as ligand; an adsorbing material MTAM03P with polystyrene resin as carrier, ligand 3 as ligand; an adsorbing material MTAM04P with polystyrene resin as carrier, ligand 4 as ligand; an adsorption material MTAM05P with polystyrene resin as carrier, ligand 5 as ligand.
Embodiment 16: The Dynamic Adsorption of Bacterial Endotoxin (LPS), Bacterial Genomic DNA (CpG DNA), Peptidoglycan (PGN), Lipoteichoic Acid (LTA), Virus ssRNA, Virus dsRNA and Zymosan by MTAM0205S as Well as MTAM0205P in Blood Plasma
16.1 Experimental Method
(49) the preparation method of embodiment 12 was employed, reaction was carried out under the same scale and condition, except that adsorbing material MTAM01S and MTAM01P were replaced by MTAM0205S and MTAM0205P, respectively.
16.2 Experimental Result
(50) MTAM0205S and MTAM0205P were capable of adsorbing various pathogen-associated molecular patterns, manifesting as stimulating activity of inflammatory cells by blood plasma significantly attenuated after adsorption (significant decrease in release of TNF-). After being filtered for 5 times, the inhibition ratio of TNF- release in inflammatory cells stimulated by pathogen-associated molecular patterns was used to represent the adsorption of pathogen-associated molecular patterns by adsorbing material, results were showed in table 1.
(51) TABLE-US-00002 TABLE 1 the detection of adsorption capacity of MTAM02~-05S as well as MTAM02~05P Pathogen-associated Adsorbing molecular patterns CpG virus virus material LPS DNA PGN LTA ssRNA dsRNA Zymosan MTAM02S 92.6% 80.4% 84.3% 76.8% 64.3% 65.9% 70.7% MTAM03S 88.7% 90.7% 96.6% 81.6% 72.2% 77.1% 72.6% MTAM04S 94.4% 85.2% 88.6% 80.1% 78.6% 70.6% 68.9% MTAM05S 82.7% 75.3% 79.4% 86.4% 67.7% 62.8% 77.6% MTAM02P 71.6% 81.5% 75.6% 85.3% 83.4% 76.2% 69.7% MTAM03P 77.8% 84.6% 84.2% 90.6% 64.4% 60.5% 79.8% MTAM04P 64.9% 69.4% 78.6% 66.5% 52.3% 60.9% 74.6% MTAM05P 83.6% 88.9% 80.2% 76.4% 70.6% 71.8% 77.1%
(52) Above-mentioned experiments showed that adsorbing material of the present invention had significant absorption effects on bacterial endotoxin, bacterial genomic DNA, peptidoglycan, lipoteichoic acid, virus RNA and zymosan in fluid such as blood plasma and the like, the stimulation effect of immune cells by blood plasma was significantly attenuated after adsorption, the adsorbing material of the present invention was suitable for blood purification of sepsis patients.