CN114939164B

CN114939164B - Hydrogel for photothermal treatment and preparation method and application thereof

Info

Publication number: CN114939164B
Application number: CN202210509900.3A
Authority: CN
Inventors: 刘江; 胡小佩; 赵行; 周蓉卉; 但红霞
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2022-05-11
Filing date: 2022-05-11
Publication date: 2023-08-04
Anticipated expiration: 2042-05-11
Also published as: CN114939164A

Abstract

The invention relates to a hydrogel for photothermal treatment, and a preparation method and application thereof, and belongs to the field of hydrogels. The invention takes guanosine and polydopamine-gold nanoparticles as raw materials, and builds the supermolecular hydrogel integrating the photo-thermal effect and the carrier by introducing dynamic boron ester bonds. The supermolecule hydrogel provided by the invention has good mechanical property, self-repairing property and injectability, and has the advantages of small toxic and side effects and good in vivo biocompatibility. The supermolecular hydrogel overcomes the defect that the traditional physical coating type hydrogel delivery system is easy to cause abrupt release, has excellent slow release effect, has excellent photo-thermal anti-tumor activity in vivo and in vitro, and has wide application prospect in preparing hydrogel biological materials and medicaments for photo-thermal treatment.

Description

Hydrogel for photothermal treatment and preparation method and application thereof

Technical Field

The invention belongs to the field of hydrogels, and particularly relates to a hydrogel for photothermal treatment, and a preparation method and application thereof.

Background

Squamous cell carcinoma of oral cavity (Oral squamous cell carcinoma, OSCC), abbreviated as squamous cell carcinoma of oral cavity, is the most common malignant tumor of orofacial, has the characteristics of shallow occurrence position, clear tumor typing and easy observation, and the incidence rate and death rate rise year by year, and postoperative recurrence and metastasis are easy, and the survival and prognosis of patients are seriously affected. Clinically, the treatment mode adopted at present is local chemical drug treatment, and the treatment method mainly enriches the drugs in tumor parts through administration modes such as external application, intratumoral injection and the like, so that the anti-tumor effect of the drugs can be effectively exerted, and the adverse reaction of the whole body is reduced. However, the traditional chemotherapeutic drugs have poor stability and rapid metabolism in vivo, which results in multiple administration and large administration dosage, and simultaneously reduces the bioavailability of the chemotherapeutic drugs, thus having low therapeutic effect. Thus, there is a need to find new and effective treatments.

Photothermal therapy (Photothermal therapy, PTT) is an emerging approach to treat superficial tumors by injecting it into the interior of the human body using materials with high photothermal conversion efficiency, focusing it near the tumor tissue using targeted recognition techniques, and converting the light energy into heat energy under irradiation from an external light source to kill cancer cells. Photothermal therapy has received much attention in tumor therapy due to advantages such as noninvasive tumor ablation and large tissue penetration depth. Polydopamine-gold nanoparticles (PDA-AuNPs) have attracted considerable attention as a known photothermal agent in the field of photothermal treatment of tumors. PDA-AuNPs are usually applied in the form of injection, but because PDA-AuNPs are easy to disperse, local injection is easy to spread to non-tumor parts after being in vivo, the administration frequency is increased, intravenous injection is easy to gather in surrounding normal tissues, obvious toxic and side effects are generated, tumor cells cannot be completely removed, and the photo-thermal treatment efficiency is reduced.

In recent years, hydrogel-based delivery systems have provided a new strategy for efficient delivery of photothermal agents due to high drug loading and targeted accumulation properties. The Chinese patent application with the application number of 202010420945.4 discloses a chiral supramolecular nucleoside hydrogel based on a boron ester bond, a preparation method and application thereof, wherein the supramolecular hydrogel is obtained by mixing guanosine and borate serving as raw materials in a solvent. The supermolecule hydrogel has excellent stability, injectability and self-repairing property, and simultaneously has good biocompatibility, and does not show obvious acute toxicity in animal bodies. However, in one aspect, this application discloses only the use of the supramolecular hydrogels as scaffold materials in the field of tissue engineering, and does not disclose that the hydrogels can be used as photothermal therapy delivery systems; on the other hand, even if the supramolecular hydrogel is used as a carrier to encapsulate a photothermal agent for photothermal treatment, the photothermal agent is liable to be suddenly released in vivo, resulting in poor photothermal treatment effect. Therefore, development of a photothermal therapeutic agent having excellent therapeutic effects on superficial tumors including OSCC with little toxic and side effects is highly demanded.

Disclosure of Invention

The invention aims to provide hydrogel for photothermal treatment with excellent treatment effect and low toxic and side effects, and a preparation method and application thereof.

The invention provides a hydrogel for photothermal therapy, which is prepared from a photothermal agent, inorganic base and B (OH) ₃ Guanosine and an aqueous solvent as raw materials; wherein, the photo-thermal agent, inorganic alkali and B (OH) ₃ The molar ratio of (2-72) is 1:1, and the ratio of the photothermal agent to guanosine is (0.6-0.9) mol (3.5-14) mg.

Further, the photothermal agent is polydopamine-gold nanoparticles; and/or, the guanosine is D-guanosine.

Further, the inorganic base is NaOH; and/or the aqueous solvent is water or an aqueous buffer solution, and the pH value of the aqueous buffer solution is preferably 7.35-7.45.

Further, the aqueous buffer is a PBS buffer.

Further, the photothermal agent, inorganic base, B (OH) ₃ The molar ratio of the photo-thermal agent to the guanosine is (12-32) 1:1, and the ratio of the photo-thermal agent to the guanosine is (0.6-0.8) mol (7-14) mg.

Further, the concentration of guanosine is 7-28mg/mL.

Further, the concentration of guanosine is 14-28mg/mL.

Further, the hydrogel contains a boric acid diester bond.

The invention also provides a method for preparing the hydrogel for photothermal treatment, which is characterized by comprising the following steps: the method comprises the following steps: photo-thermal agent, inorganic base, B (OH) ₃ Mixing in aqueous solvent, adding guanosine, heating for dissolving, and cooling.

The invention also provides application of the hydrogel in preparation of biological materials or medicines for photothermal treatment.

Further, the biological material or the medicine is the biological material or the medicine for preventing and/or treating superficial tumors.

Further, the superficial tumor is oral squamous cell carcinoma.

The invention takes guanosine and polydopamine-gold nanoparticles as raw materials, and builds a stable supermolecular hydrogel integrating the photo-thermal effect and the carrier in a chemical bonding mode by introducing dynamic boron ester bonds and controlling the specific proportion and concentration of the raw materials.

The supermolecule hydrogel provided by the invention has good mechanical properties, self-repairing property and injectability.

The supramolecular hydrogel provided by the invention has good light stability, and no obvious temperature drop is observed through three laser switch cycle tests.

The supermolecular hydrogel provided by the invention has good in vivo biocompatibility in vivo, no obvious inflammatory reaction is found at an injection site, and the toxic and side effects are small.

The supermolecular hydrogel is constructed in a chemical bonding mode, overcomes the defect that the traditional physical coating type hydrogel delivery system is easy to cause abrupt release, has excellent slow release effect, and has excellent photo-thermal anti-tumor activity in vivo and in vitro. Animal experiments prove that the supermolecular hydrogel can exert excellent in vivo photo-thermal anti-OSCC tumor activity when being used in an OSCC transplantation tumor model. The supermolecule hydrogel has wide application prospect in preparing hydrogel biological materials and medicines for photothermal treatment.

The method for preparing the supermolecule hydrogel is simple, safe and nontoxic, and is suitable for expanded production.

It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.

Drawings

Figure 1 structural characterization of gbpa hydrogels. a) Photographs of GBPA hydrogels at different concentrations (concentration: 0.35% w/v, 0.7% w/v, 1.05% w/v, 1.4% w/v and 2.8% w/v); b) 1.4% w/v GBPA hydrogel lyophilized samples, naB (OH) ₄ Solution and B (OH) ₃ Of powders ¹¹ B NMR spectrum (solvent: D) ₂ O); c) CD profile of 0.7% w/v GBPA hydrogel; d) PXRD pattern of 1.4% w/v GBPA hydrogel lyophilized sample; e) AFM image of 2.8% w/v GBPA hydrogel diluted solution (scale bar: FIG. 1 is 2 μm, FIG. 2 is 1 μm) and SEM images of 1.4% w/v GBPA hydrogel lyophilized samples (scale bar: FIG. 1 is 100 μm and FIG. 2 is 200 μm).

Figure 2 mechanical properties and injectability of gbpa hydrogels. a) Rheology detection of storage modulus G' and loss modulus g″ of GBPA hydrogels; b) Rheology detection of storage modulus G' and loss modulus g″ of GBPA hydrogels under shear stress changes; c) Rheology detection GBPA hydrogel self-healing; d) Rheologically analyzing the relationship between GBPA hydrogel viscosity and shear rate; e) Injection experiments verify the self-healing and injectability of GBPA hydrogels.

Figure 3 photo-thermal effect of gbpa hydrogel. a) G, PDA-AuNPs-20 solution and GBPA hydrogel temperature profile; b) Temperature change curves of GBPA hydrogels with different PDA-AuNPs-20 absorbance; c) Temperature change curves of GBPA hydrogel under irradiation of different laser power densities; d) Temperature change curve of GBPA hydrogel after three laser on/off cycles; e) Infrared thermogram of GBPA hydrogels; scale bar: 1cm.

Figure 4. In vitro photothermal anti-OSCC cell activity of gbpa hydrogels. The effect of GBPA hydrogels on Cal-27, UM1, HSC-3 and HSC-4 cell activity was tested by a live dead cell fluorescent staining assay; scale bar: 750 μm.

Figure 5 in vivo degradability and biocompatibility of gbpa hydrogels. a) Subcutaneous injection of 100 μlgbpa hydrogel and H in mice ₂ After O, stopping experiment at a specific time point, and observing gel degradation condition by using materials; b) H&E staining to observe histopathological conditions of the GBPA hydrogel on important organs (heart, liver, spleen, lung and kidney) of the mice at different time points; scale bar: 500 μm.

FIG. 6 GBPA hydrogel photo-thermal inhibits the growth of OSCC cell Cal-27 graft tumor. a) Schematic flow chart of animal experiment; b) Cal-27 tumor growth curves for each treatment group; c) Body weight change curves for mice of each treatment group; d) At 16 days, tumor visual images of each treatment group; e) H & E staining patterns of vital organs (heart, liver, spleen, lung and kidney) of mice for each treatment group at day 16; scale bar: 300 μm. Results are expressed as mean ± standard deviation. P <0.05, (< P <0.01, (< P < 0.001), n=5.

Figure 7. Potential mechanisms of gbpa hydrogel photothermal inhibition of OSSC graft tumor growth. a) H & E staining, TUNEL immunofluorescence, immunohistochemical staining to examine the ability of GBPA hydrogel to induce apoptosis and inhibit proliferation in OSCC tumor tissue; scale bar: FIG. 1 is 300 μm and FIGS. 2-4 are 200 μm; b-d) expression rate of apoptotic cells, ki67 and Caspase 3 positive cells in OSCC tumor tissue. Results are expressed as mean ± standard deviation. P <0.05, (< P <0.01, (< P < 0.001), n=3.

Detailed Description

The raw materials and equipment used in the invention are all known products and are obtained by purchasing commercial products.

The guanosine (G) used in examples and experimental examples was purchased from SIGMA-Aldrich, U.S.A., as D-guanosine.

The PBS buffer used in examples and experimental examples had a pH in the range of 7.35 to 7.45.

Referring to the methods reported in the prior art ([ 1] Bast U.S. NG, comen J, puntes V.kinetically controlled seeded growth synthesis of citratestabilized gold nanoparticles of up to 200nm:size focusing versus Ostwald ripening.Langmuir,2011,27:11098-11105; [2]Wu YH,Wang HB,Gao F,et al.An injectable supramolecularpolymer nanocomposite hydrogel for prevention of breast cancer recurrence with theranostic andmammoplastic functions.Adv Funct Mater,2018,28:1801000 ]), polydopamine-gold nanoparticles (PDA-AuNPs) were prepared as follows:

first, 0.097g of sodium citrate was weighed and dissolved in 150mL of triple distilled water to prepare a sodium citrate solution with a concentration of 2.2mM, which was heated to boiling. Then, 1mL of HAuCl was added ₄ (25 mM) solution, the color of the solution changed to pink within 10-15 min, indicating successful synthesis of gold seeds (. About.10 nm). After this time, the reaction was stopped and cooled to 90 ℃. Next, 1mL of sodium citrate (60 mM) and HAuCl were added sequentially (at intervals of 2 min) ₄ (25 mM) solution and stirred for 30min. Then, the above procedure was repeated three times to obtain AuNPs (-20 nm).

And (3) selecting AuNPs with the particle size of 20nm, centrifuging (9000 rpm,20 min), adding the obtained precipitate into a Dopamine (DA) solution (1 mg/mL, tris-HCl pH 8.5), vigorously stirring for 20min, centrifuging, and washing twice to obtain polydopamine-gold nanoparticles, namely PDA-AuNPs-20.

EXAMPLE 1 preparation of 0.7% w/v GBPA hydrogels of the invention

The vial was filled with aqueous PDA-AuNPs (2M), followed by aqueous NaOH (0.5M) and B (OH) respectively ₃ The aqueous solution (the concentration is 0.5M) is fully and uniformly mixed, and then is kept stand at room temperature for 5 to 10 minutes to obtain light black transparent liquid, and guanosine is weighed and added into the liquid, heated to 100 ℃ for fully dissolving, and cooled at room temperature to obtain the GBPA hydrogel with the concentration of 0.7%w/v. PDA-AuNP aqueous solution volume, naOH aqueous solution volume, B (OH) ₃ The volume of the aqueous solution and the mass of guanosine are shown in Table 1.

EXAMPLE 2 preparation of 1.05% w/v GBPA hydrogels of the invention

Referring to the preparation method of example 1, 1.05% w/vGBPA hydrogels of the invention were prepared according to the dosing amounts shown in Table 1.

EXAMPLE 3 preparation of 1.4% w/v GBPA hydrogels of the invention

Referring to the preparation method of example 1, 1.4% w/vGBPA hydrogels of the invention were prepared according to the dosing amounts shown in Table 1.

EXAMPLE 4 preparation of 2.8% w/v GBPA hydrogels of the invention

Referring to the preparation method of example 1, 2.8% w/vGBPA hydrogels of the invention were prepared according to the dosing amounts shown in Table 1.

TABLE 1 charging and naming of GBPA hydrogels of examples 1-4 and comparative example 1

The following is a control hydrogel preparation method:

comparative example 1 preparation of control hydrogels

The preparation method of reference example 1 was different only in that the amount of guanosine to be charged was changed to 1.75mg, and a control hydrogel was prepared: 0.35% w/v GBPA hydrogel.

The following experiments prove the beneficial effects of the invention.

Experimental example 1 structural characterization of hydrogels

1. Experimental method

The vials of examples 1-4 and comparative example 1 after hydrogel preparation were inverted and observed.

The GBPA hydrogels prepared in the examples are taken and respectively subjected to ¹¹ B NMR (solvent: D) ₂ O) characterization, circular Dichroism (CD) characterization, powder X-ray diffraction analysis (PXRD), atomic Force Microscope (AFM) characterization, scanning Electron Microscope (SEM) characterization.

2. Experimental results

FIG. 1a shows that sample vials at 0.7% w/v and above form stable hydrogels after inversion, whereas sample vials at 0.35% w/v do not form hydrogels after inversion, indicating a minimum gel formation concentration of GBPA hydrogels of 0.7% w/v.

As shown in FIG. 1B, B (OH) ₃ And NaB (OH) ₄ Peaks at 19.37 and 2.52ppm, respectively, while the GBPA hydrogel peaked at 6.90ppm, indicating the formation of a boronic acid diester bond in the GBPA hydrogel. The circular dichroism spectrum (Circular dichroism, CD) shows two positive absorption peaks at 220 and 339nm and one negative absorption peak at 247nm, indicating that GBPA hydrogels may have G tetramers present and they are stacked in a head-to-head and head-to-tail fashion (fig. 1 c). As shown in fig. 1d, a lyophilized sample of GBPA hydrogel was prepared at 2θ≡27.0 ° (d=3.3 °) And 2θ≡6.2 ° (d=14.2+.>) One peak was shown for each, further indicating that the GBPA hydrogel was formed from G tetramers by pi-pi stacking. Scanning electron microscopy and atomic force microscopy showed that the hydrogel exhibited a loosely porous, fragmented structure (fig. 1 e).

Experimental example 2 mechanical Properties and injectability of GBPA hydrogels

1. Experimental method

Sample to be tested: 1.4% w/v GBPA hydrogel.

The testing method comprises the following steps: 1.4% w/v GBPA hydrogel is taken, 1mL of the GBPA hydrogel is taken when the heated gel is in a liquid state, and is dripped on a parallel plate of a rheometer (Anton Paar MCR 302, germany) preheated to 80 ℃, a plate size of 50mm and a measuring gap of 1mm are selected, then excessive liquid overflowing from the edge of the plate is gently removed, and after parameters are set, a rheological experiment is started when the temperature of the parallel plate is reduced to 25 ℃.

2. Experimental results

Rheological experiments evaluate the viscoelasticity of GBPA hydrogels, and the results show that the storage modulus G' is much greater than the loss modulus G ", indicating that the samples are in the gel state with good solid-like mechanical properties (fig. 2 a). FIG. 2b shows that G' is greater than G "when the strain is less than 5.6%, GBPA is in the gel state; on the contrary, GBPA is in a sol state, and the GBPA hydrogel is proved to show good gel-sol transition behavior. Sample feedingThe high strain-low strain alternating behavior, G' and G "of the samples were essentially unaffected, and could be restored to the original state, indicating that GBPA hydrogels had good self-healing properties and rapid gel-sol and sol-gel transition behavior (fig. 2 c). Meanwhile, fig. 2d shows that the viscosity of the sample gradually decreases with increasing shear rate, indicating that GBPA hydrogels have shear thinning properties. Finally, the GBPA solution is sucked by a syringe, different letters can be written on the glass plate after the gel is formed, and H is injected at the same time ₂ The gel state was still present in O, further confirming that GBPA hydrogels have good shear thinning properties and injectability (fig. 2 e).

The results show that the GBPA hydrogel of the invention has good mechanical properties, self-repairing property and injectability.

Experimental example 3, photo-thermal Effect of GBPA hydrogel and in vitro photo-thermal anti-tumor Activity

1. Experimental method

Sample to be tested: 1.4% w/v GBPA hydrogel.

The testing method comprises the following steps: first, a 808nm laser (LWIRL 808-5W-F IRL 18090311) was used to set 2W cm ^-2 For 10min, for H ₂ The temperature changes of O, auNPs, PDA-AuNPs, guanosine aqueous solution and GBPA hydrogels were tested and recorded. GBPA hydrogels with different PDA-AuNPs absorbance (abs= 0.5,1,1.5,2,3,4) were then selected and set at different power densities (0.5, 1,1.5,2w cm) using an 808nm laser ^-2 ) The temperature change of the GBPA hydrogel was tested and recorded by irradiation for 10 min. In addition, set 2W cm ^-2 The temperature change of the GBPA hydrogel was tested and recorded by laser on/off three cycles (20 min each) to characterize the photostability of the GBPA hydrogel. Finally, a 808nm laser instrument is combined with a thermal infrared imager (LS 13D 2-0622) to set 2W cm ^-2 For 10min, for H ₂ The temperature changes of the O and GBPA hydrogels were tested and recorded with photographs.

2. Experimental results

To evaluate the photothermal effect of GBPA hydrogels, laser irradiated strips were selectedPiece (806 nm,2W cm) ^-2 ) Testing was performed. First, photothermal testing was performed on the constituent components of the GBPA hydrogel, and when the same concentration of PDA-AuNPs-20 was assembled into the hydrogel, the temperature of the GBPA hydrogel did not decrease, indicating that the GBPA hydrogel could maintain the photothermal effect of the PDA-AuNPs (fig. 3 a). As shown in FIGS. 3b, c, the temperature change of GBPA hydrogel is related to the concentration and the power of the laser, and as the PDA-AuNPs-20 concentration increases from 0.5 to 4, the power increases from 0.5 to 2W cm ^-2 The temperature of GBPA hydrogels also gradually increases. The results of the infrared thermography also demonstrate that GBPA hydrogels have good photothermal effects (fig. 3 e). The GBPA hydrogel was then tested for photostability by three laser switching cycles, no significant drop in temperature was observed, indicating good photostability (fig. 3 d). These results indicate that GBPA hydrogels possess good photothermal properties and photostability.

To facilitate biomedical applications in vivo, the photothermal antitumor activity of GBPA hydrogels was evaluated in vitro. The experiments were divided into 3 groups: h ₂ Group O (i.e., control group), GBPA hydrogel group and GBPA ^Laser A group. The photothermal antitumor activity of GBPA hydrogels without and with laser irradiation was compared by a live dead cell fluorescent staining experiment for four OSCC cell lines (Cal-27, UM1, HSC-3 and HSC-4). Fig. 4 shows that the control group of four cells all showed strong green fluorescence signal, which means that most cells were living. In contrast, GBPA and GBPA ^Laser The group showed a clear red fluorescent signal indicating partial cell death. Meanwhile, compared with GBPA group, GBPA ^Laser The group exhibited a stronger red fluorescent signal, indicating the presence of more dead cells.

The results show that the GBPA hydrogel provided by the invention has good photo-thermal property and photo-stability and excellent photo-thermal anti-tumor activity in vitro.

Experimental example 4 in vivo biocompatibility of GBPA hydrogel

1. Experimental method

Sample to be tested: 1.4% w/v GBPA hydrogel.

The testing method comprises the following steps: to assess the degradation of GBPA hydrogels in vivo, 100 μl of GBPA hydrogel was subcutaneously usedIs injected into subcutaneous tissue of the back of the mouse, H ₂ O served as a control group, and the degree of degradation was observed at various time points. By H&The E-staining experiments assess the in vivo biocompatibility of GBPA hydrogels from a histopathological point of view.

2. Experimental results

FIG. 5a shows that H after subcutaneous injection in the control group ₂ O is in the liquid state in situ, diffuses to the surroundings and disappears within a few minutes. However, GBPA hydrogels were in a gel state at the injection site, with morphology that remained essentially stable for 48h and gradually decreased until 72h were completely degraded. At the same time, the GBPA hydrogel was observed to darken gradually, indicating that its degradation was accompanied by the process of Dopamine (DA) oxidation to form PDA. In addition, with control group H ₂ As in O, no death and no apparent pathological abnormalities were observed in the GBPA hydrogel group, and no apparent inflammatory response was observed at the injection site at different time points. The results showed that, compared with the control group H ₂ Compared with O, the GBPA hydrogel has slower degradation rate and good in-vivo stability. Finally, through H&The E-staining experiments assess the in vivo biocompatibility of GBPA hydrogels from a histopathological point of view. The results showed that, compared with the control group H ₂ As with O, no significant organ damage or tissue degeneration was observed in the major organs of the GBPA hydrogel treated group (including heart, liver, spleen, lung and kidney) at different time points (fig. 5 b), confirming that GBPA hydrogels have good in vivo biocompatibility.

The results show that the GBPA hydrogel provided by the invention has good in vivo biocompatibility in vivo, no obvious inflammatory reaction is found at an injection site, and the GBPA hydrogel has small toxic and side effects and has wide prospect as an in vivo biomedical application material.

Experimental example 5 in vivo photo-thermal inhibition of OSCC graft tumor growth by GBPA hydrogel

1. Experimental method

Sample to be tested: 2.8% w/v GBPA hydrogel.

The testing method comprises the following steps: collecting OSCC cells Cal-27 in logarithmic growth phase, washing with serum-free DMEM high sugar culture medium for 3 times, re-suspending and counting, and adjusting cell concentration to 2×10 ⁶ mu.L of the solution was then thinned by subcutaneous inoculation at a volume of 100 mLThe cell suspension is injected into the back of the right side of the nude mice, and an OSCC cell Cal-27 nude mice transplantation tumor model is constructed. After seeding the cells, tumor volumes were measured every 2-3 days using a sterilized vernier caliper, the weights of the mice were weighed and recorded. The calculation formula of the tumor volume is: tv=pi/6×longest diameter× (shortest diameter) ² . When the tumor volume grows to 100mm ³ At this time, mice were randomly divided into 3 groups (n=5/group): h ₂ Group O, GBPA hydrogel group and GBPA ^Laser Group (808 nm laser, irradiation time 3 min). All treatment groups were dosed by intratumoral injection, 100 μl each for 16 days, depending on the in vivo degradation time of the GBPA hydrogel, once every 3 days. After the experiment is finished on the 16 th day after the administration, all mice are sacrificed, tumor tissues and important organs (heart, liver, spleen, lung, kidney and the like) of each group are collected, one part of the tumor tissues and the important organs are fixed in 4% paraformaldehyde for 24 hours, and then the tumor tissues and the important organs are subjected to tissue dehydration, embedding and slicing treatment, and then relevant histological analysis is carried out; the other part is stored in a liquid nitrogen tank for standby.

2. Experimental results

First, to evaluate the efficacy of GBPA hydrogel in photo-thermal inhibition of OSCC cell Cal-27 graft growth, a Cal-27 cell nude mouse graft model was established (fig. 6 a). Since the in vivo degradation time of GBPA hydrogels is about 3 days, the GBPA hydrogels are degraded in the absence or presence of laser irradiation (808 nm laser, 2W cm) ^-2 5 min), mice received H once every three days ₂ The O and GBPA hydrogels were given by intratumoral injection for 16 days. The negative control group was H ₂ The O treatment group and the positive control group are GBPA hydrogel treatment groups. As seen in FIG. 6b, H ₂ Tumor volume of O-treated mice increased dramatically during treatment, whereas GBPA and GBPA ^Laser The average tumor volume of the treated group increased slowly. After 16 days of treatment, GBPA ^Laser The treatment group inhibited tumor growth by approximately 86%, whereas the GBPA treated group had a tumor growth inhibition of 20%. In summary, compared to the GBPA hydrogel group, GBPA ^Laser The treatment group showed significantly improved tumor suppression effect. At the same time H ₂ The average tumor volumes between O and GBPA groups did not have statistical differences, indicating that GBPA hydrogels exhibit lower in vivo toxicity in vivo. Likewise, with GBPA hydrogel setsIn contrast, the average size image of the tumor further demonstrated that GBPA hydrogel had excellent photothermal treatment effect under 808nm laser irradiation (fig. 6 d). In addition, at H ₂ No significant fluctuation in body weight was observed between O and GBPA treated groups (fig. 6 c).

On day 16, vital organs (including heart, liver, spleen, lung and kidney) of mice were harvested and treated with H&E staining to assess potential in vivo toxicity. FIG. 6e shows, in combination with H ₂ GBPA and GBPA as in the O-treated group ^Laser No obvious pathological abnormality appears in the important organs of the treatment group, which indicates that the GBPA hydrogel does not cause obvious systemic toxicity and has good biocompatibility.

To explore the potential mechanism of GBPA hydrogels for photothermal therapy of OSCC in vivo in one step, the potential mechanism is demonstrated by H&E staining, immunohistochemistry (IHC) and TUNEL immunofluorescence experiments assessed histological and apoptotic or proliferative conditions of OSCC tumor tissue (fig. 7). And H is ₂ GBPA compared with the group of GBPA hydrogels ^Laser Tumor cells appear severely damaged and largely disappear in the tumor tissue of the treatment group (fig. 7 a). In addition, TUNEL experiments also demonstrated GBPA ^Laser The tumor tissue of the treatment group had more apoptotic cells with green fluorescence (fig. 7 b). In contrast, in control group H ₂ Almost no apoptotic cells were detected in the tumors of O-treated mice. The quantitative analysis result of TUNEL-stained tumor tissue apoptosis cells shows that GBPA ^Laser The average apoptosis rate of tumors in the treatment group is about 81.5%, which is far higher than that of H ₂ Tumor apoptosis rate in O and GBPA treated groups (fig. 7 c). Therefore, the above results indicate that GBPA hydrogels can have excellent photothermal treatment effect on OSCC cell transplantation tumor in vivo by inducing apoptosis of tumor cells under laser irradiation state. In addition, IHC was used to study the staining of Ki67 and Caspase 3, and the results showed that it was consistent with H ₂ GBPA compared to the O and GBPA treated groups ^Laser Expression of Ki67 was significantly reduced in tumor tissues of the treatment group, and expression level of Caspase 3 was significantly increased (fig. 7 a). The results are consistent with the statistical analysis results of the data, which show that the GBPA hydrogel can realize good photo-thermal inhibition by inhibiting the proliferation of Ki67 positive cells and promoting the apoptosis of Caspase 3 positive cellsThe effect is achieved (FIGS. 7c, d).

The results show that the GBPA hydrogel is taken as a difunctional hydrogel integrating the light and heat collecting effect and the carrier, not only has good biocompatibility, but also can remarkably inhibit the growth of OSCC transplantation tumor in vivo, thereby achieving the purpose of treating OSCC.

In summary, the invention provides a hydrogel for photothermal treatment, and a preparation method and application thereof. The invention takes guanosine and polydopamine-gold nanoparticles as raw materials, and builds the supermolecular hydrogel integrating the photo-thermal effect and the carrier by introducing dynamic boron ester bonds. The supermolecule hydrogel provided by the invention has good mechanical property, self-repairing property and injectability, and has the advantages of small toxic and side effects and good in vivo biocompatibility. The supermolecular hydrogel overcomes the defect that the traditional physical coating type hydrogel delivery system is easy to cause abrupt release, has excellent slow release effect, has excellent photo-thermal anti-tumor activity in vivo and in vitro, and has wide application prospect in preparing hydrogel biological materials and medicaments for photo-thermal treatment.

Claims

1. A hydrogel for photothermal therapy, characterized in that: the hydrogel is prepared by using a photothermal agent, inorganic alkali, B (OH) ₃ Guanosine and an aqueous solvent as raw materials; wherein, the photo-thermal agent, inorganic alkali and B (OH) ₃ The molar ratio of (12-72) is 1:1, and the ratio of the photothermal agent to guanosine is (0.6-0.9) mol (3.5-14) mg; the concentration of guanosine is 7-28mg/mL; the photothermal agent is polydopamine-gold nanoparticle, and the guanosine is D-guanosine.

2. The hydrogel of claim 1, wherein: the inorganic base is NaOH; and/or the aqueous solvent is water or an aqueous buffer solution, and the pH value of the aqueous buffer solution is preferably 7.35-7.45.

3. The hydrogel according to claim 1 or 2, characterized in that: the photo-thermal agent, inorganic base, B (OH) ₃ Is of the mole of (2)The molar ratio is (12-32) 1:1, and the ratio of the photothermal agent to guanosine is (0.6-0.8) mol (7-14) mg.

4. The hydrogel of claim 1, wherein: the concentration of guanosine is 14-28mg/mL.

5. A method for preparing the hydrogel for photothermal therapy according to any one of claims 1 to 4, characterized in that: the method comprises the following steps: photo-thermal agent, inorganic base, B (OH) ₃ Mixing in aqueous solvent, adding guanosine, heating for dissolving, and cooling.

6. Use of the hydrogel according to any one of claims 1 to 4 for the preparation of a biomaterial or a medicament for photothermal therapy.

7. Use according to claim 6, characterized in that: the biological material or the medicine is used for preventing and/or treating superficial tumors.

8. Use according to claim 7, characterized in that: the superficial tumor is oral squamous cell carcinoma.