Cross-linked recombinant collagen and preparation method and application thereof

Abstract

The application belongs to the technical field of biomedical materials, and provides crosslinked recombinant collagen as well as a preparation method and application thereof. The application combines the recombination technology and the crosslinking technology, and successfully prepares a new type of biomaterial. The biomaterial takes the recombinant collagen as a core basis, is crosslinked with gamma-PGA, forms a crosslinked product with certain mechanical properties, uses EDC and NHS as crosslinking agents, and has excellent mechanical properties and biocompatibility, and the crosslinking effect is remarkable and the residue is small. The material crosslinked by the recombinant collagen and the gamma-PGA has good elasticity and mechanical strength, is suitable for various biomedical applications, has potential market application value, and is especially suitable for medical beauty and biological engineering fields.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, and particularly relates to a cross-linked recombinant collagen, its preparation method, and its application. Background Technology

[0002] Collagen is the most abundant protein in the human body, widely found in connective tissues such as skin, bones, tendons, ligaments, and blood vessels. As a structural protein, collagen plays a crucial role in maintaining the structural integrity and function of tissues. Its unique triple helix structure endows it with excellent mechanical properties and biocompatibility, making it a promising biomaterial in the fields of aesthetics and biomedicine. However, the availability of natural collagen is limited by its sources and carries the potential risk of pathogen transmission. Therefore, the preparation of recombinant collagen through gene recombination technology not only ensures its safety but also enables large-scale production to meet the growing market demand. Furthermore, recombinant collagen exhibits high purity, good batch-to-batch consistency, and can be sequence-optimized and modified as needed to further enhance its functional properties. These advantages make recombinant collagen a promising candidate for applications in the field of biomaterials.

[0003] In the field of aesthetic medicine, dermal fillers are among the most commonly used products. While existing dermal fillers such as botulinum toxin and hyaluronic acid are widely used, they also have some significant limitations. Botulinum toxin primarily relaxes muscles by blocking nerve signals; although it effectively reduces wrinkles, its effect is relatively short-lived, requiring frequent injections, and may cause side effects such as local muscle paralysis. Hyaluronic acid, as a dermal filler, has good filling and moisturizing capabilities, but it degrades rapidly in the body, requiring frequent replenishment. Furthermore, while uncrosslinked collagen has good biocompatibility, its poor mechanical properties make it difficult to maintain the filling effect for a long time. Therefore, developing novel crosslinked recombinant collagen materials is particularly important.

[0004] The application of cross-linking technology, especially the cross-linking of γ-PGA (γ-polyglutamic acid, a biopolymer composed of glutamic acid linked by γ-amide bonds) with collagen, has significantly improved the mechanical properties, stability, and durability of materials, providing a more ideal solution for clinical applications. Among these, EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) and NHS (N-hydroxysuccinimide) cross-linking technologies have significant advantages. EDC is a water-soluble carbodiimide that can cross-link carboxyl and amino groups under mild reaction conditions to form stable amide bonds. NHS can improve the cross-linking efficiency of EDC and reduce the occurrence of side reactions. The combined use of EDC and NHS can not only significantly improve the cross-linking efficiency but also reduce byproducts and residues in the cross-linking reaction, further enhancing the biocompatibility and safety of the material. Based on the advantages of the above cross-linking technologies, this invention proposes a cross-linked recombinant collagen, its preparation method, and its applications. Summary of the Invention

[0005] The purpose of this invention is to provide a cross-linked recombinant collagen, its preparation method, and its application, in order to solve the problems mentioned in the background art.

[0006] To achieve the above objectives, this invention successfully prepared recombinant human type III collagen using gene recombination technology. Collagen prepared through recombination technology not only ensures its safety and consistency but also enables large-scale production to meet market demand. Research revealed that recombinant human type III collagen possesses a triple-helix structure similar to natural collagen. This structure not only endows collagen with excellent mechanical properties and biocompatibility but also allows it to be further assembled into collagen fibers with larger molecular weights. These collagen fibers have significant application value in tissue engineering and regenerative medicine. However, recombinant collagen still has some shortcomings in water solubility and mechanical properties, especially in in vivo application, where uncrosslinked collagen struggles to maintain its function, affecting its clinical efficacy. To address this issue, this invention further crosslinks the recombinant collagen. A crosslinked recombinant collagen material is prepared through the reaction of γ-PGA, EDC, and NHS. This invention provides the following technical solution:

[0007] A method for preparing cross-linked recombinant collagen includes the following steps:

[0008] Step 1: The amino acid sequence of recombinant human type III collagen (collagen composed of amino acid sequences from natural human type III collagen, the GXY region can form a triple helix structure, and the collagen is activated by NHS and EDC and then intermolecularly coupled in the form of covalent bonds) was designed and optimized using bioinformatics methods as shown in SEQ ID NO:1. The recombinant human type III collagen gene optimized by codon preference was inserted into the expression vector, and the recombinant vector was constructed by double digestion with EcoRI and HindIII.

[0009] Step 2: Select a suitable eukaryotic expression system and transfect the constructed expression vector into the selected eukaryotic host cells; use selection markers to screen positive clones; select high-expression candidate lines from the positive clones for shake-flask culture; after the cell density reaches the required level, transfer to a 3L fermenter; maintain cell density in the 3L fermenter by adding culture medium; after the cell density reaches the target level, cool down to express protein and detect the protein expression level in the fermentation supernatant.

[0010] Step 3: Collect the fermentation supernatant, centrifuge to remove cell debris, and purify the expressed protein; validate the purified protein using appropriate methods.

[0011] Step 4: Dialyze the purified protein into water for injection, concentrate the collagen solution using an ultrafiltration tube, and perform lyophilization, including pre-freezing, sublimation drying, and desorption drying.

[0012] Step 5: Dissolve the lyophilized collagen in 20mM phosphate buffer or 0.1M MES buffer to prepare a collagen solution with a final concentration of 1-100mg / mL, preferably 5-50mg / mL, and more preferably 35mg / mL. γ-PGA was dissolved in 0.1M MES buffer, NHS and EDC were added, and the solution was incubated at room temperature for 1 hour to activate the carboxyl groups of γ-PGA. The activated γ-PGA solution was added in equal volume to the collagen solution and mixed thoroughly. Cross-linking reaction was carried out at 4-35℃, preferably at about 4℃ or about 25℃, for 1-24 hours, preferably about 4 hours, to obtain cross-linked recombinant collagen. Dialysis was performed by changing the solution every 24 hours with 100 volumes of 20mM phosphate buffer, repeating the process 3 times at 4℃. The product after cross-linking reaction was freeze-dried to obtain a sponge-like substance. The sponge-like substance was crushed into particles and washed with 100-1000 volumes of physiological saline or water for injection, changing the solution multiple times during the washing process at 4-25℃. The washed particles were swollen in physiological saline to achieve a cross-linked recombinant collagen content of 35 mg / mL.

[0013] Furthermore, the recombinant human type III collagen may be tagged to facilitate purification, such as a His tag, Flag tag, or c-Myc tag. This tag may be omitted if subsequent requirements are met. After purification, the molecular weight of the recombinant human type III collagen is verified by NR-SDS-PAGE.

[0014] Furthermore, the recombinant human type III collagen not only has a triple helix structure, but can also be further assembled into larger collagen fibers.

[0015] Furthermore, the eukaryotic expression system employs a CHO cell or yeast expression system.

[0016] Furthermore, the freeze-drying process is as follows:

[0017] Pre-freezing: Perform pre-freezing treatment at -40℃ for 8 hours;

[0018] Sublimation drying: Sublimation drying is carried out at -15℃, at a pressure of 20 Pa, for 20 hours;

[0019] Desorption drying: Desorption drying was carried out at -5℃, pressure of 20pa, and time of 8 hours.

[0020] Furthermore, the pH of the MES buffer is 3.6.

[0021] Furthermore, the molecular weight of the γ-PGA is 70-1000 kDa, and the concentration of the γ-PGA is 10-800 mg / mL, preferably 30-400 mg / mL.

[0022] Furthermore, the ratio of NHS to EDC is 1:5 to 1:1, and the ratio of EDC to γ-PGA carboxyl groups is 1:8 to 1:1.

[0023] A cross-linked recombinant collagen prepared according to the above-described method exhibits good biocompatibility and biodegradability. Furthermore, the cross-linked recombinant collagen demonstrates good biostability in vivo, is not easily degraded, and can maintain its structure and function over a long period. It is suitable for various biomedical applications, such as dermal fillers, tissue engineering scaffolds, and wound dressings, providing long-lasting and natural filling effects, and demonstrating good safety and efficacy in application.

[0024] Furthermore, the cross-linked recombinant collagen has a stable existence period of at least 36 months at 2-8°C (stable for at least about 6 months, about 12 months, about 18 months, about 24 months, about 30 months, about 36 months, or any amount of time between any two of the above values).

[0025] Compared with the prior art, the beneficial effects of the present invention are:

[0026] This invention combines recombinant and cross-linking technologies to successfully prepare a novel biomaterial. This material uses recombinant collagen as its core base, which is cross-linked with γ-PGA to form a cross-linked compound with certain mechanical properties. Using EDC and NHS as cross-linking agents, the resulting cross-linked product exhibits excellent mechanical properties and biocompatibility, with significant cross-linking effect and minimal residue. The material cross-linked with recombinant collagen and γ-PGA demonstrates good elasticity and mechanical strength, making it suitable for various biomedical applications and possessing potential market value, particularly in the fields of medical aesthetics and bioengineering. Attached Figure Description

[0027] Figure 1 This is an SDS-PAGE electrophoresis image of the recombinant human type III collagen prepared in Example 1.

[0028] Figure 2 The image shows the freeze-dried recombinant human type III collagen sample prepared in Example 2.

[0029] Figure 3 The cross-linked recombinant collagen samples prepared in Examples 3, 4, and the control group, as well as commercially available products, are included.

[0030] Figure 4 The rheological properties of cross-linked recombinant collagen. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0032] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0033] The amino acid sequence of recombinant human type III collagen is as follows:

[0034] GERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIP

[0035] GEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGF

[0036] RGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGA

[0037] PGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGE

[0038] KGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRG

[0039] PAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPG

[0040] ERGAPGFRGPAGPNGIPGEKGPAGERGAPCGGVGAAAIAGIGGEKAGGFA

[0041] PYYGDEPMDFKINTDEIMTSLKSVNGQIESLISPDGSRKNPARNCRDLKFCH

[0042] PELKSGEYWVDPNQGCKLDAIKVFCNMETGETCISANPLNVPRKHWWTD

[0043] SSAEKKHVWFGESMDGGFQFSYGNPELPEDVLDVHLAFLRLLSSRASQNIT

[0044] YHCKNSIAYMDQASGNVKKALKLMGSNEGEFKAEGNSKFTYTVLEDGCT

[0045] KHTGEWSKTVFEYRTRKAVRLPIVDIAPYDIGGPDQEFGVDVGPVCFL.

[0046] Example 1: Preparation of recombinant human type III collagen;

[0047] 1. Gene construction: The amino acid sequence of recombinant human type III collagen was designed and optimized using bioinformatics methods. The recombinant human type III collagen gene, optimized by codon preference, was inserted into the expression vector. The recombinant vector was constructed by double digestion with EcoRI and HindIII.

[0048] 2. Expression System: CHO cells were selected as the eukaryotic expression system, and the constructed expression vector was transfected into CHO cells. The plasmid carried the GS resistance gene, and positive clones were selected using MSX. The plasmid was transfected into CHO cells by electroporation, and six high-expression candidate pool cells were selected. High-expression clones were selected for shake-flask culture. After the cell density reached the required level, the cells were transferred to a 3L fermenter. The cell density was maintained in the 3L fermenter by supplementing with culture medium. After the cell density reached the target level, the temperature was lowered for protein expression. The protein expression level in the fermentation supernatant was measured using SDS-PAGE.

[0049] 3. Protein Purification: The culture supernatant was collected, and cell debris was removed by centrifugation. The expressed recombinant type III collagen was purified using ion exchange chromatography and gel filtration chromatography. After purification, non-reducing SDS-PAGE was used for verification, confirming that it mainly existed in the form of trimers.

[0050] Figure 1 Electrophoretic bands of recombinant human type III collagen obtained by SDS-PAGE electrophoresis analysis are shown. The clear bands in the lanes indicate that the recombinant collagen exists primarily in trimer form under non-reducing conditions, demonstrating the successful formation of the designed collagen triple helix structure. This result supports the description of collagen structural stability in this invention. Lane 9 represents the finally assembled recombinant human type III collagen; based on the molecular weight reference of the electrophoretic markers, the protein in lane 9 reaches the ultra-high molecular weight region.

[0051] Example 2: Freeze-drying process of recombinant human type III collagen;

[0052] The purified recombinant human type III collagen was dialyzed into water for injection, the collagen solution was concentrated using an ultrafiltration tube, and then the collagen solution was lyophilized. The specific steps are as follows:

[0053] Pre-freezing: Perform pre-freezing treatment at -40℃ for 8 hours;

[0054] Sublimation drying: Sublimation drying is carried out at -15℃, at a pressure of 20 Pa, for 20 hours;

[0055] Desorption drying: Desorption drying was carried out at -5℃, pressure of 20pa, and time of 8 hours.

[0056] Figure 2 The images show actual samples of recombinant human type III collagen after freeze-drying (left image: un-freezed; right image: freeze-dried). The samples in the images exhibit good structural preservation, indicating that the successful freeze-drying process provides a stable basis for subsequent cross-linking reactions.

[0057] Example 3: Preparation process of cross-linked recombinant collagen;

[0058] The lyophilized collagen from Example 2 was dissolved in 0.1M MES buffer (pH 3.6) to a concentration of 70 mg / mL. γ-PGA was also dissolved in 0.1M MES buffer (pH 3.6) to a concentration of 70 mg / mL. 50 mM NHS and 200 mM EDC were added to the γ-PGA solution. The solution was incubated at room temperature for 1 hour to fully activate the carboxyl groups of γ-PGA. Then, an equal volume of the activated γ-PGA solution was added to the collagen solution and mixed thoroughly. The cross-linking reaction was carried out at 25°C for 4 hours to obtain cross-linked recombinant collagen. To remove impurities and unreacted cross-linking agents, the product was dialyzed against 100 volumes of 20 mM phosphate buffer, with the buffer changed every 24 hours, repeated 3 times, at 4°C. The product after the cross-linking reaction was lyophilized to obtain a sponge-like substance. The sponge-like substance was pulverized into particles and washed with 500 volumes of physiological saline or water for injection. The cleaning solution was changed multiple times during the cleaning process to ensure the removal of residual crosslinking agents and unreacted substances. The cleaning process was carried out at 4°C.

[0059] The cleaned particles were swollen in physiological saline to achieve a recombinant collagen content of 35 mg / mL, thus obtaining an injectable collagen filler.

[0060] Example 4: Preparation process of cross-linked recombinant collagen (II);

[0061] The lyophilized collagen from Example 2 was dissolved in 0.1M MES buffer (pH 3.6) to a concentration of 70 mg / mL. γ-PGA was dissolved in 0.1M MES buffer (pH 3.6) to a concentration of 700 mg / mL. 80 mM NHS and 400 mM EDC were added to the γ-PGA solution. The solution was incubated at room temperature for 1 hour to fully activate the carboxyl groups of γ-PGA. Then, an equal volume of the activated γ-PGA solution was added to the collagen solution and mixed thoroughly. The cross-linking reaction was carried out at 25°C for 4 hours to obtain cross-linked recombinant collagen. To remove any potentially introduced impurities and unreacted cross-linking agents, the product was dialyzed with 100 volumes of 20 mM phosphate buffer, changing the buffer every 24 hours for 3 times, at 4°C. The product after the cross-linking reaction was lyophilized to obtain a sponge-like substance. The sponge-like substance was pulverized into particles and washed with 500 volumes of physiological saline or water for injection. The cleaning solution was changed multiple times during the cleaning process to ensure the removal of residual crosslinking agents and unreacted substances. The cleaning process was carried out at 4°C.

[0062] The cleaned particles were swollen in physiological saline to achieve a recombinant collagen content of 35 mg / mL, thus obtaining an injectable collagen filler.

[0063] Figure 3 This study showcases physical examples of recombinant collagen fillers prepared via a cross-linking process. The control group did not contain recombinant collagen. Figure 3 This demonstrates the successful implementation of the cross-linking reaction and verifies that the obtained collagen filler structurally conforms to the expected design.

[0064] Example 5: Performance testing of cross-linked recombinant collagen;

[0065] 1. Mechanical Property Testing: The elastic modulus and compressive strength of the cross-linked recombinant collagen were tested to ensure that its mechanical properties met application requirements. The mechanical properties of the cross-linked recombinant collagen were characterized using a DHR rheometer at room temperature. The prepared sample was carefully placed in the middle of a parallel plate with a diameter of 25 mm, maintaining an appropriate gap. The mechanical properties of the cross-linked recombinant collagen were tested under conditions of angular frequency (ω) of 10 rad / s and strain of 0.01-100%.

[0066] Figure 4 The mechanical properties of cross-linked recombinant collagen, tested using a DHR rheometer, are demonstrated. The curves in the figure show that the elastic modulus and compressive strength of the material remain stable within a certain range as strain increases. Compared to existing products on the market, the cross-linked material exhibits higher elastic modulus and compressive strength, meaning it is more robust and less prone to deformation or damage under external forces. These results demonstrate the significant improvement in mechanical properties and stability of cross-linked recombinant collagen, indicating its suitability for practical applications.

[0067] 2. Biocompatibility Testing: In accordance with GB / T 16886.6-2022 "Biological Evaluation of Medical Devices Part 6: Implantation Testing", the implantation reactivity of cross-linked recombinant collagen in rats was evaluated. Cross-linked recombinant collagen was implanted subcutaneously into the back of rats, and local reactions and behavioral changes were observed on postoperative days 1, 7, 14, and 21. The results are shown in Table 1.

[0068] Table 1

[0069] time Redness and swelling Induration Infect tissue necrosis experimental group control group difference Day 1 none none none none 0 / 5 0 / 5 No significant difference Day 7 none none none none 0 / 5 0 / 5 No significant difference Day 14 none slight none none 1 / 5 1 / 5 No significant difference Day 21 none slight none none 1 / 5 1 / 5 No significant difference

[0070] Table 1 shows that there were no significant differences between the experimental group and the control group (injected with the same volume of physiological saline) in terms of redness, induration, infection, and tissue necrosis. This indicates that the cross-linked recombinant collagen has good biocompatibility in the rat model and did not cause obvious local inflammation or tissue damage.

[0071] 3. Stability Test: The stability of the cross-linked recombinant collagen was tested under different temperature and humidity conditions to assess its degradation during storage and use. The results are shown in Table 2.

[0072] Table 2

[0073]

[0074] The results in Table 2 show that cross-linked recombinant collagen is stable for at least 36 months at 2-8°C.

[0075] In summary, this invention provides a method for preparing cross-linked recombinant collagen. The prepared cross-linked recombinant collagen has excellent mechanical properties, biocompatibility, and stability, and is particularly suitable for applications in dermal fillers, tissue engineering, and wound dressings, showing broad application prospects.

[0076] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent.

Claims

1. A method for preparing cross-linked recombinant collagen, characterized in that, Includes the following steps: Step 1: The amino acid sequence of recombinant human type III collagen was designed and optimized using bioinformatics methods as shown in SEQ ID NO:

1. The recombinant human type III collagen gene optimized by codon preference was inserted into the expression vector, and the recombinant vector was constructed by double digestion with EcoRI and HindIII. Step 2: Select a suitable eukaryotic expression system and transfect the constructed expression vector into the selected eukaryotic host cells; screen positive clones using selection markers; select high-expression candidate lines from the positive clones for shake-flask culture; after the cell density reaches the required level, transfer to a 3L fermenter; maintain cell density in the 3L fermenter by adding culture medium; after the cell density reaches the target level, cool down to express protein and detect the protein expression level in the fermentation supernatant; the eukaryotic expression system used is a CHO cell or yeast expression system; Step 3: Collect the fermentation supernatant, centrifuge to remove cell debris, and purify the expressed protein; use appropriate methods to verify the purified protein; Step 4: Dialyze the purified protein into water for injection, concentrate the collagen solution using an ultrafiltration tube, and perform lyophilization, including pre-freezing, sublimation drying, and desorption drying. Step 5: Dissolve the lyophilized collagen in 20mM phosphate buffer or 0.1M MES buffer; dissolve γ-PGA in 0.1M MES buffer, add NHS and EDC, and incubate the solution at room temperature for 1 hour to activate the carboxyl groups of γ-PGA; add an equal volume of the activated γ-PGA solution to the collagen solution and mix well; perform a cross-linking reaction at 4-35℃ for 1-24 hours to obtain cross-linked recombinant collagen; dialyze through 100 volumes of 20mM phosphate buffer, changing the solution every 24 hours, repeating 3 times, and the process is carried out at 4℃; freeze-dry the product after the cross-linking reaction to obtain a sponge-like substance; pulverize the sponge-like substance into particles and wash it in 100-1000 volumes of physiological saline or water for injection, changing the solution multiple times during the washing process, and the washing process is carried out at 4-25℃; swell the washed particles in physiological saline to make the content of cross-linked recombinant collagen reach 35mg / mL.

2. The preparation method according to claim 1, characterized in that, The freeze-drying process is as follows: Pre-freezing: Perform pre-freezing treatment at -40℃ for 8 hours; Sublimation drying: Sublimation drying is carried out at -15℃, at a pressure of 20 Pa, for 20 hours; Desorption drying: Desorption drying was carried out at -5℃, pressure of 20pa, and time of 8 hours.

3. The preparation method according to claim 1, characterized in that, The pH of the MES buffer solution is 3.

6.

4. The preparation method according to claim 1, characterized in that, The γ-PGA has a molecular weight of 70-1000 kDa and a concentration of 10-800 mg / mL.

5. A cross-linked recombinant collagen prepared by any one of the preparation methods according to claims 1-4.

6. The use of the cross-linked recombinant collagen according to claim 5 in the preparation of dermal fillers, tissue engineering scaffolds and wound dressings.

Images