CN117582556B - Method for reducing antigenicity of biomedical material - Google Patents

Method for reducing antigenicity of biomedical material Download PDF

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CN117582556B
CN117582556B CN202410077625.1A CN202410077625A CN117582556B CN 117582556 B CN117582556 B CN 117582556B CN 202410077625 A CN202410077625 A CN 202410077625A CN 117582556 B CN117582556 B CN 117582556B
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super
biomedical
hydrophilic polypeptide
aqueous solution
carbon
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CN117582556A (en
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林永亮
何海娜
吴有陵
罗锦荣
周涛
李文龙
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Guangzhou Ruitai Biological Technology Co ltd
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Guangzhou Ruitai Biological Technology Co ltd
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Abstract

The invention belongs to the technical field of biomedical materials, and discloses a method for reducing antigenicity of a biomedical material, which comprises the steps of preparing an antigen shielding material-dopamine modified super-hydrophilic polypeptide by adopting a carbon-carbon double bond modified compound, super-hydrophilic polypeptide and 3-methacrylic dopamine, and carrying out atomization spraying treatment on the biomedical material, wherein the amino acid sequence of the super-hydrophilic polypeptide is YYTYYSYYT. By the technical scheme, the antigenicity of the biomedical material can be reduced, and the structural integrity and mechanical property of the biomedical material can be maintained.

Description

Method for reducing antigenicity of biomedical material
Technical Field
The invention belongs to the technical field of biomedical materials, and particularly relates to a method for reducing antigenicity of biomedical materials.
Background
The amniotic membrane is the innermost layer of placenta, is smooth, has no blood vessel, nerve and lymph, has certain elasticity and is about 0.02-0.5 mm thick. Amniotic membrane has a structure similar to that of conjunctival tissue of human eyes, contains substances required for growth of conjunctival cells and corneal epithelial cells, and is widely used for treating diseases in the field of ophthalmology, including conjunctiva, cornea, limbal stem cell reconstruction, glaucoma surgery and the like. The amniotic membrane in clinical use at present mainly comes from human sources, and is used as an allograft implantable medical device, and has certain antigenicity in comparison with an animal-derived implantable medical device although the amniotic membrane has lower immunogenicity.
The existing methods for removing antigenic substances generally adopt the "subtractive principle", such as Chinese patent 202080057956.9, when removing antigenic biomolecules, a reducing agent, a detergent and/or an enzyme is used to dissolve hydrophilic biomolecules in the bioprosthetic tissue, wherein the reducing agent can optionally comprise beta-mercaptoethanol, dithiothreitol and tributylphosphine; the detergent may optionally include a nonionic detergent, a zwitterionic detergent, and an ionic detergent; the enzyme may be selected from the group consisting of nucleases, lipases, carbohydrases and depolymerases. Although the use of detergents, reducing agents and/or enzymes as described above may remove antigens to a large extent from biomedical materials, it is inevitable that the biomedical materials are damaged in terms of structural integrity and mechanical properties, which may directly lead to loss of part of functional components and degradation of mechanical properties of the biomedical materials. For example, when the above-mentioned "reducing agent, detergent and/or enzyme" is used to dissolve hydrophilic biological molecules in bioprosthetic tissue, the amnion material has a relatively low thickness, and therefore has relatively low mechanical properties such as maximum uniaxial tensile fracture stress, which inevitably results in the destruction of structural integrity and mechanical properties, and also causes the loss of functional components (such as growth factors), which affects the use of the amnion biomedical material.
Therefore, there is a need to develop a novel method for reducing antigenicity of biomedical materials, so as to overcome the defects of the prior art and meet clinical demands to a greater extent.
Disclosure of Invention
The invention aims to provide a method for reducing the antigenicity of biomedical materials, which is characterized in that compared with the existing method for reducing the antigenicity of biomedical materials by adopting the subtraction principle, the method is realized by the atomization spraying treatment of an antigen shielding material-dopamine modified super-hydrophilic polypeptide, thereby realizing the effects of reducing the antigenicity of biomedical materials and maintaining the structural integrity and mechanical properties of the biomedical materials.
In order to solve the technical problems, the invention provides a method for reducing antigenicity of biomedical materials, which aims to overcome the defects of the prior art and meet clinical requirements to a greater extent. Unlike the solution (i.e., using detergents, reducing agents and/or enzymes to remove antigens from biomedical materials), the present invention provides a "non-subtractive principle" solution, thereby achieving both reduced antigenicity of biomedical materials and maintaining the structural integrity and mechanical properties of biomedical materials themselves.
The invention is realized by the following technical scheme: a method for reducing antigenicity of biomedical material comprises preparing antigen shielding material-dopamine modified super-hydrophilic polypeptide from carbon-carbon double bond modified compound, super-hydrophilic polypeptide and 3-methyl acrylamide, atomizing and spraying biomedical material,
the amino acid sequence of the super-hydrophilic polypeptide is YYTYYTYT, and the structural formula is as follows:
the biomedical material comprises an allogenic material and an animal source material, wherein the allogenic material comprises human amniotic membrane, cornea and pericardium; the animal source material comprises amniotic membrane, cornea, pericardium, small intestine mucosa, pleura, peritoneum and fat omentum of pig or cattle.
Before atomization spraying treatment, the biomedical material is subjected to radiation treatment, wherein the radiation treatment is to carry out gamma ray radiation on the biomedical material in an air atmosphere, and the radiation dosage is controlled to be 5-50 kGy.
Before atomization spraying treatment, the biomedical material is subjected to ultraviolet irradiation treatment, wherein the ultraviolet irradiation treatment is to irradiate the biomedical material for 60-120 min by adopting an ultraviolet curing lamp with the wavelength of 50-280 w and 365nm in an air atmosphere.
The preparation method of the antigen shielding material-dopamine modified super-hydrophilic polypeptide comprises the following steps:
a. preparing an aqueous solution of the super-hydrophilic polypeptide with the mass concentration of 5% and an aqueous solution of the carbon-carbon double bond modified compound with the mass concentration of 2.5%, respectively, and reacting for 22-26 hours at the temperature of 20-37 ℃ according to the volume ratio of the aqueous solution of the super-hydrophilic polypeptide to the aqueous solution of the carbon-carbon double bond modified compound being 0.6-1.2:1 to prepare the carbon-carbon double bond modified super-hydrophilic polypeptide;
b. preparing an aqueous solution of the super-hydrophilic polypeptide modified by the carbon-carbon double bond with the mass concentration of 5%, adding an aqueous solution of 3-methacryloyl dopamine with the mass concentration of 2.5%, carrying out polymerization reaction for 1-24 h at the temperature of 4-40 ℃ in the presence of an initiator, and carrying out dialysis purification and freeze-drying to obtain the polypeptide.
The carbon-carbon double bond modified compound is N-acryloyloxy succinimide.
The initiator is a mixture of ammonium persulfate and sodium bisulfite.
The dialysis purification is to soak the materials in deionized water for 3 to 5 days at room temperature by adopting a dialysis bag with the molecular weight cut-off of 2000 to 3000.
The atomization spraying treatment is to spray the surface of the biomedical material by adopting an atomization spraying machine.
Compared with the prior art, the invention has the following advantages:
(1) In the existing method for reducing the antigenicity of the biomedical material based on the subtraction principle, the structural integrity and mechanical properties of the biomedical material are affected by removing the antigen from the biomedical material by using a detergent, a reducing agent and/or an enzyme, in particular to the amniotic material with a thinner thickness. Based on the above, the invention provides a method for reducing antigenicity by adopting a non-subtractive principle, namely adopting an antigen shielding material-dopamine modified super-hydrophilic polypeptide to carry out atomization spraying treatment on a biomedical material, or adopting irradiation crosslinking or ultraviolet irradiation crosslinking to carry out crosslinking treatment on the biomedical material before the atomization spraying treatment, thereby realizing the function of reducing antigenicity of the biomedical material and keeping the component integrity and mechanical property of the biomedical material. Both treatments are based on the "non-subtractive principle" and may be used together or one of them may be used alone.
(2) When the biomedical material is processed based on the non-subtraction principle, the process of soaking treatment by the aqueous solution does not exist, so that the growth factors contained in the amniotic membrane can be effectively reserved, and the loss of effective functional components in the biomedical material can be effectively reduced compared with the conventional method of soaking treatment by the aqueous solution. For example, growth factors in the amniotic membrane for ophthalmic use are important functional components for promoting ocular surface regeneration, and the conventional way of reducing antigenicity by soaking in aqueous solution inevitably causes the loss of water-soluble growth factors.
(3) The invention adopts the special super-hydrophilic polypeptide for the first time, and the 3-methacryloyl dopamine can be combined with the antigenic determinant by the synergistic effect with the 3-methacryloyl dopamine, and the super-hydrophilic polypeptide can form a hydration layer on the surface of the antigenic determinant, thereby inhibiting antigen-antibody combination and achieving the aim of reducing the antigenicity of the biomedical material.
(4) The antigen shielding material-dopamine modified super-hydrophilic polypeptide is prepared by a carbon-carbon double bond polymerization reaction, namely, the first step is to modify the carbon-carbon double bond of the super-hydrophilic polypeptide to obtain the carbon-carbon double bond modified super-hydrophilic polypeptide so as to endow the super-hydrophilic polypeptide with a carbon-carbon double bond functional group, thereby enabling the super-hydrophilic polypeptide to have a free radical polymerization chemical reaction with 3-methacryloyl dopamine; the second step is that the super-hydrophilic polypeptide modified by carbon-carbon double bonds and 3-methyl acrylamide are polymerized together, and the chemical co-polymerization mode can ensure the stable combination of the super-hydrophilic polypeptide and dopamine, thereby being beneficial to the stable combination of the super-hydrophilic polypeptide and biomedical materials and playing the antigen shielding role.
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FIG. 1 is a schematic diagram of a chemical reaction for preparing an antigen shielding material-dopamine modified super-hydrophilic polypeptide according to the present invention;
FIG. 2 is a schematic diagram of the principle of reducing antigenicity of biomedical materials by adopting an antigen shielding material-dopamine modified super-hydrophilic polypeptide in the invention;
FIG. 3 is a graph showing the results of antigenicity test of the materials of examples 2 to 6 and comparative examples 1 to 7 according to the present invention.
Detailed Description
The objects, technical solutions and advantageous effects of the present invention will be described in further detail below.
It is noted that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed, and unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The invention aims at providing a method for reducing antigenicity of biomedical materials, which at least comprises the following schemes:
(1) The method comprises the steps of adopting an antigen shielding material-dopamine modified super-hydrophilic polypeptide to carry out atomization spraying treatment on a biomedical material;
(2) After the biomedical material is subjected to radiation treatment, the biomedical material is treated in the mode (1);
(3) After the biomedical material is subjected to ultraviolet irradiation treatment, the biomedical material is treated in the mode of (1).
In the treatment mode, the configuration transformation of the protein type antigen in the biomedical material can be realized through radiation or ultraviolet irradiation treatment, so that the antigenicity of the biomedical material is reduced. The principle of reducing antigenicity of biomedical materials by adopting the antigen shielding material-dopamine modified super-hydrophilic polypeptide is as follows: the dopamine is combined with the antigenic determinant, so that the superhydrophilic polypeptide part forms a hydration layer on the surface of the antigenic determinant, and the hydration layer is used for inhibiting antigen-antibody combination, thereby achieving the antigen shielding effect on the biomedical material and achieving the aim of reducing the antigenicity of the biomedical material.
It should be noted that, the term "simultaneously" as used herein means: the treatment processes for biomedical materials are sequentially performed in sequence, but do not mean that the two treatment processes occur simultaneously in time. Namely: firstly radiating or irradiating with ultraviolet light, and then atomizing and spraying by adopting the original shielding material-dopamine modified super-hydrophilic polypeptide.
Specifically, when the radiation treatment is performed, the biomedical material is subjected to gamma ray radiation in an air atmosphere, and the radiation dose is controlled to be 5-50 kGy. When the ultraviolet irradiation treatment is carried out, the biomedical material is irradiated for 60-120 min by an ultraviolet curing lamp with the wavelength of 50-280 w and 365nm in the air atmosphere.
The preparation method for preparing the antigen shielding material-dopamine modified super-hydrophilic polypeptide comprises the following steps:
1) Preparation of carbon-carbon double bond modified super hydrophilic polypeptide
Preparing an aqueous solution of the super-hydrophilic polypeptide with the mass concentration of 5% and an aqueous solution of the carbon-carbon double bond modified compound (namely N-acryloyloxy succinimide) with the mass concentration of 2.5%, respectively, reacting for 22-26 hours at 20-37 ℃ according to the volume ratio of the aqueous solution of the super-hydrophilic polypeptide to the aqueous solution of the carbon-carbon double bond modified compound of 0.6-1.2:1, and freeze-drying to obtain the carbon-carbon double bond modified super-hydrophilic polypeptide;
2) Preparation of antigen shielding material-dopamine modified super-hydrophilic polypeptide
Preparing the super-hydrophilic polypeptide modified by the carbon-carbon double bond into an aqueous solution with the mass concentration of 5%, adding an aqueous solution of 3-methacrylic dopamine with the mass concentration of 2.5%, adding an initiator, carrying out polymerization reaction for 1-24 h at the temperature of 4-40 ℃, then soaking in deionized water for 3-5 days at room temperature by adopting a dialysis bag with the molecular weight cutoff of 2000-3000, and freeze-drying to obtain the polypeptide.
The amino acid sequence of the super-hydrophilic polypeptide is YYTYYTYT, and the amino acid sequence corresponding to three letters is Tyr-Tyr-Thr-Ser-Tyr-Tyr-Thr, and the structural formula is as follows:
further, the chemical reaction process of the prepared antigen shielding material-dopamine modified super-hydrophilic polypeptide is shown in fig. 1, and the schematic diagram for reducing the antigenicity of the biomedical material is shown in fig. 2.
The initiator added in the invention is a mixture of ammonium persulfate and sodium bisulphite, and the mass concentration of the mixture is in the range of 0.1-1%. Preferably, the mass ratio of the ammonium sulfate to the sodium bisulfite in the initiator is 1:1, and can be used for initiating the polymerization reaction between carbon-carbon double bonds.
The present invention will be described in further detail with reference to examples, but embodiments of the present invention are not limited thereto. In the following embodiments, the super-hydrophilic polypeptide is purchased from Shanghai Yao Biotechnology Co., ltd; the model of the atomizing applicator was FS1006, available from Panxoenergy ultrasonic technology Co., ltd; the human amniotic membrane is obtained from legal and regular hospitals after the procedures of ethical examination, informed consent of a donor and the like, and the obtaining way is legal and compliant.
Example 1:
the embodiment is a preparation method of an antigen shielding material-dopamine modified super-hydrophilic polypeptide, which comprises the following steps:
preparing 5% by mass concentration of super-hydrophilic polypeptide (YYTYYTYT) aqueous solution, adding 2.5% by mass concentration of carbon-carbon double bond modified compound, namely N-acryloyloxy succinimide aqueous solution, reacting for 24 hours at 25 ℃ according to the volume ratio of the super-hydrophilic polypeptide aqueous solution to the N-acryloyloxy succinimide aqueous solution of 1:1, and freeze-drying to obtain the carbon-carbon double bond modified super-hydrophilic polypeptide.
Preparing the prepared super-hydrophilic polypeptide modified by the carbon-carbon double bond into an aqueous solution with the mass concentration of 5%, adding an aqueous solution of 3-methacryloyl dopamine with the mass concentration of 2.5%, adding an initiator to initiate polymerization for 24 hours at 37 ℃, soaking the solution in deionized water for 3 days at room temperature by adopting a dialysis bag with the molecular weight cut-off of 2000, and freeze-drying to obtain the polypeptide.
The initiator added in this example was a mixture of ammonium persulfate and sodium bisulfite at a mass concentration of 0.5%, wherein the mass concentration ratio of ammonium sulfate to sodium bisulfite was 1:1.
Example 2:
the method for carrying out atomization spraying treatment on biomedical materials by adopting the antigen shielding material-dopamine modified super-hydrophilic polypeptide prepared in the embodiment 1 comprises the following treatment steps:
preparing the freeze-dried antigen shielding material-dopamine modified super-hydrophilic polypeptide into an aqueous solution with the mass solubility of 1%, and spraying the surface of the human amniotic membrane by adopting an atomization spraying machine.
Example 3:
the embodiment is a method for treating biomedical materials by adopting radiation to realize the configuration transformation of protein type antigens in the biomedical materials so as to reduce antigenicity.
Specifically, gamma ray radiation is carried out on human amniotic membrane in an air atmosphere, and the radiation dosage is controlled at 25kGy.
Example 4:
the embodiment is a method for processing biomedical materials by ultraviolet irradiation to realize the configuration transformation of protein type antigens in the biomedical materials so as to reduce antigenicity.
Specifically, human amniotic membrane is subjected to ultraviolet irradiation treatment in an air atmosphere, and irradiation is performed for 60min by using an ultraviolet curing lamp with the wavelength of 85w and 365 nm.
Example 5:
the method for carrying out atomization spraying treatment by adopting radiation treatment and antigen shielding material-dopamine modified super-hydrophilic polypeptide simultaneously comprises the following steps:
gamma-ray radiation is carried out on human amniotic membrane in an air atmosphere, and the radiation dose is 25kGy. Then, the antigen shielding material-dopamine modified super-hydrophilic polypeptide prepared in the embodiment 1 is adopted to carry out atomization spraying treatment on the human amniotic membrane subjected to the radiation treatment by adopting an atomization spraying machine. When in spraying, the mass solubility of the adopted antigen shielding material-dopamine modified super-hydrophilic polypeptide aqueous solution is 1 percent.
Example 6:
the method for carrying out atomization spraying treatment by adopting ultraviolet irradiation treatment and antigen shielding material-dopamine modified super-hydrophilic polypeptide simultaneously comprises the following steps:
ultraviolet irradiation treatment is carried out on human amniotic membrane in an air atmosphere, and irradiation is carried out for 60min by using an ultraviolet curing lamp with the wavelength of 85w and 365 nm. Then, the antigen shielding material-dopamine modified super-hydrophilic polypeptide prepared in the embodiment 1 is adopted to carry out atomization spraying treatment on the human amniotic membrane subjected to ultraviolet irradiation treatment by adopting an atomization spraying machine. When in spraying, the mass solubility of the adopted antigen shielding material-dopamine modified super-hydrophilic polypeptide aqueous solution is 1 percent.
Comparative example 1:
comparative example 1 is fresh human amniotic membrane.
Comparative example 2:
comparative example 2 is a human amniotic membrane prepared by antigen removal using Triton X-100, DNAse I and RNAse in the reference paper "preparation of decellularized natural bone matrix for removal of antigen component of transplantation immune response" ("Chinese tissue engineering research and clinical rehabilitation", 2011,15 (08): 1355-1359 ").
The method comprises the following specific steps:
fresh human amniotic membrane was prepared, repeatedly rinsed with sterile distilled water, and placed in a jar containing protease inhibitor (0.1 mg/L Laprotin, 0.5 mg/L pepstatin A, 0.6 mg/L pepstatin A) and 0.05 mol/L Tris-HCI (pH 7.4) buffer, and then shaken at constant temperature of 4℃for 5 d. Then the buffer solution in the wide-mouth bottle is replaced by Tris-HCI (pH 7.4) buffer solution containing 3 percent TritonX-100, the protease inhibitor is also added, and the sterile distilled water is used for washing after the constant temperature oscillation is carried out for 7 d at 4C; then adding DNAseI and RNAse mixture, digesting 24h at room temperature, adding Tris-HCI (pH 7.4) buffer containing 3% Triton X-100 into the bottle again, and washing with sterile distilled water after blocking at constant temperature of 7 d at 4 ℃.
Comparative example 3:
comparative example 3 is a human amniotic membrane prepared by the antigen removal method using sodium dodecyl sulfate, trypsin, and sodium deoxycholate in the reference patent "a low antigenicity amniotic membrane tissue and its treatment method" (application No. 202010180761.5)).
The method comprises the following specific steps:
taking out the dehydrated amniotic membrane, putting back into a rotary drum, adding 500 parts by weight of sodium dodecyl sulfate solution with the mass fraction of 0.1% and the temperature of 20 ℃, treating for 30min in the rotary drum, drying the waste liquid, adding 500 parts by weight of water with the temperature of 20 ℃, cleaning for 60min, repeating for 5 times, and dehydrating by a dehydrator;
taking out the dehydrated amniotic membrane, putting back into a rotary drum, adding 500 parts by weight of trypsin solution with pH of 8 and temperature of 20 ℃, treating for 60min in the rotary drum, controlling the temperature to be in waste liquid, adding 500 parts by weight of water with temperature of 20 ℃, cleaning for 60min, repeating for 5 times, and dehydrating by a dehydrator;
taking out the dehydrated amniotic membrane, putting back into a rotary drum, adding 500 parts by weight of 1% sodium deoxycholate solution with the mass fraction of 20 ℃, treating for 120min in the rotary drum, drying the waste liquid, adding 500 parts by weight of water with the temperature of 20 ℃, cleaning for 60min, repeating for 5 times, and dehydrating by a dehydrator.
Comparative example 4:
comparative example 4 is a human amniotic membrane prepared by the method of "an absorbable medical biofilm and its preparation method" (application number 201610839636.4) in which a hypotonic and hypertonic solution interlacing treatment is used for preliminary treatment, and then the material after the preliminary treatment is placed in a surfactant solution for continuous treatment for antigen removal.
The method comprises the following specific steps:
placing fresh amniotic membrane in pure water, vibrating at room temperature 22C for 2 hours at 100rpm, then placing in 10% sodium chloride solution, vibrating at room temperature 22C at 100rpm for 2 hours, taking the amniotic membrane as a cycle, and carrying out co-cycle treatment for 1-3 times; then, the mixture was placed in 1.0% Sodium Dodecyl Sulfate (SDS), and the mixture was subjected to shaking treatment at 100rpm for 10 hours, and finally, the mixture was placed in 1.0% Triton X-200, and the mixture was subjected to shaking treatment at 100rpm for 2 hours.
Comparative example 5:
comparative example 5 is a human amniotic membrane prepared by the method of antigen removal using trypsin, disodium ethylenediamine tetraacetate and dnase in reference patent "a tissue patch and preparation method thereof" (application No. 200810150793. X).
The method comprises the following specific steps:
the preparation of decellularized amniotic membrane comprises taking placenta of healthy puerpera, peeling off the amniotic membrane under aseptic operation, washing with physiological saline, separating the amniotic membrane and chorion to obtain amniotic membrane, soaking in a mixed digestion solution containing 1-4 g/L trypsin and 0.1-0.4 g/L disodium ethylenediamine tetraacetate for at least 1 hr, washing with Phosphate Buffer (PBS), soaking in a DNase solution containing 30-50U/m 1 for more than 25 min, removing residual DNA components, reducing immunogenicity, and rinsing with PBS for later use.
Comparative example 6:
comparative example 6 is a human amniotic membrane prepared by the method of antigen removal using trypsin in the reference patent "amniotic membrane graft or covering for preventing visceral adhesion or bleeding" (application No. 95100812.9).
The method comprises the following specific steps:
the amniotic membrane was taken from a fresh human placenta taken at the time of caesarean section. The amniotic membrane was manually separated from the chorion and washed with distilled water. Clean membranes were first soaked in 10% trypsin solution for 3 hours.
Comparative example 7:
comparative example 7 is a human amniotic membrane prepared by the method of antigen removal using sodium dodecyl sulfate and trypsin in the reference patent "bio-derived amniotic membrane, composite bio-derived amniotic membrane and method of preparation thereof" (application No. 200410036792.4).
The method comprises the following specific steps:
taking fresh healthy human amniotic membrane, rinsing with normal saline for 3 times, degreasing chloroform/methanol (1:1, v/v) to obtain clear supernatant, and rinsing with normal saline for 3 times, each time for 10 minutes; 0.5% w/v SDS (sodium dodecyl sulfate) for 4 hours, and physiological saline was rinsed 3 times for 10 minutes each; 0.25% w/v trypsin was digested for 8 hours, and rinsed 3 times with physiological saline for 10 minutes each.
To further illustrate the substantial features and advantages of the present invention, the following test examples were implemented.
Test example 1: amniotic antigenicity test (human serum lgM and IgG binding test)
The test included the following materials: the amniotic membrane prepared in examples 2-6 and comparative examples 1-7.
The testing method comprises the following steps: lgM and IgG binding assays were performed with human serum. Human serum was obtained from healthy human volunteers (n=5, including all ABO blood groups) and pooled into one single human serum reagent. Fresh human amniotic membrane was used for the control sample. The slides were then washed with PBS and samples were blocked with 10% goat serum for 30 minutes at room temperature. Biotin-bound goat-derived anti-human IgM or lgG was allowed to act at room temperature (1:100 concentration) for 30 minutes to detect IgM or IgG binding. Alexa Fluor 647-conjugated streptavidin was applied as secondary antibody at room temperature and allowed to act for 30 min. Finally, images were taken by a confocal laser microscope and fluorescence intensity was calculated by Image J software. The calculation formula is as follows:
fluorescence relative Intensity = sample fluorescence Intensity (unit: intensity)/control sample (i.e., fresh human amniotic membrane) fluorescence Intensity (unit: intensity)
The experimental results are shown in table 1 and fig. 3.
TABLE 1
Human IgM binding fluorescence relative intensity (unit: Intensity/ Intensity) human IgG binds to fluorescent relative intensities (units: Intensity/ Intensity)
example 2 0.12 0.09
Example 3 0.24 0.18
Example 4 0.26 0.2
Example 5 0.08 0.04
Example 6 0.09 0.05
Comparative example 1 1 1
Comparative example 2 0.36 0.19
Comparative example 3 0.38 0.21
Comparative example 4 0.22 0.1
Comparative example 5 0.24 0.15
Comparative example 6 0.42 0.25
Comparative example 7 0.26 0.17
The experimental results (Table 1 and FIG. 3) show that examples 2-6, compared to comparative example 1 (fresh human amniotic membrane), all have low specific binding fluorescence intensity of human IgM/IgG, i.e. low antigenicity, and in particular, examples 5 and 6, all have lower antigenicity than comparative examples 2-7. Namely, the method shows that: compared with the existing 'subtraction principle' technical scheme for reducing the antigenicity of the biomedical material (namely, removing antigens from the biomedical material by using a detergent, a reducing agent and/or enzyme), the 'non-subtraction principle' technical scheme provided by the invention is that the biomedical material is crosslinked by adopting irradiation crosslinking or ultraviolet irradiation crosslinking, and/or the biomedical material is subjected to atomization spraying treatment by using the antigen shielding material-dopamine modified super-hydrophilic polypeptide, so that the technical effect of reducing the antigenicity of the biomedical material is realized, wherein the effect of reducing the antigenicity of the biomedical material is better when the two technologies are used in combination.
Test example 2: amnion mechanical strength test-maximum uniaxial tensile breaking stress
The test included the following materials: the amniotic membrane prepared in examples 2-6 and comparative examples 1-7.
The testing method comprises the following steps: and (5) testing mechanical properties of the material by using an Instron 5967 universal tester. Samples were cut into 2.5cm x 1cm rectangles, n=6 for each group, and soaked with PBS for 2 hours at 37 ℃ prior to testing. The thickness was measured at 3 positions randomly with a thickness gauge, and the average value of the thickness was taken for stress calculation. Thereafter mounted on a jig of a universal tester and preloaded with a force of 0.1N, uniaxially stretched at a constant speed of 12 mm/min until fracture failure. At the same time, stress-strain curves were recorded and the maximum uniaxial tensile break stress was calculated.
The experimental results are shown in table 2.
TABLE 2
The experimental results (table 2) show that examples 2-6 have near maximum uniaxial tensile stress at break, i.e. better mechanical strength, than comparative example 1 (fresh human amniotic membrane). In addition, the maximum uniaxial tensile breaking stress of comparative examples 2 to 7 was reduced to some extent as compared with comparative example 1. Namely, the method shows that: compared with the existing 'subtraction principle' technical scheme for reducing the antigenicity of the biomedical material (namely, removing antigens from the biomedical material by using a detergent, a reducing agent and/or enzyme), the 'non-subtraction principle' technical scheme provided by the invention is that the biomedical material is crosslinked by irradiation crosslinking or ultraviolet irradiation crosslinking, and/or the biomedical material is subjected to atomization spraying treatment by using the antigen shielding material-dopamine modified super-hydrophilic polypeptide, so that the characteristic of maintaining the self mechanical property of the biomedical material is realized.
Test example 3: measurement of functional component (growth factor) content in amniotic membrane
The test included the following materials: the amniotic membrane prepared in examples 2-6 and comparative examples 1-7.
The testing method comprises the following steps: the amniotic membrane was freeze-dried, weighed, placed in a refiner for pulverization treatment, and centrifuged at 10000g at 4℃for 15 minutes, and the supernatant was separated to measure the growth factor concentration. The two growth factor contents were analyzed using an epidermal growth factor (Epidermal growth factor, EGF) and hepatocyte growth factor (hepatocyte growth factor, HGF) ELISA kit.
The experimental results are shown in tables 3 and 4.
TABLE 3 Table 3
TABLE 4 Table 4
The results of the experiments (tables 3 and 4) show that examples 2-6 have similar concentrations of epidermal growth factor and hepatocyte growth factor compared to comparative example 1 (fresh human amniotic membrane). In addition, comparative examples 2 to 7 have a somewhat reduced concentration of both growth factors as compared to comparative example 1. Namely, the method shows that: compared with the conventional aqueous solution soaking treatment mode, the method provided by the invention effectively reduces the loss of effective functional components in the biomedical material. According to the invention, the biomedical material is crosslinked by irradiation crosslinking or ultraviolet irradiation crosslinking, and/or the antigen shielding material-dopamine modified super-hydrophilic polypeptide is subjected to the non-subtractive principle of atomization spraying treatment on the biomedical material, so that the process of soaking treatment by an aqueous solution does not exist, and therefore, the growth factors contained in the amniotic membrane can be effectively reserved.
The foregoing description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any way, and any simple modification, equivalent variation, etc. of the above embodiment according to the technical matter of the present invention fall within the scope of the present invention.

Claims (6)

1. A method of reducing antigenicity of a biomedical material, characterized by: the method comprises the following steps:
(a) Preparing an aqueous solution of the super-hydrophilic polypeptide with the mass concentration of 5% and an aqueous solution of the carbon-carbon double bond modified compound with the mass concentration of 2.5%, respectively, and reacting for 22-26 hours at the temperature of 20-37 ℃ according to the volume ratio of the aqueous solution of the super-hydrophilic polypeptide to the aqueous solution of the carbon-carbon double bond modified compound being 0.6-1.2:1 to prepare the carbon-carbon double bond modified super-hydrophilic polypeptide;
(b) Preparing an aqueous solution of the super-hydrophilic polypeptide modified by carbon-carbon double bonds, wherein the mass concentration of the aqueous solution is 5%, adding an aqueous solution of 3-methacryloyl dopamine, the mass concentration of which is 2.5%, carrying out polymerization reaction for 1-24 hours at the temperature of 4-40 ℃ in the presence of an initiator, and carrying out dialysis purification and freeze-drying to prepare the antigen shielding material-dopamine modified super-hydrophilic polypeptide;
(c) After the biomedical material is subjected to radiation treatment or ultraviolet irradiation treatment, the biomedical material is subjected to atomization spraying treatment by adopting an antigen shielding material-dopamine modified super-hydrophilic polypeptide,
the amino acid sequence of the super-hydrophilic polypeptide is YYTYYTYT, and the structural formula is as follows:
the radiation treatment is to carry out gamma ray radiation on biomedical materials in an air atmosphere, the radiation dosage is controlled to be 5-50 kGy,
the ultraviolet irradiation treatment is to irradiate biomedical materials for 60-120 min by adopting an ultraviolet curing lamp with the wavelength of 50-280 w and 365nm in an air atmosphere.
2. The method according to claim 1, characterized in that: the biomedical material comprises an allogenic material and an animal source material, wherein the allogenic material comprises human amniotic membrane, cornea and pericardium; the animal source material comprises amniotic membrane, cornea, pericardium, small intestine mucosa, pleura, peritoneum and fat omentum of pig or cattle.
3. The method according to claim 1, characterized in that: the carbon-carbon double bond modified compound is N-acryloyloxy succinimide.
4. The method according to claim 1, characterized in that: the initiator is a mixture of ammonium persulfate and sodium bisulfite.
5. The method according to claim 1, characterized in that: the dialysis purification is to soak the materials in deionized water for 3 to 5 days at room temperature by adopting a dialysis bag with the molecular weight cut-off of 2000 to 3000.
6. The method according to claim 1, characterized in that: the atomization spraying treatment is to spray the surface of the biomedical material by adopting an atomization spraying machine.
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