CN116023681B - Preparation method and application of acoustic-thermal dual-response hydrogel - Google Patents

Abstract

The invention discloses a preparation method and application of a sound-heat dual-responsive hydrogel, belonging to the field of medical materials and technology. The sound-heat dual-responsive hydrogel is mainly composed of thiolated sodium alginate (SA-SH), arginine-modified sodium alginate (SA-Arg), modified titanium dioxide nanoparticles ( _TiO2 @ _MnO2 ) and β-sodium glycerophosphate (β-GP). After ultrasonic treatment, the hydrogel undergoes sol-gel transformation and produces chemical substances such as ROS, NO and _O2 , which can stimulate the immune response activity of wound or tumor immune cells, promote apoptosis of wound or cancer cells and improve the tumor microenvironment, and produce synergistic wound healing/anti-cancer effects. The hydrogel prepared by the present invention has good biocompatibility, low cost, simple and fast preparation method, fast gelation speed, sound-heat dual responsiveness, can treat lesions deep in tissues by ultrasound and its thermal effect, and has great application potential in the fields of antibacterial, wound healing, cancer treatment, etc.

Description

Preparation method and application of acoustic-thermal dual-response hydrogel

Technical Field

The present invention relates to the field of biomedical materials, and in particular to a preparation method of an acoustic-thermal dual-response hydrogel and application thereof.

Background technique

Cancer-related research has always been one of the hot topics in the biomedical field. With the deepening of cancer research, the treatment methods for cancer are becoming more mature. At present, there are three mainstream cancer treatment methods in clinical practice: surgical treatment, radiotherapy and chemotherapy, and targeted therapy. Surgical treatment is a cancer treatment method based on surgical resection. This method is simple in principle and effective, but it may remove some normal tissues and organs in the human body during the process, thereby causing sequelae or dysfunction. In addition, surgical treatment cannot prevent the metastasis and spread of cancer, and may also lead to cancer recurrence. Radiotherapy and chemotherapy are treatment methods that use high-energy radiation (radiotherapy) or chemical drugs (chemotherapy) to kill cancer cells. They have a strong ability to eliminate cancer, but high-energy rays or drugs will also damage normal cells in the human body during treatment, causing weakness in patients. It is a "double-edged sword". No matter what treatment ideas are used, it is a huge test for the patient's financial resources and energy, so it is of great significance to develop a convenient and efficient new cancer treatment strategy.

Hydrogel is a three-dimensional porous material with excellent hydrophilicity. Its microstructure similar to human tissue gives it excellent biocompatibility. Through design, hydrogel can be applied in many biomedical fields, such as antibacterial, anti-inflammatory, wound healing and loading. In cancer treatment, hydrogel also has great application potential. Cancer adjuvant therapies such as photothermal therapy (PTT), magnetic hyperthermia therapy (MHT) and catalytic therapy based on hydrogel have been reported. For example, Cheng-Xiong Wei et al. developed an injectable composite hydrogel with chitosan/hyaluronic acid/sodium β-glycerophosphate as the matrix and carbon particles as the photothermal agent. The tumor inhibition rate of the injectable composite hydrogel in the Balb/c nude mouse osteosarcoma model under near-infrared laser (808nm) irradiation reached 98.4%. The evaluation of bone regeneration in the SD rat skull defect model showed that the composite hydrogel can promote new bone formation. After 8 weeks, the bone volume/total volume ratio was 76.2%, which was in sharp contrast to the 23.9% of the control group. (Cheng Xiong-Wei, Jin Xin, Wu Cheng-Wei, et al. Injectable composite hydrogel based on carbonparticles for photothermal therapy of bone tumor and bone regeneration[J]. Journal of Materials Science&Technology, 2022, 11864-72.) Xu Yan et al. designed an in situ magnetic hydrogel, which is composed of a triblock polymer matrix (NDP) and graphene oxide nanosheets decorated with iron oxide nanoparticles (Fe ₃ O ₄ @rGO, denoted as FG) on the surface. The hydrogel (NDP-FG) not only has a high efficiency in intraoperative hemostasis, but also can prevent tumor recurrence. The stable vascular embolization performance also shows that NDP-FG hydrogel has good application prospects in transarterial embolization treatment of liver cancer. (Xu Yan, Sun Tian-Ci, Song Yong-Hong, et al. In situ Thermal-Responsive Magnetic Hydrogel for Multidisciplinary Therapy of Hepatocellular Carcinoma[J]. Nano Letters, 2022, 22(6): 2251-2260.) However, these hydrogels are complex in materials and difficult to implement, which limits their application in tumor treatment.

Ultrasound is a mechanical wave with an extremely short wavelength. Due to its unique physical effects, non-invasiveness and safety, it is widely used in the treatment of diseases such as stones and joints. In addition, sonodynamic therapy (SDT) with ultrasound as the main triggering method has also emerged in cancer treatment and is expected to become a safe and non-invasive new cancer treatment method. For example, Min Sun et al. co-assembled polylysine with Pluronic F127 and formed a stable nanogel structure through Genipin cross-linking. Subsequently, ICAM-1 antibody was transplanted onto the nanogel (named GenPLPFT) for active tumor targeting. After in vitro ultrasound treatment, F127 was stripped from GenPLPFT, inducing nanogel expansion, reducing its stability, and accelerating drug release to treat cancer (Min Sun, Yue Tao, Wang Congyu, et al. Ultrasound-Responsive Peptide Nanogels to Balance Conflicting Requirements for Deep Tumor Penetration and Prolonged Blood Circulation [J]. ACS Nano, 2022, 16 (6): 9183-9194.). However, the preparation of such ultrasound-sensitive hydrogels is complex, and the structure is mostly microspheres, which are easily metabolized and excreted by the body and lose their therapeutic ability. In addition, the chemotherapy drugs loaded in the microspheres pose a potential risk to the patient's health. Therefore, designing an ultrasound-induced non-drug cancer treatment strategy with simple material preparation and non-loss is of great significance in the field of cancer treatment.

Summary of the invention

In view of the existing technical problems, the present invention designs an in-situ antibacterial and anticancer hydrogel which is dually triggered by ultrasound and its thermal effect based on the characteristics of ultrasound deep treatment and non-invasive treatment. The in-situ antibacterial and anticancer hydrogel is composed of thiolated sodium alginate (SA-SH), arginine-modified sodium alginate (SA-Arg), modified titanium dioxide nanoparticles ( _TiO2 @ _MnO2 , TMN) and β-sodium glycerophosphate (β-GP). After being injected into the tumor microenvironment (TME) and subjected to ultrasound treatment, the in-situ antibacterial and anticancer hydrogel undergoes sol-gel transformation and produces chemical substances such as ROS, NO and _O2 . These chemical substances have broad-spectrum antibacterial properties. While killing bacteria in the wound, they can also stimulate cellular immune response activity, promote cancer cell apoptosis and improve TME, producing synergistic antibacterial and anticancer effects. This ultrasound-sensitive hydrogel can provide a simple and efficient treatment strategy for ultrasound therapy of cancer, which is of great significance in the field of cancer treatment.

In order to achieve the above object, the present invention adopts the following technical solutions:

A method for preparing an acoustic-thermal dual-responsive hydrogel comprises the following steps: utilizing the condensation reaction of carboxyl groups and amino groups to respectively graft cysteine and arginine onto sodium alginate (SA) to prepare thiolated sodium alginate (SA-SH) and arginine-modified sodium alginate (SA-Arg); adding a certain amount of _KMnO4 solution to an aminated nano- _TiO2 aqueous solution under ultrasonic conditions to obtain _TiO2 @ _MnO2 ; dissolving SA-SH and SA-Arg together in deionized water, adding a certain amount of _TiO2 @ _MnO2 to prepare a hydrogel precursor solution, and then adding a certain amount of β-GP solution. After ultrasonic treatment, the acoustic-thermal dual-responsive hydrogel is obtained.

The method for preparing the above-mentioned acoustic-thermal dual-responsive hydrogel specifically comprises the following steps:

(1) Sodium alginate was dissolved in deionized water, NHS and EDC were added, and the mixture was activated at 25°C for 30 min. Then, cysteine was added, and the mixture was reacted at room temperature for 12 h. The resulting reactant was dialyzed and freeze-dried to obtain SA-SH. Sodium alginate was dissolved in deionized water, NHS and EDC were added, and the mixture was activated at 25°C for 30 min. Then, arginine was added, and the mixture was reacted at room temperature for 12 h. The resulting reactant was dialyzed and freeze-dried to obtain SA-Arg.

(2) Mix _TiO2 and APTES and disperse them in an alkaline solution, react at 5°C for 12 hours, filter after the reaction, wash the residue with deionized water and anhydrous ethanol, and then vacuum dry to obtain amination _TiO2 nanoparticles; disperse the amination _TiO2 nanoparticles in _KMnO4 solution and ultrasonicate for 2 hours, and vacuum dry the obtained reactant to obtain _TiO2 @ _MnO2 ;

(3) SA-SH and SA-Arg are mixed and dissolved in deionized water, and TiO ₂ @MnO ₂ solution is added to obtain a hydrogel precursor solution; β-GP solution is added to the hydrogel precursor solution, and ultrasonic treatment is performed for 10 to 30 minutes to obtain an acoustic-thermal dual-responsive hydrogel.

In the step (1), the molar ratio of sodium alginate:NHS:EDC:cysteine for preparing SA-SH is 1:1:1:1; the molar ratio of sodium alginate:NHS:EDC:arginine for preparing SA-Arg is 1:1:1:1.

In the step (2), the mass ratio of _TiO2 to APTES is 100:1.

In the step (2), the alkaline solution is a 0.01 M NaOH solution.

In the step (2), the concentration of the _KMnO4 solution is 10 mg/mL.

In the step (3), the mass ratio of SA-SH:SA-Arg:TiO ₂ @MnO ₂ in the hydrogel precursor solution is 30:30:1.

In the step (3), the volume ratio of the hydrogel precursor solution: the β-GP solution is 3.2:3.

In the step (3), the concentration of the β-GP solution is 58 wt %.

The acoustic-thermal dual-responsive hydrogel is prepared by the above preparation method.

The above-mentioned acoustic-thermal dual-responsive hydrogel is used in the preparation of drugs with antibacterial, wound healing and cancer treatment effects.

The significant advantages of the present invention are:

(1) The use of an injectable hydrogel precursor solution and ultrasound and its thermal effect as a means of gelation improves the safety, convenience, and depth of treatment.

(2) After ultrasonic treatment, the hydrogel produces ROS, NO, _O2 and other substances that improve TME and promote cancer cell apoptosis, resulting in a synergistic anticancer effect.

(3) -SH and -SS- in the hydrogel have reversible denaturation and can continuously consume H ₂ O ₂ and GSH, accelerating the death of cancer cells.

(4) Ultrasound can improve the permeability of cell membranes, making it easier for small molecules to enter cancer cells and improving the effectiveness of cancer treatment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of the sol-gel transition of hydrogel under sonicothermal action and its application in cancer treatment.

FIG2 is a reaction equation of SA-SH and SA-Arg.

FIG. 3 is an X-ray photoelectron spectroscopy (XPS) of TiO ₂ @MnO ₂ .

Figure 4 is a graph showing the oxygen production capacity of TiO ₂ @MnO ₂ .

Figure 5 is a graph of ROS production of TiO ₂ @MnO ₂ under ultrasonic treatment.

FIG. 6 is a digital picture of the hydrogel before and after gelation.

FIG. 7 is an infrared thermal imaging image of the hydrogel during ultrasound.

FIG8 is an infrared spectrum of the hydrogel.

FIG. 9 is a graph showing the antibacterial properties of the hydrogel under different conditions.

Detailed ways

The present invention discloses a hydrogel material with acoustic and thermal dual responsiveness. The preparation materials include: sodium alginate, cysteine, arginine, _TiO2 , silane coupling agent KH550 (APTES), KMnO4, sodium β-glycerophosphate (β-GP), and deionized water. The schematic diagram of the sol-gel transition of the hydrogel material under acoustic and thermal action and its application in cancer treatment is shown in FIG1.

The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention.

Embodiment 1:

Step 1: Add 1 g of sodium alginate to 100 mL of deionized water, and obtain a sodium alginate solution after complete dissolution. Then, add a certain amount of NHS and EDC to activate the carboxyl group (the molar ratio of each substance is SA: NHS: EDC = 1:1:1). The activation conditions are: activation at 25°C for 30 min, then add 0.605 g of cysteine, react at 25°C for 12 h, dialyze in deionized water through a dialysis bag with a molecular weight cutoff of 8000-14000 for 3 days, and then freeze-dry at -26°C under a vacuum condition of 0.09 MPa for 2 days to obtain SA-SH; Add 1 g of sodium alginate to 100 mL of deionized water, and then add a certain amount of NHS and EDC to activate the carboxyl group (the molar ratio of each substance is SA: NHS: EDC = 1:1:1). The activation conditions are: activation at 25°C for 30 min, then add 0.605 g of cysteine, react at 25°C for 12 h, dialyze in deionized water through a dialysis bag with a molecular weight cutoff of 8000-14000 for 3 days, and then freeze-dry at -26°C under a vacuum condition of 0.09 MPa for 2 days to obtain SA-SH. Add it into 100 mL of deionized water, and after it is completely dissolved, a sodium alginate solution is obtained. Subsequently, a certain amount of NHS and EDC are added thereto to activate the carboxyl group (the molar ratio of each substance is SA: NHS: EDC = 1:1:1). The activation conditions are: activation at 25°C for 30 minutes, and then 0.870 g of arginine is added, reacting at 25°C for 12 hours, dialyzing in deionized water through a dialysis bag with a molecular weight cutoff of 8000-14000 for 3 days, and then freeze-drying at -26°C under a vacuum condition of 0.09 MPa for 2 days to obtain SA-Arg; wherein the reaction equation involved is shown in Figure 2.

Step 2: Mix 100 mg _TiO2 and 1 mg APTES and disperse them in 100 mL of 0.01 M NaOH solution, react at 25°C for 12 hours, filter with a 200-micron filter membrane after the reaction, and wash the residue with deionized water and anhydrous ethanol for three times by vacuum filtration, and then dry it in a vacuum drying oven at 50°C for 6 hours to obtain aminated _TiO2 nanoparticles; disperse 60 mg of aminated _TiO2 nanoparticles in 100 mL of 10 mg/mL _KMnO4 solution and ultrasonically treat it at 1.5 MHz frequency, 100 W power, and 25°C for 2 hours, and dry it at 50°C under a vacuum of 0.086 MPa for 3 hours to obtain _TiO2 @ _MnO2 , which is dispersed in deionized water for later use.

Step 3: Mix and dissolve 0.06g SA-SH and 0.06g SA-Arg in 3mL deionized water, add 0.2mL 10mg/mL _TiO2 @ _MnO2 solution thereto to obtain a hydrogel precursor solution; add 3mL 58wt% β-GP solution to 3.2mL hydrogel precursor solution, and ultrasonically treat at 1.5MHz, 100W power, and 25℃ for 10min to obtain the acoustic-thermal dual-responsive hydrogel ( _TiO2 @ _MnO2 hydrogel sample) described in the present invention.

Figure 3 is the X-ray photoelectron spectrum of TiO ₂ @MnO _2. It can be seen from the figure that the main peaks of O1s, Mn 2p and Ti 2p appear on the TiO ₂ @MnO ₂ sample, indicating the presence of Mn. The main O1 peak at 529.40eV corresponds to the lattice oxygen of the Ti-O bond. The Mn 2p XPS spectrum has two peaks at 641.67eV and 653.78eV, corresponding to Mn 2p _3/2 and Mn 2p _1/2 , respectively. Calculation of the two peak areas of Mn 2p shows that the ratio of Mn ³⁺ /Mn ⁴⁺ is about 0.6, indicating that MnO ₂ and Mn ₂ O ₃ exist in the sample at the same time. In the Ti 2p region, the main peak positions of Ti 2p _2/2 and Ti 2p _2/3 of TiO ₂ @MnO ₂ appear at 458.83eV and 464.50eV, respectively. The above results demonstrate the successful synthesis of TiO2@ _MnO2 .

Figure 4 is a graph of the oxygen production capacity of TiO ₂ @MnO _2. As can be seen from the figure, compared with pure TiO ₂ , TiO ₂ @MnO ₂ has an oxygen production characteristic that is positively correlated with the concentration of the reactants, while pure TiO ₂ produces almost no oxygen, which indirectly indicates the successful loading of MnO ₂ on TiO ₂ .

Figure 5 is a graph of ROS production of _TiO2 @ _MnO2 under ultrasonic treatment. DPBF is a ROS-sensitive substance with a UV absorption peak at around 400nm, and is often used to characterize ROS. After ultrasonic treatment, the ROS generated by _TiO2 @ _MnO2 will react with DPBF, resulting in a decrease in absorbance at 400nm, which is in line with the design expectations.

Figure 6 is a digital picture of the hydrogel before and after gelation. After ultrasonic treatment, the hydrogel solution can undergo sol-gel transition within 10 minutes.

Figure 7 is an infrared thermal imaging image of hydrogel during ultrasound treatment. The temperature before ultrasound treatment was 25°C, and after ultrasound treatment, the temperature of the hydrogel increased to about 35.8°C, indicating that the hydrogel has a certain degree of ultrasound temperature sensitivity.

Figure 8 is the infrared spectrum of the hydrogel. Compared with pure SA, SA-SH has two more peaks at 1700 cm ^-1 and 2500 cm ^-1 , representing amide bonds and thiol groups, respectively, indicating the successful grafting of cysteine. After gelation, the peak at 2500 cm ^-1 disappears in SA-Gel, and the surface thiol groups react to form disulfide bonds.

Embodiment 2:

The prepared Bacillus subtilis was placed in the culture medium, sealed with a sealing film, and placed in a bacterial incubator (37°C) for 24 hours; the hydrogel sample (referred to as SA-Gel+TMN2) with 2mg TiO ₂ @MnO ₂ added was placed in a clean bench for sterilization for 30 minutes and then used; the cultured bacterial solution was taken out of the clean bench and the OD value of each bacterial solution was measured with an ELISA reader. 9mL of sterile culture solution was added to each glass test tube, and each bacterial solution was diluted to an appropriate multiple by a 10-fold dilution method according to the OD value of the bacterial solution. Four wells were selected in a 24-well plate and added with an appropriate amount of diluted bacterial solution, which were recorded as 1, 2, 3, and 4. Among them, well 1 was a blank control group, well 2 was a blank ultrasonic control group, well 3 was a SA-Gel+TMN2 group, and well 4 was a SA-Gel+TMN2 ultrasonic group. The ultrasonic time of wells 2 and 4 was 10min (1.5MHz, 100W power, 25°C). After the ultrasonic treatment, 50 μL of bacterial solution was taken from each well and placed on a clean bacterial culture medium. After culturing at 37°C for 12 hours, the antibacterial effect was observed. The antibacterial performance is shown in Figure 9. As can be seen from the figure, compared with the blank group, the number of colonies in the blank ultrasonic group has hardly changed, indicating that simple ultrasonic treatment has no antibacterial properties. In addition, the number of colonies in the SA-Gel+TMN2 ultrasonic group is less than that in the SA-Gel+TMN2 group, the blank group, and the blank ultrasonic group, indicating that the antibacterial performance of SA-Gel+TMN2 is enhanced after ultrasonic treatment, which is in line with the design expectations. In summary, SA-Gel+TMN2 after ultrasonic treatment exhibits good antibacterial properties and has the potential for antibacterial applications.

The above are all preferred implementations of the present invention and do not limit the protection scope of the present invention. All other implementation examples obtained by ordinary professionals in this technical field after reading the present invention without making any creative work are within the protection scope of the present invention.

Claims (9)

1. A preparation method of the acoustic-thermal double-response hydrogel is characterized by comprising the following steps: the method comprises the following steps:

(1) Dissolving sodium alginate in deionized water, adding NHS and EDC, activating at 25deg.C for 30min, adding cysteine, reacting at room temperature for 12 hr, dialyzing the obtained reactant, and freeze drying to obtain SA-SH; dissolving sodium alginate in deionized water, adding NHS and EDC, activating at 25deg.C for 30min, adding arginine, reacting at room temperature for 12 hr, dialyzing the obtained reactant, and freeze drying to obtain SA-Arg;

(2) Mixing and dispersing TiO ₂ and APTES into an alkaline solution, reacting for 12 hours at 25 ℃, carrying out suction filtration after the reaction is finished, washing filter residues with deionized water and absolute ethyl alcohol in sequence, and carrying out vacuum drying to obtain aminated TiO ₂ nano particles; dispersing the aminated TiO ₂ nano-particles into KMnO ₄ solution, performing ultrasonic treatment for 2 hours, and vacuum drying the obtained reactant to obtain TiO ₂@MnO₂;

(3) Mixing SA-SH and SA-Arg, dissolving in deionized water, and adding a TiO ₂@MnO₂ solution to obtain a hydrogel precursor solution; adding a beta-GP solution into the hydrogel precursor solution, and performing ultrasonic treatment for 10-30 min to obtain the acoustic-thermal double-response hydrogel;

in the step (2), the alkaline solution is a 0.01M NaOH solution.

2. The method for preparing the acoustic-thermal dual-response hydrogel according to claim 1, wherein: in the step (1), sodium alginate of SA-SH is prepared: NHS: EDC: the molar ratio of cysteine is 1:1:1:1, a step of; preparation of sodium alginate of SA-Arg: NHS: EDC: arginine in a molar ratio of 1:1:1:1.

3. The method for preparing the acoustic-thermal dual-response hydrogel according to claim 1, wherein: in the step (2), the mass ratio of TiO ₂ to APTES is 100:1.

4. The method for preparing the acoustic-thermal dual-response hydrogel according to claim 1, wherein: in the step (2), the concentration of the KMnO ₄ solution is 10mg/mL.

5. The method for preparing the acoustic-thermal dual-response hydrogel according to claim 1, wherein: in the step (3), SA-SH in the hydrogel precursor solution: sA-Arg: the mass ratio of TiO ₂@MnO₂ is 30:30:1.

6. The method for preparing the acoustic-thermal dual-response hydrogel according to claim 1, wherein: in the step (3), the hydrogel precursor solution: the volume ratio of the beta-GP solution is 3.2:3.

7. The method for preparing the acoustic-thermal dual-response hydrogel according to claim 1, wherein: in the step (3), the concentration of the beta-GP solution is 58 weight percent.

8. An acoustic-thermal dual response hydrogel made by the method of claim 1.

9. The use of the acoustic-thermal dual-response hydrogel of claim 8 in the preparation of a medicament having antibacterial, wound healing promoting and cancer treatment effects.