CN113144191B - Gold silver sulfide protein composite hydrogel and preparation method and application thereof - Google Patents
Gold silver sulfide protein composite hydrogel and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of nano materials, in particular to gold-silver sulfide protein composite hydrogel and a preparation method and application thereof. The hydrogel provided by the invention comprises: negatively charged proteins, positively charged photothermal heterogeneous nanoparticles and positively charged Chitosan (CS); wherein the photo-thermal heterogeneous nanoparticles with positive charges are gold silver sulfide hybrid nanoparticles (Ag)3AuS2NPs); the protein with negative charge is elastin-green fluorescent protein complex protein- (VPGEG)72-GFP. The hydrogel has injectability, high-efficiency photothermal treatment effect and biocompatibility, and the photothermal efficiency can reach 39.0%. The medicine is uniformly dispersed around the tumor by a peritumoral injection mode, and then generates photothermal conversion by irradiation, thereby realizing the effective killing of the tumor and reducing the recurrence of the tumor.
Description
Technical Field
The invention relates to the technical field of nano materials, in particular to gold-silver sulfide protein composite hydrogel and a preparation method and application thereof.
Background
Malignant tumors are one of the great threats to human life health. With the progress of medicine, various diseases are successfully overcome, the life of human beings is gradually prolonged, but malignant tumors are still persistent and persistent in the medical world. Tongue cancer is the most common malignant tumor in oral cavity, and has high malignant degree and high metastasis rate. The current treatment of tongue cancer is a combination of surgery-based treatments including radiation and chemotherapy. However, when the tumor is deeply invaded, a hemilingual or total-lingual resection is often required, severely damaging motor, sensory and speech functions. Chemotherapy is effective for systemic treatment, but has limited effectiveness for topical treatment. Local radiotherapy can also cause side effects such as inflammation and damage to the peripheral nerves of the mouth. Although the surgical technique and the adjuvant therapy are continuously improved, the 5-year survival rate of the tongue cancer patients is still 6.6-11.7% at present due to the difficulty of the surgery and the insensitivity of the therapy. Therefore, establishing an effective and accurate non-operative treatment strategy which can inhibit tumor proliferation and reduce side injury to nerves, blood vessels and the like important beside the head and neck tumor has clinical application significance.
Thermal ablation techniques are widely known for the treatment of cancer because of their low toxicity and few side effects. Photothermal therapy (PTT), in particular, is of great interest due to the minimal invasiveness and great penetration depth of Near Infrared (NIR) light. PTT can achieve the purposes of improving the survival quality of late-stage tumors and prolonging the survival period, and simultaneously, radical cure is realized on early-stage tumors. PTT itself is almost non-toxic, with major disadvantages of poor biocompatibility, low photothermal conversion, and long-term toxicity problems associated with photothermal agents (PTAs). Among them, the organic photothermal material has good biodegradability, but the photobleaching property is not favorable for photothermal conversion. Conventional intravenous therapy reduces the accumulation of PTAs and reduces the photothermal effects at the tumor site. In particular, multiple administrations due to circulation of body fluids often cause drug resistance. Therefore, the development of novel PTAs with in situ implantation and adequate therapeutic effect is of great importance.
Disclosure of Invention
In view of the above, the technical problem to be solved by the present invention is to provide a gold-silver sulfide protein composite hydrogel for injection, a preparation method thereof, and application thereof in tumor treatment.
The invention provides a gold-silver sulfide protein composite hydrogel which comprises the following raw materials: elastin-like-green fluorescent protein composite protein, Ag3AuS2NPs and chitosan;
the mass fraction of the elastin-like-green fluorescent protein composite protein is 0.1-10%;
the Ag is3AuS2The mass fraction of NPs is 0.1% -10%;
the mass fraction of the chitosan is 0.1-10%;
the amino acid sequence of the elastin-like protein is (VPGEG)n、(VPGDG)n、(VPGKG)nOr (VPGRG)n，n＝50～100。
In the gold silver sulfide protein composite hydrogel provided by the invention, chitosan has positive charge and Ag3AuS2NPs with positive charge, elastin-like-GreenThe chromo-fluorescent protein complex protein has negative charges. In some embodiments, the chitosan is of medium viscosity (200-400 mPas).
In the present invention, the elastin-like-green fluorescent protein complex protein is (VPGEG)72-GFP。
In some embodiments, the elastin-like-green fluorescent protein complex, Ag, is contained in a gold-silver sulfide protein complex hydrogel3AuS2The mass ratio of NPs to chitosan is 20: 1: 10.
in some embodiments, the gold silver sulfide protein composite hydrogel (VPGEG)72-GFP 4% by weight, Ag3AuS2The mass fraction of NPs is 0.2%, and the mass fraction of chitosan is 2%.
The preparation method of the gold-silver sulfide protein composite hydrogel comprises the following steps:
dissolving chitosan in an acetic acid aqueous solution to obtain a chitosan solution;
dissolving the elastin-green fluorescent protein composite protein in deionized water, and then activating carboxyl of the composite protein by EDC and NHS to obtain an activated protein solution;
mixing Ag with water3AuS2NPs are mixed with the chitosan solution and then mixed with the activated protein solution, and the mixture is stirred and reacted for 10-20 min at the temperature of 55-65 ℃ to obtain the gold-silver sulfide protein composite hydrogel.
In the present invention, the Ag is3AuS2The preparation method of the NPs comprises the following steps:
adding HAuCl4·4H2O and AgNO3Dispersing in the mixed solution of oleic acid and oleylamine, stirring and reacting for 20h at 55 ℃, adding sulfur-containing oleylamine solution, stirring and reacting for 2h at 55 ℃, and preparing the Ag with the surface modified oleic acid oleylamine ligand3AuS2A nanoparticle;
resuspending Ag surface-modified oleylamine oleate ligand in chloroform3AuS2Mixing the nano particles with a CTAB aqueous solution, stirring and reacting for 3 hours at 50 ℃, and then washing to obtain Ag with positive charges3AuS2NPs。
In some embodiments, the chitosan solution has a volume fraction of acetic acid of 1% and a mass fraction of chitosan of 3%.
In some embodiments, the elastin-like-green fluorescent protein complex protein is dissolved in deionized water to a mass fraction of 13%.
In some embodiments, the Ag3AuS2The mass ratio of NPs to chitosan is 1: 10.
In some embodiments, the volume ratio of the chitosan solution to the activated protein solution is 2: 1.
The gold-silver sulfide protein composite hydrogel or the gold-silver sulfide protein composite hydrogel prepared by the preparation method disclosed by the invention is applied to preparation of a medicine for treating malignant tumors.
In the present invention, the malignant tumor is a solid tumor; preferably the malignant tumor is a non-metastatic solid tumor; more preferably, the malignant tumor is tongue cancer.
In the invention, the medicine for treating the tumor is a photosensitizer.
The invention also provides a photosensitizer for treating tumors, which comprises the gold silver sulfide protein composite hydrogel or the gold silver sulfide protein composite hydrogel prepared by the preparation method.
The invention also provides a method for treating tumors by administering the photosensitizer for treating tumors.
The mode of administration is injection.
The method for treating the tumor comprises the steps of injecting the photosensitizer to the tumor tissue, and then irradiating by 808nm laser.
Compared with the prior art, the invention provides the injectable Ag-loaded carrier3AuS2Of NPs (VPGEG)72-a GFP-chitosan photothermal hydrogel comprising: negatively charged (VPGEG)72-GFP and positively charged chitosan and positively charged gold silver sulfide heterogeneous nanoparticles; the gold silver sulfide heterogeneous nano-particles with positive charges are Ag3AuS2NPs。
In the present invention (VPGEG)72GFP by chemical Cross-linking with ChitosanBy forming hydrogels, in addition (VPGEG)72GFP and chitosan realize the doping of photo-thermal nano particles through electrostatic interaction, finally a hydrogel material with injectability, high-efficiency photo-thermal treatment effect and biocompatibility is formed, and the photo-thermal efficiency can reach 39.0%. The medicine is uniformly dispersed around the tumor by a peritumoral injection mode, and then generates photothermal conversion by irradiation, thereby realizing the effective killing of the tumor and reducing the recurrence of the tumor. In addition, the gel has high biocompatibility and can slow down the metabolism of the nanoparticles in vivo. Compared with other photothermal materials of the same type, the photothermal therapy of tongue-associated tumors by eye placement in the research has higher photothermal conversion efficiency, higher biocompatibility and slow metabolism, thereby stably exerting the therapeutic efficacy. The treatment strategy with two advantages provides a new idea for the non-operative treatment of the tongue-related malignant tumor in clinic.
Drawings
FIG. 1 is a schematic diagram of the synthesis of gold silver sulfide heterogeneous nanoparticles in example 1 of the present invention;
FIG. 2 is the shear rheology results of injectable gold silver sulfide-protein-chitosan composite hydrogel of example 1;
fig. 3 shows the photothermal conversion efficiency of the injectable gold silver sulfide-protein-chitosan composite hydrogel of example 1;
FIG. 4 shows the photothermal conversion results of the injectable gold-silver sulfide-protein-chitosan composite hydrogel of example 1;
FIG. 5 is a graph showing the photothermal conversion effect of the gold-silver sulfide protein composite hydrogel injected into the root of the mouse tongue;
FIG. 6 is a comparison of the anti-tumor effect of injectable gold-silver sulfide protein composite hydrogel against a tongue cancer mouse model;
FIG. 7 shows the H & E staining results of the major organs.
Detailed Description
The invention provides gold silver sulfide protein composite hydrogel and a preparation method and application thereof, and a person skilled in the art can appropriately improve process parameters by referring to the content. 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 high-biocompatibility gold-silver sulfide protein composite photo-thermal hydrogel provided by the invention is prepared from proteins and glycan with opposite charges and photo-thermal heterogeneous nanoparticles with positive charges. The positive-charge photo-thermal heterogeneous nanoparticles are gold silver sulfide (Ag) heterogeneous nanoparticles3AuS2NPs). Specifically, in the invention, the hydrogel is composed of a negatively charged elastin-like-green fluorescent protein complex protein- (VPGEG)72GFP and positively charged gold silver sulfide heterogenous nanoparticles (Ag)3AuS2NPs), and positively charged Chitosan (CS).
The previous experiments were performed using various negatively charged polyelectrolyte materials, including elastin-like protein/polyacrylic acid (PAA), polyglutamic acid (γ -PGA), and the like. Experiments have shown that in the formation of hydrogels, elastin-like proteins and PAA, γ -PGA play the same role, i.e. both act as negative charge providers. But the hydrogels have different properties due to the difference in molecular weight and charge density. The resulting PAA-chitosan hydrogel is in the form of a foam; whereas the γ -PGA-chitosan hydrogel is jelly-like and easily broken. More importantly, both gels are not suitable for injection. The hydrogel synthesized by the elastin-like protein has the characteristics of shearing thinning, more uniformity, low viscosity and injectability.
In the invention, the Elastin-like proteins (ELPs) are artificially synthesized protein polymers and have good biocompatibility. The elastin-like protein is mainly composed of pentapeptide repeat sequence units (Val-Pro-Gly-Xaa-Gly, VPGXG), and the fourth amino acid X is selected from glutamic acid E or aspartic acid D, so that the fourth amino acid X has negative charge with high charge density, and the elastin-like protein can be chemically crosslinked with chitosan with a large amount of amino groups to form gel. In addition, amino acid number four may be usedX is selected from lysine K or arginine R, so that the lysine K or arginine R has positive charge with high charge density, and the gel is formed by chemical crosslinking with polysaccharide with negative charges such as a large number of carboxyl groups and the like. Studies have shown that, although the physicochemical forms of the gels formed are similar, they are comparable to other complex proteins (Xaa substituted with other amino acids, or a different number of repeat units), (VPGEG)72The antitumor effect of GFP is more advantageous.
Green Fluorescent Protein (GFP) is a commonly used fluorescent marker, and has the advantage that stable expression of its gene can be observed with the naked eye. The elastin-like protein and the green fluorescent protein are compounded for expression, so that the yield and the purification efficiency of the product protein can be obviously improved. In addition, other fluorescent proteins also have similar functions. Experiments show that the addition of GFP in the protein does not influence the anti-tumor effect. And GFP is used as a molecular chaperone, which is beneficial to improving the expression quantity of the engineering protein. By performing fusion recombination on GFP and the anionic polypeptide, the yield of protein entities is improved. Meanwhile, the low immunogenicity, electronegativity and biocompatibility of the (VPGEG)72 protein are not affected.
The invention utilizes 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to realize the rapid crosslinking of protein and chitosan, thereby forming stable hydrogel. The EDC/NHS reaction, which induces cross-linking to form a gel, is one of the most commonly used bioconjugation reactions for coupling carboxyl groups with amino groups to form amide bonds.
Research shows that in the preparation method provided by the invention, conditions such as reagent dosage, reaction temperature, reaction time and the like can influence the effect of the gel. The gel is formed by the elements of protein and glycan, which are not enough in dosage and only can be in a uniform solution but cannot form gel; if the dosage is too large, the dissolution is not complete, and the gel of the product is not uniform. The dosage of the photo-thermal material also affects the effect of photo-thermal treatment, and insufficient dosage of the photo-thermal material cannot generate enough photo-thermal energy conversion, so that the tumor cells cannot be effectively killed. The ratio between the photothermal material and the elements also needs to be controlled within a certain range to ensure that the photothermal material is effectively incorporated into the gel by electrostatic interaction and no aggregation or leakage occurs. In addition, the reaction temperature and the reaction time also have certain influence on the generation of gel products, and the preferable conditions are that the reaction temperature is controlled to be about 60 ℃ and the reaction time is 15 min. When the reaction temperature is low (e.g., room temperature), the reaction is difficult to proceed, and a gel cannot be formed. If the reaction temperature is too high, the gel is formed too quickly, and it is difficult to control the uniform dispersion of the photothermal material therein. The effect of the amount of reagent on the preparation of the photothermal gel is relatively more pronounced and more important.
In a specific embodiment, the preparation method of the injectable in-vitro chemically synthesized protein composite photothermal hydrogel provided by the invention comprises the following steps:
A) dissolving positively charged chitosan in 1% (v/v) acetic acid water solution, and dispersing to obtain chitosan solution;
B) ag with positive charge3AuS2Mixing NPs with the chitosan solution;
C) will be negatively charged (VPGEG)72GFP in deionized Water and dispersed to obtain (VPGEG)72-an aqueous solution of GFP;
D) EDC, NHS and (VPGEG)72GFP mixing so as to (VPGEG)72The carboxyl group of GFP is activated, facilitating the next step of chemical crosslinking with the amino group of chitosan;
E) mixing Ag with water3AuS2Mixed solution of NPs and chitosan with activated (VPGEG)72GFP was mixed and stirred at 60 ℃ for 15min, using the amino group of chitosan and (VPGEG)72Crosslinking of the carboxyl group of GFP and passing through (VPGEG)72-GFP and Ag3AuS2NPs electrostatic interaction realizes the loading of the NPs to form the gold-silver sulfide protein composite photo-thermal hydrogel ((VPGEG)72-GFP-Chitosan-Ag3AuS2)。
In some embodiments, the positively charged Ag3AuS2NPs were prepared by the following method:
adding HAuCl4·4H2O and AgNO3Dispersing in mixed solution of oleic acid and oleylamine, stirring at 55 deg.C for 20 hr, adding oleylamine solution of sulfur powder, stirring at 55 deg.C for 2 hr to obtain the final productAg coated with oleic oleylamine ligand3AuS2A nanoparticle;
washing off Ag by centrifugation3AuS2Resuspending Ag in chloroform with excess oleic oleylamine in nanoparticle dispersion3AuS2Mixing the nanoparticles with Cetyl Trimethyl Ammonium Bromide (CTAB) water solution, stirring at 50 deg.C for 3 hr, and washing to obtain Ag with positive charge3AuS2NPs。
The excess oleic acid oleylamine is washed for 2-3 times by using ethanol.
The Ag with positive charge3AuS2And washing the NPs for 2-3 times by using water.
The invention uses surfactant CTAB as ligand to disperse oil phase Ag3AuS2The nano particles are transferred to a water phase, so that the water solubility is enhanced, the biotoxicity is further reduced, and the biocompatibility is improved.
In the present invention, (VPGEG)72The preparation of GFP is carried out by constructing host bacterium for transforming expression vector, fermenting host bacterium and inducing protein expression (VPGEG)72-GFP. 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. Obtained by expression of this system (VPGEG)72The end of the GFP protein was also ligated with a 6 XHis tag. Specifically, (VPGEG)72The amino acid sequence of GFP is MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKELGVVGLVPRGSHMGAGPGVGVPGEGVPGEGVPGEGVPGEGVPGEGVPGEGVPGEGVPGEGVPGEGVPGVGVPGEGVPGEGVPGEGVPGEGVPGEGVPGEGVPGEGVPGEGVPGEGVPGVGVPGEGVPGEGVPGEGVPGEGVPGEGVPGEGVPGEGVPGEGVPGEGVPGVGVPGEGVPGEGVPGEGVPGEGVPGEGVPGEGVPGEGVPGEGVPGEGVPGVGVPGEGVPGEGVPGEGVPGEGVPGEGVPGEGVPGEGVPGEGVPGEGVPGVGVPGEGVPGEGVPGEGVPGEGVPGEGVPGEGVPGEGVPGEGVPGEGVPGVGVPGEGVPGEGVPGEGVPGEGVPGEGVPGEGVPGEGVPGEGVPGEGVPGVGVPGEGVPGEGVPGEGVPGEGVPGEGVPGEGVPGEGVPGEGVPGEGVPGWPHHHHHH。
The gold-silver sulfide protein composite photothermal hydrogel provided by the invention improves the biocompatibility of a photothermal nano material, slows down degradation, stably and durably exerts photothermal conversion through the loading effect of gel, and realizes effective photothermal radical treatment of malignant tumors. After the gold silver sulfide protein compound hot hydrogel with high biocompatibility is injected to the position near a tumor, cancer cells can be killed accurately and controllably and completely in combination with near-infrared illumination, and recurrence is avoided. And because the gel obstructs heat scattering, the heat which is enough to kill the tumor is quickly accumulated at the tumor part, and the adjacent normal tissues are protected. While the photothermal therapy is carried out, the side damage to the important nerves, blood vessels and the like beside the tumor is not caused.
The injectable hydrogel of the present invention uses heat generated by 808nm laser irradiation. After being injected into a living body, the gel type drug has a more stable photothermal conversion efficiency (the temperature reaches 55 ℃ in three minutes under low-power laser irradiation) because the drug effect is more slowly diluted due to blood circulation than the solution type drug, thereby effectively dissolving cell membranes and denaturing proteins, so that tumor cells are directly killed or apoptotic.
The photothermal hydrogel provided by the invention has injectability, and can be used as a tumor in-situ treatment means which is simpler and more convenient to operate. 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-green fluorescent protein composite protein and chitosan in the invention form hydrogel through chemical crosslinking, and the hydrogel and the chitosan realize the doping of photo-thermal nanoparticles through electrostatic interaction, so that a hydrogel material with injectability, high-efficiency photo-thermal treatment effect and biocompatibility is finally formed, and the photo-thermal conversion effect is 39%. After the medicine is locally injected to the tumor side, accurate and controllable treatment of tongue cancer is realized by combining near infrared light with high tissue penetration capacity. The gel is used as a load matrix, so that the biocompatibility of the photo-thermal nano material can be improved, and the degradation of the nano particles in vivo can be slowed down, thereby effectively killing tumors and reducing the recurrence of the tumors. From the experimental result, the phototherapy not only has the effect of almost radically treating the tongue cancer, but also does not cause side damage to vessels and nerves around the tongue body, thereby providing a new idea and a reference method for clinical phototherapy of the tongue cancer.
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: (VPGEG)72Expression and purification of GFP
The first step is as follows: obtaining a Stable expression (VPGEG)72Strains of GFP. Use (VPGEG)72The gene sequence of GFP protein constructs pET25b prokaryotic expression vector, then E.coli BLR (DE3) escherichia coli is transformed, and stable expression strain is obtained through induced expression screening.
The second step is that: (VPGEG)72Expression of GFP. 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: (VPGEG)72Purification of GFP. Crushing thallus under high pressure, centrifuging to obtain supernatant, filtering to remove bacteria, and purifying with protein purifier via nickel affinity chromatography column, desalting column, and Q ion exchange chromatography column to obtain (VPGEG)72GFP proteins.
Example 2: synthesis of gold silver sulfide nano-particles with positive charges
The first step is as follows: preparing gold silver sulfide nano material (Ag) with heterostructure by oil solvent heating method3AuS2NPs). The method comprises the following specific steps: taking HAuCl4·4H2O 20mg，AgNO 340 mg was added to a 100mL round bottom flask, followed by 10mL oleic acid and 20mL oleylamine, warmed to 55 ℃ and stirred for 20 h. Adding 25mg of sublimed sulfur into 10mL of oleylamine, ultrasonically mixing uniformly, adding into the solution, and stirring for 2h at 55 ℃. Washing with ethanol for 3 times to obtain heterostructure gold silver sulfide nanometer material (Ag)3AuS2NPs) were dispersed in 5mL of chloroform.
The above preparation process can be referred to FIG. 1.
The second step is that: by means of fittingsBody exchange, replacing the surface oleic acid and oleylamine molecules with cetyltrimethylammonium bromide (CTAB). The method comprises the following specific steps: adding 0.5g of CTAB into a 30ml glass bottle, adding 10ml of deionized water, heating to 50 ℃, and stirring until the CTAB is dissolved. Mixing 5mLAg3AuS2The NPs chloroform solution was added to the CTAB solution and stirred for 3h at 50 ℃. After washing 3 times with water, the solution was dispersed in 1mL of deionized water.
In the present invention, hetero-structure Ag3AuS2The nanoparticles absorb 808nm near-infrared laser light and generate photothermal conversion through surface plasmon resonance. At Ag by ligand exchange3AuS2After CTAB is coated on the surface of the NPs, the CTAB can be uniformly dispersed in an aqueous solution, and the nano material is ensured to have positive charges in the aqueous solution due to the high-density amino groups. Display of Ag by transmission electron microscope3AuS2The size of the NPs is about 12 nm. Measuring Ag per 1mg heterostructure by inductively coupled plasma emission spectrometer3AuS2The NPs contained 201.4. mu.g of Au.
Example 3: preparation of gold silver sulfide protein composite hydrogel
The first step is as follows: the positively charged chitosan (medium viscosity, 200-400 mPas) was dissolved in acetic acid 1% (v/v) aqueous solution and dispersed to give a 3% chitosan solution.
The second step is that: ag with positive charge3AuS2NPs are mixed with the chitosan solution to make Ag3AuS2The mass ratio of NPs to chitosan is 1:10, and a solution A is obtained.
The third step: will be negatively charged (VPGEG)72-GFP dissolved in deionized water to make up 13 w% (VPGEG)72-aqueous GFP solution.
The fourth step: weighing EDC and NHS at a mass ratio of 5:3, and (VPGEG)72GFP solution was mixed to obtain solution B. EDC and (VPGEG)72The mass ratio of GFP was 1: 1. The purpose of this step is such that (VPGEG)72The carboxyl group of GFP is activated to facilitate the next step of chemical crosslinking with the amino group of chitosan.
The fifth step: mixing A and B according to a volume ratio of 2:1, stirring for 15min at 60 ℃ to obtain the injectable gold silver sulfide protein composite lightHot Water gel (containing 4 w% (VPGEG)72GFP, 4 w% chitosan, 0.2 w% Ag3AuS2NPs)。
Example 4: application of gold-silver sulfide protein composite hydrogel in tongue cancer model treatment
The first step is as follows: injecting tongue cancer CAL-27 cells to the root of the tongue of a nude mouse to construct a nude mouse tongue cancer model.
The second step is that: preparing the gold silver sulfide protein composite hydrogel.
The third step: and (3) injecting the silver sulfide protein composite hydrogel to the root of the tongue of the nude mouse.
The fourth step: the root of the nude mouse tongue is irradiated with near infrared light. The laser used is 808nm laser with power of 1W cm-2The irradiation time was 3 minutes.
In the invention, the gold silver sulfide protein composite hot hydrogel can be injected for photo-thermal treatment of tongue tumor, and the treatment temperature is kept above 55 ℃. Meanwhile, the gel has high biocompatibility, and can slow down the metabolism of the nanoparticles in vivo and ensure that the nanoparticles can stably exert the treatment effect.
The results are shown in FIGS. 2-7, wherein:
FIG. 2 is a shear rheological result of the injectable gold silver sulfide protein composite hydrogel of example 3, and the storage modulus (G ') is always close to but greater than the loss modulus (G') at all shear frequencies, which is sufficient to demonstrate the hydrogel properties.
Fig. 3 shows the photothermal conversion results of the injectable gold-silver sulfide protein composite hydrogel of example 3, wherein the photothermal conversion efficiency is 39%.
Fig. 4 is a graph showing the photothermal conversion effect of the injectable gold-silver sulfide protein composite hydrogel in example 3. By changing the loading concentration of the gold silver sulfide photo-thermal nanoparticles, the hydrogel can be raised to different temperatures in unit time after being irradiated by 808nm laser. In addition, since water molecules are confined in the gel skeleton, heat generation is very limited, thereby avoiding damage to adjacent non-irradiated sites during in vivo application.
FIG. 5 is a graph showing the photothermal conversion effect of the silver sulfide protein composite hydrogel injected into the root of mouse tongue in example 4. It can be seen that under the irradiation of 808nm laser, the local temperature is rapidly increased to more than 50 ℃, which is enough to achieve the effect of tumor thermal killing.
FIG. 6 shows the experiment of tumor bearing (tongue cancer) in the mouse of example 4. 50 μ L of gold silver sulfide protein composite hydrogel ((VPGEG)72GFP4 w%, chitosan 4 w%, Ag3AuS2NPs 0.2W%) were injected peritumorally into BABL/c nude mice, followed by a single laser irradiation (808nm laser, 1.0W-cm)-2For 3 minutes). Compared with a control group, the gel group has good killing effect on tongue cancer at the root of the tongue without causing local abnormal swelling. After the treatment, the tumor gradually shrinks and finally disappears completely.
FIG. 7 is the result of H & E staining of major organs of mice treated with the tongue cancer model in example 4, and it can be seen that the photothermal hydrogel did not cause significant damage to major organs of the mice.
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.
Claims (9)
1. The gold silver sulfide protein composite hydrogel comprises the following raw materials: elastin-like-green fluorescent protein composite protein, Ag3AuS2 NPs and chitosan; wherein, the elastin-like-green fluorescent protein composite protein is Ag3AuS2 The mass ratio of NPs to chitosan is 20: 1: 10;
the mass fraction of the elastin-like-green fluorescent protein composite protein is 0.1% -10%;
the Ag is3AuS2 The mass fraction of NPs is 0.1% -10%;
the mass fraction of the chitosan is 0.1% -10%;
the amino acid sequence of the elastin-like protein is (VPGEG)n、(VPGDG)n、(VPGKG)nOr (VPGRG)n，n=50~100。
2. The gold-silver sulfide protein composite hydrogel according to claim 1, wherein the chitosan is positively charged, and Ag is Ag3AuS2 NPs have positive charges, and elastin-like-green fluorescent protein complex proteins have negative charges.
3. The gold-silver sulfide protein composite hydrogel according to claim 1 or 2, wherein the elastin-like-green fluorescent protein composite protein is (VPGEG)72-GFP。
4. The gold-silver sulfide protein composite hydrogel according to claim 3, wherein (VPGEG)72-GFP 4% by weight, Ag3AuS2 The mass fraction of NPs is 0.2%, and the mass fraction of chitosan is 2%.
5. The method for preparing the gold silver sulfide protein composite hydrogel according to any one of claims 1 to 4, which is characterized by comprising the following steps:
dissolving chitosan in an acetic acid aqueous solution to obtain a chitosan solution;
dissolving the elastin-green fluorescent protein composite protein in deionized water, and then activating carboxyl of the composite protein by EDC and NHS to obtain an activated protein solution;
mixing Ag with water3AuS2 NPs are mixed with the chitosan solution and then mixed with the activated protein solution, and the mixture is stirred and reacted for 10-20 min at the temperature of 55-65 ℃ to obtain the gold-silver sulfide protein composite hydrogel.
6. The method according to claim 5, wherein the Ag is3AuS2 The preparation method of the NPs comprises the following steps:
adding HAuCl4•4H2O and AgNO3Dispersing in the mixed solution of oleic acid and oleylamine, stirring and reacting for 20h at 55 ℃, adding sulfur-containing oleylamine solution, stirring and reacting for 2h at 55 ℃, and preparing the Ag with the surface modified oleic acid oleylamine ligand3AuS2A nanoparticle;
with chloroformResuspending Ag surface-modified oleylamine oleate ligand3AuS2 Mixing the nano particles with a CTAB aqueous solution, stirring and reacting for 3 hours at 50 ℃, and then washing to obtain Ag with positive charges3AuS2 NPs。
7. The production method according to claim 5,
the volume fraction of acetic acid in the chitosan solution is 1%, and the mass fraction of chitosan is 3%;
dissolving the elastin-like-green fluorescent protein composite protein in deionized water until the mass fraction is 13%;
the Ag is3AuS2 The mass ratio of NPs to chitosan is 1: 10;
the volume ratio of the chitosan solution to the activated protein solution is 2: 1.
8. Use of the gold-silver sulfide protein composite hydrogel according to any one of claims 1 to 4 or the gold-silver sulfide protein composite hydrogel prepared by the preparation method according to any one of claims 5 to 7 in preparation of a medicament for treating tumors.
9. A photosensitizer for treating tumor, comprising the gold-silver sulfide protein composite hydrogel according to any one of claims 1 to 4 or the gold-silver sulfide protein composite hydrogel prepared by the preparation method according to any one of claims 5 to 7.
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