A constant temperature photo-thermal system and its use

Abstract

The present application relates to a kind of constant temperature photothermal system and its application;Near-infrared photothermal molecule is obtained by one-step Knoevenagel condensation, then using amphiphilic polymer is prepared near-infrared photothermal nanoparticle by nano precipitation method;By the method of free radical polymerization, two kinds of monomers, N-isopropyl acrylamide and acrylamide are added, initiator potassium persulfate and crosslinking agent N, N-methylene bisacrylamide are added, after being mixed uniformly, polymerization is carried out at room temperature, and poly (N-isopropyl acrylamide-acrylamide) hydrogel is obtained;Near-infrared photothermal nanoparticle is dropped on infected wound, and then poly (N-isopropyl acrylamide-acrylamide) hydrogel is applied to infected wound, and constant temperature wound healing is realized under near-infrared laser irradiation.The present application is based on the constant temperature photothermal system of poly (N-isopropyl acrylamide-acrylamide) hydrogel, and can be used for designing the intelligent PTT system with built-in accurate control photothermal temperature.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to a method for constructing a constant temperature photothermal system, and in particular to the application of the constant temperature system in the healing of infected wounds. Background Technology

[0002] Bacterial infections are a major threat to human health, imposing a significant burden on individuals, societies, and economies. While the discovery of antibiotics has provided humanity with a powerful weapon against bacteria, their misuse and overuse have led to the rapid evolution of drug-resistant bacteria. Without urgent action, the antibiotic resistance crisis could eventually escalate into a global pandemic or an epidemic of multidrug-resistant bacteria. Therefore, developing effective antimicrobial therapies independent of antibiotics is of paramount importance. To address this challenge, considerable efforts have been made, providing a variety of strategies to combat bacterial infections and antibiotic resistance, including surgery, the use of nanomaterials with inherent antimicrobial activity, photodynamic therapy, and photothermal therapy (PTT).

[0003] In recent years, PTT (percutaneous transluminal therapy) has become an attractive technique for treating bacterial infections due to its unique advantages, such as non-invasiveness, high selectivity, and low probability of inducing resistance. PTT primarily utilizes the large amount of heat generated by photothermal materials under near-infrared (NIR) light to disrupt the bacterial membrane and / or denature bacterial proteins, causing bacterial cell swelling and deformation. This physically destroys the integrity of the bacterial cells, thereby inactivating the bacteria. Furthermore, during treatment, bacteria find it difficult to develop resistance to PTT by blocking or reducing absorption, increasing metabolism, or increasing drug excretion.

[0004] Despite significant advancements over the past few decades, traditional photothermal transducers (PTTs) lack a precise, self-regulating temperature control mechanism, inevitably leading to thermal damage to healthy tissues and / or organs. Due to these significant limitations, mild PTTs (below 50°C) are increasingly used to mitigate the adverse effects of uncontrolled heating by adjusting various experimental parameters such as photothermal material concentration, laser power density, and irradiation time. However, such procedures are cumbersome and offer poor repeatability of photothermal temperatures. Furthermore, neglecting adjustments to external experimental parameters can cause instantaneous overheating, resulting in irreversible thermal damage. Therefore, we are eager to design a universal isothermal photothermal system that automatically shuts off the photothermal conversion process when a preset temperature is reached, safely and effectively addressing bacterial infection issues. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention discloses a constant-temperature photothermal system and its application, with the specific technical solution as follows:

[0006] The present invention provides a isothermal photothermal system, the construction method of which includes the following steps:

[0007] 1) Near-infrared photothermal molecules were obtained by one-step Knoevenagel condensation, and then near-infrared photothermal nanoparticles were prepared by nanoprecipitation using amphiphilic polymers.

[0008] 2) By using free radical polymerization, two monomers, N-isopropylacrylamide (NIPAM) and acrylamide (AM), were added, along with the initiator potassium persulfate (KPS) and the crosslinking agent N,N-methylenebisacrylamide (MBA). After thorough mixing, the mixture was polymerized at room temperature to obtain poly(N-isopropylacrylamide-acrylamide)(P(NIPAM-AM)) hydrogel.

[0009] 3) Near-infrared photothermal nanoparticles are dropped onto the infected wound, and then P(NIPAM-AM) hydrogel is applied to the infected wound to achieve constant temperature wound healing under near-infrared laser irradiation.

[0010] In a constant-temperature photothermal system of the present invention, the near-infrared photothermal molecule has the structure shown in the compound 5-(4-(4-methoxyphenyl)diphenylamino)thiophene-2-(1,3-bis(dicyanomethylene)indane)(MeO-TSI);

[0011]

[0012] The method for preparing the near-infrared photothermal molecules of the present invention includes the following:

[0013] 1) Weigh out four compounds in a molar ratio of 1:1.5:8:0.05-1:2:10:0.1: 4-bromo-4-methoxytriphenylamine, 5-aldehyde-2-thiopheneboronic acid, potassium carbonate, and tetra(triphenylphosphine)palladium. After evacuating the system and purging with argon gas 3-4 times, add a mixed solution of tetrahydrofuran and water in a volume ratio of 4:1-6:1. Heat the mixture at 70-75℃ for 8-12 hours. After the reaction is complete, purify the mixture by thin-layer chromatography to obtain compound 5-(4-(4-methoxyphenyl)diphenylamino)thiophene-2-carboxaldehyde.

[0014] 2) Compound 5-(4-(4-methoxyphenyl)diphenylamino)thiophene-2-carboxaldehyde and 1,3-bis(dicyanomethylene)indane were dissolved in acetic anhydride at a molar ratio of 1:1.5-1:2, with 50-60 mL of acetic anhydride required for each gram of 5-(4-(4-methoxyphenyl)diphenylamino)thiophene-2-carboxaldehyde. The reaction was heated at 60-65 °C for 2-6 hours. After the reaction was completed, the compound 5-(4-(4-methoxyphenyl)diphenylamino)thiophene-2-(1,3-bis(dicyanomethylene)indane) was purified by thin-layer chromatography.

[0015] The present invention provides a constant-temperature photothermal system, wherein the method for preparing near-infrared photothermal nanoparticles includes the following steps:

[0016] 1) Dissolve compound 5-(4-(4-methoxyphenyl)diphenylamino)thiophene-2-(1,3-bis(dicyanomethylene)indane) in tetrahydrofuran to prepare a stock solution of 1 mg / mL-2 mg / mL. Under stirring and argon gas purging, add the prepared stock solution dropwise to an aqueous solution of the amphiphilic polymer poloxamer 188 (F127) of 10 mg / mL-15 mg / mL. Add 200-250 μL of stock solution per mL of water. Keep stirring and argon gas purging for 20-30 minutes until the tetrahydrofuran is completely removed.

[0017] 2) Dialyze the obtained nanoparticle dispersion in ultrapure water for 1-2 days, and freeze-dry to obtain near-infrared photothermal nanoparticles.

[0018] The present invention provides a constant-temperature photothermal system, wherein the hydrogel preparation method includes the following steps:

[0019] 1) Dissolve N-isopropylacrylamide and acrylamide in water at a mass ratio of 10:1 to 50:1, where 5 mL to 10 mL of water is needed for every 450 mg of N-isopropylacrylamide. Then add 0.5% to 1% of the mass of N-isopropylacrylamide as an accelerator, tetramethylethylenediamine, and then add 1% to 2% of the mass of N-isopropylacrylamide as a crosslinking agent, N,N-methylenebisacrylamide, and stir until fully dissolved.

[0020] 2) Add 0.1%-1% potassium persulfate by mass of N-isopropylacrylamide to the fully dissolved aqueous solution, stir to dissolve fully, pour into a mold, and polymerize at room temperature to obtain a thermosensitive poly(N-isopropylacrylamide-acrylamide) hydrogel;

[0021] The application of the constant temperature photothermal system of the present invention in the healing of infected wounds.

[0022] The application of the constant-temperature photothermal system of this invention in the healing of infected wounds involves dropping near-infrared photothermal nanoparticles onto the infected wound, and then uniformly applying the prepared thermosensitive poly(N-isopropylacrylamide-acrylamide) hydrogel onto the infected wound. Under irradiation with an 808nm near-infrared laser, the near-infrared photothermal nanoparticles continuously generate heat, causing the thermosensitive poly(N-isopropylacrylamide-acrylamide) hydrogel to undergo a phase transition and turn white. The incident light is effectively scattered, and the near-infrared photothermal nanoparticles can no longer generate heat, thereby achieving constant-temperature wound healing.

[0023] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a isothermal photothermal system based on P(NIPAM-AM) hydrogel. The change in transmittance of the P(NIPAM-AM) hydrogel during the phase transition can be used to adjust the photothermal temperature of the thermosensitive hydrogel to its cloud point temperature (T0). cp It is a reversible switch. This feature can be used to design intelligent PTT systems with built-in precise control of photothermal temperature.

[0024] Beneficial effects:

[0025] 1. This invention first provides a method for constructing a constant-temperature photothermal system, comprising:

[0026] 1) A near-infrared photothermal molecule (MeO-TSI) was synthesized, which has a strong electron donor-acceptor molecular framework and extended conjugated π bridges. This molecule exhibits strong near-infrared absorption in water, demonstrating a very strong photothermal effect;

[0027] 2) A photothermal nanoparticle (MeO-TSI@F127 NPs) was synthesized. It has good stability, can be uniformly dispersed in water, and can maintain excellent photothermal stability under laser irradiation.

[0028] 3) A thermosensitive hydrogel (P(NIPAM-AM) hydrogel) was prepared. Its synthesis is simple and reusable. By changing the ratio of the two monomers, different hydrogels with different phase transition temperatures can be obtained. Due to the property of the hydrogel turning white after phase transition, it can regulate the incident light, thus realizing the control of the PTT process temperature.

[0029] 2. The present invention further provides the application of a constant temperature photothermal system in the healing of infected wounds. During the PTT process, there is no need to precisely monitor the parameters, which is expected to avoid thermal damage to normal tissues during the traditional PTT process. Attached Figure Description

[0030] Figure 1 The photophysical properties, AIE properties, and photothermal effects of the near-infrared photothermal molecules (TSI-MeO) and near-infrared photothermal nanoparticles (TSI-MeO@F127 NPs) prepared in Examples 2 and 3 are described.

[0031] (A) Ultraviolet absorption spectrum of TSI-MeO nanoaggregates in water.

[0032] (B) A graph showing the change in fluorescence intensity of TSI-MeO with hexane content.

[0033] (C) Schematic diagram of the photothermal effect of TSI-MeO nanoaggregates of different concentrations under 808nm laser irradiation (optical power density is 1.2mW / cm²). 2 ).

[0034] (D)UV absorption spectrum of TSI-MeO@F127 NPs in water.

[0035] (E)TSI-MeO@F127 NPs particle size distribution in water.

[0036] Transmission electron micrograph of (F)TSI-MeO@F127 NPs.

[0037] Schematic diagram of particle size change of (G)TSI-MeO@F127 NPs in water over 7 days.

[0038] (H) Schematic diagram of the photothermal effect of different concentrations of TSI-MeO@F127 NPs under 808nm laser irradiation (optical power density is 1.2mW / cm²). 2 ).

[0039] (I) Schematic diagram of the photothermal stability of TSI-MeO@F127 NPs and indocyanine green (ICG) during 5 heating-cooling cycles.

[0040] Figure 2 This describes the phase transition process of the thermosensitive hydrogel P (NIPAM-AM) prepared in Example 4 and the hydrogel's temperature control.

[0041] (A) Phase transition curve of P(NIPAM-AM) as temperature increases.

[0042] (B) A photograph showing the degree of scattering of incident light as the whitening of P(NIPAM-AM) increases with the phase transition.

[0043] Isothermal temperature curve under the action of (C)P(NIPAM-AM).

[0044] Infrared thermal imaging of an isothermal process under the action of (D)P(NIPAM-AM).

[0045] Figure 3 This is an in vitro antibacterial experiment of TSI-MeO@F127 NPs prepared in Example 3.

[0046] (A) Statistical analysis of bacterial survival ability based on the order of magnitude of colony formation.

[0047] (B) Photographs of colonies formed on agar plates after different experimental groups were treated.

[0048] (C) Images of bacterial viability / dead staining analysis after treatment with different experimental groups.

[0049] (D) Scanning electron micrographs of bacteria after treatment with different experimental groups.

[0050] Figure 4 This is the application of a constant-temperature photothermal system in in vivo antibacterial experiments.

[0051] (A) A schematic diagram of the timeline of the animal experiment process.

[0052] (B) Infrared thermal imaging images of mice during different experimental group treatments.

[0053] (C) Temperature change curves at the wound site during treatment in different experimental groups.

[0054] (D) Survival rate changes of mice during the 11-day treatment cycle after different experimental groups were treated.

[0055] (E) Photographs of skin wounds in mice infected with methicillin-resistant Staphylococcus aureus (MRSA) 11 days after infection.

[0056] (F) Curve showing the change in the area of ​​skin infection in mice infected with MRSA over 11 days.

[0057] (G) Curves showing changes in mouse body weight during an 11-day treatment period after treatment in different experimental groups.

[0058] (H) Photographs of colonies formed on agar plates of different groups on days 2 and 11 of the in vivo antibacterial experiment.

[0059] (I) Statistical analysis of bacterial survival ability based on the order of magnitude of colony formation.

[0060] Figure 5 This is an H&E staining analysis of tissue sections from wounds treated with a constant-temperature photothermal system.

[0061] (A) Image of H&E staining analysis of wound tissue on the second day of the in vivo antibacterial experiment.

[0062] (B) Image of H&E staining analysis of wound tissue after the in vivo antibacterial experiment. Detailed Implementation

[0063] The present invention will be further described below through specific embodiments.

[0064] The method for preparing the near-infrared photothermal molecules of the present invention includes the following:

[0065] 1) Weigh out four compounds in a molar ratio of 1:1.5:8:0.05-1:2:10:0.1: 4-bromo-4-methoxytriphenylamine, 5-aldehyde-2-thiopheneboronic acid, potassium carbonate, and tetra(triphenylphosphine)palladium. After evacuating the system and purging with argon gas 3-4 times, add a mixed solution of tetrahydrofuran and water in a volume ratio of 4:1-6:1. Heat the mixture at 70-75℃ for 8-12 hours. After the reaction is complete, purify the compound 5-(4-(4-methoxyphenyl)diphenylamino)thiophene-2-carboxaldehyde (MeO-TS) by thin-layer chromatography.

[0066] 2) MeO-TS and 1,3-bis(dicyanomethylene)indene were dissolved in acetic anhydride at a molar ratio of 1:1.5-1:2, with 50-60 mL of acetic anhydride required per gram of MeO-TS. The reaction was carried out at 60-65 °C for 2-6 hours. After the reaction was completed, the compound MeO-TSI was obtained by thin-layer chromatography.

[0067] The reaction formula is as follows:

[0068]

[0069] The method for preparing near-infrared photothermal nanoparticles according to the present invention includes the following steps:

[0070] 1) Dissolve the compound MeO-TSI in tetrahydrofuran to prepare a stock solution of 1 mg / mL-2 mg / mL. Under stirring and argon gas purging, add the prepared stock solution dropwise to an aqueous solution of the amphiphilic polymer poloxamer 188 (F127) of 10 mg / mL-15 mg / mL. Add 200-250 μL of stock solution per milliliter of water. Keep stirring and argon gas purging for 20-30 minutes until the tetrahydrofuran is completely removed.

[0071] 2) The obtained nanoparticle dispersion was dialyzed in ultrapure water for 1-2 days and then freeze-dried to obtain near-infrared photothermal nanoparticles (MeO-TSI@F127 NPs).

[0072] The hydrogel preparation method of the present invention includes the following steps:

[0073] 1) Dissolve NIPAM and AM in water at a mass ratio of 10:1 to 50:1, where 5 mL to 10 mL of water is needed for every 450 mg of NIPAM. Then add 0.5% to 1% of the mass of NIPAM as the accelerator tetramethylethylenediamine (TEMED), and then add 1% to 2% of the mass of NIPAM as the crosslinking agent MBA. Stir until fully dissolved.

[0074] 2) Add 0.1%-1% KPS by mass of NIPAM to the fully dissolved aqueous solution, stir to dissolve fully, pour into a mold, and polymerize at room temperature to obtain a thermosensitive P(NIPAM-AM) hydrogel;

[0075] The application of a constant-temperature photothermal system of the present invention in the healing of infected wounds involves dropping MeO-TSI@F127 NPs onto the infected wound, and then uniformly applying the prepared thermosensitive P(NIPAM-AM) hydrogel onto the infected wound. Under irradiation with an 808nm near-infrared laser, the MeO-TSI@F127 NPs continuously generate heat, causing the thermosensitive P(NIPAM-AM) hydrogel to undergo a phase transition and turn white. The incident light is effectively scattered, and the MeO-TSI@F127 NPs can no longer generate heat, thereby achieving constant-temperature wound healing.

[0076] Example 1

[0077] 1) Preparation of near-infrared photothermal molecules (MeO-TSI) in the isothermal photothermal system described above:

[0078] Four compounds in a molar ratio of 1:1.5:8:0.05—MeO-TPA-Br (2.83 mmol, 1.00 g), 5-aldehyde-2-thiopheneboronic acid (4.25 mmol, 0.66 g), potassium carbonate (22.64 mmol, 3.12 g), and tetra(triphenylphosphine)palladium (0.14 mmol, 164 mg)—were placed in a 100 mL round-bottom flask, which was then evacuated and purged three times with dry argon. 24 mL of a 5:1 mixture of tetrahydrofuran and water was added, and the mixture was heated to 75 °C and stirred for 8 hours. The mixture was then cooled to room temperature, quenched with water, and extracted three times with dichloromethane. After evaporating the solvent under reduced pressure, the crude product was purified on a silica gel column using a 20:1 mixture of petroleum ether and ethyl acetate as the eluent to give compound MeO-TS in 36% yield.

[0079] The reaction formula is as follows:

[0080]

[0081] Compound MeO-TS (2.60 mmol, 1.00 g) and 1,3-bis(dicyanomethylene)indane (5.20 mmol, 1.26 g) in a molar ratio of 1:2 were dissolved in 50 mL of acetic anhydride and stirred at 65 °C for 2 hours. The mixture was then cooled to room temperature. After evaporating the solvent under reduced pressure, the crude product was purified on a silica gel column using petroleum ether and ethyl acetate in a volume ratio of 5:1 to give a near-infrared photothermal molecule (MeO-TSI) as a black-blue solid in 68% yield.

[0082] The reaction formula is as follows:

[0083]

[0084] 2) Preparation of near-infrared photothermal nanoparticles (MeO-TSI@F127 NPs) in the isothermal photothermal system described above:

[0085] First, 10 mg of F127 powder was dissolved in 1 mL of water to obtain an aqueous solution with a concentration of 10 mg / mL. Then, 200 μL of a 2 mg / mL MeO-TSI tetrahydrofuran stock solution was added under stirring. Next, the suspension was bubbled with dry argon gas for 20 minutes to remove tetrahydrofuran. Finally, the suspension was dialyzed against deionized water with a molecular weight cutoff (MWCO) of 5000 Da for 1 day, and then freeze-dried to obtain MeO-TSI@F127 NPs.

[0086] 3) Preparation of thermosensitive P(NIPAM-AM) hydrogel in the isothermal photothermal system described above:

[0087] NIPAM (3.98 mmol, 450 mg), AM (0.63 mmol, 45 mg), and MBA (0.03 mmol, 4.5 mg) at a mass ratio of 10:1 were dissolved in 5 mL of water and stirred to obtain a homogeneous solution. Next, TEMED (6 μL) at a mass ratio of 1% NIPAM and KPS (0.02 mmol, 5 mg) at a mass ratio of 1% NIPAM were added under continuous stirring and mixed evenly. The resulting solution was then poured into a mold and allowed to stand at room temperature to complete polymerization and gel formation. After 24 hours, P(NIPAM-AM) hydrogel was obtained.

[0088] Example 2

[0089] 1) Preparation of near-infrared photothermal molecules (MeO-TSI) in the isothermal photothermal system described above:

[0090] Four compounds in a molar ratio of 1:2:10:0.1—MeO-TPA-Br (2.83 mmol, 1.00 g), 5-aldehyde-2-thiopheneboronic acid (5.67 mmol, 0.88 g), potassium carbonate (28.30 mmol, 3.90 g), and tetra(triphenylphosphine)palladium (0.28 mmol, 328 mg)—were placed in a 100 mL round-bottom flask, which was then evacuated and purged three times with dry argon. 25 mL of a 4:1 mixture of tetrahydrofuran and water was added, and the mixture was heated to 70 °C and stirred for 12 hours. The mixture was then cooled to room temperature, quenched with water, and extracted five times with dichloromethane. After evaporating the solvent under reduced pressure, the crude product was purified on a silica gel column using a 30:1 mixture of petroleum ether and ethyl acetate as the eluent to give compound MeO-TS in 32% yield.

[0091] The reaction formula is as follows:

[0092]

[0093] Compound MeO-TS (2.60 mmol, 1.00 g) and 1,3-bis(dicyanomethylene)indane (3.90 mmol, 0.95 g) in a molar ratio of 1:1.5 were dissolved in 60 mL of acetic anhydride, and then stirred at 60 °C for 6 hours. The mixture was then cooled to room temperature. After evaporating the solvent under reduced pressure, the crude product was purified on a silica gel column using petroleum ether and ethyl acetate in a volume ratio of 10:1 to give a near-infrared photothermal molecule (MeO-TSI) as a black-blue solid in 74% yield.

[0094] The reaction formula is as follows:

[0095]

[0096] 2) Preparation of near-infrared photothermal nanoparticles (MeO-TSI@F127 NPs) in the isothermal photothermal system described above:

[0097] First, 15 mg of F127 powder was dissolved in 1 mL of water to obtain an aqueous solution with a concentration of 15 mg / mL. Then, 250 μL of a 1 mg / mL MeO-TSI tetrahydrofuran stock solution was added under stirring. Next, the suspension was bubbled with dry argon gas for 30 minutes to remove tetrahydrofuran. Finally, the suspension was dialyzed against deionized water with a molecular weight cutoff (MWCO) of 5000 Da for 2 days, and then freeze-dried to obtain MeO-TSI@F127 NPs.

[0098] 3) Preparation of thermosensitive P(NIPAM-AM) hydrogel in the isothermal photothermal system described above:

[0099] NIPAM (3.98 mmol, 450 mg), AM (0.21 mmol, 15 mg), and MBA (0.05 mmol, 6.75 mg) at a mass ratio of 30:1 were dissolved in 7.5 mL of water and stirred to obtain a homogeneous solution. Next, TEMED (3 μL) at a mass ratio of 0.5% of NIPAM and KPS (0.01 mmol, 2.5 mg) at a mass ratio of 0.5% of NIPAM were added under continuous stirring and mixed evenly. The resulting solution was then poured into a mold and allowed to stand at room temperature to complete polymerization and gel formation. After 24 hours, P(NIPAM-AM) hydrogel was obtained.

[0100] Example 3

[0101] 1) Preparation of near-infrared photothermal molecules (MeO-TSI) in the isothermal photothermal system described above:

[0102] Four compounds in a molar ratio of 1:1.8:9:0.08—MeO-TPA-Br (2.83 mmol, 1.00 g), 5-aldehyde-2-thiopheneboronic acid (5.09 mmol, 0.79 g), potassium carbonate (25.47 mmol, 3.51 g), and tetra(triphenylphosphine)palladium (0.23 mmol, 262.4 mg)—were placed in a 100 mL round-bottom flask, which was then evacuated and purged four times with dry argon. 28 mL of a 6:1 mixture of tetrahydrofuran and water was added, and the mixture was heated to 73 °C and stirred for 10 hours. The mixture was then cooled to room temperature, quenched with water, and extracted four times with dichloromethane. After evaporating the solvent under reduced pressure, the crude product was purified on a silica gel column using a 50:1 mixture of petroleum ether and ethyl acetate as the eluent to give compound MeO-TS in 35% yield.

[0103] The reaction formula is as follows:

[0104]

[0105] Compound MeO-TS (2.60 mmol, 1.00 g) and 1,3-bis(dicyanomethylene)indane (4.68 mmol, 1.14 g) in a molar ratio of 1:1.8 were dissolved in 55 mL of acetic anhydride, and then stirred at 62 °C for 4 hours. The mixture was then cooled to room temperature. After evaporating the solvent under reduced pressure, the crude product was purified on a silica gel column using petroleum ether and ethyl acetate in a volume ratio of 8:1 to give a near-infrared photothermal molecule (MeO-TSI) as a black-blue solid in 65% yield.

[0106] The reaction formula is as follows:

[0107]

[0108] 2) Preparation of near-infrared photothermal nanoparticles (MeO-TSI@F127 NPs) in the isothermal photothermal system described above:

[0109] First, 12 mg of F127 powder was dissolved in 1 mL of water to obtain an aqueous solution with a concentration of 12 mg / mL. Then, 225 μL of a 1.5 mg / mL MeO-TSI tetrahydrofuran stock solution was added under stirring. Next, the suspension was bubbled with dry argon gas for 25 minutes to remove tetrahydrofuran. Finally, the suspension was dialyzed against deionized water with a molecular weight cutoff (MWCO) of 5000 Da for 1.5 days, and then freeze-dried to obtain MeO-TSI@F127 NPs.

[0110] 3) Preparation of thermosensitive P(NIPAM-AM) hydrogel in the isothermal photothermal system described above:

[0111] NIPAM (3.98 mmol, 450 mg), AM (0.13 mmol, 9 mg), and MBA (0.07 mmol, 9 mg) at a mass ratio of 50:1 were dissolved in 10 mL of water and stirred to obtain a homogeneous solution. Next, TEMED (4.5 μL) at a mass ratio of 0.75% of NIPAM and KPS (0.002 mmol, 0.5 mg) at a mass ratio of 0.1% of NIPAM were added under continuous stirring and mixed evenly. The resulting solution was then poured into a mold and allowed to stand at room temperature to complete polymerization and gel formation. After 24 hours, P(NIPAM-AM) hydrogel was obtained.

[0112] Example 4

[0113] The photothermal effects of MeO-TSI and MeO-TSI@F127 NPs in the isothermal photothermal system prepared by Example 1, 2 or 3 were tested under an 808 nm laser:

[0114] The MeO-TSI prepared in Examples 1, 2, or 3 exhibited broad absorption in the 500-900 nm range, with absorption extending into the NIR region, which facilitated deep tissue penetration and resulted in less tissue damage than ultraviolet and visible light. Furthermore, MeO-TSI exhibited significant aggregation-induced emission properties in chloroform / n-hexane solvents with varying n-hexane fractions. Figure 1 B). Subsequently, the photothermal effect of MeO-TSI nano-aggregates was further verified. After laser irradiation (1.2 W / cm2) for 10 minutes, the MeO-TSI nano-aggregates effectively heated up and quickly reached a stable temperature, with the highest temperature reaching 68.5℃. Figure 1 C).

[0115] The MeO-TSI@F127 NPs prepared in Examples 1, 2, or 3 showed no significant change in absorption range and peak shape compared to MeO-TSI nanoaggregates, but only a slight redshift. Figure 1 D). Following this, the particle size and morphology of the nanoparticles were determined using dynamic light scattering and transmission electron microscopy. The results showed that the nanoparticles had a particle size of 75.3 nm and exhibited a spherical morphology. Figure 1 E and 1F). Furthermore, the nanoparticles were dispersed in water under environmental conditions for 7 days, and particle size changes were continuously tested, demonstrating that the nanoparticles possess good colloidal stability. Figure 1 G). Subsequently, the photothermal properties of the nanoparticles were tested under the same experimental conditions, showing excellent photothermal effects (G). Figure 1 H). Finally, throughout the five cycles of heating and cooling, the nanoparticles maintained good photothermal conversion capabilities; however, ICG exhibited severe photobleaching after two cycles, indicating that the nanoparticles possess excellent photothermal stability. Figure 1 I). The excellent photothermal capabilities and photothermal stability exhibited by MeO-TSI@F127 NPs are important prerequisites for the implementation of isothermal photothermal systems.

[0116] Example 5

[0117] The temperature control effect of the P(NIPAM-AM) hydrogel in the isothermal photothermal system prepared by Example 1 was tested:

[0118] like Figure 2 As shown in Figure A, when the mass ratio of NIPAM to AM is 10:1, the measured T cp The temperature was 48.9℃. Next, we evaluated the ability of the P(NIPAM-AM) hydrogel to modulate near-infrared light transmittance during the phase transition. A coverslip was placed on the top surface of a cell filled with MeO-TSI@F127NPs, ensuring physical contact between them. After covering with the P(NIPAM-AM) hydrogel, an 808 nm near-infrared laser was irradiated in a direction perpendicular to the cover layer. The presence of the coverslip and the transparent hydrogel did not affect the near-infrared penetration into the MeO-TSI@F127NPs dispersion. Figure 2 B). In this case, the photothermal conversion of MeO-TSI@F127 NPs significantly increased the system temperature, causing a phase transition in the P(NIPAM-AM) hydrogel, making it an opaque white bulk. During this process, the penetration of near-infrared light was gradually blocked. When the temperature reached T... cp At that time, the area on the hydrogel corresponding to the laser spot turns completely white, and the laser beam hardly passes through the hydrogel, automatically adjusting the photothermal equilibrium temperature to the T value of the P(NIPAM-AM) hydrogel. cpFurthermore, in the absence of P(NIPAM-AM) hydrogel, the highest photothermal temperature was 72.5℃ when the concentration of MeO-TSI@F127 NPs and the laser power density were 200 μM and 1.5 W / cm², respectively. However, in the presence of P(NIPAM-AM) hydrogel, the upper limit of the equilibrium temperature was approximately 49℃, close to the Te of the hydrogel. cp ( Figure 2 C). This comparison can also be visualized using thermal images over time, demonstrating the excellent temperature control capabilities of the P(NIPAM-AM) hydrogel. Figure 2 D). This ability to adjust the incident light transmittance through phase transition whitening is verified, which is the key to the P(NIPAM-AM) hydrogel maintaining a constant PTT process temperature.

[0119] Example 6

[0120] An in vitro bactericidal effect experiment was conducted on MeO-TSI@F127 NPs in the isothermal photothermal system prepared by Example 1, 2 or 3:

[0121] The in vitro temperature-dependent antibacterial behavior of MeO-TSI@F127 NPs against MRSA was investigated by varying the concentration of MeO-TSI@F127 NPs from 0 to 200 μM, under conditions of no near-infrared irradiation and with near-infrared irradiation. Figure 3 The concentration of MeO-TSI@F127 NPs in B, 3C, and 3D was 100 μM. Figure 3 As shown in A and 3B, no significant antibacterial activity was observed in any of the groups without near-infrared irradiation. However, under near-infrared irradiation, the antibacterial effect was approximately 99.9%. Live / dead fluorescence staining was then performed to visualize the antibacterial effect of MeO-TSI@F127 NPs at 100 μM. Figure 3 As shown in Figure C, all bacteria exhibited green fluorescence in the phosphate-buffered saline (PBS), MeO-TSI@F127 NPs, and near-infrared irradiation groups alone. In contrast, near-infrared irradiation converted almost all green-emitting bacteria in the MeO-TSI@F127 NPs-treated group to red-emitting bacteria, consistent with the results of the plate count assay. The morphological changes of the bacteria under different treatments were characterized using scanning electron microscopy. Figure 3As shown in Figure D, in the groups treated with PBS, MeO-TSI@F127 NPs, and NIR irradiation alone, almost all bacteria maintained relatively intact and smooth cell bodies. Conversely, when MeO-TSI@F127 NPs and near-infrared irradiation were applied simultaneously, significant collapse and fusion of the bacterial membrane were observed, attributed to high-temperature-mediated bacterial structural disruption. Therefore, the excellent bactericidal ability exhibited by MeO-TSI@F127 NPs is highly advantageous for the application of isothermal photothermal systems in wound healing.

[0122] Example 7

[0123] Application of the aforementioned constant-temperature photothermal system in the healing of infected wounds:

[0124] Based on in vitro antibacterial results, we applied the aforementioned isothermal photothermal system to a mouse model of bacterial infection to investigate its in vivo antibacterial behavior. Specifically, T... cp A P(NIPAM-AM) hydrogel (NIPAM:AM = 10:1) at approximately 49°C was used to ensure bactericidal properties while avoiding significant thermal damage to surrounding healthy tissues and / or organs. Specifically, each mouse had a circular skin wound on its back, which was then inoculated with MRSA to induce bacterial infection. Infected mice were randomly divided into 5 groups for different treatments. Figure 4 A) During near-infrared irradiation, an infrared camera is used to dynamically monitor the temperature, such as... Figure 4 As shown in B and 4C, compared with the group not receiving near-infrared irradiation, the PBS-treated group showed only a slight increase in temperature after near-infrared irradiation. However, the MeO-TSI@F127 NPs group and the near-infrared irradiation group (denoted as "NP / L") exhibited significant wound temperature gradients, reaching as high as 61°C after laser irradiation. Interestingly, the application of P(NIPAM-AM) hydrogel effectively unified the temperature distribution in the wound area, achieving temperature equilibrium at approximately 49°C. This is entirely attributed to the near-infrared light blocking mediated by the phase transition of P(NIPAM-AM) hydrogel in the isothermal photothermal system. Subsequently, we recorded changes in mouse survival rate, infected area, and body weight throughout the evaluation period (11 days). Figure 4 As shown in Figure D, the survival rate of mice treated in the "NP / L" group decreased to 60% on day 2, while mice in other groups survived throughout the experiment. We hypothesize that the sharp decline in survival was due to thermal damage caused by uncontrolled high temperatures, which severely affected the biological function of adjacent tissues and / or major organs. Regarding wound healing, although the infected area decreased in all groups over time, the recovery rate in the "NP / L" group was slightly slower than that in the control group. Figure 4E and 4F). Notably, the wounds in the "NP / L" group darkened on day 2 and formed heat-induced eschars on day 5, unlike the conventional eschar formation in other groups. Surprisingly, the healing of infected wounds in the "NP / L" group (denoted as "NP / H / L") was significantly better than in other groups, indicating the advantage of the isothermal photothermal system in wound healing. Throughout the experimental period, except for day 1, all mice showed a trend of increasing body weight, due to the occurrence of injury and bacterial infection ( Figure 4 G). To preliminarily evaluate the antibacterial effect, one mouse from each group was randomly selected and sacrificed on day 2, and half of the infected tissue was collected for bacterial quantification. At the end of the experiment, all surviving mice were sacrificed, and half of the infected tissue was homogenized for bacterial quantification. Figure 4 As shown in H and 4I, compared with the preliminary antibacterial results on day 2, the NIR irradiation of the "NP / L" group decreased by three orders of magnitude on both day 2 and day 11 (corresponding to a killing efficiency of 99.9%). While the "NP / H / L" group only showed a two-order-of-magnitude decrease on day 2 (corresponding to a killing efficiency of 99%), the difference in antibacterial effect between the "NP / L" and "NP / H / L" groups significantly decreased on day 11, suggesting faster wound healing. In summary, this strongly demonstrates the excellent effect of the described isothermal photothermal system on wound healing.

[0125] To gain a deeper understanding of thermal injury, we performed histological analysis of the infected wound and its adjacent tissues. On day 2, hematoxylin and eosin (H&E) staining showed that, except for the "NP / L" group, all infected wound surfaces exhibited significant inflammatory responses, with the "NP / H / L" group showing the lowest degree of inflammation. Figure 5 A). Although the apical surface of inflammation disappeared in the "NP / L" group, the dermal tissue structure was severely damaged by heat, exhibiting obvious nuclear cleavage and cell lysis. Furthermore, significant inflammatory responses were observed in adjacent normal tissue, due to unavoidable heat diffusion from the irradiation center, reaching temperatures as high as 61°C. In contrast, no significant heat damage was observed in the "NP / H / L" group in the wound area and adjacent normal tissue, indicating that the P(NIPAM-AM) hydrogel in the isothermal photothermal system has a protective effect on healthy tissue during the PTT process. After the experiment, the entire skin, including the wound and adjacent normal tissue, was collected, half of which was stained with H&E. Figure 5As shown in Figure B, although some inflammatory cells were present in all groups, the number of inflammatory cells was significantly reduced in the "NP / H / L" group, indicating the role of the isothermal photothermal system in alleviating the inflammatory response. Generally, the thinner the epidermis, the faster the epidermal regeneration. It can be clearly seen that the thickness of the new epidermis in the "NP / H / L" group was much thinner than in all other groups, even comparable to the thickness of normal skin tissue. Furthermore, the number of new hair follicles was positively correlated with complete wound healing. While only a small number of immature hair follicles were found in the PBS group, the MeO-TSI@F127 NPs group, and the NIR irradiation alone group, no hair follicles were found in the wound area of ​​the "NP / L" group. Surprisingly, the wound area of ​​the "NP / H / L" group had a large number of relatively mature hair follicles, suggesting rapid wound healing and epidermal reconstruction.

[0126] The hydrogel prepared by this invention is simple to synthesize and can be reused. In in vitro and mouse model experiments, the temperature can be stably controlled near the hydrogel cloud point temperature (Tcp), which effectively avoids thermal damage to normal tissues caused by high temperature. Moreover, this method is applicable to most photothermal therapy processes.

[0127] The technical solutions disclosed and proposed in this invention can be implemented by those skilled in the art by appropriately modifying the conditions and routes, etc. Although the methods and preparation techniques of this invention have been described through preferred embodiments, those skilled in the art can obviously modify or recombine the methods and technical routes described herein without departing from the content, spirit, and scope of this invention to achieve the final preparation technique. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included within the spirit, scope, and content of this invention.

Claims

1. A constant-temperature photothermal system, characterized in that, The construction method includes the following steps: 1) Near-infrared photothermal molecules were obtained through one-step Knoevenagel condensation, and then near-infrared photothermal nanoparticles were prepared by nanoprecipitation using amphiphilic polymers. 2) By using free radical polymerization, two monomers, N-isopropylacrylamide and acrylamide, were added, along with potassium persulfate as an initiator and N,N-methylenebisacrylamide as a crosslinking agent. After thorough mixing, the mixture was polymerized at room temperature to obtain poly(N-isopropylacrylamide-acrylamide) hydrogel. 3) Near-infrared photothermal nanoparticles are dropped onto the infected wound, and then poly(N-isopropylacrylamide-acrylamide) hydrogel is applied to the infected wound to achieve constant temperature wound healing under near-infrared laser irradiation. The near-infrared photothermal molecule has the structure shown in the compound 5-(4-(4-methoxyphenyl)diphenylamino)thiophene-2-(1,3-bis(dicyanomethylene)indane) (MeO-TSI); ； The hydrogel preparation method includes the following steps: 1) Dissolve N-isopropylacrylamide and acrylamide in water at a mass ratio of 10:1-50:1, where 5-10 mL of water is needed for every 450 mg of N-isopropylacrylamide. Then add 0.5%-1% of tetramethylethylenediamine as an accelerator by mass of N-isopropylacrylamide, and then add 1%-2% of N,N-methylenebisacrylamide as a crosslinking agent by mass of N-isopropylacrylamide. Stir until fully dissolved. 2) Add 0.1%-1% of potassium persulfate by mass of N-isopropylacrylamide to the fully dissolved aqueous solution, stir to dissolve fully, pour into a mold, and polymerize at room temperature to obtain a thermosensitive poly(N-isopropylacrylamide-acrylamide) hydrogel.

2. The isothermal photothermal system as described in claim 1, characterized in that, The preparation method of the near-infrared photothermal molecule includes the following: 1) Weigh out four compounds in a molar ratio of 1:1.5:8:0.05-1:2:10:0.1: 4-bromo-4-methoxytriphenylamine, 5-aldehyde-2-thiopheneboronic acid, potassium carbonate, and tetra(triphenylphosphine)palladium. After evacuating the vacuum and purging with argon gas 3-4 times, add a mixed solution of tetrahydrofuran and water in a volume ratio of 4:1-6:

1. Heat the mixture at 70-75°C for 8-12 hours. After the reaction is complete, purify the mixture by thin-layer chromatography to obtain compound 5-(4-(4-methoxyphenyl)diphenylamino)thiophene-2-carboxaldehyde. 2) The compounds 5-(4-(4-methoxyphenyl)diphenylamino)thiophen-2-carboxaldehyde and 1,3-bis(dicyanomethylene)indane in a molar ratio of 1:1.5-1:2 were dissolved in acetic anhydride, wherein 50-60 mL of acetic anhydride was required for each gram of 5-(4-(4-methoxyphenyl)diphenylamino)thiophen-2-carboxaldehyde. The reaction was carried out at 60-65°C for 2-6 hours. After the reaction was completed, the compounds were purified by thin-layer chromatography to obtain the compound 5-(4-(4-methoxyphenyl)diphenylamino)thiophen-2-(1,3-bis(dicyanomethylene)indane).

3. The isothermal photothermal system as described in claim 1, characterized in that, The method for preparing near-infrared photothermal nanoparticles includes the following steps: 1) Dissolve compound 5-(4-(4-methoxyphenyl)diphenylamino)thiophene-2-(1,3-bis(dicyanomethylene)indane) in tetrahydrofuran to prepare a stock solution of 1 mg / mL-2 mg / mL. Under stirring and argon gas purging, add the prepared stock solution dropwise to an aqueous solution of the amphiphilic polymer poloxamer 188 (F127) of 10 mg / mL-15 mg / mL. Add 200-250 μL of stock solution per milliliter of water. Keep stirring and purging with argon gas for 20-30 minutes until the tetrahydrofuran is completely removed. 2) Dialyze the obtained nanoparticle dispersion in ultrapure water for 1-2 days, and freeze-dry to obtain near-infrared photothermal nanoparticles.

4. Application of the constant-temperature photothermal system of claim 1 in the manufacture of infected wound healing products; Near-infrared photothermal nanoparticles are dropped onto the infected wound, and then the prepared thermosensitive poly(N-isopropylacrylamide-acrylamide) hydrogel is uniformly applied to the infected wound. Under irradiation with an 808nm near-infrared laser, the near-infrared photothermal nanoparticles continuously generate heat, causing the thermosensitive poly(N-isopropylacrylamide-acrylamide) hydrogel to undergo a phase transition and turn white. The incident light is effectively scattered, and the near-infrared photothermal nanoparticles can no longer generate heat, thereby achieving constant-temperature wound healing.

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