A preparation method and application of collagen microspheres

Abstract

The present invention discloses a preparation method and application of collagen microspheres, and belongs to the field of genetic engineering technology. Two histidines are respectively inserted into the N-terminus and the C-terminus of the B-domain amino acid sequence of collagen, and the high polymer self-assembly of collagen is promoted to form a three-dimensional network structure by metal chelation, and a histidine is further inserted into the middle of the collagen to achieve the transformation of the three-dimensional morphology of the collagen assembly and form a loose and porous microsphere structure. It has been verified by experiments that the present invention can control the three-dimensional morphology of collagen and form collagen microspheres by controlling the number and position of His residues in the B-domain amino acid sequence of collagen expressed by microbial cells; the prepared collagen microspheres can significantly promote cell adhesion, extension and migration.

Description

Technical Field

[0001] The present invention relates to the field of gene engineering technology, and in particular to a preparation method of collagen microspheres and application thereof. Background Art

[0002] Collagen microspheres are small particles made of collagen. They are widely used in biomedicine and beauty fields due to their good biocompatibility, degradability and biofunctionality. As the most abundant protein in the human body, collagen is commonly found in skin, bones and connective tissues. The microsphere form helps support structure, promote cell growth and tissue repair. Common preparation methods include emulsification-solvent evaporation, freeze drying, spray drying and self-assembly, which can control particle size, morphology and drug load to meet different needs. In the field of drug delivery, collagen microspheres prolong drug efficacy and reduce the frequency of administration through controlled release characteristics, and can be surface modified to achieve targeted delivery. In tissue engineering, it is widely used in the repair of tissues such as cartilage, bone, and skin, and as a cell culture scaffold to promote regeneration and wound repair. In the field of beauty, it is used as a skin filler and anti-aging material to improve skin elasticity and reduce wrinkles.

[0003] Natural collagen is widely used in the biomedical field due to its excellent biocompatibility, degradability and biofunctionality, but its complex properties limit structural regulation and may cause immunogenicity issues. To solve this problem, chemically synthesized small molecule collagen peptides (CMPs) can fold into a triple helical structure similar to natural collagen and promote high polymer assembly through metal-ligand interactions. [1, 2] Recent studies have shown that metal-ligand interactions contribute to the formation of CMP cages for biopolymer delivery. [3] In addition, through metal-histidine coordination, the study revealed that the morphology of CMP assemblies is closely related to the number and position of histidine residues, and supramolecular structures with higher research value can be generated through programming design. [4, 5] Although chemically synthesized peptides have strong controllability and high purity, they are expensive, limited in length, and not suitable for large-scale production.

[0004] To this end, more and more studies have used hosts such as bacteria and yeast to achieve efficient heterologous expression of collagen, which can be correctly folded into a collagen triple helix structure, with the characteristics of low cost and high expression, but most recombinant collagen still lacks the self-assembly driving force to form a higher-order structure, which limits its application in drug delivery and tissue engineering. Barbara Brodsky et al. found that by growing the Streptococcus pyogenes collagen Scl2, its self-assembly ability can be enhanced, but the fiber morphology is poor and has no three-dimensional morphology. [6]Molly M. Stevens et al. added hyaluronic acid and chondroitin sulfate binding sites to the Scl2 sequence and used MMP7 sensitive peptide cross-linking to form a biodegradable hydrogel, which is expected to be used for articular cartilage regeneration [7] .

[0005] 1.Pires, MM and J. Chmielewski, Self-assembly of collagen peptides into microflorettes via metal coordination. Journal of the American ChemicalSociety, 2009. 131(7): p. 2706-2712.

[0006] 2.Przybyla, DE and J. Chmielewski, Metal-triggered radial self-assembly of collagen peptide fibers. Journal of the American ChemicalSociety, 2008. 130(38): p. 12610-12611.

[0007] 3.Pires, MM, D. Ernenwein, and J. Chmielewski, Selective Decoration and Release of His-tagged Proteins from Metal-Assembled Collagen PeptideMicroflorettes. Biomacromolecules, 2011. 12(7): p. 2429-2433.

[0008] 4.Hsu, W., YL Chen, and JC Horng, Promoting self-assembly ofcollagen-related peptides into various higher-order structures by metal-histidine coordination. Langmuir the Acs Journal of Surfaces & Colloids, 2012. 28(6): p. 3194-9.

[0009] 5.Pires, MM, et al., Controlling the morphology of metal-promotedhigher ordered assemblies of collagen peptides with varied core lengths. Langmuir the Acs Journal of Surfaces & Colloids, 2012. 28(4): p. 1993-1997.

[0010] 6.Yoshizumi, A., et al., Self-association of Streptococcus pyogenescollagen-like constructs into higher order structures. Protein Science, 2009.18(6): p. 1241-1251.

[0011] 7.Cosgriff-Hernandez, E., et al., Bioactive hydrogels based onDesigner Collagens. Acta Biomaterialia, 2010. 6(10): p. 3969-3977. Summary of the invention

[0012] The purpose of the present invention is to provide a preparation method and application of collagen microspheres to solve the problems existing in the above-mentioned prior art. The present invention inserts two histidines into the N-terminus and the C-terminus of collagen respectively, utilizes metal chelation to promote the high-polymer self-assembly of collagen to form a three-dimensional network structure, and further mutates a residue in the collagen sequence to histidine, thereby realizing the transformation of the three-dimensional morphology of the collagen self-assembly body and forming a loose and porous microsphere structure; the formed collagen microspheres can promote cell adhesion, extension and migration.

[0013] To achieve the above object, the present invention provides the following solutions:

[0014] The present invention provides a collagen protein, wherein the protein chain structure of the collagen protein is shown in any one of the following:

[0015] (1) From the N-terminus to the C-terminus, it is (His)2, B-domain, and (His)2, wherein the B-domain is a plurality of repeated collagen triplet amino acids GXY, and the amino acid sequence is shown in SEQ ID NO.2;

[0016] (2) A derivative protein having the same function as (1) in which X and / or Y in the amino acid sequence of the B-domain is deleted, substituted or mutated based on (1).

[0017] Optionally, the derivative protein includes a mutant protein obtained by mutating TQ at positions 47 and 48 in the B-domain amino acid sequence to PH based on (1). The mutation method of the present invention is not limited to the above method, and X or Y at other positions can also be replaced with histidine. By controlling the number and position of His residues in the B-domain amino acid sequence in the collagen molecule, the three-dimensional morphology of collagen can be controlled.

[0018] Optionally, the N-terminus of the B-domain further includes a V-domain, and the V-domain is connected to (His)2 via a connecting peptide, and the amino acid sequence of the connecting peptide is LVPR.

[0019] Optionally, the N-terminus of the V-domain further includes a 6×His tag.

[0020] The present invention also provides a gene encoding the collagen.

[0021] The present invention also provides a recombinant plasmid or host bacteria containing the gene. The plasmid includes but is not limited to: pColdIII series and pET series plasmids.

[0022] In one embodiment, the host bacteria is Escherichia coli, including but not limited to E. coli BL21 and E. coli BL21 (DE3).

[0023] The present invention also provides the use of the collagen or the gene or the plasmid or the host bacteria in any of the following:

[0024] (1) Application in the preparation of collagen microspheres;

[0025] (2) Application in the preparation of collagen products that promote cell adhesion, extension and / or migration.

[0026] More specifically, the invention can be applied to the preparation of products for promoting cell adhesion, extension and migration in the fields of biology, chemical industry, food, medicine, biomaterials, tissue engineering and cosmetics.

[0027] The present invention also provides a collagen microsphere, which is formed by self-assembly of the collagen through metal ion chelation and high polymerization.

[0028] The present invention also provides a method for preparing collagen microspheres, comprising the following steps:

[0029] The collagen and the metal ion solution are mixed at equal concentrations, stirred at 25°C for 4 hours, and then allowed to stand at 2°C-37°C for 7 days to obtain collagen microspheres.

[0030] The present invention also provides a collagen product for promoting cell adhesion, extension and / or migration, which contains the collagen microspheres. The product includes but is not limited to medicines, biomedical materials, cosmetics, etc.

[0031] The present invention also provides a method for controlling the three-dimensional morphology of collagen, wherein the method controls the number and position of His residues in the B-domain amino acid sequence of collagen expressed by microbial cells to achieve control of the three-dimensional morphology of collagen.

[0032] The present invention discloses the following technical effects:

[0033] (1) The present invention uses cold shock expression of Escherichia coli to introduce a collagen sequence containing histidine to prepare collagen microspheres that can self-assemble to form a highly polymerized structure. The process is simple, the cost is low, and large-scale production is possible. The present invention provides a preparation method and a sequence design pattern for preparing collagen microspheres. The collagen region of the sequence is replaceable and expandable, providing a platform for research and application based on collagen microspheres, and has broad prospects in the application of biomaterials.

[0034] (2) The present invention also regulates the microstructure of the assembly by adjusting the number and position of histidine in the collagen region. By inserting two histidines at the N-terminus and the C-terminus of the collagen respectively, the metal chelation is used to promote the high polymer self-assembly of the collagen to form a three-dimensional network structure, and further inserting a histidine in the middle of the collagen, the three-dimensional morphology of the collagen assembly is transformed to form a loose and porous microsphere structure.

[0035] (3) The collagen microspheres prepared by the present invention are prepared by Zn 2+ Induced self-assembly aggregation formation, the prepared microspheres except Zn 2+ and collagen, without any other substances, with a single ingredient, simple preparation process, no cytotoxicity, and can promote cell adhesion, extension and migration. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0037] Figure 1 Design strategy for metal chelated collagen self-assembly; (a) H2BH2 sequence pattern; (b) schematic diagram of histidine and metal ion chelation; (c) schematic diagram of metal-induced H2BH2 self-assembly; (d) schematic diagram of metal-induced H2BH2-H1 self-assembly;

[0038] Figure 2 For collagen identification; (a) is the nucleic acid electrophoresis diagram for verification of double enzyme digestion of recombinant plasmid; (b) is the SDS-PAGE identification diagram of collagen after purification and enzyme digestion; (c) circular dichroism secondary structure determination; (d) MALDI-TOF molecular weight identification;

[0039] Figure 3 are scanning electron micrographs of collagen aggregates; (ac) scanning electron micrographs of B collagen aggregates; (df) scanning electron micrographs of H2BH2 collagen aggregates; (gi) scanning electron micrographs of H2BH2-H1 collagen aggregates;

[0040] Figure 4 Verification of the biocompatibility and bioactivity of the designed collagen; (a) cell proliferation; (b) cytotoxicity; (c) cell adhesion; (d, f) cell area quantification and cell morphology; (e) live and dead cell staining; (g, h) cell migration and quantification;

[0041] Figure 5 Schematic diagram of the three-segment chimeric sequence (a) and the amino acid arrangement of the protein single chain (b). DETAILED DESCRIPTION

[0042] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0043] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0044] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0045] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0046] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0047] The main materials and methods involved in the following embodiments of the present invention are:

[0048] (1) Culture medium:

[0049] LB solid medium: 15 g / L agar, 10 g / L tryptone, 5 g / L yeast extract powder and 10 g / L NaCl, pH 7.0.

[0050] LB liquid medium: 10 g / L tryptone, 5 g / L yeast extract powder and 10 g / L NaCl, pH 7.0.

[0051] TB liquid medium: 12 g tryptone, 24 g yeast extract powder, 4 mL glycerol, 2.31 g KH2PO4 and 12.54 g K2HPO4, pH 7.5, make up to 1 L.

[0052] (2) Cultivation methods:

[0053] Seed culture conditions: The single colony grown on the plate was inoculated into LB liquid culture medium with a liquid volume of 10%. The culture was carried out in a 250 mL shake flask at 37°C for 10 h and a rotation speed of 200 rpm.

[0054] Fermentation culture conditions: TB medium was used with a medium filling volume of 20% and an inoculation size of 1%. A 500 mL shake flask was used for culture. After culture at 37°C for 24 h, IPTG was induced with a final concentration of 1 mM. The culture was induced at 25°C for 10 h, then at 15°C for 14 h, with a rotation speed of 200 rpm.

[0055] Example 1 Sequence design and sample preparation

[0056] The design was carried out according to the structure shown in VHBH, where V represents V-domain, which is a globular domain that guides the collagen region to fold correctly, H represents 2 × His, i.e., two histidines, and B represents the B-domain collagen region. The morphology of the assembly was changed by introducing His in the middle of the B-domain to form collagen microspheres. The sequence design schematic is shown in the figure. Figure 1 The specific steps are:

[0057] (1) The N and C termini of the sequence are His motifs, and the middle is the B-domain collagen region, resulting in a three-segment chimeric sequence (abbreviated as H2BH2) (see Figure 5 In a), an additional His is inserted in the middle of the B-domain to obtain H2BH2-H1. In this embodiment, the B-domain uses an amino acid sequence (amino acid sequence as shown in SEQ ID NO.2, nucleotide sequence as shown in SEQ ID NO.6) extracted from collagen Scl2 (Genbank ID: AAL50184.1) from Streptococcus pyogenes, and an integrin binding site is inserted in the middle of the collagen sequence to achieve its biological function.

[0058] (2) The globular domain V-domain (amino acid sequence as shown in SEQ ID NO.1, nucleotide sequence as shown in SEQ ID NO.5) derived from Scl2 is inserted at the N-terminus of the sequence to guide the correct folding of the collagen triple helix, the protease cleavage site LVPR is inserted between the fixed sequence unit of the globular domain and the collagen region, and 6×His is inserted at the N-terminus of the sequence for purification. Thus, three protein single chains are coiled around a common central axis to form a triple helical structure. The amino acid arrangement of the protein single chains is shown in Figure 5 Middle b.

[0059] The nucleotide and amino acid sequences involved in forming the collagen structure are shown in Table 1.

[0060] Table 1 Sequence

[0061]

[0062] The gene encoding the above amino acid sequence was synthesized, wherein the nucleotide sequences encoding B, H2BH2 and H2BH2-H1 are shown in SEQ ID NO.5-7; the above nucleotide sequences respectively contain 5' Nco I restriction site, 5' flanking sequence GC and 3' Bam HI restriction site. The synthesized genes were respectively inserted between NcoI and BamHI of pColdM plasmid to obtain the corresponding recombinant collagen plasmid. The double enzyme digestion verification results are shown in Figure 2 a, and then transforming the recombinant plasmids into E. coli BL21 (DE3) competent cells by the CaCl2 method, inoculating single colonies in LB liquid medium overnight, and then transferring to TB liquid medium at a 1% inoculation rate, culturing at 37°C for 24 h, adding IPTG, inducing at 25°C for 10 h, and then inducing at 15°C for 14 h to obtain a recombinant strain for preparing hybrid collagen; the pColdM plasmid uses pColdM-S: 5'-CTCGAGGGATCCGAATTCA-3' (shown in SEQID NO.8) and pColdM-A: 5'-GAGCTCCATGGGCACTTTG-3' (shown in SEQ ID NO.9) as primers to mutate the pColdIII plasmid to introduce an Nco I site.

[0063] After the recombinant strain was induced to ferment, the fermentation broth was centrifuged at 8000 rpm for 5 minutes to collect the bacteria. The bacteria were resuspended in 10 mM phosphate buffer, the cells were broken by a high-pressure homogenizer, and then centrifuged at 4°C and 10000 rpm for 20 minutes to remove cell debris, and then filtered with a 0.45 μm microporous filter to remove impurities. The sample was injected into a 5 mL His-Trap HP affinity chromatography column installed on a protein purifier, washed with washing solution for 8 column volumes, and then eluted with an elution buffer containing an imidazole gradient (140 mM, 400 mM), and the peak protein was collected for SDS-PAGE electrophoresis analysis. Then, trypsin with a final concentration of 0.05 mg / mL was used for enzyme digestion at 25°C for 8 h to remove the globular guide folding domain, followed by dialysis desalting and freeze-drying to obtain collagen B and H2BH2 freeze-dried powder. A small amount of freeze-dried powder was dissolved in water and identified by SDS-PAGE and MALDI-TOF. The results are as follows Figure 2 As shown in b, the results show that the purified protein presents a single band on SDS-PAGE. Figure 2 Figure d in the figure shows that the molecular weight obtained by mass spectrometry is consistent with the theoretical molecular weight, proving that the obtained collagen molecular weight is correct.

[0064] Example 2 Determination of collagen secondary structure

[0065] The collagen prepared in Example 1 was prepared to a concentration of 2 mg / mL. Then it was allowed to stand at 4°C for more than 24 h, and a 1 mm cuvette was used to perform a full wavelength scan of circular dichroism at 4°C, with wavelengths ranging from 190 nm to 260 nm, with a wavelength interval of 1 nm, and a stay of 5 s at each wavelength. The thermal change experiment was measured at 220 nm, with temperatures ranging from 4°C to 80°C, with each temperature balancing for 8 s, and a temperature increase rate of 1°C / 6 min. The typical CD spectrum of the triple helix structure of collagen shows a positive absorption peak at 225 nm.

[0066] like Figure 2 As shown in Figure c, under full wavelength scanning, the protein designed in Example 1 has a characteristic absorption peak near 220 nm; the results of the thermal change experiment show that as the temperature increases, the characteristic absorption value at 220 nm changes suddenly between 23 and 30°C, which is manifested as the destruction of the secondary structure of collagen, that is, the unwinding of the triple helix. Both the CD full spectrum and the thermal change test results show that the collagen designed in Example 1 can correctly fold to form a collagen triple helix structure and has a high thermal stability.

[0067] Example 3 Internal three-dimensional structure of collagen aggregates

[0068] The collagen prepared in Example 1 was prepared into a solution with a concentration higher than 0.5 mmol / L, and an equal proportion of ZnCl2 was added. After stirring at 25°C for 4 h, the solution was allowed to stand at 2°C to 37°C for 7 days to obtain a collagen polymer. After freezing with liquid nitrogen, the sample was placed in a freeze dryer and its internal structure was observed using a scanning electron microscope after gold was sprayed on the cross section. The results are as follows: Figure 3 shown.

[0069] The results showed that B was loose, H2BH2 was a loose and porous mesh cross-linked structure, and H2BH2-H1 was a loose and porous microsphere structure. The results showed that by inserting two histidines at the N-terminus and C-terminus of collagen, metal chelation was used to promote the high-polymer self-assembly of collagen to form a three-dimensional network structure, and further inserting a histidine in the middle of collagen, the three-dimensional morphology of the collagen assembly was transformed to form a loose and porous microsphere structure.

[0070] Example 4 Verification of the biocompatibility and bioactivity of the designed type I collagen

[0071] The lyophilized collagen B, H2BH2 and H2BH2-H1 prepared in Example 1 were prepared into a solution with a final concentration of 40 ng / μL, and bovine serum albumin (BSA) and rat tail type I collagen (Type I) of the same concentration were used as negative and positive controls. 100 μL was taken and added to a 96-well plate, three parallels in each group, and the solution was aspirated after adsorption at 4°C for 24 h, and then sterilized by UV irradiation for 30 min after drying overnight, and finally blocked with 5% BSA for 2 h. Mouse fibroblasts (NIH-3T3) were resuspended in DMEM medium containing 10% FBS at a density of 2000 per well, and 100 μL was inoculated on the cell culture plate, and the cell viability was determined using the CCK8 kit at 0, 1, 3 and 5 days respectively.

[0072] like Figure 4 As shown in Figure a, the designed collagen has no significant effect on cell proliferation. Figure 4 As shown in b, different concentrations of collagen substrate had no significant effect on cell activity, proving that it has good cell compatibility. In the same way, collagen was adsorbed onto a 96-well plate, 10,000 cells were seeded, and after 6 h of culture, the cells were washed three times with 10 mM PBS, and then the cell viability was measured using the CCK8 kit. Figure 4 As shown in Figure c, compared with BSA, synthetic collagen showed a significant promoting effect on cell adhesion, but not as good as natural rat tail type I collagen. Figure 4 As shown in Figures d and f, when the same method was used to evaluate the cell spreading area and phalloidin was used for cytoskeleton staining, cells adhered to the H2BH2 matrix showed a larger spreading area and more obvious cytoskeleton extension than BSA, but not as good as native rat tail type I collagen. Figure 4 In Figure e, the biocompatibility of the synthesized collagen was further confirmed by live-dead cell staining. Figure 4 As shown in Figures g and h, synthetic collagen H2BH2 and H2BH2-H1 substrates have a significant promoting effect on cell migration, even slightly better than rat tail type I collagen.

[0073] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A collagen protein, characterized in that The protein chain structure of the collagen is: From N-terminus to C-terminus, it is (His)2, B-domain, (His)2, wherein the amino acid sequence of the B-domain is shown in SEQ ID NO.4; The N-terminus of the B-domain further includes a V-domain, and the V-domain is connected to (His)2 via a connecting peptide, and the amino acid sequence of the connecting peptide is LVPR; The N-terminus of the V-domain also includes a 6×His tag; The amino acid sequence of the V-domain is shown in SEQ ID NO.

1.

2. A gene encoding the collagen according to claim 1.

3. A recombinant plasmid or host bacteria comprising the gene according to claim 2.

4. Use of the collagen according to claim 1, the gene according to claim 2, or the plasmid or host bacteria according to claim 3 in any of the following: (1) Application in the preparation of collagen microspheres; (2) Application in the preparation of collagen products that promote cell adhesion, extension and / or migration.

5. A collagen microsphere, characterized in that: The method is formed by self-assembly of the collagen described in claim 1 through metal ion chelation.

6. A method for preparing collagen microspheres, characterized in that: The following steps are involved: The collagen described in claim 1 is mixed with a metal ion solution at equal concentrations, stirred at 25° C. for 4 hours, and then allowed to stand at 2° C. to 37° C. for 7 days to obtain collagen microspheres.

7. A collagen product that promotes cell adhesion, extension and / or migration, characterized in that: It contains the collagen microspheres according to claim 5.

Images