Protein-drug coupled supramolecular artificial skin with synergistic effects of diminishing inflammation and promoting growth and preparation method of protein-drug coupled supramolecular artificial skin

Abstract

The embodiment of the invention discloses a protein-drug coupled supramolecular artificial skin with a synergistic effect of diminishing inflammation and promoting growth and a preparation method of the protein-drug coupled supramolecular artificial skin. The method comprises the following steps: constructing a fusion protein containing ferredoxin and an epidermal growth factor; ferredoxin and dexamethasone are subjected to a coupling reaction, and a coupling product is obtained; and dissolving the fusion protein and the coupling product in a guanidine hydrochloride solution, adding ammonium persulfate and a photoinitiator, and carrying out a UV irradiation reaction to obtain the supramolecular artificial skin. The supramolecular artificial skin capable of diminishing inflammation and promoting growth, provided by the invention, can be used as a candidate of artificial skin due to good toughness and biocompatibility, achieves dual functions of diminishing inflammation and promoting growth, and has a relatively good application prospect.

Description

Technical Field

[0001] Embodiments of the present invention relate to the technical field of functional materials, and particularly to a protein-drug conjugate supramolecular artificial skin with synergistic anti-inflammatory and growth-promoting effects and a preparation method thereof. Background Art

[0002] The skin is the largest organ of the human body and has multiple functions, such as protecting internal organs from external injuries, regulating body temperature, perceiving external stimuli, and participating in immune responses. Skin injuries, such as burns, wounds, diabetic ulcers, etc., often lead to the loss of skin barrier function and may trigger complications such as chronic inflammatory reactions and infections. During the wound healing process, although there are natural healing mechanisms, for large-area, deep wounds and chronic wounds, the effects of traditional treatment methods are still limited. Traditional artificial skins mainly rely on synthetic materials (such as polylactic acid, collagen, etc.) or biological materials (such as acellular dermal matrix) to provide mechanical support and barrier functions. However, these materials have limited functions in promoting wound healing, anti-inflammation, and antibacterial, and are difficult to meet the needs of complex wound repair. Therefore, developing an artificial skin with biological activity that can promote skin regeneration and inhibit inflammatory reactions has become an important research direction in the current biomedical field.

[0003] Currently, there are various artificial skin or wound dressing products on the market, such as hydrogels, synthetic polymer membranes, natural polysaccharide substrates, etc. Most of these products focus on protecting the wound, controlling humidity, and promoting wound surface healing. However, the existing products have limitations in anti-inflammation and tissue regeneration. Many artificial skin materials fail to effectively relieve the inflammatory reaction of chronic wounds in practical applications, or perform poorly in promoting the proliferation and migration of skin cells. This results in significant differences in the long-term healing effects of traditional wound dressings, especially when dealing with complex or infected wounds. After skin injury, inflammatory reactions and cell proliferation are key links in the repair process. Excessive inflammation can lead to tissue damage and delayed healing, while insufficient cell proliferation affects wound closure. In the prior art, anti-inflammatory drugs (such as glucocorticoids, non-steroidal anti-inflammatory drugs) and growth factors (such as EGF, VEGF) are often used alone, but lack synergistic effects, and there are problems such as uncontrollable drug release and low bioavailability.

[0004] In recent years, the research on bioactive molecules has provided new ideas for wound healing. For example, growth factors (such as epidermal growth factor EGF and fibroblast growth factor FGF) have been proven to promote the proliferation and regeneration of skin cells. In addition, anti-inflammatory molecules (such as steroids and non-steroidal anti-inflammatory drugs) are also widely used clinically to reduce the inflammatory response of wounds. The application of these active molecules can not only promote skin regeneration but also effectively reduce the inflammatory response at the wound site and the risk of infection. Protein-drug conjugation technology realizes the targeted delivery and controlled release of drugs by binding drugs to functional proteins. This technology has made significant progress in the field of cancer treatment, but its application in skin repair is still in the early stage. In the existing technology, the stability and biological activity of protein-drug conjugates still need to be optimized, and there is a lack of multifunctional designs for skin repair. How to effectively load these bioactive molecules into artificial skin materials and ensure their continuous release during the wound repair process remains an important challenge. Summary of the Invention

[0005] Therefore, the present invention proposes a growth-promoting and anti-inflammatory artificial skin, which combines biomaterials, active molecule carrier technology, and composite function design, aiming to solve the deficiencies of existing materials in anti-inflammatory and tissue regeneration. The artificial skin of the present invention can not only effectively cover and protect the wound surface but also continuously release growth factors and anti-inflammatory molecules, thereby promoting wound healing while inhibiting the inflammatory response, reducing scar formation, and reducing the risk of infection. Research shows that through appropriate material design and the loading of active molecules, the skin repair effect can be significantly improved. In addition, compared with traditional artificial skin, the artificial skin of the present invention has better biocompatibility, stronger anti-inflammatory effects, and more significant tissue regeneration-promoting functions.

[0006] For this reason, the embodiments of the present invention provide a protein-drug conjugate supramolecular artificial skin with a synergistic effect of anti-inflammatory and growth promotion and a preparation method thereof. The supramolecular artificial skin of the present invention has good toughness and biocompatibility, making it a candidate for artificial skin, achieving the dual functions of both anti-inflammatory and growth promotion.

[0007] To achieve the above object, the embodiments of the present invention provide the following technical solutions:

[0008] According to the first aspect of the embodiments of the present invention, the present invention provides a preparation method of a protein-drug conjugate supramolecular artificial skin with a synergistic effect of anti-inflammatory and growth promotion, and the method includes:

[0009] Construct a fusion protein containing ferredoxin and epidermal growth factor;

[0010] Perform a conjugation reaction between ferredoxin and dexamethasone to obtain a conjugate product;

[0011] Dissolve the fusion protein and the conjugate product in a guanidine hydrochloride solution, add ammonium persulfate and a photoinitiator, and carry out a UV irradiation reaction to obtain the supramolecular artificial skin.

[0012] Furthermore, the nucleotide sequence encoding the fusion protein is as shown in SEQ ID NO: 1.

[0013] Furthermore, the conjugation reaction is carried out in the presence of EDC and NHS, the final concentration of EDC is 2 mM, and the final concentration of NHS is 5 mM;

[0014] The conditions for the conjugation reaction are: the final concentration of ferredoxin is 100 mg / ml, the final concentration of dexamethasone is 100 μg / ml, and stir at room temperature for 2 h.

[0015] Furthermore, the concentration of the guanidine hydrochloride solution is 7 M;

[0016] The final concentration of the fusion protein is 90 mg / ml, the final concentration of the conjugate product is 10 mg / ml, the final concentration of ammonium persulfate is 50 mM, and the dosage of the photoinitiator is 1%.

[0017] Furthermore, the photoinitiator includes photoinitiator 2959.

[0018] Furthermore, the conditions for the UV irradiation reaction are: irradiate at 365 nm for 15 - 30 min.

[0019] According to the second aspect of the embodiments of the present invention, the present invention provides a protein-drug conjugate supramolecular artificial skin having a synergistic effect of anti-inflammatory and promoting growth, which is prepared by the preparation method described in any one of the above.

[0020] The embodiments of the present invention have the following advantages:

[0021] The present invention provides a supramolecular artificial skin having anti-inflammatory and growth-promoting effects. Its good toughness and biocompatibility enable it to be a candidate for artificial skin, achieving the dual functions of both anti-inflammatory and growth-promoting. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only exemplary, and for those of ordinary skill in the art, without creative efforts, other implementation drawings can also be obtained according to the provided drawings.

[0023] Figure 1 It is the plasmid map construction and protein purification diagram provided by the present invention;

[0024] Figure 2 1H NMR spectrum of the conjugate provided by the present invention;

[0025] Figure 3 Photograph of the supramolecular artificial skin provided by the present invention;

[0026] Figure 4 Electron micrograph of the supramolecular artificial skin provided by the present invention;

[0027] Figure 5 Comparison chart of stress-strain curves of the supramolecular artificial skin provided by the present invention and ferredoxin, where A is strain and B is stress;

[0028] Figure 6 CCK8 experiment for detecting the effect of the supramolecular artificial skin on cells provided by the present invention;

[0029] Figure 7 Live / dead staining for detecting the effect of the supramolecular artificial skin on cells provided by the present invention;

[0030] Figure 8 Scratch test results provided by the present invention;

[0031] Figure 9 Western Blot test results provided by the present invention. Detailed implementation manners

[0032] The following specific embodiments illustrate the implementation manners of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of them. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall within the scope of protection of the present invention.

[0033] Example 1

[0034] This example provides a protein-drug conjugate supramolecular artificial skin with a synergistic effect of anti-inflammatory and growth promotion, and its preparation method includes the following steps:

[0035] (1) Construction of the fusion protein

[0036] Design the sequence to link ferredoxin (FL) and epidermal growth factor (EGF) together, as shown in Figure 1 (A). Design primers and perform PCR. Link the constructed plasmid with the vector, transform it, pick monoclonal colonies for expression in Escherichia coli, perform protein purification, and lyophilize it using a freeze dryer after purification.

[0037] Using gene editing technology, the FL sequence and epidermal growth factor sequence were input into the SnapGene software for primer design. After synthesizing the primers, OVERLAP PCR was performed. After gel extraction of the obtained product, the gel-extracted product was ligated with the pet-28a vector, followed by transformation and plating. After screening, monoclonal colonies were obtained. After enzyme digestion identification, monoclonal colonies were picked for shaking culture and amplification culture.

[0038] Pick monoclonal colonies and add them to 5 ml of LB medium containing kanamycin (100 μg / ml). Incubate overnight at 37°C with 200 rpm shaking, then transfer to 1.5 L of LB medium and place it back in the incubator for shaking culture until the OD600 is between 0.6 and 0.8. Add IPTG (final concentration 300 mM), place it in a shaker at 16°C with 200 rpm shaking, and harvest the bacteria after 48 h.

[0039] Collect the bacterial liquid. Resuspend the collected bacterial pellet with Binding Buffer and place it on ice. Then use a low-temperature ultra-high pressure cell crusher to initially break the bacteria. After setting the instrument parameters, break the bacteria multiple times. Finally, use an ultrasonic cell disruptor to ultrasonically disrupt the broken bacterial liquid. Set the parameters as ultrasonic for 3 s, off for 6 s, power 20%, and total time 15 min. After completion, transfer the bacterial liquid to several 50 mL centrifuge tubes, centrifuge at 8000 rpm and 4°C for 40 min using a multi-functional tabletop high-speed refrigerated centrifuge. Centrifuge again in 50 mL sterile centrifuge tubes and collect all the supernatant after centrifugation for subsequent experiments. Subsequently, perform NI column protein separation and purification, elute with different buffers to obtain the target fusion protein. The protein gel electrophoresis pattern is shown in Figure 1 (B).

[0040] The nucleotide sequence encoding the fusion protein is as follows:

[0041]

[0042] (2) Preparation of the conjugate product

[0043] Ferredoxin is linked to the drug dexamethasone (DEX) through a chemical reaction, and the reaction formula is as follows:

[0044]

[0045] The specific reaction process is as follows:

[0046] 2.1. Equilibrate EDC and NHS to room temperature. Take EDC (final concentration 2 mM) and NHS (final concentration 5 mM) and add them to the MES solution containing dexamethasone (100 μg / ml), and stir at room temperature for 15 min.

[0047] 2.2. Add 2-mercaptoethanol (final concentration 20 mM) to the system of step (2.1) to quench EDC.

[0048] 2.3. Add ferredoxin (final concentration 100 mg / ml) to the system of step (2.2), and react at room temperature (25 °C) for 2 h.

[0049] 2.4. After the reaction, centrifuge to collect the precipitate, and place it in a freeze dryer for lyophilization to obtain the conjugate product.

[0050] (3) Preparation of the supramolecular artificial skin

[0051] Add the fusion protein obtained in step (1) and the conjugate product obtained in step (2) to a 7 M guanidine hydrochloride solution, where the final concentration of the fusion protein is 90 mg / ml and the final concentration of the conjugate product is 10 mg / ml. Then add ammonium persulfate (APS) with a final concentration of 50 mM and 1% photoinitiator 2959 (W / V). After mixing, irradiate with UV at a wavelength of 365 nm for 20 min to obtain the protein-drug conjugate supramolecular artificial skin with the synergistic effects of anti-inflammation and promoting growth.

[0052] Test Example 1

[0053] 1. Characterization of the supramolecular artificial skin

[0054] The composite hydrogel prepared in the present invention is formed under UV irradiation and can be made into different shapes according to requirements, with excellent plasticity ( Figure 3 ). Use a scanning electron microscope to perform scanning imaging on the composite hydrogel. As Figure 4 shown, the hydrogel has a relatively regular arrangement, showing a homogeneous spatial network structure and having a certain structural stability.

[0055] 2. Performance detection of the supramolecular artificial skin

[0056] To further explore the mechanical properties of the supramolecular artificial skin, a tensile tester was used to detect its tensile strength. The results showed that ( Figure 5 ), compared with simple ferredoxin, the supramolecular artificial skin provided by the present invention has good mechanical properties, being both hard and tough, meeting the performance requirements of artificial skin.

[0057] 3. CCK-8 assay

[0058] To further determine the effect of the supramolecular artificial skin on the metabolism of L929 cells, the Cell Counting Kit-8 (CCK8) experiment was used for detection. The experiment was divided into a blank control group (Control) and a supramolecular artificial skin treatment group. The results showed that ( Figure 6 ), the supramolecular artificial skin with a concentration of 5% has an inhibitory effect on cell proliferation. Therefore, a concentration of 3% was selected for subsequent experiments.

[0059] 4. Live / dead staining assay

[0060] After determining the concentration, the live / dead staining method was used to detect the effect of the supramolecular artificial skin on cell viability. The experiment was divided into a blank control group (Control) and a supramolecular artificial skin treatment group (3%). The results showed that ( Figure 7 ), the supramolecular artificial skin is non-toxic to cells, can improve cell viability, and has good biocompatibility.

[0061] 5. Scratch assay

[0062] To determine whether the supramolecular artificial skin has the effect of promoting cell proliferation, the experiment was set up as a blank control group (Control), a ferredoxin group, and a supramolecular artificial skin treatment group. The results showed that ( Figure 8 ), the supramolecular artificial skin treatment group can effectively promote cell proliferation with the increase of time, indicating that the supramolecular artificial skin provided by the present invention has the effect of promoting growth.

[0063] 6. Anti-inflammatory efficacy detection

[0064] The anti-inflammatory function of the supramolecular artificial skin prepared by the present invention was detected. The experiment was divided into a blank control group (Control), an LPS treatment group, and an LPS + supramolecular artificial skin treatment group. After LPS induced mouse fibroblast L929 cells to establish an inflammation model, the cells were treated with the soaking solution containing the supramolecular artificial skin, and proteins were extracted. Using the Western Blot experiment, the key proteins in the inflammatory signaling pathway were detected. The results showed that ( Figure 9 ), the supramolecular artificial skin can effectively reduce the phosphorylation of the inflammatory signal NF-κb protein content, indicating that the supramolecular artificial skin provided by the present invention has an anti-inflammatory function.

[0065] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made thereto based on the present invention, which will be obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention fall within the scope of the present invention claimed.

[0066]

[0067]

[0068]

Claims

1. A preparation method of a protein-drug conjugate supramolecular artificial skin with synergistic anti-inflammatory and growth-promoting effects, characterized in that, The method includes: Constructing a fusion protein containing ferredoxin and epidermal growth factor; Performing a coupling reaction between ferredoxin and dexamethasone to obtain a coupling product; Dissolving the fusion protein and the coupling product in a guanidine hydrochloride solution, adding ammonium persulfate and a photoinitiator, and performing a UV irradiation reaction to obtain the supramolecular artificial skin.

2. The preparation method of the protein-drug conjugate supramolecular artificial skin having the synergistic effects of anti-inflammation and growth promotion according to claim 1, characterized in that The nucleotide sequence encoding the fusion protein is as shown in SEQ ID NO:

1.

3. The preparation method of the protein-drug conjugate supramolecular artificial skin having the synergistic effects of anti-inflammation and growth promotion according to claim 1, characterized in that The coupling reaction is carried out in the presence of EDC and NHS, the final concentration of EDC is 2 mM, and the final concentration of NHS is 5 mM; The conditions of the coupling reaction are: the final concentration of ferredoxin is 100 mg / ml, the final concentration of dexamethasone is 100 μg / ml, and stirring is carried out at room temperature for 2 h.

4. The preparation method of the protein-drug conjugate supramolecular artificial skin having the synergistic effects of anti-inflammation and growth promotion according to claim 1, characterized in that The concentration of the guanidine hydrochloride solution is 7 M; The final concentration of the fusion protein is 90 mg / ml, the final concentration of the coupling product is 10 mg / ml, the final concentration of ammonium persulfate is 50 mM, and the dosage of the photoinitiator is 1%.

5. The preparation method of the protein-drug conjugate supramolecular artificial skin with the synergistic effect of anti-inflammatory and growth promotion according to claim 1, characterized in that, The photoinitiator includes photoinitiator 2959.

6. The preparation method of the protein-drug conjugate supramolecular artificial skin with the synergistic effect of anti-inflammation and growth promotion according to claim 1, characterized in that, The conditions of the UV irradiation reaction are: irradiation at 365 nm for 15 - 30 min.

7. A protein-drug conjugate supramolecular artificial skin with a synergistic effect of anti-inflammatory and growth promotion, characterized in that, It is prepared by the preparation method described in any one of claims 1 - 6.

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