CN114712330A - Bioengineering protein plaster and preparation method and application thereof - Google Patents

Bioengineering protein plaster and preparation method and application thereof Download PDF

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CN114712330A
CN114712330A CN202210054157.7A CN202210054157A CN114712330A CN 114712330 A CN114712330 A CN 114712330A CN 202210054157 A CN202210054157 A CN 202210054157A CN 114712330 A CN114712330 A CN 114712330A
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protein
plaster
val pro
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gly val
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刘凯
王帆
位政
张洪杰
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Changchun Institute of Applied Chemistry of CAS
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
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    • A—HUMAN NECESSITIES
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    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
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    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
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    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
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    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/70—Web, sheet or filament bases ; Films; Fibres of the matrix type containing drug
    • A61K9/7015—Drug-containing film-forming compositions, e.g. spray-on
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Abstract

The invention relates to the technical field of biological materials, in particular to a bioengineering protein plaster and a preparation method and application thereof. Experiments show that the bioengineering protein plaster is uniformly coated on the surface of the tumor, and then generates photothermal conversion through irradiation, so that the tumor can be effectively killed, and the tumor recurrence is reduced. Compared with the existing clinical tumor non-operative treatment means, the treatment strategy has the advantages of good anti-tumor effect, low tumor recurrence and metastasis probability, simple and quick treatment method and easy operation, and provides a new thought for the clinical skin-related malignant tumor non-operative treatment.

Description

Bioengineering protein plaster and preparation method and application thereof
Technical Field
The invention relates to the technical field of biological materials, in particular to a bioengineering protein plaster and a preparation method and application thereof.
Background
Melanoma is the most lethal malignancy of skin cancers and has become one of the most rapidly growing tumors in recent years. Due to the invasive and migratory capacity of malignant melanoma, traditional surgical therapies have difficulty in removing the entire tumor tissue and are unable to avoid the high risk of tumor recurrence. For emerging targeted therapies and immunotherapies, low drug resistance and immunotherapeutic activity are major challenges.
Photothermal therapy (PTT) has attracted considerable attention in cancer treatment because of its minimal damage to tissues and low invasiveness. Near Infrared (NIR) light based photothermal therapy has the potential advantage of large tissue penetration depths, particularly for skin tumors like melanoma. However, traditional photothermal therapy often requires intravenous injection of photothermal agents, such as organic agents and inorganic nanoparticles. These photothermal agents, however, exhibit low biocompatibility and low cumulative concentrations at the tumor site. Injectable hydrogels have recently shown great promise due to their biocompatibility, permeability to oxygen and nutrients, and porous structure that allows loading with therapeutic agents. However, external injections remain inconvenient for routine procedures and increase the incidence of complications. There is a need to develop new photothermal materials for safer and more convenient treatment of melanoma.
Here we have developed a novel engineered protein plaster for non-invasive photothermal tumor therapy. Due to the composite structure formed by the protein, the aromatic surfactant and the gold nanorods, the prepared plaster has excellent biocompatibility, bonding performance and super-strong photo-thermal effect. The adhesive shows super-strong adhesive performance, and the adhesive strength of the adhesive on common materials such as iron sheets, aluminum sheets, glass and the like can be comparable to that of commercial 502 glue. More importantly, the plaster has excellent adhesion performance on the skin surface, greatly improves the adhesion strength after laser irradiation and can be firmly attached to the surface of skin tumor. The local treatment of melanoma is realized by virtue of the excellent photothermal effect of the gold nanorods and the adhesive property of the adhesive. The in situ melanoma model demonstrated that melanoma can be effectively eradicated by local photothermal therapy with no tumor recurrence during the observation period. By taking advantage of the excellent photothermal effect and adhesive property, the plaster has great potential in treating tumors related to the skin.
Disclosure of Invention
In view of this, the invention provides a bioengineering protein plaster and a preparation method and application thereof. The bioengineering protein plaster of the invention can show excellent adhesive property on the surfaces of iron sheets, aluminum sheets, glass materials and skin, has higher photo-thermal conversion efficiency, can realize effective killing of tumors, reduces the recurrence and metastasis probability of the tumors to a certain extent, and has good biocompatibility.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a bioengineering protein preparation, which is prepared from elastin-like protein with positive charge, alkyl anionic surfactant and gold nanorods.
Preferably, the sequence of the elastin-like protein comprises (VPGKG) n or C- (VPGKG) n-C, wherein n is 36-144. Further preferably, the elastin-like protein is C- (VPGKG)144the-C is abbreviated as K144cys, and the amino acid sequence of the-C is shown as SEQ ID NO. 1.
Preferably, the alkyl anionic surfactant comprises sodium dodecylbenzene sulfonate and/or sodium dodecyl sulfate.
Preferably, the mass ratio of the positively charged elastin-like protein to the alkyl anionic surfactant is (100-300): (35-420); the mass ratio of the elastin-like protein with positive charges to the gold nanorods is (200-600) to (0.07-0.56).
The invention also provides a preparation method of the bioengineering protein, which comprises the following steps:
A) adding the elastin-like protein with positive charge into deionized water, and uniformly mixing to obtain a homogeneous protein solution;
B) adding a negatively charged anionic surfactant into deionized water, and uniformly mixing to obtain a homogeneous surfactant solution;
C) and adding the gold nanorod solution into the protein solution, vibrating and mixing uniformly, then adding the surfactant solution, vibrating and centrifuging to obtain the bioengineering protein plaster.
Preferably, the concentration of the protein solution is 100-300 mg/mL;
the concentration of the sodium dodecyl benzene sulfonate solution is 100-300 mg/mL;
the concentration of the gold nanorod solution is 0.07-0.14 mg/mL;
the volume ratio of the protein solution to the GNRs solution is 2 (1-4);
the volume ratio of the protein solution to the anionic surfactant solution is 10 (3.5-14).
Preferably, the GNRs are GNRs with surface modified CTAB ligands, and the preparation method comprises the following steps:
adding HAuCl4·4H2O and AgNO3Dispersing in a mixed solution of CTAB and sodium oleate, stirring and reacting for 1h 30min at 30 ℃, adding an Au seed solution, stirring and reacting for 30s at 30 ℃, and standing in an oil bath at 30 ℃ for 12h to obtain the gold nanorods with the CTAB ligand modified on the surface.
The invention also provides application of the bioengineering protein plaster in preparing a medicament for treating tumors.
The invention also provides a photosensitizer for treating tumors, which comprises the bioengineering protein plaster.
Compared with the prior art, the biological engineering protein plaster for smearing provided by the invention is prepared from elastin-like molecules with positive charges, gold nanorods and alkyl surfactants with negative charges. The gold nanorods have high-efficiency photothermal conversion performance in a near-infrared region. The invention uses elastin-like molecules with positive charges (such as K144cys, gold nanorods with light-heat conversion performance and alkyl surfactants with negative charges (such as sodium dodecyl benzene sulfonate), the bioengineering protein plaster with strong adhesive property, high-efficiency photo-thermal treatment effect and biocompatibility is formed by electrostatic force action, pi-cation action, hydrogen bond action, hydrophobic interaction and covalent interaction, experiments show that the bioengineering protein plaster is uniformly coated on the surface of tumor, then the radiation generates the photo-thermal conversion, can realize the effective killing of the tumor and reduce the tumor recurrence, compared with the prior clinical tumor non-operative treatment means, the anti-tumor effect is good, the tumor recurrence and metastasis probability is low, the treatment method is simple, quick and easy to operate, and the treatment strategy provides a new idea for non-operative treatment of the malignant tumor related to the clinical skin.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
Fig. 1 is a schematic diagram of the synthesis and treatment of the bioengineered protein plaster of the invention, wherein a is a flow chart of expression and purification of K144cys in example 1, B is a flow chart of preparation of the bioengineered protein plaster in example 3, and C is a procedure of a treatment test of melanoma model in example 7;
FIG. 2 shows the adhesion results of the bioengineered protein plaster on iron sheet, aluminum sheet and glass (A) and on in vitro pig skin (B-D);
FIG. 3 shows the adhesion effect of the bioengineered protein plaster on the skin of the rat back;
FIG. 4 is a diagram showing the photo-thermal conversion effect of the bio-engineered protein plaster of the present invention;
FIG. 5 shows melanoma treatment in groups of mice according to example 7 of the present invention;
FIG. 6 shows the H & E staining results of the tumors in example 7 of the present invention;
Fig. 7 shows the results of the adhesion performance test on iron sheets for different bioengineered protein plasters.
Detailed Description
The invention provides a bioengineering protein plaster and a preparation method and application thereof. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The bioengineering protein plaster provided by the invention is prepared from elastin-like protein with positive charge, alkyl anionic surfactant and photo-thermal nanoparticles. The photo-thermal nanoparticles are Gold Nanorods (GNRs).
In the invention, the Elastin-like proteins (ELPs) are artificially synthesized protein polymers and have good biocompatibility. The elastin-like protein is preferably composed of pentapeptide repeat sequence units (Val-Pro-Gly-Xaa-Gly, VPGXG); further, the fourth amino acid Xaa is preferably lysine K, the elastin-like protein is composed of pentapeptide repeat units (Val-Pro-Gly-K-Gly, VPGKG), and the elastin-like protein has positive charges with high charge density, so that the elastin-like protein can form an adhesive through electrostatic interaction with an anionic surfactant (such as sodium dodecyl benzene sulfonate) with a large amount of sulfonic negative charges. In the present invention, the elastin-like protein is more preferably C- (VPGKG) 36-144-C, in particular C- (VPGKG)144-C。
Research shows that compared with the elastin-like protein formed by other repeating units, the elastin-like protein plaster formed by the pentapeptide repeating sequence unit (Val-Pro-Gly-K-Gly, VPGKG) has more excellent adhesive property and biocompatibility, so that after being smeared on the surface of a tumor, the elastin-like protein plaster can be more firmly adhered to the surface of the tumor, cancer cells can be accurately and controllably completely killed by combining near-infrared illumination, the treatment effect is more remarkable, and relapse is avoided.
In the present invention, the alkyl anionic surfactant is negatively charged, and is preferably at least one of sodium dodecylbenzenesulfonate and/or sodium dodecylsulfate. In one embodiment of the present invention, the alkyl anionic surfactant is sodium dodecylbenzenesulfonate.
In the invention, the mass ratio of the elastin-like protein with positive charge to the alkyl anionic surfactant is (100-300): (35-420), specifically 100:420, 100:35, 100:70, 300:35 or 300: 420.
In the invention, the mass ratio of the elastin-like protein with positive charges to the gold nanorods is (200-600): (0.07-0.56), specifically 200:0.007, 200: 0.56, 600: 0.07 or 600: 0.56.
The preparation method of the biological engineering protein plaster capable of being smeared provided by the invention comprises the following steps:
A) adding the elastin-like protein with positive charges into deionized water, and uniformly mixing to obtain a homogeneous protein solution;
B) adding a negatively charged anionic surfactant into deionized water, and uniformly mixing to obtain a homogeneous surfactant solution;
C) adding the gold nanorod solution into the protein solution, shaking and mixing uniformly, then adding the surfactant solution, shaking and centrifuging to obtain the bioengineering protein plaster.
In the above preparation method provided by the present invention, the types of elastin-like and alkyl surfactants are the same as those mentioned above, and are not described herein again.
Further, the concentration of the elastin-like protein solution is preferably 100-300 mg/mL, and specifically can be 100mg/mL, 200mg/mL or 300 mg/mL.
The concentration of the GNRs solution is preferably 0.07-0.14 mg/mL, and specifically can be 0.07mg/mL or 0.14 mg/mL;
the concentration of the anionic surfactant solution is preferably 100-300 mg/mL, and specifically can be 100mg/mL, 200mg/mL or 300 mg/mL.
According to the invention, the volume ratio of the elastin-like solution to the GNRs solution is 2 (1-4), and specifically can be 2:1 or 2: 4.
In the invention, the volume ratio of the elastin-like solution to the alkyl anionic surfactant solution is 10 (3.5-14), and specifically 10:3.5 or 10: 14.
In the invention, the GNRs are surface modified CTAB ligand GNRs, and are prepared by the following method:
adding HAuCl4·4H2O and AgNO3Dispersing in a mixed solution of CTAB and sodium oleate, stirring and reacting for 1h 30min at 30 ℃, adding an Au seed solution, stirring and reacting for 30s at 30 ℃, standing in an oil bath at 30 ℃ for 12h, and preparing the gold nanorod with the CTAB ligand modified on the surface.
In the invention, the gold nanorod with the surface modified with the CTAB ligand is prepared after standing for 12h in an oil bath at 30 ℃, and then the step of washing the gold nanorod with deionized water is further included, so that excessive CTAB is washed away; the number of washing is preferably 2 to 3.
In the invention, the preparation of K144cys adopts a genetic engineering mode, firstly a host bacterium for transforming an expression vector is constructed, and then the K144cys is obtained by fermenting the host bacterium and inducing protein expression. In the examples of the present invention, Escherichia coli was used as a host bacterium. The backbone vector of the expression vector is pET25 b. The end of the K144cys protein obtained by expression of the system is also linked with a 6 XHis tag. Specifically, the amino acid sequence of K144cys is shown as SEQ ID NO.1, and the sequence is specifically:
MCGAGPGVGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGVGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGVGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGVGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGVGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGVGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGVGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGVGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGVGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGVGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGVGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGVGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGVGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGVGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGVGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGWPHHHHHHC。
The bioengineering protein plaster improves the biocompatibility of a photo-thermal nano material and stabilizes and sustainedly photo-thermal conversion, and realizes effective photo-thermal radical treatment of malignant tumors. After being coated on the surface of a tumor, the spreadable high-biocompatibility bioengineering protein plaster can be firmly adhered to the surface of the tumor, can accurately and controllably kill cancer cells completely by combining with near-infrared illumination, and has no recurrence. And because the adhesive obstructs heat scattering, the adhesive not only quickly accumulates heat enough to kill the tumor at the tumor part, but also plays a role in protecting adjacent normal tissues.
Based on the results, the invention also provides the application of the bioengineering protein plaster in preparing the medicine for treating the tumor.
The invention also provides a photosensitizer for treating tumors, which comprises the bioengineering protein plaster.
The bioengineering protein plaster of the invention utilizes the heat generated by 808nm laser irradiation. The temperature exceeds 55 ℃ within 5 minutes under low-power laser irradiation, thereby effectively dissolving cell membranes and denaturing proteins, resulting in direct death or apoptosis of tumor cells.
In addition, studies have shown that the amount of GNRs also affects the effect of photothermal therapy, and insufficient amount of GNRs cannot generate sufficient photothermal energy conversion, and thus effective killing of tumor cells cannot be achieved. Preferably, the GNRs content is 0.04%.
The bioengineering protein plaster provided by the invention can be directly smeared and can be used as a tumor in-situ treatment means with simpler and more convenient operation. Compared with conventional drug treatment, the traditional Chinese medicine composition ensures effective drug concentration and reduces toxic and side effects of the drug on normal tissues in the treatment process.
The elastin-like protein and sodium dodecyl benzene sulfonate in the invention form an adhesive through electrostatic interaction, and the gold-sulfur interaction between the gold nanorods and the elastin-like protein realizes the doping of photo-thermal nanoparticles, so that an adhesive material with excellent adhesive property, high-efficiency photo-thermal treatment effect and biocompatibility is finally formed, and the photo-thermal conversion effect is 22.0%. After being smeared on the surface of a tumor, the melanoma can be accurately and controllably treated by combining near infrared light with high tissue penetration capacity. The adhesive is used as a load matrix, so that the biocompatibility of the photo-thermal nano material can be improved, the tumor can be effectively killed, and the tumor recurrence can be reduced. From the experimental result, the traditional Chinese medicine preparation not only has the effect of almost radically treating melanoma, but also does not cause side injury to normal tissues, thereby providing a new idea and a reference method for clinical melanoma phototherapy.
The test materials adopted by the invention are all common commercial products and can be purchased in the market.
The invention is further illustrated by the following examples:
example 1: expression and purification of K144cys
The first step is as follows: obtaining the strain which stably expresses the K144 cys. The gene sequence of K144cys protein is used to construct pET25b prokaryotic expression vector, which is then transformed into E.coli BLR (DE3) colibacillus, and through inducing expression and screening, stable expression strain is obtained.
The second step is that: expression of K144 cys. The strain is cultured by an LB culture medium, then inoculated to a TB culture medium, after reaching the exponential growth phase, IPTG is added to induce protein expression for 12h at 28.5 ℃, and the strain is centrifugally cleaned and collected, and is preserved at minus 80 ℃.
The third step: and (5) purifying K144 cys. And (3) crushing the thalli under high pressure, centrifuging to obtain a supernatant, filtering for sterilization, and purifying by a protein purifier through a nickel affinity chromatography column, a cation ion exchange chromatography column and a desalting column to finally obtain the K144cys protein.
Example 2: synthesis of gold nanorods
The first step is as follows: preparing a gold nanorod growth solution as follows: 7g CTAB and 1.234g sodium oleate were weighed into a 1L glass flask, 250mL deionized water was added, and the mixture was heated to 50 ℃ in an oil bath and dissolved with stirring. After the dissolution, the temperature of the oil bath kettle is adjusted to 30 ℃ for stabilization. Preparation of 24mLAgNO 3An aqueous solution (concentration: 4mM) was added to the above glass flask, and allowed to stand for 15 min. Prepare 250mL of HAuCl4The aqueous solution (concentration 1mM) was placed in a volumetric flask and added to the above glass flask and stirred for 1h 30 min.
The second step is that: preparing a gold seed solution, which comprises the following specific steps: 5mL of CTAB aqueous solution (concentration: 0.2mM) was prepared in a 100mL glass flask, and placed in a 30 ℃ oil bath pan for preservationAnd (4) warming. Prepare 5mL of HAuCl4An aqueous solution (concentration: 0.5mM) was charged into the above 100mL glass flask, and vigorously stirred. Preparation of 0.01MNaBH40.6mL of the aqueous solution was diluted to 1mL, and the diluted aqueous solution was put into the above 100mL glass flask, and after 2min, stirring was stopped, and aging was carried out for 30 min.
The third step: A1L glass flask was charged with 1.5mL of HCl and stirred for 15 min. 0.064M ascorbic acid was prepared, and 1.25mL of the mixture was added thereto with stirring and stirred for 30 seconds. 0.4mL of the gold seed solution was added to a 1L glass flask and allowed to stand in an oil bath at 30 ℃ for 12 hours. Centrifuged at 12000rpm for 30min, washed 3 times with water, and dispersed in 50mL of deionized water.
In the present invention, the GNRs absorb 808nm near-infrared laser light and generate photothermal conversion by surface plasmon resonance. Transmission electron microscopy shows that the major diameter of the GNRs is 69.5 +/-3.5, and the minor diameter is 19.2 +/-2.5 nm. The moisture was completely removed by vacuum oven and weighed to give a GNRs solution concentration of 0.07 mg/mL.
Example 3: preparation of bioengineering protein plaster
The first step is as follows: the positively charged elastin-like protein obtained in example 1 was dissolved in deionized water to give 100mg/mL solution A.
The second step is that: the gold nanorod solution was denoted as solution B.
The third step: the sodium dodecyl benzene sulfonate with negative charge is dissolved in deionized water to prepare a solution C with 100 mg/mL.
The fourth step: mixing A and B according to a volume ratio of 2: 1, uniformly shaking, then adding a solution C, wherein the volume ratio of the solution A to the solution C is 10:7, and uniformly mixing to obtain an adhesive solution.
The fifth step: and centrifuging the adhesive solution, and removing supernatant to obtain the bioprotein plaster.
Example 4: adhesion strength test of bioengineering protein plaster
Hard bottom sheets (glass, iron and aluminum sheets) shear adhesion test: after the adhesive was evenly applied to one backsheet, a second backsheet was placed on top of the first to create a shear bond joint with an overlap area of 3mm x 5 mm. Followed by curing at room temperature for 4 days. The control group was a commercial 502 glue,the operation steps are the same as above. All samples were tested in a universal materials testing machine at 10 mm. min-1The speed of (2) was tested.
Testing the adhesion performance of the in-vitro pigskin:
Shear adhesion test a patch of bioengineered protein was spread evenly on one of the pig skins and then the other pig skin was placed symmetrically on the bioengineered protein patch (adhesion area: 3.5mm x 9 mm). Two sheets of pigskin were irradiated alternately with laser light (interval 30 s). The control was cured at room temperature for 1 hour.
Butt-bonding test two pieces of pigskins were butt-bonded together with a bioengineered protein plaster (bonding area: 2 mm. times.8.5 mm). Two pig skins were irradiated with laser light alternately from the front and back directions (interval 30 s). The control was cured at room temperature for 1 hour.
In-vitro wound closure test, the pigskin is cut off by a scalpel, and in-vitro wound injury is simulated. The cut pigskins were realigned and the bioengineered protein plaster was applied to the wound surface (adhesive area: 3mm x 8 mm). Then irradiating the bioengineering protein plaster by 808nm laser. The control was cured at room temperature for 1 hour. All samples were tested in a universal materials testing machine at 10 mm. min-1The speed of (2) was tested.
FIG. 2 shows the adhesion results of the bioengineered protein plaster of example 3. Wherein, FIG. 2A shows the adhesive properties of the plaster (the bioengineered protein plaster of example 3) on iron sheet, aluminum sheet and glass, and the shear adhesive strength is 15-20MPa, comparable to that of the commercial 502 glue. Fig. 2B, C and D show the adhesive effect of the plaster on the excised pigskin, and it can be seen that the adhesive strength of the pigskin is significantly improved after laser irradiation compared to the control group cured at room temperature.
Example 5: application of bioengineering protein plaster in wound adhesion
The first step is as follows: linear wound modeling was performed on the back of the rat with a scalpel, the wound length being 1 cm.
The second step is that: bioengineered protein plasters were prepared according to the procedure of example 3.
The third step: the bioengineered protein plaster of example 3 was applied to the wound and laser irradiation was given. Due to its very strong adhesive properties, the plaster can firmly adhere wounds together.
The treatment temperature is kept at about 60 ℃. The experiment was set up in the following groups, with 3 rats per group:
blank control group: no treatment was done.
And (3) sewing group: the wound was aligned and sutured with a medical surgical suture.
Medical adhesive group: the wound is aligned and a medical adhesive is dropped on the wound.
Adhesive + laser group: the wound was aligned, and the bioengineered protein plaster prepared in example 3 was applied and laser irradiation was given.
The results are shown in FIG. 3.
FIG. 3 is the adhesion test of the bio-engineered protein plaster of example 3 on the skin of the rat's back. 20mg of plaster was applied to the surface of the wound and subsequently the pictures taken after laser irradiation. The wounds of the rats of the adhesive + laser group were completely healed after 9 days, and no scar was left, compared to the control group.
Example 6: influence of different gold nanorod contents on photo-thermal conversion effect of biological engineering protein plaster
To evaluate the photothermal conversion performance of bioengineered protein plasters, 5mg of bioengineered protein plasters containing different mass fractions of GNRs (0%, 0.02%, 0.04% and 0.08%) were coated on the lid of a 1.5mL centrifuge tube. The irradiation was carried out with 808nm laser for 5min and the temperature was recorded.
The results are shown in FIG. 4. Fig. 4 is a graph showing the photothermal conversion effect of the bioengineered protein plaster of example 3. By changing the loading concentration of the gold nanorods, after irradiation of laser with the wavelength of 808nm, the plaster can be heated to different temperatures in unit time, and the temperature is higher along with the increase of the content of the gold nanorods, wherein the bioengineering protein plaster with the GNRs of 0.04 percent by mass can be heated to 70 ℃. Considering the requirement of treatment temperature, a bioengineering protein plaster with the GNRs mass fraction of 0.04% is selected in tumor treatment experiments.
Example 7: application of bioengineering protein plaster in melanoma model treatment
The first step is as follows: melanoma models were constructed by injecting melanoma B16F10 cells into the backs of C57BL/6 mice.
The second step is that: bioengineered protein plasters were prepared according to the procedure of example 3.
The third step: the bioengineering protein plaster of example 3 is applied on the surface of melanoma, and can be firmly adhered on the surface of the tumor due to the super-strong viscosity of the plaster.
The fourth step: irradiating the bioengineering protein plaster by near infrared light. The laser used is 808nm laser with power of 0.6W cm-2The irradiation time was 15 minutes, and irradiation was performed on the first three days (irradiation interval 24h), respectively.
The treatment temperature is kept above 55 ℃. The results are shown in FIGS. 5 to 6. The experiment was set up in groups of 5 mice each:
blank control group: without any treatment
Photo-thermal adhesive group: the bioengineering protein plaster of example 3 is smeared on the surface of tumor
Laser group: the tumor surface is irradiated with near infrared light. The laser used is 808nm laser with power of 0.6W cm-2The irradiation time was 15 minutes, and irradiation was performed on the first three days (irradiation interval 24h), respectively.
Photothermal adhesive + laser group: the bioengineering protein plaster prepared in example 3 was applied to the tumor surface and irradiated with laser.
The results are shown in FIGS. 5 to 6.
As can be seen from FIG. 5, 20mg of the plaster was applied to the surface of melanoma and then three laser irradiations (808nm laser, 0.6W. cm) were applied on the first three days (24 h intervals) -2For 15 minutes). Compared with a control group, the adhesive and laser group has a good killing effect on melanoma, and the tumor gradually shrinks after being treated and finally completely disappears.
FIG. 6 shows that the H & E staining result of tumor tissue after treatment shows that the bioengineering protein plaster of the invention effectively kills tumor cells.
Example 8: adhesion strength comparison test of different bioengineering protein plasters
Two other elastin-like proteins were prepared according to the method of example 1 except that their sequences included (VPGKG)36Or (VPGKG)72And both ends of the protein do not include cys. Bioengineered protein plasters, named K36-SDBS-GNRs and K72-SDBS-GNRs, were prepared according to the method described in example 3, respectively. It was tested for adhesion to the bioengineered protein plasters of example 3 (K144cys-SDBS-GNRs) on iron sheets. As shown in FIG. 7, the results show a significant increase in the adhesive strength of the K144cys-SDBS-GNRs compared to the K36-SDBS-GNRs and the K72-SDBS-GNRs.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.
Sequence listing
<110> Changchun applied chemistry research institute of Chinese academy of sciences
<120> bioengineering protein plaster and preparation method and application thereof
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Met Cys Gly Ala Gly Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro
1 5 10 15
Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly
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Lys Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly Lys
35 40 45
Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly
50 55 60
Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val
65 70 75 80
Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro
85 90 95
Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly
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Lys Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly Lys
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Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly
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Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Val
145 150 155 160
Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro
165 170 175
Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly
180 185 190
Lys Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly Val
195 200 205
Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly
210 215 220
Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val
225 230 235 240
Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro
245 250 255
Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly
260 265 270
Lys Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly Lys
275 280 285
Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly
290 295 300
Val Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val
305 310 315 320
Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro
325 330 335
Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly
340 345 350
Lys Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Lys
355 360 365
Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly
370 375 380
Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val
385 390 395 400
Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro
405 410 415
Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly
420 425 430
Lys Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly Lys
435 440 445
Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly
450 455 460
Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val
465 470 475 480
Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro
485 490 495
Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly
500 505 510
Lys Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly Lys
515 520 525
Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly
530 535 540
Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly Val
545 550 555 560
Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro
565 570 575
Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly
580 585 590
Lys Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly Lys
595 600 605
Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly
610 615 620
Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val
625 630 635 640
Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro
645 650 655
Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly
660 665 670
Lys Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly Lys
675 680 685
Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly
690 695 700
Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val
705 710 715 720
Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro
725 730 735
Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly
740 745 750
Val Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly Lys
755 760 765
Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly
770 775 780
Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val
785 790 795 800
Pro Gly Trp Pro His His His His His His Cys
805 810

Claims (10)

1. A bioengineering protein plaster is characterized by being prepared from elastin-like protein with positive charges, alkyl anionic surfactant and gold nanorods.
2. The bioengineered protein plaster of claim 1, wherein the elastin-like sequence comprises (VPGKG) n and/or C- (VPGKG) n-C, n-36-144.
3. The bio-engineered protein plaster of claim 2, wherein the elastin-like protein is C- (VPGKG)144-C, the elastin-like protein further comprises a His tag, and the amino acid sequence of the elastin-like protein is shown as SEQ ID NO. 1.
4. The bioengineered protein plaster of claim 1, wherein the alkyl anionic surfactant comprises sodium dodecylbenzene sulfonate and/or sodium dodecyl sulfate.
5. The bioengineered protein plaster of claim 1, wherein the mass ratio of the positively charged elastin-like and alkyl anionic surfactant is (100-300): (35-420); the mass ratio of the elastin-like protein with positive charges to the gold nanorods is (200-600) to (0.07-0.56).
6. A method for preparing a bioengineered protein plaster according to any one of claims 1 to 5, comprising the steps of:
A) adding the elastin-like protein with positive charges into deionized water, and uniformly mixing to obtain a homogeneous protein solution;
B) adding a negatively charged anionic surfactant into deionized water, and uniformly mixing to obtain a homogeneous surfactant solution;
C) adding the gold nanorod solution into the protein solution, shaking and mixing uniformly, then adding the surfactant solution, shaking and centrifuging to obtain the bioengineering protein plaster.
7. The preparation method according to claim 6, wherein the concentration of the protein solution is 100-300 mg/mL;
the concentration of the sodium dodecyl benzene sulfonate solution is 100-300 mg/mL;
the concentration of the gold nanorod solution is 0.07-0.14 mg/mL;
the volume ratio of the protein solution to the GNRs solution is 2 (1-4);
the volume ratio of the protein solution to the anionic surfactant solution is 10 (3.5-14).
8. The method of claim 6, wherein the GNRs are GNRs with surface modified CTAB ligands, and the method comprises:
adding HAuCl4·4H2O and AgNO 3Dispersing in a mixed solution of CTAB and sodium oleate, stirring and reacting for 1h 30min at 30 ℃, adding an Au seed solution, stirring and reacting for 30s at 30 ℃, standing in an oil bath at 30 ℃ for 12h, and obtaining the gold nanorods of which the surfaces are modified with CTAB ligands.
9. Use of the bioengineered protein plaster according to any one of claims 1 to 5 or the bioengineered protein plaster prepared by the preparation method according to any one of claims 6 to 8 for the preparation of a medicament for the treatment of tumors.
10. A photosensitizer for treating tumor, which is characterized by comprising the bioengineering protein plaster of any one of claims 1 to 5 or the bioengineering protein plaster prepared by the preparation method of any one of claims 6 to 8.
CN202210054157.7A 2022-01-18 2022-01-18 Bioengineering protein plaster and preparation method and application thereof Pending CN114712330A (en)

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CN110373152A (en) * 2019-08-22 2019-10-25 中国科学院长春应用化学研究所 A kind of protein binding rare earths adhesive of high intensity and preparation method thereof
CN110452662A (en) * 2019-08-22 2019-11-15 中国科学院长春应用化学研究所 A kind of bioadhesive and preparation method thereof based on bioengineered proteins
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