CN115947958A - Preparation method of collagen micropowder with low immunogenicity - Google Patents

Abstract

The invention provides a method for preparing collagen micropowder with low immunogenicity, which belongs to the technical field of medical biological materials. The collagen sponge extracted by acid enzyme method is dissolved in ionic liquid according to a certain proportion, and then the solution is dropped into the precipitate After centrifugation and washing, the regenerated collagen micropowder is cross-linked, centrifuged, washed, freeze-dried, and sieved to obtain collagen micropowder; the collagen micropowder skin filling material obtained by the present invention effectively reduces the terminal peptide Antibodies, helical region antibodies and central region antibodies have immunogenic responses, and maintain the triple helical structure and biological activity of collagen.

Description

Preparation method of collagen micropowder with low immunogenicity

technical field

The invention belongs to the technical field of medical biomaterials, in particular to a method for preparing collagen micropowder with low immunogenicity.

Background technique

Collagen is an important component of human skin. Collagen provides important structural support for the skin and adjacent soft tissues. The reduction of its content will lead to skin thinning, atrophy and aging. Collagen products are important filler materials in injectable cosmetic surgery. In 1974, the first collagen dermal filler product was introduced. In 1978, human and bovine collagen were used as dermal filler materials for the treatment of depressed scars, subcutaneous atrophy and wrinkles. In 1981, collagen was approved by the FDA as a dermal filler material for nasolabial fold filling. Currently, dermal fillers are used in almost all areas of the face, including the eyes, chin, and cheeks. Most of these procedures are safe and predictable with minimal adverse outcomes.

The success of collagen as a biomaterial is largely due to its low antigenicity and low immunogenicity. Nonetheless, understanding the mechanisms underlying human immunity to collagen and its clinical implications remain critical for the design and application of novel biomedical devices. However, adverse events may occur with the use of fillers, ranging from minor edema and bruising to infection, blindness, ischemia, and stroke. Skin allergy test must be done before injecting collagen products. Positive reactions generally include swelling, induration, erythema, itching, etc. More serious reactions include excessive growth of granulation, severe inflammation, skin necrosis, etc., and the positive rate reaches 3%. For patients with negative skin tests, the incidence of allergic reactions is generally less than 1%.

Generally speaking, the immunoreactive substances caused by collagen materials are mainly cell residues, DNA residues, and miscellaneous proteins that were not completely removed during the extraction process. Among them, the α-Gal antigen on the cell surface is the main target antigen for immune rejection caused by animal tissues or organs, and the combination of α-Gal antigen and anti-α-Gal antibody in the human body will cause a series of adverse inflammatory reactions in the human body . The detection of residual Gal antigen content in animal-derived biological materials is one of the important indicators for controlling product quality. CpG inhibition in DNA unique to vertebrates is an important immune mechanism for the recognition of self and foreign DNA. When exogenous DNA enters the body, the immune system responds by recognizing the characteristic structure of CpG. The detection of residual DNA is one of the important indicators for controlling the risk of immunogenicity of biologically derived materials. In addition, during the preparation of collagen materials, the content of cell residues, DNA residues, and foreign proteins can be effectively reduced by changing purification processes such as enzymatic hydrolysis, salting out, and dialysis.

The acid enzymatic method removes the telopeptide, which can control the content of C-telopeptide and N-telopeptide to eliminate the immunogenicity caused by the telopeptide structure; use hypertonic salt compound surfactant and H ₂ O ₂ treatment to remove residual tissue Cell and surface antigens, control the content of miscellaneous proteins to less than 0.1%; control protein purity through purification, and the residual DNA content is less than 1 μg/mL; use surfactant-alkali treatment-low pH incubation to inactivate virus in animal tissue materials Treatment; low pH incubation combined with γ-rays can remove bacterial lipopolysaccharides and control immunogenicity caused by exogenous microorganisms, especially viruses and bacterial lipopolysaccharides.

However, the collagen molecule itself also has certain antigenic components, some of which cannot be effectively reduced during the extraction process. Antigenic determinants in collagen molecules can be divided into three categories: helical region antibodies, recognition is dependent on the 3D conformation (i.e., the presence of a complete triple helix); central region antibodies, located in the triple helix portion of native collagen, but recognition is based only on amino acid sequence , rather than the 3D conformation; the terminal peptide region antibody, located in the non-helical terminal region of the molecule (terminal peptide). Among them, the immunogenicity of type I collagen is mainly distributed in the C and N terminals of the molecular chain, the telopeptide region composed of short, non-helical amino acid sequences, and the telopeptide is the source of the immunogenicity of natural I collagen, so The removal effect of telopeptide greatly affects the immunogenicity of atelopeptide type Ⅰ collagen. During the extraction process of collagen, it is necessary to remove the immune components in the collagen products, so as to avoid the presence of impurities in the collagen-based biomaterials after implantation into the human body, which may cause adverse phenomena such as allergies, pyrogenicity, or foreign body reactions.

However, the triple helix structure of collagen is a necessary condition for maintaining the biological activity of collagen materials. During the extraction process of collagen, the integrity of the triple helix structure of collagen should be kept as much as possible. Therefore, although the extraction and purification process of collagen basically eliminates cell residues, DNA residues, miscellaneous proteins, and C and N-terminal peptide chains, it effectively reduces the immunogenicity of collagen products. However, antibodies in the helical region and central region of collagen are wrapped and wound by collagen molecular chains, and the antibody components located in the helical region and central region of collagen molecular chains have not been effectively cleared and inactivated. Solvents such as trifluoroethanol (TFE) and hexafluoroisopropanol (HFIP) can be used to dissolve collagen because of their ability to capture hydrogen bonds, electrovalent bonds or destroy hydrophobic bonds, fully dissociate the triple helix structure of collagen, and effectively expose the helix Region antibody and central region antibody are beneficial for subsequent antibody removal treatment. However, once dissolved in TFE or HFIP, collagen will lose its unique triple helical structure and lose its proper biological activity forever, which is no different from gelatin. Currently, there is no regeneration of collagen after treatment with these two solvents technology. The denaturation behavior in these two solvents violates its original intention as a biomimetic material, and thus hinders its high-value utilization in spinning, medical biomaterials, food health care, and cosmetics. Moreover, this type of fluoroalcohol solvent has a large saturated vapor pressure, is volatile, and is highly corrosive and toxic.

Contents of the invention

In view of this, the present invention aims to propose a method for preparing collagen micropowder with low immunogenicity, so as to alleviate the above-mentioned technical problems.

In order to achieve the above object, technical solution of the present invention is achieved in that way:

A method for preparing collagen micropowder with low immunogenicity, comprising the steps of:

S1: Pulverize animal tissues and perform virus inactivation treatment;

S2: Obtain collagen sponge from the above materials using acid enzyme method;

S3: dissolving the collagen sponge in the ionic liquid to obtain a mixed solution;

S4: drop the mixed solution into a precipitant, and obtain regenerated collagen micropowder after precipitation and centrifugation;

S5: The regenerated collagen micropowder is crosslinked with a crosslinking agent, centrifuged, washed, freeze-dried, and finally pulverized and sieved to obtain collagen micropowder.

Further, step S2 includes:

Extract the material in step S1 with pepsin acid solution, wash with hypertonic salting out, dialyze with deionized water and freeze-dry to obtain collagen sponge.

Further, the animal tissue in step S1 is one or more than two of animal skin, Achilles tendon and bovine pericardium.

Further, in step S1, peracetic acid or hydrogen peroxide is used for virus inactivation treatment.

Further, the crosslinking agent includes glutaraldehyde, hexamethylene diisocyanate, epoxy compound, genipin, carbodiimide and dialdehyde polysaccharide.

Further, the precipitating agent is a mixture of ethanol, methanol or acetone and deionized water.

Further, the ionic liquid is imidazolium acetate-type ionic liquid, 1-butyl-3-methylimidazolium chloride-type ionic liquid, bromo 1-ethyl-3-methylimidazolium salt-type ionic liquid, chole chloride A kind of in alkali · 2ZnCl ionic liquid and 1-allyl-3-methylimidazolium chloride salt ionic liquid.

Further, step S5 includes soaking with amino acid or low molecular weight polypeptide to remove unreacted cross-linking agent between cross-linking treatment and centrifugal washing.

Further, bagging dialysis is included between centrifugal washing and freeze-drying in step S5.

Further, after step S5, it includes sterilizing by gamma ray irradiation.

Compared with the prior art, the preparation method of a kind of low immunogenicity collagen micropowder provided by the present invention has the following advantages:

1. The present invention aims to prepare a collagen micropowder material with lower immunogenicity. The micropowder is mainly used for skin filling. This technical method effectively reduces the immunogenicity of the terminal peptide antibody, the helical region antibody and the central region antibody. , which further reduces the possible immunogenic reaction caused by collagen materials, and maintains the triple helical structure and biological activity of collagen, making it have a wider range of applications.

2. In view of the above-mentioned technical defects and the characteristics of ionic liquids, the present invention utilizes ionic liquid-precipitating agent to rearrange the triple helical structure of collagen, which reduces the tightness of the triple helical structure to a certain extent, and effectively exposes the antibodies in the helical region and the antibody in the central region , Through the subsequent cross-linking process, the reduction or inactivation of the antibody in the helical region and the antibody in the central region is achieved, which effectively reduces the immunogenicity of the collagen, and the biological activity of the collagen is effectively maintained.

Description of drawings

The drawings constituting a part of the present invention are used to provide a further understanding of the present invention, and the schematic embodiments and descriptions of the present invention are used to explain the present invention, and do not constitute an improper limitation of the present invention. In the attached picture:

Fig. 1 is a schematic diagram of three kinds of antibodies in collagen according to the present invention; specifically, helical region antibody, central region antibody, and telopeptide antibody; proco l l agens.I nt Rev Connect T i issue Res 1976; 7:61–99];

Fig. 2 is the circular diphasic chromatograms of the three materials described in the specific examples described in the present invention, i.e. the CD figure, wherein A is collagen raw material, B is collagen after ionic liquid treatment, and C is ionic liquid treatment+ Collagen after cross-linking treatment.

Detailed ways

The embodiments of the present invention are described in detail below. This embodiment is implemented on the premise of the technical solution of the present invention, and detailed implementation methods and specific operating procedures are provided, but the protection scope of the present invention is not limited to the following implementation example.

The present invention will be described in detail below in conjunction with examples.

A method for preparing collagen micropowder with low immunogenicity, comprising the steps of:

S1: Pulverize animal tissues and perform virus inactivation treatment;

S2: Obtain collagen sponge from the above materials using acid enzyme method;

S3: dissolving the collagen sponge in the ionic liquid to obtain a mixed solution;

S4: drop the mixed solution into a precipitant, and obtain regenerated collagen micropowder after precipitation and centrifugation;

S5: The regenerated collagen micropowder is crosslinked with a crosslinking agent, centrifuged, washed, freeze-dried, and finally pulverized and sieved to obtain collagen micropowder.

More preferably, step S2 includes:

Extract the material in step S1 with pepsin acid solution, wash with hypertonic salting out, dialyze with deionized water and freeze-dry to obtain collagen sponge.

More preferably, the animal tissue in step S1 is one or more of animal skin, Achilles tendon and bovine pericardium.

More preferably, peracetic acid or hydrogen peroxide is used for virus inactivation treatment in step S1.

More preferably, the crosslinking agent includes glutaraldehyde, hexamethylene diisocyanate, epoxy compound, genipin, carbodiimide and dialdehyde polysaccharide.

More preferably, the precipitating agent is a mixture of ethanol, methanol or acetone and deionized water.

More preferably, the ionic liquid is imidazolium acetate type ionic liquid, 1-butyl-3-methylimidazolium chloride type ionic liquid, bromo 1-ethyl-3-methylimidazolium salt type ionic liquid, chlorine One of choline 2ZnCl ionic liquid and 1-allyl-3-methylimidazolium chloride ionic liquid.

More preferably, step S5 includes soaking with amino acid or low molecular weight polypeptide to remove unreacted cross-linking agent between cross-linking treatment and centrifugal washing.

More preferably, bagging dialysis is included between centrifugal washing and freeze-drying in step S5.

More preferably, after step S5, it includes sterilizing by gamma ray irradiation.

Specifically, the types of collagen used include: type I collagen, type II collagen, type III collagen, type V collagen and type VII collagen. For specific embodiments, it may be a single type of collagen, or a combination of two or more types.

The used treating agent ionic liquid includes: imidazolium acetate type ionic liquid, 1-butyl-3-methylimidazolium chloride salt type ionic liquid, bromo 1-ethyl-3-methylimidazolium salt type ionic liquid, chlorine Choline·2ZnCl ionic liquid and 1-allyl-3-methylimidazolium chloride ionic liquid. Each can be used alone for the treatment of collagen, but at different temperatures.

After ionic liquid treatment, the precipitant used is ethanol, methanol or acetone; or a mixture of these reagents and deionized water.

The crosslinking agents used include glutaraldehyde, hexamethylene diisocyanate, epoxy compounds, genipin, carbodiimide, and dialdehyde polysaccharides.

specific embodiment

As shown in Figure 2: the three materials have two absorption peaks at 200nm and 220nm respectively, indicating that the three materials all maintain a three-dimensional helical structure, but with the progress of the processing process, the integrity of the triple helix of collagen is slightly reduced. The tightness of the helical structure showed a downward trend. The change of the collagen helical structure will lead to the decrease of the peak intensity in the CD spectrum, and the ratio (Rpn) of the peak intensity appearing at the long wavelength (about 223nm) to the peak intensity appearing at the short wavelength (about 200nm) will decrease. The Rpn value of collagen after [BMI M]Ac dissolved and regenerated decreased from 0.152 to 0.127, indicating that the integrity of the triple helix of regenerated collagen was slightly reduced, but the Rpn value of collagen after [BMI M]Cl dissolved and regenerated decreased to 0.05, indicating that the triple-helix structure of collagen after heating and dissolving at 75°C was partially deconstructed. Obviously, a higher dissolution temperature is not conducive to the retention of the triple-helix conformation of collagen.

Using Gal antigen-deficient mice, collagen raw material (Col), ionic liquid-treated collagen (I-Col), ionic liquid-treated + cross-linked collagen (I-Co 1 ) were subcutaneously implanted. l-C) Immunological evaluation study (6 rats in each group). At the same time, a blank control group (CON) and a positive control group (POS) were set up. The CON group underwent "sham" operation, and the mice in the POS group were subcutaneously injected with BSA-complete Freund's adjuvant suspension on the back. Four weeks after the implantation of the test product, immunological index detection was carried out. The results showed that the serum total IgG and IgM contents of the POS group were significantly higher than those of the CON group, and there was a statistically significant difference (P<0.05); the total IgG and IgM contents of the other groups had no statistics compared with the CON group. There were statistically significant differences (P>0.05). The anti-Gal IgG and IgM antibodies in the Col group were slightly higher than those in the I-Col and I-Col groups. It shows that after modification, the immunogenicity of the material tends to decrease. The results of serum cytokine levels in mice are shown in Table 1. Compared with the CON group, the serum IFN-γ and IL-6 contents of the mice in the POS group were significantly increased (P<0.05); the values of the Col group, I-Col group and I-Col-C group showed a downward trend as a whole , among which I-Col-C group compared with Col group, IL-6, IL-10 were statistically significant (marked #), indicating that after modification, the immunogenicity of the material showed a decreasing trend.

Table 1 Mouse immunological evaluation test results

The above are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection scope of the present invention within.

Claims (10)

1. a preparation method of collagen micropowder with low immunogenicity, is characterized in that, comprises the steps: S1: Pulverize animal tissues and perform virus inactivation treatment; S2: Obtain collagen sponge from the above materials using acid enzyme method; S3: dissolving the collagen sponge in the ionic liquid to obtain a mixed solution; S4: drop the mixed solution into a precipitant, and obtain regenerated collagen micropowder after precipitation and centrifugation; S5: The regenerated collagen micropowder is crosslinked with a crosslinking agent, centrifuged, washed, freeze-dried, and finally pulverized and sieved to obtain collagen micropowder. 2. the preparation method of low immunogenicity collagen micropowder according to claim 1, is characterized in that, step S2 comprises: Extract the material in step S1 with pepsin acid solution, wash with hypertonic salting out, dialyze with deionized water and freeze-dry to obtain collagen sponge. 3. The method for preparing collagen micropowder with low immunogenicity according to claim 1, characterized in that: the animal tissue in step S1 is one or more of animal skin, Achilles tendon and bovine pericardium. 4. The method for preparing collagen micropowder with low immunogenicity according to claim 1, characterized in that: in step S1, peracetic acid or hydrogen peroxide is used for virus inactivation treatment. 5. the preparation method of low immunogenicity collagen micropowder according to claim 1 is characterized in that: crosslinking agent comprises glutaraldehyde, hexamethylene diisocyanate, epoxy compound, genipin, carbodiimide and bis Aldehyde polysaccharides. 6. The method for preparing collagen micropowder with low immunogenicity according to claim 1, characterized in that: the precipitating agent is a mixture of ethanol, methanol or acetone and deionized water. 7. the preparation method of low immunogenicity collagen micropowder according to claim 1 is characterized in that: ionic liquid is imidazole acetate type ionic liquid, 1-butyl-3-methylimidazolium chloride type ionic liquid , bromo 1-ethyl-3-methylimidazolium salt ionic liquid, choline chloride·2ZnCl ionic liquid and 1-allyl-3-methylimidazolium chloride ionic liquid. 8. The method for preparing collagen micropowder with low immunogenicity according to claim 1, characterized in that: step S5 includes soaking with amino acid or low molecular weight polypeptide to remove unreacted cross-linking agent between cross-linking treatment and centrifugal washing . 9. The method for preparing collagen micropowder with low immunogenicity according to claim 1, characterized in that: bagging dialysis is included between centrifugal washing and freeze-drying in step S5. 10 . The method for preparing collagen micropowder with low immunogenicity according to claim 1 , characterized in that: after step S5 , sterilization by gamma ray irradiation is included. 11 .

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