A step-by-step laser-stimulated multi-active oxygen-releasing wound dressing and its preparation method and application

Abstract

The present invention discloses a step-by-step laser-stimulated multi-active oxygen species-releasing wound dressing, as well as its preparation method and application. The wound dressing is prepared by chemically grafting protoporphyrin and upconversion nanoparticles onto a carboxylated cellulose nanofiber matrix to produce smart nanofibers responsive to white light and 980nm lasers. The nanofibers are then reacted with a thermosensitive reagent through hydrogen bonding and electrostatic interactions to form fiber balls. The spherical fibers are then simultaneously loaded with indocyanine green, sodium nitroprusside, and doxorubicin in the cavities of the balls to produce the step-by-step laser-stimulated multi-active oxygen species-releasing wound dressing. The wound dressing can respond gradually to three lasers with different penetration depths and release different levels of reactive oxygen species.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomass fiber modification, and in particular relates to a stepped laser-excited multi-active oxygen-releasing wound dressing, a preparation method thereof, and an application thereof. Background Art

[0002] The skin is the body's first line of defense. Healing after skin damage is a systematic and dynamic process involving four phases: hemostasis, inflammation, proliferation, and re-epithelialization. While most skin defects can heal spontaneously within one or two weeks, deep, full-thickness wounds or penetrating wounds often lack this ability. Furthermore, during the healing period, bacteria from the environment can easily invade the exposed wound bed, leading to bacterial infection and even colonization, forming a bacterial biofilm. Furthermore, the microenvironment of some complex wounds may harbor other issues that hinder the healing process. For example, ulcerative wounds in diabetic patients often harbor a high blood glucose microenvironment, multiple bacterial infections, and capillary defects. Wounds after skin tumor surgery often harbor residual tumor cells, which are prone to proliferation and recurrence. These issues further impair the regulation of the wound microenvironment, delaying the tissue remodeling phase and thus hindering healing. Traditional wound dressings, such as gauze and films, only provide a physical barrier and fail to provide effective therapeutic intervention to promote healing. Drug therapy is a conventional treatment method in clinical practice, but the use of drugs to treat chronic wounds can easily lead to drug dependence, resulting in the emergence of drug-resistant bacteria, and cannot effectively target problems in the superficial epidermis and deep tissues of full-thickness wounds at the same time. Summary of the Invention

[0003] The purpose of the present invention is to provide a stepped laser-excited multi-active oxygen-releasing wound dressing and a preparation method thereof. The wound dressing can respond gradiently to three lasers with different penetration depths and release different active oxygen species. It can achieve long-term resistance to the invasion of external bacteria into the wound in complex wounds while killing bacteria or tumor cells colonized deep in the tissue, promoting the formation of capillaries deep in the tissue, and accelerating wound healing.

[0004] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0005] A method for preparing a stepped laser-excited multi-active oxygen-releasing wound dressing comprises the following steps:

[0006] S1. Preparation of cavitated carboxylated cellulose nanofibers: Carboxylated cellulose nanofibers were prepared using a two-step oxidation process. First, the hydroxyl groups at C2 and C3 of the cellulose structural units of pulp fibers were selectively oxidized to aldehyde groups using sodium periodate to produce dialdehyde fibers. Then, the hydroxyl groups at C6 of the cellulose structural units were oxidized to carboxyl groups using a TEMPO / NaBr / NaClO oxidation system to obtain carboxylated cellulose nanofibers having a carboxyl group density of 0.5-1.6 mmol / g, a fiber length of 20-100 nm, and a fiber diameter of 2-15 nm. Cavitation of the carboxylated cellulose nanofibers was achieved by ultrasonication.

[0007] S2. Preparation of 980nm laser-responsive smart fibers: prepared by amidation reaction of cavitated carboxylated cellulose nanofibers with amino-upconversion nanoparticles under alkaline conditions; the 980nm laser-responsive smart fibers can convert 980nm laser light into 520-560nm white light;

[0008] S3. Preparation of white light-responsive smart fibers: Protoporphyrin and cavitated carboxylated cellulose nanofibers were prepared by esterification reaction in the dark at room temperature; the excitation wavelength of the white light-responsive smart fibers was 635 nm;

[0009] S4. Preparation of a stepped laser-stimulated multi-reactive oxygen species-releasing wound dressing: After uniformly dispersing white light-responsive smart fibers and 980nm laser-responsive smart fibers in water, they are reacted with poloxamer reagent through hydrogen bonding and electrostatic interaction to obtain fiber balls, and then the spherical fiber cavities are used to synchronously load indocyanine green, sodium nitroprusside and doxorubicin to prepare the wound dressing; the stepped laser-stimulated multi-reactive oxygen species-releasing wound dressing releases singlet oxygen under 635nm and 808nm laser excitation, and releases nitric oxide under 980nm laser irradiation.

[0010] Furthermore, the preparation of the cavitated carboxylated cellulose nanofibers in step S1 mainly comprises the following steps: reacting pulp fiber with sodium periodate at 25 to 35° C. for 4 to 5 hours to obtain dialdehyde fiber; then sequentially adding TEMPO, sodium hypochlorite and sodium bromide to the dialdehyde fiber, adjusting the pH of the reaction system to 10 using sodium hydroxide and oxidizing for 16 to 18 hours, adding ethanol to quench the reaction, and then washing and drying to obtain carboxylated cellulose nanofibers; and then ultrasonicating the carboxylated cellulose nanofibers at a power of 800 W / h and a 4° C. ice water bath for 1 to 2 hours to obtain cavitated carboxylated cellulose nanofibers; the mass ratio of the pulp fiber to sodium periodate is 1:1 to 2, the mass ratio of the dialdehyde fiber to TEMPO is 400:5 to 7, and the mass ratio of the dialdehyde fiber to sodium bromide is 1:1.312.

[0011] Furthermore, the preparation of the 980nm laser-responsive smart fiber in step S2 is mainly carried out as follows: YCl3, YbCl3, ErCl3, oleic acid and octene are uniformly mixed and placed at 140-160°C for stirring and reacting for 30-40 minutes, and then a methanol solution of NaOH and NH4F is added, and the mixture is stirred and reacted for 10 minutes. After removing the methanol at 100°C, the mixture is heated to 320°C for reaction for 1-3 hours, and NaYF4:Yb,Er particles are obtained after cooling, washing and drying. The NaYF4:Yb,Er particles are then dispersed in water, and hexadecyltrimethylammonium bromide, ammonia water with a mass concentration of 30wt% and tetramethoxybenzaldehyde are added in sequence. The method comprises the following steps: adding 1-aminopropyltriethoxysilane, stirring and reacting at 25°C for 20 to 24 hours, then adding 3-aminopropyltriethoxysilane and reacting for 12 to 16 hours, washing and drying to obtain amino upconversion nanoparticles; then uniformly dispersing the amino upconversion nanoparticles and cavitated carboxylated cellulose nanofibers in water at a mass ratio of 1 to 2.5:50, then adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in sequence, stirring and reacting at pH = 8 to 10 for 18 to 24 hours, washing until the supernatant is neutral, and freeze-drying the precipitate to obtain 980nm laser responsive smart fibers.

[0012] Furthermore, the main operation of preparing the white light responsive smart fiber in step S3 is: adding a dimethyl sulfoxide solution of protoporphyrin with a concentration of 0.5 mg / mL to an aqueous dispersion of cavitated carboxylated cellulose nanofibers with a mass fraction of 0.05 to 0.5 wt% at a volume ratio of 0.4 to 1:1, and then adding 4-dimethylaminopyridine and N-hydroxysuccinimide in sequence, reacting for 12 to 14 hours in the dark, at room temperature and under stirring conditions, and then dialyzing in water for 20 to 24 hours using a MWCO1000D dialysis bag, and freeze-drying to obtain the white light responsive smart fiber.

[0013] Furthermore, the preparation of step S4 mainly comprises the following steps: uniformly dispersing the white light responsive smart fiber and the 980 nm laser responsive smart fiber in water, ultrasonically treating for 10 minutes, adding a poloxamer reagent, stirring at 4°C for 10 minutes to obtain fiber balls, and then adding indocyanine green, sodium nitroprusside and doxorubicin, stirring at 4°C and in the dark for 2 to 3 hours to obtain a step-by-step laser-excited multi-active oxygen-releasing wound dressing; the mass ratio of the white light responsive smart fiber, 980 nm laser responsive smart fiber, poloxamer reagent, indocyanine green, sodium nitroprusside and doxorubicin is 20:10 to 20:450:1 to 3:2:1.

[0014] The present invention uses ultrasonically cavitated carboxylated cellulose nanofibers as the matrix. The ultrasonic cavitation effect can increase the specific surface area of the fibers, thereby improving the fiber's adsorption capacity for active drugs. The wound dressing is assembled from fiber balls formed by the interweaving of white light-responsive smart fibers, 980nm laser-responsive smart fibers and poloxamer reagents. The spherical cavity can further provide loading space for drugs, achieving simultaneous and efficient loading of multiple active drugs (indocyanine green, sodium nitroprusside and doxorubicin).

[0015] The stepped laser-excited multi-active oxygen-releasing wound dressing prepared by the present invention can be used to treat complex wounds, such as wounds infected with super bacteria, wounds infected with bacteria after skin tumor resection, or diabetic ulcer wounds. After the dressing is applied to the wound, it can release singlet oxygen under 635nm and 808nm laser excitation, thereby achieving long-term resistance to the invasion of external bacteria into the wound while killing bacteria or tumor cells colonized deep in the tissue; under 980nm laser irradiation, it releases nitric oxide, which promotes the formation of capillaries deep in the tissue and accelerates wound healing.

[0016] The present invention has the following beneficial effects:

[0017] (1) The stepped laser-excited multi-active oxygen-releasing wound dressing prepared by the present invention has gradient laser stimulation responsiveness. It uses 635nm, 808nm and 980nm lasers with different penetration depths to excite the wound dressing to release singlet oxygen and nitric oxide at different layers of the wound, respectively. It can achieve long-term antibacterial effects in the superficial skin layer of the wound, anti-bacterial colonization in the deep tissue, kill tumor cells, and promote the formation of capillaries deep in the tissue. By treating the entire layer of the wound, it achieves a healing effect, overcoming the problem that traditional treatment methods cannot effectively treat both the superficial epidermis and deep tissues of the wound at the same time.

[0018] (2) The present invention uses ultrasonically cavitated carboxylated cellulose nanofibers as the matrix. The ultrasonic cavitation effect can increase the specific surface area of the fibers, thereby improving the fiber's adsorption capacity for active drugs. The fiber balls, which are formed by hydrogen bonding and electrostatic interaction between white light-responsive smart fibers, 980nm laser-responsive smart fibers, and poloxamer reagents, further provide drug loading space, achieving simultaneous and efficient loading of multiple active drugs (indocyanine green, sodium nitroprusside, and doxorubicin). DETAILED DESCRIPTION

[0019] Example 1

[0020] S1. Preparation of cavitated carboxylated cellulose nanofibers: Take 2 g of absolutely dry bagasse pulp fiber in a conical flask, add 200 mL of sodium phosphate buffer solution (0.05 M, pH = 6.8), then add 2.0 g of sodium periodate, wrap it with tin foil, stir it at 25 ° C for 5 hours, and then add 10 mL of ethanol to terminate the reaction. The product is filtered, washed, and dried to obtain dialdehyde fiber. To 2 g of dialdehyde fiber, 200 mL of sodium phosphate buffer solution (0.05 M, pH = 6.8) was added, followed by sealed stirring at 500 rpm and 55°C. 0.025 g of TEMPO was then added, followed by 1.69 M sodium hypochlorite solution (1.183 mL), and finally 2.6246 g of sodium bromide. The pH of the reaction system was adjusted to 10 with sodium hydroxide and then oxidized for 18 h. 5 mL of ethanol was added for quenching, followed by washing and drying to obtain carboxylated cellulose nanofibers with a carboxyl group density of 0.5-1.6 mmol / g, a fiber length of 20-100 nm, and a fiber diameter of 2-15 nm. 2 g of carboxylated cellulose nanofibers were placed in a glass beaker, 300 mL of sodium phosphate buffer solution (0.05 M, pH = 6.8) was added, and the mixture was ultrasonicated at 800 W / h in a 4°C ice-water bath for 1 h. The reaction was then centrifuged and dried to obtain cavitated carboxylated cellulose nanofibers.

[0021] S2.980nm laser responsive smart fiber: 0.0156g YCl3, 0.0502g YbCl3, 0.0058g ErCl3, 12mL oleic acid and 15mL octene were added to a three-necked flask, and then reacted at 600rmp and 140℃ for 40min. After cooling to room temperature, 15mL of NaOH and NH4F methanol solution were added and stirred for 10min. The mass ratio of NaOH, NH4F and methanol was 1:1:10. After heating to 100℃ to remove methanol, it was heated to 320℃ for 1h, cooled, washed and dried to obtain NaYF4:Yb,Er particles. 0.5g NaYF4:Yb,Er particles were dispersed in 20mL water, and 0.1000g hexadecyltrimethylammonium bromide, 0.1mL mass fraction were added in sequence. Ammonia water with a mass of 30 wt% and 0.04 mL of tetramethoxysilane were reacted at 1000 rpm and 25 ° C for 24 hours, and then 32 μL of 3-aminopropyltriethoxysilane was added to react for 12 hours, washed, and dried to obtain amino upconversion nanoparticles; then 0.0400 g of amino upconversion nanoparticles and 2.0000 g of cavitated carboxylated cellulose nanofibers were uniformly dispersed in 300 mL of water, and then 1.0000 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 1.0000 g of N-hydroxysuccinimide were added in sequence, and the pH of the reaction system was adjusted to 8 using NaOH solution (0.01 M), and the reaction was stirred for 24 hours, washed until the supernatant was neutral, and the precipitate was freeze-dried to obtain 980 nm laser-responsive smart fibers.

[0022] S3. Preparation of white-light-responsive smart fibers: 20 mL of a 0.5 mg / mL DMSO solution of protoporphyrin was added to 50 mL of a 0.05 wt% aqueous dispersion of cavitated carboxylated cellulose nanofibers, followed by the addition of 30 mg of 4-dimethylaminopyridine and 0.5 g of N-hydroxysuccinimide. The mixture was reacted in the dark at room temperature with stirring for 14 h, then dialyzed in water for 20 h using a MWCO1000D dialysis bag, and freeze-dried to obtain white-light-responsive smart fibers.

[0023] S4. Preparation of step-by-step laser-stimulated multi-reactive oxygen species-releasing wound dressing: 0.2 g of white light-responsive smart fibers and 0.1 g of 980 nm laser-responsive smart fibers were evenly dispersed in 30 mL of water, ultrasonically treated for 10 min, and then 4.5 g of poloxamer F127 was added. The mixture was stirred at 4 °C for 10 min to obtain fiber balls. Then, 10 mg of indocyanine green, 20 mg of sodium nitroprusside and 10 mg of doxorubicin were added, and the mixture was stirred at 4 °C in the dark for 2 h to obtain a step-by-step laser-stimulated multi-reactive oxygen species-releasing wound dressing.

[0024] Example 2

[0025] S1. Preparation of cavitated carboxylated cellulose nanofibers: Take 2 g of absolutely dry bagasse pulp fiber in a conical flask, add 200 mL of sodium phosphate buffer solution (0.05 M, pH = 6.8), then add 2.5 g of sodium periodate, wrap it with tin foil, stir it at 30°C for 4.5 hours, then add 10 mL of ethanol to terminate the reaction, filter and wash the product, and dry it to obtain dialdehyde fiber. To 2g of dialdehyde fiber, 200mL of sodium phosphate buffer solution (0.05M, pH=6.8) was added, followed by sealed stirring at 500rpm and 60°C. Then, 0.028g of TEMPO was added, followed by 1.69M sodium hypochlorite solution (1.183mL), and finally 2.6246g of sodium bromide. The pH of the reaction system was adjusted to 10 with sodium hydroxide and oxidized for 17h. 5mL of ethanol was added for quenching, washing, and drying to obtain carboxylated cellulose nanofibers with a carboxyl group density of 0.5-1.6mmol / g, a fiber length of 20-100nm, and a fiber diameter of 2-15nm. 2g of carboxylated cellulose nanofibers were placed in a glass beaker, 300mL of sodium phosphate buffer solution (0.05M, pH=6.8) was added, and the mixture was ultrasonicated at 800W / h in a 4°C ice-water bath for 1.5h. The reaction was centrifuged and dried to obtain cavitated carboxylated cellulose nanofibers.

[0026] S2.980nm laser responsive smart fiber: 0.0165g YCl3, 0.0585g YbCl3, 0.0050g ErCl3, 10mL oleic acid and 12mL octene were added to a three-necked flask, and then reacted at 600rmp and 150℃ for 35min. After cooling to room temperature, 17mL of NaOH and NH4F methanol solution were added and stirred for 10min. The mass ratio of NaOH, NH4F and methanol was 1:1.2:10. After heating to 100℃ to remove methanol, it was heated to 320℃ for 2h, cooled, washed and dried to obtain NaYF4:Yb,Er particles. 0.5g of NaYF4:Yb,Er particles were dispersed in 20mL water, and 0.1200g hexadecyltrimethylammonium bromide, 0.25mL mass ion were added in sequence. Ammonia water with a molar fraction of 30wt% and 0.05mL tetramethoxysilane were reacted at 1000rmp and 25℃ for 22h, and then 30μL 3-aminopropyltriethoxysilane was added to react for 14h, washed, and dried to obtain amino upconversion nanoparticles; then 0.0600g of amino upconversion nanoparticles and 2.0000g of cavitated carboxylated cellulose nanofibers were uniformly dispersed in 300mL of water, and then 1.5000g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 1.5000g of N-hydroxysuccinimide were added in sequence, and the pH of the reaction system was adjusted to 9 using NaOH solution (0.01M), and the reaction was stirred for 20h, washed until the supernatant was neutral, and the precipitate was freeze-dried to obtain 980nm laser-responsive smart fibers.

[0027] S3. Preparation of white-light-responsive smart fibers: 35 mL of a 0.5 mg / mL DMSO solution of protoporphyrin was added to 50 mL of a 0.1 wt% aqueous dispersion of cavitated carboxylated cellulose nanofibers, followed by the addition of 50 mg of 4-dimethylaminopyridine and 1 g of N-hydroxysuccinimide. The mixture was reacted in the dark at room temperature with stirring for 13 h, then dialyzed in water for 22 h using a MWCO1000D dialysis bag and freeze-dried to obtain white-light-responsive smart fibers.

[0028] S4. Preparation of step-by-step laser-stimulated multi-reactive oxygen species-releasing wound dressing: 0.2 g of white light-responsive smart fibers and 0.15 g of 980 nm laser-responsive smart fibers were evenly dispersed in 30 mL of water, ultrasonically treated for 10 min, and then 4.5 g of poloxamer F127 was added. The mixture was stirred at 4 °C for 10 min to obtain fiber balls. Then, 20 mg of indocyanine green, 20 mg of sodium nitroprusside and 10 mg of doxorubicin were added, and the mixture was stirred at 4 °C in the dark for 2.5 h to obtain a step-by-step laser-stimulated multi-reactive oxygen species-releasing wound dressing.

[0029] Example 3

[0030] S1. Preparation of cavitated carboxylated cellulose nanofibers: Take 2 g of absolutely dry bagasse pulp fiber in a conical flask, add 200 mL of sodium phosphate buffer solution (0.05 M, pH = 6.8), then add 4.0 g of sodium periodate, wrap it with tin foil, stir it at 35 ° C for 4 hours, and then add 10 mL of ethanol to terminate the reaction. The product is filtered, washed, and dried to obtain dialdehyde fiber. To 2 g of dialdehyde fiber, 200 mL of sodium phosphate buffer solution (0.05 M, pH = 6.8) was added, followed by sealed stirring at 500 rpm and 65°C. Then, 0.035 g of TEMPO was added, followed by 1.69 M sodium hypochlorite solution (1.183 mL), and finally 2.6246 g of sodium bromide. The pH of the reaction system was adjusted to 10 with sodium hydroxide and then oxidized for 16 h. 5 mL of ethanol was added for quenching, washing, and drying to obtain carboxylated cellulose nanofibers with a carboxyl group density of 0.5-1.6 mmol / g, a fiber length of 20-100 nm, and a fiber diameter of 2-15 nm. 2 g of carboxylated cellulose nanofibers were placed in a glass beaker, 300 mL of sodium phosphate buffer solution (0.05 M, pH = 6.8) was added, and the mixture was ultrasonicated at 800 W / h in a 4°C ice-water bath for 2 h. The reaction was centrifuged and dried to obtain cavitated carboxylated cellulose nanofibers.

[0031] S2.980nm laser responsive smart fiber: 0.0180g YCl3, 0.0600g YbCl3, 0.0035g ErCl3, 5mL oleic acid and 10mL octene were added to a three-necked flask, and then reacted at 600rmp and 160℃ for 30min. After cooling to room temperature, 20mL of NaOH and NH4F methanol solution were added and stirred for 10min. The mass ratio of NaOH, NH4F and methanol was 1:1.5:10. After heating to 100℃ to remove methanol, it was heated to 320℃ for 3h, cooled, washed and dried to obtain NaYF4:Yb,Er particles. 0.5g of NaYF4:Yb,Er particles were dispersed in 20mL water, and 0.1500g hexadecyltrimethylammonium bromide, 0.5mL mass ion were added in sequence. Ammonia water with a molar fraction of 30wt% and 0.1mL tetramethoxysilane were reacted at 1000rmp and 25℃ for 20h, and then 25μL of 3-aminopropyltriethoxysilane was added and reacted for 16h, washed, and dried to obtain amino upconversion nanoparticles; then 0.100g of amino upconversion nanoparticles and 2.0000g of cavitated carboxylated cellulose nanofibers were uniformly dispersed in 300mL of water, and then 2.0000g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 2.0000g of N-hydroxysuccinimide were added in sequence, and the pH of the reaction system was adjusted to 10 using NaOH solution (0.01M), and the reaction was stirred for 18h, washed until the supernatant was neutral, and the precipitate was freeze-dried to obtain 980nm laser-responsive smart fibers.

[0032] S3. Preparation of white-light-responsive smart fibers: 50 mL of a 0.5 mg / mL DMSO solution of protoporphyrin was added to 50 mL of a 0.5 wt% aqueous dispersion of cavitated carboxylated cellulose nanofibers, followed by the addition of 50 mg of 4-dimethylaminopyridine and 2 g of N-hydroxysuccinimide. The mixture was reacted in the dark at room temperature with stirring for 12 h, then dialyzed in water for 24 h using a MWCO1000D dialysis bag, and freeze-dried to obtain white-light-responsive smart fibers.

[0033] S4. Preparation of step-by-step laser-stimulated multi-reactive oxygen species-releasing wound dressing: 0.2 g of white light-responsive smart fibers and 0.2 g of 980 nm laser-responsive smart fibers were evenly dispersed in 30 mL of water, ultrasonically treated for 10 min, and then 4.5 g of poloxamer F127 was added. The mixture was stirred at 4 °C for 10 min to obtain fiber balls. Then, 30 mg of indocyanine green, 20 mg of sodium nitroprusside and 10 mg of doxorubicin were added, and the mixture was stirred at 4 °C in the dark for 3 h to obtain a step-by-step laser-stimulated multi-reactive oxygen species-releasing wound dressing.

[0034] Performance test of the stepped laser-stimulated multi-active oxygen species-releasing wound dressing prepared in Examples 1, 2, and 3

[0035] ① The stepped laser-excited multi-active oxygen-releasing wound dressings prepared in Examples 1, 2, and 3 were excited by 635nm, 808nm, and 980nm lasers with different penetration depths, respectively. The results showed that the dressings were all able to release active oxygen, that is, the white light-responsive smart fibers prepared in Examples 1, 2, and 3 could release singlet oxygen under 635nm laser excitation, with release amounts of 1.57μmol / g, 1.98μmol / g, and 3.41μmol / g, respectively. Indocyanine green only released active oxygen under 808nm laser excitation, with release amounts of 3.25μmol / g, 5.63μmol / g, and 6.26μmol / g, respectively. Only when sodium nitroprusside and 980nm laser-responsive smart fibers coexisted could nitric oxide be released under 980nm laser excitation, with release amounts of 0.5μmol / g, 1.9μmol / g, and 2.6μmol / g, respectively. This allows the dressing to release different active oxygen species under light-controlled conditions to treat wounds on demand.

[0036] ② The biocompatibility, antibacterial, and antitumor properties of the stepped laser-excited multi-reactive oxygen species-releasing wound dressings prepared in Examples 1, 2, and 3 were studied. The test results showed that the dressings all showed good biocompatibility, antibacterial, anti-infective, and antitumor properties. After 72 hours of co-incubation with mouse fibroblasts, the cell viability of the mouse fibroblasts was 86.3%, 89.6%, and 93.4%, respectively. The dressings showed no toxicity to mouse fibroblasts and were non-irritating or non-sensitizing to mouse skin. Under 635nm, 808nm, and 980nm laser excitation at different penetration depths, the dressings exhibited antibacterial properties against Escherichia coli, Staphylococcus aureus, and drug-resistant Staphylococcus aureus, with antibacterial rates exceeding 99%. The dressings also promoted the healing of infected wounds in mice over a 14-day treatment period, with a healing rate of up to 99%. The dressing also has a strong killing effect on skin cancer cells under 635nm or 808nm laser excitation. After three excitations with 635nm laser, the cell survival rates of cancer cells dropped to 45.3%, 42.2% and 38.7%, respectively, while after three excitations with 808nm laser, the cell survival rates of cancer cells dropped to 8.3%, 5.2% and 4.7%, respectively.

Claims

1. A method for preparing a stepped laser-excited multi-active oxygen species-releasing wound dressing, characterized in that: The following steps are involved: S1. Preparation of cavitated carboxylated cellulose nanofibers: Carboxylated cellulose nanofibers were prepared using a two-step oxidation process. First, sodium periodate was used to selectively oxidize the hydroxyl groups at C2 and C3 of the cellulose structural units of pulp fibers to aldehyde groups, thereby producing dialdehyde fibers. Then, a TEMPO / NaBr / NaClO oxidation system was used to oxidize the hydroxyl groups at C6 of the cellulose structural units to carboxyl groups, thereby obtaining carboxylated cellulose nanofibers having a carboxyl group density of 0.5-1.6 mmol / g, a fiber length of 20-100 nm, and a fiber diameter of 2-15 nm. Cavitation of the carboxylated cellulose nanofibers was achieved by ultrasonication. S2. Preparation of 980nm laser-responsive smart fibers: prepared by amidation reaction of cavitated carboxylated cellulose nanofibers with amino-upconversion nanoparticles under alkaline conditions; the 980nm laser-responsive smart fibers can convert 980nm laser light into 520-560nm white light; S3. Preparation of white light-responsive smart fibers: Protoporphyrin and cavitated carboxylated cellulose nanofibers were prepared by esterification reaction in the dark at room temperature; the excitation wavelength of the white light-responsive smart fibers was 635 nm; S4. Preparation of a stepped laser-stimulated multi-reactive oxygen species-releasing wound dressing: After uniformly dispersing white light-responsive smart fibers and 980nm laser-responsive smart fibers in water, they are reacted with poloxamer reagent through hydrogen bonding and electrostatic interaction to obtain fiber balls, and then the spherical fiber cavities are used to synchronously load indocyanine green, sodium nitroprusside and doxorubicin to prepare the wound dressing; the stepped laser-stimulated multi-reactive oxygen species-releasing wound dressing releases singlet oxygen under 635nm and 808nm laser excitation, and releases nitric oxide under 980nm laser irradiation.

2. The preparation method according to claim 1, characterized in that The preparation of the cavitated carboxylated cellulose nanofibers in step S1 mainly comprises the following steps: reacting pulp fiber with sodium periodate at 25-35° C. for 4-5 hours to obtain dialdehyde fiber; then sequentially adding TEMPO, sodium hypochlorite, and sodium bromide to the dialdehyde fiber, adjusting the pH of the reaction system to 10 using sodium hydroxide, and then oxidizing for 16-18 hours. After adding ethanol to quench the reaction, the reaction is washed and dried to obtain carboxylated cellulose nanofibers; and then ultrasonicating the carboxylated cellulose nanofibers at a power of 800 W / h and in a 4° C. ice water bath for 1-2 hours to obtain cavitated carboxylated cellulose nanofibers. The mass ratio of the pulp fiber to sodium periodate is 1:1-2, the mass ratio of the dialdehyde fiber to TEMPO is 400:5-7, and the mass ratio of the dialdehyde fiber to sodium bromide is 1:1.

312.

3. The preparation method according to claim 1, characterized in that The main operations of preparing the 980nm laser responsive smart fiber in step S2 are as follows: uniformly mix YCl3, YbCl3, ErCl3, oleic acid and octene, place the mixture at 140-160°C and stir for 30-40 minutes, then add a methanol solution of NaOH and NH4F, stir for 10 minutes, remove the methanol at 100°C and then heat to 320°C for 1-3 hours, cool, wash and dry to obtain NaYF4:Yb,Er particles, then disperse the NaYF4:Yb,Er particles in water, and add hexadecyltrimethylammonium bromide, ammonia water with a mass concentration of 30wt% and tetramethoxybenzylamine in sequence. The method comprises the following steps: adding 1-aminopropyltriethoxysilane, stirring and reacting at 25°C for 20-24h, then adding 3-aminopropyltriethoxysilane and reacting for 12-16h, washing and drying to obtain amino upconversion nanoparticles; then uniformly dispersing the amino upconversion nanoparticles and cavitated carboxylated cellulose nanofibers in water at a mass ratio of 1-2.5:50, then adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in sequence, stirring and reacting at pH=8-10 for 18-24h, washing until the supernatant is neutral, and freeze-drying the precipitate to obtain 980nm laser responsive smart fibers.

4. The preparation method according to claim 1, characterized in that The main operation of preparing the white light responsive smart fiber in step S3 is as follows: adding a dimethyl sulfoxide solution of protoporphyrin with a concentration of 0.5 mg / mL to an aqueous dispersion of cavitated carboxylated cellulose nanofibers with a mass fraction of 0.05-0.5 wt% at a volume ratio of 0.4-1:1, then adding 4-dimethylaminopyridine and N-hydroxysuccinimide in sequence, reacting in the dark at room temperature with stirring for 12-14 hours, then dialyzing in water with a MWCO1000D dialysis bag for 20-24 hours, and freeze-drying to obtain the white light responsive smart fiber.

5. The preparation method according to claim 1, characterized in that The preparation of step S4 mainly comprises the following steps: uniformly dispersing the white light-responsive smart fiber and the 980 nm laser-responsive smart fiber in water, ultrasonically treating the mixture for 10 minutes, adding a poloxamer reagent, stirring the mixture at 4° C. for 10 minutes to obtain fiber balls, and then adding indocyanine green, sodium nitroprusside, and doxorubicin, stirring the mixture at 4° C. in the dark for 2 to 3 hours to obtain a step-by-step laser-excited multi-active oxygen-releasing wound dressing; the mass ratio of the white light-responsive smart fiber, the 980 nm laser-responsive smart fiber, the poloxamer reagent, the indocyanine green, the sodium nitroprusside, and the doxorubicin is 20:10 to 20:450:1 to 3:2:

1.

6. A step-by-step laser-excited multi-active oxygen species-releasing wound dressing prepared by the preparation method according to any one of claims 1 to 5.

7. Use of the step-wise laser-excited multi-active oxygen species-releasing wound dressing prepared by the preparation method according to any one of claims 1 to 5 in the preparation of complex wound dressing.