Photo-thermal hydrogel drug carrier as well as preparation method and application thereof

Abstract

According to the photo-thermal hydrogel drug carrier as well as the preparation method and the application thereof, artificial protein is prepared by designing a recombinant artificial protein sequence, so that the artificial protein has a good temperature response function, and a photo-thermal agent can be linked with a protein peptide chain, so that the photo-thermal hydrogel drug carrier which has the temperature response function and can be used for preparing the photo-thermal hydrogel drug carrier is obtained. The photo-thermal hydrogel which can be firmly combined with a photo-thermal agent gold nanorod can achieve synchronous carrying of macromolecular drugs and small molecular drugs and drug release of temperature response, has good biocompatibility and wide application potential, can achieve treatment of in-situ / far-end tumors of mice, and can also be applied to photo-thermal sterilization.

Description

Technical Field

[0001] The present invention relates to the technical field of preparation of hydrogel drug carriers, in particular to a photothermal water gel drug carrier and a preparation method and application thereof, and in particular to a preparation method of a photothermal water gel drug carrier that can be used for in situ / distant tumor treatment and sterilization. Background Art

[0002] Cancer has become one of the most concerned diseases in the world today due to its high incidence and high mortality rate. Common clinical treatments for cancer include surgical resection, chemotherapy, and radiotherapy. These traditional methods have shown certain limitations. For example, clinical surgery is often limited by the location of tumor growth and is highly risky. At the same time, incomplete surgical resection and intraoperative bleeding will lead to a higher tumor recurrence rate. Chemotherapy and radiotherapy have low tumor specificity, which is reflected in the patient's systemic adverse reactions. Among them, the use of chemotherapeutic drugs may also cause tumor cells to develop multidrug resistance (multi-drug resistance, MDR), which further increases the difficulty of curing cancer patients.

[0003] In recent years, on the basis of traditional therapies, new therapies such as photothermal therapy (PTT), immunotherapy and photodynamic therapy (PDT) have emerged, providing a wider and richer choice for the new generation of cancer treatment. Among them, photothermal therapy is a non-invasive, safe and tumor-specific treatment method using laser as energy, which has great potential for clinical application. By irradiating the target tumor site with active or passive targeted enriched photothermal agents, local hyperthermia with minimal damage to normal tissue can be achieved. Compared with the high-temperature ablation effect of traditional photothermal therapy (>50°C), recent studies have shown that warm photothermal therapy implemented at ~45°C can not only reduce the damage caused by high temperature to the organism, but also induce immunogenic death of tumor cells and enhance vascular permeability, thereby increasing the infiltration of immune cells in the tumor site and realizing the body's own immune response against tumor cells. However, warm photothermal therapy also has certain limitations: although near-infrared light has been widely used to implement photothermal therapy, its penetration into tissues is still limited, making photothermal therapy more suitable for treating tumors on the surface of the human body (such as skin cancer, breast cancer, oral cancer, etc.); the distal effect of photothermal therapy is weak, and it cannot target tumor metastases or other metastatic tumor cells; photothermal therapy is limited by the continuous irradiation of external light sources, and once the light source is left, it will not have a continuous killing effect on in situ tumor cells. The above limitations greatly limit the clinical application prospects of photothermal therapy, and there is an urgent need to find a new photothermal therapy system to implement more superior cancer treatment.

[0004] Cancer synergistic therapy implemented by combining multiple therapies is one of the new concepts for achieving efficient tumor treatment and has become the main development trend of cancer therapy. By integrating two or more treatment methods into the same system, the super-additive effect of "1+1>2" can be achieved. At present, a large number of studies have been devoted to the development of controllable integrated drug delivery systems to deliver small molecule drugs, macromolecular protein drugs or nucleic acid drugs to the tumor target site simultaneously, which can not only reduce the biological toxicity of free drugs to the body, but also achieve synergistic effects between different drugs, thereby achieving efficient and low-toxic cancer treatment. In order to deliver multiple drugs simultaneously, integrate multiple therapies, and implement synergistic combination therapies, it is necessary to design new drug controlled release materials for building more effective drug delivery systems. Nature provides a large number of sources of materials and design concepts for designing suitable drug controlled release systems. For example, bio-based materials from animals and plants, including proteins and polysaccharides, often have high biocompatibility and are sustainable green biomaterials. Among them, natural structural proteins often have specific amino acid sequences and ordered structures, which can be used to construct drug delivery carriers, skin care products, and wound dressings. For example, silk fibroin extracted from silkworm cocoons is one of the most studied natural fibrous proteins. It has a characteristic repeating peptide segment GAGAS, which can self-assemble to form a β-folded secondary structure. On a macro scale, it manifests itself as high mechanical strength and toughness of silk fibroin-based materials. It has been widely used in many fields such as tissue engineering, biosensors and soft robotics.

[0005] However, structural proteins lack dynamic responsiveness to external stimuli and targeting of tumor cells, and can only be obtained by extracting small amounts from natural samples. Therefore, there is a lack of controllability in drug release, which greatly limits the application of natural proteins in cancer treatment.

[0006] CN116687836A provides a photothermal hydrogel containing gold nanoparticles, but the hydrogel does not have the responsiveness to external environmental stimuli, does not have the controllability of the release of delivered drugs in the body, and is difficult to achieve a synergistic effect with photothermal therapy for cancer.

[0007] Therefore, it is necessary to update material production technology, improve and design new artificial protein drug carriers, and ultimately obtain a new generation of biomaterials that can respond to external stimuli and achieve simultaneous and controllable delivery of multiple drugs. Summary of the invention

[0008] In order to solve the above problems, the present invention provides a photothermal water gel drug carrier and a preparation method and application thereof. The artificial protein is prepared by designing a recombinant artificial protein sequence so that it has a good temperature response function and can realize the linking of the photothermal agent with the protein peptide chain, thereby obtaining a photothermal water gel with a temperature response function and capable of firmly combining with the photothermal agent gold nanorods. The photothermal water gel can realize the synchronous loading of macromolecular drugs and small molecule drugs and the temperature-responsive drug release, has good biocompatibility and wide application potential, and can not only realize the treatment of in situ / distant tumors in mice, but also can be applied to photothermal sterilization.

[0009] On the one hand, the present invention provides an artificial protein, which includes a photothermal agent-linked short peptide, a silk-like domain and an elastin-like domain, and the photothermal agent-linked short peptide, the silk-like domain and the elastin-like domain have the amino acid sequences shown in SEQ ID NOs: 1 to 3, respectively.

[0010] As a rapidly developing discipline, synthetic biology integrates and intersects with multiple disciplines, providing new biotechnology and means for the development of the next generation of silk-based biomaterials. The present invention uses synthetic biology to design a recombinant artificial protein derived from natural protein, cysteine-containing silk-elastin-like proteins (cSELPs), which mainly contains three components: a photothermal agent-linked short peptide, a silk-like domain, and an elastin-like domain. Among them, gold nanorods can form a stable Au-S bond with the thiol group on the photothermal agent-linked short peptide, thereby realizing the link between the photothermal agent and the protein peptide chain; the artificial protein contains a silk-like domain (GAGAGS) derived from silk protein, which can self-assemble to form a β-folded secondary structure based on the abundant alanine (Alanine, A), glycine (Glycine, G) and serine (Serine, S), providing a physical cross-linking site for the preparation of hydrogel drug carriers; the elastin-like domain is derived from the representative sequence of elastin (GVGVP), providing an adjustable stimulus responsiveness and chemical cross-linking site for the artificial protein. Using the enzyme cross-linking method, photothermal artificial protein hydrogels carrying photothermal agents can be prepared. They have both temperature responsiveness and a microporous structure, and can achieve simultaneous loading of macromolecular drugs and small molecule drugs and temperature-responsive drug release.

[0011] Photothermal therapy requires active or passive targeted enrichment of photothermal agents by irradiating the target tumor site to achieve local hyperthermia with minimal damage to normal tissues. However, free photothermal agents may cause a series of negative reactions in the body. The present invention enhances the binding ability of photothermal agents and peptide chains through sequence design, thereby designing a photothermal agent connecting peptide segment (SEQ ID NO: 1).

[0012] Silk fibroin material is a common structural protein, but it itself is not responsive to external environmental stimuli. Therefore, if silk fibroin is directly used to prepare drug delivery carriers, the drug lacks controllability in the body.

[0013] The present invention seamlessly connects the characteristic sequence of silk fibroin and the characteristic sequence of elastin, and the obtained silk-like elastin is stimulus-responsive. The drug delivery carrier based on silk-like elastin can respond to external stimuli, and the external stimuli can be used as a switch to accurately control the release of drugs.

[0014] The present invention uses temperature as a switch for drug release. Silk-like elastic protein undergoes a recoverable phase transition as the temperature changes. When the temperature rises, the hydrogel will squeeze out the drug and release it in large quantities, while at body temperature, the drug will be retained in the hydrogel or released slowly. The temperature change is achieved by the photothermal conversion of the photothermal agent under near-infrared light irradiation.

[0015] The drug will be released explosively during photothermal therapy, but slowly when not undergoing treatment. There are two main benefits: 1. It increases the retention time of the drug in the body, avoiding the need for frequent dosing due to instantaneous loss of the drug, and can also simultaneously reduce the side effects that free drugs may bring to the body; 2. The explosive release of the drug during light exposure can synergize with photothermal therapy to enhance the therapeutic effect; 3. When not undergoing photothermal therapy, the amount of drug released is small, which can also bring some therapeutic effects.

[0016] Furthermore, the artificial protein has an amino acid sequence as shown in any one of SEQ ID NOs: 4 to 7.

[0017] The present invention designs a variety of different artificial protein sequences, and compares their phase transition temperature differences based on the silk: elastin ratio. It also designs silk-like elastin with / without photothermal agent-linked peptide segments, in order to compare the differences in their binding abilities with gold.

[0018] Furthermore, the artificial protein has an amino acid sequence as shown in SEQ ID NO:5.

[0019] After a large number of screenings of artificial protein sequences, the present invention selects the protein sequence most suitable for preparing photothermal water gel.

[0020] On the other hand, the present invention provides a photothermal water gel carrier, which is formed by cross-linking the artificial protein and gold nanorods as described above.

[0021] In another aspect, the present invention provides a method for preparing a photothermal water gel carrier, the method comprising the following steps:

[0022] (1) preparing an artificial protein, wherein the artificial protein comprises a photothermal agent-linked short peptide, a silk-like domain, and an elastin-like domain;

[0023] (2) connecting the artificial protein with the gold nanorods to prepare an artificial protein solution carrying a photothermal agent;

[0024] (3) Artificial protein solution was cross-linked by enzyme to prepare photothermal hydrogel carrier.

[0025] The preparation method of the photothermal water gel drug carrier provided by the present invention mainly designs the amino acid sequence of artificial protein from scratch through synthetic biology, connects it with the photothermal agent after expression and purification, and then prepares it by enzyme catalytic cross-linking.

[0026] In some embodiments, the preparation method comprises the following steps:

[0027] (1) Using synthetic biology techniques to design artificial protein sequences and express the artificial protein cS2E8Y (c2Y);

[0028] (2) connecting the photothermal agent gold nanorods with the purified artificial protein to obtain a uniform c2Y@Au solution, i.e., an artificial protein solution carrying the photothermal agent;

[0029] (3) Preparation of c2Y@Au photothermal gel.

[0030] In some embodiments, the expression vector used in the synthetic biology technology described in step (1) is pET-19b3, and the expression strain is prokaryotic Escherichia coli BL21Star (DE3).

[0031] It is understandable that any expression system that can express recombinant proteins can be used as the expression scheme of the present invention.

[0032] Furthermore, the artificial protein has an amino acid sequence as shown in SEQ ID NO: 5; the preparation method of the artificial protein in step (1) comprises: designing an artificial protein sequence and expressing the artificial protein, and purifying the artificial protein by an ITC method.

[0033] Synthetic biology uses a prokaryotic expression system to insert the coding DNA of the target amino acid sequence into the multiple cloning site of the Escherichia coli plasmid, then transform the expression vector into the Escherichia coli expression strain, and use bacterial fermentation technology to cultivate and induce Escherichia coli to express the exogenous gene.

[0034] Then, the present invention uses an inverse temperature transition cycling (ITC) method to purify the protein, and simply mixes the purified protein with the photothermal agent gold nanorods to obtain a uniform photothermal artificial protein solution.

[0035] The ITC method is a protein purification scheme based on the temperature responsiveness of elastin-like peptides. Elastin-like peptides exhibit high solubility below their transition temperature (Tt), but when the temperature rises above Tt, they undergo aggregation of hydrophobic domains of hydrated polypeptide chains, which manifests as phase separation. After the elastin-like peptides undergo transformation at high temperature, the target protein can be simply collected by centrifugation or filtration. At the same time, this phase transition is highly reversible, and the collected target protein can be re-dissolved at a temperature below Tt without causing protein denaturation. Based on its simplicity and rapidity, this purification scheme has been widely used in the purification of a variety of elastin-like peptides. At the same time, the characteristic sequence of elastin peptides is used as a "control handle" to recombinantly fuse with other natural proteins, thereby giving artificial proteins temperature responsiveness and providing a simpler purification scheme for the purification of artificial proteins.

[0036] Furthermore, the connection of the artificial protein with the gold nanorods in step (2) refers to mixing the purified artificial protein with the gold nanorods from which CTAB has been washed overnight to form Au-S bonds.

[0037] The present invention can significantly increase the binding ability of artificial protein to the gold surface by introducing a photothermal agent-binding peptide segment at the front end of the artificial protein. By simply blending artificial protein and gold nanorods without surfactant, pure protein-wrapped gold nanorods, i.e., c2Y@Au aqueous solution, can be obtained. The gold nanorods are uniformly and stably dispersed in the artificial protein aqueous solution, which can maximize the reduction of the potential toxicity of free gold nanoparticles to the human body.

[0038] Furthermore, the gold nanorods described in step (2) are clean gold nanorods from which the surfactant has been washed away. The surfactant cetyltrimethylammonium bromide (CTAB), which is toxic to the human body, is often used in the synthesis of gold nanorods and the dispersion and protection of gold nanorods. Before combining the artificial protein with the gold nanorods, it is necessary to ensure that the CTAB has been washed away.

[0039] Furthermore, the enzymatic cross-linking in step (3) is: using horseradish peroxidase and hydrogen peroxide to catalyze the artificial protein solution to form a hydrogel in an enzymatic cross-linking manner.

[0040] The enzyme cross-linking method used in the present invention mainly uses horseradish peroxidase and hydrogen peroxide to catalyze free radical reactions, so that the tyrosine in the elastin domain can form a double tyrosine bond with the adjacent tyrosine. At the same time, since the silk fibroin domain can self-assemble to form a beta fold to provide physical cross-linking, under the combined action of physical and chemical cross-linking, a temperature-responsive photothermal hydrogel drug carrier with a porous structure can be obtained. The pores of the hydrogel have a high water content and can carry macromolecular drugs and small molecule drugs simultaneously.

[0041] Any scheme that can induce free radical cross-linking reaction of tyrosine to produce dityrosine bonds can be applied to the present invention.

[0042] On the other hand, the present invention provides an artificial protein for preparing a reagent for improving the temperature responsiveness of a photothermal water gel carrier for photothermal therapy and improving the binding ability with gold, wherein the artificial protein has an amino acid sequence as shown in SEQ ID NO: 5.

[0043] On the other hand, the present invention provides a use of a photothermal water gel carrier for preparing an agent for inhibiting the growth of distal and / or in situ tumors based on photothermal therapy, characterized in that the photothermal water gel carrier is cross-linked by an artificial protein and gold nanorods, and the artificial protein has an amino acid sequence as shown in SEQ ID NO: 5; the agent for inhibiting the growth of distal tumors based on photothermal therapy includes the photothermal water gel carrier and an anti-tumor drug.

[0044] The photothermal water gel carrier drug-carrying treatment system provided by the present invention can not only kill the in situ tumor, but also simultaneously inhibit the growth of distal tumors, breaking through the limitations of traditional photothermal therapy. The reason may be that the treatment system mobilizes the body's own immune response: first, photothermal therapy and chemotherapy drugs can kill a part of the in situ tumor cells and produce immunogenic death of tumor cells. In this case, tumor-associated antigens will be released, which can induce the body to produce effector toxic T lymphocytes against tumor cells, thereby inhibiting the growth of distal tumors.

[0045] Furthermore, the artificial protein is prepared by designing the amino acid sequence from scratch through synthetic biology, connecting with a photothermal agent after expression and purification, and then using an enzyme-catalyzed cross-linking method.

[0046] The photothermal water gel carrier and the preparation method thereof provided by the present invention have the following beneficial effects:

[0047] 1. Provides a design concept of artificial protein that seamlessly links photothermal agents with short peptides, natural-like proteins and elastin-like proteins, as well as a processing method for preparing hydrogels by combining artificial proteins with photothermal agents and using physical and chemical simultaneous cross-linking;

[0048] 2. Protein sequences with better temperature responsiveness and higher surface binding ability to gold were screened to prepare recombinant proteins for use in the preparation of photothermal water gel drug carriers;

[0049] 3. The prepared photothermal water gel drug carrier can simultaneously carry and controllably deliver macromolecular drugs and small molecules. Due to its excellent temperature responsiveness, it can better achieve the synergistic treatment of in situ / remote tumors and work synergistically with photothermal therapy.

[0050] 4. The prepared photothermal water gel drug carrier also has a very good in vitro bactericidal effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 This is a schematic diagram of the photothermal water gel drug carrier in Example 1;

[0052] Figure 2 is the SDS-PAGE image of different artificial proteins purified in Example 2;

[0053] Figure 3 The DSC heat flow diagrams of the three artificial proteins and c2Y@Au in Example 2, wherein the upper figure is the DSC heat flow diagrams of cY, c2Y, and c4Y, and the lower figure is the DSC heat flow diagrams of cY@Au and c2Y@Au and the statistical diagrams of the lower critical phase transition temperatures of artificial proteins of different sequences;

[0054] Figure 4 This is a QCM-D comparison diagram of c2Y and 2Y on the gold chip surface in Example 3;

[0055] Figure 5 Comparative SEM images of the cross-sections of c2Y and c2Y@Au hydrogels at 25°C in Example 4;

[0056] Figure 6 Comparative SEM images of the cross-section of the c2Y@Au hydrogel at different temperatures in Example 4;

[0057] Figure 7 The performance comparison diagram of c2Y and c2Y@Au hydrogel in Example 4, wherein the left figure is a comparison diagram of the shrinkage of c2Y and c2Y@Au hydrogel at three temperatures of 4, 37 and 45°C, and the right figure is a comparison diagram of the Fourier infrared spectra of c2Y and c2Y@Au hydrogel;

[0058] Figure 8 The in vitro photothermal conversion capability of the c2Y@Au hydrogel in Example 5 is characterized, wherein the left and middle figures are respectively the in vitro photothermal conversion capability of the c2Y@Au hydrogel with different gold nanorod contents under the same near-infrared light intensity, and the in vitro photothermal conversion capability of the c2Y@Au hydrogel with different near-infrared light intensities under the same gold nanorod content, and the right figure is the photothermal conversion capability after three ON and OFF near-infrared light irradiations;

[0059] Fig. 9 Characterization of the controlled release ability of c2Y@Au hydrogel for small molecule drug DOX in Example 6, where the left figure is the release curve of c2Y@Au hydrogel for small molecule drug DOX at different temperatures, and the right figure is the release curve of c2Y@Au hydrogel for small molecule drug DOX during three ON and OFF near-infrared light irradiation processes;

[0060] Fig.10 Characterization of the controlled release ability of the c2Y@Au hydrogel for the macromolecular drug IgG in Example 6;

[0061] Fig.11 This is a blood routine chart of BALB / C mice 10 days after the c2Y@Au hydrogel was subcutaneously injected into the body in Example 7;

[0062] Fig.12 This is a flow chart of the combined treatment of mouse orthotopic tumors by c2Y@Au hydrogel in Example 7;

[0063] Fig.13 This is a schematic diagram of the combined therapeutic effect of c2Y@Au hydrogel on mouse in situ tumors in Example 7, wherein the left figure is a schematic diagram of the effect of different injection methods on mouse tumor volume, and the right figure is a schematic diagram of the effect of different injection methods on mouse body weight.

[0064] Fig.14 This is a flow chart of the combined treatment of distal tumors in mice by c2Y@Au hydrogel in Example 7;

[0065] Fig.15 Schematic diagram of the combined therapeutic effect of c2Y@Au hydrogel on distal tumors in mice in Example 7;

[0066] Fig.16 This is a characterization of the bactericidal effect of c2Y@Au hydrogel in Example 7 on Escherichia coli in vitro. DETAILED DESCRIPTION

[0067] The preferred embodiments of the present invention are further described in detail below in conjunction with the accompanying drawings. It should be noted that the embodiments described below are intended to facilitate the understanding of the present invention and do not have any limiting effect on it. The raw materials and equipment used in the specific embodiments of the present invention are all known products and are obtained by purchasing commercially available products.

[0068] Example 1 Preparation of the photothermal water gel drug carrier provided by the present invention

[0069] The preparation method of the photothermal water gel drug carrier provided in this embodiment comprises the following steps:

[0070] a. Construction and transformation of silk-elastin-like plasmid: First, the three BanII restriction sites of pET-19b plasmid (Novagen, Inc.) were subjected to site-directed mutagenesis, and a DNA linker sequence was inserted into the multiple cloning site to construct the target plasmid pET-19b3 with a single BanII restriction site in the multiple cloning site (preparation method reference: Xia XX, Xu Q, Hu X, et al. Tunable self-assembly of genetically engineered silk-elastin-like protein polymers [J]. Biomacromolecules, 2011, 12 (11): 3844-50). The SELP monomer gene (SEQ ID NO: 8) fragment digested with BanII restriction endonuclease was reacted with T4 ligase at 16°C for 16 hours to increase the number of SELP copies in a single protein, and then the enzyme-digested pET-19b3 plasmid vector was added and ligated at 16°C for 8 hours. The obtained pET- h The SELP vector (SEQ ID NO: 9) will be transformed into the BL21Star (DE3) expression strain (purchased from Shanghai Weidi Biotechnology Co., Ltd., model EC1005S).

[0071] b. Expression of silk elastin: Add 100 mL of 2.5% liquid LB medium to a 500 mL small shake flask, add 0.1 mg / mL ampicillin solution (Amp), and inoculate the pET- h The E. coli carrying the SELP vector was cultured in an incubator at 37°C and 220 rpm for 12-16 hours to prepare a seed solution. Then, a 5L large shake flask was used to pour the seed solution into 1L 2.5% liquid LB medium containing the same concentration of Amp. After culturing in an incubator at 37°C and 250 rpm for about 2 hours, the OD value was measured. When the OD value reached 1.2-1.4, isopropylthiogalactoside (IPTG) was added to induce protein expression, and the bacteria were harvested after continuous culture for 6 hours.

[0072] c. Purification of silk-like elastic protein (ITC method): The bacterial solution collected after expression is divided into centrifuge bottles and centrifuged at 8500rpm and 4℃ for 15 minutes. Discard the supernatant and place the precipitate in a -20℃ refrigerator overnight to ensure the death of E. coli. Add 90mL of phosphate buffered saline (PBS) to the precipitate produced by each 1L of bacterial solution, and vortex until the solid matter in the bottle is completely dissolved. Add lysozyme at a ratio of 20mg of lysozyme per 100mL of PBS, and stir magnetically at room temperature for 2h. After the bacterial solution is broken by an ultrasonic instrument (40% power, work for 10 seconds and rest for 10 seconds, work for 2 minutes for every 90ml of bacterial solution), centrifuge at 8500rpm and 4℃ for 15min. The precipitate was discarded, and TN buffer (12.114 g Trizma (tris (hydroxymethyl) aminomethane) and 58.44 g sodium chloride were added to 1 L of water to dissolve, and the pH was adjusted to 8.0 with concentrated hydrochloric acid) was added to the supernatant according to the volume ratio of PBS solution: TN buffer solution of 1:1, and then the solution was water bathed at 70°C for 4 hours. After the solution was centrifuged at 5000 rpm and 40°C for 3 minutes, the supernatant was discarded. The weight of the precipitate was weighed, and 40 mL of deionized water was used to dissolve every 7 g of the precipitate, and stirred in a refrigerator at 4°C overnight. After the turbid solution was fully dissolved, it was centrifuged at 8500 rpm and 4°C for 15 minutes, and the precipitate was discarded. The supernatant was dialyzed in a refrigerator at 4°C for 24 hours, and the water was changed every hour in the initial stage. The protein solution obtained after centrifugation and dialysis at 8500 rpm and 4°C was a finished protein solution, and the solution was freeze-dried for 72 hours for long-term storage at room temperature.

[0073] d. Linking of artificial protein and photothermal agent gold nanorods: CTAB-stabilized gold nanorods were synthesized by seedless growth method. 5 mL HAuCl4·3H2O (1.0 mM) was mixed with 5 mL CTAB (0.2 M) at room temperature, and then 250 μl AgNO3 (4.0 mM) and 8 μl HCl (12.1 M) were added, followed by 70 μl L (+)-ascorbic acid, and the solution was gently shaken. After the solution became clear, 15 μl pre-cooled NaBH4 (0.01 M) was immediately added. The solution was placed in a water bath at 25°C overnight (synthesis method reference: Ali MRK, Snyder B, El-Sayed MA. Synthesis and Optical Properties of Small Au Nanorods Using a Seedless Growth Technique [J]. Langmuir, 2012, 28 (25): 9807-9815.). 2 mL, 5 mL and 10 mL of CTAB@Au (CTAB-stabilized gold nanorods) were taken, centrifuged at 14000 rpm for 20 min, washed twice with deionized water, and then mixed with 100 μL of c2Y protein solution to prepare c2Y@Au solution.

[0074] e. Preparation of photothermal water gel drug carrier: 12 μL 40 mg mL -1 HRP (horseradish peroxidase) solution was added to 200 μL of 5wt% c2Y@Au solution. Then, 0.4 μL of 30wt% H2O2 solution was added to the mixture to start crosslinking. The protein:HRP:H2O2 mixture was left at room temperature for 1 hour to form a stable enzyme-crosslinked hydrogel.

[0075] The preparation of photothermal hydrogels carrying macromolecular / small molecule drugs can be achieved by directly adding the corresponding drugs into the protein:HRP:H2O2 mixture solution (flow chart as shown in the figure). Figure 1 shown).

[0076] Example 2 Effect of sequence design on the properties of photothermal water gel drug carrier

[0077] In this example, four different artificial protein sequences cY (SEQ ID NO: 4), c2Y (SEQ ID NO: 5), c4Y (SEQ ID NO: 6) and 2Y (SEQ ID NO: 7) were designed respectively. They were prepared and purified according to the method provided in Example 1. The SDS-PAGE results of the four artificial proteins cY, c2Y, c4Y and 2Y obtained after purification are as follows: Figure 2 shown.

[0078] After step c, the freeze-dried protein was dissolved back to compare the effect of sequence design on the properties of the photothermal hydrogel drug carrier. Among them, cY, c2Y and c4Y have different ratios of silk domain to elastin domain (1:8, 1:4 and 1:2).

[0079] First, by using nano-DSC (differential scanning calorimetry) tests, we compared the three different sequence designs of cY, c2Y and c4Y to see if they would have a significant effect on the temperature responsiveness of artificial proteins. The test results are as follows: Figure 3 As shown by Figure 3It can be seen that c2Y@Au protein is used for subsequent experiments because it is temperature responsive and its phase transition temperature is between room temperature 25°C and body temperature 37°C, which is the best choice after comprehensive consideration (during the heating and cooling process, the heat absorption and release of the protein indicates that the protein undergoes a recoverable phase transition. It can be found that c4Y has basically no temperature response, and the phase transition temperature is higher than 37°C, so it is excluded. After combining cY and c2Y with gold nanorods respectively and testing the temperature responsiveness, it can be found that although cY@Au has the strongest temperature response, that is, the highest peak appears, its phase transition temperature is too far from 37°C, even lower than room temperature, and cannot remain stable at room temperature; c2Y@Au also has relatively obvious temperature responsiveness, and its phase transition temperature is higher than cY@Au, falling between room temperature 25°C and body temperature 37°C. Finally, c2Y@Au is selected comprehensively). Therefore, c2Y protein and gold nanorods are finally selected to be blended, and the obtained c2Y@Au can remain stable at room temperature, and in vivo due to temperature responsiveness, stimulate the continuous release of drugs.

[0080] Example 3 Effect of silk-like elastin with / without photothermal agent linked peptide on the properties of photothermal water gel drug carrier 2Y does not contain a photothermal agent-binding short peptide (silk domain: elastin domain = 1:4), c2Y contains a photothermal agent-binding short peptide (silk domain: elastin domain = 1:4).

[0081] The two different sequences, c2Y and 2Y, were compared, and the binding ability of the two artificial proteins on the gold chip surface was tested using QCM-D (dissipative quartz crystal microbalance). The test results are as follows: Figure 4 As shown. Figure 4 It can be seen that c2Y significantly improves the surface binding ability of artificial protein to gold compared with 2Y (the lower the frequency in the figure, the higher the binding ability), which proves that the introduction of photothermal agent binding peptide significantly increases the surface binding ability of artificial protein to gold.

[0082] Example 4 Preparation of porous hydrogel using physical and chemical simultaneous crosslinking

[0083] In this example, c2Y protein solution and c2Y@Au solution were taken respectively. After the c2Y group was purified according to the method provided in Example 1, horseradish peroxidase and hydrogen peroxide in the same ratio were added after step c to catalyze the formation of hydrogel; c2Y@Au group was prepared according to the method provided in Example 1, and horseradish peroxidase and hydrogen peroxide in the same ratio were added after step d to catalyze the formation of hydrogel. The microscopic morphology of the hydrogel was observed using a scanning electron microscope ( Figure 5), and found that both c2Y and c2Y@Au hydrogels have cross-linked porous networks at 25°C, indicating that after adding gold nanorods to the hydrogel, the same cross-linking method can still form a porous network structure, which is suitable for drug delivery. At the same time, based on the temperature responsiveness of artificial proteins, c2Y@Au hydrogels show significantly changed pore sizes at different temperatures ( Figure 6 ), indicating that c2Y@Au hydrogel has very good temperature responsiveness.

[0084] The weight changes of c2Y and c2Y@Au hydrogels at three temperatures (4, 37, and 45 °C) were detected. Figure 7 Left), it can be found that with the increase of temperature, both c2Y and c2Y@Au hydrogels shrink and squeeze out the contained water, and the difference in shrinkage between the two is very small, proving that the addition of gold nanorods has little effect on the drug delivery of hydrogels at high temperatures). Furthermore, the Fourier transform infrared spectra of c2Y and c2Y@Au hydrogels ( Figure 7 The right figure shows that with the addition of gold nanorods, c2Y@Au hydrogel forms more β-fold physical crosslinks compared to c2Y hydrogel, indicating that the addition of gold nanorods changes the hydrogel system, forming a denser crosslinked network, which is also consistent with Figure 5 The difference in pore size corresponds to the

[0085] Example 5 Photothermal conversion capability of photothermal water gel drug carrier

[0086] In this example, the method provided in Example 1 was used to prepare a photothermal hydrogel drug carrier. First, different contents (0.0168wt%, 0.0406wt%, 0.0610wt%) of gold nanorods were added after step c to characterize the effect of the content of gold nanorods on the photothermal conversion ability of the photothermal hydrogel. It can be seen that under the same near-infrared light irradiation time, the photothermal conversion ability of the hydrogel is positively correlated with the content of gold nanorods ( Figure 8 (left figure), and considering the need to ensure that the hydrogel can reach the target temperature (45°C) in the body, it is also necessary to reduce the amount of gold nanorods added as much as possible, so the most preferred concentration of gold nanorods in the system is 0.0406wt%.

[0087] Furthermore, by comparing the light intensity at different levels (0.3 W cm -2 , 0.5W cm -2 , 0.6W cm -2 、0.9W cm -2 ) Photothermal conversion capacity of hydrogel containing 0.0406wt% gold nanorods ( Figure 8 The optimal near-infrared light intensity is 0.9 W cm -2.

[0088] The c2Y@Au hydrogel was irradiated with three ON and OFF near-infrared light ( Figure 8 (right image) It was observed that the hydrogel still had stable photothermal conversion ability after multiple irradiation cycles.

[0089] Example 6: Ability of photothermal water gel drug carrier to control drug release

[0090] This example uses the method provided in Example 1 to prepare a photothermal hydrogel drug carrier. First, after step d, a small molecule drug doxorubicin hydrochloride (DOX) and a macromolecule drug derived from human serum IgG protein are added respectively. The same proportion of horseradish peroxidase and hydrogen peroxide are added to both groups to catalyze the formation of hydrogels. The purpose is to compare the controlled release ability of the photothermal hydrogel drug carrier for macromolecule drugs and small molecule drugs. Fig. 9 As shown in Figure 2, the release curve of the small molecule drug DOX from c2Y@Au hydrogel is positively correlated with the ambient temperature. Under the excitation of near-infrared light, the release of the drug can be greatly accelerated (see Fig. 9 In the right figure, ON means turning on the near-infrared light, and the drug release rate is significantly increased), which indicates that c2Y@Au hydrogel has a near-infrared light-stimulated controlled release effect on small molecule drugs. Fig.10 As shown in the figure, the release of macromolecular drug IgG by c2Y@Au hydrogel also showed a release curve related to ambient temperature. In summary, c2Y@Au hydrogel has good temperature-responsive sustained release ability for both macromolecular drugs and small molecule drugs.

[0091] Example 7 Use of the photothermal water gel drug carrier obtained under optimal conditions for in situ / remote tumor treatment and sterilization

[0092] This example uses the method provided in Example 1 to prepare the photothermal hydrogel drug carrier c2Y@Au hydrogel. Fig.11 As shown in the figure, the blood routine test of BALB / C mice 10 days after subcutaneous injection of c2Y@Au hydrogel shows that the artificial protein photothermal hydrogel has good biocompatibility for mice. The orthotopic 4T1 tumor model of triple-negative breast cancer was used to verify the therapeutic potential of this patented photothermal hydrogel drug carrier in a multimodal treatment system. Photothermal therapy, chemotherapy and immunotherapy were selected to design a three-combination treatment system. The small molecule chemotherapy drug DOX and the macromolecular drug anti-mouse PD-L1 (aPD-L1, immune checkpoint inhibitor) were loaded into the c2Y@Au hydrogel and injected in situ into the tumor site of the mouse (see the flowchart). Fig.12) to quickly form an in situ hydrogel drug library, and seven groups of control experiments were conducted at the same time: 1. Direct injection of PBS buffer; 2. Direct injection of PBS+NIR (PBS buffer + near-infrared light irradiation); 3. Injection of c2Y@Au hydrogel; 4. Injection of c2Y@Au hydrogel + near-infrared light irradiation; 5. Direct injection of DOX+aPD-L1; 6. Injection of c2Y@Au hydrogel carrying DOX+aPD-L1; 7. Injection of c2Y@Au hydrogel carrying DOX+aPD-L1+near-infrared light irradiation. The effects of different treatment methods on the tumor volume of mice were investigated ( Fig.13 The effect of different treatments on the body weight of mice ( Fig.13 (right image) by Fig.13 It can be seen that the c2Y@Au / DOX / aPD-L1+NIR triple combination treatment group showed the most significant tumor inhibition, indicating the effectiveness of the c2Y@Au hydrogel drug delivery system and the effectiveness of the triple combination synergistic treatment.

[0093] Furthermore, this example also investigated the therapeutic effect of the c2Y@Au / DOX / aPD-L1+NIR triple combined treatment system on distal tumors in mice ( Fig.15 ), the flowchart is shown in Fig.14 .like Fig.15 As shown in the figure, the c2Y@Au / DOX / aPD-L1+NIR triple combined treatment system can not only kill the in situ tumor, but also simultaneously inhibit the growth of the distal tumor, breaking through the limitations of traditional photothermal therapy. The main reason is that the c2Y@Au / DOX / aPD-L1+NIR triple combined treatment system mobilizes the body's own immune response: first, photothermal therapy and chemotherapy drugs can kill a part of the in situ tumor cells and produce immunogenic death of tumor cells. In this case, tumor-associated antigens will be released, which can induce the body to produce effector toxic T lymphocytes against tumor cells.

[0094] In addition to in situ / distant tumor treatment in vivo, the photothermal water gel of the present invention can also be used for photothermal sterilization. Fig.16 As shown in the figure, after incubation with c2Y@Au hydrogel for 2 hours, the survival rate of Escherichia coli dropped significantly to 0.03%. At the same time, no bacterial colonies were observed in the c2Y@Au hydrogel group and the ampicillin group after near-infrared irradiation for 30 minutes. It can be seen that even without the drug, c2Y@Au hydrogel can achieve a very good photothermal sterilization effect.

[0095] Although the present invention is disclosed as above, the present invention is not limited thereto. For example, it can be expanded according to its application range in medicine. Any person skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the scope defined by the claims.

Claims

1. An artificial protein, characterized in that It comprises a photothermal agent-connected short peptide, a silk-like domain and an elastin-like domain, wherein the photothermal agent-connected short peptide, the silk-like domain and the elastin-like domain respectively have amino acid sequences shown in SEQ ID NOs: 1 to 3.

2. The artificial protein according to claim 1, characterized in that The artificial protein has an amino acid sequence as shown in any one of SEQ ID NOs: 4-7.

3. The artificial protein according to claim 2, characterized in that The artificial protein has an amino acid sequence as shown in SEQ ID NO:

5.

4. A photothermal water gel carrier, characterized in that: It is formed by cross-linking an artificial protein and gold nanorods, and the artificial protein is as described in any one of claims 1 to 3.

5. A method for preparing a photothermal water gel carrier, characterized in that: The following steps are involved: (1) preparing an artificial protein, wherein the artificial protein comprises a photothermal agent-linked short peptide, a silk-like domain, and an elastin-like domain; (2) connecting the artificial protein with the gold nanorods to prepare an artificial protein solution carrying a photothermal agent; (3) Artificial protein solution was cross-linked by enzyme to prepare photothermal hydrogel carrier.

6. The preparation method according to claim 5, characterized in that: The artificial protein has an amino acid sequence as shown in SEQ ID NO: 5; the preparation method of the artificial protein in step (1) comprises: designing an artificial protein sequence and expressing the artificial protein, and purifying the artificial protein by an ITC method.

7. The preparation method according to claim 6, characterized in that: The step (2) of connecting the artificial protein with the gold nanorods refers to mixing the purified artificial protein with the gold nanorods from which the CTAB has been washed overnight to form Au-S bonds.

8. The preparation method according to claim 7, characterized in that: The enzymatic cross-linking in step (3) is to use horseradish peroxidase and hydrogen peroxide to catalyze the artificial protein solution to form a hydrogel in an enzymatic cross-linking manner.

9. Use of an artificial protein for preparing a reagent for improving the temperature responsiveness of a photothermal water gel carrier for photothermal therapy and improving its binding ability with gold, characterized in that: The artificial protein has an amino acid sequence as shown in SEQ ID NO:

5.

10. A use of a photothermal water gel carrier for preparing an agent for inhibiting the growth of distal tumors based on photothermal therapy, characterized in that: The photothermal water gel carrier is formed by cross-linking artificial protein and gold nanorods, and the artificial protein has an amino acid sequence as shown in SEQ ID NO: 5; the reagent for inhibiting the growth of distal tumors based on photothermal therapy includes the photothermal water gel carrier and an anti-tumor drug.

Images