Preparation method of a composite medical sponge conjugated with interferon

Abstract

The present application discloses a preparation method of a composite medical sponge conjugated with interferon, which has a double network composed of hyaluronic acid and collagen-like protein as the main structure, and conjugates interferon through Spy chemistry. The composite medical sponge dressing provided by the present application has more prominent hemostatic and wound-healing effects, excellent biocompatibility and adhesiveness compared with the prior art, can meet the requirements of actual production and application, and has great application prospects as a dressing for chronic diabetic wounds.

Description

Technical Field

[0001] The present invention relates to the field of medical dressings, and particularly to a preparation method of a composite medical sponge conjugated with interferon. Background Art

[0002] A chronic wound refers to a wound that cannot heal within more than 90 days. As one of the typical chronic wounds, chronic diabetic wounds have become one of the serious complications of diabetic patients. In addition, chronic diabetic wounds are difficult to recover through the normal wound repair process. If such chronic wounds are not treated in time, a series of special physiological microenvironments will exacerbate the deterioration of the wounds. At present, the treatment of diabetic wounds mainly focuses on methods such as blood glucose control and debridement. However, due to the high treatment cost, complex operation and easy infection, patients still face huge pressure economically and mentally.

[0003] At present, common hemostatic sponges include gelatin-based, hyaluronate-based, chitosan-based, oxidized cellulose-based, starch-based, etc. Among them, gelatin-based and chitosan-based hemostatic sponges have good hemostatic effects. However, such products have potential virus sources. Hemostatic sponge products mainly composed of cellulose-based, hyaluronate-based, etc. do not have biological activity, but concentrate the effective components in the blood by absorbing the water in the blood or block the bleeding wound surface, resulting in poor hemostatic effects and inability to quickly promote wound healing.

[0004] Interferon (IFN) plays a crucial role in the regulation of the immune system. Especially type I interferons (including IFN-α, IFN-β, and IFN-κ) play a key role in antiviral effects and diabetic wound repair. During the process of diabetic wound repair, type I interferon IFN-α coordinates wound healing by activating repair cells and promoting tissue regeneration. However, the rapid degradation and short life cycle of IFN-α severely limit its wide application in wound repair. In addition, integrating bioactive cytokines such as IFN-α into the extracellular matrix sponge to promote wound repair is a technical challenge because the biological activity of these factors depends on their intact protein structure, and direct integration may affect their stability.

[0005] In summary, there is an urgent clinical need for a medical hemostatic sponge with good biocompatibility, self-repair ability, and the function of promoting wound healing. Summary of the Invention

[0006] In view of this, the present application provides a preparation method of a composite medical sponge conjugated with interferon, including the following steps:

[0007] (a) Expressing the specific collagen in cells by means of genetic engineering using a vector containing the specific collagen gene, and then separating and purifying to obtain the specific collagen;

[0008] (b) By means of genetic engineering, a vector containing a specific interferon gene is expressed in cells to express the specific interferon, and then the specific interferon is isolated and purified;

[0009] (c) The specific collagen and the specific interferon prepared in step (a) and step (b) are chemically coupled at a ratio of 1:10 to 10:1, and the collagen coupled with interferon is obtained through incubation;

[0010] (d) Hyaluronic acid and the collagen coupled with interferon prepared in step (c) are mixed at a ratio of 1:10 to 10:1, and then 1,4-butanediol diglycidyl ether is added. After incubation, freezing, and lyophilization, a composite medical sponge coupled with interferon is prepared.

[0011] In some embodiment schemes, the amino acid sequence of the above-mentioned specific collagen is as shown in SEQ ID NO: 01.

[0012] In some embodiment schemes, the amino acid sequence of the above-mentioned specific interferon is as shown in SEQ ID NO: 02.

[0013] In some embodiment schemes, in the above step (c), the specific collagen and the specific interferon are chemically coupled at a ratio of 2:8, 4:6, 5:5, 6:4, or 8:2.

[0014] In some embodiment schemes, in the above step (c), the specific collagen and the specific interferon are chemically coupled at a ratio of 4:6.

[0015] In some embodiment schemes, the cells in the above step (a) and step (b) are Escherichia coli, and the vector is pET-28a.

[0016] In some embodiment schemes, in the above step (c), the incubation temperature is 20 - 30 °C, and the incubation time is 8 - 16 hours.

[0017] In some embodiment schemes, in the above step (c), the incubation temperature is 25 °C, and the incubation time is 12 hours.

[0018] In some embodiment schemes, the hyaluronic acid in the above step (d) has a concentration of 2% and is prepared by dissolving in 1% NaOH solution.

[0019] In some embodiment schemes, the concentration of 1,4-butanediol diglycidyl ether in the above step (d) is 1%.

[0020] In some embodiment schemes, in the above step (d), the hyaluronic acid and the collagen coupled with interferon are mixed at a ratio of 4:6.

[0021] In some embodiments, step (d) above is to incubate at 40 °C for 5 hours, freeze at -80 °C for 2 hours, and then perform lyophilization to prepare a composite medical sponge conjugated with interferon.

[0022] This application uses two methods: one is to connect interferon and collagen through gene recombination, and the other is to conjugate interferon and collagen through gene coding plus Spy chemistry. The medical sponge integrating IFN-α through Spy chemistry coupling technology is significantly superior to the medical sponge of recombinant IFN-α in terms of cell viability, migration, and protein expression. In vivo studies further confirmed that compared with blank medical sponges and saline-treated controls, the IFN-α-HA-CLP medical sponge conjugated by gene-coded coupling chemistry significantly promoted wound healing. Compared with direct gene coding, the medical sponge integrating IFN-α through Spy chemistry coupling technology had better wound healing effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 : Molecular weight characterization of recombinant collagen in Example 1 of this application

[0024] Figure 2 : Scanning electron microscope structure of recombinant collagen in Example 2 of this application

[0025] Figure 3 : Microrheological experiment of recombinant collagen in Example 3 of this application

[0026] Figure 4 : Effects of recombinant collagen treatment on mammalian cell viability and migration in Example 4 of this application

[0027] Figure 5 : Role of recombinant collagen in promoting diabetic wound healing in Example 5 of this application

[0028] Figure 6 : Immunoblot analysis of protein contents at the wound site by recombinant collagen in Example 6 of this application

[0029] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0030] TERMS

[0031] Unless otherwise stated, each of the following terms shall have the meaning set forth below.

[0032] In this article, the terms "comprises", "comprising" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device that includes a series of elements includes not only those elements, but also includes other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of more restrictions, an element defined by the sentence "comprising a ..." does not exclude the presence of other identical elements in the process, method, article or device that includes the element.

[0033] In this specification, some embodiments may be disclosed in a format of being in a range. It should be understood that this description of "being in a range" is only for convenience and brevity, and should not be interpreted as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and independent numerical values ​​within this range. For example, the description of the range 1-6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within this range, such as 1, 2, 3, 4, 5 and 6. Regardless of the breadth of the range, the above rules apply.

[0034] It should be noted that, as used herein and in the claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. For example, a nucleic acid molecule refers to one or more nucleic acid molecules. Thus, the terms "a", "a", "one or more", and "at least one" can be used interchangeably. Similarly, the terms "comprising", "including", and "having" can be used interchangeably and should generally be understood to be open-ended and non-restrictive, e.g., not excluding other unlisted elements or steps. DETAILED DESCRIPTION

[0035] Example 1: Obtaining recombinant collagen and recombinant collagen fused with interferon

[0036] The Spy Tag (ST) sequence was inserted into the C-terminus of the collagen HA-CLP containing the hyaluronic acid binding site to form the HA-CLP-ST recombinant protein, and the Spy Catcher (SC) sequence was inserted into the N-terminus of interferon IFNα to form the SC-IFNα recombinant protein. The collagen HA-CLP containing the hyaluronic acid binding site was recombined with the interferon IFNα sequence to form the HA-CLP-IFNα recombinant collagen. The protein sequences are shown in Table 1.

[0037] Table 1. Amino acid sequences of various recombinant collagens and interferon-containing fusion proteins

[0038] Name Sequence Number HA-CLP-ST ADEQEEKAKVRTELIQELAQGLGGIEKKNFPTLGDEDLDHTYMTKLLTYLQEREQAENSWRKRLLKGIQDHALDGGPCPPCRYPISRPRKRGPKGEQGPQGLPGKDGEAGAQGPAGPMGPAGEQGEKGEPGTQGAKEDRGETGPKGPKGERGEAGPAGKDGEPGPVGPAGPKGEQGPQGLPGKDGEAGAQGPAGPMGPAGEQGEKGEPGTQGAKEDRGETGPKGPKGERGEAGPAGKDGEPGPVGPAGPKGEQGPQGLPGKDGEAGAQGPAGPMGPAGEQGEKGEPGTQGAKEDRGETGPKGPKGERGEAGPAGKDGEPGPVGPAGGPCPPCRGDAHIVMVDAYKPTK SEQ ID NO: 01 SC-IFNα VDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHIGGSGGCDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKE SEQ ID NO: 02 HA-CLP-IFNα MADEQEEKAKVRTELIQELAQGLGGIEKKNFPTLGDEDLDHTYMTKLLTYLQEREQAENSWRKRLLKGIQDHALDGGPCPPCRYPISRPRKRGPKGEQGPQGLPGKDGEAGAQGPAGPMGPAGEQGEKGEPGTQGAKEDRGETGPKGPKGERGEAGPAGKDGEPGPVGPAGPKGEQGPQGLPGKDGEAGAQGPAGPMGPAGEQGEKGEPGTQGAKEDRGETGPKGPKGERGEAGPAGKDGEPGPVGPAGPKGEQGPQGLPGKDGEAGAQGPAGPMGPAGEQGEKGEPGTQGAKEDRGETGPKGPKGERGEAGPAGKDGEPGPVGPAGGPCPPCRGDGGSGGCDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKE* SEQ ID NO: 03 Spy Tag (ST) AHIVMVDAYKPTK SEQ ID NO: 04 Spy Catcher (SC) VDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHI SEQ ID NO: 05 IFNα CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKE SEQ ID NO: 06

[0039] Using conventional molecular cloning techniques and Overlap PCR technology, the nucleotide sequences of the above recombinant proteins were cloned into the vector pET-28a respectively. Then, through double digestion identification with EcoR I and Hind III and DNA sequencing, the recombinant vectors pET-28a-HA-CLP-ST, pET-28a-SC-IFN-α, and pET-28a-HA-CLP-IFN-α were successfully constructed and transformed into BL21(DE3) for expression. When the OD value reached 0.6, isopropyl β-D-1-thiogalactopyranoside (IPTG) with a concentration of 0.3 mM was added, and the cells were cultured for 20 - 24 hours. The cells were collected by centrifugation at 8000g for 20 minutes at 4°C.

[0040] The cells were lysed by ultrasonic treatment, and the proteins in the Escherichia coli lysate were purified using an immobilized metal affinity chromatography Ni-NTA column. The purified proteins were identified by Coomassie brilliant blue staining through SDS-PAGE, and the size of the recombinant proteins was further determined using protein mass spectrometry. The relevant results are as Figure 1 shown.

[0041] Example 2: Preparation of hydrogel by crosslinking recombinant collagen with hyaluronic acid

[0042] Recombinant collagens HA-CLP-ST and SC-IFNα were concentrated using ultrafiltration centrifugal tubes (Millipore). HA-CLP-ST and SC-IFNα were mixed at different ratios of 2:8, 4:6, 5:5, 6:4, and 8:2 respectively, and incubated at 25°C for 12 hours. Identification by Coomassie brilliant blue staining through SDS-PAGE showed that the chemical coupling effect was optimal when the ratio of HA-CLP-ST:SC-IFNα was 4:6. This chemically coupled collagen (HA-CLP-ST:SC-IFNα = 4:6, SPH) was used for hydrogel preparation. At the same time, blank control HA-CLP (Control) and directly gene-encoded positive control HA-CLP-IFNα (RPH) were used for hydrogel preparation.

[0043] The above three different recombinant collagens HA-CLP (Control), HA-CLP-IFNα (RPH), and HA-CLP-ST+SC-IFNα (SPH, ratio 4:6) with the same concentration were respectively mixed with 2% hyaluronic acid to prepare hydrogels. Take 0.4 g of hyaluronic acid and dissolve it in 10 mL of 1% NaOH solution. Hyaluronic acid and collagen were mixed in a ratio of 4:6 (w / w), and 1% of 1,4-butanediol diglycidyl ether (BDDE) reagent was added and mixed. The mixture was incubated at 40 °C for 5 hours, then frozen in a -80 °C refrigerator for 2 hours, and finally freeze-dried in a freeze dryer. The freeze-dried powder was dissolved in ddH2O or culture medium to prepare a hydrogel, and the internal structure of the hydrogel was observed using a Hitachi S-4800 scanning electron microscope as Figure 2 shown.

[0044] Example 3: Microrheological experiment of collagen

[0045] To confirm and quantify the strength of the hydrogel, a TA Instruments DHR-1 rheometer was used to measure the storage modulus (G′) and loss modulus (G″). The changes of G' and G'' with time reflect the viscoelastic properties of the hydrogel. G' characterizes the elastic behavior and reflects the ability of the hydrogel to store deformation energy, while G'' reflects the viscous behavior and shows the characteristics of energy dissipation over time.

[0046] The freeze-dried different hydrogels were redissolved with the same ddH2O. An appropriate amount of the sample was placed on the sample stage and loaded into the test fixture of the rheometer. The temperature was stabilized at 37 °C. The strain modulus curve was measured with a strain range of 0.01 - 1000%, logarithmically sampled, and the frequency was set to 1 HZ. Rheological studies were carried out in an oscillatory mode with a frequency of 1 HZ. The sample underwent a multi-step process: 10% strain for 60 seconds, 800% strain for 60 seconds, 10% strain for 60 seconds, 800% strain for 60 seconds, 10% strain for 60 seconds.

[0047] The results are as Figure 3 shown. Compared with the control group hydrogel, RPH and SPH showed higher storage modulus and loss modulus.

[0048] Example 4: In vitro cytological detection of recombinant collagen fused with interferon

[0049] Mouse fibroblasts NIH-3T3 and human immortalized epidermal cells HaCat were cultured in DMEM medium containing 10% FBS (Fetal Bovine Serum Gibco, 10100154) and 1% double antibiotics (penicillin-streptomycin solution) in a constant temperature incubator at 37°C with CO2. Different hydrogels (RPH, SPH) with 0.1%, 0.2%, and 0.3% dry content were immersed in the medium, and the cells were cultured with the leachate to detect the effect of the hydrogels on cell toxicity by live / dead staining.

[0050] The experimental results are as Figure 4 shown. When NIH-3T3 cells and HaCat cells were treated with hydrogels prepared at 0.1%, 0.2%, and 0.3% concentrations, cell death was not significantly induced. Moreover, as the concentration of the hydrogel increased, the cell density also increased, indicating that the hydrogel has good cytocompatibility with the cells.

[0051] Cell proliferation and toxicity detection experiments were carried out by the CCK8 method to detect the effect of different hydrogels on the viability of NIH-3T3 cells and HaCat cells. All tests were repeated three times, and the cytotoxicity of the hydrogels was evaluated. To evaluate the cell proliferation and toxicity of the hydrogels, NIH-3T3 cells and HaCat cells were cultured in DMEM medium containing 10% FBS and 1% double antibiotics. Different hydrogel materials with 0.1%, 0.2%, and 0.3% lyophilized content were immersed in the complete medium. The cultured NIH-3T3 cells and HaCat cells were plated on a 96-well plate and cultured in a constant temperature incubator at 37°C with CO2. After culturing, the old medium was aspirated, and new leachate medium was added. The cells were cultured in the cell incubator for 20 h, CCK8 reagent was added, and the cells were placed in the incubator for 30 min. The optical density (OD) at 450 nm was measured and normalized to the medium control. All tests were repeated three times, and the cytotoxicity of different hydrogels was evaluated.

[0052] The experimental results are as Figure 4 shown. The treatment groups with different concentrations of RPH and SPH hydrogels showed higher cell viability compared to the PBS control group. As the concentration of the hydrogel increased, the cell viability also increased. Among them, the cell viability of the SPH treatment group was significantly higher than that of the RPH treatment group.

[0053] The NIH-3T3 cells were cultured in DMEM medium containing 10% FBS and 1% penicillin-streptomycin. Different freeze-dried hydrogel materials at 0.1%, 0.2%, and 0.3% were soaked in the complete medium. The cultured NIH-3T3 cells were seeded, inoculated into six-well plates, and placed in an incubator at 37°C with CO2. When the cells reached approximately 80%, the old medium was discarded, and fresh leachate cell medium was added for a scratch assay to detect the effect of the hydrogel on cell migration.

[0054] The experimental results are as Figure 4 shown. The SPH treatment group exhibited a faster migration rate than the RPH treatment group and the PBS control group.

[0055] Example 5: Detection of the effect of recombinant collagen fused with interferon on wound healing in mice

[0056] Male Balb / c mice (20 - 25 g) were purchased and kept in an SPF facility. After one week of housing in the mouse house, they were induced to model diabetes using streptozotocin (STZ). The diabetic models were divided into five groups: (Saline, STZ+Saline, STZ+Control, STZ+RPH, STZ+SPH). After the mice were anesthetized with a respiratory anesthesia machine (RWD, R510IP), a 1-cm diameter wound was cut on the back of the mice. Different hydrogels were applied to the wound sites. The wound healing was photographed at 0, 4, 8, and 12 days. The wound area was calculated using Fiji software, and the wound healing rate was calculated using the formula: Wound healing rate (%) = (S0 - Sn) / S0 × 100, where S0 and Sn represent the wound areas on the 0th day and the nth day after treatment.

[0057] The experimental results are as Figure 5 shown. The wound healing rate of the STZ+SPR treatment group was significantly higher than that of the other groups (STZ+Saline, STZ+control, STZ+RPH), even exceeding the healing level of normal mice treated with saline.

[0058] Example 6: Immunoblot analysis of the wound sites in mice treated with recombinant collagen fused with interferon

[0059] The mice in Example 5 were sacrificed to obtain the wound skin. Proteins were extracted from the excised wound skin using a whole protein extraction kit (Solarbio, SKUBC3710-100T), and the concentration of the extracted proteins was measured. Then, Western blot analysis was performed to evaluate the expression levels of specific proteins. The relevant protein samples were loaded and separated by SDS-PAGE, and then transferred onto a nitrocellulose membrane. The membrane was blocked to prevent non-specific antibody binding, and then incubated overnight at 4°C with specific antibodies against collagen 1α (CAS: Ab270993), CK14 (CAS: Ab119695), anti-α-SMA, IL-6, and TNF-α to detect the presence and relative amounts of these proteins in the wound skin samples.

[0060] The experimental results are as Figure 6 shown. The hydrogel treatment group significantly upregulated the expression of α-SMA, Collagen 1α, and CK-14. The expression of α-SMA, Collagen 1α, and CK-14 in the SPH treatment group was further enhanced compared to other groups, which may be related to the enhanced inflammatory response induced by the Spy-chemistry method of loading IFN-α, thus accelerating the collagen deposition and epithelialization processes. Further evaluation of the expression of IL-6 and TNF-α found that the levels of IL-6 in the wounds of normal mice and diabetic mice treated with SPH were both low, in contrast to the previous report that STZ-induced hyperglycemia delays wound healing by increasing IL-6. The significant decrease in IL-6 after SPH treatment may be due to the anti-inflammatory effect of IFN-α, which inhibits the release of pro-inflammatory factors. In addition, the loading of IFN-α significantly increased the local expression of TNF-α in the wound, which may promote wound healing by activating the MAPK signaling pathway.

[0061] Although embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art can understand that various substitutions, changes, and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the scope of the present invention is not limited to the content disclosed in the embodiments.

Claims

1. A method for preparing a composite medical sponge conjugated with interferon, comprising the following steps: (a) Expressing the specific collagen in cells by means of genetic engineering using a vector containing a specific collagen gene, and then separating and purifying to obtain the specific collagen; (b) Expressing the specific interferon in cells by means of genetic engineering using a vector containing a specific interferon gene, and then separating and purifying to obtain the specific interferon; (c) Chemically conjugating the specific collagen and the specific interferon prepared in steps (a) and (b) in a ratio of 1:10 to 10:1, and incubating to obtain collagen conjugated with interferon; (d) Mixing hyaluronic acid and the collagen conjugated with interferon prepared in step (c) in a ratio of 1:10 to 10:1, then adding 1,4-butanediol diglycidyl ether, and incubating, freezing, and freeze-drying to prepare a composite medical sponge conjugated with interferon; The amino acid sequence of the specific collagen is as shown in SEQ ID NO: 01; the amino acid sequence of the specific interferon is as shown in SEQ ID NO:

02.

2. The preparation method according to claim 1, wherein In step (c), the specific collagen and the specific interferon are chemically conjugated in a ratio of 2:8, 4:6, 5:5, 6:4, or 8:

2.

3. The preparation method according to claim 2, wherein In step (c), the specific collagen and the specific interferon are chemically conjugated in a ratio of 4:

6.

4. The preparation method according to claim 1, wherein The cells in steps (a) and (b) are Escherichia coli, and the vector is pET-28a.

5. The preparation method according to claim 1, wherein, The incubation temperature in step (c) is 20-30 °C, and the incubation time is 8-16 hours.

6. The preparation method according to claim 1, characterized in that, The hyaluronic acid in step (d) has a concentration of 2% and is prepared by dissolving it in a 1% NaOH solution.

7. The preparation method according to claim 1, wherein In step (d), the hyaluronic acid and the collagen conjugated with interferon are mixed in a ratio of 4:

6.

8. The preparation method according to claim 1, characterized in that, The concentration of 1,4-butanediol diglycidyl ether in step (d) is 1%.

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